Journal of Pharmacognosy and Phytochemistry 2019; 8(3): 4840-4843

E-ISSN: 2278-4136
P-ISSN: 2349-8234
JPP 2019; 8(3): 4840-4843 Antibacterial activity of Withania somnifera leaf
Received: 15-03-2019
Accepted: 17-04-2019 extracts against Gram-positive bacteria
Bhavna Dwivedi
Research Scholar, Center for Bhavna Dwivedi, Sherendra Kumar Sahu and Skand Kumar Mishra
Biotechnology and Microbiology
Studies, APS University, Rewa, Abstract
Madhya Pradesh, India Antibacterial property of different medicinal plants are well established and now- a- days they are using
as an alternative medicine. In this experiment, the methanolic and aqueous leaf extract of Withania
Sherendra Kumar Sahu
somnifera, commonly known as ashwagandha were evaluated for antibacterial activity against gram
Faculty, Center for
positive Staphylococcus aureus. High performance thin layer chromatography (HPTLC) based purified
Biotechnology and Microbiology
Studies, APS University, Rewa, and characterized extracts were subjected to analysis disk diffusion inhibitory test and oxidative stress
Madhya Pradesh, India marker-reactive oxygen species (ROS) generation in bacteria. Results obtained indicated that extracts
contained significant amount of phenolic, flavonoids and other phytoconstituents like alkaloid, tannin,
Skand Kumar Mishra terpenoids, steroids. However, the calculated minimum inhibitory concentration of extract was 100±7.5
Professor and Head, Department µg. Further, a significant increased of ROS level was observed in treated bacteria concentration range
of Botany, MSG College, Rewa, from 20-800 µg/ml respectively (835±65, 1500.75±126.55, 2000±250.65, 4600±137.55, 4100±830.75,
Madhya Pradesh, India 4500±1200.59 with respect to control). Thus, the results suggested that extracts contained phenolic and
flavonoid compounds which could be responsible for oxidative stress and bacterial death.

Keywords: Withania somnifera, antibacterial activity, flavonoids, methanolic extract

Introduction
The beneficiary effects of plants are well explained in Hindu mythology. Depending upon
uses, plant kingdom is classified into medicinal and non-medicinal plants. Medicinal plants
have been used to treat health problems and to prevent diseases from thousands of years.
Secondary metabolites of plants are responsible for the biological properties of plant species
[1]
. India has rich diversity of plant species in a broad range of ecosystems. There are around
8000 of species which are considered as medicinal plant and give resources for villagers
particularly tribal communities and Ayurveda for medicinal use [2].
Withania somnifera (WS) is well known important medicinal plant belongs under the
Solanaceae family, commonly known as Indian ginseng, winter cherry and ashwagandha. In
India, the plant has been reported to grow in Himachal, Jammu and Punjab and it is
commercially cultivated in Madhya Pradesh, Rajasthan, Andhra Pradesh and Uttar Pradesh.
Specific parts of plant like roots, stems, leaves etc. contain active constituents
(phytochemicals) that used for the treatment of large number of human diseases. This
medicinal plant exhibit anti-inflammatory, anti-oxidative, antimicrobial, anti-anxiety,
immunomodulation, antihyperglycaemic, anticancer, cardiovascular protection, diuretic,
adaptogenic, anti-stress and antiarthritis properties [3]. In pathogenic diseases there are a lot
number of health issues caused by bacteria, fungus and other microbial agents. Root extracts of
ashwagandha can be used as natural drug for the treatment of several infectious diseases
caused by bacteria and fungi and leaf extract have potential as an antibacterial agent and
effective in inhibiting the antibiotic resistant S. aureus strains.
This study distributed into these heads after collection of plant materials firstly extraction with
different solvent then characterization and purification of fractions via high performance
authenticate analytical techniques and further we evaluated the antibacterial activity (here, we
used extreme pathogenic bacteria Staphylococcus aureus) of different plant extracts.

Materials and Methods

Collection of plant Material
We have used medicinal plant Withania Somnifera for this study. This plant is well described
in different samhitas like Charak Samhita, Sushruta Samhita etc. for their medicinal values.We
Correspondence
Bhavna Dwivedi have collected the plant-leaf from Rewa, M.P. by taking help of expert botanist. The plant
Research Scholar, Center for materials were cleaned with running tap water and sterilized by using commercially available
Biotechnology and Microbiology volatile disinfectant agent.
Studies, APS University, Rewa,
Madhya Pradesh, India
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Preparation of extracts 50 mg of lyophilized (different extract) powder was dissolved

Leaves were again cleaned and air dried under laminar into 500 µl of n-hexane: ethyl acetate: water (7:2:1) ratio and
airflow. Total 500 gm of leaves were grinded mechanically carried out for further steps. We used 5 x 10 cm aluminium
(by using laboratory grade grinder: speed 1000 to 5000 rpm TLC plate which was pre coated with 0.2 mm silica gel
for 10 minutes). Powder was further dried under vacuum 60F254 layer (E. Merck Ltd, Darmstadt, Germany) and kept
dryer. The dried powder (100-200 gm) subjected to different in desiccators. Sample application was performed by spotting
solvents like water and methanol and kept for 12 hours at 55- and drying method (spray technique) with a Hamilton micro
600C under constant rotating condition in multimode syringe (Switzerland) which controlled by an automatic
extractor. Thereafter, the solution was filtered by using applicator (Linomat-V). The command was fixed according to
whatman no.-1 filter paper. Suspensions were centrifuged at experimental design in WIN CATS software (Version 1.3.0).
5000 rpm for 20 minutes. The clear supernatant collected and For the light source, we used deuterium light for λmax 254 and
dried under rota evaporator to get different solvent extracted 366 nm and Tungsten light source for λmax 620 nm and the slit
powder and stored at -200C until used for further analysis. dimensions were 6.00 Х 0.45 mm. Here, we used solvent
mixture; n-hexane, ethyl acetate and water (7:2:1) as a mobile
Qualitative analysis of phytochemicals phase to separate the plant analyses. After spot development,
Leaf extracts were subjected for phytoconstituents evaluation plate was kept into solvent saturated twin trough glass
by adopting standard methods. The following chamber (10 x 10 cm) and allowed to migrate. Then plate was
phytochemicals: alkaloids, tannins, phenols, terpenoids, scanned (UV light: λ254 nm) in camag TLC Scanner 3
steroids and flavonoids were analysed. equipped with digital camera SNR-Lens (DXA252:
223971607) and also with win CATS Software. Bands were
Alkaloid test excised from plate under UV light. Then, silica containing
To evaluate the presence of nitrogenous-alkaloids compounds excised matters were dissolved into methanol and centrifuged
in extract, we adopted Meyer’s reagent based precipitation at 10,000 × g for 30 minutes. Supernatant of different bands
method. Briefly, fractions were dissolved in 0.5M HCl in were collected and evaporated into rota evaporator for further
boiling water bath and filtered. Three drops of HCl containing experiments.
extract solution was added into 1 ml of Meyer’s solution and
allowed to precipitate. Antibacterial activity
We used Staphylococcus aureus pathogenic bacteria, obtained
Steroid test from institute of microbial technology, Chandigarh, India. We
To identify presence of steroids in extract, 100 mg of each followed the proper safety and others guideline (MTCC: 737)
sample was dissolved into 2 ml of glacial acetic acid and then to maintain and perform the experiments in standard
added 2 ml of concentrated H2SO4. The color changes from laboratory conditions. To test the anti-bacterial property of
violet to greenish and/or blue denoting steroids. different fractions, we performed disc-diffusion antibiotic
sensitivity test on S. aureus uniformed layer. One mg of
Tannin test different fractions were dissolved into 100% DMSO and
To know the presence of tannins in extract, we measured 100 marked as stock solution. Serial dilution (20, 40, 100, 400 and
mg of extract and added into 10 ml of hot water, mixed well 800 µg/ml) of fractions were prepared from stock solution.
and followed by membrane filtration. After that, 2-3 drops of Small round shape whatman filter paper-II was incubated with
0.1% ferric chloride solution was added into filtrate and the diluted solution and followed by air drying. Fraction
observed to appear as brownish green color formation. soaked-filter papers were placed on the layer of S. aureus
(grown on nutrient agar plate-90 mm) and incubated at 370C
Terpenoid test (Salkowski test) for 12-24 hours. After 24 hours incubation, plates were
Extract (10 mg) was dissolved into 10 ml of Mili Q water. observed and calculated the distance and diameter of inhibited
Three ml of extract was added into 2ml of chloroform and 3 zone. MIC was calculated on the basis on the minimum
ml of concentrated H2SO4 and allowed to form a brown layer. concentration able to inhibit bacterial growth. Along with this,
This colorful layer is the indication of terpenoids. we also measured the ROS generation (responsible for
oxidative stress) in bacteria. Staining was done by DCF-DA
Test for phenolics (3µM/ml). After staining, fluorescence was measured by
To know the presence of phenolics in different extract ferric spectrofluorometer (excitation=435 nm and emission=535
chloride method were used. Briefly, a 100 µl of sample was nm). Results were expressed as relative fluorescence unit.
added into 500 µl of 5% ferric chloride solution and mixed
well. The solution was kept at room temperature for 30 Results
minutes to develop colour (indicates presence of phenol). Phytochemical analysis of water and methanolic extract
suggested that they contained alkaloids, steroids, tannins and
Test for flavonoids terpenoid along with phenol and flavonoids. Yellow
100 µl of sample was added into 500 µl of dilute ammonia precipitate in extracts confirmed the presence of alkaloids,
solution. Mixture was gently shaken and concentrated H2SO4 similarly, formation of greenish color, brownish color and
(500 µl) was slowly added and incubated for at least 3-4 hours brown ring indicated the presence of steroid, tannin and
for development of yellow colour which indicated presence of terpenoid.
flavonoids. HPTLC investigation of both extracts confirmed the one or
more flavonoid and phenolic compounds present in
HPTLC based profiling of leaf extracts multiple samples respectively (Figure: 1). It was observed that
We performed HPTLC based separation and purification for methanolic extract (Which mentioned as Fraction-A and C)
further experiments. Here, we used an advanced HPTLC was the most endowed extract, consisting of ascorbic acid,
machine (CAMAG, HPTLC) to perform the experiments. A gallic acid, hesperidin and rutin equivalent bands. In the same
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images, it was observed that aqueous extract contained a conducted disk diffusion method based minimum inhibitory
significant amount of rutin, hesperidin and ascorbic acid concentration (MIC) calculation. Fraction-A only exhibited
equivalent bands (Fraction B: Figure: 1). However, both different degree of antibacterial activity with compare to
extract did not show any band with quercetin equivalent. The control (Figure: 2). Table: 1 specifies the zones of inhibition
quantitative analysis also has shown that aqueous extracts in millimeter (mm) and corresponding MIC values in
contained bulky shoulder peaks in the region of Rf values micrograms (µg). Further, we evaluated mechanisms of
from 0.05 to 0.14 which are the characteristic peaks of rutin, antibacterial activity of Fraction-A. Reactive oxygen species
hesperidin and ascorbic acid respectively. Similar peaks (ROS) is well known to kill pathogen. It can be observed that,
observed in a methanolic extract with an extra Rf value 0.74 Fraction-A showed an increasing trend for ROS generation in
which collaborates with gallic acid. S. aureus, incrementing over a concentration range of 20 to
To evaluate antibacterial property of fraction-A, B and C, we 800 μg/ml of the fraction (Table: 2).

Fig 1: Analysis of methanolic and aqueous extract by HPTLC. A and C bands represented the fraction of methanolic extract having different Rf
values (0.05, 0.1, 0.14 and 0.74). B band represented the fraction of aqueous extract having same Rf value, except 0.74.

Fig 2: Different concentration of disks (white colour) shown zone of inhibition indicated as red arrow and compared with untreated control plate
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Table 1: Diameters of inhibition zones (in mean (mm) ± SEM) after are only presents in high amount in this fraction are
treatment with Fraction-A, B, C; MIC values (in mean (µg) ± SEM) responsible for positive anti-bacterial function.
Fractions Inhibition diameter and MIC values
A 3.5±0.2 mm**, 100±7.5 µg** Conclusion
B 0.5±0.02 mmns, 0±0 µgns Ashwagandha is already a well known medicinal plant.
C 0±0 mmns, 0±0 µgns Results of this study concluded that the methanolic extracts of
Control 0±0 mm, 0±0 µg leaf of this plant showed a considerable antibacterial activity
against S. aureus. Therefore, these fractions could be used as
Table 2: Increased level of relative fluorescence intensity of a potential drug candidate (phytomedicine) and need higher
Fraction A with compare to untreated control. Data are expressed as mammalian study for better resolution.
mean ± SEM, **p< 0.05.
Fraction A (Concentrations Relative fluorescence References
µg/ml) intensity 1. Dar RA, Shahnawaz M, Qazi PH. General overview of
Control (Untreated) 835±65 medicinal plants: A review. The Journal of
20 1500.75±126.55 Phytopharmacology. 2017; 6(6):349-351.
40 2000±250.65** 2. Singh R. Medicinal plants: A review. Journal of Plant
100 4600±137.55** Sciences. 2015; 3(1):50-55.
400 4100±830.75** 3. Singh N, Bhalla M, Jager De P, Gilca M. An overview on
800 4500±1200.59** Ashwagandha: A Rasayana (Rejuvenator) of Ayurveda.
Afr J Tradit Complement Altern Med. 2011; 8(5):208-
Discussion 213.
Plants have medicinal properties because of the presence of 4. Wadood A, Ghufran M, Jamal SB, Naeem M, Khan A,
phytochemical constituents. Phytochemicals also known as Ghaffar R et al. Phytochemical Analysis of Medicinal
secondary metabolites naturally synthesize and stored in Plants Occurring in Local Area of Mardan. Biochem
different plant parts such as leaves, vegetables, roots and Anal Biochem. 2013; 2(4):1-4.
stems. Apart from these, phytochemicals have defense 5. Khoo HE, Azlan A, Kong KW, Ismail A. Phytochemicals
mechanism, protect plant from various diseases and and Medicinal Properties of Indigenous Tropical Fruits
environmental hazards [4]. Very interestingly, while these with Potential for Commercial Development. Evid Based
compounds are used in mammalian system then they act as Complement Alternat Med. 2016; 2016:1-20.
the antifungal, antimicrobial, antiatherosclerotic, 6. Mathur V, Vats S, Jain M, Bhojak J, Kamal R.
antileukemic, anticlastogenic, stimulate immune system, Antimicrobial Activity of Bioactive Metabolites Isolated
modulate hormone metabolism and strongly influences from Selected Medicinal Plants. Asian J. Exp. Sci. 2007;
cellular homeostasis [5]. In present investigation we were also 21(2):1-6.
able to identify several flavonoids, phenolic, alkaloid, tannin, 7. El-Boshy MES, Abdalla OM, Risha A, Moustafa F.
steroid and terpenoid compounds presents in leaf extract on Effect of Withania somnifera extracts on Some Selective
the basis of biochemical characterization. HPTLC results Biochemical, Hematological, and Immunological
reveal the presence of flavonoid and phenolic like molecules. Parameters in Guinea Pigs Experimental Infected with E.
As, these components are very common phytoconstituents and coli. ISRN Veterinary Science. 2013; 2013:1-6.
present in most of the medicinal plants and we also observed 8. Jain P, Varshney R. Antimicrobial activity of aqueous
the similar components in ashwagandha leaf. Thus we and methanolic extracts of Withania somnifera
expected anti-bacterial property of leaf extract. Several (Ashwagandha). J Chem. Pharm. Res. 2011; 3(3):260-
reports suggested that methanolic root extracts of W. 263.
somnifera inhibit growth of Escherichia coli and
Enterococcus [6]. Similarly, we also observed the anti-
bacterial activity of leaf extract against Staphylococcus
aureus. Fraction A only exhibit a different degree of
antibacterial activity compare to others and 100 μg/ml
concentration is most effective against S. aureus strain.
The mechanisms what we have been identified to kill bacteria
that molecules presents in extracts induce level of reactive
oxygen species and thus it elevated levels of oxidative stress.
It is also reported that a constant elevation of ROS leads to
damage macromolecules and created pore in bacterial
membrane and thus death occur. In this study, we also
observed the similar components presents in leaf extract, thus
it could be expected that the suppression of glycolysis in
staphylococcus aureus occurred after treatment and induced
death and considered as another reason. However, W.
somnifera root extract exhibited antibacterial effect by
creating membrane pore against Escherichea coli and
Salmonella typhimureum which tested in vitro and in vivo in
Guinea pigs [7, 8]. As Fraction-A can exhibited the antibacterial
activity with compare to other fractions. The probable reasons
behind of these are the some kind of active flavonoids which

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