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Production method of rare ginsenoside Rh2

A technology of ginsenoside and production method, which is applied in the directions of biochemical equipment and method, botanical equipment and method, enzyme, etc., can solve the problems of high cost, poor stereoselectivity, complex synthesis route and the like

Inactive Publication Date: 2017-01-25
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the chemical method of glycosylation of protopanaxadiol to produce ginsenoside Rh2 has technical problems such as complex synthetic routes, high cost, low conversion rate, poor stereoselectivity, and a large number of by-products.

Method used

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  • Production method of rare ginsenoside Rh2
  • Production method of rare ginsenoside Rh2
  • Production method of rare ginsenoside Rh2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Preparation of Glycosyltransferase

[0049] Bacillus subtilis was extracted using a bacterial genomic DNA extraction kit ( Bacillus subtilis 168), and then using the extracted Bacillus subtilis genomic DNA as a template, according to the primer sequences in Table 1, use the primer pair YjiC-F and YjiC-R and the primer pair YojK-F and YojK-R to amplify respectively The glycosyltransferase genes YjiC and YojK were obtained. The PCR amplification system is: Q5 Buffer (5×) 10 μL, dNTP (2.5 mM) 3 μL, genomic DNA template: 1 μL, primers (10 μM) 2.5 μL each, Q5 high-fidelity polymerase 0.5 μL, complement Double distilled water to 50 μL. The conditions of the PCR reaction were pre-deformation at 98°C for 3 minutes (1 cycle), deformation at 98°C for 10 seconds, annealing for 20 seconds (the annealing temperature was 56°C), extension at 72°C for 1 minute (31 cycles), extension at 72°C for 2 minutes (1 cycle).

[0050] Table 1 Primer sequences

[0051]

[0052]...

Embodiment 2

[0055] Example 2 Glycosyltransferase YjiC is used to produce ginsenosides Rh2 and F12

[0056] The glycosylation reaction system includes 25 mM / L Tris-HCl (pH8.0), 1 mM / L protopanaxadiol or ginsenoside Rh2, 3 mM / L UDP-glucose, 10 mM / L MgCl 2. Add glycosyltransferase YjiC crude enzyme solution to the reaction solution at a ratio of 5% (v / v), react at 35°C, 120 rpm, for 4 h.

[0057] After the glycosylation reaction was completed, ethyl acetate was added to extract the glycosylated product, and the extraction was repeated 4 times. Then the glycosylated products extracted for a total of 5 times were combined, concentrated by a rotary evaporator, and then chromatographic methanol was added to redissolve the extracted glycosylated products, and the glycosylated products were detected and quantified by liquid chromatography-mass spectrometry. The liquid chromatograph is Agilent 1260, the chromatographic column is the C18 chromatographic column of Yuexu Technology (Shanghai) Co., L...

Embodiment 3

[0058] Example 3 Glycosyltransferase YojK catalyzes the production of ginsenoside Rh2 from protopanaxadiol

[0059] The glycosylation reaction system includes 25 mM / L Tris-HCl (pH8.0), 1 mM / L protopanaxadiol, 3 mM / L UDP-glucose, 10 mM / L MgCl 2 . Add glycosyltransferase YojK crude enzyme solution to the reaction solution at a ratio of 10% (v / v), react at 35°C, 120 rpm, for 4 h.

[0060] After the glycosylation reaction was completed, ethyl acetate was added to extract the glycosylated product, and the extraction was repeated 4 times. After combining all the extracted products, the extracted products were dried using a rotary evaporator, and then chromatographic methanol was added to dissolve the extracted glycosylated products, and the glycosylated products were detected and quantified by liquid chromatography-mass spectrometry. The liquid chromatograph is Agilent 1260, the chromatographic column is the C18 chromatographic column of Shanghai Yuexu Technology (model: XDB-C18, ...

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Abstract

The invention discloses a production method of rare ginsenoside Rh2 and belongs to the field of enzymatic synthesis of ginsenoside. The production method includes that protopanoxadiol is used as a substrate, glycosyltransferase YjiC or YojK screened from bacillus subtilis is used as a catalyst, and the glycosylation of the C3 hydroxyl of the protopanoxadiol is efficiently catalyzed through in-vitro enzyme reaction to generate the ginsenoside Rh2. The production method is cheap in raw materials, high in ginsenoside Rh2 yield, low in production cost and capable of achieving large-scale production of the precious and rare ginsenoside Rh2.

Description

technical field [0001] The present invention relates to a method for producing ginsenoside Rh2 from protopanaxadiol, more specifically, to a method for producing ginsenoside Rh2 by glycosylation of the C3 hydroxyl group of protopanaxadiol using glycosyltransferase YjiC or YojK Methods. Background technique [0002] Ginsenoside Rh2 (ginsenoside Rh2), commonly known as life-protecting element, is named for its life-protecting and prolonging effect. Ginsenoside Rh2 is one of the most important anti-tumor active substances in ginseng. It has the functions of inhibiting tumor cell growth, inhibiting the activity of tumor cell telomerase, inducing tumor cell apoptosis, anti-tumor cell metastasis, and reversing abnormal differentiation of tumor cells. , which has good anti-tumor and prevention and treatment of tumor recurrence and metastasis. Clinically, the combination of ginsenoside Rh2 with radiotherapy and chemotherapy can enhance the effect of radiotherapy and chemotherapy i...

Claims

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Application Information

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IPC IPC(8): C12P33/20C12N9/10C12N15/54
CPCC12P33/20C12N9/1051
Inventor 孙媛霞戴隆海朱玥明曾艳门燕杨建刚
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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