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Inter J Curr Trends Sci Tech, 1(4): 232236 (2010) Manuscript: IJCST010405K ISSN (Online): 0976 9730 ISSN (Print): 0976 9498 EVALUATION OF ANTIBACTERIAL GRANATUM PEELS EXTRACT ACTIVITY OF PUNICA

MV Pereira*, DK Sandhu, AS Samleti Sinhgad Institute of Pharmaceutical Sciences, Lonavala, Pune- 410401 (M.S.), India. Correspondence author E mail: melisa.pereira@rediffmail.com Punica granatum L., belongs to the family Punicaceae, which is originating from the Middle East, extending throughout the Mediterranean, eastward to China and India, and on to the American Southwest, California and Mexico in the New World. Punica granatum has been used extensively as a traditional medicine in many countries for the treatment of dysentery, diarrhea, helminthiasis, acidosis, hemorrhage and respiratory pathologies. In addition, P. granatum is reported to have antioxidant, anti-atherosclerotic, antibacterial and antiviral properties. Current work reveals the evaluation of Antibacterial activity of methanolic extract of PG peels by disc diffusion method against some common gram positive and negative bacteria. The dried pomegranate peels (20gms) was extracted (cold maceration for 72 hrs) using methanol (250 ml). The Methanolic Extract of PG (MEPG) was active against the tested bacteria. MEPG demonstrated promising antibacterial activity against tested gram positive and gram negative bacteria. The E. coli and P. aeruginosa shows more susceptibility to MEPG. INTRODUCTION Punica granatum L. belongs to the family Punicaceae, which is originating from the Middle East, extending throughout the Mediterranean, eastward to China and India, and on to the American Southwest, California and Mexico in the New World1. Punica granatum has been used extensively as a traditional medicine in many countries2 for the treatment of dysentery, diarrhoea, helminthiasis, acidosis, hemorrhage and respiratory pathologies3,4. In addition, P. granatum is reported to have antioxidant5, 6, anti-atherosclerotic7, 8, antibacterial9, 8 and antiviral11 properties. Punica granatum peel is used to treat infections found in human sexual organs as well as mastitis, acne, folliculitis, pile, allergic dermatitis, tympanitis, scalds and as an antioxidant13. The constituents of P. granatum include gallocatechins, delphinidin, cyanidin, gallic acid, ellagic acid, pelargonidin and sitosterol, which are very well known for their therapeutic properties12.
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CONTENTS Material Erythromycin standard antibiotic discs and standard nutrient agar were procured from Microlab, Mumbai. Micro-Organisms All the authentic cultures of test micro-organism have been procured from National Collection of Industrial Microorganism (NCIM), a division of National Chemical Laboratory (NCL), Pune. The test microorganism were Bacillus subtilis(ATCC633),Bacillus cereus(ATCC 10703),Staphylococcus aureus(ATCC 9144),Pseudomonas aeruginosa (ATCC 19429),E. coli(ATCC 8739) and Klebsiella pneumoniae(NCIM 2957). Prepration of Extract 20 gm of fresh peels were extracted with 250 ml of pure methanol by cold maceration for 72 hrs. The extract was filtered using Whatman filter paper no 1 and filter was then evaporated under reduced pressure and dried using rotary evaporator of 60C to get semisolid residue. The extract was stored in labeled sterile screw capped bottle at 4C till further use14-16. METHOD Antibacterial activity of MEPG was screened using the disc diffusion method (NCCLS, 1993). Inoculum was prepared with fresh cultures of bacterial strains, cultured on Nutrient agar (Hi-media) for 18 h at 37oC with physiological saline. Inoculum density of each bacterial suspension was adjusted to reach an optical comparison to that of a 0.5 McFarland standard, resulting in a suspension containing approximately 1 to 2 x 108 CFU/ml (18). Nutrient agar plates were inoculated by streaking the swab over the entire sterile agar surface. This procedure was repeated by streaking 2 more times, rotating the plate approximately 60o each time to ensure even distribution of the Inoculum. The Inoculum was allowed to dry at room temperature. The different concentrations of MEPG were loaded on 6-mm sterile discs. The plates were allowed to stand at room temperature for 30min for extract to diffuse into the agar and then they were incubated at 37oC for 24 h. Subsequently, the plates were examined for bacterial growth inhibition and IZD was measured. DETERMINATION OF MIC The MIC was determined by micro-broth dilution method17. The reconstituted extract was serially diluted 2-fold in Nutrient broth (Hi-media) medium. Duplicate tubes of each dilution (12.5, 25.0, 50.0 and 100.0 mg/ml) were inoculated with the test bacterial suspension adjusted to optical density to that of 0.5 McFarland standard and tubes were incubated at 37oC for 24 h. MIC was taken as the highest dilution (least concentration) of extract showing no detectable growth. MEPG demonstrated promising antibacterial activity against tested gram positive and gram negative bacteria. The E. coli and P.
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aeruginosa shows more susceptibility to MEPG. All the bacteria showed susceptibility to the standard antibiotic discs of Erythromycin (15 g). The results indicated that standard antibiotic Erythromycin has stronger activity than the plant extract. Table 1: MIC values of MEPG against tested bacteria. Bacteria MIC (mg/ml) S. aureus (ATCC 9144) 12.5 B. cereus (ATCC 10703) 12.5 B. subtilis (ATCC 633) 12.5 K. pneumoniae (NCIM 2957) 25 E. coli (ATCC 8739) 25 P. aeruginosa (ATCC 19429) 12.5 Table: 2 Antimicrobial activity results of MEPG (Inhibition Zone Diameter in mm) Microorganism MEPG (50mg/disc ) ERTCN (15g) S. aureus (ATCC 9144) 29 30 B. cereus (ATCC 10703) 12 24 B. subtilis (ATCC 633) 20 26 K. pneumoniae (NCIM 16 23 2957) E. coli (ATCC 8739) 18 28 P. aeruginosa (ATCC 32 34 19429)

Figure 1: Antibacterial Activity results of MEPG (Inhibition Zone Diameter in mm) RESULTS AND DISCUSSIONS The E. coli and P. aeruginosa shows more susceptibility to MEPG. All the bacteria showed susceptibility to the standard antibiotic discs of Erythromycin (15 g). The results indicated that standard antibiotic
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Erythromycin has stronger activity than the plant extract. The MIC of MEPG extract against the tested microorganisms varied between 12 mg/ml to 25 mg/ml. MEPG demonstrated promising antibacterial activity against tested gram positive and gram negative bacteria. REFERENCES 1. 2. Fink SL, Cookson BT. Pyroptosis and host cell death responses during Salmonella infection. Cell Microbial, 2007, 9, 256270 Chidambaram MKN, Jayaprakasha GK, Singh RP. Studies on antioxidant activity of pomegranate (Punica granatum) peel extract using in vivo models. J Agric Food Chem, 2002, 50, 47915. Ricci D, Giamperi L, Bucchini A, Fraternale D. Antioxidant activity of Punica granatum fruits. Fitoterapia, 2006, 77, 31012. Sanchez A, Fonseca G, Fuentes JL, Cozzi R, Cundari E, Fiore M, et al. Assessment of the genotoxic risk of Punica granatum L. (Punicaceae) whole fruit extracts. J Ethnopharmacol, 2008, 115, 41622. Related A, LinksHeber D, Seeram NP, Wyatt H, Henning SM, Zhang Y, et al. Safety and antioxidant activity of a pomegranate ellagitannin-enriched polyphenol dietary supplement in overweight individuals with increased waist size. J Agric Food Chem, 2007, 55, 100504. Parmar HS, Kar A. Medicinal values of fruit peels from Citrus sinensis, Punica granatum and Musa paradisiaca with respect to alterations in tissue lipid peroxidation and serum concentration of glucose, insulin and thyroid hormones. J Medicinal Food, 2008, 11, 37681. Aviram M, Rosenblat M, Gaitini D, Nitecki S, Hoffman A, Dornfeld L, et al. Pomegranate juice consumption for 3 years by patients with carotid artery stenosis reduces common carotid intima-media thickness, blood pressure and LDL oxidation. Clin Nutr, 2004, 23, 42333. Parmar HS, Kar A. Protective role of Citrus sinensis, Punica granatum and Musa paradisiaca peels against diet induced atherosclerosis. Nutrition Res, 2007, 27, 71018. Braga LC, Shupp JW, Cummings C, Jett M, Takahashi JA, Carmo LS, et al. Pomegranate extract inhibits Staphylococcus aureus growth and subsequent enterotoxin production. J Ethnopharmacol, 2005, 96, 3359.

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5.

6.

7.

8.

9.

10. Naz S, Siddiqi R, Ahmad S, Rasool SA, Sayeed SA. Antibacterial activity directed isolation of compounds from Punica granatum. J Food Sci, 2007, 72, 3415. 11. Zhang J, Zhan B, Yao X, Gao Y, Shong J. Antiviral activity of tannin from the pericarp of Punica granatum L. against genital Herpes virus in vitro. Zhongguo Zhong Yao Za Zhi, 1995, 20, 5568.

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12. Singh RP, Chidambaram MKN, Jayaprakasha GK. Studies on the antioxidant activity of pomegranate (Punica granatum) peel and seed extracts using in vitro models. J Agric Food Chem, 2002, 50, 816 13. Lansky EP, Newman RA. Punica granatum (pomegranate) and its potential for prevention and treatment of inflammation and cancer. J Ethnopharmacol, 2007, 109, 17706. 14. Khandelwal K.R Practical Pharmacognosy: Techniques and experiment. Nirali Prakashan, Pune. Ed-14, 2005, 153-155. 15. Shah C.S. Quadry J.S.A textbook of Pharmacognosy, Ed 11th.B.S.Shah Prakashan, Ahmedabad, 1995, 157-160. 16. NCCLS, Performance standards for antimicrobial disc susceptibility test. Approved standard NCCLS Publication M2-A5, Villanova, PA, USA, 1993. 17. Slinkard K. and Singleton V.L.Total phenol analysis: automation and comparison with manual method. 18. Bassam AS, Ghaleb A, Nasser J, Awani A.Kamel A, Antimicrobial Activity of Four plant Extract Used in Palestine in Folkloric against Methicillin-Resistance Staphylococcus aureus, Turkish Journal of Biology, 30, 195-198 (2006).

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