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In Vitro Antibacterial and Antifungal Activity of Borreria Articularis

Razia Sultana
Department of Microbiology, University of Chittagong, Chittagong 4331, Bangladesh

M Nazrul Islam Bhuiyan
Bangladesh Council of Scientific & Industrial Research (BCSIR), Chittagong Cantonment, Chittagong 4220, Bangladesh

Jaripa Begum
Bangladesh Council of Scientific & Industrial Research (BCSIR), Chittagong Cantonment, Chittagong 4220, Bangladesh

M Nural Anwar
Department of Microbiology, University of Chittagong, Chittagong 4331, Bangladesh

Abstract/ Executive Summary:

Petroleum ether extract, chloroform extract, ethyl acetate extract, ethyl alcohol extract and a pure compound 6-methyl-5-cyclodecen-1-ol obtained from aerial parts of Borreria articularis were studied for their antimicrobial activities against eleven human pathogenic bacteria and four human pathogenic fungi using disc diffusion and poisoned food method respectively. Ethyl acetate extract, ethanol extracts and the pure compound 6-methyl-5-cyclodecen-1-ol exhibited good antibacterial and antifungal activity against all the pathogens tested herein. The ethanol extract and 6-methyl-5-cyclodecen-1-ol exhibited the largest zone of inhibition (20 mm) at a concentration of 2,000 μg/disc against Escheichia coli and Vibrio cholerae respectively. In case of fungi, the crude extract of ethanol and the pure compound exhibited the highest inhibition 53.5 and 52.0% of fungal radial mycelial growth (with 100 μg/ml medium) against Aspergillus ustus and A. ochraceus respectively. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) of the pure compound 6-methyl-5-cyclodecen-1-ol were determined by broth macrodilution method. The lowest MIC (500 μg/ml) and MBC (1,000 μg/ml) were determined against V. cholerae. However, for fungi, the lowest MIC (750 μg/ml) and MFC (1,500 μg/ml) were recorded against A. ochraceus. The results suggest that active antimicrobial agent(s) present in the extracts of B. articularis may have potential for the treatment of bacterial and fungal infections.

Keywords: Antimicrobial activity, Borreria articularis, Crude extracts, 6-Methyl-5 cyclodecen-1-ol

Location: Department of Microbiology, University of Chittagong

Start Date:

End Date:

Program Area: Pest Management

Commodity: Diseases

Objectives:

To study the extracts and pure compound of B. articularis against various pathogenic bacterial and fungal strains.

Methodology:

Collection and extraction of plant material - Arial parts of Borreria articularis were collected in fresh condition from Chittagong University campus, Chittagong, Bangladesh.The collected and cleaned samples were cut into small pieces (1-2 cm), dried in air to make it suitable for grinding. The samples were ground to fine powder mechanically and then 20 g of the dried powder were kept steeped 72 h in petroleum ether, chloroform and ethyl acetate separately. On the other hand 250 g of the dried powder were kept steeped 72 h in ethyl alcohol. The extracts thus obtained separately were filtered, centrifuged at 2,000 rpm for 20 min and concentrated to a gummy material under reduced pressure at 50°C by rotary vacuum evaporator. The gummy materials were then collected in a small vial, dried as usual. Thus crude extracts were obtained. Purification of crude extract - Solvent-solvent partitioning of concentrated crude ethanol extract was done using the standard protocol. All the fractions were collected separately, dried as usual and tested for any of their antimicrobial efficacy. Of these, only the chloroform extract fraction showing high antimicrobial activity was subjected to fractionation by column chromatography using silica gel (200-300 mesh) as the adsorbent. From it a total of 21 fractions were collected separately using mixtures containing different proportions of ethyl acetate and methanol with increasing polarity as the eluants. The column-separated fractions thus obtained were examined using thin layer chromatography (TLC) technique and ultraviolet light (254 and 366 nm) to detect the presence of any fluorescent compound. The Rf-values for each were measured as usual. Fractions showing same Rf-value were mixed together and grouped into six fractions (A-F). Selection and identification of active fraction - All the fractions (A to F) were tested against three sensitive bacteria (Escherichia coli, Bacillus cereus and Salmonella typhi) and a fungus (Aspergillus ochraceus). Of these, the highly antibacterial and antifungal fraction B, eluted in petroleum ether and ethyl acetate mixture of 20: 80, yielded 1.3 g of brownish material. GC-MS analysis - The pure compound B isolated from ethanolnic extract of B. articularis was analyzed by gas chromatography-mass spectrometry (GC-MS) electron impact ionization (EI) method on GC-17A gas chromatograph (Shimadzu, Japan) coupled to a GCMS QP 5050A Mass spectrometer (Shimadzu, Japan); fused silica capillary column (30 m x 2.5 mm; 0.25 μm film thickness), coated with DB-5 ms (J&W); column temperature 100^oC (2 min) to 250^oC at the rate of 3^oC/min; carrier gas, helium at constant pressure of 90 kPa. Acquisition parameters full scan; scan range 40-350 amu. Sample was injected by splitting and the split ratio 1:20. Identification of the compounds - Compound identification was done by comparing the NIST library data of the peaks with those reported in literature, mass spectra of the peaks with literature data. Percentage composition was computed from GC peak areas on DB-5 ms column without applying correction factors. Test organisms - The crude extracts and pure compound obtained from Borreria articularis were tested for their antibacterial activity against eleven human pathogenic bacteria, viz., Shigella dysenteriae AE 14396, S. sonnei CRL.(ICDDR,B), Salmonella typhi AE 14612, S. paratyphi AE 14613, Bacillus subtilis BTCC 17, B. cereus BTCC 19, B. megaterium BTCC 18, Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa CRL(ICDDR,B), Escherichia coli ATCC 25922 and Vibrio cholerae AE 14748, and four human pathogenic fungi, viz., Aspergillus niger BTCC 504, A. ochraceus BTCC 515, A. ustus BTCC 503 and Candida albicans BTCC 493. Determination of antimicrobial activity - The in vitro antibacterial and antifungal activities of the plant materials were determined by disc diffusion method7 and poisoned food technique8 respectively. Mueller-Hinton (agar and broth) medium was used for culture of bacteria and Sabouraud (agar and broth) medium was used for culture of fungi. 5% ethanolic solution of the crude extracts and pure compound were used as the test material. All the results were compared with the standard antibacterial antibiotic Ampicillin and antifungal antibiotic nystatin. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) values of the crude extracts and pure compound were determined by macrodilution broth technique.

Source of Information: Bangladesh J Microbiol, Volume 25, Number 2, December 2008, pp 95-98

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Achievement/Findings:

From this study, it was found that B. articularis has antibacterial and antifungal properties. The pure compound 6-methyl-5- cyclodecen-1-ol isolated from B. articularis may be used as a novel natural antibacterial and antifungal agent against a wide variety of infectious pathogens after confirming its cytotoxicity. Therefore, further phytochemical studies are needed to identify other constituents responsible for antimicrobial activity.

Knowledge Management: Journal