Research Article

ANTIVIRAL ACTIVITY OF CASSIA ALATA AND COSMOS CAUDATUS LEAF

EXTRACT TO DENGUE VIRUS SEROTYPE-2 NEW GUINEA C STRAIN
IN HUH-7IT1 CELL LINE

SEPTIAN IKA PRASETYA1, ADRIAN2, SHIERLY RATNASARI2, HIDAYATI DESTI3,

MELVA LOUISA4,MARISHA ANGELINA5, TJAHJANI MIRAWATI SUDIRO3, BETI Formatted: Superscript
ERNAWATI DEWI3 Formatted: Superscript
1
2 Formatted: Left
3Department of Microbiology, Medical Faculty Universitas y of Indonesia-Cipto Formatted: Superscript
Mangunkusumo Hospital Jalan Pegangsaan Timur no 16, , Central Jakarta, -10320,
Jakarta, Indonesia.
4Department of Pharmacology, Medical Faculty Universitas Indonesia-Cipto

Mangunkusumo Hospital Jalan Salemba Raya 6, Jakarta, , Indonesia

5Research Center for Chemistry, Indonesian Institute of Sciences. Kawasan Puspitek Formatted: Superscript
Serpong, Indonesia 15416
E-mail: betied@yahoo.com

ABSTRACT

Objective: The experiment was aimed at performing in-vitro studyobjective of this study to
evaluate antiviral activity of leaf extract of Cassia alata and Cosmos caudatus against dengue
virus serotype-2 (DENV-2) in vitoHuh7it-1 cell culture.

Methods: We used Huh7-it-1 cell line and DENV2 strain New Guinea C. The tested
concentrationvarious concentration of each separate leaf extract were 10, 20, 40, 80, 160, and
320 g/mL Extracts inhibitory capability and toxicity were evaluated by focus assay and MTT
assay, respectively. Focus assay measured the decrement of virus titer after DENV2 were treated
with various concentration of the extracts and then were added to Huh7it-1 monolayer to
generate concentration-mean infectivity percentage curves. Meanwhile, MTT assay was
performed by exposing various concentration of the extracts to the cell culture and then
quantified cells viability by absorbance reading to obtain concentration-mean percentage of cell
viability. Nonlinear regression analysis was performed upon concentration-effect curves of
infectivity and viability to determine half-inhibitory concentration (IC50) and half-cytotoxic
concentration (CC50), respectively.

Results: The Cassia alata leaf extract had the IC50, CC50 and SI of <10 g/mL, 323.45 g/mL,
and >32.3 respectively. Meanwhile, the IC50, CC50, and SI of Cosmos caudatus leaf extract were
12.2 g/mL, 187.1g/mL and 15.4 respectively.

Conclusion: Given their high selectivity indices, Cassia alata and Cosmos caudatus leaf extract
showed potent antiviral activity against DENV2. Further studies are required to to identify active
compound of Cassia alata and Cosmos caudatus leaf that have potency as antiviral for DENV and
their mechanism of inhibition.

Keywords: antiviral activity, Cassia alata, Cosmos caudatus, dengue, half-inhibitory

concentration, half-cytotoxic concentration

INTRODUCTION
Dengue virus (DENV) infection is still a major health problems worldwide, especially in tropical
and sub-tropical countries. WHO estimated that the number of dengue infection reached 390
million cases (95% CI, 284 - 528 million) with 95 million (95%CI, 67-136 million) clinically-
manifested cases in 2010 globally [1]. Meanwhile in Indonesia, according to Ministry of Health
Republic of Indonesia, there were more than 100.000 people diagnosed with dengue fever and
the disease have appeared in more than 80% of nations region [2].

In line with extensive studies for the development of dengue vaccine, the search of substances Formatted: Space After: 12 pt, Line spacing: At least
17 pt, No widow/orphan control, Don't adjust space
for a breakthrough of novel antiviral drug against dengue virusDENV has been attempted. To between Latin and Asian text, Don't adjust space
between Asian text and numbers
date, there is no clinically-approved DENV dengue antiviral drug to treat as the treatment for
broad spectrum of DENVdengue infection disease. The treatment is essentially
supportive. severity is predominantly supportive. Formatted: Font: (Default) Times, Font color: Black

More attention has been given to natural sources especially herbal medicinemedicinal plants in
the search of novel substances for drug development considering its abundant availability, lower
cost, and potentially lower toxicity hence lower risk of adverse effects. To mention, artemisinin
which initially was discovered from herb rooting from Chinese traditional medicine was a
notable success story of the search of drug materials from traditional medicinal plants. Numerous
herbs with potential health benefits grow well in Indonesia yet their potentials have not been
studied extensively. Cosmos caudatus is an edible plant popular as a dish in Malayan and is
believed having various health benefits. Recent study discovered that it had high antioxidant
contents [3] and some other reviews stated that it had antibacterial, antifungal, antiosteoporotic,
antidiabetic, and blood-pressure lowering effects [4]. On the other hand, Cassia alata has long
been used as source of traditional remedy for various ailments in many regions of the world
especially in Asian and African countries. Recent studies found that extract of Cassia alata had
immunostimulant [5], antirotaviral [6] antiinflamatory, and antioxidant activity [7]. The purpose
of this study is to investigate antiviral property of leaf extract of Cassia alata and Cosmos
caudatus for dengue virus serotype-2 (DENV2 ) strain New Guinea C in Huh7it-1 cell. culture.

MATERIALS AND METHODS

Preparation of extracts
The botanical identity of Cassia alata and Cosmos caudatus plant were identified and
authenticated by botanist in Research Center for Biology, Indonesian Institute of Science (LIPI).
After taxonomical identification of each plant, the leafplants were washed and cleaned prior to
air drying at room temperature. The leafves were then ground until turned into a fine powder.
The crude extracts were sequentially extracted using n-hexane, ethyl acetate, butanol and water.
The antiviral candidate from ethyl acetate fraction was subjected to silica gel column
chromatography using n-hexane and ethyl acetate at equal volume for the diluents. The major
fraction was monitored in TLC and all fractions were tested for its antiviral effects to DENV2.
Furthermore, the candidate of tested substance was partially purified using Sepadex LH-20
column with 50%, 60% and 80% of methanol. The physical and spectral characterization of
extracts were performed using one and two dimensional nuclear magnetic resonance 500MHz
(JEOL-JNM-ECA, Japan) and liquid chromatography-mass spectrometer (Mariner
Biospectrometry). All of the extracts were diluted at concentration of 100 mg/ml in dimethyl
sulfoxide (DMSO) (Sigma Aldrich, USA). Centrifugation was performed to separate insoluble
material. The stock solution was further diluted with culture medium until reached the desired
concentration for the assays. Stock was stored at -20oC until further use.

Preparation of DENV2 and Huh7-it cell culture

High glucose Dulbeccos Modified Eagles Medium (DMEM) (GIBCO, UK) was used to
maintain of Huh-7 it-1 cell line in this study. Sodium bicarbonate was added to the medium to
maintain the pH. For maintaining of the cells we used DMEM with and 10% of fetal bovine
serum (FBS) (GIBCO) was added later. Cells were then incubated at 37C with 5% CO2 for 4-5
days until confluent. After reaching 80-100% confluence, the cells were sub-cultured. The sub-
culturing process was initiated by removing the used medium followed by rinsing the cells twice
with phosphate buffer solution (PBS) (Gibco). Then, 1 mL of 0.25% trypsin-EDTA (GIBCO,
Canada) was added and incubated for 5 to 10 minutes at 37C. After addition with trypsin, 1 mL
of fresh medium with 10% FBS was added and mixed. The mixture was then centrifuged for 10
minutes at 1500 rpm. The supernatant was discarded while the pellet was resuspended with 2 mL
of fresh medium and redistributed into new tissue culture flask for further maintenance.

We used DENV serotype 2 strain NGC adapted in HuH7it-1 human hepatocellular carcinoma
cells1. A monolayer of Huh7it-1cell in T-75 flasks were infected with DENV-2 NGC at MOI of
0.5 PFU/cell and then the flask was incubated at 37oC with 5% CO2 for 5-7 days. During the
viral propagation period, the FBS concentration of the cell culture medium was reduced to 2%.
Supernatant was harvested and centrifuged at 1000 g for 5 minutes and then were filtered using a
syringe driven 0.22 mm (Millipore Co. Bedford USA). Culture supernatant was stored at -80oC
and checked for the titer of dengue virus by focus assay as previously performed by Igarashi et
al, 1995[8].

Determination of half-cytotoxic concentration

In vitro cytotoxicity of each extract was determined by MTT assay that quantified the percentage
viability of Huh-7 cells after treated with a certain concentration of extract compared with
negative control (DMSO) 0.1%). In 96 well flat-bottom plates (Corning, USA), cell culture were
added as much as 2 104 cells/well and then it was incubated at 37oC, 5% CO2 for 24 hours. The
cells were then treated with various concentration of extract ranging from 10, 20, 40, 80,160
until 320 g/mL and were then incubated at 37oC, 5% CO2. After 48 hours of incubation, 20L
of 3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) (Promega) salt
solution was added into each well and incubated for 4 hours according to the manufacturers
instruction. The absorbance reading of each well was measured using micro plate reader at 490
nm. The percentage of cell viability and toxicity was further determined based on the absorbance
readings. The absorbance was measured at 620 nm. Background absorbance of the micro plate
was removed by subtraction of blank wells. Theoretical percentage toxicity of each concentration
was determined by dividing the mean blanked sample optical densitiy (ODs) by the mean
blanked control ODs for each sample. The resulting percentage toxicity values of each
concentration that was tested in triplicate was calculated for its mean and standard deviation and
then the mean percentage was plotted to corresponding concentration to generate concentration-
mean percentage of viability curve.A nonlinear regression equation was derived from the curve
to calculate the half-cytotoxic concentration (IC50) of each leaf extract.

Determination of half-inhibitory concentration

A total of 2104 cells/well were seeded into 96-well plate and the plate were incubated at 37C
with 5% CO2. After 24 hours, the cells were infected with DENV-2 with MOI of 1 FFU/cell.
Various concentration of extracts ranging from 10, 20, 40, 80, 160, and 320 g/mL were added
shortly afterwards. After 2 hours of infection, a mixture of 100 l of DMEM+2% FBS and
various concentration of Cassia alata and Cosmos caudatus extracts were added. The tested
concentration of each plant extracts were 10, 20, 40, 80, 160, and 320 g/mL which were tested
in triplicate. Plates were further incubated at 37C for 3 days. Next, viruses were harvested and
its titer were determined by focus assay (Igarashi, 1995) [6]. Briefly, the products of 10-fold
serial dilution of the supernatant was inoculated onto Huh-7 it-1 cell monolayer in triplicate
wells. Absorption was carried out at 37oC in 5% CO2 for 2 hours with agitation at 30 minutes
interval. Methylcellulose 1.5% overlay medium was added to the cell and incubated at 35oC in
5% CO2 for 3 days. The infected cells were stained according to previous publication by Payne et
al,2006 [9].
) [7] with slight modification. First, infected cells were fixed with 10% formaldehyde in PBS and
were incubated at room temperature for an hour. The cells were washed with PBS for three
times. To make the cells permeable, a non-indent P40 1% were added 100 l/well and then the
plate was incubated at room temperature for 30 minutes. Next, the cells were blocked with 5%
skim milk in PBS and incubated at room temperature for an hour. After cell washing, human
IgG-anti dengue were added to each well 1/1000 and incubated at room temperature for 1 hour.
For the second antibody. 1/1000 antihuman IgG label HRP was used. After washed using PBS,
substrate for horseradish peroxidase were added and cells were observed for its brownish color.
Number of foci formed in each well including in negative control well was counted manually
using microscope after staining. Each concentration of extracts were tested in triplicate hence the
mean value of percentage of infectivity for each concentration was calculated. Number of foci in
each treatment well was compared to that of negative control well to obtain percentage of
infectivity of each well. The mean value of percentage of infectivity for each concentration
triplicate was calculated and then those values were plotted against corresponding concentration
to generate concentration-percentage of inhibition curve. The half-inhibitory concentration (IC50)
was obtained from nonlinear regression equation of concentration-effect curves.

Data analysis
Mean difference of percentage of cytotoxicity and infectivity between treatments group and
negative control was analyzed using One-way ANOVA using SPSS version 23 with p value less
than 0.05 (p<0.05) considered as statistically significant difference. The value of CC 50 and IC50
were determined using simple arithmetical calculation on regression equations obtained from
concentration-percentage of viability and concentration-percentage of inhibition. Then,
selectivity index for each extract was derived from the ratio of CC50 to IC50.

RESULTS
Cytotoxicity of Cassia alata and Cosmos caudatus leaf extract on HuH7it-1 cell culture
Cytotoxicity of leaf extract of Cassia alata and Cosmos caudatus on Huh7it-1 cells were
measured using MTT assay. The mean percentage of Huh7it-1 viability for each concentration
that were tested in triplicate were calculated. A nonlinear regression curve of each extracts
concentrations and cells viability was made and then the graph formula was made using
Microsoft Excel 2013. Half-cytotoxic concentration (CC50) of Cassia alata and Cosmos caudatus
were determined from the equations obtained from respective graph. Half-cytotoxic
concentration is defined as extracts concentration that gives rise to the decrement of cell culture
viability by 50% with respect to the negative control cells percentage of viability.

Table 1: Mean percentage of Huh7it-1 cell culture viability post-treatment with

corresponding extracts concentration
Huh7it1 viability
Concentration (g/ml)
Cassia alata (%) Cosmos caudatus (%)
320 49.6 29.4
160 118.6 54
80 151.3 70.7
40 162.1 91.2
20 168.5 103.1
10 170.5 103.3
DMSO 0,1% * 100 100
*DMSO 0.1 % was used as the negative control as well as the denominator to which optical
density of each well containing a certain amount of extract compared which generated
percentage of viability of each well.
 (a) (b)
Fig. 1: concentration-mean percentage of Huh7it-1 cells viability regression curve post-
treatment of (a) Cassia alata; (b) Cosmos caudatus. From the regression equation obtained
from corresponding curve, the CC50 of Cassia alata and Cosmos caudatus were 323.9 g/ml and
187.1 g/ml respectively

Inhibitory effects of Cassia alata and Cosmos caudatus leaf extract against DENV2
Antiviral activity of Cassia alata and Cosmos caudatus leaf extracts were evaluated by focus
assay. Number of foci formed in each well including in negative control well was counted
manually using microscope after staining. Each concentration of extracts were tested in triplicate
hence the mean value of percentage of infectivity for each concentration was calculated. The
difference of number of foci formed in each test well and control well were calculated and then
its value were compared to absolute number of foci in control well to obtain the percentage of
DENV2 inhibition by that respective extract concentration. The mean percentage of DENV2
inhibition was for each concentration triplicate was then calculated. Finally, a regression curve of
extract concentration-DENV2 inhibition percentage was made using Microsoft Excel 2013 and
formula for the graph was generated. Half-inhibitory concentration of each extract was
calculated from its respective formula. Half-inhibitory concentration is defined as extracts
concentration that brings about inhibition of viral replication by 50% with relative to the negative
control cells percentage of inhibition (0%).

Table 2: Mean percentage of DENV2 inhibition post-treatment with corresponding

extracts concentration

DENV2 inhibition (%)

Extract concentration (g/ml)
Cassia alata Cosmos caudatus

320 100
100
 160 100
100
80 100
98.8
40 98.7 91.8
20 92
62.6
10 87.5 48.9
0
DMSO 0,1%* 0
*DMSO 0.1% was used as the negative control to which number of foci formed on each well
containing a certain amount of extract compared to obtain the value of inhibition percentage.

(a) (b)
Fig. 1: Regression curve for concentration-mean percentage of DENV2 post-treatment of
(a) Cassia alata; (b) Cosmos caudatus. From the regression equation obtained from
corresponding curve, the iC50 of Cassia alata and Cosmos caudatus were 0,04 g/ml and 12.2
g/ml respectively

DISCUSSION
One-way ANOVA with Tukeys multiple comparison showed that percentage of viability
differed significantly (*p <0,05) one another for every concentration of the extract except for
among concentration of 10, 20, and 40 g/mL and control. Meanwhile, mean difference of
percentage of inhibition did not show statistical significance. The regression formula for Cassia
alata is y = 6.3628 ln(x) + 73.277 thus if we substitute y with 50 to earn concentration that will
bring about half-inhibitory concentration, we will get x=0.04 which is a very low value. Thus,
we could only confidently say that the IC50 value of Cassia alata was below 10 g/mL as the
lowest concentration tested yielded more than 50% inhibition. Given that the CC50 and IC50 of C.
alata were 323.45 g/mL and <10 g/mL, hence the selectivity index of Cassia alata was >32.3.
Meanwhile, the CC50, IC50, and selectivity index of Cosmos caudatus is 187.1g/mL,
12.2g/mL, and 15.4 respectively.
In this study, the half-cytotoxic concentration of Cassia alata leaf was 323.45 g/mL. In a study
by Shaheen et al using similar MTT assay, the methanol, chloroform, butanol, and aqueous
fraction of Cassia alata gave no toxic effect on MA 104 cells with CC50 exceeded 1000 g/ml
while its ethyl acetate extract showed slight toxicity with CC 50 of 826 g/ml. The half-inhibitory
concentration of its methanol fraction was 74, hence the selectivity index of C. alata against
rotavirus was 22.8 [6]. Besides, root parts of Cassia alata Linn. contains considerable amount of
phenolic compounds, anthraquinons (rhein, chrysophanol, and aloe-emodin), and flavonoid
(kaempferol)[14]. Qualitative estimation of cassia alata partially purified flower extract found
that it contained considerable amount of flavonoids (72.9+0,061), phenols (49.4+0.04), and
tannins (18.12+0.30) [15]. The high bioflavonoids and phenolic compounds contents of Cassia
alata probably associates with its antiviral properties against DENV2.

Cosmos caudatus leaf contains flavonoids (such as cathecin, quercetin) and phenolic acids
(ferulic acid, caffeic acid, chlorogenic acid, etc) while its whole plants contains the other
flavonoids (naringenin, rutin, formic acid, kaempferol), monoterpenes, sesquiterpenes,
diterpenes, carbohydrates, quaternary ammonium salts, and carboxylic acids [4]. Study of
antiviral activity against DENV2 NGC strain in C6/36 cell culture of three compounds (rutin,
ellagic acid, and quercetin) obtained from extraction of Spondias mombin and Spondias tuberosa
resulted in finding that the CC50 values of all those compounds exceeded 1000 g/mL while
their IC50 were 362.68 g/mL for rutin and 500 g/mL for quercetin [10]. Although the
selectivity indices of rutin and quercetin were relatively low, we hypothesized that those
bioflavonoids contributed to antiviral activity of C. caudatus leaf against DENV2.

Several studies had evaluated antiviral activity of a certain kind of substance or extract of
medicinal plant with similar methodology as this study. Study of antiviral activity of three
compounds (rutin, ellagic acid, and quercetin) contained within Spondias mombin and Spondias
tuberosa against DENV2 NGC strain in C6/36 cell culture resulted in finding that the CC50
values of all those compounds exceeded 1000 g/mL while their IC50 were 362.68 g/mL for
rutin and 500 g/mL for quercetin[10]. Study by Zandi et al assessed antiviral activity of a
bioflavonoid substance, baicalein against DENV2 in Vero cells. The selectivity index of
baicalein was 21.3 (IC50 5.39) when the compound was added 5 hours before were treated before
viral infection and given continuously up to 4 days post-infection while the SI after viral
adsorption to the cells was 17.8 (IC50 6,46 g/mL). Baicalein possibly acted as direct virucidal
agent (IC50 of
1.55 g/mL) as well as anti-adsorption agent (IC50 of 7.14 g/mL) against DENV-2 and exerted
its effect by inactivating important structure and/or non-structural protein(s) of DENV-2 [11].
Another study with identical methodology that evaluate antiviral activity of quercetin showed
that the substance had the selectivity index of 35.7 g/mL when the substance was given right
after viral adsorption and of 28.9 g/mL for continuous administration from 5 hours before virus
infected the cell up to 4 days post-infection [12]. In a study conducted by Talarico et al with the
method plaque reduction and virus yield inhibition assays, two sulfated polysaccharides obtained
from the red seaweeds Gymnogongrus griffithsiae and Cryptonemia crenulata showed inhibitory
capability against DENV-2 in Vero cells with IC50 as low as 1 g/mL and selectivity index
exceeded 1,000 but it showed no antiviral activity against DENV1 and only moderate activity
against DENV 3 and DENV4. It is hypothesized that those sulfated compounds targeted virus
adsorption and internalization during virus binding to the host cell [13]. Compared to the other
substances which had been evaluated in several studies that have been mentioned above, the
selectivity indices of C. alata and C. caudatus were considered as high.Therefore, Both C. alata
and C. caudatus leaves had a potent antiviral properties in vitro for DENV2 with relatively low
toxicity against Huh7it-1 cells.

CONCLUSION
The leaf extract of Cassia alata and Cosmos caudatus had shown potent antiviral acitivity to
DENV2 in Huh7it-1 cell culture with half-inhibitory concentration and selectivity index of <10
g/mL and >32.3 for Cassia alata as well as of 12.2g/mL and 15.4 for Cosmos caudatus.
Further studies are needed to be done to confirm and utilize the findings for the development of
dengue antiviral agent. Among those required studies are in vitro study using the remainder
serotypes of DENV, in vivo study, and study to explore the bioactive compounds as well as
inferring its mechanism of action based on the treatment design of the research.
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