CN106148449B

CN106148449B - Preparation method of icariside I

Info

Publication number: CN106148449B
Application number: CN201510129279.8A
Authority: CN
Inventors: 孟坤; 汤城; 袁贤达; 李海东; 孟宪军
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: 2020-12-25
Anticipated expiration: 2035-03-24
Also published as: CN106148449A

Abstract

The invention provides a preparation method of icariside I, which comprises the following steps: firstly, carrying out enzymolysis reaction on an epimedium extract under the action of beta-glycosidase; filtering the enzymolysis reaction product obtained in the step a; and recrystallizing the filtered filter cake twice by using ethanol to obtain the icariside I pure product. The yield of icariside I in the invention relative to the epimedium extract reaches 15%, and the purity of the icariside I reaches 99%.

Description

Preparation method of icariside I

Technical Field

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

Background

Icariside I, also called Icariside I, is an effective monomer extracted and separated from traditional Chinese medicine epimedium, and the structure of the monomer is shown as the following formula (I):

icariside I, one of the total flavones of epimedium, has the effects of invigorating kidney yang, strengthening muscles and bones, and dispelling wind-damp. Can be used for treating kidney yang deficiency, sexual impotence, nocturnal emission, tendons and bones flaccidity, rheumatalgia, numbness and contracture, etc. Modern pharmacology finds that the substances have the effects of resisting tumors, resisting oxidation, resisting bacteria, diminishing inflammation and protecting cardiovascular and cerebrovascular systems.

The preparation method of icariside I is disclosed in the patent document CN 101891782A. The method comprises the steps of taking Korean epimedium leaves as a raw material, carrying out reflux extraction by ethanol, adding concentrated hydrochloric acid into a crude extract, filtering, adding distilled water into a filtrate to separate out a precipitate, washing the precipitate by ethanol, and drying to obtain icariside I. The content of icariside I in different epimedium varieties is low, generally below 0.1%, and the main flavone component in most epimedium medicinal materials is icariin. The method adopts hydrochloric acid treatment, and aims to hydrolyze icariin in epimedium extract into icariside I by concentrated hydrochloric acid. However, under the action of hydrochloric acid, only a small part of icariin is hydrolyzed into icariside I, and most part of icariin can be completely hydrolyzed to remove all glycosyl groups, so that icaritin is generated. On the other hand, the icariin is hydrolyzed by hydrochloric acid, which destroys isopentenyl group on flavone ring, resulting in much impurity in hydrolysate. Therefore, the method can only obtain the icariside I pure product by a chromatography technology, so that the process is not suitable for industrial production.

The patent document with publication number CN101899077A discloses the extraction method and application of icariside I. In the method, icariside I is obtained by extracting icariin leaves with ethanol under reflux, adding concentrated hydrochloric acid to the extract, heating and filtering, and adding distilled water to the filtrate to precipitate out a precipitate. However, the method has the defect that the icariside I obtained by hydrolyzing the epimedium extract by using concentrated hydrochloric acid can destroy isopentenyl on a flavone ring, so that the icariside I has more impurities and low purity.

Therefore, a method for obtaining icariside I with high purity is required.

Disclosure of Invention

An object of the present invention is to provide a method for preparing icariside I, which has the advantages of high conversion rate, high purity and less impurities.

The invention provides a preparation method of icariside I on one hand, which comprises the following steps:

a. firstly, carrying out enzymolysis reaction on an epimedium extract under the action of beta-glycosidase;

b. filtering the enzymolysis reaction product obtained in the step a;

c. and crystallizing the filtered filter cake by using ethanol to obtain the icariside I.

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

Preferably, in the step a, the epimedium extract is dispersed in a buffer solution with the pH value of 3.5-7.5, and the enzymolysis reaction temperature is 35-65 ℃.

Preferably, the buffer solution is one or more selected from the group consisting of disodium hydrogen phosphate-potassium dihydrogen phosphate, acetic acid-sodium acetate, phosphoric acid-sodium phosphate, citric acid-sodium citrate and disodium hydrogen phosphate-citric acid buffer solution with a pH value of 5.0-6.0.

Most preferably, the ratio of the volume/L of the buffer solution to the mass/Kg of the epimedium extract is 5-15: 1.

Preferably, the enzymolysis reaction time in the step a is 1-20 hours.

More preferably, the enzymolysis reaction time is 10-20 hours.

Most preferably, the enzymatic reaction time is 15-20 hours.

Preferably, the weight ratio of the epimedium extract to the beta-glycosidase in step a is 1-50: 1.

More preferably, the weight ratio of the epimedium extract to the beta-glucosidase in step a is 2-20: 1.

Most preferably, the weight ratio of the epimedium extract to the beta-glucosidase in step a is 5-10: 1.

Preferably, in said step c, the filtered filter cake is dried first, then crushed, and then the crushed filter cake is dispersed in ethanol for reflux dissolution, and filtered, and the volume/L of ethanol in the filtrate is 10-100 times of the mass/Kg of the crushed filter cake.

Preferably, in the step c, the crude product of the first crystallization is added into ethanol for reflux dissolution, the solution is filtered, the filtrate is cooled for recrystallization, and the volume/L of the refluxed ethanol is 20 to 300 times of the mass/Kg of the crude product of the crystallization.

Preferably, in the step a, the epimedium extract is prepared by extracting epimedium Chinese medicinal materials with an ethanol solvent.

Preferably, the ethanol solvent is an ethanol-water mixed solvent, and the extract obtained by extracting the epimedium traditional Chinese medicinal material by using the ethanol-water mixed solvent is firstly concentrated, then is absorbed by a macroporous resin column, and is eluted by using the ethanol-water solvent with the volume concentration of 30-100% to obtain the epimedium extract for the enzymolysis reaction.

The invention has the beneficial effects that: 1. the invention takes the epimedium extract as the raw material, selectively converts the icariin in the extract into icariside I, the conversion rate reaches 70 percent, and the medicinal material resources are fully utilized. 2. The invention adopts an enzymolysis method to remove part of glycosyl in icariin, has mild reaction conditions and less impurities, can obtain icariside I with the purity of 99 percent only by a recrystallization mode, and is convenient for industrial mass production.

Drawings

FIG. 1, upper panel, shows a high performance liquid chromatogram of total flavonoids in ethanol extract through step 1.1 of example 1.

FIG. 1 is a high performance liquid chromatogram of total flavonoids in an ethanol extract purified in step 1.2 of example 1.

FIG. 2 shows a high performance liquid chromatogram of a pure product of icariside I.

FIG. 3 is the mass spectrum of icariside I [ M-H ]]^-。

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 "bed volume" herein refers to the total volume of the packing in the macroporous resin column, unless otherwise specified.

The term "β -glucosidase" herein is a β -glucosidase from Japan wild company, under the trade name Aromase H2, unless otherwise specified.

Example 1

1. Preparation of epimedium extract

1.1 extraction

In this example, 400Kg of the stem-removed arrow-leaf-and-epimedium leaf were extracted in several times, crushed, and extracted with 14 times and 10 times (v/w) of 40% ethanol aqueous solution by volume for 1 hour each time for 2 times. Recovering ethanol until the extractive solution has no alcohol smell, and detecting total flavone in the extractive solution by High Performance Liquid Chromatography (HPLC), as shown in the upper half of figure 1.

1.2 macroporous resin purification

And (3) concentrating the ethanol extract obtained in the step (1.1) to remove ethanol, and purifying by using macroporous resin. Firstly, eluting the macroporous resin column by using distilled water with the volume 8 times of that of a macroporous resin column bed, wherein the flow rate of the distilled water elution is 1 time of column volume/hour, and discarding the eluent after the elution is finished;

washing the macroporous resin column bed with 30% ethanol aqueous solution with volume concentration 6 times the volume of the macroporous resin column bed, and discarding the eluent after the elution is finished;

washing the macroporous resin column in the step 2 by using an ethanol aqueous solvent with the volume concentration of 60%, wherein the volume of the ethanol aqueous solvent is 4 times of that of the bed, adjusting the flow rate of the ethanol aqueous solution to be 1 time of the bed volume/hour, and collecting the eluent. Recovering ethanol, concentrating to obtain soft extract, and spray drying to obtain herba Epimedii extract 9.8 Kg. The flavone component of the herba Epimedii extract purified by macroporous resin obtained by the above three steps is detected by high performance liquid chromatography, as shown in the lower half of FIG. 1.

The macroporous resin in the example is purchased from Hebei Baen adsorption material Co., Ltd, and the model is HPD-300.

High Performance Liquid Chromatography (HPLC) conditions: a chromatographic column: an Agela-C18 column; mobile phase: acetonitrile-water (containing 0.0125% by volume of trifluoroacetic acid), elution gradient as shown in table 1 below, elution time 60min, flow rate: 1.0mL/min, column temperature: 35 ℃ is carried out. The detection wavelengths of the ultraviolet detector are 254 nm, 272 nm and 320nm respectively.

TABLE 1 solvent gradient elution Table

The result shows that except that the peak appearing in 3 minutes is a solvent peak, the peaks appearing in 22 minutes, 24 minutes, 46 minutes and 52 minutes are all the peaks of flavone components, and the total flavone map of the medicinal materials detected by the method is compared with the map (HPLC-UV) of the total flavone in the epimedium extract, so that the total flavone components in the epimedium extract are effectively enriched after the epimedium extract is purified by macroporous resin, wherein the mass yield of the total flavone in the purified epimedium extract is 2.0-10.0 percent relative to the epimedium medicinal materials.

2. Preparation of icariside I by enzymolysis reaction

1.0Kg of the above-mentioned epimedium extract purified by macroporous resin was weighed and added to a 15L1.0mol/L acetic acid-sodium acetate buffer solution having a pH of 5.9, and dissolved with stirring at 100 rpm. When the temperature of the system is constant at 60 ℃, 0.1Kg of beta-glycosidase is added and stirred for reaction. After reacting for 3 hours at constant temperature, stopping stirring, and cooling the reaction solution to 30 ℃.

3. Purification of icariside I

And after the enzymolysis reaction is finished, centrifuging the reaction solution, collecting a filter cake, drying the filter cake to obtain 0.70Kg, crushing the filter cake, adding 35Kg of ethanol, heating, refluxing, stirring for 1 hour, filtering while the mixture is hot, slowly cooling the filtrate to 25 ℃, preserving the temperature for 10 hours, filtering, collecting the filter cake, and drying the filter cake to obtain 0.21Kg of a primary icariside I crystal product. Pulverizing the first-time crystal product of icariside I, adding 16Kg of ethanol, heating and refluxing for 1 hr, filtering, slowly cooling the filtrate to 25 deg.C, maintaining the temperature for 10 hr, filtering, collecting the filter cake, and drying to obtain 0.12Kg of icariside I recrystallized product, wherein the liquid chromatogram and mass spectrum are shown in FIG. 2 and FIG. 3, the peak of icariside I appears at 6.037min retention time in FIG. 2, and the peak of 529.4 of nucleoplasm ratio appears in FIG. 3, which is [ M-H]^-Therefore, it is shown by the above figure that icariside I obtained by the method of the present invention has a high purity.

Example 2

1. Preparation of icariside I by enzymolysis reaction

Commercially available epimedium extract 1Kg was weighed, added to 15L of 0.8mol/L disodium hydrogenphosphate-citric acid buffer solution having pH of 6.0, and dissolved with stirring at 100 rpm. When the temperature of the system is constant at 55 ℃, 0.2Kg of beta-glycosidase is added and stirred for reaction. After reacting for 6 hours at constant temperature, stopping stirring, and cooling the reaction liquid to 25-30 ℃.

2. Longspur sheepPurification of hoproside I

Centrifuging the reaction solution, collecting a filter cake, drying to obtain 0.86Kg of filter cake, crushing, adding 60.2Kg of ethanol, heating, refluxing, stirring for 1 hour, filtering while hot, slowly cooling the filtrate to 25 ℃, keeping the temperature for 10 hours, filtering, collecting the filter cake, and drying to obtain 0.26Kg of icariside I primary crystal product. Pulverizing the primary crystal product of icariside I, adding 20Kg of ethanol, heating and refluxing for 1 hour, filtering, slowly cooling the filtrate to 25 ℃, keeping the temperature for 10 hours, filtering, collecting the filter cake, and drying to obtain the final product of icariside I recrystallization, wherein the total amount of the final product is 0.15 Kg.

Claims

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

a. firstly, carrying out enzymolysis reaction on an epimedium extract under the action of beta-glycosidase, wherein the mass content of icariin is at least 15%;

b. filtering the enzymolysis reaction product obtained in the step a;

c. drying the filtered filter cake, then crushing, dispersing the crushed filter cake in ethanol for refluxing and dissolving, filtering, wherein the volume/L of the ethanol in the filtrate is 10-100 times of the mass/Kg of the crushed filter cake, and crystallizing the filtered filter cake by using the ethanol to obtain the icariside I, wherein the beta-glycosidase is beta-glycosidase of Japan Tianye company and is called as Aromase H2 under the trade name.

2. The method as claimed in claim 1, wherein in the step a, the epimedium extract is dispersed in a buffer solution having a pH value of 3.5-7.5, and the enzymatic hydrolysis reaction temperature is 35-65 ℃.

3. The method of claim 2, wherein the buffer solution is selected from one or more of disodium hydrogen phosphate-potassium dihydrogen phosphate, sodium acetate, phosphoric acid-sodium phosphate, citric acid-sodium citrate, and disodium hydrogen phosphate-citric acid buffer solution with pH value of 5.0-6.0.

4. The method as claimed in claim 3, wherein the ratio of volume/L of the buffer solution to mass/Kg of the Epimedium extract is 5-15: 1.

5. The method of claim 3, wherein the enzymatic reaction time in step a is 1-20 hours.

6. The method of claim 5, wherein the enzymatic reaction time is 10-20 hours.

7. The method of claim 6, wherein the enzymatic reaction time is 15-20 hours.

8. The method as claimed in claim 1 or 2, wherein the weight ratio of the epimedium extract to the beta-glucosidase in step a is 1-50: 1.

9. the method as claimed in claim 8, wherein the weight ratio of epimedium extract to beta-glucosidase in step a is 2-20: 1.

10. the method as claimed in claim 9, wherein the weight ratio of epimedium extract to beta-glucosidase in step a is 5-10: 1.

11. the method as claimed in claim 1, wherein in step c, the crude crystals obtained from the first crystallization are added into ethanol for reflux dissolution, the mixture is filtered, the filtrate is cooled for recrystallization, and the volume/L of the refluxed ethanol is 20-300 times of the mass/Kg of the crude crystals.

12. The method as claimed in claim 1, wherein in the step a, the epimedium extract is prepared by extracting epimedium Chinese medicinal material with ethanol solvent.

13. The method as claimed in claim 12, wherein the ethanol solvent is ethanol-water mixed solvent, and the extract obtained by extracting the epimedium traditional Chinese medicinal material with the ethanol-water mixed solvent is firstly concentrated, then is absorbed by a macroporous resin column, and is eluted with ethanol-water solvent with volume concentration of 30-100% to obtain the epimedium extract for enzymolysis reaction.