WO2008138243A1 - A preparation method of icaritin - Google Patents

Abstract

Provided is a preparation method of icaritin, comprising treating the icariin with β-glucosidase in ethanol-water solution, then obtaining the icaritin by centrifugation and recrystallization. Also provided is a preparation method of icariin, comprising absorbing the concentrated barrenwort aqueous extract by using of D101 macroporous resin, eluting the resin with different concentrations of ethanol, followed by separating the 50% ethanol eluting fraction by silica gel chromatography and obtaining the icariin by recrycatllization.

Description

 Preparation method of icariin

Technical field

 The invention relates to the field of traditional Chinese medicine, in particular to a method for extracting the active ingredient icariin from Chinese traditional medicine Epimedium.

Background technique

 Epimedium is a commonly used Chinese medicinal material. It is a dry part of the plants of the genus Epimedium genus of the Berberridaceae family. The Pharmacopoeia of the People's Republic of China (2000 edition) contains the following five kinds of Epimedium medicinal herbs. Epimedium brevicornum Maxim) Epimedium sagittatum, Epimedium pubescens Epimedium wushanenes Epimedium koreanum Nakai. Epimedium has the effects of invigorating the kidney and strengthening the yang, strengthening the muscles and strengthening the bones, and removing the phlegm and dehumidification. It is often used to treat impotence, urinary dripping, waist and knee weakness, coronary heart disease, chronic bronchitis, neurasthenia and poliomyelitis. Modern pharmacological studies have shown that Epimedium contains special chemical components and significant biological activity. Currently, the chemical component has been isolated from Epimedium and is known as icariin. The molecular structure of icariin is:

 The chemical name is: 3, 5, 7-trihydroxy-4' methoxy-8-isopentenyl flavonoid-L-pyran rhamnose β-D-glucopyranoside.

The study found that because icariin contains two glycosyl groups, namely the glucosyl group at the 3-position and the rhamnosyl group at the 7-position, its activity is poor, and the degasting product of icariin is lascivious. The activity of alizarin is very high. Most of the domestic acid hydrolysis methods are used. However, hydrochloric acid hydrolysis easily forms an addition with the double bond on the 8-substituent, resulting in a product with a low yield and no use value. However, sulfuric acid hydrolysis can only remove the 7-position glycosyl group. , and the sugar on the 3-position The base is not easily hydrolyzed, and as a result, only low-glycosyl icariin can be obtained, and its activity is higher than that of icariin, but it is still far from the icariin. Patent application (Application No. 031336353) "Method for preparing low-sugar epimedium or aglycone by enzymatic hydrolysis of epimedium glycoside", which adopts fermentation-producing enzyme inducer of Epimedium as a strain, and extracts after fermentation The enzyme was used for the hydrolysis of epimedium sugar, but the results showed that the enzymatic hydrolysate was a low-sugar, sugar-removing, differently substituted epimedium or aglycone, indicating that the deglycation efficiency was not very good.

Summary of the invention

 The invention provides a preparation method of icariin, which has complete deglycation reaction and high yield, simple operation of the whole process, convenient treatment after reaction and simple purification.

 A method for preparing icariin, which is characterized in that icariin is obtained by enzymatic hydrolysis using β-glucosidase.

 The weight ratio of the β-glucosidase to icariin is 1 : 1-10, and the enzymatic reaction conditions are carried out in an alcohol-water solution at 40-60 ° C for 20-30 hours.

 Preferably, the weight ratio of β-glucosidase to icariin is 1:5, and the enzymatic reaction conditions are carried out in an alcohol-water solution at 50 ° C for 24 hours, and the alcohol is ethanol.

 The post-treatment of the reaction mixture after the enzymatic hydrolysis is carried out by a centrifugal treatment method in which the reaction mixture is centrifuged to discard the supernatant, dissolved in acetone, centrifuged, filtered, discarded, and the filtrate is steamed. dry.

 The preparation method of the icariin further comprises purification of icariin, which is recrystallized by acetone-water.

 The preparation method of icariin further comprises the steps of preparing icariin, wherein the preparation step of icariin comprises extracting total flavonoids of Epimedium and purifying icariin, and the total flavonoids of Epimedium Extraction steps include

(1) Providing water extract of Epimedium, (2) After concentration, it is adsorbed by D101 macroporous resin column, and then eluted with water, 30% ethanol, 50% ethanol, and then rinsed back with 90% ethanol and water respectively. (3) Charge 50% ethanol, concentrate, and dry.

 The concentrated solution content was 0.2 g of Epimedium Herbs / ml.

 The D101 macroporous resin is used in an amount of 1-2 times that of the epimedium raw material, the upper column flow rate of the concentrated liquid is 5 column volumes/h, and the eluent is used in an amount of 3-4 column volumes.

The purification of the icariin comprises a separation and purification method using a silica gel column, which includes using 10:0, 9:1, 8:1, 7:1, 6:1 chloroform-methanol gradient elution; Concentration of fractions containing icariin by TLC.

 The purification of the icariin further comprises recrystallizing the concentrate obtained above with methanol, and the filtrate is washed successively with acetone and methanol.

 From icariin to icariin, the key is to hydrolyze the sugar substituents on the 3-, 7- icariin. The inventors have made long-term research and exploration and found that β-glucosidase ( β-Glycosidase can hydrolyze the sugar substituents in the above two positions in one step, and the enzymatic hydrolysis yield is high (up to about 55%), and the subsequent treatment is simple, the whole enzymatic hydrolysis process is simple, and industrial production is easy.

 The ratio of the amount of β-glucosidase added to the weight of the reactant is 1:1-10, and the enzymatic reaction conditions are carried out in an alcohol-water solution at 40-60 ° C for 20-30 hours, since the icariin can be dissolved. In the alcohol, an aqueous alcohol solution is used as the reaction liquid, and the alcohol is selected from commonly used ethanol, which has low cost and low pollution. Since the hydrolysis reaction is carried out in ethanol, the enzyme is relatively easy to be inactivated, so the reaction time should not be too long, so the selection time is 20-30 hours, at which time the glycolytic reaction is substantially completed. Preferably, the weight ratio of β-Glycosidase to icariin is 1:5, and the enzymatic reaction conditions are carried out in an alcohol-water solution at 50 ° C for 24 hours.

 After the enzymatic hydrolysis reaction, the reaction mixture is centrifuged to discard the supernatant, then dissolved in acetone, centrifuged, filtered, and the filter residue is discarded. The filtrate is evaporated to dryness to obtain icariin, which is very simple and convenient, and is easy to industrialize. . Icariin can also be further purified by recrystallization to obtain a product of high purity, and the recrystallization solvent is an acetone-water mixed solvent. The purified ampicillin obtained in the present invention has a yield of 55%.

 The invention can be used for enzymatic hydrolysis of icariin, and can also be self-extracted from Epimedium medicinal materials. The advantage of self-extraction is that it can save cost, and can also monitor impurity content, thereby adopting various kinds of targeted Way to remove some impurities.

The preparation of icariin comprises the extraction of total flavonoids of Epimedium and the purification of icariin, and the extraction step of the total flavonoids of Epimedium includes boiling the extract of Epimedium with water, and then concentrating the extract to 0.2 g. Astragalus medicinal herbs/ml, adsorbed with D101 macroporous resin column (D101 macroporous resin column should be cleaned and repacked with some organic solvents adsorbed on it before use. D101 macroporous resin is used for epimedium raw materials. 1-2 times, the upper column flow rate of the concentrate is 5 column volumes / h, the amount of eluent is 3-4 column volumes, so that the separation effect is better.), eluted with an ethanol-water gradient. Wash with water until the eluate is light yellow, elute with 30% ethanol, elute with about 4 columns, then 50% ethanol. The eluate is the total flavonoid product of Epimedium, collected and concentrated. The yield is about 2% (that is, 100 g of Epimedium can obtain about 2 g of total flavonoids of Epimedium). The D101 macroporous resin column was rinsed back with 90% ethanol and water. The total flavonoids of Herba Epimedii are detected by HPLC and contain about 25% of icariin. Further purification is required for enzymatic hydrolysis.

 The purification of icariin was carried out by silica gel column separation and purification using a 10:0, 9:1, 8:1, 7:1, 6:1 gradient of chloroform-methanol; The developing solvent is chloroform: methanol: formic acid = 8: 2: 0.2, Rf value is between 0.5 and 0.6, and the icariin fraction is collected and concentrated. The obtained icariin was further purified, recrystallized from methanol, and the filtrate was washed with acetone and methanol. The purity of the product icariin thus obtained is about 95%, which fully satisfies the requirements of the next enzymatic reaction.

 The invention adopts β-glucosidase to carry out enzymatic hydrolysis reaction of icariin, and has complete deglycosylation and high yield, and adopts self-designed extraction of icariin from Epimedium medicinal material, and the whole process operation It is simple, easy to handle after reaction, and the purity of the product is in full compliance with the medicinal standards.

DRAWINGS

Figure 1 HPLC analysis of icariin (Epimedin) prepared in Example 1

Fig. 2 HPLC image of icariin prepared in Example 2

Figure 3 Total flavonoids of Herba Epimedii HPLC map

Figure 4 Icariin HPLC map

Figure 5 Example 3 HPLC of Epimedhasin

detailed description

The present invention will be further described in detail below with reference to the embodiments.

 (Examples 1 and 2 are commercially available products, and the purity is 98% or more. Example 3 is a self-made product, and β-glucosidase (β-Glycosidase) is an Amano enzyme preparation company. Product.) Example 1

 1. Enzymatic hydrolysis of icariin 100 g of icariin, adding 5000 ml of 20% ethanol, stirring and dissolving, adding 10 g of β-glucosidase, and hydrolyzing at 60 ° C for 20 hours.

 2. Post-treatment of the reaction solution: Centrifuge the reaction solution and discard the supernatant. The precipitate was dissolved in 2,500 ml of acetone, and the solution was centrifuged. The supernatant was taken out, filtered, and the insoluble matter was discarded, and the supernatant was evaporated to dryness.

3. Icariin (glycosides) Pure preparation:

Add acetone-water (1:1) 1500ml to recrystallize, place, crystallization, filter, dry, then obtain icariin Pure 29g.

The purity was over 98% by HPLC. (Fig. 1) HPLC conditions were acetonitrile - 1% aqueous glacial acetic acid 70:30, detection wavelength 273 nm. Melting point: Above 250 °C.

Total yield: 53.3%. Example 2

 1. Enzymatic hydrolysis of icariin 100 g of icariin, adding 5000 ml of 20% ethanol, stirring and dissolving, adding 100 g of P-glucosidase, and hydrolyzing at 40 ° C for 30 hours.

 2. Post-treatment of the reaction solution: Centrifuge the reaction solution and discard the supernatant. The precipitate was dissolved in 2,500 ml of acetone, and the solution was centrifuged. The supernatant was taken out, filtered, and the insoluble matter was discarded, and the supernatant was evaporated to 25 ml per gram (relative to icariin).

 3 Preparation of icariin aglycone:

 The concentrated supernatant was taken, and the same amount of water was recrystallized, placed, crystallized, filtered, and dried to obtain 30.2 g of pure aglycone. The purity was over 98% by HPLC. (Fig. 2) HPLC conditions were acetonitrile - 1% aqueous glacial acetic acid 70:30, detection wavelength 273 nm.

The total yield is 55.5 %.

Since the solvent acetone is used in the post-treatment and the solvent acetone is used in the recrystallization, steps can be saved and the process time can be saved. Example 3

 3.1 Extraction of total flavonoids from Herba Epimedii

 3.1.1 Crushing: 10,000 g of the dried medicinal herbs of Epimedium is crushed and placed in an extraction tank.

 3.1.2 Extraction: Add 16 times the amount of water to cook for 3 times, the first 2h, and the next 1.5h. The extracts were combined 3 times, filtered, and concentrated under reduced pressure to a solution of 0.2 g of crude drug /ml.

 3.1.3 Resin Purification: The extract was adsorbed with total flavonoids through a pre-treated D101 macroporous resin column (resin amount was approximately 1.5 times that of the medicinal material). The flow rate of the column on the extract was 5 column volumes / h. After adsorption, elute with water until the water eluent is pale yellow, about 4 column volumes, and then treated with 30% ethanol, about 4 column volumes, 50% ethanol, about 4 column volumes, 90% ethanol. Washed for use.

3.1.4 Concentration: Concentrate 50% ethanol elution under reduced pressure, vacuum dry at 60 ° C, pulverize, then get 200g -^n mouth

Wide mouth.

 3.1.5 Determination: The yield of total flavonoids is about 2% (that is, 2 g of total flavonoids can be obtained from 100 g of medicinal materials), and the content of icariin is about 25% by HPLC. See Figure 3. (For the determination method, see the first edition of the 2005 Chinese Pharmacopoeia, page 229)

3.2 Purification of icariin

 3.2.1 Sample: Epimedium 50% ethanol extract 200g (product of step 3.1), dissolved in methanol-water, mixed with 60-100 mesh silica gel (sample (g): silica gel (g) =1: 2) , dry, research into fine powder for use.

3.2.2 Packing: Wet packing. The ratio of filler to sample is about 1:10 (weight ratio), soak the silica gel with chloroform, put it into the column, and balance the bed to the height of the bed.

3.2.3 Loading: Dry loading.

 3.2.4 Elution: Gradient elution with chloroform-methanol (10: 0, 9: 1, 8: 1, 7: 1, 6: 1), TLC tracking to detect whether icariin eluted. Among them, 10: 0, 9: 1 two gradients, each gradient elutes 10 column volumes. The 8:1 gradient began to detect icariin and eluted 20 column volumes. The 7: 1 and 6: 1 gradients were each eluted until 30 column volumes were eluted.

 3.2.5 Further purification: by TLC, chloroform-methanol-formic acid 8 : 2: 0.2 was developed, Rf value was 0.6, and the fraction containing icariin was combined, evaporated to dryness under reduced pressure, and the solid was transferred to the triangle with hot methanol. In the bottle, place until the pale yellow powder is precipitated and suction filtration. The obtained solid was washed successively with acetone and methanol to obtain a crude product of about 95% of about 95% of icariin.

The results are shown in Fig. 4. HPLC conditions: acetonitrile - 1% aqueous glacial acetic acid 28:72. Melting point: 230 to 232 ° C.

The yield of icariin in this step is about 10%. (ie 100g total flavonoids can get 10g icariin) 3.3 Preparation of icariin

3.3.1 Icariin enzymatic hydrolysis:

20g of icariin, 50 times of 20% ethanol, stirred at 50 °C for 1h, β-glucosidase (1:5 enzyme dosage and substrate dosage), hydrolysis reaction at 50 °C for 24h .

3.3.2 Post-treatment of the reaction solution:

The reaction solution was centrifuged, and the supernatant was discarded. The precipitate was dissolved in 25 times of acetone, and the solution was centrifuged. The supernatant was taken out, filtered, and the insoluble matter was discarded, and the supernatant was evaporated to 25 ml per gram (relative to icariin). 3.3.3 Preparation of pure extract of icariin:

The concentrated supernatant was taken, and an equal amount of water was added to recrystallize, placed, crystallized, filtered, and dried to obtain 6 g of icariin.

The liquid phase test results are shown in Fig. 5. HPLC conditions: acetonitrile - 1% aqueous glacial acetic acid 70:30, detection wavelength 280 nm

Melting point: above 25 CTC. Yield of this step: The yield of this step is 55%. The self-made icariin has a purity of only 95% or more, and the enzymatic hydrolysate is also completely complete, and the obtained icariin can fully meet the medicinal standard. The overall yield of the process was 2% X 10% X 55% = 0.11%.

Claims

Rights request  A method for preparing icariin, which comprises the steps of: enzymatically reacting icariin with β-glucosidase.  2. The method for preparing icariin according to claim 1, wherein the weight ratio of β-glucosidase to icariin is 1:3-8, and the enzymatic reaction condition is 40-60°. The reaction is carried out in an alcohol-water solution of C for 20-30 hours.  The method for preparing icariin according to claim 2, wherein the weight ratio of the β-glucosidase to the icariin is 1:5, and the enzymatic reaction condition is an alcohol-water solution at 50 °C. The reaction was carried out for 24 hours, and the alcohol was ethanol.  The method for preparing icariin according to claim 3, wherein the post-treatment of the reaction mixture after the enzymatic hydrolysis is carried out by a centrifugal treatment method, wherein the reaction mixture is centrifuged to discard the supernatant. The solution is dissolved in acetone, centrifuged, filtered, and the residue is discarded, and the filtrate is evaporated to dryness.  The method for producing icariin according to claim 1, which further comprises purification of icariin, which is recrystallized using acetone-water.  6. The method for preparing icariin according to claim 1, further comprising the step of preparing icariin, wherein the step of preparing icariin comprises extracting total flavonoids of icariin and icariin Purification, the extraction process of the total flavonoids of Epimedium includes (1) providing epimedium water extract, (2) concentrating and adsorbing with D101 macroporous resin column, eluting with water, 30% ethanol, 50% ethanol Then, rinse it back with 90% ethanol and water, and (3) charge 50% ethanol, concentrate, and dry.  The method for producing icariin according to claim 6, wherein the concentrated solution content is 0.2 g of Epimedium medicinal material/ml.  The method for preparing icariin according to claim 7, wherein the amount of the macroporous resin of D101 is 1-2 times that of the medicinal material of Epimedium, and the flow rate of the upper column of the concentrated solution is 5 column volume/h. The eluent is used in an amount of 3-4 column volumes.  9. The method for preparing icariin according to claim 6, wherein the purification of the icariin comprises a separation and purification method using a silica gel column, which comprises using 10:0, 9:1, 8:1. 7: 1, 6:1 chloroform-methanol gradient elution; Concentration of fractions containing icariin by TLC.  The method for producing icariin according to claim 9, wherein the purification of the icariin further comprises recrystallizing the concentrate with methanol, and the filtrate is washed successively with acetone and methanol.