CN106148454B

CN106148454B - Preparation method of baohuoside I

Info

Publication number: CN106148454B
Application number: CN201510128968.7A
Authority: CN
Inventors: 孟坤; 徐更; 张永圣; 刘丙征; 尚勇
Original assignee: Beijing Shenogen Biomedical Co ltd
Current assignee: Beijing Shenogen Biomedical Co ltd
Priority date: 2015-03-24
Filing date: 2015-03-24
Publication date: 2021-01-26
Anticipated expiration: 2035-03-24
Also published as: CN106148454A

Abstract

The invention provides a preparation method of baohuoside I, which comprises the steps of carrying out enzymolysis reaction on icariin under the action of pectinase to obtain a crude baohuoside I product, and then crystallizing twice to obtain a pure baohuoside I product. The baohuoside I obtained by the method has the mass yield of more than 40 percent relative to 90 percent of icariin, and the purity of more than 99 percent.

Description

Preparation method of baohuoside I

Technical Field

The invention relates to a preparation method of baohuoside I, belonging to the fields of food, health care products, cosmetics and medicines.

Background

Baohuoside I, also called Icariside II (Icariside II), is a new effective monomer obtained by enzyme conversion of icariin which is a main active ingredient extracted and separated from epimedium herb, and the structural formula of the monomer is shown as the following formula (I):

the existing research shows that baohuoside I has the functions of improving cardiovascular function, regulating endocrine and enhancing immunity.

The preparation process of the compound is disclosed in 200780034447.9, and the process includes catalytic hydrolysis of icariin with enzyme capable of eliminating glucose or microbe capable of producing the enzyme, and purifying the reaction product with silica gel column chromatography to obtain baohuoside I product. In the case of silica gel column chromatography, hexane and chloroform were used as solvents, respectively.

In the chinese patent with application number 201110186207.9, it is disclosed that an epimedium herb extract is subjected to enzymolysis by using β -glucosidase or cellulase, because the baohuoside I obtained by enzymolysis has a high content and is hardly soluble in water, tween needs to be added into the extract to increase the solubility of baohuoside I in water, thereby avoiding precipitation. Separating and purifying by macroporous resin chromatographic column to obtain baohuoside I with purity higher than 98%.

In the Chinese patent with the application number of 201210345102.8, a preparation method of baohuoside I is disclosed, and the method provides a method for simultaneously extracting and separating icariin and baohuoside I from epimedium herb.

In the method provided by the Chinese patent with the application number of 200780034447.9, chloroform with high toxicity is used for extraction when baohuoside I is purified; in the method provided in chinese patent application No. 201110186207.9, tween needs to be used to increase the solubility of baohuoside, thus increasing the preparation cost; the method described in chinese patent application No. 201210345102.8 is to extract baohuoside I directly from epimedium herb, because baohuoside I has a very low content in epimedium herb, generally not more than 0.2%, the direct extraction method will result in a lower yield, and the waste of the herb is serious, which is not suitable for industrial production.

Disclosure of Invention

The invention aims to provide a preparation method of baohuoside I, and the baohuoside I prepared by the method has the advantages of high purity and high yield.

The invention provides a preparation method of baohuoside I, which comprises the following steps:

a. carrying out enzymolysis reaction on the epimedium extract under the action of pectinase;

b. dissolving the enzymolysis reaction product in a weak polar organic solvent, filtering, and collecting filtrate;

c. adding polar solvent into the filtrate, and crystallizing to obtain baohuoside I.

Preferably, the icariin content in the epimedium extract is at least 15% by mass.

Preferably, the weak polar organic solvent is one or more selected from ethyl acetate, acetone, dichloromethane and methyl tert-butyl ether.

Preferably, the weakly polar organic solvent is acetone.

Preferably, the polar solvent is one or more of methanol, ethanol and water.

Preferably, the polar solvent is water.

Preferably, a step d is further included between the steps a and b: and centrifuging an enzymolysis product obtained by enzymolysis reaction, and dissolving a precipitate obtained by centrifugation in a weak-polarity organic solvent.

Preferably, in said step c, distilled water is added to the filtrate, reflux is performed, and the liquid obtained by reflux is cooled and crystallized.

Preferably, the volume ratio of the filtrate to the distilled water is 1-5:1, the reflux temperature is 50-100 ℃, and the cooling crystallization temperature is 0-25 ℃.

More preferably, the volume ratio of the filtrate to the distilled water is 1-2: 1, the reflux temperature is 50-80 ℃, and the cooling crystallization temperature is 10-20 ℃.

Preferably, the conditions of the enzymatic hydrolysis reaction are as follows: preparing herba Epimedii extract and phosphate buffer solution into substrate solution with pH of 4.0-8.0, adding pectase into the substrate solution, wherein the mass of pectase is 2-20 times of that of herba Epimedii extract, the enzymolysis temperature is 40-60 deg.C, and the substrate solution contains 0-25% ethanol of total volume.

More preferably, the mass of the pectinase is 5-20 times of that of the epimedium extract, the pH value of the substrate solution is 4.0-6.0, and the enzymolysis temperature is 45-55 ℃.

Most preferably, the mass of the pectinase is 15-20 times of that of the epimedium extract, and the substrate solution contains 10-20% of ethanol in the total volume.

Preferably, the pH of the substrate solution is adjusted by means of a phosphate buffer.

More preferably, the phosphate buffer is a phosphate-citrate buffer or a disodium hydrogen phosphate-potassium dihydrogen phosphate buffer.

The invention has the beneficial effects that: the invention provides a preparation method of baohuoside, which is different from the existing preparation method of baohuoside. The yield of the epimedium extract hydrolyzed by the pectinase is high, and compared with the epimedium extract containing 90% icariin, the mass yield of baohuoside I can reach more than 40%; the epimedium herb extract is hydrolyzed by pectinase, the selectivity is good, the generated impurities are few, the baohuoside I with the purity of more than 99 percent can be obtained only by a crystallization process, and the industrial mass production is easy to realize.

Drawings

FIG. 1 shows a high performance liquid chromatogram of an enzymatic reaction carried out for 12 hours.

FIG. 2 shows a high performance liquid chromatogram of 36 hours after the enzymatic reaction.

FIG. 3 shows a high performance liquid chromatogram of baohuoside I standard.

FIG. 4 shows a high performance liquid chromatogram of purified baohuoside I.

Detailed Description

Unless otherwise indicated, the term "enzymatic reaction" herein refers to a hydrolysis reaction under the action of an enzyme.

The term "epimedium extract" herein refers to an extract obtained by extracting epimedium with an aqueous ethanol solvent, which contains flavonoid components including icariin, as well as other components, unless otherwise specified.

The term "icariin" herein refers to an extract from dried stems and leaves of epimedium herb, as a single component, having the following molecular structure, unless otherwise specified:

the term "pectinase" as used herein is commercially available from Impeman under the trademark RAPIDASE pectinase as the enzyme for fermentation, product number 984, unless otherwise indicated.

Example 1

1. Preparation of baohuoside I by enzymolysis reaction

Dissolving 10g of epimedium extract containing 90% of icariin in 140mL of potassium dihydrogen phosphate-disodium hydrogen phosphate buffer solution with the pH value of 5.9 and the pH value of 1/15M, adding 60mL of 95% ethanol and 100mL of pectinase solution under the stirring condition, starting timing when the temperature is constant at 52-55 ℃, stopping the reaction after 48 hours of reaction, and cooling the reaction solution to 30 ℃.

2. Purification of baohuoside I

After the enzymolysis reaction is finished, centrifuging the reaction solution, collecting a filter cake, drying the filter cake to obtain a weight of 7.55g, crushing the filter cake, adding 176g of acetone, stirring the mixture for 1 hour, filtering the mixture, adding 129g of purified water into an acetone solution, and heating and refluxing the mixture until the solution is clear. And then slowly cooling the solution to 14 ℃, preserving the temperature for 2 hours, filtering, collecting a filter cake, and drying to obtain 6.04g of a primary crystalline product of the baohuoside I. Pulverizing the primary crystallized product of the baohuoside I, adding 165g of acetone, stirring for 1 hour, filtering, adding 120g of purified water into an acetone solution, and heating and refluxing until the solution is clear. Slowly cooling the solution to 14 ℃, then preserving heat for 2 hours, filtering, collecting filter cakes and drying to obtain 4.1g of a secondary crystal product of the baohuoside I, wherein the mass yield of the product relative to the epimedium extract is 54.3%.

Example 2

1. Preparation of baohuoside I by enzymolysis reaction

Dissolving 3.75kg of epimedium extract containing 94% of icariin in 52L of 1/15M potassium dihydrogen phosphate-disodium hydrogen phosphate buffer solution with the pH value of 5.9, adding 24L of 95% ethanol and 75L of pectinase solution under stirring, uniformly mixing, timing when the temperature is constant at 52-55 ℃, stopping reaction after reacting for 48 hours, and cooling the reaction solution to 30 ℃. The HPLC reactions for 12 hours and 36 hours are shown in FIG. 1 and FIG. 2, respectively. From a comparison of fig. 1 and fig. 2, and a comparison of fig. 3, it can be seen that the concentration of icariin and other substrates relative to baohuoside I decreases with the increase of the enzymatic hydrolysis time, indicating that the icariin and other substrates are converted into baohuoside I with the increase of the enzymatic hydrolysis time.

2. Purification of baohuoside I

After the enzymolysis reaction is finished, the reaction solution is centrifuged, filter cakes are collected, the weight of the filter cakes after drying is 2.8Kg, 66Kg of acetone is added after the filter cakes are crushed, the mixture is stirred for 1 hour and then filtered, 48Kg of purified water is added into the acetone solution, and the mixture is heated and refluxed until the solution is clear. Then slowly cooling the solution to 14 ℃, preserving the temperature for 2 hours, filtering, collecting a filter cake and drying to obtain 2.2Kg of a primary crystalline product of the baohuoside I. Crushing the primary crystallized product of the baohuoside I, adding 60Kg of acetone, stirring for 1 hour, filtering, adding 44Kg of purified water into the acetone solution, and heating and refluxing until the solution is clear. The solution is slowly cooled to 14 ℃ and then is kept warm for 2 hours, and then is filtered, and a filter cake is collected and dried, so that 1.5Kg of secondary crystallized product of the baohuoside I is obtained, the mass yield of the extract of epimedium is 40%, and the purity of the extract is 99%, as shown in figure 4. As can be seen in fig. 1 to 4, the substrate is gradually converted from icariin to baohuoside I by the enzymatic hydrolysis reaction along with the time extension, the enzymatic hydrolysis product containing baohuoside I is produced by the reaction, the baohuoside I content in the enzymatic hydrolysis product can be increased to 99% after acetone-water two-time crystallization operation, and the baohuoside I yield can reach over 40% compared with the enzymatic hydrolysis substrate.

Claims

1. A preparation method of baohuoside I comprises the following steps:

a. carrying out enzymolysis reaction on the epimedium extract under the action of pectinase, wherein the enzymolysis reaction time is 36 hours, and the conditions of the enzymolysis reaction are as follows: preparing herba Epimedii extract and phosphate buffer solution into substrate solution with pH of 4.0-8.0, adding pectase into the substrate solution, wherein the mass of pectase is 2-20 times of that of herba Epimedii extract, the enzymolysis temperature is 40-60 deg.C, and the substrate solution contains 10-25% ethanol of total volume;

b. filtering and collecting filtrate;

c. adding water into the filtrate, crystallizing to obtain baohuoside I, and further comprising a step d' between the steps a and b: centrifuging enzymolysis product obtained by enzymolysis reaction, and dissolving precipitate obtained by centrifugation in weak polar organic solvent, wherein the content of icariin in herba Epimedii extract is at least 15%, and pectase is RAPIDASE pectase available from Dismann company under trademark as enzyme for fermentation with product number of 984.

2. The method according to claim 1, wherein the weakly polar organic solvent is selected from one or more of ethyl acetate, acetone, dichloromethane and methyl tert-butyl ether.

3. The method of claim 2, wherein the less polar organic solvent is acetone.

4. The method according to claim 1, wherein in the step c, distilled water is added to the filtrate, reflux is performed, and the liquid obtained by reflux is cooled and crystallized.

5. The method according to claim 4, wherein the volume ratio of the filtrate to the distilled water is 1 to 5:1, the reflux temperature is 50-100 ℃, and the cooling crystallization temperature is 0-25 ℃.

6. The method of claim 5, wherein the volume ratio of the filtrate to the distilled water is 1-2: 1, the reflux temperature is 50-80 ℃, and the cooling crystallization temperature is 10-20 ℃.

7. The method of claim 1, wherein the mass of the pectinase is 5-20 times of the mass of the epimedium extract, the pH value of the substrate solution is 4.0-6.0, and the enzymolysis temperature is 45-55 ℃.

8. The method of claim 7, wherein the mass of the pectinase is 15-20 times that of the epimedium extract.

9. The method according to claim 8, wherein the pH of the substrate solution is adjusted by means of a phosphate buffer.

10. The method of claim 9, wherein the phosphate buffer is a phosphate-citrate buffer or a disodium phosphate-potassium dihydrogen phosphate buffer.