The Isolation For Compounds With Inhibitory Activity Against Alpha-glucosidase From A Sponge-associated Bacillus Sp.

The microorganisms in the marine environment have specific metabolic pathways and defense mechanisms, which are with great novelty and diversity in comparison with those in land and freshwater. The difference leads to the distinctive chemical structures in their metabolites. In the exploitation of the marine environment and biological resources, the screening and isolation of natural active substances with specific chemical structures from marine microorganisms are the important directions. Alpha-glucosidase inhibitors, as the first-line drugs recommended in diabetes prevention and treatment guidelines in China, can inhibit the activity of alpha-glucosidase, slow down glucose production and absorption. Thereby, it reduces the postprandial glucose, adjusts blood sugar levels, reduces the stimulation of blood glucose on the pancreas, improves insulin sensitivity, and protects the function of the pancreas. The usage of alpha-glucosidase inhibitors can effectively prevent the occurrence and development of complications. However, alpha-glucosidase inhibitors are insufficiently developed so far, which results in few candidate compound available.In this study, alpha-glucosidase inhibitory activity of the crude extract from a marine bacterium, HY95, was measured. The classification and identification of the strains, optimization of fermentation conditions, the extraction from fermentation culture, and the stability of active products were examined. Moreover, the active compounds with inhibitory effects against alpha-glucosidase were purified through the activity guided isolation. The main findings in this study are as follows:(1) The optimization for in vitro screening model of alpha-glucosidase inhibitory activity.The in vitro screening model of alpha-glucosidase inhibitory activity was optimized, to solve the instability of the measurement and the poor reproducibility of the samples. In this study, the in vitro screening model of alpha-glucosidase inhibitory activity was optimized as floowing: for treatment group,82μL PBS,40μL PNPG and3μL sample were mixed in the well on the 96-well plate, and were incubated at37℃for5min before25μL alpha-glucosidase solution was added. The optical densities on405nm of each testing mixture at incubation time of0,5,10,15,20,25,30, and40min were recorded. At45min,50μL Na2CO3was added into each well to stop the reaction. For controls,3μL acarbose instead of the sample was used as positive one while3μL DMSO instead of the sample was used as negative one. For blank group, only147μL PBS and3μL of the sample were mixed in the well. Inhibition rate (IR) was calculated based on the OD values of respective incubation time. The9inhibition rates at different incubation times were plotted to create the curve of inhibition rate vs. incubation time. The average of IR20min, IR25min, and IR30min was used as the IR of tested sample.Verification experiments showed that the inhibition rate after the optimization achieved better stability and reproducibility. For the curve of inhibition rate vs. incubation time, it not only reflects the activity changes with extention of incubation time, but also indicates activity stability of the tested samples, which could be one of the standards to judge the development potential of the samples, to decide whether it is deserved for further separation and purification. In addition, this curve is a new pattern to indicate the activity characteristic of a tested sample, and also a guide for marking the isolation and purification of the crude extracts.(2) The classification and identification of the active strain HY95.The test strain HY95was identified as Brevibacillus borstelensis based on the16S rDNA sequence analysis, cell morphology, and colony characteristics.(3) Fermentation conditions of strain HY95.In this study, four factors (incubation time, inoculum, incubation temperature, rotation speed) of culture conditions were examined in single factor test to study the growth conditions. And then, based on the alpha-glucosidase inhibitory activity and the production of the crude extract, the culture conditions were optimized.According to the experimental results, optimal culture conditions for strain HY95are as following:inoculum ratio is2.5%(V/V), shaker speed is130rpm, culture temperature is28℃and cultured time is60h. The culture medium based on Marine Broth (MB) was optimized as following:peptone5.00g/L, yeast extract1.50g/L, sodium chloride9.725g/L, magnesium chloride5.90g/L, magnesium sulfate (MgSO4·7H2O)3.24g/L, calcium chloride1.80g/L, potassium chloride0.55g/L, iron citrate0.10g/L, and the initial pH of7.5. (4) Isolation and identification of the active compounds produced by strain HY95.We launched a large-scale fermentation of strain HY95and obtained its crude extracts. Through bioassay guided separation and purification of the crude extract, a purified active compound was obtained. According to the NMR and mass profile, the compound was identified as the Cyclo(Tyr-Phe). The remarkable activity of the pure compound was verified with inhibition rate of53.72%±4.92%.