European Journal of Scientific Research ISSN 1450-216X Vol.30 No.2 (2009), pp.260-264 EuroJournals Publishing, Inc. 2009 http://www.eurojournals.com/ejsr.

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Antimicrobial, Cytotoxic and Antioxidant Activity of Centella asiatica

M.Obayed Ullah Corresponding Author Senior Lecture, Department of Pharmacy Southeast Univerisity, Banani, Dhaka 1213, Bangladesh E-mail: oidf_um@yahoo.co.in Tel: +8801918693161 Shapna Sultana Lecture Department of Pharmacy, Southeast Univerisity Banani, Dhaka 1213, Bangladesh E-mail: pharmashapna@yahoo.com Tel: +8801732791185 Afroza Haque Lecture Department of Pharmacy, Southeast Univerisity Banani, Dhaka 1213, Bangladesh E-mail: afroza98@gmail.com Tel: +8801715061776 Saira Tasmin Department of Human Ecology, School of Health University of Tokyo, Tokyo, Japan E-mail: rimzim1612@yahoo.com Abstract The n-hexane, carbon tetrachloride, chloroform soluble fractions of methanol extract from the plant Centella asiatica (Apiaceae) was subjected to antioxidant, antimicrobial and brine shrimp lethality bioassay. All the fractions showed moderate to potent antioxidant activity, of which the chloroform and aqueous soluble fraction demonstrated the strongest antioxidant activity with the IC50 value of 4.0 g/ml and 7.0 g/ml, respectively. In case of antimicrobial screening, crude extracts showed notable antibacterial and antifungal activity against sixteen microorganisms. The n-hexane, carbontetrachloride, chloroform and aqueous soluble partitionates of the methanoic soluble fractions showed average zone of inhibition ranged from 7-15 mm, 8-12 mm, 8-16 mm and 8-13 mm, respectively, at a concentration of 400 g/disc. However, in the brine shrimp lethality bioassay, all the crude extracts possessed considerable cytotoxic activity. It was evident that, the n-hexane, carbon tetrachloride, chloroform and aqueous soluble fractions have significant cytotoxic potentials having LC50 1.254, 0.826, 3.866 and 5.366g/ml respectively.

Antimicrobial, Cytotoxic and Antioxidant Activity of Centella Asiatica Keywords: Apiaceae, disc diffusion, brine shrimp, free radical scavenging

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1. Introduction
Centella asiatica L. Urban (syn. Hydrocotyle asiatica L.) belongs to the family of Apiaceae (Umbelliferae). In India, the plant is commonly known as Mandukaparni. In Srilanka and Indonesia it is given the name Thankuni Sak. In classical Indian Ayurveda literature, it is considered to be one of the Rasayana (rejuvenator) drugs. (Jayashree et al, 2003) In common with most traditional phytotherapeutic agents, C. asiatica is claimed to possess a wide range of pharmacological effects, being used for human wounds healing, mental disorders, atherosclerosis, fungicidal, antibacterial, antioxidant and anticancer purposes. C. asiatica has also been reported to be useful in the treatment of inflammations, diarrhea, asthma, tuberculosis and various skin lesions and ailments like leprosy, lupus, psoriasis and keloid. In addition, numerous clinical reports verify the ulcer-preventive and antidepressive sedative effects of C. asiatica preparations, as well as their ability to improve venous insufficiency and microangiopathy (Zheng and Qin, 2007). Previously triterpenoid acids, volatile and fatty oils, alkaloids, glycosides, flavonoids, and steroids have been isolated from the different parts of the plant. (Jayashree et al, 2003). We herein, report the preliminary antioxidant, cytotoxic and antimicrobial activities of the extractives of C. asiatica.

2. Material and methods

2.1. Plant Material The plant, C. asiatica were collected from Dhaka in the month of September 2007. A voucher specimen (DACB 33537) for this collection has been deposited in the Bangladesh National Herbarium, Mirpur, Dhaka, for future reference. 2.2. Extraction The dried powdered whole plants part of C. asiatica (500gm) was soaked in 2.5 L methanol for 15 days and filtered through a cotton plug followed by Whatman filter paper number 1. The extract was then concentrated by using a rotary evaporator. A portion (12 g) of the concentrated methanol extract was fractionated by the modified Kupchan partitioning method (Van Wagenen et al, 1993) into nhexane, carbon tetrachloride, chloroform and aqueous soluble fractions. The methanol extract (12 g) was dissolved in 100 ml of 10% aqueous methanol and extracted three times with n-hexane (100 ml X 3). The remaining aqueous phase was then increased in polarity to 20% H2O and extracted three times with carbon tetrachloride (100 ml X 3). The remaining aqueous phase was increased further in polarity to 40% H2O and extracted three times with chloroform (100 ml X 3). The subsequent evaporation of solvents afforded n-hexane (HX, 900 mg), carbon tetrachloride (CT, 450 mg), chloroform (CF, 600 mg) and aqueous soluble (AQ, 0.8 mg) crude materials. 2.3. Antimicrobial Screening The antimicrobial activity of the crude extracts was determined by the disc diffusion method, (Bauer et al, 1966, Rahman et al, 2008) against the microbial strains listed in Table 1. These were collected as pure cultures from the Institute of Nutrition and Food Science (INFS), University of Dhaka, Bangladesh. The extracts were dissolved separately in chloroform and applied to sterile discs at a concentration of 400g/disc and carefully dried to evaporate the residual solvent. Here, Kanamycin (30 g/disc) was used as the standard These plates were then kept at low temperature (4C) for 24 hours to allow maximum diffusion of the test materials and kanamycin. The plates were then incubated at 37C for 24 hours to allow maximum growth of the organisms. The test material having antimicrobial activity inhibited the growth of the microorganisms and a clear, distinct zone of inhibition was visualized surrounding the discs. The

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M.Obayed Ullah, Shapna Sultana, Afroza Haque and Saira Tasmin

antimicrobial activity of the test agents was determined by measuring the diameter of zone of inhibition expressed in mm. 2.4. Brine shrimp lethality bioassay Brine shrimp lethality bioassay (Meyer et al, 1982; Rahman et al, 2008; Hossain et al, 2004) technique was applied for the determination of general toxic property of the plant extractives. DMSO solutions of the samples were applied against Artemia salina in a 1-day in vivo assay. For the experiment, 4 mg of each of the petroleum ether, carbon tetrachloride and chloroform soluble fractions were dissolved in DMSO and solutions of varying concentrations (400, 200, 100, 50, 25, 12.50, 6.25, 3.125, 1.563, 0.781 g/ml) were obtained by serial dilution technique using DMSO for each extract. Vincristine sulphate was used as positive control. 2.5. Antioxidant Activity The antioxidant activity (free radical scavenging activity) of the extracts on the stable radical 1, 1diphenyl-2-picrylhydrazyl (DPPH) was determined by the method of Brand-Williams. et al (1995). In the experiment, 2.0 mg of each of the extracts was dissolved in methanol. Solution of varying concentrations such as 500 g/ml, 250 g/ml, 125 g/ml, 62.50 g/ml, 31.25 g/ml, 15.62 g/ml, 7.8125 g/ml, 3.91 g/ml, 1.95 g/ml and 0.98 g/ml were obtained by serial dilution technique. 2 ml of a methanol solution of the extract of each concentration was mixed with 3 ml of a DPPH-methanol solution (20 g/ml) and was allowed to stand for 20 minutes for the reaction to occur. Then the absorbance was determined at 517 nm and from these values the corresponding percentage of inhibitions were calculated by using the following equation: % inhibition = [1- (ABSsample / ABScontrol)] x 100. Then % inhibitions were plotted against respective concentrations used and from the graph IC50 was calculated by using tert-butyl-1hydroxytoluene (BHT), ascorbic acid potential antioxidant, were used as positive control.

3. Results and Discussion

The aqueous (AQ) and carbon tetrachloride (CT) extracts showed moderate antibacterial activity with the average zone of inhibition of 8-13 mm and 8-12mm, respectively, at 400g/disc. The chloroform (CF) extract showed the highest activity against the growth of S. aureus and V. parahemolyticus having the zone of inhibition of 16 mm. Besides this, the extract showed good activity against the growth of S. typhi (15 mm), E. coli (15 mm), P. aeruginosa (14 mm) and S.boydii (14 mm).In case of fungi, the average zone of inhibition was found to be 12-15mm. The n-hexane (HX) extract strongly inhibited the growth of B. megaterium and V. mimicus having zone size 15 mm for each. It showed moderate activity against S. aureus (12 mm) and V. parahemolyticus (12 mm). Among the tested fungi, the growth of A. niger (14 mm) was strongly inhibited. Following the procedure of Meyer, the lethality of the n-hexane (HX), carbon tetrachloride (CT), chloroform (CF) and aqueous soluble (AQ) crude materials extracts to brine shrimp were evaluated on A. salina after 24 hours of exposure the samples and the positive control, vincristine sulphate (VS). The LC50 were found to be were 0.3229, 1.254, 0.826, 3.866 and 5.366g/ml for VS, HX, CT, CF and AQ soluble partitionates respectively. The cytotoxicity exhibited by the crude extracts was promising and this clearly indicates the presence of potent bioactive compounds. (Meyer, et al 1982, Rahman, et al 2008, Hossain et al., 2004). In case of antioxidant screening (Table-3), the IC50 value of BHT and ascorbic acid was obtained 26.0 g/ml and 5.0 g/ml respectively. In this investigation, the chloroform extract (CF) of the plant showed the highest antioxidant activity with IC50 value of 4.00 g/ml. Aqueous soluble fraction (AQ) of the methanol extract also revealed potent antioxidant activity (IC50=7 g/ml). On the other hand, the carbon tetrachloride (CT), n-hexane soluble fraction (HX) showed moderate

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antioxidant activity with the IC50 value of 40 and 298 g/ml respectively. These results denote the presence of antioxidant principles in the extractives.

4. Conclusion
The n-hexane(HX), carbon tetrachloride (CT), chloroform(CF) and aqueous (AQ) soluble fractions of methanol extract of C. asiatica showed significant antioxidant, antimicrobial and cytotoxic activities, which supports the traditional use of this plant in various diseases. The plant can be further screened against various diseases in order to find out its unexplored efficacy and can be a potential source of biologically important drug candidates.
Table 1: Antimicrobial activity of extractives of Centella asiatica and Kanamycin
Test microorganisms HX Diameter of zone of inhibition (mm) CT CF AQ

Kan Gram positive bact. Bacillus cereus 8 8 11 8 25 Bacillus megaterium 15 8 12 13 30 Bacillus subtilis 10 7 12 10 23 Staphylococcus aureus 12 16 8 26 Sarcina lutea 11 12 11 24 Gram negative bact. Escherichia coli 8 15 12 22 Pseudomonas aeruginosa 10 11 14 9 20 Salmonella paratyphi 07 10 13 12 25 Salmonella typhi 11 12 15 10 25 Shigella boydii 10 11 14 9 25 Shigella dysenteriae 08 10 25 Vibrio mimicus 15 11 8 8 28 Vibrio parahemolyticus 12 16 11 25 Fungi Candida albicans 11 12 15 9 25 Aspergillus niger 14 11 13 11 25 Sacharomyces cerevaceae 08 11 12 8 20 - Indicates no activity. Here HX: n-Hexane soluble fraction of the methanolic extract; CT: carbon tetrachloride soluble fraction of the methanolic extract; CF: chloroform soluble fraction of the methanolic extract; AQ: aqueous fraction of the methanolic extract.

Table 2:

LC50 data of test samples of Centella asiatica and Vincristine sulphate

Samples LC50 (g/ml) VS 0.3229 HX 1.254 CT 0.826 CF 3.866 AQ 5.366 Here VS: vincristine sulphate (Std.); HX: n-Hexane soluble fraction of the methanolic extract; CT: carbon tetrachloride soluble fraction of the methanolic extract; CF: chloroform soluble fraction of the methanolic extract; AQ: aqueous fraction of the methanolic extract.

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Table 3:

M.Obayed Ullah, Shapna Sultana, Afroza Haque and Saira Tasmin

IC50 values of Centella Asiatica extractives and tert-butyl-1-hydroxytoluene and ascorbic acid

Samples IC50 (g/ml) BHT 26.0 AS 5.0 HX 298.0 CT 40.0 CF 4.0 AQ 7.0 Here BHT: tertbutyl-1-hydroxytoluene (Std.), AS: ascorbic acid (Std); HX: n-Hexane soluble fraction of the methanolic extract; CT: carbon tetrachloride soluble fraction of the methanolic extract; CF: chloroform soluble fraction of the methanolic extract; AQ: aqueous fraction of the methanolic extract.

References
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