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Application of α-glucosidase in screening active natural medicine
α-Glucosidase is also called α-glucosidase (EC.18.104.22.168), and the system is named α-D-glucosidase. It has dual functions of hydrolysis and transglycosidation in the catalytic reaction of sugar. Hydrolysis can cleave α-1,4 glycosidic bonds from the non-reducing ends of α-glucoside, oligosaccharides and glucans to release glucose; transglycosidation can convert free glucose residues to α-1 , 6 glycosidic bonds are transferred to another glucose or maltose substrate to obtain non-fermentable isomaltose (referred to as IMO).
Since Japan screened α-glucosidase production strains from Aspergillus niger in the 1980s and was able to industrially produce enzyme preparations, α-glucosidase has played an increasingly important role in basic research and industrial production. At present, most of the α-glucosidase produced abroad are pure enzymes with high enzyme activity; while domestically, the main enzyme solution is crude enzyme solution with low enzyme activity. Moreover, there are no commercial α-glucosidase enzyme preparations for sale in China. The enzymes used in the production are all from a few foreign enzyme preparation factories, and the import prices are expensive, which leads to the high production cost of IMO, which restricts my country to a certain extent. The development of the IMO industry.
α-Glucosidase can catalyze the hydrolysis of α-1.4-glycosidic bonds and hydrolyze oligosaccharides such as maltose and sucrose in the small intestine. Inhibiting the activity of α-glucosidase can slow down the production and absorption of glucose, reduce the peak blood glucose after a meal, adjust the blood glucose level, and regulate the production of fat while regulating sugar metabolism. Alpha-glucosidase inhibitor drugs have become a research hotspot in medicinal chemistry in recent years. The traditional method of screening α-glucosidase inhibitors is to use free α-glucosidase and its substrate (4-nitrophenyl-α-D-glucopyranoside (PNPG)) and the screened inhibitor simultaneously Agent role.
The application of α-glucosidase in screening active natural medicines:
1. Immobilization of α-glucosidase: using trimethylol phosphate as a cross-linking agent and chitosan as a carrier to immobilize α-glucosidase;
2. Making inhibitor screening model: Load the above-mentioned immobilized α-glucosidase α-glucosidase into a chromatography column with a diameter of 0.8 cm and a length of 8 cm, and add potassium phosphate with pH 6.8 to the column Buffer, stored at 4°C to form a screening model;
3. Validation of the model: Use the representative α-glucosidase inhibitor acarbose to verify the validity of the screening model. Insert the lower end of the column tightly into a small tube with a piston, and insert a syringe into the upper end of the column. After pressing the buffer solution in the column to about 0.4cm from the top of the column, add 50μl of 4-nitrophenyl-α-D-glucopyranoside (0.116mol/L) and a certain amount of acarbose solution (250mg /L), shake gently to mix the solution at the top of the column, then continue to press the solution until the liquid level is level with the top of the column, close the bottom piston, and then put the column into the water bath and incubate at 37°C for 10 minutes After the reaction is complete, wash the column with 5ml pH6.8 potassium phosphate buffer solution, collect the washing liquid, dilute the eluent to a volume of 10ml, and measure the absorbance value at 400nm with an ultraviolet spectrophotometer; and add the above 50μl 4- Nitrobenzene-α-D-glucopyranoside, acarbose solution and 5ml buffer solution are diluted to 10ml as a blank, and the inhibition rate of inhibitor acarbose is calculated according to the absorbance data of the obtained reaction solution;
4. Calculation of inhibition rate I(%): I(%)=(ΔAE-ΔA)/ΔAE×100%, ΔAE=fix α-glucosidase and 4-nitrobenzene-α-D before adding inhibitor -The absorbance value after the reaction of glucopyranoside (deduct the corresponding blank); ΔA= the absorbance value after the reaction of immobilized α-glucosidase and 4-nitrophenyl-α-D-glucopyranoside ( Subtract the corresponding blank); C is the inhibitor concentration, C is the abscissa, and the inhibition rate I% is the ordinate. Make the regression curve of the inhibitor concentration C and the corresponding inhibition rate I%, and get the half inhibition rate at 50% inhibition rate Concentration IC50.
5. Use the immobilized α-glucosidase screening model to screen the α-glucosidase inhibitory activity of the water-soluble parts of the traditional Chinese medicine Rhizoma vulgaris, and screen the water soluble parts of Rhizoma tigris.
Desheng produces a variety of enzyme preparation products. The enzyme preparation products provided are mainly used as test materials for kits. In addition to α-glucosidase, there are glucose dehydrogenase and glucose oxidase, cholesterol esterase, cholesterol oxidase, and purine nucleosides. Phosphorylase, lactate dehydrogenase, etc. Friends in need can consult us and provide samples for customers.