Asian Pacific Journal of Tropical Biomedicine (2012)S601-S604 S601

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Asian Pacific Journal of Tropical Biomedicine

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Document heading

Phytochemical screening, antibacterial and free radical scavenging effects

of Artemisia nilagirica, Mimosa pudica and Clerodendrum siphonanthus
-An in-vitro study
Arokiyaraj S1*, Sripriya N1, Bhagya R1, Radhika B1, Prameela L1, Udayaprakash NK2
1
Department of Biotechnology, Vel Tech High Tech Dr.Rangarajan Dr.Sakunthula Engineering College, Avadi, Chennai, India
2
Research and Development, Vel Tech Dr.RR Dr.SR Technical University, Avadi, Chennai, India

ARTICLE INFO ABSTRACT

Article history: Objective: To evaluate methanolic extracts of leaves of Artemisia nilagirica, Mimosa pudica and
Received 18 May 2012 Clerodendrum siphonanthus for phytochemical analysis, antibacterial activity and free radical
Received in revised form 21 May 2012 scavenging activity. Methods: Antibacterial activity was performed by disc diffusion method
Accepted 15 August 2012
against two gram positive and four gram negative strains. Free radical scavenging potential was
Available online 28 August 2012
evaluated using total antioxidant activity (thiocyanate method) and diphenyl-picryl-hydrazyl
(DPPH) assay. Results: Results of the present study showed that Clerodendrum siphonanthus
Keywords: exhibited significant antibacterial effect against Klebsiella pneumoniae (30 mm), Proteus mirabilis
(16 mm), Salmonella typhi (16 mm), Staphylococcus aureus (12 mm), Escherichia coli (11.5 mm)
Antibacterial activity
Artemisia nilagirica and Bacillus subtilis (10 mm). Mimosa pudica and Artemisia nilagirica showed good antibacterial
Clerodendrum siphonanthus effects. Clerodendrum siphonanthus was found to be extremely effective in scavenging lipid
Free radical peroxide (IC50 8 mg/mL) and DPPH radicals (IC50 7 mg/mL), whereas Artemisia nilagirica and
Mimosa pudica Mimosa pudica showed moderate activity. Phytochemical analysis of these plants revealed
Phytochemicals presence of tannins, alkaloids, flavanoids, terpenoids and glycosides. Conclusions: This study
showed that Artemisia nilagirica, Mimosa pudica and Clerodendrum siphonanthus may serve as a
potential agent for new therapeutics.

1. Introduction researchers have always felt the need for scientific

screening of the plants, which may help the pharmacologists
A ccording to the W orld H ealth O rganization, 80 % of and phytochemists. In drug discovery, random screening
Asian and African population still depends on traditional as a tool in identifying new biologically active molecules
medicine for primary health care. Globally, India has been has been the most productive. Free radicals are generated
acknowledged as a major resourceful area in traditional by both internal (cellular respiration etc ) and external
medicine. The primary benefits of using plant derived ( alcohol, pollution, smoking etc ) sources. T hese free
medicine are that they are relatively safer than synthetic radicals can damage all cellular macromolecules (proteins,
alternatives, offering profound therapeutic benefits and carbohydrates, lipids and nucleic acids) and attribute to
affordable treatment. Many commercially proven drugs cancer and atherosclerosis[2]. However, these radicals are
used in modern medicine are from traditional medical controlled by antioxidants, which can safely interact and
plants, with ethnobotanical and ethnomedical knowledge[1]. terminate the chain reaction before vital molecules are
A ntimicrobial substances present in tissues of higher damaged. There are several enzyme systems (catalase,
plants have long been regarded as important factors in superoxide dismutase etc) within our body that scavenge
the resistance of higher plants to various bacteria. Hence, free radicals. In addition, micronutrients such as vitamin E,
beta-carotene and vitamin C from dietary sources can act
as antioxidants[3,4].
*Corresponding author: Dr. Arokiyaraj S, Assistant Professor , D e p a r t m e n t o f
Biotechnology, Vel Tech High Tech Dr.Rangarajan Dr.Sakunthula Engineering College, Worldwide, more than hundreds of plants are used as
Avadi, Chennai, India. traditional medicine for the treatment of bacterial infections
E-mail: [email protected]
Foundation project: It is supported by Chairman, Veltech Group of Institutions, Avadi, and other diseases[5,6]. Although many have been treated
Chennai f (G.NO. HTS 728).
S602 Arokiyaraj S et al ./Asian Pacific Journal of Tropical Biomedicine (2012)S601-S604

by conventional pharmaceutical approaches, there is extracts of A. nilagirica, M. pudica and C. siphonanthus

growing interest in the use of natural products by the (5 mg/disc). The loaded discs were placed on the surface
general public. In the present study, three medicinal plants of the medium and were left to diffuse for 30 min at room
namely Artemisia nilagirica (Asteraceae)(A. nilagirica), temperature. Negative control was prepared using respective
Mimosa pudica (Fabaceae)(M. pudica) and Clerodendrum solvents. Streptomycin (15毺g/disc) was used as positive
siphonanthus (Lamiaceae)(C. siphonanthus) were selected. control. The plates were incubated at 37 曟 for 24 h. The zone
Traditionally, these plants have been acknowledged to of inhibition was recorded in millimeters and the experiment
reduce inflammation and also to treat lung diseases, cough was performed thrice.
and cold[7]. The objective of the study is to evaluate the
phytochemical analysis, antibacterial and free radical 2.4. Determination of Total Antioxidant Activity
scavenging activities of the selected medicinal plants.
The antioxidant activities of A. nilagirica, M. pudica and
C. siphonanthus were determined using the thiocyanate
2. Materials and methods method[10]. Four different concentrations (5, 10, 25 and 50
mg/mL) of the extracts were prepared in methanol and were
2.1 Plant materials and extraction added to linoleic acid emulsion (2.5 mL, 40 mM, pH 7.0)
and phosphate buffer (2 mL, 40 mM, pH 7.0). The linoleic
Leaves of A. nilagirica, M. pudica and C. siphonanthus acid emulsion was prepared by mixing 0.280 4 g linoleic
were collected from Nilgiri district and Chenglepet, Tamil acid with 0.280 4 g Tween-20 as an emulsifier in 50 mL 40
Nadu, India, in April 2010, which was authenticated by mM phosphate buffer[11]. The final volume was adjusted
the Department of Plant Biology & Biotechnology, Loyola to 5 mL using 40 mM phosphate buffer at p H 7.0, after
College, Chennai, India. A voucher specimen was deposited. homogenization. The mixed samples were then incubated
Leaves of A. nilagirica, M. pudica and C. siphonanthus at 37oC in a glass flask for 60 h, to accelerate the oxidation
were shade dried, powdered and used for the extraction. process. One milliliter of the incubated sample was removed
About 1 kg of dry powder was taken in an aspirator bottle to after 12 h, to which 0.1 mL 20 mM FeCl2 and 0.1 mL 30%
which 3 l of methanol was added for the extraction. This was ammonium thiocyanate were added. The absorbance of
shaken occasionally for 48 h and the extract was filtered with this was measured at 500 nm, using a spectrophotometer
Whatman filter paper No.1. This procedure was performed (DU640i, Beckman), with BHA as the reference compound.
thrice and all the extracts were decanted and pooled. To eliminate the solvent effect, the control sample, which
The extracts were filtered before drying, using Whatman contained the same amount of solvent added to the
filter paper No. 2 on a Buchner funnel and the solvent was linoleic acid emulsion in the test sample and the reference
removed by vacuum distillation in a rotary evaporator at 40曟 compound, was used. All data reported are the average of
[8]. triplicate analysis. Percentage inhibition of lipid peroxide
generation was calculated using the following formula.
2.2. Bacterial inoculum preparation
Control absorbance - Test absorbance
% Inhibition = 暳 100
Gram positive bacterial (such as Staphylococcus aureus Control absorbance
MTCC 96, Bacillus subtilis MTCC 121) and Gram negative
bacterial (such as Escherichia coli MTCC 443, Klebsiella
pneumoniae MTCC 109 , Proteus mirabilis MTCC 425 , 2.5. DPPH radical scavenging activity
Salmonella typhi MTCC 531) cultures were obtained from
M icrobial T ype C ulture C ollection, C handigarh, I ndia. In this assay, 1 mL of varying concentrations (5, 10, 25,
B acterial inoculums were prepared in M ueller H inton and 50 mg/mL) of the methanolic extracts of A. nilagirica,
Broth (Himedia) and maintained for 24 h at 37 曟. The cell M. pudica and C. siphonanthus was mixed with 1 mL of
suspensions were diluted with sterile MHB to provide an methanolic solution of DPPH (0.2 mM). The mixture was
initial cell count of about 106 CFU/mL. vortexed and incubated for 30 min. The optical densities of
the solutions were measured at 517 nm using Hitachi 2050
2.3. Disc diffusion method spectrophotometer, using BHA as the standard reference[12].

Antibacterial activity was carried out using disc diffusion Control absorbance - Test absorbance
Radical scavenging activity (%) = 暳100
method[9]. Petri plates were prepared with 20 mL of sterile Control absorbance
Mueller Hinton Agar (Himedia, Mumbai). The test cultures
(100 毺L of suspension containing 10 CFU/mL) were swabbed 2.6. Phytochemical analysis
8

over the solidified media and the plates were further allowed
to dry for 10 min. Sterile discs were loaded with methanolic Phytochemical analysis (flavonoids, terpenoids, steroids,
 Arokiyaraj S et al ./Asian Pacific Journal of Tropical Biomedicine (2012)S601-S604
S603

alkaloids, tannins and glycosides ) was performed by significant antibacterial effect against Bacillus subtilis (10
following Edeogal Method[13]. mm), Staphylococcus aureus (12 mm), Klebsiella pneumoniae
(30 mm), Proteus mirabilis (16 mm), Escherichia coli (11.5 mm)
2.7. Statistical analysis and Salmonella typhi (16 mm). The highest zone of inhibition
was recorded against Klebsiella pneumoniae. M. pudica and
Comparison between the control and the extract treated A. nilagirica inhibited the bacterial growth (Table 1).
groups was analyzed by SPSS software package, Version 11.5
with Student t-test. P<0.05 was considered to be significant. 3.2. Total antioxidant activity

Total antioxidant activities of A. nilagirica, M. pudica and

3. Results C. siphonanthus were determined using the ammonium
thiocyanate method. C. siphonanthus (IC50 7 mg/mL) was
3.1. Antibacterial assay found to be effective in scavenging lipid peroxide radicals.
M. pudica (IC50 10 mg/mL) scavenged the generated free
The methanol extracts of A. nilagirica, M. pudica and C. radicals significantly at concentrations >10 mg/mL (Figure 1).
siphonanthus were screened against two gram positive and Moderate activity was observed in A. nilagirica (IC50 22 mg/
four gram negative bacteria. C. siphonanthus exhibited mL).
Table 1.
Antibacterial activity of the methanol extracts of A. nilagirica, M. pudica and C. siphonanthus.
Bacteria Zone of inhibition (mm)
A. nilagirica (5 mg/disc) M. pudica (5 mg/disc) C. Siphonanthus(5 mg/disc) Streptomycin (30 毺g/disc)
Bacillus subtilis 12.0 16.0 10.0 15.0
Staphylococcus aureus 12.0 15.0 12.0 15.0
Klebsiella pneumoniae 16.0 20.0 30.0 16.0
Proteus mirabilis 12.0 11.0 16.0 15.0
Escherichia coli 11.5 12.0 11.5 15.0
Salmonella typhi 15.5 14.5 16.0 15.0
Experiments were performed in triplicates.

Table 2.
Phytochemical analysis of A. nilagirica, M. pudica and C. Siphonanthus methanolic extracts.
Plant Flavonoids Terpenoids Steroids Alkaloids Tannins Glycosides
A. nilagirica + + - + + -
M. pudica + - - + - +
C. Siphonanthus + - + - - +

+ = Present - = Absent.

120 100
inhibition of lipid peroxidation free

90
100
inhibition of DPPH radicals

80

80 70
A.nilagirica 60
radicals

A.nilagirica
60 C.siphonantus 50
C.siphonantus
M.pudica 40
40 M.pudica
BHA 30
BHA
20 20
%

10
%

0
5 10 25 50 0
5 10 25 50
Concentration(mg/mL)
Concentration(mg/mL)
Figure 1. Scavenging activity of BHA (control), A. nilagirica, M. Figure 2. Scavenging activity of BHA (control), A. nilagirica, M.
pudica and C. Siphonanthus on lipid peroxidation (n = 3). pudica and C. Siphonanthus on DPPH free radicals (n = 3).

from 10 mg/mL (Figure 2).

3.3. Inhibition of DPPH radical
3.4. Phytochemical analysis
C. siphonanthus (IC50 7 mg/mL) and M. pudica (IC50 9 mg/mL)
showed significant free radical scavenging activity generated Results of phytochemical analysis of A. nilagirica, M.
by DPPH. Since more than 50% of DPPH radical inhibition pudica and C. Siphonanthus are shown in Table 2.
is considered to be significant, the inhibition was observed
S604 Arokiyaraj S et al ./Asian Pacific Journal of Tropical Biomedicine (2012)S601-S604

in conducting the study.

4. Discussion
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