| Diabetes is one of the most serious chronic disease of the world. Postprandial hyperglycemia is the major risk factors of diabetes, so the most effective method preventing diabetes deterioration is to control the glucose level of blood. The main function of ?-glucosidase is to hydrolyze polysaccharides; hence, inhibiting the activity of ?-glucosidase may be an effective way to treat pre-diabetic and slow down the progress of diabetes. Several synthetic ?-glucosidase inhibitors have been developed and applied in the past years. Due to the side effects of these synthetic ?-glucosidase inhibitors, such as digestion disorders which related to trypsin, it is urgent to discover or design new inhibitors. At present, with the hot research of natural products, polyphenol compounds has become a hot topic of many scholars based on its good free radicals scavenging and antioxidant functions.Based on this, the work used inhibition assay, inhibition kinetic experiments and fluorescent spectrum scanning experiments and computer simulations to study the mechanism of a series of phenolic acids and tannic acid with trypsin and ?-glucosidase, to find the characteristic changes of enzymes and inhibitors and to explore the interaction between them. The main research contents and corresponding results are as follows:(I) We obtained the half inhibitory concentration of 16 kinds of phenolic acids through inhibition assay. The inhibitory effect of gallic acid, caffeic acid and 3, 5-2 nitro salicylic acid were relatively good. The inhibition types of gallic acid, caffeic acid, 3, 5-2 nitro salicylic acid, the aminosalicylic acid and 5-sulfosalicylic acid to trypsin were non competitive. It was found that the binding of the five phenolic acids and trypsin could cause the fluorescence quenching of trypsin, and both of them were static quenching. In the process of interaction between the test phenolic acids and trypsin, Gly38, Ser39, Arg66, Ile72, Asp73, Ile89, Thr241, Asn245, and Phe81 played important roles in the docking site.(II) Through inhibition rate experiments, we obtained 14 kinds half inhibitory concentrations of phenolic acids on ?-glucosidase, and the inhibitory effect of aminosalicylic acid, caffeic acid and p-coumaric acid were better, especially the aminosalicylic acid. The inhibition types of phenolic acids to ?-glucosidase were mixed inhibition type or non competitive inhibition type. The fluorescence spectra showed that the interaction between caffeic acid, ferulic acid, 2, 4-dihydroxy benzoic acid and ?-glucosidase could cause the fluorescence quenching of ?-glucosidase, and both of them were static quenching. Quantum calculation indicated that the test phenolic acid formed strong hydrogen bonding interaction with Trp391 and Asp392. And the test phenolic acid interacted with Trp391, Trp710, Trp715, Trp789, Phe385, Phe389, Phe444 and Phe786 by hydrophobic effect.III We showed that the anti-?-glucosidase activity of tannic acid was higher than that of acarbose, while its anti-trypsin activity was significantly lower than that of the trypsin inhibitor from soybean. The inhibitory pattern of tannic acid toward two tested enzymes was a mixed competitive and noncompetitive inhibition tapy. Tannic acid could bind the enzymes to form new complexes, presenting there were a strong static fluorescence quenching. The thermodynamic parameters indicated that the main driving forces between tannic acid and both the enzymes were the hydrophobic interaction followed by the electrostatic interaction.With the above research results, it could be said that the inhibitory effect of tannic acid on ?-glucosidase was much better than the tested phenolic acids, and the inhibitory effect of tannic acid on trypsin was almost the same with the tested phenolic acids. Now, people generally focused on phenolic acids and flavonoids this small molecule polyphenols, and neglected tannic acid this structure complexed polyphenols. Through this work, people re-recognized the functions of tannic acid, and tannic acid based on its widely existence in diary food and medicinal plants, plus its strong anti-?-glucosidase ability and low inhibitory activity for trypsin, it was expected to become the composition of drugs to treat diabetes. |
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