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Application of High Speed ​​Countercurrent Chromatography in Biotransformation of Glycosides

A high-speed counter-current chromatography and glycoside technology, applied in the field of glycoside conversion, can solve the problems of undisclosed high-speed counter-current chromatography, cumbersome steps, etc., and achieve the effects of eliminating product inhibition, improving reaction efficiency, and maintaining activity and concentration.

Active Publication Date: 2021-01-01
SHANDONG ANALYSIS & TEST CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, enzymes are usually added to glycosides to complete the biotransformation reaction, and then the reacted aglycones are separated by high-speed countercurrent chromatography. The steps are relatively cumbersome, and the technical scheme of using high-speed countercurrent chromatography directly as a bioenzyme conversion reactor has not yet been disclosed.

Method used

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  • Application of High Speed ​​Countercurrent Chromatography in Biotransformation of Glycosides
  • Application of High Speed ​​Countercurrent Chromatography in Biotransformation of Glycosides
  • Application of High Speed ​​Countercurrent Chromatography in Biotransformation of Glycosides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] The biotransformation of embodiment 1 genistin

[0067] 1. Preparation of two-phase solvent and sample solution

[0068] Put the ethyl acetate / buffer solution (1:1, v / v) solvent system in the separatory funnel according to the ratio, shake it fully, let it stand for stratification, and wait for the two phases to reach equilibrium, take the upper and lower phases respectively, and ultrasonically remove them. Reserve after gas.

[0069] Configure the following three solutions according to the requirements of the countercurrent chromatography bioreactor: (1) Take part of the upper phase solution as the mobile phase of the countercurrent chromatography, add genistin, and ultrasonically dissolve it fully to prepare a 62.5mg / L substrate solution (2) The lower phase is a stationary phase, add β-glucosidase, fully mix, and be mixed with a 0.75 mg / mL bioreactor enzyme solution; (3) get part of the upper phase solution as a blank mobile phase, and set aside. The above three sol...

Embodiment 2

[0080] The biotransformation of embodiment 2 polydatin

[0081] 1. Solvent system selection

[0082] According to the conversion of polydatin, the following two-phase solvent system was selected for the countercurrent chromatographic enzyme reactor in this example, namely n-hexane / buffer solution (1:1, v / v), ethyl acetate / buffer solution (1:1 , v / v), chloroform / buffer solution (1:1, v / v) and n-butanol / buffer solution (1:1, v / v) solvent system, using HPLC method to determine polydatin (substrate) and resveratilla Partition coefficient (K D ) value, the method is as follows: according to the proportion of the solvent system, prepare 20mL solvent, shake fully, and let stand to separate layers. First take 5 mL of the lower phase in a test tube, add about 1 mg of polydatin and resveratrol samples, and measure the peak area of ​​each target peak in the lower phase by HPLC, denoted as A 1 , and then add an equal volume of the upper phase, fully shake, let stand for stratification,...

Embodiment 3

[0098] The conversion of embodiment 3 daidzein

[0099] 1. Solvent system selection

[0100] For the conversion of daidzein, the following two-phase solvent system was selected for the countercurrent chromatographic enzyme reactor in this example, that is, n-hexane / buffer solution (1:1, v / v), ethyl acetate / buffer solution (1:1 , v / v), chloroform / buffer solution (1:1, v / v) and n-butanol / buffer solution (1:1, v / v) solvent system, using HPLC method to determine total soybean isoflavone glycosides (substrate) and the partition coefficient (K D ) value, the method is as follows: according to the proportion of the solvent system, prepare 20mL solvent, shake fully, and let stand to separate layers. First take 5 mL of the lower phase in a test tube, add about 1 mg of each of the substrate and the product, and use HPLC to measure the peak area of ​​each target peak in the lower phase, which is recorded as A 1 , and then add an equal volume of the upper phase, fully shake, let stand ...

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Abstract

The invention relates to the technical field of glycoside conversion, in particular to the application of a high-speed countercurrent chromatograph in the biotransformation of glycoside components. In the present invention, the high-speed countercurrent chromatograph is used as a reactor for the bioenzyme conversion reaction of glycoside components, and the difference in the distribution of glycosides and aglycones between the two-phase solvents is used to add glycoside hydrolase to the aqueous phase solution as a stationary phase in advance. The glycoside substrate to be converted is added to the mobile phase. When countercurrent chromatography is running, the enzyme is located in the stationary phase. Since the substrate glycoside is more soluble in aqueous solution, it will enter the stationary phase from the mobile phase during the flow process and undergo a sufficient enzymatic hydrolysis reaction with the glycoside hydrolase to generate small molecular aglycones. Flow out with the mobile phase to complete the biotransformation and separation process. The invention uses a high-speed countercurrent chromatograph as a biotransformation reactor, which can complete the transformation and separation at one time, can eliminate the inhibitory effect in the enzymolysis reaction process, and increase the conversion rate, and has remarkable technical effects and economic significance.

Description

technical field [0001] The invention relates to the technical field of glycoside transformation, in particular to the application of a high-speed countercurrent chromatograph as an enzymolysis reactor to bioconvert glycosides into aglycones. Background technique [0002] Glycosides, also known as glycosides, are composed of weakly polar ligands and strongly polar sugar groups, and the non-sugar parts are called aglycones. Therefore, the molecules of glycoside compounds have the characteristics of high polarity, poor fat solubility, and high molecular weight, and are not easily absorbed through the intestinal wall mucosa. Due to the long residence time of glycosides in the intestine, some glycosides are transformed under the action of enzymes secreted by specific intestinal flora, and the polarity of the transformed products is reduced, and the fat solubility is increased, so it is easier to enter the blood circulation through the intestinal wall, and then Play a medicinal e...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P17/06C12M1/42C12M1/00
CPCC12M21/18C12M27/02C12P17/06
Inventor 王岱杰王晓耿岩玲赵恒强崔莉闫慧娇
Owner SHANDONG ANALYSIS & TEST CENT
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