The culture was centrifuged (9000 rpm/20 min) and the supernatant used for extraction of secondary metabolites. The supernatant collected in a 250 ml flask was extracted by mixing 10% diaion HP-20 (Sigma) and shaking for 30 min on a magnetic stirrer. Then the flask contents were packed GW-572016 order in a glass column and washed with 15 ml distilled water. Finally, the
metabolites on diaion were eluted with 20 ml methanol. The collected methanol fractions were evaporated in a rotary evaporator (Heidolph, Germany), dissolved in DMSO and stored at – 20°C. Inhibition assay An enzyme agar solution containing β-glucosidase was prepared in 7 ml sodium acetate buffer with 0.07 g of agar powder dissolved at 80-100°C; followed by the addition of 1.2 ml of FeCl3 solution and 40 μl of enzyme β-glucosidase at 60°C (0.01 U/ml). The final volume was adjusted to 10 ml with the acetate buffer. An aliquot of 8–10 ml solution was poured into petri plates and allowed to set. The samples
selleck chemical – 5 μl of the extract – were spot inoculated with a micropipette on the surface of the agar plate and blow dried or air dried. Alternately, the samples can be loaded on sterile filter paper discs, dried and placed on the agar plate. The plates were incubated at room temperature for 15 min for primary reaction between the enzyme and inhibitor. Later on, 6–7 ml of esculin solution was added to cover the surface of agar and again incubated at room temperature for 30 min for enzyme-substrate reaction. In case, paper discs are used they have to be removed before adding esculin. Conduritol β-epoxide, an irreversible inhibitor, in concentrations 2.5, 1, 0.75, 0.50, 0.25, 0.10 and 0.05 μg was used as a positive control and DMSO without extract as negative control. 1-(3-aminopropyl)-imidazole
and 2-aminobenzimidazole were used as reversible inhibitor control, in concentrations 2000, 1000, 500, 100 and 50 μg. Clear zones of inhibition were recorded by measuring the zone size. A subset of 31 samples was also compared using this agar plate learn more method and TLC autographic method with or without developing the TLC plate. These experiments were repeated thrice with some extracts to check the reproducibility of the method. Acknowledgements The authors are thankful to Council of Scientific and Industrial Research (CSIR, India) for funding the work and Director CSIR-IMMT for the infrastructure Montelukast Sodium support. We gratefully acknowledge Dr. Tapan Chakrabarti, former Head and founder of Microbial Type Culture Collection – an International Depository Authority, Chandigarh, India, for critically examining the manuscript. We heartily thank Dr. B.P. Bag, Senior Scientist, CSIR-IMMT, Bhubaneswar, for providing us the imidazole derivatives used in the experiments. References 1. Asano N: Glycosidase inhibitors: updates and perspectives on practical use. Glycobiology 2003, 13:93R-104R.PubMedCrossRef 2. de Melo EB, Gomes AS, Carvalho I: α- and β-Glucosidase inhibitors: chemical structure and biological activity.