Raja sekhar reddy Athoor et al. / Journal of Pharmacy Research 2012,5(1),391-393 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Evaluation of anti-microbial and anti-cancer activity of secondary metabolites from marine actinomycetes

Raja sekhar reddy Athoor1* and A. Janardhan2 1 Department of Molecular Biology, School of Life Sciences, University of Skovde, Sweden 2 Department of Virology, College of Sciences, Sri Venkateswara University, Tirupati, India Received on:20-09-2011; Revised on: 15-10-2011; Accepted on:10-12-2011

ABSTRACT The actinomycetes culture was isolated from the marine environment and the secondary metabolites from this strain were tested for antibacterial activity. All the pathogenic strains used in the study were inhibited at 50µl of crude extract, among the strains E.coli (1.8cm) showed the maximum zone of inhibition and the Pseudomonas (0.6cm) showed minimum zone of inhibition at 50µl concentration. The anticancer activity against the cancer cell lines was also checked. The anticancer activity of the secondary metabolite of actinomycetes was evaluated on HeLa (human cervical cancer) cell line. To determine in vitro cytotoxicity, different concentrations of secondary metabolite extract were tested on HeLa cancer cell line by 3-(4, 5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. Secondary metabolite showed a significant antiproliferative activity and a dose dependent effect was observed. Minimum inhibition of 19% was shown by secondary metabolite at concentration 1µg/ml and maximum inhibition (98%) was observed at 9 µg/ml. The in vitro cytotoxicity assay on human cervical cancer cell line (HeLa) revealed that the secondary metabolite had the strongest cytotoxicity with IC50 of 4.9 µg/ml.

Key words: Antibacterial activity, actinomycetes, HeLa cell line, MTT, and cytotoxicity.

INTRODUCTION Some microorganisms produce secondary metabolites which may have shown morphology of the cells. Spore chain morphology was identified by cover antimicrobial activity acting against certain pathogenic microorganisms. slip culture technique. Actinomycetes isolates were biochemically charac- These metabolites, otherwise known as bioactive substances, are profoundly terized by carbohydrate fermentation test [7]. used as and may be effective against infectious diseases such as HIV-1[1]; conditions of multiple bacterial infections (penicillin, Production and extraction of secondary metabolites cephalosporines, , and vancomycin); or neural tube defects The actinomycetes strains were cultured in fermentation broth (Starch 10g, and neuropsychiatric sequelae [2, 3]. Some drugs have also been found to be Yeast extract 4g, Peptone 2g and sea water 1L) for production of secondary useful against carcinomas (, , and metabolites. Flasks were incubated on rotatory shaker at 220rpm for 7days staurosporin), risk of coronary heart disease, or may act as immune- at 250C. The broth was then centrifuge at 10000rpm for 15min to remove suppressants (cyclosporin) to aid in organ transplantation [4] thus making the actinomycetes culture and the secondary metabolites in the broth were the microbial secondary metabolites an enormous source of pharmaceutical extracted with ethyl acetate. These extract was subjected to rota evaporator importance. In the present study we mainly focused on the cancer studies. to remove the ethyl acetate followed by lyophilization. Finally crystallas Cancer is one of the most life-threatening diseases with more than 100 were formed, these crystals were dissolved in de ionized water and used to different types. Due to lack of effective drugs, expensive cost of chemo- check the antimicrobial activity and anti-cancer activity. therapeutic agents and side effects of anticancer drugs, cancer can be a cause of death. Therefore efforts are still being made for the search of effective Antimicrobial activity naturally occurring anticarcinogen that would prevent, slow or reverse can- The antibacterial activity was tested against Pseudomonas, Escherichia cer development. Plants have a special place in the treatment of cancer [5]. coli, Klebsiella, Staphylococcus, and Bacillus. The above bacterial strains Cancer is the cause of more than six million deaths each year in the world. In isolated from clinical samples. Their cultural characteristics and morpho- 2001, about 1,268,000 new cancer cases and 553,400 deaths were reported logical features were reconfirmed and also subjected to standard biochemi- in the United States [6]. cal tests [8, 9] before experimentation. The test organisms were maintained on nutrient agar slants. The isolated extracts were screened for antibacterial MATERIALS AND METHODS: activity by agar-well diffusion method with a standard disk. Dif- ferent concentrations of curde extract (5, 10, 20, 30, 40, 50µl) was added to Source of Actinomycetes the wells to check the antimicrobial activity. The actinomycetes strain was isolated from the mangrove soil collected from Kakinada, Andhra Pradesh, India. Starch casein agar was used for the Cytotoxicity assays isolation of actinomycetes. HeLa cervical cancer cell line was used for the determination of cytotoxic activity NOVEL compound. The HeLa cells were grown in a 96-well plate Identification of in Delbucco’s Minimum essential medium (DMEM/F12) (Gibco Laborato- The colony of actinobacteria isolates were observe under a high power ries) supplemented with 10% fetal bovine serum(FBS) (Gibco Laborato- magnifying lens and colony morphology was noted with respect to colour, ries) and antibiotics (streptomycin, penicillin-G, kanamycin, amphotericin mycelium, size, shape, pigment. Gram staining was performed to check the B). Cells were seeded in 96-well plates at the density of 1000 cells/well in

100 µl medium and incubated at 37ºC for 24 hour in 5% CO2 for the forma- *Corresponding author. tion of confluent monolayer. The monolayer of cells in the plate was ex- Raja Sekhar Reddy Athoor posed to various dilutions of the secondary metabolite extract (20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1µg) and incubated at 37ºC for 24 hour in 5% CO2. After Department of Molecular Biology 24 h incubation, 20 µl of MTT reagent was added to each well and incu- School of Life Sciences bated for additional 4 h. Then 100 µl of DMSO solution was added to each University of Skovde,Sweden well to solubilize the formazan crystals. The cell viability was measured using MTT assay (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) with MTT (5 mg/ml) and DMSO. This tetrazolium salt is meta-

Journal of Pharmacy Research Vol.5 Issue 1.January 2012 391-393 Raja sekhar reddy Athoor et al. / Journal of Pharmacy Research 2012,5(1),391-393 bolically reduced by viable cells to yield a purple insoluble Formozan [10] product measured at 570nm spectrophotometrically. Controls were main- tained throughout the experiment (untreated wells as negative control). The assay was performed in triplicate for each of the extracts. The mean of the cell viability values was compared to the control to determine the effect of the extract on cells and % cell viability was plotted against concentration of the novel drug. Table 1 Characterization of Actinomycetes Results: S.No Characterization Result

1 Aerial mycelia colour Orange Source of actinomycetes 2 Reverse side pigment White The strains obtained from the 3 Grams staining Gram Positive Fig 2A Hela cell before drug treatment Fig 2B Formozan crystals in control. 4 Carbon Utilization test mangrove soil were identified as actinomycetes by Nanomura Xylose Positive Rhamnose Positive key (Table 1). Glucose Positive Fructose Positive Antimicrobial Activity Sucrose Positive All the bacterial strains shows zone of inhibition at 50µl con- centration of crude extract. The Escherichia shows the maxi- mum zone of inhibition fol- lowed by Staphylococcus, Ba- cillus, Klebsiella, and Pseudomonas (Fig 1 A, B, C, Fig 2C Formozan crystals in 4.9 µg drug Fig 2D Complete cell growth inhibition of HeLa cell lines at 10, 15µg drug treated well. D, E and Table 2) treated well.

Fig.1A Zone of Inhibition of Pseudomonas

Fig.1B Zone of Inhibition of Klebsiella Fig.1C Zone of Inhibition of Bacillus Fig 2E Formozan crystals with HeLa cells. was differing with respect to the concentration of secondary metabolite added to the wells (Fig 2 A, B, C, D, E).

Fig.1D Zone of Inhibition of Stahpylococcus Fig.1E Zone of Inhibition of E.coli Table 2 Zone of inhibitions of different organisms at different concentrations of compounds

S.No Organism Zone of inhibition (in Cm) Zone of inhibition* Concentration of compound (in Cm) 5µl 10µl 20µl 30µl 40µl 50µl 50µl concentration

1 Pseudomonas 0.0 0.0 0.0 0.0 0.0 0.6 0.5 2 E.coli 0.5 0.7 0.9 1.2 1.4 1.8 2.0 3 Klebsiella 0.0 0.0 0.0 0.0 0.0 0.8 1.0 4 Staphylococcus 0.4 0.7 0.8 1.1 1.2 1.3 1.3 5 Bacillus 0.0 0.0 0.0 0.0 0.0 1.0 1.2

*standard antibiotic

Cytotoxic assay The in vitro cytotoxicity assay on human cervical cancer cell line (HeLa) revealed that the secondary metabolite had the strongest cytotoxicity with Graph 1. Plot of percentage of cell viability Vs concentration of drug in (µg/ml) shows the

IC50 of 4.9 µg/ml (Graph-1). The amount Formozan formation in each well IC50 at 4.9 µg.

Journal of Pharmacy Research Vol.5 Issue 1.January 2012 391-393 Raja sekhar reddy Athoor et al. / Journal of Pharmacy Research 2012,5(1),391-393 DISCUSSION 5. Philip, Deepa, Kaleena P K, and K Valivittan.. “In vitro cytotoxicity This study evaluated the antibacterial activities and cytotoxicity of the and anticancer activity of sansevieria roxburghiana.” 2011, 3(3): 2-4. secondary metabolites of actinomycetes. The secondary metabolites from 6. Izevbigie EB. Discovery of Water-Soluble Anticancer Agents (Edotides) our actinomycetes shown antibacterial activity against some bacterial patho- from a Vegetable Found in Benin City, Nigeria. Exp. Biol. and Med. gens isolated from the human fluid samples. These results correlated with 2003, 228:293-298. the previous findings in which the antimicrobial activity of Streptomyces 7. Nonomura, H.. Key for classification and identification of 458 species isolated from soil [11]. Earlier some marine actinomycetes isolated from Bay of the Streptomycetes included in ISP. J. Ferment. Technol., 1974, of Bengal (Coast of Inida) were screened for antagonistic and antimicrobial 52(2) : 78 - 92. activity against pathogenic bacterial and fungi [12]. Antimicrobial activities 8. Noel R. Krieg & John H. Holt, Bergey’s Manual of Systematic Bacteri- of mairne from the water of South East India were also studied by ology, 1984, 4 volumes. Anand et al 2006 [13]. The in vitro cytotoxicity of secondary metabolites 9. Peter H. and Sneath A., Bergeys manual of systematic Bacteriology, showed a significant antiproliferative activity on HeLa (human cervical 1986. 2:1104-1154. 10. Mosmann T. Rapid colorimetric assay for cellular growth and survival: cancer) cell line and a dose dependent effect was observed (IC50 - 4.9 µg/ml). Some Bioactive compounds were isolated and found selectively cytotoxic application to proliferation and cytotoxicity assays. J. Immun. Meth- against lung and colon cancer cell lines as well as melanoma. Interestingly, ods. 1983, 65 (1-2): 55–63. the compound exerts preferential antiproliferative effects in colon cancer 11. Sahin N, Ugur A. Investigation of the antimicrobial activity of some cell lines with defective p53 systems [14]. In recent studies of the biological Streptomyces isolates. Turk. J. Biol. 2003, 27:79-84. activity compounds revealed that a few strains had anticancer activity: 12. Gandhimathi R, Arunkumar M, Selvin J, Thangavelu T, Sivaramakrishnan Eb6, with an IC50 value of 2.8 µg/ml; Cc1 , with an IC50 value of 6.4 µg/ S, Kiran GS, Shanmughapriya S, Nataraj aseenivasan K. Antimicrobial ml[15] and Sm6, with an IC50 value of 5.5 µg/m4 l against colon cancer cells potential of sponge associated marine actinomycetes. J. Mycol. Méd. (HCT-116) [16] 2008, 18:16-22. 13. Anand Prem T, Bhat AW, Shouche YS, Roy U, Siddharth J, Sarma SP. REFERENCES Antimicrobial activity of marine bacteria associated with sponges from 1. Cragg, G.M.; Newman, D.J. Medicinals for the millennia: the historical the waters off the coast of South East India. Microbiol. Res. 2006, record. Ann. N.Y. Acad. Sci. 2001, 953, 3–25. 161:252-262. 2. Finglas, P.M.; Wright, A.J.; Wolfe, C.A.; Hart, D.J.; Wright, D.M.; 14. E. Erba, D. Bergamaschi, S. Ronzoni, “Mode of action of thiocoraline, Dainty, J.R. Is there more to folates than neural-tube defects? Proc. a natural marine compound with anti-tumour activity,” British Journal Nutr. Soc. 2003, 62,591–598. of Cancer, 1999, 80, 971–98. 3. Berdy, J. Bioactive microbial metabolites. A personal view. J. Antibiot. 15. Matz C, JS Webb, PJ Schupp, SY Phang, A Penesyan, S Egan, P Steinbert 2005, 58, 1–26. & S Kjelleberg.. Marine Biofilm bacteria evade eukaryotic predation by 4. Ruiz, B.; Chávez, A.; Forero, A.; García-Huante, Y.; Romero, A.; Sánchez, targeted chemical defense. PLoS ONE 2008, 3(7), 2744 M.; Rocha, D.; Sánchez, B.; Rodríguez-Sanoja, R.; Sánchez, S.; Langley, 16. Hellio C, D De La Broise, L Dufossé, Y Le Gal & N Bourgougnon. E. Production of microbial secondary metabolites: regulation by the Inhibition of marine bacteria by extracts of macroalgae: potential use carbon source. Crit. Rev. Microbiol. 2010, 36, 146–67. for environmentally friendly antifouling paints. Marine Environmen- tal Research, 2001, 52(3): 231-247.

Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.5 Issue 1.January 2012 391-393