The objective of the present study was screening of 3-hydroxy-3- methyl glutaryl Co A (HMG CoA) reductase inhibitor producing marine actinomycetes. A total of 65 morphologically different actinomycetes were screened for HMG CoA reductase inhibitor production in a two stage submerged fermentation and evaluated for HMG CoA reductase inhibitor activity by agar diffusion and thin layer chromatography technique using lovostatin as a control. Among 65 marine Actinomycete strains, only one strain produced HMG Co A reductase inhibito

SRILAKSHMI, SANKAR, PRABHAKAR,LAKSHMINARAYANA, SRINU, PHANI BHUSHAN,MOGES,

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Asian Journal of Pharmaceutical Research and Health Care (AJPRHC)

JPRHC Research Article JPRHC       January 2010        Volume 2             Issue 1           66 -74      SCREENING OF HMG CO A REDUCTASE INHIBITOR PRODUCING  MARINE ACTINOMYCETES  *1M. SRINU, 1G.V.PHANI BHUSHAN.,1FELEKE MOGES, 1J. SRILAKSHMI, 1G SANKAR, 1T. PRABHAKAR, 2 K.LAKSHMINARAYANA.  For author affiliations, see end of text  This paper is available online at www.jprhc.com    ABSTRACT:  The objective of the present study was screening of 3-hydroxy-3- methyl glutaryl Co A (HMG CoA) reductase inhibitor producing marine actinomycetes. A total of 65 morphologically different actinomycetes were screened for HMG CoA  reductase inhibitor production  in a two stage submerged fermentation and evaluated for HMG CoA  reductase inhibitor activity  by agar diffusion and thin layer chromatography technique using lovostatin as a control. Among 65 marine Actinomycete strains, only one strain produced HMG Co A reductase inhibitor.  Key words:    Lovastatin, Marine actinomycetes, HMG Co A reductase inhibitor                    JPRHC Research Article JPRHC       January 2010        Volume 2             Issue 1           66 -74     INTRODUCTION  Hypercholesterolemia involves heterogeneous disorders of lipid metabolism characterized by elevated levels of plasma total cholesterol and low density lipoprotein (LDL) derived cholesterol. Cholesterol enters the body in two ways i,e. absorption from diet or endogenous biosynthesis. The interference of either process would provide an effective means of lowering plasma total cholesterol. 3-Hydroxy- 3 -methyl glutaryl Co enzyme A (HMG Co A) reductase, a rate limiting enzyme in cholesterol biosynthetic pathway, was considered a promising target of inhibition. Structurally related compounds such as compactin (ML 236B)  and mevinolin (monacolin K) were discovered from fungal culture broths as potent and specific inhibitors of HMG Co A reductase 2,3,4,5 Since then, the derivatives  lovastatin, pravastatin and mevinolin have been used clinically for the treatment of hypercholesterolemia 6  .  Statins are a class of organic molecules which posses polyketide structure and a hydroxyl naphthalene ring system with different side chains. Statins are produced as secondary metabolites by microorganisms like bacteria 9(Bacillus spp.), Fungi10, 11,4,12 (Aspergillus spp, Monascus spps.)And Actinomycete spp.13, 14 (Streptomyces spp, Actinomadura spp). The aim of the present study is to isolate actinomycetes from marine environment and screen for HMG Co A reductase inhibitor activity.                               MATERIALS AND METHODS   Isolation of marine actinomycetes: Marine water and sediment samples were collected from different places of Bay of Bengal, Visakhapatnam. Samples were processed on the same day of collection. One gram or one milliliter (1ml) of sample was suspended in 10 ml of sterile 5mM phosphate buffer (pH=7.0) and stirred for 1 min in a super mixer. A 10-fold serial dilution of the suspension was made. One ml of the suspension was spread on starch casein agar plate (g/l: Starch 19, Casein 0.3,KNO3  2, NaCl  2, K2HPO4  2 ,MgSO4.7H2O  0.05,CaCo3 0.02,FeSO4.7H20  0.01,  agar  20)   and  Oat meal agar (g/l: Oat meal 20, agar 20, trace salt solution 1ml) plate and incubated at 280C for 21 days. Starch casein agar and Oat meal agar were supplemented with Cycloheximide 50 µg/ml and 5µg/ml rifampicin to inhibit fungal and bacterial contamination respectively.  Identification of Actinomycetes  Actinomycetes were recognized by their characteristic tough leathery colonies that adhered to the agar surface, branched vegetative mycelia, and when present, aerial mycelia and spore formation. Actinomycete colonies were characterized morphologically and physiologically following methods given in the International streptomyces Project (ISP) .Actinomycete isolate SS16/4 was maintained on yeast extract-malt extract agar slants at 280C.            Screening of HMG Co A reductase inhibitors  The selected isolate SS16/ 4 was   grown on YEME (ISP 2 media) agar slant for 5-7 days. 5 ml of a spore suspension  was added  to a 100 ml shake flask JPRHC Research Article JPRHC       January 2010        Volume 2             Issue 1           66 -74    containing 20 ml of the seed medium (for every liter: glucose 10 g, peptone 2g, soy protein 4 g and KH2Po4 1 g ,PH 7.0 +_0.2). The seed culture was incubated at 270 C for 24 to 48 hrs in a rotary shaker at 200 to 220 rpm.  2to 5 ml of the seed culture was  transferred to a 250ml shake flask containing 50 ml of the fermentation medium (per liter: glucose 15 g, peptone 5 g, corn steep powder (C.S.P.) 5 g and soybean meal 4 g, KH2PO4  1 g, PH 7.0+.0.2). The flasks were incubated at 270C for 5-7days on a rotary shaker at 200 rpm   Extraction After incubation, production media was centrifuged at 1500 rpm for 15 min at 100C in cooling centrifuge, to separate fermented broth (supernatant) and mycelium.  Before extraction , supernatant  obtained by centrifugation was acidified to  different  pH  values  of 3,5,7,9 with 1N HCl and extracted with ethyl acetate 15,12  The mycelia mass obtained by centrifugation was stirred with ethyl acetate at room temparture for 1hr, acidified to PH 3 with 1N HCl and extracted with ethyl acetate15, 12.  Extracted product was centrifuged at 1500 rpm and the organic phase was collected, and concentrated in rotary evaporator (Heidolph). Concentrated product was used for further studies16.  Thin Layer Chromatography(TLC)  TLC was used to detect the presence of HMG Co A reductase inhibitor in the extract.30 µl of concentrated product was applied to precoated silica gel plate. After the chromatogram was developed in the mobile phase (dicholarmethane and ethyl acetate 70:30 v/v) it was stained with iodine vapour and observed under UV light.  Three lovastatin standard spots were applied for comparison of the Rf values 16,17. The lovastatin standard compound was provided by Dr.Reddys laboratories. The lovastatin stock standard was prepared from the pure lactone form of the compound and   dissolved in acetonitrile at a concentration of 500µg/ml. 1:10 dilution of the stock solution in the  same diluent  was used as working standard 16. Bio-assay of HMG Co A reductase inhibitor using Neurospora crassa:-. Neurospora crassa was grown for 7-10 days on PDA slants at 280C; spores were harvested with sterile water. Twenty µl of spore suspension  of Neurospora crassa was transferred into a 40×40 mm size sterilized petri plate with PDA medium ( g/l : Potatoes  200g ,Dextrose  20g  ,agar  15g ). After solidification, wells were made using 8mm diameter borer18. Bio-assay of HMG Co A reductase inhibitor using Candida albicans: - Candida albicans   was grown for 12 hrs on PDA slants at 280C, spores were harvested with sterile water. 20µl of spore suspension of Candida albicans was taken into 40×40 mm size sterilized petri plate with PDA medium.  After solidification wells made by 8mm diameter borer19.     RESULTS AND DISCUSSION  The aim of study was to screen the HMG Co A reductase inhibitor producing marine actinomycetes. A total of 65 marine actinomycetes were screened for HMG Co A reductase inhibitor activity.   The fermentation broths obtained from 65 marine isolates were   examined by TLC. Lovastatin was used as the standard compound having HMG Co A reductase inhibitor activity. Only one of the isolate SS16/4 was detected by chromatography to contain spots comparable to the standard, shown in table-1.   JPRHC Research Article JPRHC       January 2010        Volume 2             Issue 1           66 -74    Table -1.  Rf values of standard and test samples in different solvents       Solvent       Rf of  standard  Test Organism       (Lovastatin)     SS16/4                 A            0.67       0.67                  B.              0.55       0.55                C            0.50                              .         0.50     .   A: Dichloromethane: ethyl acetate (70:30v/v) B.Ethyl acetate: Hexane: acetic acid (70:30:6) C.Acetonitrile:hexane.acetic acid (30:70:6)  Bio-assay method for determing of HMG Co A reductase inhibitor activity:     Figure-1  Bio-assay using   Neurospora crassa as test organism   Zone of inhibition of the SS16/4  mycelial  extract against N.crassa JPRHC Research Article JPRHC       January 2010        Volume 2             Issue 1           66 -74    The wells in the Petri plates were bored sufficiently apart to prevent coalescence of the zones of inhibition of the extract and lovastatin. Ethyl acetate was used as the control and it did not show any inhibition zone. The assay was carried out for the broth (extra cellular) and mycelium (intracellular) separately and inhibition zones were recorded. Zones of inhibition for SS16/4 are shown in fig 1. The optimum amount of lovastatin required to produce a zone of 10mm was 50µl(1mg/ml) and these  concentrations were  used to validate each N.crassa bioassay method. The period of incubation of N.crassa was critical for this test, as incubation beyond the specified period resulted in overgrowth and the boundaries of inhibition zone could not be clearly measured. Best results were obtained with plates incubated for 16-18 hrs at 280C. The zones of inhibition of the test extracts and standard were measured and recorded, as shown in table2.  Standard lovastatin inhibition zone    = 10 mm Ethyl acetate inhibition zone          = 0 mm  Table-2. The activity of mycelial  pellet and culture filtrate of SS16/4 against     N. crassa       Isolate        Inhibition zones (mm)  PH 3(Mycelium) PH 3(broth) SS16/4       20     10                 In the bioassay plate method using C.albicans as the test organism, lovastatin causes inhibition of growth at a concentration of 50µl (1mg/ml). The broth (extra cellular) and mycelium (intracellular) extracts of SS16/4 were assayed separately .The plates were incubated for 10-16 hrs and zones of inhibition were measured19 as shown in fig2 and table3. Standard lovastatin inhibition zone   = 15 mm Ethyl acetate inhibition zone          = 0 mm    JPRHC Research Article JPRHC       January 2010        Volume 2             Issue 1           66 -74      Figure-2 Bio-assay using Candida albicans as test organism      Table-3. The activity of mycelial pellet and culture filtrate of SS16/4 against                         C.albicans    Isolate      Inhibition zones in ( mm)                                  PH 3 (Mycelium) PH 3 (Broth) SS16/4          22     10               Zone of inhibition of the SS16/4 mycelial extract against C.albicans   JPRHC Research Article JPRHC       January 2010        Volume 2             Issue 1           66 -74       DISCUSSION  The aim of study was to screen HMG Co A reductase inhibitor producing marine actinomycetes. A total of 65 different marine actinomycetes were screened .Among them, only one strain SS16/4 showed positive results in TLC which was later confirmed by bioassay plate method.  Disregarding small modifications in fermentation conditions this study differs from those of Shindia 20 and Gunde Cimerman et al 21 in which they used methanol extraction of acidified broth followed by TLC analysis. Where as ethyl acetate extracts of acidified fermentation broth and mycelium were used for TLC and Bio-assay plate technique in our study.  By using ethyl acetate to extract HMG Co A reductase inhibitor from acidified fermentation broth and mycelium it was possible to extract a significant amount of the inhibitior. Further it was observed that when the mycelium was extracted at PH 3 large inhibition zones were observed against N.crassa and C.albicans (Figure-1 and 2). However, when the broth was extracted at different PH   values of  3,5,7,9, only small zones of inhibition wa observed for the broth with PH 3 (Table-2 and 3).  The nutritional and cultural conditions employed in this study were appropriate for screening HMG Co A reductase inhibitor production by the isolate SS16/4.Optimizing the medium may be necessary for improving the yields of the inhibitor for its characterization. ACKNOWLEDGMENTS  The authors are thankful to DBT, New Delhi for the financial support to carry out this work.                    JPRHC Research Article JPRHC       January 2010        Volume 2             Issue 1           66 -74      REFERENCES:  1. Takizawa.M., Colwell, R.R. and Hell, R.T.Isolation and diversity of actinomycetes in the Cheshapeake Bay .Applied and environmental Microbiology.1993; 59; 997-1002.   2. A.Endo, M Kuroda, Y.Ysujota; ML -236A, ML-236B and ML-236C, new inhibitors of Cholesterogenesis produced by Penicillium citrinin.J.Antibiot.29:1346-1348, 1976.  3. AG Brown, TC Smale, TJ King, R.Hassenkamp,RH Thompson. Crystal and Molecular structure of Compactin,a new antifungal metabolite from Penicillium brevicompactum.J Chem Soc Perkin trans  1976:1165-1169.  4. A Endo, Monacolin K,a new hypercholesterolemia agent produced by a Monascus species. J.Antibiot 32:852-854, 1979.  5.AW Alberts, J.Chen, G.Kuron,V.Hunt, J.Huff, .Mevinolin,a high potent competitive inhibitor of methyl glutaryl Coenzyme A reductase and Cholesterol lowering agent. Proc Natl Acad sci (USA) 77:3957-3961.  6. A Endo, The discovery and development of HMG Co A reductase inhibitors       .J.lipid. Res .33:1569-1582, 1992.  7. M Hanefeld, JP Deslypere, L Ose, PN Durrington. Efficacy and safety of   300 micrograms and 400 micrograms Cerivastatin once daily in patients with primary hypercholesterolaemia: amulticentre, randomized, double blind, placebo controlled study. J.Int.Med.Res 27:115-129, 1999.  8. S Omura.Splendid Gifts from microorganisms..2nd Ed .Tokyo research Center for biological function, the kitasato Institute, 1998.  9. Process for the preparation of HMG Co A reductase inhibitors from Bacillus species. US Patent.  10 Endo, A.1985, Compactin (ML-236B) and related compounds as Potential Cholesterol lowering agents that inhibit HMG Co A reductase.J.Med.Chem.28:401-405.  11. Matilde Manzoni, Mannela Rollini. Production and purification of statins from Aspergillus terreus strains. J.Biotechnology Techniques, 12:529-532, 1998.  12.. Hide taka Hatori,Bunji sato, Ikukosato,FR 901512, a novel HMG Co A reductase inhibitors produced by an Agonomycetous fungi No:14919. J.Antibio. 55:390-393, 2004  13.  Strains of Saccharothrix, Process for producing Pravastatin using the strains and isolation of HMG Co A reductase.US Patent.  14. Strains of Micropolyspora roseoalba CGMCC 0624 producing Pravastatin US Patent.  15. Matilde Manzoni, Manuela Rollini, Silvia Bergomi, Production and purification of statins from Aspergillus tereus strains, J.Biotechnology Techniques,12:529-532,1998.  16 Siamak M.Samiee, Nasrin Moazami, Saeid Haghighi Screening of Lovastatin Production by Filamentous fungi, Iran .Biomed.J.7 (1) :29-33,2003.  JPRHC Research Article JPRHC       January 2010        Volume 2             Issue 1           66 -74    17. Jacob Yashphe, Joseph davis, Yulin Peng, New microorganisms which convert Compactin to pravastatin, J.Actinomycetol 11:20-25, 1997.  18 .M.Sitaram Kumar, Pallapothu Mahendra Kumar, A rapid technique for screening of lovastatin producing strains of Aspergillus tereus by Agar plug and Neurospora crassa bioassay, J.Microbiological methods 40,99-104,2000.  19 .Rapid screening of Aspergillus terreus mutants for overproduction of lovastatin,World Journal of Microbiology and Biotechnology 21:123-125,2005.  20. Shindia, A.A. (1997) Mevinolin production by some fungi. Folia Micrbiol.   42:477-480  21. Gunde-Cimerman, N., Friedrich,J., Cimerman, A.Benicki,N.,(1973) Screening of   fungi for the production of an inhibitor of HMG CoA  reductase; Production of mevinolin by the fungi of the genus Pleurotus. FEMS Microbiol Lett.111:203-206.  AUTHOR AFFILIATIONS:  1Pharmaceutical Biotechnology Division, A.U. College of Pharmaceutical Sciences, Andhra University. 2Department of Botany, College of Science and Technology, Andhra University, Visakhapatnam-530003, Andhra Pradesh, India.  ADDRESS FOR CORRESPONDENCE:  Tel.:+919989547028, Email-srinumeesala78@gmail.com     

SCREENING OF HMG CO A REDUCTASE INHIBITOR PRODUCING MARINE ACTINOMYCETES

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SCREENING OF HMG CO A REDUCTASE INHIBITOR PRODUCING MARINE ACTINOMYCETES

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