Patturajan Rajeshbabu et al.

/ Journal of Pharmacy Research 2011,4(7),2223-2225

Research Article ISSN: 0974-6943

Available online through www.jpronline.info

In vitro antibacterial activity of Piper betel L. and Black betel CV. Kammar leaves against Staphylococcus aureus and Streptococcus pneumoniae
Patturajan Rajeshbabu 1, Muniappan Ayyanar1, Shaik Khaja Rasool 2, Mehboob Azrar Sheriff2 and Thangavel Sekar 1*
1

Post Graduate and Research Department of Botany, Pachaiyappas College, Chennai - 600030, Tamil Nadu, India ; 2Department of Biotechnology, The New College, Royapettah, Chennai - 600014, Tamil Nadu, India

Received on: 12-04-2011; Revised on: 18-05-2011; Accepted on:21-06-2011 ABSTRACT

A comparative study on the antibacterial activity and nutritive value of the different extracts of the leaves of Piper betel and Black betel CV. Kammar was carried out using disc diffusion and agar well diffusion method against two human pathogenic bacteria namely Staphylococcus aureus and Streptococcus pneumoniae. Leaf extracts of P. betel showed significant activity by disc diffusion method and black betel leaf extracts showed significant activity by well diffusion method against the tested organisms. Analytical study on the biochemical parameters indicated that total fat, protein and crude fibre along with calcium are higher in black betel leaves while carbohydrates, ash, alkalinity, sodium and potassium are higher in P. betel leaves. Thus it could be concluded that the traditional wisdom of chewing betel leaves would certainly useful in bacterial infections of mouth, throat and lung. Key words: Antimicrobial activity, Medicinal uses, Piper betel, Black betel.

INTRODUCTION
Piper betel L. (Betel, Piperaceae) is a semi-woody dioecious creeper, the native of Malaysia and cultivated very extensively in the warm and moist parts of south India and Sri Lanka for its leaves which is used for chewing. Betel is one of the important plants in the Asiatic region which ranks second to coffee and tea in terms of daily consumption [1]. The cultivated betel in India is usually the male plant selected from certain races and consequently does not fruit [2]. Several types of betel are grown in different parts of India showing variation in size, shape and colour of the leaves, their taste and aroma. Betel leaf is aromatic, stimulant, carminative, astringent, aphrodisiac and antiseptic, essential oil extracted from the leaf is also reported to have aromatic and astringent properties [3]. Betel has been extensively used in traditional herbal remedies in India, China, Taiwan, Thailand and many other countries [4]. Betel leaves has been reported for various pharmacological activities such as antimicrobial, antioxidant, antimutagenic, anticarcinogenic, antiinflammatory, antioxidant, radioprotective, antiallergic, antiamoebic, antifertility and antiplatelet activities [5]. The practice of chewing the betel leaves for its stimulating qualities is indulged in between a quarter and a tenth of the worlds population, which makes it one of the most popular of all psychoactive substances[6]. The betel leaves also acts as a stimulant, breath freshener, carminative, sialagogue, cardiac tonic, pain killer in joint pain, aphrodisiac, astringent, antiseptic, digestive and pancreatic lipase stimulant and wound healing agent. In India, betel leaves are used as a masticatory (the taste being warm, aromatic and bitter), together with scraped arecanut [6]. In Chinese folk medicine betel leaves are used for the treatment of various disorders and claimed to have detoxication, antioxidation and antimutation properties [1]. Previously, various solvent extracts of leaves of P. betel showed antimicrobial activity against Diplococcus pneumoniae, Klebsiella aerogenes, Staphylococcus aureus, S. mutans, S. faecalis, Candida albicans, Vibrio cholerae ogawa and etc[6-12]. However report on comparative study on the betel leaves and its variety on the antimicrobial activity is lacking. This study aims to find out the antibacterial activity of P. betle leaves and its variety Kammar against two human pathogenic bacteria Staphylococcus aureus and Streptococcus pneumonia by agar disc and well diffusion method. MATERIALS AND METHODS Plant collection Fresh leaves of Piper betel and Black betel (Piper betel. Cv Kammar) were collected from the local market at Poonamalle in Thiruvallur district of Tamil Nadu, India. Preparation of extract Fresh leaves of Piper betel and Black betel variety were washed in running tap water to remove impurities. The leaves shade dried and pulverized leaf materials were soaked in different solvents (ethanol, chloroform and ethyl acetate) for sequential extraction. They were filtered using standard Whatman No.1 filter paper on a Buchner funnel and the solvents were removed by rotary vacuum evaporator to remove excess of solvents and the extracts were subjected to qualitative test using standard procedures to identify various plant constituents. The dry extracts were stored at 4C until further use. Test organisms The pathogenic microorganisms, Streptococcus pneumoniae and Staphylococcus aureus used in the study were obtained from the Department of Microbiology, Christian Medical College, Vellore, Tamil Nadu, India. Antimicrobial Susceptibility test The bacterial associates were obtained from a standard diagnosis laboratory and were subcultured in nutrient agar slants for 4 hours at 37C in an incubator. After 24 hours the bacterial culture was further used for testing antibacterial activity. A McFarland 0.5 standard was prepared and quality controlled prior to beginning susceptibility testing. The McFarland standard was used to adjust the turbidity of the inoculum for the susceptibility test. Disc Diffusion method The assay was conducted by agar disc diffusion[13]. The test organisms were grown overnight at 37C in an incubator on a Mueller Hinton Agar (MHA) (Hi-media, Mumbai) at a pH of 7.4. After incubation, it was compared to the prepared 0.5 McFarland standard. The loaded discs were placed on the surface of the medium and left for 30 min at room temperature for compound diffusion. The sterile discs purchased from Hi-media were used for impregnation of betel extracts. The tests were conducted at five different concentrations of betel leaf extracts by dissolving in DMSO at a concentration of 1mg/ml. The loaded discs were then dried in an oven at 37C and were placed on the surface of the medium with the help of a sterile forceps and left for 30 min at room temperature for compound diffusion. Finally the discs were gently pressed with forceps to make complete contact with the surface of the medium. The plates were allowed to stand at room temperature for 30 minutes. Inoculated plates were then incubated at 37C over night in an incubator and the inhibition zones on each plates were recorded.

*Corresponding author.
Thangavel Sekar Post Graduate and Research Department of Botany, Pachaiyappas College, Chennai - 600030, Tamil Nadu, India Tel.: +91 44 26412844; Fax: +91 44 26426900 E-mail:tsekar_bot@yahoo.com

Journal of Pharmacy Research Vol.4.Issue 7. July 2011

2223-2225

Patturajan Rajeshbabu et al. / Journal of Pharmacy Research 2011,4(7),2223-2225

Well Diffusion method The assay was conducted by agar well diffusion[14]. The bacterial strains grown on nutrient agar at 37C for 18 hours were suspended on saline solution (0.85% NaCl) and the turbidity was adjusted to 0.5 McFarland standards (108 CFU/ml). Wells of diameter 5mm were punched in the agar plates using a sterile gel puncture. Wells of 5mm diameter was punched off into agar medium with sterile cork borer and filled with different concentrations (10l, 15l, 20l, 25l and 30l per well) of ethanol, chloroform and ethyl acetate extracts of betel leaves by micro pipette in each well in aseptic condition. Plates were then kept in a refrigerator to allow pre-diffusion of extract for 30minutes and further incubated in incubator at 370C for 24hrs. In both well and disc diffusion methods, DMSO (Dimethyl sulphoxide) was used as a negative control and penicillin was used as a positive control. Antibacterial activity was evaluated by measuring the zone of inhibition formed around the discs and measured with transparent ruler in millimeter. The studies were performed in triplicate against each organism. RESULTS Results of antibacterial activity of the betel leaf extracts were shown in table 1 and 2. Leaf extracts of P. betel showed significant activity by disc diffusion method and black betel leaf extracts showed significant activity by well diffusion method.
Table 1. Antibacterial activity of Piper betel and black betel by Disc Diffusion Method
Solvents Conc. (l) Zone of inhibition (mm) Piper betel S. aureus S. pneumoniae 12 14 15 17 5 8 9 12 14 9 7 6 6 9 9 11 6 7 8 9

Well Diffusion method Black betel leaf extracts showed more activity against Staphylococcus aureus and Streptococcus pneumonia in all the tested extracts, where P. betel leaf extracts does not showed activity except a modest activity of ethanol extract against S. aureus. Ethanol and ethyl acetate extract of black betel leaves showed more activity against Staphylococcus aureus with 19 and 20mm of zone of inhibition respectively at a concentration 30l and Streptococcus pneumoniae showed maximum zone of inhibition of 11mm at a concentration 30l. Nutritive value of Betel leaves and its variety Analytical study on the biochemical parameters indicated that total fat, protein and crude fibre along with calcium are higher in black betel leaves while carbohydrates, ash, alkalinity, sodium and potassium are higher in P. betel leaves (Table 3). DISCUSSION The use of chewing the leaves of betel starts right from the buccal cavity (maintaining oral hygiene) through direct introduction in the blood stream via the buccal mucosa (cardiotonic) and continues till it is ingested and assimilated
Table 3. Analysis of nutritive value of leaves of Piper betel and black betel (100g)
Parameters Moisture (%) Total Fat (%) Protein (%) Crude Fibre (%) Total Carbohydrates (%) Ash content (%) Ash insoluble in dil.HCL (%) Alkalinity (%) Calcium (mg) Sodium (mg) Potassium (mg) Iron (mg) Energy (Calories) Piper betel 9.89 2.20 19.80 14.89 38.50 14.72 1.55 4.51 1307.56 5440.67 3954.81 7.64 253.00 Black betel 9.14 3.67 21.90 15.30 36.19 13.80 1.50 2.25 1735.90 364.56 2438.48 7.67 265.00

Black betel S. aureus 9 11 13 16 18 6 8 9 11 12

S. pneumoniae 8 9 10 -

Chloroform

Ethyl acetate

Ethanol

10 15 20 25 30 10 15 20 25 30 10 15 20 25 30

g gram; mg milligram; % - percentage

S. aureus - Staphylococcus aureus; S. pneumoniae - Streptococcus pneumoniae Table 2. Antibacterial activity of Piper betel and black betel by Well Diffusion Method
Solvents Conc. (l) Zone of inhibition (mm) Piper betel S. aureus S. pneumoniae 6 7 9 11 11 Black betel S. aureus 8 9 10 11 12 13 15 16 17 20 15 16 16 18 19

(effect on digestive system, other pharmacological activities) within the human body. The bacteria that are most closely associated with caries development are the mutans Streptococci, which are known to possess high acidogenic and aciduric properties, together with their ability to synthesize extracellular glucans from sucrose catalysed by GTFs[15]. Aqueous extract of Piper betel inhibits adherence of early plaque settlers, which include Streptococcus mitis, Streptococcus sanguinis and Actinomyces sp. to saliva coated glass surfaces[11, 16]. Fathilah et al. [17] reported that the crude aqueous extract of P. betel leaves exhibits antibacterial activity towards Streptococcus mitis, Streptococcus sanguis and Actinomyces viscosus, some of the early colonizers of dental plaque. Leaf extracts, essential oil and its phenolic constituents of P. betel have been reported to have antimicrobial activity against a number of oral bacteria and the extracts have also been active against the important pathogens like Vibrio cholerae ogawa, Diplococcus pneumoniae and Klebsiella aerogenes[6- 7, 9-11, 18 ]. The higher the alkaline content and the presence of more amounts of calcium and potassium influence the digestive process and moreover the neutrality of the buccal cavity is probably serving as a buffer influencing the digestion of carbohydrates. The passage of these juices into the stomach may also sooth the sensitive gastric nerves, which would be exposed to acidic secretions during the digestive process. Because of the presence of above components, chewing of betel leaves produce a sense of well-being, increased alertness, sweating, salivation, hot sensation and energetic feeling with exhilaration [19]. It also increases the capacity to exercise physical and mental functions more efficiently for a longer duration but it may produce a kind of psychoactive effect causing a condition of mild addiction leading to habituation and withdrawal symptoms[20]. Thus it could be concluded that the traditional wisdom of chewing betel ( Piper betel or Black betel depending where they are available) leaves would certainly useful in bacterial infections of mouth, throat and lung. Extracts of Piper betel leaves do not seem to be very effective through the agar well diffusion method as seen in the case of Black betel leaves. The demonstration of broad spectrum of antibacterial activity by betel leaves may help to discover new chemical classes of antibiotic substances that could serve as selective agents for infectious disease chemotherapy and control. The information provided in the study will contribute to a better understanding of the antimicrobial activity of the plant. The effect of this plant on more pathogenic organisms and toxicological investigations and further purification studies of active molecules needs to be carried out.

S. pneumoniae 5 6 7 8 7 9 10 11 11 5 7 8 10 11

Chloroform

Ethyl acetate

Ethanol

10 15 20 25 30 10 15 20 25 30 10 15 20 25 30

S. aureus - Staphylococcus aureus; S. pneumoniae - Streptococcus pneumoniae

Disc diffusion method All the extracts showed moderate inhibition against the tested organisms. Among the investigated extracts the ethyl acetate extract exhibited the highest antibacterial effect followed by the methanol and ethanol extracts. Black betel leaf extracts showed more activity against Staphylococcus aureus than Streptococcus pneumonia when compared to P. betel. The most pronounced activity with inhibition zones of more than 14.0 mm was shown by ethyl acetate (inhibition zone 18.0 mm against S. aureus at a concentration of 30l) and hexane extract (inhibition zone 16.0mm against S. aureus at a concentration of 25l) of black betel leaves. None of the black betel leaf extracts showed inhibition zone against S. pneumonia except chloroform extract. Chloroform and ethyl acetate extract of P. betel leaves showed the highest zone of inhibition of 17mm and 14mm respectively at a concentration 30l against S. aureus and ethanol extract showed zone of inhibition of 9mm at a concentration of 30l. Ethyl acetate extract of leaves of P. betel have no activity against S. pneumonia , where as chloroform and ethanol extracts showed moderate zone of inhibition.

Journal of Pharmacy Research Vol.4.Issue 7. July 2011

2223-2225

Patturajan Rajeshbabu et al. / Journal of Pharmacy Research 2011,4(7),2223-2225

ACKNOWLEDGEMENTS The authors are thankful to the Management and Principal of The New College and Pachaiyappas college, Chennai,for providing necessary facilities to do the work. REFERENCES
1. 2. Kumar N, Misra P, Dube A, Bhattacharya S, Dikshit M, Ranade S, Piper betle L. a maligned Pan-Asiatic plant with an array of pharmacological activities and prospects for drug discovery. Curr Sci, 99, 2010, 922 932. The Wealth of India, 1969. A dictionary of Indian raw materials and industrial products, Vol. VIII. NISCAIR press, National Institute of Science Communication and Information Resources, (CSIR), DR.K.S.Krishnan Mag, Pusa, New Delhi, India. Pp. 84 - 94. Nadkarni AK, 1927. Indian Materia Medica, Volume I, Popupar Prakashan Pvt Ltd, Mumbai. Pp. 960 964. Ali N, Khan FG, Suri KA, Gupta BD, Satti NK, Dutt P, Afrin F, Qazi GN, Khan IA, In vitro antifungal activity of hydroxychavicol isolated from Piper betle L. Annals Clin Microb Antimicrob, 9, 2010,7 (http://www.ann-clinmicrob.com/content/9/1/7). Srimani P, Mandal G, Ganguly S, Saha P, Sen R, De R, Chatterjee A, Bhattacharyya M, Chaudhuri U, Bandyopadhyay SK, Chatterjee M, Antioxidant aeffect of ethanolic extract of Piper betle L. (Paan) on erythrocytes from patients with HbE-beta thalessemia. Indian J Biochem Biophys, 46, 2009, 241 246. Bissa S, Songara D, Bohra A, Traditions in oral hygiene: Chewing of betel (Piper betle L.) leaves. Curr Sci, 92, 2007, 26-28. Shitut S, Pandit V, Mehta BK, The antimicrobial efficiency of Piper betle L. leaf (stalk) against human pathogenic bacteria and phytopathogenic fungi. Cent Eur J Public Health, 7, 1999, 137139. Kumar N, Tripathi R, Putative role of betel (Piper betle L.) in oral hygiene. Plant Perox Newslett, 15, 2000, 4548. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. Ramji N, Ramji N, Iyer R, Chandrasekaran S, Phenolic antibacterials from Piper betle in the prevention of halitosis. J Ethnopharmacol, 83, 2002, 149152. Razak FA, Rahim ZHA, The antiadherence effect of Piper betel L. and Psidium guajava extracts on the adhesion of early settlers in dental plaque to saliva coated glass surfaces. J Oral Sci, 45, 2003, 201 206. Nalina T, Rahim ZHA, The Crude Aqueous Extract of Piper betle L. and its Antibacterial Effect towards Streptococcus mutans. Am J Biotech Biochem, 3, 2007, 10-15. Gupta S, Kumar N, Gupta SM, Antibacterial and antifungal activity in extract and oil of Piper betle (Linn) landrace Bangla Mahoba. Adv Zool, 31, 2009, 1620. Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolke RH, Manual of Clinical Microbiology, vol. 6. ASM, Washington, DC.1995. Perez C, Paul M, Bazerque P, An antibiotic assay by the agar well diffusion method. Acta Bio Med Exp, 15, 1990, 113-115. Sturr MG, Marquis RE, Comparative acid tolerances and inhibitor sensitivities of isolated F ATPases of oral lactic acid bacteria. Appl Environ Microbiol, 58, 1992, 2287 2291. Nalina T, Rahim ZHA, Effect of Piper betel L. leaf extract on the virulence activity of Streptococcus mutans an in vitro study. Pak J Biol Sci, 9, 2006, 1470 1475. Fathilah AR, Othman Y, Rahim ZHA, The effect of Piper betle and Psidium guajava extracts on the cell-surface hydrophobicity of selected early settlers of dental plaque. J Oral Sci 48, 2006, 71-75. Sharma S, Khan IA, Ali I, Ali F, Kumar M, Kumar A, Johri RK, Abdullah ST, Bani S, Pandey A, Suri KA, Gupta BD, Satti NK, Dutt P, Qazi GN, Evaluation of the antimicrobial, antioxidant, and anti-inflammatory activities of hydroxychavicol for its potential use as an oral care agent. Antimicrob Agents Chemother, 53, 2009, 216222. Chu NS, Effects of betel chewing on the central and autonomic nervous systems. J Biomedical Sci, 2001, 8, 229- 236. Garg SC, Jain R, Chavicol rich essential oil of Piper betle L. Cutivar Sagar Bangla. Euro Cosmet, 1996, 5, 27- 28.

3. 4.

5.

6. 7. 8.

19. 20.

Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.4.Issue 7. July 2011

2223-2225