Application of Chinese chive seed extract in preparation of medicine for treating oligoasthenospermia

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of a Chinese chive seed extract in preparation of a medicine for treating oligoasthenospermia. Based on network pharmacology prediction and animal experiment verification, it is found that by activating a PI3K / AKT signal channel, testicular cell apoptosis is inhibited, spermatogenesis disorder is improved, and therefore the sperm number and vitality of an oligoasthenospermia model rat are remarkably improved. Clinical treatment research in combination with levocarnitine oral liquid proves that the traditional Chinese medicine composition can reduce the sperm DFI level and improve the semen quality. Animal research proves that the compound can regulate the level of serum testosterone (T), reduce the levels of follicle stimulating hormone (FSH) and luteinizing hormone (LH), repair pathological damage of testis tissues, and antagonize testis cell apoptosis induced by tripterygium glycosides by up-regulating protein expression of p-PI3K / PI3K, p-AKT / AKT and Bcl-2.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and particularly relates to application of a leek seed extract in preparing a medicine for treating oligoasthenospermia. Background Art

[0002] Oligospermia (OAS) refers to a decrease in sperm count and / or motility. Symptoms typically include low sperm count, abnormal sperm morphology, and impaired motility. It is a common male reproductive system disorder. In recent years, the incidence of infertility has been increasing annually due to a variety of factors, including environmental, social, and lifestyle factors. Approximately 15% of couples of reproductive age experience infertility, with male factors accounting for over 50% of these cases. Oligospermia and asthenospermia are now recognized as the leading causes of male infertility, although the etiology and mechanisms of male infertility remain unclear. Currently, Western medicine treatments for aging include medication, surgical intervention, lifestyle adjustments, and assisted reproductive technologies tailored to individual etiology and clinical circumstances. Aromatase inhibitors, primarily letrozole and anastrozole, block the aromatase enzyme to increase testosterone levels in the testicles and promote sperm production. However, clinical studies have shown that they have side effects on the nervous system, musculoskeletal system, and liver and kidney function. In Traditional Chinese Medicine, OAS falls under the categories of "infertility", "thin sperm", and "cold sperm". Its causes and pathogenesis include kidney qi deficiency, qi and blood deficiency, etc.

[0003] Chinese chive seeds (Semen Allii tuberosum) are the dried, mature seeds of the lily family plant Allium tuberosum Rottl. Ex Spreng., first described in the Ming Yi Bie Lu (Famous Doctors' Prescriptions), and are believed to warm and nourish the liver and kidneys, strengthen yang, and consolidate essence. They are commonly used to treat liver and kidney deficiency, low back and knee pain, impotence, spermatorrhea, enuresis, frequent urination, and leukorrhea. Studies have shown that the main chemical components of Chinese chive seed porridge (or Chinese chive seed tea, or Chinese chive seed wine) include steroidal saponins, alkaloids, sulfides, and flavonoids. Modern pharmacological studies have demonstrated that Chinese chive seed porridge (or Chinese chive seed tea, or Chinese chive seed wine) possesses kidney-warming, yang-strengthening, immune-boosting, antioxidant, anti-aging, and antibacterial properties. However, the mechanism by which Chinese chive seed porridge (or Chinese chive seed tea, or Chinese chive seed wine) improves OAS remains unclear.

[0004] Network pharmacology can explain the mechanism of action of drugs in improving diseases from multiple targets and pathways. Based on the combination of network pharmacology and molecular docking, combined with animal experiments, this paper explores the mechanism of action of leek seeds in improving oligoasthenospermia, providing a theoretical basis for the clinical application of leek seeds in traditional Chinese medicine prescriptions. Summary of the Invention

[0005] The application aims to provide an application of leek seed extract in preparation of a medicine for treating oligoasthenospermia.

[0006] The application aims to provide an application of leek seed extract in preparation of a medicine for treating oligoasthenospermia.

[0007] The application provides an application of leek seed extract in preparation of a medicine for treating oligoasthenospermia, and the leek seed extract can activate a PI3K / AKT signal path, improve pathological damage of testis tissue, increase sperm quantity and activity, and further treat oligoasthenospermia.

[0008] Further, after the leek seed extract is treated in combination with levocarnitine oral liquid, sperm DFI is significantly reduced compared with that before entering the group; sperm volume, liquefaction state, sperm forward movement percentage (PR), total activity (PR+NP), sperm concentration and total sperm number in overall semen parameters are all significantly increased compared with those before treatment

[0009] Further, the preparation method of the leek seed extract comprises the following steps: taking salted leek seeds as raw materials, heating and refluxing extraction three times by using 70% ethanol at a proportion of 1:6 (w / v), collecting n-butanol extract liquid after n-butanol extraction three times on the refluxed filtrate, and drying in a 65 DEG C oven after pressure reduction distillation, so that the extract containing saponins and nucleosides is obtained.

[0010] Further, the activation of the PI3K / AKT signal path is to improve the expression levels of p-PI3K / PI3K, p-AKT / AKT protein and Bcl-2 expression in testis tissue, inhibit testis cell apoptosis, and improve sperm quality of oligoasthenospermia model rats.

[0011] Further, the improvement of the pathological damage of testis tissue is to promote the arrangement of spermatogenic cells to be neat, the structure of seminiferous tubule lumen to be restored, and the widening of testis seminiferous tubule interstitium to be reduced.

[0012] Further, the medicine also plays a treatment role by regulating serum hormone levels.

[0013] Further, the regulation of serum hormone levels is to increase testosterone level and reduce follicle stimulating hormone and luteinizing hormone levels.

[0014] Further, the medicine also improves metabolic abnormalities of oligoasthenospermia by correcting metabolic pathways including linoleic acid metabolic disorder, influence on phenylalanine / tyrosine / tryptophan biosynthesis, arginine / proline metabolism and steroid hormone biosynthesis.

[0015] Furthermore, the oligoasthenozoospermia is caused by abnormal testicular cell apoptosis and spermatogenesis disorder due to inhibition of the PI3K / AKT signaling pathway.

[0016] Furthermore, the dosage form of the medicine includes leek seed porridge, leek seed tea or leek seed wine.

[0017] Furthermore, the drug exerts its therapeutic effects through multiple components, multiple targets, and multiple pathways. The core targets of the drug include PTGS2, PPARG, BCL2, CASP3, and key molecules in the PI3K / AKT signaling pathway.

[0018] Beneficial effects:

[0019] The present invention randomly divided 60 patients with idiopathic oligoasthenospermia (kidney deficiency and sperm deficiency) who met the diagnostic criteria into two groups of 30 each. The patients were divided into a control group of positive Western medicine L-carnitine oral solution and a group of leek seed porridge (or leek seed tea, or leek seed wine) combined with L-carnitine oral solution. After three months of treatment, both the Western medicine L-carnitine oral solution group and the leek seed porridge (or leek seed tea, or leek seed wine) combined with L-carnitine oral solution group showed changes in sperm concentration, sperm motility, forward motility, and dilation factor (DFI) (P < 0.05), with the changes in the leek seed porridge (or leek seed tea, or leek seed wine) combined with L-carnitine oral solution group being more significant (P < 0.05).

[0020] According to the present invention, after three months of treatment, 11 patients in the western medicine L-carnitine oral liquid group were clinically cured, and 15 patients in the leek seed porridge (or leek seed tea, leek seed wine) combined with L-carnitine oral liquid group had significant results. 9 patients in the western medicine L-carnitine oral liquid group had significant results, and 10 patients in the leek seed porridge (or leek seed tea, leek seed wine) combined with L-carnitine oral liquid group had significant results. 5 patients in the western medicine L-carnitine oral liquid group had effective results, and 3 patients in the leek seed porridge (or leek seed tea, leek seed wine) combined with L-carnitine oral liquid group had effective results. 5 patients in the L-carnitine oral liquid group had no effect, and 2 patients in the leek seed porridge (or leek seed tea, leek seed wine) combined with L-carnitine oral liquid group had no effect. The total effective rate of the western medicine L-carnitine oral liquid group was 83.33%, and the total effective rate of the leek seed porridge (or leek seed tea, leek seed wine) combined with L-carnitine oral liquid group was 93.33%.

[0021] The present invention has verified through animal experiments that leek seed extract can significantly improve testicular quality, sperm count, and sperm motility in rats with oligoasthenozoospermia. This finding indicates that leek seed extract has significant efficacy in treating oligoasthenozoospermia, effectively improving sperm count and quality, thereby enhancing fertility. Furthermore, leek seed extract can improve testicular histopathological findings in rats with oligoasthenozoospermia, reduce spermatogenic cell disorganization and loss, and protect the normal structure of testicular tissue. This helps restore testicular spermatogenesis and further promotes sperm production and maturation.

[0022] Through in-depth research, the present invention found that leek seed extract can regulate the PI3K / AKT signaling pathway and reduce the apoptosis rate of testicular tissue. The PI3K / AKT signaling pathway plays an important role in cell proliferation, differentiation and apoptosis. By activating this pathway, leek seed extract helps protect testicular cells from apoptosis, thereby maintaining the normal physiological function of the testicles. Network pharmacology prediction results show that leek seed extract contains multiple active ingredients, which work together to improve oligoasthenozoospermia by acting on multiple targets and pathways. This multi-component, multi-target, and multi-pathway therapeutic feature makes leek seed extract have a more comprehensive and integrated effect in treating oligoasthenozoospermia. Compared with existing treatment methods, such as drug therapy and surgical intervention, leek seed extract, as a natural medicine, has the advantages of significant efficacy and small side effects. Animal experimental results show that leek seed extract improves the symptoms of oligoasthenozoospermia without observing obvious side effects, indicating that it has good safety and tolerability.

[0023] The present invention provides a new drug option for the treatment of oligoasthenozoospermia. As a traditional Chinese medicinal material, leek seed extract has broad application prospects and market demand. By developing it into a drug for the treatment of oligoasthenozoospermia, it can provide new treatment options and hope for a wide range of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 The figures are as follows: A is the blank group; B is the model group; C is the positive control group; D is the improvement group of leek seed porridge (or leek seed tea, leek seed wine);

[0026] Figure 2The sperm morphology of rats in different groups in the present invention is shown in Figure 2, where A is the blank group; B is the model group; C is the positive control group; D is the improved group of leek seed porridge (or leek seed tea, leek seed wine);

[0027] Figure 3 The electrophoresis diagram of the expression of p-PI3K, PI3K, p-AKT, Akt, and Bcl-2 proteins in the testicular tissue of OAS rats treated with leek seed porridge (or leek seed tea, leek seed wine) extract of the present invention is shown, wherein A is a blank group; B is a model group; C is a positive control group; D is an improvement group treated with leek seed porridge (or leek seed tea, leek seed wine);

[0028] Figure 4 The figure shows the effect of the leek seed porridge (or leek seed tea, leek seed wine) extract on testicular apoptosis in each group of rats in the present invention, wherein TUNEL, ×400, A is the blank group; B is the model group; C is the positive control group; D is the leek seed porridge (or leek seed tea, leek seed wine) improvement group;

[0029] Figure 5 This is a graph showing the results of multivariate pattern recognition analysis using PCA in the present invention;

[0030] Figure 6 This is a graph showing the results of multivariate statistical analysis in the present invention;

[0031] Figure 7 This is a graph showing the differential expression results of different markers in the present invention;

[0032] Figure 8 This is a graph showing the expression results of different differential metabolites in the present invention. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] The experimental methods used in the following implementations, unless otherwise specified, are commonly used experimental methods in this field;

[0039] Unless otherwise specified, the experimental materials used in the following examples are all commonly used experimental materials in this field.

[0040] Example 1

[0041] 1. Materials

[0042] 1.1 Animals: SPF male Sprague-Dawley rats, 6–8 weeks old and weighing 180–200 g, were purchased from the Animal Experiment Center of Ningxia Medical University and housed in the SPF animal room of Ningxia Medical University. The temperature was maintained at (22 ± 2)°C, the relative humidity was 45%–60%, the light intensity was 12 h per day, and the rats were well ventilated. They were fed a standard rat pellet diet and had free access to food and water. This study complied with the animal care standards in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH), animal license number SCXK(Ning)2020-0001, and was approved by the Animal Ethics Committee of Ningxia Medical University (ethics approval number BUCM-42019061002-2040). National Natural Science Foundation of China ethics number: 2016-007; Medications: leek seed porridge (or leek seed tea, leek seed wine) (Ningxia Kangtailong Chinese Medicine Pieces Co., Ltd., batch number: 2307001); L-carnitine oral solution (Northeast Pharmaceutical Group, batch number 231105, specification: 10 ml: 1 g); Tripterygium wilfordii polyglycosides tablets (Zhejiang Deende Pharmaceutical Co., Ltd., batch number: Z33020422, specification: 10 mg).

[0043] 1.2 Reagents: electrophoresis buffer (Wuhan Sewell Biotechnology Co., Ltd., catalog number G2081-1L); BCA protein quantification kit (Keygen Biotechnology Co., Ltd., catalog number 20240308); ELISA kit (Nanjing Boyan Biotechnology Co., Ltd., FSH: catalog number BY-ER334438; LH: catalog number BY-ER330447, T: catalog number BY-JZF0458); β-actin (Beijing LABLEAD Co., Ltd., catalog number A0101), PI3K (Beijing Immunoway Co., Ltd., catalog number YT6156), p-PI3K (Shanghai Abmar Biotechnology Co., Ltd., catalog number YT6156), t Company, Catalog No. TA32425), AKT (Beijing Immunoway Company, Catalog No. YT0185), p-AKT (Beijing Immunoway Company, Catalog No. YP0006), Bcl-2 (Wuhan Abclonal Company, Catalog No. A20777); TUNEL kit (Nanjing Novizan Biotechnology Co., Ltd., Catalog No. A112-02);

[0044] 1.3 Instruments: microplate reader (Bio-Rad, USA, model: Epoch), paraffin slicer (LEICA, Germany, model: RM2235), electronic analytical balance (Huazhi (Fujian) Electronic Technology Co., Ltd., model: PTX-FA220S), high-speed refrigerated centrifuge (Eppendorf, Germany, model: 5417R), inverted fluorescence microscope (LEICA, Germany, model: DM2000 / DFC450C).

[0045] 2 Methods

[0046] 2.1 Effective ingredients and potential target genes of leek seed porridge (or leek seed tea, leek seed wine) in network pharmacology

[0047] 2.1.1 Screening of active ingredients in leek seed porridge (or leek seed tea, leek seed wine)

[0048] The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP, http: / / www.tcmsp-e.com / tcmsp.php) was used to screen for active ingredients in leek seed porridge (or leek seed tea, or leek seed wine) based on oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.1. A corresponding active ingredient database was constructed. Target genes corresponding to the active ingredients in leek seed porridge (or leek seed tea, or leek seed wine) were retrieved to construct a target gene database for leek seed porridge (or leek seed tea, or leek seed wine). The targets were converted into gene symbols using the UniProt database. This database was constructed using the Cytoscape 3.10.0 platform.

[0049] 2.1.2 OAS disease target acquisition

[0050] According to the keywords "oligospermia", "deficiency of sperm motility", "spermatogenic dysfunction", "asthenospermia", search and download data of disease targets through the database (https: / / www.genecards.org / ) in the Genecards database, Herb database, DisGeNET database, Drug Bank database and OMIM database, delete duplicate values, screen all target points related to disease characteristics, and after sorting, obtain target genes related to the disease. Use the webpage to make a Venn diagram (Venn diagram) to obtain drug-disease corresponding data and obtain common targets of the disease and drugs.

[0051] 2.1.3 Analysis of interactions between targets (PPI analysis)

[0052] The target points obtained by the Venn diagram are imported into the String platform (https: / / www.string-dborg / ), the species selection is set to "human (Homo sapiens)", the minimum confidence is set to 0.4, and the option of hiding free points is checked. Build a PPI network. Then import the potential target gene data with a comprehensive score > 0.4 into Cytoscape3.10.0 software for graphical display, and apply CytoNCA and CytoHubba plugins to screen the top 10 target genes in the comprehensive centricity ranking as the core target points of leek seed porridge (or leek seed tea, leek seed wine) for improving oligospermia.

[0053] 2.1.4 Construction of "key target- active ingredient- traditional Chinese medicine" network

[0054] The effective components of leek seed porridge (or leek seed tea, leek seed wine) and the component-related target gene data collected in "2.1.1" are imported into Cytoscape software to construct a drug-effective component-target disease network, and the connection between nodes represents the interaction between factors.

[0055] 2.1.5 Gene ontology (GO) function enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis

[0056] Through GO and KEGG enrichment analysis, explore the biological processes and main signal pathways that leek seed porridge (or leek seed tea, leek seed wine) may improve aging. Set the threshold value P < 0.05, use the micro-signal visualization platform to perform GO function enrichment analysis and KEGG pathway enrichment analysis on the core target genes, and draw a column chart and a bubble chart.

[0057] 2.2 Animal experiments

[0058] 2.2.1 Modeling, drug administration and treatment

[0059] After 7 days of acclimatization, the model was established. Forty SPF male Sprague-Dawley rats were randomly divided into four groups based on body weight: a blank group, a model group, a leek seed porridge (or leek seed tea, or leek seed wine) group, and a L-carnitine group. After acclimatization, the model and improvement groups were gavaged with 40 mg / kg / day of Tripterygium wilfordii polyglycosides for four weeks to establish an oligoasthenozoospermia rat model. The L-carnitine group received 100 mg / kg / day of the leek seed porridge (or leek seed tea, or leek seed wine) tablets, and the improvement group received 237.5 mg / mL / day of the leek seed porridge (or leek seed tea, or leek seed wine) tablets for 28 days. The normal control group received an equivalent dose of normal saline. The general condition of the rats in each group was observed.

[0060] 2.2.2 Preparation of leek seed porridge (or leek seed tea, leek seed wine) extract

[0061] Salt-roasted leek seeds were heated under reflux three times with 70% ethanol (1:6 w / v). The reflux filtrate was extracted three times with n-butanol. The n-butanol extract was then collected and distilled under reduced pressure and dried in an oven at 65°C to obtain a leek seed extract containing saponins and nucleosides. The leek seed extract was diluted with distilled water, solubilized by ultrasonication, and filtered through a 0.45 μm filter to obtain a 40 mg / mL leek seed extract solution.

[0062] 2.2.3 Semen quality measurement

[0063] The right epididymis of rats was uniformly selected and cut into pieces to allow the semen to flow out. Rinse with preheated 37°C saline, filter dry with filter paper and weigh, then place in an Eppendorf tube containing 2mL of PBS preheated to 37°C, cut into pieces with surgical scissors, shake well, and incubate at 37°C for 15 minutes to allow the spermatogenic cells stored in the epididymis to fully dissociate into the culture medium. After the incubation, stir evenly with a glass rod to prepare a sperm suspension for sperm quality testing. Sperm quality analysis was performed using the SSA-II automatic sperm analysis system. The sperm density was n×10 6 / mL, and sperm quality was graded.

[0064] 2.2.4 HE staining to observe the pathological structure of testicular tissue

[0065] Rat testicular tissue was isolated and fixed with 4% paraformaldehyde solution for at least 24 hours, embedded in paraffin, and then prepared for sectioning. The sections were baked at 60°C for 2 hours, dewaxed in xylene and various concentrations of ethanol until hydrated, and then rinsed in distilled water for 5 minutes. The sections were stained with hematoxylin for 1 minute, rinsed with phosphate-buffered saline (PBS, pH 7.2-7.6), and then blued. The sections were stained with eosin for 0.5 minutes and then rinsed. The sections were dehydrated with graded ethanol (95%-100%). The sections were then placed in xylene for 10 minutes twice, mounted in neutral gum, and the structural integrity of the rat testis was observed under a light microscope.

[0066] 2.2.5 Sperm morphology observation

[0067] After the semen specimen is completely liquefied, it is washed and smeared. After the slide is dried, it is dehydrated with graded ethanol. The specimen is stained using the Paxton method, mounted, and observed under a microscope.

[0068] 2.2.6 ELISA detection of T, LH, and FSH levels in rat testicular tissue

[0069] Rat testicular tissue was homogenized and the levels of T, LH, and FSH were detected by ELISA in strict accordance with the instructions of the kit.

[0070] 2.2.7 Western blot analysis of p-PI3K, PI3K, p-AKT, AKT, Bcl-2 and other protein expressions in rat testis

[0071] 50 mg of testicular tissue from each group of rats was lysed, centrifuged, and protein concentration was measured with BCA assay. PAGE-SDS electrophoresis was performed, followed by transfer to a membrane, and blocking. The cells were incubated overnight at 4°C with primary antibodies against p-PI3K, PI3K, p-AKT, AKT, and Bcl-2 (1:1000) using β-actin as an internal control. After washing with TBST, the cells were incubated with secondary antibodies (1:10000) for 1 hour. After washing, the cells were developed with a high-sensitivity ECL chemiluminescent solution. Bands were visualized using a chemiluminescence imager and quantified using ImageJ 1.8.0 software.

[0072] 2.2.8 TUNEL detection of rat testicular cell apoptosis

[0073] Testicular tissue sections were stained using conventional methods. DAPI stains all cell nuclei blue, and apoptotic cells produce green fluorescence. The sections were then photographed and analyzed using a fluorescence microscope at a wavelength of 488 nm.

[0074] 2.2.9 Statistical analysis

[0075] Statistical Methods: Data were analyzed using SPSS 25.0 statistical software. Continuous data were expressed as x ± s, and one-way analysis of variance or nonparametric tests were used depending on whether the data were normally distributed. Enumeration data were analyzed using the chi-square test or nonparametric tests depending on whether the data were ordinal. P < 0.05 indicated statistical significance. Graphpad Prism 8.0 software was used to generate graphs.

[0076] 3 Results

[0077] 3.1 Acquisition of effective active ingredients and target prediction of leek seed porridge (or leek seed tea, leek seed wine)

[0078] A total of 7 active ingredients of leek seed porridge (or leek seed tea, leek seed wine) were obtained through TCMSP database search.

[0079] 3.2AS disease targets

[0080] 2199 targets were obtained from the GeneCards database. The aforementioned disease targets were intersected with the 2199 predicted targets obtained in "2.1.2" and a Venn diagram was drawn. Finally, a total of 44 target genes for improving oligoasthenozoospermia were obtained using leek seed porridge (or leek seed tea, leek seed wine).

[0081] 3.3 Target site interaction analysis (PPI)

[0082] The 44 target genes obtained were imported into the String platform to construct a PPI network of shared genes. Data with a comprehensive score > 0.4 were then imported into Cytoscape software for graphical display. Simultaneously, the CytoNCA and CytoHubba plug-ins were called to screen the top 10 target genes ranked by comprehensive centripetality as the core targets for leek seed porridge (or leek seed tea, leek seed wine) to improve oligoasthenozoospermia. These genes include prostaglandin endoperoxide synthase 2 (PTGS2), peroxisome proliferator-activated receptor PPARG, beta-actin (ACTB), B lymphocytoma-2 gene (BCL2), caspase 3 (CASP3), peroxisome proliferator-activated receptor (PPARA), oncogene (MYC), brain-derived neurotrophic factor (BDNF), tyrosine kinase receptor 2 (ERBB2), and nuclear receptor subfamily 3C member 1 (NR3C1).

[0083] 3.4GO enrichment analysis and KEGG pathway enrichment analysis

[0084] The intersecting targets were subjected to GO enrichment analysis and KEGG pathway enrichment analysis. GO enrichment analysis: Using "P value < 0.05, Q value < 0.01" as the screening criteria, a total of 10 entries were obtained, including 249 biological processes (BP), 40 cellular components (CC), and 64 molecular functions (MF). The top 10 entries in each group were selected to generate a three-in-one visual bar chart.

[0085] 3.5 Animal Experiment Verification Results

[0086] 3.5.1 Effects on body weight, testicular mass, sperm count, and motility in OAS rats

[0087] Compared with the normal group, the body weight of the rats in the model group was reduced, but the difference was not statistically significant. The testicular mass was significantly reduced (P<0.05), and the sperm motility and count were significantly decreased (P<0.05). Compared with the semen quality comparison groups of the rats in each group after model intervention, the sperm count and motility of the rats in the leek seed porridge (or leek seed tea, leek seed wine) improvement group and the positive control group were significantly improved, and the difference was statistically significant (P<0.05). The body weight increased, and the testicular mass did not change significantly. See Table 1 for details.

[0088] Table 1 Effects of leek seed porridge (or leek seed tea, leek seed wine) extract on body weight, testicular mass, sperm count and motility in OAS rats

[0089]

[0090] Note: Compared with the blank group 1) p < 0.05; compared with the model group 2) p < 0.05 (same as Table 2-Table 4)

[0091] 3.5.2 Effects of leek seed porridge (or leek seed tea, leek seed wine) extract on testicular tissue structure in OAS rats

[0092] HE staining results showed that the spermatogenic cells in the testicular tissue of the blank group rats were arranged neatly, and the lumen structure of the seminiferous tubules was normal; the lumen of the seminiferous tubules in the testicular tissue of the model group rats was narrowed, and the spermatogenic cells were disordered, reduced in number or disappeared; the spermatogenic cells in the testicular tissue of the leek seed porridge (or leek seed tea, leek seed wine) group and the positive control group were arranged more neatly, as shown in Figure 3. Figure 1 .

[0093] 3.5.3 Effects on Serum Index Levels in OAS Rats

[0094] Compared with the blank group, the T level in the serum of each group was decreased, the FSH level was increased, and the LH level was increased (P<0.05). Compared with the model group, the T level in the serum of the positive control group was increased, the FSH level was decreased, and the LH level was decreased (P<0.05); the T level in the serum of the leek seed porridge (or leek seed tea, leek seed wine) improvement group was increased, the FSH level was decreased, and the LH level was decreased (P<0.05), as shown in Table 2.

[0095] Table 2 Effect of leek seed porridge (or leek seed tea, leek seed wine) extract on testicular serum indexes of OAS rats

[0096]

[0097] 3.5.4 Effect of leek seed porridge (or leek seed tea, leek seed wine) extract on sperm quality of OAS rats

[0098] The normal rat sperm head was curved, in the shape of a sickle, smooth in surface, with an unclear boundary between the acrosome and the post-acrosomal region, connected to the ventral side at the head-neck junction, long and slender at the neck, uniform in thickness at the midpiece, long and gradually tapering at the principal piece, and without angular bending. The sperm morphology of the positive control and leek seed porridge (or leek seed tea, leek seed wine) groups was not different from that of the normal sperm, and the sperm of the model group was abnormal, mainly with broken heads, curling, and folded tails. The study showed that the leek seed porridge (or leek seed tea, leek seed wine) group had an improvement effect on the damage to aging sperm, as shown in Figure 2 .

[0099] 3.5.5 Effect of leek seed porridge (or leek seed tea, leek seed wine) extract on expression of p-PI3K, PI3K, p-AKT, Akt, and Bcl-2 in testicular tissue of OAS rats

[0100] Compared with the blank group, the expression levels of p-PI3K, p-Akt, and Bcl-2 proteins in the testicular tissue of the model group were significantly decreased (P<0.05); the expression levels of P-PI3K, p-AKT, and Bcl-2 proteins in the positive control group were decreased (P<0.05), and the expression levels of p-PI3K, p-AKT, and Bcl-2 proteins in the leek seed porridge (or leek seed tea, leek seed wine) improvement group were slightly decreased (P<0.05); compared with the model group, the expression levels of p-PI3K, p-Akt, and Bcl-2 proteins in the positive control group were increased, and the expression levels of p-PI3K, p-Akt, and Bcl-2 proteins in the leek seed porridge (or leek seed tea, leek seed wine) improvement group were increased (P<0.05), as shown in Figure 3 , Table 3.

[0101] Table 3 Effects of leek seed porridge (or leek seed tea, leek seed wine) extract on the expression of p-PI3K, PI3K, p-AKT, Akt, and Bcl-2 proteins in the testicular tissue of OAS rats

[0102]

[0103] 3.5.6 Effect of leek seed porridge (or leek seed tea, leek seed wine) extract on TUNEL expression in testicular tissue of OAS rats

[0104] Compared with the normal group, the apoptosis of testicular tissue cells in the model group was significantly increased, and the difference was statistically significant (P<0.05); compared with the model group, the apoptosis of cells in the positive control group and the improvement group of leek seed porridge (or leek seed tea, leek seed wine) was significantly reduced, and the difference was statistically significant (P<0.05), as shown in Table 4. Figure 4 .

[0105] Table 4 Effects of leek seed porridge (or leek seed tea, leek seed wine) on testicular apoptosis in rats of each group

[0106]

[0107] 4.1 Principal Component Analysis (PCA)

[0108] In order to study the effect of leek seed porridge (or leek seed tea, leek seed wine) extract on the metabolic profile of testicular tissue samples of oligoasthenozoospermia model rats, the metabolic profiles of each group of samples were analyzed by multivariate pattern recognition using PCA, thereby effectively highlighting the overall distribution trend of metabolomics data and the differences in metabolites between groups and the degree of intra-group variation coefficient of each group. Figure 5 As can be seen, the testicular tissue samples of the model group and the leek seed improvement group can be separated in two-dimensional and three-dimensional space, indicating that there are differences in the metabolic profiles between the two groups of samples.

[0109] 4.2 Based on multivariate statistical analysis, leek seed porridge (or leek seed tea, leek seed wine) improved serum metabolic disorders in OAS rats

[0110] To further explore the changes in serum metabolites in OAS rats after administration of leek seed porridge (or leek seed tea, leek seed wine), we used the OPLS-DA model in multivariate statistical analysis to predict the sample categories of each group. The two groups were significantly separated from the ESI+ and ESI modes, and the difference samples between the model group and the leek seed porridge (or leek seed tea, leek seed wine) group were perceived ( Figure 6The permutation test showed that the intercepts of the Q2 regression lines were all less than 0, indicating that the OPLS-DA model was not overfitting. These data suggest that leek seed porridge (or leek seed tea, leek seed wine) can correct the metabolic disorders in OAS rats.

[0111] 4.3 Potential biomarker screening

[0112] A total of 292 candidate metabolites were detected in the two groups, and metabolites with a vip value of 1.0 were considered potential biomarkers. Finally, a total of 28 metabolites were considered potential biomarkers. The expression levels of each marker are as follows Figure 7 shown.

[0113] 4.4 Analysis of metabolic pathways of OAS after improvement by leek seed porridge (or leek seed tea, leek seed wine)

[0114] Metabolic Analyzer 5.0 was used to analyze the metabolic processes of rats with OAS by visualizing the effects of leek seed porridge (or leek seed tea, or leek seed wine). The results showed that leek seed porridge (or leek seed tea, or leek seed wine) treatment had an effect on the linoleic acid metabolism of OAS.

[0115] After screening, there were 11 pathways (biosynthesis of phenylalanine, tyrosine and tryptophan, arginine and proline metabolism, tryptophan metabolism, arginine biosynthesis, glycine, serine and methionine metabolism, biosynthesis of steroid hormones, biosynthesis of unsaturated fatty acids, primary bile acid biosynthesis, arachidonic acid metabolism, fatty acid degradation, fatty acid elongation). By comparing the potential hub targets and pathways found in network pharmacology with the potential biomarkers and metabolites found in metabolomics analysis, the important biomarkers, targets and pathways were finally verified. We have established a "pathway-metabolism-target-compound" network. Figure 8 As shown in the results, in addition to these 11 pathways, a total of 21 differential metabolites were identified [among which glutamate, TXB2, retinoic acid, l-carnitine, serotonin and glutathione (oxidation) were significantly differential metabolites], and 19 core targets (TNF, NOS2, MAPK14, CCND1, STAT1, SRC, TP53, IL6, HIF1A, AK T1, STAT3, HSP90AA1, JAK2, CDKN1A, MAPK3, EGFR, MYC, FOS and RELA) were also considered to play a key role in the improvement of OAS by leek seed porridge (or leek seed tea, leek seed wine).

[0116] 5 Discussions

[0117] Male infertility is one of the common diseases in men's health. In the past 20 years, due to changes in the natural and social environment and the influence of many adverse factors, the incidence of male infertility has been on the rise. Oligozoospermia refers to the condition in which the total number of sperm in a single ejaculation is less than 39×10 6 (or concentration lower than 15×10 6 / mL) is a common cause of male infertility. Currently, the pathogenesis and causes of most male infertility cases are not fully understood, and Western medicine is ineffective in improving idiopathic male infertility. However, the mechanism by which leek seed porridge (or leek seed tea or wine) improves aging remains unclear.

[0118] Traditional Chinese Medicine considers OAS to fall under the categories of "thin sperm" and "infertility," with deficiency syndromes being the most common. Kidney yang deficiency and dysfunction of the warming function can lead to low and cold sperm. Spleen yang deficiency impairs the transport and transformation of grain essence, preventing the acquired from nourishing the innate, resulting in low and weak sperm. Chronic illness can lead to both qi and blood deficiency, as essence and blood share the same origin, leading to sperm deficiency and impaired reproductive function. According to the Compendium of Materia Medica, leek seed porridge (or leek seed tea, or leek seed wine) nourishes the liver and kidneys, warms the waist and knees, boosts yang, and strengthens essence. It is primarily used to treat symptoms such as impotence, premature ejaculation, nocturnal emission, enuresis, frequent urination, soreness and pain in the waist and knees, coldness, and excessive vaginal discharge. Previous studies have shown that leek seed porridge (or leek seed tea, or leek seed wine) primarily warms the liver and kidneys, strengthens yang, and strengthens essence. This study used network pharmacology to predict the effective ingredients, potential target genes and related pathways of leek seed porridge (or leek seed tea, leek seed wine) in improving OAS, and combined it with animal experiments for verification, to provide a direction for in-depth exploration of the mechanism by which leek seed porridge (or leek seed tea, leek seed wine) can improve OAS.

[0119] The present invention uses TCMSP to extract the active ingredients of leek seed porridge (or leek seed tea, or leek seed wine). The obtained targets are then intersected with aging disease targets to construct a PPI network. The results reveal that PTGS2, PPARG, ACTB, BCL2, CASP3, PPARA, MYC, BDNF, ERBB2, and NR3C1 are the top 10 targets. KEGG pathway analysis shows that the PI3K / Akt signaling pathway is the most enriched.

[0120] A model of oligoasthenozoospermia was established using Tripterygium wilfordii polyglycosides. After model establishment, the number and motility of sperm in the cauda epididymis of the model group were significantly lower than those in the control group, and serum testosterone levels were significantly lower than those in the control group. Microscopically, the overall structure of the testicular tissue was abnormal, with disorganized spermatogenic cells in the seminiferous tubules, some spermatogenic cells detaching and necrotic, a sharp decrease in sperm count in the seminiferous tubules, and a dramatic widening of the seminiferous tubule interstitial tissue. These findings met the diagnostic criteria for oligoasthenozoospermia, indicating successful model establishment. Following drug administration, sperm count, motility, and serum testosterone levels in all improvement groups improved to some extent. Furthermore, oral administration of Tripterygium wilfordii polyglycosides induced excessive apoptosis in the rat testis, as observed by apoptosis-related markers and TUNEL staining. Compared with the model group, all improvement groups demonstrated varying degrees of reduced apoptosis and alleviated testicular tissue damage.

[0121] PI3K controls proliferation, differentiation, programmed cell death, and migration by activating Akt. The PI3K / Akt signaling pathway has been shown to be closely associated with aging. PI3K can significantly improve sperm motility, and its mechanism of action may be related to significantly increasing cAMP levels in sperm cells. AKT, a downstream protein of PI3K, plays a crucial role in the PI3K / Akt signaling pathway. PI3K phosphorylation activates the PI3K / Akt signaling pathway, increasing Bcl-2 expression, thereby inhibiting testicular cell apoptosis and protecting testicular cells. Western blot results showed that the protein expression levels of p-PI3K / PI3K and p-AKT / AKT in the testicular tissue of the model group were significantly lower than those in the blank group, indicating that oral administration of Tripterygium wilfordii polyglycosides inhibited the PI3K / AKT signaling pathway in rat testicular tissue. Compared with the model group, the protein expression levels of p-PI3K / PI3K and p-AKT / AKT in each improvement group were increased to varying degrees. The results of animal experiments are consistent with the results of network pharmacology predictions, indicating that leek seed porridge (or leek seed tea, leek seed wine) can activate the PI3K / AKT signaling pathway, reduce the level of apoptosis, improve the testicular tissue pathological changes of oligoasthenospermia rats caused by tripterygium wilfordii polyglycosides, increase sperm count and motility, and reduce testicular damage.

[0122] In summary, the present invention has conducted multi-faceted and multi-angle research on the traditional tonic Chinese medicine leek seed porridge (or leek seed tea, leek seed wine), and preliminarily explained the mechanism of leek seed porridge (or leek seed tea, leek seed wine) in combating oligospermia and asthenospermia. The active ingredient-gene target-aging network of leek seed porridge (or leek seed tea, leek seed wine) was used to predict the pharmacological material basis of leek seed porridge (or leek seed tea, leek seed wine) in improving aging, which plays an important role in the PI3K / AKT signaling pathway. This shows that leek seed porridge (or leek seed tea, leek seed wine) plays an important role in improving aging through multiple components, multiple targets, and multiple pathways, which can provide a certain reference basis for later mechanism research and clinical application.

[0123] Example 2

[0124] The therapeutic effect of the leek seed porridge (or leek seed tea, leek seed wine) in Example 1 was explored as an effective ingredient in combination with the L-carnitine oral solution. The specific steps were as follows:

[0125] 1. Observation time and observation indicators

[0126] 1.2 Observation time

[0127] Patients were interviewed before treatment, 90±15 days after treatment, and followed up 3-6 months after the end of treatment.

[0128] 1.3 Observation indicators

[0129] 1.3.1 General Information

[0130] (1) Demographic data: patient's age, marriage, childbearing, physical examination, and specialist examination;

[0131] (2) General clinical information: allergy history, medical history, treatment history, comorbidities and medication, etc.

[0132] 1.3.2 Efficacy indicators

[0133] (1) Main efficacy indicators: sperm DFI, semen routine parameters and patient-couple pregnancy rate.

[0134] (2) Secondary efficacy indicators: changes in efficacy rate.

[0135] (3) Safety indicators: During each visit, the patient's vital signs such as blood pressure, heart rate, pulse, and the occurrence of related adverse reactions should be observed. If any adverse reactions occur, they should be recorded in detail and an adverse event form should be filled out. Based on the specific content of the patient's adverse events, they should be reported to the senior physician to decide whether to conduct subsequent trials.

[0136] 1.3.3 Criteria for determining efficacy

[0137] Formulated with reference to the World Health Organization Manual of Standardized Examination and Diagnosis and Treatment of Male Infertility and the Guiding Principles for Clinical Research of New Chinese Medicines:

[0138] (1) Cure: The patient's spouse becomes pregnant (the patient will be followed up during the medication period and 3-6 months after the end of treatment);

[0139] (2) Significantly effective: Although the patient did not conceive, the DFI was significantly reduced after treatment (DFI before treatment - DFI after treatment ≥ 10%).

[0140] (3) Effective: After treatment, at least one of the following indicators, including DFI, sperm concentration or total sperm count, total sperm motility, or progressive motility, increased by >30%;

[0141] (4) Ineffective: The patient's sperm DFI did not change significantly before and after treatment, or even increased.

[0142] The final results are shown in Tables 5 and 6.

[0143] Table 5 Effects of leek seed porridge (or leek seed tea, leek seed wine) combined with L-carnitine oral solution on sperm concentration, motility, forward motility, and DFI index in patients with oligoasthenospermia

[0144]

[0145] Note: Compared with the group before treatment, 1) P<0.05; compared with the L-carnitine group, 2) P<0.05

[0146] Table 6 Comparison of comprehensive Western medicine results between the two groups after treatment

[0147]

[0148] Note: Compared with L-carnitine group, 1) P<0.05

[0149] As shown in Tables 5 and 6 above, after treatment with leek seed extract combined with L-carnitine oral solution, sperm DFI was significantly reduced compared with before enrollment; the overall semen parameters such as semen volume, liquefaction state, sperm forward motility percentage (PR), total motility (PR+NP), sperm concentration and total sperm count were significantly increased compared with before treatment.

[0150] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. An application of a leek seed extract in preparing a drug for treating oligoasthenospermia, characterized in that: The leek seed extract can activate the PI3K / AKT signaling pathway, improve testicular tissue pathological damage, increase sperm count and motility, and thus treat oligoasthenospermia.

2. The use according to claim 1, characterized in that After treatment with the leek seed extract combined with L-carnitine oral solution, sperm DFI was significantly reduced compared with before enrollment; overall semen parameters including semen volume, liquefaction state, sperm forward motility percentage (PR), total motility (PR+NP), sperm concentration and total sperm count were significantly increased compared with before treatment.

3. The use according to claim 1, characterized in that The preparation method of the leek seed extract comprises: using salt-roasted leek seeds as raw materials, using 70% ethanol at a ratio of 1:6 (w / v) to heat and reflux extract three times, extracting the reflux filtrate with n-butanol three times, collecting the n-butanol extract, and drying it in an oven at 65°C after reduced pressure distillation to obtain an extract containing saponins and nucleoside components.

4. The use according to claim 1, characterized in that The activation of the PI3K / AKT signaling pathway is achieved by increasing the expression levels of p-PI3K / PI3K, p-AKT / AKT proteins and Bcl-2 expression in testicular tissue, thereby inhibiting testicular cell apoptosis and improving the sperm quality of oligoasthenozoospermia model rats.

5. The use according to claim 1, characterized in that The improvement of testicular tissue pathological damage is to promote the neat arrangement of spermatogenic cells, restore the lumen structure of seminiferous tubules, and reduce the widening of the testicular seminiferous tubule interstitial.

6. The use according to claim 1, characterized in that The drug also exerts a therapeutic effect by regulating serum hormone levels, such as increasing testosterone levels and reducing follicle-stimulating hormone and luteinizing hormone levels.

7. The use according to claim 1, characterized in that The drug also improves metabolic abnormalities of oligoasthenospermia by correcting metabolic pathways including linoleic acid metabolic disorders, affecting phenylalanine / tyrosine / tryptophan biosynthesis, arginine / proline metabolism and steroid hormone biosynthesis.

8. The use according to claim 1, characterized in that The oligoasthenozoospermia is caused by abnormal testicular cell apoptosis and spermatogenesis disorder due to inhibition of the PI3K / AKT signaling pathway.

9. The use according to claim 1, characterized in that The dosage form of the medicine includes leek seed porridge, leek seed tea or leek seed wine.

10. The use according to claim 1, characterized in that The drug exerts its therapeutic effects through multiple components, multiple targets and multiple pathways. The core targets of the drug include PTGS2, PPARG, BCL2, CASP3 and key molecules in the PI3K / AKT signaling pathway.

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