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synergistic activities of Withania somnifera extracts
Fitoterapia 75 (2004) 385–388
Short report
The in vitro antibacterialysynergistic activities of Withania somnifera extracts S. Arora*, S. Dhillon, G. Rani, A. Nagpal Department of Botanical Sciences, Guru Nanak Dev University, Amritsar 143 005, India Received 24 November 2003; accepted 22 January 2004
Abstract The methanol, hexane and diethyl ether extracts from both leaves and roots of Withania somnifera were evaluated for the antibacterialysynergistic activity by agar plate disc-diffusion assay against Salmonella typhimurium and Escherichia coli. Different concentrations of Tibrim, a combination of rifampicin and isoniazid, were tested to find out the minimum inhibitory concentration (MIC), which came out to be 0.1 mgyml for S. typhimurium and E. coli. From the six extracts tested, only methanol and hexane extracts of both leaves and roots were found to have potent antibacterial activity. A synergistic increase in the antibacterial effect of Tibrim was noticed when MIC of Tibrim was supplemented with these extracts. 䊚 2004 Elsevier B.V. All rights reserved. Keywords: Withania somnifera; Antimicrobial activity; Synergistic effects
Plant. Withania somnifera (L.) Dunal (Solanaceae), leaves and roots collected from 3-year-old seed-raised plants growing in the Botanic Garden of Guru Nanak Dev University, Amritsar (India) on 10 October 1997. A voucher specimen has been deposited at the Herbarium, Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar, India. Use in traditional medicine. W. somnifera possesses therapeutic value against a large number of ailments, such as mental diseases, asthma, inflammation, arthritis, *Corresponding author. Tel.: q91-0183-2451048; fax: q91-0183-2258819-2258820. E-mail address: [email protected] (S. Arora). 0367-326X/04/$ - see front matter 䊚 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2004.01.002
386
Table 1 Antibacterial and synergistic activity of extracts of W. somnifera on growth of TA100 strain of S. typhimurium and DH5a Strain of E. coli Treatment
MIC of Tibrimqextract Tibrimqmethanol Tibrimqhexane Tibrimqdiethyl ether
Roots Diameter of zone of inhibition (mm)
TA100
DH5a
TA100
DH5a
13.00"0.71a 5.44"0.53b 5.00"0.71b F(d.f. 2,24)s427.130* HSDs0.768
8.67"0.50b 12.55"0.73a 5.44"0.53c F(d.f. 2,24)s324.316* HSDs0.698
5.22"0.44b 5.67"0.71b 6.78"0.67a F(d.f. 2,24)s15.219* HSDs0.725
7.00"0.50b 9.44"0.53a 7.22"0.67b F(d.f. 2,24)s50.743* HSDs0.669
20.00"0.41a 9.00"0.24b 7.55"0.24c F(d.f.Ø2,24)s494.813* HSDs1.080
8.78"0.28b 13.55"0.29a 7.00"0.24c F(d.f.Ø2,24)s157.352* HSDs0.954
19.00"0.44a 7.00"0.24b 6.55"0.29b F(d.f.Ø2,24)s444.477* HSDs1.182
8.00"0.29b 18.00"0.44a 7.55"0.29b F(d.f.Ø2,24)s287.322* HSDs1.229
Disc diameters4 mm; concentration of extractss100 mgyml. MIC of Tibrims0.10 mgyml for both TA100 strain of S. typhimurium and DH5a strain of E. coli. Concentration of Tibrimqextracts0.10 mgymlq0.10 mgyml. Data shown are Mean"S.E. of three experiments; each experiment consisted of three replicates. *Significant at PF0.05. a,b,c Means followed by the same letter are not significantly different using HSD multiple comparison test.
S. Arora et al. / Fitoterapia 75 (2004) 385–388
Extract Methanol Hexane Diethyl ether
Leaves Diameter of zone of inhibition (mm)
S. Arora et al. / Fitoterapia 75 (2004) 385–388
387
rheumatism, tuberculosis, infections, fever, male sexual disorders and a variety of other diseases, including cancer w1,2x. Even though some studies on antibacterial effects of different extracts and purified compounds from Withania have been done w3–9x, this property of Indian Ginseng has not been studied in detail. Previously isolated classes of constituents. Alkaloids from roots w10,11x, withanolides from the leaves. Several chemotypes exist that differ in their withanolide content w12x. The principal withanolides in Indian W. somnifera are withaferin A and withanolide D w13x. Tested material. Methanol, hexane and diethyl ether extracts obtained from leaves and roots of W. somnifera, and MIC of Tibrim in combination of rifampicin and isoniazid added to extracts. Studied activity. Antibacterial using disc-diffusion method w14x. Used microorganisms. Bacterial strain TA100 of Salmonella typhimurium and DH5a strain of Escherichia coli. Results. Summarized in Table 1. Conclusions. Methanol extract of both leaves and roots in TA100 strain of S. typhimurium and hexane extract of roots in DH5a strain of E. coli showed more or less synergistic effect. Other extracts enhanced the antibacterial activity of MIC of Tibrim, but the effect was not even additive. The antibacterialysynergistic effect of extracts can be attributed to the presence of withaferin A and withanolide D. This investigation supports the idea of using plant metabolites as antimicrobial and synergistic agents, which can help mankind to curb the evolution of drug-resistant strains of microbes. Acknowledgments This paper is dedicated to the fond memories of the late Prof. I.S. Grover, our guide and former Head, Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar. References w1x Asthana R, Raina MK. Ind Drug 1989;26:199. w2x Singh S, Kumar S. Withania somnifera. The Indian ginseng ashwagandha. Lucknow: Central Institute of Medicinal and Aromatic Plants (CIMAP), 1998. p. 293.
388 w3 x w4 x w5 x w6 x w7 x w8 x w9 x w10x w11x w12x w13x w14x
S. Arora et al. / Fitoterapia 75 (2004) 385–388 Kurup PA. Curr Sci 1956a;25:57. Kurup PA. J Sci Ind Res 1956b;15C:153. Kurup PA. Antibiot Chemother 1958;8:511. Attia MS, Ahmad S, Zaidi SAH, Ahmed Z. Pak J Sci Ind Res 1972;15:199. Bhatnagar SS, Santapau H, Desa JDH, Maniar AC, Ghadially NC, Solomon MJ, Vellore S, Rao TNS. Ind J Med Res 1961;49:799. Ikram M, Haq I. Fitoterapia 1980;51:231. Jaffer HJ, Jawad ALM, Saber HS, Al-Naib A. Fitoterapia 1988;59:497. Schwarting AE, Bobbitt JM, Rother A, Atal CK, Khanna KJ, Leary JD, Walter WG. Llyodia 1963;26:258. Schroter HB, Neumann D, Katritzky AR, Swinbourne FJ. Tetrahedron 1966;22:2895. Glotter E, Kirson I, Abraham A, Lavie D. Tetrahedron 1973;29:1353. Kirson I, Glotter E, Lavie D, Abraham A. J Chem Soc Sect C Org Chem 1971;11:2032. Bauer SW, Kirby WM, Sherris JC, Thurck M. Am J Pathol 1966;45:493.
Short report
The in vitro antibacterialysynergistic activities of Withania somnifera extracts S. Arora*, S. Dhillon, G. Rani, A. Nagpal Department of Botanical Sciences, Guru Nanak Dev University, Amritsar 143 005, India Received 24 November 2003; accepted 22 January 2004
Abstract The methanol, hexane and diethyl ether extracts from both leaves and roots of Withania somnifera were evaluated for the antibacterialysynergistic activity by agar plate disc-diffusion assay against Salmonella typhimurium and Escherichia coli. Different concentrations of Tibrim, a combination of rifampicin and isoniazid, were tested to find out the minimum inhibitory concentration (MIC), which came out to be 0.1 mgyml for S. typhimurium and E. coli. From the six extracts tested, only methanol and hexane extracts of both leaves and roots were found to have potent antibacterial activity. A synergistic increase in the antibacterial effect of Tibrim was noticed when MIC of Tibrim was supplemented with these extracts. 䊚 2004 Elsevier B.V. All rights reserved. Keywords: Withania somnifera; Antimicrobial activity; Synergistic effects
Plant. Withania somnifera (L.) Dunal (Solanaceae), leaves and roots collected from 3-year-old seed-raised plants growing in the Botanic Garden of Guru Nanak Dev University, Amritsar (India) on 10 October 1997. A voucher specimen has been deposited at the Herbarium, Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar, India. Use in traditional medicine. W. somnifera possesses therapeutic value against a large number of ailments, such as mental diseases, asthma, inflammation, arthritis, *Corresponding author. Tel.: q91-0183-2451048; fax: q91-0183-2258819-2258820. E-mail address: [email protected] (S. Arora). 0367-326X/04/$ - see front matter 䊚 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2004.01.002
386
Table 1 Antibacterial and synergistic activity of extracts of W. somnifera on growth of TA100 strain of S. typhimurium and DH5a Strain of E. coli Treatment
MIC of Tibrimqextract Tibrimqmethanol Tibrimqhexane Tibrimqdiethyl ether
Roots Diameter of zone of inhibition (mm)
TA100
DH5a
TA100
DH5a
13.00"0.71a 5.44"0.53b 5.00"0.71b F(d.f. 2,24)s427.130* HSDs0.768
8.67"0.50b 12.55"0.73a 5.44"0.53c F(d.f. 2,24)s324.316* HSDs0.698
5.22"0.44b 5.67"0.71b 6.78"0.67a F(d.f. 2,24)s15.219* HSDs0.725
7.00"0.50b 9.44"0.53a 7.22"0.67b F(d.f. 2,24)s50.743* HSDs0.669
20.00"0.41a 9.00"0.24b 7.55"0.24c F(d.f.Ø2,24)s494.813* HSDs1.080
8.78"0.28b 13.55"0.29a 7.00"0.24c F(d.f.Ø2,24)s157.352* HSDs0.954
19.00"0.44a 7.00"0.24b 6.55"0.29b F(d.f.Ø2,24)s444.477* HSDs1.182
8.00"0.29b 18.00"0.44a 7.55"0.29b F(d.f.Ø2,24)s287.322* HSDs1.229
Disc diameters4 mm; concentration of extractss100 mgyml. MIC of Tibrims0.10 mgyml for both TA100 strain of S. typhimurium and DH5a strain of E. coli. Concentration of Tibrimqextracts0.10 mgymlq0.10 mgyml. Data shown are Mean"S.E. of three experiments; each experiment consisted of three replicates. *Significant at PF0.05. a,b,c Means followed by the same letter are not significantly different using HSD multiple comparison test.
S. Arora et al. / Fitoterapia 75 (2004) 385–388
Extract Methanol Hexane Diethyl ether
Leaves Diameter of zone of inhibition (mm)
S. Arora et al. / Fitoterapia 75 (2004) 385–388
387
rheumatism, tuberculosis, infections, fever, male sexual disorders and a variety of other diseases, including cancer w1,2x. Even though some studies on antibacterial effects of different extracts and purified compounds from Withania have been done w3–9x, this property of Indian Ginseng has not been studied in detail. Previously isolated classes of constituents. Alkaloids from roots w10,11x, withanolides from the leaves. Several chemotypes exist that differ in their withanolide content w12x. The principal withanolides in Indian W. somnifera are withaferin A and withanolide D w13x. Tested material. Methanol, hexane and diethyl ether extracts obtained from leaves and roots of W. somnifera, and MIC of Tibrim in combination of rifampicin and isoniazid added to extracts. Studied activity. Antibacterial using disc-diffusion method w14x. Used microorganisms. Bacterial strain TA100 of Salmonella typhimurium and DH5a strain of Escherichia coli. Results. Summarized in Table 1. Conclusions. Methanol extract of both leaves and roots in TA100 strain of S. typhimurium and hexane extract of roots in DH5a strain of E. coli showed more or less synergistic effect. Other extracts enhanced the antibacterial activity of MIC of Tibrim, but the effect was not even additive. The antibacterialysynergistic effect of extracts can be attributed to the presence of withaferin A and withanolide D. This investigation supports the idea of using plant metabolites as antimicrobial and synergistic agents, which can help mankind to curb the evolution of drug-resistant strains of microbes. Acknowledgments This paper is dedicated to the fond memories of the late Prof. I.S. Grover, our guide and former Head, Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar. References w1x Asthana R, Raina MK. Ind Drug 1989;26:199. w2x Singh S, Kumar S. Withania somnifera. The Indian ginseng ashwagandha. Lucknow: Central Institute of Medicinal and Aromatic Plants (CIMAP), 1998. p. 293.
388 w3 x w4 x w5 x w6 x w7 x w8 x w9 x w10x w11x w12x w13x w14x
S. Arora et al. / Fitoterapia 75 (2004) 385–388 Kurup PA. Curr Sci 1956a;25:57. Kurup PA. J Sci Ind Res 1956b;15C:153. Kurup PA. Antibiot Chemother 1958;8:511. Attia MS, Ahmad S, Zaidi SAH, Ahmed Z. Pak J Sci Ind Res 1972;15:199. Bhatnagar SS, Santapau H, Desa JDH, Maniar AC, Ghadially NC, Solomon MJ, Vellore S, Rao TNS. Ind J Med Res 1961;49:799. Ikram M, Haq I. Fitoterapia 1980;51:231. Jaffer HJ, Jawad ALM, Saber HS, Al-Naib A. Fitoterapia 1988;59:497. Schwarting AE, Bobbitt JM, Rother A, Atal CK, Khanna KJ, Leary JD, Walter WG. Llyodia 1963;26:258. Schroter HB, Neumann D, Katritzky AR, Swinbourne FJ. Tetrahedron 1966;22:2895. Glotter E, Kirson I, Abraham A, Lavie D. Tetrahedron 1973;29:1353. Kirson I, Glotter E, Lavie D, Abraham A. J Chem Soc Sect C Org Chem 1971;11:2032. Bauer SW, Kirby WM, Sherris JC, Thurck M. Am J Pathol 1966;45:493.