Production of Bioactive Metabolites from Marine Fungi Collected from Coastal Area of Andhra Pradesh, India

journal of pharmacy research 6 (2013) 663e666

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Original Article Isolation, identiﬁcation & production of bioactive metabolites from marine fungi collected from coastal area of Andhra Pradesh, India

Jangala Swathi, Katta Meera Sowjanya, Kumara Narendra, K.V.N. Rathnakar Reddy, Alapati Krishna Satya*

Department of Biotechnology, Acharya Nagarjuna University, Guntur 522510, Andhra Pradesh, India article info abstract

Article history: Aim: To explore antibacterial active compounds from Curvularia sp. and Aspergillus sp. Received 17 March 2013 isolated from marine source. Accepted 29 April 2013 Method: To increase the bioactive metabolite production and to search novel compound, in Available online 19 June 2013 my on-going research we collected the source sample from marine water. By the serial dilution method, different dilutions of sample are spread on Rose Bengal medium to isolate Keywords: pure colonies. Further characterization is done by the microscopic and macroscopic Antibacterial activity studies. Based on morphological and microscopic studies organisms were identified as Microscopic observation Curvularia species, and Aspergillus species. These fungal cultures were grown in potato Morphological observation dextrose agar for 5e6 days for metabolite production. Crude metabolite was extracted TLC purification using Rota vapor. Metabolite is tested for biological activities and further step TLC was performed to investigate the number of fractions present in crude extract. Results: Marine fungi organisms were isolated and screened by serial dilution method from Rose Bengal medium. Pure colony was isolated and identified as Curvularia sp. and Asper- gillus sp. by slide culture technique. Individual organisms were inoculated in potato dextrose broth for production of secondary metabolites for a period of 6e8 days. The collected crude extract of Curvularia sp. showed 27 mm diameter zone against Enterococcus faecalis and Bacillus megaterium at 100 mg and Aspergillus sp. showed 12 mm diameter zone and best activity against B. megaterium and Xanthomonas campestris at 40 mg. TLC of Curvu- laria sp. crude extract showed 2 fractions and Aspergillus sp. crude extract showed 5 fractions. Conclusion: Hence we conclude that the compound has high efficiency of antibacterial activity, which needs extensive attention in terms of new therapy. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.

* Corresponding author. Tel.: þ91 863 2346358, þ91 9490639577. E-mail address: [email protected] (A. Krishna Satya). 0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2013.04.052 664 journal of pharmacy research 6 (2013) 663e666

1. Introduction for an incubation of 3e4 days in room temperature. After the time of incubation isolated colonies were observed and pure The review noticed that mortality due to infections is cultures were maintained for each strain. increasing in developing countries. There is a need of developing new and useful compounds to provide assistance and 3.3. Macroscopic studies relief pain in all aspects of human conditions in future. Since 3.8 billion years microorganisms are being evolved and are The selected strain with full loop is placed at the center of producing more and more evolved metabolites as a mecha- Sabouraud dextrose agar and incubated to obtain colony for nism of defense for their survival. In search of bioactive com- morphological identification. pounds most of the work has been done on metabolites from algae, bacteria and protozoan isolates. Marine fungi are 3.4. Microscopic studies-slide culture preparations worldwide ecological group, but distinct in their geographical distribution and the substratum on which they grow.1 Fungi In order to accurately identify fungi it is essential to study the isolated from marine environments have recently been microscopic organism by slide culture technique.6 recognized as a rich source of biologically active metabolites. Hence fungi can be excellent source for new medicines as well.2 3.5. Production of metabolites and extraction of As going on search for new pharmaceutical compounds mycelium from marine fungi, after isolating number of organisms while studying we knew that there is no much report on Curvularia The selected fungi were inoculated in each 500 ml Erlenmeyer sp.. Even though Aspergillus sp. work is reported but even we flask containing 200 ml of potato dextrose broth medium. The still reported the antibacterial activity efficiently at low con- flask was incubated in at 28c for a week. After the metabolite centrations. So, we screened and studied Curvularia sp. and production, equal volume of ethyl acetate is added to each Aspergillus sp.. with efficient antibacterial activities. flask and incubated for few hours. Finally cell filtrate is separated by filtration using filter paper. The broth and solvent were separated using separating funnel. The organic phase is 2. Review collected and solvent is separated by condensed method using Rota vapor. Finally obtained crude extract is weighed and Number of past reviews has focused the attention of re- dissolved in 10% DMSO for antimicrobial studies.7 searchers on the tremendous treasure of the marine microbial environment. Although a diversified range viz., antibiotic, 3.6. Antibacterial activity antifungal, cytotoxic, neurotoxic, antimitotic, antiviral, anti- neoplastic and antiprotozoal activity is known, extensive Antibacterial activity of fungal extracts was performed using studies are still needed. Comparatively marine environment is standard disc diffusion method. Six bacteria were used as very dynamic and vast, thereforeincreasing interestin studying indicator targets. Assay was done with different concentra- marine fungi producing biological active compounds.3 There is tions. After the incubation of bacterial cultures with fungal no much more work on antimicrobial investigation of Curvu- extracts for 24 h the antibacterial assay was evaluated by laria sp., reported previously. However only the metabolites measuring the diameter of growth inhibition zones using were identified as 11-a-methoxycurvularin (fraction 1) and (S )- diameter measuring scale. The inhibition radii means the 5-ethyl 8,8-dimethyl nonanal (fraction 2) were isolated from of clear zone in which the tested micro organism did not grow, Curvularia oryzae (MTCC 2605).4 The literature of Aspergillus sp., DMSO is taken as control for activities.8 shows antibacterial and anticancer activity. The compound of Aspergillus shows antibacterial activity with n-butane, water, 3.7. Thin layer chromatography chloroform, and acetone.5 TLC is performed to analyze the fractions (compounds) present in the crude extract. Separation of the compound de- 3. Materials and methods pends on the usage of solvents. Silica gel is prepared in slurry form and evenly spread on glass plate. Crude extract prepared 3.1. Sample source with a concentration of 1 mg/ml was placed on the TLC plate and dried. After running with Hexane and Ethyl acetate sol- Marine water samples were collected from coastal belt vents at different proportions, spots were identified with covering Krishna, Guntur & Prakasam Dist of Andhra Pradesh iodine crystal vapors.9 covering over an area of 960 km. The water samples were collected in sterile tight bottles and transferred to the labo- ratory in 24 h of duration. 4. Results

3.2. Fungi isolation 4.1. Macroscopic observation

The water sample is diluted with different dilution rates. An Curvularia sp., is a filamentous fungi which grows rapidly on equal proportion of volume is spread on Rose Bengal medium potato dextrose agar at 27 C and produces woolly colonies journal of pharmacy research 6 (2013) 663e666 665 which later turn dark brown to black. The hyphae are septate 4.8. Systematic positioning of Aspergillus sp. and produce brown conidiophores which bear pyriform conidia. Domain: Eukaryota Kingdom: Fungi 4.2. Microscopic observation Phylum: Ascomycota Subphylum: Pezizomycotina After incubation of slide culture, slides are stained with Lac- Class: Eurotiomycetes tophenol blue for microscopic examination. First the slides are Order: Eurotiales observed under light microscope at 10 resolution and then at Family: Trichocomaceae 45 and 100 (oil immersion) for morphology of fungi. Fungi Genus: Aspergillus are identified by using the reference book on “Illustrated Genera of Imperfect Fungi” fourth edition by H. L. Barnett and Antibacterial activity of Curvularia sp., e Table 1 Barry B. Hunter. Based on the mycelium and spore Antibacterial activity of Aspergillus sp., e Table 2 morphology studies the isolate was identified as Curvularia sp.

4.3. Systematic positioning of Curvularia sp. 5. Discussion & conclusion

Kingdom: Fungi The main aim of this work is to study the marine bioactive Phylum: Ascomycota compounds. Fungi are more efficient group of organisms to be Class: Euascomycetes explored for the drug discovery purpose. Especially fungi had Order: Pleosporales provided mankind with numerous different bioactive sec- Family: Pleosporaceae ondary metabolites. In recent years marine fungi have Genus: Curvularia explored more intensely to obtain novel and biologically active compounds. In search of biologically active natural products the present study deals with screening, isolation, production as 4.4. Amount of secondary metabolite extracted from well as investigating the antimicrobial activities of desired Curvularia sp. per batch crude extract that were collected from selected strain. After the morphology and microscopic observation, isolates are identified as Curvularia sp., and Aspergillus sp. The Volume of the Amount of compound crude extract collected was prepared in low concentrations. media inoculated (L) obtained (mg) Curvularia sp. crude extract was prepared at 25 mg, 50 mg, 75 mg 1 L 200 mg and 100 mg. Zone of inhibition was highest at 100 mg concentration (27 mm diameter) for Enterococcus faecalis and Bacillus megaterium. Aspergillus sp., crude extract was prepared at 4.5. Morphological observation of Aspergillus sp. 10 mg, 20 mg, 30 mg and 40 mg. Among these concentrations 40 mg (12 mm diameter) showed best activity against B. megaterium Aspergillus sp., is a conidiophores producing fungi which and Xanthomonas campestris. Further the crude extract is grows rapidly on potato dextrose agar at 27 C and produces analyzed with TLC to know the number of fractions present in wooly colonies in which initial white color is converted into the compound. Curvularia sp., obtained a single fraction at green and finally appears as dark black. 4:6(Hexane: Ethyl acetate) and Aspergillus sp., showed 5 fractions at 2:8 (Hexane: Ethyl acetate). These fractions are yet to 4.6. Microscopic observation of Aspergillus sp. be purified by column chromatography for further analyses. Earlier reports on Curvularia sp. showed antimicrobial ac- m m 4 Aspergillus has septate hyphae. Conidia are arranged in chain tivity at 50 g and 100 g/ml but our isolated species of Curvu- form, carried on elongated cells called sterigmata produced on laria was found to have a good antimicrobial activity at even less the ends of conidiophores. Fungi are identified by using the reference book on “Illustrated Genera of Imperfect Fungi” fourth Edition by H. L. Barnett and Barry B. Hunter. Consid- Table 1 e Antibacterial activity of the crude extract of ering all these characters isolated organism was identified as Curvularia sp. Aspergillus sp. Zone of inhibition in mm m m m m 4.7. Amount of secondary metabolite extracted from Concentration 25 g50g75g 100 g Aspergillus sp., per batch Staphylococcus aureus 14 16 20 22 Streptococcus mutans 14 16 16 18 Lactobacillus casei 10 11 11 12 Lactobacillus acidophilus 17 18 18 20 Volume of the Amount of compound Enterococcus faecalis 23 25 26 27 media inoculated (L) obtained (g) Bacillus megaterium 20 22 23 27 Xanthomonas campestris 12 12 13 14 2L 1g Escherichia coli 10 11 12 12 666 journal of pharmacy research 6 (2013) 663e666

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