In vitro Evaluation of Antibacterial Activities of Crude Extracts of Withania somnifera (Ashwagandha) to Bacterial Pathogens

INTRODUCTION

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).

Table 1:	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.

RESULTS

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).


Fig. 1:	Antibacterial activity of ethyl acetate extract of Withania somnifera at different concentration from 10 to 40 mg mL^-1


Fig. 2:	Antibacterial activity of ethanol extract of Withania somnifera at different concentration from 10 to 40 mg mL^-1


Fig. 3:	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 (Fig. 4).

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.


Fig. 4:	Antibacterial activity of hexane extract of Withania somnifera at different concentration from 10 to 40 mg mL^-1

DISCUSSION

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.

CONCLUSION

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.