Isolation,purification And Enzyme Kinetics Of Glucosidase Inhibitory Components From Chlorella Active Substances

Based on the current changes in living conditions and the increase in work stress,the number of people with diabetes continues to increase,especially in many aspects of type 2 diabetes,which is also a challenge.Therapies for treating diabetes are becoming more and more innovative.In addition to the nutritional and health benefits of chlorella,the use of chlorella as a raw material for bioenergy is also receiving attention in the field of renewable energy.With the gradual deepening of human research,its potential nutritional and medicinal value will be fully explored,and the development and utilization of chlorella resources will have a bright future.Studies have shown that the active substances in chlorella are beneficial to the human body,so the correlation activity against ?-glucosidase is studied.The experimental content mainly involves the crude extraction method of the active substance in the chlorella,the high-pressure hot water extraction,centrifugation,extraction,rotary evaporation,filtration,etc.,and the analysis of the crude extract sample containing the active substance.After purification by silica gel plate thin layer chromatography,the two phases and ratios of the developing agent are continuously explored to give a band and to determine the band containing the activity.In the separation and extraction of the active substance of chlorella,the ratio of the organic solvent and the extraction time were optimized.When performing thin layer chromatography separation,the selection and ratio of the developing agent are optimized.An initial test for the activity of the in vitro model enzyme inhibition reaction was carried out on the crude chlorella extract.High-performance liquid phase analysis of the chlorella active substance,and four compounds were isolated from the substance.The four compounds w ere tested for enzyme inhibitory activity,and one of the compounds having an a-glucosidase inhibitor was collected and tested for purity.The final target product has a purity of 96.5% and can be used for nuclear magnetic analysis.The results showed that the four compounds had different degrees of inhibitory activity on ?-glucosidase;the obtained C,H spectrum binding and mass spectrometry analysis indicated that the target product was partially degraded or contained other compounds.A method for quantitative analysis of substrates and their characteristic metabolites is established based on high performance liquid chromatography techniques to measure the rate of substrate consumption and product formation at different initial concentrations.The enzyme kinetic parameters Kmand Vmax were calculated by linear regression and curve fitting.The best conditions for the sample concentration,substrate concentration,enzyme concentration and reaction time in the enzyme reaction system are explored.The inhibito ry reaction of the active substance on the enzyme in the prepared chlorella sample is reversible,and the reaction of the sample to the chlorella is reversible.It is further explored whether this type of inhibition is a competitive or non-competitive type of reaction in a reversible reaction.According to the formula,the Km of the reaction between the enzyme and the substrate,and the Km of the reaction of the enzyme with the chlorella active substance and the substrate are determined.Studies in this experimental subject have shown that the active substance of chlorella contains an inhibitor of ?-glucosidase.The isolated and purified inhibitor inhibits the activity of the enzyme in an in vitro assay.However,the structure of the obtained target product remains to be further studied.