WO2000078323A1 - A use of icariin in preparing the medicine for preventing and treating sexual disorder and vasoconstrictive diseases - Google Patents

Abstract

A use of Icariin in preparing the medicine for preventing and treating sexual disorder and vasoconstrictive diseases.

Description

 Use of icariin in the preparation of a medicament for the prevention or treatment of sexual dysfunction and vasoconstriction-related diseases

Field of invention

 The present invention relates to the use of icariin in the preparation of a medicament for the prevention and treatment of sexual dysfunction, as well as the use in improving diseases related to vasoconstriction.

Background technique

 Male sexual functions, including libido, penile erection, ejaculation, and sexual pleasure, are complex physiological reflections of the penile vascular system under the regulation of the neuro-endocrine system. Therefore, if an abnormality occurs in a certain process of the system, it may cause diseases such as hyposexuality, penile erectile dysfunction, ejaculation disorder, or penile weakness, and this disease may appear alone or in combination with each other. The incidence of male sexual dysfunction accounts for approximately 30-60% of adult males and tends to increase with age. Traditional medicine generally refers to sexual dysfunction as impotence and premature ejaculation. Until the last decade, most of the causes of sexual dysfunction were considered to be only psychological reasons. However, with the development of modern science and technology, it has been clear that more than 50% of patients with sexual dysfunction have neurological, vascular, endocrine diseases, and organic causes such as hypertension, diabetes, and taking various drugs. .

 The sexual function of men is mainly in the presence of various sexual stimuli, under the regulation of the neuro-endocrine system, through the complex physiological response of the vascular system to make the penis erect. This penile erection process first depends on the dilatation of the penile arterioles to increase the penile blood flow, and the penile cavernous smooth muscles relax and the cavernous pores swell, which promotes a further increase in blood flow and guides the increase in penile sponge internal pressure. Compression of the small veins under the white membrane of the penis blocks blood flow, which results in blood filling in the penile cavernous body while the penis has a hard erection that can be inserted into the vagina and maintains an erection long enough to reach orgasm and ejaculate.

The penile corpus cavernosum smooth muscle plays a key role in this penile erection process, and the penile erection process is regulated by the neuroendocrine system. Animal experiments show that many local distribution factors affect penile erectile function such as norepinephrine (NA), neuropeptide Y (NPY), Acetylcholine (Ach), prostaglandin (PGE), serotonin (5-HT), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), endothelin (EDRF), etc.

 Recent studies have shown that nitric oxide (NO) plays an important role in penile erection. When sexually stimulated, NO is released from parasympathetic nerves and non-adrenergic non-acetylcholinergic nerve (NANC) terminals distributed in the penis, and NO is also released in endothelial cells distributed in the vascular endothelial layer and penile sea surface body sinusoidal endothelial cells. NO can be activated Guanylate cyclase in smooth muscle cells increases guanosine cyclic guanosine (cGMP) synthesis and also activates protease G (PKG). PKG can close calcium channels and open potassium channels. Both effects can cause the surface of the penis. Body smooth muscle relaxation leads to penile erections. Cyclophosphate guanosine (cGMP) is inactivated by tablet acid diesterase (PDE).

 Sexual dysfunction can be divided into hyposexuality, erectile dysfunction, orgasmic disorder and ejaculation disorder. The causes of sexual dysfunction include vascular neuropathy, endocrine, psychological lesions, diabetes, hypertension, spinal nerve injury, smoking and other factors can cause sexual dysfunction.

 The traditional view is that sexual dysfunction is mainly caused by psychological reasons. For treatment, oral drugs such as the tranquilizer trazodone and yohimbine are used. However, the mechanism of action of this drug is not clear, its efficacy is not high, and it has side effects of varying degrees.

 In recent years, with the elucidation of the penile erection mechanism, various drugs that have a relaxing effect on the smooth muscle of the penile cavernosum have been used to diagnose and treat male sexual dysfunction. At present, vasodilators such as aparvarin, fento Drugs such as phentolamine and prostajlandin El are directly injected into the penile cavernous body to treat penile erectile dysfunction to obtain satisfactory results. However, this therapy can cause side effects such as localized pain, persistent penile erections, and penile fibrosis, so it is often restricted in clinical use.

Recently, the oral drug phosphodiesterase V inhibitor (Viagra) invented by the United States for the treatment of erectile dysfunction has attracted worldwide attention. As a chemical prosthesis, the above-mentioned drugs are only used to induce penile erection at one time, and have different degrees of side effects. Redness, headaches, visual impairments, hypotension, etc., especially the safety of patients with cardiovascular disease are controversial worthy of attention. Therefore, safe and effective drugs for improving the human body's instinctual erectile function need to be researched and developed, and recent research has sought to develop safe and effective NO donors for the treatment of sexual dysfunction. .

 The crude drug Epimedium is often used as an ingredient in the prescriptions of nourishing and strengthening agents in the traditional medical field, but its mechanism of action has not yet been clarified. Recent research on the effective ingredients of Epimedium has proved that Epimedium contains many ingredients, such as icariin, volatile oil, wax alcohol, predane, plant alcohol, tannin, linoleic acid, etc. Recently, a small amount of magnoflorin, icariin A, epimedin A, B, C, quercetin, dehydrated icariin -3-0-rhamnoside, etc. have been isolated. Among them, icariin is currently only used as a standard reagent for the analysis of icariin. Some studies have reported that Epimedium has anti-fatigue effects, improves immune function, and improves liver function. But so far, there are no reports about the use of icariin monomers to treat sexual dysfunction.

Object of the invention

 The object of the present invention is to find and develop new medical uses of icariin

Brief description of the invention

 The inventors found through research that icariin has an unexpectedly strong relaxing effect on the smooth muscle of the penile cavernous body and a mild relaxing effect on aortic smooth muscle. Its pharmacological effect and the nitric oxide (NO)-cGMP pathway in smooth muscle cells Related to enhanced activity. It can be used to improve and enhance the erectile function of the penis and improve the symptoms of diseases related to vasoconstriction.

 Therefore, the present invention relates to the use of icariin in the preparation of a medicament for the prevention of the treatment of sexual dysfunction and the use in the improvement of diseases related to vasoconstriction.

Detailed description of the invention

 The present invention relates to the next test. The present invention relates to the use of icariin in the preparation of a medicament for the prevention and treatment of sexual dysfunction, and the use of the medicament for the preparation and improvement of vasoconstriction-related diseases.

The present invention relates to a method for preventing or treating sexual dysfunction and vasoconstriction-related diseases. A pharmaceutical composition comprising icariin as an active ingredient and a pharmaceutically acceptable carrier and excipient. The present invention relates to a method for preventing or treating sexual dysfunction and vasoconstriction-related diseases, which comprises preventing or treating an effective amount of icariin or icariin-containing Pharmaceutical composition.

Icariin is a flavonoid glycoside monomer isolated from the crude drug Epimedium. Its chemical structural formula is C ₃₃ H ₄ . 0 ₁₅ , molecular weight: 676. 67, melting point: 225-230 ° C.

 According to the present invention, icariin used in the present invention may be used alone or in the form of a pharmaceutical composition. The pharmaceutical composition includes icariin as an active ingredient and a pharmaceutically acceptable carrier.

 According to the present invention, the pharmaceutical composition of the present invention can be prepared according to methods known in the art and can be administered orally or parenterally. Oral preparations include, for example, tablets, chews, capsules, suspensions, solutions, etc., and parenteral preparations include, for example, injection solutions. Topical preparations include, for example, creams, ointments, patches, sprays and the like.

 According to the present invention, icariin is obtained from the icariin drug by an organic solvent or a mixture of an organic solvent and water. Organic solvents that can be used include alcohols such as methanol, ethanol, etc., alkanes such as dichloromethane, chloroform, ethers such as diethyl ether, ketones such as acetone, lipids such as methyl acetate, ethyl acetate, and hydrocarbons such as hexane Wait.

The following examples are used to illustrate the present invention in more detail, but it is not meant to limit the present invention thereto. Example 1 Preparation of Icariin

 Extraction method (1)

Take 500 grams of dried Epimedium (ground part) and 10 liters of MeOH, and heat for 3 days for 3 times. The solvent was recovered by centrifugation and concentrated under reduced pressure to obtain a MeOH extract. The MeOH extract was suspended in water and extracted with the same amount of hexane for a total of 4 times. The aqueous layer was concentrated to 1/3. In this concentrated water layer, 5% citric acid was suspended (pH = 5.5), and then extracted with the same amount of CHC1 ₃ , and then an appropriate amount of NH ₄ 0H was suspended (pH = 10), and then the same amount of CHC1 was used. ₃ extract. This process is repeated 3 times. Finally, the alkaline aqueous layer was repeatedly extracted three times with the same amount of butanol to obtain a crude icari in rich fraction (IRF) (the content of icariin was 8% by HPLC analysis).

 Extraction method (2)

 Take dry Epimedium (above ground) 500 grams plus 10 litres of EtOH, 80C. Heating confluence extraction 3 times, 1 hour each time, 3 times in total. Filtration and recovery of ethanol under reduced pressure were concentrated to a slurry. Add 4 times the amount of water to disperse and let stand for 3 hours to remove chlorophyll and a few waxes. Take 4 times of n-butanol and combine the filtrates and concentrate under reduced pressure. It was adsorbed on a 40-60 mesh polyamide column and eluted with 30% ethanol until it was colorless. The eluent was concentrated under reduced pressure. Add 70% ethanol (1:20) to dissolve, and let stand for 8 hours, then filter to obtain the total icariin. Epimedium total glycosides and absolute ethanol (1: 15) were repeatedly eluted and recrystallized to obtain crude epimedium total glycosides. The total glycosides of Epimedium is 52%.

 Icariin isolation

The crude icariin or total icariin is dissolved in MeOH, adsorbed on silica gel and dried, and then added to a prepared silica gel column (8x60cm) with CHC1: MeOH = 5: 1, 4: 1, 3: 3: 1, It dissolves in the order of 2: 1, and each eluted liquid is concentrated, and then the mixture is mixed with MeOH / H ₂ 0 to precipitate pale yellow crystals.

 The crystal was colored in UV, and the Pauly reagent was positive. The Rf value of the TLC analysis was 0.6, and the melting point was 239. C. It was confirmed to be pure icariin (icariin, the content of icariin was 99.8% by HPLC analysis).

Lab 1 Safety Lab

According to the rules of acute toxicity test, 5 male and female SD rats were divided into 1 group, divided into 4 groups. The oral dose of the first group was icariin 3g / kg, the second group was 2g / kg, and the third group was lg. / kg, the third group is 0.5g / kg. Observe the results on the 7th after the administration, LD ₅ . Male is 1. 88g / kg, and female is 1. 95g / kg. Experiment 2 Pharmacodynamics and Mechanism

Experimental principle The sexual function of men is mainly to stimulate penile erection through the complex physiological response of the vascular system under the regulation of the neuro-endocrine system under various sexual stimuli. This penile erection process first depends on the dilatation of the penile arterioles and the smooth muscle relaxation of the corpus cavernosum, and the expansion of the corpus cavernosal sinuses promotes an increase in blood flow to the corpus cavernosum, which leads to an increase in the pressure of the corpus cavernosum and compresses the white membrane The lower veins block blood flow, which causes the blood in the penile cavernous body to fill and the penis to harden and erect. During this penile erection, the relaxation of the smooth muscle of the corpora cavernosa plays a key role. Therefore, drugs with a relaxing effect on the smooth muscle of the penis sponge body can help the erectile function of the penis. The relaxing effect of penile cavernosal smooth muscle is also regulated by non-adrenergic noncaproylcholine (NANC) ergic nerve and endothelial cell relaxing factor (EDRF). Nitric oxide (NO) plays an important role in this EDRF. NO or NO-containing substances diffuse into smooth muscle cells, activate soluble guanylate cyclase, increase cGMP synthesis, induce relaxation of penile cavernosal smooth muscle, and promote penile erection. The cGMP in the penile cavernous body is again inactivated by the tablet type V enzyme. Therefore, drugs that promote the increase in NO production in the smooth muscle of the penis sponge body, or promote the activity of guanylate cyclase, or inhibit the activity of phosphodiesterase, can increase the synthesis or activity of cGMP, and promote sexual erectile dysfunction. Therapeutic effect. It is an effective experimental method to study the erectile function of the drug penis by using in vitro experiments on the tissues of the sponge body of the penis to observe the effect of the drug on its tonicity, or to determine the effect of the drug on the diabolase.

2. 1. Icariin relaxes the smooth muscle of penile sea surface in rabbits:

Male rabbits (New Zealand rabbits, body weight: 2.5-3.0 kg) were used to isolate penile sea surface smooth muscle, and the pharmacological and pharmacodynamic experiments of icariin were performed in vitro. A total of 60 rabbits were used. The effects of different concentrations of icariin on the tension of smooth muscle in the penile sea surface were observed, and the effect of icariin on the tension of abdominal aorta smooth muscle was observed. In order to study the pharmacological mechanism of the effect of icariin on the relaxation of penile sea surface smooth muscle, atropine, a prostaglandin E1 inhibitor, indomethacin, a prostaglandin E1 inhibitor, was observed. Reagents for NO production or NO activity N ^w -nitric- L-arginine (L-NNA) and pyrogallol, methylene blue, inhibitor of ornithine cyclase activity, 1H- [1, 2, 4] Effects of oxadiazolo [4, 3, -a] quinoxaline-1-one (0DQ) on the effect of icariin on the smooth muscle relaxation of the penile sea surface body. experimental method

 5 ° C Rabbits were anesthetized with sodium pentobarbital (30-50mg / kg) after resection of the penis and abdominal aorta, and continued to supply 95% oxygen and 5% carbon dioxide mixed gas and filled with Tyrode nutrient solution and maintained a constant temperature of 37.5 ° C In the container, the penis cavernosa was separated into muscle strips of 2x2x6mm size, and 4 bands wide arterial rings were separated in the aorta. Each of them contained 10 ml of Tyrode solution and continuously supplied 95% oxygen and 5% carbon dioxide mixed gas. In the glassware tube bath, one end is fixed at the bottom, and the other end is connected to the force displacement transducer. Changes in the tension of the penile cavernous body strips and arterial rings were measured and recorded using a multi-channel recorder (TSD 105, Biopac Systems, Santa Barbara, CA, USA). The icariin extract and icariin were observed. Effects caused by tension in the cavernous strips of the penis and the aortic ring.

In order to induce the tension of the penis sponge strip and the arterial ring in the quiet state at first, the penis sponge and the arterial ring were pulled to a tension of 2 grams, which was effective for 1.5-2 hours. After the contractile response was induced by using stupid paralin (PHE; 5 X 10 ⁶ M), it was washed away with Typhoon's solution to return to the baseline. This method is repeated three or more times. If the difference between the three systolic response amplitudes is within 10%, it is determined to be the tension of the penile sponge and the arterial ring in the quiet state, and the next step is to analyze the efficacy. During the experiment, each smooth muscle strip or ring was kept at an interval of 30 minutes in order to restore the tension of the penile sponge strip and the arterial ring to a quiet state before proceeding to the next experiment. a-Experiment on the effects of Epimedium extract and Icariin on the tension of the penile cavernous body strips and aortic rings

In order to observe the effects of epimedium extract and icariin on the tension of the penile cavernous body and aortic ring, the penile cavernous body and aortic ring were induced with benzylline (PHE; 5 χ 10 ⁶ Μ) When shrinking and holding the platform, the icariin extract and icariin are concentrated Increasing amounts (Epimedium extract l-50m _g / ml, icariin 1 (Γ.- 10- ⁴ M) are sequentially input, and to observe all of the smooth muscle relaxation effect concentration. Each drug concentration of at least Observe the drug effects and statistical analysis of muscle strips or muscle rings in 8 animals b. Experiment on the mechanism of relaxation effect of icariin on penile cavernous strips

b-1. g7 concept has choline (NANC) nerves antagonist atropine (10- ⁶ M) Effect of relaxing effects of icariin corpus cavernosum due to the article

b-2. ^ j of prostaglandin E1 observed inhibitor indomethacin (10- ⁴ Μ) Effect of relaxing effects of icariin corpus cavernosum due to the article

b-3. N ^w -nitric-L-arginine (L-gift)

Effect of (ισ ^ ^^^ ασ) on the relaxation effect of Epimedium ring on the corpus cavernosum

b-4. Methyl blue (10 ⁴ ), 1H- [1, 2, 4] dipyrido [4, 3,-] quinoxaline-1-one (TO) was observed (1 (TM) Effect of Icariin on the Relaxation of Penile Sea-Plane Smooth Body

In order to observe the effects of the above-mentioned various agents on the relaxation effect of icariin on the penile cavernous body strips, the penile cavernous body strips were induced by phenylephrine (PZE; 5 X 10—). While maintaining the platform, the various agents were used in accordance with after each concentration were put, wait 20 minutes, and then the male beans saponin in an amount of increasing concentrations (icariin 10- ^1Q - 1 (ΤΜ) are sequentially input, and to observe all of the smooth muscle concentration icariin Relaxing effect. Compared with the degree of relaxation before the penetration of various agents, analyze its effect on the relaxing effect of icariin on the penile cavernous strips of the penis. Observe the drug effects of at least 5 muscle strips or muscle rings of each animal and For statistical analysis.

Benfulin hydrochloride, acetylcholine chloride, atropine sulfate, stilbenol, indomethacin, N ^m -nitric-L-arginine, 1H- [1, 2, 4] oxadiazolo [4, 3 , -A] quinoxaline-1-one

Replacement page (by-law 26 糸) Products from Sigma Chemical Co. (St. Louis, MO, USA), and methylene blue from Mallinckrodt Chemical Co. (St. Louis, MO, USA). Composition of Tyrode solution (mEq eight) Na ⁺ (153. 6), Γ (5. 3), Ca ⁺⁺ (3. 0), Mg ⁺⁺ (1. 2), CI "(157. 2), H ₂ P0 ₄ —— (0. 6), S0 ₄ —— (1. 2), HC0 ₃ (7. 1), glucose (11. 4). Statistical analysis

 The data were analyzed by computer (Acqkowlege 3.2 program, Biopac systems, USA) and all relaxation responses were expressed as the percentage of maximum relaxation response. Maximum relaxation refers to the difference between the maximum contraction point induced by PHE and the maximum downward deviation in the curve in all experiments. distance. Statistical analysis was performed using Student's test. Changes in smooth muscle tone at different drug concentrations were determined by log attenuation analysis. If the p-value is less than 0.05, it is considered statistically significant. All values are expressed as mean standard error. Experimental results

 C. Effect of Epimedium Extract and Icariin on Tension of Penile Cavernous Strips and Aortic Rings

Observe the results of the effects of Epimedium extract and Icariin on the tension of the penile cavernous body strips and the aortic ring. The results are shown in Tables 1 and 2. Table 1 and Table 2 can be seen in the data, and Epimedium extract of icariin using phenylephrine ^{(PHE; 5 X 10- 6 M} ) to induce contraction of the aorta strip and penis rings, display The effect of concentration-dependent relaxation was shown (p <0.001). EC50 icariin (EC _5.) And the penis arteries are ring ^{4. 67x10- 4 M, 2. 71xlO_ 3} M. This result indicates that relaxation of the corpus cavernosum icariin on relaxation of aortic rings than the strong ^5.8-fold (p <0. 001). Table 1. Epimedium extract (IRF) for using phenylephrine ^{(PHE; 5 X 10- 6 M} ) induced contraction of the corpora cavernosa bars and relaxation of the aortic ring exhibit concentration dependence. Icariin concentration 0. 1 0. 5 1. 0 5. 0 10. 0 15. 0 P value

(mg / ml)

 Penis sponge body loose 1 o + 2. 24 9. 86 18. 74 44. 24 87. 45 94. 23 <0. 001 o

 Relaxation effect (%) (CO O

 n = 8) ± 0. 8200 ± 1 · 27 ± 3. 25 ± 4. 35 ± 4. 12 ± 4. 36 aortic ring relaxation effect 1. 34 9. 45 23. 05 36. 45 <0. 001 should ( %) (N = 8) ± 0. 98 ± 3. 24 ± 4. 21 ± 4. 35

P value <0. 001 <0. 001 <0. 001 <0. 001 <0. 001 Table 2. Of icariin using phenylephrine ^{(PHE; 5 X 10- 6 M} ) induced contraction of the corpora cavernosa bars and relaxation of the aortic ring exhibit concentration dependence.

Crude icariin (IRF) and half effective concentration of icariin (EC ₅ )

The change of smooth muscle tension under different drug concentrations was analyzed by log attenuation. The half effective concentrations (EC ₅ ) of icariin crude in the corpora cavernosa and arterial rings were 4. 08mg / ml, 22. 4mg / ml. The half effective concentrations (EC ₅ ) of icariin in corpora cavernosa and arterial rings are 4. 67x10 ⁴ M, 2. 83x10. Calculated according to the content of icariin in the crude icariin (IRF) 8% can be obtained in terms of a concentration of icariin ^{4. 08x10- 4 M, 2. 64xlO "} 3 M, this results EC50 of icariin _{(EC 50) 4. 67xlO" M} , 2. 83xl0 3 M is almost the same. Explain that Epimedium Ring is in the component of Epimedium It is a major medicinal ingredient and has a relaxing effect on the penile cavernous body. The arterial ring strength is 5.5 times or more. An experimental study on the mechanism of the effect of icariin on the corpus cavernosum

1. After observing the effect of atropine (NANC) nerve blocker atropine (10 ^-6 M) on the relaxing effect of icariin on the penile cavernous corpuscle, the results showed that icariin on atropine The relaxation effect caused by the pre-treated cavernous body of the penis was not significantly different from that of the control group (p> 0.05), which indicated that this relaxation effect was not affected by atropine.

2. The results of observing the effect of prostaglandin E1 inhibitor indomethacin (10_ ⁴ M) on the relaxing effect of icariin on the penile cavernous corporal strips indicate that icariin on the penis pretreated with indomethacin The relaxation effect caused by the cavernous body strip was not significantly different from the control group (p> 0.05), which indicates that this relaxation effect is not affected by indomethacin. The results are shown in Table 3.

3. N ^w -nitric- L-arginine (L-NNA) (10— ⁴ M) and diphenyltriamidine (10— ⁴ M), which are inhibitors of NO production or NO activity, were observed. The effect of the relaxation effect caused by the penile cavernous body strips indicates that icariin has been used in the treatment of N ^w -nitric-L-arginine (L-NNA) (10 ⁴ M) and pyrogallol (10— ⁴ M) The relaxation effect caused by the pre-treated penile cavernous body strip was significantly suppressed compared with the control group (p <0.001), which indicates that this relaxation effect is related to NO production or NO activation. The results are shown in Table 3.

4. activity observed in cyclic guanosine monophosphate alcohol inhibitor methylene blue ^{(10- 4 M), 1H- [} 1, 2, 4] oxadiazole and [4, 3, -a] quinoxalin-1-one ^{(ODQ) (10 "4 M} ) results icariin smooth muscle relaxant effect of the penile body shows that sea, of icariin by methylene blue ^{(10- 4 M), 1H- [} 1, 2, 4] The oxadiazolo [4, 3, -a] quinoxaline-1-one (0DQ) (1 (TM) pre-treated penile cavernous body strips had a significantly less relaxing effect than the control group (ρ <0. 001), the results are shown in Table 4. The above experimental results show that the relaxation effect of icariin on smooth muscle and nitric oxide

(NO) -Bird Phosphocyclase (CGMP) activation is involved. The results are shown in Table 3.

Table 3 endothelial cell damage, even stupid three hope, methylene blue, 0DQ, atropine, pretreatment after indomethacin, icariin stupid to use phenylephrine; penis (PHE 5 X 10- ⁶ M) induced contraction Effects of strip relaxation effects.

Table 4 Responses of different concentrations of 0DQ pretreatment to the relaxing effect of icariin on pre-exposure to corpora cavernosa muscle strips (PHE; 5x10 ⁶ Μ)

Experiment 3 Effect of Epimedium Saccharum Ring on Esterase Activity

 experimental method

 Isolation of phosphodiesterase from human platelets

Phosphodiesterase V is distributed not only in the corpora cavernosa, but also in platelets. Use Phosphodiesterase type V is isolated from platelets, and analyzing the effect of drugs on its activity is a routine experimental method. The method is to centrifuge fresh human blood (360 gm) for 10 minutes (360 g) to obtain platelet-rich plasma (PRP), and then centrifuge PRP (1400 g) for 10 minutes to precipitate platelets. Take 1ml of precipitated platelets, mix with water-cooled buffer (Hepes 20nM, Sucrous 0. 25M, EDTA ImM, benzylsulfonyl fluoride (PMSF) ImM, pH 7. 2) and float, then use an ultrasonic shaker (Cell distuptor, Burkard Scientific, Uxbridge, Middlesex, UK). The cell lysate was separated by telecentricity (799 g) for 10 minutes, and then subjected to continuous centrifugation (165.000 g) for 60 minutes. The supernatant was taken and the complex enzyme was separated by Pharmacia FPLC (Pharmacia LTD., Milton Keynes, UK) for use.

 In order to purify the gallate diesterase by Mono-Q ion exchange chromatography, a Mono-Q column (5ml X 50mm), 5ml of buffer A and 10ml of buffer B were repeatedly washed, and telecentric separation (165. 000g) was obtained. 10 ml of the supernatant was placed in a Mono-Q column and the flow rate was set at 1 ml / min. Then, the active ingredients of the complex enzyme were passed through a linear gradient salt (0-0. 5M NaCl) at a flow rate of 1 ml / min to analyze various enzyme components. The activity of each of the obtained enzyme components was measured, and the active enzyme components were collected and put into a liquid nitrogen bottle for later use.

 2. Screening of active substances that inhibit enzymes

The phosphodiesterase activity analysis was performed at a final reaction volume of 0.5 ml, and preparations were made on water from the time the enzyme was added to the start of the reaction. Analytical buffer (Tris 40mM pH 8. 0, MgCl ₂ 10 mM, BSA 0.125mg / ml, 2-mercaptoethanol 3. 75 mM) Each time a freshly prepared buffer is used, the reactant [2H ] cGMP (ca. 28 ci / mmol; ca. 100, 000 οριη / 100μΙ) is prepared in advance with analytical buffer. The PDE V activity detection method is a two-step analysis method using [3H] cGMP labeled isotopes. The [3H] cGMP 200 μΐ was added to the measurement cuvette, the test substance is added (icariin 10- ^1Q M- 10- ⁴ Μ) or control with the same amount of DMS0) 100 μΐ and purified diester monument Enzyme 100 μΐ, add buffer to 0.5 ml, and react at 30 ° C for 20 minutes. Immerse in boiling water to stop the reaction. After the reaction was completed, 100 L (lmg / ml) of snake venom (Ophiophagus Hannah; Sigma Chemical Co) was added to the test tube and incubated at 30 ° C for 10 minutes at constant temperature to convert [3H] 5-GMP to [3H] guanosine. The converted [3H] guanosine was analyzed in an AG1-X2 resion (Bio-Rad) reactor using NaOH (0. 1 N) in ion exchange column chromatography to analyze the eluted sample. The radioactivity was measured to analyze phosphoric acid. The activity of diesterase V,

 3. Experimental results

The experimental results have icariin on phosphodiesterase V concentration-dependent inhibition of phosphatase activity ^{(10- 8 M, 0. 06%} ; 10- 7 M, 32. 7%; 10 "6 M, 56. 2% ^{; 10 "5, 83. 7%} ; 10- 4 M, 99. 9%), EC50 is 0. 43μΜ. See the table below. Inhibition of icariin on phosphodiesterase V activity (%).

Concentration 10 " ⁴ Μ 10— ⁵ Μ 10— ⁶ Μ 10— ⁷ Μ 10" ⁸ Μ 10 " ⁹ Μ

 Icariin 99. 7 87. 9 63. 2 42 2 0 It has been experimentally proven that icariin has a stronger relaxation effect on the corpora cavernosa than the aortic ring, and the relaxation of icariin on the smooth muscle of the corpora cavernosa The mechanism of action is to prevent cGMP inactivation by increasing NO production and bird phosphorylase activation and inhibiting the activity of phosphodiesterase, increasing the concentration of cGMP in the smooth muscle of the penile cavernous body and relaxing the penile cavernous body, Induces penile erections. Experiment 4. Clinical effect

In order to understand the clinical effects of icariin, 56 placebo-controlled and voluntary subjects were used as subjects to perform a placebo control using the International Index for Sexual Function (IIEF-V). A double-double-blind preliminary clinical trial. The average age of the subjects was 39 to 6.1 years, and the onset period was 5.4 to 3.7 years. There were 17 cases of diabetes, 9 cases of hypertension, and 8 cases of heart disease. The experimental drug (containing icariin 50mg / tablet) is the same as the placebo package, and the experiment was performed without the drug informant knowing the drug information. The selected volunteers were not randomly divided into two groups, given placebo or experimental drugs (2 tablets / time, 3 times / day), orally

1 month. Patients were asked to write sexual function evaluation questionnaires (a total of 5 items each with a 5 degree score) before and after taking the drug. The changes in sexual function before and after the administration were compared by Student t test analysis to evaluate the efficacy.

The experimental results showed that the scores of the sexual function of the subjects before treatment were 1.82 ± 0.54 for self-confidence in maintaining erection and erectile maintenance, and the frequency of erectile penis insertion into the vagina was 1.89 ± 0.67. Penis insertion during intercourse The frequency of vaginal and maintaining erections was 1.82 ± 0.65, the penile erection and difficulty in maintaining erections during intercourse were 1.89 ± 0.61, and the satisfaction of sexual life was 1.79 ± 0.62. The sexual function scores after treatment were respectively: the icariin group was 3.32 ± 0.39, the placebo was 2.04 ± 0.63, and the erection frequency of the penis could be evaluated in the erection of the erection. The glucoside group was 3.11 ± 0.32, the placebo was 2.07 ± 0.88, and the frequency of penile insertion and maintenance of erections during intercourse was 3.18 ± 0.47 in the icariin group, 2.17 ± 0.97 in placebo, and penile erection and erection during intercourse. In the maintenance difficulty score, the icariin group was 3.25 ± 0.43, the placebo was 2.18 ± 0.92, and the sexual satisfaction score was 3.36 ± 0.47, the placebo was 2.14 ± 0.95. The score of the glycoside tablet group was significantly higher than the placebo (p <0.01). The overall clinical effect was 75.4% and 35.4% in the icariin tablet group and placebo, respectively. No significant side effects were found except for 4 patients with mild gastrointestinal reactions. There were no significant differences in clinical outcomes and side effects in patients with diabetes, hypertension, and heart disease. The results are shown in the table below. IIEF Score of Icariin on Treatment of Sexual Dysfunction

On the penis

When penis-to-penis pair meets age ^ confidence. ^ ^ M- ^ m ^. Degree

 .

 Treatment treatment treatment treatment treatment treatment treatment treatment treatment treatment

(n = 280 0.54 0.39 0.67 0.32 0.65 0.47 0.61 0.47 0.62 0.47

• ¾ ^ 'J (n = 28) 2.04 ± 2.07 soil 2.17 ± 2.18 soil 2.14 ±

 0.63 0.88 0.97 0.92 0.95

P value 0.002 0.001 0.01 0.002 0.001

Claims

Rights request

1. Use of icariin in the manufacture of a medicament for the prevention or treatment of diseases related to sexual dysfunction and vasoconstriction.

 2. A pharmaceutical composition for the prevention or treatment of sexual dysfunction and vasoconstriction-related diseases, which comprises epimedium ring as an active ingredient and a carrier and an excipient to be used.

 3. The use according to claim 1 or the pharmaceutical composition according to claim 2, wherein said medicine or pharmaceutical composition contains 10-500 mg of icariin.

 4. The use or pharmaceutical composition according to claims 1-3, wherein said medicine or pharmaceutical composition is a tablet, pill, capsule, suspension, solution, injection, cream, ointment, spray, chewing Use in the form of a patch.

 5. The use or pharmaceutical composition according to claims 1-3, wherein the drug administration route can be oral, sublingual, transdermal, transmuscular or subcutaneous, skin mucosa, urethra, vagina, or vein.