Application of abnormal wickerhamia in biofabrication of baohuoside I and application of baohuoside I in prevention and / or treatment of reproductive damage

Abstract

The application provides an application of abnormal Wickerham yeast in bio-manufacturing of baohuoside I and an application of baohuoside I in preventing and / or treating reproductive damage. The abnormal Wickerham yeast can efficiently convert Epimedium monomer into baohuoside I, and a product with a purity of greater than or equal to 96 % is obtained through separation and purification. Meanwhile, animal experiments show that the baohuoside I can significantly improve male kidney-reproductive double damage induced by a chemotherapeutic drug, improve the body weight, testis / epididymis weight and sperm quantity of a model animal, regulate the levels of serum testosterone, luteinizing hormone and follicle-stimulating hormone, reduce blood urea nitrogen and serum creatinine, and repair testicular, epididymal and kidney tissue pathological damage. The application provides a green and efficient baohuoside I bio-manufacturing process and an application of the baohuoside I in preparing a drug for resisting chemotherapy-induced reproductive damage and protecting reproductive and kidney functions, and has clinical value and industrialization prospect.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical manufacturing technology, specifically relating to the application of abnormal Wickham yeast in the biomanufacturing of cyproheptadine I and the application of cyproheptadine I in the prevention and / or treatment of reproductive damage. Background Technology

[0002] Chemotherapy drugs are an important tool in the treatment of malignant tumors and in the pretreatment of hematopoietic stem cell transplantation. However, while killing tumor cells, they also produce significant toxic side effects on normal tissues, especially the reproductive system and kidneys. Clinical studies have shown that many chemotherapy drugs can cause severe damage to testicular spermatogenesis; at the same time, some chemotherapy drugs also have nephrotoxicity, which can be accompanied by kidney damage. Currently, there is a lack of safe, effective, and highly targeted protective drugs against reproductive and kidney damage caused by chemotherapy drugs. For example, busulfan, a commonly used alkylating agent chemotherapy drug, has particularly prominent reproductive system and kidney toxicity, making it a typical representative of research on reproductive damage caused by chemotherapy drugs.

[0003] Epimedium is a traditional Chinese medicine used to tonify the kidneys and enhance male virility. Plutoside I is its core active flavonoid component, possessing pharmacological effects such as antioxidant and anti-inflammatory properties. Current methods for preparing Plutoside I mainly involve plant extraction and chemical semi-synthesis, which suffer from problems such as low extraction rates, high costs, harsh reaction conditions, and environmental pollution. Microbial transformation offers advantages such as mild conditions, high selectivity, environmental friendliness, and suitability for industrial production; however, a standardized biomanufacturing process for efficiently converting epimedium monomers into high-purity Plutoside I is currently lacking. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide the application of *Epimedium anomala* in the bio-production of cymosin I. *Epimedium anomala* can efficiently convert epimedium monomers to produce high-purity cymosin I, and has the characteristics of simple operation, low cost, and suitability for industrial production.

[0005] This invention provides abnormal Wickham yeast ( Wickerhamomyces anomalus Application of ) in the bio-manufacturing of cyproheptadine I.

[0006] This invention provides a method for the biosynthesis of icariin I, using icariin monomer as a substrate, biotransformation with Wickham yeast, and separation and purification of the transformed product to obtain icariin I.

[0007] Preferably, the biotransformation involves inoculating the abnormal Wickham yeast into an icariin monomer solution; Preferably, the inoculum size of the *Wickham's abnormal* yeast culture is 0.1% to 5%. Preferably, the effective viable cell concentration of the abnormal Wickham yeast culture is ≥1×10⁻⁶. 8CFU / mL.

[0008] Preferably, during the biotransformation, the biotransformation system further includes a carbon source; the added carbon source accounts for 2% to 8% of the total weight of the biotransformation system.

[0009] Preferably, the separation and purification method includes: removing bacterial cells from the transformation product, collecting the liquid phase, and extracting it with an extractant; removing the extractant from the obtained extract and performing column chromatography, eluting, collecting the eluent containing cyproheptadine I, and then purifying it to obtain cyproheptadine I.

[0010] This invention provides the use of cyproheptadine I in the preparation of medicaments for the prevention and / or treatment of reproductive damage.

[0011] Preferably, the reproductive injury includes chemotherapy-induced male kidney-reproductive injury.

[0012] Preferably, the reproductive injury includes at least one of the following: oxidative stress injury of reproductive organs, decreased reproductive organ weight, decreased sperm count, hypothalamic-pituitary-gonadal axis disorder, decreased renal function, and abnormal renal-reproductive tissue morphology.

[0013] Preferably, the hypothalamic-pituitary-gonadal axis disorder includes at least one of elevated luteinizing hormone levels, elevated follicle-stimulating hormone levels, and decreased testosterone levels.

[0014] Preferably, the decline in renal function includes elevated blood urea nitrogen and / or serum creatinine levels.

[0015] This invention provides the application of *Icaryopsis anomala* in the bioproduction of icariin I. The invention uses *Icaryopsis anomala* as a biocatalyst and icariin monomer as a substrate for microbial transformation. The transformation conditions are mild, requiring no strong acids, strong bases, or organic catalysts. The fermentation process is green and environmentally friendly, generating no toxic or harmful waste. After separation and purification, the HPLC purity of icariin I can reach over 96%, meeting the quality requirements of pharmaceutical-grade raw materials. Compared with traditional plant extraction and chemical semi-synthesis methods, this invention has significant advantages such as simple operation, low cost, environmental friendliness, high product purity, and suitability for industrial scale-up, providing a novel green manufacturing route for the large-scale production of icariin I.

[0016] This invention also provides an application of cyproheptadine I in renal-reproductive protection. Addressing the clinical challenge of lacking effective protective drugs against chemotherapy-induced damage to the male kidney and reproductive system, such as busulfan, this invention demonstrates through animal experiments that the obtained cyproheptadine I can significantly increase testicular / epididymal weight and sperm count in model animals, regulate serum testosterone, luteinizing hormone, and follicle-stimulating hormone levels, reduce blood urea nitrogen and serum creatinine, repair pathological damage to testicular, epididymal, and kidney tissues, and alleviate oxidative stress by regulating the Keap1 / Nrf2 / HO-1 / GPX4 signaling pathway, thereby achieving dual protection of the kidney and reproductive system. Therefore, this invention not only establishes a green and efficient new method for the biomanufacturing of cyproheptadine I, but also provides a new protective strategy for chemotherapy-induced reproductive and renal damage, possessing significant clinical application value and industrialization prospects. Attached Figure Description

[0017] Figure 1 The image shows the HPLC chromatogram of the process of converting icariin into icariin by *Saccharomyces cerevisiae*. The red line represents the mixed standard of icariin and cypermethrin I, the yellow line represents the icariin raw material, and the pink line represents the 21-day fermentation system of icariin by *Saccharomyces cerevisiae*. Figure 2 The results show the effects of cyproheptadine I on body weight, testicular weight, epididymal weight, and sperm count in mice treated with busulfan; Figure A represents body weight; Figure B represents testicular weight; Figure C represents epididymal weight; Figure D represents sperm count; compared with the control group: #### indicates p <0.0001, ### indicates p <0.001, ## indicates p <0.01, # indicates p <0.05; compared with the model group: express p <0.0001, express p <0.001, express p <0.01, express p <0.05; ns indicates no statistically significant difference; Figure 3 The results show the effects of cyproheptadine I on serum reproductive hormones and renal function indicators in mice treated with busulfan; where A represents testosterone (T), B represents luteinizing hormone (LH), C represents follicle-stimulating hormone (FSH), D represents blood urea nitrogen (BUN), and E represents serum creatinine (SCr). Figure 4Representative hematoxylin and eosin staining results of testicular tissue in each group are shown in the figure. Scale bar: 50 μm. Among them, Control is the blank control group, Model is the model group, WZY is the positive drug Wuzi Yanzong Wan group, YBL is the low-dose Baohuogan I group, and YBH is the high-dose Baohuogan I group. Figure 5 Representative hematoxylin-eosin staining results of epididymal tissues from each group. Scale bar: 100 μm; Figure 6 Representative hematoxylin-eosin staining results of kidney tissue from each group, scale bar: 50 μm; Figure 7 The effect of cyproheptadine I on the expression of Keap1, Nrf2, HO-1, and GPX4 proteins in testicular tissue was investigated. Among them, A represents the protein expression band results, B represents the relative expression level of Keap1 protein, C represents the relative expression level of Nrf2 protein, D represents the relative expression level of HO-1 protein, and E represents the relative expression level of GPX4 protein. Detailed Implementation

[0018] This invention provides the application of an abnormal Wickham yeast strain in the conversion of icariin monomers into cymosin I. In the embodiments of this invention, the abnormal Wickham yeast used was purchased from Beijing Beina Chuanglian Biotechnology Research Institute, strain number BNCC195349. The abnormal Wickham yeast described in this invention is not limited to the specific strain used in the embodiments; those skilled in the art can obtain abnormal Wickham yeast with the same function through conventional strain screening or commercial purchase, without any inventive effort.

[0019] This invention provides a method for the biomanufacturing of cymosin I, using icariin monomer as a substrate and employing *Saccharomyces cerevisiae* for biotransformation. The resulting transformation product is then separated and purified to obtain cymosin I. The icariin monomer is preferably icariin monomer with a purity ≥90%, and commercially available products are acceptable. In the embodiments of this application, icariin monomer with a purity ≥95% is used. High purity of the icariin monomer is beneficial for transformation and subsequent purification. *Saccharomyces cerevisiae* is commonly used in food fermentation to add flavor or in agricultural biocontrol. This application is the first to propose the use of *Saccharomyces cerevisiae* in the biomanufacturing of cymosin I. The *Saccharomyces cerevisiae* used can efficiently transform icariin to cymosin I. This transformation process is characterized by mild conditions, high selectivity, and few byproducts. It does not require the use of strong acids, strong bases, or organic catalysts, and has the advantages of being green, environmentally friendly, low-cost, and suitable for industrial production.

[0020] In this invention, the inoculation amount of the abnormal Wickham yeast culture is preferably 0.1% to 5%, and can be 1%, 2%, or 3%. The effective viable cell concentration of the abnormal Wickham yeast culture is preferably ≥1×10⁻⁶. 8 CFU / mL, which can be 5 × 10 8 CFU / mL, 1×10 9 CFU / mL or 5×10 9 CFU / mL. This effective viable bacteria concentration ensures a sufficient amount of bacteria in the biotransformation system, which can accelerate the transformation process and improve the transformation rate.

[0021] In this invention, during the biotransformation, the biotransformation system preferably further includes a carbon source, and the added carbon source preferably accounts for 2% to 8% of the total weight of the biotransformation system. The carbon source can be at least one of the following: glucose, sucrose, fructose, and soluble starch, etc. In this embodiment, glucose is used as the carbon source. The addition of the carbon source can provide the energy required for cell growth and prolong enzyme activity. The amount of carbon source added can be 4%, 5%, or 6%. In this embodiment, the added amount is 5% of the total weight of the fermentation system. Those skilled in the art can select the appropriate amount based on the scale of the fermentation system, the metabolic requirements of the strain, and the conversion efficiency of the target product.

[0022] In this invention, the preferred conditions for biotransformation are shaking fermentation at 25-30°C for 18-23 days. The biotransformation temperature can be 26°C, 27°C, or 28°C. The fermentation time can be 20 days, 21 days, or 22 days. In this embodiment, a shaking fermentation method at 27°C for 21 days is used. Those skilled in the art can make adaptive adjustments based on strain activity, substrate concentration, target yield of transformation product, and actual culture environment.

[0023] In this invention, the separation and purification method preferably includes: removing bacterial cells from the transformation product, collecting the liquid phase, and extracting it with an extractant; after removing the extractant from the obtained extract, performing column chromatography, eluting, collecting the eluent containing cyproheptadine I, and then purifying it to obtain cyproheptadine I.

[0024] In this invention, the method for removing bacterial cells preferably includes centrifugation, static sedimentation, filtration, or flocculation sedimentation; the method for collecting the liquid phase preferably includes supernatant or filtrate. In this embodiment of the invention, centrifugation is used to remove bacterial cells and collect the supernatant.

[0025] In this invention, the extractant is preferably an organic solvent immiscible with water, selected from one or more of ethyl acetate, chloroform, dichloromethane, or n-butanol. The volume ratio of the extractant to the liquid phase is preferably 1:3 to 3:1, more preferably 1:2 to 2:1, and most preferably 1:1 (equal volumes). In this embodiment, ethyl acetate is used as the extractant, and extraction is performed three times with equal volumes, and the organic phases are combined. Pogostemonidine I is a moderately polar flavonoid compound with limited solubility in water but good solubility in organic solvents. Therefore, the extractant can effectively extract pogostemonidine I from the liquid phase to the organic phase, achieving preliminary separation and enrichment. Those skilled in the art can adjust the volume ratio and number of extractions appropriately according to the emulsification during extraction, as long as effective extraction is achieved.

[0026] In this invention, the preferred method for removing the extractant is to evaporate or sublimate the extractant by means of heating, depressurization, freeze drying, etc., leaving a crude extract containing the target product. In the embodiments of this application, anhydrous sodium sulfate is used for drying, and the crude extract containing cymosin I is obtained by depressurization concentration.

[0027] In this invention, the column chromatography is preferably silica gel column chromatography, but it can also be macroporous adsorption resin column chromatography, polyamide column chromatography, or reversed-phase column chromatography. This column chromatography can effectively separate cymoxanil I from unconverted icariin, bacterial metabolites, pigments, and other impurities, significantly improving the purity of the target product. In the embodiments of this application, 200-300 mesh silica gel column chromatography is used. This mesh size range provides sufficient specific surface area and resolution, ensuring good separation of each component during elution.

[0028] In this invention, the eluent is preferably a solvent with good polarity matching and solubility for cyproheptadine I, such as a combination of dichloromethane-methanol, chloroform-methanol, or petroleum ether-acetone. In an embodiment of this invention, a mixture of dichloromethane and methanol is used as the eluent. The eluent can selectively elute cyproheptadine I adsorbed on the column chromatography packing from impurities, achieving separation of the target product from impurities. The elution method is preferably gradient elution, but isocratic elution can also be used. In an embodiment of this invention, linear gradient elution is used, with the volume ratio of dichloromethane to methanol gradually changing from 10:1 to 3:1. The gradient elution can elute weaker impurities first based on the difference in polarity of each component, with cyproheptadine I eluting concentratedly when the polarity of the eluent increases to a suitable range, while strongly polar impurities remain on the column, thereby obtaining better separation effect and higher purity of the target product.

[0029] In this invention, the purification method is preferably a commonly used purification technique in the art, such as preparative high-performance liquid chromatography (HPLC), recrystallization, medium-pressure preparative chromatography, or high-speed countercurrent chromatography (HSCLC). In this embodiment, thin-layer chromatography (TLC) is used to detect and combine identical eluents containing cyproheptadine I, followed by HPLC purification. The purification steps of this invention can further remove trace impurities remaining after column chromatography, achieving a purity of cyproheptadine I of over 96%, meeting pharmaceutical-grade quality requirements, while ensuring the accuracy and reproducibility of pharmacological experimental results.

[0030] This invention provides the use of cyproheptadine I in the biomanufacturing of medicaments for the prevention and / or treatment of reproductive damage.

[0031] In this invention, the reproductive injury preferably includes chemotherapy-induced male kidney-reproductive injury. The chemotherapy drugs include alkylating agents and / or platinum-based drugs, preferably one or more of busulfan, cyclophosphamide, ifosfamide, cisplatin, and carboplatin. While killing tumor cells, these chemotherapy drugs produce significant toxic side effects on normal tissues, especially the reproductive system and kidneys. In this application's embodiments, busulfan-treated mice are used as a reproductive injury model for illustration.

[0032] In this invention, the reproductive damage preferably includes at least one of the following: oxidative stress damage to reproductive organs, decreased reproductive organ weight, decreased sperm count, hypothalamic-pituitary-gonadal axis disorder, decreased renal function, and abnormal renal-reproductive tissue morphology.

[0033] In this invention, the oxidative stress damage to the reproductive organs includes abnormal changes in the expression of Keap1, Nrf2, HO-1, and GPX4 proteins in testicular tissue. Keap1, Nrf2, HO-1, and GPX4 are core proteins in the anti-oxidative stress signaling pathway. In embodiments of this invention, experiments show that cyproheptadine I can inhibit Keap1 expression, release and activate Nrf2, and subsequently upregulate HO-1 and GPX4, thereby enhancing the body's ability to scavenge reactive oxygen species, alleviating busulfan-induced oxidative stress damage, and ultimately protecting testicular tissue from the toxic effects of chemotherapy drugs.

[0034] In this embodiment of the invention, compared with normal mice, the reproductive injury model mice showed a significant decrease in body weight, testicular weight, epididymal weight, and sperm count, indicating that chemotherapy drugs caused reproductive organ atrophy and spermatogenesis dysfunction. However, after treatment with cyproheptadine I, body weight, testicular weight, epididymal weight, and sperm count all showed a significant increase, indicating that cyproheptadine I can effectively antagonize male reproductive damage caused by chemotherapy drugs, promote the repair of reproductive organ tissues, restore spermatogenesis function, and increase the number of mature sperm, thereby playing a significant reproductive protective role.

[0035] In this invention, the hypothalamic-pituitary-gonadal axis disorder preferably includes at least one of elevated luteinizing hormone (LH) levels, elevated follicle-stimulating hormone (FSH) levels, and decreased testosterone levels. In an embodiment of this invention, a reproductive injury model mouse exhibited a significant decrease in testosterone accompanied by a significant increase in LH and FSH levels. This indicates that chemotherapy drugs damaged testicular interstitial cells, reducing their ability to synthesize testosterone. The decrease in testosterone weakens the negative feedback to the pituitary gland, causing it to compensate by secreting more LH and FSH, thus disrupting the hypothalamic-pituitary-gonadal axis. Pogostemonidine I can repair testicular tissue damaged by chemotherapy drugs, restoring the interstitial cells' ability to synthesize testosterone and partially restoring the spermatogenic tubules' spermatogenic function, thereby rebuilding the negative feedback homeostasis of the hypothalamic-pituitary-gonadal axis and restoring these three hormones to normal levels.

[0036] In this invention, the decline in renal function preferably includes elevated levels of blood urea nitrogen and / or serum creatinine. Blood urea nitrogen and serum creatinine are core blood biochemical indicators for assessing renal function. Blood urea nitrogen is a product of protein metabolism, generated in the liver and excreted through glomerular filtration; serum creatinine is a metabolite of creatine and phosphocreatine in muscle tissue, also excreted through glomerular filtration and not reabsorbed by the renal tubules. Significant elevations in these two indicators indicate a decline in glomerular filtration function. In the embodiments of this application, after intervention with cyproheptadine I, the levels of blood urea nitrogen and serum creatinine in the reproductive injury model mice significantly decreased, indicating that renal function was restored.

[0037] In this invention, the abnormal renal-reproductive tissue morphology preferably includes atrophy of the seminiferous tubules in the testes, a reduction in the number of spermatogenic cell layers, and a scarcity of mature sperm in the lumen; disordered epididymal tubular structure and a sharp decrease in the number of sperm in the lumen; glomerular atrophy, and vacuolization or necrosis of renal tubular epithelial cells. In the embodiments of this invention, the above-mentioned abnormal renal-reproductive tissue morphology was observed in mice with reproductive injury models. After treatment with cymosin I, the above-mentioned tissue morphology abnormalities were significantly improved: the seminiferous tubule structure of the testes in the high-dose group of mice tended to be intact, the spermatogenic cells were arranged neatly and with increased layers, and a large number of mature sperm were visible in the lumen; the epididymal tubular structure returned to normal, and the number of sperm in the lumen increased significantly; the morphology of the glomeruli and renal tubules in the kidney tissue was significantly improved, and the lesion area was reduced. This indicates that cymosin I can effectively repair chemotherapy-induced renal-reproductive tissue damage, restore testicular spermatogenesis, epididymal sperm storage capacity, and renal filtration function, and confirm its dual protective effect on renal-reproductive tissue from a histological morphology perspective.

[0038] In this embodiment of the invention, Wuzi Yanzong Wan was used as a positive control and compared with the low-dose group and the high-dose group of Baohuogan I. The results showed that Wuzi Yanzong Wan could partially improve the reproductive damage induced by busulfan, but its effect was weaker than that of the high-dose group of Baohuogan I. Specifically, the low-dose group of Baohuogan I was comparable to or slightly better than the Wuzi Yanzong Wan group in terms of weight recovery, testicular / epididymal weight increase, and sperm count increase; while the high-dose group of Baohuogan I was significantly better than the Wuzi Yanzong Wan group in all the above indicators, and showed stronger efficacy in regulating serum testosterone, luteinizing hormone, and follicle-stimulating hormone levels, reducing blood urea nitrogen and serum creatinine, and repairing pathological damage to testicular, epididymal, and kidney tissues. The above results indicate that the efficacy of Baohuogan I prepared in this invention at high doses far exceeds the protective effect of the classic kidney-tonifying traditional Chinese medicine Wuzi Yanzong Wan, and has a significant advantage in dual protection of the kidney and reproductive system. Meanwhile, through comparative experiments with different doses, the present invention further determined that the preferred effective dose range of cyproheptadine I in the treatment of chemotherapy-induced male renal-reproductive damage is ≥20 mg / kg. Within this dose range, cyproheptadine I can significantly counteract busulfan-induced renal-reproductive dual damage, and no obvious toxic side effects were observed.

[0039] This invention is the first to discover that *Anomala virosa* yeast has the ability to convert icariin into cyproterone I, and establishes an efficient and environmentally friendly biomanufacturing method. Through the preparation method provided by this invention, icariin can be efficiently converted to produce high-purity (≥96%) cyproterone I. This application also provides the application of baicalin I in renal-reproductive protection. Through a busulfan-induced male-mouse renal-reproductive injury model, it was confirmed that baicalin I can significantly improve reproductive injury indicators such as weight loss, testicular / epididymal weight reduction, sharp decrease in sperm count, decrease in serum testosterone, and compensatory increase in luteinizing hormone / follicle-stimulating hormone in model mice. At the same time, it reduces blood urea nitrogen and serum creatinine levels and repairs histopathological damage to the testes, epididymis, and kidneys. Its effects are superior to the classic positive control drug Wuzi Yanzong Wan in many indicators. Moreover, it exerts an antioxidant stress effect by regulating the Keap1 / Nrf2 / HO-1 / GPX4 signaling pathway, thus achieving dual protection of male renal-reproductive function induced by chemotherapy drugs for the first time. It has clear industrialization prospects and clinical translational value.

[0040] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, illustrates the application of the abnormal Wickham yeast provided by the present invention in the biomanufacturing of cyproheptadine I and the application of cyproheptadine I in the prevention and / or treatment of reproductive damage. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0041] Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods in the art.

[0042] Example 1 Preparation of Pogostemon I by Biotransformation of Abnormal Wickham Yeast 1. Strain activation The *Wickham's abnormal* yeast used in this application was purchased from Beijing Beina Chuanglian Biotechnology Research Institute, strain number BNCC195349. *Wickham's abnormal* yeast strain preserved in glycerol tubes was inoculated onto PDA medium plates and activated at 30℃ for 3 days. Single colonies were then picked and inoculated into PDB liquid medium, shaken, and cultured for 24 hours to prepare an activated bacterial suspension with a viable cell concentration of 1×10⁻⁶. 8 CFU / mL and above.

[0043] 2. Substrate preparation Weigh 20 mg of icariin monomer with a purity ≥95%, place it in a 150 mL Erlenmeyer flask, add 40 mL of sterile distilled water, add glucose at a weight percentage of 5% of the biotransformation system, mix thoroughly, autoclave at 121℃ for 20 min, and cool to room temperature.

[0044] 3. Biotransformation Under aseptic conditions, activated bacterial solution was added to the substrate solution at an inoculum rate of 1%, and fermented in a constant temperature shaker at 27°C and 180 rpm for 21 days until fermentation was completed.

[0045] 4. Separation and purification The fermentation broth was centrifuged at 10,000 rpm for 10 min to remove bacterial cells, and the supernatant was collected. The supernatant was extracted three times with equal volumes of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude extract. The crude extract was then subjected to 200-300 mesh silica gel column chromatography with a linear gradient elution of dichloromethane and methanol, varying the dichloromethane:methanol volume ratio from 10:1 to 3:1. TLC analysis was performed, and the combined fractions were purified by HPLC to obtain the final product. The obtained product was a pale yellow powder.

[0046] 5. Analysis and identification The molecular ion mass-to-charge ratio of the product, determined by electrospray ionization mass spectrometry, was 514.52, similar to that of cymoxanol I (molecular formula C). 27 H 30 O 10 The theoretical molecular weight (514.52) is consistent with that of the other two molecules.

[0047] The obtained product was characterized by nuclear magnetic resonance (NMR), and the results of the NMR spectrum analysis are as follows: 1H NMR (600 MHz, MeOD) δ: 12.51 (1H, s, OH-5), 7.84 (2H, d, J=8.8 Hz, H-2', 6'), 7.10 (2H, d,J=8.8 Hz, H-3', 5'), 6.30 (1H, s, H-6), 5.26 (1H, brs, Rha-H-1), 5.14 (1H, t,J=6.6 Hz, H-2"), 3.84 (3H, s, OCH3-4'), 1.61 (3H, s, H-4"), 1.63 (3H, s, H-5"), 0.78 (3H, d, J=6.0 Hz, Rha-H-6). 13C NMR (150 MHz, MeOD) δ: 154.87 (C-2), 135.46 (C-3), 179.09 (C-4), 162.24 (C-5), 99.32 (C-6), 162.61 (C-7), 106.94(C-8), 157.78 (C-9), 105.26 (C-10), 22.12 (C-1"), 123.20 (C-2"), 132.70 (C-3"), 26.41 (C-4"), 18.76 (C-5"), 123.48 (C-1, 131.68 (C-2', 6'), 115.05 (C-3', 5'), 159.87 (C-4'), 56.49 (4'-OMe).Rha:102.08 (C-1), 71.54 (C-2), 71.88 (C-3), 72.96 (C-4), 71.57 (C-5), 18.53 (C-6). The above data are basically consistent with the literature report (Zhou Lu. Study on chemical constituents and bioactivity of Epimedium sagittatum and Morus alba root bark [D]. Shandong University, 2023). Therefore, the compound was identified as 3,5,7-trihydroxy-4'-methoxy-8-isopentenylflavonoid-3-O-α-L-rhamnopyranoside, i.e., baohuo glycoside I.

[0048] HPLC analysis showed that the purity of the product, cymoxanil I, was ≥96%. The HPLC results are shown in the table below. Figure 1 .

[0049] Example 2 Pharmacodynamic study of phorbol I in improving busulfan-induced reproductive damage 1. Animal grouping and modeling SPF-grade male ICR mice were randomly divided into 5 groups: control group, model group, positive control drug Wuzi Yanzong Wan group (WZY), low-dose baicalein I group (YBL), and high-dose baicalein I group (YBH). Except for the control group, male reproductive injury model was established in all other groups by intraperitoneal injection of busulfan. After modeling, each group was administered the following drugs by gavage for 30 consecutive days: 2 g / kg for the positive control drug Wuzi Yanzong Wan group, 5 mg / kg for the low-dose baicalein I group, 20 mg / kg for the high-dose baicalein I group, and the control group and model group were given the same volume of solvent.

[0050] 2. Detection indicators Thirty days after administration, mice in each group were tested. The tests included: body weight; testicular and epididymal weight; sperm count; serum reproductive hormone levels, including testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH); renal function indicators, including blood urea nitrogen (BUN) and serum creatinine (Scr); histopathological examination using hematoxylin-eosin staining; and Western blot (WB) detection of relevant protein expression.

[0051] Compared with the blank control group, the body weight, testicular weight, epididymal weight, and sperm count of mice in the model group were significantly reduced, indicating that busulfan successfully induced reproductive organ atrophy and spermatogenic dysfunction. Compared with the model group, the above indicators in the low-dose group of baicalin I showed some recovery; the high-dose group showed further significant improvement, with better results than the positive control drug Wuzi Yanzong Wan group. The test results are shown below. Figure 2 .

[0052] Serum reproductive hormone levels were measured, showing that serum T levels in the model group mice were significantly lower than those in the blank control group, while LH and FSH levels increased significantly in a compensatory manner. Compared with the model group, both low- and high-dose groups of baicalin I significantly increased T levels and decreased LH and FSH levels, with the high-dose group showing better results, restoring levels to near normal. The positive control drug Wuzi Yanzong Wan group also showed improvement, but the effect was weaker than that of the high-dose baicalin I group. Results are shown below. Figure 3 A, B, and C.

[0053] The results of renal function tests showed that BUN and Scr were significantly higher in the model group mice than in the blank control group, indicating impaired renal function. Compared with the model group, the low-dose group of baicalin I reduced BUN and Scr, while the high-dose group significantly reduced them to near-normal levels. The positive control drug Wuzi Yanzong Wan group only partially reduced BUN and Scr, and its effect was not as good as the high-dose group of baicalin I. The test results are shown below. Figure 3 D and E in the text.

[0054] Histopathological examination of testicular tissue showed that: in the model group, the seminiferous tubules were significantly atrophied, the number of spermatogenic cell layers was reduced, and sperm were rare in the lumen; in the low-dose group of baicalin I, the seminiferous tubule structure was partially restored, and a small number of sperm were visible; in the high-dose group, the seminiferous tubule structure was intact, the spermatogenic cells were neatly arranged, and the lumen was filled with a large number of mature sperm, close to the blank control group; the positive control group showed better improvement than the model group but not as good as the high-dose group of baicalin I, see details below. Figure 4 .

[0055] Histopathological examination of the epididymis revealed that the model group exhibited disordered epididymal tubular structure and sparse sperm count; the high-dose baicalein I group showed clear tubular structure and a significantly increased sperm count, approaching that of the blank control group. (See details...) Figure 5 .

[0056] Histopathological examination of the kidney tissue showed that in the model group, glomerular atrophy, vacuolar degeneration of renal tubular epithelial cells, and interstitial inflammatory infiltration were observed; in the high-dose group of baicalin I, the morphology of glomeruli and renal tubules was significantly improved, and the lesions were significantly alleviated. (See details...) Figure 6 .

[0057] The WB test results are shown below. Figure 7 The results showed that, compared with the blank control group, the expression of Keap1 protein was increased and the expression of Nrf2, HO-1, and GPX4 proteins was decreased in the testicular tissue of the model group mice. Compared with the model group, both low- and high-dose groups of cyproheptadine I significantly reduced Keap1 expression and increased the expression of Nrf2, HO-1, and GPX4, with the high-dose group showing a more significant effect. The positive control group also showed a similar trend, but the intensity was weaker.

[0058] The above results indicate that cyproheptadine I improved busulfan-induced renal-reproductive dysfunction in mice in a dose-dependent manner.

[0059] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Abnormal Wickham yeast ( Wickerhamomyces anomalus Application of ) in the bio-manufacturing of cyproheptadine I.

2. A method for the biosynthesis of cyproheptadine I, characterized in that, Using icariin monomer as a substrate, biotransformation was performed using *Wickhamia lanceolata* yeast. The resulting transformation product was then separated and purified to obtain icariin I.

3. The method according to claim 2, characterized in that, The biotransformation involves inoculating the abnormal Wickham yeast into an Epimedium monomer solution; The inoculum size of the abnormal Wickham yeast was 0.1% to 5%. The effective viable cell concentration of the abnormal Wickham yeast culture is ≥1×10⁻⁶. 8 CFU / mL.

4. The method according to claim 2, characterized in that, During the biotransformation, the biotransformation system also includes a carbon source; the added carbon source accounts for 2% to 8% of the total weight of the biotransformation system.

5. The method according to claim 2, characterized in that, The separation and purification method includes: removing bacterial cells from the transformation product, collecting the liquid phase, and extracting it with an extractant; after removing the extractant from the extract, performing column chromatography, eluting, collecting the eluent containing cyproheptadine I, and then purifying it to obtain cyproheptadine I.

6. Application of cyproheptadine I in the preparation of drugs for the prevention and / or treatment of reproductive damage.

7. The application according to claim 6, characterized in that, The reproductive damage includes male kidney-reproductive damage induced by chemotherapy drugs.

8. The application according to claim 6, characterized in that, The reproductive damage includes at least one of the following: oxidative stress damage to reproductive organs, decreased reproductive organ weight, decreased sperm count, hypothalamic-pituitary-gonadal axis disorder, decreased renal function, and abnormal renal-reproductive tissue morphology.

9. The application according to claim 8, characterized in that, The hypothalamic-pituitary-gonadal axis disorder includes at least one of elevated luteinizing hormone (LH) levels, elevated follicle-stimulating hormone (FSH) levels, and decreased testosterone levels.

10. The application according to claim 8, characterized in that, The decline in renal function includes elevated blood urea nitrogen and / or serum creatinine levels.

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