Isolation And Purification Of ?-glucosidase Inhibitor Produced By HBUM 174625

Diabetes Mellitus is a common,chronic metabolic disease.With the improvement of living standards and changing lifestyles,the number of diabetic patients has increased extremely.Alpha-glucosidase inhibitors?AGIs?are a class of oral hypoglycemic drugs that are used to delay the absorption of intestinal sugars and to treat diabetes.The aim of our research is to discover new?-glucosidase inhibitor from endophytic actinomycetes.The endophytic actinomycetes strain HBUM 174625 of medicinal plants,which was obtained from the early stage screening and possessed higher inhibition rate of alpha-glucosidase,was used to this study.The results of amplification of the polyketone enzyme?PKS-II?gene and the non-ribosomal peptide synthesis enzyme?NRPS?gene indicate that the strain 174625 has the potential to synthesize polyketides and non-ribosomal peptide compounds.Four kinds of fermentation media were used to determine which one is fit for production of?-glucosidase inhibitor by strain HBUM 174625 and the best one is 1%of glucose,2%of soluble starch,1%of soybean powder,1%of yeast powder,KH₂PO₄ 0.1%,MgSO₄ 0.05%,CaCO₃ 0.1%,pH 7.0-7.5.The fermentation conditions of HBUM 174625 were further optimized by response surface methodology.Two factors which had significant effect on?-glucosidase inhibitor production were determined as soluble starch and yeast powder by Plackett-Burman design.The Optimal medium is 1%of glucose,1.7%of soluble starch,1%of soybean powder,1.7%of yeast powder,KH₂PO₄ 0.1%,MgSO₄ 0.05%,CaCO₃ 0.1%,pH7.0-7.5.The alpha-glucosidase inhibitory activity of the strain HBUM 174625 in the optimized fermentative medium was increased by 35%over the initial medium.Thirty litres whole broth of strain HBUM 174625 were obtianed by batch fermented with optimum medium and was added equal volume of methanol and centrifuged after 12 hr.The supernatant was concentrated under reduced pressure to afford an aq.soln,and the aq.soln was extracted three times with isovolumetric ethyl acetate.The ethyl acetate phase was concentrated to obtain 20g of crude extract.Isolation and purification were carried out using normal phase silica gel column chromatography,Sephadex LH-20 gel column chromatography and Biotage Flash chromatography.Finally,two compounds,named D3-1and F2-1,were obtained.Compound D3-1 and F2-1 were identified to uracil and 4',5,7-trihydroxy isoflavones?genistein?by ¹H-NMR?¹³C-NMR?HMQC?HMBC and ESI-MS.The inhibition rate of?-glucosidase of compound F2-1 was 50%at the concentration of0.42mg/mL.