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In Vitro Antimicrobial Activities and Cytotoxicity of Ethyl Acetate Extract from Streptomyces Maritimus

M. Abdul Alim Al-Bari
Department of Pharmacy, University of Rajshahi, Rajshahi-6205, Bangladesh.

M. Abu Sayeed
Department of Pharmacy, University of Southeast, Banani, Dhaka, Bangladesh.

Alam Khan
Department of Pharmacy, University of Rajshahi, Rajshahi-6205, Bangladesh.

M. Robiul Islam
Department of Agronomy and Agricultural Extension, University of Rajshahi, Rajshahi-6205, Bangladesh.

Proma Khondokar
Department of Pharmacy, University of Rajshahi, Rajshahi-6205, Bangladesh.

M. M. Sazedur Rahman
Institute of Biological Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh.

M. Anwar Ul Islam
Department of Pharmacy, University of Rajshahi, Rajshahi-6205, Bangladesh.

Abstract/ Executive Summary:

Ethyl acetate extract from new actinomycetes, Streptomyces maritimus, showed good antibacterial and antifungal activities against a total of 14 bacteria (5 Gram positive plus 9 Gram negative) and 8 fungi. The Minimum Inhibitory Concentrations (MIC) were determined and found to be 16 μg mL^-1 against Bacillus subtilis, Staphylococcus aureus, Shigella dysenteriae and Aspergillus flavus while 32 μg mL^-1 against Salmonella typhi, Candida albicans and Aspergillus niger.

Keywords: Streptomyces maritimus, Antimicrobial activity, Cytotoxicity

Location: Pharmaceutical Microbiology Laboratory, Department of Pharmacy, Faculty of Science, University of Rajshahi, Rajshahi-6205, Bangladesh.

Start Date: 00-01-2004

End Date: 00-06-2004

Program Area: Pest Management

Commodity: Extract (plant, seed)

Objectives:

To study the antimicrobial activities of the ethyl acetate extract from its cultural broth against a number of bacteria (both Gram positive and Gram negative) and fungi.

Methodology:

The present study was carried out during January-June 2004 in Pharmaceutical Microbiology Laboratory, Department of Pharmacy, Faculty of Science, University of Rajshahi, Rajshahi-6205, Bangladesh. The organism was isolated from soil sample, collected from a cultivated land of Northern part of Bangladesh, at a depth of 1 m; by using crowded plate technique. The organism was identified as Streptomyces maritimus strain BD26T (GenBank accession number AF233338) previously described on the basis of its similar morphological, physiological, biochemical, antimicrobial activities and 16S rDNA sequencing analysis data. The maximum secretion of metabolites from the strain was found at the 14th day of incubation in yeast extract sucrose agar (alkaline pH8.6) medium at 37.5°C by maintaining all the physicochemical factors at optimum level for the culture. Test pathogenic microorganisms employed for in vitro antimicrobial assay were obtained from the Institute of Nutrition and Food Science (INFS), University of Dhaka and International Center for Diarrhea Disease and Research, Bangladesh (ICDDRB) Dhaka. The antimicrobial assay of ethyl acetate extract was performed against a various pathogenic bacteria (Gram positive and Gram negative) and a number of fungi by standard disc diffusion technique. The sample solution of the extract to be tested was prepared by dissolving a definite amount of material in ethyl acetate to attain the desired concentrations (200, 20 μg^-1 disc for fungi and 100,30 μg^-1 disc for bacteria). To compare the antibacterial and antifungal activities, kanamycin (30 μg disc^-1) and nystatin (20 μg disc^-1) were used as standard antibiotics respectively. Briefly, in this study, the test discs, standard discs and blank discs were placed in a petridish with a particular bacteria or fungi and then left in a refrigerator at 4°C for 12-18 h in order to diffuse the material from the discs to the surrounding media in the petridishes. The petridishes were then incubated at 37°C for overnight to allow the bacterial growth and 48-72 h for fungal growth. The antibacterial and antifungal activities of the extract were then determined by measuring the respective zones of inhibition in mm. The Minimum Inhibitory Concentrations (MICs) of the crude extract against Bacillus subtilis, Staphylococcus aureus, Salmonella typhi, Shigella dysenteriae, Candida albicans, Aspergillus flavus and Aspergillus niger were determined by serial dilution technique. The cytotoxity activity of the ethyl acetate extract was determined by brine shrimp lethality bioassay. In this method, the eggs of the brine shrimp, Artemia salina Leach, were collected from an aquarium shop (Dhaka, Bangladesh) and hatched for 48 h to mature shrimp. The test sample extract was prepared by dissolving them in DMSO (not more than 50 μL in 5 mL solution) plus sea water (3.8% NaCl in water) to attain concentrations-5, 10, 20, 40 and 80 μg mL^-1 A vial containing 50 μL DMSO diluted to 5 mL was used as a control. Then about 10 brine shrimp nauplii were applied to each of all experimental vials and control vial. The number of the nauplii that died after 24 h was counted. The findings were presented graphically by plotting log of concentration versus percentage of mortality of nauplii from which LC₅₀ was determined by extrapolation. Statistical analyses of the antibacterial and antifungal activities of compounds with different concentrations of each (20, 30, 100 and 200 μg disc^-1) was performed using Kruskal-Wallis test (Debnath and Shil 2001). Individual antibacterial and antifungal activity differences of the tested compounds was examined using post hoc Nemenyi’s test following Kruskal-Wallis test. A significance level of 5% was considered as significance (p<0.05) in all cases. Probit analysis was used to determine the LD₅₀ values from the mortality data using Probit software. The cytotoxicity of the novel ethyl acetate extract was compared with the standard gallic acid and also with the anticancer agent vincristine sulfate. Determination of LD₅₀ by probit analysis allowed the ranking of the extract with respect to their biocidal activity.

Source of Information: Biotechnology, 6(1): 81-85, 2007, ISSN 1682-296X

Article Link (Online Journal): DOI: 10.3923/biotech.2007.81.85, URL: http://scialert.net/abstract/?doi=biotech.2007.81.85

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The ethyl extract displayed poor antibacterial activity at the concentration of 30 μg disc^-1, but gave promising activity at concentrations of 100 μg disc^-1. The MIC values of this complex against the tested organisms indicated their noticeable antibacterial and antifungal potencies compared with standard antibiotic, kanamycin and nystatin respectively. The different antibacterial activity of the complexes indicated their different mechanism of biocidal property and further studies are required to explore the exact mechanism of antibacterial potency. It was concluded that the extract possesses substantial antibacterial activity with a minimum inhibitory concentration and moderate cytotoxicity. Further, acute toxicity and other pharmacological tests are necessary to utilize the extract as a potential therapeutic agent.

Knowledge Management: Journal