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. ARTICLE INFO ABSTRACT Article history: Received on: 06/03/2013 Revised on: 22/03/2013 Accepted on: 05/04/2013 Available online: 27/04/2013 Antimicrobial activity of the aqueous and organic solvent extracts of the leaves of Pavetta indica were tested against Bacillus subtilis, Escherichia coli and Saccharomyces cerevisiae using disc diffusion assay. Most of the leaf extracts showed bactericidal activity against B. subtilis. None of the extracts exhibited any activity against E. coli and S. cerevisiae. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), 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 were found to be stable despite thermal treatment. Phytochemical analysis of aqueous extract revealed the presence of flavonoids, saponins and carbohydrates. Methanol extract was found to be positive for saponin and cardiac glycosides. TLC and bioautography were also done to identify the active fractions responsible for the 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. Key words: Pavetta indica, Antibacterial activity, Phytochemicals, TLC, Bioautography INTRODUCTION Pavetta indica Linn. belongs to the family Rubiaceae. It is widely distributed from the Andaman Islands, India and the north-western Himalayas to southern China and southwards throughout Malaysia to northern Australia. P. indica is a shrub or small tree of 3-5 m in height, with opposite branches having leaves that are membranous and variable in shapes and sizes. P. indica leaves are used in the treatment of liver disease, pain from piles, urinary diseases and fever (Kirtikar and Basu, 1975; Thabrew et al., 1987). It is a medicinally important plant having antiinflammatory activities (Mandal et al., 2003). Golwala et al. (2009) reported analgesic activity of leaf extract of P. indica. Its root extract also have diuretic and purgative activity (Kumar, 2006). Other species of Pavetta also showed different biological activities. P. gardeniifolia, P. pyroides and P. crassipes have been reported to possess anti-malarial activity (Sandra et al., 2009; Gbeassory et al., 1989). Schistosomicidal properties of ethanol and acetone extracts of P. owariensis have been reported by Balde et al., (1989). . * Corresponding Author Department of Biotechnology, Visva-Bharati University Santiniketan-731235, West Bengal, India Telefax: +91-3463-261101 Amos et al. (2004) have shown that the extracts of P. crassipes dose-dependently decreased spontaneous motor activity (SMA) in mice and attenuated amphetamine-induced hyperactivity and the different episodes of stereotypic behavioral patterns induced by amphetamine. The antimicrobial activities have been very briefly mentioned in some other species of Pavetta (Balde et al., 1990; Balde et al., 2010; Anago et al., 2011) but to our knowledge it has not been characterized adequately to assess its potential as future therapeutic source for combating microbes. In this report we describe preliminary studies to show the existence of bactericidal (Gram-positive) and bacteriostatic (Gram-negative) activities in the leaf extracts of Pavetta indica Linn. for the first time. The phytochemicals produced by the plants for their self protection have been demonstrated to protect human against a number of diseases. Antimicrobial activities of phytoconstituents such as phenol, tannins, terpenoids, essential oils, alkaloids, saponins and flavonoids have been reported by several authors (Weimann et al., 1997; Atindehou et al., 2002; Edeoga et al., 2005; Ahmed et al., 2012; Kamal et al., 2010) in other plant systems. We have done preliminary experiments to obtain the phytochemical profiles in leaf extracts of this species for correlating the observed antibacterial activities with these phytoconstituents. © 2013 Vinod Kumar Gupta et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercialShareAlike Unported License (http://creativecommons.org/licenses/by-nc-sa/3.0/). Gupta et al. / Journal of Applied Pharmaceutical Science 3 (04); 2013: 078-082 MATERIALS AND METHODS Collection and identification of plant materials Fresh leaves of P. indica were collected from Santiniketan, West Bengal, India. The plant Pavetta indica Linn., was identified by Prof. K. N. Bhattacharya, Botany Department, Visva-Bharati University, India and the dried specimen was deposited in the Herbarium of the Botany Department, VisvaBharati University, India. Preparation of plant extracts Fresh leaves of P. indica were air dried under shade and then ground into fine powder with the help of an electric grinder. For extraction of the dried powder, aqueous and organic solvents i.e., methanol, chloroform, benzene and hexane were used. Airdried 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 days. After this period, the samples were filtered, supernatants were collected and solvents were evaporated to dryness. This process was repeated two more times and final dry extracts were stored at 4 °C until use. Growth and maintenance of microorganisms The dried aqueous and solvent extracts dissolved in dimethylsulphoxide (DMSO) were screened against Bacillus subtilis (MTCC 121), Escherichia coli (MTCC 484) and Saccharomyces cerevisiae. Stock cultures of B. subtilis and E. coli were maintained on slants or plates of LB (Luria-Bertani)-agar whereas S. cerevisiae on YPD (Yeast extract powder-Mycological peptone-Dextrose)-agar at 4 °C. Inoculums of healthy cultures for experiments were prepared by transferring a single colony from the stock culture plate to 1.5 ml of LB broth or YPD broth and incubated at 180 rpm for 18 h at 30 °C (for B. subtilis and S. cerevisiae) or at 37 °C (for E. coli). Appropriate quantities of these inoculums were then directly used for larger liquid cultures or diluted suitably in 0.9% saline before use for antimicrobial screening. The general microbial techniques and compositions of LB, YPD broth and LB or YPD-agar are described by Sambrook and Russel (2001). Antimicrobial activity screening Screening of antimicrobial activities of the extracts were performed using the disc diffusion method (Bauer et al., 1966) and has already been described in Gupta et al. (2010). The plates were prepared by pouring 25 to 30 ml of media into sterile 90 mm Petri dishes. After adjusting the turbidity of the inoculums prepared according to the method described above, a sterile cotton swab was dipped into the suspension and was spread uniformly on agar 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 dried extract (dissolved at 250 mg/ml in DMSO) was spotted on each of the filter papers. After this, the plates were then incubated at respective ambient temperatures for 18 h (for B. subtilis and E. coli) and upto 36 h (for S. cerevisiae). Subsequently, the zone of 079 inhibition formed around the discs was measured. Ampicillin (250 μg/ml) and commercial Fluconazole (10 mg/ml) were used as positive controls for all such experiments. Minimum inhibitory concentration (MIC) determination MIC was performed by Guerin-Faublee et al. (1996) method and has been described by Gupta et al. (2010). It was done by carrying out the disc diffusion tests with discs spotted with serially diluted concentrations of aqueous and methanol extracts. For making serial dilutions, the dried crude extracts were dissolved in DMSO at a concentration of 250 mg/ml and serially diluted (1:1) with media to concentrations of 125, 62.5, 31.3, 15.6, 7.8, 3.9, 1.95 and 0.98 mg/ml for use. The lowest concentration of a particular extract that inhibited the growth of B. subtilis was noted as the MIC value of that extract for the B. subtilis. Minimum Bactericidal Concentration (MBC) determination The method of Greenwood (1989) was used to determine the MBC of the aqueous and methanol extracts. Serial dilutions of these crude extracts were made with sterile LB broth in test tubes 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 these test tubes containing various concentrations of the crude extracts as mentioned above and incubated at 30 °C for 24 h. At the end of this incubation period, the contents of the tubes were plated on LB-agar and grown for 18-20 h at 30 °C to determine the bactericidal activities in extracts. The concentration of crude extract at which no microbial growth occurred (as evidenced from absence of subsequent microbial growth in the LB-agar) was recorded as the MBC of that crude extract. Thermal stability test To determine the effect of temperature on the stability of leaf extracts, these extracts in 1.5 ml microfuge tubes, each with 250 mg/ml concentration of the crude extracts in DMSO, were treated at 40, 60, 80, and 100 °C and autoclaved at 15 psi, all for 15 min, separately. The samples were cooled to room temperature and the residual antibacterial activities were determined against the target organisms with the help of disc diffusion method described above. Preliminary phytochemical studies Qualitative phytochemical tests of P. indica leaf extracts were carried out for detecting the presence of saponins (by foaming test), carbohydrates (by Fehling’s test), cardiac glycosides, flavonoids etc. and were detected using colour development tests as described by Harborne (1998) and Khandelwal (2000). Thin Layer Chromatography (TLC) TLC silica gel 60 F254 plates (Merck), 20 X 20 cm2 and 0.2 mm thick, were used for TLC. Water and methanol extracts of leaf at a concentration of 50 mg/ml (6 µl; dissolved in methanol) were applied on TLC plates. The chromatograms (in duplicate) 080 Gupta et al. / Journal of Applied Pharmaceutical Science 3 (04); 2013: 078-082 were run using benzene: ethanol: ammonia (BEA, 18:2:0.2) or ethyl acetate: methanol: water (EMW, 10:1.35:1) as running solvents. At the end of the run, the TLC plates were subjected to bioautography as detailed below. Bioautography Bioautography technique of Nostro et al. (2000) was used with some modifications for the detection of antimicrobial components present in the leaf extracts, after separating these components on TLC plates as described above. At the end of TLC run, 1% inoculums of B. subtilis or E. coli containing LB-agar were poured on TLC plates. After solidification of the LB-agar medium on the TLC plates, they were incubated for 24 h at 30 °C and 37 °C for B. subtilis and E. coli respectively. Subsequently, the bioautogram was sprayed with a 1% aqueous solution of 3(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) based on the reduction of MTT by mitochondrial dehydrogenase of viable cells resulting in a blue formazan product after incubation. It was then incubated at 30 °C for few minutes. Zones in which bacterial growth was inhibited failed to take the bluish stain and indicated the presence of active compounds in that area of TLC plate. carried out by treating the extracts dissolved in DMSO at 40, 60, 80, 100 °C and by autoclaving (121 °C) for 15 min. The results obtained against B. subtilis have demonstrated very little or no loss in these activities in comparison to the untreated samples (Table 3). Phytochemical analyses revealed the presence of saponins, carbohydrates and flavonoids in aqueous extract and also saponins and cardiac glycosides in methanol extracts (Table 4). Table. 3: Thermal stability test of P. indica leaf extracts against B. subtilis. Antimicrobial activity (mm) Temperature (°C) Aqueous extract Methanol extract 40 7.75 7.75 60 7.75 7.75 80 7.75 7.75 100 7.75 7.75 Autoclaved 6.75 7.75 DMSO No No Extracts used at 750 µ g/disc. Inhibition zone diameter including disc diameter of 6 mm. No = No detectable inhibition. (-) = Not tested. DMSO was used as negative control. Table. 4: Phytochemical analysis of P. indica leaf extracts. Phytochemicals Aqueous extract Methanol extract Saponins +++ + Carbohydrates + Cardiac glycosides + Flavonoids +++ + = Present in low amounts; +++ = Present in high amounts; - = absent RESULTS Disc diffusion tests of water and organic (methanol, chloroform and benzene) extracts of P. indica leaves showed appreciable antimicrobial activity against B. subtilis, the Grampositive bacteria. But none of these extracts exhibited any growth inhibitory effects on E. coli, the Gram-negative bacteria or S. cerevisiae, the fungus (Table 1). Table. 1: Antimicrobial activity of P. indica leaf extracts against test microorganisms by disc diffusion method. Zone of inhibition (mm) Extracts/ controls B. subtilis E. coli S. cerevisiae Aqueous 7.5 No No Methanol 7.5 No No Chloroform 6.5 No No Benzene 6.5 No No Hexane No No No DMSO No No No Ampicillin 28.0 13.0 --11 Fluconazole 18.5 All the extracts used at 750 µg/disc. Inhibition zone diameter including disc diameter of 6 mm. No = No detectable inhibition. (-) = Not tested. DMSO was used as negative control. Ampicillin (250 µg/ml) and Fluconazole (10 mg/ml) were used as positive controls at 3 µl/disc. Table. 2: MIC and MBC values of aqueous and methanolic extracts of P. indica leaf against B. subtilis. Extracts MIC (mg/ml) MBC (mg/ml) Aqueous 3.91 - 7.81 3.91 - 7.81 Methanol 1.95 – 3.91 1.95 - 3.91 MIC and MBC results of water and methanol extracts of leaves are shown in Table 2 which indicate that these extracts have bactericidal activities. Both MIC and MBC values ranged between 1.95-7.81 mg/ml. The thermal stability tests of the antibacterial activities found in aqueous and methanolic crude extracts were Fig. 1: TLC-bioautography of methanol and aqueous extract of leaf of P. indica in different running solvents. Lanes A, B, E and F = Bioautography against B. subtilis. Lanes C, D, G and H = Bioautography against E. coli. Lanes A, B, C and D were run under BEA and E, F, G and H were run under EMW running solvents system respectively. The Rf values of different components are shown in the margins. Gupta et al. / Journal of Applied Pharmaceutical Science 3 (04); 2013: 078-082 TLC separation of different phytochemicals present in the leaf extracts of P. indica followed by bioautography using B. subtilis demonstrate that under polar running solvent system (EMW), the anti-gram positive bactericidal activity resolves into several areas on the TLC plate (shown by arrows in Fig. 1 lane E ) where killing of the B. subtilis is clearly visible in the form of colourless spots; these spots are areas on TLC plates that failed to take MTT stain due to absence of live bacteria in there. When the bioautography was performed using E. coli for identification of the active fraction on the TLC plates, it was seen that the zones of inhibition are not as clear as in the case of B. subtilis but diffused which suggests that while the compounds that are present in these spots are not able to kill E. coli (i.e. are not bactericidal for E. coli), they certainly are acting as growth inhibitors against E. coli (shown by arrows in Fig. 1 lanes C, D, G and H). It is also to be noted that these spots did not resolve well (figure 1 lanes A, B, C and D) when TLC was carried out in non-polar solvent system (BEA). DISCUSSION Genus Pavetta has about 200 species (Mouly et al., 2009); among them P. indica is known to be used in ethnomedicine for the treatment of microbial infections (Nandagopalan et al., 2011). Another species P. crassipes has antimicrobial activity against Klebsiella pneumoniae, Proteus species, Pseudomonas aeruginosa and Staphylococcus aureus (Mustapha et al., 2007; Aliyu et al., 2008) etc. Ibekwe et al. (2012) have found that P. crassipes leaf extracts have antimicrobial activity against Mycobacterium tuberculosis. Bello et al. (2011) isolated a flavonoid (quercetin-3-O-rutinoside) from P. crassipes leaves that was found to be responsible for antimicrobial activity against E. coli, Streptococcus pyogenes, Corynebacterium ulcerans, Klebsiella pneumoniae, Neisseria gonorrhoeae and Pseudomonas aeruginosa. P indica has not been reported to possess any anti-microbial activities earlier. Our studies described above suggest that P. indica does possess appreciable antimicrobial activities against Gram-positive bacteria. In disc diffusion tests, the extracts of P. indica leaves showed bactericidal activities against the Gram-positive B. subtilis bacteria but no detectable activity against Gram-negative bacteria (E. coli). The Gram-negative bacteria are generally regarded more resistive due to the presence of lipopolysaccharides in their outer membranes that tend to prevent the entry of inhibitors (Nikaido and Vaara, 1985). MIC results indicate that the antimicrobial components extract similarly in water and methanol; however, MBC results indicate that while these components possess clear bactericidal effects against B. subtilis they extract somewhat better in methanol than in water (as evidenced by lower MBC values of methanol extracts). The heat stability exhibited by these antibacterial activities indicate the plant’s leaf tissues as valuable source for extraction of antimicrobial compounds, having application as preservatives in food-processing industries to inhibit the microbial 081 growth in processed food products. Presence of flavonoids in phytochemical testing is interesting as some of the antimicrobials characterized from other plant sources have been found to be flavonoids in nature (Harborne and Baxter, 1999; Alarcón et al., 2008; Kanwal et al., 2009; Galeotti et al., 2008; Sathiamoorthy et al., 2007). Our TLC-bioautography results seem to suggest that antibacterial compounds may be polar in nature as several components in the leaf extracts resolved into areas on TLC plates that exhibited clear bactericidal zones (Fig. 1) when the TLC experiments were carried out in polar solvent system such as ethyl acetate : methanol : water (EMW). In contrast when the same extracts were run in a non-polar solvent (BEA) system on TLC, these bands did not migrate well supporting the view that the antimicrobial components may be more polar in nature. It is not possible to determine at this point whether the different spots (i.e. zones of killing/growth inhibition) found on TLC-bioautography experiments are the parts of the same antimicrobial molecule at different stages of metabolic synthesis or there are multiple compounds of antimicrobial nature in the leaf extracts. Further studies are needed to answer such questions. CONCLUSION Based on the results described above, we conclude that the extracts of leaves of this plant Pavetta indica possess clear bactericidal activity on Gram-positive bacteria and growth inhibitory activity on Gram-negative bacteria. 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