Pharmaceutical composition composed of cistanche deserticola, prepared rehmannia root, schisandra chinensis and semen cuscutae, preparation method of pharmaceutical composition and application of pharmaceutical composition in prevention and treatment of oligospermia and asthenospermia

Abstract

The invention discloses a pharmaceutical composition consisting of cistanche, prepared rehmannia root, schisandra chinensis and semen cuscutae, wherein the mass ratio of the cistanche to the prepared rehmannia root to the schisandra chinensis to the semen cuscutae is (2-6): (1-5): (1-4): (0.1-2). In in-vivo experiments of SD rats with oligoasthenospermia induced by tripterygium glycosides, the pharmaceutical composition disclosed by the invention can be used for remarkably increasing sperm quantity, sperm motility and sperm motility rate, reducing sperm malformation rate, partially reversing pathological changes of testis and epididymis tissues, improving ultrastructural damage of spermatogonium cells, repairing spermatogonium stem cell (SSCs) pools, and improving the curative effect of the spermatogonium cells. The levels of various sex hormones are callback to be normal. The pharmaceutical composition of the present invention has been demonstrated to promote spermatogenesis by acting on the source of spermatogenesis-SSCs and on the start of meiosis-spermatoblasts (GC-2).

Description

Technical Field

[0001] The invention relates to the technical field of medicine, and in particular to a pharmaceutical composition consisting of Cistanche deserticola, Rehmannia glutinosa, Schisandra chinensis and Cuscuta australis, a preparation method thereof, and use thereof in preventing and treating oligoasthenospermia. Background Art

[0002] The clinical manifestation of oligospermia is a decrease in sperm density, and the diagnostic standard is a sperm concentration analysis of less than 15×10 6 / mL; Asthenospermia is characterized by decreased sperm motility, and the diagnostic criteria are that the proportion of fast, straight-line forward-moving sperm is less than 25% or the proportion of forward-moving sperm is less than 50%; the two often appear together clinically, namely oligoasthenospermia, which is the main cause of difficulty in conceiving or infertility for couples.

[0003] The pharmaceutical composition composed of Cistanche deserticola, Rehmannia glutinosa, Schisandra chinensis and Cuscuta australis, the preparation method thereof and the use thereof for preventing and treating oligoasthenospermia are not known in the prior art. Summary of the invention

[0004] Based on this, the present invention provides a pharmaceutical composition consisting of Cistanche deserticola, Rehmannia glutinosa, Schisandra chinensis and Cuscuta chinensis, wherein the mass ratio of the Cistanche deserticola, Rehmannia glutinosa, Schisandra chinensis and Cuscuta chinensis is (2-6): (1-5): (1-4): (0.1-2).

[0005] Furthermore, the mass ratio of the Cistanche deserticola, the Rehmannia root, the Schisandra chinensis and the Cuscuta chinensis is (3-5): (2-4): (1-3): (0.5-2).

[0006] Furthermore, the mass ratio of the Cistanche deserticola, the Rehmannia root, the Schisandra chinensis and the Cuscuta chinensis is (3.5-4.5): (2.5-3.5): (1.5-2.5): (0.8-1.5).

[0007] Furthermore, the mass ratio of the Cistanche deserticola, the Rehmannia root, the Schisandra chinensis and the Cuscuta chinensis is about 4: about 3: about 2: about 1.

[0008] Further, the mass ratio of the Cistanche deserticola, the Rehmannia glutinosa, the Schisandra chinensis and the Cuscuta chinensis is not about 2.958: about 4.226: about 1.268: about 1.55.

[0009] According to another aspect of the present invention, there is provided a method for preparing the above-mentioned pharmaceutical composition, the method comprising the following steps:

[0010] (1) Weigh appropriate amounts of Cistanche deserticola, Rehmannia glutinosa, Schisandra chinensis, and Cuscuta australis, add the first water for soaking, boil over high heat until boiling, boil over low heat for a period of time, filter, and obtain a first filtrate and a filter residue;

[0011] (2) adding a second amount of water to the filter residue, boiling over high heat, boiling over low heat for a period of time, filtering, and obtaining a second filtrate; and

[0012] (3) combining the first filtrate and the second filtrate, drying and concentrating to obtain the pharmaceutical composition.

[0013] Furthermore, the mass ratio of the Cistanche deserticola, the Rehmannia root, the Schisandra chinensis and the Cuscuta chinensis is about 4: about 3: about 2: about 1.

[0014] Furthermore, the water is deionized water.

[0015] Furthermore, the ratio of the sum of the masses of the Cistanche deserticola, Rehmannia glutinosa, Schisandra chinensis and Cuscuta australis to the mass / volume (g / ml) of the first water is 0.01-1, for example, about 0.1.

[0016] Furthermore, the ratio of the sum of the masses of the Cistanche deserticola, Rehmannia glutinosa, Schisandra chinensis and Cuscuta australis to the mass / volume (g / ml) of the second water is 0.01-1, for example, about 0.1.

[0017] Furthermore, the soaking time is 20 to 40 minutes, for example, about 30 minutes.

[0018] Furthermore, the slow-cooking time is 0.5 to 2 hours, for example, about 1 hour.

[0019] Furthermore, the drying is vacuum drying.

[0020] Furthermore, the mass of the Cistanche deserticola is in the range of 1 to 3 g, for example, about 2 g.

[0021] Furthermore, the mass range of the Rehmannia glutinosa is 1 to 3 g, for example, about 1.5 g.

[0022] Furthermore, the mass of the Schisandra chinensis is in the range of 0.5 to 2 g, for example, about 1 g.

[0023] Furthermore, the mass of the dodder seeds ranges from 0.1 to 1 g, for example, about 0.5 g.

[0024] According to another aspect of the present invention, there is provided a pharmaceutical preparation comprising the above pharmaceutical composition and one or more optional pharmaceutically acceptable excipients.

[0025] Furthermore, the auxiliary material is selected from one or more of the following: diluent, wetting agent, binder, disintegrant, inclusion agent, flavoring agent, sustained-release agent, glidant, lubricant, dispersant, plasticizer, opacifier and antioxidant.

[0026] Furthermore, the dosage form of the pharmaceutical preparation is powder, tablet, pill, capsule, film, lozenge, granule, injection or oral solution.

[0027] Furthermore, the pharmaceutical preparation further comprises one or more other drugs for preventing and / or treating oligospermia and / or asthenospermia.

[0028] Furthermore, the drug is selected from one or more of the following: antioxidants, anti-infective drugs, gonadotropin therapeutic drugs, pancreatic kallikreinase and pentoxifylline.

[0029] Furthermore, the antioxidant is selected from one or more of the following: N-acetylcysteine, vitamin E, vitamin C, coenzyme Q10 and L-carnitine.

[0030] Furthermore, the gonadotropin therapeutic drug includes recombinant follicle-stimulating hormone and urinary gonadotropin.

[0031] According to another aspect of the present invention, there is provided a use of the above-mentioned pharmaceutical composition or the above-mentioned pharmaceutical preparation in the preparation of a medicine for preventing and / or treating oligospermia and / or asthenospermia.

[0032] Furthermore, the pharmaceutical composition or the pharmaceutical preparation prevents and / or treats oligospermia and / or asthenospermia in one or more of the following ways: improving spermatocyte motility, improving sperm density, improving sperm motility, increasing sperm survival rate, reducing sperm deformity rate, improving organ structural abnormalities, improving spermatogonia ultrastructure abnormalities, repairing spermatogonial stem cell pools, and improving abnormal serum biochemical indicators.

[0033] Furthermore, the improvement of sperm motility is to increase sperm dynamics parameters.

[0034] Furthermore, the sperm dynamics parameter is selected from one or more of the following: sperm curve velocity, sperm linear velocity, sperm path velocity, sperm movement angle, sperm lateral swing amplitude and sperm whipping frequency.

[0035] Furthermore, the organ structural abnormality includes testicular structural abnormality and / or epididymal structural abnormality.

[0036] Furthermore, the testicular structural abnormality includes one or more of the following: a decrease in the number of epithelial cells in the seminiferous tubules, a disordered arrangement of epithelial cells in the seminiferous tubules, a decrease in the thickness of the seminiferous epithelium, and a decrease in the number of sperm in the lumen of the seminiferous tubules.

[0037] Furthermore, the epididymal structural abnormality includes enlarged epididymal duct space and / or decreased number of sperm in the epididymal duct lumen.

[0038] Furthermore, the spermatogonia ultrastructural abnormality is spermatogonia chromatin condensation.

[0039] Furthermore, the spermatogonial stem cell pool is repaired to increase the number of spermatogonial stem cells.

[0040] Furthermore, the abnormal biochemical indexes include one or more of the following: decreased testosterone level, decreased gonadotropin-releasing hormone level, increased follicle-stimulating hormone level and increased luteinizing hormone level.

[0041] Beneficial effects of the present invention:

[0042] In the in vivo experiment of oligoasthenospermia SD rats induced by Tripterygium wilfordii polyglycosides of the present invention, the pharmaceutical composition of the present invention can significantly increase the number of sperm, sperm motility and sperm motility, reduce the sperm deformity rate, partially reverse the pathological changes of testicular and epididymal tissues, improve the ultrastructural damage of spermatogonia, repair the spermatogonial stem cell (SSCs) pool, and adjust the levels of various sex hormones to normal. The pharmaceutical composition of the present invention has been confirmed to promote spermatogenesis by acting on the source of spermatogenesis - SSCs and the initiation of meiosis - GC-2. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0044] Figure 1 It is a schematic diagram of the effects of the pharmaceutical composition of the present invention in various compatibility ratios on the viability of GC-2 cells.

[0045] Figure 2 The figure is a schematic diagram showing the effect of the No. 5 pharmaceutical composition of the present invention on the sperm concentration, total motility, deformity rate and viability of oligoasthenospermia rats.

[0046] Figure 3 This is a schematic diagram of the effect of the No. 5 pharmaceutical composition of the present invention on the sperm motility of oligoasthenospermia rats.

[0047] Figure 4 This is a schematic diagram of the effect of the No. 5 pharmaceutical composition of the present invention on the sperm deformity rate of oligoasthenospermia rats.

[0048] Figure 5 This is a schematic diagram of the effect of the No. 5 pharmaceutical composition of the present invention on the pathological changes in testicular tissue of oligoasthenospermia rats.

[0049] Figure 6 It is a schematic diagram of the effect of the No. 5 pharmaceutical composition of the present invention on the epithelial thickness of the seminiferous tubules in the testis of oligoasthenospermia rats.

[0050] Figure 7This is a schematic diagram of the effects of the No. 5 pharmaceutical composition of the present invention on the pathological changes in the epididymal tissue of oligoasthenospermia rats.

[0051] Figure 8 The figure is a schematic diagram showing the effect of the No. 5 pharmaceutical composition of the present invention on the ultrastructure of testicular spermatogonia in oligoasthenospermia rats.

[0052] Fig. 9 The figure is a schematic diagram showing the effect of the No. 5 pharmaceutical composition of the present invention on the expression of testicular spermatogonial stem cell markers PLZF and OCT-4 in oligoasthenospermia rats.

[0053] Fig.10 This is a schematic diagram of the effect of the No. 5 pharmaceutical composition of the present invention on the proportion of testicular spermatogonial stem cells in the seminiferous epithelium of oligoasthenospermia rats.

[0054] Fig.11 This is a schematic diagram of the effects of the No. 5 pharmaceutical composition of the present invention on serum T, LH, FSH and GnRH in oligoasthenospermia rats. DETAILED DESCRIPTION

[0055] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0056] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the field of the invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0057] The present invention is intended to encompass all alternatives, variations and equivalents that may be included in the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0058] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0059] In the present invention, the term "comprising" is synonymous with "including". As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus comprising the listed elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0060] As described in the background technology section, the pharmaceutical composition composed of Cistanche deserticola, Rehmannia glutinosa, Schisandra chinensis and Cuscuta chinensis and its preparation method and use for preventing and treating oligoasthenospermia of the present invention are not found in the prior art. In order to solve the above problems, the present invention provides a pharmaceutical composition composed of Cistanche deserticola, Rehmannia glutinosa, Schisandra chinensis and Cuscuta chinensis, wherein the mass ratio of Cistanche deserticola, Rehmannia glutinosa, Schisandra chinensis and Cuscuta chinensis is (2-6): (1-5): (1-4): (0.1-2).

[0061] The Cistanche in the present invention nourishes kidney yang and blood, while Rehmannia root nourishes blood and yin, nourishes essence and fills marrow. Schisandra chinensis is astringent, replenishes qi and produces body fluid, nourishes kidney and calms the mind. Cuscuta chinensis nourishes liver and kidney, consolidates essence and reduces urine.

[0062] In the present invention, when mass ratio, ratio, mass, time or other value or parameter is expressed as a range, preferred range, or a series of upper preferred values ​​and lower preferred values, it should be understood that all ranges formed by any pairing of any range upper limit or preferred value and any range lower limit or preferred value are specifically disclosed, regardless of whether the range is disclosed separately. For example, when the range "2 to 6" is disclosed, the described range should be interpreted as including the range "2 to 6", "2 to 5", "2 to 4", "2 to 3", "3 to 6", "3 to 5", "3 to 4", "4 to 6", "4 to 5", "5 to 6" and the like. When a numerical range is described in this article, unless otherwise stated, the range is intended to include its end value and all integers and fractions within the range.

[0063] In a preferred embodiment, the mass ratio of the Cistanche deserticola, the Rehmannia glutinosa, the Schisandra chinensis and the Cuscuta australis is (3-5): (2-4): (1-3): (0.5-2).

[0064] In a preferred embodiment, the mass ratio of the Cistanche deserticola, the Rehmannia glutinosa, the Schisandra chinensis and the Cuscuta chinensis is (3.5-4.5): (2.5-3.5): (1.5-2.5): (0.8-1.5).

[0065] In a preferred embodiment, the mass ratio of the Cistanche deserticola, the Rehmannia glutinosa, the Schisandra chinensis and the Cuscuta chinensis is about 4: about 3: about 2: about 1.

[0066] The present invention uses modern research methods to screen the best formula of the pharmaceutical composition of the present invention and verify its efficacy on male oligoasthenospermia. 6 (6 4 ) The optimal compatibility screening experiment of 6 ratios obtained by uniform design showed that No.5 (Cistanche: Rehmannia root: Schisandra chinensis: Cuscuta australis = 4:3:2:1) was the best compatibility ratio.

[0067] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 4" includes ±5% of 4, or from 3.8 to 4.2; "about 3" includes ±5% of 3, or from 2.85 to 3.15; "about 2" includes ±5% of 2, or from 1.9 to 2.1; "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0068] In a preferred embodiment, the mass ratio of the Cistanche deserticola, the Rehmannia glutinosa, the Schisandra chinensis and the Cuscuta chinensis is not about 2.958: about 4.226: about 1.268: about 1.55.

[0069] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2.958" includes ±5% of 2.958, or from 2.8101 to 3.1059; "about 4.226" includes ±5% of 4.226, or from 4.0147 to 4.4373; "about 1.268" includes ±5% of 1.268, or from 1.2046 to 1.3314; "about 1.55" includes ±5% of 1.55, or from 1.4725 to 1.6275.

[0070] According to another aspect of the present invention, there is provided a method for preparing the above-mentioned pharmaceutical composition, the method comprising the following steps:

[0071] (1) Weigh appropriate amounts of Cistanche deserticola, Rehmannia glutinosa, Schisandra chinensis, and Cuscuta australis, add the first water for soaking, boil over high heat until boiling, boil over low heat for a period of time, filter, and obtain a first filtrate and a filter residue;

[0072] (2) adding a second amount of water to the filter residue, boiling over high heat, boiling over low heat for a period of time, filtering, and obtaining a second filtrate; and

[0073] (3) combining the first filtrate and the second filtrate, drying and concentrating to obtain the pharmaceutical composition.

[0074] In a preferred embodiment, the mass ratio of the Cistanche deserticola, the Rehmannia glutinosa, the Schisandra chinensis and the Cuscuta chinensis is about 4: about 3: about 2: about 1.

[0075] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 4" includes ±5% of 4, or from 3.8 to 4.2; "about 3" includes ±5% of 3, or from 2.85 to 3.15; "about 2" includes ±5% of 2, or from 1.9 to 2.1; "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0076] In a preferred embodiment, the water is deionized water.

[0077] In a preferred embodiment, the ratio of the sum of the masses of the Cistanche deserticola, Rehmannia glutinosa, Schisandra chinensis and Cuscuta australis to the mass / volume (g / ml) of the first water is 0.01-1, such as about 0.1.

[0078] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 0.1" includes ±5% of 0.1, or from 0.095 to 0.105.

[0079] In a preferred embodiment, the ratio of the sum of the masses of the Cistanche deserticola, Rehmannia glutinosa, Schisandra chinensis and Cuscuta australis to the mass / volume (g / ml) of the second water is 0.01-1, such as about 0.1.

[0080] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 0.1" includes ±5% of 0.1, or from 0.095 to 0.105.

[0081] In a preferred embodiment, the soaking time is 20 to 40 minutes, such as about 30 minutes.

[0082] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes ±5% of 30, or from 28.5 to 31.5.

[0083] In a preferred embodiment, the slow-cooking time is 0.5 to 2 hours, such as about 1 hour.

[0084] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0085] In a preferred embodiment, the drying is vacuum drying.

[0086] In a preferred embodiment, the mass of the Cistanche deserticola is in the range of 1 to 3 g, such as about 2 g.

[0087] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2" includes ±5% of 2, or from 1.9 to 2.1.

[0088] In a preferred embodiment, the mass of the Rehmannia glutinosa is in the range of 1 to 3 g, such as about 1.5 g.

[0089] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1.5" includes ±5% of 1.5, or from 1.425 to 1.575.

[0090] In a preferred embodiment, the mass of Schisandra chinensis is in the range of 0.5 to 2 g, such as about 1 g.

[0091] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0092] In a preferred embodiment, the mass of the dodder seeds ranges from 0.1 to 1 g, such as about 0.5 g.

[0093] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.5" includes ±5% of 0.5, or from 0.475 to 0.525.

[0094] According to another aspect of the present invention, there is provided a pharmaceutical preparation comprising the above pharmaceutical composition and one or more optional pharmaceutically acceptable excipients.

[0095] In a preferred embodiment, the auxiliary material is selected from one or more of the following: diluents, wetting agents, binders, disintegrants, inclusion agents, flavoring agents, sustained-release agents, glidants, lubricants, dispersants, plasticizers, opacifiers and antioxidants.

[0096] In a preferred embodiment, the dosage form of the pharmaceutical preparation is powder, tablet, pill, capsule, film, lozenge, granule, injection or oral solution.

[0097] In a preferred embodiment, the pharmaceutical preparation further comprises one or more other drugs for preventing and / or treating oligospermia and / or asthenospermia.

[0098] In a preferred embodiment, the drug is selected from one or more of the following: antioxidants, anti-infective drugs, gonadotropin therapeutic drugs, kallikrein and pentoxifylline.

[0099] In a preferred embodiment, the antioxidant is selected from one or more of the following: N-acetylcysteine, vitamin E, vitamin C, coenzyme Q10 and L-carnitine.

[0100] In a preferred embodiment, the gonadotropin therapeutic drug includes recombinant follicle stimulating hormone and urinary gonadotropin.

[0101] According to another aspect of the present invention, there is provided a use of the above-mentioned pharmaceutical composition or the above-mentioned pharmaceutical preparation in the preparation of a medicine for preventing and / or treating oligospermia and / or asthenospermia.

[0102] In a preferred embodiment, the pharmaceutical composition or the pharmaceutical preparation prevents and / or treats oligospermia and / or asthenospermia in one or more of the following ways: improving spermatocyte motility, improving sperm density, improving sperm motility, increasing sperm survival rate, reducing sperm deformity rate, improving organ structural abnormalities, improving spermatogonia ultrastructure abnormalities, repairing spermatogonial stem cell pools, and improving abnormal serum biochemical indicators.

[0103] In a preferred embodiment, the improvement of sperm motility is to increase sperm kinetic parameters.

[0104] In a preferred embodiment, the sperm dynamics parameter is selected from one or more of the following: sperm curve velocity, sperm linear velocity, sperm path velocity, sperm movement angle, sperm lateral swing amplitude and sperm whipping frequency.

[0105] In a preferred embodiment, the organ structural abnormality includes testicular structural abnormality and / or epididymal structural abnormality.

[0106] In a preferred embodiment, the testicular structural abnormality includes one or more of the following: a decrease in the number of epithelial cells in the seminiferous tubules, a disordered arrangement of epithelial cells in the seminiferous tubules, a decrease in the thickness of the seminiferous epithelium, and a decrease in the number of sperm in the lumen of the seminiferous tubules.

[0107] In a preferred embodiment, the epididymal structural abnormality includes an enlarged epididymal duct space and / or a decreased number of sperm in the epididymal duct lumen.

[0108] In a preferred embodiment, the spermatogonia ultrastructural abnormality is spermatogonia chromatin condensation.

[0109] In a preferred embodiment, the repairing of the spermatogonial stem cell pool is to increase the number of spermatogonial stem cells.

[0110] In a preferred embodiment, the abnormal biochemical indexes include one or more of the following: decreased testosterone level, decreased gonadotropin-releasing hormone level, increased follicle-stimulating hormone level and increased luteinizing hormone level.

[0111] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions or conditions recommended by the manufacturer.

[0112] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0113] The above features mentioned in the present invention or the features mentioned in the embodiments can be combined in any way. All the features disclosed in this patent specification can be used in combination with any combination form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0114] Embodiment 1

[0115] 1. Experimental materials and instruments

[0116] 1.1 Experimental cells

[0117] GC-2 (P5) was purchased from the Cell Bank of Type Culture Collection Committee of the Chinese Academy of Sciences (SCSP-5055).

[0118] 1.2. Drugs and Reagents

[0119] Cistanche deserticola YCMa. (Beijing Bencao Fangyuan Pharmaceutical Group Co., Ltd., 22021102); Rehmannia glutinosa Libosch., Schisandra chinensis (Turcz.) Baill. and Cuscuta chinensis Lam. (Beijing Tairenkang Pharmaceutical Development Co., Ltd., 23052801, 23032601 and 230251); Cell proliferation and activity detection (CCK-8) kit (Punose, P-CA-001).

[0120] 1.3 Experimental instruments

[0121] Electronic balance (Sartorius Scientific Instruments Co., Ltd., BS224S); Nucleic acid protein analyzer (EppendorfAG, 6133000044); Applied Biosystems VeritiPro PCR instrument (Thermo Fisher Scientific, A48141); QuantStudio TM3Real-Time PCR System (Thermo Fisher Scientific, A28137); desktop high-speed refrigerated centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd., TGL-20M); inverted microscope (Shanghai Princeton Biotechnology Development Co., Ltd., IX71-A21PH); capped multi-manifold freeze dryer (Zhengzhou Huachen Instrument Co., Ltd., ZLGJ-18); fully automatic microplate reader (Thermo, MK3).

[0122] 2. Experimental methods

[0123] 2.1 Drug preparation

[0124] Cistanche deserticola, Rehmannia glutinosa, Schisandra chinensis and Cuscuta chinensis were all identified by Zhu Jingjing, a researcher at the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences. After mixing the medicinal materials, add ten times the volume of deionized water and soak for 30 minutes, then boil at high temperature until boiling, keep it slightly boiling for 1 hour and filter it, and filter it while hot. Repeat the extraction once, and combine the two filtrates. The filtrate is vacuum dried and concentrated to obtain anhydrous powder, i.e., the pharmaceutical composition of the present invention, which is weighed and the extraction rate is calculated, and refrigerated for use.

[0125] 2.2 Four-factor six-level uniform design experiment

[0126] In this study, U 6 (6 4 ) table (as shown in Table 1), with the four components of Cistanche deserticola, Rehmannia glutinosa, Schisandra chinensis and Cuscuta chinensis as the investigation factors, quadratic polynomial stepwise regression analysis was used. Considering the interaction between the components, 6 levels were set for each factor, that is, 6 different doses with increasing gradients (the daily dosage ranges of the four drugs were confirmed according to the 2020 edition of the Pharmacopoeia standard dosage combined with clinical practice) (as shown in Table 2), and finally 6 different compatibility combinations were obtained.

[0127] Table 1 Uniform design table U6(6 4 )

[0128]

[0129] Table 2 Comparison table of each factor level

[0130]

[0131] 2.3. GC-2 cell administration and grouping

[0132] This experiment involves a total of 6 compatible drugs, and each drug concentration is set at 0μg / mL and 1μg / mL, and each concentration is set at 4 replicates. The cells were plated in a 96-well plate at 8000 cells / well (except the edge wells). After the cells adhered to the wall for 24 hours, the culture medium was discarded, 100μL of each drug-containing culture medium was added for 24 hours, and 100μL of complete culture medium was added to the edge wells. Subsequently, the culture medium was discarded, 100μL of CCK-8 working solution was added in the dark, and the OD value was measured at 450nm using an enzyme reader after incubation for 2 to 3 hours, and the relative cell viability (OD value of each group / average OD value of the control group) was calculated.

[0133] 2.4 Statistical Analysis

[0134] GraphPad Prism 8.0 software was used for statistical analysis, and the experimental data were The two-sided Student T-test was used for pairwise comparison between the groups, and P < 0.05 indicated statistically significant differences.

[0135] 3. Experimental results

[0136] 3.1. Uniform design results

[0137] According to the uniform design principle, the following formula configuration table was obtained in the DPS7.05 statistical software (as shown in Table 3). The compatibility ratios of Cistanche deserticola, Rehmannia glutinosa, Schisandra chinensis and Cuscuta australis in the 6 experimental drugs were No.1 (12:12:15:10), No.2 (15:36:18:8), No.3 (6:2:1:3), No.4 (21:30:9:11), No.5 (4:3:2:1), and No.6 (9:24:6:7).

[0138] Table 3 Sample configuration table for formulation test

[0139]

[0140] 3.2. Effect of the pharmaceutical composition of the present invention on GC-2 cell viability

[0141] like Figure 1 As shown, compared with the control group, the pharmaceutical composition of the present invention with No.5 ratio showed a significant promoting effect on the viability of GC-2 cells (P < 0.05), and there was no significant difference in cell viability between the other ratio groups and the control group (P > 0.05). Therefore, this experiment selected the No.5 ratio as the optimal compatibility of the pharmaceutical composition of the present invention and used it in subsequent experiments.

[0142] Example 2 Pharmacodynamics experiment of No.5 pharmaceutical composition of the present invention

[0143] 1. Experimental materials and instruments

[0144] 1.1 Experimental Animals

[0145] Sixty SPF-grade 6-week-old male SD rats, weighing (160±10) g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., animal license number SCXK (Beijing) 2019-0008. The animals were kept in the Experimental Animal Center of the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, with constant temperature and humidity, natural circadian rhythm lighting, free movement, drinking water, and eating. The experiment began after 7 days of adaptive feeding. The animal experiment process passed the ethical review of experimental animal welfare of the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences (ethical approval number: 2023B270).

[0146] 1.2. Drugs and Reagents

[0147] Tripterygium wilfordii polyglycosides tablets (Zhejiang Deende Pharmaceutical Co., Ltd., batch number 171118); Clomiphene citrate tablets (Gate Pharmaceutical Co., Ltd., batch number R1210); Hematoxylin and eosin (HE) staining kit (Biyuntian, batch number C0105S); glutaraldehyde fixative (Shanghai Yuanye Biotechnology Co., Ltd., batch number R41014); Calcein / PI cell viability and cytotoxicity detection kit (Biyuntian, batch number C2015S); PLZF antibody (GeneTex, USA, batch number GTX111046); OCT-4 antibody (Cell Signaling Technology, USA, batch number 75463); goat anti-rabbit / mouse secondary antibody (Earthox, USA, batch numbers E030120, E030110); DAPI anti-fluorescence quenching mounting medium (Biyuntian Biotechnology, P0131); T, LH, FSH and GnRH ELISA kit (Shanghai ELISA Biotechnology Co., Ltd., batch numbers ml103518, ml104147, ml059034, ml003038).

[0148] 1.3 Experimental instruments

[0149] Inverted fluorescence microscope (Shenzhen Sinico Optical Instrument Co., Ltd., batch number XK-SW40FLT); BSA224S balance (Sartorius Scientific Instrument Co., Ltd.); BX60 upright microscope (Olympus, Japan); FV1000 (40X-100X) laser confocal microscope (Olympus, Japan); Varioskan LUX multifunctional microplate reader (Thermo, USA).

[0150] 2. Experimental methods

[0151] 2.1 Grouping and Dosing

[0152] 60 rats were randomly divided into 6 groups: blank group (Control), model group (Model), clomiphene group (CC), low dose group (CRW-L) of the pharmaceutical composition No.5 of the present invention, medium dose group (CRW-M) of the pharmaceutical composition No.5 of the present invention, and high dose group (CRW-H) of the pharmaceutical composition No.5 of the present invention, with 10 rats in each group. Tripterygium wilfordii polyglycosides, clomiphene tablets and the aqueous extract of the pharmaceutical composition No.5 of the present invention were all prepared with normal saline. Except for the control group, all other groups were gavaged with 20 mg / kg of Tripterygium wilfordii polyglycosides (the clinical dosage for humans is 1 mg / kg) every morning to establish a model of oligoasthenozoospermia caused by deficiency of kidney essence. The control group was gavaged with the same amount of normal saline in the morning and evening, and the model group was gavaged with the same amount of normal saline in the evening; the low, medium and high dose groups of the pharmaceutical composition No.5 of the present invention were gavaged with 1.215, 2.43 and 4.86 g·kg in the evening, respectively. -1 The aqueous extract of the No. 5 pharmaceutical composition of the present invention (daily dosage of adult pills is 27 g / d); the clomiphene group was gavaged 4.5 mg·kg every evening -1 ·d -1 Clomiphene tablet suspension (clinical dosage for adults: 50 mg / time / day). Each group of rats was gavaged at 1 ml / 100 g for 30 days. (The dosage was determined based on the body surface area of ​​humans and rats)

[0153] 2.2. Detection of sperm concentration and sperm dynamic parameters

[0154] Freshly separated rat epididymal tail was taken, placed in HBSS (preheated at 37°C), cut into pieces, blown appropriately, incubated in a 37°C water bath for 5 min, 10 μl of sperm suspension was dropped on a glass slide, and computer assisted sperm analysis (CASA) technology was used to detect and record sperm concentration, total sperm motility, curve speed, average path speed, linearity, forwardness, swinging and whipping frequency.

[0155] 2.3. Sperm motility test

[0156] According to the instructions of Calcein / PI Cell Viability and Cytotoxicity Assay Kit, prepare the assay working solution, and take a sample containing approximately 1×10 6 An appropriate amount of fresh sperm suspension per sperm was mixed with an appropriate amount of detection working solution, incubated at 37°C in the dark for 30 minutes, and placed under a fluorescence microscope to observe and record the fluorescence expression at the corresponding wavelength. Only green fluorescence or both green and red fluorescence indicate that the sperm is alive, and only red fluorescence indicates that the sperm is dead. The percentage of sperm showing green fluorescence was calculated.

[0157] 2.4. Sperm deformity rate detection

[0158] Pipette an appropriate amount of sperm suspension onto the slide and spread evenly, then let it stand and dry. Then immerse and fix it in methanol for 10 minutes, let it stand and dry, then stain it with eosin for 2 minutes, rinse it with running water, let it stand and dry, permeabilize it with xylene, let it dry, drop the sealing agent, and cover the slide with a coverslip. Count the number of abnormal sperm in every 200 sperm in different fields of view under a microscope.

[0159] 2.5 Analysis of pathological damage of rat testis and epididymis

[0160] The testicles were fixed with tissue fixative for 48 h, embedded in paraffin, sectioned, dewaxed and hydrated, and stained with HE. The pathological changes of testicular tissue in each group were observed under an optical microscope. The thickness ratio of the seminiferous epithelium of 30 seminiferous tubules in each testis was randomly measured, and the average value was calculated and statistically analyzed.

[0161] 2.6 Ultrastructural analysis of testicular spermatogonia

[0162] Fresh tissues were fixed in 2.5% glutaraldehyde at 4°C for 6 h, then fixed in 1% osmium acid for 2 h, dehydrated in acetone gradient, infiltrated, embedded, polymerized, sectioned, stained, and observed under a microscope.

[0163] 2.7 Immunofluorescence detection of changes in the number of spermatogonial stem cells

[0164] The paraffin sections of testicular tissue were dewaxed, hydrated, antigen retrieved, blocked with 10% goat serum, permeabilized with 0.5% Triton-100, incubated with PLZF (1:200) and OCT-4 (1:250) antibodies at 4°C overnight, incubated with the corresponding secondary antibodies (all 1:400) at room temperature for 2 h, and mounted with anti-fluorescence quenching mounting medium containing DAPI, and observed and images were collected under a fluorescence microscope.

[0165] 2.8. ELISA kits to detect serum T, LH, FSH, and GnRH levels

[0166] The serum was taken out from the -80°C refrigerator, thawed on ice, and the hormone level was detected according to the instructions of the ELISA kit.

[0167] 2.9 Statistical Analysis

[0168] GraphPad Prism 8.0 software was used for statistical analysis, and the experimental data were ANOVA analysis of variance was used for comparison of means among multiple groups, and two-sided Student's T-test was used for comparison between groups. P < 0.05 indicated statistically significant differences.

[0169] 3. Experimental results

[0170] 3.1. Effects of the No.5 pharmaceutical composition of the present invention on various sperm parameters of oligospermic and asthenospermic rats

[0171] like Figure 2 , 3 , 4 and Table 4, Tripterygium wilfordii polyglycosides tablets significantly reduced the sperm concentration, sperm motility, sperm motility and various sperm dynamic parameters of rats (P < 0.05, P < 0.01, P < 0.001), and significantly increased the sperm deformity rate (P < 0.001). All doses of the No. 5 pharmaceutical composition of the present invention can significantly alleviate the sperm toxicity of Tripterygium wilfordii polyglycosides to varying degrees (P < 0.05, P < 0.01, P < 0.001). Clomiphene can significantly reduce sperm deformity and increase sperm motility caused by Tripterygium wilfordii polyglycosides (P < 0.001), and can also increase sperm density and motility, but not significantly.

[0172] Table 4 Effect of the No.5 pharmaceutical composition of the present invention on sperm dynamic parameters of oligoasthenozoospermia rats ( n=6)

[0173]

[0174]

[0175] Note: Compared with the control group, ## Indicates P < 0.01, ### indicates P < 0.001, compared with the model group, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0176] 3.2. Effect of the No. 5 pharmaceutical composition of the present invention on pathological damage of the testicles and epididymis of rats induced by Tripterygium wilfordii polyglycosides tablets

[0177] like Figure 5 , 6 As shown in the figure, the lumen structure of the seminiferous tubules in the control group was complete, neatly and tightly arranged, with clear layers, and a large number of sperm cells in the lumen. Tripterygium wilfordii polyglycosides modeling caused the distance between the seminiferous tubules in the testis to widen, a large number of spermatogenic epithelial cells to fall off, and the arrangement was disordered. The number of sperm cells in the lumen decreased, and the thickness of the seminiferous epithelium decreased significantly (P < 0.001). The lumen structure lesions of the seminiferous tubules in the rats of each Chinese medicine administration group were significantly improved, and a few epithelial cells fell off, but the number of tubular epithelial cells increased significantly and were neatly arranged, and the number of sperm in the lumen also increased significantly. The thickness of the seminiferous epithelium in the medium-dose group and the high-dose group was significantly thickened (P < 0.05, P < 0.001). The distance between the seminiferous tubules in the rats in the clomiphene group became smaller, and the seminiferous epithelium was partially thickened, but the arrangement of spermatogenic cells was still relatively disordered and partially fell off.

[0178] like Figure 7As shown in the figure, the epididymal ducts of the control group were arranged tightly and regularly, with complete tissue structure, regular arrangement of epithelial cells, and a large number of sperm in the lumen, which were evenly distributed. The lumen tissue of the epididymal ducts of the model group was loosely arranged, the intertubular spaces became larger, and the number of sperm cells decreased significantly. Compared with the model group, the epididymal lesions in the Chinese medicine group were significantly improved, the tubules were compactly arranged, and the amount of normal sperm in the lumen increased and was evenly distributed; the epididymal lesions of the rats in the clomiphene group were not significantly improved.

[0179] 3.3 Effect of the No.5 pharmaceutical composition of the present invention on ultrastructural damage of rat testicular spermatogonia induced by Tripterygium wilfordii polyglycosides tablets

[0180] like Figure 8 As shown, the spermatogonia in the control group (A) were oval, with rich and evenly distributed chromatin. However, the administration of Tripterygium wilfordii polyglycosides (B) caused damage to spermatogonia, and the chromatin was severely condensed (red arrow). Low (C), medium (D), and high (E) doses of the No. 5 pharmaceutical composition water extract of the present invention and clomiphene (F) can alleviate the damage of Tripterygium wilfordii polyglycosides to spermatogonia. Among them, as the dose of the Chinese medicine administration group increased, the chromatin distribution in the spermatogonia became more even.

[0181] 3.4. Effect of the No. 5 pharmaceutical composition of the present invention on the testicular spermatogonial stem cell pool of oligospermic and asthenospermic rats

[0182] like Fig. 9 , 10 As shown, the number of SSCs in the testis of the control group rats was abundant and evenly dispersed. Tripterygium wilfordii polyglycosides tablets caused a significant depletion of the SSCs pool in the model group rats (P < 0.001), and the medium and high doses of the No. 5 pharmaceutical composition water extract of the present invention could significantly restore the rat testis SSCs pool (P < 0.05, P < 0.01), and the low doses of the No. 5 pharmaceutical composition of the present invention and clomiphene also promoted the recovery of the SSCs pool, but there was no statistical significance.

[0183] 3.5. Effect of the No.5 pharmaceutical composition of the present invention on serum T, LH, FSH and GnRH levels in oligospermic and asthenospermic rats

[0184] like Fig.11 As shown, compared with the control group, the serum T and GnRH levels of the model group rats were significantly reduced (P < 0.001), and the FSH and LH levels were significantly increased (P < 0.001). Compared with the model group, the serum T and GnRH levels of the No. 5 pharmaceutical composition group of each dose of the present invention and the clomiphene group were significantly increased (P < 0.05, P < 0.01, P < 0.001), and the FSH and LH levels were significantly reduced (P < 0.05, P < 0.01, P < 0.001).

[0185] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A pharmaceutical composition consisting of Cistanche deserticola, Rehmannia glutinosa, Schisandra chinensis and Cuscuta australis, characterized in that: The mass ratio of the Cistanche deserticola, the Rehmannia root, the Schisandra chinensis and the Cuscuta chinensis is (2-6): (1-5): (1-4): (0.1-2).

2. The pharmaceutical composition according to claim 1, characterized in that The mass ratio of the Cistanche deserticola, the Rehmannia glutinosa, the Schisandra chinensis and the Cuscuta chinensis is (3-5): (2-4): (1-3): (0.5-2); Preferably, the mass ratio of the Cistanche deserticola, the Rehmannia glutinosa, the Schisandra chinensis and the Cuscuta chinensis is (3.5-4.5): (2.5-3.5): (1.5-2.5): (0.8-1.5); More preferably, the mass ratio of the Cistanche deserticola, the Rehmannia glutinosa, the Schisandra chinensis and the Cuscuta chinensis is about 4: about 3: about 2: about 1; Also preferably, the mass ratio of the Cistanche deserticola, the Rehmannia glutinosa, the Schisandra chinensis and the Cuscuta chinensis is not about 2.958: about 4.226: about 1.268: about 1.

55.

3. A method for preparing the pharmaceutical composition according to claim 1 or 2, characterized in that: The method comprises the following steps: (1) Weigh appropriate amounts of Cistanche deserticola, Rehmannia glutinosa, Schisandra chinensis, and Cuscuta australis, add the first water for soaking, boil over high heat until boiling, boil over low heat for a period of time, filter, and obtain a first filtrate and a filter residue; (2) adding a second amount of water to the filter residue, boiling over high heat, boiling over low heat for a period of time, filtering, and obtaining a second filtrate; as well as (3) combining the first filtrate and the second filtrate, drying and concentrating to obtain the pharmaceutical composition.

4. The method according to claim 3, characterized in that The mass ratio of the Cistanche deserticola, the Rehmannia glutinosa, the Schisandra chinensis and the Cuscuta chinensis is about 4: about 3: about 2: about 1; Preferably, the water is deionized water; More preferably, the ratio of the sum of the mass of the Cistanche deserticola, Rehmannia glutinosa, Schisandra chinensis and Cuscuta australis to the mass / volume (g / ml) of the first water is 0.01 to 1, for example, about 0.1; More preferably, the ratio of the sum of the mass of the Cistanche deserticola, Rehmannia glutinosa, Schisandra chinensis and Cuscuta australis to the mass / volume (g / ml) of the second water is 0.01 to 1, for example, about 0.1; More preferably, the soaking time is 20 to 40 minutes, for example about 30 minutes; More preferably, the slow-cooking time is 0.5 to 2 hours, for example, about 1 hour; Also preferably, the drying is vacuum drying.

5. The method according to claim 3, characterized in that: The mass of the Cistanche deserticola is in the range of 1 to 3 g, for example, about 2 g; Preferably, the mass range of the Rehmannia glutinosa is 1 to 3 g, for example, about 1.5 g; Preferably, the mass of Schisandra chinensis is in the range of 0.5 to 2 g, for example, about 1 g; Preferably, the mass of the dodder seeds is in the range of 0.1 to 1 g, for example, about 0.5 g.

6. A pharmaceutical preparation, characterized in that A pharmaceutical composition comprising the pharmaceutical composition of claim 1 or 2, and one or more optional pharmaceutically acceptable excipients; Preferably, the excipient is selected from one or more of the following: diluent, wetting agent, binder, disintegrant, inclusion agent, flavoring agent, sustained-release agent, glidant, lubricant, dispersant, plasticizer, opacifier and antioxidant; Preferably, the dosage form of the pharmaceutical preparation is powder, tablet, pill, capsule, film, lozenge, granule, injection or oral solution; Preferably, the pharmaceutical preparation further comprises one or more other drugs having the function of preventing and / or treating oligospermia and / or asthenospermia; More preferably, the drug is selected from one or more of the following: antioxidants, anti-infective drugs, gonadotropin therapy drugs, pancreatic kallikrein and pentoxifylline; Preferably, the antioxidant is selected from one or more of the following: N-acetylcysteine, vitamin E, vitamin C, coenzyme Q10 and L-carnitine; Also preferably, the gonadotropin therapeutic drug includes recombinant follicle-stimulating hormone and urinary gonadotropin.

7. Use of the pharmaceutical composition according to claim 1 or 2 or the pharmaceutical preparation according to claim 6 in the preparation of a medicament for preventing and / or treating oligospermia and / or asthenospermia.

8. The use according to claim 7, characterized in that The pharmaceutical composition or the pharmaceutical preparation prevents and / or treats oligospermia and / or asthenospermia in one or more of the following ways: improving spermatocyte motility, improving sperm density, improving sperm motility, increasing sperm survival rate, reducing sperm deformity rate, improving organ structural abnormalities, improving spermatogonia ultrastructure abnormalities, repairing spermatogonial stem cell pools, and improving serum biochemical index abnormalities.

9. The use according to claim 8, characterized in that Improving sperm motility means increasing sperm dynamics parameters; Preferably, the sperm dynamics parameter is selected from one or more of the following: sperm curve velocity, sperm linear velocity, sperm path velocity, sperm movement angle, sperm lateral swing amplitude and sperm whipping frequency; Preferably, the organ structural abnormality includes testicular structural abnormality and / or epididymal structural abnormality; More preferably, the testicular structural abnormality includes one or more of the following: a decrease in the number of epithelial cells in the seminiferous tubules, a disordered arrangement of epithelial cells in the seminiferous tubules, a decrease in the thickness of the seminiferous epithelium, and a decrease in the number of sperm in the lumen of the seminiferous tubules; More preferably, the epididymal structural abnormality includes an increase in the epididymal ductal space and / or a decrease in the number of sperm in the epididymal ductal cavity; More preferably, the spermatogonia ultrastructural abnormality is spermatogonia chromatin condensation; More preferably, the repairing of the spermatogonial stem cell pool is to increase the number of spermatogonial stem cells.

10. The use according to claim 8, characterized in that The abnormal biochemical indexes include one or more of the following: decreased testosterone level, decreased gonadotropin-releasing hormone level, increased follicle-stimulating hormone level and increased luteinizing hormone level.

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