INTRODUCTION:

Microbes are the cause of an infectious diseases pose critical health problems and they are one of the main causes of morbidity and mortality. Bacterial infection from virtually any site (respiratory tract, digestive tract, nervous system, urinary tract) in the body can pass into the blood stream and cause sepsis. Fever, rigors, hypotension, endocarditis, coma and death can result if sepsis is not quickly and properly treated. Thus global prevalence of microbial infections is estimated to increase, every year where shocking hospital infection statistics reports that 2 million patients get health care associated infections, 100,000 deaths and $30 billion spent to treat health care/hospital infections each year. Usually human gut contains 1013–1014 bacteria, which is 10-fold more than the total human cells in the body and these are exposed to selection pressure whenever antibiotics are used (17). The National Nosocomial Infections Surveillance System (NNIS) has reported that the prevalence of multidrug-resistant gram-negative bacteria is increasing every year.

For example, the prevalence of imipenem-resistant P. aeruginosa, quinolone-resistant P. aeruginosa, third-generation cephalosporin–resistant P. aeruginosa, and third-generation cephalosporin–resistant Klebsiella pneumoniae have increased by greater than 20% in 2003, compared with prevalence in 1998–2002 (24). Thus antibiotics conflict a delicate balance between humans and bacterial strains. Moreover, antibiotics are not only used in human medicine but also for treatment of mass prevention and growth promotion in animals, with resistant bacteria passed to humans via the food chain (21; 35). Finally, results in antibiotic-resistant strains and become untreatable with currently available antibiotics, emerges a major burden to the medical community in developed and especially in developing countries. Gentamicin and tetracycline are broad-spectrum antibiotics, where gentamicin was found to be more effective in treating gram-negative bacterial infections (4) and tetracycline have been widely used in human and veterinary medicine, as growth promotors in animal husbandry (13). Not surprisingly this antibiotic resistance is prevalent in a diverse range of bacteria (29). Thus, reducing antibiotic use doesn’t compensate/lowered the multidrug-resistant problem formed part of a positive response on the part of the United Kingdom government to the House of Lords report, 1998.

In order to uproot this problem world Health Organization, has listed 21,000 medicinal plants around worldwide for understanding the efficacy, safety and stabilization of medicinal plants, in which 2500 species are in India, out of which 150 species are used commercially on large scale. Therefore India is crowned as botanical garden of the world (31). In recent years there has been an increasing interest in the field of herbal medicine and their drugs are gaining popularity both in national and international levels, because of their natural origin and enhanced therapeutic value with nil/less side effects (16). In order to over ride this problem, ecologically and economically safe drug should be replaced.

W. somnifera is a most valued Indian medicinal plant commonly known as Ashwagandha, belonging to the family Solanaceae, is a member of GRAS (Generally Regarded as Safe) plants and a popular home remedy in the Indian pharmacopoeia. It is widely used in Indian traditional system as well as all over the world in many indigenous drug preparations. W. somnifera root extract has been shown to possess anti-inflammatory, anticancer, immunostimulatory, cardiovascularprotective, anti-tumour, adaptogenic, antiperoxidative, radiosensitizing and thyroregulatory effects (20; 23; 8; 19; 14; 33; 26). In addition, W. somnifera root extract has shown to have immunomodulatory activity (2), antistress effect (5) and has strong antifungal activity against murine aspergillosis (10; 36). Among the Indian medicinal plants, thirteen positive alkaloids and till date around 138 withanolides have been reported from W. somnifera (18).

Many toxicology studies confirmed that various active compounds of the plant appear to be safe. Till-date no herb-herb or herb–drug interactions have been reported in the literature with W. somnifera (6). Previous report shows that methanolic extracts of W. somnifera protected rats against hepatic, renal and skin pathology induced by fungicide (carbendazim) and DMBA (dimethyl benzanthracene) (3; 27). Moreover the methanolic root extract of W. somnifera is a rich source of steroidal lactones called withanolides (15). Hence the present study, we investigated a comparative analysis of ethanol and methanol root extracts of W. somnifera with standard antibiotics to explore its inhibition efficacy against bacterial strains.

MATERIALS AND METHODS:

Antibiotic Discs

Gentamicin and tetracycline antibiotic discs were purchased from Himedia laboratories Pvt. limited, Mumbai, India.

Plant materials

The dried roots of W. somnifera were collected from Chidambaram, Cuddalore District, Tamil Nadu, India. The root was identified and authenticated at the Herbarium of Botany Directorate in Annamalai University. A voucher specimen (No: 2934) was deposited in the Botany Department of Annamalai University.

Preparation of ethanol and methanol extracts of W. somnifera

Dried root of W. somnifera was ground in a miller and the dried powder was stored at 4 °C. A portion of the root powder was extracted with 95% ethanol (1:10 W/V) and 70% methanol separately for 2 days with constant stirring. Suspensions were filtered through Whatman No.1 filter paper to retain the clear solution. The residue was extracted again and pooled extract was vacuum evaporated below 50°C. The dried extracts were stored at 4°C.

Bacterial strains

Bacillus subtilis, Escherichia coli, Klebsiella pnuemoniae, Pseudomonas aeruginosa and Salmonella typhi strains were used to test the antimicrobial activity of ethanol and methanol root extracts of W. somnifera. All these bacterial strains were obtained from the Dept. of Agricultural Microbiology and Dept. of Raja Muthiah Medical College and Hospital, Annamalai University, Annamalai nagar, Tamil Nadu, India.

Preparation of Luria–Bertaini (LB) Medium and inoculum of bacterial strains

All bacterial strains were maintained and sub cultured in LB medium (containing Sodium chloride 10g/L, Bactotryptone 10g/L, Yeast extract 5g/L and agar 15g/L). Then the bacterial strains were inoculated into LB broth and kept for 2-5 hrs incubation at 37º C in an incubator. Growth can be identified by the turbidity in broth.

Antibiotic sensitivity Assay (Kirby-Bauer Method)

Agar medium prepared was poured into the petridish and allowed to solidify. After this a 100µl of 5 hour culture was poured into the media by the micropipette and it was uniformly swapped on the agar medium. Commercially available gentamicin and tetracycline antibiotics discs (7) were placed on the medium with the help of sterile forceps. Sterilized Whatman No.1 filter paper discs were dipped on the ethanol and methanol root extracts of W. somnifera and placed on the same plates (34). These plates were incubated at 37ºC for 24 hours. After the incubation, the diameters of the inhibition zones were measured.

Table 1. Antibacterial activity of Withania somnifera extracts

S.No	Organism	Zone of inhibition (mm)
S.No	Organism	Ethanol extract	Methanol extract
1	Bacillus subtilis	14	16
2	Escherichia coli	18	20
3	Klebsiella pnuemoniae	17	19
4	Pseudomonas aeruginosa	19	22
5	Salmonella paratyphi	18	21

Table 2. Antibiotic sensitivity patterns of microorganisms for commercial antibiotics

S.No	Organism	Zone of inhibition (mm)
S.No	Organism	Gentamicin	Tetracycline
1	Bacillus subtilis	11	14
2	Escherichia coli	17	18
3	Klebsiella pnuemoniae	13	19
4	Pseudomonas aeruginosa	21	16
5	Salmonella paratyphi	21	20

Table 3. Comparative Analysis of Antibacterial activities of Withania somnifera extracts and Commercial Antibiotics

S.No	Organism	Zone of inhibition (mm)		Maximum Zone of inhibition (mm)
S.No	Organism	Ethanol extract	Methanol extract	Gentamicin/ Tetracycline
1	Bacillus subtilis	14	16	14	Tetracycline
2	Escherichia coli	18	20	18	Tetracycline
3	Klebsiella pnuemoniae	17	19	19	Tetracycline
4	Pseudomonas aeruginosa	19	22	21	Gentamicin
5	Salmonella paratyphi	18	21	21	Gentamicin

Fig.1. Antibacterial activity of ethanol and methanol root extracts of W.somnifera and commercial antibiotics (gentamicin and tetracycline)

T-Tetracycline G - Gentamicin M-Methanolic Extract of W.somnifera E- Ethanolic Extract of W.somnifera

A - Antibacterial activity of ethanol and methanol root extracts of W.somnifera and tetracycline against Klebsiella pnuemoniae.

B - Antibacterial activity of ethanol and methanol root extracts of W.somnifera and tetracycline against Escherichia coli.

C - Antibacterial activity of ethanol and methanol root extracts of W.somnifera and tetracycline against Bacillus subtilis.

D - Antibacterial activity of ethanol and methanol root extracts of W.somnifera and tetracycline against Pseudomonas aeruginosa.

E - Antibacterial activity of ethanol and methanol root extracts of W.somnifera and tetracycline against Salmonella paratyphi.

F - Antibacterial activity of ethanol and methanol root extracts of W.somnifera and gentamicin against Klebsiella pnuemoniae.

G - Antibacterial activity of ethanol and methanol root extracts of W.somnifera and gentamicin against Escherichia coli.

H - Antibacterial activity of ethanol and methanol root extracts of W.somnifera and gentamicin against Bacillus subtilis.

I - Antibacterial activity of ethanol and methanol root extracts of W.somnifera and gentamicin against Pseudomonas aeruginosa.

J - Antibacterial activity of ethanol and methanol root extracts of W.somnifera and gentamicin against Salmonella paratyphi.

RESULTS:

The results rendered significant antibacterial activity of commercial antibiotics gentamicin and tetracycline, ethanol as well as methanol extracts of W. somnifera root against the bacterial strains (Bacillus subtilis, Escherichia coli, Klebsiella pnuemoniae, Pseudomonas aeruginosa and Salmonella paratyphi) was shown in Fig1.The inhibition of the bacterial strains was more prominent with methanol root extract as compared to ethanol root extract of W. somnifera (Table 1). Antibiotic sensitivity pattern of the commercial antibiotic discs such as gentamicin and tetracycline against these bacterial strains was shown in Table 2. Comparative analysis of antibacterial activities of ethanol and methanol root extracts of W. somnifera and commercial antibiotics was shown in Table 3.

DISCUSSION:

Antibiotics exert serious untoward effects to the host tissues leading to the systemic toxicity (9). Global alarming of antimicrobial resistance began to appear in the middle of the last century. In recent reports, the risks of excessive/inappropriate use of antibiotics in clinical medicine and of the use of antibiotics in animal feed as growth promoters emerged as the main reason of antibiotic resistance. Knowing the value of bioactive compounds of medicinal plants, as well as to get over the multidrug-resistant problem, research institutes and multinational drug companies pay their attention in isolating effective drugs (12).

W. somnifera was one of the leading medicinal plant for chemical and biological investigations (30). Previous report confirms that both aqueous as well as methanolic extract of leaf and root of W. somnifera possess alkaloids and protein (confirmed by alkaloid specific tests and routine protein estimation assay) as possible factor for their strong antibacterial properties against Escherichia coli Staphylococcus aureus and Salmonella typhimurium. Toxicity studies revealed that the active compounds of the plant appear to be safe (25). Our study reports showed that methanol root extract of W. somnifera showed greater zone of inhibition against Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa and equal zone of inhibition against Klebsiella pnuemoniae and Salmonella paratyphi as compared to commercial antibiotics, whereas on the other hand the ethanol extracts show equalent / less activity as compared to commercial antibiotics. This might be due to the ethanol and methanol root extracts of W. somnifera, as a rich source of many pharmacologically and medicinally important compounds such as steroidal compounds including steroidal lactones, withaferin A, withanolides A-y, withasomniferin-A, withasomidienone, withasomniferols A-C, withanone etc., and various useful alkaloids. Apart from these contents ethanol and methanolic root extracts of W. somnifera also contain various bioactive compounds such as withaniol, acylsteryl glucosides, starch, reducing sugar, hantreacotane, ducitol, a variety of amino acids (including aspartic acid, proline, tyrosine, alanine, glycine, glutamic acid, cystine, tryptophan) and high amount of iron (11). Methanol root extract of W. somnifera has more potent antibacterial activity as compared to its ethanolic root extract. This may be due to methanol root extracts of W. somnifera, containing numerous bioactive compounds known as withanolides (steroidal lactones with ergostane skeleton) (11), b-sitosterol, stigmasterol, b-sitosterol glucoside, stigmasterol glucoside, α + β glucose, Viscosa lactone B and the rare compound 16 β -acetoxy-17(20)-ene and 6 α -hydroxy-5,7 α -epoxy (more thermostable withanolide A) (22) as compared to ethanol root extract; this might be eminent in activating the immune components of the host, leading to the observed increase in phagocytosis and intracellular killing by peritoneal macrophages (32; 10). However the mechanism through which the active constituents of methanol root extract of W. somnifera participate to inhibit these bacterial strains is still unclear and further studies have to be carried out.

From our study, we conclude that methanol root extracts of W. somnifera with its well-powered bioactive compounds can counteract these bacterial strains, which direct the human life towards the valuable traditional medicine against the drug resistant microbes.

REFERENCES:

1. Abraham A, Kirson I, Glotter E and Lavie D. A chemotaxonomical study of W. somnifera (L) Duna, Phytochemistry. 7;1968: 957-962.

2. Agarwal R, Diwanay S, Patki P and Patwardhan B. Studies on immunomodulatory activity of W. somnifera (Ashwagandha) extracts in experimental immune inflammation. Journal of Ethnopharmacology. 67;1999: 27-35.

3. Akbarsha MA, Vijendrakumar S, Kadalmani B, Girija R and Faridha A. Curative property of W. somnifera Dunal root in the context of carbendazin-induced histopathological changes in the liver and kidney of rat. Phytomedicine. 7; 2000: 499-507.

4. Ali BH. Agents ameliorating or augmenting experimental gentamicin nephrotoxicity: some recent research. Food Chem. Toxicol. 41; 2003: 1447-1452.

5. Archana R and Namasivayam A. Antistressor effect of W. somnifera. Journal of Ethnopharmacology. 64;1999: 91- 93.

6. Arseculeratne SN, Gunatilaka AA and Panabokke RG. Studies of medicinal plants of SriLanka Part14 Toxicity of some traditional medicinal herbs. Journal of Ethnopharmacology. 13;1985: 323-335.

7. Bauer RW, Kirby MDK, Sherris JC and Turck M. Antibiotic susceptibility testing by standard single disc diffusion method. Am. J. Clin. Pathol. 45;1966: 493-496.

8. Bhattacharya SK and Muruganandam AV. Adaptogenic activity of W. somnifera an experimental study using a rat model of chronic stress. Pharmacol. Biochem. Behav. 75;2003:547-555.

9. Devi PU. W. somnifera dunal (ashwagandha): Potential plant source of a promising drug for cancer chemotherapy and radiosensitisation. Indian J. Exp. Biol. 34;1999: 927-932.

10. Dhuley JN. Therapeutic efficacy of ashwagandha against experimental aspergillosis in mice. Immunopharmacol. Immunotoxicol. 20; 1998: 191-198.

11. Elsakka M, Grigorescu E, Stanescu U, Stanescu U and Dorneanu V. New data referring to chemistry of W. somnifera species. Rev. Med. Chir. Soc. Med. Nat. Iasi. 94; 1990: 385-387.

12. Evans WC. Trease and Evan’s pharmacognosy 14th edition. WB Sounders London PP780, 1996.

13. Falkiner FR. The consequences of antibiotic use in horticulture. J.Antimicrob. Chemother. 41; 1998: 429-431.

14. Ferrari CK. Functional foods, herbs and neutrceuticals: towards biochemical mechanisms of healthy aging. Biogerontology. 5; 2004: 275-285.

15. Ganzera M, Choudhary MI and Khan IA. Quantitative HPLC analysis of withanolides in W. somnifera. Fitoterapia. 74; 2003: 68-76.

16. Grover JK, Yadav S and Vats V. Medicinal plants of India with antidiabetic potential. Journal of Ethnopharmacology. 81; 2002:81-100.

17. Guarner F and Malagelada JR. Gut flora in health and disease. Lancet 361; 2003: 512-519.

18. Gupta GL and Rana AC. W. somnifera (Ashwagandha): A Review. Pharmacognosy Reviews.1: Issue 1, 2007.

19. Iuvone T, Esposito G, Capasso F and Izzo AA. Induction of nitric oxide synthase expression by W. somnifera in macrophages. Life Science. 72; 2003:1617-1625.

20. Keifer D and Pantuso T. Panax Ginseng. Am Fam Physician. 68; 2003:1539-1542.

21. Levy SB. Active efflux, a common mechanism for biocide and antibiotic resistance. Journal of Applied Microbiology. 92; 2002: 65-71.

22. Misra L, Mishra P, Pandey A, Sangwan RS, Sangwan NS and Tuli R. Withanolides from W. somnifera roots. Phytochemistry. 69; 2008: 1000-1004.

23. Mohan R, Hammers HJ, Bargagna MP, Zhan XH, Herbstritt CJ, Ruiz A, Zhang L, Hanson AD, Conner BP, Rougas J and Pribluda VS. Withaferin A is a potent inhibitor of angiogenesis. Angiogenesis. 7; 2004:115-122.

24. National Nosocomial Infections Surveillance System (2004) National Nosocomial Infections Surveillance (NNIS) System Report, data summary from January 1992 through June 2004, issued October 2004. Am. J. Infect. Control. 32:470– 485.

25. Owais M, Sharad KS, Shehbaz A and Saleemuddin M. Antibacterial efficacy of W. somnifera (ashwagandha) an indigenous medicinal plant against experimental murine salmonellosis. Phytomedicine.12; 2005: 229-235.

26. Panda S and Kar A. Effects of root extract of Ashwagandha, Withania somnifera, on function of thyroid in cockerel. Ind. J. Anim. Sci. 67; 1997: 575-576.

27. Prakash J, Gupta SK and Dinda AK. W. somnifera root extract prevent DMBA-induced quamous cell carcinoma of skin in Swiss albino mice. Nutr. Cancer. 42; 2002: 91-97.

28. Resistance to antibiotics and other antimicrobial agents. Report of the House of Lords Select Committee on Science and Technology. London: Stationery Office, 1998.

29. Roberts M.C. Tetracycline resistance determinants: mechanisms of action, regulation of expression, genetic mobility, and distribution. FEMS Microbiol. Reviews 9; 1996: 1-24.

30. Sangwan RS, Chaurasia ND, Misra LN, Lal P, Uniyal GC, Sharma R, Sangwan NS, Suri KA, Qazi GN and Tuli R. Phytochemical variability in commercial herbal products and preparations of W. somnifera (Ashwagandha). Curr. Sci. 86; 2004: 461-465.

31. Seth SD and Sharma B. Medicinal plants of India. Indian J. Med. Res. 120; 2004: 9-11.

32. Threlfall EJ, Frost JA, Ward LR and Rowe B. Increasing spectrum of resistance in multi-resistant Salmonella typhimurium. Lancet. 347; 1996:1053-1054.

33. Tripathy AK, Shukla YN and Kumar S. Ashwagandha W. somnifera. Dunal Solanaceae. A status report. Med. Arom. Plant Sci. 18; 1996: 46-62.

34. Ulubelen A, Topcu G, Eris C, Sonmez U, Kartal M, Kurucu S and Bozok-Johansson C. Terpenoids from Salvia sclarea. Phytochemistry. 36; 1994: 971-974.

35. Walsh C and Fanning S.Antimicrobial resistance in foodborne pathogens--a cause for concern?. Curr. Drug Targets. 9; 2008: 808-815.

36. Ziauddin M, Phansalkar N, Patki P, Diwanay S and Patwardhan B. Studies on immunomodulatory activity of ashwagandha. Journal of Ethanopharmocology. 50; 1996: 69-76.

Received on 04.05.2013 Accepted on 20.05.2013

Asian J. Pharm. Res. 3(2): April- June 2013; Page 98-102