CN115894585A

CN115894585A - Preparation method of epimedium extract, and epimedium extract and application thereof

Info

Publication number: CN115894585A
Application number: CN202211661958.6A
Authority: CN
Inventors: 李斯文; 李珏泉; 李玉婷
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2022-12-23
Filing date: 2022-12-23
Publication date: 2023-04-04

Abstract

The invention discloses a preparation method of epimedium extract, the epimedium extract and application thereof, wherein the preparation method comprises the following steps: carrying out ultrasonic extraction on epimedium in a solvent, collecting an organic phase and a water phase after extraction, concentrating the organic phase under reduced pressure, and drying to obtain an epimedium extract A; extracting water phase with water saturated n-butanol, collecting n-butanol phase, concentrating under reduced pressure, and drying to obtain n-butanol phase herba Epimedii extract B; carrying out enzymolysis on the extract A to obtain epimedium enzymolysis products; carrying out acidolysis on the extract B to obtain an epimedium acidolysis substance; and uniformly mixing the enzymolysis product and the acidolysis product to obtain the epimedium extract C. The extract A prepared by the invention mainly contains icariin, and the extract B mainly contains epimedin C; the yield of icariside II in the zymolyte is 40.19-86.25%, the yield of anhydroicaritin in the acidolysis product is 61.27-77.51%, and the epimedium extract C has obvious effects of resisting fatigue and treating erectile dysfunction.

Description

Preparation method of epimedium extract, and epimedium extract and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a preparation method of an epimedium extract, and the epimedium extract and application thereof.

Background

Penile Erectile Dysfunction (ED) is one of the common sexual dysfunctions, which is characterized by an inability to erect or maintain a sufficient period of time for the penis. At present, most of the erectile dysfunction is treated clinically by western medicines, such as sildenafil citrate, which is a cyclic guanosine monophosphate (cGMP) -specific phosphodiesterase type 5 (PDE 5) selective inhibitor. But it has low selectivity for PDE6, and is liable to cause visual abnormalities such as blurred vision, blue/green vision, diplopia, achromatopsia, etc. (Wang Jianjun, shi Yili. Adverse reaction and safety monitoring of sildenafil [ J ]. Journal of clinical drug therapy, 2007 (02): 54-57.).

3238 Zxft 3238 (3262 Zxft 3262, pharmacological efficacy and action mechanism of icariin for preventing and treating erectile dysfunction. Beijing, first Hospital, beijing university, 2011-05-01.) points out that constructing an ED rat model through iliac artery ligation and testis extraction, and continuously irrigating icariin, can obviously improve the water average of ICP, iNOS and eNOS, and improve the ratio of smooth muscle/myofibril of the corpus cavernosum penis, and shows that icariin can effectively treat ED. Meanwhile, the effect of icariin on inhibiting PDE1-PDE11 enzyme is researched, and the result shows that icariin has selective inhibition effect on PDE5, can be combined with enzyme protein functional region of PDE5 in competition with cGMP, specifically blocks the degradation effect of PDE5 on cGMP, and proves that the icariin is similar to the action mechanism of sildenafil. And icariside II is dose-dependent and can improve the penile erection function of rats, and the action effect is stronger than that of icariin.

A large number of patents and literatures carry out relevant researches on metabolites of icariin, for example, CN107964555B discloses a method for realizing biological enrichment of icariside II by using trichoderma viride. And then the application of the compound in erectile dysfunction (CN 108392487A), reproductive dysfunction prevention (CN 105079013B) and kidney disease prevention (CN 105079014B) is verified. CN102417504A discloses an S-icaritin compound, a synthesis method and an application, wherein icariin is used as a raw material, dehydrated icaritin is obtained after naringinase treatment, a group capable of releasing hydrogen sulfide under the action of carboxylesterase in vivo is connected, and ED can be effectively treated under the dual actions of the dehydrated icaritin and the group. (Dell' Agli M. J Nat prod.2008Sep;71 (9): 1513-7) indicate that the IC50 values of icariin, icariside II, anhydroicaritin, and sildenafil for PDE5A1 were 5.9. Mu.M, 0.16. Mu.M, 2.2. Mu.M, and 0.075. Mu.M, respectively, indicating that the inhibitory effects of icariside II and anhydroicaritin against the PDE5 enzyme were all stronger than those of icariin. The above documents all show that icariin metabolites such as icariside II and anhydroicaritin have higher activity than icariin.

At present, a plurality of patents disclose methods for preparing icariside II and preparing anhydroicaritin by total synthesis by using icariin as a raw material. For example, CN106148454B discloses a method of hydrolyzing icariin by enzyme, and obtaining icariside II pure product by two subsequent crystallization methods; CN106755214B discloses a method for obtaining icariside II by taking icariin as a raw material and performing two-phase enzymatic hydrolysis; CN109776559B discloses a method for preparing dehydrated icaritin through total synthesis. However, no patent is published at present, and epimedin C and icariin are obtained by extraction and separation from epimedium herb and are further processed respectively to obtain dehydrated icaritin and icariside II, so that the effective components in the epimedium herb are utilized to the maximum extent.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the fact that only icariin in epimedium and metabolites thereof are subjected to relevant research in the prior art, but the epimedin C is ignored, so that the epimedin C is rich in content and can be developed into a component of a medicament for treating ED. The preparation method can realize the relative enrichment of icariin and epimedin C, and further processes the icariin and epimedin C respectively to obtain icariside II and anhydroicaritin with higher activity so as to more fully play the application of the epimedium in the fields of fatigue resistance and ED treatment.

The invention also provides an epimedium extract and application thereof.

The technical scheme is as follows: in order to achieve the above purpose, the preparation method of the epimedium extract comprises the following steps:

(1) Pulverizing herba Epimedii into powder, sieving, and mixing to obtain powder to be extracted;

(2) Putting the epimedium herb to-be-extracted powder obtained in the step (1) into an extraction solvent containing water and ethyl acetate, and performing ultrasonic extraction;

(3) Standing and layering the extracting solution obtained in the step (2), and separating an ethyl acetate phase and a water phase;

(4) Concentrating the ethyl acetate phase obtained in the step (3) under reduced pressure, and drying to obtain herba Epimedii extract A;

(5) Adding the water phase obtained in the step (3) into water saturated n-butanol for extraction, collecting the n-butanol phase, concentrating under reduced pressure, and drying to obtain herba Epimedii extract B;

(6) Putting the extract A obtained in the step (4) into a solvent containing hydrolase for enzymolysis, centrifuging the suspension, collecting the precipitate, and drying to obtain epimedium enzymolysis product;

(7) Putting the extract B obtained in the step (5) into an acid solution for acidolysis, adjusting the pH value to be neutral after acidolysis, centrifuging the suspension, collecting the precipitate, and drying to obtain an epimedium acidolysis substance;

(8) And (5) uniformly mixing the epimedium herb zymolyte and the acidolysis product obtained in the steps (6) and (7) to obtain the epimedium herb extract C.

Wherein the screened screening grain size in the step (1) is 80-100 meshes.

Wherein, the extraction solvent in the step (2) is water and ethyl acetate with the volume ratio of 1:1-1:3ml/ml of mixed solvent, wherein the material-liquid ratio of the powder to be extracted to the extraction solvent is 1:12-1:40g/mL, the ultrasonic extraction time is 30-90min, and the ultrasonic power is 100-200W.

Wherein the volume ratio of the water phase to the water-saturated n-butanol in the step (5) is 1:1-1:3ml/ml, extraction time of 5-30min, and extraction times of 1-3 times.

Wherein the hydrolase in the step (6) comprises cellulase, naringinase or helicase, the solvent is phosphate buffer solution, the dosage of the enzyme is 0.5-1.5% of the mass of the solvent, the enzymolysis temperature is 30-50 ℃, the enzymolysis time is 30min-2h, the ratio of the extract A to the solvent containing the hydrolase is 1:10-1:20g/mL.

Preferably, the hydrolase is helicase.

Wherein the acid solution in the step (7) is sulfuric acid, nitric acid or hydrochloric acid, the concentration is 1-3 mol/L, the acidolysis temperature is 70-80 ℃, the acidolysis time is 30min-2h, and the ratio of the extract B to the acidolysis solvent is 1:10-1:20g/mL.

The epimedium extract prepared by the preparation method of the epimedium extract is provided.

Wherein, the epimedium extract A in the epimedium extract contains icariin 75.31-127.59mg/g, the extract B contains epimedin C229.52-267.23mg/g, the yield of icariside II in zymolyte is 40.19-86.25%, and the yield of anhydroicaritin in acidolysis substance is 61.27-77.51%.

The epimedium extract is applied to preparing the medicines for resisting fatigue and treating erectile dysfunction.

In the invention, epimedin C, icariin, icariside II and anhydroicaritin are key indexes in the processes of epimedium extraction, enzymolysis and acidolysis, and the extraction conditions under different conditions are reflected by measuring the content of the epimedin C, the icariin, the icariside II and the anhydroicaritin.

The mechanism is as follows: under the action of a composite solvent of water and ethyl acetate, the invention can simultaneously and fully extract two effective components of icariin with poor water solubility and epimedin C with good water solubility, and can realize the primary separation of the icariin and the epimedin C. The separated water phase can further realize effective extraction of the epimedin C under the action of water-saturated n-butyl alcohol. The epimedin C and icariin have the same aglycone, namely anhydroicaritin, the main secondary glycosides of the epimedin C comprise rhamnosyl icariside II and anhydroicaritin, and the main secondary glycosides of the icariin comprise icariside I, icariside II and anhydroicaritin. Wherein the literature reports that icariside II, anhydroicaritin and icariin (Dell' Agli M.J Nat prod.2008Sep;71 (9): 1513-7)) have the inhibitory effects on PDE5 enzymes from large to small, and the literature indicates that the inhibitory effects of icariside II and anhydroicaritin on PDE5A1 are respectively 5.9 mu M, 0.16 mu M, 2.2 mu M and 0.075 mu M, which indicate that the inhibitory effects of icariside II and anhydroicaritin on PDE5 enzymes are all stronger than that of icariin; there is no report on epimedin C and some Toxicity (The toxicty and Metabolism Properties of Herba Epimedii Flavonoids on Laval and Adult Zebraphish). Meanwhile, a large number of documents report that Icariin is metabolized into related secondary glycosides through intestinal enzymes when absorbed into blood through intestines after being orally taken in vivo, and the secondary glycosides are fast in absorption, short in peak time and long in half-life, and are more favorable for the effect of treating ED (Liu C, biomed Pharmacother.2017Oct; 94.

Therefore, the extracted epimedin C and icariin need to be further processed. Compared with icariin, epimedin C has a rhamnosyl group connected with the rhamnosyl group at the alpha-L position of the 3 position, and common enzymes for hydrolyzing rhamnose bonds such as helicase, naringinase and alpha-L rhamnosidase can only hydrolyze the rhamnose bonds at the alpha-L position. The activity of epimedin C is influenced by the plurality of rhamnosyl groups, and the effect of rhamnosyl icariside II generated by enzymolysis on ED is rarely reported, so that more severe reaction conditions are needed to enable the rhamnosyl icariside II to generate dehydrated icaritin to exert the drug effect. Under such conditions, icariin tends to produce a series of secondary glycosides and unwanted by-products, and thus two different reaction conditions are required for treating epimedin C and icariin.

Meanwhile, because icariside II only enzymatically degrades glucose group at position 7 and retains rhamnosyl group at position 3 has stronger inhibitory effect on PDE5, the enzymatic hydrolysis condition of icariin also needs to be controlled, and the drug effect is prevented from being insufficiently improved due to excessive enzymatic hydrolysis into dehydrated icariin.

The invention creatively realizes the separation and relative enrichment of icariin and epimedin C in the extraction stage aiming at the situations, and carries out different reactions aiming at different extraction parts, so that the two most main effective components with the highest content in epimedium are reacted into the corresponding secondary glycoside forms which are easy to absorb and have higher activity. The icariin is enzymolyzed into icariside II, the epimedin C is acidolyzed into dehydrated icaritin, and the enzymolysis product and the acidolysis product are mixed uniformly to be used as a final epimedium extract, so that the medicinal effect of the epimedium on treating ED is improved to the maximum extent. Meanwhile, most of documents report that the activity of icariin on PDE5 is about one tenth of that of sildenafil, and the drug effects of icariside II and anhydroicaritin are several times of that of icariin, so that the dosage can be obviously reduced.

The epimedium extract obtained by the preparation method is rich in icariside II and anhydroicaritin, and the epimedin C and icariin with high content in epimedium are fully utilized, so that the drug effect of the epimedium is improved.

The method of the invention effectively realizes the extraction and conversion of the component which has the same mother nucleus, 2 to 3 times of icariin content and basically has no activity on the premise of realizing the conversion of the icariin into the secondary icariside II with the highest activity, namely the epimedin C and the icariin. The prior art only considers the extraction conversion of icariin, and basically does not pay attention to the potential value of epimedin C.

In fact, epimedin C is inherently high in epimedium crude drugs, but may have some toxicity, and has no anti-ED activity. Meanwhile, the epimedin C has certain activity after being acidolyzed or being subjected to other treatment methods to remove the flavonoid aglycone of rhamnosyl and glucosyl, namely the anhydroicaritin, so the invention realizes the conversion of the epimedin C-anhydroicaritin by adopting an acidolysis method. However, icariin cannot be converted into the structural form with the highest activity, namely icariside II under the harsh condition of acidolysis, so that the invention designs and realizes the relative separation and enrichment of icariin and epimedin C in the extraction stage and adopts the forms of enzymolysis and acidolysis respectively to ensure that the icariin and the epimedin C are converted into the theoretically highest active structures, namely the icariside II and the dehydrated icariin.

The invention effectively realizes the full utilization of two main components in the epimedium herb, namely icariin and epimedin C, and exerts the medicinal effect of the epimedium herb to the maximum extent. In the extraction stage, epimedin C and icariin are extracted respectively and converted into more active forms respectively. Wherein: 1. the epimedin C does not inhibit the activity of PDE5 enzyme per se, and because a rhamnosyl group is connected to the rhamnosyl group at the alpha-L position of the 3 position, but common enzymes for hydrolyzing rhamnose bonds, such as helicase, naringinase, alpha-L rhamnosidase and the like, can only hydrolyze the rhamnose bonds at the alpha-L position, so that more severe reaction conditions are needed to generate dewatered icariin to play the drug effect, and the icariin cannot obtain the secondary glycoside form with the highest activity of icariside II under the condition. 2. Icariin can be subjected to preliminary enzymolysis to form icariside II and can also be further subjected to enzymolysis to form dehydrated icaritin, and the icariside II is reported to be higher in activity in a literature, so that the reaction process needs to be controlled. The invention realizes the relative separation and enrichment of icariin and epimedin C by early-stage extraction, and obtains the secondary glycoside form with the highest theoretical activity by respectively treating with enzymolysis/acidolysis methods, thereby maximally improving the drug effect of epimedium.

The invention separates the epimedin C and the icariin for the first time and then respectively processes the separated epimedin C and the icariin, and the optimal active form is obtained by adopting different processing methods aiming at the epimedin C and the icariin. The chemical structure of epimedin C is that a rhamnosyl group is connected to the rhamnosyl group at the alpha-L position of the 3-position, and common enzymes for hydrolyzing rhamnose bonds such as helicase, naringinase, alpha-L rhamnosidase and the like can only hydrolyze the rhamnose bonds at the alpha-L position, so that more severe reaction conditions are needed to ensure that the anhydroicaritin is generated to play the drug effect. Actually, epimedin C has one more rhamnosyl group than icariin, so that the icariin has no activity, meanwhile, a part of glucose in icariin can be removed by conventional enzyme to improve the activity, but rhamnosyl icariside 2 obtained by treating the epimedin C with the enzyme still has no activity report. Furthermore, epimedin C needs to be converted into anhydroicaritin to be active under a severe condition, but icariin cannot be converted into the highest active form under the condition.

Has the beneficial effects that: compared with the prior art, the invention has the following advantages:

(1) The raw materials selected by the invention are screened in the early stage, crude medicinal materials with higher contents of epimedin C and icariin are selected, and after corresponding enzymolysis and acidolysis treatment, the absorption of the epimedium extract in the body is greatly improved, and the medicinal effect is more favorably exerted. A great deal of literature currently shows that Icariside can be absorbed in intestines after being taken orally and can be absorbed into blood to play a role only by being hydrolyzed into secondary glycoside forms such as Icariside I, icariside II and anhydroicaritin (Liu C, biomed Pharmacother.2017Oct; 94. Icariside II and anhydroicaritin not only have better drug effect than icariin, but also need to be metabolized by enzyme conversion when absorbed in vivo, so the invention is favorable for directly realizing the conversion of icariin in vitro.

(2) The invention realizes the separation and relative enrichment of icariin and epimedin C in the extraction stage, and carries out different reactions aiming at different extraction parts, so that the two most main effective components with the highest content in epimedium react into the corresponding secondary glycoside forms which are easy to absorb and have higher activity. The literature generally reports that epimedium contains epimedium and icariin as main active ingredients, and The content of epimedium C is approximately 3-5 times of that of icariin (effective bioconversion of epizoon C to icariin by a fungal enzyme from Aspergillus nidulans), but epimedium C has certain Toxicity (The Toxicity and Metabolism Properties of herbaceous Epimedium flavone and Adult Zebraphis). Therefore, in order to maximally utilize the effective components in epimedium, further treatment of conventional epimedium extracts is required. Meanwhile (Dell' Agli M. J Nat prod.2008Sep;71 (9): 1513-7) indicates that IC50 of icariin, icariside II, anhydroicaritin, and sildenafil on PDE5A1 are respectively 5.9. Mu.M, 0.16. Mu.M, 2.2. Mu.M, and 0.075. Mu.M, which indicates that the inhibitory effects of icariside II and anhydroicaritin on PDE5 enzyme are all stronger than that of icariin.

(3) The invention respectively carries out different treatments on the epimedin C and the icariin obtained by extraction and separation to obtain the respective optimal pharmacodynamic structure forms. Compared with icariin, epimedin C is additionally connected with a rhamnosyl group on the alpha-L position of the 3-position, and common enzymes for hydrolyzing rhamnoside bonds, such as helicase, naringinase and alpha-L rhamnosidase, can only hydrolyze the rhamnoside bonds on the alpha-L position. The active of epimedin C is influenced by the more rhamnosyl, and the effect of rhamnosyl icariside II generated by enzymolysis on ED is rarely reported, so that more severe reaction conditions are needed to enable the rhamnosyl icariside II to generate dehydrated icaritin to exert the drug effect. Under the condition, icariin generates a series of secondary glycosides and redundant byproducts, so that two different reaction conditions are required for treating epimedin C and icariin. Meanwhile, because icariside II only enzymatically degrades glucose group at position 7 and retains rhamnosyl group at position 3 has stronger inhibitory effect on PDE5, the enzymatic hydrolysis condition of icariin also needs to be controlled, and the drug effect is prevented from being insufficiently improved due to excessive enzymatic hydrolysis into dehydrated icariin.

(4) The invention establishes the simultaneous detection of epimedin C, icariin, rhamnosyl icariside II, icariside II and anhydroicaritin, and the analysis method has high separation degree, good peak shape and short detection time, and can complete the analysis of one sample injection within 30 minutes. On the basis of the detection method, the simultaneous detection of the extracts, zymolytes and acidolytics of the epimedium crude drugs prepared by different extraction, enzymolysis and acidolysis methods can be realized, the analysis error is greatly reduced, the detection efficiency is improved, and the subsequent related development is facilitated.

(5) The epimedium extract A prepared by the invention contains icariin 75.31-127.59mg/g, the extract B contains epimedin C229.52-267.23mg/g, the yield of icariside II in zymolyte is 40.19-86.25%, and the yield of anhydroicaritin in acidolysis substance is 61.27-77.51%, thus having obvious effects of resisting fatigue and treating erectile dysfunction.

Drawings

FIG. 1 is an HPLC chart of crude extract of Epimedium herb prepared in example 14 of the present invention.

FIG. 2 is an HPLC chart of Epimedium extract A prepared in example 3 of the present invention;

FIG. 3 is an HPLC chart of Epimedium extract B prepared in example 5 of the present invention;

FIG. 4 is a HPLC chart of the epimedium herb zymolyte prepared in example 7 of the present invention;

FIG. 5 is an HPLC chart of an acid hydrolyzate of Epimedium herb prepared in example 10 of the present invention;

FIG. 6 is an HPLC chart of epimedium herb zymolyte group B prepared in example 11 of the present invention;

FIG. 7 is a HPLC chart of acid hydrolysate group B of Epimedium herb prepared in example 12 of the present invention;

FIG. 8 is an HPLC chart of Epimedium herb extract group C prepared in example 13 of the present invention.

Detailed Description

In order that the present invention may be more readily understood, the present invention is further described below by reference to specific examples, which are intended to be illustrative only and not to be limiting in any way, the scope of the present invention being limited only by the claims, and any modifications or changes that can be easily made by one skilled in the art to the present invention without departing from the technical solution of the present invention will fall within the scope of the claims of the present invention.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The experimental procedures in the examples, which do not specify specific conditions, are generally carried out under conventional conditions or conditions recommended by the manufacturer.

Epimedium was purchased from Tongrentang.

The hydrolase is cellulase, helicase and naringinase from Aladdin (product number c 140864), source leaf (product number S10083) and Jiangsu Xin Rui Biotechnology limited, wherein the cellulase is 10000u/g and the helicase wall breaking rate is 90%.

In the embodiment, epimedin C, icariin, icariside II and anhydroicaritin in epimedium are simultaneously measured, and the chromatographic conditions are improved by referring to Chinese pharmacopoeia, which is concretely as follows:

chromatographic conditions

The chromatographic instrument is as follows: agilent 1260; the column was an Agilent zobax C18 (4.6 mm. Times.50 mm. Times.3.5 μm); the column temperature is 30 ℃; the flow rate is 1ml/min; the sample amount is 10 mul; the detection wavelength is 270nm; acetonitrile was used as mobile phase a and water as mobile phase B, and gradient elution was performed as specified in the table below.

Example 1

Herba Epimedii is used as raw material, and herba Epimedii extract A is prepared by the following process

(1) Pulverizing herba Epimedii as raw material with a pulverizer, sieving with 80 mesh sieve, and mixing to obtain powder to be extracted;

(2) Mixing epimedium powder to be extracted according to a material-liquid ratio of 1:20g/mL of the feed volume ratio of 1:1, performing ultrasonic extraction for 30min in a water-ethyl acetate solution under the condition of power of 150W;

(3) Standing the extractive solution obtained in step (2) for layering, separating ethyl acetate phase and water phase, concentrating the upper ethyl acetate phase under reduced pressure, and drying to obtain herba Epimedii extract A containing icariin 75.31mg/g.

Example 2

(1) Pulverizing herba Epimedii with a pulverizing machine, sieving with 80 mesh sieve, and mixing to obtain powder to be extracted;

(2) Mixing epimedium powder to be extracted according to the material-liquid ratio of 1:12g/mL of the mixture in a volume ratio of 1:2, performing ultrasonic extraction for 60min under the condition of 100W of power;

(3) Standing the extractive solution obtained in step (2) for layering, separating ethyl acetate phase and water phase, concentrating the upper ethyl acetate phase under reduced pressure, and drying to obtain herba Epimedii extract A containing icariin 105.29mg/g.

Example 3

(2) Mixing epimedium powder to be extracted according to a material-liquid ratio of 1:40g/mL input volume ratio 1:3, performing ultrasonic extraction for 90min in a water-ethyl acetate solution under the condition of 200W of power;

(3) Standing the extractive solution obtained in step (2) for layering, separating ethyl acetate phase and water phase, concentrating the upper ethyl acetate phase under reduced pressure, and drying to obtain herba Epimedii extract A containing icariin 127.59mg/g. (HPLC chromatogram is shown in FIG. 2)

Example 4

Epimedium extract B was prepared from the aqueous phase obtained in example 3 as the starting material by the following procedure

(1) The water phase obtained in example 3 was mixed in a volume ratio of 1: adding 1mL/mL of the mixture into water-saturated n-butanol, extracting for 20min in a separating funnel by shaking (100 rpm), standing, separating to obtain two phases, continuously extracting the upper n-butanol phase for three times;

(2) Combining the n-butanol phases obtained in the step (1), concentrating under reduced pressure, and drying to obtain herba Epimedii extract B containing epimedin C243.87mg/g.

Example 5

(1) The water phase obtained in example 3 was mixed in a volume ratio of 1: adding 2mL/mL of the extract into water-saturated n-butanol, extracting for 10min in a separating funnel by shaking (100 rpm), standing to separate two phases, continuously extracting the upper n-butanol phase, and totally extracting twice;

(2) Combining the n-butanol phases obtained in the step (1), concentrating under reduced pressure, and drying to obtain herba Epimedii extract B containing epimedin C267.23 mg/g. (HPLC profile is shown in FIG. 3)

Example 6

Epimedium extract B was prepared from the aqueous phase obtained in example 3 as the starting material by the following process

(1) The water phase obtained in example 3 was mixed in a volume ratio of 1: adding 3mL/mL of the mixture into water-saturated n-butanol, extracting in a separating funnel by shaking (100 rpm) for 30min, standing, separating two phases, and extracting once;

(2) Concentrating the n-butanol phase obtained in step (1) under reduced pressure, and drying to obtain herba Epimedii extract B containing epimedin C229.52 mg/g.

Example 7

Herba Epimedii extract A obtained in example 3 is used as raw material, and herba Epimedii zymolyte is prepared according to the following process

(1) Taking the epimedium extract A obtained in the embodiment 3 as a raw material, and mixing the raw material and the liquid according to a material-liquid ratio of 1:10g/ml of the solution is put into phosphate buffer solution containing helicase, the dosage of the enzyme is 1.0 percent of the mass of the solvent, the enzymolysis temperature is 50 ℃, and the enzymolysis time is 1h;

(2) Centrifuging the suspension obtained in the step (1), collecting the precipitate, and drying to obtain the epimedium herb zymolyte, wherein the yield of icariside II is 86.25%, and the content is 83.68mg/g. (HPLC profile is shown in FIG. 4)

Test example 1

The preparation method of example 7 was adopted, and different hydrolases helicase, cellulase, naringinase, icariside II content and anhydroicaritin content in step (1) were used as shown in Table 1.

TABLE 1 yield of icariside II and anhydroicaritin after enzymatic hydrolysis

The investigation result of the test example of the invention shows that the snailase is more advantageous to be used as the hydrolase. As shown in Table 1, the enzymolysis efficiency of cellulase is low, the yield of icariside II is low, naringinase is easy to remove rhamnosyl on the 3-position of icariin, the reaction process is not easy to control, the activity of inhibiting PDE5 enzyme is not obviously improved, the conversion efficiency of helicase is high, the yield of icariside II is high, and the helicase is less and completely enzymolyzed into dehydrated icariin, so the method has more advantages.

The yield of icariside II = the amount of icariside II obtained by actual enzymolysis/theoretically the amount of icariside II obtained by all enzymolysis is multiplied by 100%

The yield of the dehydrated icaritin = the amount of the dehydrated icaritin obtained by actual enzymolysis/the amount of the dehydrated icaritin obtained by theoretical total enzymolysis is multiplied by 100 percent

Test example 2

By adopting the preparation method of the embodiment 7, the epimedium extract A obtained in the embodiment 3 is used as a raw material, and the ratio of the raw material to the liquid is 1: throwing 10mg/ml of the solution into phosphate buffer solution containing helicase, wherein the dosage of the helicase is 0.5-1.5 percent of the mass of the solvent, the enzymolysis temperature is 30-50 ℃, and the enzymolysis time is 30min-2h;

the results are shown in tables 2-3.

TABLE 2 factors and levels for enzymatic investigations are listed in the table

TABLE 3 results of orthogonal experiments

Note: k is the mean factor level

According to the analysis of the range R, the influence sequence of the three factors on the enzymolysis efficiency of the epimedium extract A is as follows: enzymolysis temperature > enzymolysis time > enzyme dosage, and the best combination is A according to an orthogonal table ₃ B ₂ C ₃ . Namely, the epimedium extract A obtained in example 3 is most preferably combined as a raw material, and the ratio of the raw materials to the liquid is 1: adding 10mg/ml of the above extract into phosphate buffer solution containing snailase with solvent mass of 1.5%, performing enzymolysis at 50 deg.C for 1 hr, centrifuging the obtained suspension, collecting precipitate, and drying to obtain herba Epimedii zymolyte. However, considering that the influence of the enzyme dosage of 1.0% and 1.5% on the yield of icariside II is not large, the increase of the enzyme dosage in the actual production increases the cost on one hand and may cause the condition of excessive enzymolysis on the other hand. Comprehensively considering, the epimedium extract A obtained in example 3 is used as a raw material, and the ratio of material to liquid is 1: adding 10mg/ml of the extract into phosphate buffer solution containing helicase with the solvent mass of 1.0%, and performing enzymolysis extraction at 50 deg.CAnd centrifuging the obtained suspension, collecting the precipitate, and drying to obtain epimedium enzymolysis product.

Example 8

The epimedium acidolysis product is prepared by adopting the epimedium extract B obtained in the example 5 as a raw material according to the following process

(1) Taking the epimedium extract B obtained in the example 5 as a raw material, and mixing the raw materials according to a material-liquid ratio of 1:15g/ml of the mixture is put into 2mol/L sulfuric acid, and acidolysis is carried out for 1h at 75 ℃;

(2) Adjusting the pH of the acidolysis solution obtained in the step (1) to be neutral by using sodium hydroxide with the mass fraction of 10%, centrifuging the suspension, collecting the precipitate, and drying to obtain the epimedium acidolysis product, wherein the yield of the dehydrated icaritin is 61.27%.

The yield of the anhydroicaritin = the amount of anhydroicaritin obtained by actual acidolysis/the amount of icariside II available by theoretical total acidolysis × 100%.

Example 9

(1) Taking the epimedium extract B obtained in the example 5 as a raw material, and mixing the raw materials according to a material-liquid ratio of 1:10g/ml of the mixture is put into 3mol/L sulfuric acid, and acidolysis is carried out for 0.5h at 80 ℃;

(2) Adjusting the pH of the acidolysis solution obtained in the step (1) to be neutral by using sodium hydroxide with the mass fraction of 10%, centrifuging the suspension, collecting the precipitate, and drying to obtain the epimedium acidolysis product, wherein the yield of the dehydrated icaritin is 69.11%.

Example 10

Herba Epimedii extract B obtained in example 5 is used as raw material to prepare herba Epimedii acidolysis product by the following process

(1) Taking the epimedium extract B obtained in the example 5 as a raw material, and mixing the raw materials according to a material-liquid ratio of 1:20g/ml of the mixture is put into 1mol/L sulfuric acid, and acidolysis is carried out for 2h at 70 ℃;

(2) Adjusting the pH of the acidolysis solution obtained in the step (1) to be neutral by using sodium hydroxide with the mass fraction of 10%, centrifuging the suspension, collecting the precipitate, and drying to obtain the epimedium acidolysis product, wherein the yield of the dehydrated icaritin is 77.51%, and the content of the dehydrated icaritin is 92.74mg/g. The reason for the higher yield under the condition is probably related to the higher solvent dosage and the lower sulfuric acid concentration and temperature, and when the condition is too severe, the epimedin C can be acidolyzed into the anhydroicaritin and can be further damaged. (HPLC spectrum is shown in FIG. 5)

Example 11

The epimedium enzymolysis product B is prepared according to the method of the embodiment 7 by taking equivalent epimedium extract A and epimedium extract B obtained in the embodiment 3 and the embodiment 5, and contains rhamnosyl icariside II 91.23mg/g, icariside II 41.27mg/g and anhydroicaritin 1.47mg/g. (HPLC profile is shown in FIG. 6)

Example 12

Equivalent epimedium extract A and epimedium extract B obtained in example 3 and example 5 are taken to prepare epimedium acidolysis product B according to the method of example 10, and the product B contains rhamnosyl icariside II 4.12mg/g, icariside II 2.17mg/g and anhydroicaritin 45.13mg/g. (HPLC spectrum is shown in FIG. 7)

Example 13

Equal amount of epimedium enzymolysis products and epimedium acidolysis products obtained in the embodiment 7 and the embodiment 10 are taken and mixed evenly to obtain epimedium extract C, icariside II 42.36mg/g and anhydroicaritin 47.35mg/g.

(HPLC spectrum is shown in FIG. 8)

The above examples 11-13 show that the medicinal effects of epimedium herb are not fully applied when only a zymolyte or only an acidolysis product is used. I.e., icariin and epimedin C are not all converted to the most potent form.

Example 14

Herba Epimedii is used as raw material, and the crude extract is prepared by the following process

(2) Mixing epimedium powder to be extracted according to a material-liquid ratio of 1:40g/mL of the mixture is put into a reactor with a volume ratio of 1:3, performing ultrasonic extraction for 90min in a water-ethyl acetate solution under the condition of 200W of power;

(3) Concentrating the extracting solution under reduced pressure and drying to obtain the epimedium crude extract. (HPLC profile is shown in FIG. 1)

Test example 3

Pharmacodynamic experiment

1. The anti-fatigue experimental scheme is as follows:

a. grouping and raising animals

96 ICR mice were divided into 8 groups of 12 mice each, male, with a body weight of 20 + -2 g. Each group of mice is blank group, herba Epimedii extract A group, herba Epimedii extract B group, herba Epimedii zymolyte group, herba Epimedii acidolysis group, herba Epimedii zymolyte A group, herba Epimedii acidolysis group B group, and herba Epimedii extract C group.

The dry paste powders (i.e. the final products of the concentration and drying in each example) of the same amount in examples 3, 5, 7, 10, 11, 12 and 13 are respectively taken to prepare liquid medicines with certain concentration by using phosphate buffer as the epimedium extract A group, the epimedium extract B group, the epimedium zymolyte group, the epimedium acidolysis group, the epimedium zymolyte B group, the epimedium acidolysis group and the epimedium extract C group, and the phosphate buffer as the blank group. The administration was made by gavage of each group at a dose of 150mg/kg mouse body weight for 30 consecutive days, once a day.

b. Liver glycogen assay

After 30min of the last administration, 6 mice were randomly selected from each group, the mice were immediately sacrificed, the liver tissues were separated, rinsed with normal saline and blotted with filter paper, 100mg of liver tissues were accurately weighed, and the measurement was performed according to the liver/muscle glycogen measurement kit (Nanjing institute of bioengineering).

c. Weight bearing swimming experiment

After 30min of the last administration, the tail root weight-bearing lead skin (the weight of the lead skin is 5% of the body weight) of each group of the remaining mice was placed in a swimming box (the water temperature is 25.0 ℃ +/-1.0 ℃, and the water depth is 30 cm), and the time from the beginning of swimming to exhaustion of the mice (the heads of the mice are all immersed in water and cannot float out of the water surface for 10 s) is recorded, namely the time of the weight-bearing swimming of the mice.

The experimental results are as follows:

after the mice are administrated for a long time, the glycogen amount and the swimming time are greatly improved compared with those of a blank group, and the liver glycogen amount and the swimming time are sequentially divided into an epimedium extract C group, an epimedium zymolyte group, an epimedium acidolysis group, an epimedium zymolyte B group, an epimedium extract A group, an epimedium acidolysis group and an epimedium extract B group from large to small, wherein the epimedium extract B group has a certain anti-fatigue effect, and the epimedin C has no activity.

The anti-fatigue effects of the epimedium zymolyte group B, the epimedium acidolysis product group B and the epimedium extract group A are similar, and the fact that only the icariin and the epimedin C are subjected to the same enzymolysis or acidolysis treatment can lead to limited improvement of the drug effect because the rhamnosyl icariside II or the icariin with unobvious drug effect is generated and is easy to damage under the strong acid environment. Compared with the simple icariin, the icariin zymolyte group and the icariin acidolysis group have obviously improved drug effects, and show that icariside II and the product of anhydroicaritin obtained after the icariin and the epimedin C are respectively treated in the embodiments 7 and 10 have more obvious anti-fatigue effect compared with the icariin. Meanwhile, the epimedium extract C group obtained by mixing the two has the highest drug effect, which shows that icariside II and anhydroicaritin have potential synergistic action, and the synergistic action is related to overhigh administration dosage of the icariside II in a simple epimedium zymolyte group. The literature reports (Liu C, biomed Pharmacother.2017Oct; 94. The epimedium zymolyte and the epimedium acidolysis prepared by the invention can fully utilize high-content components in epimedium, including icariin and epimedin C with the content of about 2-3 times of the icariin, and are respectively developed into high-activity component forms, namely icariside II and anhydroicaritin.

TABLE 4 Effect of different drugs on mouse liver glycogen and swimming time

P < 0.05 compared to model control

2. Mating behavior assay protocol was as follows:

a. grouping and raising animals

Eight weeks old Wistar rats were collected in 96 groups of 6 males each with half male. Each group of mice is blank group, herba Epimedii extract B group, herba Epimedii extract A group, herba Epimedii zymolyte group, herba Epimedii acidolysis group, herba Epimedii zymolyte A group, herba Epimedii acidolysis group B group, and herba Epimedii extract C group.

Taking proper amount of dry paste powder in the

embodiments

3, 5, 7, 10, 11, 12 and 13, respectively, preparing liquid medicine with certain concentration by phosphate buffer solution as an epimedium extract A group, an epimedium extract B group, an epimedium zymolyte group, an epimedium acidolysis group, an epimedium zymolyte B group, an epimedium acidolysis group and an epimedium extract C group, and taking the phosphate buffer solution as a blank group. Each group was administered by gavage at a dose of 75mg/kg rat body weight for 30 consecutive days, once a day.

b. Establishment of female mouse estrus model

Before the mating test, female rats were acclimatized in a mating test cage every night and day, and were subjected to acclimatization training. At 48h before the experiment, 0.75. Mu.g/g estradiol benzoate was injected subcutaneously for each, and 10. Mu.g/g progesterone was injected 4h before.

c. Observation of mating behavior

Experiments were performed at night, keeping the ambient environment quiet, with slightly dark light, using a clear polypropylene observation cage, and recording relevant behavioral indicators. 1 male rat is put into a cage independently for 10min to adapt to the environment, then 1 female rat in estrus is put into the cage, and erection latency, ejaculation latency and mating latency are observed and recorded.

Wherein, the period from the co-location of male and female rats in the latent period of erection to the first erection of the male rats, the tail end of the penis is congested, and the penis body appears as erection; ejaculation latency refers to the time required for ejaculation after the penis is inserted into the vagina; the mating latency refers to the time required for the male and female rats to begin mating after penile erection.

TABLE 5 Effect of different drugs on sexual behavior in rats

P < 0.05 compared to model control group

The experimental result shows that the sexual capacity of a mouse is greatly improved after long-time administration, and the epimedium herb extract C prepared by the invention obviously shortens the erection latency and the mating latency of a rat, shows that the sexual desire of the rat is greatly improved, obviously prolongs the ejaculation latency, enhances the sexual capacity of the rat and promotes the erection of the penis of the rat. The degree of improving sexual function is sequentially herba Epimedii extract C group, herba Epimedii zymolyte group, herba Epimedii acidolysis group, herba Epimedii zymolyte B group, herba Epimedii extract A group, herba Epimedii acidolysis group B group, and herba Epimedii extract B group from large to small. Wherein the herba Epimedii zymolyte group B, herba Epimedii acidolysis product group B and herba Epimedii extract group A have similar effects; compared with the simple icariin, the epimedium enzymolysis product group and the epimedium acidolysis product group have obviously improved drug effect; meanwhile, the epimedium extract C group obtained by uniformly mixing the two has the highest medicinal effect. The above results and the results of the anti-fatigue experiments of mice show that according to the method of the invention, the icariin and the epimedin C are separated firstly, and then the zymolyte and the acidolysis product obtained after the respective treatment have very strong anti-fatigue and erectile dysfunction treatment effects, and meanwhile, the epimedium extract C prepared after the uniform mixing has higher drug effect and can fully utilize the main effective components in the crude medicinal materials of the epimedium.

In addition, it is known from the above experiments that the activity of icariside II is greater than that of anhydroicaritin, whereas that of anhydroicaritin is greater than that of icariin, and that epimedin C and rhamnosyl icariside II are inactive. The epimedium is subjected to preliminary enzymolysis to icariside II and further subjected to enzymolysis to dehydrated icaritin, and the epimedin C is subjected to preliminary acidolysis to rhamnosyl icariside II and further subjected to acidolysis to dehydrated icaritin. Therefore, the medicinal effect epimedium extract C group is similar to the epimedium zymolyte group, but the zymolyte group needs to consume 2 parts of icariin, the extract C group consumes 1 part of icariin, and the other part is provided by acidolysis of epimedin C, which indicates that the extract group can fully utilize the main effective components of epimedium.

Meanwhile, the epimedium extract B group and the acidolysis object group indicate that the epimedin C has no activity and the dehydrated epimedium element has activity after acidolysis; the extract A group and the zymolyte group show that the icariside II activity is greater than that of icariin; the epimedium zymolyte group and the zymolyte group B show that rhamnosyl icariside II prepared by enzymolysis of epimedin C has no activity; the acid hydrolysate group and the acid hydrolysate group B show that icariin is easy to be destroyed by acid hydrolysis, and the comparison with the epimedium extract group C further shows that the icariin and the epimedin C can obtain the maximum drug effect after being respectively treated after being separated.

Claims

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

(8) And (4) uniformly mixing the epimedium enzymolysis product and the acidolysis product obtained in the steps (6) and (7) to obtain an epimedium extract C.

2. The method for preparing epimedium extract according to claim 1, wherein the sieved grain size of the sieve of step (1) is 80-100 mesh.

3. The method for preparing epimedium extract according to claim 1, wherein the extraction solvent of step (2) is water and ethyl acetate in a volume ratio of 1:1-1:3, the ratio of the powder to be extracted to the extraction solvent is 1:12-1:40g/mL, the extraction time of ultrasonic extraction is 30-90min, and the ultrasonic power is 100-200W.

4. The method for preparing epimedium extract according to claim 1, wherein the volume ratio of the aqueous phase to the water-saturated n-butanol of step (5) is 1:1-1:3, the extraction time is 5-30min, and the extraction times are 1-3.

5. The preparation method of epimedium extract according to claim 1, wherein the hydrolase in step (6) comprises cellulase, naringinase or helicase, the solvent is phosphate buffer, the amount of the enzyme is 0.5-1.5% of the mass of the solvent, the enzymolysis temperature is 30-50 ℃, the enzymolysis time is 30min-2h, the ratio of extract A to solvent containing hydrolase is 1:10-1:20g/mL.

6. The method for preparing epimedium extract according to claim 1, wherein the acid solution in step (7) is sulfuric acid, nitric acid or hydrochloric acid, the concentration is 1mol/L to 3mol/L, the acid hydrolysis temperature is 70 ℃ to 80 ℃, the acid hydrolysis time is 30min to 2h, the ratio of extract B to acid hydrolysis solvent is 1:10-1:20g/mL.

7. An epimedium extract prepared by the preparation method of the epimedium extract of claim 1.

8. The epimedium extract according to claim 7, wherein the epimedium extract A contains icariin 75.31-127.59mg/g, the extract B contains epimedin C229.52-267.23mg/g, the yield of icariside II in zymolyte is 40.19-86.25%, and the yield of anhydroicaritin in acidolysis substance is 61.27-77.51%.

9. An application of the epimedium extract of claim 7 in preparing the medicines for resisting fatigue and treating erectile dysfunction.