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KMITL Sci. J. Vol.8 No.

2 (Section A) July December, 2008

A STUDY ON ANTIBACTERIAL ACTIVITY OF CRUDE EXTRACTS OF ASIATIC PENNYWORT AND WATER PENNYWORT AGAINST STAPHYLOCOCCUS AUREUS
Duangkamol Taemchuay1,21, Theera Rukkwamsuk3, Thavajchai Sukpuaram4 and Nongluck Ruangwises5 1 Center for Agricultural Biotechnology, Kasetsart University, Kamphaengsaen Campus, Nakhon-Pathom 73140, Thailand 2 Center for Agricultural Biotechnology (AG-BIO/PERDO-CHE) 3 Department of Large Animal and Wildlife Clinical Science, Faculty of Veterinary Medicine, Kasetsart University, Kamphaengsaen Campus, Nakhon-Pathom 73140, Thailand 4 Department of Veterinary Public Health and Diagnostic Services, Faculty of Veterinary Medicine, Kasetsart University, Kamphaengsaen Campus, Nakhon-Pathom 73140, Thailand 5 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Mahidol University, Bangkok 10400, Thailand

ABSTRACT
Crude extracts of Asiatic Pennywort (Centella asiatica (Linn.) Urban) and Water Pennywort (Hydrocotyle umbellata Linn.) extracted with three kinds of solvent; hexane, ethanol and water, were tested for antibacterial activity against S. aureus ATCC 25923. From TLC results, asiatic acid was detected in crude extract of Asiatic Pennywort extracted with hexane and ethanol. Asiaticoside was detected in crude extract of Asiatic Pennywort extracted with ethanol and water. Neither asiatic acid nor asiaticoside was detected in crude extract of Water Pennywort. From disc diffusion results, water crude extract of Centella asiatica demonstrated that average inhibition zones ranged from 6.54-17.72 mm in diameter. Determination of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were performed using agar dilution method. The MIC and MBC values were 3 and 4 mg/ml in C. asiatica leave powder extracted with water and 2 and 3 mg/ml in C. asiatica leaves extracted with water. In conclusion, crude extract of Asiatic Pennywort, particularly extracted with water, had a promising antibacterial effect against Staphylococcus aureus. KEYWORDS : Mastitis, S. aureus, Centella asiatica, antibacterial activity

1. INTRODUCTION
In dairy farming, mastitis plays an important role in economic loss to dairy farmers. Udder infection is predominantly caused by bacteria, resulting in intramammary infection. The infection induces inflammation of the mammary gland resulting in decreased milk production and impaired milk quality. Staphylococcus aureus is one of the major pathogens, it can infect udders, and spread from cow to cow during the milking process. Treatment of S. aureus infection in bovine mastitis is intramammary infusion of antibiotic into inflamed quarters of the udder[3]. However, the success rate of treatment is low because the bacteria usually penetrate the mammary gland tissue, form abscesses and finally form scar tissues. This result may impair penetration of antibiotics to the infected tissue of the udder[1,5]. The conventional antibiotic treatment has been incriminated as a catalyst for resistance in bacteria isolated from treated animals, other animals within the herd, and food derived from cattle for human consumption[2] and as a cause of illegal antibiotic residue in market milk. Currently, the problem of antibiotic resistance in bacteria is increasing. Therefore, the use of medical herbs may be an alternative treatment for the disease remedy as to replace antibiotics, which is preferable for health of animals and humans. It is also an option to reduce cost of treatment, and to promote the use of available natural sources within the country. Asiatic Pennywort (Centella asiatica (Linn.) Urban) is one of the herbs of interest for treatment of bacterial infections. The herb consists of madecassic acid, asiatic acid and asiaticoside, which are astringent
1

Corresponding Author. E-mail : kkduangk@kmitl.ac.th

KMITL Sci. J. Vol.8 No.2 (Section A) July December, 2008 and stimulant for tissue repairment. It also inhibits growth of Staphylococcus spp. and reduces inflammation[4]. Water Pennywort (Hydrocotyle umbellata Linn.) is characteristically similar to Asiatic Pennywort, which is believed to have the same active constituents and probably good antibacterial activities. This study was aimed at evaluating antibacterial activity of crude extracts of Asistic Pennywort and Water Pennywort against Staphylococcus aureus.

2. MATERIALS AND METHODS


2.1 Preparation of crude extracts
Asiatic Pennywort and Water Pennywort (from known sources of free-herbicides) were collected from Nakhon-Pathom and Ratchaburi Province in April, 2008. For preparation of crude extracts, fresh leaves of plants were dried at 50C in the hot air oven for 24 hours and finely ground into a powder by cutting mill machine. The leave powder was extracted with two kinds of solvent (n-hexane and 95% ethanol) and macerated at room temperature for 72 hours. The extract was hand-squeezed through a thin cloth and filter pass Whatman filter paper No.1, respectively. The extract was concentrated and n-hexane or 95% ethanol was removed under vacuum by using the rotary evaporators (BUCHI Rotavapor R-205, Switzerland) to obtain the crude extract. The extract was kept at -20C until use. Preparation of water crude extract, the fresh leaves of plants was boiled in distilled water under reflux for one hour. The solution was then filtered and freeze-dried (FTS system) to obtain the powder which was re-dissolved in sterile distilled water immediately before use.

2.2 Analytical TLC


The triterpenoid compound of plants was identified by Thin Layer Chromatography (TLC) on silica gel plates. A combination of chloroform, methanol and water (15:7:1) as the mobile phase was found to be appropriate in separating these compounds from the extract. The plates were visualized by spraying with anisaldehyde solution, followed by heating at 110C for 5 minutes.

2.3 S. aureus culture


S. aureus ATCC 25923 was cultured and used as a reference strain. The reference strain was touched with a needle loop and the growth was transferred to blood agar and incubated at 37 C 18 to 24 hours. The strain was confirmed using coagulase solution and mannitol salt agar (MSA, Merck).

2.4 Antibacterial activity test


The Antimicrobial susceptibilities were tested by the disc diffusion test, the agar dilution method for determining the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) according to the guidelines recommended by the CLSI[6]. Disc diffusion test was performed using sterile 6 mm-diameter filter paper discs (Whatman, Maidstone, UK). The discs were prepared using 10 L of crude extract diluted in the solvent to a concentration of 1,000 mg/mL, 800 mg/mL, 400 mg/mL, 200 mg/mL, 100 mg/mL and 50 mg/mL respectively; thus each disc contained 10 mg, 8 mg, 4 mg, 2 mg, 1 mg and 0.5 mg respectively. All discs were dried at room temperature overnight. At least three isolated colonies of the same morphological type were selected from blood agar culture. The top of each colony was touched with a wire loop and the growth transferred to a tube containing 5 mL of Mueller-Hinton broth (MHB, Merck). The broth culture was incubated at 37 C (usually 3 to 5 hours) until the turbidity of the 0.5 McFarland standard, this resulted in a suspension containing approximately 1 to 2 108 CFU/mL. The 0.5 McFarland suspension should be 150 L in 5 mL of Mueller-Hinton agar (MHA, Merck) and mixed to pour plate, the final inoculum on the agar would be approximately 106 CFU/mL. The discs were placed on the surface of the inoculum MHA and incubated at 37 C 18 to 24 hours. The disc diffusion test was determined by measuring the diameter of the inhibition zone. Experiments were performed in triplicate and the mean of the diameters of the inhibition zones calculated. The agar dilution method for determining the minimum inhibitory concentration (MIC) used 1 L of bacterial strain in the 0.5 McFarland suspension (containing 104 CFU/mL.) that was spotted to MHA supplemented with the crude extracts at a concentration of 10, 9, 8, 7, 6, 5, 4, 3 and 2 mg/mL. The plates were incubated at 37 C 18 to 24 hours, the results were expressed as the lowest concentration of crude extract that inhibited colony growth. The minimum bactericidal concentration (MBC) was determined by removing the agar from MIC plate and placing it on a Mannitol Salt Agar (MSA, Merck) and incubated at 37 C 18 to 24 hours. Unchange of media colour was defined as the lowest concentration of crude extract with no bacterial growth. The yellow colour of media showed S. aureus growth.

KMITL Sci. J. Vol.8 No.2 (Section A) July December, 2008

3. RESULTS AND DISCUSSION


The result of chromatogram is presented in Figure 1, asiatic acid was detected in crude extract of Asiatic Pennywort extracted with hexane and ethanol. Asiaticoside was detected in crude extract of Asiatic Pennywort extracted with ethanol and water. Neither asiatic acid nor asiaticoside was detected in crude extract of Water Pennywort.

Asiatic acid

Asiaticoside

10 11

12

Figure 1. Chromatogram of the isolated compounds and the crude extract from C. asiatica and H. umbellata.
1,7 = standard asiaticoside 2,8 = standard Asiatic acid 3 = C. asiatica leave powder extracted with n-hexane (CaPH) 4 = C. asiatica leave powder extracted with 95% ethanol (CaPE) 5 = C. asiatica leave powder extracted with water (CaPW) 6 = C. asiatica leaves extracted with water (CaW) 9 = H. umbellata leave powder extracted with n-hexane (HuPH) 10 = H. umbellata leave powder extracted with 95% ethanol (HuPE) 11 = H. umbellata leave powder extracted with water (HuPW) 12 = H. umbellata leaves extracted with water (HuW) Antibacterial activity of inhibition zone of crude extracts against S. aureus ATCC 25923 is presented in Table 1, the inhibition zones ranged from 6.54-17.72 mm. The 95% ethanol extract of C. asiatica showed inhibition zone of 6.44 mm in diameter at 4 mg and 6.49 mm at 10 mg of crude extract. The water extract of leave powder of C. asiatica showed inhibition zone of 6.54 mm at 4 mg, 10.22 mm at 8 mg and 10.24 mm at 10 mg of crude extract. The water extract of C. asiatica had inhibition zone of 6.98 mm at 0.5 mg, 9.99 mm at 1 mg, 12.59 mm at 2 mg, 15.06 mm at 4 mg, 17.72 mm at 8 mg and 17.16 mm at 10 mg of crude extract. In C. asiatica leave powder extracted with water, inhibition zone at 4 mg was lower than at 8 and 10 mg of crude extract. In C. asiatica leaves extracted with water, inhibition zone at 0.5 mg was lower than at 1, 2, 4, 8 and 10 mg of crude extract. In C. asiatica leave powder extracted with 95% ethanol, inhibition zone at 8 mg did not differ from 10 mg of crude extract. C. asiatica extracted with nhexane and H. umbellata extracted with three solvents did not inhibit S. aureus ATTC 25923. The MIC and MBC values were 3 and 4 mg/ml in C. asiatica leave powder extracted with water and 2 and 3 mg/ml in C. asiatica leaves extracted with water.

Table 1.

Antibacterial activity [(measured as mean ( s.d.) of inhibition zone (mm)] of crude extracts against S. aureus ATCC 25923.

KMITL Sci. J. Vol.8 No.2 (Section A) July December, 2008

Plants Extract Contro l CaPH CaPE CaPW CaW HuPH HuPE HuPW

Concentration of crude extracts (mg/disc) 0.5 6.00 0.00a 6.00 0.00a 6.00 0.00a 6.00 0.00a 6.98 2.05*a 6.00 0.00a 6.00 0.00a 6.00 0.00a 1 6.00 0.00a 6.00 0.00a 6.00 0.00a 6.00 0.00a 9.99 1.39*b 6.00 0.00a 6.00 0.00a 6.00 0.00a 2 6.00 0.00a 6.00 0.00a 6.00 0.00a 6.00 0.00a 12.59 2.93*c 6.00 0.00a 6.00 0.00a 6.00 0.00a 4 6.00 0.00a 6.00 0.00a 6.00 0.00a 6.54 1.10*a 15.06 1.00*c 6.00 0.00a 6.00 0.00a 6.00 0.00a 8 6.00 0.00a 6.00 0.00a 6.44 0.73*a 10.22 3.95*b 17.72 1.22*d 6.00 0.00a 6.00 0.00a 6.00 0.00a 10 6.00 0.00a 6.00 0.00a 6.49 0.73*a 10.24 1.11*b 17.16 1.47*d 6.00 0.00a 6.00 0.00a 6.00 0.00a

HuW 6.00 0.00a 6.00 0.00a 6.00 0.00a 6.00 0.00a 6.00 0.00a 6.00 0.00a * shows significant differences between each extract and control. a, b, c, d different superscripts within the same row show significant differences between concentrations in each plant extract.

4. CONCLUSIONS
In this study, C. asiatica extracted with water significantly inhibited S. aureus ATCC 25923. The active compound in crude extracts consisted of asiaticoside in triterpines group, which had an antibacterial effect against S. aureus ATCC 25923 Reference strain as indicated by inhibition zones between 6.54 17.72 mm in diameter and the MIC and MBC values of 2 and 3 mg/ml. However, this study was a preliminary report of antibacterial activity of crude extracts from C. asiatica and H. umbellata. The crude extracts tested in an in vitro study of S. aureus field strains needed further research.

5. ACKNOWLEDGEMENTS
This research is funded by the Kasetsart University Research and Development Institute and the Center of Excellence for Agricultural Biotechnology, Postgraduate Education and Research Development Office, Commission on Higher Education, Ministry of Education.

REFERENCES
[1] Belschner, A.P., J.W. Hallberg, S.C. Nickerson, and W.E. Owens. 1996. Staphylococcus aureus mastitis therapy revisited in Proceedings. 35th Annual. Meeting., National Mastitis Council., Madison, WI, pp. 116-122. [2] National Mastitis Council Research Committee Report. 2004. Bovine Mastitis Pathogens and Trends in Resistance to Antibacterial Drugs. NMC Annual Meeting proceedings. pp 400-414. [3] Barkema, H.W., Y.H. Schukken and R.N. Zadoks. 2006. Invited Review: The Role of Cow, Pathogen and Treatment Regimen in the Therapeutic Success of Bovine Staphylococcus aureus Mastitis. J Dairy Sci. 89: 1877-1895. [4] Department of Pharmaceutical Botany, Faculty of Pharmacy, Mahidol University. 1996. Medicinal Plants in Thailand: Volume I. Amarin Printing and Publishing Public Co. Ltd., Bangkok. Page 65. [5] Erskine, R.J., S.A. Wagner and F.J. DeGraves. 2003. Mastitis therapy and pharmacology. Vet. Clin. North Am. Food Anim. Pract. 19: 109-138. [6] CLSI. 2007. Performance Standards for Antimicrobial Susceptibility Testing; Seventeenth Informational Supplement. CLSI document M100-S17. Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania, USA.

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