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Optimization method of hydrolysis conditions of [beta]-glucosidase on glycoside drugs

A technology of glucosidase and optimization method is applied in the field of optimization of hydrolysis conditions of glycosides by β-glucosidase, which can solve the problems of high market price, influence on biological activity, low content and the like, so as to improve the conversion of glycosides. rate effect

Inactive Publication Date: 2018-11-30
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Because the content of genipin, arctigenin and polydagenin in plants is very low, it is difficult to realize the direct extraction method, so the market price is very high, and the traditional acid hydrolysis method is easy to make the iridoid structure of genipin Destroyed by acid, affecting its biological activity

Method used

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  • Optimization method of hydrolysis conditions of [beta]-glucosidase on glycoside drugs
  • Optimization method of hydrolysis conditions of [beta]-glucosidase on glycoside drugs
  • Optimization method of hydrolysis conditions of [beta]-glucosidase on glycoside drugs

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Keep the three substrate concentrations of geniposide, arctiin, and polydatin constant, the reaction temperature is constant, and the reaction time is constant:

[0037] (1) Prepare different pH buffers

[0038] Liquid A is citric acid solution, weigh 2.101g of citric acid into 100ml of water and stir until dissolved; Liquid B is disodium hydrogen phosphate solution, weigh 5.361g of disodium hydrogen phosphate into 100ml of water and stir until dissolved, the following are liquids A and B Table of ph value of liquid;

[0039]

[0040] (2) Weigh 0.02g of β-glucosidase and 0.01g of the three substrates of geniposide, arctiin and polydatin into 1.5ml ep tubes;

[0041] (3) Take 1ml of the prepared buffer solution with different pH to dissolve the enzyme and substrate, and then react with 50μl enzyme and μl50 substrate of the same pH respectively;

[0042] (4) After reacting at 50°C for 1 hour, add 300 μl DNS, cook in boiling water for 10 minutes, dilute, and measure the...

Embodiment 2

[0051] Keep the pH of the buffer solution constant, the concentrations of the three substrates of geniposide, arctiin and polydatin constant, and the reaction temperature constant:

[0052] (1) Weigh 0.02g of β-glucosidase and 0.01g of the three substrates of geniposide, arctiin and polydatin into 1.5ml ep tubes, and add 1ml of sodium acetate buffer to dissolve;

[0053] (2) Take 50 μl of substrate and 50 μl of enzyme into ep tubes, do five reactions for each drug, and carry out the reactions in a 50°C water bath;

[0054] (3) At 1h, 2h, 4h, 6h, 8h, and 10h after the reaction, take out a reaction for each drug, and measure the absorbance value;

[0055] (4) After the reaction is over, add 300 μl of DNS to the reaction solution to terminate the reaction, then boil in boiling water for 10 minutes;

[0056] (5) Take 100 μl of the boiled reaction solution into a test tube, add 4900 μl of distilled water to dilute, and measure the absorbance value at A520nm;

[0057] Table of A52...

Embodiment 3

[0065] Keep the pH value of the buffer constant, the reaction time constant, and the reaction temperature constant:

[0066] (1) Weigh 0.02g of enzyme and 0.02g of substrate into ep tubes, and dissolve them with 1ml of sodium acetate buffer;

[0067] (2) Dilute the substrate concentration to 2%, 1%, 0.5%, 0.1%, and 0.005% respectively;

[0068] (3) Take 50 μl of enzyme and 50 μl of substrates of different concentrations, react at 50°C for 1 hour, add 300 μl of DNS, boil in boiling water for 10 minutes, dilute 50 times and measure the absorbance at A520nm;

[0069] Table of A520nm of geniposide reaction as a function of substrate concentration

[0070]

[0071] The table of A520nm of arctiin reaction as a function of substrate concentration

[0072]

[0073] Table of A520nm of polydatin reaction with substrate concentration

[0074]

[0075] Depend on Figure 8 , Figure 9 , Figure 10 It can be seen that the optimum substrate concentrations of geniposide, arctii...

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Abstract

The invention discloses an optimization method of hydrolysis conditions of [beta]-glucosidase on glycoside drugs. According to the optimization method, the [beta]-glucosidase can hydrolyze a non-reduced [beta]-glycosidic bond so as to release a corresponding ligand and glucose; such glycoside substances as geniposide, arctiin, polydatin and the like are hydrolyzed; an enzymolysis effect can be achieved on the [beta]-glucosidase on the basis of substrate concentration, reaction temperature, pH and reaction duration of such glycoside substances as geniposide, arctiin, polydatin and the like; thethree glycosides, namely geniposide, arctiin and polydatin, are converted by virtue of the [beta]-glucosidase; and by determining the optimum temperature, pH, substrate concentration and reaction time of the glycoside substances during hydrolysis, an optimum reaction combination of a hydrolysis reaction is confirmed, and a glycoside substance conversion rate is improved.

Description

technical field [0001] The invention belongs to the technical field of extracting glycoside drugs, and in particular relates to a method for optimizing the hydrolysis conditions of glycoside drugs by β-glucosidase. Background technique [0002] Geniposide is a kind of iridoid glucoside extracted from the fruit of Gardenia jasminoides of the Rubiaceae plant. It is easily soluble in water and the content in Gardenia is about 5%. Genipin is mostly present in plants as It exists in the more stable form of geniposide. At present, the plants reported to have geniposide are mostly medicinal plants, such as Gardenia, Eucommia, Churula, Cistanche, etc. Among them, the content of geniposide in the traditional medicinal material Gardenia in my country is 3%-5 %, genipin is produced under the catalysis of β-glucosidase in Gardenia, with a content of 0.005%-0.01%[1]. The hydrolyzate of geniposide is genipin, which is a good natural bio-crosslinking agent, capable of cross-linking with s...

Claims

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Application Information

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IPC IPC(8): C12P19/14C12P19/02
CPCC12P19/02C12P19/14
Inventor 刘新育林晖陈红歌
Owner HENAN AGRICULTURAL UNIVERSITY
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