α-glycosidase Inhibitor Derived From Marine Sponge-associated Microorganisms

Marine sponge-associated microorganisms are the important source of nature products for Pharmaceuticals. It is a potential way for developing new diabetes drug (α-glycosidase inhibitor).In this study, the α-glucosidase inhibitory activity screening model is built-up to search for the active crude extract from the microorgainism metabolite samples prepared from marine sponges in Friday harbor of US. The reaction system of the α-glucosidase inhibitory activity screening is performed on a96wells plates. The reaction condition, including the concentration of enzymes, is set at0.008U/mL, pH6.8,37℃, and wavelength405nm. Moreover, the screening model was optimized with reactant PNPG concentration10mmol/L, reaction time of25min and the solvent DMSO content of2%(V/V).Used Acarbose as a positive controlled sample,212crude extracts samples from marine sponge assiciated microorganisms were screened with the above optimized model. The results indicated that metabolites of19strains had high inhibitiory activity(>50%). Among them,5strains showed inhibitory effect higher than70%against α-glucosidase at concentration0.4,0.6,0.8mg/mL. The inhibitory effects was higher than that of Acarbose (58%) at concentration1.6mg/mL. As result, the strain HY936, with high inhibitory activity (79.3%) at concentration0.4mg/mL, was identified as Bacillus firmus by the means of16S rDNA sequence.The cultivating condition of strains was optimized for a higher production of active components. Single factor experimental design was performed to analysis the influence of cultivating time, rotating speed, inoculation volume. The orthogonal test L9(34) took peptone, yeast extract, and NaCl concentrations in the culture media as well as the pH value into account. After the optimization of culture conditions, the fermentation process using strains of Bacillus firmus was conducted to accumulate2.8g crude extract, which was seperated and purified through silica collumn of TLC or HPLC. Fr.1and Fr.4were obtained by using silicagel column and they have high inhibitory activity against α-glucosidase, the inhibitary rates of35.5%and19.6%, respectively. Then subfraction Ⅱ was isolated from Fr.1showed inhibitary rate of82.38%. Therefore, sub fraction II was subjected to GC-MS analysis and11kinds of compounds were found, including cyclic dipeptide and piperidone as the major components. The two kinds of substances provides potential clues for developing new a-glycosidase inhibitors. Attentions should be paid on the study of them in further.