阅读说明：本技术 酶转化高效制备淫羊藿苷元的方法 (Method for efficiently preparing icariin through enzyme conversion ) 是由 金凤燮 鱼红闪 于 2020-12-16 设计创作，主要内容包括：本发明公开一种酶转化高效制备淫羊藿苷元的方法,首先制备耐有机溶剂淫羊藿黄酮苷酶,将耐有机溶剂淫羊藿黄酮苷酶与主要的淫羊藿黄酮苷(朝藿定A或朝藿定B或朝藿定C或淫羊藿苷)在含有有机溶剂的反应体系中进行酶反应,可有效减少中间产物,提高淫羊藿苷元转化得率至90%以上。反应后的耐有机溶剂淫羊藿黄酮苷酶可以回收利用,降低了淫羊藿苷元的制备成本,具有操作简单、成本低、适合大批量生产等特点。(The invention discloses a method for preparing anhydroicaritin with high efficiency by enzyme conversion, which comprises the steps of firstly preparing organic solvent-resistant anhydroicaritin, and carrying out enzyme reaction on the organic solvent-resistant anhydroicaritin and main anhydroicaritin (epimedin A, epimedin B, epimedin C or icariin) in a reaction system containing an organic solvent, so that intermediate products can be effectively reduced, and the anhydroicaritin conversion yield can be improved to more than 90%. The organic solvent-resistant icariin after the reaction can be recycled, the preparation cost of the icariin is reduced, and the method has the characteristics of simple operation, low cost, suitability for mass production and the like.)

1. A method for efficiently preparing icariin by enzyme conversion is characterized in that: preparing organic solvent-resistant epimedium flavonoid glycosidase, and reacting the organic solvent-resistant epimedium flavonoid glycosidase with epimedium flavonoid glycoside in a reaction system containing an organic solvent to prepare the icariin.

2. The method for preparing icariin with high efficiency through enzymatic conversion according to claim 1, which is characterized by comprising the following steps in sequence:

a. adding 5-20% by mass of epimedium herb, liquorice stem and leaf or sophora flower serving as an enzyme production inducer into the dry culture medium, and fermenting aspergillus or aspergillus niger or yeast for 5-8 days by using a liquid fermentation method or a solid fermentation method to obtain a raw enzyme liquid, wherein the raw enzyme liquid is a fermentation liquid obtained by the liquid fermentation method or a leaching liquor of the culture medium obtained by the solid fermentation method;

b. centrifuging the raw enzyme solution to collect supernatant, adding 3 times of methanol or ethanol into the supernatant, standing overnight, centrifuging and collecting enzyme protein precipitate; dissolving the zymoprotein precipitate in 0.01-0.06M, pH 4-6 acetic acid buffer solution or phosphoric acid buffer solution or citric acid buffer solution with the volume of 1/3-1/10 of raw enzyme solution, and centrifuging to remove impurities to obtain organic solvent-resistant epimedium flavonoid glycosidase solution;

c. mixing an organic solvent-resistant epimedium flavonoid glycosidase solution with a methanol or ethanol solution of epimedium flavonoid glycoside with the mass concentration of 0.5-14%, and reacting at the temperature of 30-60 ℃ for 6-36 hours to obtain a reaction solution, wherein the epimedium flavonoid glycoside is at least one of epimedin A, epimedin B, epimedin C or icariin, and the volume concentration of the methanol or the ethanol is 5-40%;

d. centrifuging the reaction solution to collect precipitate A, washing the collected precipitate A with water, dissolving in tetrahydrofuran, and filtering; adding a methanol or ethanol solution with the volume concentration of 20-55% into the filtrate, and centrifuging and collecting a precipitate B; washing the precipitate B with ice-cold methanol or ethanol solution with volume concentration of 20-55%, and drying to obtain the icariin.

3. The method for efficiently preparing icariin through enzymatic conversion according to claim 2, wherein the d step of centrifuging collects the precipitate B and collects a supernatant, and the supernatant is concentrated and dried under reduced pressure to obtain icariin.

4. The method for efficiently preparing icariin by enzymatic conversion according to claim 2 or 3, wherein the d step is to centrifuge the reaction solution and then collect the precipitate A and the supernatant respectively, add 3 times volume of methanol or ethanol to the collected supernatant, collect the enzyme protein precipitate overnight and centrifuge; and d, dissolving the collected enzyme protein precipitate in 70-90% volume of the original enzyme solution obtained in the step a, and carrying out centrifugation to remove impurities so as to obtain a recovered enzyme solution, wherein the pH value of the solution is 4-6 acetic acid buffer solution or phosphoric acid buffer solution or citric acid buffer solution, and the pH value of the solution is 0.02-0.04M.

Technical Field

The invention relates to a preparation method of icariin, in particular to a method for efficiently preparing icariin through enzyme conversion.

Background

Herba epimedii is a common traditional Chinese medicine, and stems and leaves of the herba epimedii are used as medicines, so that the herba epimedii has the efficacies of tonifying kidney and strengthening yang, strengthening muscles and bones, dispelling wind and dampness, treating impotence and spermatorrhea, rheumatalgia and the like. The epimedium herbs which are found at present belong to more than 50 types of epimedium herbs, wherein the medicinal epimedium herbs which are collected in the Chinese pharmacopoeia 2015 edition are as follows: epimedium herb of berberidaceae (Epimedium brevicornum Maxim.)Epimedium brevicornumMaxim); epimedium sagittatum (Maxim.) MaximEpimedium sagittatumMaxim); epimedium pubescens (Epimedium pubescensMaxim); korean epimedium (Epimedium koreanumNakai) and Epimedium wushanense(Epimedium wushanenseT.s.ying), and the like. The main effective component of epimedium herb (stem and leaf) is epimedium flavonoid glycoside, and the structure is as follows:

common icariin flavonoid glycosides are shown in the following table.

However, more than 90% of epimedium flavonoid glycosides in epimedium Chinese medicinal materials are icariin with 2 glycosyl groups, epimedin A, epimedin B, epimedin C and the like with 3 glycosyl groups, and the icariin can not be directly absorbed by human bodies and has low physiological activity. After oral administration, icariin and glycosyl groups of epimedin A, epimedin B and epimedin C are degraded into anhydroicaritin without sugar group under the action of enzymes of intestinal digestive system and intestinal microorganisms, and the drug effect is absorbed. However, the transformation of the digestive system of the human body is very limited, so that most of the icariin is difficult to absorb. The icaritin has high absorptivity, and has good effects in resisting tumor, thrombi, hepatic fibrosis, osteoporosis, neuroprotection, improving prostate function, enhancing immunity, resisting inflammation, resisting oxidation, and resisting skin aging.

In order to obtain low-glycosyl icariin and aglycone, the Chinese invention patent No. ZL0313363.53 entitled method for preparing low-glycosyl icariin or aglycone by enzymatic hydrolysis of icariin glycosyl discloses that an enzyme produced by bacteria, molds and yeasts gradually hydrolyzes icariin-1 (epimedin A) or icariin-2 (epimedin B) or icariin-3 (epimedin C) glycosyl to form icariin-7 (icariin A) or icariin-8 (icariin B) or icariin-9 (icariin C); further hydrolyzing to icariin-15 (icariside II or baohuoside I), and finally becoming icaritin. The icariin (icariin-1) is gradually hydrolyzed by enzyme produced by bacteria, mold and yeast to become icariin-15 (icariside II or baohuoside I) or icariin-16 (icariside I), and finally becomes icariin aglycone. However, the method has many intermediate products, the yield of the icariin is very low and is less than 10 percent.

The following steps are provided: li Hui Ling, Chen hong Ji, Lin Chen, Ye De Xiao and Zhou jin Lin, etc., published in 2020 "research on process conditions for preparing icaritin by biological enzyme method", journal of biological chemical engineering, No. 2 months in 2020, No. 6 (No. 1), pages 62-64, and 68. The paper mainly researches 5 factors such as temperature, pH, buffer solution multiple, substrate-enzyme ratio, enzymolysis time and the like in the process of converting beta-glucosidase and enzymolysis icariin glycosyl into icariin (aglycon) so as to improve the method for converting the icariin (aglycon). However, the enzymatic conversion of epimedin a, epimedin B and epimedin C is not disclosed, and only the study of icariin as an enzyme reaction substrate has disadvantages of low reaction concentration, large reaction vessel, high industrial cost and the like.

So far, no report is available about the efficient preparation of icariin by using the reaction of icariin-ketolase and icariin (epimedin A or epimedin B or epimedin C or icariin) which are resistant to organic solvents in a reaction system added with organic solvents.

Disclosure of Invention

The invention aims to solve the technical problems in the prior art and provides a method for efficiently preparing icariin through enzyme conversion.

The technical solution of the invention is as follows: a method for efficiently preparing icariin by enzyme conversion is characterized in that: preparing organic solvent-resistant epimedium flavonoid glycosidase, and reacting the organic solvent-resistant epimedium flavonoid glycosidase with epimedium flavonoid glycoside in a reaction system containing an organic solvent to prepare the icariin.

The preparation method sequentially comprises the following steps:

a. adding 5-20% by mass of epimedium herb, liquorice stem and leaf or sophora flower serving as an enzyme production inducer into the dry culture medium, and fermenting aspergillus or aspergillus niger or yeast for 5-8 days by using a liquid fermentation method or a solid fermentation method to obtain a raw enzyme liquid, wherein the raw enzyme liquid is a fermentation liquid obtained by the liquid fermentation method or a leaching liquor of the culture medium obtained by the solid fermentation method;

b. centrifuging the raw enzyme solution to collect supernatant, adding 3 times of methanol or ethanol into the supernatant, standing overnight, centrifuging and collecting enzyme protein precipitate; dissolving the zymoprotein precipitate in 0.01-0.06M, pH 4-6 acetic acid buffer solution or phosphoric acid buffer solution or citric acid buffer solution with the volume of 1/3-1/10 of raw enzyme solution, and centrifuging to remove impurities to obtain organic solvent-resistant epimedium flavonoid glycosidase solution;

c. mixing an organic solvent-resistant epimedium flavonoid glycosidase solution with a methanol or ethanol solution of epimedium flavonoid glycoside with the mass concentration of 0.5-14%, and reacting at the temperature of 30-60 ℃ for 6-36 hours to obtain a reaction solution, wherein the epimedium flavonoid glycoside is at least one of epimedin A, epimedin B, epimedin C or icariin, and the volume concentration of the methanol or the ethanol is 5-40%;

d. centrifuging the reaction solution to collect precipitate A, washing the collected precipitate A with water, dissolving in tetrahydrofuran, and filtering; adding a methanol or ethanol solution with the volume concentration of 20-55% into the filtrate, and centrifuging and collecting a precipitate B; washing the precipitate B with ice-cold methanol or ethanol solution with volume concentration of 20-55%, and drying to obtain the icariin.

And d, centrifugally collecting the precipitate B and collecting supernatant, and concentrating and drying the supernatant under reduced pressure to obtain the icariin.

D, centrifuging the reaction solution, collecting precipitate A and supernatant respectively, adding 3 times of methanol or ethanol into the collected supernatant, standing overnight, centrifuging and collecting zymoprotein precipitate; and d, dissolving the collected enzyme protein precipitate in 70-90% volume of the original enzyme solution obtained in the step a, and carrying out centrifugation to remove impurities so as to obtain a recovered enzyme solution, wherein the pH value of the solution is 4-6 acetic acid buffer solution or phosphoric acid buffer solution or citric acid buffer solution, and the pH value of the solution is 0.02-0.04M.

The invention firstly prepares the organic solvent-resistant epimedium flavonoid glycosidase, and carries out enzyme reaction on the organic solvent-resistant epimedium flavonoid glycosidase and main epimedium flavonoid glycoside (epimedin A, epimedin B, epimedin C or icariin) in a reaction system containing an organic solvent, thereby effectively reducing intermediate products and improving the conversion yield of icariin to more than 90 percent. The organic solvent-resistant epimedium flavonoid glycosidase can be recycled, the preparation cost of the icariin is reduced, and the method has the characteristics of simple operation, low cost, suitability for mass production and the like.

Detailed Description

The analysis method adopted by the embodiment of the invention is as follows:

firstly, detecting epimedin A, B, C and icariin by a High Performance Liquid Chromatography (HPLC): the chromatograph, a Waters 2695 high performance liquid chromatography analyzer, a Waters 2996 diode array detector and an Empower chromatographic workstation. Chromatography column, Zhonghui Kromasil C18 chromatography column (5 μm, 4.6 mm. times.250 mm); mobile phase, acetonitrile (a) water (B): 17-27A for 0-8 min; 8-32 min, 27A and the like; 32-60 min, and 27% -85% A linear gradient; gradient of 60-70 min, 85A and the like; 70-80 min, 85-90A; sample size, 10L; column temperature, 35 ℃; flow rate, 1.0 mL/min; detection wavelength, 273 nm. The reference standard substance epimedin A, B, C or icariin and the sample to be detected are respectively added with 2 mg, 10 mL of 50% ethanol is respectively added to prepare a standard substance solution of 0.2 mg/mL and the sample to be detected.

Secondly, detecting icariin by college liquid chromatography (HPLC): the chromatograph, a Waters 2695 high performance liquid chromatography analyzer, a Waters 2996 diode array detector and an Empower chromatographic workstation. A chromatographic column: zhonghui Kromasil C18 column (5 μm, 4.6 mm. times.250 mm); mobile phase: methanol-tetrahydrofuran-0.1% phosphoric acid water (35: 26: 39), isocratic elution for 40 min. Sample size, 10 μ L; column temperature, 35 ℃; the volume flow rate is 1.0 mL/min; detection wavelength, 273 nm. Dissolving 1 mg standard substance icariin in 10 mL of methanol to prepare a solution with the concentration of 1 mg/mL; filtering with 0.45 μm filter membrane for use. A sample to be tested: precisely weighing 1 mg of icariin product, dissolving in 10 mL of methanol to obtain 0.1 mg/mL solution, and adding 1.5 mL of methanol into 1 mL of the solution to obtain 0.04 mg/mL sample solution. Filtering with 0.45 μm filter membrane for use.

Thirdly, determining the epimedium flavonoid glycosidase reactant by a Thin Layer Chromatography (TLC) method: the enzyme reactant is spotted on a silica gel plate, and the developing agent is ethyl acetate: butanone: methanol: water = 8: 7: 1: 1, developing the developed TLC plate under ultraviolet 275 nm, and taking a picture; the proportion of each epimedium flavonoid glycoside of TLC version is determined by scanning with Bandscan software.

Example 1:

the method for efficiently preparing the icariin through enzyme conversion sequentially comprises the following steps of:

a. aspergillus [ 2 ]Aspergullussp.39g bacteria, reference: dongming Wang et al, Process Biochemistry 47 (2012) 133-138 page]Inoculating the strain into 1000 ml of liquid culture medium containing 5% corn steep liquor and 1% herba Epimedii powder, and culturing at 29 deg.C under stirring for 7 days to obtain fermentation liquid, i.e. stock enzyme solution;

b. centrifuging the raw enzyme solution to remove impurities and collecting supernatant, adding 3 times of methanol solution into the supernatant, standing overnight, centrifuging and collecting enzyme protein precipitate; dissolving the zymoprotein precipitate in citric acid buffer solution with volume of 1/10, pH of 5.0 and 0.04M of raw zymolyte solution, and centrifuging to remove impurities to obtain organic solvent resistant icariin flavosidase solution of Aspergillus;

c. mixing 70 ml of organic solvent-resistant epimedium flavonoid glycosidase solution with 70 ml of methanol solution (volume concentration is 30%) of commercial icariin with the mass concentration of 8.57%, and reacting at the temperature of 50 ℃ for 28 hours to obtain reaction solution; detecting almost all icariin in the reaction solution by TLC to be converted into icariin;

d. centrifuging the reaction solution, respectively collecting precipitate A and supernatant, adding 3 times of methanol into the collected supernatant, standing overnight, centrifuging, and collecting zymoprotein precipitate; and (b) dissolving the collected enzyme protein precipitate in 70% volume of 0.02M pH5 citric acid buffer solution of the original enzyme solution obtained in the step a, centrifuging to remove impurities to obtain a recovered enzyme solution, wherein the enzyme activity of the recovered enzyme solution is equal to that of the original enzyme solution obtained in the step a, and the recovered enzyme solution can be repeatedly used.

Washing the collected precipitate A with 10 ml of water for 2-3 times, dissolving in 25 ml of tetrahydrofuran, and filtering; adding 50 ml of 40% methanol solution in volume concentration into the filtrate, and centrifuging to collect precipitate B; washing the precipitate B with 6 ml of ice-cold 40% methanol for 2-3 times, and drying at 60 ℃ to obtain 3.0 g of icariin. HPLC detection shows that the purity of the icariin product reaches 98 percent, and the theoretical yield of the icariin is 91.8 percent.

The supernatant was collected while collecting the precipitate B by centrifugation, and the supernatant was concentrated under reduced pressure and dried to obtain 0.24 g of 91.4% icaritin (containing 1.94% icariside II and 1.42% icariside I).

Replacing icariin with at least one of commercially available epimedin A, epimedin B or epimedin C with the mass concentration of 0.5-14% and replacing methanol with the volume concentration of 30% in the step C with methanol or ethanol with the volume concentration of 5-40% by the method of the embodiment 1, and the icariin product with the theoretical yield of more than 90% can be prepared.

Example 2:

a. preparing the epimedium flavonoid glycosidase resistant to the organic solvent: aspergillus niger [ 2 ]Aspergullus nigerg.848, reference: yongkun Xiao et al, J.ginseng Res.43 (2019) page 195-]The strain was inoculated into 400 g (dry matter) of a solid medium containing 10% licorice stem-leaf powder, and cultured (occasionally stirred) at 30 ℃ for 8 days; then leaching the enzyme in the solid culture medium by using 2000 ml of normal saline, separating supernatant, and filtering to obtain filtrate, namely raw enzyme solution;

b. centrifuging the raw enzyme solution to remove impurities and collect supernatant, adding ethanol with volume concentration of 95% of 3 times of that of the supernatant, standing overnight, centrifuging and collecting zymoprotein precipitate; dissolving the zymoprotein precipitate in a phosphate buffer solution with the volume of 1/5, 0.04M and the pH value of 5.0 of the original zymogen solution, and centrifuging to remove impurities to obtain an organic solvent resistant epimedium flavonoid glycosidase solution of aspergillus niger;

c. mixing 100 ml of organic solvent-resistant epimedium flavonoid glycosidase solution with 100 ml of ethanol solution of commercial epimedin A with the mass concentration of 6% (the volume concentration of the ethanol is 30%), and reacting for 20 hours at the temperature of 50 ℃ to obtain reaction solution; detecting all the epimedin A to be converted into epimedium aglycone by TLC;

d. centrifuging the reaction solution, respectively collecting precipitate A and supernatant, adding 3 times of ethanol into the collected supernatant, standing overnight, centrifuging, and collecting zymoprotein precipitate; and (b) dissolving the collected enzyme protein precipitate into 80 volume percent of the original enzyme solution obtained in the step a and 0.03M, pH5 phosphate buffer solution, centrifuging to remove impurities to obtain a recovered enzyme solution, wherein the enzyme activity of the recovered enzyme solution is equal to that of the original enzyme solution obtained in the step a, and the recovered enzyme solution can be repeatedly used.

Washing the collected precipitate A with 10 ml of water for 2-3 times, dissolving in 25 ml of tetrahydrofuran, and filtering; adding 50 ml of 50% ethanol solution in volume concentration into the filtrate, and centrifuging to collect precipitate B; washing the precipitate B2-3 times with 6 ml of ice-cold ethanol with the volume concentration of 30%, and drying at 60 ℃ to obtain 2.41 g of icariin. HPLC detection shows that the purity of the icariin product reaches 98 percent, and the theoretical yield of the icariin is 91.1 percent.

By the method of example 2, an icariin aglycone product with a theoretical yield of 90% or more can be prepared by replacing epimedin a with at least one of commercially available icariin with a mass concentration of 1-14%, or epimedin B or epimedin C.

Example 3:

a. preparing the epimedium flavonoid glycosidase resistant to the organic solvent: candida was inoculated into 400 g (dry matter amount) of a wheat bran solid medium containing 15% of sophora flower powder, and cultured (occasionally stirred) at 30 ℃ for 8 days; then leaching the enzyme in the solid culture medium by using 2000 ml of 0.02 mol and pH =5.0 acetic acid buffer solution, separating supernatant, and filtering to obtain filtrate, namely raw enzyme solution;

b. centrifuging the raw enzyme solution to remove impurities and collect supernatant, adding methanol with 3 times of volume into the supernatant, standing overnight, centrifuging and collecting zymoprotein precipitate; dissolving the zymoprotein precipitate in acetic acid buffer solution with the volume of the original zymolyte solution of 1/8, 0.04M and the pH value of 5.0, and centrifuging to remove impurities to obtain the organic solvent-resistant epimedium flavonoid glycosidase solution of the candida;

c. mixing 100 ml of organic solvent-resistant epimedium flavonoid glycosidase solution with 100 ml of ethanol solution of commercial epimedin C with the mass concentration of 6% (the volume concentration of the ethanol is 25%), and reacting for 24 hours at the temperature of 50 ℃ to obtain reaction solution; detecting all epimedin C by TLC to be converted into icariin;

d. centrifuging the reaction solution, respectively collecting precipitate A and supernatant, adding 3 times of ethanol into the collected supernatant, standing overnight, centrifuging, and collecting zymoprotein precipitate; and (b) dissolving the collected enzyme protein precipitate in 70 volume percent of the original enzyme solution obtained in the step (a) of 0.04M acetic acid buffer solution with pH5, centrifuging to remove impurities to obtain a recovered enzyme solution, wherein the enzyme activity of the recovered enzyme solution is equal to that of the original enzyme solution obtained in the step (a), and the recovered enzyme solution can be reused.

Washing the collected precipitate icariin A with 10 ml of water for 2-3 times, dissolving in 25 ml of tetrahydrofuran, and filtering; adding 50 ml of 50% ethanol solution, centrifuging and collecting the precipitate icariin B; washing the precipitate B2 times with 6 ml of ice-cold 30% ethanol, and drying at 60 deg.C to obtain 2.41 g icariin. HPLC detection shows that the purity of the icariin product reaches 98 percent, and the theoretical yield of the icariin is 91.3 percent.

By the method of example 3, the epimedin aglycone product with the theoretical yield of more than 90 percent can be prepared by replacing epimedin C with at least one of commercial icariin with the mass concentration of 0.5 to 14 percent, epimedin A or epimedin B, and the volume concentration of ethanol used in the step C is 10 to 40 percent.