Preparation method of epimedium extract and epimedium extract and application thereof

Abstract

The invention discloses a preparation method of an epimedium extract and the epimedium extract and application thereof. The preparation method comprises the following steps: ultrasonically extracting epimedium in a solvent, collecting an organic phase and an aqueous phase after the extraction, and drying the organic phase after decompression concentration to obtain an epimedium extract A; adding water-saturated n-butanol to the aqueous phase for extraction, collecting the n-butanol phase after the extraction, and drying the n-butanol phase epimedium extract B; enzymolyzing the extract A to obtain an epimedium enzymolysate; acid-hydrolyzing the extract B to obtain an epimedium acid hydrolysate; and uniformly mixing the enzymolysis product and the acid hydrolysate to obtain an epimedium extract C. The extract A prepared by the invention mainly contains icariin, and the extract B mainly contains epimedin C; the yield of icariin II in the enzymatic hydrolysate is 40.19-86.25%, and the yield of dehydrated icariin in the acid hydrolysate is 61.27-77.51%. The epimedium extract C has obvious anti-fatigue and erectile dysfunction treatment effects.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and in particular relates to a preparation method of an epimedium extract and the epimedium extract and application thereof. Background Art

[0002] Erectile dysfunction (ED) is one of the common sexual dysfunctions, which is manifested by the inability of the penis to erect normally or maintain a sufficient erection for a sufficient period of time. At present, most of the clinical treatments for erectile dysfunction are Western medicine, such as sildenafil citrate, a selective inhibitor of phosphodiesterase type 5 (PDE5) specific for cyclic guanosine monophosphate (cGMP). However, it is not very selective for PDE6 and can easily cause visual abnormalities such as blurred vision, blue / green vision, diplopia, and color blindness (Wang Jianjun, Shi Yili. Adverse reactions and safety monitoring of sildenafil [J]. Journal of Clinical Drug Therapy, 2007(02):54-57.).

[0003] Xin Zhongcheng (Xin Zhongcheng, Pharmacological efficacy and mechanism of action of icariin in the prevention and treatment of erectile dysfunction. Beijing, Peking University First Hospital, 2011-05-01.) pointed out that the ED rat model was established by internal iliac artery ligation and testicular removal, and continuous gavage of icariin could significantly increase the levels of ICP, iNOS, and eNOS, and improve the ratio of smooth muscle / myofibril in the corpus cavernosum, indicating that icariin can effectively treat ED. At the same time, the article studied the inhibitory effect of icariin on PDE1-PDE11 enzymes. The results showed that it had a selective inhibitory effect on PDE5, could compete with cGMP and combine with the enzyme protein functional region of PDE5, and specifically blocked the degradation of cGMP by PDE5, proving that its mechanism of action was similar to that of sildenafil. In addition, icariin II improved the erectile function of rat penis in a dose-dependent manner, and its effect was stronger than icariin.

[0004] A large number of patents and literature have conducted relevant research on the metabolites of icariin, such as CN107964555B, which discloses a method for bio-enrichment of icariin II using Trichoderma viride. Its application in erectile dysfunction (CN108392487A), prevention and treatment of reproductive dysfunction (CN105079013B), and prevention and treatment of kidney disease (CN105079014B) was subsequently verified. CN102417504A discloses an S-icariin compound, a synthesis method, and its use. Icariin is used as a raw material, and dehydrated icariin is obtained after treatment with naringinase, and a group that can release hydrogen sulfide under the action of carboxylesterase in the body is attached. Under its dual action, it can effectively treat ED. (Dell'Agli MJ Nat Prod. 2008 Sep; 71(9): 1513-7) pointed out that the IC50 values ​​of icariin, icariin II, dehydrated icariin, and sildenafil on PDE5A1 were 5.9 μM, 0.16 μM, 2.2 μM, and 0.075 μM, respectively, indicating that icariin II and dehydrated icariin have stronger inhibitory effects on PDE5 enzyme than icariin. The above literatures all show that the metabolites of icariin, such as icariin II and dehydrated icariin, have higher activity than icariin.

[0005] At present, many patents have disclosed methods for preparing icariin II using icariin as raw material and preparing dehydrated icariin by total synthesis. For example, CN106148454B discloses a method of enzymatic hydrolysis of icariin under the action of pectinase, and then obtaining pure icariin II by two crystallizations; CN106755214B discloses a method of obtaining icariin II by biphasic enzymatic hydrolysis using icariin as raw material; CN109776559B discloses a method for preparing dehydrated icariin by total synthesis. However, there is no patent that discloses a method for extracting and separating epimedin C and icariin from epimedium, and further treating them separately to obtain dehydrated icariin and icariin II, thereby maximizing the utilization of the effective ingredients in epimedium. Summary of the invention

[0006] Purpose of the invention: The prior art only studies icariin and its metabolites in epimedium, while ignoring epimedin C, which is more abundant and can also be developed into a component of drugs for treating ED. The preparation method of the present invention can achieve the relative enrichment of icariin and epimedin C, and further process them to obtain more active icariin II and dehydrated icariin, so as to give full play to the application of epimedium in the fields of anti-fatigue and treatment of ED.

[0007] The invention also provides an epimedium extract and application thereof.

[0008] Technical solution: In order to achieve the above-mentioned purpose, the preparation method of the epimedium extract of the present invention comprises the following steps:

[0009] (1) Using Epimedium as a raw material, grinding, sieving, and mixing to obtain a powder to be extracted;

[0010] (2) putting the epimedium powder to be extracted obtained in step (1) into an extraction solvent containing water and ethyl acetate, and performing ultrasonic extraction;

[0011] (3) allowing the extract obtained in step (2) to stand for stratification, and taking out an ethyl acetate phase and an aqueous phase;

[0012] (4) concentrating the ethyl acetate phase obtained in step (3) under reduced pressure and then drying to obtain an epimedium extract A;

[0013] (5) adding the aqueous phase obtained in step (3) into water-saturated n-butanol for extraction, collecting the n-butanol phase, concentrating under reduced pressure and then drying to obtain epimedium extract B;

[0014] (6) putting the extract A obtained in step (4) into a solvent containing a hydrolase for enzymatic hydrolysis, centrifuging the suspension, collecting the precipitate and drying it to obtain an epimedium hydrolysate;

[0015] (7) putting the extract B obtained in step (5) into an acid solution for acid hydrolysis, adjusting the pH to neutral after acid hydrolysis, collecting the precipitate after centrifugation of the suspension and drying it to obtain an epimedium acid hydrolysate;

[0016] (8) The enzymatic hydrolysate and acid hydrolysate of epimedium obtained in step (6) and step (7) are mixed evenly to obtain epimedium extract C.

[0017] Wherein, the sieving particle size of the sieving in step (1) is 80-100 mesh.

[0018] Wherein, the extraction solvent in step (2) is a mixed solvent of water and ethyl acetate in a volume ratio of 1:1-1:3 ml / ml, the ratio of the powder to be extracted to the extraction solvent is 1:12-1:40 g / mL, the ultrasonic extraction time is 30-90 min, and the ultrasonic power is 100-200 W.

[0019] Wherein, the volume ratio of the aqueous phase to water-saturated n-butanol in step (5) is 1:1-1:3 ml / ml, the extraction time is 5-30 min, and the number of extractions is 1-3 times.

[0020] Wherein, the hydrolase described in step (6) includes cellulase, naringinase or snailase, the solvent is phosphate buffer, the enzyme dosage is 0.5%-1.5% of the solvent mass, the enzymatic hydrolysis temperature is 30°C-50°C, the enzymatic hydrolysis time is 30min-2h, and the ratio of extract A to the solvent containing the hydrolase is 1:10-1:20g / mL.

[0021] Preferably, the hydrolase is snail enzyme.

[0022] Wherein, the acid solution in step (7) is sulfuric acid, nitric acid or hydrochloric acid, the concentration is 1 mol / L-3 mol / L, the acid hydrolysis temperature is 70°C-80°C, the acid hydrolysis time is 30min-2h, and the ratio of extract B to acid hydrolysis solvent is 1:10-1:20g / mL.

[0023] The epimedium extract prepared by the preparation method of the epimedium extract of the present invention.

[0024] Among them, the epimedium extract A contains icariin 75.31-127.59 mg / g, the extract B contains epimedin C 229.52-267.23 mg / g, the yield of icariin II in the enzymatic hydrolysate is 40.19-86.25%, and the yield of dehydrated icariin in the acid hydrolysate is 61.27-77.51%.

[0025] The invention discloses an application of the epimedium extract in preparing drugs for resisting fatigue and treating erectile dysfunction.

[0026] In the present invention, epimedin C, icariin, icariin II and dehydrated icariin are key indicators in the process of epimedium extraction, enzymatic hydrolysis and acid hydrolysis, and the extraction conditions under different conditions can be reflected by measuring their contents.

[0027] Mechanism: Under the action of a composite solvent of water and ethyl acetate, the present invention can fully extract two effective components, icariin with poor water solubility and epimedin C with good water solubility, at the same time, and can achieve preliminary separation of icariin and epimedin C. The separated aqueous phase can further achieve effective extraction of epimedin C under the action of water-saturated n-butanol. Epimedin C and icariin have the same aglycone, i.e., dehydrated icariin. The main secondary glycosides of epimedin C include rhamnosyl icariin II and dehydrated icariin, while the main secondary glycosides of icariin include icariin I, icariin II and dehydrated icariin. Among them, the literature reports that the inhibitory effect on PDE5 enzyme is in descending order of activity: icariin II, dehydrated icariin, and icariin ((Dell'Agli MJ Nat Prod. 2008 Sep; 71(9): 1513-7)). The literature points out that the IC50 of icariin, icariin II, dehydrated icariin, and sildenafil on PDE5A1 are 5.9 μM, 0.16 μM, 2.2 μM, and 0.075 μM, respectively, indicating that icariin II and dehydrated icariin have stronger inhibitory effects on PDE5 enzyme than icariin; while there is no relevant report on epimedin C, and it has certain toxicity (The Toxicity and Metabolism Properties of Herba Epimedii Flavonoids on Laval and Adult Zebrafish). At the same time, a large number of literature reports show that after oral administration, icariin is absorbed into the blood through the intestine and often needs to be metabolized into related secondary glycosides by intestinal enzymes. The secondary glycosides are absorbed quickly, have a short peak time, and a long half-life, which is more conducive to the treatment of ED (Liu C, Biomed Pharmacother. 2017 Oct; 94: 1048-1056.) (Comparative Pharmacokinetics Study of Icariin and Icariside II in Rats) (Progress in Research on the Pharmacological Action and Mechanism of Icariside II Chinese Journal of New Drugs and Clinical Medicine (Chin J New Drugs Clin Rem), September 2018, Vol. 37, No. 9).

[0028] Therefore, it is necessary to further process the extracted epimedin C and icariin. Compared with icariin, epimedin C has another rhamnose group attached to the α-L rhamnose group at the 3rd position, and the commonly used enzymes for hydrolyzing rhamnosidic bonds, such as snail enzyme, naringinase, α-L rhamnosidase, etc., can only hydrolyze the α-L rhamnosidic bond. This extra rhamnose group affects the activity of epimedin C, and the rhamnosyl icariin II produced by its enzymatic hydrolysis has rarely been reported to have an effect on ED. Therefore, more intense reaction conditions are required to generate dehydrated icariin to exert its pharmacological effect. Under this condition, icariin often generates a series of secondary glycosides and excess by-products, so two different reaction conditions are required to treat epimedin C and icariin.

[0029] At the same time, since icariin II, which only hydrolyzes the glucose at position 7 and retains the rhamnosyl at position 3, has a stronger inhibitory effect on PDE5, it is also necessary to control the enzymatic hydrolysis conditions of icariin to prevent excessive enzymatic hydrolysis into dehydrated icariin, resulting in insufficient improvement in efficacy.

[0030] In view of the above situation, the present invention innovatively realizes the separation and relative enrichment of icariin and epimedin C in the extraction stage, and performs different reactions on different extraction parts, so that the two most important effective ingredients in epimedium are reacted into their corresponding secondary glycoside forms that are easy to absorb and more active. That is, icariin is enzymatically hydrolyzed into icariin II, epimedin C is acidolyzed into dehydrated icariin, and the enzymatic hydrolysis product and the acidolysis product are mixed as the final epimedium extract, thereby maximizing the efficacy of epimedium in treating ED. At the same time, most literature reports that the activity of icariin on PDE5 is about one-tenth of sildenafil, while the efficacy of icariin II and dehydrated icariin is several times that of icariin, which can significantly reduce the dosage.

[0031] The epimedium extract obtained by the preparation method of the invention is rich in icariin II and dehydrated icariin, fully utilizes epimedin C and icariin with high content in epimedium, and improves the medicinal effect of epimedium.

[0032] The method of the present invention effectively realizes the extraction and conversion of epimedin C, which has the same parent nucleus as icariin and has a content of 2-3 times that of icariin, but has basically no activity itself, while realizing the conversion of icariin into epimedin II, the most active secondary glycoside. The prior art often only considers the extraction and conversion of icariin, and basically does not pay attention to the potential value of epimedin C.

[0033] In fact, the content of epimedin C in raw materials of Epimedium is high, but it may have certain toxicity and no anti-ED activity. At the same time, the flavonoid aglycone of epimedin C, i.e., dehydrated epimedin, which is free of rhamnosyl and glucosyl groups by acid hydrolysis or other treatment methods, has certain activity, so the present invention uses acid hydrolysis to achieve the conversion of epimedin C-dehydrated epimedin. However, icariin cannot be converted into icariin II, the most active structural form, under such harsh conditions as acid hydrolysis, so the present invention is designed to achieve the relative separation and enrichment of icariin and epimedin C in the extraction stage, and adopts the forms of enzymatic hydrolysis and acid hydrolysis respectively, to ensure that icariin and epimedin C are converted into the theoretically highest active structures, i.e., icariin II and dehydrated epimedin.

[0034] The present invention effectively realizes the full utilization of the two main components of epimedium, icariin and epimedin C, and maximizes the efficacy of epimedium. Epimedin C and icariin are extracted separately in the extraction stage and converted into more active forms respectively. Among them: 1. Epimedin C itself does not inhibit the activity of PDE5 enzyme, and because the α-L rhamnose group at the 3rd position is connected with another rhamnose group, and the commonly used enzymes for hydrolyzing rhamnosidic bonds, such as snail enzyme, naringinase, α-L rhamnosidase, etc., can only hydrolyze the α-L rhamnosidic bond, so more intense reaction conditions are required to generate dehydrated icariin to exert its efficacy, and icariin often cannot obtain the most active secondary glycoside form of icariin II under this condition. 2. Icariin can be initially hydrolyzed into icariin II, and can also be further hydrolyzed into dehydrated icariin. It is often reported in the literature that icariin II has higher activity, so the reaction process needs to be controlled. The present invention achieves the relative separation and enrichment of icariin and epimedin C through early extraction, and uses enzymatic hydrolysis / acid hydrolysis methods to obtain the secondary glycoside form with the highest theoretical activity, thereby maximizing the efficacy of epimedium.

[0035] The present invention separates epimedin C and icariin for the first time and then treats them separately. One of the keys of the present invention is to adopt different treatment methods for the two to obtain the best active form. The chemical structure of epimedin C has another rhamnose group connected to the α-L rhamnose group at the 3rd position, while the commonly used enzymes for hydrolyzing rhamnosidic bonds, such as snail enzyme, naringinase, α-L rhamnosidase, etc., can only hydrolyze the rhamnosidic bond at the α-L position, so more intense reaction conditions are required to generate dehydrated icariin to exert its efficacy. In fact, epimedin C has one more rhamnose group than icariin, which makes it inactive. At the same time, conventional enzymes can remove a part of glucose from icariin to increase its activity, but rhamnosyl icariin 2 obtained by enzymatic treatment of epimedin C still has no activity reports. Moreover, epimedin C needs drastic conditions to be converted into dehydrated icariin to become active, but under these conditions, icariin cannot be converted into the most active form.

[0036] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0037] (1) The raw materials used in the present invention have been screened in advance, and raw materials with high contents of epimedin C and icariin are selected. After corresponding enzymatic hydrolysis and acid hydrolysis treatment, the absorption of epimedium extract in the body is greatly improved, which is more conducive to exerting its medicinal effect. At present, a large number of literatures show that after oral administration, icariin is often absorbed in the intestine and needs to be hydrolyzed by enzymes into icariin I, icariin II, dehydrated icariin and other glycoside forms before it can be absorbed into the blood and exert its effects (Liu C, Biomed Pharmacother. 2017 Oct; 94: 1048-1056.) (Comparative Pharmacokinetics Study of Icariin and Icariside II in Rats), and the plasma drug peak time of icariin II is short (about 40 minutes) and the half-life is long (about 3.5 hours) (Progress in Research on the Pharmacological Action and Mechanism of Icariin II Chinese Journal of New Drugs and Clinical Medicine (Chin J New Drugs Clin Rem), September 2018, Vol. 37, No. 9). Icariin II and dehydrated icariin not only have better efficacy than icariin, but also require enzyme conversion and metabolism for absorption in the body. Therefore, the present invention is advantageous for directly realizing the conversion of icariin in vitro.

[0038] (2) The present invention achieves the separation and relative enrichment of icariin and epimedin C in the extraction stage, and performs different reactions on different extraction parts, so that the two most important active ingredients in epimedium are reacted into their corresponding secondary glycoside forms that are easily absorbed and more active. Literature generally reports that the main active ingredients in epimedium are epimedin and icariin, and the content of epimedin C is roughly 3-5 times that of icariin (Efficient bioconversion of epimedin C to icariin by a glycosidase from Aspergillus nidulans), but epimedin C has certain toxicity (The Toxicity and Metabolism Properties of Herba EpimediiFlavonoids on Laval and Adult Zebrafish). Therefore, in order to maximize the utilization of the active ingredients in epimedium, it is necessary to further process the conventional epimedium extract. At the same time (Dell'Agli MJ Nat Prod. 2008 Sep; 71 (9): 1513-7) pointed out that the IC50 of icariin, icariin II, dehydrated icariin and sildenafil on PDE5A1 were 5.9 μM, 0.16 μM, 2.2 μM and 0.075 μM, respectively, indicating that icariin II and dehydrated icariin have stronger inhibitory effects on PDE5 enzyme than icariin.

[0039] (3) The present invention treats the extracted and separated epimedin C and icariin differently to obtain their respective optimal pharmacodynamic structures. Epimedin C has an additional rhamnose group attached to the α-L rhamnose group at the 3rd position compared to icariin, while commonly used enzymes for hydrolyzing rhamnosidic bonds, such as snailase, naringinase, α-L rhamnosidase, etc., can only hydrolyze the α-L rhamnosidic bond. This extra rhamnose group affects the activity of epimedin C, and the rhamnosyl icariin II produced by its enzymatic hydrolysis has rarely been reported to have an ED effect. Therefore, more intense reaction conditions are required to generate dehydrated icariin to exert its pharmacological effect. Under these conditions, icariin often generates a series of secondary glycosides and excess by-products, so two different reaction conditions are required to treat epimedin C and icariin. At the same time, since icariin II, which only hydrolyzes the glucose at position 7 and retains the rhamnosyl at position 3, has a stronger inhibitory effect on PDE5, it is also necessary to control the enzymatic hydrolysis conditions of icariin to prevent excessive enzymatic hydrolysis into dehydrated icariin, resulting in insufficient improvement in efficacy.

[0040] (4) The present invention establishes the simultaneous detection of epimedin C, icariin, rhamnosyl epimedin icariin II, icariin II, and dehydrated icariin. The analytical method has high separation, good peak shape, and short detection time. The analysis of one injection can be completed within 30 minutes. On the basis of this detection method, we can realize the simultaneous detection of extracts, enzymatic hydrolysates, and acid hydrolysates prepared from raw epimedium medicinal materials under different extraction, enzymatic hydrolysis, and acid hydrolysis methods, which greatly reduces the analysis error and improves the detection efficiency, which is conducive to subsequent related development.

[0041] (5) The epimedium extract A prepared by the present invention contains icariin 75.31-127.59 mg / g, the extract B contains epimedin C 229.52-267.23 mg / g, the yield of icariin II in the enzymatic hydrolysate is 40.19-86.25%, and the yield of dehydrated icariin in the acid hydrolysate is 61.27-77.51%, and has obvious anti-fatigue and erectile dysfunction treatment effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the HPLC chart of the crude extract of Epimedium prepared in Example 14 of the present invention.

[0043] Figure 2 HPLC chart of the Herba Epimedii Extract A prepared in Example 3 of the present invention;

[0044] Figure 3 HPLC chart of the Herba Epimedii Extract B prepared in Example 5 of the present invention;

[0045] Figure 4 This is the HPLC chart of the epimedium hydrolysate prepared in Example 7 of the present invention;

[0046] Figure 5 HPLC chart of the epimedium acid hydrolysate obtained in Example 10 of the present invention;

[0047] Figure 6 This is the HPLC chart of Epimedium hydrolysate Group B prepared in Example 11 of the present invention;

[0048] Figure 7 This is the HPLC chart of Epimedium acid hydrolysate group B prepared in Example 12 of the present invention;

[0049] Figure 8 This is the HPLC chart of Epimedium extract group C prepared in Example 13 of the present invention. DETAILED DESCRIPTION

[0050] In order to make the present invention easier to understand, the present invention is further illustrated below in conjunction with specific embodiments, which do not limit the present invention in any way. These embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Without departing from the technical solution of the present invention, any changes or modifications made to the present invention that are easily implemented by ordinary technicians in the field will fall within the scope of the claims of the present invention.

[0051] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Experimental methods without specific conditions specified in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0052] Epimedium was purchased from Tong Ren Tang.

[0053] Cellulase, snail enzyme and naringinase in the hydrolase were purchased from Aladdin (item number c140864), Yuanye (item number S10083) and Jiangsu Xinrui Biotechnology Co., Ltd., respectively, with cellulase at 10000 u / g and snail enzyme having a cell wall breaking rate of 90%.

[0054] In the embodiment, epimedin C, icariin, icariin II, and dehydrated icariin in epimedium were simultaneously determined, with reference to the Chinese Pharmacopoeia and improved chromatographic conditions, as follows:

[0055] Chromatographic conditions

[0056] The chromatographic instrument was Agilent 1260; the chromatographic column was Agilent zobax C18 (4.6 mm × 50 mm × 3.5 μm); the column temperature was 30°C; the flow rate was 1 ml / min; the injection volume was 10 μl; the detection wavelength was 270 nm; acetonitrile was used as mobile phase A and water was used as mobile phase B, and gradient elution was performed as specified in the following table.

[0057]

[0058] Example 1

[0059] Using Epimedium as raw material, the Epimedium extract A was prepared according to the following process:

[0060] (1) Using Epimedium as raw material, grinding with a powdering machine, passing through an 80-mesh sieve, and mixing to obtain powder to be extracted;

[0061] (2) the epimedium powder to be extracted was added into a water-ethyl acetate solution with a volume ratio of 1:1 at a solid-liquid ratio of 1:20 g / mL, and ultrasonic extraction was performed for 30 min at a power of 150 W;

[0062] (3) The extract obtained in step (2) was allowed to stand for stratification, and the ethyl acetate phase and the aqueous phase were separated. The upper ethyl acetate phase was concentrated under reduced pressure and then dried to obtain an epimedium extract A containing icariin 75.31 mg / g.

[0063] Example 2

[0064] Using Epimedium as raw material, the Epimedium extract A was prepared according to the following process:

[0065] (1) Using Epimedium as raw material, grinding with a powdering machine, passing through an 80-mesh sieve, and mixing to obtain powder to be extracted;

[0066] (2) the powder of Epimedium to be extracted was put into a water-ethyl acetate solution with a volume ratio of 1:2 at a solid-liquid ratio of 1:12 g / mL, and ultrasonic extraction was performed for 60 min at a power of 100 W;

[0067] (3) The extract obtained in step (2) was allowed to stand for stratification, and the ethyl acetate phase and the aqueous phase were separated. The upper ethyl acetate phase was concentrated under reduced pressure and then dried to obtain an epimedium extract A containing icariin 105.29 mg / g.

[0068] Example 3

[0069] Using Epimedium as raw material, the Epimedium extract A was prepared according to the following process:

[0070] (1) Using Epimedium as raw material, grinding with a powdering machine, passing through an 80-mesh sieve, and mixing to obtain powder to be extracted;

[0071] (2) the powder of Epimedium to be extracted was put into a water-ethyl acetate solution with a volume ratio of 1:3 at a solid-liquid ratio of 1:40 g / mL, and ultrasonic extraction was performed for 90 min at a power of 200 W;

[0072] (3) The extract obtained in step (2) was allowed to stand for stratification, and the ethyl acetate phase and the aqueous phase were separated. The upper ethyl acetate phase was concentrated under reduced pressure and then dried to obtain an epimedium extract A containing icariin 127.59 mg / g. (HPLC spectrum see Figure 2 )

[0073] Example 4

[0074] Using the aqueous phase obtained in Example 3 as raw material, the epimedium extract B was prepared according to the following process:

[0075] (1) The aqueous phase obtained in Example 3 was added to water-saturated n-butanol at a volume ratio of 1:1 ml / mL, and extracted in a separatory funnel with shaking (100 rpm) for 20 min. The two phases were then separated by standing, and the upper n-butanol phase was further extracted for a total of three extractions;

[0076] (2) The n-butanol phase obtained in step (1) was combined, concentrated under reduced pressure and then dried to obtain an epimedium extract B containing epimedin C243.87 mg / g.

[0077] Example 5

[0078] Using the aqueous phase obtained in Example 3 as raw material, the epimedium extract B was prepared according to the following process:

[0079] (1) The aqueous phase obtained in Example 3 was added to water-saturated n-butanol at a volume ratio of 1:2 ml / mL, and extracted in a separatory funnel with shaking (100 rpm) for 10 min. The two phases were then separated by standing, and the upper n-butanol phase was further extracted for a total of two extractions;

[0080] (2) The n-butanol phase obtained in step (1) was combined, concentrated under reduced pressure and then dried to obtain an epimedium extract B containing epimedin C267.23 mg / g. (HPLC spectrum see Figure 3 )

[0081] Example 6

[0082] Using the aqueous phase obtained in Example 3 as raw material, the epimedium extract B was prepared according to the following process:

[0083] (1) The aqueous phase obtained in Example 3 was added to water-saturated n-butanol at a volume ratio of 1:3 ml / mL, and extracted in a separatory funnel with shaking (100 rpm) for 30 min. The two phases were then separated by standing, and the extraction was performed once in total;

[0084] (2) The n-butanol phase obtained in step (1) was concentrated under reduced pressure and then dried to obtain an epimedium extract B containing epimedin C229.52 mg / g.

[0085] Example 7

[0086] The epimedium extract A obtained in Example 3 was used as a raw material to prepare the epimedium hydrolysate according to the following process:

[0087] (1) The Epimedium extract A obtained in Example 3 was used as a raw material and added into a phosphate buffer solution containing snail enzyme at a material-liquid ratio of 1:10 g / ml, the enzyme dosage was 1.0% of the solvent mass, the enzymatic hydrolysis temperature was 50° C., and the enzymatic hydrolysis time was 1 h;

[0088] (2) The suspension obtained in step (1) was centrifuged, and the precipitate was collected and dried to obtain an epimedium hydrolysate, wherein the yield of epimedin II was 86.25% and the content was 83.68 mg / g. (HPLC spectrum see Figure 4 )

[0089] Test Example 1

[0090] The preparation method of Example 7 was adopted, and different hydrolases including snailase, cellulase and naringinase were used in step (1). The contents of icariin II and dehydrated icariin were shown in Table 1.

[0091] Table 1. Yields of icariin II and dehydrated icariin after enzymatic hydrolysis

[0092]

[0093] The results of the test examples of the present invention show that it is more advantageous to use snail enzyme as the hydrolase. As shown in Table 1, the enzymatic hydrolysis efficiency of cellulase is not high, and the yield of icariin II is low, while naringinase is easy to remove the rhamnosyl group at the 3rd position of icariin, which is not conducive to controlling the reaction process, resulting in the inhibition of PDE5 enzyme activity. Snail enzyme has high conversion efficiency, high yield of icariin II, and less complete enzymatic hydrolysis into dehydrated icariin, so it is more advantageous to use snail enzyme in the present invention.

[0094] Icariside II yield = the actual amount of icariside II obtained by enzymatic hydrolysis / the theoretical amount of icariside II that can be obtained by all enzymatic hydrolysis × 100%

[0095] Yield of dehydrated icariin = the amount of dehydrated icariin obtained by actual enzymatic hydrolysis / the theoretical amount of dehydrated icariin obtained by complete enzymatic hydrolysis × 100%

[0096] Test Example 2

[0097] The preparation method of Example 7 was adopted, and the epimedium extract A obtained in Example 3 was used as a raw material, and was added into a phosphate buffer solution containing snail enzyme at a material-liquid ratio of 1:10 mg / ml, the enzyme dosage was 0.5%-1.5% of the solvent mass, the enzymolysis temperature was 30°C-50°C, and the enzymolysis time was 30min-2h;

[0098] The results are shown in Table 2-3.

[0099] Table 2. Factors and levels investigated by enzymatic hydrolysis

[0100]

[0101] Table 3. Orthogonal experiment results

[0102]

[0103]

[0104] Note: k is the average factor level

[0105] From the range R analysis, it can be seen that the order of influence of the three factors on the enzymatic hydrolysis efficiency of the epimedium extract A is: enzymatic hydrolysis temperature > enzymatic hydrolysis time > enzyme dosage, and from the orthogonal table, it can be seen that the best combination is A3B2C3. That is, the most preferred combination is that the epimedium extract A obtained in Example 3 is used as a raw material, and a phosphate buffer solution containing 1.5% of the solvent mass of snail enzyme is added according to a solid-liquid ratio of 1:10 mg / ml, and enzymatic extraction is carried out at 50°C for 1h. After the obtained suspension is centrifuged, the precipitate is collected and dried to obtain the epimedium hydrolysate. However, considering that the enzyme dosage of 1.0% and 1.5% has little effect on the yield of epimedin II, and at the same time, in actual production, the increase in the enzyme dosage increases the cost on the one hand, and may lead to excessive enzymatic hydrolysis on the other hand. After comprehensive consideration, the epimedium extract A obtained in Example 3 was used as the raw material, and was added into a phosphate buffer solution containing 1.0% snail enzyme by mass of the solvent at a solid-liquid ratio of 1:10 mg / ml. The enzymatic extraction was carried out at 50°C for 1 hour. After the obtained suspension was centrifuged, the precipitate was collected and dried to obtain the epimedium hydrolysate.

[0106] Example 8

[0107] The epimedium extract B obtained in Example 5 was used as a raw material to prepare epimedium acid hydrolysate according to the following process:

[0108] (1) The Epimedium Extract B obtained in Example 5 was used as a raw material, and was added into 2 mol / L sulfuric acid at a material-liquid ratio of 1:15 g / ml, and acid-hydrolyzed at 75° C. for 1 h;

[0109] (2) The pH of the acid hydrolyzate obtained in step (1) was adjusted to neutral with 10% by mass sodium hydroxide, the suspension was centrifuged, the precipitate was collected and dried to obtain an epimedium acid hydrolyzate, and the yield of dehydrated icariin was 61.27%.

[0110] The yield of dehydrated icariin = the amount of dehydrated icariin actually obtained by acid hydrolysis / the theoretical amount of icariin II that can be obtained by total acid hydrolysis × 100%.

[0111] Example 9

[0112] The epimedium extract B obtained in Example 5 was used as a raw material to prepare epimedium acid hydrolysate according to the following process:

[0113] (1) The Epimedium Extract B obtained in Example 5 was used as a raw material, and was added into 3 mol / L sulfuric acid at a material-liquid ratio of 1:10 g / ml, and acid-hydrolyzed at 80° C. for 0.5 h;

[0114] (2) The pH of the acid hydrolyzate obtained in step (1) was adjusted to neutral with 10% by mass sodium hydroxide, the suspension was centrifuged, the precipitate was collected and dried to obtain an epimedium acid hydrolyzate, and the yield of dehydrated icariin was 69.11%.

[0115] Example 10

[0116] The epimedium extract B obtained in Example 5 was used as a raw material to prepare epimedium acid hydrolysate according to the following process:

[0117] (1) The Epimedium Extract B obtained in Example 5 was used as a raw material, and was added into 1 mol / L sulfuric acid at a material-liquid ratio of 1:20 g / ml, and acid-hydrolyzed at 70° C. for 2 h;

[0118] (2) The pH of the acid hydrolyzate obtained in step (1) was adjusted to neutral with 10% by mass sodium hydroxide, and the suspension was centrifuged and the precipitate was collected and dried to obtain an acid hydrolyzate of epimedium, with a dehydrated icariin yield of 77.51% and a content of 92.74 mg / g. The high yield under this condition may be due to the relatively high amount of solvent used, the relatively low sulfuric acid concentration and temperature. When the conditions are too severe, epimedin C will not only be acidolyzed into dehydrated icariin but may also be further destroyed. (HPLC spectrum see Figure 5 )

[0119] Embodiment 11

[0120] Take the same amount of the epimedium extract A and epimedium extract B obtained in Example 3 and Example 5, and prepare epimedium hydrolysate B according to the method of Example 7, which contains 91.23 mg / g rhamnosyl icariin II, 41.27 mg / g icariin II, and 1.47 mg / g dehydrated icariin. (HPLC spectrum see Figure 6 )

[0121] Example 12

[0122] Take equal amounts of the epimedium extract A and epimedium extract B obtained in Example 3 and Example 5, and prepare epimedium acid hydrolysate B according to the method of Example 10, containing 4.12 mg / g of rhamnosyl epimedin II, 2.17 mg / g of epimedin II, and 45.13 mg / g of dehydrated epimedin. (HPLC spectrum see Figure 7 )

[0123] Embodiment 13

[0124] Equal amounts of the enzymatic hydrolysate of epimedium and the acid hydrolysate of epimedium obtained in Example 7 and Example 10 were mixed to obtain the epimedium extract C of the present invention, containing 42.36 mg / g of icariin II and 47.35 mg / g of dehydrated icariin.

[0125] (HPLC spectrum see Figure 8 )

[0126] The above Examples 11-13 illustrate that when only enzymatic hydrolysates or only acid hydrolysates are used, the medicinal effects of Epimedium are not fully utilized, that is, icariin and epimedin C are not all converted into the most effective form.

[0127] Embodiment 14

[0128] Using Epimedium as raw material, the crude extract of Epimedium was prepared according to the following process

[0129] (1) Using Epimedium as raw material, grinding with a powdering machine, passing through an 80-mesh sieve, and mixing to obtain powder to be extracted;

[0130] (2) the powder of Epimedium to be extracted was put into a water-ethyl acetate solution with a volume ratio of 1:3 at a solid-liquid ratio of 1:40 g / mL, and ultrasonic extraction was performed for 90 min at a power of 200 W;

[0131] (3) The extract is concentrated under reduced pressure and then dried to obtain a crude extract of Epimedium. (HPLC spectrum see Figure 1 )

[0132] Test Example 3

[0133] Pharmacodynamics experiments

[0134] 1. The anti-fatigue test plan is as follows:

[0135] a. Animal grouping and feeding

[0136] 96 ICR mice, male, weighing 20±2g, were divided into 8 groups, with 12 mice in each group. The mice in each group were blank group, epimedium extract group A, epimedium extract group B, epimedium enzymatic hydrolysate group, epimedium acid hydrolysate group, epimedium enzymatic hydrolysate group A, epimedium acid hydrolysate group B, and epimedium extract group C.

[0137] Take an appropriate amount of the dry paste powder of Example 3, Example 5, Example 7, Example 10, Example 11, Example 12, and Example 13 (i.e., the final product after concentration and drying of each example), and prepare a certain concentration of liquid with phosphate buffer as Epimedium extract group A, Epimedium extract group B, Epimedium hydrolysate group, Epimedium acid hydrolysate group, Epimedium hydrolysate group B, Epimedium acid hydrolysate group B, and Epimedium extract group C, and phosphate buffer as a blank group. Each group was intragastrically administered, and the dosage was 150 mg / kg mouse body weight, once a day for 30 consecutive days.

[0138] b. Liver glycogen determination

[0139] 30 minutes after the last administration, 6 mice were randomly selected from each group and killed immediately. The liver tissue was separated and rinsed with physiological saline and dried with filter paper. 100 mg of liver tissue was accurately weighed and measured according to the liver / muscle glycogen determination kit (Nanjing Jiancheng Bioengineering Institute).

[0140] c. Weighted swimming test

[0141] 30 minutes after the last administration, the remaining mice in each group were placed in a swimming box (water temperature 25.0℃±1.0℃, water depth 30 cm) with lead weights (the weight of the lead weight was 5% of the body weight) at the base of their tails. The time from the start of swimming to exhaustion (the head of the mouse was completely submerged in water and could not float to the surface for 10 seconds) was recorded, which was the weighted swimming time of the mice.

[0142] Experimental results:

[0143] After long-term administration of the drug, the amount of liver glycogen and swimming time of mice were mostly significantly improved compared with the blank group, from large to small, they were epimedium extract C group, epimedium hydrolysate group, epimedium acid hydrolysate group, epimedium enzymatic hydrolysate group B, epimedium extract A group, epimedium acid hydrolysate group B, and epimedium extract B group. Except for epimedium extract B group, all of them had a certain anti-fatigue effect, indicating that epimedin C itself has no activity.

[0144] Among them, the anti-fatigue effects of epimedium enzymatic hydrolysate group B, epimedium acid hydrolysate group B and epimedium extract group A are similar, indicating that simply subjecting icariin and epimedin C to the same enzymatic or acid hydrolysis treatment will result in limited improvement in efficacy due to the generation of rhamnosyl epimedin II or icariin, which has insignificant efficacy, being easily destroyed in a strong acidic environment. Compared with simple icariin, the efficacy of the epimedium enzymatic hydrolysate group and the epimedium acid hydrolysate group is significantly improved, indicating that the products of icariin II and dehydrated epimedin obtained after icariin and epimedin C are treated in Example 7 and Example 10, respectively, have more obvious anti-fatigue effects than icariin. At the same time, the epimedium extract group C of the present invention obtained by mixing the two has the highest efficacy, indicating that icariin II and dehydrated icariin may have potential synergistic effects, and this synergistic effect may be related to the excessive dosage of icariin II in the simple epimedium hydrolysate group. Literature reports (Liu C, Biomed Pharmacother. 2017 Oct; 94: 1048-1056.) Icariin and snail enzyme are made into enteric-coated capsules and then taken orally. Compared with the simple icariin group or osteoporosis group, its comprehensive bioavailability is significantly improved, and the area under the drug-time curve of dehydrated icariin is greater than that of icariin II, indicating that it often needs to be enzymatically hydrolyzed into icariin II and dehydrated icariin by intestinal enzymes before absorption in the body. The epimedium enzymatic hydrolysate and epimedium acid hydrolysate prepared by the present invention can make full use of high-content components in epimedium, including icariin and epimedin C with a content of about 2-3 times that of icariin, and develop them into high-activity component forms, namely icariin II and dehydrated icariin, respectively. Moreover, under the dosage of the present invention, the activity of the simple high-content epimedium enzymatic hydrolysate, namely icariin II, is slightly inferior to that of the epimedium extract C prepared by the present invention, and the reason may be related to the decrease in the absorption rate of icariin II in the body under high dosage.

[0145] Table 4. Effects of different drugs on liver glycogen and swimming time in mice

[0146]

[0147]

[0148] Compared with the model control group, *P<0.05

[0149] 2. The mating behavior assay protocol is as follows:

[0150] a. Animal grouping and feeding

[0151] 96 eight-week-old Wistar rats were selected, half of which were male and half were female, and the males were divided into 8 groups, with 6 rats in each group. The mice in each group were blank group, epimedium extract group B, epimedium extract group A, epimedium enzymatic hydrolysate group, epimedium acid hydrolysate group, epimedium enzymatic hydrolysate group A, epimedium acid hydrolysate group B, and epimedium extract group C.

[0152] Take appropriate amount of dry powder of Example 3, Example 5, Example 7, Example 10, Example 11, Example 12, and Example 13 respectively, and prepare a certain concentration of liquid with phosphate buffer as Epimedium extract group A, Epimedium extract group B, Epimedium hydrolysate group, Epimedium acid hydrolysate group, Epimedium hydrolysate group B, Epimedium acid hydrolysate group B, and Epimedium extract group C, and phosphate buffer is used as a blank group. Each group is intragastrically administered, and the dosage is 75 mg / kg rat body weight, once a day for 30 consecutive days.

[0153] b. Establishment of female mouse estrus model

[0154] Before the mating experiment, the female rats were placed in the mating cage every evening to adapt to the environment and undergo adaptive training. 48 hours before the formal experiment, each rat was subcutaneously injected with 0.75 μg / g estradiol benzoate, and 4 hours before the formal experiment, 10 μg / g progesterone was injected.

[0155] c. Observation of mating behavior

[0156] The experiment was conducted at night, keeping the surrounding environment quiet and slightly dim, using a transparent polypropylene observation cage to record relevant behavioral indicators. First, a male rat was placed in a cage alone for 10 minutes to adapt to the environment, and then an estrus female rat was put in to observe and record the erection latency, ejaculation latency and mating latency.

[0157] The erection latency period refers to the time from the beginning of coexistence of male and female rats to the first erection of the male rat. Erection occurs when the end of the glans penis becomes engorged with blood and the body of the penis appears. The ejaculation latency period refers to the time from the insertion of the penis into the vagina to ejaculation. The mating latency period refers to the time required for male and female rats to begin mating after the penis becomes erect.

[0158] Table 5. Effects of different drugs on sexual behavior in rats

[0159]

[0160] Compared with the model control group, *P<0.05

[0161] The above experimental results show that the sexual ability of mice is greatly improved after long-term administration. The epimedium extract C prepared by the present invention significantly shortens the erection latency and mating latency of rats, indicating that their sexual desire is greatly improved, and at the same time significantly prolongs the ejaculation latency, enhances the sexual ability of rats, and promotes the erection of their penis. The degree of improving sexual ability is from large to small, which is epimedium extract C group, epimedium hydrolysate group, epimedium acid hydrolysate group, epimedium hydrolysate B group, epimedium extract A group, epimedium acid hydrolysate B group, and epimedium extract B group. Among them, the effects of epimedium enzymolysis product group B, epimedium acid hydrolyzate group B and epimedium extract group A are similar; and the epimedium enzymolysis product group and epimedium acid hydrolyzate group have significantly improved efficacy compared with simple icariin; at the same time, the epimedium extract group C of the present invention obtained by mixing the two has the highest efficacy. The above results and the results of the anti-fatigue experiment on mice all show that according to the method of the present invention, the enzymolysis product and acid hydrolyzate obtained by first separating icariin and epimedin C and then treating them separately have strong anti-fatigue and erectile dysfunction effects, and the epimedium extract C obtained after mixing has higher efficacy and can make full use of the main active ingredients in the raw epimedium medicinal material.

[0162] In addition, it can be seen from the above experiment that the activity of icariin II is greater than that of dehydrated icariin, and the activity of dehydrated icariin is greater than that of icariin, and epimedin C and rhamnosyl icariin II are inactive. Epimedium is initially hydrolyzed into icariin II, and further hydrolyzed into dehydrated icariin, epimedin C is initially acid-hydrolyzed into rhamnosyl icariin II, and further acid-hydrolyzed into dehydrated icariin. Therefore, the above-mentioned medicinal effect of epimedium extract group C and epimedium hydrolysate group are similar, but the enzymatic hydrolysate group needs to consume 2 parts of icariin, while the extract group C consumes 1 part of icariin and the other part is provided by the acid hydrolysis of epimedin C, indicating that the extract group of the present invention can make full use of the main active ingredients of epimedium.

[0163] At the same time, the epimedium extract group B and the acid hydrolyzate group showed that epimedin C was inactive, and the dehydrated icariin after acid hydrolysis was active; the extract group A and the enzymatic hydrolyzate group showed that the activity of icariin II was greater than that of icariin; the epimedium enzymatic hydrolyzate group and the enzymatic hydrolyzate group B showed that rhamnosyl icariin II hydrolyzed from epimedin C was inactive; the acid hydrolyzate group and the acid hydrolyzate group B showed that icariin was easily destroyed by acid hydrolysis. Compared with the epimedium extract group C, the above further showed that the maximum efficacy could be obtained by separating icariin and epimedin C and treating them separately.

Claims

1. A method for preparing an epimedium extract, characterized in that: The steps include: (1) Using Epimedium as raw material, grinding, sieving, and mixing to prepare the powder to be extracted; (2) putting the Epimedium powder to be extracted obtained in step (1) into an extraction solvent containing water and ethyl acetate, and performing ultrasonic extraction; (3) allowing the extract obtained in step (2) to stand for stratification, and taking out an ethyl acetate phase and an aqueous phase; (4) concentrating the ethyl acetate phase obtained in step (3) under reduced pressure and then drying to obtain an epimedium extract A; (5) adding the aqueous phase obtained in step (3) into water-saturated n-butanol for extraction, collecting the n-butanol phase, concentrating under reduced pressure and then drying to obtain epimedium extract B; (6) adding the extract A obtained in step (4) into a solvent containing a hydrolase for enzymatic hydrolysis, centrifuging the suspension, collecting the precipitate and drying it to obtain an epimedium hydrolysate; (7) putting the extract B obtained in step (5) into an acid solution for acid hydrolysis, adjusting the pH to neutral after acid hydrolysis, collecting the precipitate after centrifugation of the suspension and drying it to obtain an epimedium acid hydrolysate; (8) uniformly mixing the enzymatic hydrolysate and acid hydrolysate of epimedium obtained in step (6) and step (7) to obtain epimedium extract C; The extraction solvent in step (2) is a mixed solvent of 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 1:12-1:40 g / mL, the extraction time of ultrasonic extraction is 30-90 min, and the ultrasonic power is 100-200 W; The volume ratio of the aqueous phase to water-saturated n-butanol in step (5) is 1:1-1:3, the extraction time is 5-30 min, and the number of extractions is 1-3 times; The hydrolase in step (6) comprises cellulase, naringinase or snailase, the solvent is phosphate buffer, the enzyme dosage is 0.5%-1.5% of the solvent mass, the enzymatic hydrolysis temperature is 30°C-50°C, the enzymatic hydrolysis time is 30 min-2 h, and the ratio of extract A to the solvent containing the hydrolase is 1:10-1:20 g / mL; The acid solution in step (7) is sulfuric acid, nitric acid or hydrochloric acid, with a concentration of 1 mol / L-3 mol / L, an acid hydrolysis temperature of 70°C-80°C, an acid hydrolysis time of 30 min-2 h, and a liquid ratio of extract B to acid hydrolysis solvent of 1:10-1:20 g / mL.

2. The method for preparing the epimedium extract according to claim 1, characterized in that: The sieving particle size in step (1) is 80-100 mesh.

3. An epimedium extract prepared by the method for preparing the epimedium extract according to claim 1.

4. The epimedium extract according to claim 3, characterized in that The epimedium extract A contains icariin 75.31-127.59 mg / g, the extract B contains epimedin C 229.52-267.23 mg / g, the yield of icariin II in the enzymatic hydrolysate is 40.19-86.25%, and the yield of dehydrated icariin in the acid hydrolysate is 61.27-77.51%.

5. Use of the epimedium extract according to claim 3 in the preparation of anti-fatigue and erectile dysfunction treatment drugs.

Images