In vitro Evaluation of Antibacterial Activities of Crude Extracts of Withania somnifera (Ashwagandha) to Bacterial Pathogens
The present aim of study is to detect the antibacterial property in the extracts of Withania somnifera. The antibacterial activity of Withania somnifera was tested on clinically isolated bacterial pathogens, i.e., Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis causing infections in human beings. Different solvents as ethanol, ethyl acetate, dichloromethane, hexane (from higher polarity to lower polarity) extracts was used for the study of antibacterial activity, by agar well diffusion method. The bacterial plates were prepared by nutrient agar. The extracts at different concentration from 10 to 40 mg mL-1 were loaded in the wells prepared in nutrient agar. Zone of inhibition was measured around the wells to check the antibacterial activity of extracts. Results showed the polar solvents to have higher antibacterial property in comparison to the nonpolar solvents. Relatively higher Minimum Inhibitory Concentration (MIC) were obtained for both gram positive bacteria S. aureus, B. subtilis and gram negative bacteria, E. coli and P. aeruginosa, with polar extract; however, less inhibitory effect was noted for nonpolar extracts. Ethyl acetate extract possesses great inhibitory activity for gram positive bacteria, S. aureus followed by B. subtilis. Among gram negative bacteria, highest inhibitory effect was observed with P. aeruginosa followed by E. coli. Antimicrobial activity of crude extract of W. somnifera were carried out to validate the use of traditional medicinal herbal and the results of this study tend to give credence to the common use of W. somnifera plant.
Multi-drug resistance is a world-wide problem, attributed to the extensive
use of antibiotics, selection pressure on bacterial strains and lack of new
drugs, vaccines and diagnostic aids. These shortcomings lead to an urgent global
call for new antimicrobial drugs, particularly from natural resources. Majority
of medicinal plant species are rich in biomolecule contents which can cope with
health hazards and recently, antibacterial activity of many plant species have
been reported Pandey and Mishra (2010). The genera Withania
somnifera plays an important role in the indigenous medicine of South East
Asia, e.g., in the Unani and Ayurvedic systems. The twenty-three known Withania
species are widely distributed in the drier parts of tropical and subtropical
zones, ranging from the Canary Islands, the Mediterranean region and northern
Africa to Southwest Asia (Mirjalili et al., 2009).
Withania somnifera has been used as an antioxidant, adaptogen, aphrodisiac,
liver tonic, anti-inflammatory agent and astringent and more recently as an
antibacterial, antihyperplycemic and antitumoral, as well as to treat ulcers
and senile dementia (Rastogi and Mehrotra, 1998). Most
of its biological activities have been attributed to the presence of group of
compounds referred as withanolides. The roots and leaves of Withania are
used as drugs (Khanna et al., 2006). Various
withanolides have been isolated from W. somnifera. Withaferin A and 3-β-hydroxy-2,
3 dihydro withanolide F show promising antibacterial, antitumour, immunomodulating
and anti-inflammatory properties. It also possesses adaptogenic, cardiotropic,
cardioprotective and anticoagulant properties (Rasool and
Varalakshmi, 2006). Withania somnifera is widely claimed to have
potent aphrodisiac, sedative, rejuvenative and life prolonging properties. The
plant was traditionally used to promote youthful vigor, endurance, strength
and health, nurturing the time elements of the body and increasing the production
of vital fluids, muscle fat, blood, lymph, semen and cells. The similarity between
these restorative properties and those of ginseng roots has led to ashwagandha
roots being called Indian ginseng. It also helps counteract chronic fatigue,
weakness, dehydration, bone weakness, loose teeth, thirst, impotency, premature
ageing, emaciation, debility and muscle tension. The leaves of the plant are
bitter in taste and used as an antihelmenthic (Mirjalili
et al., 2009). Bruised leaves and fruits are locally applied to tumors
and tubercular glands, carbuncles and ulcers. The roots are used as a nutrient
and health restorative in pregnant women and old people. The roots are also
used in constipation, senile debility, rheumatism, general debility, nervous
exhaustion, loss of memory, loss of muscular energy and spermatorrhoea (Harikrishnan
et al., 2008).
Withania somnifera contains active ingredients like steroidal alkaloids
and lactones known as withanolides. Withaferin A and withanolide D are the two
main withanolides that contribute to most of the biological activities of Withania
somnifera (Mirjalili et al., 2009; Harikrishnan
et al., 2008). In this study, we determine the antimicrobial activities
of W. somnifera extract. Although a lot of work has been carried out
on the medicinal applications of W. somnifera, there is still little
information on the uses of the stem and leaves (Elsakka
et al., 1990; Arora et al., 2004;
Rajendran and Ramakrishnan, 2009). This study, therefore,
provides information on the antibacterial activity (against the microorganisms
causing skin, upper respiratory tract, gastrointestinal and urinogenital tract
infection) of W. somnifera extract.
MATERIALS AND METHODS
The present study was done from March to May, 2010 in the Plant Biotechnology Laboratory, Centre for Biotechnology, University of Allahabad, Allahabad. Plantlets of W. somnifera was collected from the nursery in Allahabad.
Test organisms: Pure bacterial cultures (Table 1)
were obtained from National Collection of Industrial Microorganisms (NCIM) National
Chemical Laboratory, Pune and were maintained on nutrient agar (Rajendran
and Ramakrishnan, 2009).
Extraction from W. somnifera: Whole plant of W. somnifera
was washed thoroughly under running tap water, dried on paper towel, then
kept in oven at 60°C for proper drying and finally crushed to fine powder
in mixer grinder. The dried powder of the plant (10 g) was dissolved in 100
mL of ethyl acetate in soxhlet apparatus. The extract was collected after three
days, filtered and kept in sterilized dark bottle. This procedure is repeated
three times for proper extraction. The extracts were evaporated to dryness using
rotary evaporator. A semisolid or dried crude extracts of whole plant so obtained
was resuspended in dimethyl sulphoxide (DMSO) to determine minimum inhibitory
concentration (Ghosh et al., 2008).
|| List of bacterial pathogens isolated from patients (1)
It was stored at 4°C for further studies. The residue was dried and the
above procedure was followed for ethanol, dichloromethane and hexane also. The
extractive values of ethyl acetate, ethanol, dichloromethane and hexane plant
extracts were analysed for antimicrobial activity.
Determine minimum inhibitory concentration: MIC was determined by the
broth dilution method (NCCLS, 2000). Different concentrations
of whole plant in ethyl acetate, ethanol, dichloromethane and hexane (ranging
from 10 mg mL-1 to 40 g mL-1) were tested separately for
each bacterium and inhibition zone of microbial growth in the plates containing
tested solutions was judged by comparison with blank control plates. Minimum
inhibitory concentration is defined as the lowest concentration of test samples
that result in a complete inhibition of visible growth. Experiments were carried
out in triplicate.
Antibacterial activities: The antibacterial susceptibility tests were
carried out using agar diffusion method (Rajendran and Ramakrishnan,
2009; Mahesh and Satish, 2008; Perez
et al., 1990; Kambizi and Afolayan 2008;
Lokhande et al., 2007) which is routinely used
in hospitals to test antimicrobial susceptibility for antibiotic-resistant bacteria,
followed by the dilution method for products which possess a bioactivity. Plant
extracts were delivered into well form lower to higher concentration and plates
were incubated at 37°C for 24 h. The presence of zone of inhibition was
regarded as the indicator of antimicrobial action and antimicrobial activity
was expressed in terms of average diameter of the zone of inhibition measured
in millimeter. Each test was carried out in triplicate.
Whole plant extracts of W. somnifera (ethyl acetate) tend to inhibit gram positive bacteria, S. aureus and B. subtilis. However, the inhibitory activity was very low in hexane extract (15 mm and no zone of inhibition for 10 mg mL-1) in comparison to ethanol extract (no zone of inhibition-10 mm), dichloromethane extract (15 mm-no zone of inhibition). Same pattern was also observed with gram negative bacteria, E. coli and P. aeruginosa. Relatively higher MIC concentrations were obtained for gram negative bacteria P. aeruginosa with ethyl acetate extract.
Surprisingly, no inhibitory effect has been noted for hexane, this could be
attributed to the extraction of active component of W. somnifera in ethyl
acetate rather than hexane. Results show that ethyl acetate extracts possess
great inhibitory effect for gram positive bacteria, S. aureus followed
by B. subtilis (Fig. 1).
||Antibacterial activity of ethyl acetate extract of Withania
somnifera at different concentration from 10 to 40 mg mL-1
||Antibacterial activity of ethanol extract of Withania somnifera
at different concentration from 10 to 40 mg mL-1
||Antibacterial activity of dichloromethane extract of Withania
somnifera at different concentration from 10 to 40 mg mL-1
Among gram negative bacteria highest inhibitory effect was observed with P.
aeruginosa, followed by E. coli (Fig. 1). Ethanolic
extracts possess great inhibitory effect for gram positive bacteria, S. aureus
whereas no zone of inhibition appeared for B. subtilis (Fig.
2). Among gram negative bacteria highest inhibitory effect was observed
with P. aeruginosa, followed by E. coli (Fig. 2).
In case of dichloromethane great inhibitory effect for gram positive bacteria
S. aureus followed by B. subtilis and among gram negative bacteria
highest inhibitory effect was observed with P. aeruginosa, followed by
E. coli (Fig. 3) and in case of hexane extract zone
of inhibition only appeared in S. aureus whereas no zone of inhibition
appeared in case of E. coli, P. aeruginosa and B. subtilis
In this manuscript, we have reported that ethyl acetate extract of Withania somnifera plant has high antibacterial activity for gram negative as well as gram positive bacteria with a very low MIC.
||Antibacterial activity of hexane extract of Withania somnifera
at different concentration from 10 to 40 mg mL-1
The present study strongly demonstrated that W. somnifera plant has
potent antibacterial property. The above results indicate that both alcoholic
as well as ethyl acetate extracts possessed strong antibacterial activity while
hexane and dichloromethane fraction was not as effective against bacteria which
shows the compounds were extracted in the polar solvents rather than nonpolar
solvents (Owais et al., 2005). The methanolic
extract was also inhibiting the growth of bacteria P. aeruginosa, E. coli
and S. aureus than aqueous extract (Rajendran
and Ramakrishnan, 2009). According to Mirjalili et
al. (2009) the important compounds withaferin and withanolides were
isolated from the methanolic extraction of the root W. somnifera. According
to Arora et al. (2004), the methanolic extract
of both leaves and root shows antibacterial activity, whereas, only root extract
in hexane shows antibacterial activity. The previous findings also shows that
the aqueous extract of Withania inhibit the growth of gram negative bacteria
N. gonorrhoea, which also supports the result because water is the most
polar solvent and the withanolides are extracted in the water properly (Kambizi
and Afolayan, 2008). The methanolic extract of the W. somnifera also
inhibit the growth of B. subtilis, E. coli, P. fluorescens and
S. aureus (Mahesh and Satish, 2008). According to
Choudhary et al. (1995), they used ethanol to
extract withanolides and fraction it and did it spectroscopic studies to isolate
the steroidal lactones that is withanolides. This finding also shows that the
withanolides, steroidal lactones, are extracted in ethanol, methanol and ethyl
acetate range of polar solvents which are potent inhibitor of bacterial growth.
The results of the viability assay have proved W. somnifera to hold excellent potential as an antibacterial agent. W. somnifera has withanolides which are steroidal lactones in nature and withaferin which makes a mucilaginous layer around the urinogenital, gastrointestinal and respiratory tract when consumed orally. The layers trap the microbial flora and make them unable to invade the system. Therefore, the bacteria cannot grow in the media containing W. somnifera extract. Thus, from the above investigation it can be concluded that the plant W. somnifera is a potential candidate for antimicrobial agent to treat diseases. Thus, further work can be carried out to isolate the exact active moiety responsible for the biological activity, characterize it and commercialize it.
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