Antimicrobial and Phytochemical Analysis of Centella asiatica (L.) R. Perumal Samy*,1, Ignacimuthu 2, Vincent TK Chow 1 1 Infectious Diseases Programme, Department of Microbiology, Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597 2 Entomology Research Institute, Loyola College, Chennai - 600 034, India. Abstract In vitro antibacterial studies were carried out using hexane, dichloromethane and methanol extract of leaves of Centella asiatica by disc-diffusion method against gram-positive and gram-negative bacteria. The methanol and dichloromethane extracts of leaf showed a broad spectrum antibacterial activity. Thus the results substantiate the traditional usage of this plant as a medicine. Keywords: Centella asiatica; antibacterial activity; medicine *Corresponding author. Tel..: +65 6516 6200; fax: +65 6776 6872. E-mail address: micramar@nus.edu.sg (R.Perumal Samy).
Plant and collection Centella asiatica (L.) Urban belongs to the family Umbeliferae also known as Neer Brahmi. Brahmi is one of the most important medicinal herbs described by Charaka as an antiaging plant. It is mainly found in the marshy areas in throughout India, Madagascare, Africa and Sri Lanka and grow in an altitude of 650 meter etc. Centella asiatica plant material (leaves) was collected during January 1998 from Kolli hills in India and authenticated by S.P. Subiramani. The plant was identified by the author, in the Department of Plant Biology and Biotechnology, Loyola College, Chennai - 600 034, India. A voucher specimen (PBB 0120) has been deposited at the department. Uses in traditional medicine and reported activities The leaves are used to treat wounds, skin, leprosy, ulcerous, abnormalities, mental and neurological disturbances, hypertension [1], improvement in mentally retard children [2], sedative and antioxidant properties have been reported for the leaf. In addition, brain tonic and its efficacy for the treatment of tuberculosis, syphilis, amoebic dysentery and common colds are documented in the literature. Previously isolated classes of constituents Several phytochemical constituents were reported in C. asiatica such as triterpenoid saponins [3], asiaticoside (0.1-0.6%), asiatic acid (0.1-0.5%), madecassic acid (0.5-0.18%), aglycones [4], saponins-asiaticoside B, brahminoside [5], centelloside, indcentelloside [6], thankuniside, isothankuniside [7], brahamic acid, betulic acid [8] and alkaloid hydrocotyline [9], madecassic acid, glucose, rhamnose, sitosterol,
stigmasterol, fatty oil contains glycerides of pamitic acid, stearic acid, linoleic acid, ascorbic acid, others calcium, phosphate, and iron etc. Tested material Different fractions were obtained by partitioning of MeOH soxhlet extracts of dried plant material (leave; yields: 3.15%, 6.54% and 8.23% respectively). The phytochemical screening gave positive result [10]. Micro-organisms Bacterial organisms used in this assay listed in Table 2 and obtained from the Department of Medical Microbiology, Dr. A.L. Mudhaliar Post Graduate Institute of Basic Medical Sciences, Taramani, Chennai, India. Antibacterial activity was studied by disc diffusion method [11,12]. Results In this study, in vitro results reported in Table 1 and 2 which methanolic and dichlormethane extract of C. asiatica showed significant and higher rate of antimicrobial activity against various bacteria used in study. While hexane extract exhibited only a moderate activity. Conclusions The leaves of C. asiatica are exhibited a wide range of activity against the selected bacteria. Phytochemical screening of different fractions gradually were influenced the level of antibacterial activity, the methanol fraction of the leaf extract showed
maximum activity. Phytochemical screening of methanol extracts being the most active, Acknowledgement The authors are grateful to Dr. S. J. Kingsley and all the staff members in the department of Plant Biology and Biotechnology, Loyola College, Chennai - 600 034 References [1] Kartning T. Clinical Application of Centella asiatica (L.) Urb. in Craker L.E. and Simon J.E (eds.), Herbs Spices and Medicinal Plants. Vol. II. Oryx Press, Phoemix, AZ. 1988:145. [2] Sharma R, Jaiswal AN, Kumar S, Chaturvedic C, Tewari PV. J Res Edu Ind Med 1985;1:55. [3] Singh B, Rastogi RP. Phytochemistry 1968;7:1385. [4] Rastogi RP, Sankar B, Dhar ML. J Sci Ind Res 1960;19B:252. [5] Sahu NP, Roy SK, Mahato SB. Phytochemistry 1999;28:2852. [6] Bhattacharya SC. J Ind Chem Soc 1956;893. [7] Datta T,Basu UP. Bull Nat Inst India 1968;37:178 [8] Prum N, Ilel B, Raynand. J Pharmazie 1983;38:423. [9] Basu NK, Lamsal PP. Quatr J Pharm and Phamacol 1947;20:135. [10] Harbone JB. Phytochemical Methods. 2 nd ed; London: Chapamn and Hall, 1984;55, 84:120. [11] Bauer AW, Kirby WMM, Sherries JC, Truck M. Am J Clin Path 1966;45:493. [12] Jagtap NS, Khadabadi SS, Ghorpade DS, Banarase NB, Naphade SS. Research J. Pharm. and Tech. 2, 2009;327-330.
Table 1. Phytochemical screening of Centella asiatica leaf extracts. Test for compounds Hexane Dichloromethane Methanol Alkaloids - + + Coumarins - - - Flavones - - - Lignins - - + Phenols + + + Proteins + + + Saponins - - + Starch - - + Steroids + + + Sugar - - - Tanins - - - Triterpenoid + + + +, Present; -, Absent
Table 2. Antibacterial activity of different crude extracts of leaves of Centella asiatica Concentration of the extracts (Zone of inhibition 9 mm in diameter) Drugs Hexane (mg/ml), Dichloromethane (mg/ml), Methanol (mg/ml), Chl Str Bacteria 3.5 7.25 15 30 60 3.5 7.25 15 30 60 3.5 7.25 15 30 60 36 31 Bacillus subtilis - - 14 15 18 10 12 15 18 20 12 15 18 20 25 28 33 Bacillus cereus - 14 16 18 20 11 13 17 20 21 10 12 15 17 26 24 22 Escherichia coli - - 10 14 15-12 14 15 18 10 12 12 14 26 31 24 Klebsiella aerogenes - 14 17 19 20 14 17 20 22 23 12 15 19 20 28 32 26 Proteus vulgaris - 11 13 14 16 12 14 17 20 21 10 11 13 16 25 24 23 Proteus mirabilis 10 14 15 16 19 13 16 18 20 20 12 15 18 19 23 27 29 Pseudomonas aeruginosa - 13 15 16 20-13 14 18 19 10 14 16 18 22 30 32 Staphylococcus aureus 12 17 19 20 21 11 13 14 16 23 16 19 20 23 28 34 29 Salmonella typhii 10 15 17 19 22 10 15 17 18 22 15 17 20 22 27 26 30 Bacteria: Bacillus subtilis, Bacillus cereus, Escherichia coli, Klebsiella aerogenes, Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus and Salmonella typhi, (-) Activity, (-) Absent ;Reference drugs: Chloramphenicol (30 µg/disc), Streptomycin (30 µg/disc)