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Method for screening alpha-amylase inhibitor

An amylase inhibitor and amylase technology, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of unclear active ingredients, difficult screening, time-consuming and laborious, etc., and achieve intuitive screening results, fast screening, Effect with high sensitivity and specificity

Active Publication Date: 2016-08-10
SHANGHAI UNIV OF T C M
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the various in vivo and in vitro experimental screening methods adopted in the prior art have relatively high requirements on the purity and dosage of the test compound, and there are defects such as difficulty in screening and high cost.
In particular, the active ingredients of natural substances are usually not clear enough, and most of the active effects are produced by the synergy of multi-components and multi-targets. When screening α-amylase inhibitors derived from natural substances using existing screening methods , there will be more difficulties, not only time-consuming and laborious, but also complicated and inefficient

Method used

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  • Method for screening alpha-amylase inhibitor
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  • Method for screening alpha-amylase inhibitor

Examples

Experimental program
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Effect test

Embodiment 1

[0034] 1. Solution preparation

[0035] Preparation of enzymatic reaction buffer: Dissolve 302.5mg tris and 330mg anhydrous calcium chloride in water, adjust the pH to 7.5 with hydrochloric acid, and then add water to make the volume up to 100mL, and the pH is 7.5, three The enzymatic reaction buffer with a molar concentration of hydroxymethylaminomethane of 25 mmol / L is stored in a refrigerator at 2 to 4°C for later use.

[0036] Preparation of substrate solution: 312mg NaH 2 PO 4 ·2H 2 O and 716mg Na 2 HPO 4 ·12H 2 Dissolve O in 100mL of deionized water and adjust the pH to 6.9 to obtain a phosphate buffer solution with a pH of 6.9 and a phosphate concentration of 20mmol / L; take 1g of soluble starch and dissolve it in 100mL of the above phosphate buffer solution and place it in a 95℃ water bath After heating for 4 hours, let it cool and centrifuge at 8000 rpm for 10 minutes. The supernatant obtained is the substrate solution of phosphate buffer solution containing 1wt% soluble st...

Embodiment 2

[0043] The difference between this embodiment and embodiment 1 is that the substrate solution used is a phosphate buffer solution with a pH of 6.9 and containing 0.05 wt% soluble starch and a phosphoric acid with a pH of 6.9 and 1 wt% soluble starch. The salt buffer solution, and the rest of the solutions are the same as described in Example 1.

[0044] Spot the test solution on two thin-layer plates (abbreviated as: A plate and B plate), and each plate was spotted at 3 points in parallel, and the amount of sample was 1 μL; the methanol solvent was evaporated and immersed in In the above substrate solution; take out, evaporate the solvent, and then immerse in the α-amylase solution, carry out the enzymatic reaction at 37°C for 30 minutes; after the reaction is completed, take out the thin-layer plate, evaporate the solvent, and then immerse in the color development Color reaction in the reagent solution; inspection under visible light, the inspection result is as figure 2 Shown:...

Embodiment 3

[0046] This embodiment is different from Embodiment 1 only in that the iodine content in the developer solution used is 0.1 mg / mL and 20 mg / mL, respectively, and the rest of the solutions are the same as those described in Embodiment 1.

[0047] Spot the test solution on two thin-layer plates (abbreviated as: A plate and B plate), and each plate was spotted at 3 points in parallel, and the amount of sample was 1 μL; the methanol solvent was evaporated and immersed in In the substrate solution; remove and evaporate the solvent, then immerse in the α-amylase solution, and carry out the enzymatic reaction at 37°C for 30 minutes; after the reaction is completed, remove the thin-layer plate, evaporate the solvent, and then immerse in the above-mentioned color development Color reaction in the reagent solution; inspection under visible light, the inspection result is as image 3 Shown: all sample points of the test solution on the A and B plates have blue spots on a white or yellow or b...

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Abstract

The invention discloses a method for screening an alpha-amylase inhibitor. The method comprises: preparing an enzymatic reaction buffer solution, a substrate solution, an alpha-amylase solution, a color-developing reagent solution, and a to-be-tested product solution for later use; performing sample application of the to-be-tested product solution on a thin layer plate; allowing a solvent to be volatilized or using a proper developing solvent for developing, performing air-drying, and soaking the obtained product in the substrate solution; taking the obtained product out, allowing the solvent to be volatilized, soaking the obtained product in the alpha-amylase solution, and carrying out an enzymatic reaction at 5-50 DEG C for 10-60 min; after the reaction is completed, taking the obtained thin layer plate out, allowing the solvent to be volatilized, soaking the obtained product in the color-developing reagent solution for a color-developing reaction; and viewing the obtained thin layer plate in visible light. Blue spots which appear in the white or yellow or brown background are the alpha-amylase inhibitor. The invention provides the method which enables the alpha-amylase inhibitor to be conveniently fast screened and provides an ideal approach for screening active ingredients containing the alpha-amylase inhibitor from natural substances.

Description

Technical field [0001] The present invention relates to a method for screening α-amylase inhibitors, in particular, to a method for screening α-amylase inhibitors using thin-layer chromatography-bioautography technology. Background technique [0002] Diabetes is an endocrine and metabolic disease caused by insufficient absolute or relative secretion of insulin in the body. It has the characteristics of high fatality rate, high disability rate and high medical expenses. It is the third most serious threat to human beings after tumors and cardiovascular and cerebrovascular diseases. Healthy chronic non-communicable diseases. At present, clinical hypoglycemic drugs include sulfonylureas, biguanides, insulin sensitizers, non-sulfonylurea insulin secretagogues, glucosidase inhibitors, etc. Among them, α-glucosidase inhibitors inhibit the small intestinal epithelium by competitive The action of glycosidase on the chorion reduces the degradation of carbohydrates, delays the digestion a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/90
Inventor 谷丽华江悦侴桂新王峥涛
Owner SHANGHAI UNIV OF T C M
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