Method for modifying flavonoid glycoside compounds with galactosy transferase

A technology of flavonoids and compounds, applied in the field of biopharmaceuticals, can solve the problems of low solubility of natural flavonoid glycoside compounds, achieve the effects of improving bioavailability, easy acquisition, and improving water solubility

Inactive Publication Date: 2009-11-11
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] The purpose of the present invention is to solve the problem of low solubility of natural flavonoid glycoside compounds, and provide a method for modifying flavonoid glycoside compounds by galactosyltransferase

Method used

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  • Method for modifying flavonoid glycoside compounds with galactosy transferase
  • Method for modifying flavonoid glycoside compounds with galactosy transferase
  • Method for modifying flavonoid glycoside compounds with galactosy transferase

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preparation example Construction

[0039] Preparation of enzyme extract:

[0040]Construct the expression plasmid containing β-1,4-galactosyltransferase gene, transfer it into Escherichia coli BL21 (DE3) strain, induce the expression of the target protein with isopropylthiogalactopyranoside (IPTG), sonicate the cells, After Ni column affinity purification and ultrafiltration concentration, the enzyme extract with high purity is obtained. The molecular weight and purity of the target protein were tested by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the concentration of the enzyme extract was determined by BIOFORD method. The obtained enzyme extract was added with 50% glycerol in the total volume of the enzyme extract and DTT with a final molar concentration of 0.01mmol / L, and stored at -20°C for future use. Avoid repeated freezing and thawing to avoid the decrease in enzyme activity.

[0041] This method is applicable to natural flavonoid glycoside compounds, especially flavonoid ...

Embodiment 1

[0042] Example 1: Synthesis of Glycosylated Puerarin

[0043] Prepare fresh enzymatic reaction buffer according to the method in the above embodiment, weigh Hepes 0.952g, BSA 0.4g, MnCl 2 4H 2 O 0.00475g, after being fully dissolved in distilled water, dilute to 200mL. Then prepare 9.12mg / mL puerarin solution and 4.00mg / mL UDP-galactose solution with enzymatic reaction buffer respectively.

[0044] Put 5 mL of puerarin solution and 5 mL of UDP-galactose solution into the reaction vessel, the molar final concentration ratio is 10:3; then add 200 μL of enzyme extract (enzyme extract is prepared according to the method in the above embodiment); Make up the volume of reaction buffer to 50mL and mix gently. React at 37°C for 30 minutes. After the reaction was completed, the reaction vessel was placed in a boiling water bath for 5 minutes to remove the protein in the reaction solution. After cooling to room temperature, centrifuge at 12,000 rpm for 10 minutes, and discard the p...

Embodiment 2

[0051] Embodiment 2: the synthesis of glycosylated naringin

[0052] Prepare fresh enzymatic reaction buffer according to the method in the above embodiment, weigh Hepes 0.952g, BSA0.4g, MnCl 2 4H 2 O 0.00475g, after being fully dissolved in distilled water, dilute to 200mL. Then prepare 1.45mg / mL naringin solution and 29.3mg / L UDP-galactose solution with enzymatic reaction buffer respectively.

[0053] Add 42mL of naringin solution and 5mL of UDP-galactose solution into the reaction vessel, and the molar final concentration ratio is 7:16; then add 200 μL of enzyme extract (enzyme extract is prepared according to the method in the above embodiment); Accelerate the reaction buffer to make up the volume to 50mL, and mix gently. React at 37°C for 30 minutes. After the reaction was completed, the reaction vessel was placed in a boiling water bath for 10 minutes to remove the protein in the reaction solution. After cooling to room temperature, centrifuge at 10,000 rpm for 10 m...

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Abstract

The invention discloses a method for modifying flavonoid glycoside compounds with galactosy transferase. The flavonoid glycoside compounds are ubiquitous secondary metabolites in the nature and one of main active ingredients of medicinal plant, and have physiological activities of antibacterium, cancer resistance, antioxidation and the like. However, natural flavonoid glycoside compounds frequently have the defects of poor water solubility, poor drug effect and the like. The method solves the defects of relatively low solubility of the natural flavonoid glycoside compounds and takes the natural flavonoid glycoside compounds as a forerunner to improve the water solubility thereof by a glycosylation reaction, thereby improving the bioavailability thereof. The technical essential of the method is as follows: an enzymatic reaction is applied to the synthesis of polyglycoside of flavonoid compounds. Compared with chemical synthesis methods, the method has higher synthesis efficiency and lower cost. After glycosylation modification, part of drugs can improve drug effect and reduce toxic side effect; therefore, the method provides a new approach for the development of new drugs of the flavonoid compounds.

Description

【Technical field】: [0001] The invention belongs to the technical field of biopharmaceuticals. It specifically relates to a method for modifying flavonoid glycosides by using galactosyltransferase. This method is based on the natural active lead of flavonoid glycosides, and by analyzing the relationship between its chemical structure and biological activity, the glycosides of flavonoids are glycosides. Kylation modification is one of the effective ways to develop new drugs. 【Background technique】: [0002] Flavonoids, also known as bioflavonoids or plant flavonoids, are a large class of phenolic substances that exist in nature. Yellow in color and known as bioflavonoids. [0003] Flavonoids are a class of substances with 2-phenylchromone as the mother nucleus. Two aromatic rings (A ring, B ring) are connected by a three-carbon chain, with a C6-C3-C6 basic configuration. The three rings are labeled A, B, and C, respectively. [0004] [0005] Basic structure of flavonoi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/60C12P19/44
Inventor 张连文马晓峰王鹏
Owner NANKAI UNIV
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