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Hepatoprotective effect of bisbenzylisoquinoline alkaloid tiliamosine from

Tiliacora racemosa in high fat diet/diethylnitrosamine induced non-alcoholic
steatohepatitis

Article in Biomedicine & Pharmacotherapy · September 2018

DOI: 10.1016/j.biopha.2018.09.116

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 Biomedicine & Pharmacotherapy 108 (2018) 963–973

Contents lists available at ScienceDirect

Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Hepatoprotective effect of bisbenzylisoquinoline alkaloid tiliamosine from T

Tiliacora racemosa in high-fat diet/diethylnitrosamine-induced non-
alcoholic steatohepatitis
S. Sylvester Darvina, Erenius Toppoa, S. Esakkimuthua, T.P. Ajeesh Krishnab, S. Antony Ceasarb,
A. Stalina, K. Balakrishnaa, N. Muniappane, N. Pazhanivelf, R. Mahaprabhuf, M. Gabriel Paulraja, ,
⁎

P. Pandikumara, , S. Ignacimuthua,b,c, , N.A. Al-Dhabid

⁎ ⁎

a
Division of Ethnopharmacology, Entomology Research Institute, Loyola College, Chennai, Tamil Nadu, 600 034, India
b
Division of Biotechnology, Entomology Research Institute, Loyola College, Chennai, Tamil Nadu, 600 034, India
c
International Scientific Partnership Programme, King Saud University, Riyadh 11451, Saudi Arabia
d
Addiriyah Chair for Environmental Studies, Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
e
Department of Veterinary Pharmacology and Toxicology, Madras Veterinary College, Chennai, 600 007, India
f
Department of Veterinary Pathology, Madras Veterinary College, Chennai, 600 007, India

ARTICLE INFO ABSTRACT

Keywords: Non-alcoholic steatohepatitis (NASH) is one of the aggressive forms of non-alcoholic fatty liver disease (NAFLD)
Bisbenzylisoquinoline alkaloids and is a potential risk factor of HCC. This study reports the curative effect of tiliamosine on NASH. Tiliamosine
HepG2 cells was isolated from Tiliacora racemosa Colebr. (Menispermaceae) and its structure was confirmed by studying the
NASH physical and spectroscopic data. The effects of tiliamsoine on lipid accumulation and lipotoxicity were evaluated
NAFLD
using palmitate-oleate induced steatosis in HepG2 cells. The in vivo efficacy of tiliamosine was evaluated using
High fat diet
HFD fed, DEN induced non-alcoholic steatohepatitis Wistar rats. In HepG2 cells, tiliamosine did not affect the
Diethylnitrosamine
cell viability up to 100 μM concentration and showed GI25 value of 264.28 μM. The treatment with tiliamsoine
significantly lowered the ORO concentration by 44.17% and triglyceride accumulation by 69.32% at 50 μM
concentration (P < 0.005). It also reduced the leakage of LDH and transaminases in PO-BSA induced HepG2
cells. The treatment with tiliamsoine significantly decreased the plasma levels of transaminases, phosphatase
and LDH (P < 0.05) in HFD-DEN induced steatohepatitis. The histology and the immunohistochemistry of the
hepatic sections were in accordance with the biochemical findings. Preliminary molecular analysis indicated that
the hepatic FXR expression was upregulated and TNFα expression was downregulated by the treatment with
tiliamsoine. This study provided preliminary evidence on the use of tiliamosine for the treatment of NASH.

1. Introduction factors such as lipotoxicity, inflammation, oxidative stress and insulin

resistance were proposed as the causative factors [2]. In the last two
Non-alcoholic Fatty Liver Disease (NAFLD) is one of the chronic decades, the incidence of NAFLD has increased alarmingly in South
liver diseases and its prevalence is rising globally. NAFLD comprises a Asian countries by 30% due to the epidemic of metabolic syndrome
wide spectrum of health conditions and Non-alcoholic steatohepatitis among the younger population [3]. Among Indians, NAFLD was re-
(NASH) represents an aggressive form of NAFLD, which leads to cir- ported as one of the important causes of HCC [4]. In India, HCC is the
rhosis and hepatocellular carcinoma (HCC). NASH is characterised by leading gastrointestinal cancer; the age adjusted incidence rate ranges
the presence of steatosis, inflammation and ballooning injury in the from 0.7 to 7.5 for men and for women 0.2 to 2.2 per 100,000 persons
hepatocytes [1]. Progression of NASH is poorly known and various per year [5]. Various existing pharmacological interventions such as

Abbreviations: BSA, bovine serum albumin; CMC, carboxymethylcellulose; BBI, bisbenzyl isoquinoline alkaloids; DMSO-d6, deuterated dimethyl sulfoxide; GI,
growth inhibition concentration; HFD, high fat diet; MDA, malondialdehyde; lit.mp., melting point given in the literature; BMI, body mass index; mp, melting point;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NaCl, sodium chloride; NAFLD, non-alcoholic fatty liver disease; NASH, nonalcoholic steato-
hepatitis; ORO, oil red - O; PBS, phosphate buffered saline; POBSA, palmitate, oleate, BSA complex; DEN, diethylnitrosamine; Tc, total cholesterol; Tg, triglyceride
⁎
Corresponding authors at: Division of Ethnopharmacology, Entomology Research Institute, Loyola College, Chennai, Tamil Nadu, 600 034, India.
E-mail addresses: [email protected] (M.G. Paulraj), [email protected] (P. Pandikumar), [email protected] (S. Ignacimuthu).

https://doi.org/10.1016/j.biopha.2018.09.116
Received 10 July 2018; Received in revised form 18 September 2018; Accepted 19 September 2018
0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
S. Sylvester Darvin et al. Biomedicine & Pharmacotherapy 108 (2018) 963–973

thiazolidinediones, glucagon‐like peptide‐1 receptor agonists and novel

interventions such as obeticholic acid have been tried for the man-
agement of NAFLD/ NASH. NASH is considered as the potential risk
factor for the development of fibrosis and cirrhosis in 10–25% of
NAFLD subjects and hence specific pharmacological interventions to
halt NASH progression are needed [6].
Bisbenzyl isoquinoline alkaloids (BBI) are one of the major phyto-
chemicals reported from the members of Menispermaceae [7] and
various biological effects of BBI alkaloids have been reported [8–10].
Some of these alkaloids have curariform activity; Eg: (+)-tubocurarine,
was used as the prototype for the development of skeletal muscle re-
laxants [11]. Hypotensive and antimycobacterial effects of some BBI
alkaloids were reported [12–15]. Cardiovascular effects of some BBI
alkaloids were screened and daurisoline and neferine from Nelumbo
nucifera were shown to have anti-arrhythmic potential [16]. The BBI
alkaloids such as liensinine, neferine and isoliensinine exerted anti-
oxidant effect in lipopolysaccharide elicited microglial cells [17]. Tu-
mour inhibiting effects of some BBI alkaloids such as cissampareine,
thalidasine [18] and tetrandrine [19] were reported in the literature. Fig. 1. Structure of tiliamosine.
Kuroda and his co-workers also demonstrated the antitumour effect of
selected BBI alkaloids using HeLa and Ehrlich Ascites carcinoma cells Elution with chloroform-methanol (9:1) gave tiliamosine (Fig. 1) as
[20]. Marshall and his co-workers evaluated the antiplasmodial, ani- pale brown amorphous powder (yield: 10 g).
tamoebic and cytotoxic properties of selected BBI alkaloids using in
vitro methods [21]. 2.2. In silico studies
Tiliacora racemosa Colebr. (Synonym: Tiliacora acuminata Miers) is
one of the members of Menispermaceae, reported to have various BBI 2.2.1. Prediction of ADME properties and molecular docking of tiliamosine
alkaloids such as tiliamosine, tiliacorine, tiliarine, N - methyl tiliamo- with FXRα
sine, tiliaresine, tiliacorinine, acorine, mosine and nor-tiliacorinine. The pharmacokinetic properties of tiliamosine viz., absorption, dis-
Seal and Mukherjee evaluated the cytotoxic effect of five BBI alkaloids tribution, metabolism and excretion were predicted using DataWarrior
isolated from Tiliacora racemosa [22]. At our laboratory, bioassay software (Version 4.7.2) [23] and SwissADME web tool [24]. Tiliamosine
guided fractionation of Tiliacora racemosa using PO-BSA induced stea- was docked with FXRα (PDB ID: 1OSH) protein using Autodock tools
tosis in HepG2 cells yielded tiliamosine as the active constituent. This [25–32] (Supplementary data 1).
study reports the hepatoprotective effect of tiliamosine using in vitro
and in vivo models of non-alcoholic steatohepatitis.
2.3. Bioassays
2. Methodology
2.3.1. Chemicals
Dulbecco modified eagle medium (DMEM), Fetal bovine serum
2.1. Phytochemistry
(FBS) and Trypsin were procured from Gibco (India); Fenofibrate,
HEPES, MTT, ORO, sodium palmitate and sodium oleate were acquired
2.1.1. Instrumentation (Chemistry)
from Sigma Aldrich (Bangalore, India). Antibiotic mixture, gentamycin,
The UV–vis spectrum was recorded on a UV–vis SpectrumTek
LDH and MDA (EZcount™ LDH Cell Assay Kit, #CCK036) and
double beam spectrometer in MeOH in the range of 200–500 nm. IR
(EZAssay™ TBARS Estimation Kit for Lipid Peroxidation, #CCK023)
spectrum was recorded on a Perkin- Elmer FT-IR grating spectrometer
assay kits were obtained from Hi- Media chemicals (Mumbai, India).
(Spectrum Two) in KBr disc. 1H and 13C NMR were taken on Bruker
RNAlater, RNeasy mini kit and QuantiTect Reverse Transcription Kit
instrument at 500 and 125 MHz, respectively in DMSO-d6 with TMS as
were obtained from Thermo Scientific (Germany). Kits used for bio-
the internal standard. Chemical shifts values are given in δ scale.
chemical assays were purchased from Accurex biomedical Pvt Ltd
(Mumbai, India) and Agappe diagnostics (Kochi, India). All other che-
2.1.2. Plant material
micals and solvents of analytical grade were purchased from Qualigens.
Aerial parts of Tiliacora racemosa were collected from Pallavaram
and its environs, Kancheepuram District, Tamilnadu, India and its bo-
tanical identity was authenticated by PP, one of the authors of this 2.3.2. Cell culture
communication. A voucher specimen (ERI/EP/SD/01) was deposited in HepG2 cells obtained from National Centre for Cell Sciences (Pune,
the herbarium at Entomology Research Institute, Loyola College, India) were cultured in DMEM supplemented with 1% antibiotic solu-
Chennai for future reference. tion (100 U/mL penicillin, 100 μg/mL streptomycin sulfate, 0.25 μg/mL
amphotericin B) and 10% FBS. The cells were maintained in a humi-
2.1.3. Isolation of tiliamosine dified 5% CO2 incubator at 37 °C. The cells were sub-cultured at 90%
To extract the alkaloids, coarsely powdered aerial parts of T. race- confluence by total media replacement using 0.25% (w/v) trypsin in
mosa (2.5 Kg) were soaked in 5% aqueous acetic acid for 48 h and fil- 0.53 mM EDTA and they were used for the experiments. DMSO was
tered through Whatman #1 paper. The aqueous acidic solution was used to dissolve the test materials and the final concentration of the
washed with chloroform, cooled in ice and basified to pH 10 with DMSO in the media was kept as 0.01%.
aqueous ammonia. It was then extracted with chloroform (3 x 200 mL).
Combined chloroform layer was washed with water and then dried over 2.3.3. MTT assay and fixation of the doses for in vitro assays
anhydrous sodium sulphate and concentrated to yield crude alkaloid The cytotoxic effect of the tiliamosine was investigated using MTT
mixture (15 g). The crude alkaloid mixture was packed on a flash assay as described in the previously published work [33] and the con-
column (100–200 mesh silica gel; Avra) in chloroform and eluted with centrations of tiliamosine were fixed between 12.5 and 300 μM. The
chloroform-methanol mixture with increasing amounts of methanol. assays were done in triplicates; the cell population growth percentage

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S. Sylvester Darvin et al. Biomedicine & Pharmacotherapy 108 (2018) 963–973

Fig. 2. (a) Docked and (b) hydrophobic orientations of tiliamosine and depiction of corresponding amino acid residues with FXRα active site.

Fig. 3. (a) Docked and (b) hydrophobic orientations of WAY-362450 and depiction of corresponding amino acid residues with FXRα active site.

Table 1
Molecular docking of tiliamosine and WAY-362450 with FXRα protein (1OSH).
Ligand Binding amino acid residues Binding Energy Inhibition Constant VDW_HB desolv_energy Ligand efficiency
(kcal/mol) (μM) (kcal/mol)

Tiliamosine PRO`270/O, GLU`293/OE1/OE2 −6.26 25.96 −7.75 0.14

WAY-362,450 HIS`298/NE2 −8.47 623.7 −9.65 0.26

was calculated using the following formula. 2.3.4. Hepatoprotective effect of tiliamosine on PO-BSA induced
lipotoxicity in HepG2 cells
Cell viability percentage =
Abs570 nm (test material)
× 100 The experiment was carried out according to the previously pub-
Abs570 nm (control) lished procedure [33]. The concentrations of tiliamosine for this study
were fixed between 12.5 and 50 μM, on the basis of MTT assay. Fe-
GI25 value of tilimosine was calculated using linear regression nofibrate was used as the positive control and its dose was fixed as 50
analysis with GraphPad Prism (Version 5.0) and cell viability percen- μM [33]. After 24 h of treatment, the amount of lipids accumulated into
tage. the doses for further studies were kept below its GI25 value. the cells was measured using ORO staining [34]. In another set of

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S. Sylvester Darvin et al. Biomedicine & Pharmacotherapy 108 (2018) 963–973

2.3.5.2. Acute toxic effect of tiliamsoine in rats. Acute toxicity was

estimated according to OECD guideline #423 [36] without any
modification; the LD50 cut-off value was found and the dose range for
evaluating the efficacy was kept below 1/20th dose of LD50 cut-off value
[37].

2.3.5.3. Test samples. Tiliamosine was suspended in a vehicle

containing 0.9% NaCl, 0.5% CMC, 0.2% Tween-80 and 98.4%
distilled water. Sorafenib was selected as positive control and its
animal equivalent dose was fixed as 60 mg/kg on the basis of body
surface area [38]. The vehicle alone served as negative control. The
dose range for tiliamosine was fixed between 50 and 100 mg/kg. The
treatments were given through oral gavage, once daily continuously for
four weeks.
Fig. 4. Effect of tiliamosine on the viability of HepG2 cells after 24 h of treat-
ment.
2.3.5.4. Induction of non-alcoholic steatohepatitis by high fat diet and
Results are expressed as per cent viability from normal, untreated cells; Values
indicate mean ± SD of three independent experiments; Values having same
diethylnitrosamine. HFD – DEN induced Non-alcoholic steatohepatitis
alphabet did not deviate significantly (Tukey’s HSD; P ≤ 0.05). was developed according to the method of Wang and coworkers [39].
One week after acclimatization, the animals except in normal control
group were given a single intraperitoneal injection of DEN (30 mg/kg in
experiment, the cells were harvested and lysed using lysis buffer con-
PBS) and fed ad libitum with HFD for eight weeks before the start of the
taining 1% Triton X-100 in PBS. The level of Tg in the cell lysate was
treatment and throughout the experimental period. The animals in
estimated by GPO-PAP method, as per the manufacturer’s directions.
normal control group were given an intraperitoneal injection with PBS
The protein content in the cell lysate was estimated by Lowry’s method,
and fed with NFD. On the eighth week after HFD-DEN administration,
using a commercial kit. The level of Tg in the cell lysate was normalized
the animals were randomized and the treatments were given
with protein level and the results were given as mg Tg per g protein for
continuously for four weeks. Group I (normal control) consisted of
three independent experiments. The effect of tiliamosine on protection
normal animals treated with vehicle; Group II (disease control)
of hepatic cells from lipotoxicity induced by PO-BSA was assessed by
consisted of negative control animals administered with HFD-DEN
estimating the LDH and MDA levels [35] using a commercial kit as per
and treated with vehicle; Group III (positive control) consisted of
manufacturer’s instructions Likewise, the levels of transaminases in the
animals administered with HFD- DEN and treated with sorfeinb
spent media were also measured using commercial spectrophotometric
(60 mg/kg) and the animals in Group IV and V consisted of those
kits.
administered with HFD-DEN and treated with tiliamosine at 50 and
100 mg/kg concentrations, respectively. Thirty rats were used for this
2.3.5. Evaluating the in vivo hepatoprotective efficacy of tiliamosine in study and each group consisted of six animals.
HFD-DEN induced Non-alcoholic steatohepatitis
2.3.5.1. Animals. Eight weeks old male Wistar rats purchased from the 2.3.5.5. Ultrasonography. Abdominal ultrasonography of the animals
Tamil Nadu Veterinary and Animal Sciences University, Chennai were was done before (zero day) and after (25th day) the treatment using
used in this study. The animals were maintained in polypropylene cages esaote MyLab™ Gamma Ultrasound instrument at Frequency of
at 21 ± 1 °C with a relative humidity of 50 ± 5% and 12/12 light/ 10–22 MHz at the Department of Veterinary Pharmacology, Madras
dark cycles. All these protocols were approved by the Institutional Veterinary College, Chennai by NM, one of the authors of this
Animal Ethics Committee (Clearance number: IAEC-ERI-LC-06/14). manuscript.

Table 2
Effect of tiliamosine on lipid accumulation and lipotoxicity in PO-BSA induced steatotic HepG2 cells.
Groups % ORO Tg % LDH release MDA level in the cell lysates ALT AST
(mg Tg/ g protein) (nM/mg protein) (IU/L) (IU/L)

Normal control 101.3 ± 2.93a,c 22.31 ± 3.14a 4.84 ± 1.37a 5.77 ± 1.48a 32.76 ± 3.44a 19.22 ± 2.37a
Disease control 167.9 ± 18.65b 79.32 ± 6.52b 46.94 ± 5.23b 29.90 ± 1.58d 101.9 ± 8.48b 48.15 ± 2.49b
Fenofibrate (50 μM) 115.8 ± 10.54a,c 32.76 ± 1.97a,c 3.91 ± 1.38a 12.44 ± 0.60c 38.33 ± 7.22a,c 22.08 ± 1.44a
Tiliamosine(12.5 μM) 121.2 ± 1.14c 34.83 ± 4.82c 18.77 ± 1.33c 11.29 ± 0.48b,c 52.93 ± 2.58c 30.64 ± 1.24c
Tiliamosine (25 μM) 107.9 ± 5.64a,c 27.91 ± 5.60a,c 12.58 ± 1.24c 9.938 ± 0.35b,c 43.31 ± 2.37a,c 28.18 ± 1.44c
Tiliamosine (50 μM) 93.72 ± 4.85a 24.34 ± 3.70a,c 2.98 ± 1.12a 8.94 ± 0.13b 29.74 ± 8.58a 19.66 ± 1.17a

Values indicate mean ± SD of three independent experiments; values having same alphabets in a column did not vary significantly (Tukey’s HSD; P ≤ 0.05).

Table 3
Effect of tiliamosine on the masses of selected organs in animals with HFD-DEN induced non-alcoholic steatohepatitis after four weeks of treatment.
Group Liver Kidney Spleen Adipose

a a a
Normal control 3.14 ± 0.61 0.71 ± 0.20 0.25 ± 0.09 0.79 ± 0.24a,d
Disease control 6.06 ± 1.34b 1.03 ± 0.24a 0.36 ± 0.14a 1.89 ± 0.54b
Sorafenib (60 mg/kg) 3.14 ± 0.49a 0.83 ± 0.05a 0.30 ± 0.04a 1.15 ± 0.19c,d
Tiliamosine (50 mg/kg) 2.58 ± 0.56a 0.67 ± 0.19a 0.26 ± 0.07a 0.54 ± 0.08a
Tiliamosine (100 mg/kg) 2.48 ± 0.80a 0.75 ± 0.31a 0.30 ± 0.06a 0.69 ± 0.19a,c

Values indicate mean ± SD of six animals; Values having same alphabet in a column did not vary significantly (Tukey’s HSD; P ≤ 0.05).

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S. Sylvester Darvin et al. Biomedicine & Pharmacotherapy 108 (2018) 963–973

Fig. 5. Representative microphotographs of PO-BSA induced steatotic HepG2 cells treated with tiliamosine.
The cells were treated with the test materials for 24 h and the images were taken after ORO staining at 400× magnification.

MyLab™ Gamma used for the imaging of abdominal organs of the rats
used in the study. The frequency was set at 15 MHz, depth was set at
15 mm. Time Gain Control (TGC) and overall gain were adjusted time
to time to get proper image.

2.3.5.6. End of the study. Feed and water intake were recorded at 9 a.m.
every day and the body weights were measured once a week. The animals
were euthanized under CO2 anesthesia at 28th day after treatment, and blood
was collected through cardiac puncture and stored in EDTA coated tubes.
Plasma was separated from blood after centrifugation at 3500 rpm for 15 min
and stored at 4 °C for biochemical assays. Masses of liver, kidney, spleen and
Fig. 6. Effect of tiliamosine on the body weight of animals with HFD-DEN in- reteroperitoneal adipose tissue were recorded. Liver tissues were trimmed
duced non-alcoholic steatohepatitis after four weeks of treatment. and stored in RNase Later for RNA extraction and fixed in 10% neutral-
Values represent mean ± SD of six animals; Values carrying same alphabet for buffered formalin for histological examination, respectively. Tc (Agappe,
a week did not vary significantly (Tukey’s HSD; P ≤ 0.05). #11403002), Tg (Agappe, #114140002), HDL-c (Agappe, #11414004), ACP
(Accurex, #L02017), ALP (Accurex, #15417), ALT (Accurex, #L04517), AST
2.3.5.5.1. Animal preparation. Rats were anesthetised by an (Accurex, #L06217), GGT (Accurex, #03317), LDH (Accurex, #L11018),
intraperitoneal injection of xylazine (13 mg/kg) and ketamine (87 mg/kg) Albumin (Accurex, #L74716), Total protein (Agappe, #11013003) and Urea
[40]. Immobilisation and anaesthetic effects were noticed after 15 min and (Agappe, #11216001) levels were measured on the same day of euthanasia
the anaesthesia lasted up to 3–4 h post administration of anaesthetics. using commercial spectrophotometric assay kits.
Ventral abdominal areas were shaved, the animals were placed on supine
position on the ultrasound table and the ultrasound gel was applied on the 2.3.5.7. Histopathology and immunohistochemical staining. Neutral buffered,
shaved area before ultrasonography. formalin fixed and paraffin inserted liver tissues were processed and stained
2.3.5.5.2. B-mode ultrasound. The ultrasound scanner, esaote with hematoxylin and eosin. The slides were evaluated for ballooning

Table 4
Effect of tiliamosine on plasma and hepatic lipid profiles in animals with HFD-DEN induced non-alcoholic steatohepatitis after four weeks of treatment.
Group Total cholesterol Triglycerides HDL cholesterol TC/HDL-c Liver lipid
(mg/dL) (mg/dL) (mg/dL) Levels (mg/g liver)

Normal control 49.59 ± 5.22a 75.18 ± 22.32a 68.98 ± 6.61a 0.72 ± 0.13a 31.44 ± 0.83a
Disease control 128.50 ± 27.70b 189.40 ± 58.05b 68.52 ± 6.97a 1.87 ± 0.31b 55.18 ± 11.61b
Sorafenib (60 mg/kg) 98.40 ± 4.65b,d 182.30 ± 8.62b 80.19 ± 11.80a 1.24 ± 0.18a,b 40.95 ± 4.36a,b
Tiliamosine (50 mg/kg) 69.53 ± 28.47a,d 108.20 ± 29.36a 54.07 ± 16.57a 1.33 ± 0.44a,b 33.42 ± 1.72a
Tiliamosine (100 mg/kg) 58.95 ± 7.52a 80.68 ± 35.69a 50.19 ± 21.05a 1.30 ± 0.39a,b 34.60 ± 3.02a

Values indicate mean ± SD of six animals; Values having same alphabet in a column did not vary significantly (Tukey’s HSD; P ≤ 0.05).

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S. Sylvester Darvin et al. Biomedicine & Pharmacotherapy 108 (2018) 963–973

Table 5
Effect of tiliamosine on plasma hepatic marker enzymes in animals with HFD-DEN induced non-alcoholic steatohepatitis after four weeks of treatment.
Group ACP ALP ALT AST GGT LDH

a a a a a
Normal control 3.85 ± 0.97 17.37 ± 7.45 32.91 ± 9.43 53.83 ± 7.31 19.19 ± 5.86 334.1 ± 81.26a
Disease control 46.34 ± 13.80b 94.95 ± 10.85b 223.5 ± 66.88b 214.1 ± 67.22b 67.50 ± 9.85b 1711.0 ± 438.6b
Sorafenib (60 mg/kg) 8.15 ± 1.64 51.35 ± 10.58c 81.92 ± 18.78a,c 136.8 ± 23.70c 38.78 ± 6.09c 894.1 ± 213.8a
Tiliamosine (50 mg/kg) 10.90 ± 1.97a 57.67 ± 11.61c 90.60 ± 22.67c 141.6 ± 30.45c 44.55 ± 16.36c 741.4 ± 624.2a
Tiliamosine (100 mg/kg) 9.60 ± 2.62a 50.37 ± 13.69c 96.44 ± 11.27c 131.6 ± 46.09c 47.87 ± 11.73c 654.7 ± 257.6a

Values (in IU/L) indicate mean ± SD of six animals; Values having same alphabet in a column did not vary significantly (Tukey’s HSD; P ≤ 0.05).

Table 6
Effect of tiliamosine on plasma protein and urea levels in animals with HFD-DEN induced non-alcoholic steatohepatitis after four weeks of treatment.
Group Albumin Total protein Albumin/ Urea
(g/dL) (g/dL) Total Protein (mg/dL)

Normal control 1.88 ± 0.09a 6.53 ± 0.53a 0.29 ± 0.03a 15.18 ± 2.86a
Disease control 1.52 ± 0.13b 7.55 ± 1.35a 0.20 ± 0.02b 52.85 ± 2.09b
Sorafenib (60 mg/kg) 1.99 ± 0.14a 7.20 ± 0.88a 0.27 ± 0.01a 25.66 ± 4.50c
Tiliamosine (50 mg/kg) 1.87 ± 0.10a 6.75 ± 0.07a 0.27 ± 0.01a 20.18 ± 6.08a,c
Tiliamosine (100 mg/kg) 1.78 ± 0.31a 6.81 ± 0.46a 0.26 ± 0.06a 27.28 ± 8.15c

Values indicate mean ± SD of six animals; Values having same alphabet in a column did not vary significantly (Tukey’s HSD; P ≤ 0.05).

degeneration, apoptosis, fatty changes and MNC infiltration. Liver cell 3. Results
proliferation was semi-quantitatively assessed by immunostaining using
proliferating cell nuclear antigen (PCNA). Briefly tissue sections after 3.1. Phytochemistry
regular dehydration and rehydration, were incubated with 3% H2O2 for
15 min. Then, they were heated using a steamer for 20 m in 10 mM sodium Tiliamosine (Fig. 1) was isolated form aerial parts of T. racemosa as
citrate (pH 6.0) buffer to retrieve antigen. The routine biotin-streptavidin pale brown amorphous powder. It analysed for C36H36N2O6 and the
immunohistochemical method consisted of sequential incubations in goat or ESI-MS gave [M + H]+ at m/z 593. It gave positive Drangendorff’s test
horse serum blocking solution, and streptavidin conjugated to a horseradish for alkaloids. The compound was identified by its physical and spec-
peroxidase label. The liver specimens were finally treated with troscopic data; it was previously isolated from the same plant [48–50].
diaminobenzidine and then counterstained with hematoxylin. For PCNA To the best of our knowledge this is the first report on the complete
staining, a total of ten randomly chosen fields were evaluated and the assignment of the 1H and 13C NMR of tiliamosine (Supplementary data
results were expressed as the number of PCNA positive cells per 100 2).
hepatocytes.
3.2. In silico analysis of tiliamosine

2.3.5.8. Liver lipid quantification by sulphophosphovanillin In silico investigation of ADME properties of tiliamosine are given in
method. Quantification of liver lipids was done according to the supplementary Table 3 which showed tiliamosine lacked mutagenic,
method of Barnes and Blackstock [41]. One gram of liver sample was tumorigenic and irritant properties. Tiliamosine showed strong binding
homogenised in chloroform: methanol (2:1 ratio) mixture using affinity to the active site of FXRα at PRO`270/O, GLU`293/OE1/OE2
homogeniser thrice; the solvent portion was centrifuged at 3000 rpm (Fig. 2); WAY-362450 showed binding on FXRα at HIS`298/NE2
for 15 min; the supernatant was taken and made into 10 mL with (Fig. 3). The binding affinity and ligand efficiency of tiliamosine was
chloroform: methanol mixture. In a clean vial, 0.5 mL of the sample comparable with that of WAY-362450 (Table 1).
solution was taken, dried under nitrogen atmosphere, added with
0.5 mL of conc. H2SO4 and heated in a boiling water bath for 10 min. 3.3. Effect of tiliamosine on the viability of HepG2 Cells
The mixture was cooled; 5 mL of vanillin - phosphoric acid regent was
added with 0.2 mL acid digest, incubated for 30 min and the absorbance The effect of tiliamosine on the viability of HepG2 cells was ana-
was read at 530 nm. Olive oil was used as the standard and the results lysed using MTT assay and it was used for the fixation of doses for
were expressed as mg Tg/ g wet tissue. further in vitro studies. The treatment with tiliamosine did not exhibit
any toxicity up to 100 μM and it showed GI25 value of 264.28 μM. The
dose range of tiliamosine was fixed between 12.5 and 50 μM for in vitro
2.3.5.9. Preliminary assessment of mRNA expression of selected genes in studies (Fig. 4).
PO-BSA induced HepG2 cells and HFD-DEN induced rats. Primers of genes
such as PPARα, PPARγ, FXR, TNFα and β-actin were designed in 3.4. Effect of tiliamosine on lipid accumulation of PO-BSA induced steatotic
consultation with previously published literature [42–47]. The HepG2 cells
protocols and primer sequences are given in Supplementary data 3.
HepG2 cells incubated with PO-BSA complex showed 1.6 fold increment
in the ORO concentration; it indicated the increased accumulation of in-
2.3.6. Statistics tracellular Tg. The treatment with tiliamosine at all the selected con-
The values were given as mean ± SD for six animals. Statistical centrations brought down the ORO concentration. To confirm this finding,
significances of the data were assessed using one way analysis of var- intracellular Tg content was estimated; the reductions in intracellular Tg
iance followed by Tukey’s HSD (Graphpad Prism, Ver. 5.0, CA, USA) level at 25 and 50 μM concentrations were comparable with that of normal
and considered as significant at P ≤ 0.05. control and fenofibrate (Table 2). Microphotographs of HepG2 cells stained

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Fig. 7. Representative abdominal ultrasonogram of animals with HFD-DEN induced non-alcoholic steatohepatitis after four weeks of treatment with tiliamosine.
Scans were taken by B-mode ultrasound with Frequnce at 15 MHz and depth of 15 mm; (A) Normal control; (B) Negative control; (C) Sorafenib (60 mg/kg); (D)
Tiliamosine (50 mg/kg) and (E) Tiliamosine (100 mg/kg).

with ORO also substantiated the lipid lowering effect of tiliamosine in PO- also reduced the levels of transaminases in the spent media indicating
BSA induced steatosis (Fig. 5). the reduction of membrane damage. This was further confirmed by the
reductions in the MDA content in the treated cells (Table 2).

3.5. Effect of tiliamosine on PO-BSA induced lipotoxicity in HepG2 cells

3.6. Effect of tiliamosine on the expression of selected genes of HepG2 cells
Incubating HepG2 cells with PO-BSA increased the leakage of LDH
indicating the lipotoxicity by PO-BSA. The treatment with tiliamosine PO-BSA administration significantly lowered the relative expres-
significantly brought down the leakage of LDH to normal at 50 μM sions of FXR and PPARα in HepG2 cells, while it increased expression of
concentration; it indicated the reduction of lipotoxicity. The treatment PPARγ. The treatment with tiliamosine significantly improved the

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Fig. 8. Representative histological images of

liver of animals with HFD-DEN induced non-
alcoholic steatohepatitis after four weeks of
treatment with tiliamosine.
Representative micrographs of (A) normal
control; (B) negative control; (C) sorafenib
(60 mg/kg); (D) tiliamosine (50 mg/kg) and
(E) tiliamosine (100 mg/kg) were given; clear
cells are marked with red arrows; macro-vesi-
cular steatosis are marked with bold arrows;
large lipid droplets are marked with doted ar-
rows and aggregates of inflammatory cells are
marked with circles (F) Steatohepatitis Activity
Scores; ND – Not detected; values
(mean ± SD) having same alphabet did not
vary significantly (Tukey’s HSD; P ≤ 0.05).

expressions of FXR; it lowered the expression of PPARγ (Supplementary tissue mass. Kidney and spleen showed statistically insignificant inter-
data 3). group variations (Table 3).

3.7. Acute toxicity study of tiliamosine 3.9. Effect of tiliamosine on the plasma biochemistry and liver lipid levels of
animals with HFD-DEN induced non-alcoholic steatohepatitis
Tiliamosine did not show any potential toxic effects on rats up to
2 g/kg concentration and it fell in the category 5 as per the OECD The treatment with tiliamosine significantly lowered the TC and Tg
guideline #423. The LD50 cut-off value of tiliamosine fell > 2 g/kg levels four weeks after treatment. The treatment did not alter the HDL-c
concentration and the dose range was fixed between 50–100 mg/kg. levels, but it significantly reduced the TC/HDL-c ratio. The liver lipid
levels were also significantly reduced by the treatment with tiliamosine
3.8. Effect of tiliamosine on relative organ masses in animals with HFD- (Table 4). The treatment with tiliamosine significantly altered the levels
DEN induced non- alcoholic steatohepatitis of phosphatases, transaminases, GGT and LDH levels; these reductions
were comparable with that of positive control (Table 5). In the treated
At the end of the treatment period, the disease control animals animals there was an improvement in the plasma albumin levels, but
showed 35.04% loss in the body mass compared to the normal control this increment was statistically insignificant (Table 6).
animals (Fig. 6). This can be correlated with the reduction in the feed
and water intake (Supplementary data 4). The treatment with tilia- 3.10. Effect of tiliamosine on the expression of selected genes in animals
mosine significantly improved the body weight and this increment was with HFD-DEN induced non-alcoholic steatohepatitis
comparable with that of normal control and sorafenib. The treatment
further lowered the relative liver mass and reteroperitoneal adipose As in the case of PO-BSA induced HepG2 cells, liver tissues from the

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Fig. 9. Representative immunostained hepatic sections to show Proliferating Cell Nuclear Antigen (PCNA) expression from animals of HFD-DEN induced non-
alcoholic steatohepatitis after four weeks of treatment with tiliamosine.
Representative micrographs of (A) Normal control; (B) Disease control ; (C) Sorafenib (60 mg/kg); (D) Tiliamosine (50 mg/kg); (E) Tiliamosine (100 mg/kg) were
given. (F) Percentage of PCNA positive hepatocytes were counted from ten randomly selected fields at 400x magnification; Values (mean ± SD) having same
alphabet did not vary significantly (Tukey’s HSD; P ≤ 0.05).

treated animals showed the upregulation of FXR mRNA expression and mononuclear cell infiltration (Fig. 8). Hepatic sections were im-
downregulation in the mRNA expression of TNFα (Supplementary data munostained with PCNA to semi-quantitatively assess the hepatocyte
3). proliferation and this also showed that the treatment with tiliamosine
significantly reduced the number of PCNA positive cells (Fig. 9).
3.10.1. Ultrasound observation of effect of tiliamosine animal liver with
HFD-DEN induced non-alcoholic steatohepatitis 4. Discussion
Disease control animals showed mixed echogenicity and diffuse le-
sions in hepatic lobes; it indicated the presence of steatohepatitis. The NASH is one of the serious forms of NAFLD and its role on cirrhosis
animal treated with sorafenib and tiliamosine showed the homogenous and HCC was initially reported in 1990s; further studies demonstrated
echogenicity which showed the ameliorative effect of tiliamosine on that NASH as one of the risk factors of HCC [51]. So far, no specific
NASH (Fig. 7). pharmacological intervention was prescribed for the treatment of
NASH. NASH is connected with many pathways and molecules; thus
3.10.2. Effect of tiliamosine on the liver histology of animals with HFD-DEN there is a need to identify leads that target multiple pathways with
induced non- alcoholic steatohepatitis minimal adverse effects. Various natural products have been tried for
Administration of HFD and DEN to the animals induced congestion treatment of NAFLD; many phytochemicals were shown to have anti-
and hydropic degeneration of hepatocytes. The hepatocytes in the HFD- NAFLD efficacy by modulating various molecular pathways [52–54].
DEN control animals were non- eosinophilic and showed multifocal This study documented the curative effect of tiliamosine, a BBI alkaloid
mononuclear cell infiltration. The treatment with tiliamosine sig- on NASH.
nificantly improved the viability of hepatocytes and it reduced the In silico evaluation of the ADME properties of tiliamosine showed

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good bioavailability score and it lacked any mutagenic, tumorigenic Acknowledgments

and irritant properties. Molecular docking analysis showed that tilia-
mosine shared strong binding affinity on active site of the FXRα as We thank Entomology Research Institute, Loyola College, Chennai
WAY- 362450. MTT assay showed that tiliamsoine was not toxic up to for providing facilities and financial assistance. The authors extend
100 μM. Free Fatty Acid induced HepG2 cells shared morphological their appreciation to the International Scientific Partnership Program
resemblance to human steatotic hepatocytes and are widely used for (ISPP) at King Saud University for funding this research work thorough
screening molecules to treat NAFLD [55,56]. In this study, adminis- ISPP #0020. The authors sincerely thank the reviewers and editors for
tration of PO-BSA complex induced steatosis and lipotoxicity as in- their critical assessment and comments, which was helpful to improve
dicated by increased intracellular Tg levels and leakage of LDH as well the quality of the manuscript.
as transaminases. The treatment with tiliamosine significantly lowered
the accumulation of Tg and lowered the lipotoxicity indicating its anti- Appendix A. Supplementary data
NAFLD potential.
According to multiple hit hypothesis, steatosis precedes pathogen- Supplementary material related to this article can be found, in the
esis of NASH [57] and the co-administration of HFD [39] or wester- online version, at doi:10.1016/j.biopha.2018.09.116.
nized diet [58] or alcohol [59] with DEN progresses the pathogenesis of
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