Preparation method of ginsenoside Rh4 and application of ginsenoside Rh4 in medicines for treating dyszoospermia

Abstract

The invention discloses a preparation method of ginsenoside Rh4 and application of the ginsenoside Rh4 in preparation of a medicine for treating and preventing dyszoospermia. According to the method, ginseng stem and leaf total saponins are taken as a substrate, an ammonium bicarbonate or sodium bicarbonate aqueous solution is taken as a solvent, BaCl2 is taken as a catalyst, the ginsenoside Rh4 is efficiently prepared through a one-step conversion, one-step extraction and separation method, the efficient directional conversion mechanism of triol type ginsenosides Rg1-Rh4 is clarified, and the method has the advantages of being wide in reaction substrate source, simple in process, high in yield and the like. The method has the characteristics of economy, safety and high yield, and is suitable for large-scale industrial production. In-vivo and in-vitro tests prove that the ginsenoside Rh4 has a remarkable pharmacodynamic effect on treating and preventing spermatogenesis disorder caused by a series of factors such as external environment heat stress and chemistry, spermatogenesis injury caused by multiple factors can be remarkably relieved, the practicability is high, and the ginsenoside Rh4 has a remarkable development potential and is worthy of popularization and application. The efficient utilization of the panax total saponins and the application of the panax total saponins in the field of preparation of medicines and health can be effectively promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of active development and application of natural medicines, and specifically relates to a preparation method of ginsenoside Rh4 and its application in spermatogenesis disorder medicines. Background Art

[0002] In recent years, the quality of male semen has declined significantly, and the incidence of male infertility has gradually increased. However, people pay more attention to the effects of environmental pollutants and infections on male reproduction, while the effects of rising ambient temperature and lifestyle changes on male reproductive development and function have not received sufficient attention. Spermatogenesis is a temperature-dependent process. Spermatogonia play a decisive role in sperm formation and maintaining the stability of sperm count, and are extremely sensitive to temperature requirements. Studies have shown that male testicular temperature is 2-8°C lower than core body temperature, which is the best condition for maintaining spermatogenesis in the scrotum and is also one of the necessary conditions for testicular growth (see Salonia A, et al. European Association of Urology Guidelines on Sexual and Reproductive Health—2021 Update: Male Sexual Dysfunction [J]. European Urology, 2021, 80 (3): 333-357; Zaha I, et al. The Role of Oxidative Stress in Infertility [J]. Journal of Personalized Medicine, 2023, 13 (8): 1264).

[0003] Studies have shown that the binding of testosterone to the androgen receptor (AR) secreted by supporting cells is necessary to initiate spermatogenesis. Any structural and functional damage in this process may lead to spermatogenesis damage. Heat stress (HS) and di(2-ethylhexyl) phthalate (DEHP) are often exposed in modern life. The latter is one of the environmental endocrine disruptors and is widely used in pharmaceutical manufacturing and the production of plastic products. It is closely related to people's lives. Infertility, as a reproductive system disease affecting the world, has become a problem for many people of childbearing age. Compared with the past, male infertility is mainly manifested as spermatogenesis dysfunction (see: Ling Xiaohui, et al. Single-cell transcriptome study of mouse testicular supporting cells involved in spermatogenesis disorders [J]. Chinese Journal of Andrology, 2024, 38(04): 38-47; Wei Jintao, et al. Research progress on Chinese medicine regulating ferroptosis pathway to intervene in testicular spermatogenic epithelial damage [J]. Chinese Journal of Andrology, 2024, 30(05): 450-455). The spermatogenesis damage induced by HS, DEHP and their metabolites may be related to the pathological factors of male infertility. However, there are no relevant research reports on spermatogenesis disorders caused by external environment and chemical factors in the current medical and health field. Therefore, seeking natural and effective active ingredients from traditional Chinese medicine to alleviate spermatogenesis damage and improve reproductive function has good application potential.

[0004] Ginseng is a traditional Chinese herbal medicine in my country. It is widely used because of its good pharmacological effects and relatively small side effects. In recent years, the protective effect of ginseng and its saponin components on reproductive system diseases has become a focus of attention. Ginsenoside Rh4 is a relatively important active ingredient in ginseng, but due to the complex separation and purification process of ginsenoside Rh4, and the relatively serious consumption and high cost, its application in the field of medicine and health is limited. In the prior art, CN 106046096 A discloses a method for large-scale conversion and production of ginsenoside Rh4 using triol group ginsenosides, involving a fermentation tank and a series of organic acid reactions, which is not only complex in process and has safety issues, but also has low purity of ginsenoside Rh4. CN 103193846 A discloses a method for preparing cis-trans isomers of ginsenoside Rk3 and ginsenoside Rh4. Silica gel column chromatography and high-speed countercurrent separation are combined to obtain ginsenoside Rk3, Z-type ginsenoside Rh4 and E-type ginsenoside Rh4. The purity of the three compounds is greater than 95%. Although the method can effectively separate ginsenoside Rk3 and Rh4, its yield is extremely low, the process is complicated, the cost is high, and it is not suitable for industrial production. CN 118773172 A discloses a method for large-scale production of rare ginsenoside Rh4, which uses a β-glucosidase variant as a catalyst to catalyze the conversion of ginsenoside Rg1 into ginsenoside Rh4 via ginsenoside Rh1. The purity of ginsenoside Rh4 after purification is ≥95%, which is suitable for large-scale production. However, this method uses ginsenoside Rg1 as a raw material, and although ginsenoside Rg1 has a high content in ginseng, the separation process is cumbersome and easily causes waste of resources, which is not conducive to the sustainable development of ginseng. Moreover, the application of ginsenoside Rh4 in preventing and treating spermatogenesis disorders has not been reported yet.

[0005] In summary, most of the current preparation methods of ginsenoside Rh4 focus on using triol group ginsenosides, through acid conversion, and further using a series of separation means for purification. This process is not only operational, but also easy to cause waste of resources. In particular, during the conversion of ginsenosides of ginsenosides of ginsenosides into ginsenoside Rh4, byproducts such as ginsenosides Rg6, F4 and Rk3 will inevitably be produced, and the separation process is extremely cumbersome. The advantage of the present invention is that, by using a mild alkaline hydrolysis method, metal ions are used as catalysts to efficiently and directional convert ginsenoside Rh4 from ginseng stem and leaf saponins. The present invention has the characteristics of simple process, economy, safety and high yield, and in vitro and in vivo experiments have proved that the ginsenoside Rh4 prepared by the present invention has a wide range of application potential in the preparation of spermatogenesis drugs or health products. It is creatively proposed and proved that ginsenoside Rh4 is a spermatogenesis damage protective agent with great development prospects, which can be used to treat and prevent spermatogenesis problems caused by a series of factors such as heat stress and chemistry in the external environment. It has strong practicality and broad development prospects. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a method for preparing ginsenoside Rh4 and its application in drugs for spermatogenesis disorders in view of the deficiencies of the above-mentioned prior art. The present invention uses total saponins of ginseng stems and leaves as substrates, aqueous ammonium bicarbonate (NH4HCO3) solution or sodium bicarbonate (NaHCO3 solution as solvent, BaCl2 as catalyst, and a mild alkaline hydrolysis method to achieve efficient directional conversion of ginsenosides of ginsenosides, and efficiently prepares ginsenoside Rh4 through the method of "one-step conversion, one-step extraction and separation", and reveals its application in the preparation of drugs and health products for the treatment and prevention of spermatogenesis disorders, which is closely related to life and broadens the application scope of ginseng in the field of medicine and health, and has significant practicality and development potential.

[0007] In order to fully demonstrate the superiority of the present invention and solve the problems of complex separation of ginsenoside Rh4 and serious waste of resources in the current technology, the technical solution adopted by the present invention is:

[0008] A method for preparing ginsenoside Rh4, the method comprising the following steps:

[0009] S1. Using 10% to 40% NH4HCO3 aqueous solution or NaHCO3 aqueous solution as solvent, add total saponins of ginseng stems and leaves under stirring, and then add metal ions (BaCl2) as catalyst, place in a water bath at a temperature of 65 to 75°C to react for 4 to 6 hours, stand and cool to obtain a reaction solution, extract the reaction solution with a saturated n-butanol solution of water to obtain a crude product of ginsenoside Rh4;

[0010] S2. Separate and purify the crude ginsenoside Rh4 obtained in S1 by silica gel column chromatography or semi-preparative liquid chromatography to obtain high-purity ginsenoside Rh4.

[0011] Preferably, the ratio of the total saponins of ginseng stems and leaves to the NH4HCO3 aqueous solution or the NaHCO3 aqueous solution in S1 is 1:20.

[0012] Preferably, the BaCl2 in S1 accounts for 2% to 8% of the total saponins in ginseng stems and leaves.

[0013] Preferably, the content of ginsenoside Rh4 in the crude ginsenoside Rh4 obtained in S1 is 60% to 75%.

[0014] Preferably, the chromatographic conditions of the semi-preparative liquid chromatography purification in S2 are: the stationary phase is octadecyl bonded silica gel, the mobile phase is methanol and water, and isocratic elution is performed.

[0015] Preferably, the purity of ginsenoside Rh4 prepared in S2 is ≥98%.

[0016] The total saponin extract of ginseng stems and leaves in the present invention can be obtained by soaking extraction, reflux extraction, ultrasonic extraction, countercurrent extraction and supercritical extraction. The extractant used is a lower alcohol aqueous solution or a lower ketone. The lower alcohol includes methanol, ethanol and n-butanol, and the lower ketone is acetone.

[0017] The present invention also provides an application of ginsenoside Rh4 in the preparation of medicines and health products for treating or preventing spermatogenesis disorders.

[0018] When the ginsenoside Rh4 is used to prepare medicines and health products for treating or preventing spermatogenesis disorders, it can also be combined with other active ingredients or pharmaceutically acceptable carriers.

[0019] Among them, the active ingredients are one or more of licorice extract, ophiopogon extract, peppermint extract, vitamin C and its derivatives or vitamin E and its derivatives.

[0020] When the ginsenoside Rh4 of the present invention is used to prepare a drug for treating or preventing spermatogenesis disorders, it can be made into tablets, capsules, powders, injections, soft capsules, pills, syrups, granules and pastes, etc., for oral or parenteral administration.

[0021] The content of ginsenoside Rh4, the core ingredient, in the drug or health product for treating or preventing spermatogenesis disorders is 2.0% to 45.0%, and more preferably 5.0% to 20.0%.

[0022] The above-mentioned ginsenoside Rh4 content is an effective amount for treating spermatogenesis damage caused by heat stress (HS) and chemical (DEHP), and can also be further adjusted according to the nature of the administration route and preparation, the recipient's weight, age and other general conditions, as well as the nature and severity of the disease being treated.

[0023] The present invention has significant technical effects due to the adoption of the above technical solution:

[0024] 1. The present invention uses total saponins from ginseng stems and leaves as substrates, ammonium bicarbonate or sodium bicarbonate aqueous solution as solvent, BaCl2 as catalyst, and a mild alkaline hydrolysis method to clarify the efficient and directional transformation mechanism of triol ginsenoside Rg1→Rh4, realize the efficient and directional transformation of ginsenosides, and efficiently prepare ginsenoside Rh4 through the method of "one-step transformation, one-step extraction and separation", which has the following advantages: (1) The source of reaction substrate is wide, and the inexpensive and easily available total saponins of ginseng are used for direct hydrolysis, and the acidity and conversion time are strictly controlled to ensure the complete conversion of total saponins of ginseng to the maximum extent, thereby improving the yield of ginsenoside Rh4; (2) The metal ion B aCl2 is used as a catalyst to effectively reduce the amount of ammonium bicarbonate / sodium bicarbonate used, and promote the conversion efficiency of ginsenosides of ginsenosides, greatly improving the reaction efficiency and yield; (3) the process is simple, there is no need to separate diol and triol saponins in advance, reducing the use of macroporous resins and silica gel, using weak alkalinity to transform ginsenosides, promoting the conversion of ginsenosides of ginsenosides, and directly extracting ginsenoside Rh4 from the reaction solution; (4) the influence of cis-trans isomers of ginsenosides Rk3 and Rh4 on their purity can be effectively reduced, and high-purity ginsenoside Rh4 can be obtained by silica gel column chromatography or semi-preparative liquid phase efficient separation, which can effectively save the separation and purification time of ginsenoside Rh4. The preparation method of the present invention has the characteristics of economy, safety, simplicity and high yield, is suitable for large-scale industrial production, can effectively promote the efficient utilization of total saponins in non-medicinal parts of ginseng, and promote the sustainable and healthy development of ginseng.

[0025] 2. The present invention provides the use of ginsenoside Rh4 in the preparation of drugs and health products for treating or preventing spermatogenesis disorders. Through in vivo and in vitro experiments, it is fully proved that ginsenoside Rh4 has a unique pharmacological effect in treating spermatogenesis damage caused by heat stress and chemical factors, and the effect is remarkable. It is creatively proposed that ginsenoside Rh4 can be used to treat and prevent spermatogenesis disorders caused by environmental heat stress and chemical factors, which has strong practicality, and reveals that ginsenoside Rh4 is a very promising and potential spermatogenesis damage protective agent, with strong practicality and outstanding medicinal value, which can effectively promote the efficient utilization of total saponins of Panax genus and its application in the preparation of medicine and health fields.

[0026] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the HPLC analysis chart of the crude products of total saponins of ginseng stems and leaves and ginsenoside Rh4 in Example 1;

[0028] Figure 2 This is the mechanism of the directional transformation of ginsenoside Rg1→Rh4 in the present invention;

[0029] Figure 3The testicular tissue pathological analysis results of ginsenoside Rh4 on scrotal heat-stressed mice in Example 2;

[0030] Figure 4 This is the result of ginsenoside Rh4 improving the expression of testosterone synthesis-related proteins in mice induced by DEHP in Example 3. DETAILED DESCRIPTION

[0031] Example 1

[0032] This embodiment is the preparation of ginsenoside Rh4, comprising the following steps:

[0033] S1. Use 2L of 10% NH4HCO3 aqueous solution as solvent, add total saponins of ginseng stems and leaves at a solid-liquid ratio of 1:20, stir while adding, then add BaCl2 catalyst at 4% of the mass of total saponins of ginseng stems and leaves, place in a water bath at a temperature of 75°C to react for 4 hours, stand and cool to obtain a reaction solution, extract the reaction solution with a saturated n-butanol solution with water (the volume ratio of water to n-butanol is 1:1), and obtain a crude product of ginsenoside Rh4, about 7.4 g, with a yield of 7.4%.

[0034] S2. The crude ginsenoside Rh4 obtained in S1 was separated and purified by semi-preparative high performance liquid phase. The preparative liquid phase conditions were: chromatographic column Tanture C18 (150×10mm, 5μm), flow rate of 10-20mL / min, injection volume of 20μL, detection wavelength of 203nm, and mobile phase of water (A, 45%)-methanol (B, 55%) isocratic elution for 30min. After further separation and purification, the efficiently prepared ginsenoside Rh4 was obtained, and the recovery rate reached 86.7%. According to HPLC analysis, the purity of ginsenoside Rh4 reached 98.2%.

[0035] The total saponins of ginseng stems and leaves of the present invention are analyzed by HPLC, and they mainly contain Rg1, Re, Rb1, Rc, Rd and other components, with a total content of about 85% (UV method); the crude ginsenoside Rh4 after conversion mainly contains Rk3 and Rh4, etc., and contains a small amount of Rg6 and F4. The HPLC analysis chart is shown in the attached Figure 1 .

[0036] In this example, ginsenoside Rh4 was efficiently prepared by using total ginsenosides of ginseng stems and leaves as substrates, NH4HCO3 as solvent, and BaCl2 as catalyst through a "one-step conversion, one-step extraction and separation" method, clarifying the efficient and directional conversion mechanism of triol ginsenoside Rg1→Rh4. Its structural transformation is shown in the attached figure. Figure 2 As shown. Figure 2It can be seen that through the preparation method described in the present invention, the ginsenoside Rg1 will undergo a desugaring reaction (C-20 position) under the action of mild alkaline hydrolysis, and further undergo deoxyisomerization to produce ginsenosides Rk3 and Rh4. HPLC analysis shows that the ratio of ginsenoside Rk3 and Rh4 in the crude ginsenoside Rh4 is 1:3.

[0037] In addition, this example further explores the effects of different BaCl2 catalyst concentrations and other catalyst types (pyrrole, barium sulfate, snail enzyme) on the conversion rate of ginsenoside Rh4. The preparation method is the same as above, except that the 4% BaCl2 catalyst is replaced with no catalyst, 2% BaCl2, 6% BaCl2, 8% BaCl2, 8% pyrrole, 8% snail enzyme, and 8% barium sulfate. The results are shown in Tables 1 and 2.

[0038] Table 1 Effect of catalyst concentration on crude ginsenoside Rh4 conversion rate

[0039]

[0040] Table 2 Effect of different catalysts on the conversion rate of crude ginsenoside Rh4

[0041]

[0042] The results show that the BaCl2 catalyst of the present invention has great superiority and can improve the directional conversion efficiency of triol ginsenoside Rg1→Rh4, thereby improving the reaction efficiency and yield.

[0043] Example 2

[0044] This example is about the protective effect of ginsenoside Rh4 on spermatogenesis damage induced by scrotal heat stress.

[0045] 1. Materials, reagents, and instruments

[0046] Ginsenoside Rh4 (purity 98.2%, HPLC determination, provided by Example 1 of the present invention), sodium pentobarbital (C 11 H 17 N2NaO3) purity>99%.

[0047] Malondialdehyde (MDA), reduced glutathione (GSH) and superoxide dismutase (SOD) kits were purchased from Nanjing Jiancheng Bioengineering Institute; testosterone kit was purchased from R&D Company, USA. BP211D electronic balance, Sartorius Company, Germany; DK-98-1 electric constant temperature water bath, Tianjin Test Instrument Co., Ltd.; HC-2517 high-speed centrifuge, Anhui Zhongke Zhongjia Company; Epoch ultra-microplate spectrophotometer, Berton Instrument Co., Ltd., USA; FSH-2A adjustable high-speed homogenizer, Jintan Huacheng Hailong Experimental Instrument Factory.

[0048] 2. Experimental animals:

[0049] 40 male ICR mice weighing 25±2g were purchased from Changchun Yisi Experimental Animal Technology Co., Ltd. All mice were allowed free access to water and feed, and maintained under a day-night cycle at a temperature of 22±2°C and a relative humidity of 50±10%. All experiments were carried out in accordance with the Regulations on Laboratory Animal Management of the Science and Technology Committee of my country.

[0050] 3. Animal grouping and drug administration:

[0051] The drug-treated group was intragastrically administered with ginsenoside Rh4 at a dose of 10 mg / kg, 20 mg / kg and 40 mg / kg once a day for 14 consecutive days, and the blank control group and the model group were intragastrically administered with an equal volume of purified water. Six hours after the seventh day of drug administration, the mice in the model group and the drug-treated group were subjected to heat stress using a water bath (43°C, constant temperature). Each mouse was intraperitoneally injected with 40 mg / kg sodium pentobarbital for anesthesia, and the lower 1 / 3 of the body was bathed in a water bath at a temperature of 43°C for 18 minutes. After stress, it was placed on a heating pad to restore body temperature. On the 15th day, blood was collected from the eyeballs of mice in each group in turn, and serum was separated by centrifugation at 3000r / min for 10 minutes, and stored at 4°C for standby use; the testicles and epididymis were quickly dissected. After being rinsed with 4°C saline, dried with filter paper, weighed, the left testicle and epididymis were fixed in 10% formaldehyde solution, and sliced, and the right testicle and epididymis were stored in a -80°C low-temperature refrigerator.

[0052] The results of the experiment are shown in Table 3. It can be seen that after scrotal heat stress (HS), the testicles of the mice in the model group were significantly reduced, and the testicular and epididymal indexes were significantly reduced compared with the blank control group (P<0.001). After Rh4 administration, the testicles and epididymis of the mice were significantly improved, but the weight of the mice did not change significantly. The testicular and epididymal indexes of the Rh4 group (10 mg / kg, 20 mg / kg and 40 mg / kg) were significantly increased compared with the model group (P<0.001). In addition, there was no significant change in the indexes of other organs between the groups.

[0053] Table 3 Effects of ginsenoside Rh4 on testis and epididymis indexes in mice with spermatogenic cell damage induced by heat stress

[0054]

[0055] Note: Compared with the blank group ( *** P<0.001, compared with the model group ( # P<0.05, ## P<0.01, ### P<0.001).

[0056] 4. Determination of biochemical indicators in serum

[0057] Blood was collected from mice to separate serum, and testosterone was determined according to the kit method to study the effect of ginsenoside Rh4 on testosterone in mice with spermatogenic cell damage caused by HS. The results are shown in Table 4.

[0058] The results showed that heat stress significantly reduced the concentration of testosterone in serum (P<0.05). After Rh4 treatment, testosterone increased compared with the model group, restoring the level of testosterone in a dose-dependent manner.

[0059] Table 4 Effects of ginsenoside Rh4 on testosterone in mice with spermatogenic cell damage induced by HS

[0060]

[0061]

[0062] Note: Compared with the blank group ( * P<0.05, compared with the model group ( # P<0.05, ## P<0.01).

[0063] 5. Determination of oxidation indexes in testis

[0064] The left testis was weighed, 9 volumes of ice saline were added, 10% testis tissue homogenate was prepared by tissue homogenizer, and the supernatant was centrifuged. The plate was spotted according to the kit method, the OD value was measured at 450nm, and the content of malondialdehyde (MDA), the activity of reduced glutathione (GSH) and the level of superoxide dismutase (SOD) in the testis were calculated according to the formula to study the effect of ginsenoside Rh4 on testis tissue peroxidation in mice with spermatogenic cell damage caused by HS. The results are shown in Table 5.

[0065] Compared with the blank group, the MDA content in the oxidative tissue homogenate of mice in the model group was significantly increased, and the levels of GSH and SOD were significantly decreased (P<0.05), which caused the accumulation of lipid peroxidation products in the mice and reduced the antioxidant metabolism level; compared with the model group, the MDA content in the ginsenoside Rh4-treated group was significantly decreased (P<0.01), and the levels of GSH and SOD were significantly increased (P<0.001), indicating that ginsenoside Rh4 can alleviate lipid peroxidation caused by heat stress to a certain extent and regulate the antioxidant metabolism level in the body.

[0066] Table 5 Effects of ginsenoside Rh4 on testicular tissue peroxidation in mice with spermatogenic cell damage induced by HS

[0067]

[0068] Note: Compared with the blank group ( ** P<0.01, *** P<0.001, compared with the model group ( # P<0.05, ## P<0.01, ### P<0.001).

[0069] 6. Histopathological observation of testis and epididymis

[0070] The left testis and epididymis tissues were fixed in 10% neutral formaldehyde solution, dehydrated with low to high concentration alcohol, transparentized in xylene, embedded in conventional paraffin, sliced, dewaxed and stained with H&E, and the pathological changes of testis and epididymis tissues were observed under an optical microscope to study the effect of ginsenoside Rh4 on testis and epididymis tissue pathology. The results are as follows Figure 3 shown.

[0071] Through the observation of testicular and epididymal tissue pathology, the spermatogenic cells in the testicular tissue of the control mice were neatly arranged and showed normal cell morphology. The testicles of the HS model group showed severe spermatogenic cell necrosis, the cell nucleus was wrinkled, and some seminiferous tubules collapsed or atrophied due to the shedding of the germ cell layer. The testicular sections of the mice in the Rh4 administration group showed that the testicular tissue almost restored its normal structure, preserved the spermatogenic cell morphology well and contained a large number of spermatocytes and sperm cells. H&E staining showed that the testicular damage and necrosis in the HS model group were relatively serious, and the low and high doses of Rh4 groups had a certain protective effect on alleviating testicular damage.

[0072] Example 3

[0073] This example is about the improving effect of ginsenoside Rh4 on spermatogenesis disorder in mice induced by the chemical agent di(2-ethylhexyl) phthalate.

[0074] 1. Reagents

[0075] Ginsenoside Rh4 (purity 98.2%, determined by HPLC, provided by Example 1 of the present invention) and di(2-ethylhexyl) phthalate (DEHP, item number: No. P0297) were purchased from Shanghai Chemical Industry Development Co., Ltd.

[0076] Testosterone (Testosterone T), luteinizing hormone (LH), and sex hormone detection ELISA kits were purchased from Shanghai ELISA Biotechnology Co., Ltd.; BCA protein concentration determination kit was provided by Shanghai Bio-Tech Biotechnology Co., Ltd.

[0077] 2. Experimental animals and protocols

[0078] Three-week-old ICR male mice were provided by Changchun Yisi Biotechnology Co., Ltd., with a quality certificate number of SCXK(Ji)-2020-0002 (Changchun, China). Before the start of the experiment, all mice were kept under standard feeding conditions for 1 week (12h light / dark cycle, relative humidity of 60±10%, 22±2℃) to adapt to the new feeding conditions. All experimental animal studies followed the "Regulations on the Management of Experimental Animals" formulated by the Experimental Animal Ethics Committee.

[0079] All mice were randomly divided into 4 groups (12 mice in each group): (1) blank control group; (2) DEHP group (model group); (3) DEHP+Rh4 low-dose group (20 mg / kg); (4) DEHP+Rh4 high-dose group (40 mg / kg). Except for the blank group, mice in other groups were given 500 mg / kg of DEHP (dissolved in corn oil) by gavage to induce spermatogenesis disorder model (blank group was given 0.9% corn oil) for a total of 35 days. One week after modeling, Rh4 was dissolved in 9% saline and gavage was administered for 28 days. After the experiment, all mice were killed after blood sampling from the eyeballs, and part of the testicular tissue was collected and fixed in 4% paraformaldehyde solution for histopathological staining.

[0080] The results of the experiment on the effect of ginsenoside Rh4 on the testis and epididymis index of DEHP-induced spermatogenesis-impaired mice are shown in Table 6. It can be seen that after DEHP modeling, the testis of the model group (DEHP) mice was significantly reduced compared with the control group, and the testis and epididymis indexes were significantly reduced (P<0.001). After Rh4 administration, the testis and epididymis of the mice were significantly improved, but the weight of the mice did not change significantly. Compared with the model group, the testis and epididymis indexes of the Rh4 group (20 mg / kg and 40 mg / kg) were significantly increased (P<0.001). In addition, there was no significant change in other organ indexes between the groups.

[0081] Table 6 Effects of ginsenoside Rh4 on testis and epididymis indexes in DEHP-induced spermatogenesis-impaired mice

[0082]

[0083] Note: Compared with the blank group ( *** P<0.001, compared with the model group ( # P<0.05, ## P<0.01, ### P<0.001).

[0084] 3. Serum hormone level detection

[0085] The concentrations of testosterone (T) and luteinizing hormone (LH) in the testicular tissue serum of mice were determined according to the instructions of the kit, and the corresponding OD values ​​at the corresponding wavelengths were detected using an enzyme-labeled instrument, and the corresponding concentrations under the corresponding standard curves were calculated to detect changes in androgen levels.

[0086] The results are shown in Table 7. After DEHP modeling, the levels of testosterone (T) and luteinizing hormone (LH) in mouse serum decreased significantly, while this phenomenon was improved after Rh4 treatment, indicating that Rh4 can effectively improve DEHP-induced testosterone secretion disorders in mice.

[0087] Table 7 Improvement of ginsenoside Rh4 on sex hormone secretion disorders induced by DEHP in mice

[0088]

[0089] Note: Compared with the blank group ( * P<0.05, ** P<0.01, compared with the model group ( # P<0.05, ## P<0.01, ### P<0.001).

[0090] 4. Western blot analysis

[0091] To determine the changes in protein levels, total protein was extracted from testicular tissue samples using a pre-prepared lysis buffer (containing RIPA buffer and phosphatase inhibitors). After lysis, the supernatant obtained by discarding the precipitate was used to determine the protein concentration of each group using a BCA kit and normalized. The proteins were separated using a 15% SDS-PAGE gel. After the gel was transferred to a PVDF membrane, it was sealed with 5% skim milk for 1 hour, and then the target antibodies LHR (1:10000), CYP450 (1:10000), StAR (1:10000), FSHR (1:10000), AR (1:10000) and 17β-HSD (1:1000) were added. Then, the membranes were incubated overnight in a 4°C refrigerator. The next day, rabbit / mouse antibodies (1:10000) diluted in PBS were added to bind to the target antibodies. Finally, the protein expression was observed using a colorimetric solution (ECL) in a multifunctional gel imaging system.

[0092] This example further detected the expression of testosterone synthesis pathway-related proteins and the AR and FSHR proteins secreted by supporting cells by Western blotting analysis, and studied the effect of Rh4 on the DEHP-induced testosterone synthesis pathway and supporting cell secretion function-related proteins in mouse testes. Figure 4 As shown, after DEHP modeling, the expressions of testosterone synthesis-related proteins LHR, StAR, 17β-HSD and CYP450 were downregulated (p<0.01, p<0.001), and the expressions of supporting cell-secreted proteins AR and FSHR were downregulated (p<0.01, p<0.001), but these phenomena were restored after Rh4 administration.

[0093] Example 4

[0094] This embodiment is a drug tablet for treating spermatogenesis disorder, and the formula is as follows: 100g of ginsenoside Rh4 (purity 98%), 1000g of starch, 11g of talc, and 22g of magnesium stearate;

[0095] The preparation method is as follows: after evenly mixing ginsenoside Rh4 and starch, the mixture is sieved through a 120-mesh sieve, sized into granules, talcum powder and magnesium stearate are added, mixed evenly, and pressed into 1,000 tablets, wherein the ginsenoside Rh4 accounts for 8.65% of each tablet.

[0096] Dosage: Orally, 3 tablets each time, three times a day, after meals.

[0097] Example 5

[0098] This embodiment is a capsule preparation for treating spermatogenesis disorder, and the formula is as follows: 100 g of ginsenoside Rh4 (purity 98%), 1000 g of starch;

[0099] The preparation method is as follows: after fully mixing ginsenoside Rh4 and starch, passing through a 120-mesh sieve, whole grains are put into capsules to prepare capsules, and the proportion of ginsenoside Rh4 in each grain is 8.90%.

[0100] Dosage: Orally, 3 capsules each time, three times a day, after meals.

[0101] Example 6

[0102] This embodiment is a drug tablet for treating spermatogenesis disorder, and the formula is as follows: 20g of ginsenoside Rh4 (purity 98%), 40g of licorice extract, 20g of ophiopogon extract, 1000g of starch, 10.8g of talc, and 21.6g of magnesium stearate;

[0103] The preparation method is as follows: after evenly mixing ginsenoside Rh4, licorice extract, ophiopogon extract and starch, passing through a 120-mesh sieve, whole grains are formed into granules, talcum powder and magnesium stearate are evenly added, and then pressed into tablets. The proportion of ginsenoside Rh4 in each tablet is 1.76%.

[0104] Dosage: Orally, 5 tablets each time, three times a day, after meals.

[0105] Example 7

[0106] This embodiment is a capsule for treating spermatogenesis disorder, and the formula is as follows: 20g of ginsenoside Rh4 (purity 98%), 30g of Poria cocos extract, 600g of vitamin C, and 1000g of starch;

[0107] The preparation method is as follows: after mixing the above substances evenly, passing through a 120-mesh sieve, whole grains are formed into capsules, and the proportion of ginsenoside Rh4 in each grain is 1.19%.

[0108] Dosage: Orally, 5 capsules each time, three times a day, after meals.

[0109] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. The above embodiment is only a brief example of the clinical application of the present invention through the above method, and the application of ginsenoside Rh4 in the preparation of drugs for the treatment and prevention of spermatogenesis disorders is described in detail in combination with pharmacological experiments, but the present invention is not limited to the above implementation. The details and form of the technical solution of the present invention can be modified or replaced without departing from the formulation ideas and scope of use of the present invention, and the present invention can be implemented in various ways, but these modifications and replacements should be within the scope of protection of the present invention and will not be described one by one here.

Claims

1. A method for preparing ginsenoside Rh4, characterized in that: The method comprises the following steps: S1. Using 10% to 40% by mass of ammonium bicarbonate NH4HCO3 aqueous solution or sodium bicarbonate NaHCO3 aqueous solution as solvent, add total saponins of ginseng stems and leaves under stirring, and then add metal ion BaCl2 as a catalyst, place in a water bath at a temperature of 65 to 75°C to react for 4 to 6 hours, and after cooling, extract the reaction solution with a saturated n-butanol solution to obtain a crude product of ginsenoside Rh4; S2. Separate and purify the crude ginsenoside Rh4 obtained in S1 by silica gel column chromatography or semi-preparative liquid chromatography to obtain ginsenoside Rh4.

2. The method according to claim 1, characterized in that: The ratio of the total saponins of ginseng stems and leaves to the NH4HCO3 aqueous solution or the NaHCO3 aqueous solution in S1 is 1:

20.

3. The method according to claim 1, characterized in that The BaCl2 described in S1 accounts for 2% to 8% of the total saponins in the stems and leaves of ginseng.

4. The method according to claim 1, characterized in that: The content of ginsenoside Rh4 in the crude ginsenoside Rh4 obtained in S1 is 60% to 75%.

5. The method according to claim 1, characterized in that The chromatographic conditions for the semi-preparative liquid chromatography purification described in S2 are: the stationary phase is octadecyl bonded silica gel, the mobile phase is methanol and water, and isocratic elution is performed.

6. The method according to claim 1, characterized in that The purity of ginsenoside Rh4 prepared by S2 is ≥98%.

7. An application of ginsenoside Rh4 prepared by the method according to any one of claims 1 to 6, characterized in that: The ginsenoside Rh4 can be used to prepare medicines and health products for treating or preventing spermatogenesis disorders.

8. The use according to claim 7, characterized in that: When the ginsenoside Rh4 is used to prepare medicines and health products for treating or preventing spermatogenesis disorders, it can also be combined with other active ingredients or pharmaceutically acceptable carriers.

9. The use according to claim 8, characterized in that: The active ingredient is one or more of liquorice extract, ophiopogon extract, peppermint extract, vitamin C and its derivatives, and vitamin E and its derivatives.

10. The use according to any one of claims 7 to 9, characterized in that: The content of ginsenoside Rh4 in the medicine or health product is 2.0% to 45.0%.

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