Application of xanthoxylin in preparation of product for treating dyszoospermia

Abstract

The invention discloses application of xanthoxylin in preparation of a product for treating dyszoospermia, and belongs to the technical field of biological medicines. The invention provides an application of xanthoxylin in preparation of products for maintaining and recovering cryptorm testis spermatogenesis and products for improving cryptorm testis structures and repairing testis. By designing experimental groups with different administration dosages of xanthoxylin and combining with comprehensive analysis of data such as mouse sperm quantity, organ index and tissue slice, the treatment effect of LM100mg / kg dosage is most prominent, the testis tissue structure of cryptorchis mouse can be improved, the expression of spermatogenesis related markers is promoted, and the clinical application prospect is broad. Therefore, the effects of repairing testis and improving spermatogenesis capability are achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of peppermint in the preparation of products for treating spermatogenesis disorders. Background Art

[0002] With the continuous development of social industry, environmental pollution is becoming more and more serious, and humans are increasingly affected by biochemical products, which has led to a gradual decline in the quality of male sperm. According to research data, the proportion of azoospermia, oligospermia and asthenospermia has increased in male infertility patients; at the same time, sperm density, vitality and survival rate have also shown a downward trend. Some experts pointed out that infertility has become the third largest disease endangering human health after tumors and cardiovascular diseases. The impact of lifestyle on male fertility cannot be ignored. Modern society has a fast pace and high work pressure. Many men are in a state of tension and anxiety for a long time. This mental stress can cause endocrine system disorders, thereby affecting the production and quality of sperm. In addition, irregular work and rest habits can also disrupt the biological clock and further damage the health of sperm.

[0003] In addition to clear causes such as genetic factors, gonad infection, acquired testicular damage, and immune factors, about 70% of male infertility has no clear cause. Male infertility can be mainly divided into congenital, acquired, and idiopathic. The most common cause of male congenital infertility is primary testicular failure. The treatment of congenital infertility includes removing sperm from the testicles to obtain sperm for immediate use in assisted reproductive technology (ART) cycles or cryopreservation for later use. Acquired infertility is defined as the absence of sperm in the ejaculate due to obstruction of sperm transport. Some acquired diseases can be improved through product treatment, acupuncture, and intensive exercise. Idiopathic infertility is also called unexplained infertility, which refers to a type of infertility in which the cause of the infertility cannot be found using existing diagnostic methods.

[0004] Cryptorchidism is a common male infertility disease that may lead to abnormal spermatogenesis and infertility. Under current medical conditions, the treatment for cryptorchidism is mainly surgical correction. Since surgical treatment cannot completely restore testicular function, finding products that can promote cryptorchidism spermatogenesis is of great significance for improving the fertility of cryptorchid patients.

[0005] Lemairamin is a compound extracted from natural plants and is widely used in traditional medicine to treat various diseases. Lemairamin belongs to the amide class of substances and is one of the numbing substances of pepper. Its molecular formula is C 16 H 25 NO. Lemairamin has many physiological functions, such as anesthesia, excitement, antibacterial, wind and dampness removal, insecticide and analgesia.

[0006] Lemairamin is an organic compound found in Zanthoxylum plants. It is particularly abundant in Chinese peppers and has a unique numbing taste. The chemical structure of lemairamin is a derivative of vanillin, specifically one of the compounds containing a benzoate group. Lemairamin has also attracted research interest in the medical field due to its effect on TRPV1 receptors, possibly as a treatment for local pain or other related diseases.

[0007] However, there is no related technical disclosure on the safety, dosage range and long-term use effect of Lemairamin for cryptorchidism. Summary of the invention

[0008] The purpose of the present invention is to provide an application of Lemairamin in the preparation of a product for treating spermatogenesis disorder. The symptoms of spermatogenesis disorder specifically include the characteristics of testicular failure to produce sperm, low sperm count, incomplete testicular structure, and poor testicular performance. The technology provides an application of Lemairamin in the preparation of a product for maintaining and restoring cryptorchid testicular spermatogenesis, improving cryptorchid testicular structure, and testicular repair products.

[0009] The present invention is achieved through the following technical solutions:

[0010] Application of peppermint in the preparation of products for treating spermatogenesis disorders.

[0011] Application of Xanthocyanin in the preparation of a product for maintaining and restoring spermatogenesis in cryptorchid testes, and application of the product in improving sperm count.

[0012] A product for maintaining and restoring cryptorchid testicular spermatogenesis, wherein the product uses peppermint as an active ingredient; the product also contains pharmaceutically acceptable excipients to prepare a pharmaceutically corresponding dosage form.

[0013] Preferably, the dosage of the anthocyanin is 25 to 200 mg / kg.

[0014] Preferably, the dosage of Xanthan gum is 100 mg / kg.

[0015] The invention discloses an application of pepper in the preparation of a product for improving cryptorchid testicular structure and testicular repair, and an application of the product in improving the testicular organ coefficient and protecting the testicular seminiferous tubule structure.

[0016] A product for improving cryptorchid testicular structure and testicular repair, the product uses peppermint as an active ingredient; the product also contains pharmaceutically acceptable excipients to prepare a pharmaceutically corresponding dosage form.

[0017] Preferably, the dosage of the anthocyanin is 25 to 200 mg / kg.

[0018] Preferably, the dosage of Xanthan gum is 100 mg / kg.

[0019] Preferably, the products include medicines and health products.

[0020] Compared with the prior art, the present invention has at least the following technical effects:

[0021] (I) The present invention provides an application of Lemairamin in the preparation of a product for treating spermatogenesis disorder. The symptoms of spermatogenesis disorder specifically include the characteristics of testicular failure to produce sperm, low sperm count, incomplete testicular structure, and poor testicular performance. This technology provides an application of Lemairamin in the preparation of a product for maintaining and restoring cryptorchid testicular spermatogenesis, improving cryptorchid testicular structure, and testicular repair products.

[0022] (ii) The present invention uses biological experiments to measure the organ coefficient (i.e., the ratio of organ weight to the total body weight of the animal), which is a common method for evaluating the effects of a particular treatment or product on organ health and function. The higher organ coefficient of LM100 indicates that it has better testicular repair ability.

[0023] (III) In the experiment, the present invention successfully established the CO cryptorchidism model, and no sperm was detected in the cryptorchidism group mice, which verified the validity of the model. In the BO normal control group, mice showed a higher sperm count, which provided a strong basis for subsequent comparison of results.

[0024] Regarding the increase in sperm count in each group of mice treated with Lemairamin, although they did not reach the level of the BO normal control group, they still showed a certain degree of positive effect. Especially the LM100 group, they showed a significant effect in improving sperm count, exceeding the performance of other experimental groups. It is further pointed out that the product has a certain promoting effect on spermatogenesis.

[0025] (IV) In the experiment of the present invention, the testicular tissue of CO cryptorchidism model mice showed obvious pathological changes, such as enlarged seminiferous tubule cavity, disordered cell arrangement and thinning of the tubule wall. These phenomena indicate that the high temperature environment caused by the operation has a negative impact on the spermatogenesis process. However, the testicular tissue of mice treated with LM25 and LM100 of pepper showed a healthier seminiferous tubule structure, more orderly cell arrangement, smaller tubule cavity, and the presence of mature sperm, which shows that these compounds can protect the spermatogenesis process to a certain extent. The results highlight the importance of product dosage for spermatogenesis.

[0026] (V) The present invention designs experimental groups with different dosages of Lemairamin. Combined with comprehensive analysis of data such as mouse sperm count, organ index, and tissue sections, it is found that the therapeutic effect of LM100 mg / kg is the most prominent, and the repair ability of the testicles is the most significant.

[0027] The above conclusions can lay a foundation for a deeper understanding of the molecular mechanism of spermatogenesis in the future and generate new insights into the field of reproductive medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the left testis organ coefficient of each mouse group;

[0029] Figure 2 Schematic diagram of the right testicular organ coefficient of each mouse group;

[0030] Figure 3 Schematic diagram of the effect of pepper on serum hormone levels in cryptorchid mice;

[0031] Figure 4 Schematic diagram of the sperm count of mice in each group, with a scale of 20×10;

[0032] Figure 5 Schematic diagram of sperm counting of mice in each group;

[0033] Figure 6 Schematic diagram of HE staining microscope photography of each group of mice, with a scale of 20×10. DETAILED DESCRIPTION

[0034] The embodiments of the present invention will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0035] Experimental example:

[0036] 1. Experimental purpose: Based on the establishment of cryptorchid mouse model, the testicular tissue structure, spermatogenic cell proliferation and apoptosis, and sperm parameters were analyzed to comprehensively evaluate the effect of Lemairamin on spermatogenesis in cryptorchid mice.

[0037] 2. Experimental methods:

[0038] 2.1 Materials and Equipment

[0039] 2.1.1 Experimental materials: The main experimental materials are shown in Table 1

[0040] Table 1 Main experimental materials

[0041] Reagents Specification company 98% glacial acetic acid 500mL Merck Chemical Technology (Shanghai) Co., Ltd. Hematoxylin 30g Thermo Fisher Scientific 98% anhydrous ethanol 500mL Merck Chemical Technology (Shanghai) Co., Ltd. Potassium Alum 50g Bellambo Biotechnology (Hangzhou) Co., Ltd. Mercury oxide 100g Thermo Fisher Scientific Erythrocyte 50g Merck Chemical Technology (Shanghai) Co., Ltd. 98% concentrated hydrochloric acid 500mL Bio-Rad Biomedical Products 98% Formaldehyde 500mL Bio-Rad Biomedical Products Sodium dihydrogen phosphate 500g Bellambo Biotechnology (Hangzhou) Co., Ltd. Disodium hydrogen phosphate 500g Merck Chemical Technology (Shanghai) Co., Ltd. ammonia 500mL Guansen Biotech Shanghai Co., Ltd.

[0042] 2.1.2 Solution formulation

[0043] (1) Harris hematoxylin solution: 1 g of hematoxylin, 10 mL of anhydrous ethanol, 200 mL of distilled water, 1 to 2 drops of glacial acetic acid, and 1 g of Harris hematoxylin crystals. Dissolve them in 95% alcohol to a volume of about 10 mL. Add 1 to 2 drops of glacial acetic acid and adjust the pH to an acidic reaction. Filter the solution to remove insoluble matter.

[0044] (2) Water-soluble eosin stain: eosin powder: 1 g, 70% ethanol 95 mL, distilled water 5 mL, glacial acetic acid 10 mL. Add eosin powder to 70% ethanol and stir evenly. Add distilled water and glacial acetic acid and continue stirring until completely dissolved. Filter the solution to remove impurities. Store the solution in a brown bottle and protect from light.

[0045] (3) Fixative: 10 mL of paraformaldehyde solution (usually 37%) and 90 mL of phosphate buffered saline (PBS).

[0046] 2.1.3 Experimental equipment: The instruments required for this experiment are shown in Table 2.

[0047] Table 2 Instrument table

[0048] Experimental instrument name Part Number or Model Purchased Company Constant temperature and humidity incubator 9162 Merck Chemical Technology (Shanghai) Co., Ltd. Syringe Filters 0.45μm Merck Chemical Technology (Shanghai) Co., Ltd. Double ultra-clean workbench BL-100 Thermo Fisher Scientific Acidity meter Z527807 Merck Chemical Technology (Shanghai) Co., Ltd. Temperature controlled shaker MaxQ 6000 Thermo Fisher Scientific Water Purifier Milli-Q IQ 7000 Merck Chemical Technology (Shanghai) Co., Ltd. Chromatography columns 12mL Merck Chemical Technology (Shanghai) Co., Ltd. ELISA reader 130L-611E Berten Instruments, Inc. - USA Ultrasonic cell disruptor GS-150Y Guansen Biotech Shanghai Co., Ltd. Benchtop centrifuge DILITCEN 22 Suzhou Beirui Instrument Technology Co., Ltd. Vertical Pressure Steam Sterilizer SJ511C Junhe (Shanghai) Instrument Technology Co., Ltd. Ultra-low temperature storage box WD8660 Thermo Fisher Scientific Constant temperature incubator MP-100A Bellambo Biotechnology (Hangzhou) Co., Ltd. Pipette 10μL / 100μL / 1000μL Thermo Fisher Scientific Electrophoresis Apparatus PS0123 Thermo Fisher Scientific Mini gel electrophoresis tank A25977 Thermo Fisher Scientific Electric blast drying oven DHG-9023A(23L) Wuxi Marit Technology Co., Ltd. ChemiDoc MP All-in-one Imaging System 800-4-BIORAD Bio-Rad Biomedical Products

[0049] 2.2 Experimental procedures

[0050] 2.2.1 Establishment of cryptorchid mouse model

[0051] In this experiment, cryptorchidism surgery was used to construct a model in 10-day-old Kunming mice.

[0052] Anesthetized 10-day Kunming mice were placed in a wide-mouth bottle filled with ether and fixed on the operating table in a supine position. After wiping the abdomen of the mouse with 75% alcohol, the abdominal cavity of the mouse was incised, and the size of the wound was about 1cm±0.5mm. Then the fat head connected to the testicle and epididymis was sutured into the abdominal cavity, so that the testicle and epididymis were indirectly fixed in the abdominal cavity. Finally, the wound was sutured and disinfected. The mice were observed and recorded regularly after the operation.

[0053] 2.2.2 Dosage Grouping

[0054] Experimental grouping:

[0055] The mice were randomly divided into four groups, namely BO group (normal control group), CO group (cryptorchidism control group) and LMx (Xanthoxylum bungeanum group, i.e. experimental group) group (x represents dosage, which are 25 mg / kg, 50 mg / kg, 100 mg / kg and 200 mg / kg respectively).

[0056] The mice in the BO and CO groups received the same soybean oil injection treatment, and the mice in the LMx group received gradient treatment with different doses of Lemairamin.

[0057] In the BO and CO groups, the amount of soybean oil gavaged into mice was 10 μL / g, which was dynamically adjusted according to the body weight of the mice.

[0058] The dosage ratio of the LMx group is: 25 mg / kg means 25 mg is given to a 1 kg mouse; the preparation of the drug needs to be adjusted according to the method of administration.

[0059] Experimental operation process:

[0060] During the 35-day experimental period, the experimental mice were subcutaneously injected with soybean oil every day, and the BO and CO groups were injected with soybean oil, while the LMx group was injected with Lemairamin solution according to their respective dose gradients. The body weight of the mice was measured and the data were recorded at a fixed time every week.

[0061] Data processing method: All experimental data were statistically analyzed using GraphPad Prism 7.04 software.

[0062] 2.2.3 Dissection and sperm counting

[0063] Experimental steps:

[0064] On the 35th day after the end of the experiment, blood samples were collected from the eyeballs of the experimental mice, and then they were euthanized, and the weight of both testicles was weighed and recorded. One testicle was quickly frozen in liquid nitrogen and transferred to an ultra-low temperature freezer for subsequent histological analysis; the other testicle was placed in an EP tube containing warm phosphate buffered saline (PBS) for processing.

[0065] Sperm count: The collected paratesticular epididymal tissue was sheared and counted using a Myron sperm counting instrument. The data were analyzed using GraphPad Prism 7.04 software.

[0066] 2.2.4 HE staining

[0067] The testis is the reproductive gland of male animals, which has the function of producing gametes and endocrine. The testis is oval, with a layer of tunica vaginalis on the outside and a dense connective tissue on the deep part. It thickens at the posterior edge of the testis and enters the testis to form the testicular septum. The radial branches of the mediastinal connective tissue penetrate into the testicular substance and divide the substance into testicular lobules. Each lobule contains one or several winding and tortuous seminiferous tubules. The seminiferous tubules are composed of supporting cells and various germ cells and have the function of producing sperm. The interstitial cells between the seminiferous tubules have the function of producing androgens. The seminiferous tubules merge into straight seminiferous tubules at the tips of each lobule, enter the ductal network in the mediastinal connective tissue, and finally 10 to 30 testicular efferent ducts are separated from the testicular network and merge into the epididymal duct of the epididymal caput. Testicular cells are divided into spermatogenic cells and somatic cells. Somatic cells include interstitial cells, myoid cells and supporting cells, which constitute the microenvironment of the testis and are crucial for regulating normal spermatogenesis.

[0068] HE staining solution (also known as hematoxylin-eosin staining) is one of the commonly used staining methods in the field of cell biology and histology, mainly used to observe the morphology and structure of cells and tissues. The function of hematoxylin is to bind to the DNA and RNA in the cell nucleus to make the cell nucleus appear blue-purple. The function of eosin is to bind to the proteins and sugars in the cytoplasm to make the cytoplasm appear red or pink, thereby achieving the purpose of distinguishing the cell nucleus and cytoplasm, and clearly observing the structural details inside the cell. When studying testicular tissue, HE staining can help researchers observe the morphological structures of seminiferous tubules, supporting cells, interstitial cells, etc. The specific process is as follows:

[0069] (1) Fixation: Place the tissue sample in a 4% paraformaldehyde solution for fixation, usually for 24 hours.

[0070] (2) Dehydration and transparency: The fixed tissue samples need to be dehydrated in a series of alcohol solutions with increasing concentrations, generally including 70%, 80%, 90%, 95% and 100% alcohol. The soaking time of each concentration depends on the actual situation. The dehydrated tissue samples need to be transparentized with xylene.

[0071] (3) Wax immersion: Place the transparent tissue sample into melted paraffin, then cool and solidify to form a paraffin block.

[0072] (4) Sectioning: Use a microtome to cut the paraffin block into thin slices, usually with a thickness of 4-5 μm.

[0073] (5) Paste: Paste the cut tissue sections on a glass slide and gently dry them.

[0074] (6) Dewaxing: Remove the paraffin on the tissue sections, usually using xylene for dewaxing.

[0075] (7) Hydration: Dewaxed tissue sections need to be hydrated through a gradient of ethanol and water.

[0076] (8) Hematoxylin staining: Soak the tissue sections in hematoxylin solution for staining for about 5-10 minutes.

[0077] (9) Wash with distilled water: Rinse tissue sections with distilled water to remove excess hematoxylin dye.

[0078] (10) Eosin staining: Immerse the tissue sections in eosin staining solution for about 1-2 minutes.

[0079] (11) Wash with distilled water: Rinse tissue sections with distilled water to remove excess eosin dye.

[0080] (12) Differentiation: Place tissue sections in a 1% hydrochloric acid alcohol solution for differentiation. The time can be adjusted as needed, usually from a few seconds to tens of seconds.

[0081] (13) Dehydration: Tissue sections were dehydrated using a 95% and 100% ethanol series.

[0082] (14) Transparentization: Place tissue sections in xylene for transparentization.

[0083] (15) Sealing: Add sealing glue to the dehydrated and transparent slices and cover them with a coverslip.

[0084] (16) Microscopic observation: Use a microscope to observe and photograph tissue sections for further analysis and recording.

[0085] 3 Results Analysis

[0086] 3.1 Mouse body weight analysis

[0087] During the administration, the body weight of the mice was accurately measured and recorded every week.

[0088] Combined with the data in Table 3, the results show that the weight gain of mice in the LM100 group was particularly significant, significantly higher than that of other groups. In contrast, the average weight of mice in the CO group was relatively low, significantly lower than that of the BO group and the experimental group (LMx group).

[0089] The data showed that the LMx group had a significant effect on the recovery of physical signs in mice.

[0090] However, the weight gain of mice in the LM25, LM50, and LM200 groups was significantly lower than that in the CO group. The results suggested that there was an optimal dose of LMx group in promoting weight recovery in mice.

[0091] In addition, it was observed that the body weight of mice in the BO group was lower than that of mice in the LM100 group. This is because the mice in this group did not receive any product to counteract the effects of soybean oil, which resulted in limited weight gain.

[0092] The experimental results are shown in Table 3.

[0093] Table 3 Effect of Lemairamin on body weight of cryptorchid mice

[0094] Mouse grouping Dosage Weight(g) CO - 40.904±3.243 BO - 43.946±0.688 LM25 25 mg / kg 42.024±3.716 LM50 50mg / kg 41.910±4.527 LM100 100mg / kg 46.568±6.022 LM200 200mg / kg 42.166±3.001

[0095] The above Table 3 shows that the weight of mice was measured every week (n=5). BO is a normal mouse at 45 days old that was subcutaneously injected with soybean oil; CO is a cryptorchid mouse at 45 days old that was subcutaneously injected with soybean oil; LM25, LM50, LM100, and LM200 are the weights of cryptorchid mice at 45 days old that were subcutaneously injected with different doses of Lemairamin.

[0096] 3.2 Mouse testis analysis

[0097] 3.2.1 Spermatogenesis refers to the process by which sperm is produced by the seminiferous tubules in the testicles through a series of complex biological processes.

[0098] Table 4 Effect of Lemairamin on the organ coefficient of the left testis of cryptorchid mice (x±s, n=8)

[0099] Mouse grouping Dosage <![CDATA[Left testis (m*10 3 ) <!-- 6 -->]]> CO - 0.985±0.043 BO - 3.583±0.242 LM25 25 mg / kg 1.025±0.072 LM50 50mg / kg 1.031±0.110 LM100 100mg / kg 1.292±0.384 LM200 200mg / kg 1.062±0.126

[0100] Table 5 Effect of Lemairamin on the organ coefficient of right testis in cryptorchid mice (x±s, n=8)

[0101] Mouse grouping Dosage <![CDATA[Right testis (m*10 3 )]]> CO - 1.065±0.131 BO - 3.437±0.401 LM25 25 mg / kg 0.946±0.156 LM50 50mg / kg 1.181±0.043 LM100 100mg / kg 1.210±0.036 LM200 200mg / kg 1.081±0.211

[0102] The testicular organ coefficient shown in Tables 4 and 5 was calculated by the ratio of testicular weight to the body weight of the experimental mice.

[0103] The testicular organ coefficient can be used as a direct indicator of spermatogenesis ability. By analyzing the testicular organ coefficient, the development of the testicles under normal circumstances can be obtained.

[0104] like Figure 1 Shown is a schematic diagram of the left testicle organ coefficient of each mouse group.

[0105] like Figure 2 Shown is a schematic diagram of the right testicular organ coefficient of each mouse group.

[0106] The results show that Figure 1 and Figure 2 Among them, LM100 has the highest testicular organ coefficient, proving that LM100 has a strong ability to repair testicles.

[0107] The repair results of other experimental groups in this experiment were weaker, which proved that the product has a gradient property and the higher the concentration, the better the effect.

[0108] 3.2.2 Effects of pepper on serum hormone levels in cryptorchid mice

[0109] The process of spermatogenesis is regulated by multiple hormones such as gonadotropins and sex hormones.

[0110] Male reproduction is regulated by a variety of reproductive hormones in the hypothalamus-pituitary-testis axis, and changes in reproductive hormone levels can lead to damage or repair of male reproductive function. In this study, the ELISA method was used to determine the levels of testosterone (T), follicle-stimulating hormone (FSH), gonadotropin-releasing hormone (GnRH), total superoxide dismutase (T-SOD), estrogen (E), malondialdehyde (MDA), luteinizing hormone (LH) and other hormones in the serum of each group of mice.

[0111] like Figure 3 Shown is a schematic diagram of the effect of pepper on serum hormone levels in cryptorchid mice.

[0112] The results showed that cryptorchid surgery and different doses of pepper had a certain effect on hormone levels in cryptorchid mice.

[0113] Testosterone (T) levels are closely related to spermatogenesis in male mice, showing a positive correlation: the higher the testosterone level, the more active the spermatogenesis of the mice.

[0114] according to Figure 3 The data shown in A show that compared with the BO group (normal control group), the testosterone (T) level in the serum of male mice in the CO group (cryptorchidism control group) was significantly reduced (P < 0.01). This result clearly shows that cryptorchidism can lead to a decrease in the testosterone (T) level in the body.

[0115] Further observation revealed that as the concentration of anthocyanin increased, the testosterone content showed an upward trend, especially at a concentration of 100 mg / kg, where the best effect was achieved.

[0116] However, when the concentration of pepper increased to 200 mg / kg, the testosterone content showed a downward trend, indicating that within the dose range of 25-100 mg / kg, pepper intervention can effectively increase the testosterone (T) level of cryptorchid mice, and this increase effect is dose-dependent. However, when the pepper dose reaches or exceeds 100 mg / kg, its increase effect may no longer be obvious.

[0117] On the other hand, low concentrations of follicle-stimulating hormone (FSH) promote the formation of seminiferous tubules, but too high a concentration may suppress testosterone levels, thereby adversely affecting spermatogenesis.

[0118] Figure 3 The data in Figure B revealed that compared with the BO group, the level of follicle-stimulating hormone (FSH) in the serum of male mice in the CO group was slightly lower, but the difference did not reach significance (P>0.05).

[0119] This result suggests that cryptorchidism may lead to a decrease in the level of follicle-stimulating hormone (FSH) in the body. It is speculated that this may be because the mouse body attempts to further regulate the level of follicle-stimulating hormone (FSH) by increasing the formation of seminiferous tubules in order to cope with the damage caused by cryptorchidism surgery. Compared with the CO group, the FSH concentration in the P25, P50, and P200 groups (representing the 25 mg / kg, 50 mg / kg, and 200 mg / kg doses of huaxuexin treatment groups) treated with huaxuexin was significantly increased (P < 0.01). This finding suggests that within the dose range of 25-200 mg / kg, huaxuexin may promote the formation of seminiferous tubules by increasing the level of follicle-stimulating hormone (FSH), thereby helping to repair the damage to the testicles caused by cryptorchidism surgery.

[0120] Gonadotropin-releasing hormone (GnRH) plays an important role in promoting testosterone secretion and spermatogenesis.

[0121] like Figure 3 As shown in Figure C, compared with the BO group (normal control group), the gonadotropin-releasing hormone (GnRH) content in the serum of male mice in the CO group (cryptorchidism control group) was reduced, but it did not reach a significant difference (P>0.05). This result indicates that cryptorchidism surgery does not significantly affect the level of gonadotropin-releasing hormone (GnRH) in the body.

[0122] Further analysis found that compared with the CO group, the GnRH levels in the P25 and P50 groups (representing 25 mg / kg and 50 mg / kg doses of pepper treatment, respectively) that received pepper intervention were significantly decreased (P < 0.01), while no significant changes were observed in the other groups.

[0123] This finding suggests that within the dose range of 25-200 mg / kg, intervention with pepper may increase the gonadotropin-releasing hormone (GnRH) level of cryptorchid mice through some mechanism, thereby promoting the secretion of testosterone (T), which helps to repair the damage caused by cryptorchid surgery. However, it is worth noting that in the group treated with lower doses (25 mg / kg and 50 mg / kg) of pepper, the GnRH level was significantly reduced, which may be related to the specific mechanism of action of pepper and the difference in its effects at different doses, which requires further research to clarify.

[0124] High levels of estradiol (E) can adversely affect the quantity and quality of sperm, thereby reducing male fertility. Figure 4 The data shown in E show that compared with the BO group (normal control group), the estradiol (D) level in the serum of male mice in the CO group (cryptorchidism control group) was increased. Although this increase did not reach a significant difference (P>0.05), this result still suggests that the spermatogenesis process of cryptorchidism mice may be seriously affected.

[0125] Further analysis found that compared with the CO group, the levels of estradiol (E) in the P25, P50 and P100 groups (representing 25 mg / kg, 50 mg / kg and 100 mg / kg doses of cypermethrin treatment groups, respectively) treated with cypermethrin showed a certain degree of downward trend, and this trend was significantly different (P<0.05). However, no significant decrease in estradiol (E) levels was observed in the higher dose cypermethrin treatment group (≥200 mg / kg).

[0126] This result shows that within the dose range of 25-200 mg / kg, intervention with pepper may have the potential to reduce the level of estradiol (E) in mice, thereby helping to improve spermatogenesis. However, it is worth noting that this reduction seems to have a dose effect, that is, as the dose of pepper increases, its effect of reducing estradiol (E) levels does not continue to increase, but instead tends to stabilize or slightly decrease at high doses.

[0127] Malondialdehyde (MDA) is a hallmark product of lipid peroxidation. When a large number of oxygen free radicals are generated in the body, they react with the lipid part of sperm cells to produce MDA, which has a clear toxic effect on sperm cells.

[0128] according to Figure 3The data shown in E show that compared with the BO group (normal control group), the malondialdehyde (MDA) level in the serum of male mice in the CO group (cryptorchidism control group) increased, although this increase did not reach a statistically significant difference (P value close to 0.05), indicating that cryptorchidism surgery may lead to an increase in malondialdehyde (MDA) levels. This change may be that the mouse body attempts to reduce the malondialdehyde (MDA) level through some mechanism to cope with the oxidative stress damage caused by cryptorchidism surgery.

[0129] At the same time, the reduction of malondialdehyde (MDA) levels usually means a reduction in the products of lipid peroxidation, which may be due to the increase in the activity of antioxidant enzymes in the body, effectively limiting the occurrence of oxidative damage. However, after comparing the intervention groups with different doses of pepper: compared with the CO group, the MDA levels in the serum of mice in the P25, P50 and P200 groups treated with 25mg / kg, 50mg / kg and 200mg / kg pepper were increased, but these increases also did not reach significant differences (P>0.05). The other experimental groups did not show obvious changes in MDA levels.

[0130] This result shows that within a certain dosage range, pepper can increase the level of malondialdehyde (MDA) in mice, but this increase is not accompanied by a significant improvement in spermatogenesis, and there is a clear dose effect. The effect of pepper on MDA levels is not always positive, and its effect is strictly regulated by dosage.

[0131] Luteinizing hormone (LH) plays a vital role in the reproductive system. It not only promotes the secretion of testosterone (T), but also works synergistically with follicle-stimulating hormone (FSH) to ensure the normal progress of spermatogenesis.

[0132] according to Figure 3 The data shown in F show that compared with the BO group (normal control group), the luteinizing hormone (LH) level in the serum of male rats in the CO group (cryptorchidism control group) was decreased. Although this decrease did not reach a statistically significant difference (P>0.05), it still indicated that cryptorchidism surgery may have a certain inhibitory effect on the secretion of LH.

[0133] Further comparison of different doses of pepper intervention groups: Compared with the CO group, the LH level in the serum of the P100 group of mice treated with 100 mg / kg pepper was significantly reduced (P < 0.05), suggesting that pepper may have an inhibitory effect on the secretion of LH at a specific dose. However, when the pepper dose was increased to 200 mg / kg, the LH level of the P200 group of mice showed an upward trend, although this increase did not reach a significant difference (P> 0.05). In other experimental groups, the LH level did not change significantly.

[0134] This result shows that the effect of pepper on LH levels is dose-dependent, that is, different doses may show different effects. Low doses of pepper may inhibit the secretion of LH, while high doses of pepper may promote the secretion of LH, but this promoting effect did not reach statistical significance.

[0135] In summary, this study measured the serum reproductive hormone levels of mice by ELISA method and found that cryptorchidism surgery reduced testosterone (T) levels and affected spermatogenesis. The effects of pepper intervention on reproductive hormones are complex. Low doses (25-100 mg / kg) increase T and FSH levels, while high doses weaken the effect. The effect on GnRH is not in line with expectations, with low doses decreasing and high doses showing no significant changes. It can reduce estradiol (E) levels, but there is a dose effect. The effects on malondialdehyde (MDA) levels are inconsistent, with low doses possibly increasing and high doses showing no significant changes. The effects on luteinizing hormone (LH) are also dose-dependent, with low doses inhibiting and high doses promoting but not reaching significance. Cryptorchidism surgery can destroy the reproductive internal environment of mice, interfere with the secretion of multiple reproductive hormones in the male hypothalamus-pituitary-testis axis, destroy the spermatogenesis process, and change the levels of hormones related to mouse reproduction. Huajiaosu can significantly improve the changes in hormone levels caused by cryptorchidism surgery and promote spermatogenesis in mice. There is a dose effect, and 100 mg / kg may be the optimal concentration of Huajiaosu to promote spermatogenesis. 3.3 Analysis of sperm count in mice

[0136] Normal sperm should be tadpole-shaped, consisting of a head, body, and tail. Through HE staining, you can see that the acrosome is light red, the nucleus is located at the base of the acrosome, which is purple-red, and the tail is light purple-red. There are many types of abnormal sperm morphology, such as large head, small head, cone head, amorphous head, vacuolar head, double head, acrosome-free head, etc. These abnormal morphologies may be related to infection, trauma, androgen changes or chemical products and genetic factors.

[0137] During the mouse dissection, each experimental group was observed and recorded in detail. The specific operation steps included: placing the bilateral epididymis of each group of mice in a 1.5mL centrifuge tube containing 200μL phosphate buffered saline (PBS) and carefully processing it until the tissue fragments were no longer significantly visible. Subsequently, 10μL of supernatant was extracted and added to a sperm counting plate for accurate sperm count determination.

[0138] It is worth noting that no sperm could be detected in the mice of the CO cryptorchidism group, which confirmed the successful establishment of the cryptorchidism azoospermia model.

[0139] In contrast, the BO normal group showed a higher sperm count, which was significantly better than the other experimental groups.

[0140] Furthermore, it was found that the sperm count of each group of mice treated with Lemairamin increased. Although it was still lower than that of the BO normal control group, this difference revealed that the substance had a positive effect on mouse spermatogenesis to some extent.

[0141] like Figure 4 The figure shows the number of sperm in each group of mice. A: BO normal control group; B: CO cryptorchid control group; C: LM25 pepper experimental group; D: LM50 pepper experimental group; E: LM100 pepper experimental group; F: LM200 pepper experimental group. Scale: Microscope 20×10. In the figure, the red arrow marks the sperm.

[0142] like Figure 5 Shown is a schematic diagram of sperm counts in each group of mice.

[0143] like Figure 4 and Figure 5 The results showed that the sperm count in the LM100 group was particularly outstanding, exceeding that of other experimental groups.

[0144] The above conclusions not only demonstrate the effectiveness of the cryptorchidism model, but also prove the potential promoting effect of Lemairamin on spermatogenesis.

[0145] 3.4 Mouse HE staining

[0146] During the dissection, special attention was paid to changes in the mouse testicles.

[0147] The testicles of mice in each group were placed in Bouin's fixative, then embedded in paraffin and stained with HE.

[0148] like Figure 6 The following are the schematic diagrams of HE staining microscopy of each group of mice. A: BO normal control group; B: CO cryptorchid control group; C: LM25 pepper experimental group; D: LM50 pepper experimental group; E: LM100 pepper experimental group; F: LM200 pepper experimental group. The scale bar is 20×10 microscopy.

[0149] Depend on Figure 6 It can be seen that by observing and taking pictures under a microscope, subtle differences in tissues can be captured.

[0150] Observation of the testicular tissue sections of each group of mice under a light microscope revealed that:

[0151] The seminiferous tubules of the BO group were arranged regularly and had normal structures. The seminiferous tubules were composed of multiple layers of spermatogenic cells and supporting cells. From the base to the lumen, the spermatogenic cells in each layer were spermatocytes, spermatogonia, round spermatids, elongated spermatids and sperm, etc. The supporting cells were located between spermatocytes and spermatogonia, and the cells were arranged densely and regularly.

[0152] The testes of mice in the CO cryptorchidism group showed obvious abnormalities, including enlarged lumina of the seminiferous tubules, disordered cell arrangement, thinning of the tubular wall, and obvious germ cell fragments.

[0153] This suggests that the high temperature environment caused by the surgery interfered with the normal spermatogenesis process, resulting in impaired sperm production.

[0154] However, in mice treated with LM25 and LM100, the structure of the seminiferous tubules was relatively healthy, the cells were arranged more orderly, the lumen was smaller, and they contained mature sperm, showing a certain degree of protective effect.

[0155] Nevertheless, the cavities of seminiferous tubules in these treatment groups were still larger and the number of sperm was lower compared with the BO normal control group. In particular, in the Lemairamin LM50 group, the structural damage of seminiferous tubules was similar to that of the CO cryptorchidism control group, but the presence of mature sperm was still observed.

[0156] The results highlight the importance of product dosage for spermatogenesis and reveal a role for Lemairamin in maintaining or restoring spermatogenesis in mice.

[0157] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of peppermint in the preparation of products for treating spermatogenesis disorders.

2. The application of pepper in the preparation of a product for maintaining and restoring spermatogenesis in cryptorchid testes, characterized in that: Use of the product in improving sperm count.

3. A product for maintaining and restoring spermatogenesis in cryptorchid testes, characterized in that: The product uses peppermint as an active ingredient; the product also contains pharmaceutically acceptable auxiliary materials to prepare pharmaceutically corresponding dosage forms.

4. A product for maintaining and restoring spermatogenesis in cryptorchid testes according to claim 3, characterized in that: The dosage of the anthocyanin is 25 to 200 mg / kg.

5. A product for maintaining and restoring spermatogenesis in cryptorchid testes according to claim 4, characterized in that: The dosage of the peppermint is 100 mg / kg.

6. An application of pepper in preparing a product for improving cryptorchid testicular structure and testicular repair, characterized in that: The product is used in improving the testicular organ coefficient and protecting the testicular seminiferous tubule structure.

7. A product for improving cryptorchid testicular structure and testicular repair, characterized in that: The product uses peppermint as an active ingredient; the product also contains pharmaceutically acceptable auxiliary materials to prepare pharmaceutically corresponding dosage forms.

8. A product for improving cryptorchid testicular structure and testicular repair according to claim 7, characterized in that: The dosage of the anthocyanin is 25 to 200 mg / kg.

9. A product for improving cryptorchid testicular structure and testicular repair according to claim 8, characterized in that: The dosage of the peppermint is 100 mg / kg.

10. The product for improving cryptorchid testicular structure and testicular repair according to claim 8, characterized in that: The products include medicines and health products.

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