Application of mulberry extract in preparation of medicine for treating and / or preventing male infertility

Abstract

The invention relates to application of a mulberry extract in preparation of a medicine for treating and / or preventing male infertility. The mulberry extract comprises alkaloid with the weight content being 3% or above, and / or polysaccharide with the weight content being not higher than 70%, and / or flavone with the weight content being not higher than 10%, and / or amino acid with the weight content being not higher than 50%, and / or other components. The mulberry extract disclosed by the invention has a remarkable treatment effect on male infertility related to sexual male dysfunction and male reproductive dysfunction.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to the use of mulberry extract in the preparation of a medicament for treating and / or preventing male infertility. Technical Background

[0002] When a couple of childbearing age does not take contraceptive measures and has normal sexual life for more than one year without being able to conceive naturally, male infertility caused by male factors is called male infertility. It is estimated that 15% of couples of childbearing age globally have fertility problems, and male factors account for about 50% of them. Male infertility is not an independent disease. Most are the results of multiple etiologies and factors. Its main risk factors are mainly manifested in: age factors, bad habits (smoking, drinking), obesity, bad living habits (staying up late, lack of exercise), pathogen infection, varicocele, and bad mental and psychological factors, etc.

[0003] Clinically, male infertility is divided into two categories: sexual dysfunction and normal sexual function. The latter is often called "reproductive dysfunction". Clinically, sexual dysfunction and reproductive dysfunction affect and promote each other and often do not exist independently. Sexual dysfunction is clinically manifested as sexual desire disorder, erectile dysfunction, ejaculation disorder, and orgasm deficiency: sexual desire disorder is mainly related to the reduction of testosterone level or the change of its receptor function; erectile dysfunction is mainly related to the damage of cavernous smooth muscle or the disorder of endothelial cell function; ejaculation dysfunction is mainly related to the damage of smooth muscle, autonomic nerve and the reduction of testosterone level; orgasm deficiency is mainly related to androgen and pudendal sensory nerve disorders. Among them, erectile dysfunction and ejaculation disorder seriously affect the combination of sperm and fertilized eggs and are closely related to male infertility. Reproductive dysfunction is mostly caused by abnormal sperm parameters, such as reduced sperm count, poor sperm motility, etc. Clinical manifestations are mostly manifested as vas deferens obstruction, abnormal sperm quality, abnormal semen, varicocele and inflammation, etc. Among them, sperm quality is an important indicator for evaluating male infertility. According to the semen analysis results, it can be further divided into azoospermia, oligospermia, asthenospermia, asthenozoospermia and normospermic infertility.

[0004] Male reproductive dysfunction and sexual dysfunction seriously affect people's quality of life and fertility, but existing therapeutic drugs often fail to meet expectations and have poor clinical effects. Developing safe and effective drugs that can not only improve sperm quality and motility but also improve sexual dysfunction not only has important scientific significance and medical value but also has great social value. Summary of the Invention

[0005] The object of the present invention is to provide the use of mulberry extract in the preparation of a medicament for treating and / or preventing male infertility, and the mulberry extract has a significant therapeutic effect on male infertility related to sexual dysfunction and reproductive dysfunction.

[0006] In the present invention, based on the total weight percentage of the components of the mulberry extract being 100%, the mulberry extract contains alkaloids with a weight content of more than 3% (optionally containing alkaloids with a weight content of 3 - 99%, further optionally containing alkaloids with a weight content of 15 - 99%, further optionally containing alkaloids with a weight content of 30 - 99%, further optionally containing alkaloids with a weight content of 40 - 99%, further optionally containing alkaloids with a weight content of 50 - 99%, further optionally containing alkaloids with a weight content of 60 - 99%, further optionally containing alkaloids with a weight content of 35 - 70%, further optionally containing alkaloids with a weight content of 60 - 75%);

[0007] and / or contains polysaccharides with a weight content not higher than 70% (optionally containing polysaccharides with a weight content of 0.2 - 70%, further optionally containing polysaccharides with a weight content of 0.2 - 50%, further optionally containing polysaccharides with a weight content of 0.2 - 35%, further optionally containing polysaccharides with a weight content of 0.2 - 25%, further optionally containing polysaccharides with a weight content of 0.2 - 23%, further optionally containing polysaccharides with a weight content of 20 - 25%),

[0008] and / or contains flavonoids with a weight content not higher than 10% (optionally containing flavonoids with a weight content of 0.05 - 5%, further optionally containing flavonoids with a weight content of 0 - 2%, further optionally containing flavonoids with a weight content of 0.05 - 2%, further optionally containing flavonoids with a weight content of 0.5 - 1.5%, further optionally containing flavonoids with a weight content of 0 - 1%, further optionally containing flavonoids with a weight content of 0.05 - 1%),

[0009] and / or contains amino acids with a weight content not higher than 50% (optionally containing amino acids with a weight content of 0 - 30%, further optionally containing amino acids with a weight content of 0 - 25%, further optionally containing amino acids with a weight content of 0 - 20%, further optionally containing amino acids with a weight content of 0 - 5%, further optionally containing amino acids with a weight content of 3 - 25% or further optionally containing amino acids with a weight content of 5 - 20%),

[0010] and / or other components (the weight content can be optionally 0 - 25%, further optionally 0 - 20%, further optionally 0 - 15%, further optionally 0 - 11%, further optionally 2 - 20%, further optionally 4 - 8%.

[0011] Preferably, male infertility is male sexual dysfunction and / or male reproductive dysfunction.

[0012] Preferably, the male sexual dysfunction and / or male reproductive dysfunction is caused by endocrine disorder and / or metabolic disorder; preferably, the metabolic disorder is glucose and lipid metabolism disorder; preferably, the endocrine disorder is abnormal hormone level.

[0013] Preferably, the glucose and lipid metabolism disorder is selected from at least one of diabetes, hyperglycemia, overweight, obesity, hyperlipidemia; preferably, the abnormal hormone level is abnormal sex hormone level, preferably abnormal androgen level.

[0014] Preferably, the male sexual dysfunction is selected from at least one of sexual desire disorder, erectile dysfunction, ejaculation disorder, anorgasmia; preferably, the male sexual dysfunction is erectile dysfunction and / or ejaculation disorder.

[0015] Preferably, the male reproductive dysfunction is selected from at least one of vas deferens obstruction, abnormal sperm quality, abnormal semen, varicocele and inflammation; more preferably, the abnormal sperm quality is selected from at least one of azoospermia, oligospermia, asthenospermia, asthenozoospermia. Preferably, the male reproductive dysfunction is oligospermia, asthenospermia and / or oligoasthenospermia.

[0016] Preferably, the treatment and / or prevention of male infertility is specifically embodied as at least one of the following:

[0017] 1) Promote sperm production;

[0018] 2) Improve sperm motility;

[0019] 3) Improve sperm quality;

[0020] 4) Improve testicular index;

[0021] 5) Improve the structure of seminiferous tubules and the morphology and quantity of spermatogenic cells in testicular tissue;

[0022] 6) Improve the relevant sex hormone level; preferably, the sex hormone is androgen; more preferably, the androgen is testosterone (T);

[0023] 7) Improve the expression level of androgen synthase; the androgen synthase includes at least one of the rate-limiting enzyme for testosterone synthesis, steroidogenic acute regulatory protein (STAR), the important enzyme for testosterone synthesis, cytochrome P450 17α-hydroxylase (CYP17A1), 17β-hydroxysteroid dehydrogenase (17β-HSD);

[0024] 8) Improve the expression level of androgen receptor;

[0025] 9) Improve penile tissue damage and / or cell damage; preferably, the penile cells are penile cavernosal endothelial cells; preferably, improve the content of NO in penile cavernosal endothelial cells; preferably, the improvement of penile tissue damage specifically includes improving at least one of the average thickness of the cavernous scaffold, connection density, separation degree, and the average intercept length of the blood sinus lacunae.

[0026] Further preferably, the treatment and / or prevention of male reproductive dysfunction is specifically manifested as at least one of the following:

[0027] 1) Promote spermatogenesis;

[0028] 2) Improve sperm motility;

[0029] 3) Improve sperm quality;

[0030] 4) Improve testicular index;

[0031] 5) Improve the structure of seminiferous tubules in testicular tissue and the morphology and quantity of spermatogenic cells; preferably, the spermatogenic cells include at least one of spermatogonia, primary spermatocytes, secondary spermatocytes, and spermatids;

[0032] 6) Improve the levels of related sex hormones; preferably, the sex hormone is androgen; further preferably, the androgen is testosterone (T);

[0033] 7) Improve the expression level of androgen synthase; preferably, the androgen synthase includes at least one of the rate-limiting enzyme for testosterone synthesis, steroidogenic acute regulatory protein STAR, the important enzyme for testosterone synthesis, cytochrome P450 17α-hydroxylase CYP17A1, and 17β-hydroxysteroid dehydrogenase 17β-HSD;

[0034] 8) Improve the expression level of androgen receptor;

[0035] 9) Improve testicular cell damage; preferably, the testicular cells are Leydig cells;

[0036] Further preferably, the treatment and / or prevention of male sexual dysfunction is specifically manifested as at least one of the following:

[0037] 1) Improve the levels of related sex hormones; preferably, the sex hormone is androgen; further preferably, the androgen is testosterone (T);

[0038] 2) Improve the expression level of androgen synthase; preferably, the androgen synthase includes at least one of the rate-limiting enzyme for testosterone synthesis, steroidogenic acute regulatory protein STAR, the important enzyme for testosterone synthesis, cytochrome P450 17α-hydroxylase CYP17A1, and 17β-hydroxysteroid dehydrogenase 17β-HSD;

[0039] 3) Improve the expression level of androgen receptor;

[0040] 4) Improve penile tissue and / or cell damage; preferably, the penile cells are penile cavernosal endothelial cells; preferably, improve the content of NO in penile cavernosal endothelial cells; preferably, the improvement of penile tissue damage includes improving at least one of the average thickness of the cavernous scaffold, connection density, separation degree, and average intercept length of the blood sinus cavity;

[0041] 5) Improve the IIEF-15 score; preferably, the IIEF-15 score includes at least one of erectile function, satisfaction with insertion, orgasm, libido, and overall satisfaction.

[0042] On the other hand, the present invention provides the use of the mulberry extract in the preparation of a product for inhibiting the apoptosis of Leydig cells induced by high glucose in vitro;

[0043] On the other hand, the present invention provides the use of the mulberry extract in the preparation of a product for improving the endothelial cell damage induced by high glucose in vitro.

[0044] In an alternative embodiment of the present invention, based on the sum of the weight percentages of the components of the mulberry extract being 100%, the weight contents of the components in the mulberry extract are:

[0045]

[0046] Optionally, based on the sum of the weight percentages of the components of the mulberry extract being 100%, the weight contents of the components in the mulberry extract are:

[0047]

[0048] Optionally, based on the sum of the weight percentages of the components of the mulberry extract being 100%, the weight contents of the components in the mulberry extract are:

[0049]

[0050] Optionally, based on the sum of the weight percentages of the components of the mulberry extract being 100%, the weight contents of the components in the mulberry extract are:

[0051]

[0052] Optionally, based on the sum of the weight percentages of the components of the mulberry extract being 100%, the weight contents of the components in the mulberry extract are:

[0053]

[0054] Optionally, based on the sum of the weight percentages of the components of the mulberry extract being 100%, the weight contents of the components in the mulberry extract are as follows:

[0055]

[0056] Optionally, based on the sum of the weight percentages of the components of the mulberry extract being 100%, the weight contents of the components in the mulberry extract are as follows:

[0057]

[0058] Further optionally, based on the sum of the weight percentages of the components of the mulberry extract being 100%, the weight contents of the components in the mulberry extract are as follows:

[0059]

[0060] Further optionally, based on the sum of the weight percentages of the components of the mulberry extract being 100%, the weight contents of the components in the mulberry extract are as follows:

[0061]

[0062] Further optionally, based on the sum of the weight percentages of the components of the mulberry extract being 100%, the weight contents of the components in the mulberry extract are as follows:

[0063] Alkaloids 40 - 65%,

[0064] Polysaccharides 22 - 25%,

[0065] Flavonoids 0.5 - 1%,

[0066] Amino acids 5 - 25%,

[0067] Other components 5 - 10%,

[0068] Even more optionally, based on the mulberry extract, the weight contents of the components are as follows:

[0069]

[0070] Optionally, based on the mulberry extract, the weight contents of the components are as follows:

[0071]

[0072] Optionally, based on the mulberry extract, the weight contents of the components are as follows:

[0073]

[0074]

[0075] Optionally, based on the mulberry extract, the weight contents of the components are as follows:

[0076] Optionally, based on the mulberry extract, the weight contents of the components are as follows:

[0077] Optionally, based on the mulberry extract, the weight contents of the components are as follows:

[0078] Optionally, based on the mulberry extract, the weight contents of the components are as follows:

[0079] Optionally, based on the mulberry extract, the weight contents of the components are as follows:

[0080]

[0081]

[0082] Optionally, based on the mulberry extract, the weight contents of the components are as follows:

[0083] Optionally, based on the mulberry extract, the weight contents of the components are as follows:

[0084] Optionally, based on the mulberry extract, the weight contents of the components are as follows:

[0085] Optionally, based on the mulberry extract, the weight contents of the components are as follows:

[0086] Optionally, based on the mulberry extract, the weight contents of the components are as follows:

[0087]

[0088]

[0089] Optionally, based on the mulberry extract, the weight contents of the components are as follows:

[0090] Optionally, based on the mulberry extract, the weight contents of the components are as follows:

[0091] Optionally, based on the mulberry extract, the weight content of each component is as follows:

[0092] Optionally, based on the mulberry extract, the weight content of each component is as follows:

[0093] Optionally, based on the mulberry extract, the weight content of each component is as follows:

[0094] Optionally, based on the mulberry extract, the weight content of each component is as follows:

[0095] In the mulberry extract, the weight content of each component is as follows:

[0096]

[0097] In the mulberry extract, the weight content of each component is as follows:

[0098]

[0099] In the mulberry extract, the weight content of each component is as follows:

[0100]

[0101] In the mulberry extract, the weight content of each component is as follows:

[0102] In the mulberry extract, the weight content of each component is as follows:

[0103] In the mulberry extract, the weight content of each component is as follows:

[0104] In the mulberry extract, the weight content of each component is as follows:

[0105] In the mulberry extract, the weight content of each component is as follows:

[0106]

[0107]

[0108] In the mulberry extract, the weight content of each component is as follows:

[0109]

[0110] In the mulberry extract, the weight content of each component is as follows:

[0111]

[0112] In the mulberry extract, the weight content of each component is as follows:

[0113]

[0114] In the mulberry extract, the weight content of each component is as follows:

[0115]

[0116] The weight content of each component can be any combination of the above values.

[0117] Optionally, the alkaloid includes one or more of 1-deoxynojirimycin (1-deoxynojirimycin or DNJ), N-methyl-1-deoxynojirimycin, fagomine (fagomine or FAG), 3-epi-fagomine, 1,4-dideoxy-1,4-imino-D-arabinitol (1,4-dideoxy-1,4-imino-D-arabinitol or DAB), calystegin B2, calystegin C1, 2-O-(α-D-galactopyranosyl)-1-deoxynojirimycin, 6-O-(β-D-glucopyranosyl)-1-deoxynojirimycin, 1,4-dideoxy-1,4-imino-(2-O-β-D-glucopyranosyl)-D-arabinitol.

[0118] Among them, optionally, the weight percentage of DNJ is not less than 50% of the total alkaloid (optionally 60-99%).

[0119] Optionally, the heavy metal content of the mulberry extract does not exceed 10 ppm.

[0120] The drug is an oral dosage form; optionally, the drug is tablets, capsules, oral solutions, oral emulsions, pills, granules, syrups and powders.

[0121] In the present invention, the mulberry extract may be provided in the form of commercially available total alkaloids tablets from mulberry twigs (National Medicine Approval Number: Z20200002).

[0122] On the other hand, the present invention provides the use of the above-mentioned mulberry extract in the treatment and / or prevention of male infertility.

[0123] On the other hand, the present invention provides a method for treating male infertility, which includes administering a therapeutically or prophylactically effective amount of the above-mentioned mulberry extract to a patient in need thereof.

[0124] Optionally, the mulberry extract may be prepared according to the method described in CN 110393738A. In one embodiment, the preparation of the mulberry extract includes the following steps: preparing a crude extract of Moraceae plants; optionally, separating by cation resin and / or anion resin; optionally, subjecting the resin effluent to alcohol precipitation treatment and collecting the supernatant; and optionally, concentrating and / or drying the supernatant.

[0125] Optionally, in the present invention, the preparation of the mulberry extract includes the following steps: 1) preparing a crude extract of Moraceae plants; 2) separating the crude extract by cation resin and / or optional anion resin to obtain a resin effluent, and optionally step 3): subjecting the resin effluent obtained in step 2) to alcohol precipitation treatment and collecting the supernatant; 4) concentrating and / or drying the supernatant. Optionally, before subjecting the resin effluent obtained in step 2) to alcohol precipitation treatment, concentration and / or drying treatment is carried out.

[0126] Optionally, the Moraceae plant is one or more combinations selected from Morus multicaulis Perrott., Morus alba L., Morus atropurpurea Roxb, Morus mizuho Hotta, Morus wittiorum Hand Mazz., Morus laevigata Wall, Morus nigra Linn., Morus cathayana Hemsi., Morus serrata Roxb., Morus mongolica Schneid., Morus bombycis Koidz., Morus notabilis Schneid., Morus nigriformis Koidz., Morus yunnanensis Koidz., Morus australis Poir., Morus mongolica (Bur.) Schneid var. diabolica Koidz., large-leaf mulberry, weeping mulberry (Morus alba Var. Pendula Dippel), white-vein mulberry, mulberry varieties bred from the above mulberry species, and hybrid mulberries selected from within or between the above mulberry species;

[0127] Optionally, the Moraceae plant is one or more selected from Morus atropurpurea, Morus multicaulis, Morus alba, Morus serrata, Morus bombycis or hybrid mulberries, and the hybrid mulberry can be optionally Morus atropurpurea 11, Guisangyou 62 or Sangteyou 2. Various parts of the Moraceae plant such as leaves, roots, branches, barks, buds, stems, fruits, etc. can be used, and mulberry branches, mulberry leaves or mulberry white bark can be optionally used.

[0128] In the present invention, the mulberry extract can be optionally a mulberry branch extract, a mulberry white bark extract, a mulberry leaf extract or a mixed extract thereof.

[0129] In one embodiment, the mulberry extract is prepared according to the following steps: crushing the Moraceae plant, heating and refluxing for extraction with water and / or an alcohol solution or acidified water, with the solvent amount being 3 - 20 times that of the crude drug, repeating the extraction 1 - 3 times, combining the extraction solutions, concentrating, loading onto a cation exchange resin, eluting with 0.2 - 3N ammonia water, loading the eluate onto an anion exchange resin, collecting the non-adsorbed part, adding ethanol, precipitating to remove impurities, concentrating and / or drying to obtain the extract.

[0130] In one embodiment, the mulberry extract is prepared according to the following steps: pulverize the Moraceae plant, heat and reflux extract with water and / or an alcohol solution or acid water, the solvent amount being 3-20 times the amount of the original medicinal material, repeat the extraction 1-3 times, combine the extraction solutions, concentrate, load onto a cation exchange resin, elute with 0.2-3N ammonia water, load the eluate onto an anion exchange resin, collect the non-adsorbed part, concentrate and / or dry to obtain the extract.

[0131] In one embodiment, the mulberry extract is prepared according to the following steps: pulverize the Moraceae plant, heat and reflux extract with water and / or an alcohol solution or acid water, the solvent amount being 3-20 times the amount of the original medicinal material, repeat the extraction 1-3 times, combine the extraction solutions, concentrate, load onto a cation exchange resin, elute with 0.2-3N ammonia water, concentrate and / or dry the eluate to obtain the extract.

[0132] In one embodiment, the mulberry extract is prepared according to the following steps: pulverize the Moraceae plant, heat and reflux extract with water, the solvent amount being 3-20 times (optionally 4-15 times, further optionally 4-12 times) the amount of the original medicinal material, repeat the extraction 1-3 times (the extraction time can be optionally 0.5-3 h each time, further optionally 1-2 h each time), combine the extraction solutions, concentrate, load onto a cation exchange resin, elute with 0.2-3N ammonia water, load the eluate onto an anion exchange resin, collect the non-adsorbed part (i.e., the anion resin effluent), add ethanol, precipitate to remove impurities, concentrate and / or dry to obtain the extract.

[0133] Optionally, before the resin separation treatment in step 2), the concentrated crude extract can also be subjected to alcohol precipitation treatment. During the alcohol precipitation treatment, add ethanol to the crude extract, stir and mix evenly, stop stirring and let it stand for a certain time to precipitate the insoluble substances therein. Optionally, calculated in L / kg, the volume-mass ratio of the added ethanol to the plant raw material is 0.2-20 times, optionally 0.4-10 times. Further optionally, an alcohol precipitation tank is used for the alcohol precipitation treatment. Optionally, the stirring speed in the alcohol precipitation treatment is 10-600 rpm, optionally 40-500 rpm, further optionally 80-400 rpm or 300 rpm.

[0134] Optionally, after loading the cation resin into the column, activation is carried out in the order of washing with an acidic solution, washing with a basic solution, and washing with an acidic solution. Optionally, the basic solution is washed until the pH of the eluate is 8.0 - 9.5, optionally 8.5 - 9.5; optionally, the basic solution is selected from ammonia water solution, sodium hydroxide solution, potassium hydroxide solution, or sodium carbonate solution; optionally, the concentration of the basic solution is 0.5 - 4 mol / L, optionally 1 - 2 mol / L. Optionally, the acidic solution is washed until the pH of the eluate is 3.0 - 7.0, optionally 4.5 - 6.5. Optionally, the acidic solution is selected from hydrochloric acid solution, phosphoric acid solution, sodium hydrogen phosphate - citric acid buffer solution. Optionally, the concentration of the acidic solution is 0.5 - 4 mol / L, optionally 1.5 - 2 mol / L. Optionally, after the last acidic solution wash of the cation resin, it can also be rinsed with 3 - 5 times the column volume of deionized water.

[0135] Optionally, the cation resin is one or a combination of more than one of 732 strong - acid styrene - type cation exchange resin, 002SC strong - acid styrene - type cation resin, 734 strong - acid styrene - type cation exchange resin, D001 macroporous strong - acid styrene - type cation exchange resin, D113 macroporous weak - acid cation resin, and D254 macroporous strong - base quaternary ammonium - type cation exchange resin.

[0136] Optionally, the weight ratio of the dosage of the cation resin to the weight of the plant raw material input is 1:1 - 30 (optionally 1:1 - 25, 1:2 - 20, 1:2 - 15, 1:2 - 10, 1:2 - 7, 1:2 - 3).

[0137] After loading the plant crude extract onto the cation resin, the loaded cation resin is eluted with an eluent. Optionally, the eluent is a salt solution or a basic solution containing cations, optionally one or more of sodium chloride, ammonium chloride, ammonium sulfate, ammonium nitrate, ammonia water, potassium chloride, and sodium hydroxide.

[0138] Optionally, the concentration of the eluent is 0.04 - 5 mol / L (optionally 0.5 - 2.5 mol / L, 0.2 - 3 mol / L, and further optionally 0.5 - 2.5 mol / L).

[0139] Optionally, the eluent flow rate is 1 - 15 BV / h (optionally 5 - 10 BV / h, and further optionally 5 - 6 BV / h).

[0140] Optionally, the weight of the eluent used for separating the cation resin is 0.1 - 30 times the weight of the plant raw material input, and optionally, the elution is carried out with 0.5 - 10 times the weight of the plant raw material input of the eluent.

[0141] The starting point of collection can be determined according to the pH of the effluent of the cation resin. For example, when using an alkaline solution such as ammonia water for elution, the eluent is collected when the pH of the effluent of the cation column is > 7, or the starting point of collection of the effluent is determined according to the color reaction or precipitation reaction. Optionally, when the volume of the collected liquid reaches 0.1 - 10 times (further optionally, 0.2 - 5 times) the weight of the plant raw material charged, the collection is stopped, and the collected liquid is optionally purified through an anion column.

[0142] When purifying through an anion column, optionally, after loading the anion resin, activation is carried out in the order of washing with an alkaline solution, washing with an acidic solution, and washing with an alkaline solution.

[0143] Optionally, the alkaline solution is washed until the pH of the eluate is 8.0 - 9.5, optionally 8.5 - 9.5;

[0144] Optionally, the alkaline solution is selected from ammonia water solution, sodium hydroxide solution, potassium hydroxide solution or sodium carbonate solution; optionally, the concentration of the alkaline solution is 0.5 - 4 mol / L, optionally 1 - 2 mol / L.

[0145] Optionally, the acidic solution is washed until the pH of the eluate is 3.0 - 7.0, optionally 4.5 - 6.5. Optionally, the acidic solution is selected from hydrochloric acid solution, phosphoric acid solution, sodium hydrogen phosphate - citric acid buffer solution. Optionally, the concentration of the acidic solution is 0.5 - 4 mol / L, optionally 1 - 2 mol / L.

[0146] Optionally, the anion resin is one or a combination of more than one of 711 strong - basic styrene - type anion resin, 717 strong - basic styrene - type anion exchange resin, D201 macroporous strong - basic styrene - type anion exchange resin, D218 macroporous strong - basic acrylic - type anion exchange resin, D301 - G macroporous weak - acid - type styrene - type anion exchange resin and D301 macroporous weak - basic styrene - type anion exchange resin.

[0147] Optionally, the dosage ratio of the anion resin to the weight of the plant raw material charged is 1:1 - 80 (optionally, 1:1 - 64, 1:1 - 32, 1:1 - 24, 1:5 - 16, 1:3).

[0148] Collection starts when the liquid flows out of the anion resin. Optionally, when the volume of the collected liquid reaches 0.05 - 10 times (optionally, 0.1 - 5 times) the weight of the plant raw material charged, the collection is stopped.

[0149] Optionally, the weight ratio of ethanol used in the alcohol precipitation treatment to the weight of the plant raw material fed is 1:4 - 600 (optionally 1:20 - 300, further optionally 1:20 - 50, 1:40, 1:80, 1:22). In the alcohol precipitation treatment, the stirring speed is 10 - 600 rpm (optionally 40 - 500 rpm, 80 - 400 rpm). The time of the alcohol precipitation treatment is 12 - 24 h.

[0150] Further, before the alcohol precipitation treatment, it further includes the steps of centrifuging or microfiltration membrane filtering to remove impurities from the anion resin effluent, and then performing counterion osmosis membrane concentration. The specific gravity of the concentrated liquid can be 1.0 - 1.3, optionally 1.1 - 1.25.

[0151] Optionally, the drug further includes a pharmaceutically acceptable carrier. The carrier is an inactive ingredient that is non-toxic to the human body and conforms to the route of administration or the mode of administration. The carrier can be a solid or liquid excipient. Solid excipients, for example, include microcrystalline cellulose, mannitol, lactose, pregelatinized starch, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, sodium carboxymethyl starch, aspartame, calcium hydrogen phosphate, sodium lactate, poloxamer, sodium dodecyl sulfate, sodium carboxymethyl cellulose, gelatin, xanthan gum, polyvinylpyrrolidone, starch, magnesium stearate, sodium carboxymethyl starch, and talc powder; liquid excipients, for example, include water, ethanol, syrup, and glycerol.

[0152] In the present invention, the mulberry extract is abbreviated as SZ-A.

[0153] Beneficial effects:

[0154] The mulberry extract can improve infertility, including male sexual dysfunction and male reproductive disorders. The mulberry extract can increase the total number of sperm, the number of motile sperm, and improve sperm motility, can improve penile tissue damage, can significantly increase the testosterone level in the testicular tissue, significantly increase the expression of STAR, the rate-limiting enzyme for testosterone synthesis, CYP17A1, an important enzyme for testosterone synthesis, and androgen receptor AR, significantly improve the structure of the seminiferous tubules in the testis, the state of interstitial cells, reduce inflammatory cell infiltration, and increase the number and layers of spermatogenic cells at all levels, with closer connections and neater arrangements. Description of the drawings

[0155] Figure 1 are the results of (A) the total number of sperm and (B) the number of motile sperm of male SD rats in each test group of Experimental Example 1;

[0156] Figure 2 are the results of the sperm count, the number of motile sperm, and sperm motility of GAN-induced male mice in each test group of Experimental Example 2 (n = 5, the data are "mean ± standard error", and the comparison of differences between groups uses one-way ANOVA. Compared with the Model group, *P < 0.05, **P < 0.01);

[0157] Figure 3 Testosterone levels in the testicular tissues of male mice induced by GAN in each experimental group of Test Example 2 (n = 10, data are "mean ± standard error", one-way ANOVA was used for comparison of differences between groups, compared with the Model group, *P < 0.05, **P < 0.01);

[0158] Figure 4 STAR levels in the testicular tissues of male mice induced by GAN in each experimental group of Test Example 2 (n = 3, data are "mean ± standard error", one-way ANOVA was used for comparison of differences between groups, compared with the Model group, *P < 0.05, **P < 0.01);

[0159] Figure 5 CYP17A1 levels in the testicular tissues of male mice induced by GAN in each experimental group of Test Example 2 (n = 3, data are "mean ± standard error", one-way ANOVA was used for comparison of differences between groups, compared with the Model group, *P < 0.05, **P < 0.01);

[0160] Figure 6 AR levels in the testicular tissues of male mice induced by GAN in each experimental group of Test Example 2 (n = 3, data are "mean ± standard error", one-way ANOVA was used for comparison of differences between groups, compared with the Model group, *P < 0.05, **P < 0.01);

[0161] Figure 7 Morphological diagrams of HE staining of testicular tissues of male mice induced by GAN in each experimental group of Test Example 2;

[0162] Figure 8 Numbers of spermatogenic cells at all levels in the testicular tissues of male mice induced by GAN in each experimental group of Test Example 2 (data are "mean ± standard error", one-way ANOVA was used for comparison of differences between groups, compared with the Model group, *P < 0.05, **P < 0.01);

[0163] Figure 9 Body weight results of C57BL / 6J obese mice induced by HFD in each experimental group of Test Example 3;

[0164] Figure 10 Results of sperm count, motile sperm count and sperm motility of C57BL / 6J obese mice induced by HFD in each experimental group of Test Example 3; compared with the Model group, *P < 0.05, **P < 0.01;

[0165] Figure 11 Testicular index results of C57BL / 6J obese mice induced by HFD in each experimental group of Test Example 3; compared with the Model group, *P < 0.05, **P < 0.01;

[0166] Figure 12 It is the HE staining diagram of the corpus cavernosum structure of the penile tissue of the C57BL / 6J obese reproductive disorder mouse model induced by HFD diet in Test Example 3; compared with the Model group, *P<0.05, **P<0.01, ***P<0.001;

[0167] Figure 13 It is the results of the sperm count, motile sperm count and sperm motility of the mice in each test group of Test Example 4; compared with the Model group, *P<0.05, **P<0.01;

[0168] Figure 14 It is the HE staining diagram of the seminiferous tubule structure of the testicular tissue of the mice in each test group of Test Example 4;

[0169] Figure 15 It is the diagram of the number of spermatogenic cells at all levels and the total number of spermatogenic cells of the mice in each test group of Test Example 4; compared with the Model group, *P<0.05, **P<0.01;

[0170] Figure 16 It is the HE staining diagram of the corpus cavernosum structure of the penile tissue of the mice in each test group of Test Example 4; compared with the Model group, *P<0.05, **P<0.01, ***P<0.001;

[0171] Figure 17 It is the result of the effect of SZ-A on the damage of Leydig cells induced by PA (palmitic acid) in Test Example 5;

[0172] Figure 18 It is the result 1 of the effect of SZ-A on the damage of cavernous endothelial cells induced by high glucose in Test Example 6; compared with the high glucose group, *P<0.05, **P<0.01, ***P<0.001;

[0173] Figure 19 It is the result 2 of the effect of SZ-A on the damage of cavernous endothelial cells induced by high glucose in Test Example 7; compared with the high glucose group, *P<0.05, **P<0.01, ***P<0.001;

[0174] Figure 20 It is the result of observing endothelial cell mitochondria under a fluorescence microscope in Test Example 8;

[0175] Figure 21 It is the diagram of the IIEF-15 score results of the clinical trial in Test Example 9. Detailed implementation manners

[0176] The present invention will be further illustrated by specific experimental examples below. It should be understood that the following experiments are only used to illustrate the technical solutions of the present invention and do not limit the protection scope of the present invention.

[0177] The mulberry extract of the present invention can be prepared by the following method:

[0178] I. Preparation examples of mulberry extract

[0179] Preparation example 1 of mulberry extract

[0180] Take 1000 kg of fresh mulberry branches (Morus atropurpurea Roxb. cv. Yuesang 11 with serrate leaves), crush them, add 4000 L of water, extract for 2 h by heating reflux method, combine the extractive solutions, filter to remove insoluble substances to obtain a crude extractive solution. The crude extractive solution is thermally concentrated until the mass percentage of solid substances reaches 4%, and is kept at 50 °C as the sample loading solution for the cation resin column.

[0181] Use 150 kg of D113 macroporous weakly acidic styrene-based cation resin to fill the column, wash it with 2 mol / L hydrochloric acid solution until the pH of the eluate is 4.5; wash it with 1 mol / L sodium hydroxide solution until the pH of the eluate is 8.5; wash it with 2 mol / L hydrochloric acid solution until the pH of the eluate is 4.5; then rinse with 5 times the column volume of deionized water to complete activation. Load the concentrated extractive solution, then elute with 1000 L of 2.5 mol / L ammonia water at an elution rate of 6 BV / h, collect the eluate when the pH of the effluent from the cation column is > 7, stop collection when the collected liquid reaches 900 L, and directly purify the collected liquid through an anion column.

[0182] Use 62.5 kg of D218 macroporous strongly basic acrylic-based anion resin to fill the column, wash it with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0; wash it with 1.5 mol / L hydrochloric acid solution until the pH of the eluate is 3.5; wash it with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0; complete activation. Load the eluate of the cation resin collected onto the anion resin, and collect the effluent until the effluent reaches 920 L to end.

[0183] The collected liquid is centrifuged to remove impurities and then concentrated by reverse ion osmosis membrane. The specific gravity of the concentrated liquid is 1.25, and it is transferred to an alcohol precipitation tank. Add 25 L of anhydrous ethanol under a stirring speed of 500 rpm of the stirring paddle. After the addition of ethanol is completed, stop stirring, perform alcohol precipitation for 24 h, take the supernatant, and concentrate it under reduced pressure to obtain a mulberry branch extract paste (i.e., mulberry extract SZ-A).

[0184] In the mulberry branch extract paste, the mass percentage of alkaloids is 52%, the mass percentage of polysaccharides is 22%, the mass percentage of flavonoids is 0.8%, and the mass percentage of amino acids is 20%. Among the alkaloids, the content of 1-DNJ is 60%, FAG is 17%, and DAB is 15%.

[0185] Preparation example 2 of mulberry extract

[0186] Take 10 kg of fresh mulberry branches (Mulberry Special Excellence No. 2), crush them, add 150 L of water in two portions, and extract each portion by decoction for 3 h. Combine the extracts and filter to remove insoluble substances. Concentrate the extract by heating until the mass percentage of solids reaches 8%. Transfer it to an ethanol precipitation tank and add 2367.9 g of anhydrous ethanol (3 L) with a stirring paddle at 300 rpm. Stop stirring after the addition of ethanol and perform ethanol precipitation for 24 h. Take the supernatant as the sample loading solution for the cation resin column. Load a 5 kg column with 002SC strong acidic styrene-based cation resin and activate the cation resin according to the method of Preparation Example 1. Load the extract treated by concentration and ethanol precipitation, then elute with 100 L of 5 mol / L potassium chloride at an elution rate of 5 BV / h. Detect the effluent with 20% silicotungstic acid. Start collecting when white precipitate forms and stop collecting when the collected liquid reaches 25 L. Purify the collected liquid directly through an anion column.

[0187] Load a 10 kg column with 711 strong basic styrene-based anion resin and activate the anion resin according to the method of Preparation Example 1. Load the collected cation resin eluate onto the anion resin and collect the effluent until the effluent reaches 15 L. Reload the collected liquid onto the cation resin and perform separation twice more with the cation resin and anion resin in sequence according to the above method.

[0188] Centrifuge the collected liquid after three column separations to remove impurities and then concentrate it through an anti-ion permeable membrane. The specific gravity of the concentrated liquid is 1.25. Transfer it to an ethanol precipitation tank and add 125 g of anhydrous ethanol with a stirring paddle at 1000 rpm. Stop stirring after the addition of ethanol and perform ethanol precipitation for 24 h. Take the supernatant and concentrate it under reduced pressure to obtain an extract paste. Additionally, take fresh mulberry bark and mulberry leaves (Mulberry Special Excellence No. 2) for extraction, and the extraction method and parameters are the same as the above method.

[0189] In the obtained mulberry branch extract, the mass percentage of alkaloids is 98%, the mass percentage of polysaccharides is 0.2%, the mass percentage of flavonoids is 0.05%, and the mass percentage of amino acids is 0. Among the alkaloids, the content of 1-DNJ is 99%, FAG is 0.5%, and DAB is 0.4%.

[0190] In the obtained mulberry bark extract, the mass percentage of alkaloids is 95%, the mass percentage of polysaccharides is 2%, the mass percentage of flavonoids is 0.1%, and the mass percentage of amino acids is 1%. Among the alkaloids, the content of 1-DNJ is 96%, FAG is 1.5%, and DAB is 1.4%.

[0191] In the obtained mulberry leaf extract, the mass percentage content of alkaloids is 90%, the mass percentage content of polysaccharides is 4%, the mass percentage content of flavonoids is 0.1%, and the mass percentage content of amino acids is 3%. Among the alkaloids, the content of 1-DNJ is 91%, the content of FAG is 3.1%, and the content of DAB is 2.8%.

[0192] Preparation Example 3 of Mulberry Extract

[0193] Take 1000 kg of fresh mulberry branches (Guangdong mulberry), crush them, add 11500 L of water, extract by heating under reflux for 2 h, combine the extraction liquid, filter to remove insoluble substances, and obtain a crude extract. The crude extract is first centrifuged to remove impurities, and then concentrated by an anti-ion permeation membrane until the mass percentage content of solids reaches 1% to obtain the sample loading solution for the cation resin column.

[0194] Load a column with 300 kg of D001 macroporous strongly acidic styrene-based cation resin, and activate the cation resin according to the method of Preparation Example 1. Load the concentrated crude extract, elute with 5000 L of 0.04 mol / L ammonium nitrate at an elution rate of 5 BV / h, detect the effluent with 20% silicotungstic acid, start collecting when white precipitate forms, and stop collecting when the collected liquid reaches 1000 L.

[0195] Concentrate the collected liquid obtained after separation by the cation column by nanofiltration membrane and then concentrate it under reduced pressure to obtain an extract paste.

[0196] In the obtained mulberry branch extract, the mass percentage content of alkaloids is 15%, the mass percentage content of polysaccharides is 20%, the mass percentage content of flavonoids is 7%, and the mass percentage content of amino acids is 45%. Among the alkaloids, the content of 1-DNJ is 55%, the content of FAG is 23%, and the content of DAB is 10%.

[0197] Preparation Example 4 of Mulberry Extract

[0198] Take 333 kg of dry mulberry branches (Yuesang No. 11), crush them, add 4000 L of water, extract twice by the heating reflux method, reflux for 1 h each time, combine the extraction liquid, filter, and concentrate the extraction liquid to 1 kg of crude drug amount / L.

[0199] Load a column with 150 kg of D113 macroporous weakly acidic styrene-based cation exchange resin, wash it with 2 mol / L hydrochloric acid solution until the pH of the eluate is 4.5; wash it with 1 mol / L sodium hydroxide solution until the pH of the eluate is 8.5; wash it with 2 mol / L hydrochloric acid solution until the pH of the eluate is 4.5; then rinse with 5 times the column volume of deionized water to complete activation. Load the concentrated extract, then elute with 1000 L of 2.5 mol / L ammonia water at an elution rate of 6 BV / h. Collect the eluate when the pH of the effluent from the cation column is > 7. When the collected liquid reaches 900 L, stop collection and directly purify the collected liquid through an anion column.

[0200] Load a column with 125 kg of D218 macroporous strongly basic acrylic-based anion exchange resin, wash it with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0; wash it with 1.5 mol / L hydrochloric acid solution until the pH of the eluate is 3.5; wash it with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0; complete activation. Load the eluate of the collected cation resin onto the anion resin and collect the effluent with a pH greater than 8 until the effluent reaches 870 L.

[0201] Filter the collected liquid obtained after separation by the anion column through a microfiltration membrane to remove impurities, then concentrate it with an anti-ion permeation membrane. The specific gravity of the concentrated liquid is 1.1. Transfer it to an alcohol precipitation tank and add 15 kg of anhydrous ethanol under a stirring speed of 400 rpm of the stirring paddle. Stop stirring after the addition of ethanol and perform alcohol precipitation for 24 h. Take the supernatant and concentrate it under reduced pressure to obtain the extract paste of mulberry twig extract. Sample content: The mass percentage of alkaloids is 80%, the mass percentage of polysaccharides is 5%, the mass percentage of flavonoids is 0.1%, and the mass percentage of amino acids is 4%. Among the alkaloids, the content of 1-DNJ is 75%, FAG is 12%, and DAB is 10%.

[0202] Preparation Example 5 of Mulberry Extract

[0203] Take 400 kg of dry mulberry twigs (Mulberry No. 11 in Guangdong), crush them, add 4000 L of water, and extract twice by the heating reflux method for 1 h each time. Combine the extracts, filter, and concentrate the extract to 1 kg of crude drug amount per liter.

[0204] Load a column with 62.5 kg of D218 macroporous strongly basic acrylic-based anion exchange resin, wash it with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0; wash it with 1.5 mol / L hydrochloric acid solution until the pH of the eluate is 3.5; wash it with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0; complete activation. Load the concentrated extract onto the anion resin and collect the effluent.

[0205] The collected liquid obtained after separation by an anion column was filtered through a microfiltration membrane to remove impurities and then concentrated by an anti-ion permeation membrane, and further concentrated and dried under reduced pressure to obtain a mulberry twig extract paste. Sample content: The mass percentage of alkaloids is 3%, the mass percentage of polysaccharides is 70%, the mass percentage of flavonoids is 10%, and the mass percentage of amino acids is 10%. Among the alkaloids, the content of 1-DNJ is 68%, FAG is 17%, and DAB is 8%.

[0206] Preparation Example 6 of Mulberry Extract

[0207] Take 1500 kg of fresh mulberry twigs (Morus atropurpurea Roxb. cv. Yuansang 11), crush them, add 6000 L of water, extract by heating under reflux for 2 h, combine the extraction liquids, filter to remove insoluble substances to obtain a crude extract. The crude extract was thermally concentrated to a solid content mass percentage of 4% and kept at 50 °C as the loading solution for the cation resin column.

[0208] Use 100 kg of D113 macroporous weakly acidic styrene-based cation resin to fill the column, wash with 2 mol / L hydrochloric acid solution until the pH of the eluate is 4.5; wash with 1 mol / L sodium hydroxide solution until the pH of the eluate is 8.5; wash with 2 mol / L hydrochloric acid solution until the pH of the eluate is 4.5; then wash with 5 times the column volume of deionized water to complete activation. Load the concentrated extract onto the column, then elute with 1000 L of 2.5 mol / L ammonia water at an elution rate of 6 BV / h. Collect the eluate when the pH of the effluent from the cation column is >7. When the collected liquid reaches 900 L, stop collection and directly purify the collected liquid through an anion column.

[0209] Use 62.5 kg of D218 macroporous strongly basic acrylic-based anion resin to fill the column, wash with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0; wash with 1.5 mol / L hydrochloric acid solution until the pH of the eluate is 3.5; wash with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0; complete activation. Load the eluate of the cation resin collected onto the anion resin and collect the effluent until the effluent reaches 870 L. Concentrate the effluent under reduced pressure to obtain a mulberry twig extract paste, in which the mass percentage of alkaloids is 30%, the mass percentage of polysaccharides is 35%, the mass percentage of flavonoids is 2%, and the mass percentage of amino acids is 25%. Among the alkaloids, the content of 1-DNJ is 62%, FAG is 20%, and DAB is 13%.

[0210] Preparation Example 7 of Mulberry Extract

[0211] Take 1000 kg of fresh mulberry branches (Morus atropurpurea Roxb. cv. Yuesang 11 with fine teeth), crush them, add 4000 L of water, extract for 2 h by the heating reflux method, combine the extracts, filter to remove insoluble substances, and obtain a crude extract. The crude extract is thermally concentrated until the mass percentage of solids reaches 4%, and kept at 50 °C as the sample loading solution for the cation resin column.

[0212] Use 100 kg of D113 macroporous weakly acidic styrene-based cation resin to fill the column. Wash it with 2 mol / L hydrochloric acid solution until the pH of the eluate is 4.5; wash it with 1 mol / L sodium hydroxide solution until the pH of the eluate is 8.5; wash it with 2 mol / L hydrochloric acid solution until the pH of the eluate is 4.5; then rinse it with 5 times the column volume of deionized water to complete activation. Load the concentrated extract onto the column, then elute it with 1000 L of 2.5 mol / L ammonia water at an elution rate of 6 BV / h. Collect the eluate when the pH of the effluent from the cation column is > 7. When the collected liquid reaches 900 L, stop collection and directly purify the collected liquid through an anion column.

[0213] Use 62.5 kg of D218 macroporous strongly basic acrylic-based anion resin to fill the column. Wash it with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0; wash it with 1.5 mol / L hydrochloric acid solution until the pH of the eluate is 3.5; wash it with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0; complete activation. Load the eluate of the cation resin collected onto the anion resin and collect the effluent until the effluent reaches 870 L. Concentrate the effluent under reduced pressure to obtain an extract paste of mulberry branches. Among them, the mass percentage of alkaloids is 40%, the mass percentage of polysaccharides is 25%, the mass percentage of flavonoids is 0.5%, and the mass percentage of amino acids is 25%. Among the alkaloids, the content of 1-DNJ is 57%, FAG is 24%, and DAB is 16%.

[0214] Preparation Example 8 of Mulberry Extract

[0215] Take 333 kg of dry mulberry branches (Morus atropurpurea Roxb. cv. Yuesang 11), crush them, add 4000 L of water, and extract twice by the heating reflux method, with each reflux for 1 h. Combine the extracts, filter, and concentrate the extract to 1 kg of crude drug amount per liter.

[0216] The column was packed with 150 kg of D113 macroporous weakly acidic styrene-based cation exchange resin, washed with 2 mol / L hydrochloric acid solution until the pH of the eluate reached 4.5; washed with 1 mol / L sodium hydroxide solution until the pH of the eluate reached 8.5; washed with 2 mol / L hydrochloric acid solution until the pH of the eluate reached 4.5; and then rinsed with 5 times the column volume of deionized water to complete activation. The concentrated extract was loaded onto the column, and then eluted with 1000 L of 2.5 mol / L ammonia water at an elution rate of 6 BV / h. The eluate was collected when the pH of the effluent from the cation column was >7. When the collected liquid reached 900 L, the collection was stopped, and the collected liquid was directly purified through an anion column.

[0217] The column was packed with 62.5 kg of D218 macroporous strongly basic acrylic-based anion exchange resin, washed with 1.5 mol / L sodium hydroxide solution until the pH of the eluate reached 9.0; washed with 1.5 mol / L hydrochloric acid solution until the pH of the eluate reached 3.5; washed with 1.5 mol / L sodium hydroxide solution until the pH of the eluate reached 9.0; and activation was completed. The eluate of the cation resin was loaded onto the anion resin, and the effluent with a pH greater than 8 was collected until the effluent reached 870 L.

[0218] The collected liquid obtained after separation by the anion column was filtered through a microfiltration membrane to remove impurities and then concentrated by a counterion osmosis membrane. The specific gravity of the concentrated liquid was 1.1. It was transferred to an alcohol precipitation tank, and 15 kg of absolute ethanol was added under stirring at 400 rpm. After the addition of ethanol was completed, stirring was stopped, and alcohol precipitation was carried out for 24 h. The supernatant was taken and concentrated under reduced pressure to obtain the extract paste of mulberry twig extract. Sample content: The mass percentage content of alkaloids was 63%, the mass percentage content of polysaccharides was 23%, the mass percentage content of flavonoids was 1%, and the mass percentage content of amino acids was 5%. Among the alkaloids, the content of 1-DNJ was 61.9%, FAG was 16.6%, and DAB was 11.1%.

[0219] Preparation Example 9 of Mulberry Extract

[0220] 1000 kg of fresh mulberry twigs (Mulberry No. 11 in Guangdong) were taken, crushed, added with 4000 L of water, and extracted by heating under reflux for 2 h. The extract solutions were combined and filtered to remove insoluble substances to obtain a crude extract. The crude extract was thermally concentrated until the solid content reached 4%, and kept at 50 °C as the loading solution for the cation resin column.

[0221] Use 120 kg of D113 macroporous weakly acidic styrene-based cation exchange resin to fill the column, wash it with 2 mol / L hydrochloric acid solution until the pH of the eluate is 4.5; wash it with 1 mol / L sodium hydroxide solution until the pH of the eluate is 8.5; wash it with 2 mol / L hydrochloric acid solution until the pH of the eluate is 4.5; then rinse it with 5 times the column volume of deionized water to complete activation. Load the concentrated extract solution, then elute it with 1000 L of 2.5 mol / L ammonia water at an elution rate of 6 BV / h. Collect the eluate when the pH of the effluent from the cation column is > 7. When the collected solution reaches 900 L, stop collecting and directly purify the collected solution through an anion column.

[0222] Use 45 kg of D218 macroporous strongly basic acrylic-based anion exchange resin to fill the column, wash it with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0; wash it with 1.5 mol / L hydrochloric acid solution until the pH of the eluate is 3.5; wash it with 1.5 mol / L sodium hydroxide solution until the pH of the eluate is 9.0; complete activation. Load the eluate of the collected cation resin onto the anion resin and collect the effluent until the effluent reaches 870 L to end.

[0223] The collected solution obtained after separation by the anion column is filtered through a microfiltration membrane to remove impurities and then concentrated by an anti-ion permeation membrane. The specific gravity of the concentrated liquid is 1.1. Transfer it to an alcohol precipitation tank and add 15 kg of anhydrous ethanol under a stirring paddle at 300 rpm. Stop stirring after the addition of ethanol and perform alcohol precipitation for 24 h. Take the supernatant and concentrate it under reduced pressure to obtain the extract paste. Sample content: The mass percentage content of alkaloids is 70%, the mass percentage content of polysaccharides is 20%, the mass percentage content of flavonoids is 0.6%, and the mass percentage content of amino acids is 5%. Among the alkaloids, the content of 1-DNJ is 70%, FAG is 13%, and DAB is 10%.

[0224] II. Pharmacodynamic Experiments of Mulberry Extracts

[0225] Test Example 1: Effect of Mulberry Extract on Sperm Production in Normal Rats

[0226] 1. Test Sample

[0227] The mulberry extract prepared in Preparation Example 1.

[0228] 2. Preparation of Administration Solution

[0229] First, weigh a certain amount of the test substance, place it in an appropriate beaker, add an appropriate amount of solvent, and sonicate (300 W, 40 KHZ) for about 10 minutes until completely dissolved. After standing at room temperature, transfer it to a graduated cylinder to the required volume to obtain the preparation solution of the required concentration.

[0230] 3. Animal Administration and Sampling

[0231] Forty male Sprague-Dawley rats, 5 - 7 weeks old (138 - 183 g), were randomly divided into a vehicle control group and three groups administered with mulberry extracts (at the total alkaloid dosage of 100 mg / kg / d, 200 mg / kg / d, and 500 mg / kg / d respectively). They were given intragastric administration at 10 mL / kg once a day. After 4 weeks of administration, the male rats were sacrificed and the testes and epididymides were collected.

[0232] 4. Detection methods

[0233] Sperm count:

[0234] One epididymis was placed in 1 mL of 0.9% sodium chloride injection and the epididymal tissue was minced. It was incubated at 37°C to prepare a sperm suspension. The sperm suspension was flushed into a hemocytometer, and the number of sperm under the microscope was counted. Then the total number of sperm was calculated according to the dilution factor.

[0235] Sperm motility examination:

[0236] Slide preparation: The tail of the epididymis was cut open with ophthalmic scissors, and the contents of the tail of the epididymis were mixed with 0.9% sodium chloride injection to make a sperm suspension. Two drops of the sperm suspension were placed on a glass slide. The above operations were completed under the condition of maintaining a temperature of 37°C.

[0237] Observation: The swimming states of about 200 sperm were randomly recorded under a high-power microscope. The criteria for judging the swimming states of sperm: I. Good motility, the sperm swim rapidly in a straight line; II. Moderate motility, the sperm are relatively active; III. Poor motility, the sperm move slowly and rotate in place; IV. Dead sperm, with sperm morphology but no motility.

[0238] 5. Test results

[0239] Compared with the vehicle control group, the number of sperm and the number of motile sperm in male rats in each dose group of mulberry extracts increased (see Table 1 and Figure 1 ).

[0240] Table 1 Sperm function

[0241]

[0242]

[0243] Note: Dilution method: One epididymis was taken, minced and added to 1 mL of normal saline. 50 μL was taken out and diluted to 1 mL, and then 100 μL was taken out and diluted to 1 mL and counted under the microscope. The number of sperm (M) in 4 large squares around the hemocytometer was counted. Calculation formula: M / 0.4 × dilution factor × 1000 = number of sperm / mL.

[0244] Sperm motility rate = (Grade I + Grade II + Grade III) / (Grade I + Grade II + Grade III + Grade IV) × 100%.

[0245] Table 1 and Figure 1 showed that after healthy male SD rats were intragastrically administered with mulberry extract for 4 consecutive weeks, the total sperm count and the number of motile sperm increased, indicating that mulberry extract could promote sperm production in male rats.

[0246] Test Example 2: GAN-induced male mouse reproductive dysfunction model

[0247] 1. Test samples

[0248] Mulberry twig extract (SZ-A) from Preparation Example 8

[0249] 2. The experimental groups are as follows in the table:

[0250] Table 2

[0251]

[0252] Note: Gubra-Amylin NASH (GAN) is a high-fat, high-cholesterol, high-fructose diet containing 40% saturated fat, 22% fructose and 2% cholesterol.

[0253] 3. Experimental methods

[0254] Fifty healthy male C57BL / 6J mice with good health and shiny hair were selected and given maintenance feed, and were adaptively fed and observed for 1 week. After ensuring that the animals were well adapted, the model was established. The model group was given GAN (Research Diets D09100310) feed, and the control group continued with the maintenance feed for 20 weeks to construct a male reproductive dysfunction mouse model. The mice were divided into 5 groups: the control group, the model group, the low, medium, and high dose groups of mulberry twig extract (denoted as SZ-A-L, SZ-A-M, SZ-A-H respectively), with 10 mice in each group; each experimental group was administered according to the dose and administration method in the table, and the blank group and the model group were given an equal amount of normal saline. After 21 days of administration, the materials were taken for efficacy evaluation.

[0255] 4. Detection methods

[0256] 1) Sperm count detection:

[0257] After dissecting the mice, take the unilateral epididymal tissue in physiological saline (1 mL), cut the epididymis into pieces to obtain sperm, and incubate it in a shaker at 37.6 °C for 5 min, and immediately detect the sperm count with a hemocytometer.

[0258] 2) Motile sperm count detection:

[0259] Use an ophthalmic scissors to cut open the cauda epididymis, mix the contents of the cauda epididymis with 0.9% sodium chloride injection to make a sperm suspension, and take 2 drops of the sperm suspension on a glass slide. The above operations are completed under the condition of keeping warm at 37°C. Observe under a high-power microscope, randomly record the swimming states of 200 sperm, calculate the number of motile sperm and sperm motility, and sperm motility = number of motile sperm / number of sperm.

[0260] 3) Detect the testosterone (T) level by Elisa.

[0261] 4) Detect the levels of androgen synthases (STAR, CYP17A1) and androgen receptor (AR) by Western.

[0262] 5) Pathology: Observe the structure of seminiferous tubules, the morphology and quantity of spermatogenic cells in testicular tissue by HE staining, and observe sperm maturation and spermatocyte grading.

[0263] 5. Result analysis

[0264] (1) Total sperm count, number of motile sperm and sperm motility

[0265] Figure 2 The results showed that compared with the blank group, the total sperm count, number of motile sperm and sperm motility in the model group were significantly decreased. After treatment with SZ-A, the low, medium and high dose groups of SZ-A could significantly increase the sperm count, number of motile sperm and sperm motility, especially the improvement effect on the total sperm count, which was basically restored to normal.

[0266] (2) Testosterone level

[0267] Figure 3 The results showed that compared with the blank group, the testosterone level in testicular tissue of the model group was significantly decreased. After treatment with SZ-A, the low, medium and high dose groups of SZ-A could significantly increase the testosterone level in testicular tissue.

[0268] (3) Levels of STAR, CYP17A1 and AR

[0269] Figures 4 - 6 The results showed that the high dose group of SZ-A significantly increased the expression of STAR, the rate-limiting enzyme for testosterone synthesis, CYP17A1, an important enzyme for testosterone synthesis, and AR, the androgen receptor.

[0270] (4) Structure of seminiferous tubules in testicular tissue

[0271] Figure 7The results showed that the connections between the seminiferous tubules in the testes of mice in the Blank group were tight, and the spermatogenic cells at all levels were arranged neatly, and no obvious abnormalities were observed in the morphology of each cell. In the Model group, the connections between the seminiferous tubules in the testes of mice were loose, with vacuolar degeneration of interstitial cells and infiltration of inflammatory cells; the connections between the spermatogenic cells at all levels in the seminiferous tubules were loose and disordered, the number of layers of spermatogenic cells decreased, and some spermatogenic cells exfoliated. Compared with the Model group, each dose group of SZ-A could significantly improve the structure of the seminiferous tubules in the testes and the state of interstitial cells, reduce the infiltration of inflammatory cells, and increase the number of layers and the number of spermatogenic cells at all levels, with tight connections and neat arrangements.

[0272] (5) Number of spermatogenic cells

[0273] Figure 8 The results showed that compared with the blank group, the number of spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, and all spermatogenic cells in the seminiferous tubules of mice in the Model group were significantly reduced; after treatment with SZ-A, each dose group could significantly increase the number of each spermatogenic cell, and showed a dose-effect relationship. Among them, the high-dose group of SZ-A had an improvement effect on the number of spermatogenic cells close to that of the normal group.

[0274] Experimental Example 3: A mouse model of reproductive disorder induced by HFD diet in C57BL / 6J. Forty-five 6-week-old healthy male C57BL / 6J mice were randomly divided into a normal group (Chow), a high-fat model group (HFD), and a mulberry extract group (SZ-A), with 9 mice in each group. The mulberry extract was the mulberry extract prepared in Preparation Example 9. Among them, the mice in the normal group were fed with a basal diet, and the model group and the SZ-A group were fed with a high-fat diet (Research Diet, D12492, 60 kcal% Fat); after 8 weeks of feeding, each experimental group was treated with the corresponding dose of the test drug for 6 consecutive weeks. During the treatment period, the high-fat diet was continued, and the body weight and food and water intake of the animals were monitored. The model group was given normal saline. After the experiment, samples were taken for efficacy evaluation.

[0275] Table 3 Experimental grouping and drug administration methods

[0276] Type Group Dosage of Administered Drug (Calculated by Alkaloids) Normal Mice Blank 0 Model Model 0 SZ - A SZ - A 400 mg / kg / d

[0277] Detection indexes:

[0278] (1) Total number of sperm, number of motile sperm and sperm motility in mice, and the detection method referred to Experimental Example 2.

[0279] (2) Penis tissue pathology of the mouse model: HE staining was used to observe the cavernous body structure of the penis tissue, and the improvement of the average thickness of the spongy scaffold, connection density, separation degree and the average intercept length of the blood sinus cavity was evaluated.

[0280] Figures 9 - 12The results showed that the total sperm count, the number of motile sperm, and sperm motility in the model group of mice decreased, body weight increased significantly, testicular index decreased, the structure of the corpus cavernosum penis deteriorated, a large number of free-like sponge scaffolds were visible in the central part, the overall number of branches of the sponge scaffold decreased significantly, local blood sinus lacunae merged with each other, and the area increased significantly. Compared with the model group, SZ-A could significantly increase the total sperm count, the number of motile sperm, and sperm motility in mice, reduce body weight, increase testicular index, and the testicular index almost returned to the level of the normal group. The internal sponge-like scaffold of the corpus cavernosum penis showed a reticular distribution, the overall number of branches of the sponge scaffold decreased slightly, the scaffold was thick and firm, and the scaffold gap was small.

[0281] Experimental Example 4: Establishment of a diabetic sexual dysfunction model in Apoe mice induced by STZ combined with high-fat and high-cholesterol

[0282] After 1 week of adaptive feeding, 30 8-week-old Apoe mice were fed a high-fat and high-cholesterol diet (HFHC) for 5 weeks. At the 6th week, STZ (streptozotocin) (100 mg / kg / d) was injected intraperitoneally once to establish a mouse model of glycolipid metabolism disorder. At the same time, 10 8-week-old C57 mice were adaptively fed for 1 week and then fed a normal diet for 5 weeks. At the 6th week, an equal amount of solvent was injected intraperitoneally once as the normal control group.

[0283] After successful modeling, the model group was randomly divided into 3 groups, including the model group, the low (150 mg / kg / d) and high (300 mg / kg / d) dose groups of SZ-A prepared in Preparation Example 8. After administration according to the following table for 6 weeks, the samples were taken for efficacy evaluation.

[0284] Table 4 Animal grouping and administration methods

[0285]

[0286] Detection indexes:

[0287] (1) The detection methods for the total sperm count, the number of motile sperm, and sperm motility in mice refer to Experimental Example 2.

[0288] Pathology:

[0289] (2) The effect of SZ-A on testicular pathology: HE staining was used to observe the structure of seminiferous tubules, the morphology and number of spermatogenic cells in testicular tissue, and to observe sperm maturation and spermatocyte grading.

[0290] (3) HE staining was used to observe the results of the corpus cavernosum structure of penile tissue, and to evaluate the improvement of the average thickness, connection density, separation degree of its sponge-like scaffold and the average intercept length of blood sinus lacunae.

[0291] Result analysis:

[0292] (1) Figure 13The results showed that the total sperm count, the number of motile sperm, and sperm motility in the model group of mice decreased. Compared with the model group, SZ-A could significantly increase the total sperm count, the number of motile sperm, and sperm motility in mice.

[0293] (2) Figure 14 The results showed that the connections between the seminiferous tubules in the testes of mice in the Blank group were tight, and the spermatogenic cells at all levels were arranged neatly, and no obvious abnormalities were seen in the morphology of each cell. In the Model group, the connections between the seminiferous tubules in the testes of mice were loose, with vacuolar degeneration of interstitial cells and infiltration of inflammatory cells; the connections between the spermatogenic cells at all levels in the seminiferous tubules were loose and disordered, the number of layers of spermatogenic cells decreased, and some spermatogenic cells exfoliated. Compared with the Model group, each dose group of SZ-A could significantly improve the structure of the seminiferous tubules in the testes and the state of interstitial cells, reduce the infiltration of inflammatory cells, and increase the number and layers of spermatogenic cells at all levels, with tight connections and neat arrangements.

[0294] Figure 15 The results showed that compared with the Blank group, the number of spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, and total spermatogenic cells in a single seminiferous tubule of mice in the Model group decreased significantly; after treatment with SZ-A, each dose group could significantly increase the number of each spermatogenic cell, and there was a dose-effect relationship. Among them, the high-dose group of SZ-A had an improvement effect on the number of spermatogenic cells close to that of the normal group.

[0295] (3) Results of observing the corpus cavernosum structure of the penile tissue by HE staining The pathological observation results were as Figure 16 shown: The structure of the corpus cavernosum of the penis in the model group deteriorated, a large number of free-like sponge scaffolds could be seen in the central part, the overall number of branches of the sponge scaffold decreased significantly, the local blood sinus lacunae merged with each other, and the area increased significantly. Compared with the model group, the sponge-like scaffolds inside the corpus cavernosum of the penis in the SZ-A group were distributed in a reticular pattern, the overall number of branches of the sponge scaffold decreased slightly, the scaffold was thick, firm, the gap between the scaffolds was small, and the average thickness, connection density, separation degree of the sponge-like scaffold and the average intercept length of the blood sinus lacunae were all improved.

[0296] Experimental Example 5: Effect of SZ-A on PA (palmitic acid)-induced damage to testicular interstitial cells

[0297] 5*10 5 / mL TM3 cells (normal mouse testicular interstitial cells) were inoculated into 6-well plates. After culturing overnight, they were divided into a blank group, a model group, and an SZ-A dose group (the specific grouping is as follows). The model group and the SZ-A administration group were added with 400 μmol / l of PA, and the SZ-A group was given the drug. After incubation for 24 h, the drug efficacy was evaluated.

[0298] Group 1: Control group;

[0299] Group 2: Model group (PA group);

[0300] Group 3: Administration group, the drug is the mulberry twig extract in Preparation Example 1 at 200 μg / mL;

[0301] Group 4: Administration group, the drug is the mulberry twig extract in Preparation Example 2 at 200 μg / mL;

[0302] Group 5: Administration group, the drug is the mulberry twig extract in Preparation Example 3 at 200 μg / mL;

[0303] Group 6: Administration group, the drug is the mulberry twig extract in Preparation Example 4 at 200 μg / mL;

[0304] Group 7: Administration group, the drug is the mulberry twig extract in Preparation Example 5 at 200 μg / mL;

[0305] Group 8: Administration group, the drug is the mulberry twig extract in Preparation Example 7 at 200 μg / mL.

[0306] 1) Western blot was used to detect STAR, the rate-limiting enzyme for testosterone synthesis, CYP17A1, an important enzyme for testosterone synthesis, and 17β-HSD.

[0307] Figure 17 The results showed that after PA induced Leydig cells, the expressions of multiple androgen synthesis-related enzyme systems CYP17A1, STAR, and 17β-HSD in the model group decreased. After intervention with different contents of SZ-A, the expressions of androgen synthesis-related enzymes could be increased.

[0308] Experimental Example 6 Effect of SZ-A on high glucose-induced cavernous endothelial cell injury 1

[0309] RCCEC cells (cavernous endothelial cells) were seeded at 5000 cells / well in a 96-well plate and cultured overnight in serum-free medium. They were divided into a control group, an isosmotic control group, a model group, and an SZ-A dose group (the specific grouping is as follows). The control group used an endothelial cell-specific medium containing 5.55 mM glucose. The isosmotic control group was an endothelial cell-specific medium containing 5.55 mM glucose and supplemented with 24.45 mM mannitol. The model group and the SZ-A administration group were endothelial cell-specific media containing 5.55 mM glucose and supplemented with 24.45 mM glucose (i.e., 30 mM glucose). The SZ-A group was given the drug. After incubation for 72 h, the cell viability was detected using a CCK-8 reagent and the cell survival rate was calculated.

[0310] Group 1: Control group: 5.55 mM glucose

[0311] Group 2: Isosmotic control group: 5.55 mM glucose + 24.45 mM mannitol

[0312] Group 3: Model group: 30 mM glucose

[0313] Group 4: 30 mM glucose + 150 μg / ml SZ-A (calculated as the extract); SZ-A is the extract in Preparation Example 1;

[0314] Group 5: 30 mM glucose + 150 μg / ml SZ-A (calculated as the extract); SZ-A is the extract in Preparation Example 8;

[0315] Group 6: 30 mM glucose + 150 μg / ml SZ-A (calculated as the extract); SZ-A is the extract in Preparation Example 4;

[0316] Group 7: 30 mM glucose + 150 μg / ml SZ-A (calculated as the extract); SZ-A is the mulberry twig extract in Preparation Example 2;

[0317] Figure 18 The results showed that SZ-A could inhibit high glucose-induced cavernous endothelial cell injury and improve cell viability.

[0318] Test Example 7 Effect of SZ-A on High Glucose-Induced Cavernous Endothelial Cell Injury 2

[0319] At a density of 3×10 5 / mL RCCEC cells (cavernous endothelial cells) were seeded in 6-well plates. After culturing overnight without serum, they were divided into a control group, an isosmotic control group, a model group, and SZ-A dose groups (the specific grouping is as follows). The control group was endothelial cell-specific medium containing 5.55 mM glucose. The isosmotic control group was endothelial cell-specific medium containing 5.55 mM glucose and 24.45 mM mannitol. The model group and the SZ-A administration groups were endothelial cell-specific medium containing 5.55 mM glucose and supplemented with 24.45 mM glucose (i.e., 30 mM glucose). The SZ-A groups were given the drug. After incubating for 72 h, the cells were collected and the nitric oxide content in the cells was detected.

[0320] Group 1: Control group: 5.55 mM glucose

[0321] Group 2: Isosmotic control group: 5.55 mM glucose + 24.45 mM mannitol

[0322] Group 3: Model group: 30 mM glucose

[0323] Group 4: 30 mM glucose + 150 μg / ml SZ-A (calculated as the extract); SZ-A is the extract in Preparation Example 3;

[0324] Group 5: 30 mM glucose + 150 μg / ml SZ-A (calculated as the extract); SZ-A is the extract in Preparation Example 1;

[0325] Group 6: 30 mM glucose + 150 μg / ml SZ-A (calculated as the extract); SZ-A is the extract in Preparation Example 4;

[0326] Group 7: 30 mM glucose + 150 μg / ml SZ-A (calculated as the extract); SZ-A is the mulberry twig extract in Preparation Example 2;

[0327] Figure 19 The results showed that SZ-A could inhibit high glucose-induced cavernous endothelial cell injury and improve the intracellular NO content.

[0328] Test Example 8 SZ-A on the endothelial cell injury model induced by high insulin

[0329] Cell culture: Human umbilical vein endothelial cells (HUVEC) were cultured in DMEM medium (10% FBS + 1% double antibody).

[0330] Cell passage: Digested with 0.25% trypsin and inoculated at a density of 10 5 / mL into a 12-well plate and cultured in a cell culture incubator at 37 °C and 5% CO2. The culture medium was changed every 2-3 days, and once the cells occupied about 80% of the field of view, they could be passaged again.

[0331] Model construction: After taking the passaged HUVEC cells and culturing them overnight, they were randomly divided into a normal group, a model group, and an SZ-A group (the mulberry twig extract in Preparation Example 8, calculated as the total alkaloid, with the administration doses of 100 μg / mL, 200 μg / mL, 300 μg / mL, and 400 μg / mL), a total of 6 groups. Among them, the cells in the normal group were given DMEM medium (10% FBS + 1% double antibody), the model group (10% FBS + 1% double antibody + 40 U insulin), and the SZ-A group (10% FBS + 1% double antibody + 40 U insulin + the corresponding dose of SZ-A). After culturing for 24 h, the mitochondrial membrane potential was detected by the Mito-Tracker method.

[0332] Mito-Tracker Deep Red FM (mitochondrial far-infrared fluorescent probe): When the cells were cultured to a certain density in a cell culture plate or dish, the cell culture medium was removed, and the prepared Mito-Tracker Deep Red FM working solution was added. After incubating at 37 °C for 15-30 minutes, the Mito-Tracker Deep Red FM working solution was removed, and the freshly pre-warmed cell culture medium at 37 °C was added. Observation was carried out with a fluorescence microscope. At this time, bright strong fluorescence staining of mitochondria could be observed.

[0333] Results: SZ-A could increase the number of endothelial cell mitochondria and improve mitochondrial damage, suggesting that SZ-A could significantly improve endothelial cell function ( Figure 20)。

[0334] Experimental Example 9: Clinical Evaluation

[0335] 1. Experimental Protocol

[0336] 1.1 Experimental Grouping

[0337] This trial adopted a prospective, single-arm, open, multi-center trial design. Inclusion criteria: (1) Males aged 45 to 70 years old, married or unmarried but with a fixed sexual partner; (2) Subjects diagnosed with type 2 diabetes according to the WHO standard, and glycated hemoglobin ≥ 7%; (3) Clear consciousness and no cognitive abnormalities; (4) Have signed the informed consent form. Exclusion criteria: (1) History of prostate, penile or urethral surgery or trauma; (2) Congenital abnormalities of the reproductive system; (3) Use of drugs that may cause ED within the past 1 month; (4) Severe organ lesions, major somatic diseases such as malignant tumors; (5) History of drug abuse; (6) Mental illness. All have signed the informed consent form. A total of 17 people were enrolled in the study.

[0338] 1.2 Administration Method

[0339] The experimental group was given tablets orally, with the initial dose of 1 tablet each time, 3 times a day; after 4 weeks, it was increased to 2 tablets each time, 3 times a day; and the total continuous treatment was 48 weeks.

[0340] Preparation method of the tablets: Take the mulberry twig extract of Preparation Example 8, add appropriate excipients, mix evenly, add water to make soft material, granulate and dry, add magnesium stearate, mix evenly, and press tablets to obtain the mulberry twig extract preparation, each tablet containing 50 mg of total alkaloids.

[0341] 1.3 Detection and Processing of Experimental Data

[0342] 1. At week 0 (before medication) and week 48 (after medication), the International Index of Erectile Function (IIEF) was used to score the degree of ED in the subjects. IIEF includes 15 questions, all scored using a 6-point scoring method (0 - 5 points), with a total full score of 75 points. The scoring table is as follows:

[0343] Table 5

[0344]

[0345]

[0346] Note: The International Index of Erectile Function (IIEF) was first developed during the clinical research and development of sildenafil to more reliably evaluate the erectile function status and treatment effect of ED patients. Currently, IIEF-15 has become the gold standard for evaluating the efficacy of ED clinical trials and has been gradually applied to the efficacy studies of different ED etiologies, various ED comorbidities, different cultural backgrounds, or different treatment interventions. It contains 15 items and is divided into 5 parts: erectile function (questions 1-5), satisfaction with penetration (questions 6-8), orgasm (questions 9-10), libido (questions 11-12), and overall satisfaction (questions 13-15). Patients answer each item based on their sexual life in the recent 4 weeks. The lower the IIEF-15 score, the more severe the ED (the worse the erectile function).

[0347] 2. Statistical analysis

[0348] SPSS 20.0 software was used for data processing, and the t-test was used for the differences before and after treatment.

[0349] 3. Experimental results

[0350] The results showed that after SZ-A treatment, the IIEF-15 score increased, indicating that SZ-A can improve erectile function. It was found that SZ-A improved all 5 aspects of erectile function, satisfaction with penetration, orgasm, libido, and overall satisfaction ( Figure 21 ). Further analysis found that among the 17 subjects, 11 had an increased score after taking SZ-A treatment, indicating that the treatment rate of SZ-A for ED was approximately 65%.

Claims

1. Use of mulberry extract in the preparation of a medicament for treating and / or preventing male infertility, characterized in that, Based on the total weight percentage of the components of the mulberry extract being 100%, the mulberry extract contains alkaloids with a weight content of more than 3% (optionally containing alkaloids with a weight content of 3 - 99%, further optionally containing alkaloids with a weight content of 15 - 99%, further optionally containing alkaloids with a weight content of 30 - 99%, further optionally containing alkaloids with a weight content of 40 - 99%, further optionally containing alkaloids with a weight content of 50 - 99%, further optionally containing alkaloids with a weight content of 60 - 99%). and / or contains polysaccharides with a weight content not higher than 70% (optionally containing polysaccharides with a weight content of 0.2 - 70%, further optionally containing polysaccharides with a weight content of 0.2 - 50%, further optionally containing polysaccharides with a weight content of 0.2 - 35%, further optionally containing polysaccharides with a weight content of 0.2 - 25%, further optionally containing polysaccharides with a weight content of 0.2 - 23%, further optionally containing polysaccharides with a weight content of 20 - 25%). and / or contains flavonoids with a weight content not higher than 10% (optionally containing flavonoids with a weight content of 0.05 - 5%, further optionally containing flavonoids with a weight content of 0 - 2%, further optionally containing flavonoids with a weight content of 0.05 - 2%, further optionally containing flavonoids with a weight content of 0.5 - 1.5%, further optionally containing flavonoids with a weight content of 0 - 1%, further optionally containing flavonoids with a weight content of 0.05 - 1%). and / or contains amino acids with a weight content not higher than 50% (optionally containing amino acids with a weight content of 0 - 30%, further optionally containing amino acids with a weight content of 0 - 25%, further optionally containing amino acids with a weight content of 0 - 20%, further optionally containing amino acids with a weight content of 0 - 5%, further optionally containing amino acids with a weight content of 3 - 25% or further optionally containing amino acids with a weight content of 5 - 20%). and / or other components (the weight content is optionally 0 - 25%, further optionally 0 - 20%, further optionally 0 - 15%, further optionally 0 - 11%, further optionally 2 - 20%, further optionally 4 - 8%).

2. The use according to claim 1, characterized in that, The male infertility is male sexual dysfunction and / or male reproductive dysfunction.

3. The use according to claim 1 or 2, characterized in that, The male sexual dysfunction and / or male reproductive dysfunction is caused by endocrine disorders and / or metabolic disorders. Preferably, the metabolic disorder is abnormal glucose and lipid metabolism; preferably, the endocrine disorder is abnormal hormone levels. Preferably, the abnormal glucose and lipid metabolism is selected from at least one of diabetes, hyperglycemia, overweight, obesity, and hyperlipidemia.

4. The use according to any one of claims 1 to 3, characterized in that, The male sexual dysfunction is selected from at least one of sexual desire disorder, erectile dysfunction, ejaculation disorder, and anorgasmia; preferably, the male sexual dysfunction is erectile dysfunction and / or ejaculation disorder. The male reproductive dysfunction is selected from at least one of vas deferens obstruction, abnormal sperm quality, abnormal semen, varicocele and inflammation; further preferably, the abnormal sperm quality is selected from at least one of azoospermia, oligospermia, asthenospermia and asthenospermia; preferably, the male reproductive dysfunction is oligospermia, asthenospermia and / or oligoasthenospermia.

5. The use according to any one of claims 1 to 4, characterized in that, The treatment and / or prevention of male infertility is specifically embodied as at least one of the following: 1) Promote sperm production; 2) Improve sperm motility; 3) Improve sperm quality; 4) Improve testicular index; 5) Improve the structure of seminiferous tubules in testicular tissue and the morphology and quantity of spermatogenic cells; 6) Improve the relevant sex hormone levels; preferably, the sex hormone is androgen; further preferably, the androgen is testosterone; 7) Improve the expression level of androgen synthase; 8) Improve the expression level of androgen receptor; 9) Improve penile tissue damage and / or cell damage; preferably, the penile cells are penile cavernosal endothelial cells; preferably, improve the content of NO in penile cavernosal endothelial cells; preferably, the improvement of penile tissue damage includes at least one of improving the average thickness of the cavernous scaffold, connection density, separation degree and the average intercept length of blood sinus cavities; Further preferably, the treatment and / or prevention of male reproductive dysfunction is specifically embodied as at least one of the following: 1) Promote sperm production; 2) Improve sperm motility; 3) Improve sperm quality; 4) Improve testicular index; 5) Improve the structure of seminiferous tubules in testicular tissue and the morphology and quantity of spermatogenic cells; 6) Improve the relevant sex hormone levels; 7) Improve the expression level of androgen synthase; 8) Improve the expression level of androgen receptor; 9) Improve testicular cell damage; preferably, the testicular cells are Leydig cells; Further preferably, the treatment and / or prevention of male sexual dysfunction is specifically embodied as at least one of the following: 1) Improve the relevant sex hormone levels; preferably, the sex hormone is androgen; further preferably, the androgen is testosterone; 2) Improve the expression level of androgen synthase; 3) Improve the expression level of androgen receptor; 4) Improve penile tissue and / or cell damage; preferably, the penile cells are penile cavernosal endothelial cells; preferably, improve the content of NO in penile cavernosal endothelial cells; preferably, the improvement of penile tissue damage includes at least one of improving the average thickness of the cavernous scaffold, connection density, separation degree and the average intercept length of blood sinus cavities; 5) Improve the IIEF-15 score; preferably, the IIEF-15 score includes at least one of erectile function, satisfaction with insertion, orgasm, libido and overall satisfaction.

6. Use of mulberry extract in the preparation of a product for inhibiting apoptosis of Leydig cells induced by high glucose in vitro; Optionally, use of the mulberry extract in the preparation of a product for improving endothelial cell damage induced by high glucose in vitro, Based on the sum of the weight percentages of the components of the mulberry extract being 100%, the mulberry extract contains alkaloids with a weight content of more than 3% (optionally containing alkaloids with a weight content of 3 - 99%, further optionally containing alkaloids with a weight content of 15 - 99%, further optionally containing alkaloids with a weight content of 30 - 99%, further optionally containing alkaloids with a weight content of 40 - 99%, further optionally containing alkaloids with a weight content of 50 - 99%, further optionally containing alkaloids with a weight content of 60 - 99%). and / or contains polysaccharides with a weight content not higher than 70% (optionally containing polysaccharides with a weight content of 0.2 - 70%, further optionally containing polysaccharides with a weight content of 0.2 - 50%, further optionally containing polysaccharides with a weight content of 0.2 - 35%, further optionally containing polysaccharides with a weight content of 0.2 - 25%, further optionally containing polysaccharides with a weight content of 0.2 - 23%, further optionally containing polysaccharides with a weight content of 20 - 25%). and / or contains flavonoids with a weight content not higher than 10% (optionally containing flavonoids with a weight content of 0.05 - 5%, further optionally containing flavonoids with a weight content of 0 - 2%, further optionally containing flavonoids with a weight content of 0.05 - 2%, further optionally containing flavonoids with a weight content of 0.5 - 1.5%, further optionally containing flavonoids with a weight content of 0 - 1%, further optionally containing flavonoids with a weight content of 0.05 - 1%). and / or contains amino acids with a weight content not higher than 50% (optionally containing amino acids with a weight content of 0 - 30%, further optionally containing amino acids with a weight content of 0 - 25%, further optionally containing amino acids with a weight content of 0 - 20%, further optionally containing amino acids with a weight content of 0 - 5%, further optionally containing amino acids with a weight content of 3 - 25% or further optionally containing amino acids with a weight content of 5 - 20%). and / or other components (the weight content is optionally 0 - 25%, further optionally 0 - 20%, further optionally 0 - 15%, further optionally 0 - 11%, further optionally 2 - 20%, further optionally 4 - 8%).

7. The use according to any one of claims 1-6, characterized in that, Based on the sum of the weight percentages of the components of the mulberry extract being 100%, the weight content of each component in the mulberry extract is as follows: Optionally, based on the sum of the weight percentages of the components of the mulberry extract being 100%, the weight content of each component in the mulberry extract is as follows: Optionally, based on the sum of the weight percentages of the components of the mulberry extract being 100%, the weight content of each component in the mulberry extract is as follows: Optionally, based on the sum of the weight percentages of the components of the mulberry extract being 100%, the weight content of each component in the mulberry extract is as follows: Optionally, based on the sum of the weight percentages of the components of the mulberry extract being 100%, the weight content of each component in the mulberry extract is as follows: Optionally, based on the sum of the weight percentages of the components of the mulberry extract being 100%, the weight contents of the components in the mulberry extract are as follows: Optionally, based on the sum of the weight percentages of the components of the mulberry extract being 100%, the weight contents of the components in the mulberry extract are as follows: Further optionally, based on the sum of the weight percentages of the components of the mulberry extract being 100%, the weight contents of the components in the mulberry extract are as follows: Further optionally, based on the sum of the weight percentages of the components of the mulberry extract being 100%, the weight contents of the components in the mulberry extract are as follows: Further preferably, the alkaloids include one or more of 1-deoxynojirimycin (1-deoxynojirimycin or DNJ), N-methyl-1-deoxynojirimycin, fagomine (fagomine or FAG), 3-epi-fagomine, 1,4-dideoxy-1,4-imino-D-arabinitol (1,4-dideoxy-1,4-imino-D-arabinitol or DAB), calystegin B2, calystegin C1, 2-O-(α-D-galactopyranosyl)-1-deoxynojirimycin, 6-O-(β-D-glucopyranosyl)-1-deoxynojirimycin, 1,4-dideoxy-1,4-imino-(2-O-β-D-glucopyranosyl)-D-arabinitol; Optionally, the weight percentage of DNJ is not less than 50% of the total alkaloids (optionally 60-99%).

8. The use according to any one of claims 1-7, characterized in that The preparation method of the mulberry extract comprises the following steps: 1) Prepare a crude extract of a Moraceae plant; 2) Separate the crude extract through a cation resin and / or an optional anion resin to obtain the mulberry extract; Preferably, the method further comprises the following steps: 3) Perform alcohol precipitation on the resin effluent of step 2), and collect the supernatant; 4) Perform concentration and / or drying treatment on the supernatant; Optionally, it further comprises the following step: perform concentration and / or drying treatment on the resin effluent of step 2).

9. The use according to any one of claims 1-8, characterized in that, The mulberry extract acts on humans or mammals.

10. The use according to any one of claims 1-9, characterized in that, The drug is in an oral dosage form; optionally, the drug is tablets, capsules, oral solutions, oral emulsions, pills, granules, syrups, and powders.

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