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Method for preparing hypoglycosyl anemonic saponin by encyme method hydrolyzing anemonic saponin glycosyl

A technology of Pulsatilla saponins and Pulsatilla saponins, which is applied in the field of preparation of low sugar-based Pulsatilla saponins, can solve the problems of inability to hydrolyze Pulsatilla saponins, poor purpose of glycosyl hydrolysis, serious acid hydrolysis pollution, etc., and achieve elimination of hemolysis, high conversion rate, and strong purpose Effect

Inactive Publication Date: 2006-08-09
SUNFLOWER PHARM GRP (TIANJIN) DRUG RES INST CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the above acid-base hydrolysis method, the purpose of sugar base hydrolysis is poor, all the glycolipid bonds are hydrolyzed by alkali hydrolysis, the pollution of acid hydrolysis is serious, and the damage to aglycone is great
[0024] Although Chinese Patent No. 99112764.1 discloses a "method for preparing rare ginsenosides by enzymatically changing ginsenoside glycosyl groups", since ginsenosides and Pulsatilla saponins are two products with completely different configurations, the disclosed Enzyme can not hydrolyze Pulsatilla saponin at all

Method used

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  • Method for preparing hypoglycosyl anemonic saponin by encyme method hydrolyzing anemonic saponin glycosyl

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] a. With the thermoaerobic bacteria Bacillus sp.JF bacteria (document 1) containing 3% corn flour, 0.2% enzyme-producing inducer - Pulsatilla pulsatilla extract (dry matter), 0.01% MgSO 4 The culture medium is ventilated for 30-40 hours at a temperature of 60° C., centrifuged and sterilized to obtain an enzyme-containing mixture, and 50-80% ethanol is used to precipitate the enzyme protein, and freeze-dried to obtain saponin enzyme.

[0036] b. Mix 10 grams of the above-mentioned saponin enzyme, 30 grams of Pulsatilla saponin, 1500 milliliters of acetic acid buffer (0.2M, pH 5.0) and 150 milliliters of ethanol to make the concentration of the reactant 0.01-15%, and stir at a temperature of 75°C React for 4 hours.

[0037] c. Add 6000 ml of ethanol after the reaction, filter to remove the protein precipitate, and evaporate the filtrate to dryness under reduced pressure to obtain 20 g of low-glycoside saponin crude product.

[0038] More than 80% of Pulsatilla saponins ar...

Embodiment 2

[0041] a. Aspergillus niger (Aspegillus niger) was cultured with agitation and ventilation at a temperature of 28-30°C for 50- After 100 hours, centrifuge and sterilize to obtain an enzyme-containing mixed solution, use 60-75% saturated ammonium sulfate to precipitate the enzyme protein, collect the protein, and dissolve it in acetic acid buffer (0.02M, pH 5.0) of 1 / 10 of the fermentation broth volume , dialyzed to remove ammonium sulfate, and centrifuged to remove slag, which is the enzyme solution.

[0042] b. Mix 4 grams of Pulsatilla saponin, 100 milliliters of acetic acid buffer solution (0.02M, pH 5.0) and 50 milliliters of the above enzyme solution, so that the concentration of the reactant is 0.01-15%, and react for 40 hours at a temperature of 4°C.

[0043] c. adding 1 / 3 volume of n-butanol to extract the saponin three times, and evaporating to dryness under reduced pressure to obtain the crude product of low sugar-based secondary saponin.

[0044] Use the silica gel...

Embodiment 3

[0046] Others are the same as Example 2, mix with 1 gram of Pulsatilla saponin, 100 milliliters of acetic acid buffer (0.02M, pH5.0) and 50 milliliters of the above-mentioned enzyme liquid, obtain two kinds of 0.4 gram of pure low-glycoside secondary saponins and 0.1 gram of aglycone. The structure was determined by magnetic resonance method (documents 2 and 3), and its structure is Pulsatilla saponins 7 and 8.

[0047] The enzyme liquid DEAE-Cellulose ion exchange resin column method and the BioRed protein preparation chromatograph (document 6) separated and purified Pulsatilla saponin enzyme protein, and the molecular weight was determined by SDS electrophoresis method (document 7), and the molecular weight of the enzyme was 53,000.

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Abstract

The method for preparing hypoglycosyl anemonic saponin includes the following steps: using the enzyme capable of enzyme hydrolyzing anemonic saponin glycosyl, mixing said enzyme, anemonic saponin and buffer liquor and making them produce reaction for 4-40 hr., its reaction temp. is 4-75 deg.C and pH value is 3-9, so as to extract the invented hypoglycosyl anemonic saponin. Said invention has no pollution, and is high in conversion rate.

Description

Technical field: [0001] The invention relates to a method for preparing low-glycoside pulsatilla saponins, in particular to a method for treating pulsatilla saponin with an enzyme capable of hydrolyzing pulsatilla glycoside glycosides, and enzymatically hydrolyzing the pulsatilla glycoside glycosides to prepare low-sugar-based pulsatilla saponins. Background technique: [0002] Pulsatilla is a plant of the family Ranunculaceae and the genus Pulsatilla. It is a perennial herb, and its roots are often used as medicine. Mainly distributed in Northeast China, North China and Shaanxi, Anhui, Jiangsu, Hubei, Sichuan and other places. According to historical records, "Pulsatilla grows in high mountains, valleys and fields, and is collected in April. There are many kinds of Pulsatilla, and "Chinese Pharmacopoeia" only records that Pulsatilla chinensis is used as a genuine Pulsatilla, which is Pulsatilla chinensis (Bge.) Regel, a plant of Ranunculaceae. In addition, there are the fo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P33/00C12R1/07C12R1/72C12R1/66C12R1/645
Inventor 金凤燮鱼红闪叶文才
Owner SUNFLOWER PHARM GRP (TIANJIN) DRUG RES INST CO LTD
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