阅读说明：本技术 一种积雪草苷元的制备方法 (Preparation method of asiaticoside ) 是由 张贵民 陈小荣 韩廷伟 于 2020-01-19 设计创作，主要内容包括：本发明属于中药技术领域,具体涉及一种积雪草苷元的制备方法。本发明提供的积雪草苷的酶解方法收率及纯度均显著高于酸碱水解法,并且对设备要求低,具有环境污染小、无环境压力等优点,为中药积雪草的活性物质研究及开发提供了有效方法。(The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a preparation method of asiaticoside. The enzymolysis method of asiaticoside provided by the invention has the advantages of obviously higher yield and purity than those of acid-base hydrolysis methods, low equipment requirement, small environmental pollution, no environmental pressure and the like, and provides an effective method for research and development of active substances of traditional Chinese medicine centella asiatica.)

1. A preparation method of asiaticoside is characterized by comprising the following steps:

(1) and (3) carrying out enzymolysis reaction: adding asiaticoside into buffer solution, adding hydrolase, and reacting at 30-60 deg.C for 12-120 hr;

(2) and (3) suction filtration: carrying out suction filtration on the reaction liquid in the step (1), removing filtrate, dissolving a filter cake by using absolute ethyl alcohol, wherein the weight-volume ratio of asiaticoside to absolute ethyl alcohol is 1:25, carrying out suction filtration on the dissolved solution, retaining the filtrate, repeating the operation once on the obtained filter cake, and combining the two filtrates;

(3) and (3) crystallization: and (3) concentrating the filtrate obtained in the step (2) to saturation, adding water for crystallization until the alcohol content is below 20%, carrying out suction filtration on the crystallized liquid, discarding the filtrate, drying the filter cake at 80 ℃, and weighing.

2. The method of claim 1, wherein the buffer of step (1) consists of water, acetic acid and sodium acetate and has a pH of 3.0 to 6.0, preferably 5.0.

3. A process as claimed in claim 1, wherein the hydrolase enzyme in step (1) is one or more of helicase, naringinase, cellulase or hesperidinase, preferably helicase.

4. The process of claim 1, wherein the temperature of the reaction in step (1) is from 30 ℃ to 60 ℃, preferably from 40 ℃ to 50 ℃, and most preferably 45 ℃.

5. The process according to claim 1, wherein the reaction time in step (1) is 12 to 120 hours, preferably 50 to 100 hours, most preferably 84 hours.

6. The method according to claim 1, wherein the mass ratio of asiaticoside to hydrolase in the reaction system in step (1) is 1:1/8 to 1:8, preferably 1: 4.

7. The method according to claim 1, wherein the concentration of asiaticoside in the reaction system in step (1) is 1g/L to 20g/L, preferably 10 g/L.

8. The method as claimed in claim 1, wherein the method is used for enzymatic hydrolysis of total asiaticoside or madecassoside.

9. The method of claim 1, wherein said asiaticoside is asiatic acid.

10. An enzymolysis method of asiaticoside is characterized by comprising the following steps:

(1) and (3) carrying out enzymolysis reaction: adding asiaticoside into buffer solution composed of water, acetic acid and sodium acetate to make the final concentration of substrate be 10g/L, adding helicase into reaction system according to the ratio of substrate to enzyme being 1:4, and reacting at 45 deg.C for 84 h;

(2) and (3) suction filtration: carrying out suction filtration on the reaction liquid in the step (1), removing filtrate, dissolving a filter cake by using absolute ethyl alcohol, wherein the weight-volume ratio of asiaticoside to absolute ethyl alcohol is 1:25, carrying out suction filtration on the dissolved solution, retaining the filtrate, repeating the operation once on the obtained filter cake, and combining the two filtrates;

(3) and (3) crystallization: and (3) concentrating the filtrate obtained in the step (2) to saturation, adding water for crystallization until the alcohol content is below 20%, carrying out suction filtration on the crystallized liquid, discarding the filtrate, drying the filter cake at 80 ℃, and weighing.

Background

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a preparation method of asiaticoside.

Disclosure of Invention

The invention mainly aims to provide a method for efficiently obtaining asiatic acid, which is realized by the following technical scheme:

an enzymolysis method of asiaticoside comprises the following steps:

(1) and (3) carrying out enzymolysis reaction: adding asiaticoside into buffer solution, adding hydrolase, and reacting at 30-60 deg.C for 12-120 h;

(2) and (3) suction filtration: and (2) carrying out suction filtration on the reaction liquid obtained in the step (1), discarding filtrate, dissolving a filter cake by using absolute ethyl alcohol, wherein the weight-volume ratio of asiaticoside to absolute ethyl alcohol is 1:25, carrying out suction filtration on the dissolved solution, retaining the filtrate, repeating the operation once for the obtained filter cake, and combining the two filtrates;

(3) and (3) crystallization: concentrating the filtrate obtained in the step (2) to saturation, adding water for crystallization until the alcohol content is 10% -30%, carrying out suction filtration on the crystallized liquid, discarding the filtrate, drying a filter cake at 80 ℃, weighing, calculating the yield, and measuring the purity of the obtained product by a high performance liquid chromatography.

The buffer solution in the step (1) consists of water, acetic acid and sodium acetate, and the pH value is 3.0-6.0, and preferably 5.0.

The hydrolase in the step (1) is helicase, naringinase, cellulase or hesperidinase, preferably helicase;

the reaction temperature is 30-60 ℃, preferably 40-50 ℃, and optimally 45 ℃;

the reaction time is 12 to 120 hours, preferably 50 to 100 hours, and optimally 84 hours;

the ratio of asiaticoside to enzyme in the reaction system is 1:1/8-1:8, preferably 1: 4;

the concentration of asiaticoside in the reaction system is 1g/L-20g/L, preferably 10 g/L.

An enzymolysis method of asiaticoside comprises the following steps:

(1) and (3) carrying out enzymolysis reaction: adding asiaticoside into buffer solution composed of water, acetic acid and sodium acetate to make the final concentration of substrate be 10g/L, adding helicase into reaction system according to the ratio of substrate to enzyme being 1:4, and reacting at 45 deg.C for 84 h;

(2) and (3) suction filtration: carrying out suction filtration on the reaction liquid in the step (1), removing filtrate, dissolving a filter cake by using absolute ethyl alcohol, wherein the weight-volume ratio of asiaticoside to absolute ethyl alcohol is 1:25, carrying out suction filtration on the solution, retaining the filtrate, repeating the operation once on the obtained filter cake, and combining the two filtrates;

(3) and (3) crystallization: concentrating the filtrate obtained in the step (2) to saturation, adding water for crystallization until the alcohol content is below 20%, carrying out suction filtration on the crystallized liquid, discarding the filtrate, drying a filter cake at 80 ℃, weighing, calculating the yield, and measuring the purity of the product by a high performance liquid chromatography.

The method can be used for enzymolysis of total asiaticoside and madecassoside, in addition to enzymolysis of asiaticoside.

Compared with the acid-base hydrolysis method in the prior art, the enzymolysis method provided by the invention has the following advantages:

(1) the enzymolysis reaction is mild and has specificity, the byproducts are less, and the yield and the purity of the obtained product are high.

(2) Compared with acid-base reaction, the enzymolysis reaction has the advantages of less pollution, no environmental pressure and low equipment requirement.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The present invention is further illustrated below by specific examples in order to provide those skilled in the art with a full understanding of the present invention, but it should be understood by those skilled in the art that the examples of the present invention are not to be construed as limiting the present invention in any way.