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Antimicrobial Activity of Pavetta Indica Leaves

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Antimicrobial Activity of Pavetta Indica Leaves Journal of Applied Pharmaceutical Science Vol. 3 (04), pp. 078-082, April, 2013 Available online at http://www.japsonline.com DOI: 10.7324/JAPS.2013.3414 ISSN 2231-3354 Antimicrobial activity of Pavetta indica leaves Vinod Kumar Gupta, Charanjeet Kaur, Aritra Simlai and Amit Roy* Department of Biotechnology, Visva-Bharati University Santiniketan-731235, West Bengal, India. ABSTRACT ARTICLE INFO Article history: Antimicrobial activity of the aqueous and organic solvent extracts of the leaves of Pavetta indica were tested Received on: 06/03/2013 against Bacillus subtilis, Escherichia coli and Saccharomyces cerevisiae using disc diffusion assay. Most of the Revised on: 22/03/2013 leaf extracts showed bactericidal activity against B. subtilis. None of the extracts exhibited any activity against E. Accepted on: 05/04/2013 coli and S. cerevisiae. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), Available online: 27/04/2013 thermal stability and qualitative phytochemicals studies were performed. Both MIC and MBC of the aqueous and methanol extracts were found to be between 1.95 - 7.81 mg/ml. The activity of aqueous and methanol extracts Key words: were found to be stable despite thermal treatment. Phytochemical analysis of aqueous extract revealed the Pavetta indica, Antibacterial presence of flavonoids, saponins and carbohydrates. Methanol extract was found to be positive for saponin and activity, Phytochemicals, cardiac glycosides. TLC and bioautography were also done to identify the active fractions responsible for the TLC, Bioautography antimicrobial activities. Results showed the presence of a number of bactericidal components. The study suggests P. indica to be a source for isolation of antibacterial compounds for human health care and use as preservatives in food processing industry. INTRODUCTION Amos et al. (2004) have shown that the extracts of P. crassipes dose-dependently decreased spontaneous motor activity Pavetta indica Linn. belongs to the family Rubiaceae. (SMA) in mice and attenuated amphetamine-induced hyperactivity It is widely distributed from the Andaman Islands, India and the and the different episodes of stereotypic behavioral patterns north-western Himalayas to southern China and southwards induced by amphetamine. throughout Malaysia to northern Australia. P. indica is a shrub or The antimicrobial activities have been very briefly small tree of 3-5 m in height, with opposite branches having leaves mentioned in some other species of Pavetta (Balde et al., 1990; that are membranous and variable in shapes and sizes. P. indica Balde et al., 2010; Anago et al., 2011) but to our knowledge it has leaves are used in the treatment of liver disease, pain from piles, not been characterized adequately to assess its potential as future urinary diseases and fever (Kirtikar and Basu, 1975; Thabrew et therapeutic source for combating microbes. In this report we al., 1987). It is a medicinally important plant having anti- describe preliminary studies to show the existence of bactericidal inflammatory activities (Mandal et al., 2003). Golwala et al. (Gram-positive) and bacteriostatic (Gram-negative) activities in the (2009) reported analgesic activity of leaf extract of P. indica. Its leaf extracts of Pavetta indica Linn. for the first time. The root extract also have diuretic and purgative activity (Kumar, phytochemicals produced by the plants for their self protection have 2006). Other species of Pavetta also showed different biological been demonstrated to protect human against a number of diseases. activities. P. gardeniifolia, P. pyroides and P. crassipes have been Antimicrobial activities of phytoconstituents such as phenol, reported to possess anti-malarial activity (Sandra et al., 2009; tannins, terpenoids, essential oils, alkaloids, saponins and Gbeassory et al., 1989). Schistosomicidal properties of ethanol and flavonoids have been reported by several authors (Weimann et al., acetone extracts of P. owariensis have been reported by Balde 1997; Atindehou et al., 2002; Edeoga et al., 2005; Ahmed et al., et al., (1989). 2012; Kamal et al., 2010) in other plant systems. We have done preliminary experiments to obtain the phytochemical profiles in leaf * Corresponding Author extracts of this species for correlating the observed antibacterial Department of Biotechnology, Visva-Bharati University Santiniketan-731235, West Bengal, India activities with these phytoconstituents. Telefax: +91-3463-261101 © 2013 Vinod Kumar Gupta et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial- ShareAlike Unported License (http://creativecommons.org/licenses/by-nc-sa/3.0/). Gupta et al. / Journal of Applied Pharmaceutical Science 3 (04); 2013: 078-082 079 MATERIALS AND METHODS inhibition formed around the discs was measured. Ampicillin (250 μg/ml) and commercial Fluconazole (10 mg/ml) were used as Collection and identification of plant materials positive controls for all such experiments. Fresh leaves of P. indica were collected from Santiniketan, West Bengal, India. The plant Pavetta indica Linn., Minimum inhibitory concentration (MIC) determination was identified by Prof. K. N. Bhattacharya, Botany Department, MIC was performed by Guerin-Faublee et al. (1996) Visva-Bharati University, India and the dried specimen was method and has been described by Gupta et al. (2010). It was done deposited in the Herbarium of the Botany Department, Visva- by carrying out the disc diffusion tests with discs spotted with Bharati University, India. serially diluted concentrations of aqueous and methanol extracts. For making serial dilutions, the dried crude extracts were dissolved Preparation of plant extracts in DMSO at a concentration of 250 mg/ml and serially diluted Fresh leaves of P. indica were air dried under shade and (1:1) with media to concentrations of 125, 62.5, 31.3, 15.6, 7.8, then ground into fine powder with the help of an electric grinder. 3.9, 1.95 and 0.98 mg/ml for use. The lowest concentration of a For extraction of the dried powder, aqueous and organic solvents particular extract that inhibited the growth of B. subtilis was noted i.e., methanol, chloroform, benzene and hexane were used. Air- as the MIC value of that extract for the B. subtilis. dried powder of P. indica leaves (10 g) in solvents (1:10, w/v) was taken in a 250 ml conical flask and kept at room temperature for 5 Minimum Bactericidal Concentration (MBC) determination days. After this period, the samples were filtered, supernatants The method of Greenwood (1989) was used to determine were collected and solvents were evaporated to dryness. This the MBC of the aqueous and methanol extracts. Serial dilutions of process was repeated two more times and final dry extracts were these crude extracts were made with sterile LB broth in test tubes stored at 4 °C until use. in the range of 15.64, 7.82, 3.91, 1.95, 0.98 and 0.49 mg/ml. Then overnight grown test organism (10 µl) was pipetted into each of Growth and maintenance of microorganisms these test tubes containing various concentrations of the crude The dried aqueous and solvent extracts dissolved in extracts as mentioned above and incubated at 30 °C for 24 h. At dimethylsulphoxide (DMSO) were screened against Bacillus the end of this incubation period, the contents of the tubes were subtilis (MTCC 121), Escherichia coli (MTCC 484) and plated on LB-agar and grown for 18-20 h at 30 °C to determine the Saccharomyces cerevisiae. Stock cultures of B. subtilis and E. coli bactericidal activities in extracts. The concentration of crude were maintained on slants or plates of LB (Luria-Bertani)-agar extract at which no microbial growth occurred (as evidenced from whereas S. cerevisiae on YPD (Yeast extract powder-Mycological absence of subsequent microbial growth in the LB-agar) was peptone-Dextrose)-agar at 4 °C. Inoculums of healthy cultures for recorded as the MBC of that crude extract. experiments were prepared by transferring a single colony from the stock culture plate to 1.5 ml of LB broth or YPD broth and Thermal stability test incubated at 180 rpm for 18 h at 30 °C (for B. subtilis and S. To determine the effect of temperature on the stability of cerevisiae) or at 37 °C (for E. coli). Appropriate quantities of these leaf extracts, these extracts in 1.5 ml microfuge tubes, each with inoculums were then directly used for larger liquid cultures or 250 mg/ml concentration of the crude extracts in DMSO, were diluted suitably in 0.9% saline before use for antimicrobial treated at 40, 60, 80, and 100 °C and autoclaved at 15 psi, all for screening. The general microbial techniques and compositions of 15 min, separately. The samples were cooled to room temperature LB, YPD broth and LB or YPD-agar are described by Sambrook and the residual antibacterial activities were determined against the and Russel (2001). target organisms with the help of disc diffusion method described above. Antimicrobial activity screening Screening of antimicrobial activities of the extracts were Preliminary phytochemical studies performed using the disc diffusion method (Bauer et al., 1966) and Qualitative phytochemical tests of P. indica leaf extracts has already been described in Gupta et al. (2010). The plates were were carried out for detecting the presence of saponins (by prepared by pouring 25 to 30 ml of media into sterile 90 mm Petri foaming test), carbohydrates (by Fehling’s test), cardiac dishes. After adjusting the turbidity of the inoculums prepared glycosides, flavonoids etc. and were detected using colour according to the method described above, a sterile cotton swab development tests as described by Harborne (1998) and was dipped into the suspension and was spread uniformly on agar Khandelwal (2000). plates. Then sterile Whatman no. 1 filter papers (6 mm diameter) were placed on the spread surface of the Petri dishes and 3 μl of Thin Layer Chromatography (TLC) dried extract (dissolved at 250 mg/ml in DMSO) was spotted on TLC silica gel 60 F254 plates (Merck), 20 X 20 cm2 and each of the filter papers.
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