Evidence-Based Complementary and Alternative Medicine

Bioactives and Traditional

Herbal Medicine for the
Treatment of Cardiovascular/
Cerebrovascular Diseases
Guest Editors: Joen-Rong Sheu, Pitchairaj Geraldine, and Mao-Hsiung Yen
Bioactives and Traditional Herbal
Medicine for the Treatment of
Cardiovascular/Cerebrovascular Diseases
Evidence-Based Complementary
and Alternative Medicine

Bioactives and Traditional Herbal

Medicine for the Treatment of
Cardiovascular/Cerebrovascular Diseases

Guest Editors: Joen-Rong Sheu, Pitchairaj Geraldine,

and Mao-Hsiung Yen
Copyright © 2014 Hindawi Publishing Corporation. All rights reserved.

This is a special issue published in “Evidence-Based Complementary and Alternative Medicine.” All articles are open access articles
distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
Editorial Board
Mahmood Abdulla, Malaysia Jen-Hwey Chiu, Taiwan Ching-Liang Hsieh, Taiwan
Jon Adams, Australia William C. S. Cho, Hong Kong Jing Hu, China
Zuraini Ahmad, Malaysia Jae Youl Cho, Korea Gan Siew Hua, Malaysia
Ulysses Albuquerque, Brazil Seung-Hun Cho, Republic of Korea Sheng-Teng Huang, Taiwan
Gianni Allais, Italy Chee Yan Choo, Malaysia Benny Tan Kwong Huat, Singapore
Terje Alraek, Norway Ryowon Choue, Republic of Korea Roman Huber, Germany
Souliman Amrani, Morocco Shuang-En Chuang, Taiwan Angelo Antonio Izzo, Italy
Akshay Anand, India Joo-Ho Chung, Republic of Korea Kong J., USA
Shrikant Anant, USA Edwin L. Cooper, USA Suresh Jadhav, India
Manuel Arroyo-Morales, Spain Gregory D. Cramer, USA Kanokwan Jarukamjorn, Thailand
Syed Asdaq, Saudi Arabia Meng Cui, China Yong Jiang, China
Seddigheh Asgary, Iran Roberto Cuman, Brazil Zheng L. Jiang, China
Hyunsu Bae, Republic of Korea Vincenzo De Feo, Italy Stefanie Joos, Germany
Lijun Bai, China Rocı́o Vázquez, Spain Sirajudeen K.N.S., Malaysia
Sandip K. Bandyopadhyay, India Martin Descarreaux, USA Z. Kain, USA
Sarang Bani, India Alexandra Deters, Germany Osamu Kanauchi, Japan
Vassya Bankova, Bulgaria Siva Durairajan, Hong Kong Wenyi Kang, China
Winfried Banzer, Germany Mohamed Eddouks, Morocco Dae Gill Kang, Republic of Korea
Vernon A. Barnes, USA Thomas Efferth, Germany Shao-Hsuan Kao, Taiwan
Samra Bashir, Pakistan Tobias Esch, Germany Krishna Kaphle, Nepal
Jairo Kenupp Bastos, Brazil Saeed Esmaeili-Mahani, Iran Kenji Kawakita, Japan
Sujit Basu, USA Nianping Feng, China Jong Yeol Kim, Republic of Korea
David Baxter, New Zealand Yibin Feng, Hong Kong Cheorl-Ho Kim, Republic of Korea
Andre-Michael Beer, Germany Josue Fernandez-Carnero, Spain Youn Chul Kim, Republic of Korea
Alvin J. Beitz, USA Juliano Ferreira, Brazil Yoshiyuki Kimura, Japan
Yong Boo, Republic of Korea Fabio Firenzuoli, Italy Joshua K. Ko, China
Francesca Borrelli, Italy Peter Fisher, UK Toshiaki Kogure, Japan
Gloria Brusotti, Italy W. F. Fong, Hong Kong Nandakumar Krishnadas, India
Ishfaq A. Bukhari, Pakistan Romain Forestier, France Yiu Wa Kwan, Hong Kong
Arndt Büssing, Germany Joel J. Gagnier, Canada Kuang Chi Lai, Taiwan
Rainer W. Bussmann, USA Jian-Li Gao, China Ching Lan, Taiwan
Raffaele Capasso, Italy Gabino Garrido, Chile Alfred Längler, Germany
Opher Caspi, Israel Muhammad Ghayur, Pakistan Lixing Lao, Hong Kong
Han Chae, Korea Anwarul Hassan Gilani, Pakistan Clara Bik-San Lau, Hong Kong
Shun-Wan Chan, Hong Kong Michael Goldstein, USA Jang-Hern Lee, Republic of Korea
Il-Moo Chang, Republic of Korea Mahabir P. Gupta, Panama Tat leang Lee, Singapore
Rajnish Chaturvedi, India Mitchell Haas, USA Myeong S. Lee, UK
Chun Tao Che, USA Svein Haavik, Norway Christian Lehmann, Canada
Hubiao Chen, Hong Kong Abid Hamid, India Marco Leonti, Italy
Jian-Guo Chen, China N. Hanazaki, Brazil Ping-Chung Leung, Hong Kong
Kevin Chen, USA K. B. Harikumar, India Lawrence Leung, Canada
Tzeng-Ji Chen, Taiwan Cory S. Harris, Canada Kwok Nam Leung, Hong Kong
Yunfei Chen, China Thierry Hennebelle, France Ping Li, China
Juei-Tang Cheng, Taiwan Seung-Heon Hong, Korea Min Li, China
Evan Paul Cherniack, USA Markus Horneber, Germany Man Li, China
ChunGuang Li, Australia Andrea Pieroni, Italy Mei Tian, China
Xiu-Min Li, USA Richard Pietras, USA Evelin Tiralongo, Australia
Shao Li, China Waris Qidwai, Pakistan S. C. Tjen-A-Looi, USA
Yong Hong Liao, China Xianqin Qu, Australia MichaThl Tomczyk, Poland
Sabina Lim, Korea Cassandra L. Quave, USA Yao Tong, Hong Kong
Bi-Fong Lin, Taiwan Roja Rahimi, Iran K. V. Trinh, Canada
Wen Chuan Lin, China Khalid Rahman, UK Karl Wah-Keung Tsim, Hong Kong
Christopher G. Lis, USA Cheppail Ramachandran, USA Volkan Tugcu, Turkey
Gerhard Litscher, Austria Gamal Ramadan, Egypt Yew-Min Tzeng, Taiwan
Ke Liu, China Ke Ren, USA Dawn M. Upchurch, USA
I-Min Liu, Taiwan Man Hee Rhee, Republic of Korea Maryna Van de Venter, South Africa
Gaofeng Liu, China Mee-Ra Rhyu, Republic of Korea Sandy van Vuuren, South Africa
Yijun Liu, USA José Luis Rı́os, Spain Alfredo Vannacci, Italy
Cun-Zhi Liu, China Paolo Roberti di Sarsina, Italy Mani Vasudevan, Malaysia
Gail B. Mahady, USA Bashar Saad, Palestinian Authority Carlo Ventura, Italy
Juraj Majtan, Slovakia Sumaira Sahreen, Pakistan Wagner Vilegas, Brazil
Subhash C. Mandal, India Omar Said, Israel Pradeep Visen, Canada
Jeanine Marnewick, South Africa Luis A. Salazar-Olivo, Mexico Aristo Vojdani, USA
Virginia S. Martino, Argentina Mohd. Zaki Salleh, Malaysia Y. Wang, USA
James H. McAuley, Australia Andreas Sandner-Kiesling, Austria Shu-Ming Wang, USA
Karin Meissner, USA Adair Santos, Brazil Chenchen Wang, USA
Andreas Michalsen, Germany G. Schmeda-Hirschmann, Chile Chong-Zhi Wang, USA
David Mischoulon, USA Andrew Scholey, Australia Kenji Watanabe, Japan
Syam Mohan, Malaysia Veronique Seidel, UK Jintanaporn Wattanathorn, Thailand
J. Molnar, Hungary Senthamil R. Selvan, USA Wolfgang Weidenhammer, Germany
Valério Monteiro-Neto, Brazil Tuhinadri Sen, India Jenny M. Wilkinson, Australia
H.-I. Moon, Republic of Korea Hongcai Shang, China Darren Williams, Republic of Korea
Albert Moraska, USA Karen J. Sherman, USA Haruki Yamada, Japan
Mark Moss, UK Ronald Sherman, USA Nobuo Yamaguchi, Japan
Yoshiharu Motoo, Japan Kuniyoshi Shimizu, Japan Yong-Qing Yang, China
Frauke Musial, Germany Kan Shimpo, Japan Junqing Yang, China
MinKyun Na, Republic of Korea Byung-Cheul Shin, Korea Ling Yang, China
Richard L. Nahin, USA Yukihiro Shoyama, Japan Eun Jin Yang, Republic of Korea
Vitaly Napadow, USA Chang Gue Son, Korea Xiufen Yang, China
F. R. F. Nascimento, Brazil Rachid Soulimani, France Ken Yasukawa, Japan
S. Nayak, Trinidad And Tobago Didier Stien, France Min H. Ye, China
Isabella Neri, Italy Shan-Yu Su, Taiwan M. Yoon, Republic of Korea
Télesphore Nguelefack, Cameroon Mohd Roslan Sulaiman, Malaysia Jie Yu, China
Martin Offenbacher, Germany Venil N. Sumantran, India Jin-Lan Zhang, China
Ki-Wan Oh, Republic of Korea John R. S. Tabuti, Uganda Zunjian Zhang, China
Y. Ohta, Japan Toku Takahashi, USA Wei-bo Zhang, China
Olumayokun A. Olajide, UK Rabih Talhouk, Lebanon Hong Q. Zhang, Hong Kong
Thomas Ostermann, Germany Wen-Fu Tang, China Boli Zhang, China
Stacey A. Page, Canada Yuping Tang, China Ruixin Zhang, USA
Tai-Long Pan, Taiwan Lay Kek Teh, Malaysia Hong Zhang, Sweden
Bhushan Patwardhan, India Mayank Thakur, India Haibo Zhu, China
Berit Smestad Paulsen, Norway Menaka C. Thounaojam, India
Contents
Bioactives and Traditional Herbal Medicine for the Treatment of Cardiovascular/Cerebrovascular
Diseases, Joen-Rong Sheu, Pitchairaj Geraldine, and Mao-Hsiung Yen
Volume 2014, Article ID 495323, 2 pages

Modulation of Cardiac Autonomic Dysfunction in Ischemic Stroke following Ayurveda (Indian System
of Medicine) Treatment, Sriranjini Sitaram Jaideep, Dindagur Nagaraja, Pramod Kumar Pal, D. Sudhakara,
and Sathyaprabha N. Talakad
Volume 2014, Article ID 634695, 8 pages

Guanxinkang Decoction Exerts Its Antiatherosclerotic Effect Partly through Inhibiting the
Endoplasmic Reticulum Stress, Hao Wang, Zhi-Min Zheng, and Bu-Lang Gao
Volume 2014, Article ID 465640, 8 pages

Qingkailing Suppresses the Activation of BV2 Microglial Cells by Inhibiting

Hypoxia/Reoxygenation-Induced Inflammatory Responses, Lulu Mana, Shan Wang, Haiyan Zhu,
Yanwei Xing, Lixia Lou, Aiming Wu, Bin Dong, Yikun Sun, Shuo Yang, Lin Wang, and Yonghong Gao
Volume 2014, Article ID 696218, 8 pages

Time-Course of the Effects of QSYQ in Promoting Heart Function in Ameroid Constrictor-Induced

Myocardial Ischemia Pigs, Qi Qiu, Yang Lin, Cheng Xiao, Chun Li, Yong Wang, Kexu Yang, Wei Suo, Yu Li,
Wenjing Chuo, Yongxiang Wei, and Wei Wang
Volume 2014, Article ID 571076, 13 pages

Neuroprotective Effect of a Formula, Moschus Combined with Borneolum Synthcticum, from

Traditional Chinese Medicine on Ischemia Stroke in Rats, Xin-hua Xia, Qiang Li, and Mei Liu
Volume 2014, Article ID 157938, 10 pages

Anti-Inflammatory Effects of the Chinese Herbal Formula Sini Tang in Myocardial Infarction Rats,
Jiangang Liu, Karoline Peter, Dazhuo Shi, Lei Zhang, Guoju Dong, Dawu Zhang, Heimo Breiteneder,
Rudolf Bauer, Johannes Jakowitsch, and Yan Ma
Volume 2014, Article ID 309378, 10 pages

Chinese Herbal Medicine for Aspirin Resistance: A Systematic Review of Randomized Controlled
Trials, Ai-ju Liu, Hui-qin Li, Ji-huang Li, Yuan-yuan Wang, Dong Chen, Yan Wang, and Guo-qing Zheng
Volume 2014, Article ID 890950, 16 pages

Antiplatelet Activity of Morus alba Leaves Extract, Mediated via Inhibiting Granule Secretion and
Blocking the Phosphorylation of Extracellular-Signal-Regulated Kinase and Akt, Dong-Seon Kim,
Hyun Dong Ji, Man Hee Rhee, Yoon-Young Sung, Won-Kyung Yang, Seung Hyung Kim,
and Ho-Kyoung Kim
Volume 2014, Article ID 639548, 11 pages

Antihypercholesterolemic and Antioxidative Potential of an Extract of the Plant, Piper betle, and Its
Active Constituent, Eugenol, in Triton WR-1339-Induced Hypercholesterolemia in Experimental Rats,
Karuppasamy Venkadeswaran, Arumugam Ramachandran Muralidharan, Thangaraj Annadurai,
Vasanthakumar Vasantha Ruban, Mahalingam Sundararajan, Ramalingam Anandhi, Philip A. Thomas,
and Pitchairaj Geraldine
Volume 2014, Article ID 478973, 11 pages
Andrographolide, a Novel NF-𝜅B Inhibitor, Induces Vascular Smooth Muscle Cell Apoptosis via a
Ceramide-p47phox-ROS Signaling Cascade, Yu-Ying Chen, Ming-Jen Hsu, Joen-Rong Sheu, Lin-Wen Lee,
and Cheng-Ying Hsieh
Volume 2013, Article ID 821813, 10 pages

Evodiamine Induces Transient Receptor Potential Vanilloid-1-Mediated Protective Autophagy in

U87-MG Astrocytes, Ann-Jeng Liu, Sheng-Hao Wang, Sz-Ying Hou, Chien-Ju Lin, Wen-Ta Chiu,
Sheng-Huang Hsiao, Thay-Hsiung Chen, and Chwen-Ming Shih
Volume 2013, Article ID 354840, 9 pages

Exploratory Pharmacokinetics of Geniposide in Rat Model of Cerebral Ischemia Orally Administered

with or without Baicalin and/or Berberine, Linmei Pan, Wenzhe Wang, Feiyan Shi, Jing Zhou,
Meng Zhang, Huaxu Zhu, and Mingfei Zeng
Volume 2013, Article ID 349531, 7 pages

Inhibitive Effects of Mulberry Leaf-Related Extracts on Cell Adhesion and Inflammatory Response in
Human Aortic Endothelial Cells, P.-Y. Chao, K.-H. Lin, C.-C. Chiu, Y.-Y. Yang, M.-Y. Huang,
and C.-M. Yang
Volume 2013, Article ID 267217, 14 pages

Okadaic Acid, a Bioactive Fatty Acid from Halichondria okadai, Stimulates Lipolysis in Rat Adipocytes:
The Pivotal Role of Perilipin Translocation, Nen-Chung Chang, Aming Chor-Ming Lin, Cheng-Chen Hsu,
Jung-Sheng Liu, Leo Tsui, Chien-Yuan Chen, Thanasekaran Jayakumar, and Tsorng-Harn Fong
Volume 2013, Article ID 545739, 10 pages

Effects and Mechanisms of Chinese Herbal Medicine in Ameliorating Myocardial Ischemia-Reperfusion

Injury, Qing Liu, Jiqiang Li, Jing Wang, Jianping Li, Joseph S. Janicki, and Daping Fan
Volume 2013, Article ID 925625, 14 pages

Hinokitiol, a Natural Tropolone Derivative, Offers Neuroprotection from Thromboembolic Stroke In

Vivo, Thanasekaran Jayakumar, Wen-Hsien Hsu, Ting-Lin Yen, Jun-Yun Luo, Yu-Cheng Kuo,
Tsorng-Harn Fong, and Joen-Rong Sheu
Volume 2013, Article ID 840487, 8 pages
Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2014, Article ID 495323, 2 pages
http://dx.doi.org/10.1155/2014/495323

Editorial
Bioactives and Traditional Herbal Medicine for
the Treatment of Cardiovascular/Cerebrovascular Diseases

Joen-Rong Sheu,1 Pitchairaj Geraldine,2 and Mao-Hsiung Yen3

1
Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 110, Taiwan
2
Department of Animal Science, Bharathidasan University, Tiruchirappalli, Tamil Nadu 620 024, India
3
Department of Pharmacology, National Defense Medical Center, Taipei, Taiwan

Correspondence should be addressed to Joen-Rong Sheu; [email protected]

Received 11 June 2014; Accepted 11 June 2014; Published 16 June 2014

Copyright © 2014 Joen-Rong Sheu et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The cardiovascular diseases (CVDs) have been the major For instance, Chinese herbal formula Sini Tang (SNT), a
cause of morbidity and mortality in developed countries decoction that consisted of four herbs: Aconitum carmichaelii,
over the last several decades, and developing countries are Cinnamomum cassia, Zingiber officinale, and Glycyrrhiza
rapidly catching up with this epidemic disease. The under- uralensis, was reported to improve cardiac function after
lying pathology is atheromatous vascular disease, resulting myocardial infarction (MI) in rats. Similarly, another paper
in coronary artery disease (CAD), cerebrovascular disease, in this issue reports the neuroprotective effect of formula
peripheral vascular disease, and the subsequent development moschus combined with borneolum synthcticum from tra-
of heart failure and cardiac arrhythmias. There is extensive ditional Chinese medicine on ischemia stroke in rats. In this
evidence to show that drug treatment of conventional risk study it was found that this formula significantly ameliorates
factors is effective in reducing cardiovascular events. More neurobehavioral disturbances, shrinks relative infarct size,
effective treatment of CVD with various classes of antihyper- rescues neural dysfunction, and prevents neuron cells from
tensive drugs has been associated with greater benefits, but apoptosis caused by cerebral ischemia or reperfusion to
some recent studies suggest we may be reaching the optimal relieve brain damage.
level of treated blood pressure in some patient groups. The antiatherosclerotic effect of Guanxinkang (GXK)
Apart from the treatment of cardiovascular risk factors with decoction on the apoptosis, mitochondrial membrane poten-
pharmacological agents and the use of antithrombotic drugs, tial (MMP), and endoplasmic reticulum stress (ERS) of
there is growing awareness of the role of dietary factors human umbilical vein endothelial cells (HUVEC) pretreated
and herbal medicines in the prevention of CVD and the with homocysteinemia was presented in this special issue.
possibility of their use in treatment. In this special issue on A review paper in this special issue presents the effects and
bioactives and traditional herbal medicine for the treatment mechanisms of Chinese herbal medicine in ameliorating
of cardiovascular/cerebrovascular diseases, we called for myocardial ischemia-reperfusion injury. Moreover, our own
limited research and review papers on such subjects. paper describes the neuroprotective effect and mechanisms of
In spite of the major advances of neuroprotective thera- hinokitiol, a tropolone related compound found in the heart-
peutic approaches for treating ischemic stroke over the last wood cupressaceous plants, in rats against middle cerebral
decade, stroke is still a serious problem for which effective artery occlusion- (MCAO-) induced thromboembolic stroke.
drug therapy is not yet available. In the search for neu- A therapeutic effect of QSYQ, a drug commonly used
roprotective agents from natural sources, a number of plant to treat heart dysfunction in clinical practice in China, has
extracts and several natural products were isolated and been evaluated against pig myocardial ischemia (MI). This
reported to provide neuroprotection against ischemic stroke. paper addressed the issue that therapeutic QSYQ adminis-
A few papers in this special issue address the neuroprotective tration regulates vasoactive factors to improve myocardial
effects of Chinese herbal medicine and natural compounds. oxygen supply, reduce myocardial injury, improve cardiac
2 Evidence-Based Complementary and Alternative Medicine

function, and inhibit myocardial apoptosis via decreasing

the level of TNF-𝛼 and active caspase-3. Subsequently, this
special issue described the antihypercholesterolemic and
antioxidative properties of an ethanolic extract of Piper
betle and of its active constituent, eugenol, in experimental
hypercholesterolemia in Wistar rats. This paper provides
well understanding of the fact that the hypercholesterolemia-
ameliorating effect is better defined in eugenol-treated rats
than in Piper betle extract-treated rats, and it was almost as
effective as that of the standard lipid-lowering drug, lovas-
tatin. A paper in this special issue describes the antiplatelet
activity of Morus alba leaves extract mediated via inhibiting
granule secretion and blocking the phosphorylation of extra-
cellular signal-regulated kinase and Akt. Finally, a clinical
study about the effect of Ayurveda therapies on the cardiac
autonomic dysfunction was offered in this special issue.
This is the first study on positive modulation of cardiac
autonomic activity after adjuvant Ayurveda treatment in
ischemic stroke. We anticipate that this special issue presents
innovative knowledge to increase the therapeutic value of
herbal and/or Chinese medicines for treatment or prevention
of cardiovascular and ischemia-reperfusion injury-related
disorders.
Joen-Rong Sheu
Pitchairaj Geraldine
Mao-Hsiung Yen
Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2014, Article ID 634695, 8 pages
http://dx.doi.org/10.1155/2014/634695

Research Article
Modulation of Cardiac Autonomic Dysfunction in
Ischemic Stroke following Ayurveda (Indian System of
Medicine) Treatment

Sriranjini Sitaram Jaideep,1,2 Dindagur Nagaraja,3 Pramod Kumar Pal,3

D. Sudhakara,4 and Sathyaprabha N. Talakad1
1
Department of Neurophysiology, National Institute of Mental Health & Neurosciences (NIMHANS), Hosur Road,
Bangalore 560029, India
2
Clinical Research Group, School of Health Sciences, Institute of Transdisciplinary Health Sciences and Technology (FRLHT-ITDHST),
Bangalore 560106, India
3
Department of Neurology, National Institute of Mental Health & Neurosciences (NIMHANS), Hosur Road, Bangalore 560029, India
4
Advanced Center for Ayurveda in Mental Health and Neurosciences, National Institute of
Mental Health & Neurosciences (NIMHANS), Hosur Road, Bangalore 560029, India

Correspondence should be addressed to Sathyaprabha N. Talakad; [email protected]

Received 4 December 2013; Accepted 2 April 2014; Published 26 May 2014

Academic Editor: Joen-Rong Sheu

Copyright © 2014 Sriranjini Sitaram Jaideep et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.

Objectives. Cardiac autonomic dysfunction in stroke has implications on morbidity and mortality. Ayurveda (Indian system of
medicine) describes stroke as pakshaghata. We intended to study the effect of Ayurveda therapies on the cardiac autonomic
dysfunction. Methods. Fifty patients of ischemic stroke (middle cerebral artery territory) (mean age 39.26 ± 9.88 years; male 43,
female 7) were recruited within one month of ictus. All patients received standard allopathic medications as advised by neurologist.
In addition, patients were randomized to receive physiotherapy (Group I) or Ayurveda treatment (Group II) for 14 days. Continuous
electrocardiogram and finger arterial pressure were recorded for 15 min before and after treatments and analyzed offline to obtain
heart rate and blood pressure variability and baroreflex sensitivity (BRS). Results were analysed by RMANOVA. Results. Patients
in Group II showed statistically significant improvement in cardiac autonomic parameters. The standard deviation of normal to
normal intervals,and total and low frequency powers were significantly enhanced (𝐹 = 8.16, 𝑃 = 0.007, 𝐹 = 9.73, 𝑃 = 0.004,
𝐹 = 13.51, and 𝑃 = 0.001, resp.). The BRS too increased following the treatment period (𝐹 = 10.129, 𝑃 = 0.004). Conclusions.
The current study is the first to report a positive modulation of cardiac autonomic activity after adjuvant Ayurveda treatment in
ischemic stroke. Further long term studies are warranted.

1. Introduction (BPV), and baroreflex sensitivity (BRS) have been identified

as subtle markers of such cardiac dysregulation, persisting in
Stroke leads to dysfunction of the autonomic nervous system, the chronic state and associated with poor prognosis [4–8].
including cardiovascular, gastrointestinal, genitourinary, Effective strategies to modulate cardiac autonomic dysfunc-
thermoregulatory, and sudomotor systems [1]. The cardiovas- tion may hence provide better prognosis and survival. The
cular regulation is affected due to aberrant activity in the possibility of enhancing the baroreflex as a therapeutic
central areas regulating these functions or their pathways strategy in acute stroke has been proposed [9]. Though 𝛽
[2]. This has been associated with fatal complications [3]. blockers can influence baroreflex by vagal modulation [10],
Heart rate variability (HRV), blood pressure variability they are associated with potential harm [11, 12].
2 Evidence-Based Complementary and Alternative Medicine

From an Ayurveda perspective, stroke is recognized as Ksheerabala 101 orally, 5 drops BD with 15 mL warm water.
pakshaghata (hemiplegia), a disease attributed to an aber- Refer Table 3 for details of medicine used.
ration of vatadosha (a physiologic entity). Cardiac dysreg-
ulation is understood as a secondary consequence of this 2.3. Assessment Criteria. Patients were evaluated with cardiac
process [13]. The diverse treatments advocated in Ayurveda autonomic function tests which included heart rate variability
for this disease primarily harmonize the aberrant physiology. (HRV), blood pressure variability (BPV), and baroreflex
Hence, we hypothesized that the Ayurveda therapy may also sensitivity (BRS), before and after the treatment. The resting
modulate the cardiac dysregulation. The current study aimed heart rate (HR) and blood pressure (BP) recordings were
to evaluate the effect of adjuvant Ayurveda therapies on carried out in the autonomic laboratory, Department of
cardiac dysregulation in ischemic stroke using HRV, BPV, and Neurophysiology, NIMHANS under standardized conditions
BRS parameters. [14, 15]. Patients were advised to abstain from alcohol and
nicotine for 24 hours before evaluation. The tests were
2. Patients and Methods performed in a silent room maintained at a temperature of
22–26∘ C, between 8 am and 11 am. Patients were advised to
The present study was conducted in the Departments of Neu- have light breakfast two hours prior to the tests and empty
rophysiology, Neurology and Advanced Center for Ayurvedic bowel and bladder before the tests. They were familiarized
Research in Mental health and Neurosciences, National with the laboratory settings and briefed about the tests.
Institute of Mental Health and Neuro Sciences (NIMHANS), Recordings were done after 30 minutes of supine rest.
Bangalore. The study was a comparative randomized clinical
evaluation, approved by the Institute Ethics Committee, 2.3.1. Resting HRV. Lead II electrocardiogram (ECG) and
Advanced Center for Ayurvedic Research in Mental health breathing signals were conveyed through analog digital con-
and Neurosciences, NIMHANS. Patients and/or relatives verter (Power lab, 16 channels data acquisition system, AD
were explained about the nature and design of the study and Instruments, Australia) with a sampling rate of 1024 Hz. MLS
informed consent was obtained. 310 Module was used to analyze the different HRV measures.
It was ensured that patients were breathing at normal respi-
2.1. Inclusion and Exclusion Criteria. Patients diagnosed with ratory rate of 12–15 breaths/min by recording the respiratory
ischemic stroke by a neurologist and as pakshaghata (hemi- movements using stethograph. The data were stored in PC
plegia) by an Ayurveda physician by thorough clinical exam- and analyzed offline using an automatic programme that
ination and imaging were screened from casualty section, allowed visual checking of the raw ECG and breathing signals.
stroke unit, and neurorehabilitation section, NIMHANS. Fifteen-minute basal recordings were stored and later, a 5-
Patients were included if aged between 20 and 60 years, minute artifact/ectopic free segment was analyzed to obtain
of either gender; with first time ischemic stroke in middle time domain and frequency domain parameters of HRV as
cerebral artery (MCA) territory diagnosed by history, clinical per the guidelines of task force report [16].
examination and CT scan/MRI of brain, between 10 and 30
2.3.2. Resting BPV and Spontaneous BRS. Blood pressure was
days of stroke, with stable neurological status following the
recorded using the Finometer (Finapres Medical Systems
necessary acute phase treatment after stroke, motor weakness
(FMS), The Netherlands). Following height correction, phys-
of at least one limb of MRC Grade 0–3 and fulfilling the cri-
iological and return to flow calibrations, the finger arterial
teria to undergo Ayurveda therapies. Patients were excluded
pressure was recorded continuously for 15 min. The Physical
if sensorium was altered or severely aphasic to comprehend
was then turned off during the recording. The recorded data
simple instructions, associated with comorbidities such as
was downloaded offline using Finolink software provided
uncontrolled hypertension, diabetes, or other systemic dis-
by FMS and stored in a PC. All recordings were scanned
eases, pregnant or lactating. All patients were admitted in
and 5-minute artifact/ectopic free segment was analyzed
the Advanced Ayurvedic Research Institute for Mental Health
using Nevrokard cardiovascular parameter analysis (CVPA)
and Neurosciences, NIMHANS, and randomized to receive
software (version 2.1.0) to obtain the time domain and
the following for a period of 15 days.
frequency domain parameters of blood pressure variability.
Baroreflex sensitivity (BRS) values were derived by sequence
2.2. Intervention. Group I received conventional treatment and spectral methods using the same software as per guide-
including antiplatelet agents or anticoagulants, antihyperten- lines [17, 18].
sives, and medications for diabetes mellitus hyperlipidemia
along with physiotherapy and speech therapy as required. 2.4. Control Data. To assess the presence and extent of car-
Group II received conventional treatment and speech ther- diac autonomic dysfunction in the ischemic stroke patients,
apy as in Group I. In addition they received adjuvant similar ECG data was obtained from 30 clinically healthy
Ayurveda treatment consisting of (a) external therapies subjects.
such as Abhyanga (methodical massage) with Niramisha
mahamasha taila and Bhashpa svedana (steam therapy) from 2.5. Description of Ayurveda Therapies
day 1 to day 14, Matra basti with 60 mL of Balaswagandhadi
taila from day 8–14 and (b) internal medication of Ashtavarga 2.5.1. Abhyanga (Therapeutic Massage). Abhyanga was per-
kashaya orally, 15 mL TID with 15 mL warm water and formed during morning hours (8 am–12 noon). Patient was
Evidence-Based Complementary and Alternative Medicine 3

Table 1: Demographic details of the patients.

Variable Group I (𝑛 = 25) Group II (𝑛 = 25) 𝑃 value

Age (in years) 40.12 ± 9.31 38.40 ± 10.50 0.543
Gender (M : F) 20 : 5 23 : 2 0.111
Height (cm) 168.08 ± 2.04 170.84 ± 1.43 0.274
Weight (kg) 66.72 ± 2.38 67.01 ± 2.51 0.936
BMI (kg/m2 ) 24.04 ± 0.78 23.33 ± 0.76 0.519
Duration of illness (days) 14.96 ± 4.16 17.20 ± 5.74 0.121
Smoking 14 17 0.718
Alcohol 15 17 0.860
Hypertension 2 3 1.000
Diabetics 2 2 1.000
Right side infarct 10 8 —
Left side infarct 15 17 —
M: male; F: female; BMI: body mass index; values expressed as mean ± SD.

advised to empty bowel and bladder before starting the groups of patients. Independent sample “𝑡” test was carried
therapy. Patient was made to lie in supine posture over out to compare (a) the HRV variables between pakshaghata
the Droni (Abhyanga table). Requisite quantity of Niramisha patients and healthy subjects and (b) the baseline character-
Mahamasha taila (medicated oil) was warmed on a water istics of the two groups of patients. 2 × 2 repeated measures
bath and applied on the patient’s body. The entire body was analysis of variance (RMANOVA) was used to find the
then massaged by 2 masseurs with uniform pressure using significance of study parameters within and between the two
long strokes and in circular motion, as required [19]. groups of patients. The two independent factors included in
the analysis were (1) treatment (between-subjects factor) with
2.5.2. Svedana (Sudation). Bhashpa sveda variety of Svedana two levels: conventional treatment with physiotherapy and
was done. The patient was made to sit in a Bhashpa svedana conventional treatment with Ayurveda and (2) time (within-
yantra (customized wooden box) into which warm vapors subjects factor) with two levels: baseline and after 2 weeks.
were passed. This induced perspiration in the patient. This The main effects when found to be significant were further
was continued for 10–15 min depending on the patients’ tested for the direction of differences by using pairwise
tolerance level [19]. comparisons adjusted by Bonferroni’s method. Values are
represented as mean ± SEM and significance was considered
2.5.3. Basti (Therapeutic Enema). Matra basti (therapeutic if 𝑃 < 0.05.
oil enema) variety of basti was used. The procedure was
performed after noon (between 12 noon and 3 pm). Patients 3. Results
were advised to take light food and empty bowel and bladder
before starting the therapy. Local Abhyanga and svedana was 3.1. Demographic Details. Fifty patients satisfying the study
performed around the gluteal region. Following this, the criteria were recruited and allotted to receive treatment.
patient was advised to lie down in the left lateral position on Baseline evaluation of the demographic details of patients
Abhyanga table with left led stretched out and the right leg indicated the homogeneity in age, gender, height, weight,
flexed at the knee and hip joints. The patient’s left arm was body mass index, risk factors predisposing to stroke, duration
placed beneath his head. A pinch of Saindhava lavana (black of illness, and side of infarct in both the groups (Table 1). Two
salt) was mixed in 60 mL of lukewarm Balaswagandhadi taila patients in Group II were unable to stay in the hospital for
and drawn into a syringe. A rubber catheter was attached to the entire duration of treatment and hence dropped from the
enema syringe and its tip was lubricated with the same oil. study. HRV could not be analyzed in 12 patients (Group I-8,
The catheter was then introduced into the anus (up to 4 inches Group II-4) due to the presence of artifacts in the ECG. BPV
length). Patient was advised to breathe slowly and deeply and and BRS analysis was done in a subsample of 24 cases (Group
the oil was steadily pushed into the rectum. The patient was I-11, Group II-13).
then made to lie supinely while his legs were raised at the hips
for 3-4 times and the gluteal region was massaged gently for 3.2. Comparison of Ischemic Stroke Patients with Healthy Con-
few seconds. Patient was advised to retain the enema as long trols. At baseline, the stroke patients demonstrated signifi-
as possible [19]. cantly lower mean NN and higher heart rate when compared
with healthy controls. In addition, the time domain measures
2.6. Statistical Methods. Statistical analysis was performed SDNN and RMSSD and frequency domain measures TP, LF,
using SPSS. Chi-square and Fisher Exact test was used to find HF, and HFnu were significantly reduced and the LF/HF ratio
the significance of categorical data comparison between two was significantly higher (Table 2).
4 Evidence-Based Complementary and Alternative Medicine

Table 2: Comparison of heart rate variability (HRV) measures between healthy controls and ischemic stroke patients.

HRV variables Controls (𝑛 = 30) Patients (𝑛 = 38) 𝑃 value

NN interval (ms) 869.96 ± 23.15 795.67 ± 16.22 0.009∗∗
HR (bpm) 70.44 ± 1.88 76.08 ± 1.61 0.026∗
SDNN (ms) 48.01 ± 3.42 36.47 ± 2.25 0.004∗∗
RMSSD (ms) 38.06 ± 4.13 28.00 ± 2.80 0.027∗
TP (ms2 ) 2489.00 ± 337.06 1286.63 ± 148.50 0.000∗∗∗
LF (ms2 ) 527.16 ± 62.53 290.11 ± 34.87 0.001∗∗
LFnu 44.92 ± 2.71 49.01 ± 3.13 0.481
HF (ms2 ) 812.62 ± 196.77 364.71 ± 90.81 0.005∗∗
HFnu 49.18 ± 2.61 41.87 ± 3.30 0.042∗
Sympathovagal balance 1.09 ± 0.13 1.66 ± 0.20 0.043∗
NN: R-R interval (ms); HR: heart rate (bpm: beats per minute); SDNN: standard deviation of NN intervals (ms); RMSSD: square root of the mean of the sum
of squares of differences between adjacent NN intervals (ms); TP: total power (ms2 ); LF: low frequency power (ms2 ); HF: high frequency power (ms2 ); nu:
normalized units; SVB: sympathovagal balance; values expressed as mean ± SEM; ∗ 𝑃 < 0.05, ∗∗ 𝑃 < 0.01, ∗∗∗ 𝑃 < 0.001.

3.3. Effect of Treatments on HRV Measures. Significant treat- To the best of our knowledge, this is the first study
ment effect over time was observed for SDNN (𝐹 = 8.16, 𝑃 = to evaluate the effect of adjuvant Ayurveda therapies on
0.007), TP (𝐹 = 9.73, 𝑃 = 0.004), and LF power (𝐹 = 13.51, cardiac autonomic dysfunction in ischemic stroke. The study
𝑃 = 0.001). Adjusted pairwise comparisons for time showed has used a whole system treatment protocol, adapted from
improvement in these parameters in the group treated Ayurveda classics keeping in mind convenience for clinical
with adjuvant Ayurveda therapies. There was also a signi- application. This is a positive deviation from single drug clini-
ficant group effect for mean NN (𝐹 = 6.41, 𝑃 = 0.016), cal trial modules that may be ineffective to capture the essence
HR (𝐹 = 5.88, 𝑃 = 0.021), and RMSSD (𝐹 = 5.87, 𝑃 = of Ayurveda treatments [20]. However, we were limited
0.021) and adjusted pairwise comparison for group was signi- by factors including short follow-up period that prevented
ficant for RMSSD in the group treated with adjuvant evaluation of lasting effect of the treatment. Also, HRV could
Ayurveda therapies at 2 weeks (𝑃 = 0.025). The LF/HF not be analyzed in few patients (𝑛 = 12) due to the presence
ratio showed marginal group effect (𝐹 = 3.34, 𝑃 = 0.076). of artifacts. BPV and BRS analysis was done in a subsample
Pairwise adjustment for group comparisons indicated margi- of 24 cases. However, compared to the total sample, the
nally lesser LF/HF ratio (𝑃 = 0.067) in the patients treated subsample analysis was not significantly different indicating
with adjuvant Ayurveda therapies at 2 weeks. Though there that subsample is representative of the total sample.
was increase in LF power, no significant change in LFnu or In the current study, when compared with healthy con-
LF/HF ratio in the group treated with adjuvant Ayurveda trols, the patients with ischemic stroke demonstrated signif-
therapies (Figures 1(a)–1(f)). icantly lower mean NN and higher heart rate, time domain
measures (SDNN, RMSSD) and frequency domain measures
3.4. Effect of Treatments on BPV and BRS Measures. The (TP, LF, HF, and HFnu) were reduced, and the LF/HF ratio
SBP, DBP, and MBP showed a trend for decrease in both was significantly higher indicating definite cardiac autonomic
the groups but it was not statistically significant. We also did dysfunction. These findings of altered autonomic activity
not find any significant changes in the time domain and fre- with increased sympathetic activation correlate with earlier
quency domain measures of BPV following treatment in studies on ischemic stroke [6, 14, 21, 22]. The aberrant neu-
both the groups (data not shown). Among the sequence and rocardiac regulation persists even in the postacute phase [23]
spectral parameters of spontaneous baroreflex, the Up BRS and abnormal heart rate dynamics has also been associated
was significantly enhanced in patients treated with adjuvant with poststroke mortality [24].
Ayurveda therapies after pairwise adjustment for time and Following intervention, it was observed that the patients
group at 2 weeks (Figure 1(g)). receiving adjuvant Ayurveda therapies demonstrated signif-
icant enhancement of time domain (SDNN) and frequency
4. Discussion domain parameters (TP, LF power) of HRV. There was also
a significant group effect for mean NN and RMSSD and
The science of Ayurveda has evolved by in-depth observation adjusted pairwise comparison for group was significant in the
and experience of scholars. However, the potential benefits of group treated with adjuvant Ayurveda therapies. This com-
this science need to be evaluated with objectivity. The current bined with a trend for increase of the HF power and a margi-
study demonstrated that cardiac autonomic functions can be nally lesser LF/HF in this group. Though there was increase
used as a marker to detect one facet of aberrant physiology in in LF power, no significant change was observed in LFnu
pakshaghata (hemiplegia) and that a whole system Ayurveda or LF/HF ratio. Among the BPV measures, the SBP, DBP,
treatment protocol as used here was able to modulate the and MBP showed a trend for decrease in both the groups
same. but it was not statistically significant. We also did not
Evidence-Based Complementary and Alternative Medicine 5

NN (RR) interval Heart rate SDNN RMSSD

1200 100 † 50 ∗ 40 †
†
80 40
900 30
60 30

(bpm)
(ms)

(ms)
(ms)
600 20
40 20
300 10
20 10

0 0 0 0
Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post
Group I Group II Group I Group II Group I Group II Group I Group II
(a) (b) (c) (d)
Total power LF power BRS (up sequence)
2500 800 15 ∗∗
∗∗
2000 ∗∗
600

(ms/mmHg)
10
1500
(ms)2
(ms)

400
1000
5
500 200

0 0 0
Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post
Group I Group II Group I Group II Group I Group II
(e) (f) (g)

Figure 1: Effect of intervention on heart rate variability measures and baroreflex sensitivity (a) NN—R-R interval (ms); (b) HR—heart rate
(bpm—beats per minute); (c) SDNN—standard deviation of NN intervals (ms); (d) RMSSD—square root of the mean of the sum of squares
of differences between adjacent NN intervals (ms); (e) TP—total power (ms2 ); (f) LF—low frequency power (ms2 ); (g) BRS—baroreflex
sensitivity (ms/mm Hg); values expressed as Mean±SEM; ∗ 𝑃 < 0.05, ∗∗ 𝑃 < 0.01, pre versus post, within group comparison; † 𝑃 < 0.05
Group I versus Group II at 2 weeks.

find any significant changes in the other BPV parameters adjuvant Ayurveda therapies indicates a positive cardiac
following treatment in both the groups. Among the sequence autonomic regulation amongst others.
and spectral parameters of spontaneous baroreflex, the Up In the present study, we used a whole system approach
BRS was significantly enhanced and the 𝛼HF also showed combining different modes of treatment as mentioned in
a trend for increase following adjuvant Ayurveda ther- Ayurveda. These treatments are indicated for neurological
apies. illnesses generally and pakshaghata (hemiplegia) specifically.
The time domain measure SDNN and frequency domain Most of the herbs used as internal medication in the
measure of TP are robust indicators of overall variability and current study have been studied for their antioxidant and
reflect vagal activation. RMSSD too mirrors the parasympa- neuroprotective activity including Bala (Sida cordifolia) in
thetic influence on the HRV [16, 25]. The physiological impli- Ksheerabala 101 [33, 34], Devadaru (Cedrus deodara) [35],
cations of LF power have been conflicting. Earlier studies Lashuna (Allium cepa) [36, 37], Shunti (Zingiber officinale)
have demonstrated LF power to be an indicator of sympa- [38], and so forth. The release of free radicals in the brain is
thetic tone largely [16, 26, 27] while a parasympathetic con- well established in acute and chronic stages of ischemic stroke
tribution is also hypothesized [28]. Recent work in this area, [39, 40] and antioxidant therapy is seen to be beneficial in late
however, suggests that the LF component of HRV reflects the treatment of ischemic stroke too [41]. The therapeutic pro-
baroreflex gain and hence interventions that increase the LF cedures included Abhyanga (methodical massage), Svedana
power may modulate the baroreflexes in turn affecting the (sudation), and Basti (colonic administration. Different mas-
cardiac autonomic outflow [29–31]. It is also debated that sage techniques are known to enhance vagal activity [42, 43].
reduced LF power can exist despite clinically evident sym- A study on oriental massage demonstrated an improved pro-
pathetic overactivity and this can be attributed to a reduced file of cardiovascular autonomic regulation [44]. The benefits
baroreflex modulation amongst others [32]. In our study too, of svedana (sudation) may be akin to that of thermal therapy
it was observed that the LF power of the ischemic stroke in the saunas. It is observed that repeated sauna improves
patients was significantly lower than the healthy controls at cardiac function and hemodynamics [45]. Parasympathetic
baseline, indicating an initially reduced baroreflex function. activity is enhanced and sympathetic activity also reduced
However, the subsequent increase in most of the HRV on HRV analysis following application of heat and steam
components compounded by the increased BRS following generating (HSG) sheets [46, 47].
6 Evidence-Based Complementary and Alternative Medicine

Table 3: Medicines used. Table 3: Continued.

Ingredients Ingredients
Sanskrit name Latin name Sanskrit name Latin name
(1) Ashtavarga kashaya [48] Kantakari Solanum indicum
Bala Sida cordifolia Gokshura Tribulus terrestris
Salaprni Desmodium gangeticum
Sahachara Barleria prionitis
Prishnaparni Uraria picta
Eranda Ricinus communis
Jala Water
Shunti Zingiber officinale
Tila taila Gingelly oil
Rasna Vanda roxburghii Ksheera Milk
Suradruma Cedrus deodara Ashwagandha Withania somnifera
Sinduvara Vitex negundo Vidari Ipomea digitata
Lasuna Allium cepa Shatavari Asparagus racemosus
(2) Ksheerabala 101 [49] Kachora Hedychium spicatum
Balamoola Sida cordifolia Devadaru Cedrus deodara
Tila taila Gingelly oil Bala Sida cordifolia
Goksheera Milk Rasna Vanda roxburghii
Prasarini Paederia foetida
Balamoola kalka Sida cordifolia
Kushta Saussurea lappa
(3) Balaswagandhadi thailam [50]
Shatahva Anethum sowa
Ashwagandha Withania somnifera
Parushaka Grewia asiatica
Bala Sida cordifolia Bharangi Clerodendrum serratum
Tila taila Gingelly oil Punarnava Boerhavia diffusa
Lodhra Symplocos racemosa Pippali Mula Piper longum
Sarjikakshara Chitrak Plumbago zeylanica
Bidara Zizyphus jujuba Jivanti Leptadenia reticulata
Haridra Curcuma longa Sariva Hemidesmus indicus
Daruharidra Berberis aristata Mashaparni Teramnus labialis
Mudgaparni Phaseolus trilobus
Renuka seeds Vitex negundo
Guduchi Tinospora cordifolia
Kushta Saussurea lappa
Yashti Glycyrrhiza glabra
Musta Cyperus rotundus
Saindhava lavana Rock salt
Srigandha Santalum album Hingu Ferula foetida
Sariva Hemidesmus indicus
Katukarohini Piccrorhiza kurro
Shatahva Anethum sowa
A probable mode of action of the therapies involved has
Devadaru Cedrus deodara been put forth by way of evidence that we could gather from
Manjistha Rubia cordifolia contemporary scientific observations. Further critical evalu-
Yashti Glycyrrhiza glabra ation of the individual components and whole medicines is
required for more definitive claims.
Usheera Vetiveria zizanioides
Ksheera Milk
(4) Niramisha mahamasha taila [51]
5. Conclusions
Masha Phaseolus mungo Acharya Sushruta says “it is allowed to interpret concepts of a
Bilva Aegle marmelos discipline through premises of other sciences to comprehend
wider connotations with an ultimate objective of common
Agnimantha Clerodendron latifolium
good” [52]. The present study was an endeavor in this direc-
Shyonaka Oroxylum indicum tion. There is evidence from the current data and literature
Kashmiri Gmelina arborea to support the usefulness of adjuvant Ayurveda therapies
Patala Stereospermum suaveolens
in modulating cardiac autonomic dysfunction in ischemic
stroke. However, further studies that can incorporate more
Brihati Solanum xanthocarpum aspects of individualized whole system protocols of Ayurveda
Evidence-Based Complementary and Alternative Medicine 7

treatment and in a larger population are definitely warranted [12] T. Dziedzic, A. Slowik, J. Pera, and A. Szczudlik, “Beta-blockers
to enhance the benefits of the science. reduce the risk of early death in ischemic stroke,” Journal of the
Neurological Sciences, vol. 252, no. 1, pp. 53–56, 2007.
[13] A. Charaka, A. Drdhabala, and A. Chakrapanidatta, The
Conflict of Interests Charaka Samhita of Agnivesha, Munshiram Manoharlal Pub-
The authors declare that there is no conflict of interests lishers, Varanasi, India, 1992.
regarding the publication of this paper. [14] A. R. Gujjar, T. N. Sathyaprabha, D. Nagaraja, K. Thennarasu,
and N. Pradhan, “Heart rate variability and outcome in acute
severe stroke: role of power spectral analysis,” Neurocritical
Acknowledgments Care, vol. 1, no. 3, pp. 347–353, 2004.
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The authors thank the Central Council for Research in cardiac autonomic functions in patients with major depression:
Ayurveda & Siddha (CCRAS), Department of AYUSH, Min- a study using heart rate variability measures,” Journal of Affective
istry of Health & Family Welfare, Government of India, New Disorders, vol. 100, no. 1–3, pp. 137–141, 2007.
Delhi, India, for funding the project. The publication of the [16] “Heart rate variability: standards of measurement, physiological
paper was largely supported by the clinical research corpus interpretation and clinical use. Task Force of the European
to FRLHT-ITDHST, Bangalore from the Jamsetji Tata Trusts, Society of Cardiology and the North American Society of
Mumbai. Pacing and Electrophysiology,” European Heart Journal, vol. 17,
pp. 354–381, 1996.
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Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2014, Article ID 465640, 8 pages
http://dx.doi.org/10.1155/2014/465640

Research Article
Guanxinkang Decoction Exerts Its Antiatherosclerotic Effect
Partly through Inhibiting the Endoplasmic Reticulum Stress

Hao Wang,1 Zhi-Min Zheng,2 and Bu-Lang Gao2

1
Department of Traditional Chinese Medicine, The First Hospital of Shijiazhuang City, 36 Fanxi Road,
Shijiazhuang, Hebei 050011, China
2
First Hospital of Shijiazhuang and Organ Transplantation Committee in Chinese Medical Association, 36 Fanxi Road,
Shijiazhuang, Hebei 050011, China

Correspondence should be addressed to Hao Wang; [email protected] and Bu-Lang Gao; [email protected]

Received 7 November 2013; Revised 10 March 2014; Accepted 10 March 2014; Published 18 May 2014

Academic Editor: Joen-Rong Sheu

Copyright © 2014 Hao Wang et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Purpose. To investigate the antiatherosclerotic effect of Guanxinkang (GXK) decoction on the apoptosis, mitochondrial membrane
potential (MMP), and endoplasmic reticulum stress (ERS) of human umbilical vein endothelial cells (HUVEC) pretreated with
homocysteinemia (HCY). Materials and Methods. HUVEC were randomly divided into 5 groups: (1) blank control group (control),
(2) model control group (model), (3) GXK low dose group, (4) GXK medium dose group, and (5) GXK high dose group. For the
three GXK groups, HCY was given to reach the concentration of 3.0 mmol/L after HUVEC had been incubated with rabbit serum
containing GXK for two hours. At 3, 6, 12, and 24 h after HCY had been incubated with the cells, the HUVEC were collected for test
of the apoptosis rate, MMP, and GRP78 protein (reflecting ERS). Results. In the model control group, the apoptosis rate and GRP
78 protein expression of HUVEC significantly increased (𝑃 < 0.05), while MMP significantly decreased (𝑃 < 0.05) compared with
the blank control group. After GXK treatment of medium and high doses, the apoptosis rate and the GRP 78 protein expression
significantly (𝑃 < 0.05) decreased, while MMP significantly increased (𝑃 < 0.05) in a time-dependent manner compared with the
model control group. Conclusion. GXK can antagonize the injury of HUVEC caused by HCY and the antagonism effect increases
with the concentration and treatment duration of GXK, with the possible mechanism of GXK antagonism being through inhibiting
ERS caused by HCY.

1. Introduction indicated a positive correlation between an elevation of

total plasma homocysteinemia (HCY) and stroke, myocardial
Atherosclerosis is a chronic inflammatory condition in which infarction, peripheral vascular disease, and venous throm-
the arterial wall thickens because of the accumulation of boembolism [4]. HCY is a thiol-containing amino acid
cholesterol, macrophages, and smooth muscle cells (SMC), formed during the intracellular demethylation of methionine
ultimately restricting blood flow through the artery. It is and causes multifold effects through the reactivity of its
the primary pathologic condition underlying coronary artery sulfhydryl group. Increase in levels of HCY impairs cellular
and cerebrovascular diseases resulting in heart attack and function and causes loss of endothelial antithrombotic func-
stroke, respectively. Prevention of atherosclerosis can reduce tion, lipid peroxidation, impairment of platelet aggregation,
the incidence and mortality of cerebrovascular and cardio- enhanced oxidative stress, and apoptosis [5–7]. These unfa-
vascular diseases. vorable vascular effects of HCY are caused by the generation
Hyperhomocysteinemia is an independent risk factor for of reactive oxygen species (ROS) [8], which play a critical
atherosclerotic cardiovascular disease, stroke, and peripheral role in the damage and dysfunction of the endothelium
vascular disorders [1, 2] because it is a potent proinflamma- [9]. ROS acts as an upstream factor for mitochondrial
tory factor to accelerate the development of atherosclerosis membrane depolarization, and increased production of ROS
[3]. Epidemiological studies in the general population have is implicated in loss of mitochondrial membrane potential
2 Evidence-Based Complementary and Alternative Medicine

(MMP) [10] and induction of members of Bcl2 protein family. reagents were from Boster Biotech (Wuhan, China). HUVEC
Mitochondria release cytochrome-c and caspases that result was from the central laboratory of the affiliated hospital to
in eventual endothelial cell apoptosis an cardiac dysfunction Liaoning University of Traditional Chinese Medicine.
[11].
The endoplasmic reticulum (ER) is a multifunctional
2.2. Preparation of Rabbit Medicine Serum. This study proto-
intracellular organelle responsible for the synthesis and fold-
col was approved by the animal care and ethics committee
ing of proteins as well as calcium storage and signaling.
of our hospital. 30 New Zealand white rabbits were ran-
When its function is disturbed by various physiological
domly divided into five groups used for the preparation of
and pathological conditions like misfolded protein accu-
rabbit medicine serum: blank control group (control), model
mulation, hypoxia, Ca2+ depletion, or microbial infection, control group (model), GXK low dose group (GXKL), GXK
endoplasmic reticulum stress (ERS) develops, and ERS can medium dose group (GXKM), and GXK high dose group
regulate process such as cell survival and cell death [12]. Cells (GXKH). The lowest dosage of Guanxinkang of human is
alleviate ERS through the unfolded protein response, and the 10 g; according to the animal equivalent dose conversion, the
upregulation of ER chaperones, such as the glucose-regulated dosage of the rabbit is 1.45 g/kg which was used in GXKL.
protein 78 (GRP78), contributes to the repair of unfolded Then it was increased 2 times and 4 times, respectively,
proteins. However, if stress is sustained, the unfolded pro- in GXKM and GXKH, as follows: (1) blank control group
tein response causes cell death by transcriptional induction (control): the rabbits were fed no HCY nor GXK so that the
of CCAAT/enhancer-binding protein homologous protein rabbit serum contained no HCY or GXK; (2) model control
(CHOP), the caspase-12 dependent pathway, and activation group (model): the rabbits were fed as the blank control group
of the c-Jun NH2-terminal kinase dependent pathway [13]. has been fed; (3) GXK low dose group (GXKL): GXK at
GRP78 is the major ER-resident chaperone and the most 1.45 g/kg was used to feed the rabbits through gastric tube
abundant glycoprotein in the ER. It is widely used as a so that the rabbit serum contained low dose GXK; (4) GXK
biomarker of ERS because it plays a critical role in protein medium dose group (GXKM): GXK at 2.90 g/kg was used
folding and ER Ca2+ binding [14]. to feed the rabbits through gastric tube so that the rabbit
HCY-induced vascular injury may also involve ERS and serum contained medium dose GXK; (5) GXK high dose
activation of the unfolded protein response caused by incor- group (GXKH): GXK at 5.80 g/kg was used to feed the rabbits
rect protein folding [15]. HCY can cause ERS by disrupting through gastric tube so that the rabbit serum contained high
disulfide bond formation, causing misfolding of proteins dose GXK. Seven days after the rabbits were treated with
traversing the ER. HCY induces the expression of GADD153 different medicines, the rabbits were anesthetized and blood
(a basic region leucine zipper transcription factor involved was aspirated from the hearts. The blood was centrifuged for
in ERS-induced cell death), the ER chaperones GRP78 and 10 min at 2000 r/m within one hour, and the supernatant was
GRP94, and Herp (a protein involved in the degradation of used as the rabbit serum which was further filtered and kept
misfolded ER protein) [16]. for experiment.
Guanxinkang decoction (GXK) is a compound tradi-
tional Chinese herbal medicine composed of the following
six Chinese herbs of Radix Astragali, Trichosanthes, Radix 2.3. Treatment of HUVEC. The HUVEC were grown in the
Salviae Miltiorrhizae, Allium macrostemon, Pinellia tuber- DMEM containing 20% fetal bovine serum in an incubator
ifera, and Radix Puerariae. This decoction has been proved under 5% CO2 . The cells used in this study were from
to have an antiatherosclerotic effect and this effect is partly passage 3 and the cells were washed twice with the DMEM
through its interference with lipid regulation, inflammation and adjusted to the concentration of 1 × 105 /mL before
activities, and ion channels [17–20]. However, the exact effect experiment. The cells were randomly divided into 5 groups
of this decoction on the endothelial cells is not known, and which were given 10% rabbit serum containing different
this study intended to investigate its protection effect on medicines for treatment: (1) blank control group (control):
the endothelial layer of the vascular wall by studying the 10% rabbit serum from the blank control group of rabbits was
apoptosis rate, the MMP, and the GRP78 expression levels of used with no HCY or GXK being added; (2) model control
the human umbilical vein endothelial cells (HUVEC) treated group (model): 10% rabbit serum from the model control
with GXK and HCY in sequence. group of rabbits was used; then HCY was given to reach
the concentration of 3.0 mmol/L after the 10% blank control
rabbit serum was added for two hours; (3) GXK low dose
2. Materials and Methods group (GXKL): 10% rabbit serum containing low dose of GXK
was given; (4) GXK medium dose group (GXKM): 10% rabbit
2.1. Agents. Trypsin and Dulbecco’s modified Eagle’s medium serum containing medium dose of GXK was given; (5) GXK
(DMEM) were purchased from Gibco (Invitrogen, Shang- high dose group: 10% rabbit serum containing high dose GXK
hai, China), HCY and propidium iodide (PI) from Sigma- was added. For the three low-high dose GXK groups, HCY
Aldrich (China branch), and MMP detection kit (JC-1) was was given to reach the concentration of 3.0 mmol/L after the
from Beyotime Institute of Biotechnology (Shanghai). Goat HUVEC had been incubated with GXK at low, medium, and
anti-GRP78 monoclonal antibody was bought from Santa high doses for two hours. Then, the HUVEC were collected
Cruse Corporation (USA), and HRP enzyme-labeled rabbit for test at 3, 6, 12, and 24 h after HCY had been incubated with
secondary antibody against goat IgG and Western blotting the cells.
Evidence-Based Complementary and Alternative Medicine 3

2.4. Flow Cytometry. The HUVEC in every group were Table 1: Apoptosis rate under the action of GXK at different points
centrifuged and collected at 3, 6, 12, and 24 h after incubation (𝑋 ± 𝑠, 𝑛 = 3).
with HCY. The cells were washed twice with PBS and adjusted
Apoptosis rate (%)
to 1 × 106 /mL in concentration. PI was added into every Groups
3h 6h 12 h 24 h
sample group which was put in a 4C∘ refrigerator for staining
Control 2.12 ± 0.41 1.96 ± 0.49 2.03 ± 0.38 2.32 ± 0.67
in the dark for 20–30 min. Then, PI was washed off, PBS
was added, and the cells were screened using a 300-mesh Model 10.22 ± 1.43 18.74 ± 0.60∗ 24.83 ± 1.97∗ 32.15 ± 2.78∗
sieve. Flow cytometry was performed to detect the DNA GXKL 8.42 ± 1.14 14.26 ± 2.17 20.38 ± 3.10 23.51 ± 2.16#
content and the apoptosis rate of cells. The Cell Quest GXKM 6.25 ± 0.95 8.46 ± 1.79# 9.81 ± 2.45# 15.34 ± 1.81#
software (FACSCalibur, BD, USA) was used to calculate the GXKH 5.77 ± 1.09 6.74 ± 1.93# 7.92 ± 3.56# 11.35 ± 2.35#
cell apoptosis rate. Note: ∗ compared with control group, 𝑃 < 0.05; # compared with model
group, 𝑃 < 0.05; GXK: Guanxinkang; GXKL: Guanxinkang low dose group;
GXKM: Guanxinkang medium dose group; GXKH: Guanxinkang high dose
2.5. Detection of MMP. The HUVEC in every group were group.
centrifuged and collected at 3, 6, 12, and 24 h after incubation
with HCY. The supernatant was discarded. DMEM of 0.5 mL
(with no serum) was added into every tube of cells, and a GXKL group but significantly (𝑃 < 0.05) in both the GXKM
positive control was set up. The CCCP (10 mM) provided in and GXKH groups compared with the model group (Table 1
the MMP detection kit was diluted at 1 : 1000 and was added and Figure 1).
into the cell culture medium, and the medium was diluted to
10 𝜇M. The cells in the positive control group were added into 3.2. MMP Detection. Compared with the blank control
the cell culture medium for incubation of 20 min. Then, one group, the MMP decreased significantly (𝑃 < 0.05) in all the
mL JC-1 dye was added into every tube of cells and blended other groups especially in the model control group. However,
completely. The cells were incubated in an incubator at 37C∘ the MMP in all the groups treated with different concentra-
for 20 min. Then, the supernatant was aspirated; the cells were tions of GXK was higher than in the model control group,
washed twice with JC-1 dye. DMEM of 1 mL was added and and the difference was more significant with increase of
flow cytometry was performed at the conditions of excitation treatment duration. The difference was the greatest at the
480 nm and emission 520 nm using Cell Quest software. treatment point of 24 h. At the same time point, the MMP
increased insignificantly (𝑃 > 0.05) in the GXL group but sig-
2.6. Western Blot Analysis for GRP78. Twenty-four hours nificantly (𝑃 < 0.05) in both the GXKM and GXKH groups
after the HUVEC were treated with HCY, the culture medium compared with the model control group (Table 2 and
was aspirated and the cells were scraped with a cytobrush. Figure 2).
PBS was added into the cells for centrifugation, and culture
flasks and the cytobrush were washed. The rinse solution 3.3. Expression of GRP78 in HUVEC. Compared with the
together with the cells was put into the centrifugal tubes for blank control group, the expression of GRP78 protein
centrifugation at 300 g for 5 min. Then, the cell precipitate increased significantly (𝑃 < 0.05) and reached 0.36 ± 0.07
was aspirated, washed twice with PBS, and added with cell at 24 h after HCY treatment in the model control group
lysate. The cells were scraped with a cytobrush and put into (Table 3). The expression decreased in all the other groups
the Eppendorf tube. The cells were put on ice for 30 min with GXK treatment and the decrease became more apparent
for their complete degradation and then centrifuged at 4C∘ with increase of treatment duration. The decrease of the
and 12000 g for 10 min. The supernatant was collected for expression was the greatest at 24 h. At the same time point,
the total protein. Then, Western blot analysis for GRP78 was the decrease of the expression of GRP78 protein was not
performed as instructed. significant (𝑃 > 0.05) in the GXKL group but significant (𝑃 <
0.05) in both the GXKL and GXKH groups compared with
2.7. Statistical Analysis. All the data were expressed as mean ± the model control group, with the best result being obtained
standard deviation using SPSS10.0 software for the analysis of at 24 h (0.11 ± 0.07) (Figure 3).
two-factor ANOVA, with the significant 𝑃 value set at <0.05.
4. Discussion
3. Result
In this study, we investigated the role of GXK decoction
3.1. Cell Apoptosis. Compared with the blank control group, in protecting the endothelial cells and found that GXK can
the cell apoptosis rate was significantly (𝑃 < 0.05) increased significantly antagonize the effect of HCY on HUVEC. With
in all the other groups especially in the model control the treatment of GXK, the HUVEC apoptosis rate and the
group. However, the cell apoptosis rate in the groups treated expression of GRP78 protein are significantly decreased,
with different concentrations of GXK was lower than in the while the HUVEC MMP is increased, favoring a protection
model control group, and the difference was more significant effect on the endothelial cells.
with increase of treatment duration. The difference was the Atherosclerosis is the hardening and narrowing of the
greatest at the treatment point of 24 h. At the same time point, arteries and this progressive process silently and slowly
the apoptosis rate decreased insignificantly (𝑃 > 0.05) in the blocks arteries, putting blood flow at risk. Atherosclerosis is
4 Evidence-Based Complementary and Alternative Medicine

800 800

600 600
Cell number

Cell number
400 400

32.30%
200 200
2.32%
0 0
0 30 60 90 120 150 0 30 60 90 120 150
Channels (FL2-A) Channels (FL2-A)
(a) Control group at 24 h (b) Model group at 24 h

800 800

600 600
Cell number

Cell number

400 400

23.47% 14.63%
200 200

0 0
0 30 60 90 120 150 0 30 60 90 120 150
Channels (FL2-A) Channels (FL2-A)
(c) GXKL at 24 h (d) GCKM at 24 h

800

600
Cell number

400

13.25%
200

0
0 30 60 90 120 150
Channels (FL2-A)

Debris Dip G2
Aggregates Dip S
Dip G1 Apoptosis
(e) GXKH at 24 h

Figure 1: Apoptosis rate under the action of Guanxinkang at 24 h. (1) Compared with the control group, apoptosis rate of each group was
increased, especially in the model group (𝑃 < 0.05). (2) Apoptosis rate of GXKL decreased not significantly. (3) Apoptosis rate of GXKM or
GXKH decreased significantly. Compared with the model group, the difference is significant (𝑃 < 0.05).
Evidence-Based Complementary and Alternative Medicine 5

104 200

103

Mean 947.27
Counts
FL2-H
102

101

100 0
100 101 102 103 104 100 101 102 103 104
FL1-H FL2-H
(a) Control group at 24 h

104 200

103

Mean 392.34
Counts
FL2-H

102

101

100 0
100 101 102 103 104 100 101 102 103 104
FL1-H FL2-H
(b) Model group at 24 h

104 200
3

Mean 518.47
10
Counts
FL2-H

102

101

100 0
100 101 102 103 104 100 101 102 103 104
FL1-H FL2-H
(c) GXKL at 24 h
4 200
10

103
Mean 856.76
Counts
FL2-H

102

101

100 0
100 101 102 103 104 100 101 102 103 104
FL1-H FL2-H
(d) GXKM at 24 h
4 200
10

103
Mean 920.74
Counts
FL2-H

102

101

100 0
100 101 102 103 104 100 101 102 103 104
FL1-H FL2-H
(e) GXKH at 24 h

Figure 2: MMP of HUVEC under the action of Guanxinkang at 24 h. (1) Compared with the control group, the data of MMP in each group
decreased, especially in the model group (𝑃 < 0.05). (2) The data of MMP in cells under the action of GXK of different doses was significantly
higher than that in the model group, and with the effect time progressing, the difference is more obvious. (3) The data of MMP of GXKL
increased not significantly. (4) The data of MMP of GXKM and GXKH increased significantly (𝑃 < 0.05).
6 Evidence-Based Complementary and Alternative Medicine

Table 2: MMP of HUVEC under the action of GXK at different times (𝑋 ± 𝑠, 𝑛 = 3).

MMP
Groups
3h 6h 12 h 24 h
Control 1085.21 ± 12.24 992.24 ± 13.32 965.67 ± 12.67 932.43 ± 15.14
Model 712.25 ± 16.31∗ 584.69 ± 19.21∗ 498.17 ± 20.26∗ 389.56 ± 22.36∗
GXKL 737.79 ± 17.42 673.73 ± 16.91 624.29 ± 17.41 510.32 ± 20.47#
GXKM 755.22 ± 17.21 736.53 ± 17.69# 799.43 ± 16.38# 856.26 ± 15.81#
GXKH 781.27 ± 16.31 801.25 ± 16.31# 863.64 ± 14.93# 915.64 ± 16.83#
Note: GXK: Guanxinkang; GXKL: Guanxinkang low dose group; GXKM: Guanxinkang medium dose group; GXKH: Guanxinkang high dose group; MMP:
mitochondrial membrane potential; ∗ compared with control group, 𝑃 < 0.05; # compared with HCY model group, 𝑃 < 0.05.

Table 3: Level of protein GRP78 in HUVEC under the action of GXK at different points (𝑋 ± 𝑠, 𝑛 = 3).

Mean optic density

Groups
3h 6h 12 h 24 h
Control 0.06 ± 0.01 0.07 ± 0.02 0.07 ± 0.01 0.08 ± 0.03
Model 0.19 ± 0.06 0.24 ± 0.05∗ 0.27 ± 0.09∗ 0.36 ± 0.07∗
GXKL 0.16 ± 0.04 0.21 ± 0.07 0.20 ± 0.04 0.25 ± 0.02
GXKM 0.15 ± 0.05 0.17 ± 0.02# 0.16 ± 0.06# 0.13 ± 0.03#
GXKH 0.14 ± 0.03# 0.18 ± 0.08# 0.14 ± 0.09# 0.11 ± 0.07#
Note: ∗ compared with control group, 𝑃 < 0.05; # compared with HCY model group, 𝑃 < 0.05; GXK: Guanxinkang; GXKL: Guanxinkang low dose group;
GXKM: Guanxinkang medium dose group; GXKH: Guanxinkang high dose group.

1 2 3 4 5 6 7 8 9 10 Endoplasmic reticulum contains many proteins, for

78 KD GRP78 example, PERK (PKR-like eukaryotic initiation factor 2𝛼
kinase), IRE-1 (inositol requiring enzyme-1), ATF6 (acti-
vating transcription factor 6), CHOP (C/EBP homologous
42 KD 𝛽-Actin protein), GRP78 (glucose-regulated protein 78) and so on.
GRP78 protein is the major ER-resident chaperone and is
Figure 3: GRP78 of HUVEC at 24 h under the action of widely used as a biomarker of ERS [24] because it plays
Guanxinkang. (1-2): blank control; (3-4): HCY model control; (5- a crucial role in protein folding and ER Ca2+ binding. It
6): Guanxinkang low dose group; (7-8): Guanxinkang medium dose facilitates protein folding in the ER, and thus it reduces the
group; (9-10): Guanxinkang high dose group. (1) GRP78 protein is number of misfolded proteins and alleviates ERS. A common
more significantly expressed in the cells in model group (𝑃 < 0.05).
stimulus for the induction of GRP78 is the presence of mis-
(2) At 24 hours, expression of GRP78 in GXKL has not decreased
obviously. (3) It increased significantly in GXKM and GXKH (𝑃 < folded proteins in the ER15. Studies demonstrated that HCY
0.05). induced vascular injury involving ERS and the increase of
GRP78 protein [15, 16]. So only the expression of GRP78 was
observed in this experiment.
the primary cause of heart attacks, strokes, and peripheral In our study, HCY increased the apoptosis rate and
vascular diseases, which are usually called cardiovascular GRP78 protein expression but decreased MP in the model
disease, the number one killer in USA, with more than control group. GXKL reduced apoptosis and MMP (𝑃 > 0.05)
800,000 deaths in 2005 [21]. Hyperhomocysteinemia is an but showed no obvious effect on HCY-induced GRP78. This
independent risk factor for atherosclerosis [1, 2] and can cause is because apoptosis can occur directly through the mito-
apoptosis and decrease of MP of endothelial cells. Tyagi et al. chondrial pathway and also can be induced by ER stress
[7] studied the mitochondrial mechanism of microvascular [25, 26]. The treatment of GXKM and GXKH significantly
endothelial cell apoptosis caused by hyperhomocysteinemia antagonized these effects caused by HCY. GXK has been
and found that HCY-mediated ROS production promotes proved to have a protective effect on the endothelial cells
endothelial cell apoptosis in part by disturbing MP (decrease against atherosclerosis partly through its interference with
and loss of MP), which results in subsequent release of lipid regulation, inflammation activities, and ion channels
cytochrome-c and activation of caspase-9 and caspase-3, [17–20]. Our study demonstrated that GXK could also
leading to cell death. Hyperhomocysteinemia is a potent antagonize the damage effect HCY on the endothelial cells
proinflammatory factor to accelerate the development of through inhibiting ERS by decreasing the apoptosis rate and
atherosclerosis [22]. Pathophysiological levels of HCY could the expression of GRP78 protein and increasing MP.
interfere with human monocyte function by upregulating GXK is a compound traditional Chinese herbal medicine
monocyte chemoattractant protein 1 and IL-8 expression and composed of six Chinese herbs of radix astragali, Tri-
secretion via oxidative stress [23]. chosanthes, Radix Salviae Miltiorrhizae, Allium macrostemon,
Evidence-Based Complementary and Alternative Medicine 7

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Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2014, Article ID 696218, 8 pages
http://dx.doi.org/10.1155/2014/696218

Research Article
Qingkailing Suppresses the Activation of BV2
Microglial Cells by Inhibiting Hypoxia/Reoxygenation-Induced
Inflammatory Responses

Lulu Mana,1 Shan Wang,2 Haiyan Zhu,1 Yanwei Xing,3 Lixia Lou,1 Aiming Wu,1 Bin Dong,1
Yikun Sun,1 Shuo Yang,4 Lin Wang,5 and Yonghong Gao1
1
Key Laboratory of Chinese Internal Medicine of Ministry of Education and Beijing,
Dongzhimen Hospital Affiliated to Beijing University of Chinese Medicine, Beijing 100700, China
2
The Southern District of Guang’anmen Hospital, China Academy of Chinese Medical Sciences, Beijing 102618, China
3
Guang’anmen Hospital, China Academy of Chinese Medical Sciences, Beijing 100053, China
4
Institute of Information on Traditional Chinese Medicine, China Academy of Chinese Medical Sciences, Beijing 100700, China
5
Department of Preventive Health Care, China-Japan Friendship Hospital, Beijing 10029, China

Correspondence should be addressed to Yonghong Gao; [email protected]

Received 13 November 2013; Accepted 2 April 2014; Published 28 April 2014

Academic Editor: Joen-Rong Sheu

Copyright © 2014 Lulu Mana et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Qingkailing (QKL) is a well-known composite extract used in traditional Chinese medicine. This extract has been extensively
administered to treat the acute phase of cerebrovascular disease. Our previous experiments confirmed that QKL exerts an inhibitory
effect on cerebral ischemia-induced inflammatory responses. However, whether QKL suppresses the activation of microglia, the
primary resident immune cells in the brain, has yet to be determined. In this study, BV2 microglial cells were used to validate the
protective effects of QKL treatment following ischemia-reperfusion injury simulated via hypoxia/reoxygenation in vitro. Under
these conditions, high expression levels of ROS, COX-2, iNOS, and p-p38 protein were detected. Following ischemia/reperfusion
injury, QKL significantly increased the activity of BV2 cells to approximately the basal level by modulating microglial activation
via inhibition of inflammatory factors, including TNF-𝛼, COX-2, iNOS, and p-p38. However, QKL treatment also displayed dose-
dependent differences in its inhibitory effects on p38 phosphorylation and inflammatory factor expression.

1. Introduction to the stressful stimulus. Studies have revealed that activated

microglia can protect neurons via phagocytic removal of
Ischemic cerebrovascular disease (ICVD) is a notably pathogens and dead cell debris, as well as secretion of
common clinical cerebrovascular disease. The mechanisms neurotrophic factors [3–5].
underlying cerebral ischemia-reperfusion injury are quite However, additional evidence has indicated that activated
complicated. Oxidative stress and the inflammatory response microglia can also synthesize and release inflammatory medi-
are considered to be the key mediators of cerebral ischemic ators, leading to neuronal dysfunction and death, in response
injury. The injury and repair mechanisms in anoxic and to injury and stimulation [6, 7]. Abnormally activated
ischemic environments, which are mediated by the abnor- microglia can damage neurons by releasing proinflammatory
mally activated microglia, have become a recent target of cytokines, inflammation-related enzymes, and chemokines
neurological research field. Unfortunately, these mechanisms and activating NADPH oxidase to generate reactive oxygen
have yet to be fully elucidated. The specific mechanism species, induce the expression of IL-1𝛽, TNF-𝛼 and MMP-
by which microglia become activated in response to cere- 9, promote leukocyte infiltration, and weaken the blood-
bral ischemia remains controversial [1, 2]. When ischemia brain barrier [8]. Because of their rapid activation and their
occurs, microglia are rapidly activated initially in response important roles in oxidative stress and the inflammatory
2 Evidence-Based Complementary and Alternative Medicine

response, microglia may represent a new line of investigation 2.2.2. Establishment of the In Vitro BV2 Cell Hypoxia/Reoxy-
in the search for a clinical therapy for cerebral ischemia. genation Model. BV2 cells were digested into a cell suspen-
Qingkailing (QKL) is a modified extract from a tra- sion, and the cell density was adjusted to 5 × 104 cells/mL.
ditional Chinese medicine, An-Gong-Niu-Huang Pill. It is After 80% confluence, the cells were washed 3 times with
composed of Radix Isatidis, Flos Lonicerae, Concha Mar- PBS, replaced with serum-free DMEM medium, and placed
garitifera Usta, baicalin, Fructus gardeniae, cholic acid, in a three-gas incubator at 37∘ C containing 1.0% O2 to initiate
hyodeoxycholic acid, and Cornu Bubali [9]. QKL injection hypoxia, followed by reoxygenation in an incubator at 37∘ C
has been widely used clinically to treat the acute phase containing 5% CO2 .
of cerebrovascular disease and has displayed excellent effi-
cacy in improving neurological function [10, 11]. We and 2.2.3. Evaluation of the BV2 Cell Viability. The MTT method
other investigators demonstrated that QKL injection can was used to assess cell viability. Cell viability was measured
improve neurological function, decrease the expression of using an enzyme-labeling instrument at 492 nm. The hypoxia
adhesion molecules, inhibit inflammatory responses, and duration was 12 hours, while reoxygenation occurred for 12 or
partially restore BBB function in a rat model of brain 24 hours.
ischemia/reperfusion injury [12–15].
Based on the findings described above, we speculated that 2.2.4. ROS Assay Using Flow Cytometry. The BV2 cells were
QKL might modulate the inflammatory response mediated cultured in a six-well plate at a density of 5 × 104 cells/mL.
by activated microglia. BV2 microglial cells were used as a The cells were digested and harvested following exposure to
model, and in vitro hypoxia/reoxygenation was performed normoxia or 12 h of hypoxia followed by 12 h of reoxygena-
to simulate ischemia/reperfusion in vivo. The roles of the tion. Then, the cells were incubated in 1 mL of DCFH-DA
inflammatory factors TNF-𝛼, iNOS, and COX-2 and the (10 𝜇mol/L) for 30 minutes. The cells were washed three times
regulatory function of the corresponding signaling pathways with PBS and analyzed via flow cytometry (Beckman Co.,
are also examined. FC50).

2. Materials and Methods 2.3. Effects of QKL on BV2 Cell Viability after
2.1. Materials. MTT and dimethyl-sulfoxide (DMSO) were Hypoxia/Reoxygenation
obtained from Solarbio Corporation, China. The mouse 2.3.1. Cell Group. The experiment comprised six groups: a
TNF-𝛼 ELISA Kit was purchased from Neobioscience Co., control group (the cells were cultured normally in serum-
Ltd., China. Trizol reagent was purchased from Invitrogen free DMEM); the model group (12 h of hypoxia and 12 h
Corporation, USA. The Reverse Transcription System (A3500 of reoxygenation in serum-free DMEM medium); the low,
kit) was purchased from Promega Corporation, USA. The medium, and high QKL groups (a final QKL concentration
Power SYBR Green PCR Master Mix kit was purchased of 0.0625%, 0.125%, or 0.25%, resp., in serum-free DMEM
from ABI Corporation, USA. RIPA Lysis Buffer, Super was added before hypoxia); and the minocycline group
ECL Plus Detection Reagent, Prestained Protein Marker (200 nmol/L minocycline was added before hypoxia).
(10-170KD), proteinase inhibitor cocktail, PhosSTOP phos-
phatase inhibitor, and the ROS detection kit (product no:
C1300) were purchased from Applygen Technologies Co., 2.3.2. MTT Assay the Changes of Cell Viability. The hypoxia
Ltd. COX-2 (H-62) and iNOS (N-20) were purchased from duration was 12 hours, while reoxygenation lasted for 12
Santa Corporation, USA. The mouse anti-𝛽-actin and HRP- hours. The MTT method was used to assess cell viability, as
labeled goat anti-rabbit IgG antibodies were purchased from described above.
Wuhan Boster Biological Technology, Ltd. The p-p38 and
p38𝛼/𝛽 (H-147) antibodies were purchased from Beijing 2.4. Effect of QKL on the Concentration of TNF-𝛼 in the
ZhongShan-Golden Bridge Biological Technology Co., Ltd., Supernatant of BV2 Cells Subjected to Hypoxia/Reoxygenation.
China. Minocycline was purchased from Sigma Corporation, The groups assigned were the same as above. The cell
USA. QKL was purchased from Yabao Beizhongda (Beijing) supernatants were collected and stored at −80∘ C. ELISA was
Pharmaceutical Co., Ltd., China. performed to measure the TNF-𝛼 concentration in the cell
culture supernatants.
2.2. Cell Culture and the Hypoxia-Reoxygenation Injury Model
2.5. Effect of QKL on Inflammatory Cytokine (COX-2
2.2.1. Cell Culture. Mouse microglial BV2 cells were pur- and iNOS) mRNA Expression in BV2 Cells Subjected to
chased from the cell culture center at the Institute of Basic Hypoxia/Reoxygenation. Real-time quantitative polymerase
Medical Sciences (IBMS) of the Chinese Academy of Med- chain reaction (real-time PCR) was performed to evaluate
ical Sciences (CAMS). The cells were suspended in culture the mRNA expression of COX-2 and iNOS. The mRNA was
medium specialized for microglia (DMEM containing 10% extracted, reverse-transcribed, and quantified via real-time
fetal bovine serum, 100 U/mL penicillin, and 100 U/mL strep- PCR. The following PCR primers were used: COX-2 (191 bp),
tomycin). At 80% confluency, the cells were passaged using forward: 5󸀠 -GATGACTGCCCAACTCCC-3󸀠 ; reverse: 5󸀠 -
trypsin-EDTA solution (0.05% trypsin and 0.02% EDTA). AACCCAGGTCCTCGCTTA-3󸀠 . iNOS (95 bp), forward:
Evidence-Based Complementary and Alternative Medicine 3

󸀠
-CAGCTGGGCTGTACAAACCTT-3󸀠 ; reverse: 5󸀠 -CATTG- injury, for the simulation of ischemia-reperfusion injury in
GAAGTGAAGCGTTTCG-3󸀠 . 𝛽-actin (185 bp), forward: 5󸀠 - vitro.
AGGCCAACCGTGAAAAGATG-3󸀠 ; reverse: 󸀠 -TGGCGT-
GAGGGAG A GCATAG-3󸀠 . 3.2. QKL Increased Cell Viability in BV2 Microglia Exposed to
The cycling conditions were 50∘ C for 2 min and initial 12 Hours of Hypoxia and 12 Hours of Reoxygenation. Based
denaturation at 95∘ C for 10 min, followed by 40 cycles at 95∘ C on the model establishment above, according to the results of
for 30 s and 55∘ C for 30 s. The data were analyzed using the pilot experiments, the nontoxic dose range of QKL treatment
Stratagene MX3000P System. The levels of COX-2 and iNOS was chosen to be 0.0625%, 0.125%, and 0.25% (Figure 2).
mRNA normalized to that of 𝛽-actin were compared between Alternatively, 200 nmol/L minocycline, the specific inhibitor
the different groups. of microglia, was applied prior to hypoxia. The results
revealed that the 𝐴 value of the cells treated with any of
the three concentrations of QKL or minocycline, which were
2.6. Effect of QKL on Inflammatory Factor (COX-2 and iNOS)
exposed to hypoxia for 12 h and reoxygenation for 12 h, was
Protein Expression in BV2 Cells and on the p38 Signaling
significantly higher than the model group. This result con-
Pathway during Ischemia-Reperfusion Injury In Vitro. After
firmed that QKL remarkably increases BV2 cell survival and
reoxygenation, the cells were washed 3 times with PBS,
activation under ischemia-reperfusion conditions, reverting
followed by pipetting repeatedly in RIPA Lysis Buffer. Then,
them to control levels, preventing both the activation and
the cellular protein extract was collected. The proteins were
injury of BV2 cells.
separated via 10% SDS-PAGE and transferred to nitrocellu-
lose membranes, which were subsequently incubated in a
primary antibody to COX-2, iNOS, p-p38, or p38 (1 : 1000) at 3.3. QKL Decreased the Expression of TNF-𝛼 in BV2 Cell
4∘ C overnight. After the membranes were washed three times Supernatants. TNF-𝛼 is an important inflammatory factor
with TBST, they were further incubated in the appropriate that is released after microglial activation that plays an
horseradish peroxidase-conjugated secondary antibody for important role in nerve inflammation. The results (Figure 3)
2 hours at room temperature. Then, ECL visualization was revealed that compared to the control group, the concentra-
performed. NIH Image J was used to calculate the gray values. tion of TNF-𝛼 in the model group was significantly increased
The intensity of the target protein was normalized to that of an (𝑃 < 0.05). Treatment with 0.0625%, 0.125%, or 0.25% QKL
or 200 nmol/L minocycline significantly reduced TNF-𝛼 pro-
internal reference to determine the relative expression level of
duction induced by hypoxia/reoxygenation compared to the
the target protein.
model group (𝑃 < 0.05). Together, these results indicated that
ischemia-reperfusion injury activates BV2 cells to produce a
2.7. Statistical Analysis. The data were expressed as the means large quantity of TNF-𝛼, which participates in the inflamma-
± SD. Statistical analysis was performed via one-way analysis tory response induced by ischemia reperfusion. QKL blocked
of variance (ANOVA) using SPSS11.0 software. 𝑃 < 0.05 was microglial activation by inhibiting TNF-𝛼 expression.
considered to be significant.
3.4. QKL Decreased the Expression of COX-2 and iNOS.
3. Results Activated microglia can also damage neurons by releasing
3.1. Establishment of the In Vitro Hypoxia/Reoxygenation proinflammatory cytokines, inflammation related enzymes,
Model. Based on inverted phase contrast microscopy, and so on. We performed real-time PCR and Western blot
analyses to examine the changes in the expression of COX-
approximately 60–80% of the cells in the control group
2 and iNOS (Figures 4(a) and 4(b)). After hypoxia for
were well-anchored contained, a polygonal cell body;
12 hours and reoxygenation for 12 hours, the mRNA and
approximately 20–40% of the cells were suspended. As
protein expression levels of COX-2 and iNOS were signifi-
hypoxia continued, the cell body gradually became rounded, cantly increased. Treatment with 0.25% QKL or 200 nmol/L
and the attached cells became suspended and gathered into minocycline significantly decreased the mRNA expression
clusters. Hypoxia for 12 h resulted in significant changes level compared to the model group. However, treatment with
compared to the control group, and these changes were 0.25% QKL clearly decreased the protein expression of COX-
exacerbated after reoxygenation for 24 h (Figure 1(a)). MTT 2 and iNOS in BV2 cells exposed to hypoxia/reoxygenation.
colorimetry suggested that, compared to the control group, In addition, treatment with 0.125% QKL also decreased the
after hypoxia for 12 h followed by reoxygenation for 12 h protein expression of iNOS in BV2 cells. Together, these
or 24 h in an three-gas incubator, the number of surviving results demonstrate that QKL alleviates the increases in
BV2 cells in the model group were significantly reduced COX-2 and iNOS expression, protecting against further
(𝑃 < 0.01) (Figure 1(b)). As shown in Figure 1(c), the level of ischemia-reperfusion caused by protein and gene expression.
ROS in the model group (hypoxia for 12 h and reoxygenation Importantly, these results are consistent with those of the cell
for 12 h) was significantly higher than the normal group viability and TNF-𝛼 expression assays described above.
(𝑃 < 0.01).
Based on the comparisons of the cellular morphology and 3.5. QKL Inhibited p38 Phosphorylation in BV2 Cells Exposed
viability after different hypoxia durations, we chose 12 h of to Hypoxia/Reoxygenation. To further elucidate whether the
hypoxia and 12 h of reoxygenation, which caused significant p38 signaling pathway affects the expression of iNOS and
4 Evidence-Based Complementary and Alternative Medicine

1.5

1.0 ##

A value
Control Hypoxia
y 6h
##

0.5 ##

Hypoxia 12 h Hypoxia 12 h/ 0.0

H 12 h

H12h/R 12 h

H 12 h/R 24 h
reoxygenation 12 h

Control
Model
(a) (b)

50
##
61

44

40

30

ROS level
C C
20

10

0
100 101 102 103 100 101 102 103 Control Model

ROS DCF ROS DCF

Control Hypoxia 12 h/reoxygenation 12 h

(c)

Figure 1: Effects of different durations of hypoxia/reoxygenation on BV2 microglia cells. (a) BV2 cells were exposed to hypoxia for 6 or
12 hours followed by reoxygenation for 12 hours. Morphological changes were observed under an inverted microscope (×100). (b) After
exposure to hypoxia for 12 hours, the BV2 cells were reoxygenated for 12 or 24 hours. Cell viability was assed using the MTT method. Each
value indicates the mean ± SD. 𝑁 = 5. ## 𝑃 < 0.01 compared to the control group. (c) ROS level in BV2 microglial cells after hypoxia for 12
hours and reoxygenation for 12 hours based on flow cytometry. 𝑁 = 4. ## 𝑃 < 0.01 compared to the control group.

COX2 in BV2 cells, we measured the expression of phos- to the vascular endothelium, induce inflammatory responses,
phorylated p38 protein and analyzed the ratio of p-p38 to and cause tissue edema, destroying brain tissue [3–5].
p38. As shown in Figure 5, the control group expressed Previous studies have demonstrated that, whether under
a small amount of p-p38 protein, and p-38 expression the conditions of diseases or culture systems, the primary
was significantly increased in the model group. Treatment source of proinflammatory cytokines is microglia rather than
with 0.0625% QKL or 200 nmol/L minocycline significantly astrocytes. Moreover, IL-1𝛽 and TNF-𝛼 have been suggested
decreased p-p38 protein expression in BV2 cells subjected to to be the most important proinflammatory cytokines in this
hypoxia/reoxygenation compared to the model group (𝑃 < process [16]. The massive expression of TNF-𝛼 acts as a
0.01).
key mediator of cerebral ischemia-induced inflammation,
which cannot only mediate a cascade of inflammatory
4. Discussion responses but also amplify inflammation by interacting with
As one of the primary mechanisms of cerebral ischemia, other inflammatory factors, thus inducing the expression
the inflammatory response can cause secondary brain injury. of adhesion molecules on the endothelium as well as other
Inflammatory cytokines, which are released from so-called inflammatory mediators by macrophages, endothelial cells,
inflammatory cells such as neutrophils, granulocytes, lym- and spongiocytes. TNF-𝛼 induces the migration of activated
phocytes, and glia, can stimulate the adherence of leukocytes leukocytes to the ischemic area, and these cells release free
Evidence-Based Complementary and Alternative Medicine 5

0.6 iNOS and COX-2 are suggested to be important factors

∗∗
involved in microglial activation and critical mediators of
∗∗ cerebral ischemic injury caused by microglia. When reper-
∗∗
0.4
fusion occurs, a large amount of oxygen molecules gathers
## in the cerebral ischemia area, which react with NO to
A value

produce powerful oxidants that can cause irreversible oxida-

tive damage to cells [21]. An iNOS gene knock-out mouse
0.2 model exhibited a significant decrease in the infarct volume
following middle cerebral artery occlusion [22]. NADPH
is a multisubunit enzyme complex; once NADPH becomes
0.0 activated, ROS, a second messenger of cell signal transduc-
tion, is strongly produced, which leads to the activation
Control

Model

QKL 0.0625%

QKL 0.125%

QKL 0.25%

Minocycline
of the p38-MAPK signaling pathway and the expression of
proinflammatory cytokines (e.g., TNF-𝛼) and inflammation-
related enzymes (e.g., iNOS and COX-2) [23, 24]. Conversely,
NADPH is specifically activated by these cytokines, thus
Figure 2: Effects of different dosages of QKL on the viability of BV2 promoting the production of ROS and other inflammatory
microglial cells in the hypoxia/reoxygenation model. The cells were factors. In this manner, a vicious cycle of ever-increasing
treated with 0.0625%, 0.125%, or 0.25% QKL or 200 𝜇M minocycline
neurotoxins is initiated, ultimately resulting in neuronal
before hypoxia. The MTT method was performed, and the results
are expressed as the means ± SD; 𝑛 = 6. ## 𝑃 < 0.01 compared to the necrosis [25].
control group; ∗∗ 𝑃 < 0.01 compared to the model group. In our experiments, increases in the mRNA and protein
expression levels of both COX-2 and iNOS in BV2 microglia
were induced by hypoxia/reoxygenation. Treatment with the
100 low or middle concentration of QKL displayed a significant
# effect on the protein expression of COX-2. The inhibitory
effects of the high concentration of QKL on the mRNA
80 and protein expression of iNOS and COX-2 were the most
significant, which demonstrated that the inhibitory effects of
QKL on the inflammatory response became stronger as the
TNF-𝛼 (pg/mL)

60
QKL concentration increased.
∗ Studies have indicated that oxidative stress, TNF-𝛼, and
∗
40 ∗ so forth [4], via the p38 MAPK signaling pathway in
∗
conjunction with other signaling pathways, modulate the
gene expression and activity of enzymes in response to
20 cell stress, and it is hypothesized that protein phospho-
rylation is necessary for the activation of these pathways
0 and the progression of signal transduction [26]. In our
study, the low concentration of QKL significantly reduced
Control

Model

QKL 0.0625%

QKL 0.125%

QKL 0.25%

Minocycline

the expression of p-p38 protein in BV2 cells subjected to

hypoxia/reoxygenation. However, this effect is not equivalent
to its effect on the expression of levels of COX-2 and iNOS.
Together, these results indicate that QKL exerts different
Figure 3: Effect of QKL on the expression of TNF-𝛼 in BV2 cell actions at varying concentrations. The high concentration
supernatants. Cell supernatants were collected after hypoxia/reoxy- of QKL significantly inhibited microglial activation, but it
genation. ELISA was performed to measure the expression of TNF-
may also affect other signaling pathways. However, there
𝛼; the results are expressed as the means ± SD. 𝑁 = 6. ## 𝑃 < 0.01
compared to the control group; ∗∗ 𝑃 < 0.01 compared to the model were dose-dependent differences in the inhibition of p38
group. phosphorylation and inflammatory cytokine expression that
merit further examination.

radicals, causing secondary injury [17, 18]. Furthermore, Conflict of Interests

TNF-𝛼 itself can enhance the activation of microglia [19, 20].
All authors declare that there is no conflict of interests
Based on the findings of our study, after hypoxia for
regarding the publication of this paper.
12 h followed by reoxygenation for 12 h, the levels of ROS
increased and the concentration of TNF-𝛼 in the cell culture
supernatant was significantly increased, but cell viability Authors’ Contribution
decreased. Treatment with the high, middle, or low concen-
tration of QKL clearly inhibited the expression of TNF-𝛼 and Lulu Mana, Shan Wang, Haiyan Zhu, Yanwei Xing, Lixia Lou,
increased cell viability. and Aiming Wu contributed equally to this paper.
6 Evidence-Based Complementary and Alternative Medicine

6 2.0
#
#
Relative mRNA levels of COX-2

Relative mRNA levels of iNOS

1.5
4

1.0 ∗ ∗

2 ∗ ∗
0.5

0 0.0
Control

Model

QKL 0.0625%

QKL 0.125%

QKL 0.25%

Minocycline

Control

Model

QKL 0.0625%

QKL 0.125%

Minocycline
QKL 0.25 %
(a)
QingkaiLing (%)
Control Model 0.0625 0.125 0.25 Minocycline

72 kD COX-2

130 kD iNOS

42 kD 𝛽-Actin

(A)

1.5 2.0
#
Relative optimal density of COX-2

Relative optimal density of iNOS

1.5
1.0 #

∗∗ 1.0 ∗ ∗∗
∗∗ ∗∗
0.5
0.5

0.0 0.0
QKL 0.25 %
Control

Model

QKL 0.0625%

QKL 0.125%

Minocycline

Control

Model

QKL 0.0625%

QKL 0.125%

Minocycline
QKL 0.25 %

(B)
(b)

Figure 4: Hypoxia/reoxygenation-induced changes in the protein and mRNA expression levels of COX-2 and iNOS; 𝛽-actin was also
quantified as a control. (a) The mRNA expression levels of COX-2 and iNOS were determined via real-time PCR. (b) The cellular protein
expression levels of COX-2 and iNOS were analyzed via Western blot. # 𝑃 < 0.01 compared to the control group; ∗ 𝑃 < 0.05 and ∗∗ 𝑃 < 0.01
compared to the model group.
Evidence-Based Complementary and Alternative Medicine 7

1.5

Relative optimal density of p-p38

##
1.0
∗∗ ∗∗
QingkaiLing (%)
Control Model 0.0625 0.125 0.25 Minocycline
0.5
38 kD p-p38

38 kD p38
0.0

Control

Model

QKL 0.0625%

QKL 0.125%

Minocycline
QKL 0.25%
(a) (b)

Figure 5: Effect of QKL on p38 phosphorylation in BV2 microglial cells exposed to hypoxia/reoxygenation. (a) Western blot was performed
on the cell lysates using anti-p-p38 and anti-p38 antibodies. (b) The differences in the protein expression levels between the groups were
analyzed using Image J software. ## 𝑃 < 0.01 compared to the control group. ∗∗ 𝑃 < 0.01 compared to the model group.

Acknowledgments [10] F. Cheng, W. Song, S. Guo, D. Wang, and Q. Wang, “Meta-

analysis of clearing heat and removing toxicity therapy on
This study was supported by the National Natural Science ischemic stroke,” Pharmacology and Clinics of Chinese Materia
Foundation of China (Grant no. 81173229) and the indepen- Medica, vol. 27, no. 1, pp. 106–109, 2011.
dent subject of Beijing University of Chinese Medicine (no. [11] C. Li, X. Wang, and S. Chen, “Clinical study on acute upper
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Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2014, Article ID 571076, 13 pages
http://dx.doi.org/10.1155/2014/571076

Research Article
Time-Course of the Effects of QSYQ in Promoting
Heart Function in Ameroid Constrictor-Induced Myocardial
Ischemia Pigs

Qi Qiu,1 Yang Lin,1 Cheng Xiao,2 Chun Li,3 Yong Wang,3 Kexu Yang,1 Wei Suo,1
Yu Li,3 Wenjing Chuo,3 Yongxiang Wei,1 and Wei Wang3
1
Capital Medical University Beijing An Zhen Hospital, Beijing 100029, China
2
China-Japan Friendship Hospital, Beijing 100029, China
3
Beijing University of Chinese Medicine, Beijing 100029, China

Correspondence should be addressed to Yongxiang Wei; yongxiang [email protected] and Wei Wang; [email protected]

Received 24 October 2013; Revised 9 March 2014; Accepted 9 March 2014; Published 10 April 2014

Academic Editor: Joen-Rong Sheu

Copyright © 2014 Qi Qiu et al. This is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

We aim to investigate the therapeutic effects of QSYQ on a pig myocardial ischemia (MI) model and to determine its mechanism of
action. The MI model was induced by Ameroid constriction of the left anterior descending coronary (LAD) in Ba-Ma miniature pigs.
Four groups were created: model group, digoxin group, QSYQ group, and sham-operated group. Heart function, Ang II, CGMP,
TXB2 , BNP, and cTnT were evaluated before (3 weeks after operation: 0 weeks) and at 2, 4, and 8 weeks after drug administration.
After 8 weeks of administration, the pigs were sacrificed for cardiac injury measurements. Pigs with MI showed obvious histological
changes, including BNP, cTnT, Ang II, CGRP, TXB2 , and ET, deregulated heart function, and increased levels of apoptotic cells
in myocardial tissue. Treatment with QSYQ improved cardiac remodeling by counteracting those events. The administration of
QSYQ was accompanied by a restoration of heart function and of the levels of Ang II, CGRP, TXB2 , ET BNP, and cTnT. In addition,
QSYQ attenuated administration, reduced the apoptosis, and decreased the level of TNF-𝛼 and active caspase-3. In conclusion,
administration of QSYQ could attenuate Ameroid constrictor induced myocardial ischemia, and TNF-𝛼 and active caspase-3
seemed to be the critical potential target of QSYQ.

1. Introduction left ventricular ejection fraction (LVEF) and do not have

beneficial effects in the treatment of early-stage hemody-
Cardiac dysfunction caused by myocardial ischemia is namic disorders [2, 3]. In recent years, the study of traditional
the difficulty in clinical treatment. Clinical studies sug- Chinese medicine (TCM) prevention and treatment of heart
gest that diuretics and vasodilators are able to improve failure has led to a large number of basic and clinical research
the clinical symptoms of patients with cardiac insuffi- results based on complex components of many molecular
ciency. ACEI (angiotensin-converting enzyme inhibitor), 𝛽- targets [4, 5].
receptor blockers, and aldosterone receptor antagonists can Apoptosis is suggested to be a key event in ischemia-
improve the prognosis. However, positive inotropic agents reperfusion injury, resulting in LV dysfunction, remodeling,
and vasodilators can improve the clinical symptoms at an and heart failure [6, 7]. One study found that, in patients
early stage, but long-term use can lead to increased mortality, with end-stage heart failure, the incidence of myocardial
and some drugs may also decrease the survival rate [1]. apoptosis was 0.08–0.25%, while the normal myocardium
Angiotensin-converting enzyme inhibitors (ACEI) and 𝛽- apoptosis rate was 0.001-0.002% [8]. Moderate and sustained
receptor blockers, though they improve myocardial function levels of myocardial apoptosis may lead to heart failure [9].
in the long term, inhibit myocardial remodeling and increase Therefore, drugs that inhibit cardiomyocyte apoptosis in
2 Evidence-Based Complementary and Alternative Medicine

patients with residual cardiac function may be protective antibody (Abcam, USA, cat. no. Ab6671); rabbit polyclonal
and may delay and inhibit the progression of heart failure. TNF receptor I antibody (Abcam, USA, cat. no. Ab19139);
Ginseng, Carthamus tinctorius, and other traditional Chinese rabbit polyclonal active caspase-3 antibody (Abcam, USA,
medicines and their active ingredients can inhibit cardiomy- cat. no. Ab13847); rabbit polyclonal caspase-8 antibody
ocyte apoptosis after ischemia [10, 11]. Qin et al. have reported (Abcam, USA, cat. no. Ab15552); mouse monoclonal [6C5]
that inhibition of cell apoptosis significantly improves heart GAPDH-antibody (Abcam, USA, cat. no. Ab8245); and ECL
function in myocardial infarction rabbits [12]. In particular, plus (GE, USA; cat. no. 2134).
TNF-𝛼/caspase-3-mediated apoptosis is considered the main
apoptotic pathway in heart failure (HF) [13]. Therefore, in 2.2. Animal Handling Procedure. Forty male Ba-Ma minia-
vivo detection of apoptosis may not only prove clinically ture pigs (20 ± 2 kg) were purchased from the Institute of
useful in diagnosis and prognosis but also indicate that the Experimental Animals in the Fuwai Hospital Experimen-
TNF-𝛼/caspase-3 pathway is potential therapeutic target [14]. tal Animal Center. The pigs were housed under standard
Qishenyiqi (QSYQ) is a traditional Chinese medicine that laboratory conditions, fed three times a day, and given tap
has long been used for the routine treatment of coronary water ad libitum. Experimental procedures were reviewed
heart disease (CHD) and chronic heart failure (CHF) in and approved by the Animal Care and Use Committee and
China [15]. It consists of six Chinese herbs (Radix Astragali the Ethics Committee in the Fuwai Hospital before the animal
Mongolica, Salvia miltiorrhiza bunge, FlosLonicerae, Poria, experiments were carried out.
Radix Aconiti Lateralis Preparata, and Radix Glycyrrhizae)
and is widely produced in China in accordance with the
Chinese Pharmacopoeia standard of quality control [15]. Our 2.3. Surgical Protocol. After 1 week of dietary modification,
previous study found that QSYQ ameliorates myocardial all animals underwent the surgical procedure. The day
hypertrophy and remodeling by inhibiting the expression of before surgery, all animals received aspirin (325 mg orally)
AngII in LAD rats [16]. However, little is known about the and were fasted for 12 h. Prior to surgical procedure, all
exact targets of QSYQ in its effects on myocardial remodeling. animals received prophylactic antibiotics and buprenorphine
The purpose of the present study is to investigate whether (0.03 mg/kg, intramuscular) for pain control. All survival
QSYQ treats HF by improving left ventricular remodeling procedures were conducted in a sterile fashion. For all
associated with apoptosis. surgical procedures, animals were given general anesthesia
after sedation with Telazol (4 mg/kg, intramuscular), fol-
lowed by endotracheal intubation and ventilation with a
volume-cycled ventilator (North American Dragger). Gen-
2. Materials and Methods eral anesthesia was maintained with a gas mixture of oxygen
2.1. Materials. This study used the following materials: at 1.5–2 L/min and 3% isoflurane. The animal’s vital signs
Ameroid constrictor, 2.75 mm diameter (Research Instru- were recorded during surgery and throughout postopera-
ment SW, USA); anerdian (Shanghai Likang Disinfec- tive recovery. Femoral access via a percutaneous stick was
tant High-Tech limited, batch no. 20050808); ketamine achieved for arterial access, blood draws, and blood pres-
hydrochloride injection (Jiangsu Hengrui Medicine Com- sure monitoring. An Ameroid constrictor (internal diameter
pany Limited, lot no. KH050302); diazepam injection (Tian- 2.75 mm) was placed on the proximal left circumflex coronary
jin Jin Hui Amino Acid Company Limited, lot no. 0506131); artery (LCx) via small left thoracotomy through the fourth
benzylpenicillin sodium for injection (North China Phar- intercostal space (Figure 1). Sham-operated animals (𝑛 = 6)
maceutical Company Limited batch no. D0406207); Ultra- received no Ameroid ring, but other operations were identical
vist 370 iopromide injection (Ernst Schering (Guangzhou) to the model group (Figure 1).
Pharmaceutical Co., Ltd., lot no. 43617); meglumine dia- Thirty minutes prior to the end of each procedure,
trizoate injection (X-ray contrast agent) (Shanghai Asahi a dose of buprenorphine (0.03 mg/kg, intramuscular) was
Donghai Ching Pharmaceutical, batch no. 041003); apop- administered. After each procedure, the dosage of aspirin was
tosis detection kit (In Situ Cell Death Detection kit, POD continued for five days, while a fentanyl patch (4 𝜇g/kg) was
Roche, Germany, Cat. no. 11 684-910); disodium hydrogen applied for 72 h for pain control.
phosphate (Beijing Eddie Fine Chemicals Limited, lot no.
990,329); sodium dihydrogen phosphate (Beijing Hongxing 2.4. Preparation and Dose Calculation of Concentrated QSYQ.
Chemical Plant, lot no. 870,509); formaldehyde (Beijing The QSYQ used in the present study was manufactured
Chemical Reagent Company, batch no. 03030); Lys-C (Roche, by Beijing University of Chinese Medicine (Beijing, China)
Germany); modified trypsin (Roche, Germany); BCA pro- using the six constituent Chinese herbs at a composition of
tein Quantification kit, 5× SDS-protein sample preparation 460 g Radix Astragali Mongolica, 230 g Salvia miltiorrhiza
fluid, concentrated gel buffer solution, separation gel buffer bunge, 160 g Flos Lonicerae, 160 g Scrophularia, 140 g Radix
solution, 30% bis-acrylamide/acrylamide, cellulose nitrate Aconiti Lateralis Preparata, and 90 g Radix Glycyrrhizae
film, and Kodak films (Beijing Pulilai Genetic Technologies [14, 17]. Briefly, following extraction with 95% ethanol, the
Limited); the phosphatase inhibitor PMSF and protease residue of Radix Astragali Mongolica was mixed with Salvia
inhibitors (Nanjing KGI Biotechnology Developments Lim- miltiorrhiza bunge, Flos Lonicerae, Scrophularia, and Radix
ited); EasySee Western Marker (20 kDa–90 kDa) (Beijing- Glycyrrhizae, followed by extraction with hot water (twice,
Gold Biotechnology Company); rabbit polyclonal TNF-𝛼 2 h each). The water extract was then concentrated to form
Evidence-Based Complementary and Alternative Medicine 3

Sham group Model group

Figure 1: Model of Ameroid constrictor-induced MI caused cardiac dysfunction in pigs.

a paste, and ethanol was added for 24 h. We then collected 2.8. Histology. The ventricles were fixed in 4% paraformalde-
the filtrate to form the final product. In the present study, we hyde, and paraffin-embedded hearts were sectioned at
used the dose of 0.33 g/kg, which was based on the clinical 200 mm intervals from base to apex, and serial sections of
application dose of 20 g per day per 60 kg body weight. 4 mm were cut and placed on polylysine-coated glass slides.
Tissue sections were deparaffinized and stained with Masson’s
2.5. Experimental Groups and Drug Treatment. All the model trichrome reagent.
animals underwent echocardiography at the end of the third
week after operation and were randomly assigned to 3
experimental groups (𝑛 = 8): control group, a group treated 2.9. TUNEL Method. For in situ detection of DNA frag-
with digoxin (0.0123 mg/kg body weight per day, Shanghai mentation in paraffin-embedded tissue sections, the TUNEL
Pharmaceutical Group Co., Ltd., the Hsin Yi Pharmaceutical method was performed using the In Situ Cell Death Detec-
Factory, batch no. 100505), and a group treated with QSYQ tion kit, POD (Roche Molecular Biochemicals, Indianapo-
(0.33 g/kg body weight per day). The medication started on lis, IN) following the manufacturer’s instructions. Briefly,
the day after evaluation (the first day of the fourth week after sequential 4 𝜇m tissue sections were adhered to silane-coated
operation) and continued to the end of the twelfth week after slides and allowed to dry at RT. Subsequently, sections
operation (lasting 8 weeks). were deparaffinized and rehydrated. Proteins were digested
by incubating tissue sections in 20 𝜇g/mL proteinase K
(Worthington Co., Lakewood, USA) for 15 min at room
2.6. Echocardiography. Transthoracic echocardiography of temperature (RT). Endogenous peroxidase was inactivated
all the animals was performed using an Agilent SONOS 5500 with 2% H2 O2 in distilled water (dH2 O) for 5 min at RT.
before medication and at 2, 4, and 8 weeks after medication. The labeling mixture containing biotinylated dUTP in TdT
Three-dimensional biplane Simpson’s method [18] was used enzyme buffer was added to sections and incubated at 37∘ C
to measure the LV end-diastolic volume (LVEDV) and LV in a humidified chamber for 1 h. After stopping the enzymatic
end-systolic volume (LVESV). LVEDV was assumed to be reaction, sections were rinsed with PBS for 15 min, covered
the largest volume, and LVESV was chosen as the smallest with anti-digoxigenin peroxidase conjugate, and incubated
volume. LV stroke volume (LVSV) was calculated as the for 30 min at RT in a humidified chamber. Then, sections were
difference between diastolic and systolic volume, and LVEF incubated in TBS with 0.05% diaminobenzidine (DAB) plus
was calculated as ratio of LVSV to LVEDV. 3% H2 O2 until color development. Sections were rinsed in
PBS, dehydrated, and mounted. In control sections in which
2.7. Measurement of CGRP, AngII, TXB2 , ET, BNP, and the enzyme TdT was omitted from the reaction solution, no
cTnT by Radioimmunoassay (RIA). The plasma (1 mL) was stained nuclei were observed.
homogenized in saline containing enzyme inhibitor (10 𝜇L
of 0.3 M EDTA-Na, 10 𝜇L of 0.34 M 8-hydroxyquinoline, and
5 𝜇L of 0.32 M dimercaptopropanol) on ice. The homogenate 2.10. Transmission Electron Microscope Test. From each group
was centrifuged at 8000 ×g for 10 min. The supernatant was the cardiac tissues were minced into small pieces (≤1 mm3 )
used for determination of calcitonin gene-related peptide and fixed in 2.5% glutaraldehyde in 0.1 mol/L sodium cacody-
(CGRP), angiotensin II (AngII), thromboxane B2 (TXB2 ), late buffer (pH 7.3) for 2 hrs. The specimens were rinsed in
endothelin (ET), brain natriuretic peptide (BNP), and tro- buffer, postfixed in cacodylate-buffered 2% OsO4, stained en
ponin T (cTnT), using a RIA kit (Beijing Kangyuan Ruide bloc in uranyl acetate, dehydrated gradient in ethanol, and
Biotechnology Co., Ltd., Beijing, China) following the man- embedded in epoxy resin. Finally, 50–70 nm super thin slices
ufacturer’s instructions. were prepared, stained with uranyl acetate and lead citrate,
4 Evidence-Based Complementary and Alternative Medicine

and examined under an electron microscope (JEOL-1230, 3. Results

Japanese Electronics Company, Japan).
An Ameroid constrictor-induced Ba-Ma pig model of
myocardial ischemia was used in this study. Due to differ-
ences in the coronary artery diameter in Ameroid ring mod-
2.11. Heart Preparation and Protein Extraction. The heart was els, the judgment of the administration time is important.
excised and incubated in ice-cold PBS to wash out blood Our previous study found that, 3 weeks after surgery, the left
at the end of 8 weeks after medication (𝑛 = 6). Each coronary artery of model animals was 100% blocked, which
left ventricle was then carefully dissected to remove all the suggests the formation of myocardial ischemic lesions. We
necrotic/scarred zones to keep only the viable myocardium choose 4 weeks after operation to administer drugs in this
in the marginal zone of the infarct region in model animals. study, with digoxin as the control drug. Macroscopic signs of
The left ventricular myocardium below the ligation in sham the animals in each group, cardiac function, and endocrine-
animals was also dissected. The samples were then immedi- related indicators were monitored before administration and
ately frozen in liquid nitrogen and stored at −80∘ C. at 2, 4, and 8 weeks after administration.
Frozen tissue from 6 animals of each group was ground
to a powder with liquid nitrogen using a mortar and pestle.
Tissue extraction medium (40 mM Tris-HCl pH 7.4, 7 M urea, 3.1. QSYQ Improved Heart Function in Ameroid Induced CHF
2 M Thiourea, 1% w/v DTT, and 1 mM EDTA) (1 : 10) and Pigs. Time-course echocardiography was used to assess the
protease inhibitor cocktail (1 : 50) were then added to the heart function in the four groups to evaluate the role of QSYQ
powder, and the mixture was ultrasonicated for 30 s. Ten in preventing heart from myocardial ischemia. In compar-
microliters of RNase and 5 𝜇L DNase were added, and the ison with the sham group, myocardial ischemia caused a
reaction was incubated on ice for 20 min. The samples were significant decline in LVEF and an increase in LVSV and
centrifuged at 12,000 ×g at 4∘ C for 20 min to remove the insol- LVDV (Table 1), indicating an impairment of heart function.
uble material. After centrifugation, the protein concentration Treatment with QSYQ at 0.33 g/kg and digoxin protected
of each sample was quantitated using a Bradford assay and against this impairment; furthermore, the improvement of
used for subsequent analysis. EF% of QSYQ was significant when compared with digoxin
treatment.

2.12. Western Blot Analysis. Protein samples were prepared 3.2. Serum Levels of CGRP, TXB2 , AngII, ET, BNP, and cTnT.
as described above and subjected to western blot analysis. The time-course RIA analysis of CGRP, TXB2 , AngII, ET,
The samples (50 𝜇g) were separated by SDS-PAGE (12.5% or BNP, and cTnT showed that myocardial ischemia caused the
15% gel) at 100 V for 2 h. Gels were then transferred to a NC increase of serum TXB2 , AngII, cTnT, ET, and BNP (𝑃 <
membrane that had been presoaked for 10 s in transfer buffer 0.01 or 𝑃 < 0.05, Figures 2(b), 2(c), 2(d), 2(e), and 2(f)).
(25 mM Tris, pH 7.4, 192 mM glycine, and 20% methanol) at The increase of AngII, ET, BNP, and cTnT was significantly
300 mA for 1.5 h or 2 h. Each NC membrane was blocked for suppressed by QSYQ treatment (𝑃 < 0.01 or 𝑃 < 0.05,
2 h with 0.5% dried skim milk in TBS-T (20 mM Tris, 500 mM Figures 2(c), 2(d), 2(e), and 2(f)). Digoxin treatment showed
NaCl, and 0.05% v/v Tween 20) at RT, washed three times decreased trend but without significant changes. However,
for 15 min each with TBS-T, and then incubated with a spe- the opposite was true for CGRP. CHF pigs treated with
cific primary antibody (anti-TNF-𝛼, anti-TNFR1, anti-active QSYQ and digoxin had significantly lower CGRP compared
caspase-3, anti-caspase 8, and anti-GAPDH, Abcam, USA) in to model pigs (𝑃 < 0.01 or 𝑃 < 0.05, Figure 2(a)).
TBS-T with gentle shaking overnight at 4∘ C. Each membrane
was washed three times for 15 min each with TBS-T and 3.3. QSYQ Prevents Pathological Changes of Cardiac Tissue
then incubated with the secondary antibody (horseradish in MI Pigs. To investigate the roles of QSYQ in protecting
peroxidase-labeled goat anti-rabbit IgG) in TBS-T with gentle CHF, we initially examined the heart morphological changes
shaking at 37∘ C for 1 h. Each membrane was rinsed three in the different groups. Eight weeks after drug administration,
times for 15 min each with TBS-T, developed with the Super pathological examination showed that the cardiac myocytes
Enhanced Chemiluminescence Detection kit (GE healthcare, exhibited an irregular shape and arrangement, with myocar-
USA). All the membranes were exposed in and scanned by dial fibrosis (Figure 3). Masson’s trichrome staining showed
a ChemiDoc XRS+ (Bio-Rad). A semiquantitative analysis that the fibrotic area was significantly greater in the model
based on the OD values was performed by Image Lab group than in the sham group (Figure 4). Moreover, the
Software (Bio-Rad), using ANOVA to compare groups. number of cardiac myocytes was greatly reduced. Notably,
QSYQ treatment significantly suppressed these phenotypes
in the model animals and decreased the fibrotic areas, while
2.13. Statistical Analysis. Statistical analysis was performed digoxin treatment did not suppress those phenotypes.
with SPSS version 17.0. All data are presented as the mean ±
standard deviation (SD). Statistical analysis was carried out 3.4. QSYQ Treatment Inhibits Apoptosis in CHF Pigs after MI.
on three or more groups using one-way analysis of variance Apoptosis is one of the major outcomes of CHF. Our previous
(ANOVA) and Dunnett’s test. Values of 𝑃 < 0.05 were findings motivated us to further investigate the impact of
considered statistically significant. QSYQ on myocardial cell apoptosis. By TUNEL assay, we
 Table 1: The comparison of echocardiography assessment of left ventricular function at each time point in each group (𝑥̄± 𝑠).
Time Group 𝑛 LVSV mL ΔLVSV mL LVDV mL ΔLVDV mL EF% ΔEF%
Sham 4 6.51 ± 2.40 — 26.70 ± 8.27 — 76.93 ± 4.22 —
Model 6 20.05 ± 2.08∗ — 43.18 ± 4.08 — 51.85 ± 7.50∗∗ —
Before administration
Digoxin 6 27.53 ± 12.97∗∗ — 51.73 ± 21.38∗ — 47.90 ± 14.07∗∗ —
QSYQ 6 29.30 ± 7.93∗∗ — 46.77 ± 11.41∗ — 36.90 ± 12.70∗∗ —
Sham 4 5.76 ± 1.83 −0.75 ± 0.57 25.20 ± 4.06 −1.50 ± 12.33 77.68 ± 3.33 0.75 ± 0.89
Model 6 20.00 ± 5.04∗ −0.05 ± 2.963 39.40 ± 12.15 −3.78 ± 8.07 46.05 ± 7.55∗∗ −5.80 ± 0.05
Evidence-Based Complementary and Alternative Medicine

2 weeks after administration

Digoxin 6 20.67 ± 9.67∗ −6.86 ± 3.30∗△△ 46.00 ± 15.30∗ −5.73 ± 6.08 56.78 ± 15.04∗∗ 8.88 ± 0.97∗∗△△
QSYQ 6 25.53 ± 5.78∗ −3.77 ± 2.15 47.97 ± 7.82 1.20 ± 3.59 47.13 ± 3.99∗∗ 10.23 ± 8.71△△󳵳
Sham group 4 6.77 ± 2.85 0.26 ± 0.45 30.63 ± 7.12 3.93 ± 15.39 79.05 ± 5.61 10.23 ± 9.78
Model group 6 22.85 ± 5.31∗∗ 2.80 ± 3.23 50.08 ± 11.76∗ 6.90 ± 7.68 50.34 ± 7.44∗∗ −1.51 ± 0.06
4 weeks after administration
Digoxin group 6 20.83 ± 9.22∗ −6.70 ± 3.75∗△△ 44.95 ± 12.43 −6.78 ± 8.95∗∗△△ 55.23 ± 15.95∗∗ 7.33 ± 1.88∗∗△△
QSYQ 6 20.67 ± 12.65∗ −8.63 ± 4.72△ 44.97 ± 21.84 −1.80 ± 10.43∗∗△△ 55.80 ± 8.23∗∗ 18.90 ± 4.47∗△△󳵳󳵳
Sham group 4 7.10 ± 3.18 0.59 ± 0.78 28.98 ± 6.98 2.28 ± 15.25 74.88 ± 6.84 18.90 ± 12.03
Model group 6 21.88 ± 7.65∗∗ 1.83 ± 5.57 46.88 ± 13.52∗ 3.70 ± 9.44 50.71 ± 8.97∗∗ −1.14 ± 1.47
8 weeks after administration
Digoxin group 6 18.03 ± 6.20∗ −9.50 ± 6.77∗∗△△ 42.75 ± 7.67 −8.98 ± 13.71 53.98 ± 14.74∗∗ 6.08 ± 0.67∗∗△△
QSYQ 6 17.81 ± 8.49 −11.49 ± 0.56∗∗△△ 30.07 ± 10.09 −16.70 ± 1.32∗∗△△ 55.87 ± 5.02∗ 18.97 ± 7.68∗∗△△󳵳󳵳
Note: ∗ 𝑃 < 0.05 and ∗∗ 𝑃 < 0.01 compared with sham group; △ 𝑃 < 0.05 and △△ 𝑃 < 0.01 compared with model control group; and 󳵳 𝑃 < 0.05 and 󳵳󳵳 𝑃 < 0.01 compared with digoxin group.
ΔLVSV = LVSV𝑡 − LVSV before administration , ΔLVDV = LVDV𝑡 − LVDV before administration , and ΔEF% = EF%𝑡 − EF% before administration .
5
6 Evidence-Based Complementary and Alternative Medicine

300 400

300
200
CGRP (pg/mL)

∗∗

TXB2 (pg/mL)
∗∗
∗∗ ∗
200 ∗ ∗
∗󳵻 ∗ ∗
∗󳵻 󳵻 ∗
∗ ∗ ∗
100 ∗
∗∗ ∗∗ ∗ 100
∗∗ ∗∗

0 0
0 2 4 6 8 10 0 2 4 6 8 10
Time (weeks) Time (weeks)
(a) (b)
400 500

400
300 ∗∗
∗∗
∗ ∗∗ ∗
AngII (pg/mL)

∗∗ 300
ET (ng/mL)

∗ ∗∗
200
󳵻 󳵻 ∗ ∗
200 ∗∗
󳵻
∗
100
100

0 0
0 2 4 6 8 10 0 2 4 6 8 10
Time (weeks) Time (weeks)
(c) (d)
40 0.8

∗∗
30 0.6
∗ ∗∗
∗
BNP (pg/mL)

cTnT (ng/mL)

∗∗
20 ∗ ∗
∗ ∗ 0.4
󳵻󳵻

10 󳵻
0.2

0
0 2 4 6 8 10 0 2 4 6 8 10
Time (weeks) Time (weeks)

Sham Digoxin Sham Digoxin

Model QSYQ Model QSYQ
(e) (f)

Figure 2: CGRP, TXB2 , AngII, ET BNP, and cTnT in plasma as measured by RIA (𝑛 = 6). QSYQ: Qishenyiqi. ∗ 𝑃 < 0.05 compared with sham
group, ∗∗ 𝑃 < 0.01 compared with sham group, Δ 𝑃 < 0.05 compared with model group, and ΔΔ 𝑃 < 0.01 compared with model group.
Evidence-Based Complementary and Alternative Medicine 7

Sham

Model

Digoxin

QSYQ

Figure 3: Morphology (×200). HE results in different groups. Sham: sham group. Model: model group. Digoxin: digoxin group. QSYQ: QSYQ
group.

found that increased numbers of apoptotic myocardial cells Western blots showed that cardiac TNF-𝛼, TNFR1, active
were present in CHF pigs, whereas QSYQ treatment dramat- caspase-3, and caspase-8 (Figure 7) in the Control group
ically decreased the apoptosis rate (Figure 5). Digoxin did not increased (𝑃 < 0.01) compared with the sham-operated
decrease the apoptosis rate significantly when compared with group, while treatment with QSYQ for 8 weeks reduced TNF-
model group. 𝛼, active caspase-3, and caspase-8 compared with the Control
group (𝑃 < 0.05), to a level similar to sham group (Figure 7).
3.5. Transmission Electron Microscopy Results of the Heart Digoxin treatment inhibited the caspase-3 activation but did
Tissue in Different Groups. Normal cardiac ultrastructure not work on TNF-𝛼 expression.
was shown in sham group. Pyknotic mitochondria and
myogenic fragmentation are observed in model and digoxin 4. Discussions
group. QSYQ treatment prevented mitochondria pyknotic
and myogenic fragmentation, but digoxin did not (Figure 6). Improving heart function is the most important part of
treatment of CHF. Digoxin is one of the most widely
3.6. QSYQ Treatment Decreases TNF-𝛼 and Active Caspase- used drugs which functions by inhibiting the 𝛼-subunit
3. TNF-𝛼, TNFR1, active caspase-3, and caspase-8 levels are of cell-membrane Na+ , K+ -ATPases, promoting Na+ -Ca2+
critical in the prognosis for myocardial apoptosis [19, 20]. exchange, and increasing intracellular Ca2+ concentration,
8 Evidence-Based Complementary and Alternative Medicine

Sham

Model

Digoxin

QSYQ

Figure 4: Morphology (×200). Masson results in different groups. Sham: sham group. Model: model group. Digoxin: digoxin group. QSYQ:
QSYQ group.

which acts on the contractile proteins, resulting in enhanced are decreases in myocardial contractility, left ventricular
myocardial contractility [21]. In addition, digoxin can pump function, and cardiac output, resulting in inadequate
improve CHF patients’ baroreceptor sensitivity and inhibit organ perfusion. The end-diastolic volume is a reflection of
AV nodal conduction. Digoxin increases LVEF and improves both structural remodeling and diastolic filling (end-diastolic
hemodynamic parameters in a dose-dependent manner, but myocyte fiber length). The end-systolic volume is influenced
it increases the mortality of female patients [22]. Based on by both the end-diastolic volume and fiber shortening,
the exact cardiac function improvement efficacy, we chose but asymmetric contraction may make echocardiographi-
digoxin as a positive control to observe the onset time and cally derived measures of end-systolic volume inaccurate.
degree of heart function improvement of QSYQ. Although heart rate and fiber shortening both affect ejection
The present study showed that, as the treatment time fraction, ejection fraction is influenced to a far greater extent
increased, QSYQ-treated pigs showed a gradual trend of by end-diastolic volume because changes in stroke volume
recovery of heart function and significant improvement in tend to be much smaller than changes in end-diastolic
myocardial injury indicators. The main clinical manifes- volume [23]. BNP, a polypeptide consisting of 32 amino
tations of cardiac insufficiency after myocardial ischemia acids, is mainly secreted by the ventricles with the feature of
Evidence-Based Complementary and Alternative Medicine 9

(A) (B) (C) (D)

0.5

0.4 ∗∗ ∗∗

0.3 ∗󳵻
(%)

0.2

0.1

0.0
Sham Model Digoxin QSYQ

Figure 5: QSYQ inhibits apoptosis in pigs with HF. TUNEL analysis was carried out 8 weeks after drug treatments. (A) TUNEL-positive cells
(apoptotic cells) in the sham group; (B) model; (C) digoxin; (D) QSYQ. ∗ 𝑃 < 0.05 compared with sham group, ∗∗ 𝑃 < 0.01 compared with
sham group, Δ 𝑃 < 0.05 compared with model group, and ΔΔ 𝑃 < 0.01 compared with model group.

explosive synthesis and short half-life, and shows sensitivity two are mutually antagonistic in pathological states [29].
and specificity to ventricular dysfunction. As the ventricular Angiotensin II is a major peptide in the renin-angiotensin
volume and pressure load are proportional and closely related system. Its biological effects include blood vessel contraction,
to left ventricular function, BNP has become an important fibrinolysis inhibition, and tissue fibrosis promotion. It plays
biochemical indicator of the level of cardiac function in an important role in the progression of coronary heart
CHF patients [24, 25]. cTnT is a specific structural protein, disease. Myocardial ischemia and hypoxia can increase the
a subunit of troponin complexes in myofibrillar filaments activity of the RAS system and increase circulating AngII,
with calcium binding sites, that is primarily involved in the which may be an important indicator of the presence of
activation and regulation of calcium ions in the process coronary heart disease and blood stasis [30]. TXB2 is the
of muscle contraction. Approximately 95% of the cTnT is stable metabolite of TXA2 and has been used to represent
present in the muscle fiber, and 5% of cTnT is secreted from the levels of TXA2 in clinical situations. Plasma TXB2 is sig-
the myocardial cytoplasm into the blood under physiological nificantly increased in heart failure patients, and benazepril
conditions. This free cTnT in the blood significantly increases can reduce it [31]. TXA2 is a metabolite of arachidonic acid
when myocardial cells are injured [26]. The concentration and is synthesized and released by platelets. It has powerful
of cTnT in the blood and the degree of myocardial injury vasoconstrictor activity by promoting platelet aggregation
are positively correlated with each other and negatively and thrombus formation. TXA2 decreases cAMP in platelets
correlated with congestive heart failure [27]. and vascular smooth muscle cells by inhibiting AC or as
CGRP, AngII, TXB2 , and ET are vasoactive substances, a Ca2+ carrier that directly contributes to Ca2+ influx and
which can regulate vascular contraction and the myocardial dense pipeline system Ca2+ release, which promote platelet
oxygen supply. CGRP is composed of 37 amino acids and aggregation, local vasoconstriction, and endothelial damage
is a powerful arteriovenous vascular relaxant. It is more [32]. The present study showed that QSYQ can regulate the
sensitive in the small vessels and is the most powerful expression of these substances and protect the myocardium
and most enduring relaxing factor, as it increases adeny- from hypoxic injury.
late cyclase (Ac) activity and increases cAMP reserves in Another major finding in this study was that QSYQ can
vascular endothelial cells or vascular smooth muscle cells significantly decrease the apoptosis rate by downregulating
[17, 28]. ET is a potent vasoconstrictor of 21 amino acids both TNF-𝛼 and active caspase-3, but no changes of TNFR
that is synthesized and secreted by endothelial cells, and its or caspase-8 were detected in the myocardial infarct border
abnormal concentration is a sign of vascular endothelial cell zone. TNF-𝛼 is an important inflammatory factor that medi-
injury. Under physiological conditions, ET concentration is ates the development of inflammation. In the myocardial
low, and CGRP is in a state of dynamic equilibrium. The ischemia process, TNF-𝛼 can inhibit myocardial contraction,
10 Evidence-Based Complementary and Alternative Medicine

Sham Model
mito

SR
mf
SR

mf

mito

Digoxin QSYQ

SR
mito

mf
mf SR

mito

Figure 6: Ultrastructure changes. Sham: sham group. Model: model group. Digoxin: digoxin group. QSYQ: QSYQ group. SR: sarcoplasmic
reticulum; mito: mitochondria; and mf: myofibril.

promote myocardial hypertrophy, and induce apoptosis of has been used as a sign of irreversible apoptosis. In addition
cardiac cells. The TNF-𝛼/TNFR1 extrinsic pathway of apopto- to apoptosis-induced biological effects, caspase-3 can also
sis is an important sensor signal [17]. Binding of Fas ligand to shear the muscle fibers cells 𝛼-actin and troponin T [35], and
cell-surface Fas causes a conformational change to the intra- 𝛼-actin and muscle fiber rupture further reduce contractile
cellular death domain (DD) of Fas, causing it to bind to a Fas- function.
associated death domain (Fas associated via death domain,
FADD) adapter protein, to form DISC protein complexes 5. Conclusion
[33]. Activated procaspase-8 and procaspase-10 are recruited
to DISC [34], which activates procaspase-triggered apoptosis. This paper presents a multitarget pharmacological study of
Caspases have numerous important roles in the regulation the Chinese herbal formula QSYQ. This TCM with multiple
of apoptosis. Both endogenous and exogenous apoptosis chemical components targets multiple proteins, which may
pathways are activated by caspase family members, including produce greater efficacy and fewer side effects than any single
caspase-8, caspase-9, and caspase-3, while the antiapoptotic constituent. Our results also show that therapeutic QSYQ
effects of caspase inhibitors have been confirmed. Caspase- administration can regulate vasoactive factors to improve
3 plays an important role as a substrate of apoptosis and myocardial oxygen supply, reduce myocardial injury, improve
Evidence-Based Complementary and Alternative Medicine 11

TNF-𝛼

TNFR1

Active-caspase-3

Caspase-8

GAPDH
(A) (B) (C) (D)
0.8 1.5

∗∗ ∗∗
0.6
1.0

0.4
󳵻󳵻
0.5
0.2

0.0 0.0
TNF-𝛼 TNFR1

Sham Digoxin Sham Digoxin

Model QSYQ Model QSYQ

(a) (b)
0.25 0.8
∗∗

0.20
0.6

0.15
∗󳵻 ∗󳵻󳵻
0.4
0.10

0.2
0.05

0.00 0.0
Active-caspase-3 Caspase-8

Sham Digoxin Sham Digoxin

Model QSYQ Model QSYQ

(c) (d)

Figure 7: The western blot results of (a) TNF-𝛼, (b) TNFR1, (c) active caspase-3, and (d) caspase-8 in the four groups (𝑛 = 6), (A) sham
group, (B) model group, (C) digoxin group, and (D) QSYQ group. ∗ 𝑃 < 0.05 compared with sham group, ∗∗ 𝑃 < 0.01 compared with sham
group, Δ 𝑃 < 0.05 compared with model group, and ΔΔ 𝑃 < 0.01 compared with model group.
12 Evidence-Based Complementary and Alternative Medicine

TNF

TNFR1

T T
R R
A A
D D
D D
FADD YXJD

Caspase-8

Caspase-3

Apoptosis

Figure 8: Potential mechanism by which QSYQ attenuates cell death signaling pathways in cardiac dysfunction.

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Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2014, Article ID 157938, 10 pages
http://dx.doi.org/10.1155/2014/157938

Research Article
Neuroprotective Effect of a Formula, Moschus Combined with
Borneolum Synthcticum, from Traditional Chinese Medicine on
Ischemia Stroke in Rats

Xin-hua Xia,1 Qiang Li,2 and Mei Liu3

1
The First Hospital Affiliated to Guangzhou Medical University, Guangzhou 510120, China
2
The Heart Center, The First Hospital Affiliated to Lanzhou University, Lanzhou 710030, China
3
School of Chinese Material Medical of Guangzhou University of Chinese Medicine, 232 East Waihuan Road,
Guangzhou Higher Education Mega Center, Guangzhou, Guangdong 510006, China

Correspondence should be addressed to Mei Liu; [email protected]

Received 21 November 2013; Accepted 22 February 2014; Published 25 March 2014

Academic Editor: Pitchairaj Geraldine

Copyright © 2014 Xin-hua Xia et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Moschus compatible with borneolum synthcticum is a well-known herb pair in Traditional Chinese Medicine and the present study
aims to assess the neuroprotective effect of a formula composed of this herb pair on ischemia stroke in rats. The middle cerebral
artery occlusion model of focal cerebral ischemia in rat was performed by using intraluminal suture method. The behavioral scores,
infarct volume, and neuron ultrastructure of model and formula-treated rats were investigated after the 2 h of ischemia and 24 h of
reperfusion. Meanwhile the expression levels of caspase-3, caspase-9, Bcl-2, and Bax were measured by western blot analysis. The
formula treatment showed obvious neuroprotective effect according to significant decrease of the neurological scores (𝑃 < 0.01)
and the infarct volumes (𝑃 < 0.05) when compared to the MCAO group. We also observed that this formula had antiapoptosis
activity on neuron cell under electron microscope. Furthermore, our result supported the idea that pro- and postadministration of
this formula had an antiapoptosis effect by decreasing remarkably the expression of caspase-3 and caspase-9 (𝑃 < 0.05) as well as
increasing significantly the ratio of Bcl-2 to Bax (𝑃 < 0.01). All evidences demonstrated the neuroprotective effect of this formula
on ischemia stroke due to decrease of brain infract volume and modulation of the expression of apoptosis-related proteins.

1. Introduction tPA application, which is restricted to up to 3 hours after

symptom onset [5]. As a result, a report from 2008 estimated
Stroke is a cerebrovascular accident that is caused by either that only 1.8 to 2.1% of all stroke patients had been treated
the ischemia or rupture hemorrhage of an artery to the brain with tPA in the United States [6]. On the other hand, some
and results in the interruption of blood flow to the brain patients experienced disastrous outcomes in the form of fatal
[1]. Stroke is in the top three among all causes of death edema or intracranial hemorrhage following thrombolysis.
behind diseases of the heart and cancer [2]. Up to 87% of Some researchers have reported that reperfusion caused by
strokes are ischemia origin which is most attributable to tPA or surgery after a long ischemic period can induce a
the blockage of a artery by a blood clot [3]. Currently, the larger infarct area than that associated with permanent vessel
principle of clinical treatment to acute stroke is restoration occlusion in some animal stroke models [7, 8]. Thus, while
of blood supply as soon as possible for resupplying oxygen to reperfusion may reduce infarct size and improve clinical
ischemic brain tissue [4]. At present, the only US Food and outcome in some patients, in others it may exacerbate the
Drug Administration (FDA) approved treatment of tissue brain injury and produce a so-called cerebral reperfusion
plasminogen activator (tPA) within 3 h of symptom onset injury [9, 10]. With the progress made in the understanding
is available to reestablish cerebral blood flow. However, this of the mechanisms in ischemia stroke and reperfusion injury,
treatment has some limits of the short time window for an increasing number of strategies have been developed
2 Evidence-Based Complementary and Alternative Medicine

for limiting or preventing further brain damage [11–13]. analysis. With this information, we are hoping to offer some
However, the translation of these promising strategies into effective therapeutic alternatives to treat ischemia stroke,
effective therapies in humans has been disappointing. It is which will be an important theoretical significance and
urgent to find therapeutic alternatives to treat ischemic stroke application value for further treatment of ischemic stroke.
and reperfusion injury well.
Interestingly, prescriptions from Traditional Chinese 2. Materials and Methods
Medicine (TCM) are made to treat all kinds of cerebrovas-
cular diseases according to their experience and heritage 2.1. Materials and Animals. Natural moschus (batch num-
from generation to generation over thousand years. A lot of ber: 20100322) and borneolum synthcticum (batch number:
studies have reported that some TCM preparations including 20100413) were purchased from Chinese Herbal Medicine
Qing-Kai-Ling injection, An-Gong-Niu-Huang pill, Tong- Co., Ltd., of Guangdong province, which were qualified by
Xin-Luo capsule, Nao-Shuan-Tong capsule, and Tong-Luo- GC-FID according to Chinese Pharmacopeia 2010 edition.
Jiu-Nao capsule have been widely used in clinical practice 6-0 nylon monofilaments for MCAO were bought from
to treat cerebrovascular diseases and showed effectiveness for Beijing Shadong Biotech Inc. (Beijing, China); nitrocellu-
the treatment of stroke by antioxidation, anti-inflammation, lose membrane, 2,3,5-triphenyltetrazolium chloride (TTC),
and neuroprotective function against ischemic reperfusion Tween 80, formaldehyde, chloral hydrate, and heparin were
injury [14–19]. These preparations were composed of more purchased from Sigma Company (St. Louis, MO, USA).
than ten Chinese herbal medicines with a large number of Monoclonal antibodies of B-cell lymphoma 2 protein (Bcl-
complex components. Among them, the drug combination 2), Bcl-2 associated X protein (Bax), caspase-3, caspase-9, and
of moschus compatible with borneolum synthcticum was 𝛽-actin and horseradish peroxidase-conjugated IgG antibody
considered as the basic and primary unit of these prepa- were purchased from Nanjing Jianye Biotech Inc. (Nanjing,
rations from the view of TCM. Moschus is a rare Chinese Jiangsu, China).
medicine, which is dry secretions and origins from mature SD rats of SPF grade in either sex with the weight between
male moschus deer including Moschus berezovskii Flerov, 200 and 250 g (certificate number: SCXY-YUE-2003-0001)
Moschus sifanicus Przewalski, and Moschus moschiferus Lin- were purchased from Experimental Animal Center of Guang-
naeus. Moschus has been used as a common drug to dong Province. All rats were housed in SPF grade animal
awaken damaged brain in China with a long history. And room with temperature of 25 ± 2∘ C and relative humidity of
borneolum synthcticum is the resin product of Dryobalanops 60% and were feed SPF grade rat food and sterile distilled
aromatica Gaertn.f., which is also a regular refreshing and water. All animal procedures involving animals and their care
resuscitating drug commonly used for treatment of stroke, were conducted in accordance with the guidelines of Animal
phlegm, coma, or related diseases. Both of them are generally Use and Care of the National Institutes of Health (NIH
used as a drug pair in TCM, and borneolum synthcticum Pub. number: 85-23, revised 1996) and were approved by the
mostly acts as an adjuvant or excipient in prescriptions, Animal Care and Use Committee of Guangzhou University
enhancing the efficiency of other drugs like moschus. We of Chinese Medicine.
have extensively made a formula composed of moschus
combined with borneolum synthcticum to treat the acute 2.2. Middle Cerebral Ischemia Reperfusion Model and
stages of cerebrovascular disease and have acquired excellent Experimental Groups. The middle cerebral artery occlusion
result on improving neurological function in clinical practise. (MCAO) model of focal cerebral ischemia in rat brain was
Observing hundreds of cases of ischemic stroke in clinical performed by using intraluminal suture method [20]. Rats
practice, we have found that this formula used in early were fasted 12 h before experimental surgery with free access
stage of ischemic injury could improve patients’ awareness to water. Then all rats were anaesthetized with chloral hydrate
of barriers, shorten the treatment of ischemic stroke, and (0.35 g⋅kg−1 body weight, i.p.) and were allowed to breathe
reduce a series of complications caused by the disturbance of spontaneously. Rats were placed in the supine position on
consciousness. However, the specific neuroprotective phar- the rat board with removal of hair in the center of neck. The
macological action and possible mechanism of this formula right common carotid artery (CCA), external carotid artery
on ischemic stroke are still unclear and have not been (ECA), internal carotid artery (ICA), and the pterygopalatine
reported yet. Therefore, we investigated the neuroprotective artery were exposed and separated along the inner edge of
effects and possible molecular mechanism in order to offer a sternocleidomastoid. Meanwhile vascular branch was being
more effective therapeutic or alternative treatment for clinical electrocauterized to prevent bleeding during separation.
practice of ischemic stroke. The model of ischemia stroke was Then pterygopalatine artery and ECA were ligated at 1cm
performed in rats by middle cerebral ischemia reperfusion distal from the carotid bifurcation, and the distal end was
model (MCAO) method which was similar to the syndromes burned off with coagulator. The CCA was occluded with
of clinical ischemia patients. The behavioral scores, brain aneurysm clips and a small incision was made in the ECA
infarct volume, and neuron ultrastructure of modeled rats with syringe needle at about 0.5 cm away from ligation at
and formula-treated rats were investigated to find out how the proximal end, and then pulling the proximal end of the
the formula works to protect the brain function and repair external carotid artery in straight with the internal carotid
the brain damage. And In order to evaluate the antiapoptosis artery was done. A nylon filament with silicone resin-coated
activity of this formula, the expression levels of caspase-3, tip (about 220 𝜇m in diameter and 4 mm in length) was
caspase-9, Bcl-2, and Bax also were measured by western blot inserted through the incision into the ICA about 18–20 mm.
Evidence-Based Complementary and Alternative Medicine 3

Reperfusion initiated by removing the filament after two solution at 37.0∘ C for 30 min and then fixed by 10% formalin
hours of ischemia by blocking the artery, then the artery was for 24 h. Finally, the stained brain slices were photographed
ligated; meanwhile tissue and skin were sutured. The rats in in sequence with a camera after 24 hours of fixation. Areas of
sham group were conducted with the same procedure of rats red and white staining were measured using an Image-Pro
in MCAO group but with no insertion of nylon filament. Plus6.0 (Media Cybernetics, Wyoming, USA). The percent
Body temperature of rat was kept at 36.5 to 37.0∘ C from of infarction size was calculated by the equation: %Infarct
the start of the surgery until the animal was awake from volume = Infarct volume/Total volume of slice × 100.
anesthesia. Neurological function was evaluated when the
MCAO rats were awake, and those with scores less than 2 2.5. Ultrastructure Change of Neurons. The rat of each group
were excluded from the present study. was decapitated and the brain was quickly removed after
MCAO rats with neurological scores between 2 and 3 24 hours of reperfusion following MCAO injury. The right
were randomly divided into three groups (8 rats for each frontal cortex of brain tissue was cut into pieces of 1 mm3 .
group). The groups were denoted as sham group, vehicle- All pieces were rapidly immerged and prefixed in cold 2.5%
treated MCAO group (MCAO), and formula-treated group glutaraldehyde solution, then they were put and postfixed
(MCAO+MB), respectively. Besides these three groups, other in 1% osmium acid solution. After fixation, the samples
MCAO rats were divided into several subgroups (6 rats for were dehydrated in 50%, 70%, 80%, 90%, and 100% ethanol,
each group) for the dosage and time window assessment. respectively. Finally, the dehydrated brain slices were embed-
The drug injections were prepared by the First Hospital ded in phthalate propylene and then cut into 50 nm ultrathin
Affiliated to Guangzhou Medical University, which were sections. The sections were stained by uranyl acetate and lead
composed of 1 mg⋅mL−1 of moschus and 3 mg⋅mL−1 of citrate. The stained sections were observed by an electron
borneolum synthcticum. The dosages of drugs were cal- microscope.
culated as equivalent dose as the usual dosage for clin-
ical practice in hospital of TCM. The sham and MCAO
2.6. Western Blot Analysis. After 2 h of ischemia followed by
group were administrated intraperitoneally normal saline
24 h of reperfusion, the whole right hemisphere tissue was
of 10 mL⋅kg−1 body weight, the formula-treated group was lysed in lysis buffer for 15 minutes at 4∘ C and then centrifuged
administrated intraperitoneally drug injection with the con- at 2060 ×g for 10 min. The supernatants were removed for
tent of 10 mL⋅kg−1 body weight, and the subgroups for western blot analysis. The same amount of total protein
dose assessment were administrated intraperitoneally drug was isolated by sodium dodecyl sulfate polyacrylamide gel
injection with the content of 15 mL⋅kg−1 , 10 mL⋅kg−1 , and electrophoresis and then was transferred to a polyvinyli-
5 mL⋅kg−1 body weight, respectively. The subgroups for time dene fluoride membrane. This membrane was incubated
window assessment were administrated intraperitoneally with primary monoclonal antibodies of caspase-3, caspase-
drug injection with the content of 10 mL⋅kg−1 body weight. 9, Bax, Bcl-2, and 𝛽-actin overnight at 4∘ C after blocking.
Each group was administrated intraperitoneally the corre- The blots were washed with 5% dry milk in phosphate
lated injection with a dose described above once at 30 min buffered saline/0.1% Tween 20 (PBST) and incubated with
before ischemia and then repeated administration at 2 h, 12 h, secondary antibody of horseradish peroxidase-conjugated
and 24 h after ischemia. IgG at room temperature for 1 hour. The protein bands were
detected using an Amersham enhanced chemiluminescence
2.3. Neurological Deficit Evaluation. Neurological deficits detection system (GE Healthcare, Little Chalfont, UK). The
were measured after 2 h of ischemia and 24 h of reperfusion protein expression of caspase-3, caspase-9, Bax, and Bcl-2 was
based on a five-point scale system reported in Xu’s study analyzed with densitometry scans after normalization with
[21]. Zero score was made for modeled rats with no obvious the corresponding expression of 𝛽-actin.
neurological deficits, one score was made for modeled rats
with mild neurological deficits such as failure to extend 2.7. Statistical Analysis. All data are expressed as means ±
contralateral forepaw on lifting of the animal by tail, two SEM. SPSS 19.0 (SPSS, Chicago, IL, USA) was used for
score was made for modeled rats with moderate neurological statistical analysis. One-way analysis of variance was used
deficits including circling to the contralateral side but normal followed by post hoc analysis for significance with the
posture at rest, three score was made for modeled rats with Student-Newman-Keuls multiple comparison test. 𝑃 < 0.05
severe neurological deficits such as falling to the contralateral was considered statistically significant.
side at rest, and four score was made for modeled rats with
very severe neurological deficits of no spontaneous motor
activity or even death.
3. Results
3.1. Major Components of the Tested Formula Drug. The
2.4. Cerebral Infarct Volume Determination. The modeled formula used in the present study was prepared by the
rats were anaesthetized after 2 h of ischemia and 24 h of First Hospital Affiliated to Guangzhou Medical University.
reperfusion. The brain of each rat was quickly removed from The natural moschus and the geoauthentic borneolum syn-
skull, rinsed with ice-cold saline immediately, and then put thcticum exported from Indonesia and named “Meipian” in
into the ice-cold saline for 15 min and after that the brain Chinese Pinyin were used to prepare the formula injection,
was cut into 2 mm slices. Slices were incubated with 2% TTC which is qualified by GC-FID following the related standards
4 Evidence-Based Complementary and Alternative Medicine

×104 ×104
90 90
O CH3
80 Borneol 80 Borneol
Muskone
70 OH 70
Muskone
60 60

50 50

40 40

30 30

20 20

10 10
Standard reference Formula preparation
0 0
3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
(min) (min)
(a) (b)

Figure 1: The major components of the formula were determined by GC-FID method as follows. Helium at a flow rate of 1.1 mL/min was used
as the carrier gas. The initial oven temperature was 80∘ C, followed by a ramp to 180∘ C at 20∘ C/min, and then ramped to 280∘ C at 30∘ C/min
(held at 280∘ C for 5 min). The injection port and detector temperatures were at 280∘ C, respectively. The hydrogen and air flow rates in FID
40 and 430 mL/min, respectively. (a) Was the GC-FID chromatogram of the standard references mixed with muscone and borneol. (b) Was
the GC-FID chromatogram of the tested formula composed of moschus compatible with borneolum synthcticum.

protocols of CHP 2010 and satisfied the standards in CHP 1.5 times, 1 time, and 0.5 time of equivalent dose of
2010. The contents of muscone and borneol in the formula the clinical dosage. Three subgroups were administrated
injection were determined by GC-FID following the related intraperitoneally corresponding drugs with the content of
quantitative standards protocols established by our own 15 mL⋅kg−1 (MCAO+H/MB), 10 mL⋅kg−1 (MCAO+M/MB),
lab (Figure 1). The muscone was the major component of and 5 mL⋅kg−1 (MCAO+L/MB). When compared with the
moschus and its yield was of 5.04%, and the content of bor- infract size of rats from model group (Figure 3(a)), the
neol was up to 89.6% in the injection formula. This formula infarction size of rats reduced significantly by 70% (𝑃 < 0.01),
contained 50.4 𝜇g of muscone and 2.69 mg of borneol per mL. 64% (𝑃 < 0.01), and 35% (𝑃 < 0.05) in high, moderate, and
low dose groups, respectively.
3.2. Treatments Decreased Neurological Behavioral Score and The time window was also evaluated in the present
Reduced Brain Infarct Volume. Before TTC staining, the study. Injection of 10 mL⋅kg−1 formula drug was equivalent
neurological function score was calculated by behavior and effective dose of clinical dosage at reducing infarct
changes of animal with 5 levels. There were no obvious volume and improving neurological function, so this dose
neurological deficits observed in rats from sham group. was used in time window experiments. Four subgroups
While in the MCAO group more severe neurological deficits were administrated intraperitoneally with the formula drug
were observed including circling movements, severe paw injection at 2, 4, 6, and 9 h after ischemia, respectively. The
flections, and less spontaneous movements. All ischemia rats formula treatment with different time significantly reduced
suffered from neurological impairment, and the scores of cerebral infarct volume (𝑃 < 0.01 or 𝑃 < 0.05, Figure 3(b)).
other rats were significantly higher than sham-operated rats. The maximum reduction of infarct volume was statistically
The neurological scores of formula-treated group declined significantly decreased by 71% in the 2 h group compared to
significantly (𝑃 < 0.05) when compared with rats from that in model group (𝑃 < 0.01). The infarct volumes gradually
MCAO group (Figure 2(a)). As shown in Figure 2(b), the decreased significantly by 56% (𝑃 < 0.01) and 31% (𝑃 < 0.05)
image of the normal brain tissue displayed the color of rose in the 4 h and 6 h treatment groups, respectively. However,
red; meanwhile the image of the infarct area displayed a the cerebral infarct volume of rats in 9 h group was 15% lower
color of pale white after TTC staining. The area of pale white than that in model group with no significance.
shrank evidently in ischemia rats of formula-treated group.
In particular, the infract volume of rats from formula-treated 3.4. Treatments Repaired Ultrastructure Change of Neurons.
group reduced significantly by 63% (𝑃 < 0.01) of the infract The ultrastructural change of neurons was enlarged and
volume in rats from model group (Figure 2(c)). observed by the electron microscope. As shown in Figure
4(a), the double membranes of the nerve cells in brain tissue
3.3. Dose and Time Window Assessment on Infarct Vol- from the sham group were clear to distinguish, and the
ume. In the dose-effect experiments, high dose, moder- nuclear of these cells was large and clear without nuclear
ate dose, and low dose of the formula were set as the chromatin condensation. There was much endoplasmic
Evidence-Based Complementary and Alternative Medicine 5

5
Neurological scores

MCAO +
MB
3
∗
2

1 ∗∗

MCAO
0
Sham MCAO MCAO + MB
(a) (b)
30

25
Infarct volume (%)

20

15
∗∗
10

0
MCAO MCAO + MB
(c)

Figure 2: The comparison on the protective effects of treatments on the neurological deficits and the infarct volume after ischemia stroke. The
rats were administrated intraperitoneally by the tested formula drug at 30 min before ischemia and at 2 h, 12 h, and 24 h after ischemia. After
2 h of ischemia and 24 h of reperfusion, the neurological scores were evaluated according to a graded scoring system described in the method
in Section 2.3, and the infarct volume was calculated by the method described in Section 2.4. (a) Represented the different neurological deficits
scores of sham group, MCAO group, and formula-treated group; (b) was the representative images of TTC-staining brains from MCAO group
and the formula-treated group; the normal brain tissue displayed the color of rose red; meanwhile the infarct area displayed a color of pale
white; (c) showed the different infarct volumes from rats of sham group, MCAO group, and formula-treated group. Bars represent means ±
SEM of six rats; ∗ significant difference at 𝑃 ≦ 0.05. ∗∗ Remarkably significant difference at 𝑃 ≦ 0.01.

30 30

25 25
Infarct volume (%)

∗
Infarct volume (%)

20 ∗ 20

15 15
∗∗
∗∗ 10 ∗∗
10 ∗∗

5 5

0 0
MCAO

MCAO + MB-2 h

MCAO + MB-4 h

MCAO + MB-6 h

MCAO + MB-9 h
MCAO

MCAO + H/MB

MCAO + M/MB

MCAO + L/MB

(a) (b)

Figure 3: The results of dose-dependent and window time experiments: (a) the MCAO rats were administrated intraperitoneally with the
prepared formula drug at high dose of 15 mL/kg body weight, moderate dose of 10 mL/kg, and low dose of 5 mL/kg. (b) The MCAO rats were
administrated intraperitoneally 10 mL/kg at 2 h, 4 h, 6 h, and 9 h of ischemia. ∗ Significant difference at 𝑃 ≦ 0.05 and ∗∗ remarkably significant
difference at 𝑃 ≦ 0.01.
6 Evidence-Based Complementary and Alternative Medicine

(a) (b) (c)

Figure 4: The representative images of neurons in MCAO rats’ brains from different groups under electron microscope (×12 k). (a) Was
a representative image of neuron cell from sham group, (b) was an representative image of neuron cell from MCAO group, and (c) was a
representative image of neuron cell from formula-treat group.

reticulum in cytoplasm and normal mitochondria without as the most important determining factors for the fate of cells
lesions around the nucleus. Figure 4(b) showed that the nerve in response to apoptotic stimulation. The ischemic brain sam-
cells of brain tissue damaged by ischemia from the MCAO ples from all groups were used to determine the protein levels
group shrank obviously, both of the cell membrane and the of Bcl-2 and Bax by western blot analysis (Figure 6(a)). The
nuclear membrane were blurred, and there were nuclear formula treatment significantly increased expression level of
chromatin agglomerated, mitochondria swelled, rough endo- Bcl-2 (𝑃 < 0.05, Figure 6(b)) and decreased expression level
plasmic reticulum expanded, and gap around nerve cells of Bax (𝑃 < 0.05, Figure 6(c)) after ischemia. The ratio of
increased obviously observed under the electron microscope. Bcl-2/Bax was drastically increased in formula-treated rats
However, the nerve cells of brain tissue with ischemic injury (𝑃 < 0.05, Figure 6(d)).
treated by the formula drug had integral double nuclear
membrane. There were no nuclear chromatin condensa- 4. Discussion
tion and no obvious expansion of cytoplasm endoplasmic
reticulum observed under electron microscope, and little Although dramatic progress has been made to alleviate the
mitochondria swelled (Figure 4(c)). impact of stroke on public health and reduce stroke incidence
and mortality, unfortunately, most therapeutic approaches
3.5. Western Blot Analyses. Next, the molecular mechanism developed in the laboratory have failed in large clinical trials.
of neuroprotective activity induced by the formula was Therefore, translational stroke research requires a revaluation
investigated by western blot analysis in MCAO rats. More of traditional approaches and the development of a new
and more evidences have revealed that different kinds of cell conceptual framework to guide therapy. Increasing numbers
death including necrosis, necroptosis, and apoptosis occurred of stroke patients have sought TCM therapy to improve
after cerebral ischemia injury [22, 23]. We also observed the physical functions in recent years that is worth our attention
different morphologic change in neuron cells after ischemia [24]. There are more and more evidence-based studies that
and treatment. Therefore, some markers protein of cell apop- have shown TCM’s beneficial effects in stroke patients [25–
tosis like caspase-3 and caspase-9 were measured by western 27]. In this context, there is much to be gained by learning
blot analysis. After 2 h of ischemia and 24 of reperfusion, the how the traditional therapies work to treat ischemia stroke
brain tissue of rats from all groups was collected and prepared and understanding how these protective measures provide
for western blot analysis. As shown in Figure 5(a), there was useful lessons on how to best counteract ischemic brain
significant increase of caspase-3 and caspase-9 expression injury. This paper aimed to provide an effective formula
levels observed in rats brain tissue from MCAO group, with possible mechanisms and highlighted the potential
which confirmed that the neuronal cell apoptosis happened application for the future of stroke therapy. In China, TCM
during the period of ischemia stroke. The treatment of the have been used to treat stroke with a long history over 2000
formula drug reduced remarkably the expression of caspase- years. In recent decades, patent medicines of TCM (TCPM)
3 (𝑃 < 0.05, Figure 5(b)) and caspase-9 (𝑃 < 0.05, Figure and acupuncture were widely and regularly used in stroke
5(c)) indicating that this formula drug has an antiapoptosis patients in either western medicine hospitals or traditional
activity. Chinese medicine hospitals [28, 29]. Currently, there are
Furthermore, the expression levels of some apoptosis- more than 100 TCPM used for stroke and approved by the
related proteins, such as Bcl-2 and Bax which are considered Chinese State Food and Drug Administration [30].
Evidence-Based Complementary and Alternative Medicine 7

Sham MCAO + MB MCAO

Caspase-9

Caspase-3

𝛽-Actin

(a)
0.6 0.6

0.5 0.5

Ratio of caspase-3 to 𝛽-actin

Ratio of caspase-9 to 𝛽-actin

0.4 0.4

0.3 ∗ 0.3
∗
∗
0.2 0.2 ∗∗

0.1 0.1

0 0
Sham MCAO + MB MCAO Sham MCAO + MB MCAO
(b) (c)

Figure 5: The comparison on the expression levels of caspase-3 and caspase-9 in the brain tissue after ischemia. (a) Was the representative
graphs of caspase-3 and caspase-9, and 𝛽-actin was used as loading control. (b) Showed quantitative comparison on ratio of caspase-3 to
𝛽-actin among different groups; (c) showed quantitative comparison on ratio of caspase-9 to 𝛽-actin among different groups. Bars represent
means ± SEM of eight rats. ∗ Significant difference at 𝑃 ≦ 0.05. ∗∗ Remarkably significant difference at 𝑃 ≦ 0.01.

As a highly valued ingredient of Chinese medicinal polyherbal classical formulations are generally borneolum
remedies, moschus is a detoxification agent for treating synthcticum combined with other ingredients to enhance the
inflammation, relieving swelling, and killing pain, which therapeutical effect of the treatments which especially often
has been used widely in the important therapies of stroke, used in the treatments for cardiovascular and cerebrovascular
coma, and convulsion in clinical practice of the traditional diseases in the clinical practice of TCM. Many researchsd
hospitals. The administration of moschus extract has been have also approved that the borneolum synthcticum has a
recommended and listed in the CHP and the Japanese phar- beneficial effect on increasing the bioavailability, tissue dis-
macopoeia for various indications requiring cardiovascular tribution, and blood concentration of other drugs and makes
stimulation, anti-inflammatory medication, or androgenic other drugs transport through BBB easier [35]. In the present
hormone therapy [31]. Borneolum synthcticum is also a com- study, the formula tested in animal experiments originates
mon drug used often to treat coma, loose heat, brighten eyes, from An-Gong-Niu-Huang pill which contains more than
relieve swelling, and kill pain in TCM. Some reports have ten herbal agents and has very clear therapeutical effect on
shown that borneolum synthcticum was used as one of the stroke in clinical practice of TCM. The moschus compatible
most effective ingredients of the traditional medicines which with borneolum synthcticum at ratio of 1 : 1 in this patent
treat and prevent cardiovascular diseases like coronary artery drug was consider the primary drug combination and called
disease, heart stroke, and heart infarction for its properties “monarch drug” in this drug. As the basis of this ratio, we also
of antiapoptosis, anti-inflammation, and abirritation [32–34]. investigated the neuroprotective effect of different ratio on
In addition, the borneolum synthcticum is also considered as ischemia stroke and found that the ratio of 1 : 3 was the most
a great promoter or enhancer to guide other drug to disease effective to reduce infract size in animal experiments and to
position in the theory and practice of TCM. Therefore, the improve the syndromes of patients who suffered from stroke
8 Evidence-Based Complementary and Alternative Medicine

1.2
Sham MCAO + MB MCAO

1 ∗
Bcl-2
∗

Ratio of Bcl-2 to 𝛽-actin

0.8

0.6
Bax
0.4

0.2
𝛽-Actin
0
Sham MCAO + MB MCAO
(a) (b)
1.2 2
∗
1
1.6
Ratio of Bax to 𝛽-actin

Ratio of Bcl-2 to Bax

0.8 ∗
∗ 1.2
0.6 ∗

0.8
0.4

0.4
0.2

0 0
Sham MCAO + MB MCAO Sham MCAO + MB MCAO
(c) (d)

Figure 6: The comparison on the expression levels of Bcl-2 and Bax in the brain tissue after ischemia. (a) Was the representative graphs of
Bcl-2 and Bax, and 𝛽-actin was used as loading control. (b) Showed quantitative comparison on ratio of Bcl-2 to 𝛽-actin among different
groups, (c) showed quantitative comparison on ratio of Bax to 𝛽-actin among different groups, and (d) was quantitative comparison on ratio
of Bcl-2 to Bax. Bars represent means ± SEM of eight rats. ∗ Significant difference at 𝑃 ≦ 0.05. ∗∗ Remarkably significant difference at 𝑃 ≦ 0.01.

in clinical practice. Therefore, we made a formula composed 3.0%. The recoveries of two analytes were from 90.21% to
of moschus compatible with borneolum synthcticum at a 98.6%. These results indicated that the developed method was
ratio of 1 : 3, and the present study also demonstrated for accurate enough for the quality control of this injection.
the first time that this formula had neuroprotective effect on Behavioral examination of neurological function score
protecting brain function and repairing the brain damage and determination of brain infarct volume in rats after
from ischemia stroke through significantly reducing the ischemia and/or reperfusion are commonly used as the
infract volume and improving neurological deficits. indicators to evaluate the ischemia injury and the severity
This formula has been prepared for injection and named of brain injury. Considering the dose-dependent and time
“She-bing injection” by our own lab. The method for quality window studies, it is showed that the neurological function
control of this injection was established in order to guarantee impairment and infarct volume reached the peak after 2 h
its therapeutical effect and safety. The muscone and borneol of focal cerebral ischemia followed by 24 h of reperfusion.
are the major components of this formula and were chosen as Therefore, the same time was selected as time window for
the chemical markers to control the quality of this injection. this study. The brain regions with a blockage of arterial
The GC-FID was performed to simultaneously determine the blood involved in regulating movement, and therefore the
quantitative contents of muscone and borneol. The standard animals with ischemia can show movement disorder after
curves for the tested components were linear over the studied being awake. The right artery in ischemia reperfusion caused
concentration ranges with correlation coefficient ≥0.9998. the same side motion control injuries, because the cortex
The limits of qualification for muscone and borneol were had a function of cross-control, which leads to left limbs
0.021 and 0.014 𝜇g⋅mL−1 , respectively. The RSDs of analytical muscle weakness, and the rats rotated to the left when moving
accuracy and precision for two analytes are all lower than and displayed typical symptoms of rear-end and leaning to
Evidence-Based Complementary and Alternative Medicine 9

the left. Neurological function scores of the drug-treated Conflict of Interests

rats dropped significantly lower than the MCAO group. It
was indicated that the drugs could improve neurological The authors declare that there is no conflict of interests
dysfunction in focal cerebral ischemia-reperfusion rats. regarding the publication of this paper.
Furthermore, we investigated the dose and time window
of this formula based on the effect of reducing infract volume Authors’ Contribution
in the MCAO modeled rats. And the results indicated that
this formula had a broad time window with a significant Xinhua Xia was responsible for all experiment conducting,
therapeutical effect in 6 hours after ischemia in MCAO rats, data analyses, and paper writing. Mei Liu focused on the
which suggested that this formula could have more effect on experiment design and paper revision and was addressed as
those patients who were excluded from the tPA treatment the corresponding author. And Qiang Li contributed to this
because of the strict limit of symptom onset time. work equally.
This formula is an important agent affecting many central
nervous system functions in clinical practice. Its neuroprotec- Acknowledgments
tive effects have been documented in present study by lesion
model of experimentally induced ischemia. The evidences This study is supported by the following fund sources: Youth
described above now suggested that this drug formula can Science Fund Project (Grant no. 30901902) and General
significantly decrease lesion volume and improve functional Project (Grant no. 81373628) from National Natural Science
recovery after stroke. Although the concrete mechanisms Foundation of China; Science and Technology Key Project
underlying the neuroprotection against cerebral ischemia by (Grant no. 2012B031800239) from Science and Technology
this formula are not completely known, some researchers Department of Guangdong Province; and General Project
have reported that neuronal cell death in the hippocampus (Grant no. 201300000141) from Science and Information
might occur via apoptosis, a consequence of ischemia [36]. Technology Bureau of Guangzhou.
In our previous study, we also found that this formula had
a rescue effect on cerebral ischemia by decreasing neuronal References
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Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2014, Article ID 309378, 10 pages
http://dx.doi.org/10.1155/2014/309378

Research Article
Anti-Inflammatory Effects of the Chinese Herbal Formula Sini
Tang in Myocardial Infarction Rats

Jiangang Liu,1 Karoline Peter,2 Dazhuo Shi,1 Lei Zhang,1 Guoju Dong,1 Dawu Zhang,1
Heimo Breiteneder,2 Rudolf Bauer,3 Johannes Jakowitsch,4 and Yan Ma2
1
Center of Cardiology, Xiyuan Hospital, China Academy of Chinese Medical Sciences, No. 1 Xiyuan Caochang,
Haidian District, Beijing 100091, China
2
Molecular Research in Traditional Chinese Medicine Group, Department of Pathophysiology and Allergy Research,
Vienna General Hospital, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria
3
Department of Pharmacognosy, Institute of Pharmaceutical Sciences, University of Graz, Universitaetsplatz 4/I, 8010 Graz, Austria
4
Clinical Division of Cardiology, Department of Internal Medicine II, Vienna General Hospital,
Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria

Correspondence should be addressed to Dazhuo Shi; [email protected] and Yan Ma; [email protected]

Received 3 November 2013; Revised 24 January 2014; Accepted 27 January 2014; Published 3 March 2014

Academic Editor: Joen-Rong Sheu

Copyright © 2014 Jiangang Liu et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The aim of this study was to evaluate the anti-inflammatory profiling of the Chinese herbal formula Sini Tang (SNT) in myocardial
infarction (MI) rats. SNT, a decoction consisting of four herbs: Aconitum carmichaelii, Cinnamomum cassia, Zingiber officinale,
and Glycyrrhiza uralensis, was characterized as a remedy to treat syndromes corresponding to heart failure and MI in China.
Potential biomarkers, which reflect the extent of myocardial necrosis and correlate with cardiac outcomes following MI, such
as atrial natriuretic peptide (ANP), high sensitivity C-reactive protein (hs-CRP), and proinflammatory cytokines such as tumor
necrosis factor-𝛼, interleukin-6, and interleukin-1𝛽 (TNF-𝛼, IL-6, and IL-1𝛽) were determined in plasma, serum, and in myocardial
tissue of MI rats after treatment with SNT. Our data indicate that SNT decreased significantly the levels of hs-CRP, TNF-𝛼, IL-6, and
IL-1𝛽 in MI rats. SNT decreased the expression of ANP levels in plasma and increased the vascular active marker nitric oxide, which
limits vascular inflammation. In addition, SNT could decrease the expression of endothelin-1 levels in rat plasma post-MI. Our data
suggest that the Chinese herbal formula SNT has the potential to improve cardiac function after MI. SNT may be a candidate for
treating MI and its associated inflammatory responses.

1. Introduction Traditional Chinese medicine (TCM) has been used in

China for centuries for treatment of cardiac disease and is
Heart failure (HF) is a critical disease. Currently 26 million now attracting interest in Western countries as a source of
people are suffering from HF worldwide, one quarter of alternative or complementary therapies due to its reputed
them in Europe [1]. One of the major causes of HF is effectiveness, low cost, and relative absence of side effects.
myocardial infarction (MI). Every sixth man and every Previous studies provided scientific evidence to support the
seventh woman in Europe died from MI [2]. MI is associated use of Chinese herbal medicine for treating MI and HF [8–
with an inflammatory reaction, which is a prerequisite for 13]. A TCM Sini Tang decoction (SNT), which consists of four
healing and scar formation [3, 4]. Inflammatory markers Chinese medical herbs, the root of Sichuan aconite (Aconitum
such as interleukin-6 (IL-6), interleukin-1𝛽 (IL-1𝛽), high carmichaelii Debeaux, Ranunculaceae), the bark of Chinese
sensitivity C-reactive protein (hs-CRP), and tumor necrosis cinnamon (Cinnamomum cassia J. Presl., Lauraceae), the
factor-𝛼 (TNF-𝛼) reflect the extent of myocardial necrosis rhizome of ginger (Zingiber officinale Roscoe, Zingiberaceae),
and correlate with cardiac outcomes following MI [5–7]. and the root of Chinese licorice (Glycyrrhiza uralensis Fisch.
2 Evidence-Based Complementary and Alternative Medicine

Table 1: Composition of the Chinese herbal formula SNT.

Pin Yin Chinese Common Latin Amount/weight ratio

Zhi Fu Zi 制附子 Aconite Aconitum carmichaelii 6
Rou Gui 肉桂 Cinnamon Cinnamomum cassia 1
Gan Jiang 干姜 Ginger Zingiber officinale 3
Jiu Gan Cao 灸甘草 Licorice Glycyrrhiza uralensis 8

ex DC. Fabaceae) [14], was used in preliminary and previous We used 90 (50% male and 50% female) Sprague-Dawley
studies in a MI rat model [15, 16] (Table 1). rats (180–200 g) provided by the Vital Laboratory Animal
The crude aconite contains the highly toxic diester- Technology Company (Beijing, China) for this study. Rats
diterpenoid alkaloids aconitine, hypaconitine, and mesaconi- were acclimatized with a 12/12 hours light/dark cycle at a
tine. Our previous results indicated that SNT is suitable and controlled room temperature of 23–25∘ C and a humidity of
safe for the rat model and has the potential to improve 50–70%, and rats were allowed free access to food and water
early ventricular remodeling and cardiac function after MI. for seven days before use.
SNT reduced collagen matrix accumulation in the serum
and in the myocardial tissue, which is associated with a
significant improvement in systolic function. Furthermore, 2.2. Drugs and Reagents Information. Chemical standards of
SNT decreased the expression of Toll-like receptors (TLRs) aconitine, mesaconitine, and hypaconitine were purchased
levels in the myocardial tissue. TLRs belong to a group of from PhytoLab GmbH, Germany. Fosinopril sodium (FS)
type I transmembrane receptors with endogenous and exoge- (10 mg/tablet) was supplied by Sino-American Shanghai
nous ligand binding ability to stimulate innate and adaptive Squibb Pharmaceutical Co., Ltd. (China). Atrial natriuretic
immune responses and induce immune and inflammatory peptide (ANP) Kit, total nitric oxide (NO), and endothelin
cytokines IL-6, TNF-alpha, and other gene transcriptions and 1 (ET-1) were purchased from GBD Ltd., USA. Interleukin-
protein expressions [17–20]. 1𝛽 (IL-1𝛽), interleukin-6 (IL-6), high sensitivity C-reactive
In this study we aimed to confirm these preliminary protein (hs-CRP), and tumor necrosis factor-𝛼 (TNF-𝛼) Kits
observations by evaluating a number of potential biomarkers were provided by R&D systems (USA). Coomassie blue
and proinflammatory cytokines as well as atrial natriuretic protein assay Kit was offered by Nanjing Science and Technol-
peptide (ANP), the potent vasoconstrictor endothelin-1 (ET- ogy Co., Ltd. (China). The Two-step Immunohistochemical
1), and the vascular active marker nitric oxide (NO), which Detection Kit was produced by Zhongshan Golden Bridge
limits vascular inflammation and plays an important role in Biotechnology Co., Ltd. (Beijing, China).
cardiac remodeling and wound repair after MI [21–24]. ANP
conveys the cardiac protective properties of vasodilation, 2.3. Preparation of SNT Decoction. SNT (0.5 g/g
inhibition of cardiomyocyte hypertrophy, cardiac fibroblast extract/crude drug) including processed Glycyrrhiza
proliferation, collagen synthesis, and enhanced lipolysis [25]. uralensis, processed Aconitum carmichaelii, Zingiber
ANP and other natriuretic peptides are the gold standard officinale, and Cinnamomum cassia was prepared at the
biomarkers in determining the diagnosis and prognosis of HF Pharmacy Department of Xiyuan Hospital, China Academy
following MI [26]. During acute myocardial infarction, ET- of Traditional Chinese Medicine (Beijing, China). Aconite
1 enhances myocardial necrosis and arrhythmogenesis but was cut into small pieces before use. Herbs were soaked in
seems to exert a favorable effect on subsequent infarct healing sterilized drinking water (500 mL) for one hour in a clay pot
and early ventricular remodeling [27]. Thus ANP, ET-1, and at room temperature and cooked to boiling. The decoction
NO were measured and evaluated in rat plasma and serum in was performed twice by cooking gently for 30 min each. The
this study. two obtained decocts were combined, filtered, and stored at
Compared to our previous studies we optimized the 4∘ C before administration to rats [16].
dosage of administration by using a low as well as a high dose
of SNT in this study. We also used fosinopril sodium which is
indicated for the treatment of HF [28, 29] and decreases the 2.4. Quality Control of SNT by HPLC. HPLC analysis of SNT
inflammatory response after MI [30] as a positive control. decoctions was developed during this study using an agilent
1100 series LC system equipped with a Hypersil BDS RPC-18
column (100 × 3 mm; particle size 3 mm, Agilent, Germany)
2. Materials and Methods as described by Peter et al. [16]. A mixed standard solution of
aconitine (160 𝜇g), mesaconitine (150 𝜇g), and hypaconitine
2.1. Animals. All animal experiments were approved by the (230 𝜇g) in 1 mL acetonitrile/H2 O (80 : 20) was prepared. The
Administrative Committee of Experimental Animal Care mobile phase was a mixture of buffer A (an aqueous buffer,
and Use of China Academy of Chinese Medical Sciences pH3, containing 15 mM ortho-phosphoric acid and 1.5 mM
(CACMS, Permit number CACMS/20100322) and con- tetrabutylammonium hydroxide) and buffer B (0.01% formic
formed to the National Institute of Health Guide for the Care acid in acetonitrile). Buffer A and buffer B were used for
and Use of Laboratory Animals [31]. gradient elution (5–24.4% of buffer B at 0–11 min, 24.4–26%
Evidence-Based Complementary and Alternative Medicine 3

of buffer B at 11–17 min, 26–40% of buffer B at 17-18 min, 40– Table 2: The experimental rats groups. Drugs and decocts diluted
100% of buffer B at 18-18.1 min, and 100% of buffer B at 18.1– with distilled drinking water were administered orally once a day for
23 min). The injection volume was 5 𝜇L and the flow rate was 30 days starting two days after induction of AMI.
kept at 0.5 mL/min at 25∘ C. The detection wavelength was
Group Sham Model FS SNT-LD SNT-HD
set at 203 nm. The content of aconitine, hypaconitine, and
𝑁 10 10 10 10 10
mesaconitine samples was calculated according to Csupor
et al. [32]. Operation Sham AMI AMI AMI AMI
Oral administration DW DW 0.9 mg/kg 4.5 g/kg 13.5 g/kg
Sham: sham operated.
2.5. MI Model of Rats. Acute myocardial infarction (AMI)
AMI: induced acute myocardial infarction.
was induced in both male and female rats by left anterior DW: drinking water.
descending artery (LAD) ligation. The surgical procedures FS: fosinopril sodium (0.9 mg/kg).
were performed using the well-established technique [33, 34]. SNT-LD: low dose of SNT (4.5 g/kg).
These experiments were carried out strictly in accordance SNT-HD: high dose of SNT (13.5 g/kg).
with the recommendations in the national legislation of
China and performed at the Center of Animals Laboratory
of the Beijing Xiyuan Hospital, China Academy of Chinese 3000 rpm for 15 min. Plasma and sera were obtained and
Medical Sciences. All surgeries were performed under diethyl conserved at −80∘ C for further analysis. The heart tissue were
ether anesthesia and all efforts were made to minimize collected, weighed, and randomly assigned. One heart of each
suffering. Rats were anesthetized with diethyl ether and group was stored at −80∘ C for ELISA.
placed in a supine position on a table for the operations.
The left anterior descending artery (LAD) was occluded as
described by Dietl et al. [15]. To prevent infection rats were 2.9. ELISA Assay of Serum, Plasma and Heart Tissue. Five of
given penicillin (40.000 units) after the operation for 3 days. the twelve frozen heart tissue samples of each group were ran-
Sixty surviving infarct rats were randomly divided into five domly picked. The heart sections were weighed and 300 mg
groups (Table 2). The oral administration of the drugs began of each sample was cut into small pieces and 1 mL of saline
two days after AMI-induction. SNT and FS, an angiotensin solution was added. After homogenization on ice, samples
converting enzyme (ACE) inhibitor as a positive control, were were centrifuged and the supernatants were stored at −80∘ C
diluted with distilled drinking water and administered orally for analysis. ELISA tests were performed strictly according
in a volume of 10 mL/kg body weight once every morning for to the instructions. The protein concentration of each tissue
4 weeks. sample was measured with the BCA (bicinchoninic acid)
Protein Assay Kit (Thermo Fisher Scientific Inc., USA).
The following biomarkers, known to be involved in the
2.6. Echocardiography Assessment. Echocardiography was
process of cardiac remodeling, were determined by ELISA
performed 30 days after surgery according to the method
[39, 40]. Functional index ANP, proinflammatory cytokines
described by Yin et al. [35]. Ten rats from each group were
IL-6, IL-1𝛽, and TNF-𝛼, inflammatory biomarker hs-CRP,
anesthetized by intraperitoneal injection of ethyl carbamate
and vascular active markers ET-1 and NO were measured in
(6 mL/kg). After cleaning the rat chest the cardiac short
plasma, serum, and myocardial tissue samples according to
axis (papillary level), left ventricle end-diastolic dimension
the manufacturer’s instructions.
(LVDd), and left ventricle end-systolic dimension (LVDs)
were measured using an ATL HDI-5000 Diagnostic Ultra-
sound System (Philips Ultrasound Inc., China). The ejection 2.10. Statistical Analysis. Data were tabulated and presented
fraction (EF) was calculated from the left ventricle end- as mean ± standard deviation, and the significance of changes
diastolic volume (LVEDV) and the left ventricle end-systolic was assessed with one-way repeated measures analysis of
volume (LVESV) as EF% = [(LVEDV − LVESV)/LVEDV] variance (ANOVA). All results were tested on normal dis-
× 100. The data calculations were performed using a single- tribution by aid of One-Sample Kolmogorov-Smirnov Test.
blind method [36, 37]. Bonferroni’s Holm test was followed for multiple compar-
isons. One-way ANOVA Tukey HSD test was used for
2.7. Measurement of Myocardial Infarct Size (IS). The patho- pairwise multiple comparisons. A value of 𝑃 < 0.05 was
logical slice was photographed in microdistance using a considered statistically significant. Data were analyzed using
Canon IXUS 90IS digital camera. The microscopic color the Statistical Package for the Social Sciences (SPSS Inc.,
image processing system (DpxView Pro, Korea) was used Chicago, USA).
to calculate the left ventricular IS (%, myocardial infarction
area/left ventricular area × 100) by an investigator who was
blinded to the identity of the pathological slice as described
3. Results
by Takagawa et al. [38]. 3.1. Quality Control of SNT by HPLC. The retention times
of HPLC measurement of SNT decoction and the stan-
2.8. Collection of Blood and Tissue Samples. After four weeks, dards mixture of aconitine, mesaconitine, and hypaconitine
the anesthetized rats were sacrificed. Blood samples were were under the same chromatographic conditions (Figure 1).
taken from the left ventricle (LV) cavity and centrifuged at However, no aconitine, mesaconitine, and hypaconitine were
4 Evidence-Based Complementary and Alternative Medicine

600 20 1 2 3

(mAU)
(mAU)

400
10
200
0 0
0 5 10 15 20 0 5 10 15 20
(min) (min)

(a) (b)

Figure 1: Quality control and toxicity evaluation of SNT by HPLC. (a) HPLC measurement of SNT. (b) The standard solution of diester-
diterpenoid alkaloids: (1) mesaconitine, (2) hypaconitine, and (3) aconitine. mAU: milli absorption units.

Table 3: Echocardiographic parameters overview of ventricular remodeling effects.

Sham Model FS SNT-LD SNT-HD Remodeling effects (versus model)

LVDs (mm) 0.91 ± 0.20 6.44 ± 1.59∗∗ 3.81 ± 1.21∗∗󳵳󳵳 4.95 ± 1.95∗∗ 3.74 ± 1.47∗∗󳵳󳵳 ↓
LVDd (mm) 3.29 ± 0.81 6.24 ± 0.72∗∗ 4.45 ± 1.28 5.37 ± 1.78 4.87 ± 1.47 ↓
EF (%) 93.32 ± 2.94 55.48 ± 12.89∗∗ 78.03 ± 10.70∗󳵳󳵳 69.69 ± 13.91∗∗󳵳 77.83 ± 12.32∗󳵳󳵳 ↑
󳵳󳵳 󳵳
2
IS (mm ) 38.04 ± 8.35 23.91 ± 7.99 31.25 ± 10.68 27.81 ± 4.91 ↓
∗
𝑃 < 0.05, ∗∗ 𝑃 < 0.01 versus sham group; 󳵳 𝑃 < 0.05, 󳵳󳵳 𝑃 < 0.01 versus model group.

detected in the SNT decoction. SNT is suitable and safe for 3.4. Effects of SNT on ANP. In comparison with the sham
the rat model used in this study. group, the plasma ANP levels of rats in three treatment
groups were decreased after four weeks (FS: 222.26 ±
3.2. Effects of SNT on Echocardiography. Four weeks after MI, 80.25 ng/L and SNT-LD and SNT-HD: 224.82 ± 80.19 ng/L
ultrasound echocardiography showed a significant increase and 189.05 ± 49.96 ng/L versus the sham group: 250.00 ±
of the left ventricular dimension at end diastole (LVDd) and 21.32 ng/L). In comparison with the model group, the SNT-
the left ventricular dimension at end systole (LVDs) in the HD group showed more reduction of the ANP content (SNT-
model group (6.24 ± 0.72 mm versus 3.29 ± 0.81 and 6.44 ± HD versus model group: 367.34 ± 43.20, 𝑃 < 0.01). The data
1.59 mm versus 0.91 ± 0.20, 𝑃 < 0.05) compared to the are shown in Table 4 and Figure 3.
sham group. The FS and SNT treatment groups exhibited
significantly decreased LVDs versus the sham group (FS: 3.5. Effects of SNT on ET-1 and NO. Four weeks after ischemia
3.81 ± 1.21 mm, SNT-LD: 4.95 ± 1.95 mm and SNT-HD: the ET-1 levels of the treatment groups were decreased
3.74 ± 1.47 mm, 𝑃 < 0.01). The FS treatment group exhibited compared to model group (FS: 166.15 ± 9.01 ng/L, SNT-
significantly decreased LVDd (4.45 ± 1.28 mm versus 3.29 ± LD: 166.74 ± 7.70 ng/L, and SNT-HD: 158.49 ± 9.97 ng/L
0.81 mm, 𝑃 < 0.01). The SNT treatment groups also showed versus model group: 171.09 ± 13.17 ng/L). In comparison
decreased LVDd versus the sham group (SNT-LD: 5.37 ± to the model group the NO levels of all treatment groups
1.78 mm and SNT-HD: 4.87 ± 1.47 mm, 𝑃 > 0.05), but not were increased (FS: 53.78 ± 6.24 𝜇mol/L, SNT-LD: 53.06 ±
significantly. The left ventricular ejection fraction (EF) was 11.54 𝜇mol/L, and SNT-HD: 64.85 ± 8.74 𝜇mol/L versus
significantly lower in the model group compared to the sham model group: 48.18 ± 10.14 𝜇mol/L). The SNT-HD group
group (55.48 ± 12.89% versus 93.32 ± 2.94%, 𝑃 < 0.01). showed a significant difference (𝑃 < 0.05) in the NO level
The FS and SNT treatment groups showed an improved left compared to model group. The data are shown in Table 4 and
ventricular function of the EF compared to the sham (FS: Figure 3.
78.03 ± 10.70%, SNT-LD: 69.69 ± 13.9%, 𝑃 < 0.05 and
SNT-HD: 77.83 ± 12.32%, 𝑃 < 0.01) and model groups
3.6. Effects of SNT on Serum and Myocardial Tissue Levels of
(FS and SNT-HD: 𝑃 < 0.01, SNT-LD: 𝑃 < 0.05). SNT
Inflammatory Factors. The levels of inflammatory biomarker
treatment improved the cardiac function by increasing the EF
and cytokines hs-CRP, IL-6, IL-1𝛽, and TNF-𝛼 in rat serum
by 23.63% (difference between sham group: 93.32% and SNT-
and myocardial tissue were decreased in the treatment groups
LD: 69.69%), (Table 3 and Figure 2).
(Table 5 and Figure 4). The treatment groups showed a
significant difference in the level of hs-CRP in comparison
3.3. Effects of SNT on Infarct Size. The infarct size (IS) to the model group (FS: 5.02 ± 0.71 mg/L, SNT-LD: 3.94 ±
obtained using the midline length measurement is shown in 0.61 mg/L, and SNT-HD: 3.97 ± 0.77 mg/L versus model
Table 3. The IS values from the FS, SNT-LD and SNT-HD group: 6.71 ± 0.70 mg/L, 𝑃 < 0.01). The treatment groups
groups (23.91 ± 7.99 mm2 , 𝑃 < 0.01, 31.25 ± 10.68 mm2 and showed a decrease in the level of IL-6 compared to model
27.81 ± 10.33 mm2 , 𝑃 < 0.05) were significantly smaller than group (FS: 210.60 ± 38.83 ng/L, SNT-LD: 196.81 ± 22.68 ng/L,
from the model group (38.04 ± 8.35 mm2 ). and SNT-HD: 202.55 ± 37.37 ng/L versus model group:
Evidence-Based Complementary and Alternative Medicine 5

LVDs
10 8 LVDd
∗∗
∗∗
8
∗∗ 6

LVDd (mm)
LVDs (mm)

6
∗∗ ∗∗
4
4

2
2

0 0
Sham Model FS SNT-LD SNT-HD Sham Model FS SNT-LD SNT-HD
(a) (b)
Ejection fraction
Infarct size
100 50
∗ ∗
∗∗
80 40
∗∗
Infarct size (mm2 )
60 30
EF (%)

40 20

20 10

0 0
Sham Model FS SNT-LD SNT-HD Sham Model FS SNT-LD SNT-HD
(c) (d)

Figure 2: Echocardiographic parameters. (a) Echocardiographic measurements of left ventricular dimension at end systole (LVDs), (b) left
ventricular dimension at end diastole (LVDd), (c) left ventricular ejection fraction (EF), and (d) infarct size results.

Table 4: Overview of vascular functional index marker expression levels in serum or in plasma (𝑥 ± 𝑠).

Sham Model FS SNT-LD SNT-HD Expression levels (versus model)

ET (ng/L) 161.40 ± 13.95 171.09 ± 13.17 166.15 ± 9.01 166.74 ± 7.70 158.49 ± 9.97 ↓
NO (𝜇mol/L) 54.04 ± 11.88 48.18 ± 10.14 53.78 ± 6.24 53.06 ± 11.54 64.85 ± 8.74󳵳 ↑
NO/ET 3.35 2.83 3.24 3.18 4.09∗∗󳵳󳵳 ↑
ANP (ng/L) 250.00 ± 21.32 367.34 ± 43.20 222.26 ± 80.25 224.82 ± 80.19 189.05 ± 49.96󳵳 ↓
ET and ANP were measured in plasma. NO was measured in serum.
∗∗
𝑃 < 0.01 versus sham group, 󳵳 𝑃 < 0.05, 󳵳󳵳 𝑃 < 0.01 versus model group.

240.65 ± 27.80 ng/L). The levels of TNF-𝛼 were decreased 4. Discussion

significantly in FS and SNT-HD groups compared to the
model group (FS: 22.17 ± 8.66 ng/L, 𝑃 < 0.05; SNT-LD: In China, Chinese herbal medicine is widely used as an
25.61 ± 7.57 ng/L and SNT-HD: 23.94 ± 4.27 ng/L, 𝑃 < adjunct to biomedicine in treating MI [41]. Our previous
0.05 versus model group: 33.23 ± 5.78 ng/L). The levels of studies indicated that SNT is suitable and safe for the
IL-1𝛽 were decreased significantly in serum (FS: 151.17 ± animal model study and has the potential to improve early
16.62 ng/L, 𝑃 < 0.01; SNT-LD: 170.91 ± 15.09 ng/L and ventricular remodeling and cardiac function after MI [15, 16].
SNT-HD: 170.8 ± 23.30 ng/L, 𝑃 < 0.05 versus model group: Pathological H&E staining showed that the IS values from the
199.86 ± 25.44 ng/L) as well as in myocardial tissue (FS: FS and SNT groups (23.91 ± 7.99 mm2 , 31.25 ± 10.68 mm2
32.85 ± 3.64 ng/g, 𝑃 < 0.01; SNT-LD: 37.32 ± 4.89 ng/g and 27.81 ± 10.33 mm2 ) were smaller than from the model
and SNT-HD ng/g, 𝑃 < 0.05 versus model group: 41.56 ± group (38.04 ± 8.35 mm2 ). FS and SNT could decrease LVDs
6.08 ng/g). significantly in the FS and SNT-HD groups (FS: 3.81 ±
6 Evidence-Based Complementary and Alternative Medicine

ANP level in plasma Endothelin-1 level in plasma

500 190

400 180
ANP (ng/L)

ET-1 (ng/L)
300 170

200 160

100 150

0 140
Sham Model FS SNT-LD SNT-HD Sham Model FS SNT-LD SNT-HD
(a) (b)
NO level in serum
80

70
NO (𝜇mol/L)

60

50

40

30
Sham Model FS SNT-LD SNT-HD
(c)

Figure 3: Effects of SNT on ANP, ET-1 levels in plasma, and NO levels in serum. ANP (atrial natriuretic peptide) levels (a) and ET-1
(endothelin-1) levels (b) in plasma. NO (nitric oxide) levels in serum (c). ANP and NO levels of the SNT-HD group were significantly more
different than those of the model group (𝑃 < 0.05).

Table 5: Overview of inflammatory factors and cytokines levels in serum and in myocardial tissue.

Sham Model FS SNT-LD SNT-HD Expression levels (versus model)

In serum
hs-CRP (mg/L) 4.53 ± 0.66 6.71 ± 0.70∗∗ 5.02 ± 0.71󳵳󳵳 3.94 ± 0.61󳵳󳵳 3.97 ± 0.77󳵳󳵳 ↓
IL-6 (ng/L) 204.17 ± 39.83 240.65 ± 27.80 210.60 ± 38.83 196.81 ± 22.68 202.55 ± 37.37 ↓
TNF-𝛼 (ng/L) 23.38 ± 7.10 33.23 ± 5.78∗∗ 22.17 ± 8.66󳵳 25.61 ± 7.57 23.94 ± 4.27󳵳 ↓
IL-1𝛽 (ng/L) 157.15 ± 17.34 199.86 ± 25.44 151.17 ± 16.62󳵳󳵳 170.91 ± 15.09󳵳
∗∗
170.8 ± 23.30󳵳 ↓
In myocardial tissue
IL-1𝛽 (ng/g) 29.73 ± 1.53 41.56 ± 6.08∗∗ 32.85 ± 3.64󳵳 37.32 ± 4.89 30.37 ± 1.91󳵳󳵳 ↓
∗∗ 󳵳 󳵳󳵳
𝑃 < 0.01 versus sham group; 𝑃 < 0.05, 𝑃 < 0.01 versus model group.

1.21 mm, 𝑃 < 0.01; SNT-LD: 4.95 ± 1.95 mm and SNT-HD: the rats in the SNT treated groups had an improved left
3.74 ± 1.47 mm, 𝑃 < 0.01 versus the model group). FS and ventricular function. The left ventricular ejection fraction
SNT could also decrease LVDd significantly only in the FS (EF%) was significantly increased in all treatment groups
group (FS: 6.24±0.72 mm, 𝑃 < 0.05, SNT-LD: 5.37±1.78 mm, compared with the model group (𝑃 < 0.05 and 𝑃 < 0.01).
and SNT-HD: 4.87 ± 1.47 mm versus the sham group). Quality control of SNT by HPLC showed that no toxic diester-
Echocardiography the key diagnostic tool and was performed diterpenoid alkaloids of aconite were detected in the SNT
to assess LV function and volumes, valvular function, extent decoction. The present study suggests that the Chinese herbal
of myocardial damage, and to detect complications [42]. The medicine SNT might play an important role in the treatment
results of our echocardiographic evaluation indicated that of MI and its associated inflammatory reactions.
Evidence-Based Complementary and Alternative Medicine 7

hs-CRP level in serum

8 IL-6 level in serum
∗∗ 300

6
250
hs-CRP (mg/L)

IL-6 (ng/L)
4

200
2

0 150
Sham Model FS SNT-LD SNT-HD Sham Model FS SNT-LD SNT-HD
(a) (b)
𝛼 1𝛽
50

∗∗
40 ∗∗

30
𝛼

1𝛽

20

10

0
FS FS
(c) (d)
1𝛽
50
∗∗

40
1𝛽

30

20
FS
(e)

Figure 4: Effects of SNT on the levels of inflammatory factors in serum and myocardial tissue. The hs-CRP levels (a) of the FS and the SNT
treatment groups showed significant decreases in comparison to the model group (𝑃 < 0.01). The IL-6 levels in serum (b) were reduced in
the FS and SNT treatment groups. The TNF-𝛼 levels (c) of the treatment groups were reduced. FS and the SNT-HD groups showed significant
decreases in comparison to the model group (𝑃 < 0.05). The IL-1𝛽 levels in serum (d) were reduced in the FS and SNT groups (𝑃 < 0.01 and
𝑃 < 0.05 versus model group, resp.). The IL-1𝛽 levels in myocardial tissue (e) were reduced in the FS and SNT groups and significantly in the
FS and SNT-HD groups (𝑃 < 0.05 and 𝑃 < 0.01, resp.) compared to model group.

Endothelin-1 (ET-1), a potent vasoconstrictor, is synthe- smooth muscle cell contractility and myocardial oxygen
sized in the vasculature and myocardium by various cell types consumption [21]. NO acts as a key contributor to vascular
and is associated with the development of cardiac dysfunc- health due to its effects of limiting vascular inflammation,
tion. Nitric oxide (NO) plays an important role in the home- platelet aggregation, monocyte adhesion to endothelial cells,
ostasis of the cardiovascular system by regulating vascular and abnormal smooth muscle cell proliferation. Endothelial
8 Evidence-Based Complementary and Alternative Medicine

dysfunction is connected to a decreased bioavailability of and the antigenotoxic and anti-inflammatory activities of
vasodilators, especially NO [22]. The levels of ET-1 in the licorice. To fully determine the anti-inflammatory effects of
plasma were decreased in all treatment groups but did SNT thorough investigations should be performed including
not show significant differences among the groups in our an evaluation of the number of infiltrating inflammatory cells
experiments. The SNT-HD treatment group showed more and MI associated inflammatory cytokine levels in the future.
decrease than other treatment groups. Yang et al. reported
the regulating effect of SNT on blood pressure because it 5. Conclusions
adjusts the expression levels of endothelin receptor-A and
endothelial NO synthesis in the heart and the vasoactive We evaluated the anti-inflammatory profiles of the Chinese
marker levels (ET and NO) in the blood [43]. The NO levels herbal formula SNT, an ancient traditional medicinal formula
of all treatment groups were increased. In comparison to using a rat model. Our data indicate that SNT could reverse
the model group the SNT-HD group showed a significant ventricular remodeling and improve heart function. SNT has
difference (𝑃 < 0.05) in the NO level. NO was affected by the potential to decrease the levels of hs-CRP and cytokines
the treatment with SNT at high doses. TNF-𝛼, IL-6, and IL-1𝛽 after MI.
Atrial natriuretic peptide (ANP) acts as a vasodilator SNT decreased the expression of ET-1 levels in the plasma
which is released in response to increasing atrial filling and increased the vascular active marker nitric oxide. In
pressures [23]. The most important factor governing ANP addition, the SNT high-dose treatment group decreased the
secretion is mechanical stretching of the atria, which occurs expression levels of ANP in plasma significantly. These new
when extracellular fluid volume or blood volume is elevated. findings will help us to develop more successful therapies
The release of ANP in disease states such as MI and HF including SNT for treating MI and its associated inflamma-
appears to be related to both mechanical and cellular events tory responses.
[24]. The levels of ANP in the plasma of all treatment groups
were lower than in the model group, but only the high dose
group showed a significant difference (𝑃 < 0.05). ANP was Conflict of Interests
also affected by the treatment with SNT at high doses. The The authors declare that there is no conflict of interests
examination of a larger number of subjects may be necessary regarding the publication of this paper.
to discover significant differences between the experimental
groups.
Biochemical markers like collagen metabolites, proin- Authors’ Contribution
flammatory cytokines, and matrix metalloproteinases are
Jiangang Liu and Karoline Peter contributed equally to the
getting increasingly important in ventricular remodeling
work.
research and clinic cardiology. An ideal biochemical marker
of clinical cardiology should be a prognostic indicator,
should assist in the early diagnosis, reliably reflect the Acknowledgments
therapeutic response, and help in grading the risk associated
with each stage of MI [38, 39]. CRP is an inflammatory This work was supported by the Austrian Federal Ministry
biomarker which reflects the extent of myocardial necrosis of Science and Research and the Austrian Federal Ministry
and correlates with cardiac outcomes following AMI [6]. of Health (GZBMWF-402.000/3-ll/6b/2008 to Yan Ma) and
The inflammatory response and cytokines such as TNF-𝛼, China International Scientific Cooperation and Exchange of
IL-1𝛽, or IL-6 are increased soon after myocardial ischemic Special Items (S2010BR0446 to Dazhuo Shi).
injury and can acutely regulate myocyte survival or apoptosis
and trigger additional cellular inflammatory responses [7]. References
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Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2014, Article ID 890950, 16 pages
http://dx.doi.org/10.1155/2014/890950

Review Article
Chinese Herbal Medicine for Aspirin Resistance:
A Systematic Review of Randomized Controlled Trials

Ai-ju Liu,1 Hui-qin Li,1 Ji-huang Li,1 Yuan-yuan Wang,2 Dong Chen,2
Yan Wang,2 and Guo-qing Zheng1
1
Department of Neurology, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou 325027, China
2
Department of Cardiology, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou 325027, China

Correspondence should be addressed to Yan Wang; [email protected] and Guo-qing Zheng; gq [email protected]

Received 30 October 2013; Revised 15 December 2013; Accepted 16 December 2013; Published 20 February 2014

Academic Editor: Mao-Hsiung Yen

Copyright © 2014 Ai-ju Liu et al. This is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Aspirin resistance (AR) is a prevalent phenomenon and leads to significant clinical consequences, but the current evidence for
effective interventional strategy is insufficient. The objective of this systematic review is thus to assess the efficacy and safety of
Chinese herbal medicine (CHM) for AR. A systematical literature search was conducted in 6 databases until December 2012 to
identify randomized controlled trials (RCTs) of CHM for AR. As a result, sixteen RCTs with a total of 1011 subjects were identified,
suggesting that the interests of the medical profession and the public in the use of CHM for AR have grown considerably in the recent
years. Tongxinluo capsule and Danshen-based prescriptions were the most frequently used herbal prescriptions, while danshen
root, milkvetch root, Leech, and Rosewood were the most frequently used single herbs. Despite the apparent reported positive
findings, it is premature to determine the efficacy and safety of CHM for the treatment of AR due to poor methodological quality
and insufficient safety data. However, CHMs appeared to be well tolerated in all included studies. Thus, CHM as a promising
candidate is worthy of improvement and development for further clinical AR trials. Large sample-size and well-designed rigorous
RCTs are needed.

1. Introduction drug in 1763 isolated salicin, the glycoside of salicylic acid

and the active ingredient of aspirin, from the bark of a willow
1.1. Description of the Condition. Cerebral infarction and tree in England [6, 7]. Aspirin was not born until 1897 when
myocardial infarction can be fatal and seriously affect the Bayer company hired Felix Hoffman, a German chemist,
patients’ living quality, and their incidence will increase who created acetylsalicylic acid by acetylating salicylic acid’s
greatly in recent decades, especially in developing countries, hydroxyl ring resulting in well-tolerated compound [6, 8].
due to more work pressure and population aging [1, 2]. After that time, people around the world began to appreciate
Antiplatelet therapy is an important prevention measure for its fast onset and predictable relief. However, for all that
lowering the likelihood of myocardial infarction and stroke has been discovered about its beneficial effects, sometimes
in high risk vascular patients [3, 4]. Among antiplatelet drugs, aspirin’s effects are like an infatuation, eventually, they fade
acetylsalicylic acid, known as aspirin, became a cornerstone or disappear [6]. That is to say, despite the impressive efficacy
in the treatment of cardio/cerebrovascular diseases and has and safety of low dose aspirin in preventing atherothrom-
been shown to be effective in the secondary prevention of bosis, many patients who were receiving aspirin therapy
vascular events [4, 5]. The use of aspirin can be dated back still suffered significant clinical consequences such as stroke,
to early Hippocrates who used willow leaves, rich in Acetyl- myocardial infarction, and other vascular death events [9].
salicylic acid, to relieve the aches associated with multiple This clinical phenomenon was called aspirin resistance (AR).
illnesses in ancient Greece [6, 7]. Reverend Edmund Stone The reported frequency of AR was high and variable based
who progressed aspirin from folk remedy to a blockbuster on the different dosage of aspirin and biochemical method
2 Evidence-Based Complementary and Alternative Medicine

testing platelet function [9]. The study by Gum et al. [10] epinephrine or ADP; (e), rapid platelet function assay (Veri-
reported a 5% prevalence of AR in patients taking aspirin for fyNow Aspirin system): analysis of light transmission in a test
prevention of atherosclerotic events, whereas another study cartridge containing fibrinogen-coated beads in whole blood;
reported a 60% rate of AR in patients with intermittent (f), levels of serum TXB2: radioimmunoassay or ELISA;
claudication [11]. A large systematic review including 42 TXA2 is rapidly converted into the stable metabolite TXB2;
studies reported a mean prevalence of AR as 25% in patients (g): urinary 11-dehydro-TXB2: measurement using radioim-
taking aspirin for secondary prevention [9]. Therefore, the munoassay or ELISA; systemic TXB2 undergoes hepatic
intervention of AR is an important measure for the preven- transformation into 11-dehydro-TXB2; (h): flow cytometry:
tion of cardio/cerebrovascular diseases. automated laser detection of platelet activation markers (e.g.,
Aspirin resistance has been defined by clinical and/or P-selectin, CD63, changes in GP IIb/IIIa complex conforma-
laboratory criteria, but a generally accepted definition based tion) using antibodies; (i): thromboelastography: analysis of
on valid diagnostic criteria has not been established. The clot strength from formation to lysis after stimulation with
possible first description of AR has been debated since the arachidonic acid or ADP [14, 19–21].
1980s; FitzGerald et al. [12] demonstrated that adenosine
diphosphate (ADP) induced platelet aggregation was maxi-
mally inhibited by aspirin 40∼80 mg/day, but values returned 1.2. Description of the Intervention. The current evidence for
to baseline with chronic administration at higher doses of effective intervention of AR is insufficient. Clinical treatment
up to 2,600 mg/day. Unfortunately, “aspirin resistance” was of AR such as an increase in aspirin dose and the addition
not actually used until 10 years later when Helgason et al. of other antiplatelet drugs is usually not effective [21, 22].
[13] reported inadequate inhibition of platelet aggregation Aspirin doses of 75–150 mg daily were at least as effective as
by aspirin in some patients suffering from cerebrovascular higher doses daily, which was demonstrated in trials com-
disease. However, “aspirin resistance” remains yet with no paring different doses of aspirin versus no aspirin, and the
standard definition. This term used in the literature mainly addition of dipyridamole to aspirin did not reduce the inci-
included the following [14–16]. (1) Clinical “aspirin resis- dence of ischemic events significantly comparing with aspirin
tance” or “treatment failure” is the occurrence of acute throm- alone [23]. Clopidogrel, an oral thienopyridine, can selec-
botic events despite regular aspirin therapy. (2) Laboratory- tively and irreversible block the P2Y12 platelet receptor for
defined “aspirin resistance” or aspirin nonresponsiveness ADP, thereby inhibiting ADP induced platelet aggregation.
is present when in vitro platelet reactivity is not properly Aspirin plus clopidogrel can inhibit the two main pathways
blocked despite the use of aspirin, ranged from the spe- of platelet aggregation, the arachidonate/TXA2 and the ADP
cific failure to inhibit thromboxane A2 (TXA2), failure to pathways [24]. The study by Dropinski et al. [25] reported
inhibit a test of platelet function that is dependent on that clopidogrel had significant effect on AR for patients with
TXA2 production, and the very broad failure to inhibit one coronary artery disease. However, another study by Lev et
or more platelet function assays. (3) “High on-treatment al. [26] reported about 50% of the AR patients who were
residual platelet reactivity” found in vitro in patients with also resistant to the effects of clopidogrel because AR patients
antiplatelet therapy does not necessarily mean “resistance” to have a low inhibitory response to clopidogrel. Dipyridamole
the antiplatelet drug from a pharmacological point of view. is a phosphodiesterase (PDE) inhibitor and has been used
(4) Some authors distinguished types of “aspirin resistance” in antithrombotic treatment [27]. Although the addition of
[17, 18] as follows: (a), pharmacokinetic resistance: platelet dipyridamole to aspirin has been reported be more effective
aggregability was successfully inhibited by in vivo addi- in the secondary prevention of stroke than aspirin alone
tion of aspirin; (b), pharmacodynamic resistance: platelet [28], there was insufficient evidence to indicate that dipyri-
aggregability continued when in vitro aspirin was added, damole was effective for AR in patients with vascular disease
with the persistent formation of TXA2; (c), pseudoresistance [29]. Cilostazol, an inhibitor of phosphodiesterase III, has
resistance: platelet aggregability was continued even when in antiplatelet aggregation activity by inhibiting the conversion
vitro aspirin was added, but there was successful inhibition of of cyclic adenosine monophosphate (cAMP) to 5󸀠 -AMP and
TXA2 formation. eventually potentiates the glycoprotein IIb-IIIa inhibitory
The key laboratory parameters are used to examine signals to increases the level of cAMP [30, 31]. However, the
platelet responses to aspirin in a laboratory study using current evidence only showed a tendency in supporting the
measures such as: (a), bleeding time: measurement of the time additional use of cilostazol because cilostazol add-on therapy
necessary for bleeding to stop after standardized skin inci- did not reduce the rate of AR [32]. Furthermore, high doses
sion; (b): light transmission aggregometry: analysis of light of aspirin and the addition of other antiplatelet drugs can
transmission after stimulation of platelets with arachidonic increase the risk of gastrointestinal hemorrhage and other
acid, collagen, ADP, or other agonists in anticoagulated adverse events [22].
platelet-rich plasma; (c), impedance aggregation (whole Faced with the limitations of the presently available treat-
blood aggregometry): Measurement of electrical impedance ments, complementary and/or alternative medicine (CAM)
between two electrodes in whole blood after stimulation is thus increasingly being sought to treat AR worldwide.
with arachidonic acid, collagen, ADP, or other agonists; Currently, the usage of CAM has increased between the
(d), platelet function analyzer-100 (PFA-100), assessment general population and medical personnel in many countries
of aggregation under high shear; whole blood is aspi- [50]. One recent study has reported that treatment of AR
rated through an aperture coated with collagen and either patients by adding one kind of CAMs, omega-3 fatty acids,
Evidence-Based Complementary and Alternative Medicine 3

release of TXA2 in rabbit blood sample with a concentration-

Phospholipid dependent effect, and Poria(Indian bread) inhibits TXA2
receptor-mediated platelet aggregation by suppressing Gq-
mediated signaling pathway in rabbit blood sample with a
Arachidonic acid concentration-dependent effect [58]. Ilexonin A, an active
Aspirin
ingredient of Maodongqing, is a potent inhibitor of vWF
Cyclooxygenase 1
binding and vWF mediated platelet activation and aggre-
Prostaglandin H2 gation in healthy volunteer blood sample [59]. Ilexonin A
Chinese
herbal can also inhibit arachidonic acid- (AA-) induced platelet
Thromboxane synthase
medicine aggregation by interfering with TXA2 formation in rabbit
Aspirin
Thromboxane A2 blood sample with a dose-dependent effect [60]. Danshen-
resistance
based preparations have been used for hundreds of years in
Vasoconstriction; treatment of cardiovascular ischemic diseases [61] with the
platelet aggregation properties of inhibiting prostaglandin synthesis and platelet
adhesion and aggregation in diabetic patients [62] and with
suppression of both the formation and the release of TXA2
Figure 1: Mechanism of Chinese herbal medicine for aspirin in rats [63]. Thus, CHM has the pharmacological effects of
resistance. inhibiting TXA2 and consequently TXA2-dependent platelet
functions.

1.4. Why It Is Important to Do This Review. Owing to the

significant health risk of AR and the limitations of currently
can improve response to aspirin and effectively reduces available conventional therapies, there have been a number
platelet reactivity [51]. In China, the important characteristic of controlled studies over the past decade to evaluate the
of China’s national medical system is that traditional Chinese efficacy and safety of CHM for AR. However, to date, there
medicine (TCM) and Western medicine complement and was no systematic review available to determine the efficacy
cooperate with each other, being responsible together for of CHM for AR. In addition, many of the current clinical
the health care of Chinese people [52]. In the past decades, trials of CHM for AR are thought of as “not so good” studies
Chinese herbal medicine (CHM), as one form of CAMs, has according to Cochrane criteria. In a TCM reviewing process,
been widely used throughout China and elsewhere in the researchers may need to include such papers to identify
world for the treatment of AR [53]. current problems and areas worthy of improvement and
future development [64]. Therefore, the objective of present
systematic review is to assess the efficacy and safety of CHM
1.3. How the Intervention Might Work. Pharmacological for AR.
studies have found the multitarget intervention effects of
CHM on the pathophysiology of AR (Figure 1). For example,
Buyang Huanwu decoction (BHD), a well-known classic 2. Methods
CHM prescription for stroke, can inhibit platelet aggregation
by interfering with cyclooxygenase-1 (COX-1) expression on This systematic review is conducted according to the pre-
platelet and endotheliocyte in rabbits [42]. Some CHMs for ferred reporting items for systematic reviews and meta-
promoting blood circulation such as Rheum palmatum and analyses: the PRISMA statement [65].
Erigeron breviscapus and some tonic herbs such as Codonopsis
pilosula, Astragalus membranaceus, Angelica sinensis, and 2.1. Eligibility Criteria. Types of studies: studies have to be
ginsenosides play an important role in the inhibition of TXA2 randomized clinical trials (RCTs) concerning specifically
synthesis in vitro in the porcine lung microsoma as donor the effectiveness of CHM for AR, regardless of blinding,
of enzymes [54]. Ginkgolide C from Ginkgo biloba leaves publication status, or language. Studies of quasi-RCTs which
is an inhibitor of collagen stimulated platelet aggregation are allocating participants by date of birth, day of the week,
by reducing TXA2 formation in rat blood sample with a medical record number, and month of the year were excluded.
dose-dependent effect [55]. The ingredients of CHM flavones Types of participants: criteria for the diagnosis of AR
can suppress platelet aggregability through inhibiting TXA2 are as follows: >70% platelet aggregation rate induced by
receptor [44]. Panaxtrial saponins can inhibit platelet aggre- 10 𝜇mol/L ADP and >20% aggregation rate induced by 0.5 g/L
gation and TXA2 releasing induced by collagen and arachi- arachidonic acid (AA). The diagnostic criterion of aspirin
donic acid, improve vascular endothelial function, reduce semiresistance (ASR) is meeting one of the two conditions
platelet surface activity, inhibit blood platelet adhesion, above. The exclusion criteria of AR are the following: (1)
reduce blood viscosity, improve microcirculation, and resist aspirin allergy or asthma; (2) all kinds of blood disease, hem-
thrombosis formation in patients with cardiovascular disease orrhagic disease, or bleeding tendency; (3) platelet count
[56, 57]. Atractylodis lanceae rhizoma can inhibit collagen- >450 × 109 /L or <100 × 109 /L; (4) using ticlopidine, clopi-
or TXA2-induced platelet aggregation by suppressing the dogrel, dipyridamole, unfractionated heparin, low molecular
collagen-induced signal pathway, which is upstream of the heparin, and other nonsteroidal anti-inflammatory drugs
4 Evidence-Based Complementary and Alternative Medicine

during the last 2 weeks of observation period; (5) active remained. After going through the titles and abstracts, 78
gastric ulcer patient; (6) gout patient. papers were excluded on the basis that they were nonclinical
Types of interventions: any intervention using CHM trials, case report, or lack of comparison group. By reading
monotherapy or adjunct therapy for AR in the treatment the full text of the remaining 25 articles, 7 were excluded
group was included regardless of the dose, frequency, and for not being RCTs or not real RCTs; 1 paper was removed
intensity of CHM. The interventions of control groups were due to plagiarism; 1 paper was excluded as a result of not
regular low dose aspirin treatment (aspirin 100 mg/d), high carrying out random method. Ultimately, 16 eligible studies
dose aspirin treatment (aspirin 300 mg/d), no intervention, were selected out for this systematic review. The screening
or other antiplatelet drug treatment such as dipyridamole, process is summarized in a PRISMA 2009 flow diagram
clopidogrel, and cilostazol. The treatment course was at least (Figure 2).
2 weeks in these studies.
Types of outcome measures: the primary outcome mea-
surement was platelet aggregation rate induced by AA and/or 3.2. Characteristics of Included Studies. There were a total of
ADP at the end of treatment course. The secondary outcome 1011 subjects in the 16 included trials of CHM for AR pub-
measurement was the clinical efficacy rate and adverse events lished between 2006 and 2012. Among them, 398 participants
at the end of treatment course. The clinical efficacy rate was received CHM adjunct therapy; 217 participants received
defined as the decreased value of platelet aggregation rate CHM monotherapy; 396 participants received aspirin alone
before and after treatment. The standard of clinical efficacy therapy, the addition of other antiplatelet drug to aspirin
rate met any one of the following: (1) AR becomes ASR or therapy, or no treatment. The age of participants ranged from
aspirin sensitive; (2) ASR becomes aspirin sensitive; (3) the 40 to 84 years old, and the treatment duration ranged from
decreased value of platelet aggregation rate is more than 10% 2 weeks to 3 months. All of the studies were performed
and platelet active value is normal. The diagnosis criteria of in a single center and conducted in China. These studies
aspirin sensitive are as follows: ≤70% platelet aggregation rate related to a wide range of conditions including cardiovascular
induced by 10 mL ADP and ≤20% aggregation rate induced by disease [33, 37, 38, 40, 41, 43, 46–48], cerebral infarction [34,
0.5 g/L AA. 39, 45, 67], cardiocerebrovascular disease [35, 49], and high
risk hypertension [36]. In 13 studies [33–37, 39, 41, 43, 45,
2.2. Literature Search. We electronically searched CEN- 46, 48, 49, 67], the diagnostic criteria of AR are an average
TRAL, PubMed, EMBASE, Chinese National Knowledge platelet aggregation rate ≥70% with 10 𝜇mol/L ADP as an
Infrastructure (CNKI), VIP information database, and Wan- agonist and/or ≥20% induced by 0.5 g/L AA [68]. Two studies
fang Data Information Site until December 2012 to identify adopted a maximum platelet aggregation rate ≥30% induced
RCTs involving CHM for AR patients. In addition, we hand- by 0.5 g/L AA as the diagnostic criteria of AR [38, 47]. Only
searched a list of medical journals relevant to this topic and 1 study [40] adopted a platelet aggregation ≥18 ohm with
references from relevant articles. Using free text and the collage or ≥13 ohm with ADP as the diagnostic criteria of
medical subjects headings (MeSH) terms combined “aspirin AR [69]. Five studies were three-group designed study [33,
resistance” and “Chinese herbal medicine or traditional Chi- 37, 39–41]. In 2 studies, both CHM monotherapy and CHM
nese medicine or Chinese materia medicine or integration of adjunct therapy were compared with the regular aspirin
Chinese herbal and Western medicine.” treatment [37, 40]. Both CHM monotherapy and CHM
adjunct therapy was compared with no treatment in 1 study
[33]. In two studies, CHM adjunct therapy were compared
2.3. Study Selection and Data Extraction. Two investigators
with regular aspirin treatment and the addition of other
independently reviewed the titles and abstracts to select
antiplatelet drug [39, 41]. There was positive control in 5
potential references. Then, the two investigators read the
studies. Among them, 3 studies used dipyridamole [34, 45,
selected articles independently and made a final decision
67]; 1 study used clopidogrel [41]; 1 study used cilostazol [39].
for selection or not. Key data were extracted according to
All studies used platelet aggregation as primary outcome, and
standardized criteria, including patients, methods, interven-
other indexes related to platelet aggregation were also used
tions, and outcomes. Disagreements were settled through
as outcome in 7 studies [34, 36, 38, 39, 41, 48, 49]. Clinical
discussion or consultation with one correspondence author.
effective rate was observed in 4 studies [35, 47, 49, 67], but the
standard was not completely the same in each study. Adverse
2.4. Risk of Bias in Individual Studies. The methodological events were reported in 7 studies [35, 36, 38, 40, 47, 49, 67],
quality of RCTs was assessed independently by two investi- and the incidence of adverse event in treatment group was
gators using the 12-item criteria from Cochrane Back Review lower than in control group. The detailed characteristics of
Group [66]. All data were analyzed using Review Manager included studies are summarized in Table 1.
5.0 software. We are reconciling any discrepancy through
discussion with one correspondence author.
3.3. Description of the CHM and Its Prescription. Seven
3. Results Chinese herbal prescriptions were tested in 12 (72.5%) of
the 16 included studies, while the other 4 studies used
3.1. Description of Studies. We identified 106 potentially rel- single herb or active ingredients (Table 2). Tongxinluo cap-
evant articles. After removal of duplicates, 103 records sule (TXLC) [39–41, 47] and Danshen-based prescriptions,
 Table 1: Basic characteristics of the included studies.
Sample and characteristics
Included Eligibility Study Interventions Intergroup
Type of disease (𝑛) (male/female; age) Outcome index
trials criteria of AR designs differences
Trial Control Trial Control
RCT
(method
unreported)
Coronary heart Huoxue capsule
and controlled 12 13 Platelet
disease AA# and ADP# 12 pieces tid No treatment 𝑃 > 0.05
nonblinded — — aggregation rate
(37) for 3 month
three-group
Peng et al. design
2011 [33] study
Huoxue capsule
12 pieces tid + aspirin Huoxue 12
12 Platelet aggregation rate 𝑃 < 0.05
100 mg qd capsule —
for 3 month
Evidence-Based Complementary and Alternative Medicine

Huoxue capsule +
No treatment Platelet aggregation rate 𝑃 < 0.05
aspirin
RCT
Aspirin
(method Sodium ferulate 40 (M: 21, F:
100 mg qd +
Ma et al. Acute cerebral unreported) 100 mg, 40 (M: 23, F: 17) 19) (1) Platelet aggregation rate (1) 𝑃 < 0.05
AA# and ADP# dipyridamole
2012 [34] infarction (80) and controlled tid + aspirin 100 mg qd Mean age: 63 y Mean age: (2) TXB2 (2) 𝑃 < 0.05
150 mg qd
nonblinded for 4 weeks 65 y
for 4 weeks
parallel study
RCT (1) Platelet aggregation rate (1) 𝑃 < 0.05
30 (M: 13, F:
(random (2) TXB2 (2) 𝑃 < 0.05
Cardio/ Diao Xin Xue kang Aspirin 30 (M: 15, F: 15) 17)
Su 2012 AA# and ADP# number table) (3) 6-K-PGF 1𝛼 (3) 𝑃 < 0.05
Cerebrovascular 1.6 g, tid 300 mg qd Mean age: 62 y
[35] and controlled (4) TXB2/6-K-PGF 1𝛼 (4) 𝑃 < 0.05
disease (60) for 4 weeks for 4 weeks Mean age:
nonblinded (5) Clinical effective rate (5) 𝑃 < 0.05
61.2 y
parallel study (6) Adverse events (6) 𝑃 < 0.01
(1) Platelet aggregation rate (1) 𝑃 < 0.01
RCT
CDDP 270 mg tid +
(method
High risk aspirin Aspirin (2) Acute myocardial (2) 𝑃 < 0.05
Guo 2012 ADP# unreported) 50 53
hypertensive 100 mg qd 100 mg qd infarction
[36] and controlled — —
patients (103) for 1 month for 1 month (3) Cerebral infarction (3) 𝑃 < 0.05
nonblinded
parallel study
(4) Bleeding events (4) 𝑃 > 0.05
5
 6

Table 1: Continued.
Sample and characteristics
Included Eligibility Study Interventions Intergroup
Type of disease (𝑛) (male/female; age) Outcome index
trials criteria of AR designs differences
Trial Control Trial Control
RCT
(method
(1) ADP-induced platelet
unreported) 10 (M: 4, F: 6) (1) 𝑃 < 0.05
Aspirin 100 mg 10 (M: 3, F: 7) aggregation rate
Cardiovascular AA# and ADP# and controlled CDDP 10 pieces tid Mean age:
qd Mean age:
disease (30) nonblinded for 2 weeks 12.31 ±
for 2 weeks 67.70 ± 12.791 y (2) AA-induced
Chai et al. three-group 17.61 y (2) 𝑃 > 0.05
platelet aggregation rate
2008 [37] design
study
CDDP 10 pieces tid +
10 (M: 3, F: 7)
aspirin 𝑃 < 0.05 or
CDDP Mean age: Platelet aggregation rate
100 mg qd 𝑃 < 0.01
67.4 ± 14.7 y
for 2 weeks
CDDP + aspirin Aspirin Platelet aggregation rate 𝑃 < 0.01
RCT
(random Qishenyiqi pill
Qishenyiqi Pill 17 (M: 12, F: 22) (1) Platelet aggregation rate (1) 𝑃 < 0.05
Chen et al. Coronary heart number table) 0.5 g tid + aspirin
AA# 0.5 g tid Mean age: 17 (2) TG, VLDL (2) 𝑃 > 0.05
2008 [38] disease (34) and controlled 100 mg qd
for 4 weeks 53.4 ± 9.2 y (3) Adverse events (3) 𝑃 > 0.05
nonblinded for 4 weeks
parallel study
RCT
(random
number table) TXLC 4 pieces tid +
Aspirin 20 (M: 20, F:
Cerebral infarction ADP# and controlled aspirin 20 (1) Platelet aggregation rate (1) 𝑃 < 0.05
100 mg qd 40)
(60) nonblinded 100 mg qd — (2) TXB2 (2) 𝑃 < 0.05
for 1 month —
Zhang et three-group for 1 month
al. 2010 design
[39] study
Cilostazol
100 mg qd +
20 (1) Platelet aggregation rate (1) 𝑃 > 0.05
TXLC + Aspirin aspirin
— (2) TXB2 (2) 𝑃 > 0.05
100 mg qd
for 1 month
Evidence-Based Complementary and Alternative Medicine
 Table 1: Continued.
Sample and characteristics
Included Eligibility Study Interventions Intergroup
Type of disease (𝑛) (male/female; age) Outcome index
trials criteria of AR designs differences
Trial Control Trial Control
(1) Platelet aggregation rate (1) 𝑃 < 0.01
Cilostazol + aspirin Aspirin
(2) TXB2 (2) 𝑃 < 0.01
RCT
(random
number table) 29 (M: 17, F:
ADP# and Aspirin 30 (M: 19, F: 11) (1) Platelet aggregation
Coronary heart and controlled TXLC 3 pieces tid 12) Mean age: (1) 𝑃 < 0.05
COL# 100 mg qd Mean age: Rate
disease (89) nonblinded for 1 month 66.93 ± (2) 𝑃 > 0.05
for 1 month 66.69 ± 10.56 y (2) Adverse events
Yin et al. three-group 10.75 y
2010 [40] design
study
TXLC 1 piece tid +
30 (M: 3, F: 7)
aspirin (1) Platelet aggregation rate (1) 𝑃 < 0.05
Aspirin Mean age:
100 mg qd (2) Adverse events (2) 𝑃 > 0.05
67.4 ± 14.7 y
for 1 month
(1) Platelet aggregation rate (1) 𝑃 > 0.05
Evidence-Based Complementary and Alternative Medicine

TXLC + aspirin TXLC

(2) Adverse events (2) 𝑃 > 0.05
RCT
(method
unreported) TXLC 4 pieces tid +
Aspirin (1) Platelet aggregation rate (1) 𝑃 < 0.05
Acute coronary AA# and ADP# and controlled aspirin 24
100 mg qd 23 (2) TXB2 (2) 𝑃 < 0.05
syndrome (70) nonblinded 100 mg qd (M: 20, F: 50)
for 1 month (3) CRP (3) 𝑃 < 0.05
three-group for 1 month
Song et al. design
2008 [41] study
Clopidogrel
75 mg qd + (1) Platelet aggregation rate (1) 𝑃 > 0.05
23
TXLC + aspirin aspirin (2) TXB2 (2) 𝑃 > 0.05
—
100 mg qd (3) CRP (3) 𝑃 > 0.05
for 1 month
(1) Platelet aggregation rate (1) 𝑃 < 0.05
Clopidogrel + aspirin Aspirin (2) TXB2 (2) 𝑃 < 0.01
(3) CRP (3) 𝑃 < 0.05
RCT
(method Xuefuzhuyutang
Aspirin 30 (M: 38, F: 22)
Wu 2012 Cardiovascular unreported) 1 dose/d + aspirin
AA# and ADP# 100 mg qd Mean age: 30 Platelet aggregation rate 𝑃 < 0.05
[43] disease (60) and controlled 100 mg qd
for 4 weeks 35–80 y
nonblinded for 4 weeks
parallel study
7
 8

Table 1: Continued.
Sample and characteristics
Included Eligibility Study Interventions Intergroup
Type of disease (𝑛) (male/female; age) Outcome index
trials criteria of AR designs differences
Trial Control Trial Control
RCT
Dipyridamole
(method Gingko biloba 36 (M: 18, F:
150 mg qd (1) Platelet aggregation rate (1) 𝑃 < 0.01
Liu 2010 Ischemic stroke unreported) 2 pieces, tid + aspirin 36 (M: 21, F: 15) 18)
AA# or ADP# + aspirin (2) Clinical effective rate (2) 𝑃 < 0.05
[67] (72) and controlled 100 mg qd Mean age: 65 y Mean age:
100 mg qd (3) Adverse events (3) 𝑃 < 0.01
nonblinded for 1 month 67 y
for 1 month
parallel study
RCT
Zhuyu Tongmai Dipyridamole
(method 40 (M: 23, F:
capsule 150 mg qd 40 (M: 21, F:
Liu 2008 Cerebral infarction unreported) 17)
ADP# 2 pieces tid + aspirin + aspirin 19)Mean age: Platelet aggregation rate 𝑃 < 0.01
[45] (80) and controlled Mean age:
100 mg qd 100 mg qd 38–72 y
nonblinded 41–75 y
for 1 month for 1 month
parallel study
RCT
Fufang Danshen Fufang 26 (M: 14, F:
(method
Cheng et injection 20 ml + Danshen 26 (M: 14, F: 12) 12)
Chronic coronary unreported)
al. 2010 AA# and ADP# aspirin injection Mean age: Mean age: Platelet aggregation rate 𝑃 < 0.05
disease (52) and controlled
[46] 100 mg qd 20 ml 65 ± 9 y 66 ± 8 y
nonblinded
for 2 weeks for 2 weeks
parallel study
RCT
(method TXLC 4 pieces tid +
TXLC 4 pieces 32 (M: 44, F: 20) (1) Platelet aggregation rate (1) 𝑃 < 0.05
Liu et al. Coronary heart unreported) aspirin
AA# > 30% tid Mean age: 32 (2) Clinical effective rate (2) 𝑃 < 0.05
2006 [47] disease (64) and controlled 100 mg qd
for 3 weeks 69.4 ± 11.2 y (3) Adverse events (3) 𝑃 > 0.05
nonblinded for 3 weeks
parallel study
RCT
Lumbrokinase Lumbrokinase
(method 30 (M: 13, F:
enteric-coated capsules enteric-coated 30 (M: 16, F: 14)
Sun et al. Coronary heart unreported) 17)
AA# and ADP# 60 wan IU tid + aspirin capsules Mean age: Platelet aggregation rate 𝑃 < 0.01
2009 [48] disease (60) and controlled Mean age:
100 mg qd 60 wan IU tid 68.1 ± 14.7 y
nonblinded 62.31±17.61 y
for 1 month for 1 month
parallel study
RCT (1) Platelet aggregation rate (1) 𝑃 < 0.05
(method Zhilonghuoxuetongyu 30 (M: 13, F: (2) TXB2 (2) 𝑃 > 0.05
Cardio/ Aspirin 30 (M: 15, F: 15)
Luo et al. unreported) capsule 17) (3) 6-K-PGF 1𝛼 (3) 𝑃 < 0.05
Cerebrovascular AA# and ADP# 300 mg qd Mean age:
2012 [49] and controlled 1.6 g, tid Mean age: (4) TXB2/6-K-PGF 1𝛼 (4) 𝑃 > 0.05
disease (60) for 4 weeks 43–70 y
nonblinded for 4 weeks 41–70 y (5) Clinical effective rate (5) 𝑃 < 0.05
parallel study (6) Adverse events (6) 𝑃 < 0.01
AA#: arachidonic acid induced platelet aggregation rate > 20%; ADP#: adenosine diphosphate induced platelet aggregation rate > 70%; COL#: collagen induced platelet aggregation rate > 30%; CDDP: compound
Danshen dripping pill; TXB2: thromboxane B2; 6-K-PGF 1𝛼: 6-keto-prostaglandin F1a; TG: triglyceride; VLDL: very-low-density lipoprotein; CRP: C response protein; TXLC: Tongxinluo capsule.
Evidence-Based Complementary and Alternative Medicine
Evidence-Based Complementary and Alternative Medicine 9

Table 2: Chinese herbal prescription or single herb or active ingredients for aspirin resistance in the 16 reviewed studies.

Chinese herbal
prescription or Content (Chinese pinyin, English herb name, Latin herb name, Chinese patent
Reference Preparations/dosage
single herb or Family) medicine
active ingredients
Renshen (Ginseng; Radix Ginseng; Panax ginseng C. A. Mey.),
Shuizhi (Leech; Hirudo; Whitmania pigra Whitman or
Hirudo nipponica Whitman or Whitmania acranulata
Whitman), Quanxie (Scorpion; Scorpio; Buthus martensii
Karsch), Chishao (peony root; Radix Paeoniae Rubra;
Paeonia lactiflora Pall. or Paeonia veitchii Lynch), Chantui
Zhang et al.
(cicada slough; Periostracum Cicadae; Cryptotympana pustulata
2010 [39],
Fabricius), Tubiechong (ground beetle; Eupolyphaga Seu
Yin et al. 2010
Steleophaga; Eupolyphaga sinensis Walker or Steleophaga plancyi
[40], Capsule/3 or 4
Tongxinluo capsule (Boleny)), Wusong (Centipede; Scolopendra; Yes
Song et al. capsules tid
Scolopendra subspinipes mutilans L. Koch), Tanxiang
2008 [41],
(Sandalwood; Lignum Santali Albi; Santalum album L.),
Liu et al.
Jiangxiang (Rosewood; Lignum Dalbergiae Odoriferae;
2006 [47]
Dalbergia odorifera T. Chen), Ruxiang (Frankincense;
Olibanum; Boswellia carterii Birdw. or Boswellia bhaw-dajiana
Birdw or Boswellia neglecta M. Moore), Suanzaoren (spine date
seed; Semen Ziziphi Spinosae; Ziziphus jujuba Mill. var. or
spinosa (Bunge) Hu ex H. F. Chou), Bingpian (Borneol;
Borneolum Syntheticum)
Danshen (danshen root; Radix Salviae Miltiorrhizae; Salvia
Chai et al. Compound
miltiorrhiza Bge)., Sanqi (Sanqi; Radix Notoginseng; Dripping pill/10 pills
2008 [37], danshen dripping Yes
Panax notoginseng (Burk.) F. H. Chen), Bingpian (Borneol; tid
Guo 2012 [36] pill
Borneolum Syntheticum)
Danggui (Chinese angelica; Radix Angelicae Sinensis; Angelica
sinensis (Oliv.) Diels) 9 g, Shengdihuang (unprocessed
rehmannia root; Radix Rehmanniae Recens) 9 g, Taoren (peach
seed; Semen Persicae; Amygdalus persica L. or
Amygdalus davidiana (Carr.) C. de Vos ex Henry) 12 g, Honghua
(Safflower; Flos Carthami; Carthamus tinctorius L.) 9 g, Zhike
(orange fruit; Fructus Aurantii; Citrus aurantium L.) 6 g,
Chishao (peony root; Radix Paeoniae Rubra; Paeonia lactiflora
Decoction//1 dose
Xuefu Zhuyu Pall. or Paeonia veitchii Lynch) 6 g, Chuanxiong (sichuan lovage
Wu 2012 [43] qd No
Decoction rhizome; Rhizoma Ligustici Chuanxiong;
Ligusticum chuanxiong Hort.) 5 g, Chaihu (Chinese thorowax
root; Radix Bupleuri; Bupleurum chinensis DC. or
Bupleurum scorzonerifolium Willd.) 3 g, Jiegeng (platycodon
root; Radix Platycodonis; Platycodon grandiflorus (Jacq.) A.
DC.) 5 g, Niuxi (two-toothed achyranthes root; Radix
Achyranthis Bidentatae; Achyranthes bidentata Bl.) 9 g, Gancao
(liquorice root; Radix Glycyrrhizae; Glycyrrhiza uralensis Fisch.
or Glycyrrhiza inflata Bat. or Glycyrrhiza glabra L.) 3 g.
Xuefu Zhuyu decoction minor Jiegeng (platycodon root; Radix
Platycodonis;
Platycodon grandiflorus (Jacq.) A. DC.), plus Huangqi
Peng et al. Huoxue capsule (milkvetch root; Radix Astragali seu Hedysari; Capsule/2 capsules
Yes
2011 [33] Astragalus membranaceus (Fisch.) Bge. var. Mongholicus (Bge.) tid
Hsiao or Astragalus membranaceus (Fisch.) Bge.), and
Suanzaoren (spine date seed; Semen Ziziphi Spinosae;
Ziziphus jujuba Mill. var. or spinosa (Bunge) Hu ex H. F. Chou)
Huangqi (milkvetch root; Radix Astragali seu Hedysari;
Astragalus membranaceus (Fisch.) Bge. var. Mongholicus (Bge.)
Hsiao or Astragalus membranaceus (Fisch.) Bge.), Danshen
Chen et al. Qisheyiqi dripping Dripping pill/0.5 g
(danshen root; Radix Salviae Miltiorrhizae; Salvia miltiorrhiza Yes
2008 [38] pill bid
Bge.), Sanqi (Sanqi; Radix Notoginseng; Panax notoginseng
(Burk.) F. H. Chen), oil of Jiangxiang (Rosewood; Lignum
Dalbergiae Odoriferae; Dallbergia odarifera T. Chen)
10 Evidence-Based Complementary and Alternative Medicine

Table 2: Continued.
Chinese herbal
prescription or Content (Chinese pinyin, English herb name, Latin herb name, Chinese patent
Reference Preparations/dosage
single herb or Family) medicine
active ingredients
Mengchong (Tabanus; Gadfly), Shuizhi (Leech; Hirudo;
Whitmania pigra Whitman or Hirudo nipponica Whitman or
Whitmania acranulata Whitman), Taoren (peach seed; Semen
Zhuoyu Tongmai Capsule/2 capsules
Liu 2008 [45] Persicae; Amygdalus persica L. or Amygdalus davidiana (Carr.) Yes
capsule tid
C. de Vos ex Henry), Dahuang (rhubarb root and rhizome;
Radix et Rhizoma Rhei; Rheum palmatum L. or
Rheum tanguticum Maxim. ex Balf. or Rheum officinale Baill.)
Danshen (danshen root; Radix Salviae Miltiorrhizae;
Cheng et al. Fufang Danshen
Salvia miltiorrhiza Bge.), Jiangxiang (Rosewood; Lignum Injection/20 ml qd Yes
2010 [46] injection
Dalbergiae Odoriferae; Dalbergia odorifera T. Chen)
Huangqi (milkvetch root; Radix Astragali seu Hedysari;
Astragalus membranaceus (Fisch.) Bge. var. Mongolicus (Bge.)
Hsiao or Astragalus membranaceus (Fisch.) Bge.), Guizhi (cassia
twig; Ramulus Cinnamomi; Cinnamomum cassia Presl),
Daxueteng (sargentgloryvine stem; Caulis Sargentodoxae;
Luo et al. Zhilong Huoxue Sargentodoxa cuneata (Oliv.) Rehd. et Wils.), Shuizhi (Leech;
Capsule/1.6 g tid No
2012 [49] Tongyu capsule Hirudo; Whitmania pigra Whitman or Hirudo nipponica
Whitman or Whitmania acranulata Whitman), Quanxie
(Scorpion; Scorpio; Buthus martensii Karsch), Dilong
(Earthworm; Lumbricus; Pheretima aspergillum (E. Perrier) or
Pheretima vulgaris Chen or Pheretima guillelmi (Michaelsen) or
Pheretima pectinifera Michaelsen )
Sodium ferulate The sodium salt of ferulic acid. It is found in the root of
Ma et al. 2012
tablets Angelica sinensis. Ferulic acid can also be extracted from the Tablets/100 mg tid Yes
[34]
root of the Chinese herb Ligusticum chuanxiong.
Diao
Su 2012 [35] Xinxuekang A dry extract of the root of Dioscorea nipponica Makino Capsule/1.6 g tid Yes
capsule
Gingko biloba Extract of Yinxingye (ginkgo leaf; Folium Ginkgo; Ginkgo biloba
Liu 2010 [67] Tablets/2 tablets tid Yes
tablets L.)
Lumbrokinase Enteric-coated
Sun et al. A group of proteolytic enzymes derived from the earthworm
enteric-coated capsule/60,000 IU Yes
2009 [48] Lumbricus rubellus
capsules tid

including compound danshen dripping pill [36, 37], Qisheny- criteria from the Cochrane Back Review Group [66]. Risk of
iqi dripping pill [38], and Fufang Danshen injection [46], bias in included studies varied from 2/12 to 6/12, of average 3.7.
were the most frequently used standardized prescriptions. Only 4 articles [35, 38, 40, 67] reported the method of random
Danshen (danshen root; Radix Salviae Miltiorrhizae; Salvia sequences generation, although all trials claimed that they
miltiorrhiza Bge.), Huangqi (milkvetch root; Radix Astra- were RCTs. Only 1 trial [38] reported allocation concealment,
gali seu Hedysari; Astragalus membranaceus (Fisch.) Bge. and none of the included studies mentioned blinding. The
var. Mongholicus (Bge.) Hsiao or Astragalus membranaceus dropout data were not reported in 5 trials [35, 38, 40, 47,
(Fisch.) Bge.), Shuizhi (Leech; Hirudo; Whitmania pigra 67]. All the included studies have relatively small sample
Whitman or Hirudo nipponica Whitman or Whitmania sizes and do not have formal sample size calculation. The
acranulata Whitman), and Jiangxiang (Rosewood; Lignum methodological quality of included studies was summarized
Dalbergiae Odoriferae; Dalbergia odorifera T. Chen) were the in Table 3.
most frequently used single herbs, which were used for more
than 3 times in all the trials. Nine of 11 preparations were
Chinese patent medicine and have a rigorous quality control 3.5. Results of Individual Studies. Meta-analysis could not be
of herbal preparations, whereas the other 2 preparations were performed owing to the high clinical heterogeneity and low
homemade [43, 49] (Table 2). methodological quality of the included studies. Additionally,
the number of trials is too small to draw a meaningful funnel
3.4. Risk of Bias in Included Studies. The methodological plot. Therefore, we also did not conduct the funnel plot
quality of RCTs was assessed independently using 12-item analysis.
Evidence-Based Complementary and Alternative Medicine 11

Records identified through database Additional records identified through

Identification
searching other sources
(n = 105) (n = 1)

Records after removing duplicates

Screening (n = 103)

Records excluded (n = 78)

Records screened ∙ Case report or lack in comparison
(n = 103) group
∙ Not reports of clinical trials.

Full-text articles assessed Full-text articles excluded (n = 9)

Eligibility

for eligibility ∙ Not RCT or not real RCT (7)

(n = 25) ∙ Not carried out random method (1)
∙ Plagiarism (1)

Studies included in
Included

qualitative synthesis
(n = 16)

Figure 2: PRISMA 2009 flow diagram.

3.6. CHM Plus Aspirin versus No Treatment. One RCT [33] indicated the significant effects of CHM for reducing platelet
reported significant effects of CHM plus aspirin for reducing aggregation (𝑃 < 0.05). However, Luo et al. [49] indicated
platelet aggregation rate compared with no treatment (𝑃 < that AR patients receiving Zhilong Huoxue Tongyu capsule
0.05). Although platelet aggregation rate was partly reduced therapy have no significant difference in comparison with
after CHM monotherapy, there was no significant difference high dose aspirin therapy.
between CHM monotherapy group and no treatment control
group (𝑃 > 0.05), Table 1.
3.9. CHM Plus Aspirin versus Aspirin. There were 6 RCTs
3.7. CHM Monotherapy versus Aspirin 100 mg/d. There are 2 comparing CHM plus aspirin with aspirin 100 mg/d therapy.
RCTs comparing CHM monotherapy with aspirin 100 mg/d All 6 trials indicated more significant effect of CHM plus
therapy. Yin et al. [40] reported significant effect of CHM aspirin for reducing platelet aggregation rate compared with
for reducing platelet aggregation rate when compared with aspirin 100 mg/d therapy (𝑃 < 0.05) [36, 37, 39–41, 43].
aspirin 100 mg/d (𝑃 < 0.05). Chai et al. [37] showed that
CHM has significant effect in reducing platelet aggregation 3.10. CHM Plus Aspirin versus Dipyridamole or Cilostazol or
induced by ADP (𝑃 < 0.05) but has no significant effect Clopidogrel Plus Aspirin. There were 5 RCTs that compared
in reducing platelet aggregation induced by AA (𝑃 > 0.05) CHM plus aspirin therapy with dipyridamole or cilostazol
(Table 1). or clopidogrel plus aspirin therapy [34, 39, 41, 45, 67].
Three RCTs [34, 45, 67] showed significant difference in
3.8. CHM Monotherapy versus Aspirin 300 mg/d. There are 2 reduction of platelet aggregation rate when comparing CHM
RCTs comparing CHM monotherapy with high dose aspirin plus aspirin therapy with dipyridamole plus aspirin therapy
therapy group (aspirin 300 mg/d) [35, 49]. Studies of Luo et al. (𝑃 < 0.05, 𝑃 < 0.01). However, Song et al. [41] and Zhang
[49] and Su [35] showed significant difference in the reduc- et al. [39] indicated that AR patients receiving CHM plus
tion of platelet aggregation rate when CHM monotherapy aspirin therapy have no significant difference compared with
was compared with high dose aspirin therapy (𝑃 < 0.05). cilostazol or clopidogrel plus aspirin therapy based on the rate
Based on the testing of TXB2 and TXB2/6-K-PGF 1 𝛼, Su [35] of platelet aggregation and TX B2 production (𝑃 > 0.05).
12 Evidence-Based Complementary and Alternative Medicine

Table 3: The methodological quality of included studies.

A B C D E F G H I J K L Total + Total − Total ?

Peng et al. 2011 [33] ? − − − − ? ? ? + + ? + 3 4 5
Ma et al. 2012 [34] ? − − − − ? ? ? + + ? + 3 4 5
Su 2012 [35] + − − − − ? + ? + + ? + 5 4 2
Guo 2012 [36] ? − − − − ? − ? + + ? + 3 5 3
Chai et al. 2008 [37] ? − − − − ? ? ? − + ? + 2 5 5
Chen et al. 2008 [38] + + − − − ? + ? + + ? + 6 4 3
Zhang et al. 2010 [39] ? − − − − ? ? ? + + ? + 3 4 6
Yin et al. 2010 [40] + − − − − ? + ? + + + + 5 4 3
Song et al. 2008 [41] ? − − − − ? ? ? + + ? + 3 4 5
Wu 2012 [43] ? − − − − ? ? ? + + ? + 3 4 5
Liu 2010 [67] + − − − − ? + ? + + ? + 5 4 2
Liu 2008 [45] ? − − − − ? ? ? + + ? + 3 4 5
Cheng et al. 2010 [46] ? − − − − ? ? ? + + ? + 3 4 5
Liu et al. 2006 [47] ? − − − − ? + ? + + ? + 4 4 4
Sun et al. 2009 [48] ? − − − − ? ? ? + + ? + 3 4 5
Luo et al. 2012 [49] ? − − − − ? ? ? + + ? + 3 4 5
A: adequate sequence generation; B: concealment of allocation; C: blinding (patient); D: blinding (investigator); E: blinding (assessor); F: incomplete outcome
data addressed (ITT analysis); G: incomplete outcome data addressed (dropouts); H: free of selective reporting; I: similarity at baseline; J: cointerventions
constant; K: compliance acceptable; L: similar timing outcome assessments. +: yes, −: no, and ?: unclear.

3.11. CHM Plus Aspirin versus CHM. There were 7 RCTs aspirin group and 17 cases in dipyridamole plus aspirin group
which compared CHM plus aspirin therapy with CHM (𝑃 < 0.01). Su [35] reported 2 cases of adverse events in Diao
monotherapy [33, 37, 38, 40, 46–48] (Table 1). Six RCTs Xinxuekang group and 16 cases in high dose aspirin group
demonstrated that CHM plus aspirin therapy has significant (𝑃 < 0.01). Luo et al. [49] reported 2 cases of adverse events in
effect on reducing platelet aggregation rate when compared Zhilong Huowue Tongyu capsule group and 12 cases in high
with CHM monotherapy (𝑃 < 0.05, 𝑃 < 0.01) [33, 37, 38, 46– dose aspirin group (𝑃 < 0.01); see Table 1.
48], whereas 1 study [40] showed no significant difference in
platelet aggregation rate (𝑃 > 0.05).
4. Discussion
3.12. Clinical Effective Rate. Clinical effective rate was 4.1. Summary of Evidence. To our knowledge, this study is the
reported in 4 out of 16 included studies [35, 47, 49, 67]. first systematic review to determine the efficacy and safety
One RCT [47] indicated more significant effect of CHM plus of CHM for AR patients. A total of 1011 subjects in the
aspirin for improving clinical effective rate compared with 16 included trials of CHM for AR have emerged between
CHM monotherapy (𝑃 < 0.05). Liu et al. [67] reported 2006 and 2012, suggesting that the interests of the public
significant difference in improving clinical effective rate when and the medical profession in the use of CHM for AR
comparing CHM plus aspirin therapy with dipyridamole plus have grown considerably in the recent years. Most of herbal
aspirin therapy (𝑃 < 0.05). Su [35] and Luo et al. [49] reported preparations were Chinese patent medicine, which have a
better effect of CHM for improving clinical effective rate of rigorous quality control. The current evidence is insufficient
AR compared with high dose aspirin (𝑃 < 0.05). to recommend the routine use of CHMs for AR because of
the poor methodological quality and lack of safety data.
3.13. Adverse Events. Among the 16 included studies, adverse
events were reported in 7 studies [35, 36, 38, 40, 47, 49, 4.2. Limitations. Several methodological limitations in the
67]. There were no adverse events reported in 3 studies primary studies should be addressed. First, although all the
[38, 40, 47], while the left 4 studies reported the occurrence included trials claimed to be RCTs, only 4 trials [35, 38, 40, 67]
of adverse events, and statistical analysis was done. Guo et reported the method of random sequences generation. Lack
al. [36] reported adverse events such as acute myocardial of random description would lead to the possibility of bias.
infarction, cerebral infarction, or intracerebral hemorrhage, Only 1 trial [38] reported allocation concealment. Inadequate
and the results showed that CHM was less likely to have or unclear allocation concealment in trials could lead to an
adverse events (𝑃 < 0.05 or 𝑃 < 0.01). A number of mild average 18% more “beneficial” than effect estimates from
adverse events were observed in 3 studies [35, 49, 67], such trials with adequate concealment (95% CI 5% to 29%) [70].
as headache, dizziness, facial flushing, epigastric discomfort, None of the included studies mentioned blinding, which may
and nausea, which can recover with no treatment. Liu et al. result in performance bias and detection bias. In addition,
[67] reported 4 cases of adverse events in Gingko biloba plus placebo control was not used in all included trials, which
Evidence-Based Complementary and Alternative Medicine 13

will make positive result in intervention group. Second, ITT 5.2. Implications for Research. CHMs are widely used in the
analysis provides the least bias for a clinical trial [71]. CON- treatment of AR. There are too many unanswered questions,
SORT guidelines emphasized the necessity of ITT analysis on making it impossible to draw positive or negative conclu-
the reporting of randomized controlled trials [72]. However, sions. The most frequently used standardized Chinese patent
there was no trial that mentioned analyzing data based on the medicine such as TXLC and Danshen-based prescriptions
ITT principle. Moreover, only 1 trial [36] mentioned 49 cases as well as the most frequently used herbs, including dan-
of withdrawal in the course of intervention. Third, selective shen root, milkvetch root, Leech, and Rosewood that may
bias is also a methodological limitation for this review. It is contribute in composing a fundamental prescription, could
unclear if there was selective reporting in all included trials. be promising candidates for further clinical trials of AR.
High proportions of positive results were usually published Owing to serious attention to methodological quality, we
in some Asian countries including China [73]. In this review, recommend that the CONSORT 2010 statement [78], RCTs
all included trials were from China. Therefore, we cannot investigating CHM [79], and CONSORT for TCM [80],
exclude the possibility of selective bias. Fourth, members which consist of a checklist to determine study quality and
of the International Committee of Medical Journal Editors rigor, respectively, should be used as a guideline for further
stated that all clinical trials must be registered in order designing and reporting of RCTs.
to be publicated in September 2004 [74]. However, none The safety of herbal medicines has become a major
of the included trials have been registered, which would concern to both national health authorities and the general
affect the validity of trials. Our search strategy should have public. Thus, World Health Organization (WHO) published
identified the majority of the available literature. However, WHO guidelines on safety monitoring of herbal medicines
we acknowledge the possibility that the review may not be in pharmacovigilance systems in 2004 [81]. However, the
fully comprehensive. Publication bias may also exist when risks caused by drug-herb interactions, especially for those
nonsignificant findings remain unpublished, and thereby involving anticoagulant/antiplatelet drugs and CHM, are
inflating the apparent magnitude of the effect. Fifth, an often ignored or underestimated. A recent review of poten-
adequate sample size is vital to the design of randomized con- tial harmful interactions between anticoagulant/antiplatelet
trolled trials [75]. All included studies have relatively small agents and CHM indicated that the additive anticoagulant
sample sizes and do not have formal sample size calculation, or antiplatelet effects of the CHMs, especially danshen root,
which would make this review’s validity doubtable. Sixth, 12 Danggui (Chinese angelica; Radix Angelicae Sinensis; Angel-
different CHMs with great variation in terms of composition, ica sinensis (Oliv.) Diels), Jiang (ginger; Rhizoma Zingiberis
dosage, and duration of interventions were reported in 16 Recens; Zingiber officinale Rosc.), Yinxingye (ginkgo leaf;
included trials. There was a wide heterogeneity in the CHMs Folium Ginkgo; Ginkgo biloba L.), Gancao (liquorice root;
among the included studies because TCMs are composed of Radix Glycyrrhizae; Glycyrrhiza uralensis Fisch. or Gly-
more than one herb and produce different therapeutic effects cyrrhiza inflata Bat. or Glycyrrhiza glabra L.), and Jianghuang
with different concentration proportions of the constituents (Turmeric; Rhizoma Curcumae Longae; Curcuma longa L.),
[76]. This makes it difficult to recommend any specific CHM could increase bleeding risks in those patients with car-
for clinical use. dio/cerebrovascular diseases [82]. However, adverse events in
the present study were reported only in 7 studies [35, 36, 38,
40, 47, 49, 67]. Therefore, special attention should be paid
5. Concluding Remarks to antiplatelet drug-CHMs interactions due to the potential
risks of increased bleeding. Furthermore, many reports on
5.1. Implications for Practice. The available evidence from adverse drug reactions demonstrated that the toxic effects
present systematic review is insufficient to recommend the with the use of CHM could be associated with hepatotoxicity,
routine use of CHM for AR because of the poor methodologi- nephrotoxicity, nervous system damage, and so forth [83].
cal quality, high clinical heterogeneity, and lack of safety data. Therefore, adverse events should have been reported in each
However, CHMs appeared to be well tolerated in all included included trial in order to guide the clinical use of CHM for
studies. Therefore, the efficacy and safety of CHM therapy for AR. A standard reporting format for ADR that has recently
AR remain to be further determined. However, it should be been developed should be used as a guideline of reporting
remembered that a lack of scientific evidence does not neces- adverse events and ADRs in the future clinical study [84].
sarily mean that the treatment is ineffective [77]. TXLC [39–
41, 47] and Danshen-based prescriptions, including com- 6. Conclusions
pound danshen dripping pill [36, 37], Qishenyiqi dripping
pill [38], and Fufang Danshen injection [46], were the most Sixteen RCTs were identified in this systematic review for
frequently used standardized Chinese patent medicine. These evaluating the efficacy and safety of various CHMs in the
prescriptions have potentially clinical efficiency and their treatment of AR. Despite the apparent reported positive
preparations have a rigorous quality control, which should findings, it is premature to determine the efficacy and safety
be the priority for clinical use of AR. In addition, Danshen of CHM for the treatment of AR due to poor methodological
(danshen root), Huangqi (milkvetch root), Shuizhi (Leech), quality and insufficient safety data. However, CHMs appeared
and Jiangxiang (Rosewood) as the most frequently used to be well tolerated in all included studies. Therefore, CHM
single herbs may contribute in composing a fundamental as a promising candidate are worthy of improvement and
prescription for AR. development for further clinical AR trials. Large sample-size
14 Evidence-Based Complementary and Alternative Medicine

and well-designed rigorous RCTs are needed to accurately [15] R. Berent and H. Sinzinger, “‘Aspirin—resistance’? A few critical
determine the benefits and risks of CHM for AR. considerations on definition, terminology, diagnosis, clinical
value, natural course of atherosclerotic disease, and therapeutic
consequences,” Journal of Vascular Diseases, vol. 40, no. 6, pp.
Conflict of Interests 429–438, 2011.
[16] S. Sanderson, J. Emery, T. Baglin, and A.-L. Kinmonth, “Nar-
The authors have declared that no competing interests exist.
rative review: aspirin resistance and its clinical implications,”
Annals of Internal Medicine, vol. 142, no. 5, pp. 370–380, 2005.
Acknowledgments [17] A.-A. Weber, B. Przytulski, A. Schanz, T. Hohlfeld, and K.
Schrör, “Towards a definition of aspirin resistance: a typological
This project was supported by the Grant of National Nat- approach,” Platelets, vol. 13, no. 1, pp. 37–40, 2002.
ural Science Foundation of China (81173395/H2902) and [18] T. Kawasaki, Y. Ozeki, T. Igawa, and J.-I. Kambayashi,
the young and middle-aged university discipline leaders of “Increased platelet sensitivity to collagen in individuals resistant
Zhejiang province, China (2013277). to low-dose aspirin,” Stroke, vol. 31, no. 3, pp. 591–595, 2000.
[19] N. Zimmermann and T. Hohlfeld, “Clinical implications of
aspirin resistance,” Thrombosis and Haemostasis, vol. 100, no. 3,
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Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2014, Article ID 639548, 11 pages
http://dx.doi.org/10.1155/2014/639548

Research Article
Antiplatelet Activity of Morus alba Leaves Extract,
Mediated via Inhibiting Granule Secretion and Blocking
the Phosphorylation of Extracellular-Signal-Regulated
Kinase and Akt

Dong-Seon Kim,1 Hyun Dong Ji,2 Man Hee Rhee,2 Yoon-Young Sung,1 Won-Kyung Yang,1
Seung Hyung Kim,3 and Ho-Kyoung Kim1
1
Basic Herbal Medicine Research Group, Korea Institute of Oriental Medicine, Daejeon 305-811, Republic of Korea
2
College of Veterinary Medicine, Kyungpook National University, Daegu 702-701, Republic of Korea
3
Institute of Traditional Medicine & Bioscience, Daejeon University, Daejeon 300-716, Republic of Korea

Correspondence should be addressed to Ho-Kyoung Kim; [email protected]

Received 23 September 2013; Revised 21 December 2013; Accepted 30 December 2013; Published 19 February 2014

Academic Editor: Joen-Rong Sheu

Copyright © 2014 Dong-Seon Kim et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Ethnopharmacological Relevance. Morus alba L. leaves (MAE) have been used in fork medicine for the treatment of beriberi, edema,
diabetes, hypertension, and atherosclerosis. However, underlying mechanism of MAE on cardiovascular protection remains to
be elucidated. Therefore, we investigated whether MAE affect platelet aggregation and thrombosis. Materials and Methods. The
anti-platelet activity of MAE was studied using rat platelets. The extent of anti-platelet activity of MAE was assayed in collagen-
induced platelet aggregation. ATP and serotonin release was carried out. The activation of integrin 𝛼IIb 𝛽3 and phosphorylation
of signaling molecules, including MAPK and Akt, were investigated with cytofluorometer and immunoblotting, respectively. The
thrombus formation in vivo was also evaluated in arteriovenous shunt model of rats. Results. HPLC chromatographic analysis
revealed that MAE contained rutin and isoquercetin. MAE dose-dependently inhibited collagen-induced platelet aggregation. MAE
also attenuated serotonin secretion and thromboxane A2 formation. In addition, the extract in vivo activity showed that MAE at 100,
200, and 400 mg/kg significantly and dose-dependently attenuated thrombus formation in rat arterio-venous shunt model by 52.3%
(𝑃 < 0.001), 28.3% (𝑃 < 0.01), and 19.1% (𝑃 < 0.05), respectively. Conclusions. MAE inhibit platelet activation, TXB2 formation,
serotonin secretion, aggregation, and thrombus formation. The plant extract could be considered as a candidate to anti-platelet and
antithrombotic agent.

1. Introduction thrombus formation in areas of ruptured atheromatous

plaques [3, 4]. Therefore, inhibiting platelet function repre-
Cardiovascular diseases, including thrombosis, stroke, isch- sents a promising approach for preventing thrombosis [5, 6].
emic, and coronary heart diseases, are a leading cause of Antiplatelet drugs have been developed to inhibit platelet
mortality, accounting for around 30% of global deaths [1]. activity in acute thrombotic situations as well as to prevent
Especially, thrombotic diseases constitute a major cardio- adverse events and treatment of atherothrombotic disease [7].
vascular complication affecting a great number of patients. Aspirin and clopidogrel for oral administration and glycopro-
Thrombosis is closely related to activated platelet adhesion, tein IIa/IIIb antagonists (abciximab, eptifibatide, tirofiban,
aggregation, secretion functions, and activation of intrinsic etc.) for injection are commonly used antiplatelet drugs, but
and extrinsic coagulation systems, which cause blood they have several clinical disadvantages including gastroin-
coagulation and fibrin formation [2]. Most acute coronary testinal side-effects, hemorrhage and thrombocytopenia [7–
syndromes are caused by platelet aggregation and subsequent 11]. With this regards, much attention has been given to the
2 Evidence-Based Complementary and Alternative Medicine

development of dietary supplements or herbal medicines for acetate- (1.8 g), butanol- (6.7 g) and water-soluble (27.6 g)
prevention or treatment of cardiovascular diseases for their fraction, respectively.
merits in safety [12]. The extract and the fractions were dissolved in saline or
Morus alba L. (Mulberry) leaf belongs to the Moraceae phosphate-buffered saline (PBS) and then filtered through a
family, distributed mainly in the temperate and subtropical 0.22 m syringe filter for experiments.
regions in the northern hemisphere. It has been traditionally
used in China, Korea, Japan, and other Asian countries as 2.3. High Performance Liquid Chromatography Analysis. The
herbal tea as well as herbal medicine. According to the oldest sample was analyzed by reverse phase-high performance
Korean medical book (Dong-ui-bo-gam), it is called “Sang- liquid chromatography using Waters Alliance 2695 system
Yeop” and introduced to be beneficial in alleviating beriberi, (Waters Co., Milford, MA, USA), coupled with a 2996 photo-
edema and pains [13]. diode array detector. Data processing was carried out using
Recent studies have reported that it shows antiatheroscle- Empower software (Waters Co.). Phenomenex Luna C18
rosis [14], antihypertension [15, 16], antiobesity [17], antidia- column (250 mm × 4.6 mm; particle size 5 𝜇m, Phenomenex,
betic [18, 19], liver protection [20], antiviral [21] and antimi- Torrance, CA, USA) was used as stationary phase. The mobile
crobial [22] effects. This antiplatelet or antithrombotic effect phase consisted of eluent A (0.1% trifluoroacetic in water) and
of M. alba leaves has never been described before, though an eluent B (acetonitrile). The starting eluent was 90% A and
antiplatelet effect has been described with its barks [23]. 10% B. The proportion of eluent B was increased linearly to
In this study, we discovered that the M. alba leaves extract 40% from 10 min to 40 min. The column temperature was
has strong inhibitory activities on collagen-induced platelet kept at 40∘ C and the detector wavelength was set over the
aggregation in vitro and on thrombosis in vivo. range of 190 to 400 nm and recorded at 360 nm. The flow rate
was 1.0 mL/min, and the injection volume was 10 𝜇L, and the
2. Materials and Methods column temperature was kept at 40∘ C.
Identification was based on retention time, UV spectra by
2.1. Materials. Collagen was obtained from Chrono-Log comparison with commercial standards. For each compound,
Co. (Havertown, PA, USA). Aspirin, fibrinogen, plasmin peak areas were determined at the wavelength providing
and dimethyl sulfoxide (DMSO) were from Sigma Chem- maximal UV absorbance. Calibration curves of the stan-
ical Co. (St. Louis, MO, USA). Fura-2/AM was obtained dards ranging from 12.5 to 200 𝜇g/mL (5 levels) revealed
from Sigma Chemical Co. (St. Louis, MO, USA). Anti- good linearity with 𝑅2 values exceeding 0.99 (peak areas
bodies against phospho-p44/42, p44/42, phospho-p38, p38, versus concentration). Quantitation was performed based on
phospho-SAPK/JNK, phospho-PI3K (p85), phospho-Akt, external standards with a mixture of standards of known
and 𝛽-actin were acquired from Cell Signaling (Beverly, concentration that were analyzed in duplicate before and after
MA, USA). ATP assay kits were purchased from Biomedical the batch of samples, and the peak areas were used to calculate
Research Service Center (Buffalo, NY, USA). A TXB2 enzyme the sample contents of the compounds.
immunoassay (EIA) kit was from Cayman Chemical (Ann
Arbor, MI, USA). Fibrinogen Alexa Fluor 488 conjugate was
obtained from Molecular Probes (Eugene, OR, USA). HPLC- 2.4. Experimental Animals. Male Sprague Dawley rats (300∼
grade reagents, methanol and water were obtained from J. T. 350 g) were purchased from Japan SLC (Hamamatsu, Japan).
Baker (Phillipsburg, NJ, USA). All other chemicals were of The animals were acclimated for 1 week prior to the experi-
reagent grade. ments, and housed in an air-conditioned animal room with
a 12/12 h light/dark cycle at a temperature of 22 ± 1∘ C and
2.2. Preparation of the Ethanol Extract and Solvent Fractions humidity of 50 ± 10%. The animals were provided with a
from Morus alba. Morus alba leaves as a dried herb was laboratory diet and water ad libitum.
purchased from Omniherb Co. (Yeongcheon, Korea) and All experimental protocols involving the use of animals
was authenticated, based on its microscopic and macroscopic were conducted in accordance with National Institutes of
characteristics, by the Classification and Identification Com- Health (NIH) guidelines and approved by the Committee on
mittee of the Korea Institute of Oriental Medicine (KIOM). Animal Care at the Korea Institute of Oriental Medicine.
The committee consisted of nine experts in the fields of
plant taxonomy, botany, pharmacognosy, and herbology. A 2.5. Washed Rat Platelet Preparation. Blood was withdrawn
voucher specimen (MAL-20120605) was deposited at the from abdominal vein of rats and collected directly into
herbarium of the Department of Herbal Resources Research anticoagulant citrate dextrose (ACD) solution that con-
at the KIOM. tained 0.8% citric acid, 2.2% trisodium citrate, and 2%
Dried leaves of Morus alba (500 g) were extracted twice dextrose (w/v). Washed platelets were prepared as previ-
with 70% (v/v) ethanol (5 L) for 4 h under mantle-reflux. ously described [24]. Briefly, platelet-rich plasma (PRP) was
The extracts were filtered and evaporated under reduced obtained by centrifuging rabbit blood samples at 230 ×g for
pressure to give Morus alba leaves extract (MAE, 56.0 g). The 10 min. Platelets were precipitated by centrifugation of the
extract (40 g) was suspended in water (1.2 L) to be partitioned PRP at 800 ×g for 15 min and washed with HEPES buffer
subsequently with n-hexane (2 × 1.2 L), ethyl acetate (2 × (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2 , 5.6 mM glucose,
1.2 L), and then n-butanol (2 × 1.2 L) and the solvent-soluble 3.8 mM HEPES, and pH 6.5) containing 0.35% BSA and
fractions were evaporated to afford hexane- (3.9 g), ethyl 0.4 mM EGTA. The washed platelets were resuspended in
Evidence-Based Complementary and Alternative Medicine 3

HEPES buffer (pH 7.4) and the cell dilution was adjusted to 4 centrifuged at 12,000 ×g for 5 min at 4∘ C. The supernatant
× 108 cells/mL. was collected and serotonin release was measured with a
serotonin ELISA kit (Labor Diagnostika Nord GmbH &
2.6. Platelet Aggregation In Vitro Assay. Platelet aggregation Co, Nordhorn, Germany) according to the manufacturer’s
was evaluated as previously described [25]. Aggregation instructions.
was monitored by measuring light transmission with an
aggregometer (Chrono-Log, Havertown, PA, USA). The 2.10. Measurement of Thromboxane 𝐵2 Formation. Washed
washed platelets (3 × 108 /mL) were pre-incubated at 37∘ C for platelets (3 × 108 /mL) were pre-incubated with or without
2 min with either MAE or vehicle and then stimulated with MAE for 2 min at 37∘ C in the presence of 1 mM CaCl2
2.5 𝜇g/mL collagen. The mixture was further incubated for and then stimulated with 2.5 𝜇g/mL collagen for 5 min. The
5 min with stirring at 170 ×g. The vehicle concentration was reactions were terminated by adding ice-cold 2.5 mM EDTA
less than 0.1% to minimize the effect of this reagent. and 100 𝜇M indomethacin. After centrifugation at 12,000 ×g
for 3 min at 4∘ C, the supernatant was collected and the
concentration of TXB2 was measured using a TXB2 EIA kit
2.7. [Ca2+ ]i Measurement. The intracellular calcium ion con- according to the manufacturer’s protocol.
centration ([Ca2+ ]𝑖 ) was measured with Fura-2/AM as previ-
ously described [26]. Briefly, the platelets were incubated with
5 𝜇M of Fura-2/AM for 30 min at 37∘ C and washed. The Fura- 2.11. Immunoblotting. Platelet suspensions (3 × 108 /mL) were
2-loaded platelets (3 × 108 /mL) were then pre-incubated with pre-incubated with MAE or vehicle (0.1% (v/v) DMSO)
MAE for 2 min at 37∘ C in the presence of 1 mM CaCl2 and at 37∘ C for 2 min. Platelet activation was induced by the
subsequently stimulated with collagen for 5 min. Fluorescent addition of 2.5 𝜇g/mL collagen and the reaction was allowed
signals were recorded using a Hitachi F-2500 fluorescence to proceed for 5 min. After terminating the reaction, lysates
spectrofluorometer (F-2500, Hitachi, Japan). Light emission were then prepared by solubilizing and centrifuging the
was measured at 510 nm with simultaneous excitation at 340 platelets in sample buffer (0.125 M Tris-HCl, pH 6.8; 2%
and 380 nm that changed every 0.5 s. Fura-2 fluorescence SDS, 2% 𝛽-mercaptoethanol, 20% glycerol, 0.02% bromophe-
in the cytosol measured with the spectrofluorometer was nol blue, 1 𝜇g/mL phenylmethylsulfonyl fluoride (PMSF),
calculated as previously described by Schaeffer and Blaustein 2 𝜇g/mL aprotinin, 1 𝜇g/mL leupeptin, and 1 𝜇g/mL pepstatin
A). Protein concentration was determined using a BCA assay
[27] with the following formula: [Ca2+ ]𝑖 224 nM × (𝐹 −
(PRO-MEASURE; iNtRON Biotechnology, Seoul, Republic
𝐹min )/(𝐹max −𝐹), in which 224 nM is the dissociation constant
of Korea). Total cell proteins (30 𝜇g) from the platelet
of the Fura-2-Ca2+ complex, and 𝐹min and 𝐹max represent the
lysate were resolved by 10% SDS-PAGE and transferred to
fluorescence intensity levels at very low and very high Ca2+
nitrocellulose membranes in transfer buffer (25 mM Tris,
concentrations, respectively. In our experiment, 𝐹max was the
pH 8.5; 0.2 M glycine, and 20% methanol). The membranes
intensity of the Fura-2-Ca2+ complex fluorescence at 510 nm were blocked in TBS-T containing 5% nonfat dry milk
after the platelet suspension containing 1 mM of CaCl2 had and incubated with primary antibody diluted in a blocking
been solubilized with Triton X-100 (0.1%). 𝐹min was the solution. The blots were then incubated with horseradish
fluorescence intensity of the Fura-2-Ca2+ complex at 510 nm peroxidase-conjugated secondary antibody. Antibody bind-
after the platelet suspension containing 20 mM Tris/3 mM ing was visualized using enhanced chemiluminescence
of EGTA had been solubilized with Triton X-100 (0.1%). (iNtRON Biotechnology, Seoul, Republic of Korea).
𝐹 represented the intensity of Fura-2 complex fluorescence
at 510 nm after the platelet suspension was stimulated with
collagen with or without MAE in the presence of 1 mM CaCl2 . 2.12. Assessment of Fibrinogen Binding to Integrin 𝛼IIb 𝛽3 .
Fibrinogen Alexa Fluor 488 conjugate binding to washed
platelets was quantified by flow cytometry. Briefly, washed
2.8. ATP Release Assay. Washed platelets (3 × 108 /mL) were platelets (3 × 108 /mL) were pre-incubated for 2 min with
pre-incubated for 2 min at 37∘ C with various concentrations various concentrations of MAE at room temperature in the
of MAE and then stimulated with 2.5 𝜇g/mL collagen. After presence of 0.1 mM CaCl2 . The platelets were then stimulated
the reaction was terminated, the cells were centrifuged with collagen for 5 min, immediately incubated thereafter
and the supernatants were used for the assay. ATP release with fibrinogen Alexa Fluor 488 (20 𝜇g/mL) for 5 min, and
was measured in a luminometer (GloMax 20/20; Promega, finally fixed with 0.5% paraformaldehyde at 4∘ C for 30 min.
Madison, USA) using an ATP assay kit (Biomedical Research The platelets were pelleted by centrifugation at 2,000 ×g at
Service Center, Buffalo, NY, USA) according to manufac- 4∘ C and resuspended in 500 𝜇L PBS. Since the activation
turer’s instructions. of integrin 𝛼IIb 𝛽3 is largely dependent on the generation of
Ca2+ , nonspecific binding of fibrinogen to integrin 𝛼IIb 𝛽3 was
2.9. Measurement of Serotonin Secretion. The washed measured by assessing fibrinogen binding in the presence
platelets (3 × 108 /mL) were pre-incubated for 2 min at 37∘ C of the calcium chelator EGTA (1 mM). The fluorescence of
with various concentrations of MAE in the presence of each platelet sample was analyzed using a FACS Calibur
1 mM of Ca2+ . The reaction mixture was further incubated cytometer (BD Biosciences, San Jose, CA, USA), and data
with collagen (2.5 𝜇g/mL) for 5 min. After terminating were analyzed using CellQuest software (Becton Dickinson
the aggregation reaction, the mixture was immediately Immunocytometry Systems, San Jose, CA, USA).
4 Evidence-Based Complementary and Alternative Medicine

2.13. MAE Effect on Thrombus Formation in Arteriovenous molecules in platelet aggregation induced by a ligand such as
Shunt Thrombosis Model In Vivo. The in vivo antithrombotic collagen. Therefore, we have investigated whether MAE affect
activity of MAE was evaluated in a rat arterio-venous shunt the ([Ca2+ ]𝑖 ) mobilization induced by 2.5 𝜇g/mL collagen.
thrombosis model [28]. Rats were given orally administered Collagen induced the massive amount of calcium mobiliza-
400 mg/kg, 200 mg/kg, and 100 mg/kg MAE which were tion by up to 631.7 ± 46.7 nM, which was significantly and
dissolved in 0.25% carboxymethylcellulose (CMC, Sigma, concentration-dependently inhibited by MAE (Figure 3).
USA) solution at the same time of day for 3 consecutive days
by gavage. The shunt thrombosis model was tested 2 h after 3.4. Effects of MAE on the Granule Release. In the following
the last administration. For each test, different batches of six studies, we have examined whether the extract modulated
rats were used. After anaesthesia with Urethane (1.25 g/kg the secretion of granule contents such as ATP and serotonin
i.p) (Sigma, USA), an 8 cm polyethylene tube was inserted which can activate platelets themselves acting as autacoids.
between the left jugular vein and the right carotid artery. The Collagen (2.5 𝜇g/mL) considerably induced ATP release from
saline-filled shunt was assembled by connecting two cannulae dense granule by 3-fold in comparison with resting platelets.
with a slightly curved 6 cm long tygon tubing (internal diam- As depicted in Figure 4(a), MAE dose-dependently repressed
eter 2 mm) containing a 5 cm long cotton thread (diameter collagen- (2.5 𝜇g/mL) induced ATP release. In order to
0.25 mm) which had been scraped with a scalpel blade to confirm the MAE’s effect on dense granule, we have cho-
render it more thrombogenic. The extracorporeal circulation sen another marker molecule serotonin in dense contents.
was maintained for 15 min, during which time a thrombus As expected, MAE potently and concentration-dependently
adheres to the cotton thread. The shunt was then removed inhibited serotonin release which was induced by 2.5 𝜇g/mL
and the thread with its associated thrombus was withdrawn collagen (Figure 4(b)).
and immediately weighed. The thrombus wet weight was
determined by subtracting from the value obtained the
3.5. Effects of MAE on TXA2 Formation. We next investigated
weight of the dry 5 cm cotton thread determined previously.
whether MAE affected TXA2 formation in collagen-activated
platelets. TXA2 is another marker molecule in the initial
2.14. Statistics. Data were analyzed with a one-way analysis activation of ligand binding to cognate receptor. In addition,
of variance followed by a post hoc Dunnett’s test in order to it acts as an agonist against platelet own receptor, which,
measure statistical significance of the differences observed therefore, is named “autocoid.” TXB2 is stable metabolite
(SAS Institute Inc., Cary, NC, USA). All data are presented of TXA2 and thus TXB2 formation was measured. Figure 5
as the mean ± standard error of the mean (SEM). 𝑃 values of displayed that MAE significantly inhibited TXA2 formation
0.05 or less were considered to be statistically significant. in collagen-activated platelets.

3. Results 3.6. Effects of MAE on the Phosphorylation of MAPKs and

Akt. We next investigated whether MAE affected the phos-
3.1. Chromatographic Separation of M. alba Extract. As phorylation of mitogen-activated protein kinase (MAPK),
shown Figure 1, high performance liquid chromatographic including extracellular-signal regulated kinase 1/2 (ERK1/2),
(HPLC) analysis of MAE revealed rutin and isoquercetin. p38 MAPK, and c-Jun N-terminal kinase (JNK). In previous
The MAE contained 2.83 ± 0.15 mg/g for rutin and 8.18 ± studies [26, 30], we have shown that all these MAPKs
2.4 mg/g for isoquercetin, identified at a retention time of were expressed and phosphorylated by ligands such as ADP
approximately 23.8 min and 24.7 min, respectively. and collagen. Our immunoblot analysis revealed that MAE
potently inhibited collagen-induced ERK phosphorylation
3.2. Inhibitory Effect of MAE on Collagen-Induced Platelet but marginally repressed collagen-induced JNK phospho-
Aggregation. In the beginning of those studies, we have rylation (Figure 7). However, collagen-induced p38 MAPK
evaluated whether MAE affected various ligands (ADP-, phosphorylation was not affected by the extract treatment.
collagen- and thrombin) induced platelet aggregation. As In addition, ligand binding to cognate receptor has been
shown in Figure 2(a), MAE only inhibited collagen-induced shown to activate phosphatidylinositol 3-kinase (PI3K) and
platelet aggregation but not in ADP- and thrombin-induced Akt, which target the glycogen synthase kinase (GSK) 3𝛽 as
platelet aggregation. In the previous studies [29], we have an Akt effecter. Therefore, we have investigated whether MAE
found that 2.5 𝜇g/mL of collagen, 10 𝜇M of ADP, and modulated collagen-induced Akt activation. As depicted in
0.1 U/mL of thrombin induced full activation and aggrega- Figure 7(b), MAE potently and concentration-dependently
tion of rat platelet. Therefore, we have employed collagen suppressed Akt phosphorylation which was activated by
(2.5 𝜇g/mL) as a ligand to induce platelet aggregation in fol- collagen.
lowing studies. Figure 2 shows that MAE inhibited collagen-
(2.5 𝜇g/mL) induced platelet aggregation in concentration- 3.7. Effects of MAE on the Integrin 𝛼IIb 𝛽3 Activation. In order
dependent manner. to complete platelet aggregation stably, outside-in signaling
pathway should be activated, as determined with fibrinogen
3.3. Effect of MAE on Intracellular Calcium Ion Concentration. binding to active integrin 𝛼IIb 𝛽3 .
It is well known that intracellular calcium ion ([Ca2+ ]𝑖 ) takes The influence of MAE on integrin 𝛼IIb 𝛽3 activation
a pivotal role in the activation of downstream signaling was studied in collagen-stimulated platelets. As shown in
Evidence-Based Complementary and Alternative Medicine 5

0.30 0.60
0.25 0.50
0.20 R
IQ 0.40 IQ
(AU)

(AU)
0.15 0.30
0.10 0.20 R
0.05 0.10
0.00 0.00
0.00 4.00 8.00 12.00 16.00 20.00 24.00 28.00 32.00 36.00 40.00 0.00 4.00 8.00 12.00 16.00 20.00 24.00 28.00 32.00 36.00 40.00
(min) (min)
(a) (b)

Figure 1: HPLC chromatogram of standard mixture (a) and Morus alba leaves extracts at 350 nm. The chromatographic analysis was
performed as described in the “Section 2” Identification was based on retention time and UV spectra by comparison with commercial
standards. R: rutin; Q: isoquercetin.

120 120

100 100
Platelet aggregation (%)

Platelet aggregation (%)

80 80
∗∗∗
60 60

40 40 ∗∗∗

20 20

0 0
MAE (𝜇g/mL) − 100 200 Coll. (2.5 𝜇g/mL) + + + +
MAE (𝜇g/mL) − 100 200 400
Collagen
ADP
Thrombin
(a) (b)

100
Platelet aggregation (%)

400 𝜇g/mL
80

60
200 𝜇g/mL
40

20 100 𝜇g/mL
1 min
0 Vehicle

(c)

Figure 2: The inhibitory effect of Morus alba leaves extracts (MAE) on platelet aggregation induced by collagen. Platelets (3 × 108 /mL) were
preincubated with or without MAE (100–400 𝜇g/mL) in the presence of 1 mM CaCl2 for 2 min at 37∘ C. The platelet aggregation was then
induced by 2.5 𝜇g/mL collagen, 10 𝜇M ADP, and 0.1 U/mL thrombin. The extent of platelet aggregation was measured with an aggregometer.
The aggregation reaction was terminated after 5 min, and the percent aggregation rate was calculated. Each graph shows the mean ± SEM of
at least four independent experiments. ∗∗∗ 𝑃 < 0.001 compared to the agonist control.

Figure 6, collagen (2.5 𝜇g/mL) increased the fibrinogen bind- 3.8. Effect of MAE on Arteriovenous Shunt Thrombosis.
ing to active integrin 𝛼IIb 𝛽3 , whereas resting platelets did not The AV-shunt thrombosis model has been commonly used
activate the integrin. to assess antithrombotic effects. Compared to the control,
The plant extracts reduced collagen- (2.5 𝜇g/mL) induced a 3-day oral treatment with MAE decreased thrombus
fibrinogen binding to integrin 𝛼IIb 𝛽3 in concentration- weight in the rat arteriovenous shunt thrombosis model.
dependent manner (Figure 6). As shown in Figure 8, after oral administration of 100, 200,
6 Evidence-Based Complementary and Alternative Medicine

800

600

[Ca2+ ]i mobilization (nM)

400

∗∗∗
200
∗∗∗

0
Coll. (2.5 𝜇g/mL) − + + + +
MAE (𝜇g/mL) − − 100 200 400

Figure 3: The inhibitory effect of Morus alba leaves extracts (MAE) on [Ca2+ ]𝑖 increased by collagen. Washed platelets (3 × 108 /mL) were
incubated with a calcium fluorophore (5 𝜇M, Fura-2/AM) and stimulated with collagen (2.5 𝜇g/mL). [Ca2+ ]𝑖 was then measured as described
in Section 2. The results are presented as the mean ± SEM of at least three independent experiments. ∗∗∗ 𝑃 < 0.001 compared to the agonist
control.

8 100

80
6
Serotonin release (nM)
ATP release (nM)

∗∗ 60
∗∗∗
4
40
∗∗
2
20 ∗∗∗

∗∗∗
0 0
Coll. (2.5 𝜇g/mL) − + + + + Coll. (2.5 𝜇g/mL) − − + + + +
MAE (𝜇g/mL) − − 100 200 400 MAE (𝜇g/mL) − − 50 100 200 −
Aspirin (200 𝜇g/mL) − − − − − +
(a) (b)

Figure 4: Effects of Morus alba leaves extracts (MAE) on granule secretion from the collagen-stimulated platelets. Washed platelets (3 ×
108 /mL) were pre-incubated with MAE at the indicated concentrations and stirred in an aggregometer for 2 min prior to stimulation with
2.5 𝜇g/mL collagen for 5 min. The reaction was terminated, and an ATP release assay (a) and serotonin release assay (b) were carried out as
described in Materials and Methods. Bar graphs show the mean ± SEM of at least four independent experiments. ∗∗ 𝑃 < 0.01 and ∗∗∗ 𝑃 < 0.001
compared to the agonist control.

and 400 mg/kg/day, MAE significantly and dose-dependently 4. Discussion

decreased the thrombus weight by 52.3% (𝑃 < 0.001),
These results show that in vitro the ethanol extract of M. alba
28.3% (𝑃 < 0.01), and 19.1% (𝑃 < 0.05) compared to
L. leaf (MAE) inhibits collagen-induced platelet aggregation
vehicle control, respectively. Rivaroxaban which was used as in a concentration-dependent manner. In order to elucidate
a positive control is an oral anticoagulant for the preven- the mechanism of inhibitory activities of MAE extract, we
tion and treatment of thrombosis mediated conditions [31]. have analyzed downstream signaling as follows intracellu-
Rivaroxaban 5 mg/kg/day was different from control (𝑃 < lar calcium concentration, dense granule secretions, pro-
0.001) and MAE 100 mg/kg/day (𝑃 < 0.05), but not from tein phosphorylations (e.g., MAPKs and Akt), and integrin
MAE 200 mg/kg/day and 400 mg/kg/day. signaling. In addition, we next investigated whether this
Evidence-Based Complementary and Alternative Medicine 7

140

120

100

TXB2 (ng/4 × 108 )

80 ∗∗
60

40

20
∗∗∗
0
0 100 200 400 ASA 50

MAE (𝜇g/mL)

Figure 5: Effects of Morus alba leaves extracts (MAE) on TXA2 formation from the collagen-stimulated platelets. Washed platelets (3 ×
108 /mL) were pre-incubated with MAE at the indicated concentrations and stirred in an aggregometer for 2 min prior to stimulation with
2.5 𝜇g/mL collagen. The reaction was terminated at 5 min, and TXA2 formation was performed as described in Materials and Methods. Bar
graphs show the mean ± SEM of triplicate independent experiments. ∗∗ 𝑃 < 0.01 and ∗∗∗ 𝑃 < 0.001 compared to the agonist control.

400 300
350
250
300
250 200
Count
Count

P2
200 150 P2
150
100
100
50 50
0 0
1
0 10 102 103 104 105 0 10
1
102 103 104 105

FITC-H FITC-H

(A) (B)
250
250
200
200
150
Count

Count

150 P2 P2
100
100

50 50

0
100
0
1 2 3 4 5 1 2 3 4 5
0 10 10 10 10 10 0 10 10 10 10 10

FITC-H FITC-H 80
Fibrinogen binding (%)

(C) (D)

200 60
150
Count

100
P2 40

50 ∗∗
20
0
1
0 10 102 103 104 105

FITC-H 0
Coll. (2.5 𝜇g/mL) − + + + +
(E) MAE (𝜇g/mL) − − 100 200 400
(a) (b)

Figure 6: Effects of Morus alba leaves extracts (MAE) on fibrinogen binding to integrin 𝛼IIb 𝛽3 in ADP-activated platelets. The inhibitory
effects of MAE on fibrinogen binding to integrin 𝛼IIb 𝛽3 in collagen-activated platelets were measured by flow cytometry (A). Washed
platelets (3 × 108 /mL) were pretreated with vehicle (DMSO) or MAE at concentrations ranging from 100 𝜇g/mL to 400 𝜇g/mL. Collagen
was then incubated with human fibrinogen labeled with Alexa Fluor 488 (20 𝜇g/mL) for 5 min. The cells were subsequently fixed with 0.5%
paraformaldehyde at 4∘ C for 30 min. Graphs showing fluorescent intensity present the data from one experiment but are representative of
four independent trials. Data are expressed as the mean fluorescence intensity (MFI) of fibrinogen-positive platelets. Each graph presents the
results expressed as percent of gated (a). ∗∗ 𝑃 < 0.01 compared to the agonist control.
8 Evidence-Based Complementary and Alternative Medicine

Lane 1 2 3 4 5

p-ERK2

Total and ERK

1.6

ERK2, p38 MAPK, and JNK

p-JNK

phosphorylation (ratio)
1.2

Total and JNK

0.8
∗∗
p-p38
0.4 ∗∗∗
∗∗∗
Total and p38
0
Coll. (2.5 𝜇g/mL) − + + + +
MAE (𝜇g/mL) − − 100 200 400

p-ERK2
p-JNK
p-p38
(a) (b)
Lane 1 2 3 4 5 1.2
PI3-K and Akt phosphorylation

p-PI3-K 1.0

0.8
Total PI3-K
(ratio)

0.6 ∗∗
∗∗∗
p-Akt 0.4 ∗∗
∗∗∗
0.2
Total Akt

0
Coll. (2.5 𝜇g/mL) − + + + +
MAE (𝜇g/mL) − − 100 200 400

p-PI3-K
p-Akt
(c) (d)

Figure 7: Effects of Morus alba leaves extracts (MAE) on collagen-induced phosphorylation of MAPKs, PI3K, and Akt. Washed platelets (3 ×
108 /mL) were pre-incubated for 2 min with vehicle or MAE at the indicated concentration. The platelets were then stimulated with 10 𝜇M ADP
for 5 min at 37∘ C. After terminating the reactions, total cell proteins were extracted. The proteins were separated by SDS-PAGE and transferred
onto nitrocellulose membranes. The membranes were then probed with antibodies against phospho-p44/42, p44/42, phospho-p38, p38,
phospho-SAPK/JNK, and 𝛽-actin ((a), (b)) and phospho-PI3K, phospho-Akt, PKA𝛼/𝛽/𝛾 cat, and 𝛽-actin ((c), (d)). Antibody binding was
visualized by chemiluminescence. All immunoblots are representative of three or four independent experiments. ∗∗ 𝑃 < 0.01 and ∗∗∗ 𝑃 < 0.001
versus vehicle control.

efficacy of MAE in vitro is implemented in in vivo application. first report to demonstrate the effects on platelet aggregation
That is, the extract given orally to rats for 3 days dose- and thrombosis.
dependently decreased thrombus weight in a common model Upon the ligand binding to platelet membrane receptors,
of arterial thrombosis. To the best of our knowledge this is the [Ca2+ ]𝑖 plays an important role in the initial activation of
Evidence-Based Complementary and Alternative Medicine 9

300
Collagen
250
Thrombus weight (mg)

∗∗∗
200
GP VI
∗∗
150
∗ ∗∗∗
100 Src family kinase

50
MEK PI3-kinase
0
0 100 200 400 RVX
ERK2 Akt
MAE (mg/kg/day)

Figure 8: Effect of Morus alba leaves extracts (MAE) on thrombus

Granule secretion
formation in rats. The procedures for the AV-shunt model are
([Ca2+ ]i
described in the Section 2. Thrombus weight in an arteriovenous ATP release)
shunt was measured at 2 h after administration of 3-day treat-
ment with 0.25% carboxymethylcellulose solution (control), ethanol
extract of Morus alba L. leaves (MAE) 100, 200 or 400 mg/kg/day, Integrin 𝛼IIb 𝛽3
and positive control (rivaroxaban, RVX) 5 mg/kg/day. Data are
shown as mean ± S.D. ∗ 𝑃 < 0.05, ∗∗ 𝑃 < 0.01, ∗∗∗ 𝑃 < 0.001 versus
vehicle control. Inactivation by MAE

Figure 9: The summary of inhibitory effect of MAE in collagen-

induced platelet aggregation. GP VI: glycoprotein VI; MEK1
platelet and subsequent platelet aggregation. Upregulation of mitogen-activated protein kinase kinase; ERK2, extracellular-
[Ca2+ ]𝑖 is due to both calcium influx from extracellular fluid regulated kinase 2.
and calcium mobilization from intracellular pools, such as
dense tubular system and mitochondria. Although unknown
is the source of [Ca2+ ]𝑖 , collagen dramatically escalates
intracellular calcium concentration which is significantly and widely being investigated for antiplatelet and antithrombotic
concentration-dependently reduced by MAE. In addition, we agents. At the moment, we insisted that MAE could be devel-
did not determined whether MAE modulate either calcium oped as antiplatelet or antithrombotic agents or functional
influx in platelet membrane or inositol-1,4,5-trisphosphate food.
(IP3) receptor on platelet organelles. However, MAE potently On the other hand, the phosphorylation of signaling
blocked collagen-induced [Ca2+ ]𝑖 upregulation which is at molecules, including MAPKs (i.e., ERK1/2, p38 MAPK, and
least main cause of antiplatelet activity. JNK) and PI3K/Akt is also crucial phase in outside-in and
Mammalian platelets devoid of nucleus and basically inside-out signaling of platelet aggregation. In our results,
transcriptional and translational activities are limited. the collagen-induced phosphorylation of ERK and JNK was
Although platelet transcription factors such as nuclear inhibited by MAE but not p38 MAPK. Although the role
factor kappa B (NF-𝜅B) are some reported [32, 33], it is of ERK and JNK in platelet physiology is not clear, there
largely understood that new functional protein synthesis is are several evidences that they are involved in the activation
limited. Therefore, granule substances take a pivotal role in of integrin 𝛼IIb 𝛽3 and PLA2 /TXA2 pathways [34, 35]. In
the platelet activation and aggregation due to its biological addition, MAE blocked collagen-induced TXA2 production
and functional significance. For example, dense granules (Figure 5). This indicated that MAE blocked MAPK-integrin
enclosed ATP, ADP, serotonin and Ca2+ . They act as an 𝛼IIb 𝛽3 and PLA2 /TXA2 routs and thus displayed potent
autacoid (e.g., self-activator) or an important modulator in antiplatelet activity.
the downstream signaling. Therefore, the MAE’s antiplatelet On the other hand, PI3-K/Akt pathway plays important
activity might be due to the potent inhibition of granule role in the platelet activation and aggregation. The receptor
secretion (i.e., Ca2+ , ATP, and serotonin) (Figure 9). activation leads to the phosphorylation of PI3-K and sub-
The activation of integrin 𝛼IIb 𝛽3 is plays an important role sequently activation and phosphorylation of Akt. Moreover,
in the final step of platelet activation and aggregation, which it is reported that signaling routes of PI3-K are involved
is named as inside-out signaling. That is, the phosphorylation in the transient activation of integrin 𝛼IIb 𝛽3 . As shown in
of integrin 𝛼IIb 𝛽3 is able to bind to another phosphorylated Figure 7(b), MAE significantly inhibited both PI3-K and Akt
integrin 𝛼IIb 𝛽3 , which is mediated via fibrinogen-like bridge phosphorylation. However, at the moment, direct inhibition
network. Finally, strict and stable blood thrombus is being of MAE on either PI3-K or PI3-K and Akt is not clear, which
formed. Therefore, screening of pharmacological inhibitors is remains to be elucidated in the future.
10 Evidence-Based Complementary and Alternative Medicine

Blood flow disturbances at sites of atherosclerotic plaque Oriental Medicine (KIOM) to the Ministry of Science, ICT
rupture promote platelet activation and arterial thrombus & Future Planning (MSIP), Korea. Additionally, this work
formation [36, 37]. In the present study, MAE significantly was also partially supported by ICT Fusional Construction
inhibited collagen-induced platelet aggregation in vitro and of Alternative Herbal Medicine Resources (K14410), Charac-
rat carotid artery thrombus formation in vivo. Therefore, terization of Native Biological Resources and Excavation of
MAE has potential to prevent thrombotic or cardiovascular Alternative Herbal Medicine Resources (K14411), the Korea
disease. Institute of Oriental Medicine (KIOM) to the Ministry of
MAE also inhibited serotonin and ATP secretion in a Science, ICT & Future Planning (MSIP), Korea.
concentration-dependent manner. Serotonin and ATP can
cause vasoconstriction of interarterial coronary collateral
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Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2014, Article ID 478973, 11 pages
http://dx.doi.org/10.1155/2014/478973

Research Article
Antihypercholesterolemic and Antioxidative Potential of an
Extract of the Plant, Piper betle, and Its Active Constituent,
Eugenol, in Triton WR-1339-Induced Hypercholesterolemia in
Experimental Rats

Karuppasamy Venkadeswaran,1 Arumugam Ramachandran Muralidharan,1

Thangaraj Annadurai,1 Vasanthakumar Vasantha Ruban,1 Mahalingam Sundararajan,1
Ramalingam Anandhi,1 Philip A. Thomas,2 and Pitchairaj Geraldine1
1
Department of Animal Science, School of Life Sciences, Bharathidasan University, Tiruchirappalli, Tamilnadu 620024, India
2
Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli, Tamilnadu 620001, India

Correspondence should be addressed to Pitchairaj Geraldine; [email protected]

Received 31 October 2013; Accepted 16 December 2013; Published 9 January 2014

Academic Editor: Mao-Hsiung Yen

Copyright © 2014 Karuppasamy Venkadeswaran et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.

Hypercholesterolemia is a dominant risk factor for atherosclerosis and cardiovascular diseases. In the present study, the
putative antihypercholesterolemic and antioxidative properties of an ethanolic extract of Piper betle and of its active constituent,
eugenol, were evaluated in experimental hypercholesterolemia induced by a single intraperitoneal injection of Triton WR-1339
(300 mg/kg b.wt) in Wistar rats. Saline-treated hypercholesterolemic rats revealed significantly higher mean blood/serum levels of
glucose, total cholesterol, triglycerides, low density and very low density lipoprotein cholesterol, and of serum hepatic marker
enzymes; in addition, significantly lower mean serum levels of high density lipoprotein cholesterol and significantly lower
mean activities of enzymatic antioxidants and nonenzymatic antioxidants were noted in hepatic tissue samples from saline-
treated hypercholesterolemic rats, compared to controls. However, in hypercholesterolemic rats receiving the Piper betle extract
(500 mg/kg b.wt) or eugenol (5 mg/kg b.wt) for seven days orally, all these parameters were significantly better than those in saline-
treated hypercholesterolemic rats. The hypercholesterolemia-ameliorating effect was better defined in eugenol-treated than in Piper
betle extract-treated rats, being as effective as that of the standard lipid-lowering drug, lovastatin (10 mg/kg b.wt). These results
suggest that eugenol, an active constituent of the Piper betle extract, possesses antihypercholesterolemic and other activities in
experimental hypercholesterolemic Wistar rats.

1. Introduction atherogenic and toxic to vascular cells, leading to atheroscle-

rosis, hypertension, obesity, diabetes, and functional depres-
Hypercholesterolemia is a major socioeconomic problem in sion in organs such as the liver, heart, and kidneys [3]. Clinical
common individuals as well as health professionals due to the trials have shown that lowering lipids reduces the morbidity
strong correlation between cardiovascular diseases and lipid and mortality associated with cardiovascular complications
abnormalities [1]. The modern lifestyle, with a high fat diet [4]. Intensive reduction of LDL-cholesterol levels have also
and little physical activity, significantly contributes to hyperc- been found to reverse atherosclerosis and decrease the pro-
holesterolemia and cardiovascular diseases [2]. High levels of gression of cardiovascular disease [5, 6].
low-density lipoprotein (LDL) cholesterol accumulate in the Oxidative stress induced by reactive oxygen species
extracellular subendothelial space of arteries; these are highly (ROS) plays an important role in the etiology of several
2 Evidence-Based Complementary and Alternative Medicine

diseases, including atherosclerosis and coronary heart disease 2.2. Experimental Animals. Male albino rats of the Wistar
[7, 8]. Oxidative stress contributes to the development of strain (150–200 g) were housed under conditions of con-
atherosclerosis in the vascular wall through the formation of trolled temperature (25 ± 2∘ C) with a 12 h/12 h day-night
ROS [6]. Increased formation of free radicals is accompanied cycle, during which time they had free access to food and
by perturbations in antioxidant status, resulting in oxidative water ad libitum. Animals were maintained per national
damage to cellular components [8]. Hypercholesterolemia is guidelines and protocols approved by the Institutional Ani-
reported to be associated with the oxidative stress that results mal Ethical Committee (BDU/IAEC/58/2013/09.04.2013).
from the increased production of ROS or impairment of the
antioxidant system [9]. This has nurtured research interest in 2.3. Preparation of Plant Extract. The air-dried leaves of
evaluating antioxidant-rich traditional remedies and alterna- Piper betle variety (Bangala Pan) (100 g) were chopped into
tive medicines as potentially efficacious cholesterol-lowering fine pieces. These were extracted in 95% ethanol (1 L) by
therapies which have few, or no, side-effects. using a Soxhlet’s apparatus for 72 hours. Extracts were then
The Piper betle plant is widely grown in the tropical filtered through Whatman filter paper (no. 1). The solvent was
humid climate of South East Asia and its leaves, with a strong evaporated under reduced pressure at 45∘ by using a rotary
pungent and aromatic flavour, are widely consumed as a evaporator for elimination of ethanol, and the dried extract
was stored at 4∘ C until further use.
mouth freshener. The leaves of Piper betle, which are reported
to possess medicinal properties, have been widely used as a
traditional medicine for treating several diseases including, 2.4. Experimental Induction of Hypercholesterolemia. Hyper-
catarrhal and pulmonary infections [10]. In a preliminary cholesterolemia was induced experimentally in 12 h-fasted
study by Koff et al. [11], an extract of Piper betle leaves rats by a single intraperitoneal injection of Triton WR-1339
(300 mg/kg body weight (b.wt.)) dissolved in 0.89% saline
was found to contain several bioactive molecules such as
[18]. Forty-eight hours after administration of Triton WR-
polyphenols, alkaloids, steroids, saponins, and tannins. More
1339, rats exhibited elevated serum levels of total cholesterol
recently, an extract of Piper betle was reported to exhibit and triglycerides; these rats were deemed to be hypercholes-
biological capabilities of detoxification and antioxidative and terolemic and used for further investigation.
antimutagenic activities [12]. Eugenol (4-allyl-1-hydroxy-2-
methoxybenzene), a natural food-flavouring agent found in
plant extracts of Piper betle, cinnamon, clove, basil, and 2.5. Experimental Design
nutmeg, has been found to ameliorate oxidative stress by 2.5.1. Treatment Groups. The experimental rats were divided
preventing oxidative tissue damage in different experimental into five treatment groups, each comprising five rats.
models [1, 13, 14]. The efficacy of eugenol was found in
human beings to be within the permitted levels by Food Group I. Control rats (not hypercholesterolemic and did not
and Agricultural Organization/World Health Organization receive any treatment).
Expert Committee on Food Additives (maximum permitted
intake is 2.5 mg/100 g) [15]. Eugenol is reported to be present Group II. Hypercholesterolemic rats that received only saline
in a concentration of 0.32% in Piper betle [16]; conceiv- orally for 7 days.
ably, this concentration may suffice to exert antioxidant
activity. Although Piper betle leaves have been reported to Group III. Hypercholesterolemic rats that received lovastatin
(10 mg/kg b.wt./day) in an aqueous suspension orally for 7
possess antioxidative, antifungal, hypotensive, respiratory,
days.
antidepressant, antihelminthic, and antibacterial activities
[17], there is currently no information on its putative antihy- Group IV. Hypercholesterolemic rats that received the Piper
perlipidemic or antiatherogenic potential. betle extract (500 mg/kg b.wt./day) in an aqueous suspension
In the present investigation, an attempt has been made orally for 7 days.
to determine whether an ethanolic extract of Piper betle and
its active constituent, eugenol, possess putative serum lipid- Group V. Hypercholesterolemic rats that received eugenol
lowering and antioxidant activities in comparison with that (5 mg/kg b.wt./day) in 0.5% peanut oil orally for 7 days.
of lovastatin (a commercially available serum lipid-lowering
drug) in an experimental animal model of hypercholes- Saline, lovastatin, Piper betle extract, and eugenol were
administered orally by gastric intubation once daily for 7
terolemia.
days. Blood samples were collected from all experimental rats
on day 10 (7 days after start of treatment), and, subsequently,
2. Materials and Methods serum was separated for subsequent analysis of serum lipid
profile parameters and serum hepatic marker enzymes. After
2.1. Chemicals. Lovastatin and eugenol (98%) were pur- collection of the blood samples, all the animals were sacrificed
chased from Sigma Chemical Co. (St. Louis, MO, USA). by cervical decapitation; from each animal, the liver was
Triton WR-1339 and all the other chemicals and reagents used excised and stored at −80∘ C until subsequent analysis of
were of analytical grade and were obtained from Himedia antioxidant activity and the rate of lipid peroxidation in
Laboratories (Mumbai, India). hepatic tissue samples.
Evidence-Based Complementary and Alternative Medicine 3

2.5.2. Preparation of Hepatic Tissue Samples for Analysis. absorbance being read at 470 nm against blank every minute
Hepatic tissue (100 mg tissue/mL buffer) was first homoge- for 3min on a spectrophotometer. The enzyme activity was
nized in 50 mM phosphate buffer (pH 7.2); the homogenate expressed as units/mg protein.
was then centrifuged at 12,000 ×g for 15 mins and the super-
natant was used for analysis. The protein concentration in Gpx. The activity of Gpx was determined essentially as
each fraction was determined by the method of Bradford [19], described by Rotruck et al. [27], wherein the rate at which
using crystalline bovine serum albumin as a standard. glutathione is oxidised by H2 O2 (as catalysed by Gpx present
in the sample) is determined by reading the colour developed
at 412 nm on a spectrophotometer. Gpx activity was expressed
2.6. Parameters Analysed as units per milligram protein (one unit being the amount of
enzyme that converted 1 𝜇mol of reduced glutathione (GSH)
2.6.1. Blood Glucose Level and Serum Lipid Profile Parameters.
to the oxidized form of glutathione (GSSG) in the presence of
Mean levels of blood glucose were measured by the method
H2 O2 /min).
of Sasaki et al. [20]. In the same samples, mean levels of
total cholesterol, triglycerides, and high-density lipoprotein GST. The activity of GST was determined by the method
(HDL) cholesterol were determined by standard kits (BioSys- of Habig and Jakoby [28], the principle of which is that
tems, Spain) following the manufacturer’s instructions. The GSH conjugates with 1-chloro-2,4-dinitrobenzene (c-DNB; a
atherogenic index (AI) was calculated as AI = (total choles- hydrophilic substrate) which is measured spectrophotomet-
terol − HDL)/HDL. The levels of LDL cholesterol and very rically at 340 nm. GST activity was expressed as 𝜇moles of
low-density lipoprotein (VLDL) cholesterol were calculated c-DNB formed/min/mg of protein.
by Friedewald’s formula [21], the units being expressed as
milligrams per decilitre (mg/dL).
2.6.4. Levels of Nonenzymatic Antioxidants (GSH, Ascorbic
Acid, and 𝛼-Tocopherol) in Hepatic Tissue Samples
2.6.2. Activities of Hepatic Marker Enzymes in Serum Samples.
Activities of aspartate aminotransferase (AST) and alanine GSH. GSH content (𝜇g/mg protein) was estimated by the
aminotransferase (ALT) were determined by the method method of Moron et al. [29], wherein protein in the sample
of King [22] and expressed in terms of micromoles of is first precipitated out, followed by addition 4 mL of 0.3 M
pyruvate liberated per minute per milligram of protein at Na2 HPO4 (pH 8.0) and 0.5 mL of 0.04% (w/v) 5,5-dithiobis-
37∘ C. Alkaline phosphatase (ALP) activity was assayed using 2-nitrobenzoic acid to the protein-free supernatant to yield a
disodium phenyl phosphate as substrate [23] and expressed yellow colour that is read spectrophotometrically at 412 nm.
as micromoles of phenol liberated per minute per milligram
of protein. Lactate dehydrogenase (LDH) was assayed by the Ascorbic Acid (Vitamin C). Vitamin C (𝜇g/mg protein) was
method of King, [24], the principle which is that LDH con- measured by the method of Omaye et al. [30], wherein
verts lactate to pyruvate (aided by coenzyme nicotinamide ascorbate in the sample is oxidized by copper to form
adenine dinucleotide (NAD)), and pyruvate formed reacts dehydroascorbic acid which reacts with 2,4-dinitrophenyl
with dinitrophenylhydrazine in HCl to yield an orange- hydrazine to form bis-2,4-dinitrophenyl hydrazine which, in
colored hydrazone complex in alkaline medium, which is turn, undergoes further rearrangement to form a product
measured at 420 nm. with an absorption maximum at 520 nm.

𝛼-Tocopherol (Vitamin E). Vitamin E (𝜇g/mg protein) was

2.6.3. Activities of Antioxidant Enzymes in Hepatic Tissue
estimated by the method of Desai [31], the principle which
Samples. The activities of the antioxidant enzymes catalase
is that ferric ions are reduced to ferrous ions in the presence
(CAT), superoxide dismutase (SOD), glutathione peroxidase
of tocopherol, resulting in the formation of a pink colour that
(Gpx), and glutathione-S-transferase (GST) were determined
is read spectrophotometrically at 536 nm.
by standard methods.

CAT. CAT activity was determined by the method of Sinha 2.6.5. Determination of Lipid Peroxidation in Hepatic Tis-
[25], the principle which is that dichromatic acetic acid is sues. The mean concentration of malondialdehyde (MDA),
reduced to chromic acetate when heated in the presence of a measure of lipid peroxidation, was assayed in the form
hydrogen peroxide (H2 O2 ), with the formation of perchloric of thiobarbituric acid-reacting substances (TBARS) by the
acid as an unstable intermediate. The resulting green colour method of Ohkawa et al. [32]. Briefly, to 0.2 mL of 8.1%
was read at 590 nm against a suitable blank on a spectropho- sodium dodecyl sulphate, 1.5 mL of 20% acetic acid (pH 3.5)
tometer. CAT activity was expressed as units per milligram and 1.5 mL of 0.81% thiobarbituric acid aqueous solution were
protein (one unit was the amount of enzyme that utilized added in succession. To this reaction mixture, 0.2 mL of the
1 𝜇mol of H2 O2 /min). homogenate of hepatic tissue was added. The mixture was
then heated in a boiling water bath for 60 min. After cooling
SOD. SOD activity (expressed as units/mg protein) was to room temperature, 5 mL of butanol : pyridine (15 : 1, v/v)
determined by the method of S. Marklund and G. Marklund solutions were added. The mixture was then centrifuged at
[26], wherein the degree of inhibition of pyrogallol auto- 2000 ×g for 15 min. The upper organic layer was separated,
oxidation by the sample was measured with the change in and the intensity of the resulting pink colour was read
4 Evidence-Based Complementary and Alternative Medicine

Table 1: Mean levels of blood glucose and of serum lipid profile parameters∗ in Wistar rats.

Group II Group III Group IV Group V

Parameters Group I
hypercholesterolemic, hypercholesterolemic, hypercholesterolemic, hypercholesterolemic,
tested (control)
saline-treated lovastatin-treated Piper betle extract treated eugenol-treated
Glucose 84.5 ± 2.4 194.1 ± 2.1a 144.2 ± 1.1ab 149.2 ± 0.9abc 141.9 ± 1.3abd
Total
49.2 ± 2.4 134.2 ± 4.7a 61.5 ± 1.6ab 62.2 ± 2.8abc 59.2 ± 3.1abc
cholesterol
Triglycerides 44.6 ± 2.4 149.6 ± 2.7a 59.7 ± 0.9ab 63.7 ± 1.6abc 53.4 ± 2.9abc
HDL
29.4 ± 4.7 20.2 ± 2.1a 28.0 ± 0.2ab 26.7 ± 0.7ac 28.4 ± 4.2abcd
cholesterol
LDL
13.6 ± 2.4 109.7 ± 0.5a 39.4 ± 1.3ab 40.2 ± 2.7ab 22.5 ± 7.2acd
cholesterol
VLDL
4.8 ± 6.8 35.2 ± 1.9a 12.5 ± 1.0ab 14.5 ± 0.2abc 11.2 ± 0.5abd
cholesterol
A.I. 0.6 ± 0.1 4.8 ± 0.3a 2.0 ± 0.3ab 2.3 ± 0.2b 1.3 ± 0.3abcd
∗
Sampling was done 10 days after induction of hypercholesterolemia and 7 days after start of treatment.
Values represent the mean ± SD for observations made on five rats in each group.
Units: milligrams per deciliter (except for atherogenic index).
Statistical analysis: One-way analysis of variance (ANOVA), where significant, post hoc testing (least significant difference) was done for intergroup
comparisons.
HDL-C: high-density lipoprotein cholesterol, LDL-C: low-density lipoprotein cholesterol, VLDL-C: very low-density lipoprotein-cholesterol, A.I.: atherogenic
index.
a
Statistically significant difference (𝑃 < 0.05) when compared with group I values.
b
Statistically significant difference (𝑃 < 0.05) when compared with group II values.
c
Statistically significant difference (𝑃 < 0.05) when compared with group III values.
d
Statistically significant difference (𝑃 < 0.05) when compared with group IV values.

at 532 nm. Tetramethoxypropane was used as an external (group II) rats was significantly (𝑃 < 0.05) higher than that
standard. The level of lipid peroxides was expressed as nmoles in control (group I) rats. In hypercholesterolemic rats treated
of MDA formed/mg protein. with lovastatin (group III), Piper betle extract (group IV),
or eugenol (group V), significantly (𝑃 < 0.05) lower mean
2.6.6. Histopathological Studies. Conventional techniques of blood glucose levels were observed when compared to that in
paraffin-wax sectioning and haematoxylin-eosin (HE) stain- saline-treated hypercholesterolemic rats although the levels
ing were used for histological studies [33]. Slices of fresh hep- were still significantly (𝑃 < 0.05) higher than that in the
atic tissue were cut and fixed in buffered neutral formalin fix- control rats. The mean blood glucose level was significantly
ative for 24 h. Following fixation, the tissue slices were washed (𝑃 < 0.05) higher in Piper betle extract-treated hyperc-
and processed through an ascending series of alcohol (30%, holesterolemic rats than that in lovastatin-treated or eugenol-
50%, 70%, 90%, and 100%), cleared in methyl salicylate, and treated hypercholesterolemic rats. However, no significant
infiltrated with wax at 57∘ C. The hepatic tissue, thus cleared, difference was observed between the mean blood glucose
was embedded in paraffin. Sections of 6–8 𝜇m thickness were level in lovastatin-treated hypercholesterolemic rats and that
cut, stained by aqueous haematoxylin and alcoholic-eosin, in eugenol-treated hypercholesterolemic rats (Table 1).
and then examined by bright-field microscopy (200x) (Carl
Zeiss Axioskop 2 plus; Jena, Germany).
3.2. Serum Lipid Profile Parameters in Wistar Rats (Table 1).
2.6.7. Statistical Analysis. The values are expressed as mean Saline-treated hypercholesterolemic rats showed significantly
± standard deviation (SD) for five animals in each group. (𝑃 < 0.05) higher mean serum levels of total choles-
Differences between groups were assessed by one-way analy- terol, triglycerides, LDL-cholesterol, and VLDL-cholesterol,
sis of variance (ANOVA) using Statistical Package for Social a significantly higher atherogenic index and a significantly
Sciences software package for Windows (version 16.0; IBM (𝑃 < 0.05) lower mean level of HDL-cholesterol, when
Corp., Armonk, NY, USA). If one-way ANOVA yielded sig- compared to the values in control rats and in lovastatin-
nificant results, post hoc testing was performed for intergroup treated, Piper betle extract-treated, or eugenol-treated hyperc-
comparisons using the least significant difference test. Values holesterolemic rats (Table 1). However, hypercholesterolemic
were considered statistically significant when 𝑃 < 0.05. rats treated with lovastatin or Piper betle extract exhibited
significantly (𝑃 < 0.05) higher mean serum levels of
3. Results total cholesterol, triglycerides, LDL-cholesterol, and VLDL-
cholesterol, a higher atherogenic index as well as significantly
3.1. Blood Glucose Levels in Wistar Rats (Table 1). The mean (𝑃 < 0.05) lower mean serum levels of HDL-cholesterol,
blood glucose level in hypercholesterolemic, saline-treated when compared to control rats. No significant differences
Evidence-Based Complementary and Alternative Medicine 5

Table 2: Mean serum levels of hepatic marker enzymes in∗ Wistar rats.

Group II Group III Group IV Group V

Parameters Group I
hypercholesterolemic, hypercholesterolemic, hypercholesterolemic, hypercholesterolemic,
tested (control)
saline treated lovastatin treated Piper betle extract treated eugenol treated
AST 0.8 ± 0.2 1.8 ± 0.2a 1.6 ± 0.2ab 1.3 ± 0.3ab 1.2 ± 0.2bcd
ALT 1.2 ± 0.03 1.8 ± 0.3a 1.6 ± 0.2ab 1.2 ± 0.1ab 1.3 ± 0.3ab
ALP 2.0 ± 0.1 3.3 ± 0.7a 3.0 ± 0.1a 3.2 ± 0.1ab 2.8 ± 0.3ab
LDH 6.9 ± 0.4 17.2 ± 0.5a 13.4 ± 0.7ab 12.2 ± 0.4abc 12.5 ± 0.5abc
∗
Sampling done 10 days after induction of hypercholesterolemia and 7 days after start of treatment.
Values represent the mean ± SD for observations made on five rats in each group.
Units: aspartate and alanine aminotransferases: 𝜇moles × 10−2 of pyruvate liberated/min/mg protein.
Alkaline phosphatase: 𝜇moles × 10−2 of phenol liberated/min/mg protein.
Lactate dehydrogenase: 𝜇moles × 10−1 of pyruvate formed/minute/mg protein.
Statistical analysis: one-way analysis of variance (ANOVA), where significant, post hoc testing (least significant difference) done for intergroup comparisons.
AST: aspartate aminotransferase, ALT: alanine aminotransferase, ALP: alkaline phosphatase, LDH: lactate dehydrogenase.
a
Statistically significant difference (𝑃 < 0.05) when compared with group I values.
b
Statistically significant difference (𝑃 < 0.05) when compared with group II values.
c
Statistically significant difference (𝑃 < 0.05) when compared with group III values.
d
Statistically significant difference (𝑃 < 0.05) when compared with group IV values.

were observed in these parameters between hypercholes- lower than those in control rats (Table 3). In hypercholes-
terolemic rats that had been treated with Piper betle extract terolemic rats that had been treated with lovastatin, Piper
or with lovastatin (Table 1). Interestingly, eugenol-treated rats betle extract, or eugenol, significantly (𝑃 < 0.05) higher
exhibited a significantly (𝑃 < 0.05) lower mean level of total mean activities of these enzymes were noted than those
cholesterol than that in lovastatin-treated rats. In addition, in hypercholesterolemic saline-treated rats; however, these
the mean serum total cholesterol, triglyceride, and VLDL- mean enzyme activities remained significantly lower than
cholesterol levels in eugenol-treated hypercholesterolemic those in control rats (Table 3). Interestingly, the mean hepatic
rats were significantly (𝑃 < 0.05) lower than those observed Gpx activity observed in hypercholesterolemic, eugenol-
in Piper betle extract-treated hypercholesterolemic rats. A treated rats was significantly (𝑃 < 0.05) higher than that
very noteworthy finding was that the mean serum levels in lovastatin-treated hypercholesterolemic rats, while there
of triglycerides and VLDL-cholesterol in eugenol-treated were no significant differences between these two groups
hypercholesterolemic rats approximated those in normal rats in the mean hepatic activities of CAT and SOD. Also,
(Table 1). there were no significant differences in the mean hepatic
enzyme activities between hypercholesterolemic Piper betle
extract-treated and hypercholesterolemic lovastatin-treated
3.3. Activities of Hepatic Marker Enzymes in Serum of Wistar rats (Table 3).
Rats (Table 2). The mean activities of serum AST, ALT, ALP,
and LDH were found to be significantly (𝑃 < 0.05) higher
in hypercholesterolemic, saline-treated rats than those in 3.5. Concentrations of Nonenzymatic Antioxidants in Hepatic
control rats. Although hypercholesterolemic rats treated with Tissue of Wistar Rats (Table 3). The mean concentrations of
the Piper betle extract or eugenol exhibited significantly GSH, vitamin C, and vitamin E in the hepatic tissue samples
(𝑃 < 0.05) lower mean activities of these enzymes than from hypercholesterolemic, saline-treated rats were signifi-
those in hypercholesterolemic saline-treated rats, the mean cantly (𝑃 < 0.05) lower than those in control rats (Table 3).
activities of ALT, ALP, and LDH were still significantly higher However, significantly (𝑃 < 0.05) higher mean concentra-
than those in control rats (Table 2). Interestingly, the mean tions of these antioxidants were observed in hypercholes-
activity of LDH was significantly (𝑃 < 0.05) lower in terolemic rats that had been treated with lovastatin, Piper
the Piper betle extract-treated and in the eugenol-treated betle extract, or eugenol than those in hypercholesterolemic,
hypercholesterolemic rats than those in the lovastatin-treated saline-treated rats. There were no significant differences in
hypercholesterolemic rats. No significant differences were the mean values of these parameters between hypercholes-
observed in the mean serum activities of ALT, ALP, and LDH terolemic rats that had been treated with the Piper betle
between hypercholesterolemic rats that had been treated with extract and those that had been treated with eugenol. Inter-
the Piper betle extract and those that had been treated with estingly, the mean hepatic GSH concentrations in these two
eugenol (Table 2). groups of rats were found to be significantly (𝑃 < 0.05) higher
than that in the hypercholesterolemic, lovastatin-treated rats.
In hypercholesterolemic rats that received eugenol, mean
3.4. Activities of Enzymatic Antioxidants in Hepatic Tissue of hepatic tissue antioxidant concentrations approached those
Wistar Rats (Table 3). The mean activities of CAT, SOD, Gpx, in control rats; in hypercholesterolemic rats that received
and GST in the samples of hepatic tissue from hypercholes- the Piper betle extract, the mean hepatic vitamin C level
terolemic saline-treated rats were significantly (𝑃 < 0.05) approached that in control rats (Table 3).
6 Evidence-Based Complementary and Alternative Medicine

Table 3: Mean activities of enzymatic antioxidants and mean levels of nonenzymatic antioxidants and malondialdehyde in hepatic tissue
samples∗ from Wistar rats.

Group II Group III Group IV Group V

Parameters Group I
hypercholesterolemic, hypercholesterolemic, hypercholesterolemic, hypercholesterolemic,
tested (control)
saline treated lovastatin treated Piper betle extract treated eugenol treated
SOD 7.1 ± 1.4 4.5 ± 0.5a 5.0 ± 0.4ab 5.3 ± 0.2a 5.5 ± 0.3abc
CAT 53.8 ± 3.5 40.1 ± 4.0a 42.9 ± 3.2b 43.8 ± 0.2ac 44.6 ± 5.7abd
GPX 31.3 ± 5.5 13.4 ± 1.1a 20.8 ± 1.3ab 21.5 ± 2.1ab 22.4 ± 0.7ab
GST 17.0 ± 4.4 8.4 ± 1.0a 14.4 ± 1.8b 14.5 ± 0.6abc 14.7 ± 0.6abcd
GSH 3.3 ± 0.1 2.0 ± 0.1a 2.6 ± 0.1ab 2.7 ± 0.1ab 2.8 ± 0.1abd
VIT-C 2.3 ± 1.4 1.6 ± 0.6a 1.7 ± 0.8a 1.9 ± 0.7ab 1.8 ± 0.6abcd
VIT-E 1.9 ± 1.2 1.0 ± 0.4a 1.3 ± 0.6ab 1.5 ± 0.1abc 1.5 ± 0.7bcd
MDA 1.2 ± 0.1 3.8 ± 0.4a 1.8 ± 0.3ab 2.0 ± 0.2abc 1.6 ± 0.1acd
∗
Sampling done 10 days after induction of hypercholesterolemia and 7 days after start of treatment.
Values represent the mean ± SD for observations made on five rats in each group.
Units: CAT—𝜇moles of H2 O2 utilized/min/mg protein.
SOD—units/mg protein.
Gpx—𝜇moles of GSH oxidized/min/mg protein.
GST—𝜇moles of c-DNB formed/min/mg protein.
GSH—microgram of reduced glutathione/mg protein.
Vitamins C and E—micrograms/mg protein.
MDA—𝜇moles of MDA produced/mg protein.
Statistical analysis: one-way analysis of variance (ANOVA), where significant, post hoc testing (least significant difference) done for intergroup comparisons.
CAT: catalase, SOD: superoxide dismutase, Gpx: glutathione peroxidase, GST: glutathione-S-transferase, GSH: reduced glutathione, MDA: malondialdehyde,
H2 O2 : hydrogen peroxide, c-DNB: 1-chloro-2,4-dinitrobenzene.
a
Statistically significant difference (𝑃 < 0.05) when compared with group I values.
b
Statistically significant difference (𝑃 < 0.05) when compared with group II values.
c
Statistically significant difference (𝑃 < 0.05) when compared with group III values.
d
Statistically significant difference (𝑃 < 0.05) when compared with group IV values.

3.6. MDA Concentrations in Hepatic Tissues of Wistar Rats with the nucleus pushed to periphery (Figure 1(b)). In
(Table 3). The mean concentration of MDA in hepatic tissue hypercholesterolemic lovastatin-treated rats, section showed
samples from hypercholesterolemic, saline-treated rats was normal hepatocyte with darkly stained nucleus, (arrows)
significantly (𝑃 < 0.05) higher than that in control rats central vein, and wide sinusoids (Figure 1(c)). In hyperc-
(Table 3). Although the mean hepatic MDA concentrations in holesterolemic Piper betle extract-treated rats, section showed
hypercholesterolemic rats that had been treated with lovas- illustrating few small vacuolated hepatocytes with occa-
tatin, Piper betle extract, or eugenol were significantly (𝑃 < sional inflammatory cell infilteration (Figure 1(d)). In hyperc-
0.05) lower than that in hypercholesterolemic, saline-treated holesterolemic eugenol-treated rats, sections showed normal
rats, they remained significantly (𝑃 < 0.05) higher than hepatic architecture, with parenchymal structures preserved
that in control rats. Hypercholesterolemic rats treated with (Figure 1(e)).
eugenol exhibited a significantly lower mean concentration
of MDA than those treated with the Piper betle extract. Inter-
estingly, no significant differences were observed in the mean 4. Discussion
hepatic tissue concentration of MDA in eugenol-treated or
Piper betle extract-treated hypercholesterolemic rats, when Triton WR-1339, one of the well-known non-ionic detergent
compared with the mean hepatic tissue concentrations noted (oxyethylated tertiary octylphenol formaldehyde polymer),
in the lovastatin-treated hypercholesterolemic rats. that has been widely used to produce acute hyperlipidemia
in animal models. Triton WR-1339-induced hypercholes-
terolemia has been demonstrated to alter the physicochem-
3.6.1. Histopathological Examination. Sections of hepatic tis- ical properties of lipoproteins, thereby preventing the uptake
sue from the experimental groups of rats were stained by of lipoproteins from the circulation through extra hepatic
H&E and then subjected to histopathological examination tissues resulting in increased level of circulatory lipoproteins
by light microscopy (Figure 1). Sections of hepatic tissue in animal models [34]. Triton WR-1339 model being a rapid
from control rats exhibited normal hepatocytes, with normal and convenient system [18] has been extremely employed for
nuclei and sinusoidal spaces with Kupffer cells (arrows) screening natural [35–37] or chemical hypolipidemic drugs
(Figure 1(a)). In sections from hypercholesterolemic saline- [38–40] and also to delineate features of cholesterol and
treated rats, revealing loss of normal liver radiating pattern, triacylglycerol metabolism [41]. In addition, Triton WR-1339
periportal inflammation with cellular infilteration in cen- has also been used successfully to study intestinal lipoprotein
tral vein (detached line), vacuolated hepatocytes (arrows) synthesis in animal models [42]. Hence, the Triton WR-1339
Evidence-Based Complementary and Alternative Medicine 7

(a) (b)

(c) (d)

(e)

Figure 1: Histoarchitecture of hepatic tissue Wistar rats. Sections of hepatic tissue from the experimental groups of rats were stained
by H&E and then subjected to histopathological examination by light microscopy (Figure 1). Sections of hepatic tissue from control rats
showing central vein with normal hepatocyte, healthy nucleus, and sinusoidal spaces with kupffer cells (arrows) (a). In sections from
hypercholesterolemic saline-treated rats, revealing loss of normal liver radiating pattern, periportal inflammation with cellular infiltration
in central vein (Marked place), and vacuolated hepatocytes (arrows) with the nucleus pushed to periphery (b). In hypercholesterolemic
lovastatin-treated rats, section showed normal hepatocyte with darkly stained nucleus, (arrows) central vein and wide sinusoids (c).
In hypercholesterolemic Piper betle extract-treated rats, section showed illustrating few small vacuolated hepatocytes with occasional
inflammatory cell infiltration (d). In hypercholesterolemic eugenol-treated rats, sections showed normal hepatic architecture, with
parenchymal structures preserved (e).

model has been examined not only as a screening method for reduce the risk of developing ischemic heart disease or
antihyperlipidemic agents, but also as a means for elucidating the occurrence of further cardiovascular or cerebrovascu-
lipid metabolism [43]. In the present study, the putative lar complications [44]. Since hyperlipidemia is frequently
antihypercholesterolemic effects of an ethanol extract of Piper associated with hyperglycemia, an attempt was made in
betle and of one of its active constituents, eugenol, were the current study to measure the blood glucose level in
compared with those of a well-known lipid-lowering drug, hypercholesterolemic rats (Table 1). In hypercholesterolemic
lovastatin, in a rodent model of hypercholesterolemia that saline-treated rats, a significantly higher mean blood glucose
was induced by Triton WR-1339. level than that in control (normal) rats was noted (Table 1).
Hyperglycemia and hyperlipidemia are important risk However, the administration of the Piper betle extract or
factors for diabetes-accelerated atherosclerosis. The main eugenol significantly decreased the blood glucose levels in
aim of treatment in patients with hyperlipidemia is to hypercholesterolemic rats. These results are consistent with
8 Evidence-Based Complementary and Alternative Medicine

those of an earlier study in which an extract of the flower of of an extract of Piper betle, or of eugenol, to hypercholes-
Cassia auriculata significantly decreased serum glucose levels terolemic rats resulted in significantly lower mean serum
in hyperlipidemic rats [37]. triglyceride levels than the mean level seen in hypercholes-
In the present investigation, in hypercholesterolemic terolemic, saline-treated rats (Table 1). This effect may have
rats that had received the Piper betle extract or eugenol, been due to enhanced catabolism of triglycerides caused by
significantly lower mean serum levels of total cholesterol, increased stimulation of plasma lipoprotein lipase activity.
triglycerides, LDL-cholesterol and VLDL-cholesterol and Higher mean levels of HDL-cholesterol were also noted
significantly higher mean serum levels of HDL-cholesterol in hypercholesterolemic rats that had been treated with
than those in hypercholesterolemic saline-treated rats were lovastatin, Piper betle extract, or eugenol, when compared to
noted. This effect was more pronounced in rats that had the mean level in hypercholesterolemic, saline-treated rats.
received eugenol than in rats that had received the extract of The lipid-lowering effect brought about by administration of
Piper betle. The lipid-lowering effect of leaves of Piper betle the Piper betle extract and of eugenol might have been due to
and of its active component, eugenol, in an experimental reactivation of lipolytic enzymes for early clearance of lipids
animal model of hypercholesterolemia is probably mediated from the circulation in triton-induced hyperlipidemia. Our
through inhibition of hepatic cholesterol biosynthesis and results are consistent with those of Vallianou et al. [50].
reduction of lipid absorption in the intestine. In the present The atherogenic index (ratio of LDL-cholesterol to HDL-
study, the lower mean serum total cholesterol levels were cholesterol) is also a predictive indicator of cardiovascular
associated with lower levels of the LDL-cholesterol fraction. disease incidence [35]. Apparently, lowering the atherogenic
Serum LDL-cholesterol level is a major and potentially index is an important measure in reducing the risk of
modifiable risk factor of cardiovascular diseases and serves atherosclerosis. In the present study, hypercholesterolemic
as a target for numerous antihypercholesterolemic therapies. rats that had been administered Piper betle extract or eugenol
Our finding suggests that the cholesterol-lowering activity exhibited significantly lower mean atherogenic index values
of the Piper betle extract is due to enhanced catabolism of than did hypercholesterolemic, saline-treated rats.
LDL-cholesterol through hepatic receptors, as suggested by Küçükgergin et al. [51] demonstrated that hypercholes-
Khanna et al. [43]. In addition, higher mean serum levels terolemia is a primary factor contributing to oxidative
of HDL-cholesterol were noted, which is reported to have a damage to hepatocytes, leading to malfunctioning of the
preventive function against atherogenesis. An independent liver through microvesicular steatosis and intracellular lipid
inverse relationship between blood HDL-cholesterol levels accumulation. The extent of hepatic damage can be assessed
and cardiovascular risk incidence has been documented by noting the mean activities of serum transaminases and
and reported [45]. HDL-cholesterol is commonly termed alkaline phosphatase (ALP) [52]. In the present study,
as “good cholesterol,” since it facilitates the mobilisation of the mean activities of serum AST, ALT, ALP, and LDH
triglycerides and cholesterol from plasma to liver, where these were significantly higher in hypercholesterolemic, saline-
are catabolised and eliminated in the form of bile acids. treated rats than those in control rats (Table 2). However,
Elevated plasma levels of triglycerides are found to be such elevations in the mean levels of serum AST, ALT,
associated with an increased incidence of coronary artery ALP, and LDH enzymes appear to have been prevented in
disease [46]. Such higher plasma triglyceride levels have been hypercholesterolemic rats that had been treated with the
attributed mainly to an increased population of small, dense Piper betle extract or with eugenol, since the mean levels
LDL-cholesterol deposits which are very atherogenic [47] and were significantly lower than those in hypercholesterolemic,
enhanced cholesteryl ester mass transfer from apolipopro- saline-treated rats (Table 2); these observations suggest that
tein B-containing lipoproteins (VLDL-cholesterol and LDL- the Piper betle extract and eugenol were able to protect the
cholesterol) [4]. Triglycerides are also proposed to be a major hepatic tissue from hypercholesterolemia-induced oxidative
determinant of cholesterol esterification, its transfer, and stress-mediated cellular damage. These results are consistent
HDL-cholesterol remodelling in human plasma [48]. The with those of an earlier study, in which the mean serum
restoration of catabolic metabolism of triglycerides could be levels of AST, ALT, ALP, and LDH were found to be sig-
due to an increased stimulation of the lipolytic activity of nificantly lower in rats with Triton WR-1339-induced acute
plasma lipoprotein lipase. These alterations in the levels of hypercholesterolemia that had been treated with a mushroom
extract or with chrysin [53].
serum lipid peroxide and antioxidant status in subjects with
Oxygen-free radicals are found to be produced dur-
high serum total cholesterol and LDL-cholesterol levels and
ing hypercholesterolemic atherogenesis [54]. Living tissues
low HDL-cholesterol levels may increase the susceptibility of are endowed with innate antioxidant defense mechanisms
LDL-cholesterol to oxidation in the circulation. through enzymatic and nonenzymatic antioxidants that are
As enhanced lipid peroxidation leads to higher athero- involved in the quenching of superoxide anions and H2 O2
genicity, it is plausible that antioxidant status should have [55]. A reduction in the activity of these enzymes is associated
a major impact not only on the rate of LDL oxidation but with the accumulation of highly reactive free radicals, leading
perhaps on development of atherosclerosis [49]. A potential to deleterious effects such as loss of integrity and function
risk of atherosclerosis in individuals with high serum lipid of cell membranes [56]. In the present investigation, the
levels may be associated with LDL oxidation as a result of mean activities of CAT, SOD, GPx, and GST in hepatic tissue
increased levels of LDL-cholesterol and decreased antioxi- samples from hypercholesterolemic saline-treated rats were
dant enzyme activity. In the present study, administration significantly (𝑃 < 0.05) lower than those noted in control
Evidence-Based Complementary and Alternative Medicine 9

rats. However, such a decline in the mean activities of CAT, Eugenol might be effective in preventing the toxic man-
SOD, GPx, and GST appears to have been prevented in ifestations produced by increased levels of lipids induced by
hepatic tissue sample from hypercholesterolemic rats that triton WR-1339. In the present study, the oral administration
had been treated with lovastatin, the Piper betle extract or of eugenol or of the Piper betle extract to hypercholes-
eugenol, since the mean activities were significantly higher terolemic rats resulted in significantly lower mean levels of
than those in samples from hypercholesterolemic, saline- MDA than that in saline-treated hypercholesterolemic rats.
treated rats (Table 3). The antioxidant activity of eugenol, The decrease in intensity of lipid peroxidation, as inferred
which has a phenolic structure, has already been evaluated by from the lower mean levels of MDA, was possibly due to the
the extent of protection offered against free radical-mediated free radical-scavenging property of the hydroxyl groups at the
lipid peroxidation in both in vitro and in vivo studies [13]. seventh position of the eugenol molecule.
Nonenzymatic antioxidants also play a vital role in pro- Hypercholesterolemia-induced hepatic abnormalities
tecting cells from oxidative damage. GSH is an important can be further confirmed by histopathological findings.
antioxidant in living systems because it is involved in numer- In the present investigation, Triton WR-1339-induced
ous biochemical pathways within the cells. It plays a key role hypercholesterolemic rats that had been treated with saline
in liver detoxification reactions by maintaining the structural alone showed marked changes in the liver, ballooning
integrity of cell membranes [57]. 𝛼-tocopherol (Vitamin E), degeneration of the hepatocytes, and occasional collection
a nonenzymatic antioxidant, is believed to protect biological of chronic inflammatory cells (Figure 1). Deepa and
membranes from oxidative damage by its ability to quench Varalakshmi [62] observed similar fatty changes in the
lipid peroxides [58]. It is possible that elevated levels of hepatic tissue, which are consistent with the abnormal
oxygen-free radicals in hypercholesterolemia may damage biochemical parameters observed in the present study.
the myocardial cell besides affecting the coronary arteries. However, treatment with eugenol appeared to ameliorate or
Ascorbic acid (Vitamin C), the most widely recognized water- prevent the adverse effects, as suggested by the presence of
soluble antioxidant, prevents the oxidative damage to the cell only minimal or partial fatty changes. So also Sudhahar et
membrane that is induced by aqueous radicals; it also reduces al. [63] reported that the administration of lupeol and lupeol
and regenerates oxidized 𝛼-tocopherol and lipid peroxides linoleate to hypercholesterolemic rats resulted in reduction
[59]. In the present study, the mean hepatic concentrations of of fatty changes in hepatic tissue.
GSH and of vitamins C and E were found to be significantly
lower in hypercholesterolemic, saline-treated rats than those
in control rats (Table 3), possibly due to lipidemic oxidative
5. Conclusion
stress. However, the mean hepatic concentrations of GSH and In conclusion, the present investigation has demonstrated
of vitamins C and E were significantly higher in hypercholes- the putative lipid-lowering effect (by virtue of antioxidant
terolemic rats that had been treated with lovastatin, Piper activity) of an ethanolic extract of Piper betle and of eugenol,
betle extract, or eugenol than those in hypercholesterolemic, the major constituent of the Piper betle extract, in Triton WR-
saline-treated rats (Table 3). No significant differences were 1339-induced, hypercholesterolemic rats. The lipid-lowering
noted in these test parameters between lovastatin-treated, potential and antioxidant capacity of eugenol appeared to be
Piper betle extract-treated, and eugenol-treated hypercholes- more pronounced than that of the Piper betle extract and as
terolemic rats. Hence, treatment with the Piper betle extract effective as that of the standard lipid-lowering drug, lovas-
or eugenol possibly acted by reducing lipidemic oxidative tatin. Hence, eugenol may possibly be developed as an alter-
stress, therein permitting these antioxidants to be maintained native cholesterol-lowering drug; however, further molecular
at near normal levels. studies are required to investigate the mechanism underlying
In biological environments, the most favourable substrate the antihypercholesterolemic effect of this compound. Future
for lipid peroxidation is represented by polyunsaturated studies must focus on the hypolipidemic effect of eugenol
fatty acids. Hypercholesterolemia-mediated atherosclerosis is under conditions of chronic hypercholesterolemia.
associated with an increase in the level of the lipid peroxida-
tion product, malondialdehyde (MDA), which is an index of
the level of oxygen-free radicals [54, 60]; it also reacts with
Conflict of Interests
polyunsaturated fatty acids, causing free radical-mediated The authors declare that there is no conflict of interests.
tissue damage in cellular membranes. The polyunsaturated
fatty acids in the cell membrane are protected against lipid
peroxidation through endogenous antioxidants such as 𝛼- Acknowledgments
tocopherol [61]. A decrease in lipid peroxidation leads to Financial assistance provided by University Grants Commis-
a reduction in arterial wall cholesterol content. Therefore, sion-Basic Scientific Research (UGC-BSR) in the form of
reduction of atherosclerosis caused by hypercholesterolemia one time grant to the corresponding author is gratefully
is associated with a decrease in lipid peroxidation, while acknowledged. The instrumentation facility provided by
increased lipid peroxidation is a characteristic feature of the University Grants Commission-Special Assistance Pro-
hypercholesterolemia; it impairs cell membrane fluidity and gramme (UGC-SAP) of the Department of Animal Science,
alters the activity of membrane-bound enzymes and recep- Bharathidasan University, is also acknowl-edged. The authors
tors, resulting in membrane malfunction [55]. thank Dr. S. Pannerselvam, Professor and Head, Sugarcane
10 Evidence-Based Complementary and Alternative Medicine

Research Station, Sirugam-ani-Tiruchirappalli, Tamilnadu, [15] World Health Organization, “FAO/WHO expert committee
India, for providing the Piper betle variety. on food additives evaluation of certain food additives and
contaminants,” Tech. Rep. 683, WHO, Geneva, Switzerland,
1982.
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Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2013, Article ID 821813, 10 pages
http://dx.doi.org/10.1155/2013/821813

Research Article
Andrographolide, a Novel NF-𝜅B Inhibitor,
Induces Vascular Smooth Muscle Cell Apoptosis via
a Ceramide-p47phox-ROS Signaling Cascade

Yu-Ying Chen,1 Ming-Jen Hsu,2 Joen-Rong Sheu,1,2 Lin-Wen Lee,3 and Cheng-Ying Hsieh1,2
1
Graduate Institute of Medical Sciences, School of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei 11031, Taiwan
2
Department of Pharmacology, School of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei 11031, Taiwan
3
Department of Microbiology and Immunology, Taipei Medical University, 250 Wu-Hsing Street, Taipei 11031, Taiwan

Correspondence should be addressed to Lin-Wen Lee; [email protected] and Cheng-Ying Hsieh; [email protected]

Received 30 October 2013; Accepted 4 December 2013

Academic Editor: Mao-Hsiung Yen

Copyright © 2013 Yu-Ying Chen et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Atherosclerosis is linked with the development of many cardiovascular complications. Abnormal proliferation of vascular smooth
muscle cells (VSMCs) plays a crucial role in the development of atherosclerosis. Accordingly, the apoptosis of VSMCs, which
occurs in the progression of vascular proliferation, may provide a beneficial strategy for managing cardiovascular diseases.
Andrographolide, a novel nuclear factor-𝜅B inhibitor, is the most active and critical constituent isolated from the leaves of
Andrographis paniculata. Recent studies have indicated that andrographolide is a potential therapeutic agent for treating cancer
through the induction of apoptosis. In this study, the apoptosis-inducing activity and mechanisms in andrographolide-treated
rat VSMCs were characterized. Andrographolide significantly induced reactive oxygen species (ROS) formation, p53 activation,
Bax, and active caspase-3 expression, and these phenomena were suppressed by pretreating the cells with N-acetyl-L-cysteine,
a ROS scavenger, or diphenylene iodonium, a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) inhibitor.
Furthermore, p47phox, a Nox subunit protein, was phosphorylated in andrographolide-treated rat VSMCs. However, pretreatment
with 3-O-methyl-sphingomyelin, a neutral sphingomyelinase inhibitor, significantly inhibited andrographolide-induced p47phox
phosphorylation as well as Bax and active caspase-3 expression. Our results collectively demonstrate that andrographolide-reduced
cell viability can be attributed to apoptosis in VSMCs, and this apoptosis-inducing activity was associated with the ceramide-
p47phox-ROS signaling cascade.

1. Introduction death of VSMCs, which occurs in the pathogenesis and

progression of vascular proliferative disorders, such as
Atherosclerosis remains a major and increasing health con- atherosclerosis and restenosis, often represents a critical
cern in developed countries, although prevention strategies feature of blood vessel remodeling [4]. In addition, neoin-
have substantially increased. Consequently, developing novel tima development and lesion growth in VSMCs seem to
therapeutic agents for atherosclerosis patients remains a be restrained by late apoptosis [5]. The regulated balance
major research priority [1]. Aberrant vascular smooth muscle between the death and survival signals perceived by a cell is
cells (VSMCs) proliferation has been shown to play a critical used to control the initiation of apoptosis [6]. Because cell
role in the pathogenesis of atherosclerosis-related events apoptosis can inhibit the proliferation of VSMCs, inducing
including in-stent restenosis, restenosis after percutaneous apoptosis may provide a pharmacological basis for treating
transluminal angioplasty, transplant vasculopathy, and vein proliferative cardiovascular disorders.
bypass graft failure [2, 3]. Therefore, inhibition of VSMC Increased reactive oxygen species (ROS) production is
proliferation might be a major target for the treatment of known to play a vital role in VSMC proliferation and apop-
cardiovascular diseases. Apoptosis, or the programmed cell tosis and leads to the development of atherosclerosis. The
2 Evidence-Based Complementary and Alternative Medicine

apoptosis of VSMCs caused by enhanced ROS production O

affects the progression of atherosclerotic lesions and may
induce plaque rupture [7]. ROS are small, extremely reac-
HO O
tive molecules because of their unpaired valence electrons.
There are several intracellular ROS producers, including 2
main manufacturers, the mitochondria and nicotinamide
adenine dinucleotide phosphate (NADPH) oxidase. A rapidly
expanding body of experimental evidence gathered since
the first identification of VSMCs implicates that NADPH
oxidase (Nox) in vascular cells is the underlying cause of
oxidative stress in various cardiovascular diseases [8]. Nox
is a complex composed of membrane-bound (p22phox and
Nox1-4) and cytoplasmic (Rac, p47phox, and p67phox) sub-
units. When it is activated, cytoplasmic subunits connect with
HO
their membrane-bound counterparts and generate an active
complex that oxidizes NADPH, leading to the production of
ROS [9]. The Nox-dependent production of ROS is thought
to be a crucial regulator of smooth muscle cell viability and
is believed to be linked to the development and severity of OH
human atherosclerotic lesions [10].
Figure 1: Chemical structure of andrographolide (Andro).
Andrographolide (Figure 1), a novel nuclear factor-𝜅B
(NF-𝜅B) inhibitor, is the most active and critical con-
stituent isolated from the leaves of Andrographis panicu-
lata [11]. A. paniculata has long been used as a herbal from Sigma-Aldrich (St. Louis, MO, USA). The 3-O-methyl-
medicine to prevent and treat upper respiratory tract sphingomyelin (3-OMS) was purchased from Biomol
infections, diarrhea, rheumatoid arthritis, and laryngitis (Plymouth Meeting, PA, USA). Anti-caspase-3 monoclon-
in Asia and Scandinavia [11, 12]. Recent studies have al antibodies (mAbs) and anti-Bax polyclonal antibody (pAb)
indicated that andrographolide inhibits tumor growth by were obtained from cell signaling (Beverly, MA, USA); the
inducing cell cycle arrest [13, 14] or apoptosis [15, 16] anti-phospho-p47phox serine359 pAb was acquired from
in various types of cancer cells. Recently, our previous Abcam (Cambridge, MA, USA); the anti-𝛼-tubulin
study confirmed that andrographolide enhances NF-𝜅B sub- mAb was obtained from NeoMarkers (Fremont, CA,
unit p65 Ser536 dephosphorylation through neutral sph- USA). The hybond-P polyvinylidene difluoride (PVDF)
ingomyelinase (nSMase)-mediated ceramide formation in membrane, enhanced chemiluminescence (ECL) Western
VSMCs [17], involving an increase in cyclic GMP/PKG, blotting detection reagent and analysis system, horseradish
followed by the inhibition of the p38MAPK/HO− -NF-𝜅B- peroxidase (HRP)-conjugated donkey anti-rabbit immuno-
ERK2 cascade in activated platelets [18, 19]. However, andro- globulin G (IgG), and sheep anti-mouse IgG were acquired
grapholide has demonstrated antiproliferative and apop- from Amersham (Buckinghamshire, UK). Andrographolide
totic effects on various types of cancer cells, whether it was dissolved in 0.1% dimethyl sulfoxide (DMSO) and stored
induces apoptosis in VSMCs is not known. Furthermore, at 4∘ C until used.
ROS appear to mediate the apoptosis-inducing activity
of andrographolide, but the source of ROS formation in 2.2. Rat Aortic Smooth Muscle Cell Primary Culture. The
andrographolide-induced apoptosis remains unclear. In the male Wistar rats used in this study were purchased from
present study, by considering the pivotal role of abnormal BioLASCO (Taipei, Taiwan). The VSMCs were enzymatically
VSMC proliferation in the development of atherosclerosis dispersed from the male Wistar rats (250–300 g). Thoracic
and restenosis, we examined the detailed cellular signal- aortas from the Wistar rats were removed and stripped
ing events associated with andrographolide-induced VSMC of endothelium and adventitia. The VSMCs were obtained
apoptosis. using a modification of the combined collagenase and
elastase digestion method [20]. These cells were grown in
DMEM supplemented with 20 mM HEPES, 10% FBS, 1%
2. Materials and Methods penicillin/streptomycin, and 2 mM glutamine at 37∘ C in a
humidified atmosphere of 5% CO2 . The growth medium was
2.1. Materials. Dulbecco’s modified Eagle’s medium changed every 2-3 days until the cells reached confluence.
(DMEM), trypsin (0.25%), L-glutamine, penicillin/strepto- The growth medium was removed, and the monolayer was
mycin, and fetal bovine serum (FBS) were purchased from rinsed with phosphate-buffered saline (PBS). A trypsin-
Gibco (Gaithersburg, MD, USA). Andrographolide (≥98%), EDTA solution was added, and the monolayer was incubated
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro- at 37∘ C for 2 min. The culture dishes were observed under
mide (MTT), N-acetyl-L-cysteine (NAC), diphenyleneiodo- a phase-contrast microscope until the cells detached. The
nium chloride (DPI), 2,7-dichlorofluorescein diacetate cells were removed using 10 mL of DMEM and centrifuged
(DCF-DA), and dimethyl sulfoxide (DMSO) were obtained at 900 rpm for 7 min. The pellet was resuspended in DMEM
Evidence-Based Complementary and Alternative Medicine 3

in a culture dish, and cells from Passages 4–8 were used 2.7. Statistical Analysis. The experimental results are
in all experiments. The primary cultured rat aortic VSMCs expressed as the means ± standard error and are accompanied
showed the “hills and valleys” pattern, and the expression by the number of observations. Data were assessed using
of 𝛼-smooth muscle actin was confirmed (data not shown). an analysis of variance. If an analysis indicated significant
All protocols were approved by the Taipei Medical University differences among the group means, then each group was
Animal Care and Use Committee. compared with the others using the Newman-Keuls method.
Values of 𝑃 < 0.05 were considered statistically significant.
2.3. Cell Viability Assay. The VSMCs (2 × 104 cells/well) were
seeded on 24-well plates and cultured in DMEM containing 3. Results
10% FBS for 24 h. The VSMCs were pretreated with NAC
(1 mM) before being treated with andrographolide (50 𝜇M) 3.1. The Role of ROS in Andrographolide-Reduced Cell Via-
for 48 h. The cell number was measured using a colorimetric bility in Rat VSMCs. We previously determined that andro-
assay based on the ability of mitochondria in viable cells to grapholide resulted in loss of cell viability in a concentration-
reduce MTT as previously described [21]. The cell number dependent manner by using an MTT assay (unpublished
index was calculated as the absorbance of treated cells/control data). However, the detailed mechanism involved in this
cells × 100%. phenomenon remains unclear. ROS formation is known to
play a crucial role in cell apoptosis [7]. Therefore, we inves-
tigated the role of ROS in andrographolide-induced VSMC
2.4. Measurement of Intracellular ROS. The VSMCs (5 × 105 death. Figure 2(a) shows that treatment with 50 𝜇M andro-
cells/dish) were loaded with DCF-DA (20 𝜇M) for 20 min grapholide significantly induced ROS formation 1.5 ± 0.0-
and then treated following the experimental design. These fold at 10 min compared with the control group (𝑃 < 0.001,
cells were washed with PBS before trypsinization. The levels 𝑛 = 4). We subsequently preincubated with NAC (1 mM)
of intracellular ROS were detected using Coulter Epics XL an ROS scavenger, in andrographolide-treated VSMCs. Fig-
flow cytometry (Beckman Coulter, Miami, FL, USA). Data ure 2(b) shows that treatment with andrographolide reduced
were collected from 10,000 cells per experimental group. the cell viability of rat VSMCs to 46.9 ± 6.6% compared with
All experiments were repeated at least 4 times to ensure the control group (𝑃 < 0.001, 𝑛 = 4), whereas pretreatment
reproducibility. with 1 mM NAC significantly reversed the andrographolide-
induced reduction in VSMC viability (𝑃 < 0.001, 𝑛 = 4).
2.5. Immunoblot Analysis. Immunoblot analyses were per- Taken together, these data suggest that the reduction of cell
formed as described previously [20]. Briefly, the VSMCs (5 × viability in andrographolide-treated rat VSMCs was related
105 cells/dish) were treated as the experimental design. After to the cellular redox state, possibly as a consequence of ROS
the experimental period, the proteins were extracted using a formation.
lysis buffer. Lysates were centrifuged, the supernatant protein
(50 𝜇g) was collected and subjected to sodium dodecyl 3.2. Nox-Mediated Redox Signaling in Andrographolide-
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Induced ROS Formation. Coronary artery restenosis, a fre-
the separated proteins were electrophoretically transferred quent complication of angioplasty, is accompanied by an
onto 0.45 𝜇m PVDF membranes by using semidry transfer increase in Nox-generated ROS production [22]. Therefore,
(Bio-Rad, Hercules, CA, USA). The blots were blocked with we investigated the involvement of Nox-mediated signaling
TBST (10 mM Tris-base, 100 mM NaCl, and 0.01% Tween 20) in andrographolide-induced ROS formation. In Figure 3(a),
containing 5% bovine serum albumin for 1 h and then probed pretreatment with NAC (1 mM) or DPI (10 𝜇M), a Nox
with various primary antibodies. The membranes were incu- inhibitor, significantly suppressed andrographolide-induced
bated with HRP-linked anti-mouse IgG or anti-rabbit IgG ROS formation. Furthermore, growing evidence has sug-
(diluted 1 : 3000 in TBST) for 1 h. The immunoreactive bands gested that the activation of the Nox subunit p47phox is
were detected using an ECL system. Bar graphs depict the required for ROS production in vascular cells [23]. We
ratios of quantitative results obtained by scanning the reactive subsequently determined whether Nox subunit activation is
bands and quantifying the optical density by using video den- required for andrographolide-induced ROS production in
sitometry (Bio-Profil; Biolight Windows application version rat VSMCs. As shown in Figure 3(b), serine359 phosphory-
2000.01; Vilber Lourmat, France). lation of the p47phox subunits was significantly increased
1.6 ± 0.1-fold after andrographolide stimulation for 10 min
2.6. Transfection and Luciferase Reporter Assays. The cells compared with the control group (𝑃 < 0.01, 𝑛 = 3).
were transfected with PG13-luc and Renilla-luc plasmids These results suggest that phosphorylation of the Nox subunit
using the Turbofect reagent. The treated and untreated p47phox mediates ROS formation in rat VSMCs treated with
cells were harvested, and the luciferase activity level was andrographolide.
determined using the Dual-Glo luciferase assay system kit.
The luciferase activity level was normalized based on the 3.3. Effects of ROS Scavengers on Andrographolide-Stimulated
Renilla luciferase activity level. The level of luciferase activity p53 Activation, Bax, and Active Caspase-3 Expression in Rat
was quantified as the ratio of the activity of cells treated with VSMCs. It is known that oxidative stress triggers the activa-
andrographolide to that of the untreated control cells. tion and nuclear translocation of p53 [24], and p53-induced
4 Evidence-Based Complementary and Alternative Medicine

1.6 ∗∗∗ 120

1.4 ∗∗ ###
ROS production (folds of control)

100

Cell viability (% of control)

1.2
80
1.0

0.8 60 ∗∗∗
0.6
40
0.4
20
0.2

0.0 0
Andro 0 5 10 20 Andro − + +
(min) NAC − − +
(a) (b)

Figure 2: The role of ROS in andrographolide-reduced cell viability in rat VSMCs. (a) Rat VSMCs were treated with 50 𝜇M andrographolide
for the indicated periods. Cells were harvested, and the formation of ROS was examined using flow cytometric analysis of DCF-DA-
stained cells, as described in Section 2. (b) Cells were pretreated with a vehicle or 1 mM NAC for 30 min before being treated with
50 𝜇M andrographolide for 48 h; cell viability was subsequently determined using an MTT assay. The results shown are representative of
4 independent experiments. The data are presented as the mean ± SEM (error bars: ∗∗ 𝑃 < 0.01 and ∗∗∗ 𝑃 < 0.001, compared with the control
group, and ### 𝑃 < 0.001, compared with the group treated only with andrographolide).

p-p47phox

𝛼-Tubulin
1.6
∗∗∗
2.0
p47phox phosphorylation (folds of control)

1.4 ∗
ROS production (folds of control)

∗∗ ∗∗
1.2
1.5
1.0
##
0.8
###
1.0
0.6

0.4
0.5
0.2

0.0
Andro − + + + 0.0
NAC − − + − Andro 0 10 20 30
DPI − − − + (min)
(a) (b)

Figure 3: Nox-mediated redox signaling in andrographolide-induced ROS formation. (a) Cells were pretreated with a vehicle, 1 mM NAC,
or 10 𝜇M DPI for 30 min before being treated with 50 𝜇M andrographolide for 10 min, and the production of ROS was examined using flow
cytometric analysis of DCF-DA-stained cells, as described in Section 2. (b) Cells were treated with 50 𝜇M andrographolide for the indicated
periods. Cells were harvested, and the phosphorylation of p47phox was examined using immunoblotting. The data are presented as the mean
± SEM (error bars: ∗ 𝑃 < 0.05, ∗∗ 𝑃 < 0.01, and ∗∗∗ 𝑃 < 0.001, compared with the control group, and ## 𝑃 < 0.01 and ### 𝑃 < 0.001, compared
with the group treated only with andrographolide).
Evidence-Based Complementary and Alternative Medicine 5

Bax

𝛼-Tubulin

5 ∗∗ 2.0
∗∗∗
PG13-luc/renilla-luc (folds of control)

Bax expression (folds of control)

4
1.5 ###

3 #
# 1.0
2 ###

0.5
1

0 0.0
Andro − + + + Andro − + + +
NAC − − + − NAC − − + −
DPI − − − + DPI − − − +
(a) (b)

Active caspase-3

𝛼-Tubulin
Active caspase-3 expression (folds of control)

∗∗∗
3

#
2 #

0
Andro − + + +
NAC − − + −
DPI − − − +
(c)

Figure 4: Effects of ROS scavengers on andrographolide-stimulated p53 activation, Bax, and active caspase-3 expression in rat VSMCs. (a)
Cells were transiently transfected with PG-13-luc and Renilla-luc for 48 h. After transfection, the cells were pretreated with a vehicle, 1 mM
NAC, or 10 𝜇M DPI for 30 min before being treated with 50 𝜇M andrographolide for another 24 h. A PG13-luciferase assay was subsequently
conducted. Cells were pretreated with a vehicle, 1 mM NAC, or 10 𝜇M DPI for 30 min before being treated with 50 𝜇M andrographolide for
48 hr, and the expression of Bax (b) and active caspase-3 (c) was examined using immunoblotting. The data are presented as the mean ± SEM
(error bars: ∗∗ 𝑃 < 0.01 and ∗∗∗ 𝑃 < 0.001, compared with the control group, and # 𝑃 < 0.05, ## 𝑃 < 0.01, and ### 𝑃 < 0.001, compared with the
group treated only with andrographolide).

apoptosis involves the generation of ROS [25]. Therefore, whether p53 transactivation increases in cells exposed to
we used a PGl3-Luc reporter construct that contained a p53 andrographolide. As shown in Figure 4(a), cells treated
DNA-binding site linked to a basal promoter that controls with 50 𝜇M andrographolide for 24 h exhibited a 4.2 ± 0.7-
the expression of a luciferase reporter gene [26] to examine fold increase in PG13-luciferase activity level compared with
6 Evidence-Based Complementary and Alternative Medicine

the control group (𝑃 < 0.01, 𝑛 = 5). Pretreating cells involved [30]. A variation in the balance between the prolif-
with NAC or DPI apparently inhibited the andrographolide- eration and apoptosis of VSMCs is considered to play a vital
induced increase in PG13-luciferase activity 68.8% and 65.6%, role in the development of atherosclerosis and cardiovascular
respectively (𝑛 = 5) (Figure 4(a)). diseases [31, 32]. Thus, maintaining the alteration between the
It has been suggested that the activation of p53 regulated proliferation and apoptosis of VSMCs has been proposed as
and promoted discrete steps of the apoptosis cascade such as an effective therapeutic method for preventing and treating
the upregulation of Bax genes [27] and the overexpression vascular diseases, including atherosclerosis [33]. Apoptosis
of Bax accelerates apoptotic death through interaction with (programmed cell death) in a wide range of physiological
components of the permeability transition pore complex, settings is to remove discarded cells [34]. Recent studies
causing the opening and rupture of its outer mitochondrial have indicated that andrographolide inhibits tumor growth
membrane [28]. As shown in Figure 4(b), treatment with by inducing cell cycle arrest [13, 14] or apoptosis [15, 16] in
50 𝜇M andrographolide for 48 h significantly induced Bax various types of cancer cells. In the present study, andro-
expression 1.7 ± 0.1-fold compared with the control group grapholide was also observed to induce apoptosis in rat
(𝑃 < 0.001, 𝑛 = 3), whereas pretreatment with NAC or DPI VSMCs, whereas no cytotoxic effect was observed (data not
significantly inhibited andrographolide-induced reductions show), suggesting that andrographolide may be a potential
in Bax expression 59% and 18%, respectively (𝑛 = 3). therapeutic agent in VSMC-proliferation-related diseases.
The expression levels of active caspase-3, an apoptotic- The net balance between proliferation, apoptosis, and
pathway-related proapoptotic protein, were subsequently necrosis determines the extent of cell growth. A growing body
determined in andrographolide-treated VSMCs. As shown of evidence now suggests that ROS play a role in both cellular
in Figure 4(c), treatment with andrographolide for 48 h necrosis and apoptosis [35]. Andrographolide was reported to
significantly increased the levels of active caspase-3 2.8 ± 0.3- induce ROS and caspase-dependent apoptosis in lymphoma
fold at the concentration of 50 𝜇M compared with the control cell lines and in primary tumor samples [36]. Therefore, we
group (𝑃 < 0.001, 𝑛 = 4), whereas pretreatment with hypothesized that andrographolide might cause apoptosis in
NAC or DPI significantly inhibited andrographolide-induced rat VSMCs through mechanisms that involve cellular redox
reductions in active caspase-3 expression by 43% and 29%, systems. We determined that the effects of andrographolide
respectively (𝑛 = 4). These results suggest that Nox-mediated were concentration related and accompanied by ROS gener-
redox signaling induces p53 activation as well as Bax and ation (Figures 2 and 3(a)).
active caspase-3 expression in andrographolide-treated rat The proapoptotic protein Bax is known to cause apop-
VSMCs. tosis by disrupting mitochondrial integrity [37]. Yang et al.
demonstrated that andrographolide induces the expression
of Bax, activates caspases, and stimulates apoptosis in lym-
3.4. The Role of Ceramide Signaling in Andrographolide-
phoma cells [36]. Activation of p53 is known to increase the
Induced p47phox Phosphorylation, Bax, and Active Caspase-3
expression of Bax in response to selected stress signals [38].
Expression in Rat VSMCs. The precise mechanism involved
A recent study observed that andrographolide can activate
in the andrographolide-induced phosphorylation of p47phox
p53 through ROS-dependent to TRAIL-induced apoptosis
in rat VSMCs remains unclear. A previous study reported
in cancer cells [39]. Among ROS, O2 − is highly reactive
ceramide to be a critical signaling molecule that mediates
and short lived and can spontaneously or enzymatically
the activation of Nox in various cells [29]. In addition, we
dismutate to a second signaling intermediate, H2 O2 , through
demonstrated that andrographolide can activate the nSMase-
superoxide dismutase. Although the production of O2 − con-
ceramide cascade in rat VSMCs, and andrographolide-
tributes to the primary biological activity of Nox, much of
induced ceramide formation was markedly attenuated by 3-
the signaling is mediated by the dismutation product H2 O2 .
OMS, an nSMase inhibitor [17]. As shown in Figure 5(a),
H2 O2 is more stable than O2 − and is capable of crossing
pretreatment with 3-OMS (30 𝜇M) for 30 min significantly
biological membranes and inducing nucleus DNA damage to
inhibited andrographolide-induced p47phox phosphoryla-
cause a p53-dependent pathway [40, 41]. In the present study,
tion 38% (𝑃 < 0.05, 𝑛 = 3). Pretreatment with 3-OMS also
andrographolide-induced p53 activation, Bax, and active
significantly diminished andrographolide-induced Bax and
caspase-3 expressions were significantly diminished by treat-
active caspase-3 expression in rat VSMCs (Figures 5(b) and
ment with NAC and DPI. These data indicate the involvement
5(c)).
of the ROS-mediated p53-Bax-caspase apoptotic pathway in
andrographolide-induced VSMC apoptosis (Figure 4).
4. Discussion Noxs are multiprotein complexes of various composi-
tions depending on the cell type. This enzyme, originally
VSMCs represent a moving component of the vasculature and described in phagocytes, consists of 2 membrane-bound
constitute the medial layer of blood vessels. VSMCs following subunits (p22phox and Nox2) and 3 cytosolic subunits, such
pathological stimuli can adopt a “de-differentiated” pheno- as p47phox, p67phox, and Rac1 (nonphagocytes) or Rac2
type or undergo hypertrophy and synthesize excess extra- (phagocytes), which are recruited upon activation to the
cellular matrix and inflammatory cytokines, which divide membrane-bound Nox/p22phox complex. VSMCs contain
and migrate toward the intima. The abnormal proliferation several sources of ROS, among which the Nox1 and Nox2 are
and reduced apoptosis can lead to excessive accumulation of predominant. Barry-Lane et al. have suggested that p47phox
VSMCs in the intima and media of atherosclerotic lesions is the only subunit that is used specifically by Nox2 and
Evidence-Based Complementary and Alternative Medicine 7

p-p47phox Bax

𝛼-Tubulin 𝛼-Tubulin

2.0 2.0
p47phox phosphorylation (folds of control)

∗
∗∗∗

Bax expression (folds of control)

1.5 1.5

# ##
1.0 1.0

0.5 0.5

0.0 0.0
Andro − + + Andro − + +
3-OMS − − + 3-OMS − − +
(a) (b)

Active caspase-3

𝛼-Tubulin

2.5
Active caspase-3 expression (folds of control)

∗∗

2.0

##
1.5

1.0

0.5

0.0
Andro − + +
3-OMS − − +
(c)

Figure 5: The role of ceramide signaling in andrographolide-induced p47phox phosphorylation, Bax, and active caspase-3 expression in rat
VSMCs. Cells were pretreated with a vehicle or 30 𝜇M 3-OMS for 30 min before being treated with 50 𝜇M andrographolide for 10 min (a), or
48 h (b and c). The extent of p47phox phosphorylation (a), Bax (b), or active caspase-3 expression (c) was examined. The data are presented
as the mean ± SEM (error bars: ∗ 𝑃 < 0.05, ∗∗ 𝑃 < 0.01, and ∗∗∗ 𝑃 < 0.001, compared with the control group, and ## 𝑃 < 0.01 and ### 𝑃 < 0.001,
compared with the group treated only with andrographolide).

by Nox1 expressed in VSMCs [42]. Furthermore, p47phox restored ROS formation and apoptosis-inducing activity in
for oxidase activation requires a phosphorylated serine at andrographolide-treated rat VSMCs, and the incubation of
position 359 that is absolutely required for oxidase activity andrographolide apparently increased the phosphorylation
and must be phosphorylated to allow translocation [43]. of p47phox, a Nox subunit. These data collectively indicate
A functional role for p47phox has also been shown using that Nox-mediated redox signaling plays a crucial role in rat
VSMCs from p47phox knockout mice, in which the agonist VSMCs treated with andrographolide. However, the precise
stimulation of ROS was reduced [44, 45]. In the present mechanism involved in the andrographolide-induced phos-
study, we observed that DPI, a Nox inhibitor, significantly phorylation of p47phox in rat VSMCs remains unclear.
8 Evidence-Based Complementary and Alternative Medicine

Andrographolide
− O2
O2

nSMase
H 2 O2 Ceramide SM

Nox
p
p47phox

H2 O 2 Bax
Mitochondria

Caspase-3

Bax

p53

Apoptosis

Nucleus

Figure 6: Hypothetical scheme of the signal pathways in andrographolide-induced rat VSMC apoptosis. Andrographolide stimulated the
ceramide-mediated signal events, resulting in the activation of the p47phox-ROS cascade, ultimately stimulating active caspase-3 expression
and VSMC apoptosis. Nox produces superoxide (O2 − ), followed by the induction of H2 O2 . H2 O2 is capable of inducing DNA damage to cause
p53 activation, which can lead Bax and active caspase-3 expression. nSMase: neutral sphingomyelinase; SM: sphingomyelinase; Nox: NADPH
oxidase.

Ceramide, the central core lipid in the metabolism phenomenon was markedly attenuated by 3-OMS [17]. In
of sphingolipids, is produced through hydrolysis of com- the present study, we observed that 3-OMS apparently abol-
plex sphingolipids, such as sphingomyelin, by mammalian ished andrographolide-induced p47phox phosphorylation,
SMases or through the acylation of a long-chain sphingoid Bax, and active caspase-3 expression in rat VSMCs (Figure 5).
base (sphingosine) in a de novo biosynthetic pathway. The Taken together, these results indicate that andrographolide
SMases and its role in ceramide metabolism are the most increases the turnover of sphingomyelin in rat VSMCs, and
extensively studied. Recent studies have demonstrated that this lipid-signaling pathway may mediate the action of the
ceramide increased in endothelial cells exposed to death andrographolide-induced activation of Nox, resulting in O2 −
factors, including tumor necrosis factor 𝛼, interleukin 2, and production and apoptosis-inducing activity in rat VSMCs.
endostatin, and in ischemic reperfused myocardium [46, In the present study, we showed that andrographolide,
47]. In these studies, the ceramide signaling pathway has the active component of the plant A. paniculata, has the
been confirmed to be involved in the activation of Nox and ability to reduce cell viability in rat VSMCs. The in-depth
consequent O2 − production [48, 49]. Ceramide also enhances mechanism of its apoptosis-inducing activity is related to the
ROS formation in mammalian cells by straightly raising the Nox-mediated redox signaling of cells, because this signaling
permeability of mitochondrial membranes to cytochrome c is blocked by NAC and DPI. In addition, this is the first
[50] and restraining the isolated mitochondria complex III study to indicate the role of the ceramide-p47phox signal-
[51]. In addition, the accumulation of ceramides caused by the ing pathway in andrographolide-induced ROS-mediated cell
activation of SMases has been observed in response to various apoptosis (Figure 6). In conclusion, we showed that the
stimuli, such as oxidants and heat stress [52, 53]. On the ceramide-p47phox-ROS signaling cascade may contribute
other hand, our previous study found that andrographolide to andrographolide-induced VSMC apoptosis. Using this
can directly enhance ceramide level in VSMCs, and this novel natural lactone diterpenoid as a therapeutic strategy
Evidence-Based Complementary and Alternative Medicine 9

for cardiovascular disorders involving VSMC proliferation Journal of Clinical Pharmacy and Therapeutics, vol. 29, no. 1, pp.
and atherogenesis warrants further preclinical and clinical 37–45, 2004.
investigation. [13] H.-Y. Cheung, S.-H. Cheung, J. Li et al., “Andrographolide
isolated from Andrographis paniculata induces cell cycle arrest
and mitochondrial-mediated apoptosis in human leukemic HL-
Conflict of Interests 60 cells,” Planta Medica, vol. 71, no. 12, pp. 1106–1111, 2005.
The authors declare no conflict of interests. [14] J. Li, H.-Y. Cheung, Z. Zhang, G. K. L. Chan, and W.-F. Fong,
“Andrographolide induces cell cycle arrest at G2/M phase and
cell death in HepG2 cells via alteration of reactive oxygen
Acknowledgments species,” European Journal of Pharmacology, vol. 568, no. 1–3,
pp. 31–44, 2007.
This work was supported by Grants from the National Science [15] J. Zhou, S. Zhang, O. Choon-Nam, and H.-M. Shen,
Council, Taiwan (NSC97-2320-B-038-016-MY3 and NSC100- “Critical role of pro-apoptotic Bcl-2 family members in
2320-B-038-021-MY3), and Taipei Medical University (TMU- andrographolide-induced apoptosis in human cancer cells,”
R-100-03 and TMU102-AE1-B20). Biochemical Pharmacology, vol. 72, no. 2, pp. 132–144, 2006.
[16] M. T. Cheung, R. Ramalingam, K. K. Lau, W. L. Chiang, and
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Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2013, Article ID 354840, 9 pages
http://dx.doi.org/10.1155/2013/354840

Research Article
Evodiamine Induces Transient Receptor
Potential Vanilloid-1-Mediated Protective Autophagy
in U87-MG Astrocytes

Ann-Jeng Liu,1,2 Sheng-Hao Wang,3 Sz-Ying Hou,3,4 Chien-Ju Lin,3 Wen-Ta Chiu,1,5
Sheng-Huang Hsiao,2 Thay-Hsiung Chen,6,7 and Chwen-Ming Shih3,4,8
1
Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan
2
Department of Neurosurgery, Taipei City Hospital Ren-Ai Branch, Taipei, Taiwan
3
Department of Biochemistry, School of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan
4
Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan
5
Department of Neurosurgery, Taipei Municipal Wan-Fang Hospital, Taipei, Taiwan
6
Department of Surgery, College of Medicine, Taipei Medical University, Taiwan
7
Division of Cardiac Surgery, Cathy General Hospital, Taipei, Taiwan
8
Traditional Herbal Medicine Research Center, Taipei Medical University Hospital, Taipei, Taiwan

Correspondence should be addressed to Thay-Hsiung Chen; [email protected] and Chwen-Ming Shih; [email protected]

Received 21 October 2013; Accepted 23 November 2013

Academic Editor: Joen-Rong Sheu

Copyright © 2013 Ann-Jeng Liu et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Cerebral ischemia is a leading cause of mortality and morbidity worldwide, which results in cognitive and motor dysfunction,
neurodegenerative diseases, and death. Evodiamine (Evo) is extracted from Evodia rutaecarpa Bentham, a plant widely used in
Chinese herbal medicine, which possesses variable biological abilities, such as anticancer, anti-inflammation, antiobesity, anti-
Alzheimer’s disease, antimetastatic, antianoxic, and antinociceptive functions. But the effect of Evo on ischemic stroke is unclear.
Increasing data suggest that activation of autophagy, an adaptive response to environmental stresses, could protect neurons
from ischemia-induced cell death. In this study, we found that Evo induced autophagy in U87-MG astrocytes. A scavenger of
extracellular calcium and an antagonist of transient receptor potential vanilloid-1 (TRPV-1) decreased the percentage of autophagy
accompanied by an increase in apoptosis, suggesting that Evo may induce calcium-mediated protective autophagy resulting from
an influx of extracellular calcium. The same phenomena were also confirmed by a small interfering RNA technique to knock down
the expression of TRPV1. Finally, Evo-induced c-Jun N-terminal kinases (JNK) activation was reduced by a TRPV1 antagonist,
indicating that Evo-induced autophagy may occur through a calcium/c-Jun N-terminal kinase (JNK) pathway. Collectively, Evo
induced an influx of extracellular calcium, which led to JNK-mediated protective autophagy, and this provides a new option for
ischemic stroke treatment.

1. Introduction that protective autophagy is induced in a stroke mode since

blockade of autophagy results in an increase in cell death
Brain ischemia, a restriction of the blood supply to tissues, [1] suggests an agent possessing autophagy-inducing ability
causing a shortage of oxygen and glucose needed for cellular which may provide a new option for ischemic stroke treat-
metabolism, is the leading cause of death and disability ment.
worldwide. Tissue plasminogen activator (tPA) therapy is the Evodiamine (Evo) is a quinozole alkaloid isolated from
major treatment for ischemic stroke. However, the window Evodia rutaecarpa Bentham which is widely used in Chinese
for tPA treatment is within 0∼3 h after onset of a stroke, which herbal medicine with variable effects, such as anticancer,
limits its clinical use. Accumulating evidence demonstrating anti-inflammation, anti-obesity, anti-Alzheimer’s disease,
2 Evidence-Based Complementary and Alternative Medicine

antimetastatic, antianoxic, and anti-nociceptive functions 2. Materials and Methods

[2]. An in vitro study showed that Evo has an endothelium-
dependent vasodilatory effect on isolated rat mesenteric 2.1. Cell Culture, Treatment, and Chemicals. Human U87-MG
arteries [3]. Furthermore, Evo may serve as an antiatheroscle- astrocytes (obtained from a Caucasian strain) classified as
rosis agent since it can inhibit oxidative stress-induced grade IV glioblastoma were purchased from the American
production of chemokine receptor (CCR)-1, CCR2, and intra- Type Culture Collection (Manassas, VA) and grown at 37∘ C
cellular adhesion molecule (ICAM)-1 [4, 5], suggesting that in culture medium consisting of Dulbecco’s modified Eagle’s
Evo may possess anticardiovascular disease activity. It was medium (DMEM) supplemented with 10% heat-inactivated
reported that Evo protected rats from myocardial ischemia- fetal bovine serum (FBS), 200 mM L-glutamine, 100 U/mL
reperfusion injury [6]. However, the effect of Evo on ischemic penicillin, 100 𝜇g/mL streptomycin, 100 mM sodium pyru-
stroke in the brain is not well understood. vate, and 1% nonessential amino acids. The mixture was
Evo is considered a transient receptor potential vanilloid- kept in a humidified atmosphere containing 5% CO2 . For
1 (TRPV1) agonist [7]. TRPV1, a ligand-gated calcium ion the drug-response experiments, cells were either pretreated
channel activated by vanilloids, protons, and various environ- (treatment group) or not (control group) with the indicated
mental stresses, was implicated as a pain-sensing transducer inhibitor for 1 h and then incubated with 6 𝜇M Evo for the rest
and plays a key role in regulating cell death. Capsaicin, a of the experimental period [16]. DMEM, FBS, and nonessen-
well-known TRPV1 agonist, extracted from hot chili peppers, tial amino acids were purchased from Hyclone (Logan, UT),
was shown to lead to human nasopharyngeal carcinoma and phenol red-free RPMI, L-glutamine, penicillin-strepto-
cell death through mitochondrial and endoplasmic reticular my-cin, and sodium pyruvate were obtained from Gibco
(ER) stress [8]. Furthermore, capsaicin induced apoptosis (Grand Island, NY). Evo, bovine serum albumin (BSA),
in glioma cells through a p38-mediated signal pathway [9]. acridine orange (AO), Fluo-3 AM, 5,5,6,6,-tetrachloro-
However, capsazepine (CPZ), a TRPV1 antagonist potenti- 1,1,3,3,-tetraethylbenzimidazolylcarbocyanine iodide (JC-1),
ated the anticancer effect of tumor necrosis factor-related ethylene glycol tetra-acetic acid (EGTA), CPZ, and 3-
apoptosis-inducing ligand (TRAIL) through upregulation (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
of death receptors [10]. Therefore, the role of TRPV1 in (MTT) were purchased from Sigma (St. Louis, MO). Pro-
regulating apoptosis is controversial, and the effect of TRPV1 pidium iodide (PI) was purchased from Calbiochem (San
on autophagy needs to be investigated. Diego, CA). The Annexin-V-FITC reagent was supplied by
Autophagy, an evolutionarily conserved mechanism reg- Biovision (Mountain View, CA). Rabbit polyclonal anti-LC3
ulating the turnover of long-lived proteins and damaged was obtained from MBL International (Nagoya, Japan). Rab-
organelles, is also considered type II programmed cell death. bit polyclonal anti-GAPDH and anti-TRPV1 were obtained
Autophagy is characterized by the formation of double- from Cell Signaling (Beverly, MA). Secondary anti-bodies,
membrane vesicles (autophagosomes) and the processing including horseradish peroxidase- (HRP-) conjugated goat
of microtubule-associated protein 1 light chain 3 (LC3). anti-rabbit immunoglobulin G (IgG), were purchased from
Dysfunction of autophagy may lead to cancer development, Pierce (Rockford, IL). Polyvinylidene difluoride (PVDF)
bacterial and viral infections, neurodegenerative disorders, membranes were supplied by Millipore (Bedford, MA), and
and cardiovascular diseases [11, 12]. The role of autophagy the Protein Assay Dye Reagent was from Bio-Rad (Hercules,
in ischemia has been widely investigated. Rapamycin, an CA).
autophagy inducer, protected against hypoxia-induced brain
injury through induction of autophagy [13]. Superoxide
dismutase (SOD)-2 knockdown led to ischemic brain dam- 2.2. Measurement of Acidic Vesicular Organelles (AVOs).
age under a hyperglycemic condition through induction of Autophagy was analyzed by flow cytometry with AO dye
oxidative stress, which was associated with a reduction in according to published procedures [17]. After the indicated
autophagy [14]. Furthermore, melatonin-induced neuronal treatment, cells were stained with AO (1 𝜇g/mL) for a period
protection against cell death resulting from glucose-oxygen of 20 min. Trypsinized adherent cells and cells suspended
deprivation was abolished by the autophagy inhibitor, 3- in the medium were collected in phenol red-free RPMI
methyladenine (3-MA) [15]. All of the above suggest that medium. Green (510∼530 nm) and red (650 nm) fluorescence
autophagy may play a protective role in ischemia-induced emissions from 104 cells illuminated with blue (488 nm)
neuronal damage. excitation light were measured on a flow cytometer using
Human U87-MG astrocytes (obtained from a Caucasian CellQuest software (Becton Dickinson, San Jose, CA). The
strain) classified as grade IV glioblastoma were employed percentage of autophagy was summed across the upper-left
as a cell model in this study. We found that Evo induced and upper-right quadrants.
protective autophagy of astrocytes. Treatment with a TRPV1
antagonist was able to increase intracellular calcium level and 2.3. Measurement of Apoptosis. Apoptosis was evaluated by
block Evo-induced c-Jun N-terminal kinase (JNK) activation flow cytometry using two-color analysis of FITC-labeled
which results in suppression of autophagy. Combined with annexin V/PI doublestaining, as described in our previous
our previous report [16], we demonstrated that Evo induced publication [18]. Trypsinized adherent cells and suspended
an increase in intracellular calcium resulting from an influx cells in the medium were collected in HEPES buffer con-
of extracellular calcium through the TRPV1 channel, which taining 10 mM HEPES (pH 7.4), 140 mM NaCl, and 2.5 mM
led to JNK-mediated protective autophagy. CaCl2 . Subsequently, cells were stained with annexin V
Evidence-Based Complementary and Alternative Medicine 3

(1 𝜇g/mL) and PI (0.2 ng/mL) for 15 min and then ana- a hallmark of autophagy. As demonstrated in Figure 1(a), the
lyzed on a flow cytometer using CellQuest software (Becton protein level of LC3-II increased after treatment with Evo for
Dickinson). The percentage of total apoptosis was the sum 16 h. Furthermore, flow cytometry with AO dye and annexin
of primary apoptosis (annexin V+/PI−) and late apoptosis V/PI double dye was, respectively, applied to determine levels
(annexin V+/PI+). of autophagy and apoptosis. As shown in Figure 1(b), the
percentage of cells containing autophagosomes increased and
2.4. Measurement of Intracellular Calcium. After treatment, reached a maximum of 52% at 24 h. But the percentage of cells
astrocytes were harvested and incubated with 500 nM Fluo- undergoing apoptosis (indicated in the upper-right quadrant)
3 AM dye for 30 min at 37∘ C and then were immediately increased in a dose-dependent manner and reached 27% after
analyzed on a flow cytometer (530 nm) using FL-1 as a 48 h of treatment with Evo (Figure 1(c)).
detector. Relative intracellular calcium concentrations were Our previous report demonstrated that Evo induced
calculated from the ratio of the geometric mean values of release of calcium from the ER, which led to induction of
the FL-1 peak generated from Evo-treated cells against each
autophagy and apoptosis. However, the role of extracellular
respective control, as indicated in the figure legends.
calcium in Evo-induced autophagy is unclear. In this study,
a scavenger of extracellular calcium (EGTA) was used to
2.5. Western Blot Analysis. Adherent cells and suspended detect the involvement of extracellular calcium. Using flow
cells were collected and lysed with 50 𝜇L of lysis buffer cytometry with Fluo-3 AM dye, we investigated the varia-
containing 25 mM HEPES, 1.5% Triton X-100, 0.1% sodium
tion in intracellular calcium. As shown in Figure 2(a), Evo
dodecyl sulfate (SDS), 0.5 M NaCl, 5 mM EDTA, 0.1 mM
induced an elevation in intracellular calcium at 8 h, which
sodium deoxycholate, and a protease inhibitor cocktail
increased 3.1-fold compared to the control. Cells pretreated
(Roche, Boehringer Mannheim, Germany) [19]. We then
added sampling buffer (60 mM Tris-HCl at pH 6.8, 2% SDS, with EGTA were resistant to Evo, suggesting that an influx
10% glycerol, and 140 mM 𝛽-mercaptoethanol) to each lysate, of extracellular calcium was involved in the Evo-induced
and the mixture was sonicated using Microson Ultrasonic increase in intracellular calcium. Furthermore, EGTA also
Cell Disruptor (Misonix Inc, Farmingdale, NY), subsequently reduced Evo-induced autophagy accompanied by an increase
boiled for 7 min, and centrifuged at 15,000 g for 5 min. in apoptosis (Figures 2(b) and 2(c)). These results indicated
The amount of protein was determined using the Bio- that Evo induced elevation of intracellular calcium resulting
Rad Protein Assay Dye Reagent. Proteins electrotransferred from an influx of extracellular calcium, which led to the
onto PVDF membranes were immunoblotted with anti-LC3, formation of protective autophagy against apoptosis.
anti-GAPDH, and anti-TRPV1 anti-bodies. Detection was
performed with appropriate HRP-conjugated secondary anti- 3.2. Evo Induces TRPV1-Mediated Autophagy. TRPV1 is a
bodies and enhanced chemiluminescence reagent (Pierce). ligand-gated calcium ion channel, which plays a key role in
The band density was quantified by Gel-Pro Analyzer den-
regulating cell death. It was reported that Evo can activate
sitometry software (Media Cybernetics, Silver Spring, MD).
the TRPV1 channel, but the effect of TRPV1 on autophagy
is unclear. In this study, we investigated the role of TRPV1
2.6. Knockdown of TRPV1. Astrocytes were transfected with in Evo-induced autophagy. As indicated in Figure 3(a), the
50 nM of small interfering (si)RNA using the Lipofectamine Evo-induced increase in intracellular calcium was reduced
RNAiMAX reagent (Invitrogen, San Diego, CA) according to
by treatment with a TRPV1 antagonist (CPZ) in a dose-
the manufacturer’s instructions. In brief, cells were incubated
dependent manner. Furthermore, the percentage of cells that
with lipofectamine RNAimax reagent and 50 nM TRPV1
underwent autophagy was reduced in CPZ-treated astrocytes
siRNA (Santa Cruz Biotechnology, Santa Cruz, CA) for 6 h,
and then the medium was refreshed, followed by incubation (Figure 3(b)). In contrast, Evo-induced apoptosis was further
for a further 42 h. Transfected cells were treated with Evo for enhanced by treatment with CPZ at 48 h (Figure 3(c)),
the indicated time and analyzed by flow cytometry with AO suggesting that Evo-induced calcium-dependent protective
staining or annexin V/PI double-staining. autophagy may have been due to activation of the TRPV1
channel.
2.7. Statistical Analysis. Values are expressed as the mean ± To further confirm the role of TRPV1 in Evo-induced
standard deviation (SD). Statistical analysis was performed autophagy, cells were treated with siRNA against the TRPV1
using Student’s 𝑡-test (for two groups) or a one-way analysis of coding sequence. Using immunoblotting, we observed that
variance (ANOVA) followed by Duncan’s multiple-range test levels of the TRVP1 protein were significantly reduced after
(for three or more groups). 𝑃 values of <0.05 were considered being transfected with TRPV1 siRNA (Figure 4(a)). TRPV1
statistically significant. knockdown resulted in reductions in levels of intracellular
calcium and autophagy after treatment with Evo (Figures
3. Results 4(b) and 4(c)). However, the percentage of apoptosis further
increased in cells with TRPV1 knockdown after treatment
3.1. Evo Triggers Calcium-Mediated Protective Autophagy in with Evo (Figure 4(d)). Collectively, these findings suggest
Astrocytes. To investigate the effect of Evo in astrocytes, that Evo-induced protective autophagy in astrocytes is medi-
immunoblotting was applied to detect variations in LC3, ated by the TRPV1 signaling pathway.
4 Evidence-Based Complementary and Alternative Medicine

Control 24 h 48 h
0.6 50.8 31.5

FL-3
FL-1

60
b
50

40

Autophagy (%)
c
30

Time (h) 0 4 8 16 24 48 20
LC3-I
LC3-II 10
a
GAPDH 0
(a) (b)
Control 24 h 48 h
3.0 15.7 32.7
FL-2

FL-1

35

30 c

25
Apoptosis (%)

20
b
15

10

5 a

0
(c)

Figure 1: Evodiamine (Evo) induces autophagy and apoptosis in astrocytes. Cells were treated with 6 𝜇M of Evo for the indicated time and then
analyzed by Western blotting with anti-LC3 and anti-GAPDH anti-bodies. GAPDH was used as an internal control to normalize the amount
of proteins applied in each lane (a). Cells were treated with 6 𝜇M of Evo for 24 or 48 h, using flow cytometry with acridine orange staining for
autophagy (b). Data presented in the upper panel represent the results of three independent experiments, and the respective statistical results
are presented in lower panels of (b). (c) Cells were treated with 6 𝜇M Evo for the indicated time periods and then analyzed by flow cytometry
with annexin-V/PI double-staining for apoptosis. Data presented in the upper panel represent the results of three independent experiments,
and the respective statistical results are presented in lower panels of B. One-way ANOVA followed by Duncan’s multiple-range test was used
to determine whether the results for the experimental groups significantly differed from those of the respective controls. Columns not sharing
the same superscript significantly differ (𝑃 < 0.05).

3.3. Evo Induces TRPV1-Mediated JNK Activation. In our pre- activation resulted from an increase in intracellular calcium
vious report, we found that Evo could induce JNK-mediated released from the ER. However, the role of extracellular
protective autophagy since blockade of JNK resulted in an calcium was not mentioned. In this study, using a TRPV1
increase in apoptosis [16]. Furthermore, Evo-induced JNK antagonist, we examined the role of TRPV1 in Evo-induced
Evidence-Based Complementary and Alternative Medicine 5

5
60

4
50 ∗∗∗
Relative [Ca2+ ]i

3 40

Autophagy (%)
∗∗
30
2
20
1
10

0 0
Evo − + Evo − + − +
24 h 48 h
Control Control
EGTA EGTA
(a) (b)
70

60 ∗∗

50
Apoptosis (%)

40

30
∗∗
20

10

0
Evo − + − +
24 h 48 h
Control
EGTA
(c)

Figure 2: Evodiamine (Evo) induces calcium-mediated protective autophagy in astrocytes. (a) Cells were pretreated with EGTA, a scavenger
of extracellular calcium, for 1 h, and then incubated with Evo for a further 8 h. Levels of intracellular calcium were measured by flow cytometry
with Fluo-3 AM dye. The effects of EGTA on autophagy (b) and apoptosis (c) were evaluated by flow cytometry using acridine orange staining
and annexin V/PI dye, respectively. ∗∗ 𝑃 < 0.01, ∗∗∗ 𝑃 < 0.001 compared to the respective control, by Student’s 𝑡-test.

JNK activation. As demonstrated in Figure 5, Evo-induced an increase in apoptosis. An inhibitor of TRPV1 also sup-
JNK activation was reduced after treatment with CPZ, indi- pressed Evo-induced autophagy and intracellular calcium
cating that Evo-induced JNK activation was affected by both elevation accompanied by an increase in apoptosis, sug-
calcium released from the ER and an influx of extracellular gesting that Evo-induced protective autophagy may have
calcium. resulted from extracellular calcium influx through the TRPV1
channel. The same phenomena were also confirmed using
4. Discussion an siRNA technique against TRPV1. Finally, inhibition of
TRPV1 by treatment with CPZ decreased Evo-induced JNK
Ischemic stroke, a leading cause of mortality and morbidity activation. Collectively, Evo induced an influx of extracellular
worldwide, leads to severe cognitive and motor dysfunc- calcium through the TRPV1 channel, which subsequently led
tion, neurodegenerative diseases, and death [20]. The only to JNK activation and induction of protective autophagy.
therapy for acute cerebral ischemia is tPA treatment within TRPV1 is expressed by astrocytes, and Evo induced a
a 1∼3 h time window after onset of a stroke. Therefore, TRPV1-dependent increase in intracellular calcium, which
identifying novel therapeutic targets is a challenge in this was responsible for JNK-mediated protective autophagy.
field. In this study, we found that Evo induced protective Consistent with our results, capsaicin induced reactive oxy-
autophagy in astrocytes. Furthermore, a scavenger of extra- gen species (ROS)/AMPK-mediated protective autophagy
cellular calcium resulted in a reduction in autophagy and in thymocytes [21]. Furthermore, capsaicin also enhanced
6 Evidence-Based Complementary and Alternative Medicine

3.0
60
a
2.5 a
50
2.0
Relative [Ca2+ ]i

40 b

Autophagy (%)
b
a
1.5 c b
30

1.0 c b
20

0.5 10

0.0 0
Evo − + − + − +
Evo
24 h 48 h

Control Control
CPZ 3 𝜇m CPZ 3 𝜇m
CPZ 6 𝜇m CPZ 6 𝜇m
(a) (b)
40
c
b
30
Apoptosis (%)

a
20

10

0
Evo − +

Control
CPZ 3 𝜇m
CPZ 6 𝜇m
(c)

Figure 3: Evodiamine (Evo) induces TRPV1-dependent protective autophagy in astrocytes. (a) Cells were pretreated with capsazepine (CPZ),
a TRPV1 antagonist, for 1 h, and then incubated with Evo for a further 8 h. Levels of intracellular calcium were measured by flow cytometry
with Fluo-3 AM dye. (b) The effects of CPZ on autophagy were evaluated by flow cytometry using acridine orange staining. (c) Cells pretreated
with CPZ were incubated with Evo for 48 h. The percentage of apoptosis was determined using annexin V/PI double staining on a flow
cytometer. One-way ANOVA followed by Duncan’s multiple-range test was used to determine whether the results for the experimental groups
significantly differed from those of the respective controls. Columns not sharing the same superscript significantly differ (𝑃 < 0.05).

hepatic PPAR𝛿 and autophagy-related proteins to reduce apoptosis [16], indicating that Evo-induced apoptosis may
hepatic inflammatory factor in wild-type but not TRPV1−/− have been due to an imbalance in ER resulting from opening
mice [22], suggesting that TRPV1 activation may induce of a calcium channel, but not TRPV1 activation.
survival autophagy. However, it was reported that capsaicin Autophagy was found in neurons and glial cells in an
induced cell cycle arrest and apoptosis in urothelial bladder ischemia-mimicking animal model [24], suggesting that
cancer cells through TRPV1 activation [23]. In this report, glial cells may serve as one of the damage targets in stroke.
we observed that Evo induced apoptosis in a time-dependent 3-MA was shown to possess the ability to reduce the stroke-
manner, which was not reduced by treatment with CPZ. induced infarct volume, brain edema, and motor deficits
Moreover, blockade of calcium release from the ER by treat- [25, 26]. Furthermore, postischemic intracerebroventricular
ment with an IP3 R channel inhibitor reduced Evo-induced injections of 3-MA reduced the lesion volume after initiation
Evidence-Based Complementary and Alternative Medicine 7

3.5

3.0

2.5 ∗∗∗

Relative [Ca2+ ]i
2.0

1.5

1.0
TRPV1
0.5
GAPDH
0.0
siRNA Control TRPV1 Evo − +

Control siRNA
TRPV1 siRNA
(a) (b)
60 35
∗∗
50 30

25
40
Autophagy (%)

Apoptosis (%)
∗∗∗
20
30
15
20
10
10 5

0 0
Evo − + − + − + − +
Evo
24 h 48 h 24 h 48 h

Control siRNA Control siRNA

TRPV1 siRNA TRPV1 siRNA
(c) (d)

Figure 4: Reduction of evodiamine-(Evo)-induced protective autophagy by silencing the TRPV1 gene. (a) Cells were transfected with control
or TRPV1 siRNA using the lipofectamine RNAimax reagent for 48 h, and the level of TRPV1 protein was measured by an immunoblot assay
using anti-TRPV1 and anti-GAPDH anti-bodies to monitor the efficiency of the siRNA. GAPDH was used as an internal control to normalize
the amount of proteins applied in each lane. (b) The elevation of cytosolic calcium induced by Evo was reduced by knockdown of TRPV1.
Cells with or without knockdown of TRPV1 were treated with Evo for 8 h and then analyzed by Fluo-3 AM staining using flow cytometry.
After transfection, cells were treated with Evo for another 24 or 48 h, trypsinized, and collected to determine percentages of autophagy (c) and
apoptosis (d), respectively, using acridine orange staining and annexin V/PI staining. ∗∗ 𝑃 < 0.01, ∗∗∗ 𝑃 < 0.001 compared to the respective
control, by Student’s 𝑡-test.

of ischemia [27]. However, rapamycin and a GSK-3𝛽 inhibi- may be a possible target for application in treating acute
tor both activated autophagy to reduce ischemia-induced cerebral ischemia, but the detailed molecular mechanism and
neuroinflammation and necrosis of neurons [13, 28]. An its significance in the clinic remain to be further investigated.
autophagy inhibitor reduced melatonin-mediated neuropro- Disturbance of intracellular calcium levels was shown to
tection [15], suggesting that autophagy may play a survival be involved in regulating cell death. Our previous reports
role in response to ischemia-induced environmental stress. demonstrated that Evo could induce the release of calcium
Therefore, the role of autophagy in stroke is controversial. The from the ER, which led to autophagy and apoptosis, since
reasons for this difference may have been due to the chemical blockade of the calcium channel of ER resulted in reductions
agents used, such as 3-MA and rapamycin, both of which in apoptosis and autophagy [16]. In this study, we found
are nonspecific inhibitors of autophagy. Thus, the effects that a scavenger of extracellular calcium and an inhibitor
resulting from blockade of autophagy using these agents must of TRPV1 both reduced Evo-induced intracellular calcium
be carefully considered. In this study, we found that a natural elevation and autophagy but increased the levels of apop-
compound, Evo, could activate JNK-mediated protective tosis, suggesting that Evo-induced elevation of intracellular
autophagy in astrocytes, suggesting that activation of TRPV1 calcium may be due to release of calcium by ER and
8 Evidence-Based Complementary and Alternative Medicine

Relative folds 1.0 0.8 0.5 4.9 2.4 2.2 [4] W.-Q. Rang, Y.-H. Du, C.-P. Hu et al., “Protective effects of
p-JNK1 evodiamine on myocardial ischemia-reperfusion injury in rats,”
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[5] C.-P. Hu, L. Xiao, H.-W. Deng, and Y.-J. Li, “The cardiopro-
t-JNK1
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tion. Cells treated with or without capsazepine (CPZ) were incu- “Evodiamine and rutaecarpine inhibit migration by LIGHT via
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[8] S. W. Ip, S. H. Lan, H. F. Lu et al., “Capsaicin mediates
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in intracellular calcium led to the induction of autophagy, wal, “Capsazepine, a TRPV1 antagonist, sensitizes colorectal
which may be an adaptive response to protect astrocytes from cancer cells to apoptosis by TRAIL through ROS-JNK-CHOP-
mediated upregulation of death receptors,” Free Radical Biology
cell death resulting from ER stress.
and Medicine, vol. 53, no. 10, pp. 1977–1987, 2012.
Currently, there is no effective therapy for cerebral ische- [11] T. Shintani and D. J. Klionsky, “Autophagy in health and disease:
mia, which causes cognitive and motor dysfunction, neu- a double-edged sword,” Science, vol. 306, no. 5698, pp. 990–995,
rodegenerative diseases, and even acute death. In this study, 2004.
we found that Evo, extracted from E. rutaecarpa, has the [12] M. M. Hippert, P. S. O’Toole, and A. Thorburn, “Autophagy in
ability to induce survival autophagy in astrocytes through cancer: good, bad, or both?” Cancer Research, vol. 66, no. 19, pp.
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option for ischemia treatment. [13] S. Carloni, G. Buonocore, and W. Balduini, “Protective role of
autophagy in neonatal hypoxia-ischemia induced brain injury,”
Neurobiology of Disease, vol. 32, no. 3, pp. 329–339, 2008.
Conflict of Interests [14] S. L. Mehta, Y. Lin, W. Chen et al., “Manganese superoxide
dismutase deficiency exacerbates ischemic brain damage under
The authors declare that there is no conflict of interests hyperglycemic conditions by altering autophagy,” Translational
regarding the publication of this paper. Stroke Research, vol. 2, no. 1, pp. 42–50, 2011.
[15] Y. Guo, J. Wang, Z. Wang, Y. Yang, X. Wang, and Q. Duan,
“Melatonin protects N2a against ischemia/reperfusion injury
Acknowledgments through autophagy enhancement,” Journal of Huazhong Univer-
sity of Science and Technology, vol. 30, no. 1, pp. 1–7, 2010.
This study was sponsored by Grants from the National [16] A. J. Liu, S. H. Wang, K. C. Chen et al., “Evodiamine, a
Science Council (NSC99-2320-B08-008-MY3) and Cathy plant alkaloid, induces calcium/JNK-mediated autophagy and
General Hospital (101CGH-TMU-05), Taipei, Taiwan, Chwen calcium/mitochondria-mediated apoptosis in human glioblas-
to Ming Shih. toma cells,” Chemico-Biological Interactions, vol. 205, no. 1, pp.
20–28, 2013.
[17] S. H. Wang, Y. L. Shih, W. C. Ko, Y. H. Wei, and C. M. Shih,
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Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2013, Article ID 349531, 7 pages
http://dx.doi.org/10.1155/2013/349531

Research Article
Exploratory Pharmacokinetics of Geniposide in
Rat Model of Cerebral Ischemia Orally Administered
with or without Baicalin and/or Berberine

Linmei Pan,1 Wenzhe Wang,1 Feiyan Shi,1 Jing Zhou,1

Meng Zhang,1 Huaxu Zhu,1 and Mingfei Zeng2
1
Separation Engineering of Chinese Traditional Medicine Compound, Nanjing University of Chinese Medicine, Nanjing 210028, China
2
Thermo Fisher Scientific, Shanghai, China

Correspondence should be addressed to Linmei Pan; [email protected]

Received 18 August 2013; Revised 22 October 2013; Accepted 23 October 2013

Academic Editor: Joen-Rong Sheu

Copyright © 2013 Linmei Pan et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Huang-Lian-Jie-Du-Tang (HLJDT), a classical Chinese prescription, has been clinically employed to treat cerebral ischemia for
thousands of years. Geniposide is the major active ingredient in HLJDT. The aim is to investigate the comparative evaluations
on pharmacokinetics of geniposide in MCAO rats in pure geniposide, geniposide : berberine, and geniposide : berberine : baicalin.
Obviously, the proportions of geniposide : berberine, geniposide : baicalin, and geniposide : berberine : baicalin were determined
according to HLJDT. In our study, the cerebral ischemia model was reproduced by suture method in rats. The MCAO rats were
randomly assigned to four therapy groups and orally administered with different prescription proportions of pure geniposide,
geniposide : berberine, geniposide : baicalin, and geniposide : berberine : baicalin, respectively. The concentrations of geniposide
in rat serum were determined using HPLC, and main pharmacokinetic parameters were investigated. The results indicated that
the pharmacokinetics of geniposide in rat serum was nonlinear and there were significant differences between different groups.
Berberine might hardly affect the absorption of geniposide, and baicalin could increase the absorption ability of geniposide.
Meanwhile, berberine could decrease the absorption increase of baicalin on geniposide.

1. Introduction apoptosis. Geniposide has a strong antioxidative capacity

and endothelial cell protective effect, which can reduce the
Huang-Lian-Jie-Du-Tang (HLJDT, or Oren-gedoku-to in endothelial cells of the vascular oxidation injury (Figure 1)
Japanese), an important multiherb remedy in China and [8]. In addition, geniposide had a certain analgesic and anti-
other Asian countries, has been used clinically to treat cere- inflammatory effect [9]. At present, researchers have studied
bral ischemia for decades [1–4]. It consists of four medicines the effects of some other ingredients on the pharmacokinetics
including Rhizome coptidis (9 g), Radix scutellariae (6 g), and pharmacodynamics of geniposide [10–12].
Cortex phellodendri (6 g), and Fructus gardenia (9 g). Modern There are two other major constitutes, alkaloids (berber-
pharmacological studies have shown that HLJDT decoction ine) and flavonoids (baicalin), existing in HLJDT (Figure 1).
can prevent cerebral ischemia and antioxidation [5, 6]. The So it is necessary to investigate the pharmacokinetics of
Gardenia contains iridoid glycosides, in which geniposide has geniposide in different groups. The aim of this study is
the highest content (content of which is up to 6%). Genipo- to explore whether the berberine and baicalin can affect
side has a protective effect from loss of cerebral ischemia and the pharmacokinetic behavior of geniposide. It valued the
reperfusion injury [7], but the mechanism is not clear. Geni- formulation optimization design and clinical application of
poside inhibited focal cerebral ischemic injury and induced HLJDT. According to the proportion of pharmacodynamic
HIF-1𝛼 and HIF-1, which depends on apoptosis and expres- ingredients of HLJDT, the study had comparatively evaluated
sion of related gene RTP801mRNA, thereby reduced neuronal the pharmacokinetics of geniposide in MCAO rats after oral
2 Evidence-Based Complementary and Alternative Medicine

COOCH3
O
COOH O
O
O
CH2 OH OH O O
HOH2 C HO
OO HO N+
HO OH H3 CO
OH OH O OCH3
OH
Baicalin Berberine
Geniposide

Figure 1: The structures of geniposide, baicalin, and berberine.

administration of geniposide, geniposide : berberine, genipo- 2.4. Animals. Male Sprague-Dawley (SD) rats were pur-
side : baicalin, and geniposide : baicalin : berberine. The phar- chased from the slaccas experiment animal company (Shang-
macokinetics parameters were analyzed by software kinetica hai, China). Animal welfare and experimental procedures
version 4.4 (Innaphase, MA, USA). The validated method is were strictly in accordance with the Guide for the Care and
successfully applied to control the quality of geniposide and Use of Laboratory Animals (US National Research Council,
investigate interaction among other ingredients of HLJDT 1996) and the related ethics regulations of Nanjing University
decoction in cerebrovascular disease. of Chinese Medicine.
Experiments were conducted in 290 ± 10 g SD rats under
2. Experiment chloral hydrate anesthesia and fixed the rats on the operating
table. A ventral midline incision was made in the neck. The
2.1. Materials and Reagents. Geniposide and paeoniflorin omohyoid muscle was separated longitudinally and retracted
were purchased from National Institute for the Control of laterally and isolated. A loose ligature was then placed around
Pharmaceutical and Biological Products (Beijing, China). the external cartid artery. The external artery was perma-
The extracts of geniposide, baicalin, and berberine were nently ligated rostrally. The vessel was ransected between the
purchased from Zelang Biotechnology Company (Nanjing, ligatures, and the remaining stump was reflected caudally. A
China). Methanol used for HPLC was chromatographic grade piece of monofilament suture material was inserted into the
(Han bang Company, Jiangsu, China). Deionized water was lumen of the stump and advanced into the internal carotid
prepared in a Mill-Q academic water purification system artery. When completely advanced, the tip of the monofila-
ment should block the blood flowing into the middle cerebral
(Millipore, Bedford, MA, USA). Glacial acetic acid was ana-
artery. The silk ligature was tied around the stump to secure
lytical grade, which was provided by Jiuyi Chemical Reagent
the piece of monofilament. The skin irrcision was closed
Company (Shanghai, China). Heparin sodium was purchased
using wound clips. Four hours after surgery, as a successful
from Sigma. Acetonitrile, HPLC grade was purchased from replication of the model of MCAO, the body temperature
Merck. will rise over 0.8∘ C, and rats showed visible symptoms of
neurological deficit which are characterized by severe left-
2.2. Apparatus and Chromatographic Conditions. The High
sided hemiparesis and right Horner’s syndrome [13, 14]. It
Performance Liquid Chromatography (HPLC) system con- was mainly shown in reducing the activities, such as apathy,
sisted of a Waters 515 pump, DAD detector (Waters Asso- dumping to the right side when walking, keeping on rotating,
ciation, Milford, MA, USA); N2000 LC chromatography and neurobiology score significantly increase. These were
workstation (Zhejiang University, China). The mobile phase criteria for evaluating the ischemic insult. Rats, which did
was Acetonitrile (A) and 0.1% H3 PO4 (B) with isocratic not show behavioral deficit, were excluded from the MCAO
eluting (A : B = 12 : 88, v/v) at a flow rate of 1.0 mL/min. The group, and the rest were divided into four subgroups ran-
detector operated at 238 nm. The injection volume was 20 𝜇L domly.
and the column temperature was 30∘ C.
2.5. Biosample Collection. The MCAO rats were divided
2.3. Sample Treatment. The experiment mainly studied the into four groups randomly, pure geniposide subgroup, geni-
influence of baicalin and berberine on the pharmacokinetic poside : berberine subgroup, geniposide : baicalin subgroup,
behavior of geniposide. According to the preliminary exper- and geniposide : baicalin : berberine subgroup. Pure genipo-
iments, the average contents of geniposide, baicalin, and side was dissolved in 0.5% CMC-Na aqueous solution and
berberine were 22.82, 40.02, and 25.78 mg/g in HLJDT. Pure was oral to the pure geniposide subgroup rats (containing
geniposide, geniposide : beberine, geniposide : baicalin, and 142.28 mg geniposide/kg according to body weight). The
geniposide : beberine : baicalin were weighed accurately on geniposide : berberine subgroup, geniposide : baicalin sub-
the basis of proportion in HLJDT, and then dissolved in group, and geniposide : baicalin : berberine subgroup also
0.50% carboxymethyl cellulose sodium (CMC-Na) aqueous received gavages of geniposide dissolved in 0.5% CMC-Na
solution before use. aqueous solution at the dosage of containing 142.28 mg
Evidence-Based Complementary and Alternative Medicine 3

geniposide/kg according to body weight. Blood samples Table 1: Precision and accuracy of the determination of geniposide
(0.5 mL) were collected from the abdominal vein before in rat serum (interday 𝑛 = 5; intraday 𝑛 = 5).
dosing (to serve as a control) and at 0.083, 0.25, 0.5, 0.75, 1.0,
Intraday Interday
1.5, 2.0, 4.0, 6.0, 8.0, 12.0, and 24.0 h after drug administration, Concentration (𝜇g/mL)
then immediately transferred into EP tube and centrifuged RSD (%) RSD (%)
for 5 min at 5000 rpm in 4∘ C to separate serum. The 2.32 1.34 6.68
serum was stored at −70∘ C after separation until assayed as 0.58 2.74 5.06
described below. 0.15 3.37 6.31
Serum samples (200 𝜇L) were acidified with approxi-
mately 100 𝜇L of 0.01 mol/L acetic acid added to each of them.
Then, each portion was vortexed with 800 𝜇L of methanol for
numerical variables among groups were analyzed with one-
1 min and centrifuged at 5000 rpm for 10 min. The organic
way analysis of variance (ANOVA) LSD-t and SNK-q. A 𝑃
layer was transferred into an empty tube and was dried at
value of less than 0.05 was considered statistically significant.
40∘ C under a nitrogen stream. The residue was dissolved in
100 𝜇L methanol. After centrifugation at 800 rpm for 10 min,
fifty microliters supernatant was analyzed with HPLC. 3. Results
2.6. Preparation of Standard Solutions and Quality Control 3.1. Selectivity. The selectivity of the method was evaluated
Samples. The geniposide reference standards were accurately by analyzing blank serum samples prior to administration.
weighed and dissolved in methanol, and then diluted to The chromatograms were free of interfering peak at the
appropriate concentration ranges for the establishment of retention time of geniposide (13.3988 min); the retention time
calibration curves in rat serum. The concentration of refer- paeoniflorin (8.392 min) of Figure 2 showed the represen-
ence solution was 9.26 𝜇g/mL. The paeoniflorin as internal tative chromatograms of blank serum sample (a), serum
standard was prepared as the same of geniposide and its sample spiked with geniposide and serum sample spiked
concentration was 1.20 𝜇g/mL. These solutions were stored with paeoniflorin (b), and serum samples 1 h after oral
at 4∘ C. The geniposide reference standard solutions at seven administration of geniposide (c).
different concentrations 0.15, 0.29, 0.58, 1.16, 2.32, 4.63, and
9.26 𝜇g/mL were prepared by spiking 100 𝜇L blank serum 3.2. Linearity and Lower Limit of Quantification. Each cali-
with appropriate volumes of the standard stock solution. bration curve was constructed with six different concentra-
Recovery of the liquid-liquid extraction procedure was tions by plotting the peak areas ratios of geniposide versus
evaluated at low (0.15 𝜇g/mL), medium (0.58 𝜇g/mL), and the concentration of geniposide using linear regression.
high (2.32 𝜇g/mL) concentrations for geniposide. It was Good linearity was obtained from 0.15 to 9.26 𝜇g/mL. The
calculated by comparing the mean peak area (𝑛 = 5 at lower limit of quantification (LLOQ) for geniposide was
each concentration) of the extracted quality control sample 0.15 𝜇g/mL.
with that of the unextracted standard solution containing the
equivalent amount of analyses.
3.3. Accuracy and Precision. The intra- and interday precision
Quality control (QC) samples were used for the study
and accuracy were determined by replicate analyses of QC
of intraday and interday accuracy and precision; extraction
samples continuously on the same day (intraday) for 5 days
recovery and stability were prepared in the same way as above
(inter-day), respectively. The intra-and interday precision and
and were prepared from blank serum at concentrations of
accuracy were shown in Table 1. The precision of geniposide
0.15, 0.58, and 2.32 𝜇g/mL; five replicates were analyzed in
calculated as the relative standard deviation (RSD) at various
each of the three analytical runs. The accuracy was expressed
concentrations was lower than 15% for intra- and interday
by the relative error (RE) and the precision was evaluated by
assays, and the accuracy was within 15% for QC samples. The
the relative standard deviation (RSD).
results demonstrated that the precision and accuracy of this
The stability of geniposide was evaluated under mim-
method were acceptable.
icking conditions likely to be encountered during sample
storage and the analytical process by analyzing five replicates
of QC samples for the analysis. The freeze-thaw stability was 3.4. Recovery. The mean (±SD) recovery for geniposide from
determined after one freeze and thaw cycle. The QC samples rat serum was 85.34 ± 2.04%, 81.92 ± 2.23%, 78.75 ± 3.69% at
were stored at −20∘ C for 24 h and thawed unassisted at room 2.32, 0.58, 0.15 𝜇g/mL, respectively., 0.15, 0.58, and 2.32.
temperature.
3.5. Stability. The stability of geniposide was determined by
2.7. Data Analysis. The different serum concentrations of analyzing QC samples at three concentrations exposed to
geniposide were expressed as means ± standard deviation encounter during sample storage. QC samples were frozen
(SD) and the mean concentration-time curve was plotted. and stored at −20∘ C for a week. Freeze-thaw stability was
All data were analyzed by software kinetica version 4.4 determined after one cycle of freezing and thawing, the cor-
(Innaphase, MA, USA) to obtain the relative pharmacoki- responding relative errors were less than ±10% for samples at
netic parameters. Statistical analysis was performed using the the concentrations of 4.36, 3.45, and 8.79 𝜇g/mL, respectively.
SPSS Version 13.0 (SPSS Inc., Chicago, IL). Differences in Geniposide showed good stability in one week after storage
4 Evidence-Based Complementary and Alternative Medicine

800 7
700
6
600

Geniposide (𝜇g/mL)
5
500
4
(mV)

400
300 3
200 2
100
1
0
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
−1
(min)
0 5 10 15 20 25
(a) Time (h)
800 Geniposide (𝜇g/mL)
700 Geniposide : berberine (𝜇g/mL)
600 Geniposide : baicalin (𝜇g/mL)
Geniposide : baicalin : berberine (𝜇g/mL)
500
(mV)

400 Figure 3: The serum concentration-time curve of geniposide in rats

300 after oral administration of pure geniposide, geniposide : berberine,
Paeoniflorin geniposide : baicalin, and geniposide : baicalin : berberine to MCAO
200
Geniposide rats.
100
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 in rat serum after oral administration of pure genipo-
(min) side, geniposide : berberine, geniposide : baicalin, and geni-
(b) poside : baicalin : berberine. The mean serum concentration-
800 time curve profiles were illustrated in Figure 3 and its phar-
700 macokinetics data were shown in Table 2. The concentration-
time profile demonstrated bimodal phenomenon; the first
600
peak occurred at about 0.75 h and the second at 8 h after
500
oral administration of pure geniposide. Compared with pure
(mV)

400 geniposide, bimodal phenomenon of geniposide : berberine,

300 geniposide : baicalin, and geniposide : baicalin : berberine in
200 Paeoniflorin Geniposide MCAO rats serum also existed, but two peak times were
100 advanced, and the values of AUC(0−𝑡) , AUC(0−∞) , and
0 AUMC(0−𝑡) , 𝐶max were significantly increased after oral
administration of geniposide : baicalin (𝑃 < 0.01). These
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
results indicated that baicalin increased the absorption of
(min) geniposide. Pharmacokinetic comparison of geniposide on
(c) MCAO rats after administration of geniposide and geni-
poside : berberine illustrated that the existence of berberine
Figure 2: Typical chromatograms for the determination of geni- might hardly affect the pharmacokinetic behavior of genipo-
posides: (a) chromatogram of a blank serum sample, (b) chro- side.
matograms of a serum sample spiked with paeoniflorin and geni-
In order to observe the pharmacokinetic behavior of
poside, and (c) chromatogram of the serum sample of an MCAO rat
taken 1 h after the oral administration of the geniposide.
geniposide in combination with baicalin and berberine,
the pharmacokinetics of geniposide with pure geniposide,
geniposide : baicalin, and geniposide : baicalin : berberine in
MCAO rats were compared. The serum concentrations of
geniposide were determined and pharmacokinetic param-
at −20∘ C for 7 days, the concentration of geniposide in serum eters were estimated (Table 2). As shown in Figure 3 and
would deviate to less than ±15% from those in freshly spiked Table 2, the serum profile demonstrated that the absorption
serum. ability of geniposide in geniposide : baicalin : berberine group
was higher than pure geniposide group and lower than
3.6. Pharmacokinetics Study. HLJDT contains effective parts geniposide : baicalin group. The lower concentration and
of three categories: alkaloids, flavonoids, and iridoid gly- decreased AUC(0−∞) of geniposide were found in MCAO
cosides, three active ingredients: berberine, baicalin, and rats given geniposide : baicalin : berberine compared to geni-
geniposide, and so forth [15]. The developed and vali- poside : baicalin. These results suggested that berberine
dated HPLC method was used to determine geniposide decreased the absorption of geniposide : baicalin.
Evidence-Based Complementary and Alternative Medicine 5

Table 2: Pharmacokinetic differences of oral administration of geniposide, geniposide : baicalin, geniposide : berberine, and genipo-
side : baicalin : berberine to MCAO rats.
Parameters Geniposide Geniposide : baicalin Geniposide : berberine Geniposide : baicalin : berberine
AUC(0–𝑡) (mg/L∗h) 14.59 ± 4.24 49.59 ± 2.68∗∗ 16.04 ± 4.61 26.88 ± 13.83
AUC(0–∞) (mg/L∗h) 23.63 ± 13.02 59.00 ± 10.59∗∗ 21.32 ± 9.36 30.61 ± 14.56
AUMC(0–𝑡) 136.73 ± 54.16 435.39 ± 48.12∗∗ 135.39 ± 39.93 203.81 ± 116.58
AUMC(0–∞) 235.03 ± 196.14 589.97 ± 73.48 188.91 ± 55.26 297.80 ± 137.63
MRT(0–𝑡) (h) 9.15 ± 1.38 8.77 ± 0.72 8.47 ± 1.24 7.42 ± 0.97∗
MRT(0–∞) (h) 22.52 ± 17.38 13.13 ± 4.83 15.81 ± 6.91 11.76 ± 4.95
VRT(0–𝑡) (h2 ) 49.40 ± 7.29 43.48 ± 10.59 53.26 ± 20.10 41.71 ± 15.44
VRT(0–∞) (h2 ) 770.40 ± 1189.48 177.17 ± 164.06 316.07 ± 248.52 188.61 ± 178.69
𝑡1/2𝑧 (h) 15.11 ± 12.73 8.47 ± 3.45 10.41 ± 6.17 8.42 ± 4.45
𝑇max ( h) 0.96 ± 0.53 0.55 ± 0.19 0.88 ± 0.65 0.60 ± 0.39
CL𝑧/𝐹 (L/h/kg) 8.05 ± 4.76 2.47 ± 0.37 7.55 ± 2.52 5.38 ± 2.05
V𝑧/𝐹 (L/kg) 119.31 ± 41.06 28.72 ± 6.09∗∗ 97.81 ± 49.63 66.23 ± 43.19
𝐶max (mg/L) 1.48 ± 0.19 5.68 ± 1.19∗∗ 2.99 ± 0.87∗∗ 3.99 ± 1.08∗∗
Values are given as means ± SD of 6 rats.
∗
P < 0.05, compared with pure geniposide group.
∗∗
P < 0.01, compared with pure geniposide group.

4. Discussion geniposide (14.59 ± 4.24, 23.63 ± 13.02, and 1.48 ± 0.19) and
lower than that of geniposide : baicalin : berberine (26.88 ±
Huang-Lian-Jie-Du-Tang, the representative medicine for 13.83, 30.61±14.56, and 3.99±1.08) and geniposide : baicalin
heat-clearing and detoxicating, has been used to treat cerebral (49.59±2.68, 59.00±10.59, and 5.68±1.19). The data demon-
ischemia for thousands of years in China. In the past few strated that coadministration of geniposide with baicalin,
years, geniposide was confirmed as the main pharmaco- the bioavailability of geniposide, increased significantly in
dynamic ingredient anticerebral ischemia in HLJDT [16]. MCAO rats body. It might be because baicalin inhibited the
After comparing of an organic solvent such as acetonitrile, hydrolysis of geniposide. Berberine showed no significant
ethyl acetate, chloroform, methanol [17–22] and through influence on behaviors in the pharmacokinetics of geniposide
comprehensive comparison recovery, impurity interference in MCAO rats, but it decreased the bioavailability of coad-
and other factors, it is ultimately found that methanol can ministration of geniposide with baicalin. It might be because
basically comply with the requirements of the experiment, so berberine partly neutralized baicalin and the hydrolysis on
the selection of the organic solvent of methanol precipitates geniposide was enhanced.
protein. Geniposide in rat serum determination investigated At the same time, we studied the pharmacokinetics of
geniposide content in the serum level of the HPLC method; geniposide with or without baicalin and/or berberine in
the method provides highly sensitive, specific, reliable data. normal rats. The results had some differences from that of the
The requirements were applicable to the determination of MCAO rats. Compared with normal rats, in MCAO rat, the
biological samples researching HLJDT pharmacokinetics, time to peak (𝑡max ) was shorter, apparent volume of distribu-
providing a reliable method. tion (𝑉) and concentration to peak (𝐶max ) were higher, the
It investigated pharmacokinetic differences of coadmin- dwell time (MRT) was longer, clearance rate (CL) was lower,
istration geniposide with baicalin and/or berberine changing and so on, indicating that the absorption effects of geniposide
by time in cerebral ischemia rats. Using kinetica version in MCAO rats were better than that of normal rats. Hou et
4.4 and SPSS Version 13.0 to process serum concentra- al. [28] considered that the geniposide could be hydrolyzed
tion data and using the method of statistical moments we by sulfatase, and 𝛽-glucuronidase held that the hydrolyzed
calculated pharmacokinetic parameters [23–25]. As shown enzymes might be reduced in MCAO rats. However, in the
in Figure 3 and Table 2, the concentration-time profile future, more experiments such as tissue distribution and
of geniposide : berberine, geniposide : baicalin, and genipo- metabolic pathway of geniposide in the body should be
side : baicalin : berberine in MCAO rats serum demonstrated taken out for the purpose of exploring its absorption and
bimodal phenomenon. The bimodal phenomenon might be metabolism mechanism.
resulted from some circulations such as hepatoenteral circu-
lation which was consistent with other published conclusions
[26, 27]. The peak times of geniposide : baicalin and geni- 5. Conclusions
poside : baiclin : berberine were advanced, and the advanced
times of geniposide : baicalin were longer than that of genipo- The animal study showed significant comparative pharma-
side : baiclin : berberine. The values of AUC(0 − 𝑡) , AUC(0 − ∞) , cokinetics of geniposide alone or in combination with bai-
and 𝐶max of geniposide : berberine were 16.04 ± 4.61, 21.32 ± calin and/or berberine in rat model of cerebral ischemia.
9.36, and 2.99 ± 0.87, which were similar to those of pure Coadministration of geniposide with baicalin could
6 Evidence-Based Complementary and Alternative Medicine

significantly increase the AUC(0−𝑡) , AUC(0−∞) , AUMC(0−𝑡) , [12] M. Li, B. Wang, Z. S. Tang, J. P. Hou, and H. R. Li, “Mechanism
and 𝐶max . In the meantime, coadministration of geniposide of protective effect on cerebral ischemia of Baicalin and genipo-
with berberine could not significantly affect the pharma- side,” Pharmacology and Clinics, vol. 28, pp. 34–36, 2012.
cokinetic parameters. However, the pharmacokinetic para- [13] J. B. Bederson, L. H. Pttis, M. Tsuji, M. C. Nishimura, R. L. Davis,
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examination,” Stroke, vol. 17, no. 3, pp. 472–476, 1986.
[14] Q. S. Wang, H. Fu, and H. K. Zeng, “Suture method improve-
Conflict of Interests ment and evaluation of rat model of focal cerebral ischemia,”
There is no potential conflict of interests involved with this South Journal of Medical Sciences, vol. 40, pp. 291–294, 2012.
work. [15] X. F. Xiao, X. L. Qiao, L. Gao et al., “Huanglian three ingredients
in rats in vivo pharmacokinetic study,” Chinese Medicine, vol. 5,
pp. 13–16, 2008.
Acknowledgments [16] J. Zhou, B. H. Qiu, L. M. Pan, H. X. Zhu, and L. W. Guo, “Study
This research was supported by the priority academic pro- on pharmacokinetics- pharmacodynamics of geniposide in
HuanglianJieduTang active fraction (HLJDTAF),” Chinese Jour-
gram development of Jiangsu Higher Education Institu-
nal of Hospital Pharmacy, vol. 32, pp. 487–491, 2012.
tions, PADA (YS2012ZYX310), and also a Project Funded by
[17] C.-M. Chen and H.-C. Chang, “Determination of berberine in
Jiangsu Province Natural Science Foundation of Education
plasma, urine and bile by high-performance liquid chromatog-
(BK2012855). raphy,” Journal of Chromatography B, vol. 665, no. 1, pp. 117–123,
1995.
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Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2013, Article ID 267217, 14 pages
http://dx.doi.org/10.1155/2013/267217

Research Article
Inhibitive Effects of Mulberry Leaf-Related Extracts on
Cell Adhesion and Inflammatory Response in Human Aortic
Endothelial Cells

P.-Y. Chao,1 K.-H. Lin,2 C.-C. Chiu,2 Y.-Y. Yang,3 M.-Y. Huang,4 and C.-M. Yang4
1
Department of Nutrition and Health Sciences, Chinese Culture University, Taipei 11114, Taiwan
2
Graduate Institute of Biotechnology, Chinese Culture University, Taipei 11114, Taiwan
3
Graduate Institute of Applied Life Science, Chinese Culture University, Taipei 11114, Taiwan
4
Research Center for Biodiversity, Academia Sinica, Nankang, Taipei 106, Taiwan

Correspondence should be addressed to P.-Y. Chao; [email protected]

Received 2 August 2013; Revised 5 November 2013; Accepted 5 November 2013

Academic Editor: Joen-Rong Sheu

Copyright © 2013 P.-Y. Chao et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Effects of mulberry leaf-related extracts (MLREs) on hydrogen peroxide-induced DNA damage in human lymphocytes and on
inflammatory signaling pathways in human aortic endothelial cells (HAECs) were studied. The tested MLREs were rich in flavonols,
especially bombyx faces tea (BT) in quercetin and kaempferol. Polyphenols, flavonoids, and anthocyanidin also abounded in
BT. The best trolox equivalent antioxidant capacity (TEAC) was generated from the acidic methanolic extracts of BT. Acidic
methanolic and water extracts of mulberry leaf tea (MT), mulberry leaf (M), and BT significantly inhibited DNA oxidative damage
to lymphocytes based on the comet assay as compared to the H2 O2 -treated group. TNF-𝛼-induced monocyte-endothelial cell
adhesion was significantly suppressed by MLREs. Additionally, nuclear factor kappa B (NF-𝜅B) expression was significantly reduced
by BT and MT. Significant reductions were also observed in both NF-𝜅B and activator protein (AP)-1 DNA binding by MLREs.
Significant increases in peroxisome proliferator-activated receptor (PPAR) 𝛼 and 𝛾 DNA binding by MLREs were also detected in
M and MT extracts, but no evidence for PPAR 𝛼 DNA binding in 50 𝜇g/mL MT extract was found. Apparently, MLREs can provide
distinct cytoprotective mechanisms that may contribute to its putative beneficial effects on suppressing endothelial responses to
cytokines during inflammation.

1. Introduction and liver X receptors negatively modulate inflammatory

responses by downregulation of AP-1 and NF-𝜅B [6–8].
Atherosclerosis is a chronic inflammatory process char-
PPARs are transcription factors activated by fatty acids and
acterized by increased oxidative stress [1]. The resulting
fatty acid-derived eicosanoids [9]. Three isotypes (𝛼, 𝛽/𝜎,
adhesion of monocytes to the vascular endothelium and
subsequent migration into the vessel wall are the pivotal and 𝛾) expressed in macrophages control the inflammatory
early events in atherogenesis [2]. Inflammatory cytokines, status and regulate cholesterol metabolism [10]. A number of
tumor necrosis factor-alpha (TNF-𝛼), nuclear factor kappa phytochemicals commonly used in research have antioxidant
B (NF-𝜅B), and activator protein (AP)-1 are the major activity that can protect cells from ROS-mediated DNA dam-
redox-sensitive eukaryotic transcription factors that regulate age that results in mutation and subsequent carcinogenesis
the expression of adhesion molecules [3, 4]. Because the [11]. It is evident that quercetin metabolites are distributed in
activation of NF-𝜅B and AP-1 can be inhibited to various human atherosclerotic lesions [12]. The specific target should
degrees by different antioxidants, endogenous reactive oxy- therefore be taken into account when evaluating the antiox-
gen species (ROS) may play an important role in these redox- idant activity of dietary flavonols in vivo. Several herbs are
sensitive transcription pathways in atherogenesis [1, 5]. In consumed to protect against common and serious diseases
macrophages, nuclear receptors such as glucocorticoid, estro- such as cardiovascular and cerebrovascular events, cancer,
gen, peroxisome proliferator-activated receptors (PPARs), and other age-related degenerative diseases as well [13].
2 Evidence-Based Complementary and Alternative Medicine

These protective effects are considered, in large part, to be provided by the owner of Quan-Ming Silkworm Farm located
related to the various antioxidants contained in them. Several in Miaoli, Taiwan. Harvested mulberry leaves were divided
studies have shown that polyphenolic and flavonol substances into eight individual batches and lyophilized by freeze drying
are the most common compounds in herbs with strong in a Freeze Dryer (FD-5060, Panchum Scientific Corp.,
antioxidant activity [14–16]. The bioactive components of Taipei, Taiwan), grinding to powder, and storing at −80∘ C
herbaceous plants may be responsible for anticancer effects until use. Extracts were produced from 200 mg aliquots
through growth inhibition and apoptosis in human chronic of MLRC powder with a 50-fold volume of methanol at
myeloid leukemia K562 cells [17]. 4∘ C extract overnight and then filtered through Whatman
Morus, a genus of flowering plants in the family Moraceae, grade no.1 qualitative filter paper. Extracts were concentrated
comprises 10–16 species of deciduous trees commonly known by rotary vacuum evaporator (R205, Buchi, Switzerland),
as mulberries that grow wild and under cultivation in many resuspended in 99.5% ethanol to 1 mL, and then stored at
temperate regions of the world. Mulberries are widespread −20∘ C until HAECs treatment.
and important crops for fruit, timber, and silkworm feeding, The edible portion of each MLRC leaf was weighed,
as well as being excellent amenity trees. Morus leaves are lyophilized, ground to powder, and extracted by distilled
also recognized as excellent animal food. Mulberry leaf deionized (dd)H2 O [25]. The extraction mixture was heated
extracts contain rutin, isoquercetin, and various derivatives to 90∘ C in a steam bath and refluxed for 2 h, cooled in
of kaempferol and quercetin glycosides that can scavenge 1,1- the refrigerator, sonicated for 5 min, and diluted to 50 mL
with ddH2 O to form the final extract solution. Individual
diphenyl-2-picryl-hydrazyl (DPPH) radical [18] and inhibit
water extracts were used for the comet assay. Ten mL of
the formation of conjugated dienes and thiobarbituric acid-
62.5% aqueous methanol containing BHT (2 g/L) was added
reacting substances (TBARS) by copper-induced oxidative to 1.25 g lyophilized samples, followed by adding 5 mL 6 M
modification of rabbit and human low-density lipoproteins HCl to bring the total volume up to 12.5 mL. The final
[19]. In addition to antioxidant activity, mulberry leaves and mixture consisted of 1.2 M HCl in 50% aqueous methanol.
extracts have antimicrobial activity [20], induce apoptosis Acid hydrolysates methanolic extracts were analyzed for their
[18], are antiinflammatory and anti-hyperglycemic, attenuate antioxidant composition, antioxidant activity, and the comet
atherosclerotic lesions in animal models, and lower blood assay.
lipids in mild hyperlipidemic patients [21–24]. Although a
variety of medicinal herbs including mulberry are known
to be potent sources of polyphenols and flavonols, studies 2.2. Polyphenol Assay. Polyphenol content was determined
on the protective effects of antioxidant activity in mulberry according to the method of Taga et al. [26]. Briefly, standard
leaf-related extracts (MLREs), especially bombyx faces tea gallic acid and an aliquot of the acidic methanolic extract
and mulberry leaf tea, on DNA damage and cell adhesion were diluted with an ethanol/water (60 : 40, v/v) solution
are either scarce or little known. The objectives of this containing 0.3% HCl. Two mL of 2% Na2 CO3 were mixed into
study were to isolate, identify, and evaluate the antioxidant each sample of 100 𝜇L and allowed to equilibrate for 2 min
components, antioxidant activity, and extent to which acidic before adding 50% Folin-Ciocalteu reagent. Absorbance at
methanolic hydrolysate and water extracts of mulberry leaves 750 nm was measured at room temperature. The standard
can protect DNA in human lymphocytes from oxidative curve for gallic acid was used to calculate polyphenol levels.
damage induced by H2 O2 . The effects of MLREs, including
bombyx faces tea (BT), mulberry leaf tea (MT), and mulberry 2.3. Determination of Total Flavonoids. Total flavonoid con-
leaf (M) methanolic extracts, on endothelial cell-monocyte tents were determined using the method of Ordoñez et al.
adhesion and the inflammatory response were assessed in [27]. A 0.5 mL 2% AlCl3 -ethanol solution was added to
human aortic endothelial cells (HAEC). Moreover, the effects 0.5 mL of acidic methanolic extract, and absorbance was
of MLREs on intracellular redox-sensitive transcriptional measured at 420 nm after standing for 1 h at room temper-
pathways such as NF-𝜅B, AP-1, signal transducers, and ature. Extract samples were evaluated at a final concentration
activators of transcription (STAT)3, and PPAR 𝛼 and 𝛾, of 0.1 mg/mL. Total flavonoid content was calculated as a
which may contribute to leukocyte recruitment and vascular quercetin equivalent (mg/g).
inflammation in atherogenesis, were examined by western
blot analysis and electrophoretic mobility shift assays. Our 2.4. Determination of Total Flavonols. Total flavonols in plant
study explores the relationship between the composition extracts were estimated using the method of Kumaran and
and content of flavonols and polyphenols having antioxidant Joel Karunakaran [28]. Two mL of 2% AlCl3 ethanol and
efficiency and the prevention of DNA oxidative damage 3.0 mL (50 g/L) sodium acetate were added to 2.0 mL acidic
afforded by mulberry leaf-related compounds. The results of methanolic extracts. The absorption at 440 nm was read
our study may help determine the antioxidant activity and after 2.5 h at 20∘ C. Sample extracts were evaluated at a final
anti-inflammatory effects of MLREs. concentration of 0.1 mg/mL. Total flavonoid content was
calculated as a quercetin equivalent (mg/g).
2. Materials and Methods
2.5. Flavonols Analysis by HPLC. One mL of acid hydrolysa-
2.1. Mulberry Leaf-Related Compounds Extract Preparation. tes methanolic extract was filtered through 0.45 𝜇m filter
Mulberry leaf-related compounds (MLRC) were generously prior to 20 𝜇L injection into a high-performance liquid
Evidence-Based Complementary and Alternative Medicine 3

chromatograph (HPLC). Samples were analyzed with a Spec- and herbaceous plants [34], the DNA damage was induced
traSystem UV6000LP Photodiode Array Detection System by exposing the lymphocytes to 10 𝜇M H2 O2 and shown the
(Thermo Separation Products, San Jose, USA) and an ODS protection effects. Consequently, these concentrations of the
column (250 × 4.6 mm, 5 𝜇m; YMC, ODS-A, YMC Co., extracts were chosen for the effect treatments.
Kyoto, Japan). The mobile phase consisted of methanol-water
(30 : 70, v/v) with 1% formic acid and 100% methanol. The 2.10. Cell-Viability Testing. After culturing, lymphocytes were
gradient was 25–74% methanol in 40 min at a flow rate exposed to each MLREs. Each lymphocyte solution was used
of 0.75 mL/min. the spectrum was recorded at 365 nm for at three concentrations (25, 50, and 100 𝜇g/mL) for 30 min at
flavonol determination [25]. 37∘ C. DNA damage was induced by exposing lymphocytes to
H2 O2 (10 𝜇M) for 5 min on ice. Treatment on ice minimized
2.6. ABTS/HRP–H2 O2 Assay of Total Antioxidant Activity. the possibility of cellular DNA repair after H2 O2 injury. Cells
The total antioxidant capacity of hydrophilic and lipophilic were centrifuged at 100 g for 10 min, washed, and resuspended
antioxidants was determined using the horseradish perox- in the same medium as the comet assay. All experiments were
idase catalyzed oxidation of 2,2-azino-bis-(3-ethylbenzot- carried out in triplicate. Cell viability was tested using the
hiazoline-6-sulfonic acid) (ABTS) [29]. The reaction mixture tetrazolium/formazan (MTT) assay [35] prior to and after
contained 0.5 mL of 1000 𝜇M ABTS (in ddH2 O) and 3.5 mL treatment with MLRC extract or H2 O2 .
of 100 𝜇M H2 O2 (in 0.1 M PBS). The reaction was started
by the addition of 0.5 mL of 44 U/mL peroxidase (in 0.1 M 2.11. DNA Single Strand Break Damage Estimation Using the
PBS). After 1 h, 0.05 mL of MLREs was added to the mixture. Comet Assay. The comet assay was performed as described
Absorbance was measured at 730 nm after 5 min. Trolox was in Szeto et al. [36] with acidic methanolic hydrolysate and
used as a standard and the total antioxidant capacity of water extracts from tested plants. Cultured lymphocytes
mulberry extracts was measured as mM Trolox equivalent. (105 cells/mL) were embedded in 75 𝜇L of 1% low-melting-
point agarose on a microscope slide (precoated with agarose)
2.7. Scavenging Activity on 1,1-Diphenyl-2-picryl-hydrazyl at 37∘ C. The gel was allowed to set at 4∘ C and cells were lysed
(DPPH) Radical. The scavenging activity on DPPH radical of for a period of at least 2 h in lysis buffer at 4∘ C. Cells were
MLRC acidic methanolic extracts was determined according then alkaline-unwound, following which electrophoresis was
to the method of Shimada et al. [30]. Briefly, an aliquot of carried out using the electrophoresis buffer at 4∘ C for 15 min
0.4 mL of acidic methanolic extracts with series dilution was at 25 vDC and 300 mA. All steps were conducted under
added to 0.8 mL of 1 mM DPPH freshly prepared in methanol, dim light to prevent additional DNA damage. Following
mixed well, and left to stand for 30 min before measuring electrophoresis, slides were dipped into a neutralization
absorbance at 517 nm. The scavenging effect percentage was buffer and stained with ethidium bromide. The comet-like
calculated as [1 − (OD517 nm )/(control OD517 nm) ] × 100. The images resulting from the extension of DNA were scored as
IC50 of the scavenging effect percentage was then calculated. a reflection of the single strand breaks under a fluorescence
microscope (Zeiss-Axiovert 100, Zeiss, Germany). Triplicate
2.8. Isolated Human Peripheral Blood Lymphocytes. Blood slides were prepared for each experimental point sample,
samples were obtained from six donors, four male and and 50 comet-like images selected at random per slide to
two female healthy nonsmokers, that were 24 to 48 years determine average DNA damage values. A computerized
old. Fresh whole blood (20–30 mL) from volunteers was image analysis system (VisCOMET 1.6, Impuls GmbH, Ger-
taken with informed consent, and lymphocytes were isolated many) was employed to analyze two comet parameters, DNA
using a separation solution kit supplemented with Ficoll- damage by tail DNA percentage [(total brightness of tail
Paque Plus lymphocyte isolation sterile solution (Pharmacia area/total brightness of total area) × 100%] and tail moment
Biotech, Sweden) [31]. Cells were harvested within 1 day (tail length × tail DNA%). The inhibition percentage of tail
of blood samples being taken and cultured with AIM V DNA% and tail moment were calculated relative to the 10 𝜇M
medium containing serum-free lymphocyte medium (Gibco H2 O2 treated group.
Invitrogen, USA) in a humidified atmosphere of 5% CO2 in
air at 37∘ C for 24 h. 2.12. Cell Cultures and Treatment. HAECs (Clonetics Corp.)
were grown in Medium 200 (GIBCO Invitrogen) supple-
2.9. Dosage Selected for the Comet Assay. Previously, we mented with 1% low serum growth supplement (LSGS;
demonstrated that the effect of lymphocyte exposure to 10 𝜇M GIBCO Invitrogen) and 10% FBS (GIBCO Invitrogen) in
H2 O2 on DNA single-strand break damage was 60-fold an atmosphere of 95% air and 5% CO2 at 37∘ C in plastic
greater than the control and with 99.4% cell viability [32]. flasks in an incubator (Astec Co.) as described by Vielma
However, when lymphocytes were exposed to 50 𝜇M H2 O2 , et al. [37]. The human monocytic cell line, U937 (Amer-
the oxidative damage increased significantly compared to the ican Type Culture Collection) was grown in a suspension
control under 98.2% cell viability. Therefore, 10 𝜇M H2 O2 culture in RPMI-1640 (GIBCO Invitrogen) containing 10%
for the treatment dosage was selected in the current study. FBS (Sigma) and 1% antibiotic-antimycotic mixture (Sigma)
Furthermore, when lymphocytes were treated with three under an atmosphere of 95% air and 5% CO2 at 37∘ C.
concentrations (25, 50, and 100 𝜇g/mL) of acidic methanolic After incubation with MLREs and aspirin, or TNF-𝛼, cell
and water extracts from indigenous purple vegetables [33] viability was assessed using the tetrazolium/formazan (MTT)
4 Evidence-Based Complementary and Alternative Medicine

assay [35]. The adhesion assays were performed as described GGC CTT-5󸀠 ), STAT3 (5󸀠 -GAT CCT TCT GGG AAT TCC
by Zhu and Loft [38]. Briefly, U937 cells were labeled TAG ATC-3󸀠 and 3󸀠 -CTA GGA AGA CCC TTA AGG ATC
with a fluorescent dye, incubated with 10 𝜇mol/L 2,7-bis (2- TAG-5󸀠 ) (Sigma), PPAR-𝛼 (5󸀠 -AAA AAC TGG GCC AAA
carboxyethyl)-5 (6)-carboxyfluorescein acetoxymethyl ester GGT CT-3󸀠 ), and PPAR-𝛾 (5󸀠 -TGA AAC TAG GGT AAA
at 37∘ C for 1 h in RPMI-1640 medium and subsequently GTT CA-3󸀠 ) (Sigma), 1 𝜇L poly (dI⋅dC), and 1 𝜇L nuclear
washed by centrifugation. Confluent HAECs in 24-well plates extract (as prepared previously). The mixture was incubated
were incubated with U937 cells (106 cells/mL) at 37∘ C for at room temperature for 20 min in the dark. After 1x Orange
1 h. Nonadherent leukocytes were removed. The numbers of Loading Dye (LI-COR Biosciences) was added, the binding
adherent leukocytes were determined by photographing and reaction was loaded onto a native 4% polyacrylamide gel and
counting four randomly chosen fields per well at 100x using separated by electrophoresis at 90 vDC for 40 min. The gels
a Zeiss Axio Mager Z1 Upright Fluorescence Microscope. were scanned using an Odyssey Infrared Imaging System (LI-
Experiments were performed in triplicate and repeated three COR Biosciences) [40].
times. HAECs were incubated with 25 𝜇g and 50 𝜇g of MLREs
or 10 𝜇M aspirin for 18 h, followed by treating with 2 ng/mL 2.16. Statistical Analysis. Data are expressed as the mean ±
TNF-𝛼 for 6 h in the NF-𝜅B p65, AP-1, and STAT-3 expression standard deviation, and statistical significance was analyzed
assays. using one-way ANOVA followed by the Tukey’s Range Test
at the 0.05 significance level. Pearson’s linear correlation was
2.13. Nuclear Protein Isolation. Protein extracts were pre- also determined. The means of three replicates are reported.
pared as described by Min et al. [39]. Briefly, after cell
activation for the indicated times, cells were washed in 1 mL
ice-cold PBS, centrifuged at 400 g for 5 min, resuspended
3. Result
in 400 𝜇L ice-cold hypotonic buffer (10 mM HEPES, 1.5 mM 3.1. Antioxidant Composition and Antioxidant Activity. Poly-
MgCl2 , 0.1 mM EDTA, 10 mM KCl, 1 mM DTT, 0.5 mM phenols, flavonoids, and flavonols were abundant in BT
PMSF, pH 7.9), incubated on ice for 10 min, vortexed, and cen- at levels of 34.65 ± 0.82 mg/g DW gallic acid, 84.83 ±
trifuged at 15,000 g for 30 sec. The supernatant was collected 8.68 mg/g DW quercetin equivalent, and 25.47 ± 3.86 mg/g
and stored at −70∘ C for cytosolic protein analysis. Pelleted DW quercetin equivalent, respectively (Table 1). Anthocy-
nuclei were gently resuspended in 44.5 𝜇L ice-cold extraction anidins were abundant in BT and ML at levels of 143.32 ±
buffer (20 mM HEPES, pH 7.9 with 1.5 mM MgCl2 , 0.42 M 1.81 and 135.82 ± 0.31 units/g DW, respectively. Table 2 shows
NaCl, 0.2 mM EDTA, and 25% glycerol) with 5 𝜇L of 10 mM that quercetin and kaempferol were both rich in BTat levels
DTT and 0.5 𝜇L of Protease Inhibitor Cocktail (Sigma), incu- of 2853.33 ± 180.37 and 646.67 ± 50.33 𝜇g/g DW, respectively,
bated on ice for 20 min, vortexed, and centrifuged at 15,000 g while the levels of M were 1666.67 ± 189.03 and 426.67 ±
for 5 min at 4∘ C. Aliquots of the supernatant containing 46.19 𝜇g/g DW, respectively. However, no myricetin or morin
nuclear proteins were frozen in liquid nitrogen and stored at was detected in the MLRC (Table 2). Thus, these MLREs
−70∘ C. displayed variations in their antioxidant substances levels.
MLREs showed antioxidant activity, proving their capac-
2.14. Western Blot Analysis. Nuclear lysates were subjected to ity to scavenge the ABTS radical cation. The antioxidant
12% SDS-PAGE (Bio-Rad Laboratories), after which proteins activity in acidic methanolic hydrolysate sample extracts was
were transferred to a PVDF membrane (Bio-Rad). Mem- expressed in trolox equivalent antioxidant capacity (TEAC)
branes were probed with a mouse monoclonal NF-𝜅B p65 (Table 3). BT showed a significantly higher TEAC value
antibody (BD Biosciences). After incubation in a secondary (155.14 ± 2.90 𝜇g/g DW) than MT (102.39 ± 0.95 𝜇g/g DW)
antibody solution consisting of IRDye 700CW-conjugated and M (76.54 ± 1.61 𝜇g/g DW). TEAC values were positively
goat anti-mouse IgG (LI-COR Biosciences) for 60 min at and significantly (𝑟 = 0.41, 𝑃 = 0.034) correlated with
room temperature with gentle shaking, the membrane was the content of quercetin among MLREs (Table 4). Thus,
washed four times for 5 min each at room temperature in PBS different quercetin contents displayed various levels of anti-
with 0.1% Tween-20 and while being gently shaken. After a oxidant activity. However, M had a significantly high effi-
final rinse, the membrane was scanned using AlphaEaseFC cacy for DPPH radical scavenging activity (IC50 188.83 ±
image analysis software (Alpha Innotech Corporation) to 3.61 𝜇g/mL) compared to BT (IC50 669.01 ± 7.23 𝜇g/mL) and
analyze spot density. The internal control was set at 100% to MT (IC50 836.40 ± 7.08 𝜇g/mL) (Table 3). Hence, each tested
determine the relative density of protein expression [40]. sample showed significant differences in scavenging of DPPH
radical at IC50 .
2.15. Electrophoretic Mobility Shift Assay for NF-𝜅B, AP-1, and
STAT3. The binding reaction consisted of 1 𝜇L 10× binding 3.2. Effects of Acidic Methanolic and Water Extracts from
buffer (100 mM TRIS, 500 mM NaCl, 10 mM DTT, pH 7.5), MLRC on H2 O2 -Induced DNA Damage to Lymphocytes. The
5 𝜇L H2 O, 2 𝜇L 25 mM DTT/2.5% Tween-20, 1 𝜇L IRDye 700- effects of MLRC extracts on cell cytotoxicity were determined
labeled EMSA oligonucleotides specific for NF-𝜅B (5󸀠 -AGT by the MTT assay. Lymphocytes were exposed to each of three
TGA GGG GAC TTT CCC AGG C-3󸀠 and 3󸀠 -CGC TTG different MLRC extracts at three concentrations (25, 50, and
ATG ACT CAG CCG GAA-5󸀠 ), AP-1 (5󸀠 -CGC TTG ATG 100 𝜇g/mL) for 30 min at 37∘ C. DNA damage was induced
ACT CAG CCG GAA-3󸀠 and 3󸀠 -GCG AAC TAC TGA GTC by exposing lymphocytes to H2 O2 (10 𝜇M) for 5 min on ice.
Evidence-Based Complementary and Alternative Medicine 5

Table 1: The contents of various antioxidant substances in MLRC.

Polyphenol Flavonoids mg quercetin Flavonol mg quercetin Athocyanidin
mg gallic acid/g DW equivalent/g DW equivalen/g DW unit/g DW
BT 34.65 ± 0.82a 84.83 ± 8.68a 25.47 ± 3.86a 143.32 ± 1.81a
MT 23.23 ± 0.54b 55.99 ± 10.55b 13.26 ± 2.66b 88.58 ± 4.12b
M 19.87 ± 0.61c 49.60 ± 5.41b 14.22 ± 1.48b 135.82 ± 0.31a
All values are means ± S.D. (𝑛 = 3).
Means within a column with different superscripts (a∼c) are significantly different, 𝑃 < 0.05.

Table 2: The contents of flavonols in acidic methanolic extracts of 14.62% of tail DNA% compared to treatment only with H2 O2 .
MLRC. MT had better inhibition efficacy (84.39%) than M and BT
Flavonols (𝜇g/g DW) extracts at the 100 𝜇g/mL level (Figure 1(a)). Tested plants
showed at least 1342.63 of tail moment in BT extract at the
Samples Myricetin Morin Quercetin Kaempferol
25 𝜇g/mL level, while the BT extract at 100 𝜇g/mL had the
BT ND ND 2853.33 ± 180.37a 646.67 ± 50.33a
highest tail moment (3281.93) compared to the rest of the
MT ND ND 373.33 ± 23.09c ND
M ND ND 1666.67 ± 189.03b 426.67 ± 46.19b
acidic methanolic extract samples (Figure 1(b)). Moreover,
the trend in tail moment in all samples continued climbing
ND: not detected.
up from 25–100 𝜇g/mL.
Table 3: TEAC values and the IC50 of DPPH radical scavenging The effects of various water extracts of MLRC H2 O2 -
activity of acidic methanolic hydrolysates of MLRC. induced DNA damage to lymphocytes are presented in
Figure 2. At lower concentrations, all tested samples had
Sample TEAC DPPH radical scavenging activity lower tail DNA%, indicating better inhibition efficacies
(Trolox mM)∗ IC50 (𝜇g/mL) (Figure 2(a)). BT and M had the best inhibition efficacies,
BT 155.14 ± 2.90a 669.01 ± 7.23b with both exhibiting values of 14.22 and 14.69 tail DNA%
MT 102.39 ± 0.95b 836.40 ± 7.08a at 25 𝜇g/mL, respectively. Moreover, MT had a higher tail
M 76.54 ± 1.61c 188.83 ± 3.61c DNA% than the other extracts at the same dose, and the
∗ highest tail DNA% was observed in 32.12% of MT at a
TEAC value at sample 100 𝜇g/mL concentration.
concentration of 100 𝜇g/mL. The tail moment of all samples
Table 4: Correlation between antioxidant substances and TEAC continued to increase up to the 100 𝜇g/mL level (Figure 2(b)).
values treated with acid hydrolysates of MLRC. The minimum tail moment occurred in M at 1536.74 in the
25 𝜇g/mL extracts. Moreover, the DNA damage induced by
Flavonols H2 O2 was significantly higher than the DNA damage treated
Myricetin Morin Quercetin Kaempferol with extracts, and H2 O2 treated had 87.26 in tail DNA% and
TEAC∗ 𝑟 = 0.41 8328.84 in tail moment.
— — —
𝑃 = 0.034
—: no significant correlation. 3.3. Effects of MLREs on Monocyte-Endothelial Cell Adhe-
∗
TEAC values at 100 𝜇g/mL of sample concentration. sion. As shown in Figure 3, pretreatment of HAECs with
100 𝜇g/mL MLREs for 18 h significantly suppressed the adhe-
No mulberry extracts were cytotoxic at the concentrations sion of U937 monocytes to TNF-𝛼-stimulated HAECs similar
used, with >98% of cells remaining viable [41]. The comet to that of 10 𝜇M aspirin (positive control). Data also show that
assay was performed to determine the DNA damaging 25 and 50 𝜇g/mL BT, MT, and M methanol extracts inhib-
activity of the plants because it is a sensitive method for ited the adhesion of U937 monocytes to TNF-𝛼-stimulated
monitoring single strand DNA breaks at the single cell HAECs by 81.98% and 81.87% (BT), 74.49% and 83.31% (MT),
level. Any DNA damage that occurred is represented as a and 80.11% and 75.59% (M), respectively.
tail length (tail migration) of the DNA strand. The effects
of pretreatment with acidic methanolic extracts of MLRC 3.4. Effects of MLREs on NF-𝜅B Activity, and MLREs Inhibits
on 10 𝜇M H2 O2 -induced DNA oxidative damage in human the Binding of NF-𝜅B, AP-1, and STAT3. Figure 4 demon-
lymphocytes are presented in Figure 1, indicating that there strates that MLREs regulated the DNA-binding activity and
was a protective effect of pretreatment on lymphocytes with expressions of NF-𝜅B in HAECs. In untreated HAECs, NF-
each of the MLRC extracts at the lowest dose (25 𝜇g/mL). 𝜅B p65 was only found within the cytosol; however, its
Lymphocytes pretreated with 25–100 𝜇g/mL extracts of MT nuclear translocation was observed upon treatment with
extracts experienced a greater level of protection against TNF-𝛼. Compared to the TNF-𝛼 group, pretreatment with
H2 O2 exposure than lymphocytes exposed to other tested BT and MT methanol extracts significantly decreased the
compounds and in a dose-dependent manner. BT had better expression of NF-𝜅B p65 in the nuclear compartment, while
inhibition efficacy at lower concentration (25 𝜇g/mL), and the M methanol extract did not reduce the expression
the maximum protective effect of lymphocyte pretreatment of NF-𝜅B p65 activity. Furthermore, an EMSA analysis
was observed with pretreatment by 25 𝜇g/mL BT, exhibiting was carried out to determine if the reduced expression of
6 Evidence-Based Complementary and Alternative Medicine

Control H 2 O2

BT25 BT50 BT100

100

80

MT25 MT50 MT100

Tail DNA (%)

60

40 ∗ a∗ a∗
∗
∗ ∗ ∗ b
∗
20 ∗
M25 M50 M100
0
25 50 100
Mulberry extracts (𝜇g/mL)
H 2 O2 MT
BT M
(a) (b)
10000

8000
Tail moment

6000

4000 a∗ a∗
b∗
∗
a c∗
∗ ∗ ab∗
2000 ∗

0
25 50 100
Mulberry extracts (𝜇g/mL)
H2 O 2 MT
BT M
(c)

Figure 1: Effects of various acidic methanolic extracts of MLRC on H2 O2 -induced DNA damage to lymphocytes. The comet-like images
resulting from the extension of DNA (a) were observed using a fluorescence microscope. Tail DNA% (b) and tail movement (c) were exposed
to test mulberry leaf-related extracts at 25, 50, and 100 𝜇g/mL and treated with H2 O2 at 10 𝜇M. Values with different letters differ significantly
with regard to oxidative damage when comparing different MLRC extracts. ∗ 𝑃 < 0.05 refers to differences in oxidative damage as compared
with 10 𝜇M H2 O2 -alone treatment.
Evidence-Based Complementary and Alternative Medicine 7

Control H2 O 2

BT25 BT50 BT100

100

80

MT25 MT50 MT100

Tail DNA (%)

60

40 ab∗
b∗ b∗
∗
ab
∗ b∗ b∗
20 b∗ a b∗
M25 M50 M100
0
25 50 100
Mulberry extracts (𝜇g/mL)
H 2 O2 MT
BT M
(a) (b)

10000

8000
Tail moment

6000

4000 ∗
∗ ∗
ab∗ a∗ ∗
∗
a ab ∗ ab
2000 bc∗

0
25 50 100
Mulberry extracts (𝜇g/mL)
H2 O 2 MT
BT M
(c)

Figure 2: Effects of various water extracts of MLRC on H2 O2 -induced DNA damage to lymphocytes. The comet-like images resulting from
the extension of DNA (a) were observed using a fluorescence microscope. Tail DNA% (b) and tail movement (c) were exposed to test mulberry
leaf-related extractsat 25, 50, and 100 𝜇g/mL and treated with H2 O2 at 10 𝜇M. Values with different letters differ significantly with regard to
oxidative damage when comparing different MLRC extracts. ∗ 𝑃 < 0.05 refers to differences in oxidative damage as compared with 10 𝜇M
H2 O2 -alone treatment.

NF-𝜅B in response to treatment with MLREs resulted in binding of AP-1 with TNF-𝛼 resulted from the treatment of
reduced binding of NF-𝜅B to DNA. The treatment of HAECs HAECs; however, pretreatment of HAECs with BT, MT, and
with TNF-𝛼 led to increased binding of NF-𝜅B; however, M significantly decreased DNA-bound AP-1 (Figure 5(b)).
pretreatment of HAECs with BT, MT, and M significantly STAT3 DNA-binding activities to its cognate recognition
decreased DNA-bound NF-𝜅B (Figure 5(a)). The increased site were remarkably reduced compared to TNF-𝛼 treatment
8 Evidence-Based Complementary and Alternative Medicine

(A)
(A) (B)

200 𝜇m 200 𝜇m

(C) (D)

200 𝜇m 200 𝜇m

(E) (F)

200 𝜇m 200 𝜇m

(G) (H)

200 𝜇m 200 𝜇m

(I)

200 𝜇m

(a)
180
160 #
Monocyte adherence (cells/HPF)

140
120
100
80
60
∗ ∗
40 ∗ ∗
∗ ∗ ∗
20
0
Control

TNF-𝛼

Aspirin

BT25

BT50

MT25

MT50

M25

M50

(b)

Figure 3: Mulberry leaf related extracts (MLREs) reduce tumor necrosis factor (TNF)-𝛼-induced adhesion of monocytes to human aortic
endothelial cells (HAECs). (a) The number of adherent U937 cells (marked by arrow) was monitored by fluorescence microscopy under
control (a), 2 ng/mL TNF-𝛼 (b), co-treated with TNF-𝑎 and aspirin (10 𝜇M) (c), cotreated with TNF-𝛼 and BT (25 𝜇g/mL) (d), cotreated with
TNF-𝛼 and BT (50 𝜇g/mL) (e), cotreated with TNF-𝛼 and MT (25 𝜇g/mL) (f), cotreated with TNF-𝛼 and MT (50 𝜇g/mL) (g), cotreated with
TNF-𝛼 and M (25 𝜇g/mL) (h), and cotreated with TNF-𝛼 and M (50 𝜇g/mL) (i). (b) HAECs were pretreated with MLREs for 18 h, followed
by stimulating with TNF-𝛼 for 6 h. Fluorescence-labeled monocytic U937 cells were added to HAECs and allowed to adhere for 2 h. Data are
expressed as a percentage of TNF-𝛼-induced expression (mean ± S.D.) of three experiments. Mean ± S.D. ∗ 𝑃 < 0.05, BT, MT, and M at 25
and 50 𝜇g/mL versus TNF-𝛼; # 𝑃 < 0.05, control versus TNF-𝛼.
Evidence-Based Complementary and Alternative Medicine 9

Control TNF-𝛼 Aspirin BT25 BT50 MT25 MT50 M25 M50

10 𝜇M 𝜇g/mL 𝜇g/mL 𝜇g/mL 𝜇g/mL 𝜇g/mL 𝜇g/mL
TNF-𝛼 − + + + + + + + +
NF-𝜅B p 65
65 kDa

𝛽-Actin
43 kDa
(a)
1.0

# # #
0.8 ∗
∗
Ratio (nuclear/𝛽-actin)

#
#
0.6
∗
∗ ∗ ∗ #
0.4

0.2

0.0
Control

TNF-𝛼

Aspirin

BT25

BT50

MT25

MT50

M25

M50
(b)
Figure 4: Effects of MLREs on NF-𝜅B activity. After HAECs were pretreated with the indicated samples and incubated with TNF-𝛼, nuclear
extracts were prepared, and the expression of NF-𝜅B p65 was assessed by western blot analysis. A representative image of three similar results
is shown (a). Actin served as the loading control for the nuclear compartment. Semiquantitative analysis of three independent experiments
are also shown (b). # indicates a significant difference between the TNF-𝛼 treated or experimental treatment groups and the control, 𝑃 < 0.05.
∗ indicates a significant difference between the TNF-𝛼 and experimental treatment groups, BT, MT, and M at 25 and 50 𝜇g/mL.

(lane 3) in the nuclear fraction, indicating that MLREs at expression, at least in part, by increasing the DNA binding of
50 𝜇g/mL inhibited STAT3-binding activities. In addition, the transcription factors PPAR 𝛼 and 𝛾.
results in Figure 5(c) (lanes 4–10) show that aspirin, MLREs at
50 𝜇g/mL, and BT at 25 𝜇g/mL remarkably decreased STAT3
levels compared to TNF-𝛼 (lane 3); however, both MT and
4. Discussion
M at 25 𝜇g/mL increased STAT3-binding activities. These 4.1. Antioxidant Composition and Antioxidant Activity. BT
results indicate that only 50 𝜇g/mL MLREs, 25 𝜇g/mL BT, contains high levels of polyphenols, flavonoids, flavonols,
and aspirin block STAT3 activation and might downregulate anthocyanidin, quercetin, and kaempferol (Tables 1 and 2).
TNF-𝛼-induced expressions of inflammatory signaling path- BT also had a higher TEAC value (155.14 mM, Table 3) than
ways. the TEAC values of 4.7 mM [42] and 6.4 mM [43] reported
for quercetin, as well as 2.42 mM reported for quercetin-3-
3.5. MLREs Regulated the DNA-Binding Activity and Expres- rutinoside [44]. Furthermore, González-Paramás et al. [45]
sions of PPAR 𝛼 and PPAR 𝛾 in HAECs. PPAR 𝛼 and 𝛾 demonstrated that flavonol contents were significantly and
effectively bound to PPAR 𝛼 and 𝛾 oligo-IRDye 700 as highly correlated (𝑟 = 0.8) with TEAC values. Kim et al. [46]
indicated in lanes 3–10 of Figure 6. Supershifts are bands from reported that mulberry leaves contain abundant quercetin
the binding of each specific antibody. The binding activities and inhibit lipid peroxidation at concentrations of 0.1 and
of PPAR 𝛼 and 𝛾 were detected at baseline. The 25 𝜇g/mL 0.01 mg/mL. In our study, the amount of quercetin was
MT extract (lane 7) and 25 and 50 𝜇g/mL M extracts (lanes significantly correlated (𝑟 = 0.41, Table 4) with TEAC value.
9-10) increased activity compared to incubating with TNF- However, no correlation was found among myricetin, morin,
𝛼 alone (lane 3). However, BT at both 25 and 50 𝜇g/mL kaempferol, and TEAC, which may be due to the structure
extracts showed reduced effects on PPAR 𝛼 and 𝛾 expression. required to reinforce the free radical scavenging activity that
Moreover, the 50 𝜇g/mL MT extract had no effect on PPAR 𝛼 varies with the type of free radical. Naowaratwattana et al.
binding activity but increased the binding activity of PPAR [18] reported that a 50% methanolic extract of mulberry
𝛾 in comparison to incubation with TNF-𝛼 alone. These leaf contained rutin, isoquercetin, and various derivatives of
data suggest that MT and M both promoted PPAR 𝛼 and 𝛾 kaempferol and quercetin glycosides, while a water extract
10 Evidence-Based Complementary and Alternative Medicine

DNA Control TNF-𝛼 Aspirin BT25 BT50 MT25 MT50 M25 M50
Oligo 10 𝜇M 𝜇g/mL 𝜇g/mL 𝜇g/mL 𝜇g/mL 𝜇g/mL 𝜇g/mL
TNF-𝛼: − + + + + + + + +
NF-𝜅B

Free probe

(a)

DNA Control TNF-𝛼 Aspirin BT25 BT50 MT25 MT50 M25 M50
Oligo 10 𝜇M 𝜇g/mL 𝜇g/mL 𝜇g/mL 𝜇g/mL 𝜇g/mL 𝜇g/mL
TNF-𝛼: − + + + + + + + +
AP-1

Free probe

(b)

DNA Control TNF-𝛼 Aspirin BT25 BT50 MT25 MT50 M25 M50
Oligo 10 𝜇M 𝜇g/mL 𝜇g/mL 𝜇g/mL 𝜇g/mL 𝜇g/mL 𝜇g/mL
TNF-𝛼: − + + + + + + + +
STAT3

Free probe

(c)

Figure 5: MLREs inhibits the binding of (a) NF-𝜅B, (b) AP-1, and (c) STAT3 to target DNA sequence. NF-𝜅B, AP-1, and STAT3 transcription
factor-DNA interactions were assessed by EMSA.

contained primarily chlorogenic acid and caffeoylquinic acid extracts. The amount of quercetin was especially positively
derivatives. The IC50 of DPPH radical scavenging was 79.4 correlated with the observed TEAC values.
and 204.2 𝜇g/mL for 50% methanolic and water extracts,
respectively [18]. In our study, M extracts contained quercetin
4.2. Estimation of DNA Single Strand Break Damage from
and kaempferol (Table 2), which may account for part of Exposure to Acidic Methanolic and Water Extracts. One of
the highly scavenged DPPH radical in comparison to the the antioxidant activity tests, scavenging of DPPH radi-
rest of MLREs. Khan et al. [47] demonstrated that the IC50 cal, was selected due to its quick and simple, but it is a
was 220.23 𝜇g/mL, which is much less efficacious than the less biologically relevant assay. Previously, we reported that
mulberry leaf acidic methanolic extract IC50 of 188.8 𝜇g/mL with 2 𝜇g⋅mL−1 of methanolic and water extracts from the
(Table 3). However, the IC50 of in vitro free radical scav- indigenous vegetables [33] and herb plants [48] significantly
enging activity of mulberry leaves from Naowaboot et al. prolonged lag phase of low-density lipoprotein (LDL). The
[22] was 61.7 ± 2.1 𝜇g/mL, which is more efficacious than LDL oxidation assay is considered to be one of the most
the IC50 concentration derived in the present study. Taken biologically relevant assays [49]. Both studies [33, 48] also
together, the polyphenol-rich MLREs exhibited distinct cell- showed that the concentration of the tested extracts for
free antioxidant activity (i.e., TEAC) as validated by their scavenging DPPH radical was in higher levels at 10 mg of
levels of polyphenols and flavonols, with distinct antioxidant tested extracts with 2–53% inhibition. Lower concentration
activity strongly accounting for the antioxidant activity of the was then chosen for the treated dosage in the present study.
Evidence-Based Complementary and Alternative Medicine 11

DNA Control TNF-𝛼 Aspirin BT25 BT50 MT25 MT50 M25 M50
Oligo 10 𝜇M 𝜇g/mL 𝜇g/mL 𝜇g/mL 𝜇g/mL 𝜇g/mL 𝜇g/mL
TNF-𝛼: − + + + + + + + +

PPAR 𝛼

Free probe

(a)
DNA Control TNF-𝛼 Aspirin BT25 BT50 MT25 MT50 M25 M50
Oligo 10 𝜇M 𝜇g/mL 𝜇g/mL 𝜇g/mL 𝜇g/mL 𝜇g/mL 𝜇g/mL
TNF-𝛼: − + + + + + + + +
PPAR𝛾

Free probe

(b)

Figure 6: Effect of treatments on the expression of PPAR 𝛼 (a) and PPAR 𝛾 (b) in TNF-𝛼-stimulated HAECs by EMSA.

In addition, lower concentration also could avoid the toxicity vascular endothelium and subsequent migration into the
effect of higher level of extracts on lymphocytes. Quercetin vessel wall are early events in atherogenesis [2]. Our results
was found to protect against H2 O2 -induced DNA damage in are consistent with previous research showing that dietary
human lymphocytes at 10 𝜇M [50] and at 3.1 to 25 𝜇M [51]. polyphenols such as catechin and quercetin significantly
Quercetin was also reported to induce DNA strand breaks in reduce the binding of monocytes to HAECs [53, 54]. The
various cell types, but only at higher doses at 100 𝜇M or above reduced cellular adhesion may be due to the inhibition
[50]. Duthie et al. [50] proposed that the dihydroxy structure of cellular adhesion molecule expression, as purple sweet
of quercetin and myricetin might be essential in protecting potato leaf extract (PSPLE) reduced both VCAM-1 and
DNA against hydrogen peroxide. No such hydroxyl groups ICAM-1 expression [40]. Shibata et al. [55] demonstrated
are present in the tocopherol molecule. Noroozi et al. [52] that pretreatment of cultured bovine aortic endothelial cells
demonstrated that, in addition to quercetin, kaempferol (BAECs) with mulberry leaf aqueous fractions inhibited
could also inhibit H2 O2 -induced DNA strand breaks in TNF-𝛼- and LPS-induced expression of Lectin-like Ox-LDL
human lymphocytes. In our study, both BT and M were rich
receptor-1 (LOX-1), a cell-surface receptor for atherogenic
in quercetin and kaempferol (Table 2), which may account for
Ox-LDL (oxidized low-density lipoprotein). It also appears
the protective roles of both extracts in preventing DNA strand
to mediate Ox-LDL-induced inflammation, which may be
breaks from human lymphocytes.
crucial in atherogenesis. Furthermore, mulberry leaf aqueous
fractions inhibited the TNF-𝛼-induced activation of NF-𝜅B
4.3. MLREs Regulated the DNA-Binding Activity and Expres- and phosphorylation of inhibitory factor of NF-𝜅B-alpha
sions of NF-𝜅B, AP-1, STAT3, and PPARs in HAECs. (I𝜅B-𝛼). Thus, mulberry leaf aqueous fractions suppress
Because MLREs exhibited free radical scavenging, the anti- TNF-𝛼- and LPS-induced LOX-1 gene expression mediated
inflammatory effects of MLREs on TNF-𝛼-induced cell by inhibiting NF-𝜅B activation.
adhesion and adhesion molecule expression were determined Chan et al. [56] demonstrated that mulberry leaf extract
in the present study. Our results suggest that MLREs and (MLE) was rich in polyphenols. MLE can effectively inhibit
aspirin significantly inhibited TNF-𝛼-induced monocyte- the migration of vascular smooth muscle cells (VSMC) by
endothelial cell adhesion (Figure 3). Significant reductions blocking small guanosine triphosphatases (small GTPase)
in NF-𝜅B expression and DNA binding by aspirin, MT and Akt/NF-𝜅B signals. The Rho family of small GTPases
extract (25 𝜇g/mL), and BT extract (both 25 and 50 𝜇g/mL) such as Rho, Rac, and Cdc42 acts as a molecular switch to
were also observed. The adhesion of monocytes to the regulate the actin cytoskeleton. The Rho family of GTPases
12 Evidence-Based Complementary and Alternative Medicine

is also reported to be involved in the regulation of NF-𝜅B- might downregulate intracellular redox-dependent signaling
dependent transcription [56]. MLE has been shown to pos- pathways in HAECs upon TNF-𝛼 stimulation, which might
sess hypoglycemic effects in an insulin-dependent diabetes prevent ROS-mediated endothelial cell dysfunction. MLRCs
mellitus (type 1 diabetes) animal model, and potential hypo- have anti-inflammatory effects through the modulation of
glycemic compounds in mulberry leaves were suggested to be NF-𝜅B, AP-1, STAT3, and PPARs signaling. These chemi-
2-O-𝛼-D-galactopyranosyl-DNJ and fagomine [57]. Konno cals appear to provide distinct cytoprotective mechanisms
et al. [58] further reported that mulberry latex contains that might contribute to their putative beneficial effects
alkaloidal sugar-mimic glycosidase inhibitors with antidia- in suppressing endothelial responses to cytokines during
betic activities such as 1,4-dideoxy-1,4-imino-D-arabinitol, 1- inflammation. These results may benefit further in vivo
deoxynojirimycin, and 1,4-dideoxy-1,4-imino-D-ribitol. Kim studies to assess the therapeutic potential of phytochemicals
et al. [59] recently showed that treated type 2 diabetes mellitus as anti-inflammatories for use in cytokine-induced vascular
(T2D) with a 1 : 1 : 1 mixture of ginseng root, mulberry leaf disorders, including atherosclerosis.
water extract, and banana leaf water extract (6 g/d) for 24
weeks significantly decreased plasma intracellular adhesion
Acknowledgment
molecule-1 (ICAM-1) and vascular cell adhesion molecule-
1 (VCAM-1) levels. This indicates suppressed inflammatory This work was supported in part by Grants from the National
responses in T2D but no significant differences in glucose Science Council of Taiwan (NSC 95–2320-B-034-001 and
homeostasis control measurement changes. Rigamonti et al. NSC 96–2320-B-034-001).
[8] reported that both PPAR 𝛼 and 𝛾 may be thought of as sup-
pressing the activity of NF𝜅B and AP-1 pathways that reduce
inflammatory mediator production in the macrophages of
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Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2013, Article ID 545739, 10 pages
http://dx.doi.org/10.1155/2013/545739

Research Article
Okadaic Acid, a Bioactive Fatty Acid from
Halichondria okadai, Stimulates Lipolysis in Rat Adipocytes:
The Pivotal Role of Perilipin Translocation

Nen-Chung Chang,1 Aming Chor-Ming Lin,2

Cheng-Chen Hsu,3 Jung-Sheng Liu,4 Leo Tsui,4 Chien-Yuan Chen,4
Thanasekaran Jayakumar,5 and Tsorng-Harn Fong3,4
1
Division of Cardiovascular, Department of Internal Medicine, School of Medicine, College of Medicine,
Taipei Medical University Hospital, Taipei, Taiwan
2
Department of Emergency, Shin Kong Wu Ho-Su Memorial Hospital, Taipei 11101, Taiwan
3
Department of Anatomy, School of Medicine, College of Medicine, Taipei Medical University, No. 250, Wu-Hsing Street,
Taipei 11031, Taiwan
4
Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan
5
Department of Pharmacology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan

Correspondence should be addressed to Tsorng-Harn Fong; [email protected]

Received 21 August 2013; Accepted 7 October 2013

Academic Editor: Joen-Rong Sheu

Copyright © 2013 Nen-Chung Chang et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

Lipid metabolism in visceral fat cells is correlated with metabolic syndrome and cardiovascular diseases. Okadaic-acid, a 38-carbon
fatty acid isolated from the black sponge Halichondria okadai, can stimulate lipolysis by promoting the phosphorylation of several
proteins in adipocytes. However, the mechanism of okadaic acid-induced lipolysis and the effects of okadaic acid on lipid-droplet-
associated proteins (perilipins and beta-actin) remain unclear. We isolated adipocytes from rat epididymal fat pads and treated
them with isoproterenol and/or okadaic acid to estimate lipolysis by measuring glycerol release. Incubating adipocytes with okadaic
acid stimulated time-dependent lipolysis. Lipid-droplet-associated perilipins and beta-actin were analyzed by immunoblotting and
immunofluorescence, and the association of perilipin A and B was found to be decreased in response to isoproterenol or okadaic
acid treatment. Moreover, okadaic-acid treatment could enhance isoproterenol-mediated lipolysis, whereas treatment of several
inhibitors such as KT-5720 (PKA inhibitor), calphostin C (PKC inhibitor), or KT-5823 (PKG inhibitor) did not attenuate okadaic-
acid-induced lipolysis. By contrast, vanadyl acetylacetonate (tyrosine phosphatase inhibitor) blocked okadaic-acid-dependent
lipolysis. These results suggest that okadaic acid induces the phosphorylation and detachment of lipid-droplet-associated perilipin
A and B from the lipid droplet surface and thereby leads to accelerated lipolysis.

1. Introduction cardiovascular disease [2, 3]. Many hormones and drugs

have been identified to participate in the regulation of lipid
Adipose tissues play critical roles in energy storage, lipid metabolism, and various hormones and drugs lead to lipoly-
metabolism, and glucose homeostasis. The development of sis through distinct lipolytic pathways [4]. The most studied
obesity may be due to excess adipose tissues accumulation or lipolytic pathway is the cAMP-dependent protein kinase
abnormal lipid metabolism. Obesity increases the risk of A (PKA) pathway in adipocytes, in which catecholamines
cardiovascular diseases and diabetes mellitus type 2, comor- bind to beta-adrenoreceptors and activate membrane-bound
bidities often detected in metabolic syndrome [1], and visceral adenylyl cyclases and thereby increase intracellular cAMP
fat is linked to metabolic disorders and increased risk of formation [5]. Subsequently, the elevated cAMP levels
2 Evidence-Based Complementary and Alternative Medicine

enhance PKA activity, leading to the phosphorylation and In this study, we used okadaic acid to induce lipolysis in
activation of hormone-sensitive lipase (HSL) [6] and lipid- primary cultures of isolated rat adipocytes. We report that
droplet-associated perilipins [7]. Activated HSL and per- glycerol release was increased in a time-dependent manner
ilipins elicit the hydrolysis of triacylglycerol stored in lipid after incubation of cells with okadaic acid. We also ana-
droplets and the release of free fatty acids and glycerol from lyzed lipid-droplet-associated perilipin A/B and beta-actin by
adipocytes [8]. immunoblotting and immunofluorescent labeling and found
Okadaic acid, a polyether derivative of a 38-carbon fatty that treatment with okadaic acid induced the detachment
acid extracted from the black sponge Halichondria okadai, is a of perilipin A and B but not of beta-actin from the surface
potent inhibitor of protein phosphatases-1 and -2A (PP1 and of lipid droplets. Furthermore, using specific inhibitors of
PP2A) [9, 10] and a known tumor promoter [11]. Okadaic protein kinase A (KT-5720), protein kinase G (KT-5823),
acid has been used to study various cellular processes such protein kinase C (calphostin C), or tyrosine phosphatases
as the cell cycle [12], apoptosis [13], nitric oxide metabolism (vanadyl acetylacetonate), we examined the signaling path-
[14], and calcium signaling [15]. Okadaic acid can stimulate ways through which okadaic acid induces lipolysis in primary
protein phosphorylation rapidly and induce a variety of rat adipocytes.
metabolic processes in diverse cell types [16, 17]. Treating
adipocytes with okadaic acid markedly increases the phos- 2. Materials and Methods
phorylation of many proteins and stimulates basal lipolysis
[16]. Okadaic acid has been reported to stimulate lipolysis by 2.1. Reagents. Okadaic acid (≥90%), isoproterenol, collagen-
inducing the translocation of phosphorylated and activated ase (type II), poly-L-lysine, leupeptin, benzamidine, sodium
HSL from the cytosol to the lipid-droplets to accelerate fluoride (NaF), ethylenediaminetetraacetic acid (EDTA),
lipolysis [18]. beta-mercaptoethanol, Triton X-100, Tween-20, dimethyl
In addition to catalytic lipases, perilipins, a family of pro- sulfoxide (DMSO), Free Glycerol Determination Kit,
teins that coat the surface of lipid droplets [7, 19], have been diaminobenzidine (DAB), n-propyl gallate, KT-5720, KT-
proposed to regulate lipid metabolism in adipocytes [8, 20]. 5823, calphostin C, vanadyl acetylacetonate, bovine serum
Perilipin is produced as distinct isoforms that are generated albumin (BSA), tris[hydroxymethyl]aminomethane (Tris),
through differential splicing. In adipocytes, perilipin A is the N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid
major isoform and perilipin B is the less abundant and shorter (TES), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
variant. Both proteins arise from alternative splicing of bromide (MTT), glutaraldehyde, rabbit polyclonal anti-
mRNA transcripts and share a common N-terminal domain perilipin A/B antibodies, mouse monoclonal anti-beta-actin
[21]. Phosphorylation of perilipin A is required for the antibody, and FITC-conjugated anti-rabbit IgG were all
translocation of HSL during PKA-stimulated lipolysis [22]. purchased from Sigma-Aldrich (St. Louis, MO). Biotin-
In adipocytes from perilipin-null animals, basal lipolysis level conjugated anti-rabbit IgG and biotin-conjugated anti-mouse
is higher and stimulated lipolysis is lower than in adipocytes IgG that was preabsorbed with rat serum were purchased
from wild-type mice. These findings suggest that perilipins from Vector Labs (Burlingame, CA). Streptavidin conjugated
serve as gatekeeper proteins that are involved in regulating with peroxidase was purchased from DAKO (Copenhagen,
lipid storage and protecting lipids against the hydrolysis by Denmark). DMEM/F12 medium (phenol-red-free) was
lipases. Phosphorylated perilipins facilitate lipase function purchased from GIBCO (Grand Island, NY), and materials
following lipolytic stimulation [20]. Okadaic acid treatment required for sodium dodecyl sulfate-polyacrylamide gel
can promote perilipin phosphorylation in adipocytes [23], electrophoresis (SDS-PAGE) were purchased from Bio-Rad
but the underlying molecular mechanism remains unclear. (Hercules, CA).
Previously, we showed that globular beta-actin was associ-
ated with intracellular lipid droplets in adipocytes [24] and 2.2. Animals. Adult male Wistar rats with body weights in
examined how okadaic acid affected lipid-droplet-associated the 200–300 g range were used for the experiments. Rats were
beta-actin. housed under a 12:12 h light/dark daily cycle at 23∘ C and were
provided with standard laboratory rat chow and water. All
The clinical implications of okadaic acid have been well
animal care was approved under guidelines established by the
documented as okadaic acid administered orally to rats Taipei Medical University Ethical Committee for Laboratory
causes intestinal damage, diarrhea, and death but has no Animals.
detectable effect on the liver [25]. On the other hand, it has
been reported that intravenous administration of okadaic 2.3. Preparation and Incubation of Isolated Adipocytes. Rats
acid causes little effect on the intestinal function but severely were injected intraperitoneally with sodium pentobarbital
affects the liver [26]. Apart from these acute effects, the (40 mg/kg body weight) and adipocytes were isolated from
okadaic acid group of toxins seems to have some important the sacrificed rats using procedures described by Rodbell [30]
chronic effects. These toxins have been found to be potent with minor modifications. Briefly, epididymal fat pads were
tumor promoters [27], and the possibility that they are also removed immediately after sacrificing animals and were
tumor inducers has been suggested [28]. Although existing washed and incubated in DMEM/F12 medium (phenol-red-
observations of human populations are not conclusive, there free). The fat pads were minced with scissors and placed in
are epidemiologic lines of evidence that associate these toxins glass vials with medium, and the fat tissue was digested using
with digestive cancer [29]. a collagenase solution (3.3 mg/mL of type-II collagenase in
Evidence-Based Complementary and Alternative Medicine 3

DMEM/F12 medium, pH 7.4, with 3 𝜇M glucose, 4% BSA) 1 h, washed with TBS, and then incubated with peroxidase-
with constant shaking at 75 rpm for 30 min at 37∘ C. Subse- conjugated streptavidin at room temperature for 1 h to
quently, fat cells were filtered through a nylon mesh and cen- enhance the signals of immunoreactive bands. After washing
trifuged for 5 min at 1000 rpm, after which the supernatant again with TBS, immunoreactive bands were visualized by
layer of cells was washed thrice with medium to eliminate exposing membranes to a diaminobenzidine solution (5%
collagenase. The packed fat cells were resuspended in buffer diaminobenzidine and 0.02% H2 O2 in TBS).
A (25 mM TES, pH 7.4, 135 mM NaCl, 5 mM KCl, and 1 mM
MgCl2 ) at 37∘ C and adjusted to a density of 1 × 105 cells/mL. 2.8. Immunofluorescent Labeling of Isolated Intracellular Lipid
Droplets. Isolated intracellular lipid droplets were placed on
2.4. Drug Treatment. Adipocytes were incubated in a total 10% poly-L-lysine-coated slides for 20 min at room temper-
volume of 500 𝜇L in the presence or absence of various ature for adhesion. After washing with phosphate-buffered
reagents (as shown in the figures); 450 𝜇L of fat cells was saline (PBS: 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2 PO4 ,
supplemented with BSA (2.5%, w/v) and incubated with and 8 mM Na2 HPO4 , pH 7.4), the isolated lipid droplets were
50 𝜇L of the drug solution (okadaic acid or isoproterenol) for fixed using 2% glutaraldehyde in PBS for 5 min. After washing
the indicated periods at 37∘ C in a CO2 incubator. The final with PBS, the lipid droplets were incubated with 5% non-
concentrations of okadaic acid and isoproterenol were 1 𝜇M fat milk in PBS for 30 min at room temperature to block
and 10 𝜇M, respectively. After incubation, the reaction mix- nonspecific binding sites. The samples were next incubated
ture was mixed well and then centrifuged at 1000 rpm for with rabbit polyclonal anti-perilipin A/B antibodies at 4∘ C
5 min to separate the medium and the fat cells, which were overnight. After washing with PBS, the samples were incu-
used to assay for glycerol release and to isolate intracellular bated with FITC-conjugated anti-rabbit IgG for 1 h at room
lipid droplets, respectively. temperature. After washing again with PBS, the samples were
mounted with 2% n-propyl gallate and 60% glycerol in PBS
2.5. Measurement of Lipolysis Based on Glycerol Release. (pH 8.0), sealed in place using nail polish, and examined
Lipolysis was measured by determining the amount of glyc- using a Nikon epifluorescence microscope (Nikon, Tokyo,
erol released into the medium that was collected after drug Japan).
treatment and centrifugation; glycerol release was measured
using a Free Glycerol Determination Kit. The glycerol content 2.9. Statistical Analysis. Data are presented as the mean ±
was calculated from absorbance at 540 nm according to SE from at least 3 independent experiments. The significance
the manufacturer’s instructions, and glycerol release was of differences between experimental and control groups was
expressed as a percentage of the vehicle control (0.1% DMSO). assessed using Student’s 𝑡-test; 𝑃 < 0.05 was considered
statistically significant.
2.6. Isolation of Intracellular Lipid Droplets and SDS-PAGE.
Intracellular lipid droplets were isolated from fat cells accord-
ing to the method of Okuda et al. [31] with minor modifica- 3. Results
tion. Briefly, isolated fat cells were washed thrice with normal 3.1. Okadaic Acid Induces Lipolysis in a Time-Dependent Man-
saline to remove BSA from the reaction medium and then ner. We investigated the effect of okadaic acid on lipolysis in
incubated in lysis buffer (5 mM Tris buffer, pH 7.4, 0.025% rat visceral fat cells. Okadaic acid (1 𝜇M) increased glycerol
Triton X-100, 1 mM EDTA, 50 mM NaF, 10 𝜇g/mL leupeptin, release by cultured adipocytes in a time-dependent manner
and 1 mM benzamidine) on ice for 15 min. The samples were (Figure 1(a)): the amounts of glycerol released were 15.5 ±
vortexed and then centrifuged at 13000 ×g for 15 min at 4∘ C. 0.2, 21.6 ± 0.8, 29.5 ± 0.2, 47.9 ± 0.1, and 74.9 ± 2.3 𝜇g/mL
The floating fat-cake fractions were collected and mixed of packed cells following treatment with okadaic acid for 0,
with equal volumes of 2X sample buffer (62.5 mM Tris, pH 15, 30, 60, and 120 min, respectively. During incubation for
6.8, 5% beta-mercaptoethanol, 2% SDS, and 10% glycerol). 15, 30, 60, and 120 min, lipolysis increased 1.4-, 1.9-, 3.1-, and
The samples were heated to 95∘ C for 5 min and clarified by 4.8-fold, respectively (Figure 1(b)).
centrifugation at 10000 rpm for 5 min before using them for
SDS-PAGE. Equal amounts of fat-cake extracts were loaded 3.2. Okadaic Acid Treatment Diminishes the Association of
and resolved by SDS-PAGE on 7.5% polyacrylamide slab gels. Perilipins A and B with Lipid Droplets. To explore the role of
lipid-droplet-associated perilipins in okadaic-acid-induced
2.7. Western Blotting. After electrophoresis, proteins were lipolysis, we used immunoblotting to examine perilipins
transferred to nitrocellulose membranes and blocked with during the 120 min treatment with okadaic acid. Almost
5% nonfat milk in Tris-buffered saline (TBS; 50 mM Tris no lipid-droplet-associated perilipin A was detected after
and 200 mM NaCl, pH 7.5), containing 0.05% Tween-20 at cells were treated for 30 min with okadaic acid (data not
room temperature for 1 h and then incubated overnight at shown), and, therefore, we tested shorter incubation times.
4∘ C with rabbit polyclonal anti-perilipin A/B antibodies or To investigate the short-term effects of okadaic acid on
mouse monoclonal anti-beta-actin antibody diluted in TBS. lipolysis, fat cells were treated with 1 𝜇M okadaic acid for
After washing with TBS, the membrane strips were incubated 0, 5, 10, 15, 20, and 30 min. Glycerol release increased only
with biotin-conjugated anti-rabbit IgG or biotin-conjugated slightly after incubation with okadaic acid for 5 min but
anti-mouse IgG (rat serum preabsorbed to avoid nonspecific increased substantially after incubation for 10, 15, 20, and
binding between mouse and rat) at room temperature for 30 min (Figure 2(a)). Furthermore, we used immunoblotting
4 Evidence-Based Complementary and Alternative Medicine

Lipolysis (glycerol mg/mL packed cells)

0.1 600

Glycerol release (% of control)

∗∗∗
∗∗∗ 500
0.08
400
0.06 ∗∗∗ ∗∗∗
300
0.04 ∗∗∗ ∗∗∗
∗ 200 ∗
0.02 100

0 0
0 15 30 45 60 75 90 105 120 0 15 30 60 120
Incubation time (min) Incubation time (min)
(a) (b)

Figure 1: Time course of okadaic-acid-induced glycerol release in rat adipocytes. Fat cells were incubated with okadaic acid (1 𝜇M, 37∘ C)
for the indicated periods. After incubation, media were collected and glycerol release was measured as described in Materials and Methods.
Glycerol released into the media was assayed as an index of lipolysis and expressed as mg/mL of packed cell volume (a). Glycerol release
data are expressed as percentages of the values obtained after incubation for 0 min (b). Each point represents the mean ± SE of three separate
experiments. ∗ 𝑃 < 0.05 and ∗∗∗ 𝑃 < 0.001 compared with values of 0 min incubation.

to examine lipid-droplet-associated perilipins during the and okadaic-acid-treated group (Figures 3(g) and 3(h)). This
30 min incubation with okadaic acid. The rabbit polyclonal result suggested that most of the perilipins detached from the
anti-perilipin A/B antibodies can label native perilipin A surface of lipid droplets after cells were incubated with the
(62 kD), phosphorylated perilipin A (65 kD), and perilipin drugs.
B (46 and 48 kD). We detected phosphorylated perilipin A
(65 kD) after 5 min treatment with okadaic acid and observed 3.4. Okadaic Acid Enhances Isoproterenol-Induced Lipolysis.
that the levels of lipid-droplet-associated perilipin A (62 kD Isoproterenol (10 𝜇M) or okadaic acid (1 𝜇M) has been
and 65 kD) and perilipin B (46 kD and 48 kD) decreased shown previously to induce lipolysis [16]. To compare the
gradually following okadaic acid treatment (Figure 2(b)). signal transduction pathways in the lipolysis induced by
Quantification of these results demonstrated that total per- okadaic acid and isoproterenol, we combined the 2 drugs
ilipin A decreased only by 0.98-fold after 5 min but decreased and stimulated fat cells. Incubation of cells with isoproterenol
significantly after 10, 15, 20, and 30 min incubation by 0.56-, (10 𝜇M) for 120 min stimulated glycerol release that was 3.0-
0.47-, 0.22-, and 0.18-fold, respectively (Figure 2(c)). The fold higher than in the buffer-A group, and incubation with
levels of total perilipin B decreased significantly after 5, 10, okadaic acid (1 𝜇M) for 120 min stimulated glycerol release
15, 20, and 30 min incubation by 0.73-, 0.65-, 0.51-, 0.26-, and that was 4.1-fold higher than in the DMSO group; however,
0.35-fold, respectively (Figure 2(d)). By contrast, the level of incubation of cells with both isoproterenol and okadaic
lipid-droplet-associated beta-actin (42 kD) did not decrease acid for 120 min stimulated 5.6-fold higher glycerol release
noticeably during the 30 min incubation with okadaic acid compared to the DMSO group (Figure 4). These results
(Figure 2(b)). demonstrated that okadaic acid treatment can enhance
isoproterenol-induced lipolysis in rat adipocytes.
3.3. Okadaic Acid and Isoproterenol Induce Detachment of
Perilipins from Lipid Droplets. We used immunofluorescent 3.5. A Tyrosine-Phosphatase Inhibitor but Not Inhibitors
labeling to investigate morphologically how isoproterenol of PKA, PKC, and PKG Regulates Okadaic-Acid-Induced
and okadaic acid affect lipid-droplet-associated perilip- Lipolysis. To determine whether PKA, PKG, and PKC affect
ins. Labeling with the polyclonal anti-perilipin antibodies okadaic-acid-induced lipolysis in adipocytes, we used KT-
revealed bright fluorescence along the circumference of the 5720 (specific inhibitor of PKA), KT-5823 (specific inhibitor
isolated intracellular lipid droplets in the buffer-A group (Fig- of PKG), and calphostin C (specific inhibitor of PKC). Basal
ures 3(a) and 3(b)) and in the 0.1%-DMSO group (Figures 3(c) level of glycerol release was not affected by treatment with
and 3(d)). This result indicated that perilipins are not only these 3 inhibitors, and preincubating cells with the inhibitors
coisolated with intracellular lipid droplets but also associated did not abolish or attenuate okadaic-acid-induced lipolysis
with the surface of lipid droplets. (Figure 5). By contrast, preincubation with the tyrosine-
When we incubated fat cells with isoproterenol (10 𝜇M) or phosphatase blocker vanadyl acetylacetonate (300 𝜇M)
okadaic acid (1 𝜇M) for 120 min and then isolated intracellu- potently inhibited okadaic-acid-induced lipolysis (Figure 6).
lar lipid droplets for immunofluorescent labeling, only weak The data suggested that tyrosine phosphatases, but not PKA,
fluorescence was detected surrounding the lipid droplets PKG, or PKC, are involved in okadaic-acid-induced lipolysis
in the isoproterenol-treated group (Figures 3(e) and 3(f)) in rat visceral fat cells.
Evidence-Based Complementary and Alternative Medicine 5

160
∗∗
Glycerol release (% of control)

140 ∗
∗ ∗
120
100
80
65 kD
Perilipin A 62
60 kD

40 Perilipin B 48 kD
46 kD
20
Actin 42 kD
0
0 5 10 15 20 30 0 5 10 15 20 30
Incubation time (min) Incubation time (min)
(a) (b)
1.2 1.2

1 1
Perilipin A (folds/basal)

Perilipin B (folds/basal)
0.8 0.8 ∗∗ ∗∗
∗∗
∗∗ ∗∗
0.6 0.6
∗∗
0.4 ∗∗ 0.4 ∗∗∗
∗∗
0.2 0.2

0 0
0 5 10 15 20 30 0 5 10 15 20 30
Incubation time (min) Incubation time (min)
(c) (d)

Figure 2: Effects of okadaic acid on lipid-droplet-associated perilipin A and B in rat adipocytes. Cells were incubated with okadaic acid (1 𝜇M,
37∘ C) for the indicated periods. After incubation, each reaction mixture was centrifuged to separate the medium from the fat cells. Glycerol
release was measured as described in Materials and Methods, and glycerol release data are expressed as percentages of the values obtained
after incubation for 0 min (a). After incubation, fat cells were homogenized and centrifuged and the proteins of the fat cake fraction were
separated using SDS-PAGE (7.5% gels) and subjected to immunoblotting with a primary antibody against perilipins. Immunoblots showing
perilipin A (62 kD and 65 kD) and B (46 kD and 48 kD) or beta-actin (42 kD) from okadaic-acid-treated cells (b). Immunoreactive perilipin
A (c) and perilipin B (d) were quantified as a percentage relative to the density in unstimulated fat cells. Each point represents the mean ±
SE of three separate experiments; ∗ 𝑃 < 0.05, ∗∗ 𝑃 < 0.01, and ∗∗∗ 𝑃 < 0.001 compared with values of 0 min incubation. The shift in the
molecular weight of perilipin A from 62 kD to 65 kD could be observed after 5 min stimulation with okadaic acid.

4. Discussion and PP2A potently [16, 17]. When adipocytes are incubated
with 1 𝜇M okadaic acid, which is sufficient for inhibiting
Our results have demonstrated that treatment of adipocytes PP1 and PP2A, the phosphorylation of many proteins is
with okadaic acid can induce lipolysis in a time-dependent increased and glycerol release is stimulated in adipocytes
manner. Perilipin A (62 kD) and B (46 and 48 kD) and beta- [16, 23]. PP1 and PP2A are abundant in rat adipocytes and
actin (42 kD) were abundant in quiescent fat cells and were also the major phosphatases in these cells [32]. PP2A is
associated with lipid droplets isolated from these cells. After the principal phosphatase responsible for dephosphorylating
incubating adipocytes for 5 min with okadaic acid, phos- HSL in adipocytes [33]. Conversely, PP1 is the main phos-
phorylated perilipin A (65 kD) was detected, and following phatase that dephosphorylates perilipin in adipocytes [34].
okadaic acid treatment for 10 min, the amounts of perilipin Thus, treatment with okadaic acid could inhibit both PP1
A and B associated with lipid droplets decreased and glyc- and PP2A to enhance the phosphorylation of both perilipin
erol release increased substantially. Furthermore, okadaic- and HSL and stimulate lipolysis. In our study, treatment
acid-induced lipolysis was suppressed by an inhibitor of with okadaic acid (1 𝜇M, for 2 h) increased the release of
tyrosine phosphatases but not by inhibitors of PKA, PKG, glycerol 4.8-fold in freshly prepared fat cell suspensions (1
or PKC. These results suggest that treatment with okadaic × 105 cells/mL). Previously, glycerol release was increased by
acid activates tyrosine phosphatases and leads to perilipin A approximately 11-fold in freshly prepared fat cell suspensions
phosphorylation, which results in the detachment of perilipin (3 × 105 cells/mL) incubated with 2 𝜇M of okadaic acid for 3 h
A and B from the surface of lipid droplets and leads to [35]. Thus, okadaic acid might induce lipolysis in a concen-
lipolysis and glycerol release in rat visceral adipocytes. tration- and time-dependent manner.
Okadaic acid, a polyether derivative of fatty acid, can Lipid-droplet-associated perilipin is considered to act as a
penetrate the plasma membrane readily and inhibit PP1 barrier or gatekeeper with critical roles in regulating cellular
6 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(a) (b)

(c) (d)

(c) (d)

(e) (f)

(e) (f)

(g) (h)

(g) (h)

Figure 3: Immunofluorescent labeling of perilipin A/B in isolated intracellular lipid droplets. Adipocytes were incubated with buffer A,
DMSO, isoproterenol (10 𝜇M), or okadaic acid (1 𝜇M) for 2 h at 37∘ C. Perilipins were assembled as a bright ring surrounding the surface of
lipid droplets in buffer A control (a and b) and DMSO (c and d), but labeling for perilipins was weak on lipid droplets in the isoproterenol-
stimulated group (e and f) and absent on lipid droplets in the okadaic-acid-stimulated group (g and h). Figures (a), (c), (e), and (g) show
phase-contrast images of figures (b), (d), (f), and (h), respectively. Arrows indicate isolated intracellular lipid droplets. Bar = 20 𝜇m.

lipid metabolism [8, 20]. Perilipin A (62 kD) is a prominent (65 kD), and two perilipin B bands (46 and 48 kD) on
phosphoprotein that becomes phosphorylated after isopro- immunoblots. The 65/67 kD doublet of phosphorylated per-
terenol treatment, after which the protein migrates as a ilipin A was not identified here possibly because we did not
65/67 kD doublet in SDS-PAGE and is also the most heavily use radiolabels or because of the limitations of the anti-
radiolabeled protein in the cell [7]. The anti-perilipin A/B perilipin antibody.
polyclonal antibodies we used in this study can recognize Treatment with isoproterenol can stimulate the phospho-
native perilipin A (62 kD), phosphorylated perilipin A rylation and translocation of perilipin from the lipid-droplet
Evidence-Based Complementary and Alternative Medicine 7

0.1 0.1
Lipolysis (glycerol mg/mL packed cells)

††

Lipolysis (glycerol mg/mL packed cells)

++
0.08 0.08 ∗∗∗

###
0.06
0.06
∗∗∗
0.04
0.04 ∗
###
0.02
0.02
0
Buffer A DMSO ISO OA ISO + OA
0
Figure 4: Okadaic acid enhances isoproterenol-induced glycerol DMSO + − − −
release. Adipocytes were incubated for 2 h with isoproterenol
(10 𝜇M) or okadaic acid (1 𝜇M) or both. Glycerol release was Vanadate − + − +
assayed (in triplicate) as an index of lipolysis and expressed as Okadaic acid − − + +
mg/mL of packed cell volume. Data are the mean ± SE of three
separate experiments. ∗∗∗ 𝑃 < 0.001 compared with buffer A Figure 6: Effect of tyrosine phosphatase inhibition on okadaic-
acid-induced lipolysis in rat adipocytes. Cells were incubated
group, ### 𝑃 < 0.001 compared with DMSO-treated group, ++ 𝑃 <
with okadaic acid (1 𝜇M) in the absence or presence of vanadyl
0.01 compared with isoproterenol-treated group, and †† 𝑃 < 0.01
acetylacetonate (300 𝜇M) for 2 h at 37∘ C. The medium was collected
compared with okadaic acid-treated group. ISO: isoproterenol; OA:
and glycerol release was determined. ∗ 𝑃 < 0.01 and ∗∗∗ 𝑃 < 0.001
okadaic acid.
compared to the DMSO-treated group, ### 𝑃 < 0.001 compared to
the okadaic-acid-treated group. Vanadate: vanadyl acetylacetonate.
0.1
###
Lipolysis (glycerol mg/mL

∗∗∗ +++ †††

0.08
packed cells)

0.06 does not increase the catalytic activity of HSL but induces
the translocation of HSL to the lipid droplets. Phosphorylated
0.04 HSL translocates from the cytosol to the surface of lipid
droplets [37, 38], and phosphorylated perilipins might detach
0.02 from the surface of droplets to expose the triacylglycerol
stored in lipid droplets and then facilitate lipid hydrolysis by
0 HSL [20, 22].
On the other hand, we found that when adipocytes were
Buffer A + − − − − − − − −
− + − − − − − − −
incubated with okadaic acid or isoproterenol, at different
DMSO
KT-5720 − − + − − − + − − incubation periods (0–30 min), the expression levels of beta-
KT-5823 − − − + − − − + − actin in lipid droplets remain unchanged. The cytoskeleton
Calphostin C − − − − + − − − + proteins associated with lipid droplets seem to be resistant
Okadaic acid − − − − − + + + + to drug treatment. For example, formation of vimentin
intermediate filamentous cages enclosed and protected the
Figure 5: Effects of inhibitors of PKA, PKG, and PKC on okadaic nascent lipid droplets of 3T3-L1 adipocytes from disruption of
acid-induced lipolysis in rat adipocytes. Cells were incubated with
colchicine treatment [39]. Hence, we suggested that the lipid-
inhibitor alone or okadaic acid (1 𝜇M) in the absence or presence of
inhibitors of PKA (KT-5720; 0.3 𝜇M), PKG (KT-5823; 3 𝜇M), or PKC
droplet-associated beta-actin might be a stable structural
(calphostin C; 0.5 𝜇M) for 2 h at 37∘ C; media were then collected protein on the surface of intracellular lipid droplet.
and glycerol release was measured. ∗∗∗ 𝑃 < 0.001 compared to the In this study, isoproterenol-induced lipolysis was
DMSO-treated group, ### 𝑃 < 0.001 compared to the KT-5720- enhanced by okadaic acid treatment, which agrees with the
treated group, +++ 𝑃 < 0.001 compared to the KT-5823-treated previous results [16]. Isoproterenol-stimulated lipolysis, but
group, and ††† 𝑃 < 0.001 compared to the calphostin-C-treated not okadaic-acid-induced lipolysis, was antagonized by insu-
group. lin in a previous study [35], and we found that okadaic-
acid-induced lipolysis was not inhibited by a PKA blocker
(KT-5720). These findings suggest that okadaic acid and
surface to the cytosol in rat adipocytes [36, 37]. We observed isoproterenol stimulate lipolysis through distinct signaling
here that okadaic acid treatment of rat adipocytes also leads pathways.
to the detachment of perilipin A and B from the surface of PKA is involved in catecholamine- or isoproterenol-
lipid droplets. Thus, phosphorylation may trigger perilipin induced lipolysis, and PKG is responsible for lipolysis stim-
disassociation from the surface of lipid droplets. Moreover, ulated by atrial natriuretic peptide [4]. Because the specific
Morimoto et al. [18] reported that okadaic acid treatment inhibitors of PKA (KT-5720) and PKG (KT-5823) did not
8 Evidence-Based Complementary and Alternative Medicine

abolish or attenuate okadaic-acid-induced glycerol release in Authors’ Contribution

our study, we suggest that neither PKA nor PKG is involved
in okadaic-acid-induced lipolysis in rat adipocytes. Okadaic Dr. Nen-Chung Chang and Dr. Aming Chor-Ming Lin con-
acid has been reported to inhibit PP2A and activate protein tributed equally to this work.
kinase B (PKB) [40] and then promote the translocation of
glucose transporter 4 (GLUT4) from cytosol to cell mem- Acknowledgments
brane and increase glucose uptake in adipocytes [41]. In the
case of okadaic acid treatment, glucose uptake and GLUT4 This study was supported by research Grants from Taipei
translocation in rat adipocytes and 3T3-L1 preadipocytes Medical University Hospital (100-TMU-TMUH-03) and Shin
might be mediated through the activation of PKC-zeta and/or Kong Wu Ho-Su Memorial Hospital (SKH-TMU-93-33).
PKC-lambda [42]. These reports suggest that PKB and PKC
are responsible for okadaic-acid-induced glucose uptake.
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Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2013, Article ID 925625, 14 pages
http://dx.doi.org/10.1155/2013/925625

Review Article
Effects and Mechanisms of Chinese Herbal Medicine in
Ameliorating Myocardial Ischemia-Reperfusion Injury

Qing Liu,1,2 Jiqiang Li,2 Jing Wang,2 Jianping Li,1 Joseph S. Janicki,1 and Daping Fan1
1
Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC 29208, USA
2
The Second Clinical School of Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510405, China

Correspondence should be addressed to Daping Fan; [email protected]

Received 24 July 2013; Revised 26 August 2013; Accepted 4 September 2013

Academic Editor: Joen-Rong Sheu

Copyright © 2013 Qing Liu et al. This is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Myocardial ischemia-reperfusion (MIR) injury is a major contributor to the morbidity and mortality associated with coronary
artery disease, which accounts for approximately 450,000 deaths a year in the United States alone. Chinese herbal medicine,
especially combined herbal formulations, has been widely used in traditional Chinese medicine for the treatment of myocardial
infarction for hundreds of years. While the efficacy of Chinese herbal medicine is well documented, the underlying molecular
mechanisms remain elusive. In this review, we highlight recent studies which are focused on elucidating the cellular and molecular
mechanisms using extracted compounds, single herbs, or herbal formulations in experimental settings. These studies represent
recent efforts to bridge the gap between the enigma of ancient Chinese herbal medicine and the concepts of modern cell and
molecular biology in the treatment of myocardial infarction.

1. Introduction formulations, has been widely used in traditional Chinese

medicine for the treatment of MI for hundreds of years.
Myocardial infarction (MI) and the accompanying acute The purpose of this review is to highlight recent studies that
loss of viable myocardium is the leading cause of death in experimentally address the mechanistic effects of extracted
industrialized countries. Even if the patient survives the acute compounds, single herbs, or herbal formulations on several
phase of MI, the subsequent adverse myocardial remod- factors and pathways known to be involved in MIR injury.
eling and impairment of cardiac function severely impact
their quality of life and 5-year survival. Early restoration 2. Myocardial Ischemia-Reperfusion Injury
of blood flow to the ischemic myocardium is a common
treatment strategy aimed at limiting myocardial infarct size. 2.1. Oxidative Stress. Reactive oxygen species (ROS) have
However, reperfusion can cause additional cell death and, in both a physiological and pathological role in cellular and
many cases, paradoxically increase infarct size, a situation tissue adaptation to environmental factors. Normally, low
referred to as myocardial ischemia-reperfusion (MIR) injury. levels of oxygen radicals and oxidants are present in cells and
MIR is characterized by a rapid increase in cytokines and are important in maintaining cellular homeostasis, mitosis,
chemokines and an influx of leukocytes into the vulnera- differentiation, and signaling [2]. However, during MIR, ROS
ble region bordering the infarcted site. This inflammatory formation is markedly increased and cellular injury occurs
response not only results in cardiomyocyte apoptosis during (Figure 1). Although mammalian cells express endogenous
the acute phase, but also results in an adverse myocar- free radical scavenging enzymes, such as superoxide dis-
dial remodeling that further compromises cardiac func- mutase (SOD), catalase (CAT), and glutathione peroxidase
tion. Therefore, limiting ischemia-reperfusion (I/R) induced (GPx), these antioxidative defenses are insufficient during
myocardial inflammation may not only lower the acute death MIR [3, 4]. Oxidative stress during MIR injury contributes
rate, but also improve long term survival and quality of life to a vicious cycle as it promotes mitochondrial dysfunction,
[1]. Chinese herbal medicine, especially combined herbal excitotoxicity, lipid peroxidation, and inflammation [5–7].
2 Evidence-Based Complementary and Alternative Medicine

Ischemia Reperfusion
Palmatine, Forsythoside B, (1) Antioxi Cardiomyocyte
dation
SiNi Decoction, etc. Anaerobic metabolites
(2) Anti-inflammation −O2
Free radicals +O2

Tanshinone IIA,
Cytokines, chemokines, (7) Inhibiting Astragaloside IV,
ROS
Schisandrin B, ShuMai
adhesion molecules Ca2+ overload Lycium barbarum,
(MCP1, HSP, HMGB)
Decoction, etc. Ca2+ overload Acanthopanax
TNFR Senticosus
TLRs ILR injection, etc.
MPTP
AMPK, JNK, NF-𝜅B
TLRs
Inflammatory pathways
cells Bax,
Caspase
poptosis Bcl
Salidroside, Tyrosol, (3) Anti-a Mitochondrion
Cardiotonic Pill, etc. CXCR4 Apoptotic
Tissue damage
SDF-1 cell death
(4) Protecting mito
VEGF chondrial function
Angiogenesis
BMSCs migration
(5) Increasing
BMSCs migration (6) Promoting angiogenesis Herba Cistanches,
Tanshinone IIA, Radix et Rhizoma Rhodiolae Kirilowii, ShuMai Cistanche, Guanxin II,
Salvianolic acid B, etc. Decoction,TongXinLuo Superfine, etc. etc.

Figure 1: Effects and mechanisms of Chinese herbal medicine in myocardial ischemia-reperfusion (MIR) injury. During ischemia, oxygen is
not available to accept the electrons in the metabolic degradation of substrates, and consequently anaerobic metabolites become important
in the preservation of myocardial viability. However, free radicals and reactive oxygen species (ROS) formation is markedly increased in
this procedure. Reperfusion also generates high ROS levels which have an adverse impact on specific signal transduction systems, thereby
predisposing the heart to further oxidative cell damage. Damaged cell debris, fibrinogen, cytokines, and chemokines will activate the receptors,
including TLRs, TNFR, and ILR, in the host inflammatory cells as well as the cardiomyocytes. This sterile inflammatory process leads to
the formation of a vicious circle, whereby the cardiomyocyte TLRs, TNFR, and ILR are activated by inflammatory cell-generated ligands.
Typically, this has an adverse impact on specific signal transduction systems (e.g., AMPK, JNK, and NF-𝜅B pathways), thereby activating the
caspase cascade. Elevated ROS levels also result in intracellular Ca2+ overload which adversely affects mitochondrial function by opening
the mitochondrial permeability transition pore (MPTP). As a result, the balance between Bax and Bcl is interrupted and the caspase cascade
is further activated, leading to apoptotic cell death and myocardial tissue damage. Injured tissue expresses SDF-1, which interacts with its
specific receptors (e.g., CXCR4) to facilitate the trafficking, adhesion, and infiltration of bone marrow derived stem cells (BMSCs). BMSCs
produce high levels of the endothelial cell-specific angiogenic factor, VEGF, which is a critical regulator of angiogenesis that includes the
stimulation of proliferation, migration, and proteolytic activity of endothelial cells and eventually leads to an increase in vessel density and
the facilitating of myocardial regeneration and remodeling. During the MIR injury process, there are seven target areas where Chinese herbal
medicine can exert protective effects on cardiomyocyte. Examples are as follows: (1) anti-oxidation actions of Palmatine, Forsythoside B,
and SiNi Decoction; (2) anti-inflammatory properties of Tanshinone IIA, Schisandrin B, and ShuMai Decoction; (3) anti-apoptosis ability
of Salidroside, Tyrosol, and Cardiotonic Pill; (4) protection of mitochondrial function by Herba Cistanches, Cistanche, and Guanxin II; (5)
increasing BMSCs migration by Tanshinone IIA and Salvianolic acid B (6) promoting angiogenesis by Radix et Rhizoma Rhodiolae Kirilowii,
ShuMai Decoction and TongXinLuo Superfine; and (7) inhibiting Ca2+ overload by Astragaloside IV, Lycium barbarum, and Acanthopanax
senticosus injection.

2.2. Sterile Inflammation. Ischemia and reperfusion cause inflammation during the acute phase determines the extent to
sterile inflammation. Nevertheless, the consequences of MIR which cardiac function is compromised during the following
share many phenotypic parallels with activation of a host myocardial remodeling phase.
immune response directed toward invading microorganisms During the sterile inflammation phase of MIR, TLRs play
[8]. This sterile inflammation is mainly triggered by the detrimental roles as demonstrated by extensive experimental
interactions between toll-like receptors (TLRs) and their evidence [11]. To date, 11 TLRs (TLR1–TLR11) have been
endogenous ligands generated in ischemic and reperfused identified in mammals. It should be noted that, during MIR,
myocardium, such as apoptotic cell debris, fibrinogen, high the expression of TLR4 is significantly increased in both the
mobility group box (HMGB) 1, and heat shock proteins failing myocardium, and infiltrated macrophages and thus
(HSPs) [9]. The activation of immune cell and cardiomyocyte TLR4 is thought to be a central mediator of inflammation
TLR and other signaling pathways results in a vicious cycle and cardiac injury. TLR4 has been identified as a mediator
of inflammatory response in the I/R region and causes of inflammation and organ injury in several models of
significant cardiomyocyte apoptosis (Figure 1). Following the sterile tissue injury including MIR, and a soluble inhibitor
acute I/R period, the cardiac function is further compromised of TLR4 was able to prevent contractile dysfunction in wild-
by adverse myocardial remodeling [10]. The magnitude of the type cells [12]. Using a temporary left anterior descending
Evidence-Based Complementary and Alternative Medicine 3

(LAD) artery occlusion model, Oyama et al. first observed oxidative stress, mitochondrial dysfunction, and cell death
myocardial infarct size reductions in 2 distinct strains of [30]; it is a key modulation event in cell death medi-
mice that lack functional TLR4 signaling, accompanied with ated by reactive oxygen and nitrogen species [31]. JNK is
reduced neutrophil infiltration in the affected myocardium also required for TNF-𝛼-stimulated ROS production and
[13]. TLR2, which is expressed in cardiomyocytes and many cytochrome c-mediated cell death; Bcl-2 family members
other cell types, also contributes to the pathogenesis of are essential components of this mitochondrial apoptotic
cardiac dysfunction during MIR [14, 15]. Activation of TLR2, machinery. Studies have suggested that blockage of JNK
TLR4, and TLR5 increases the myocardial level of the inflam- mitochondrial translocation or JNK inhibition prevents ROS
matory cytokines, chemokines, and cell surface adhesion production and mitochondrial dysfunction and may be an
molecules [16]. Given the known role of TLR4 and TLR2 in effective treatment for I/R-induced cardiomyocyte death
MIR, inhibition of TLR4 and TLR2 signaling is a promising [32–35]. The nuclear factor kappa B (NF-𝜅B) also modu-
approach to reduce morbidity and mortality in MI patients. lates apoptosis during ischemia and reperfusion [36]. TLR
There are a variety of TLR ligands generated during signaling pathway leads to translocation of NF-𝜅B to the
MIR. For example, heat shock proteins (HSPs) are a class nucleus and thus up-regulation of expression of proinflam-
of molecular chaperones that promote intracellular protein matory cytokines. However, there is the possibility that a
folding. They may be released into the extracellular space crosstalk between the TLR/NF-𝜅B and PI3K/Akt signaling
after cell trauma and interact with adjacent cells or distant pathways and modulation of the crosstalk could protect the
cells via bloodstream delivery [17]. Extracellular HSP60 myocardium from I/R injury [37].
induced apoptotosis via the activation of TLRs [18]. Another Within the mitochondria dependent intrinsic apoptosis
example is HMGB1 which is a damage-associated molecular pathway, which has an important function in cardiac cell
pattern (DAMP) protein secreted by injured cells [19]. It injury under various pathological conditions [38], mitochon-
plays a major role in early MIR by binding to TLRs and drial permeability transition pore (MPTP) opening plays a
the receptor for advanced glycation end products (RAGE), pivotal role [39]. The event of MPTP opening is affected by
resulting in the activation of proinflammatory pathways and various factors including intracellular Ca2+ , oxidative radi-
enhanced myocardial injury [20]. In fact, a prerequisite for cals, ATP levels and the levels of Bcl-2 family proteins [40].
neutrophil-mediated tissue damage is the “priming” effect
of various pro-inflammatory stimuli generated by dam-
aged tissue during MIR, such as HSP60 and HMGB1 [21]. 2.4. Bone Marrow Stem Cell Migration. Bone marrow mes-
Cytokines released by TLR-activated cells such as tumor enchymal stem cells (BMSCs) are multipotent cells that
necrosis factor-alpha (TNF-𝛼) and IL-1 can elicit neutrophil secrete angiogenic factors. Injured tissues express specific
polarization and upregulation of cell-surface glycoproteins receptors, such as CXCR4, and/or their ligands including
such as macrophage adhesion molecule-1 (Mac-1) [22]; Mac- stromal cell-derived factor-1 (SDF-1), to facilitate trafficking,
1 upregulation in peripheral neutrophils is a very early event adhesion, and infiltration of BMSCs. During MIR, BMSCs are
in MIR [23]. preferentially attracted to and retained in the ischemic tissue
[41, 42]. As a result of the hypoxic microenvironment, these
2.3. Apoptosis and Mitochondrial Function. MIR leads to BMSCs produce high levels of vascular endothelial growth
the activation of cell death programs, including apoptosis, factor (VEGF), leading to an increase in vessel density and
autophagy-associated cell death, and necrosis [24]. Apoptosis facilitating myocardial regeneration and remodeling [43, 44]
involves an orchestrated caspase signaling cascade, including (Figure 1).
caspase-3 and caspase-9, which induces a self-contained
program of cell death, characterized by the shrinkage of 2.5. Angiogenesis. Angiogenesis refers to the sprouting,
the cell and its nucleus, with plasma membrane integrity bridging, intussusception, and/or enlargement of capillaries.
persisting until late in the process [25]. The balance between In the late stage of MI repair, enhancement of blood flow
apoptotic factors Bcl-2 and Bax has been found altered in car- to ischemic myocardium can result from either true angio-
diomyocytes during ischemia [26]. Autophagy is stimulated genesis or the recruitment of preexisting coronary collat-
by nutrient starvation and growth factor deprivation when erals [45]. VEGF is an endothelial cell-specific angiogenic
cells are unable to take up external nutrients. Autophagy is factor and also a critical regulator of angiogenesis that
also activated by decreases in ATP in order for the cell to stimulates proliferation, migration, and proteolytic activity of
maintain energy homeostasis and survival. Autophagy may endothelial cells [46]. Ischemia or coronary artery occlusion
serve primarily to maintain energy production during acute induces myocardial VEGF expression, which leads to an
ischemia but switch to clear up damaged organelles during angiogenesis-induced restoration of tissue blood flow and the
chronic ischemia or reperfusion [27]. prevention of further tissue damage (Figure 1). In addition,
Multiple cell signaling pathways, such as the AMPK, VEGF is a potent survival factor during physiological and
JNK, and NF-𝜅B pathways, have been shown to be involved tumor angiogenesis, and has been shown to induce expres-
in MIR-induced cardiomyocyte apoptosis (Figure 1). AMPK sion of anti-apoptotic proteins in endothelial cells [47, 48].
orchestrates the regulation of energy-generating and energy-
consuming pathways; its activation has been shown to protect
the heart against ischemic injury [28, 29]. Activated JNK 2.6. Other Factors. The activation of ATP-sensitive potassium
signaling, especially in mitochondria, is associated with (KATP) channel subunits and ATPase, and calcium (Ca2+ )
4 Evidence-Based Complementary and Alternative Medicine

overload are also involved in MIR (Figure 1). Ischemia- tissues, as well as the COX-2 and iNOS expressions in
reperfusion may activate some ion channels that do not open MIR myocardium of rats [49]. Jiang et al. reported that
under normal physiological conditions. One such channel is the MDA content and MPO activity in ischemic myocardial
the KATP channel, whose activation facilitates potassium ion tissue of rats treated with Forsythoside B, a compound
efflux, hyperpolarization, and action potential repolarization. derived from the Chinese herb, Lamiophlomis rotate (Benth.)
The resulting shortening of the action potential duration Kudo, were both significantly reduced. These reductions
decreases the total influx of sodium and calcium, which were accompanied by a significantly improved recovery
alleviates overloading of intracellular calcium (Ca2+ ) which in myocardial function [50]. Hwa et al. reported that 2-
in turn weakens myocardial contraction force and reduces Methoxycinnamaldehyde (2-MCA), a compound derived
myocardial oxygen consumption. Therefore, the opening of from the Chinese herb, Cinnamomum cassia, significantly
KATP channels plays an active role in protecting the heart increased HO-1 induction by promoting the translocation of
against MIR injury. Nrf-2 from cytosol to nucleus in endothelial cells in an MIR
model [51]. In addition, Hu et al. demonstrated that cyclovi-
robuxine D, a compound derived from the Chinese herb,
3. Effects and Mechanisms of Chinese Herbal Buxus microphylla, significantly protected rat aorta endothe-
Medicine in MIR lial cells against hypoxia-induced injury and enhanced nitric
oxide (NO) release from endothelial cells; these effects were
The typical symptoms of cardiovascular diseases induced inhibited by nitric oxide synthase (NOS) inhibitor N-nitro-
by MIR have been recorded in several ancient books of L’argininemethyl ester (L-NAME) [52]. Das et al. studied
Traditional Chinese Medicine (TCM), such as Inner Canon the effects of a single herb, Makhana, and demonstrated
of Huangdi and Treatise on Febrile Diseases. In TCM, Qi that the cardioprotective properties of Makhana were linked
(energy) and Blood (material) are the main components to its ability to scavenge ROS [53]. Some decoctions and
compromised in MIR, whereby the principal mechanism is patent drugs made up of Chinese herbs have also been
considered to be a disorder or deficiency of Qi and a disorder shown to exert the anti-oxidative effects on MIR. Zhao et
of the circulation (blood stasis) that results in severe pain al. found that SiNi Decoction (SND), composed of Chinese
and even death. Therefore, the main aims of Chinese herbs herbs, Aconite, Ginger and Licorice, could enhance the activity
and herbal formulations in MIR treatment are to regulate of myocardial and myocyte mitochondrial SOD and reduce
or replenish Qi, and to unblock circulation or resolve blood MDA by increasing the expression of Mn-SOD mRNA [54].
stasis. In Tables 1–4, we list four categories of Chinese herbal Wang et al. reported that in rats treated with Acanthopanax
medicine that have been used in the practice of TCM and/or Senticosus Injection (ASI) at doses of 25, 50, and 100 mg/kg
recent research, including compounds extracted from herbs via femoral vein infusion 30 min after coronary occlusion, the
(Table 1), single herbs (Table 2), decoctions (Table 3), and content of myocardial MDA was decreased significantly and
patent drugs made up of Chinese herbs (Table 4). All of the dose-dependently and the activities of myocardial SOD and
abbreviations used in these tables are listed at the end of the GSH-Px were increased dramatically [55].
paper, and the main mechanisms and the representatives of
Chinese herbal medicine in MIR treatment are schematized
in Figure 1. In the following sections, these herbal medicines
3.2. Anti-Inflammation. The manifestation of MIR shares
are grouped according to their efficacy in TCM terminol-
many phenotypic similarities with the activation of a host
ogy, and the underlying cellular and molecular mechanisms
immune response directed toward invading microorganisms.
demonstrated by experimental investigations are discussed.
HSPs and HMGB1 are both involved in the initiation of host
defense and tissue repair. Molecules derived from immune
3.1. Anti-Oxidation. Many Chinese herbal medicines, cells and cardiomyocytes have been utilized as biomarkers
including extracted compounds, single herbs, decoctions, to evaluate the anti-inflammatory effects of Chinese herbal
and patent drugs, exert their beneficial effects on MIR medicine on MIR, including IL-6, MCP-1, TGF-𝛽1, TNF-𝛼,
via their anti-oxidative activity. A number of biomarkers CRP, IL-1𝛽, VCAM-1, ICAM-1, HMGB1, HSP25 and Hsp70,
have been used to evaluate the antioxidative effects of macrophage adhesion molecule-1 (Mac-1), troponinT (Tn-T),
these Chinese herbal medicines, such as ROS, SOD, phosphorylated p38, activated MAPK, and tissue inhibitor of
GPx, CAT, nitric oxide synthase (NOS), malondialdehyde matrix metalloproteinase (TIMP)-1.
(MDA), myeloperoxidase (MPO), heme oxygenase (HO)-1, Ren et al. indicated that Tanshinone IIA (Tan IIA), a
superoxide anion, GOT, 15-F2t-isoprostane (15-F2t-IsoP), compound extracted from the Chinese herb, Salvia mil-
ET-1, cycloxygenase-2 (COX-2), thioredoxin-1 (Trx-1), tiorrhiza Bunge, attenuated expression of MCP-1, TGF-𝛽1,
thioredoxin-related protein-32 (TRP32), redox-sensitive and TNF-𝛼 as well as macrophage infiltration in rats when
PKC𝜀/mKATP pathway, glutathione (GSH), oxidized administered intragastrically at a dose of 60 mg/kg/day [56].
glutathione (GSSG), glutathione reductase (GRD), CuZn- Jiang et al. reported that treatment with Forsythoside B
superoxide dismutase (CuZn-SOD), and Mn-SOD. significantly decreased the levels of TNF-𝛽, IL-6, and HMGB1
Through in vivo and in vitro experiments, Kim et al. in a rat MIR model [24, 50]. Results of a study by Chiu
revealed that palmatine, a compound extracted from the and Ko indicated that the reduction of Hsp25 and Hsp70
Chinese herb, Coptidis rhizome, markedly reduced serum expression by Schisandrin B (Sch B), a compound extracted
MDA level, and the activity of SOD and CAT in the cardiac from Chinese herb, Schisandra chinensis, in MIR rats resulted
 Table 1: Efficacy and mechanisms of Chinese herb-derived compounds in the treatment of MIR.
Mechanism of action in
Plant Compound Mechanism Biomarker/Targets In vivo/In vitro References
TCM terminology
Anti-inflammation MCP-1, TGF-𝛽1, TNF-𝛼, NF-𝜅B In vivo [56]
Antioxidant VEGF, HIF-1𝛼; MDA, SOD, GPx Both [68, 72]
Tanshinone IIA Antiapoptosis Bcl-2/Bax, caspase-3 Both [72]
Promote angiogenesis VEGF, HIF-1𝛼 In vivo [68]
Promote BMSCs migration SCF-1, CXCR-4 In vivo [67]
Sodium tanshinone IIA sulfonate Anti-apoptosis LDH, JNK, p38 In vivo [73]
Magnesium tanshinoate B Anti-apoptosis p-JNK, cytochrome c, caspase-3 In vitro [74]
Salvianolic acid A Activate calcium channels I-CaL In vivo [75]
Salvia miltiorrhiza Salvianolic acid B Promote angiogenesis VEGF In vivo [76]
Antioxidant 15-F2t-IsoP, ET-1, CK-MB In vivo [77]
Salvianolic acids
Reduce ME CK In vitro [78]
Tonifying Qi (energy) to Tanshinone combined with Inhibit of intracellular calcium,
ICAM-1, Ca2+ In vivo [79]
activate circulation and salvianolic acids and anti-apoptosis, antioxidants
Evidence-Based Complementary and Alternative Medicine

enrich Blood Tanshinone IIA combined with CAT, L-arginine, eNOS, AMPK,
Antioxidant In vivo [80]
salvianolic acid B Akt
Danshensu Antioxidant, reduce ME SOD, MDA; CKMB, LDH In vivo [81]
MDA, SOD, and GPx; LDH, CK,
Salvia miltiorrhiza extract Antioxidant, reduce ME In vivo [82, 83]
GOT
Aqueous extracts of Salvia CK-MB and cTnT,
Reduce ME, promote angiogenesis In vivo [84]
miltiorrhizae 6-keto-PGF-1𝛼/TXB-2
Salvia miltiorrhizae Antioxidant COX-2; TXB2, 6-keto-PGF1-𝛼 In vivo [85]
Inhibit Ca2+ overload, up-regulate
Saponin of red ginseng Ca2+ ; KATP; cTnI; PI3K Both [86, 87]
KATP
Radix Ginseng
Total ginsenosides Antioxidant, anti-apoptosis Ca2+ ; eNOS, iNOS, GR; PI3K, Akt In vivo [87, 88]
Radix Ginseng extracts Antioxidant, reduce ME NO, eNOS; CK, LDH In vivo [88]
Up-regulate KATP channel KATP channel subunits Kir6.1,
Astragalus membranaceus Astragaloside IV In vivo [70]
subunits, facilitate KATP currents Kir6.2, SUR2A, SUR2B
Rhodiola Salidroside, tyrosol Anti-apoptosis caspase-3, p-JNK, cytochrome c In vitro [60]
17-Methoxyl-7-hydroxy-benzene- Antioxidant, anti-inflammation, MDA; TNF-𝛼; NF-𝜅B p65,
Millettia pulchra Both [89]
furanchalcone and anti-apoptosis Bcl-2-associated X protein
Anti-inflammation and Hsp25, Hsp70 In vivo [57]
Schisandra chinensis Schisandrin B
Antioxidant cytochrome P-450 In vivo [90]
Increase Na+ -K+ -ATPase and Na+ -K+ -ATPase, Ca2+ -ATPase;
Lycium barbarum Lycium barbarum polysaccharides 2+ In vivo [71]
Ca -ATPase, anti-apoptosis Bax, Bcl-2
5
 6

Table 1: Continued.
Mechanism of action in
Plant Compound Mechanism Biomarker/Targets In vivo/In vitro References
TCM terminology
Tetramethylpyrazine Antioxidant, inhibit neutrophil HO-1; Migrated neutrophil In vivo [91]
Ligusticum wallichii
Aqueous extracts of Rhizoma CK-MB, cTnT;
Reduce ME, promote angiogenesis In vivo [84]
Chuanxiong 6-keto-PGF-1𝛼/TXB-2
ROS, MDA, SOD; CRP, TNF-𝛼,
Carthamus tinctorius L. Extracts of Carthamus tinctorius Antioxidant, anti-inflammation Both [92, 93]
Moving Qi and IL-1𝛽; PI3K
activating circulation to Extracts of Panax notoginseng Antioxidant, anti-inflammation MDA, SOD; CRP, TNF-𝛼, IL-1𝛽 In vivo [92]
Panax notoginseng
resolve stasis Notoginsengnosides Reduce ME CK In vitro [78]
SOD, GOT, GPx, MDA; CK-MB,
Antioxidant, reduce ME, and
LDH, cTnT; ICDH, MDH, In vivo [94]
Dipsacus asper Asperosaponin VI protect mitochondrial function
𝛼-KGDH, ATP, Ca2+
Bcl2/Bax, caspase-3; LDH, CREB,
Anti-apoptosis, reduce ME In vitro [95]
PI3K
Pyrolae Flavonoid of Herba Pyrolae Antioxidant, reduce ME SOD, MDA; CK, LDH In vivo [96]
MDA, MPO, SOD, GPx;Tn-T,
Lamiophlomis rotata Forsythoside B Antioxidant, anti-inflammation TNF-𝛼, IL-6, HMGB1; IkBa, In vivo [50]
NF-𝜅B
Hydroalcoholic extract of Sida
Sida cordifolia L. Antioxidant, reduce ME SOD, CAT; LDH, CK-MB In vivo [24]
cordifolia L.
Desmodium Desmodium gangeticum Stimulate muscarinic receptors Muscarinic receptor In vivo [97]
3,5-Dimethoxy-4-(3-(2-carbonyl-
ethyldisulfanyl)-propionyl)-
Anti-apoptosis Caspase-3, Bcl-2/Bax, Akt In vitro [61]
Leonurus benzoic acid
Inducing Diuresis to 4-Guanidino-butyl ester
resolve stasis Inhibit Ca2+ overload,
4-Guanidino-n-butyl syringate Ca2+ ; Bcl-2, Bax, LDH In vivo [98]
antiapoptosis
2+
Acorus gramineus Acori graminei Rhizoma Inhibit calcium overload Ca In vivo [99]
Phytolacca Oleanolic Acid Anti-apoptosis AMPK, p38, FOXO3 In vitro [100]
Tetrandra Tetrandrine Inhibit neutrophil, antioxidant neutrophil adhesion, Mac-1; ROS In vivo [23]
Baicalensis Botanical Flavonoids Antioxidant ROS, NO, SOD, CAT, GPx Both [101, 102]
Cooling Blood to stop
Coptidis rhizoma Palmatine Antioxidant, reduce ME SOD, MDA, COX-2; LDH, CK In vivo [49]
bleeding
KATP channel opening; NO, ROS,
Buxus microphylla Cyclovirobuxine D Antioxidant, reduce ME In vivo [52, 103]
SOD, MDA; CPK, LDH, FFA
Tonifying Qi to Cinnamon 2-Methoxycinnamaldehyde Antioxidant, anti-inflammation VCAM-1,TNF-𝛼, HO-1 In vivo [51]
invigorate Yang A semipurified fraction of Herba Protect mitochondrial function, ATP-generation, mitochondrial
Herba Cistanches Both [65]
Cistanches antioxidant, and anti-apoptosis uncoupling; GSH; caspase-3
Regulating Qi and Corydalis Corydalis yanhusuo extract Anti-apoptosis Bax, Bcl-2 In vivo [104]
moving Qi MPO, superoxide anion; migrated
Magnolia officinalis Magnolol Antioxidant; inhibit neutrophil In vivo [105]
neutrophil
Evidence-Based Complementary and Alternative Medicine
Evidence-Based Complementary and Alternative Medicine 7

Table 2: Efficacy and mechanism of single Chinese herbs in the treatment of MIR.

Mechanism of action in In vivo/In vitro References

Herb Mechanism Biomarker/Targets
TCM terminology
VEGFR (Flt-1, KDR, and In vivo [106]
Rhodiola Promote angiogenesis
Replenishing and moving Tie-2)
Qi Euryale ferox (Makhana) Antioxidant TRP32, ROS, and Trx-1 Both [53]
Perfusion pressure,
Recovery of contractile In vivo [107]
Aurantii Fructus aortic flow, and coronary
dysfunction
flow

in cardioprotection [57]. Shen et al. reported that neutrophils the Chinese herbs, Safflower, red peony, salvia, Chuanxiong,
from MIR animals displayed a significant morphological and Dalbergiae Odoriferae, tilted the balance between Bax
change and Mac-1 up-regulation, both of which could be and Bcl-2 toward an anti-apoptotic state, decreased mito-
prevented by Tetrandrine (TTD), a compound extracted chondrial cytochrome c release, reduced caspase-9 activation,
from the Chinese herb, Stephania tetrandra [23]. and attenuated subsequent caspase-3 activation and postis-
Decoctions and patent drugs made up of Chinese herbs chemic myocardial apoptosis in rats [63, 64].
have also been demonstrated to exert anti-inflammatory
effects in MIR. Yin et al. showed that a significant reduction
in TIMP-1 and TNF levels and improved cardiac function in 3.4. Protecting Mitochondrial Function. MPTP has been
MIR rats were achieved by treatment with ShuMai Decoction used as a target for protecting mitochondrial function
consisting of Astragalus mongholicus Bunge, Salvia miltior- by Chinese herbal medicine in the treatment of MIR.
rhiza Bge, and Eupolyphaga sinensis, in a dose-dependent ATP-generation capacity, mitochondrial uncoupling, cAMP
manner [58]. Zhang et al. studied the patent drug Xiong- response element-binding protein (CREB), cytochrome c,
shao Capsule (XSC), comprised of Chinese herbs, Rhizoma cytochrome P-450, mitochondrial glutathione (GSH), mito-
Chuanxiong and Radix Paeoniae Rubra, and found that it chondrial Ca2+ , and mitochondrial MDA have been used as
reduced levels of MCP-1 and TNF-𝛼 as well as inflammatory biomarkers to evaluate the effects of Chinese herbal medicine.
cell infiltration (ICI) in the ischemic myocardium [59]. Wong and Ko reported that a semipurified fraction
of Herba Cistanches (HCF1) increased mitochondrial ATP-
generation capacity and ADP-stimulated state respiration
3.3. Anti-Apoptosis. Alterations of pro and antiapoptotic in H9c2 cardiomyocytes during MIR. HCF1 pretreatment
signaling pathways, including changes in the levels of could protect against MIR injury in rats presumably mediated
apoptosis-modulating molecules and induction of caspases, by the induction of glutathione antioxidant [65]. Siu and
have been used to examine the anti-apoptotic effects of Ko studied the single Chinese herb, Cistanche, and found
Chinese herbal medicine in MIR. Levels and/or activities it enhanced mitochondrial glutathione status, decreased
of caspase-3, caspase-9, Bcl-2/Bax, p-JNK, p-AMPK, p-p38,
mitochondrial Ca2+ level, and increased the mitochondrial
phosphatidylinositol 3-kinase (PI3 K), Akt, p-I𝜅B-𝛼, NF-𝜅B,
membrane potential and respiration rate in rat hearts [66].
p65, Bcl-2-associated X protein, cytochrome c, and forkhead
Others reported that the patent drug, Guanxin II, decreased
transcription factor 3 (FOXO3) are among the commonly
mitochondrial cytochrome c release and attenuated caspase-3
used biomarkers.
activation in rat MIR myocardium [63, 64].
Sun et al. revealed that Salidroside and Tyrosol, two
compounds extracted from the Chinese herb, Rhodiola,
separately or in combination, significantly reduced caspase- 3.5. Increasing BMSCs Migration. Bone marrow mesenchy-
3 activity, cytochrome c release, and JNK activation in an in mal stem cells (BMSCs) are preferentially attracted to and
vitro study [60]. Liu et al. reported that 3,5-Dimethoxy-4- retained in ischemic tissue. SDF-1 and CXCR4 have been
(3-(2-carbonyl-ethyldisulfanyl)-propionyl)-benzoic acid 4- used as targets for increasing BMSC migration to protect
guanidino-butyl ester, derived from the Chinese herb, Leonu- cardiomyocytes against MIR.
rus, inhibited apoptosis by increasing the ratio of Bcl-2/Bax, Tong et al. studied the effect of Tan IIA on MIR both
decreasing the level of cleaved-caspase-3, and enhancing the in vitro and in vivo. Their data showed that combination
phosphorylation of Akt [61]. An in vivo study by Jiang et al. treatment with Tan IIA and BMSCs significantly reduced the
demonstrated that rats treated with Forsythoside B showed infarct size and improved cardiac function after MI, which
a significant recovery in myocardial function due to down- primarily resulted from Tan IIA induced increase of the
regulated phosphorylation of IkB-𝛼 and NF-𝜅B [50]. migration of BMSCs to ischemic region [67].
Ling et al. studied the effects of the patent drug, Car-
diotonic Pill (CP) combined with the Chinese herb, Salvia
miltiorrhiza, and found that CP treatment (50 mg/mL) sig- 3.6. Promoting Angiogenesis. Angiogenesis limits MIR dam-
nificantly inhibited TNF-𝛼-induced apoptosis in cardiomy- age by restoring tissue blood flow. Related molecules such
ocytes through activating Akt signaling [62]. Others have as VEGF, von Willebrand factor (vWF), hypoxia-inducible
showed that Guan xin er hao (Guanxin II), which consists of factor 1𝛼 (HIF-1𝛼), VEGFR (Flt-1, KDR, and angiopoietin
 8

Table 3: Efficacy and mechanisms of Chinese herb decoctions in the treatment of MIR.
Mechanism of action in TCM
Decoction Constituent herbs Mechanism Biomarker/Targets In vivo/In vitro References
terminology
Antioxidant, protect
DangGui BuXue Decoction Astragali and Angelica roots GSH, GSSG, GRD In vivo [108]
Tonifying Qi to enrich Blood mitochondrial function
Astragalus, angelica, red peony, LDH, CK, AST;
BuYang HuanWu Decoction Reduce ME In vivo [109]
earthworm, and so forth CD40-CD40L
TNF-𝛼, p38, MAPK,
Replenishing Qi to activate Blood Astragalus mongholicus, Salvia Anti-inflammation In vivo [58]
ShuMai Decoction TIMP-1
and recover circulation miltiorrhiza, Eupolyphaga,
VEGF, PDGF-BB,
Wallich and Hirudo nipponica Promote angiogenesis In vivo [110]
PI3K, Akt
Whitman, Moschus berezovskii
Invigorating Yang to recover Antimitochondrial SOD, MDA, MnSOD
Sini Decoction Aconite, ginger, and licorice In vivo [54]
circulation oxidation mRNA
Dan-Chuan-Hong Salvia, Rhizoma Chuanxiong, and
Moving Qi to activate circulation Anti-apoptosis TUNEL In vivo [111]
Decoction safflower
Enriching Blood to engender Radix Salvia miltiorrhiza, and Radix Redox-sensitive
DanShen GeGen Decoction Antioxidant In vivo [112]
fluid Puerariae lobatae PKC𝜀/mKATP pathway
Radix et Rhizoma vWF, VEGF, HIF-1𝛼,
Enrich Qi and cool Blood Radix, Rhizoma Rhodiolae kirilowii Promote angiogenesis In vivo [69]
Rhodiolae Kirilowii HIF-1𝛽
Evidence-Based Complementary and Alternative Medicine
 Table 4: Efficacy and mechanism of patent drugs made up of Chinese herbs in the treatment of MIR.
Mechanism of action in TCM
Patent drug name Main ingredient Mechanism Biomarker/Targets In vivo/In vitro References
terminology
Salvia, arrowroot, woody, hawthorn, Antioxidant, promote
XinKeShu Tablet eNOS; VCAM-1 In vivo [113]
Activating Blood to resolve stasis, panax angiogenesis
and moving Qi to relieve pain Ginseng, leeches, scorpion, Eupolyphaga, Antioxidant,
TongXinLuo Superfine NO, eNOS; vWF, Hhcy In vivo [114]
centipede, et al proangiogenesis
ShuMai Capsule Peanut shells Promote angiogenesis vWF, VEGF In vivo [115]
Caspase-3, Caspase-9,
GuanXin ErHao (Guanxin Safflower, red peony, salvia, Chuanxiong, Anti-apoptosis, protect
Moving Qi to activate circulation Bcl-2/Bax, cytochrome In vivo [63, 64]
II) and so forth mitochondrial function
and relieve pain c, Akt
Rhizoma Chuanxiong, Radix Paeoniae
XiongShao Capsule Anti-inflammation TNF-𝛼, MCP-1, ICI In vivo [59]
Rubra
Evidence-Based Complementary and Alternative Medicine

GSH, 𝛼-TOC,
CuZn-SOD,
Radix Ginseng, Cornu Cervi, Cordyceps, Antioxidant, protect Ca2+ -induced
Vigconic 28 (VI-28) In vivo [116]
Replenishing Qi to activate Radix Salviae, Semen Allii, and so forth mitochondrial function permeability,
circulation, and moving Qi to mitochondrial MDA,
relieve pain Ca2+ , cytochrome c
Acanthopanax Senticosus Antioxidant, inhibit
Acanthopanax SOD, MDA, GPx; Ca2+ In vivo [55]
Injection Ca2+ overload
ShuangShen NingXin Ginseng total saponins, total salvianolic,
Antioxdiant SOD, MDA In vivo [117]
Capsule corydalis
ShuangShen TongGuan Ginseng, astragalus, Atractylodes, and so NF-𝜅B, p65, TNF-𝛼,
Anti-inflammation In vivo [118]
Recipe forth ICAM-1, MJIC-Cx43
Antioxdiant, reduce SOD, GPx; LDH, CK;
Replenishing Qi and invigorating
ShenFu Injections Red ginseng, Monkshood ME, Na+ -K+ -ATP and In vivo [119]
Yang
up-regulate-ATPase Ca2+ -ATP

Replenishing Blood and DanHong Injection Salvia, safflower Antioxidant SOD, MDA In vivo [120]
activating circulation Cardiotonic Pill Salvia miltiorrhiza Anti-apoptosis Caspase-3, Akt In vivo [62]
9
10 Evidence-Based Complementary and Alternative Medicine

receptor (Tie-2)), platelet-derived growth factor (PDGF- GSH: Glutathione

BB), and phosphatidylinositol 3-kinase (PI3K) have been GPx: Glutathine peroxidease
used as the targets for angiogenesis promotion to protect MPO: Myeloperoxidase
cardiomyocytes against MIR. CAT: Catalase
Xu et al. found that the compound, Tanshinone IIA, COX-2: Cycloxygenase-2
elicited a significant cardioprotective effect by up-regulating GOT: Glutamic oxalacetic transaminase
VEGF expression in MI rats and enhancing HIF-1𝛼 expres- ME: Myocardial enzymes
sion [68]. Experiments of Gao et al. showed that the expres- TRP32: Thioredoxin-related protein-32
sions of vWF, HIF-1𝛼, HIF-1𝛽, and VEGF were significantly GSH: Glutathione
increased in myocardium treated with Radix et Rhizoma GSSG: Oxidized glutathione
Rhodiolae Kirilowii Decoction [69]. GRD: Glutathione reductase
CuZn-SOD: CuZn-superoxide dismutase
PI3K: Phosphatidylinositol 3-kinase
3.7. Up-Regulating KATP Channel Subunits and ATPase, and HMGB1: High-mobility group box1
Inhibiting Calcium Overload. KATP channel subunits Kir6.1, HSP: Heat shock protein
Kir6.2, SUR2A and SUR2B, Na+ -K+ -ATPase, Ca2+ -ATPase TIMP: Tissue inhibitor of matrix
and intracellular calcium (Ca2+ ), and L-type calcium current metalloproteinase
(I-CaL) have been used to assess the effects of Chinese herbal ICI: Inflammatory cell infiltration
medicine in protecting cardiomyocytes against MIR. LDH: Lactate dehydrogenase
Han et al. examined the effects of Astragaloside IV (As CK: Creatine kinase
IV), a compound extracted from the Chinese herb, Astra- CK-MB: Creatine kinase isoenzyme-MB
galus membranaceus. They found that As IV significantly TXB2: Thromboxane B2
up-regulated mRNA and protein levels of KATP channel VEGF: Vascular endothelial growth factor
subunits Kir6.1, Kir6.2, and SUR2A and SUR2B [70]. Lu HIF-1a: Hypoxia-inducible factor 1a
and Zhao reported that Lycium barbarum polysaccharides, Vwf: Von Willebrand factor
extracted from the Chinese herb, Lycium barbarum, signifi- SDF-1: Stromal cell-derived factor-1
cantly increased Na+ -K+ -ATPase and Ca2+ -ATPase activities SCF-1: Stem cell factor-1
in myocardium of ischemia-reperfusion rats [71]. CXCR4: CXC chemokine receptor 4
I-CaL: L-type calcium current
15-F2t-IsoP: 15-F2t-isoprostane
4. Summary and Perspective 6-keto-PGF1-a: 6-keto-prostaglandin F1alpha
GR: Glucocorticoid receptor
In summary, significant progress has been made regarding CREB: cAMP response element-binding protein
the mechanistic research into the efficacy of Chinese herbal Tn-T: TroponinT
medicine for the treatment of MIR. However, much work FOXO3: Forkhead transcription factor 3
remains. Most clinical studies were of limited extrapolatable Mac-1: Macrophage adhesion molecule-1
value because of the small sample sizes and/or incomplete HO-1: Heme oxygenase-1
data. Experimental studies have focused mainly on single Tie-2: Angiopoietin receptor
compounds extracted from Chinese herbs. Studies of Chinese TRP32: Thioredoxin-related protein-32
decoctions or formulations are relatively scarce, although Trx-1: Thioredoxin-1
decoction and formulations are the main forms of therapy GSSG: Oxidized glutathione
in TCM practice. Capitalization of the interactions between GRD: Glutathione reductase
the different components and herbs is the essence of TCM. PDGF-BB: Platelet-derived growth factor
Many herbs are paired together to attenuate toxicity as well Hhcy: Hyperhomocysteinemia
as to enhance efficacy. Encouragingly, the number of studies MJIC-Cx43: Myocardial junction intercellular
on patent Chinese herbs has been gradually increasing. These communication connexin 43.
studies help us to understand the mechanisms underlying
the use of Chinese herbs and formulations for the treatment
of MIR. Accordingly, there is a strong likelihood that such
ongoing research will lead to novel therapies for the treatment Conflict of Interests
of myocardial ischemia and reperfusion injury using Chinese The authors declare that they have no conflict of interests
herbs and herbal formulations. regarding the publication of this paper.

Abbreviations
Acknowledgment
ROS: Reactive oxygen species
NOS: Nitric oxide synthase This work was supported by Grants from the National
MDA: Malondialdehyde Institute of Health nos. R21AT006767 and R01HL116626 (to
SOD: Superoxide dismutase D. Fan).
Evidence-Based Complementary and Alternative Medicine 11

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Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2013, Article ID 840487, 8 pages
http://dx.doi.org/10.1155/2013/840487

Research Article
Hinokitiol, a Natural Tropolone Derivative, Offers
Neuroprotection from Thromboembolic Stroke In Vivo

Thanasekaran Jayakumar,1 Wen-Hsien Hsu,2 Ting-Lin Yen,1 Jun-Yun Luo,1

Yu-Cheng Kuo,1 Tsorng-Harn Fong,3 and Joen-Rong Sheu1
1
Department of Pharmacology, Graduate Institute of Medical Sciences, Taipei Medical University, 250 Wu-Hsing Street,
Taipei 110, Taiwan
2
Department of Surgery, Wan-Fang Hospital, Taipei Medical University, 111 Hsing-Long Road, Taipei 110, Taiwan
3
Department of Anatomy, College of Medicine, Taipei Medical University, Taipei 110, Taiwan

Correspondence should be addressed to Tsorng-Harn Fong; [email protected] and Joen-Rong Sheu; [email protected]

Received 17 July 2013; Accepted 9 September 2013

Academic Editor: Mao-Hsiung Yen

Copyright © 2013 Thanasekaran Jayakumar et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.

Hinokitiol (𝛽-thujaplicin), a tropolone-related compound found in the heartwood cupressaceous plants, is widely used in hair
tonics, tooth pastes, cosmetics, and food as an antimicrobial agent. Increasing evidence has confirmed that hinokitiol exhibits
anticancer activity in a variety of cancers through inhibition of cell proliferation. In the present study, we have investigated
the neuroprotective effect and mechanisms of hinokitiol in rats against middle cerebral artery occlusion (MCAO)-induced
thromboembolic stroke. Treatment with hinokitiol (0.2 and 0.5 mg/kg; intraperitoneally) 30 min before MCAO dose dependently
attenuated cerebral ischemia and improved neurobehavioral deficits in cerebral ischemic rats. Intraperitoneal administration of
hinokitiol significantly reduced infarct size compared to that in control rats. MCAO-induced focal cerebral ischemia was associated
with increased expressions of hypoxia-inducible factor (HIF)-1𝛼, inducible nitric oxide synthase (iNOS), tumor necrosis factor
(TNF)-𝛼, and active caspase-3 in ischemic regions. However, these expressions were obviously inhibited by hinokitiol (0.2 and
0.5 mg/kg) treatment. This study demonstrates for the first time that in addition to being originally considered as an agent against
microbes and variety of cancers, hinokitiol possesses potent neuroprotective activity. This activity is mediated, at least in part, by
inhibition of inflammatory responses (i.e., HIF-1𝛼, iNOS expression) and apoptosis (i.e., TNF-𝛼, active caspase-3), resulting in a
reduction of infarct volume and improvement in neurobehavior in rats with cerebral ischemia. Therefore, the therapeutic potential
of hinokitiol may lead to novel role for treatment or prevention of ischemia/reperfusion injury-related disorders.

1. Introduction less than 5% patients due to its narrow therapeutic window

[5]. Furthermore, clinical data showed that whatever its
Stroke is the third leading cause of death, ranking after administration time, rt-PA increases the risk of hemorrhagic
heart disease and cancer and the primary cause of adult transformation (HT) [6]. Clinical practice also showed that
disability worldwide [1, 2]. It is also a major health concern rt-PA does not induce recanalization in all ischemic patients.
in the industrialized countries. In spite of major advances of Therefore, research has been directed at finding alternative
neuroprotective therapeutic approaches for treating ischemic therapies that target neurons downstream of the site of the
stroke over the last decade, stroke is still a serious problem thrombosis in an effort to salvage them from the vicious
for which effective drug therapy is not yet available [3]. The circle of inflammation, necrosis, and apoptosis, all of which
recombinant tissue plasminogen activator (rt-PA), a throm- are implicated in ischemic stroke [7]. Natural products are a
bolytic agent, has long been used to improve the outcomes prolific source of bioactive agents of different structure and
of acute ischemic stroke patients by restoring cerebral blood varying biological activity. In the search for neuroprotective
flow (CBF) [4]. Nevertheless, its use remains limited to agents from natural sources, a number of plant extracts
2 Evidence-Based Complementary and Alternative Medicine

and several natural products isolated from them have been

reported to provide neuroprotection against ischemic stroke O
[8].
Hinokitiol, also known as 𝛽-thujaplicin, is a tropolone
derivative found in the heartwood of cupressaceous plants
[9]. As an iron-chelating compound, it triggers apoptosis OH
via activation of caspase-3 [10] and exerts a spectrum of
biological effects including differentiation-inducing [11], anti- (a)
inflammatory [12], antibacterial [13], antifungal [14], and 4
antioxidant [15] capacities, as well as antitumor activity [16]. ∗∗∗
Hinokitiol has also been widely used in hair tonics, tooth ∗∗∗
pastes, cosmetics, and food as an antimicrobial agent [17]. 3

Neurobehavioral scores
Hinokitiol has been shown to suppress tumor growth by ###
inhibiting cell proliferation and inducing apoptosis in various 2
carcinoma cell lines [16]. Hinokitiol regulates immune cell
function by inhibiting the production of TNF-𝛼 from LPS-
stimulated macrophages via inhibition of NF-𝜅B activity [9]. 1
Our recent study also clearly demonstrates that hinokitiol
possesses antiplatelet activity by inhibiting the PLCg2 and/or 0
PKC cascades, and hydroxyl radical formation, followed by
suppressing the activation of MAPKs and Akt [18]. Despite
such wide range of roles in signaling pathways, there is no 0 1 24
report about the direct evidence of the neuroprotective effect Time after MCAO (hours)
of hinokitiol. The purpose of this contribution is thus to
demonstrate whether hinokitiol has a neuroprotective effect Sham Hinokitiol (0.2 mg/kg)
Control Hinokitiol (0.5 mg/kg)
against thromboembolic stroke in rats.
Vehicle
(b)
2. Materials and Methods
Figure 1: (a) Chemical structure of hinokitiol (C10 H12 O2 , MW:
2.1. Materials. Hinokitiol (Figure 1(a), 99%), collagen 164.2). (b) Effects of hinokitiol on the MCAO-induced neurobehav-
(type I), cremophor EL, 5,5-dimethyl-1 pyrroline N-oxide ioral deficits scores. Neurobehavioral scores were recorded at 1 and
(DMPO), and bovine serum albumin (BSA) were purchased 24 h after MCAO. Data are expressed as the means ± SEM (𝑛 = 10).
∗∗∗ ###
from Sigma (St. Louis, MO). Hinokitiol was dissolved in 𝑃 < 0.001 compared to sham-operated group; 𝑃 < 0.001
a solvent (cremophor : ethanol : DMSO at 1 : 1 : 4) for the compared to solvent-treated group.
present study.

2.2. Animals. Male Wistar rats (250∼300 g) were used in 2.4. Thromboembolic Occlusion of the Middle Cerebral Artery
this study. All animal experiments and care were performed (MCA) in Rats. Animals were anesthetized with a mixture
according to the National Research Council Guide for the Care of 75% air and 25% O2 gases containing 3% isoflurane. The
and Use of Laboratory Animals and were approved by the rectal temperature was maintained at 37 ± 0.5∘ C. The right
Institutional Animal Care and Use Committee (IACUC) of MCA was occluded with a blood clot as an embolus. The
Taipei Medical University (number LAC-98-0088). Before method of embolus preparation and surgical procedures were
undergoing the experimental procedures, all animals were slightly modified from a previous description by Krueger and
clinically normal, free of apparent infection or inflammation, Busch [19]. Briefly, arterial blood (0.6 mL) was withdrawn
from a femoral catheter in a 1 mL syringe. The blood was
and showed no neurological deficits.
immediately injected into PE-50 tubes. The tubes were kept
at 4∘ C for approximately 22 h, and the thread-like clots were
2.3. Experimental Groups. Animals were divided into 5 removed and placed in a phosphate-buffered saline (PBS)-
groups: (1) a sham-operated group; (2) a group orally treated filled dish. The clots were then washed to remove blood
with an isovolumetric solvent (distilled water) for 14 days, cells. Washed portions of the clots were transferred to fresh
followed by thromboembolic occlusion; (3) a group orally dishes, and the washing process was repeated until the PBS
treated with solvent (cremophor : ethanol : DMSO at 1 : 1 : 4) remained clear. These clot sections were cut into 30 mm-long
for 14 days, followed by thromboembolic occlusion; 4 and fragments and then drawn up with the PBS solution into a
5 groups treated with a single dose of hinokitiol (0.2 and PE-50 catheter.
0.4 mg/kg, resp.), followed by thromboembolic occlusion. The common carotid artery (CCA) was identified, and
Rats received the isovolumetric normal saline, solvent, or approximately 1 cm of the external carotid artery (ECA)
hinokitiol (0.2 or 0.5 mg/kg) 30 min before MCAO was was ligated and cut. Subsequently, the pterygopalatine artery
performed. (PA) was clamped with a 10 mm microaneurysm clamp, and
Evidence-Based Complementary and Alternative Medicine 3

the CCA was similarly clamped before the carotid bifurca- The supernatant (50 𝜇g protein) was subjected to sodium
tion. The internal carotid artery (ICA) was then clamped dodecylsulfate polyacrylamide gel electrophoresis (SDS-
between the carotid bifurcation and the PA. Next, the PE- PAGE) and electrophoretically transferred to polyvinyli-
50 catheter containing the clot was introduced approximately dene difluoride (PVDF) membranes (0.45 𝜇m, Hybond-
5 mm into the previously cut ECA and tied in place with P, Amersham). After incubation in blocking buffer and
sutures. The ICA clamp was removed, and the clot was flushed being washed three times with TBST buffer (10 mM Tris-
into the ICA over a period of approximately 5 s. The PA clamp base, 100 mM NaCl, and 0.1% Tween 20; pH 7.5), blots
was removed, and the rat was left in this condition for 1 h. were treated with an anti-HIF-1𝛼 polyclonal antibody (pAb,
At the end of this period, the catheter was removed from 1 : 1000; R&D, Minneapolis, MN), an anti-iNOS monoclonal
the ECA stump, an unperturbed portion of the ECA close to antibody (mAb; 1 : 3000, BD Biosciences, San Jose, CA), an
the bifurcation was tied off, and the incision was closed. After anti-TNF-𝛼 pAb (1 : 1000; Cell Signaling, Beverly, MA), and
closure of the operative sites, the animals were allowed to an antiactive caspase-3 pAb (1 : 250; Biovision, Mountain
wake from the anesthesia. An observer blinded to the identity View, CA) or an anti-𝛼-Tubulin mAb (1 : 2000; Santa Cruz
of the groups assessed the neurological deficits at 1 and
Biotechnology, Santa Cruz, CA) in TBST buffer overnight.
24 h after reperfusion (before being euthanized) by forelimb
Blots were subsequently washed with TBST and incubated
akinesia (also called the postural tail-hang) test, whereas
with a secondary horseradish peroxidase (HRP)-conjugated
a spontaneous rotational test was used as a criterion for
goat anti-mouse mAb ordonkey anti-rabbit immunoglobulin
evaluating the ischemic insult [20]. Animals not showing any
G (IgG) (Amersham) for 1 h. Blots were then washed, and
behavioral deficits at the above time points after reperfusion
the immunoreactive protein was detected using film exposed
were excluded from the study. On the other hand, reperfusion
to enhanced chemiluminescence (ECL) detection reagents
was also ensured by an improvement in ipsilateral local blood
(ECL+ system; Amersham). The bar graph depicts the ratios
flow to at least 60% of the baseline following an initial sharp
of semiquantitative results obtained by scanning reactive
decrease to about 50%∼60% of the baseline caused by MCA
bands and quantifying the optical density using videodensit-
occlusion as determined using a continuous laser Doppler
ometry (Bio-1D vers. 99 image software).
flow meter (LDF; Oxford Array, Oxford Optronix, Oxford,
UK) with a standard needle probe (pp-051).
Rats were euthanized by decapitation after 24 h of reper- 2.6. Statistical Analysis. Experimental results are expressed
fusion. The brains were cut into 2 mm coronal slices starting as the mean ± SEM and are accompanied by the number of
1 mm from the frontal pole. Each stained brain (2% 2,3,5- observations. The experiments were assessed by the method
triphenyltetrazolium; TTC) slice was drawn using a com- of analysis of variance (ANOVA). If this analysis indicated
puterized image analyzer (Image-Pro plus). The calculated significant differences among the group means, then each
infarct areas were then compiled to obtain the infarct vol- group was compared using the Newman-Keuls method. A 𝑃
ume (mm3 ) per brain. Infarct volumes were expressed as a value of <0.05 was considered statistically significant.
percentage of the contralateral hemisphere volume using the
formula (the area of the intact contralateral [left] hemisphere 3. Results
− the area of the intact region of the ipsilateral [right]
hemisphere) to compensate for edema formation in the 3.1. Effects of Hinokitiol on Neurological Deficit Score and
ipsilateral hemisphere [21]. Ischemic Brain Damage. Following stroke, animals subse-
quently exhibit a variety of neurological deficits. It is very
significant to evaluate neurological function outcome after
2.5. Expressions of HIF-1𝛼, iNOS, TNF-𝛼, and Active Caspase-
stroke. The Bederson scale is a global neurological assessment
3 in Thromboembolic Occlusion-Insulted Brain. Expressions
that was developed to measure neurological impairments
of HIF-1𝛼, iNOS, TNF-𝛼, and active caspase-3 in the ischemic
following stroke [23]. Our results revealed that hinokitiol
brain at 24 h after thromboembolic occlusion-reperfusion
could improve neurological behavior disturbance based on
injury were analyzed by immunoblotting as described by
neurological deficit scores.
Rodrigo et al. [22], with minor modifications. Thromboem-
bolic occlusion-insulted and sham-operated rats were anes- The neurological deficit of vehicle-treated, hinokitiol-
thetized with chloral hydrate (400 mg/kg, i.p.), and then the treated, and sham-operated rats, evaluated 24 h after MCAO,
apex of the heart was penetrated with a profusion cannula are shown in Figure 1(b). The neurological scores were sig-
inserted through the left ventricle into the ascending aorta. nificantly increased (𝑃 < 0.001) after 24 hr of ischemia as
Perfusion with ice-cold PBS was performed, and an incision compared to sham-operated rats and after 1 hr of ischemia.
was made in the right atrium for venous drainage. Brains Treatment of hinokitiol (0.2 and 0.5 mg/kg) significantly
were freshly removed and sectioned coronally into four improved the neurological deficit in MCAO-induced rats
sequential parts from the frontal lobe to the occipital lobe. when compared to vehicle-treated rats. Moreover, the neu-
The third of four parts of the right hemisphere was separately rological scores were significantly (𝑃 < 0.001) effected by
collected, snap-frozen in liquid nitrogen, and stored at −70∘ C. hinokitiol treatment at a dose of 0.5 mg/kg than that of the
The frozen tissues were placed in homogenate buffer and solvent and 0.2 mg/kg hinokitiol-treated groups (Figure 1(b)).
homogenized and then sonicated for 10 s three times at 4∘ C. No score was found in the sham-operated rats or in the
The sonicates were subjected to centrifugation (10,000 ×g). hemisphere contralateral to the ischemic side.
4 Evidence-Based Complementary and Alternative Medicine

100

80

Infarct volume (%)

60
∗∗∗ ∗∗
40 ∗∗∗ ∗∗∗

20 ∗∗
∗∗
0

−20

−40
0 3 5 7 9 11 13 15 17
Coronal section (mm from the frontal pole)

Solvent
Hinokitiol (0.5 mg/kg)
0.2 0.5 Figure 3: Effects of hinokitiol on the infarct volume of brain coronal
Sham Control Vehicle
Hinokitiol (mg/kg)
section. Brains were dissected 24 h from reperfusion and sectioned
(a) at 2 mm thickness in the region from 1 mm to 15 mm of distance to
60
the frontal pole. Data are expressed as means ± SEM. ∗∗𝑃 < 0.01 and
∗∗∗
∗∗∗ 𝑃 < 0.001 compared to the solvent-treated group.
50
###
Infarct volume (%)

40 infarct volume (white area) compared to the solvent-treated

group in a dose-dependent manner (Figures 2(a) and 2(b)).
30 ### Figure 3 confers statistical results of the infarct areas of
solvent- and hinokitiol- (0.5 mg/kg) treated groups at various
20 distances from the frontal pole. The infarct area of the 3rd
section was the largest in both groups than others. As shown
10 in Figure 3, hinokitiol (0.5 mg/kg) decreased the area of
infarction in the coronal section of MCAO-induced ischemic
0
Sham Control Vehicle 0.2 0.5 injury.
Hinokitiol (mg/kg)
(b)
3.3. Hinokitiol Treatment Downregulates the Protein Expres-
sions of HIF-1𝛼 and iNOS in Thromboembolic Cerebral Tissues.
Figure 2: (a) Effects of hinokitiol in ischemia/reperfusion brain In order to investigate the effect of hinokitiol treatment
injury induced by MCAO in rats. Digital photographs show the on the inflammatory reaction in the ischemic brain, we
infarct region in brain sections stained by 2% TTC 24 h after MCAO. measured the expression of HIF-1𝛼 and iNOS in throm-
(b) Dose-response effect of hinokitiol in ischemia/reperfusion brain boembolic occlusion-insulted cerebral tissues. As shown
injury induced by MCAO in rats. Rats were injected with solvent in Figure 4, HIF-1𝛼, detected 24 h after thromboembolic
or hinokitiol (0.2 or 0.5 mg/kg, i.p.) at the time of 30 min before
occlusion-reperfusion injury, was more pronounced than
the onset of MCAO compared to sham control. Data are presented
as percentage of contralateral hemisphere and expressed as means
the level obtained in the corresponding area of the sham-
± SEM. ∗∗∗𝑃 < 0.001 compared with sham group; ###𝑃 < 0.001 operated group. HIF-1𝛼 expression was significantly dimin-
compared with vehicle control group (𝑛 = 10∼15). ished by the treatment of hinokitiol at doses of 0.2 mg/kg
(𝑃 < 0.05) and 0.5 mg/kg (𝑃 < 0.001) compared to the
solvent-treated rats.
3.2. Effects of Hinokitiol on MCAO-Induced Focal Cerebral NO generated by the inducible form of NO synthase
Infarction Volume in Embolic Occlusion-Induced Rats. The (iNOS) has been implicated in many pathophysiological
most commonly used tools for measuring the efficacy of states leading to myocardial dysfunction. In the present study,
putative neuroprotective compounds are TTC staining. In Figure 5 shows that the protein levels of iNOS in the brain
the present study, the cerebral infarction was examined using of MCAO-injured group were higher compared to those in
2 mm-thick slices of the cerebrum 24 h after thromboembolic the sham groups. However, at doses of 0.2 mg/kg (𝑃 < 0.05)
occlusion-induced reperfusion using 2% TTC staining. Typ- and 0.5 mg/kg (𝑃 < 0.001) hinokitiol treatment significantly
ical photographs of the infarct region of TTC stained brain inhibited iNOS protein expressions in the brain (Figure 5).
sections in embolic rats treated with hinokitiol are shown
in Figure 2(a), where an intraperitoneal administration of 3.4. Hinokitiol Inhibits TNF-𝛼 Expressions in Thromboembolic
hinokitiol at doses of 0.2 and 0.5 mg/kg significantly reduced Cerebral Tissues. Tumor necrosis factor-𝛼 (TNF-𝛼) is one
Evidence-Based Complementary and Alternative Medicine 5

HIF-1𝛼 iNOS

𝛼-Tubulin 𝛼-Tubulin

2.0 2.5
1.8 ∗∗∗

Relative expression of iNOS (folds)

Relative expression of HIF-1𝛼 (folds)

1.6 2.0 ∗∗∗

1.4 #
1.2 ## 1.5
1.0
0.8 1.0 ###
0.6
0.4 0.5
0.2
0.0 0.0
Sham Control Vehicle 0.2 0.5 Sham Control Vehicle 0.2 0.5
Hinokitiol (mg/kg) Hinokitiol (mg/kg)

Figure 4: Effects of hinokitiol on the protein expression of HIF-1𝛼 in Figure 5: Effects of hinokitiol on the protein expression of iNOS
MCAO-reperfusion-induced cerebral homogenates. Rats were pre- in MCAO-reperfusion-induced cerebral homogenates. Rats were
treated with hinokitiol (0.2 mg/kg or 0.5 mg/kg) before ischemia and pretreated with hinokitiol (0.2 mg/kg or 0.5 mg/kg) before ischemia
compared with vehicle or sham control. All groups are represented and compared with the vehicle group or the sham group. All groups
as ipsilateral hemisphere. Data are expressed as the means ± SEM are represented as ipsilateral hemisphere. Data are expressed as the
(𝑛 = 5). ∗∗∗𝑃 < 0.001 compared with sham control group, ###𝑃 < means ± SEM (𝑛 = 5). ∗∗∗𝑃 < 0.001 compared with sham control
0.001 compared with vehicle control group. group; #𝑃 < 0.05 and ###𝑃 < 0.001 compared with vehicle control
group.

of the most typical proinflammatory cytokines with both on inhibiting the activation of caspase-3 in thromboembolic
beneficial and destructive properties of the central nervous occlusion-induced rats.
system. Increasing lines of evidence have demonstrated
that TNF-𝛼 plays an important role in the development
of ischemic stroke. In the present study, MCAO-induced
4. Discussion
ischemia and reperfusion resulted in 1.23-fold elevation in the A central delayed mechanism beginning within hours from
expression of TNF-𝛼 in the brain tissues. However, treatment the onset of ischemia is the robust inflammatory response in
with hinokitiol at doses of 0.2 and 0.5 mg/kg significantly the ischemic tissue [24]. There is increasing evidence showing
decreased the expressions of TNF-𝛼 by 𝑃 < 0.05 and 𝑃 < a detrimental effect of the postischemic inflammatory reac-
0.001, respectively (Figure 6). tion. Therefore, therapeutic strategies targeting the delayed
inflammatory response could inhibit the progression of the
3.5. Hinokitiol Treatment Downregulates the Expression of tissue damage providing an extended therapeutic window for
Active Caspase-3 in Thromboembolic Cerebral Tissues. To neuroprotection. Hinokitiol is a tropolone-related compound
further examine the molecular mechanisms underlying the found in various natural sources such as the heartwood
neuroprotective effect of hinokitiol on thromboembolic of several cupressaceous plants. Hinokitiol has been widely
occlusion-reperfusion, we investigated caspase-3 protein, used in hair tonics, tooth pastes, cosmetics, and food as an
which is implicated in apoptotic death. Caspase-3 is the antimicrobial agent [17]. It has also been shown to suppress
most abundant cysteine protease in the brain and is acutely tumor growth by inhibiting cell proliferation and inducing
cleaved and activated in neurons in the early stages of reper- apoptosis in various carcinoma cell lines. Our recent study
fusion, leading to cell apoptosis. In this study, a significant also demonstrates that hinokitiol has antiplatelet activity via
increase in the expression of active caspase-3 was observed inhibiting the PLCg2 and/or PKC cascades, and hydroxyl
in the injured hemisphere of the transient thromboembolic radical formation, followed by suppressing the activation of
occlusion-induced rats as compared to the level obtained MAPKs and Akt [18]. In the present study, it is demonstrated,
in the corresponding area of the sham-operated group for the first time, that intraperitoneal administration of
(Figure 7). Hinokitiol treatment at a dose of 0.5 mg/kg shows hinokitiol conceals thromboembolic stroke in rats by reduc-
a significant inhibition (𝑃 < 0.05) of active caspase-3 ing the infarct volume, improves neurological outcome, and
expression in cerebral ischemic tissues (Figure 7). However, inhibits MCAO-reperfusion-induced expressions of TNF-
at a dose of 0.2 mg/kg, hinokitiol treatment was not effective 𝛼, HIF-1𝛼, iNOS, and active caspase-3 protein expressions.
6 Evidence-Based Complementary and Alternative Medicine

Our recent study shows that hinokitiol exhibits antiplatelet

TNF-𝛼
activity ex vivo and anti-thrombogenic activity in vivo [18].
Hypoxia inducible factor-1 is a heterodimeric transcrip-
𝛼-Tubulin tion factor that plays a pivotal role in regulating cellular O2
homeostasis. It is composed of an oxygen-regulated HIF-1𝛼
subunit and a constitutively expressed HIF-1𝛽 subunit. Under
2.5 hypoxic conditions, hypoxia inhibits HIF-1𝛼 hydroxylation
∗∗ and allows its translocation to the nucleus, where it binds
Relative expression of TNF-𝛼 (folds)

to HIF-1𝛽 to form an active complex HIF-1 and initiates

2.0 the transcription of an array of target genes which are vital
for cellular adaption to hypoxia [25]. Recently, it has been
#
1.5 found that many nonhypoxic stimuli, such as cytokines,
free radicals, growth factors, and hormones, can activate
HIF-1𝛼 under normoxic conditions. Degradation HIF1-𝛼 is
1.0 ### reported to be inhibited during hypoxia, allowing its rapid
accumulation and binding to hypoxia responsive elements
0.5 and thus activating the expression of hypoxia-responsive
genes [26], many of which have a neuroprotective effect in
the ischemic brain [27]. Because the iNOS gene contains
0.0 the hypoxia-responsive enhancer (HRE) sequence to which
Sham Control Vehicle 0.2 0.5
HIF-1𝛼 binds [28], results from primary neuronal cultures of
Hinokitiol (mg/kg) cells demonstrated that HIF-1𝛼 binds to the iNOS promoter
Figure 6: Effects of hinokitiol on the protein expressions of TNF- gene under hypoxic conditions. Such binding is associated
𝛼 in MCAO-reperfusion injury in rat cerebral homogenates. Rats with an increase in iNOS expression [29]. Furthermore, HIF-
were pretreated with hinokitiol (0.2 mg/kg or 0.5 mg/kg) before 1𝛼 combined with p53 may promote apoptotic cell death in
ischemia and compared with vehicle or sham control. All groups ischemic areas [28].
are represented as ipsilateral hemisphere. Data are expressed as the A study was demonstrated that iNOS knock-out mice
means ± SEM (𝑛 = 5). ∗∗∗𝑃 < 0.01 compared with sham control showed reduced brain damage after ischemia, because
group; #𝑃 < 0.05 and ###𝑃 < 0.001 compared with vehicle control an increased expression of iNOS may also contribute to
group. enhanced neuronal injury [30], and there is an evidence
that iNOS plays a role as a mediator in the reduction of
infarct size via late preconditioning [31]. A recent study also
Active caspase-3 suggests that iNOS may be involved in the inflammatory
reaction that follows cerebral ischemia, and iNOS mRNA and
𝛼-Tubulin enzymatic activity are expressed in brain after permanent
MCA occlusion [32]. Treatment with the selective iNOS
inhibitor is reported to be reduced infarct volume, suggesting
2.5 that iNOS activity contributes to ischemic brain damage
Relative expression of caspase-3 (folds)

[33]. In the present study, it has been demonstrated that

2.0 ∗ the treatment of hinokitiol in MCAO-induced embolic rats
significantly reduced the expression of HIF-1𝛼 and iNOS,
harmful to the postischemic brain, and may be of worth in
1.5
the treatment of cerebral ischemia.
#
Tumor necrosis factor-𝛼 (TNF-𝛼) is one of the key
1.0 immunomodulatory and proinflammatory cytokines upreg-
ulated during brain ischemia [34]. TNF-𝛼 was clinically
correlated with acute hematoma enlargement, edema devel-
0.5
opment, and poor patient outcome following spontaneous
intracerebral hemorrhage (ICH) [35]. Similarly, increased
0.0 TNF-𝛼 expression was observed in several different species
Sham Control Vehicle 0.2 0.5 and in multiple experimental models of ICH. Several studies
Hinokitiol (mg/kg) have also observed a functional association between peri-
hematomal TNF-𝛼 expression and the development brain
Figure 7: Effects of hinokitiol on the protein expression of caspase-
3 in MCAO-induced rat ipsilateral brain hemisphere. Rats are pre-
edema and neurological injury after ICH [36, 37]. Moreover,
treated with hinokitiol (0.2 mg/kg or 0.5 mg/kg) before ischemia and based on experimental evidence, it has also been reported
compared with vehicle or sham control. All groups are represented that higher circulating levels of TNF-𝛼 are associated with
as ipsilateral hemisphere. Data are expressed as the means ± SEM increased risks of stroke [38], and its administration during
(𝑛 = 5). ∗𝑃 < 0.05 compared with sham control group; #𝑃 < 0.001 ischemic brain insult was shown to augment injury, as evi-
compared with vehicle control group. denced by increased tissue damage and neurological deficits
Evidence-Based Complementary and Alternative Medicine 7

[39]. Consistently, in this study, increased expression of TNF- Hospital, Taipei Medical University (100TMU-WFH-01-2),
𝛼 is directly correlated with neurological deterioration of and Taipei Medical University (TMU-R-100-03).
brain tissues in MCAO-induced embolic rats. Therefore,
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