ANTI-MICROBIAL ACTIVITIES OF BETEL NUT (ARECA CATECHU LINN) SEED EXTRACTS Ajima Karphrom 1, Suthisak Suknaisilp 2, Pasawadee Pradeepasaena 2, Sumate Tantratian 2 * ABSTRACT Seeds of betel nuts grown in Nakornchaisri, Nakornbathom province and Banpaew, Samuthprakarn province which had round shaped, immature and mature were extracted with 4 different solvents. They were water, 95% alcohol, acetone and ethylacetate. These crude extracts were tested for ability to inhibit the growth (MIC) of the pathogenic bacteria; Bacillus cereus ATCC 1729, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 and Salmonella Typhimurium ATCC 13811. It was found that the components in the betel nut seeds were best dissolved in water, as the water extraction also yielded the highest amount. It was found that different location affected the % yield and ability to inhibit the test microorganisms. The betel nut seeds from Nakornchaisri provided higher extract yield and better ability to inhibit the test organisms. These extracts were able to inhibit both gram positive and negative bacteria but prefer to inhibit gram positive, while the water and acetone extracts showed the higher inhibition activities. The minimum inhibitory concentration (MIC) of betel nut extracts on all four test organisms were about 0.78 mg/ml, when tested with paper disc diffusion technique. Keywords; betel nut extracts, anti-microbial activities 1 Biotechnology Program, Faculty of Science, Chulalongkorn, BKK 10330 2 Food Technology, Faculty of Science, Chulalongkron, BKK 10330 *Correspondence should be addressed; e-mail: Sumate.T@chula.ac.th - 209 -
INTRODUCTION Betel nut is a seed of Areca palm (Areca catechu), which grows in the tropical Pacific, Asia and parts of east Africa. This nut is known in many names, in Thailand is known as Mahk. The betel nut was grown and used as chewing session along with some calcium hydroxide, betel leaves and tobacco for a very long time. Chewing of betel nut continues to be widespread in South Asian population (Gupta and Ray, 2004). In Thailand, chewing of Mahk was popular, but declined gradually in the last decades. Betel palm is still grown and its nut is still commercial valuable. The nut was commercially available in dry, cured and fresh forms. Betel palm is grown mostly in the southern and central part of Thailand. It bares fruits through out the year. The husk is green when young and turn yellow when ripen. The nuts in the fruits turn to orange when ripen. Betel nuts contained hydrolysable tannin and condense tannin, some alkaloids and fats. Both tannins are water dissolved and red in color. The alkaloids content was 0.3-0.6% mainly composed of arecoline, arecaine and guvacine. The fats content in betel nuts was reported about 14%. The betel nuts were used in leather tanning as ability to denature protein of tannins. In pharmaceuticals, betel nuts were reported as possible beneficial effect on schizophrenia and for stroke recovery. The components in betel nut, such as arecoline and arecaine, have been reported to affect on central nervous system and heart stimulator. The phenolic compounds in betel nuts was reported to have affect on anti-virus and anti-microorganisms (Buhler and Miranda, 2000; Rodriguez, et al., 2005). The ethanol extract of betel nuts was reported for the ability to inhibit growth of Helicobacter pylori (Wang and Huang, 2005) and Staphylococcus aureus, Salmonella spp., Neisseria sp., Yersinia enterocolitica and Listeria monocytogenes (Yang and Chou, 1997). As the components in betel nuts showed benefit in anti-microbial activities, this study was aimed to determine the anti-microbial activities on some food borne pathogenic bacteria as the potential for an application in food industries. MATERIALS AND METHODS Betel nut extraction Betel fruits grown in Nakornchaisri, Nakorn Pathom Province and Banpaew, Samuthprakarn Province were collected from local markets during October and December, 2006. The fruits were grouped by the maturity, immature and ripe, and appearance, round and oval shape. The fruits were de-husking. The nuts were sliced in small pieces and dried at 60oC for 3 days. The dried nuts were blended in a blender to fine powder. The powders were extracted with distilled water, 95% ethanol, acetone and ethyl acetate. Water, ethanol, acetone or ethyl acetate extraction was accomplished by mixing 75g of betel powder to 150g of desired solvent. The water based suspension was shaken in a bath shaker at 80oC, - 210 -
100 rpm for 2 hr. Others were shaken at 200 rpm for 48 hr on a rotary shaker. Then they were filtered through a Whatman No. 1 and No. 42, respectively. The clear supernatant was collected to dry in an oven at 60-80oC for 5 days. The solid matters were grinded to powder and kept in a desiccator until the experiment. The ethyl acetate extracts were not able to dry to solid form. They demonstrated as liquid at 28oC, which we kept them in a container and refrigerated. The yield of extracts were calculated as equation (1) Yield (%) = (dry weight of betel nut extract/dry weight of betel nut powder)*100(1) Anti-bacterial activities The cultures of Bacillus cereus ATCC 1729, Staphylococcus aureus ATCC 25923,Escherichia coli ATCC 25922 and Salmonella Typhimurium ATCC 13811 were grown in MHB (Muller Hinton Broth, Himedia, Mumbai, India) for 24 hr. The cultures were adjusted the absorbance (A625) to 0.1 with an UV-Visible Spectrophotometer to obtain cell concentration at 108 CFU/ml. The betel nut extracts 200 mg, were mixed with 1% DMSO (Dimethyl Sulfoxide) 1 ml and vortexed to dissolve. The two fold serial dilutions of the mixtures were prepared until the final concentration was 0.01 mg/ml, in sterile test tubes. In case of liquid form extract, 1.5 ml of the extract was applied, and the final concentration was 15 l/ml. The Minimum Inhibitory Concentration (MIC) with paper disc diffusion method which modified from the method described by NCCLS (1999) was applied. The 5 l of prepared extract solution was transferred to 0.6 cm diameter paper disc. Then paper disc was placed on MHA (Muller Hinton Agar) that was spread with a test culture and incubated at 37oC for 18+2 hr. The paper disc contained 10 g ampicillin, 30 g chloramphinicol and Vancomycin 30 g were used as positive controls. The lowest concentration of the extract that showed inhibition zone on MHA was reported as MIC. RESULTS AND DISCUSSION The betel fruits collected from 2 different plantation locations could be divided in two shapes as round and oval as shown in Figure 1. With some difficulties in collection of the betel fruits, we were not able to collect enough betel fruits from Banpaew for all experiments. Figure 1. The betel fruits with round shape (a) and oval shape (b) - 211 -
The extract powders from betel nuts were yellowish in ethyl acetate extraction and light brown color for the extract from immature fruits to dark brown color from mature fruits. The extracts yield of the betel nuts from Nakornchaisri and Banpaew were shown in Figure 2. The highest yield was found in water and ethanol extractions. We found that extract yields of betel nut from Banpaew were less than those from Nakornchaisri. We decided to show only data of the betel nuts from Nakornchaisri for the further experiment. There were significant (p>0.05) different in the extract yield in water extraction, while no significantly (p<0.05) different in yield when extracted with other solvents. The mature and round shape fruits provided the highest extract yield of 36.81+6.47%, which was almost 2 times in amount of other water extractions. (a) (b) Figure 2. The yield of betel nuts from Nakornchaisri (a) and Banpaew (b), extracted with different solvents The anti-bacterial activities of the betel nut extracts were shown in Table 1. The betel nut extracts were able to inhibit both gram positive and negative with different MIC. The different in maturity, plantation location and variety of betel nut provided different antimicrobial activities (Mathew and Gaovndarajan, 1964). The acetone extract of the mature oval shaped fruits could inhibit all four bacteria at the concentration of 0.78 mg/ml. The ethyl acetate extractions inhibit all test bacteria with the higher concentration than other extracts, while water and alcohol extracts could inhibit the test bacteria at about the same concentration. The water extracts inhibited the gram positive bacteria, B. cereus and S. aureus, better than gram negative bacteria, E. coli and S. typhimurium. The MICs of water extract of betel nut in our experiment were much lower than Panutat and Vatanyoopaisarn (2009). They reported the inhibitory concentration of betel nut extracts at 125 and 62.5 mg/ml for B. cereus and S. aureus, repectively, when inhibitory zones had to be greater than 10 mm. - 212 -
The best anti-bacterial activities of the betel nuts from Banpraew also got from water extraction of the round shaped and mature fruits, with MIC at 0.78 mg/ml for all four test organisms. Other extracts did not have anti-bacterial activities better than extracts of Nakornchaisri betel nuts (data were not shown). Table 1. The minimal inhibitory concentration (MIC) of betel nut extracts on some food borne pathogenic bacteria The mature and oval shaped betel fruits that extracted with water and acetone had the highest potential to be used as anti-microbial agents that could be used in food industries. CONCLUSION The extraction of betel nuts from round and mature fruits with water provided the highest yield at 38%. All extracts from betel nuts inhibited both gram negative and positive bacteria. The best inhibition activities were found in the acetone extracts of oval shaped and mature fruits was at 0.78 mg/ml. REFERENCES: Buhler, D.R. and Miranda,C. 2000. Antioxidant activities of flavonoids. [online]available from: http://lpi.oregonstate.edu/f-woo/flavonoid.html [21/01/2009. Gupta, P.C. and Ray, C.S. 2004. Epidemiology of betel quid usage. Ann. Acad.Med. Singap. 33(4)31-36. Mathew, A.G. and Gaovndarajan, V.S. 1964. Polyphenolic substances of areca nut I. Changes during maturation and ripending. J. Phytochem. 3:657-665. NCCLS. Methods for Determining Bactericidal Activity of Antimicrobial Agents;Approved Guideline. 1999. Methods of Determining Bactericidal Activity ofantimicrobial, Vol 19(18) Pennsylvania, USA. - 213 -
Panutat, P. and Vatanyoopaisarn, S. 2009. Inhibitory effects of Thai herbs and spices on some food borne bacteria. King Mongkut Institute of Technology North Bangkok, Faculty of Applied Science, Department of Agro-Industrial Technology Rodriguez Vaquero, M.J., Alberto, M.R. and Manca de Nadra, M.C. 2005.Antibacterial effect of phonolic compounds from different wines. J. Food Control 18:93-101. Wang, Y.C. and Huang, T.L. 2005. Screening of anti-helicobacter pylori herbsderiving from Taiwanese folk medicinal plants. J. FEMS Immun. Med.Microbilogy 43:295-300. Yang, J.C. and Chou, C.C. 1997. Antimicrobial activity of various solventextracts of betel quid ingredients (In Chinese). [online] Available from;www.drugs.corn/npp/betl-nut.html [May 2009]. - 214 -