Use of a proteus bacteria in the preparation of a product for improving male fertility and preventing, ameliorating and / or treating alcoholic liver damage

Abstract

The present application provides the use of the Duganella in the preparation of a product for improving male reproductive capacity and preventing, improving and / or treating alcoholic liver injury. The present inventors have found that the Duganella, especially Duganella australiense, can significantly improve the decrease in male reproductive capacity caused by alcohol and alcoholic liver injury. In addition, the present application also provides a product comprising Duganella and Cistanche extract. The present inventors have found that the combined use of Duganella, especially Duganella australiense, and Cistanche extract has a significant synergistic effect in improving male reproductive capacity and treating alcoholic liver injury, and therefore has important clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and / or food. Specifically, this invention relates to the use of Donorubicin in the preparation of products for improving male fertility and preventing, improving, and / or treating alcoholic liver injury. Background Technology

[0002] In recent years, male reproductive health issues have become increasingly prominent. Alcohol is a widespread social beverage, but its negative impact on male fertility has been confirmed by numerous studies. Long-term heavy drinking can damage the male reproductive system, primarily manifested as reduced sperm count, decreased sperm motility, and an increase in abnormal sperm, even leading to azoospermia. Furthermore, alcohol can interfere with the hypothalamic-pituitary-gonadal axis, leading to decreased testosterone levels and increased gonadotropin levels, thereby affecting sperm production and maturation.

[0003] Although existing research has revealed the mechanisms by which alcohol damages male reproductive capacity, current technologies for improving this damage still have many shortcomings. For example, some studies have attempted to alleviate alcohol-induced oxidative stress by supplementing with antioxidants (such as lutein), but the effectiveness of these methods is limited.

[0004] Furthermore, alcoholic liver disease (ALD) is liver damage caused by long-term heavy alcohol consumption. Its clinical types include alcoholic fatty liver, alcoholic hepatitis, and liver fibrosis, and can even progress to cirrhosis and liver cancer. In recent years, the global incidence and mortality rates of ALD have been rising annually, becoming a significant public health issue. Acetaldehyde and reactive oxygen species (ROS) produced during alcohol metabolism are the main factors leading to liver damage. In addition, ALD not only affects patients' quality of life but also increases the medical burden and social costs. Therefore, developing effective treatment and prevention methods is of great significance for improving patients' quality of life and reducing the social burden.

[0005] However, there are currently no effective drugs for treating alcoholic liver injury in clinical practice. Commonly used drugs such as N-acetylcysteine ​​(NAC) and glucocorticoids have limited efficacy in treating alcoholic liver injury. Furthermore, the suitable populations and long-term effects of these drugs still require further validation.

[0006] In conclusion, developing a safe and effective drug to address alcohol-induced male reproductive impairment and alcoholic liver damage is of significant scientific and clinical value. Summary of the Invention

[0007] To address the above problems, the present invention aims to provide a product for improving male fertility and preventing, improving, and / or treating alcoholic liver injury. The inventors have discovered that the *Dubosiella* strain provided in this invention, particularly *Dubosiella newyorkensis* (DN), can significantly improve alcohol-induced decreased male fertility and alcoholic liver injury. Therefore, this invention provides the use of *Dubosiella* in the preparation of a product for improving male fertility and preventing, improving, and / or treating alcoholic liver injury.

[0008] The above-mentioned objective of the present invention is achieved by providing the following technical solution:

[0009] In a first aspect, the present invention provides the use of Donorubicin in the preparation of products for enhancing male fertility.

[0010] Preferably, the present invention provides the use of Donorum and Cistanche deserticola extracts in the preparation of products for improving male fertility.

[0011] According to some embodiments of the present invention, the ratio of Donorella vulgaris and Cistanche deserticola extract in the product is 2 × 10⁻⁶. 8 CFU: 1-50 mg, preferably 2×10 8 CFU: 1-10 mg, more preferably 2×10 8 CFU: 2-6 mg.

[0012] According to some embodiments of the present invention, the Cistanche deserticola extract is a water extract of Cistanche deserticola and / or an alcohol extract of Cistanche deserticola.

[0013] According to some embodiments of the present invention, the enhancement of male reproductive capacity is to enhance normal male reproductive capacity and / or prevent, improve and / or treat damage to male reproductive capacity.

[0014] According to some embodiments of the present invention, the male reproductive capacity impairment is caused by drugs, unhealthy lifestyle (such as drinking alcohol, high-fat diet) and / or environmental pollution (such as pesticides, environmental pollutants, microplastics), preferably male reproductive capacity impairment caused by alcohol; more preferably, the male reproductive capacity impairment caused by alcohol is selected from one or more of oligospermia, asthenospermia and testicular abnormalities.

[0015] Secondly, the present invention provides the use of Donorella vulgaris in the preparation of products for the prevention, improvement and / or treatment of alcoholic liver injury.

[0016] Preferably, the present invention provides the use of Donorum and Cistanche deserticola extracts in the preparation of products for the prevention, improvement and / or treatment of alcoholic liver injury.

[0017] According to some embodiments of the present invention, the ratio of Donorella vulgaris and Cistanche deserticola extract in the product is 2 × 10⁻⁶. 8 CFU: 1-50 mg, preferably 2×10 8 CFU: 1-10 mg, more preferably 2×10 8 CFU: 2-6 mg.

[0018] According to some embodiments of the present invention, the Cistanche deserticola extract is a water extract of Cistanche deserticola and / or an alcohol extract of Cistanche deserticola.

[0019] Thirdly, the present invention provides the use of Donorubicin in the preparation of products for providing adjunctive protection against alcoholic liver injury.

[0020] Preferably, the present invention provides the use of Donorum and Cistanche deserticola extracts in the preparation of products for providing adjunctive protection against alcoholic liver injury.

[0021] According to some embodiments of the present invention, the ratio of Donorella vulgaris and Cistanche deserticola extract in the product is 2 × 10⁻⁶. 8 CFU: 1-50 mg, preferably 2×10 8 CFU: 1-10 mg, more preferably 2×10 8 CFU: 2-6 mg.

[0022] According to some embodiments of the present invention, the Cistanche deserticola extract is a water extract of Cistanche deserticola and / or an alcohol extract of Cistanche deserticola.

[0023] According to some embodiments of the first, second and third aspects of the present invention, the Cistanche extract is derived from Cistanche deserticola and / or Cistanche tubulosa.

[0024] According to some embodiments of the first, second, and third aspects of the present invention, the viable count of *Dunaliella salina* in the product is not less than 1.0 × 10⁻⁶. 9 CFU / g, preferably 1.0 × 10⁻⁶ 9 -5.0×10 9 CFU / g.

[0025] According to some embodiments of the first, second, and third aspects of the present invention, the *Dunaliella* is *Dunaliella nymph* and / or *Dubosiella muris*, preferably *Dunaliella nymph*; preferably, the *Dunaliella nymph* has the accession number DSMZ No. 103457.

[0026] According to some embodiments of the first and second aspects of the present invention, the product is selected from one or more of pharmaceuticals, food, and health products; preferably, the food is a health food.

[0027] According to some embodiments of the first, second, and third aspects of the present invention, the dosage form of the product is selected from one or more of powders, suppositories, tablets, capsules, granules, oral liquids, and emulsions.

[0028] Fourthly, the present invention provides a product comprising Donorubicin and Cistanche deserticola extract.

[0029] According to some embodiments of the present invention, the ratio of Donorella vulgaris and Cistanche deserticola extract in the product is 2 × 10⁻⁶. 8 CFU: 1-50 mg, preferably 2×10 8 CFU: 1-10 mg, more preferably 2×10 8 CFU: 2-6 mg.

[0030] According to some embodiments of the present invention, the Cistanche deserticola extract is a water extract of Cistanche deserticola and / or an alcohol extract of Cistanche deserticola.

[0031] According to some embodiments of the present invention, the viable count of Donorella vulgaris in the product is not less than 1.0 × 10⁻⁶. 9 CFU / g, preferably 1.0 × 10⁻⁶ 9 -5.0×10 9 CFU / g.

[0032] According to some embodiments of the present invention, the *Dunaliella* is *Dunaliella nymph* and / or *Duncas murineis*, preferably *Dunaliella nymph*; preferably, the *Dunaliella nymph* has the accession number DSMZ No. 103457.

[0033] According to some embodiments of the present invention, the product is selected from one or more of pharmaceuticals, food, and health products; preferably, the food is a health food.

[0034] According to some embodiments of the present invention, the dosage form of the product is selected from one or more of powder, suppository, tablet, capsule, granule, oral liquid and emulsion.

[0035] According to some specific embodiments of the first to fourth aspects of the present invention, based on the total weight of the Cistanche deserticola extract, the Cistanche deserticola extract comprises: echinacoside 0.2-50% by weight and verbascoside 0.1-20% by weight.

[0036] Preferably, based on the total weight of the Cistanche deserticola extract, the content of echinacoside in the Cistanche deserticola extract is 1-10% by weight, preferably 1-5% by weight.

[0037] Preferably, based on the total weight of the Cistanche deserticola extract, the content of verbascoside in the Cistanche deserticola extract is 0.1-5% by weight, preferably 0.5-1% by weight.

[0038] According to some specific embodiments of the first to fourth aspects of the present invention, the Cistanche deserticola extract is prepared by a method comprising the following steps:

[0039] Cistanche deserticola was extracted with an extraction solvent, and then the obtained extract was concentrated under reduced pressure.

[0040] Preferably, the extraction includes: adding the pulverized Cistanche deserticola to the extraction solvent, heating and refluxing 2-5 times, each time for 1-4 hours, and combining the extracts.

[0041] Preferably, the extraction solvent is water and / or an aqueous solution of ethanol with a volume fraction of 10-50%.

[0042] Preferably, the weight ratio of the Cistanche deserticola to the volume of the extraction solvent is 1 kg: (2-20) L, and more preferably 1 kg: (6-10) L.

[0043] Preferably, the extract after compression at 40-60°C has a relative density of 1-2 after vacuum concentration, and more preferably, the extract after compression at 45-55°C has a relative density of 1-1.2.

[0044] In this invention, the term "relative density" refers to the ratio of the density of the analyte to the density of water at a given temperature.

[0045] The present invention has at least the following beneficial effects:

[0046] This invention provides novel uses of Donorubicin, particularly DN, in the preparation of products for improving male fertility and for the prevention, improvement, and / or treatment of alcoholic liver injury.

[0047] Specifically, the inventors have found that DN bacteria can significantly improve weight loss, decreased sperm count and motility, and abnormal testicular structure caused by excessive alcohol consumption. In addition, DN bacteria can significantly improve testicular oxidative stress, elevated inflammatory factors, and hormonal imbalances caused by excessive alcohol consumption. Furthermore, in normal mice, DN bacteria can effectively improve sperm motility, motility, and whipping frequency.

[0048] In addition, DN bacteria can significantly improve liver damage caused by excessive drinking, inhibit the elevation of transaminase caused by alcoholic liver damage, improve liver function, and reduce lipid deposition in the liver.

[0049] Furthermore, the inventors discovered that the combined use of DN bacteria and Cistanche deserticola extract has a significant synergistic effect in improving male reproductive capacity and treating alcoholic liver injury, thus showing important clinical application prospects. Attached Figure Description

[0050] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0051] Figure 1 This is the process for constructing the mouse alcohol model in the examples;

[0052] Figure 2 The figures show the body weight of mice in each group in the examples; where ns represents no significant difference, **p<0.01, ****p<0.0001;

[0053] Figure 3 The values ​​represent the sperm count, motility, and various motor abilities of mice in each group in the examples; where ns represents no significant difference, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001;

[0054] Figure 4 The images show the HE staining results of testicular tissue from mice in each group in the examples; the scale bar of the left image is 200 µm, and the scale bar of the right image is 100 µm.

[0055] Figure 5 The values ​​represent the levels of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the testicular tissue of mice in each group in the examples; where ns represents no significant difference, *p<0.05, **p<0.01, ****p<0.0001;

[0056] Figure 6 The values ​​represent the levels of tumor necrosis factor (TNF-α) and interleukin-1β (IL-1β) in the testicular tissue of mice in each group in the examples; where ns represents no significant difference, *p<0.05, **p<0.01, ***p<0.001;

[0057] Figure 7 The values ​​represent the levels of free testosterone (FT) in the testicular tissue of mice in each group and the levels of estrogen (E) and luteinizing hormone (LH) in their serum; where ns represents no significant difference, *p<0.05, **p<0.01, ***p<0.001;

[0058] Figure 8 The images show the HE staining results of mouse liver tissues from each group in the examples; the scale bar is 100 µm.

[0059] Figure 9 The values ​​represent the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and triglycerides (TG) in the liver tissue of mice in each group in the examples; where ns represents no significant difference, *p<0.05, ***p<0.001, and ****p<0.0001. Detailed Implementation

[0060] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are provided only to illustrate the invention and are not intended to limit its scope. Some of the experimental materials and calculation methods used in the following embodiments are as follows:

[0061] Experimental materials

[0062] DN bacteria: purchased from the German Center for Microbial and Cell Culture Collection (DSMZ), accession number DSMZ No. 103457.

[0063] PBS buffer: Na2HPO4 (disodium hydrogen phosphate): 8 mM; KH2PO4 (potassium dihydrogen phosphate): 2 mM; NaCl (sodium chloride): 137 mM; KCl (potassium chloride): 2.7 mM.

[0064] The water extract of Cistanche deserticola was obtained by the following method: 5 kg of Cistanche deserticola slices were crushed, 40 L of water was added and heated under reflux for 2 hours each time. The filtrates were combined and then concentrated under reduced pressure until the relative density reached 1.15 (measured at 50℃). The yield of the extract was calculated to be 38.5% by weight.

[0065] The contents of echinacoside and verbascoside were determined by high performance liquid chromatography. The results showed that, on a dried basis, the product contained 4.12% echinacoside by weight and 0.65% verbascoside by weight.

[0066] The ethanol extract of Cistanche deserticola was obtained by the following method: 5 kg of Cistanche deserticola slices were crushed and added to 40 L of 30% ethanol aqueous solution. The solution was heated under reflux for 2 hours each time. The filtrates were combined and then concentrated under reduced pressure until the relative density reached 1.15 (measured at 50℃). The yield of the extract was calculated to be 40.6% by weight.

[0067] The contents of echinacoside and verbascoside were determined by high performance liquid chromatography. The results showed that, on a dried basis, the product contained 4.25% by weight of echinacoside and 0.89% by weight of verbascoside.

[0068] Method for calculating the synergy index of drug combination

[0069] The Jin Zhengjun q-value method is adopted, where q = P A+B / (PA +P B −P A ×P B );

[0070] In the formula, P A is the effect of drug A when used alone; P B is the effect of drug B when used alone; P A+B is the effect of the combined use of drug A and drug B; wherein P A , P B and P A+B are calculated as: (experimental result of the corresponding experimental group − experimental result of the model group) / (experimental result of the normal group − experimental result of the model group);

[0071] Wherein, q<1 indicates that an antagonistic effect is produced after the combined use of the two drugs; q>1 indicates that a synergistic effect is produced after the combined use of the two drugs, and q=1 indicates that an additive effect is produced after the combined use of the two drugs.

[0072] In addition, the "%" of ethanol in the following examples refers to volume fraction.

[0073] Examples

[0074] 1 Experimental method

[0075] 1.1 Bacterial culture

[0076] Dissolve 36.5 g of solid GAM medium (Qingdao Haibo Biotechnology Co., Ltd.) in 1000 mL of double distilled water, sterilize under high temperature and high pressure, then dispense into sterile tubes in an anaerobic incubator, inoculate 200 μL of DN bacterial solution into every 10 mL of medium, and incubate at a constant temperature of 37°C for 48 h until a large amount of white precipitate is produced at the bottom of the sterile tube. Centrifuge the cultured DN bacterial solution at 4°C and 4000 g for 30 min, discard the supernatant, wash twice with sterile PBS, then add sterile PBS for resuspension, and finally obtain a bacterial solution with a DN bacterial concentration of 1×10 9 CFU / mL for monobacterial colonization.

[0077] 1.2 Animal modeling method and sample collection

[0078] Male C57BL / 6 mice (6-8 weeks old, 21±2 g) were purchased from the Laboratory Animal Center of Peking University Health Science Center. The production license number is SCXK (Beijing) 2023-0013. The animals are kept in a clean-grade environment, the temperature of the animal room is 25±2°C, and the interval between light and dark is 12 h. After adaptive feeding for 3 days, the control liquid diet (i.e., Lieber-Decarli control liquid mouse diet, article number: Lieber-C) was administered on days 4-8 to make the animals adapt to the liquid feeding mode.

[0079] Experiment 1: Animals were randomly divided into 6 groups: control group (N), model group (M), normal animals given DN bacteria group (ND), normal animals given inactivated DN bacteria group (NM), model animals given DN bacteria group (MD), and model animals given inactivated DN bacteria group (MM), with 12 animals in each group. The dosage of DN bacteria was 10... 9 CFU / mL, 0.2mL / animal / day.

[0080] Experiment 2: Animals were randomly divided into 7 groups: control group (N), model group (M), model animals given DN bacteria (MD), model animals given Cistanche deserticola water extract (MW), model animals given DN bacteria and Cistanche deserticola water extract (MD+MW), model animals given Cistanche deserticola alcohol extract (MA), and model animals given DN bacteria and Cistanche deserticola alcohol extract (MD+MA), with 12 animals in each group. The dosage of DN bacteria was 10... 9 CFU / mL, 0.2mL / animal / day; the dosage of Cistanche deserticola water extract and Cistanche deserticola alcohol extract is 200 mg / kg body weight / day.

[0081] Starting on day 9, the control group was fed a control liquid diet, while the model group was fed a control liquid diet containing 5% ethanol (i.e., Lieber-Decarli alcohol liquid rat food). Mouse weight and food intake were recorded daily to ensure consistent feed volume per cage, thus controlling daily caloric intake. On day 19, the model group underwent an alcohol overdose in the early morning using a 31.5% ethanol aqueous solution administered by gavage at a rate of 20 μL / mg body weight. The control group received the same amount of ultrapure water via gavage. Subsequently, the concentration of the ethanol aqueous solution used for gavage in the model group was adjusted to between 3% and 5% based on the mice's condition. The liquid diet was maintained until day 43 (see the animal model establishment procedure for details). Figure 1 Except for the control group and the model group, the other groups were given medication from day 9 until the end, for a total of 35 days.

[0082] Animal samples were collected on day 44 of the experiment. Blood was collected in EP tubes by enucleation, followed by cervical dislocation and euthanasia. Bilateral testes and epididymis of the mice were dissected, weighed and preserved, and liver and testicular tissue samples were frozen at -80°C.

[0083] Serum preparation: Blood samples collected by enucleation were allowed to stand for 2-4 hours, then centrifuged at 4000 rpm for 10 minutes at room temperature, and the supernatant was used as the serum sample.

[0084] Preparation of testicular tissue homogenate: Weigh 15-20 mg of mouse testicular tissue, add physiological saline at a ratio of 1 mg / 9 μL, homogenize in a benchtop homogenizer at a speed of 6 m / s for 40 seconds, and centrifuge at 15000 rpm for 10 min at 4℃. Collect the supernatant as testicular tissue homogenate.

[0085] Preparation of liver tissue homogenate: Weigh 15-20 mg of mouse liver tissue, add physiological saline at a ratio of 1 mg / 10 μL, homogenize in a benchtop homogenizer at 6 m / s for 40 seconds, and centrifuge at 15000 rpm for 10 min at 4℃. Collect the supernatant as liver tissue homogenate.

[0086] 1.3 Pathological evaluation

[0087] Three mice were randomly selected from each group as samples. Unilateral testis and liver tissue were harvested, washed in physiological saline, and immediately fixed in 4% (mg / mL) paraformaldehyde pre-cooled to 4°C for HE staining. The extent of damage to the testicular and liver tissues was evaluated by observing the morphology of the tissue cells in the sections.

[0088] 1.4 Determination of biochemical factor levels in biological samples

[0089] Testicular homogenate samples from 6-8 mice in each group were randomly selected. The levels of FT (Beijing Femoro Biotechnology Co., Ltd., FM3306A-96T), SOD (Jiangsu Kaiji Biotechnology Co., Ltd., KGT00150), GSH-Px (Nanjing Jiancheng Biotechnology Institute, C009-2-1), IL-1β (EcoSy Biotechnology Co., Ltd., EM001) and TNF-α (EcoSy Biotechnology Co., Ltd., EM008) in the testicular homogenate were detected according to the kit instructions below.

[0090] Serum samples from 6-8 mice were randomly selected in each group. The levels of LH (China, Beijing Femoro Biotechnology Co., Ltd., FM3318A-96T) and E (China, Beijing Femoro Biotechnology Co., Ltd., FM43282-96T) in the serum samples were detected according to the instructions of the following kits.

[0091] Liver homogenate samples from 6-8 mice were randomly selected in each group, and the levels of ALT, AST and TG were detected according to the instructions of each kit (China, Nanjing Jiancheng Biotechnology Institute, C009-2-1, C010-2-1, A110-1-1).

[0092] 1.5 Determination of mouse sperm motility based on computer-aided sperm analysis

[0093] Immediately after cervical dislocation, the epididymal tail of mice was placed in 1 mL of preheated physiological saline (SLYS, Beijing Solarbio Science & Technology Co., Ltd., China), chopped, and thoroughly mixed. The tube was then incubated in a water bath at 37°C for 5 min to allow all sperm to migrate. 10 μL of HTF culture medium containing sperm was transferred to a glass slide for observation under a microscope. At least five fields of view were selected, and computer-assisted sperm analysis (CASA) was used to analyze various parameters of mouse sperm. During the analysis, the solution was maintained at 37°C to avoid interfering with sperm motility or damaging sperm morphology.

[0094] This experiment used computer-aided sperm analysis technology to detect sperm count, percentage of total active sperm (Grade A+B), percentage of Grade A sperm (Grade A), sperm motility curve velocity (VCL), linear velocity (VSL), average path velocity (VAP), percentage of forward sperm motility (STR), sperm whipping frequency (BCF), and sperm head lateral displacement amplitude (ALH) to evaluate and analyze sperm count and motility in mice.

[0095] 1.6 Statistical Analysis

[0096] Experimental data were statistically analyzed using GraphPad Prism 8 software. Data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used to compare data among multiple groups, and p < 0.05 was considered statistically significant.

[0097] 2. Results of Experiment 1

[0098] 2.1 Digestive nephritis (DN) improves weight loss caused by excessive alcohol consumption.

[0099] Alcohol consumption caused mice to exhibit poor coat coloration, trembling, lethargy, and other symptoms. Furthermore, if... Figure 2 As shown, compared with group N mice, group M mice had significantly lower body weight, group MD mice had significantly recovered body weight, while group MM mice supplemented with inactivated DN bacteria showed no significant change in body weight. Furthermore, for normal mice, supplementation with either DN bacteria or inactivated DN bacteria had no significant effect on mouse body weight.

[0100] 2.2 Digestive nephritis (DN) bacteria improve decreased sperm count and motility in mice caused by excessive alcohol consumption.

[0101] The sperm count and sperm motility of mice were assessed by detecting the following parameters in each group of mice: sperm count, percentage of total active sperm, percentage of grade A sperm, sperm motility curve velocity, linear velocity, average path velocity, percentage of forward-moving sperm, sperm whipping frequency, and sperm head lateral displacement amplitude.

[0102] Test results as follows Figure 3 As shown, in the model group mice, supplementation with DN bacteria can effectively increase sperm count, motility, and sperm motility, and improve oligoasthenospermia caused by excessive alcohol consumption. It is noteworthy that in normal mice, DN bacteria supplementation can also effectively improve sperm motility, motility, and whiplash frequency.

[0103] 2.3 DN bacteria improve testicular structural abnormalities caused by excessive alcohol consumption

[0104] HE staining microscopic images of testicular histopathological sections ( Figure 4 The results showed that the testicular tissue of the control group mice was normal, with clear cell morphology and multiple layers of normal spermatogenic cells visible in the seminiferous tubules, and no large areas of vacuoles in the lumen. In contrast, the spermatogenic cells in the testicular tissue of the model group mice were damaged and atrophied, the cell layers of the seminiferous tubules were arranged in a disordered manner, and vacuoles appeared in the lumen. Compared with the model group, after administration of DN bacteria, the number of spermatogenic cells in the testicular tissue of mice increased, the damaged state was improved, the arrangement was more orderly, and the lumen of the seminiferous tubules recovered to a dense morphology. However, for normal mice, administration of DN bacteria or inactivated DN bacteria had no significant effect on the testicular tissue.

[0105] 2.4 Dihydrotestosterone (DN) significantly improved oxidative stress, elevated inflammatory factors, and hormonal imbalances in the testes of mice induced by excessive alcohol consumption.

[0106] The results of the detection of SOD and GSH-Px levels in mouse testicular homogenate samples are as follows: Figure 5 As shown, compared with the control group, the activities of SOD and GSH-Px in the testicular tissue of model group mice were significantly reduced; compared with the model group, the activities of SOD and GSH-Px in testicular tissue were significantly increased after administration of DN bacteria, while no significant changes were observed after administration of inactivated DN bacteria. This indicates that DN bacteria can regulate oxidative stress in mouse testes, increase the activity of antioxidant enzymes in testicular tissue, enhance antioxidant capacity, and thus reduce oxidative damage to the testes.

[0107] In addition, such as Figure 6 As shown, compared with group N, the levels of TNF-α and IL-1β in the testes of group M were significantly increased; compared with group M, the levels of IL-1β and TNF-α in group MD were significantly decreased, while the levels of IL-1β and TNF-α in group MM were not significantly changed. These results indicate that administration of DN bacteria can significantly reduce inflammatory factors in testicular tissue, inhibit the inflammatory response, and exert a protective effect.

[0108] The results of the detection of FT levels in mouse testicular homogenate samples and LH and E levels in serum samples are as follows: Figure 7 As shown, compared with group M, group MD showed significantly increased FT levels and significantly decreased LH and E levels. These results indicate that administration of DN bacteria can significantly improve hormonal imbalances related to the male reproductive system caused by excessive alcohol consumption. Furthermore, compared with the normal group, the FT levels in testicular homogenate samples from normal mice (ND group) administered DN bacteria were significantly increased.

[0109] 2.5 Digestive nephrotic syndrome (DN) improves liver damage caused by excessive alcohol consumption.

[0110] HE staining results of mouse liver tissue are as follows Figure 8 As shown, hepatocytes in the normal group were neatly arranged, structurally intact, and had dense intercellular spaces; hepatocytes in the model group had disordered structure, irregular arrangement, loose intercellular spaces, and vacuoles. The morphology of hepatocytes in the model group significantly improved after administration of DN bacteria, while the morphology of the livers in the group given inactivated DN bacteria (MM) showed no improvement. This indicates that DN bacteria can protect hepatocyte morphology and reduce structural damage to the liver in ALD mice. Furthermore, in normal mice, administration of either DN bacteria or inactivated DN bacteria had no significant effect on the liver tissue.

[0111] Biochemical tests for ALT and AST showed that the ALT and AST levels in the model group mice were significantly higher than those in the normal group. In the model group, administration of DN bacteria significantly reduced ALT and AST levels, while administration of inactivated DN bacteria did not show significant changes. This indicates that DN bacteria can inhibit the increase in transaminases caused by alcoholic liver injury and improve liver function. Figure 9 In addition, such as Figure 9 As shown, compared with the normal group of mice, the TG content in the liver of the model group mice was significantly increased, while the TG level in the MD group was significantly decreased, indicating that DN bacteria can reduce lipid deposition in the liver of mice with alcoholic liver injury.

[0112] 3. Results of Experiment 2

[0113] 3.1 Synergistic effect of DN bacteria and Cistanche deserticola extract in improving decreased sperm count and motility in mice induced by excessive alcohol consumption.

[0114] The number of sperm and the percentage of total active sperm in each group of mice were detected using CASA. The experimental results are shown in the table below.

[0115] Table 1 Comparison of sperm count in different groups of mice

[0116]

[0117] Note: Compared to N groups, # p<0.05; compared to group M, *p<0.05.

[0118] Table 2 Comparison of the percentage of total active sperm in different groups of mice

[0119]

[0120] Note: Compared to N groups, # p<0.05; compared to group M, *p<0.05.

[0121] As shown in the table above, compared with the model group, administration of DN bacteria, Cistanche deserticola water extract, or Cistanche deserticola alcohol extract alone significantly improved the decrease in sperm count and motility in mice caused by excessive alcohol consumption. Further research revealed that simultaneous administration of DN bacteria and Cistanche deserticola water extract, or simultaneous administration of DN bacteria and Cistanche deserticola alcohol extract, exhibited a synergistic effect, more effectively improving the decrease in sperm count and motility in mice caused by excessive alcohol consumption, and effectively alleviating the negative effects of excessive alcohol consumption.

[0122] 3.2 Synergistic effect of DN bacteria and Cistanche deserticola extract in improving oxidative stress, elevated inflammatory factors, and hormonal imbalance in mouse testes induced by excessive alcohol consumption.

[0123] Table 3 Comparison of SOD levels in the testes of different groups of mice

[0124]

[0125] Note: Compared to N groups, # p<0.05; compared to group M, *p<0.05.

[0126] The results of SOD level detection in mouse testicular homogenate samples are shown in the table above. The results indicate that, compared with the model group, the SOD level in mouse testicular tissue was significantly increased after administration of DN bacteria, Cistanche deserticola water extract, or Cistanche deserticola alcohol extract alone. Further research revealed that simultaneous administration of DN bacteria and Cistanche deserticola water extract, or simultaneous administration of DN bacteria and Cistanche deserticola alcohol extract, exhibited a synergistic effect, more effectively increasing the SOD level in mouse testicular tissue and significantly enhancing the antioxidant stress capacity of mouse testes.

[0127] Table 4 Comparison of TNF-α levels in the testes of different groups of mice

[0128]

[0129] Note: Compared to N groups, # p<0.05; compared to group M, *p<0.05.

[0130] The results of TNF-α level detection in mouse testicular homogenate samples are shown in the table above. The results indicate that, compared with the model group, administration of either *Dendrobium nobile*, *Cistanche deserticola* aqueous extract, or *Cistanche deserticola* alcohol extract significantly reduced TNF-α levels in mouse testicular tissue. Further research revealed that simultaneous administration of *Dendrobium nobile* and *Cistanche deserticola* aqueous extract, or simultaneously administration of *Dendrobium nobile* and *Cistanche deserticola* alcohol extract, exhibited a synergistic effect, more effectively reducing TNF-α levels in testicular tissue and inhibiting the inflammatory response.

[0131] Table 5 Comparison of testicular FT levels in different groups of mice

[0132]

[0133]

[0134] Note: Compared to N groups, # p<0.05; compared to group M, *p<0.05.

[0135] The results of FT level detection in mouse testicular homogenate samples are shown in the table above. The results indicate that, compared with the model group, administration of either DN bacteria, Cistanche deserticola water extract, or Cistanche deserticola alcohol extract significantly increased FT levels in mouse testicular tissue. Further research revealed that simultaneous administration of DN bacteria and Cistanche deserticola water extract, or simultaneous administration of DN bacteria and Cistanche deserticola alcohol extract, exhibited a synergistic effect, further increasing FT levels in mouse testicular tissue and more effectively improving hormonal imbalances related to the male reproductive system caused by excessive alcohol consumption.

[0136] 3.3 Synergistic effect of DN bacteria and Cistanche deserticola extract in improving liver damage caused by excessive alcohol consumption

[0137] Table 6 Comparison of liver ALT levels in different groups of mice

[0138]

[0139] Note: Compared to N groups, # p<0.05; compared to group M, *p<0.05.

[0140] Table 7 Comparison of liver AST levels in different groups of mice

[0141]

[0142] Note: Compared to N groups, # p<0.05; compared to group M, *p<0.05.

[0143] The results of ALT and AST detection in mouse liver tissue are shown in the table above. The results indicate that, compared with the model group, administration of either DN bacteria, Cistanche deserticola water extract, or Cistanche deserticola alcohol extract significantly reduced ALT and AST levels in mouse liver tissue. Further research revealed that simultaneous administration of DN bacteria and Cistanche deserticola water extract, or simultaneous administration of DN bacteria and Cistanche deserticola alcohol extract, exhibited a synergistic effect, further reducing ALT and AST levels in mouse liver tissue, more effectively inhibiting the elevation of transaminases caused by alcoholic liver injury, and improving liver function.

[0144] The above descriptions are merely several exemplary embodiments of the present invention and are not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any equivalent or related embodiments obtained by those skilled in the art through some modifications or variations made to the above-disclosed technical content without departing from the scope of the present invention are within the scope of the present invention.

Claims

1. Use of Donorubicin in the preparation of products intended to enhance male fertility.

2. Use of Donorum and Cistanche deserticola extracts in the preparation of products for enhancing male fertility.

3. The use according to claim 2, wherein, The ratio of Donorella vulgaris and Cistanche deserticola extract in the product is 2 × 10⁻⁶. 8 CFU: 1-50 mg, preferably 2×10 8 CFU: 1-10 mg, more preferably 2×10 8 CFU: 2-6 mg; Preferably, the Cistanche deserticola extract is a water extract of Cistanche deserticola and / or an alcohol extract of Cistanche deserticola.

4. The use according to any one of claims 1 to 3, wherein, The improvement of male reproductive capacity refers to improving normal male reproductive capacity and / or preventing, improving and / or treating damage to male reproductive capacity; Preferably, the male reproductive capacity impairment is alcohol-induced male reproductive capacity impairment; More preferably, the alcohol-induced male reproductive impairment is selected from one or more of oligospermia, asthenospermia, and testicular abnormalities.

5. Use of Donorubicin in the preparation of products for the prevention, improvement and / or treatment of alcoholic liver injury.

6. Use of Donorum and Cistanche deserticola extracts in the preparation of products for the prevention, improvement and / or treatment of alcoholic liver injury.

7. The use according to claim 6, wherein, The ratio of Donorella vulgaris and Cistanche deserticola extract in the product is 2 × 10⁻⁶. 8 CFU: 1-50 mg, preferably 2×10 8 CFU: 1-10 mg, more preferably 2×10 8 CFU: 2-6 mg; Preferably, the Cistanche deserticola extract is a water extract of Cistanche deserticola and / or an alcohol extract of Cistanche deserticola.

8. Use of Donorubicin in the preparation of products intended to provide adjunctive protection against alcoholic liver injury.

9. Use of Donorum and Cistanche deserticola extracts in the preparation of products that provide adjunctive protection against alcoholic liver injury.

10. The use according to claim 9, wherein, The ratio of Donorella vulgaris and Cistanche deserticola extract in the product is 2 × 10⁻⁶. 8 CFU: 1-50 mg, preferably 2×10 8 CFU: 1-10 mg, more preferably 2×10 8 CFU: 2-6 mg; Preferably, the Cistanche deserticola extract is a water extract of Cistanche deserticola and / or an alcohol extract of Cistanche deserticola.

11. The use according to any one of claims 1 to 10, wherein, The viable count of Donorella vulgaris in the product is not less than 1.0 × 10⁻⁶. 9 CFU / g, preferably 1.0 × 10⁻⁶ 9 -5.0×10 9 CFU / g; Preferably, the Durococcus is Durococcus nystomatus and / or Durococcus mutans, more preferably Durococcus nystomatus; more preferably, the Durococcus nystomatus has the accession number DSMZ No. 103457; Preferably, the product is selected from one or more of pharmaceuticals, food, and health products; more preferably, the food is a health food. Preferably, the dosage form of the product is selected from one or more of powder, suppository, tablet, capsule, granule, oral liquid and emulsion.

12. A product comprising Donepezil and Cistanche deserticola extract.

13. The product according to claim 12, wherein, The ratio of Donorella vulgaris and Cistanche deserticola extract in the product is 2 × 10⁻⁶. 8 CFU: 1-50 mg, preferably 2×10 8 CFU: 1-10 mg, more preferably 2×10 8 CFU: 2-6 mg; Preferably, the Cistanche deserticola extract is a water extract of Cistanche deserticola and / or an alcohol extract of Cistanche deserticola; Preferably, the viable count of Donorella vulgaris in the product is not less than 1.0 × 10⁻⁶. 9 CFU / g, preferably 1.0 × 10⁻⁶ 9 -5.0×10 9 CFU / g; Preferably, the Durococcus is Durococcus nystomatus and / or Durococcus mutans, more preferably Durococcus nystomatus; more preferably, the Durococcus nystomatus has the accession number DSMZ No. 103457; Preferably, the product is selected from one or more of pharmaceuticals, food, and health products; more preferably, the food is a health food. Preferably, the dosage form of the product is selected from one or more of powder, suppository, tablet, capsule, granule, oral liquid and emulsion.

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