Application of fructus lycii-rhizoma polygonati extract in preparation of medicine for improving spermatogenesis dysfunction

Abstract

The invention relates to the field of biological medicine, in particular to application of a Chinese wolfberry-rhizoma polygonati extract in preparation of a medicine for improving spermatogenesis dysfunction. The invention provides application of three LB-PK extracts, namely a wolfberry-polygonatum kingianum aqueous extract, a wolfberry-polygonatum kingianum alcohol extract and wolfberry-polygonatum kingianum crude polysaccharide, in preparation of a medicine for improving spermatogenic dysfunction. Experimental results show that the LB-PK extract can restore the normal regulation effect of hypothalamus-pituitary-testicular axis by reversing the problems of blood glucose, dyslipidemia, sex hormone disorder and the like caused by HFD; the LB-PK extract can improve the mechanism of spermatogenic dysfunction, can improve ERS and testosterone related synthetic pathways caused by HFD, and can reduce the expression level of inflammatory factor protein in mouse testis to restore sperm generation, thereby improving the reproductive dysfunction caused by HFD. The invention explores the effect and potential mechanism of the LB-PK extract on the male reproductive disorder, and provides a new idea for treating the male reproductive disorder.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to the application of wolfberry-polygonatum extract in the preparation of a drug for improving spermatogenic dysfunction. Background Art

[0002] A high-fat diet (HFD) is one of the main factors inducing obesity. An HFD not only causes obesity, but also gives rise to various chronic diseases, thus having a huge impact on people's physical health. As an important inducement of obesity, due to the excessive intake of saturated fatty acids and trans fatty acids, an HFD has many adverse effects on human health, causing a series of adverse physiological changes, including weight changes, visceral fat accumulation, intestinal flora disorder and intestinal barrier damage, lipid metabolism and other metabolic process disorders. These physiological changes can not only lead to diseases of multiple organs and systems such as the cardiovascular and cerebrovascular system, liver, pancreas and kidney, but also induce the occurrence of obesity, various chronic diseases and even cancer. Studies have found that the health damage caused by a long-term diet pattern lacking carbohydrates, dietary fiber and excessive intake of sugar and saturated fatty acids may even be inherited by offspring. More and more evidence shows that such an unhealthy diet pattern as an HFD is one of the risk factors for triggering various diseases, and thus should arouse people's high attention.

[0003] At present, research points out that there is a close association between HFD and male reproductive health. Epidemiological studies have confirmed the negative impact of a high-energy diet and obesity on male reproductive health. At the mechanism level, it has been found that long-term intake of an HFD may cause mitochondrial damage and kinetic changes through oxidative stress, resulting in a decline in sperm quality and male fertility. In addition, an HFD also directly or indirectly causes male reproductive disorders through various pathways, such as an increase in the levels of inflammatory mediators and reactive oxygen species (ROS), intestinal flora disorder, testicular microenvironment damage, sperm DNA remodeling and epigenetic changes. The combined action of multiple factors ultimately causes damage to male reproductive ability.

[0004] In the field of reproductive health, obesity, as the most obvious consequence of a high-fat diet (HFD), is considered one of the important factors affecting male reproductive health. With the continuous increase in the global obesity rate, the decline in male sexual function and reproductive ability caused by HFD has attracted wide attention. At the mechanistic level, obesity caused by HFD may lead to male infertility by participating in multiple physiological mechanisms such as endocrine regulation, semen production, and reproductive development. Problems such as excessive peripheral adipose tissue, abnormal blood pressure, blood sugar, blood lipids, and endocrine disorders caused by HFD can interfere with the normal regulatory function of the hypothalamic-pituitary-testicular axis and have a continuous impact on male reproductive function. Testosterone, as the main male sex hormone, is of great significance to the reproductive system. It is not only responsible for maintaining normal germ cell differentiation, promoting the emergence of secondary sexual characteristics in men, but also maintaining normal reproductive function. However, obesity caused by HFD can affect the testosterone level in men. Research shows that there is a negative correlation between body mass index (BMI) and testosterone level, and a negative correlation between body mass index and semen parameters (such as sperm density, total amount, and motility). The decrease in testosterone caused by obesity not only affects the proliferation and apoptosis of spermatogenic cells but also leads to a decrease in sperm production and motility. Overweight and obesity increase the likelihood of men suffering from oligospermia, asthenospermia, and azoospermia by interfering with the development of the male reproductive system and the regulation of gonadal hormones. Obesity may lead to an increase in DNA damage and oxidative stress response of sperm, thereby affecting sperm quality. These problems may lead to a decrease in embryo quality, embryo developmental disorders, and an increase in the abortion rate and malformation rate. Obesity also leads to a decrease in libido, a decrease in the frequency of sexual intercourse, and even erectile dysfunction. In addition, obesity may also cause adverse emotions such as anxiety and depression, which in turn affect male sexual function and lead to reproductive disorders. Currently, the therapeutic methods for obesity-related male infertility have limited efficacy. The success rate of conventional treatment is less than 30%, and the underlying molecular mechanisms have not been resolved. Therefore, in-depth exploration of the impact of HFD on male reproductive health, especially through the path of improving obesity, is of great significance for promoting male reproductive health and increasing fertility. Summary of the Invention

[0005] The object of the present invention is to provide the application of Lycium barbarum-Polygonatum sibiricum extract in the preparation of a drug for improving spermatogenic dysfunction, so as to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides the application of Lycium barbarum-Polygonatum sibiricum extract in the preparation of a drug for improving spermatogenic dysfunction, and the Lycium barbarum-Polygonatum sibiricum extract includes one or several of Lycium barbarum-Polygonatum sibiricum water extract, Lycium barbarum-Polygonatum sibiricum alcohol extract, and Lycium barbarum-Polygonatum sibiricum crude polysaccharide.

[0008] Preferably, the preparation method of the Lycium barbarum-Polygonatum sibiricum water extract includes the steps of mixing Lycium barbarum and Polygonatum sibiricum, decocting and concentrating to obtain the Lycium barbarum-Polygonatum sibiricum water extract;

[0009] The preparation method of the wolfberry-polygonatum alcohol extract comprises the steps of mixing wolfberry and polygonatum, followed by alcohol extraction, concentration and drying to obtain the wolfberry-polygonatum alcohol extract;

[0010] The preparation method of the wolfberry-polygonatum crude polysaccharide comprises the steps of water extraction, concentration, precipitation with ethanol, filtration and drying of the remaining residue from the preparation of the wolfberry-polygonatum alcohol extract to obtain the wolfberry-polygonatum crude polysaccharide.

[0011] Preferably, when preparing the wolfberry-polygonatum water extract, the mass ratio of wolfberry to polygonatum is 1:1; the number of decoction times is 3 times, the decoction time for each time is 1 h, and the solvent is water; when performing the first decoction, the amount of the solvent is 8 times the total mass of wolfberry and polygonatum; when performing the second decoction, the amount of the solvent is 6 times the total mass of wolfberry and polygonatum; when performing the third decoction, the amount of the solvent is 4 times the total mass of wolfberry and polygonatum.

[0012] Preferably, when preparing the wolfberry-polygonatum alcohol extract, the mass ratio of wolfberry to polygonatum is 1:1; the number of alcohol extraction times is 3 times, and the extractant is an ethanol solution with a volume percentage content of 80%; when performing the first alcohol extraction, the amount of the extractant is 8 times the total mass of wolfberry and polygonatum, and the extraction time is 60 min; when performing the second alcohol extraction, the amount of the extractant is 6 times the total mass of wolfberry and polygonatum, and the extraction time is 40 min; when performing the third alcohol extraction, the amount of the extractant is 4 times the total mass of wolfberry and polygonatum, and the extraction time is 30 min.

[0013] Preferably, when preparing the wolfberry-polygonatum crude polysaccharide, the number of water extraction times is 3 times, the water extraction time for each time is 2 h, and the extractant is water; when performing the first water extraction, the amount of the extractant is 8 times the mass of the remaining residue; when performing the second water extraction, the amount of the extractant is 6 times the mass of the remaining residue; when performing the third water extraction, the amount of the extractant is 4 times the mass of the remaining residue.

[0014] Preferably, the spermatogenic dysfunction includes spermatogenic dysfunction caused by a high-fat diet.

[0015] Preferably, the drug achieves the effect of improving spermatogenic dysfunction by improving blood glucose abnormality and insulin resistance, improving sex hormone disorder, improving sperm motility and quantity, and reducing the sperm malformation rate.

[0016] The present invention provides a drug for improving spermatogenic dysfunction, and the drug comprises Lycium barbarum - Polygonatum sibiricum extract; the Lycium barbarum - Polygonatum sibiricum extract comprises one or more of Lycium barbarum - Polygonatum sibiricum water extract, Lycium barbarum - Polygonatum sibiricum alcohol extract, and Lycium barbarum - Polygonatum sibiricum crude polysaccharide.

[0017] Preferably, the preparation method of the Lycium barbarum - Polygonatum sibiricum water extract comprises the steps of mixing Lycium barbarum and Polygonatum sibiricum, decocting and concentrating to obtain the Lycium barbarum - Polygonatum sibiricum water extract;

[0018] The preparation method of the Lycium barbarum - Polygonatum sibiricum alcohol extract comprises the steps of mixing Lycium barbarum and Polygonatum sibiricum, performing alcohol extraction, concentrating and drying to obtain the Lycium barbarum - Polygonatum sibiricum alcohol extract;

[0019] The preparation method of the Lycium barbarum - Polygonatum sibiricum crude polysaccharide comprises the steps of performing water extraction, concentrating, precipitating with ethanol, filtering and drying on the remaining residue obtained by preparing the Lycium barbarum - Polygonatum sibiricum alcohol extract to obtain the Lycium barbarum - Polygonatum sibiricum crude polysaccharide.

[0020] Preferably, the drug comprises pharmaceutically acceptable excipients.

[0021] More preferably, when preparing the Lycium barbarum - Polygonatum sibiricum water extract, the mass ratio of Lycium barbarum to Polygonatum sibiricum is 1:1; the number of decocting times is 3 times, the decocting time for each time is 1 h, and the solvent is water; when performing the first decocting, the dosage of the solvent is 8 times the total mass of Lycium barbarum and Polygonatum sibiricum; when performing the second decocting, the dosage of the solvent is 6 times the total mass of Lycium barbarum and Polygonatum sibiricum; when performing the third decocting, the dosage of the solvent is 4 times the total mass of Lycium barbarum and Polygonatum sibiricum.

[0022] More preferably, when preparing the Lycium barbarum - Polygonatum sibiricum alcohol extract, the mass ratio of Lycium barbarum to Polygonatum sibiricum is 1:1; the number of alcohol extraction times is 3 times, and the extractant is an ethanol solution with a volume percentage content of 80%; when performing the first alcohol extraction, the dosage of the extractant is 8 times the total mass of Lycium barbarum and Polygonatum sibiricum, and the extraction time is 60 min; when performing the second alcohol extraction, the dosage of the extractant is 6 times the total mass of Lycium barbarum and Polygonatum sibiricum, and the extraction time is 40 min; when performing the third alcohol extraction, the dosage of the extractant is 4 times the total mass of Lycium barbarum and Polygonatum sibiricum, and the extraction time is 30 min.

[0023] More preferably, when preparing the Lycium barbarum - Polygonatum sibiricum crude polysaccharide, the number of water extraction times is 3 times, the water extraction time for each time is 2 h, and the extractant is water; when performing the first water extraction, the dosage of the extractant is 8 times the mass of the remaining residue; when performing the second water extraction, the dosage of the extractant is 6 times the mass of the remaining residue; when performing the third water extraction, the dosage of the extractant is 4 times the mass of the remaining residue.

[0024] Further preferably, the spermatogenic dysfunction includes spermatogenic dysfunction caused by a high-fat diet.

[0025] Further preferably, the drug improves spermatogenic dysfunction by improving blood glucose abnormalities and insulin resistance, improving sex hormone disorders, improving sperm motility and quantity, and reducing the malformation rate of sperm.

[0026] The present invention discloses the following technical effects:

[0027] The present invention provides the application of three Lycium barbarum-Polygonatum sibiricum extracts (LB-PK extracts), namely water extract of Lycium barbarum-Polygonatum sibiricum, alcohol extract of Lycium barbarum-Polygonatum sibiricum, and crude polysaccharide of Lycium barbarum-Polygonatum sibiricum, in the preparation of drugs for improving spermatogenic dysfunction. The experimental results show that: the LB-PK extract contains 140 compound derivatives that can improve testicular function and semen quality; the LB-PK extract can effectively improve obesity caused by HFD and a series of metabolic problems caused thereby, that is, the LB-PK extract can restore the normal regulatory function of the hypothalamus-pituitary-testicular axis by reversing blood glucose, blood lipid abnormalities, and sex hormone disorders caused by HFD; the mechanism by which the LB-PK extract improves spermatogenic dysfunction can improve ERS caused by HFD, the testosterone-related synthesis pathway, and reduce the protein expression level of inflammatory factors in the testis of mice to restore sperm production, thereby improving reproductive dysfunction caused by HFD (the LB-PK extract can effectively inhibit obesity caused by HFD and reverse blood glucose and blood lipid abnormalities caused by HFD, improve sex hormone disorders, decreased sperm parameters, abnormal sperm flagellar mitochondria, and fertility of mice caused by HFD). Moreover, Lycium barbarum and Polygonatum sibiricum, as natural resources for both medicine and food, should be combined with modern pharmacological research to broaden the functional applications of the LB-PK extract. The present invention explores the effects and potential mechanisms of the LB-PK extract on male reproductive disorders, providing new ideas for the treatment of male reproductive disorders. Description of the Drawings

[0028] Figure 1 It is the total ion current chromatogram of LB-PKE combined with high-resolution mass spectrometry; wherein, A is the positive ion mode; B is the negative ion mode;

[0029] Figure 2 It is the structural formula of some compounds analyzed by UPLC-Q-Exactive Orbitrap MS that can improve testicular function and semen parameters;

[0030] Figure 3Effects of HFD on body weight, fertility and F1 of mice at different feeding weeks; among them, A-C show the body weight, fertility and the number of F1 mice of the Control group and the HFD group at 8 weeks; D-F show the body weight, fertility and the number of F1 mice of the Control group and the HFD group at 10 weeks; G-I show the body weight, fertility and the number of F1 mice of the Control group and the HFD group at 12 weeks; compared with the blank group, ##P<0.01, P<0.001; the F0 generation is 2 female mouse cubs;

[0031] Figure 4 Effects of LB-PK on serum physiological and biochemical indexes of mice with spermatogenic disorder induced by HFD; among them, A is TC; B is TG; C is LDL-C; D is HDL-C; compared with the blank group, P<0.001; compared with HFD: *P<0.05, **P<0.01, ***P<0.001;

[0032] Figure 5 Effects of HFD on oral glucose tolerance and insulin tolerance of mice; among them, A shows the blood glucose level of HFD mice during OGTT with LB-PK extract; B shows the area under the blood glucose curve (AUC) during OGTT; C shows the blood glucose level of HFD mice during ITT with LB-PK extract; D shows the area under the blood glucose curve during ITT; compared with the blank group, P<0.001; compared with HFD: ***P<0.001;

[0033] Figure 6 Effects of LB-PK extract on fertility of mice with spermatogenic disorder induced by HFD; among them, A is the fertility rate; B is the number of F1 mice; compared with the blank group, P<0.001; compared with HFD, *P<0.05, **P<0.01, ***P<0.001;

[0034] Figure 7 Effects of LB-PK extract on body weight of mice with spermatogenic disorder induced by HFD; among them, a is LB-PKD; b is LB-PKE; c is LB-PKP; the left is the trend chart of mouse body weight change, and the right is the picture of mouse body shape;

[0035] Figure 8 Effects of LB-PK extract on testicular weight, testicular index and epididymal fat hypertrophy index of mice with spermatogenic disorder induced by HFD; among them, a is testicular weight; b is testicular index; c is epididymal fat hypertrophy index; compared with the blank group, #P<0.05, P<0.001; compared with HFD: *P<0.05, **P<0.01, ***P<0.001;

[0036] Fig. 9Effects of LB-PK extract on sex hormones in HFD-induced spermatogenesis disorder mice; among them, A is serum testosterone; B is serum estradiol; C is serum follicle-stimulating hormone; compared with the blank group, P<0.001; compared with HFD: *P<0.05, **P<0.01, ***P<0.001;

[0037] Fig.10 Effects of LB-PK extract on sperm parameters in HFD-induced spermatogenesis disorder mice; among them, A is sperm motility; B is sperm concentration; C is the sperm morphology of each group after administration of LB-PK extract (scale bar is 50μm), a is Control, b is HFD, c is HFD+DL, d is HFD+DM, e is HFD+DH, f is HFD+EL, g is HFD+EM, h is HFD+EH, i is HFD+PL, j is HFD+PM, k is HFD+PH; compared with the blank group, P<0.001; compared with HFD: *P<0.05, ***P<0.001);

[0038] Fig.11 Effects of LB-PK extract on testicular histology in HFD-induced spermatogenesis disorder mice; among them, a is Control, b is HFD, c is HFD+DL, d is HFD+DM, e is HFD+DH, f is HFD+EL, g is HFD+EM, h is HFD+EH, i is HFD+PL, j is HFD+PM, k is HFD+PH; scale bar is 50μm;

[0039] Fig.12 Results of oil red staining of testis after administration of LB-PK extract; among them, a is Control, b is HFD, c is HFD+DL, d is HFD+DM, e is HFD+DH, f is HFD+EL, g is HFD+EM, h is HFD+EH, i is HFD+PL, j is HFD+PM, k is HFD+PH; taking a as an example, the scale bars from top to bottom are 500μm, 100μm and 20μm in turn;

[0040] Fig.13 Quantitative results of oil red staining; compared with the blank group, P<0.001; compared with HFD: ***P<0.001;

[0041] Fig.14Effect of LB-PK extract on sperm ultrastructure of HFD-induced spermatogenic disorder mice; among them, a is Control, b is HFD, c is HFD+DL, d is HFD+DM, e is HFD+DH, f is HFD+EL, g is HFD+EM, h is HFD+EH, i is HFD+PL, j is HFD+PM, k is HFD+PH; taking a as an example, the scales from top to bottom are 1μm and 500nm in turn;

[0042] Fig.15 Representative bands of the effects of LB-PKD and LB-PKE on testosterone synthesis, testicular inflammatory factors and protein expression related to the PERK-CHOP pathway (A), and representative bands of the effects of LB-PKP on testosterone synthesis, testicular inflammatory factors and protein expression related to the PERK-CHOP pathway (B);

[0043] Fig.16 Statistical charts of the relative protein expression levels of LB-PK extract on testosterone synthesis and testicular inflammatory factors CYP17A1, StAr, 3β-HSD, IL-1β, IL-6, TNF-α, CHOP, P-PERK (A and B); n = 3; data are expressed as mean ± SD, ##P<0.01, P<0.001 vs Control group, *P<0.05, **P<0.01, ***P<0.001 vs HFD group. Detailed implementation mode

[0044] Example 1

[0045] The present invention adopts the water decoction method, ethanol reflux method and water extraction and alcohol precipitation method to prepare the water decoction of LB-PK (Lycium barbarum-Polygonatum sibiricum) (LB-PKD, water extract of Lycium barbarum-Polygonatum sibiricum), ethanol extract of LB-PK (LB-PKE, alcohol extract of Lycium barbarum-Polygonatum sibiricum) and crude polysaccharide of LB-PK (LB-PKP) respectively. The components of LB-PKD and LB-PKE are analyzed by UPLC-Q-Exactive Orbitrap MS, and non-targeted metabolomics analysis is carried out.

[0046] 1. Materials and instruments

[0047] Medicinal materials: Lycium barbarum and Polygonatum sibiricum (processed with wine), both purchased from Xinfafa Pharmaceutical Co., Ltd. in Huaguoyuan Chinese Medicinal Materials Market, Guiyang City, Guizhou Province, and identified as Lycium barbarum L. by Professor Sun Qingwen of Guizhou University of Traditional Chinese Medicine; Polygonatum kingianum Collett & Hemsl, voucher numbers are LB2023-1 and PK2023-1 respectively, stored in Room 502, Building B of the Natural Product Research Center.

[0048] Drugs and Reagents: The main reagents involved are shown in Table 1.

[0049] Table 1 Main Reagents for Experiments

[0050] Raw materials or reagents Manufacturer Analytical grade anhydrous ethanol Tianjin Fuyu Fine Chemical Co., Ltd. Distilled water Hangzhou Wahaha Group Co., Ltd. Chromatographic grade methanol Tianjin Concord Technology Co., Ltd. Chromatographic grade acetonitrile Tianjin Concord Technology Co., Ltd. Chromatographic grade formic acid Tianjin Concord Technology Co., Ltd.

[0051] Main Equipment and Instruments: The main equipment and instruments involved are shown in Table 2.

[0052] Table 2 Main Equipment and Instruments for Experiments

[0053] Instrument Name Manufacturer EYELAN-1100 Rotary Evaporator Japan EYELA Company UV-1800 UV-Visible Spectrophotometer Shimadzu Instruments (Suzhou) Co., Ltd. FA2004N Electronic Analytical Balance Shanghai Jinghai Instrument Co., Ltd. DK-98-Ⅱ Electric Constant Temperature Water Bath Tianjin Test Instrument Co., Ltd. Quadrupole-Electrostatic Field Orbital Trap High Resolution Mass Spectrometer Thermo Fisher Scientific Inc.

[0054] 2. Methods

[0055] 2.1 Preparation of Different LB-PK Drugs (LB-PK Extracts)

[0056] 2.1.1 Preparation of LB-PK Decoction (LB-PK Water Extract, LB-PKD)

[0057] Take equal amounts of dry wolfberry and polygonatum powder and decoct them 3 times; for the first decoction, the amount of deionized water used is 8 times the total mass of dry wolfberry and polygonatum powder; for the second decoction, the amount of deionized water used is 6 times the total mass of dry wolfberry and polygonatum powder; for the third decoction, the amount of deionized water used is 4 times the total mass of dry wolfberry and polygonatum powder; the decoction time for each time is 1 h, and then combine the decoctions obtained from the 3 times of decoction, and concentrate under reduced pressure (pressure is 150 mbar) until the content of the medicinal materials is 1700 mg / 10 mL to obtain LB-PK decoction (LB-PKD), and store it at 4 °C for standby.

[0058] 2.1.2 Preparation of LB-PK Ethanol Extract (LB-PK Alcohol Extract, LB-PKE)

[0059] Take equal amounts of dry wolfberry and polygonatum powder and perform 3 times of reflux extraction; for the first reflux extraction, the amount of 80% (V / V) ethanol used is 8 times the total mass of dry wolfberry and polygonatum powder, and the reflux extraction time is 60 min; for the second reflux extraction, the amount of 80% (V / V) ethanol used is 6 times the total mass of dry wolfberry and polygonatum powder, and the reflux extraction time is 40 min; for the third reflux extraction, the amount of 80% (V / V) ethanol used is 4 times the total mass of dry wolfberry and polygonatum powder, and the reflux extraction time is 30 min; combine the extracts obtained from the 3 times of reflux extraction, concentrate under reduced pressure (pressure is 50 mbar) to 50 mL, and then freeze-dry at -30 °C for 36 h to obtain LB-PK alcohol extract (LB-PKE).

[0060] 2.1.3 Preparation of LB-PK Polysaccharide Extract (LB-PK Crude Polysaccharide, LB-PKP)

[0061] The residue after reflux extraction with 80% (V / V) ethanol (the traditional Chinese medicine residue remaining from the step of "2.1.2. Preparation of ethanol extract of LB-PK") was successively added with 8, 6, and 4 times the amount of deionized water and extracted 3 times at 80 °C in a water bath for 2 h each time. The extraction solutions obtained from the 3 extractions were combined and concentrated under reduced pressure (pressure: 50 mbar) to 50 mL. Ethanol was added to make the final ethanol concentration 80% (V / V). It was left to stand overnight at 4 °C, filtered, and the filter residue was freeze-dried at -30 °C for 36 h to obtain the LB-PK polysaccharide extract (LB-PKP).

[0062] 2.2. UPLC-MS / MS analysis of LB-PKD and LB-PKE

[0063] 2.2.1. Sample extraction procedure

[0064] (1) LB-PKE and LB-PKD were placed in a freeze dryer (Scientz-100F) and vacuum freeze-dried for 63 h; (2) ground to a powder using a grinder (MM 400, Retsch) (30 Hz, 1.5 min); (3) 50 mg of the sample powder was weighed using an electronic balance (MS105DM), and 1200 μL of -20 °C pre-cooled 70% (V / V) methanol aqueous internal standard extraction solution was added. The internal standard extraction solution was prepared by dissolving 1 mg of 2-chlorophenylalanine in 1 mL of 70% (V / V) methanol aqueous solution to prepare a 1000 μg / mL stock solution, and the 1000 μg / mL stock solution was further diluted with 70% (V / V) methanol to prepare a 250 μg / mL internal standard solution;

[0065] (4) Vortexed once every 30 min for 30 s each time, for a total of 6 times;

[0066] (5) After centrifuging at 12000 rpm for 3 min, the supernatant was aspirated, the sample was filtered through a microporous membrane (0.22 μm pore size), and stored in a sample vial for UPLC-MS / MS analysis.

[0067] 2.2.2. Chromatographic and mass spectrometric acquisition conditions

[0068] The data acquisition instrument system mainly includes ultra-high performance liquid chromatography (UPLC) (ExionLC TM AD, https: / / sciex.com.cn / ) and tandem mass spectrometry (MS / MS).

[0069] The liquid phase conditions mainly include: (1) Chromatographic column: Agilen SB-C 18, 1.8 μm, 2.1 mm × 100 mm; (2) Mobile phase: Phase A is ultrapure water (added with 0.1% (V / V) formic acid), and Phase B is acetonitrile (added with 0.1% (V / V) formic acid); (3) Elution gradient: The proportion of Phase B is 5% (V / V) at 0.00 min, linearly increases to 95% (V / V) within 9.00 min, and is maintained at 95% (V / V) for 1 min. From 10.00 - 11.10 min, the proportion of Phase B drops to 5% (V / V) and is equilibrated to 14 min at 5% (V / V); (4) Flow rate is 0.35 mL / min; Column temperature is 40 °C; Injection volume is 2 μL.

[0070] 2.2.3, Mass spectrometry conditions

[0071] It mainly includes: The temperature of the electrospray ionization (ESI) source is 500 °C; The ion spray voltage (IS) is 5500 V (positive ion mode) / - 4500 V (negative ion mode); The ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) are set to 50, 60, and 25 psi respectively, and the collision-induced ionization parameter is set to high. The QQQ scan uses the MRM mode, and the collision gas (nitrogen) is set to medium. Through further optimization of the declustering potential (DP) and collision energy (CE), the DP and CE of each MRM ion pair are completed. According to the metabolites eluted in each period, a specific set of MRM ion pairs is monitored in each period.

[0072] 2.2.4, Principles of metabolite qualitative and quantitative analysis

[0073] Based on the self-built database MWDB (Metware Database) of Metware Biotechnology, substances are qualitatively analyzed according to the secondary spectrum information. Isotope signals, duplicate signals containing K + , Na + , NH4 + , and duplicate signals of fragment ions that are themselves fragments of other larger molecular weight substances are removed during analysis.

[0074] Metabolite quantification was completed by analyzing using the multiple reaction monitoring (MRM) mode of triple quadrupole mass spectrometry. In the MRM mode, the quadrupole first screens the precursor ions (parent ions) of the target substance, excluding the ions corresponding to other molecular weight substances to preliminarily exclude interference; the precursor ions are fragmented after being induced to ionize in the collision cell to form many fragment ions, and then a characteristic fragment ion needed is selected through the triple quadrupole filter, excluding the interference of non-target ions, making the quantification more accurate and the repeatability better. After obtaining the mass spectrometry analysis data of different samples, the peak areas of all substance chromatographic peaks are integrated, and the mass spectrometry peaks of the same metabolite in different samples are corrected for integration.

[0075] 3. Results and Analysis

[0076] The components of LB-PKD and LB-PKE were analyzed by UPLC-MS / MS method, and non-targeted metabolomics detection was carried out. A total of 1409 secondary metabolites were identified, among which 140 compounds and their derivatives that can improve testicular function and semen quality were detected (details are shown in Table 4), such as Figure 2As shown, there are 5 phenolic acid chlorogenic acid derivatives (chlorogenic acid can improve spermatogenic function of the testis by reducing mitochondrial damage and inhibiting the activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (cGAS-STING) axis, inhibiting the activation of the NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome, and improving inflammatory damage of the testis); 97 flavonoids, including 15 apigenin derivatives (apigenin can increase sperm density, motility and MMP, significantly reduce sperm ROS and MDA levels, improve sperm ultrastructure and testicular tissue damage, and reduce spermatogenic cell apoptosis); 8 luteolin derivatives (naturally occurring flavonoids such as luteolin can maintain the normal range of testosterone levels); 25 kaempferol derivatives (kaempferol can improve the weights of male reproductive organs and sperm quality in a cyclophosphamide-induced asthenospermia mouse model, and reduce testicular tissue damage); 34 quercetin derivatives (quercetin promotes testosterone production and enhances testicular function by inhibiting cyclooxygenase-2 (COX-2), reducing inflammation, increasing cyclic adenosine monophosphate / protein kinase A (cAMP / PKA) signaling, upregulating steroidogenic acute regulatory protein (StAR protein), and inhibiting DAX-1, etc.); 8 naringenin derivatives (naringenin is beneficial for the continuous maturation of spermatogonia and the sperm formation process); 2 catechin derivatives (epigallocatechin-3-gallate (EGCG) can restore arsenic-induced testicular damage in mice, such as testicular spermatogenic degeneration, reduced epithelial height, and decreased sperm in seminiferous tubules); 2 morin derivatives (morin reduces cadmium-induced testicular damage by stimulating testosterone secretion and germ cell proliferation in mice); 3 hesperetin derivatives (hesperetin reduces testicular damage in diabetic rats, possibly by inhibiting apoptosis, oxidative stress, inflammation, and upregulating endogenous antioxidants), 3 alkaloid betaine derivatives (betaine can significantly restore testicular pathological damage induced by tripterygium glycosides (TWGs), and can also significantly reverse the apoptosis of spermatogenic cells), 30 steroid diosgenin derivatives (diosgenin can reverse the damage to seminiferous tubules), 3 other types, including 2 apigenin derivatives, 1 lignan, and 1 sesquiterpene. The remaining other types, lignins, and sesquiterpenes are all secondary metabolites of icariin, icariside (icariin inhibits pyroptosis and insulin resistance of NLRP3 inflammasome activation, reduces the expression of inflammatory mediators, enhances insulin sensitivity, promotes testicular hormone synthesis and secretion, restores the function of Leydig cells, and improves testicular spermatogenesis in obese mice). In addition, as Figure 1As shown in Table 3, there are 22 unique compounds in LB-PKE, including 3 flavonoids, 7 terpenoids, 7 steroids, 2 phenolic acids, 2 other types, and 1 alkaloid. Among them, there are 4 diosgenins and their derivatives with reported reproductive activity, 2 quercetin derivatives, and 1 kaempferol derivative. There are 480 compounds with content differences between LB-PKE and LB-PKD, among which 408 compounds are up-regulated and 72 are down-regulated. Among the compounds at level 1, 152 are up-regulated and 16 are down-regulated (see Table 5 for details); among the compounds at level 2, 93 are up-regulated and 7 are down-regulated; among the compounds at level 3, 179 are up-regulated and 49 are down-regulated.

[0077] Table 3 Chemically unique components and reported bioactive compounds in LB-PKE identified by UPLC-Q-Exactive Orbitrap MS

[0078]

[0079]

[0080] Note: "+" indicates detected, "NA-" indicates not detected.

[0081] Table 4 Compounds and their derivatives with improved testicular function and semen quality

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088] Table 5 Level 1 differential substances between LB-PKD and LB-PKE

[0089]

[0090]

[0091]

[0092]

[0093]

[0094] In summary, there are 480 changes in the component content between LB-PKE and LB-PKD, among which 408 compounds are up-regulated and 72 are down-regulated. The components of LB-PKD and LB-PKE were analyzed by UPLC-Q-Exactive Orbitrap MS, and non-targeted metabolomics detection was performed. A total of 1409 secondary metabolites were identified, among which 140 compound derivatives that can improve testicular function and semen parameters were detected. It can be seen that the LB-PK drug pair has great potential in the treatment of male reproductive disorders. As natural resources for both medicine and food, Chinese wolfberry and polygonatum sibiricum should be combined with modern pharmacological research to broaden the functional applications of the LB-PK drug, explore the effects and potential mechanisms of LB-PK on male reproductive disorders, and provide new ideas for the treatment of male reproductive disorders.

[0095] Example 2

[0096] 1. Materials and Instruments

[0097] Experimental animals: C57BL / 6 mice, male, SPF grade, weighing 18 - 20 g, 8 weeks old, purchased from Beijing SPF Biotechnology Co., Ltd., license number: SCXK (Beijing) 2019 - 0010. They were housed in the animal house of Guizhou Center for Natural Products Research, at a room temperature of 22 - 25 °C and a humidity of 50 - 60%. They were fed and given water ad libitum. All animal experiments were carried out strictly in accordance with international ethical guidelines and the Guide for the Care and Use of Laboratory Animals.

[0098] Experimental drugs: The main drugs involved are shown in Table 6.

[0099] Table 6 Manufacturers of main experimental drugs

[0100]

[0101]

[0102] Experimental instruments and equipment: The main instruments and equipment involved are shown in Table 7.

[0103] Table 7 Main experimental instruments and manufacturers

[0104] Instrument Name Manufacturer BEION S3 Sperm Quality Analyzer Shanghai Jumu Medical Equipment Co., Ltd. JB-P5 tissue embedding machine Wuhan Junjie Electronics Co., Ltd. RM2016 semi-automatic paraffin slicer Shanghai Leica Instruments Co., Ltd. GFL-230 Oven Tianjin Lapore Instrument Equipment Co., Ltd. KD-P tissue slicer Zhejiang Jinhua Kedi Instrument Equipment Co., Ltd. 10212432C Cover glass Jiangsu Shitai Experimental Equipment Co., Ltd. NIKON ECLIPSE E100 upright microscope Nikon Japan Centrifuge 5427R Shanghai Aibende International Trade Co., Ltd. Toledo Electronic Balance Mettler-Toledo AG EPOCH Multifunctional Microplate Reader Thermo Corporation ChemiScope 3000mini gel imaging analysis system Shanghai Qinxiang Scientific Instrument Co., Ltd. 902GP-ULTS Ultra Low Temperature Freezer Thermo Corporation SCI-VS Turbine Oscillator BioSky Biotech 1645050 Electrophoresis apparatus, electrophoresis tank Bio-Rad Corporation IMS-50 Flake Ice Machine IMS-50Biobase SWE-FP Grinder Wuhan Sewell Biotechnology Co., Ltd. PLUS-E3-20TH trace analysis ultrapure water machine Nanjing E-Tech Technology Development Co., Ltd.

[0105] 2. Experimental Methods

[0106] 2.1 Establishment of a spermatogenic dysfunction model in mice induced by HFD

[0107] Thirty-two C57BL / 6J mice were randomly divided into a blank group (Control, 8 mice) and a model group (HFD, 24 mice). The mice in each group were adaptively fed for one week at 22±2°C with a 12-h light / dark cycle, during which they were normally given feed and drinking water. After one week, except for the blank group, the feed of the mice in the other groups was replaced with the D12492 high-fat obesity model feed for 8, 10, and 12 weeks of modeling. To evaluate the damage to the fertility of male mice caused by HFD, at 8, 10, and 12 weeks of modeling, 3 mice with a body weight >120% of the blank group in the HFD group were taken out, and each mouse was caged with two 8-week-old female mice for one week. During this period, except for the blank group, the male mice in the other groups were fed a high-fat diet. The female mice with vaginal plugs were moved to another cage and observed until the pups were born. The number of pregnant female mice and the number of offspring were recorded.

[0108] 2.2. Animal grouping and drug administration

[0109] Eighty-eight C57BL / 6J mice were adaptively fed for one week at 22±2°C with a 12-h light / dark cycle, during which they were normally given feed and drinking water. After one week, except for the blank group (Control, 8 mice), the feed of the mice in the other groups was replaced with a high-fat diet for 10 weeks of modeling, denoted as HFD. The administered doses were converted from the human doses shown in the pharmacopoeia. For example, for an adult with a body weight of 60 kg, the daily dose of LB-PK should be 10 g. According to the conversion method of human and animal drug dosages, the medium dose was the human dose, and the ratios of the low, medium, and high doses were 1:2:4. The mice with a body weight >120% of the blank group in the HFD group were divided into the HFD group, the LB-PKD low-dose group of 850 mg / kg (HFD+DL), the LB-PKD medium-dose group of 1700 mg / kg (HFD+DM), the LB-PKD high-dose group of 3400 mg / kg (HFD+DH), the LB-PKE low-dose group of 335 mg / kg (HFD+EL), the LB-PKE medium-dose group of 670 mg / kg (HFD+EM), the LB-PKE high-dose group of 1340 mg / kg (HFD+EH), the LB-PKP low-dose group of 45 mg / kg (HFD+PL), the LB-PKP medium-dose group of 90 mg / kg (HFD+PM), and the LB-PKP high-dose group of 180 mg / kg (HFD+PH); there were 8 mice in each treatment. Prepared according to the concentration, the mice in each group were gavaged at a dose of 0.01 mL / g of body weight and continuously gavaged for 5 weeks, and the body weight of the mice was recorded every 7 days during this period. Calculate the body weight change rate at the end point of the intervention according to the following formula;

[0110] Body weight change rate (%) = body weight at the end point of the intervention (g) / initial body weight before modeling (g) × 100%.

[0111] 2.3. Oral Glucose Tolerance Test and Insulin Tolerance Test

[0112] The oral glucose tolerance test (OGTT) was performed 6 days before the end of the experiment. All mice were fasted for 14 h and then gavaged with a 2 g / kg glucose solution (the solution concentration was 2 g glucose / 10 mL sterile water). The blood glucose levels of the mice after gavage were measured using a SANNUO stable blood glucose meter. The sampling time points were 0, 15, 30, 60, 90, and 120 min after the administration of the glucose solution. The insulin tolerance test (ITT) was performed 3 days before the end of the experiment. After fasting for 6 h, 0.75 U / kg sterile insulin was injected intraperitoneally, and the blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 min after the injection of insulin. The areas under the curves (AUC) of OGTT and ITT were calculated respectively.

[0113] 2.4. Determination of reproductive ability

[0114] The method for establishing the mouse spermatogenic dysfunction model induced by HFD was the same as that in the step "2.1. Establishment of the mouse spermatogenic dysfunction model induced by HFD" of this example.

[0115] 2.5. Sample processing

[0116] Twelve hours after the last administration, blood was collected from the retro-orbital venous sinus of the mice, and the serum was separated for further analysis. After the mice were euthanized by overdose anesthesia, a midline abdominal incision was made, and the left and right testes, epididymides, and epididymal fat were isolated, rinsed with pre-cooled saline, and blotted dry with filter paper. The weights were measured. The left testes of three randomly selected mice in each group were fixed in testicular fixative, and after 24 h, they were fixed in 75% (V / V) ethanol. The testes of the remaining mice were weighed and homogenized in a saline grinder at a ratio of 1:9. The collected blood and tissue homogenates were centrifuged at 4 °C and 3500 rpm for 10 min, and the supernatants were taken for subsequent detection. The testicular, epididymal, and epididymal fat indices of each group were calculated according to the following formula for comparison;

[0117]

[0118] 2.6. Sperm parameter detection

[0119] Take the cauda epididymidis of the right mouse, chop it up in physiological saline, incubate it at 37°C for about 30 minutes until spermatozoa are released from the epididymis. Cut the cauda epididymidis into small pieces and place them in a 24-well plate, with 200 μL of physiological saline in each well, and incubate at 37°C for 30 minutes. Aspirate 10 μL of the sperm suspension and drop it onto a glass slide, and use the Testsimplets staining method for glass slides to observe the sperm morphology of mice at different modeling cycles. Aspirate 10 μL of the sperm suspension and drop it onto a sperm counting chamber, and use a sperm quality analyzer to count and analyze the motility. Centrifuge the sperm suspension at 1500 rpm for 5 minutes, discard the supernatant, and resuspend and fix it with an electron microscope fixative.

[0120] 2.7 Detection of sex hormones and biochemical indicators

[0121] Detect the contents of sex hormones testosterone (T), estradiol (E2), follicle-stimulating hormone (FSH), TG, TC, HDL-C, and LDL-C in the serum obtained in the step "2.5 Sample processing" according to the kit instructions; set at least 3 replicate wells for each group of mouse samples, and calculate the average value of the results for statistical analysis.

[0122] 2.8 H&E staining of the testis

[0123] For the fixed testis, infiltrate it with paraffin, embed the paraffin blocks, and complete the embedding of the paraffin sections after the paraffin solidifies again.

[0124] (1) Dewax the paraffin sections to water: sequentially place the tissue sections into environmental protection type dewaxing solution I for 20 minutes - environmental protection type dewaxing solution II for 20 minutes - absolute ethanol I for 5 minutes - absolute ethanol II for 5 minutes - 75% (V / V) alcohol for 5 minutes, and wash with tap water. (2) Pretreatment: Immerse the sections in a high-definition constant staining pretreatment solution for 1 minute. (3) Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 3 - 5 minutes, wash with tap water, differentiate with a differentiating solution, wash with tap water, blue with a bluing solution, and rinse with running water. (4) Eosin staining: Immerse the sections in 95% (V / V) alcohol for dehydration for 1 minute, and stain in eosin staining solution for 15 seconds. (5) Dehydration and mounting: Immerse the sections sequentially into absolute ethanol I for 2 minutes - absolute ethanol II for 2 minutes - absolute ethanol III for 2 minutes - n-butanol I for 2 minutes - n-butanol II for 2 minutes - xylene I for 2 minutes - xylene II for 2 minutes for clearing, and mount with neutral gum. (6) Microscopic examination and image acquisition and analysis.

[0125] 2.9 Oil Red O staining of the testis

[0126] (1) Fixation of frozen sections: Take out the frozen sections from the -20°C refrigerator and let them return to room temperature. Fix them with tissue fixative for 15 min, wash with tap water, and air dry. (2) Oil red staining: Thoroughly mix 6 parts of saturated oil red O staining solution with 4 parts of distilled water, let it stand overnight at 4°C. The next day, filter it through qualitative filter paper, place it at 4°C for 24 h and filter it a second time to obtain the oil red O working solution. Immerse the sections in the oil red staining solution in the dark for 8 - 10 min. (3) Background differentiation: Take out the sections, wait for 3 s and then immerse them successively in two tanks of 60% (V / V) isopropanol for differentiation for 3 s and 5 s respectively. Immerse the sections successively in 2 tanks of pure water for washing, 10 s each. (4) Hematoxylin staining: Take out the sections, wait for 3 s and then immerse them in hematoxylin for counterstaining for 3 - 5 min. Wash them in 3 tanks of pure water for 5 s, 10 s, and 30 s respectively. Differentiate with the differentiating solution for 2 - 10 s, wash them in 2 tanks of distilled water for 10 s each, blue return for 1 s. Gently immerse the sections in 2 tanks of tap water for washing, 5 s and 10 s respectively, and then examine the staining effect under the microscope. (5) Sealing: Seal the sections with glycerin gelatin mounting medium. (6) Microscopic examination and image acquisition and analysis.

[0127] 2.10. Observation of the effect of LB-PK on the sperm ultrastructure of HFD-induced spermatogenesis disorder mice by transmission electron microscopy

[0128] (1) Sperm collection: The cauda epididymidis of mice was minced and placed in an appropriate culture medium and incubated at 37 °C for 30 min to promote the full release of sperm from the cauda epididymidis. (2) Sperm fixation: Centrifugation was performed, and the supernatant was discarded. The sperm was resuspended and fixed using a fixative (such as glutaraldehyde) to maintain the morphology and structure of the sperm. (3) Rinsing: The fixed sperm was rinsed multiple times with a buffer to remove the residual fixative. (4) Post-fixation: The sperm was fixed with 1 wt% osmium tetroxide prepared with 0.1 M phosphate buffer PB (pH 7.4) in the dark at room temperature for about 2 h. It was rinsed 3 times with 0.1 M phosphate buffer PB (pH 7.4), 15 min each time. (5) Dehydration at room temperature: The sperm was sequentially dehydrated in 30 (V / V)% - 50 (V / V)% - 70 (V / V)% - 80 (V / V)% - 95 (V / V)% - 100 (V / V)% - 100 (V / V)% alcohol, 20 min each time, and then into 100 (V / V)% acetone twice, 15 min each time. (6) Infiltration and embedding: Acetone: 812 embedding agent = 1:1, infiltrated at 37 °C for 2 - 4 h, acetone: 812 embedding agent = 1:2, infiltrated overnight at 37 °C, and pure 812 embedding agent infiltrated at 37 °C for 5 - 8 h. The sample was inserted into an embedding plate containing pure 812 embedding agent and incubated in an oven at 37 °C overnight. (7) Polymerization: The embedding plate was placed in an oven at 60 °C for polymerization for 48 h, and the resin block was taken out for standby. (8) Localization: The resin block was cut into 1.5 μm semi-thin sections using a semi-thin microtome, stained with toluidine blue, and then placed under a light microscope for localization. (9) Ultra-thin sectioning: The resin block was cut into 60 - 80 nm ultra-thin sections using an ultra-thin microtome, and the sections were picked up with a 150-mesh Fanghua film copper grid. (10) Staining: The copper grid was placed in a saturated alcohol solution of 2 (V / V)% uranyl acetate and stained in the dark for 8 min; washed 3 times with 70 (V / V)% alcohol; washed 3 times with ultrapure water; stained with 2.6 wt% lead citrate solution in the dark to avoid carbon dioxide for 8 min; washed 3 times with ultrapure water, and gently blotted dry with filter paper. The copper grid sections were placed in a copper grid box and dried overnight at room temperature. (11) Observation under a transmission electron microscope and image acquisition for analysis.

[0129] 2.11 Detection of testicular protein expression levels in HFD-induced spermatogenesis disorder mice by Western Blot

[0130] (1) Protein extraction

[0131] For protein extraction, 100 mg of testicular tissue from each group was weighed into an EP tube, 1 mL of RIPA lysis buffer (RIPA lysis buffer: PMSF = 100:1) was added, and homogenization was performed using a homogenizer. After mashing the tissue, it was left standing on ice for 30 min and then centrifuged at 4 °C and 12,000 rpm for 5 min in a centrifuge. The protein supernatant was taken into a new 1.5 mL EP tube and stored for subsequent WB use.

[0132] (2) Determination of protein concentration

[0133] Dilute the protein standard BSA at 5 mg / mL with 0.9% PBS to a protein standard solution of 0.5 mg / mL, store at -20 °C for later use. According to the instructions of the BCA protein concentration assay kit, calculate the amount of BCA detection reagent based on the number of samples, and prepare the BCA working solution by mixing solution A and solution B in the BCA kit at a ratio of 49:1; add the standard products at 0, 5, 10, 20, 40, 60, 80, 100 μL to 0.5 mL EP tubes, and make up to 100 μL with 0.9% PBS. Add 20 μL of the protein standard and the diluted sample to each well of the 96-well plate, add 200 μL of the BCA working solution to each well, place it in an oven at 37 °C for 30 min, measure the absorbance value of each sample with an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 562 nm, draw a standard curve, and calculate the concentration of the protein to be measured through the standard curve.

[0134] (3) Denaturation of protein samples

[0135] Calculate the 5× protein loading buffer and PBS diluent according to the protein concentration required for loading. The addition ratio is protein total amount: protein loading buffer = 4:1, dilute with PBS, denature at 100 °C in a metal bath for about 5 min, and keep the sample in a -20 °C refrigerator after cooling. The mass of each sample loading is 20 μg and the volume is 10 μL.

[0136] (4) Preparation of separating gel and stacking gel

[0137] The preparation methods of separating gel and stacking gel with different ratios involved in the experiment are shown in the following table:

[0138] Table 8 Preparation method of 6% separating gel (unit: mL)

[0139] 6% separation gel 5 10 15 20 Distilled water 2.0 4.0 6.0 8.0 30% glue solution 1.0 2.0 3.0 4.0 1.5MTris 1.9 3.8 5.7 7.6 6% separation gel 5 10 15 20 10% SDS 0.05 0.1 0.15 0.2 10% AP 0.05 0.1 0.15 0.2 TEMED 0.004 0.008 0.012 0.016

[0140] Table 9 Preparation method of 8% separating gel (unit: mL)

[0141]

[0142]

[0143] Table 10 Preparation method of 15% separating gel (unit: mL)

[0144] 15% separation gel 5 10 15 20 Distilled water 0.5 1.0 1.5 2.0 30% glue solution 2.5 5.0 7.5 10.0 1.5MTris 1.9 3.8 5.7 7.6 10% SDS 0.05 0.1 0.15 0.2 10% AP 0.05 0.1 0.15 0.2 TEMED 0.002 0.004 0.006 0.008

[0145] Table 11 Preparation method of 5% stacking gel (unit: mL)

[0146] 5% stacking gel 2 4 6 8 Distilled water 2.1 2.7 4.1 5.5 30% glue solution 0.5 0.67 1.1 1.3 1MTris 0.38 0.5 0.75 1.0 10% SDS 0.03 0.04 0.06 0.08 10% AP 0.03 0.04 0.06 0.08 TEMED 0.003 0.004 0.006 0.008

[0147] (5) Gel casting and sample loading

[0148] Take a 1.5-mm-thick glass plate and align it with the thick glass plate along the edge. Clamp it with a gel clamp. Prepare the separating gel according to step (4) in "2.11. Western Blot Detection of Testicular Protein Expression Levels in HFD-Induced Spermatogenic Disorder Mice". After adding TEMED, immediately pipette and mix well, then slowly pipette it in. When the liquid level reaches the engraved line of the thin glass plate, stop pouring the gel. Aspirate deionized water and slowly pipette it in to press the gel. Let it stand at room temperature for more than 30 minutes. When there is an obvious line between the gel liquid level and the deionized water, discard the deionized water for pressing the gel. Prepare the stacking gel according to step (4) in "2.11. Western Blot Detection of Testicular Protein Expression Levels in HFD-Induced Spermatogenic Disorder Mice". After adding TEMED, quickly pipette and mix well, then aspirate the stacking gel and add it on top of the separating gel. Insert the comb obliquely. After the stacking gel solidifies, place the glass plate in the electrophoresis tank, hold both ends of the comb, and pull it out vertically upward. Add the prepared electrophoresis buffer. Aspirate 2.5 μL of the rainbow marker and add it to the first well. Thaw the protein sample in a metal bath at 37 °C in advance. After vortexing and mixing well, then aspirate 10 μL of the protein sample and add it to the corresponding well by sticking to the wall of the well.

[0149] (6) Electrophoresis and Transfer

[0150] Connect the electrophoresis tank according to the positive and negative poles. Adjust the voltage of the electrophoresis buffer to a constant voltage of 90 V and run the upper stacking gel. When the protein sample forms a straight line, adjust the voltage to 120 - 150 V. When the electrophoresis is almost over, take out the PVDF membrane cut to an appropriate size and activate it in methanol for 10 - 30 s. Put it in the electrotransfer buffer to wash away the methanol and mark the primary antibody to be incubated for later use. Electrophorese at a constant voltage until the protein sample runs to the bottom of the glass plate. Turn off the electrophoresis instrument, remove the glass plate, and cut the target band according to the corresponding indication of the molecular weight of the target protein and the marker. Place it in the "sandwich" structure clip in the electrotransfer buffer, that is, from bottom to top: sponge pad, filter paper. According to "black gel and white membrane", that is, when the black clip surface is at the bottom, first place the gel and then place the membrane correspondingly; when the white clip surface is at the bottom, first place the membrane and then place the gel correspondingly. Then carefully cover it with 3 layers of filter paper to prevent the gel and the membrane from shifting, and the sponge pad. Connect it according to the red as the positive pole and the black as the negative pole, set the current to 300 mA, and the transfer time is 2 times the molecular weight.

[0151] (7) Blocking and Antibody Incubation

[0152] Take out the membrane, place it in an antibody incubation box on a shaker, and wash the membrane 3 times with 1×TBST for 10 minutes each time; then place it in 5% skim milk powder and incubate at room temperature for 2 hours; after the blocking is completed, wash the membrane 3 times with 1×TBST for 10 minutes each time. Put the strip into a centrifuge tube containing the prepared primary antibody and incubate overnight on a shaker at 4°C. The primary antibodies include anti-β-actin (1:1000), 3β-HSD (1:500), CYP17A1 (1:500), StAR (1:500), IL-6 (1:1000), IL-1β (1:1000), TNF-α (1:1000), CHOP (1:1000), PERK (1:1000), P-PERK (1:1000). After 15 - 16 hours, take out the PVDF membrane, wash the membrane 3 times with 1×TBST for 10 minutes each time, add the secondary antibody at 1:10000, incubate at room temperature for 1 hour, and wash the membrane three times on a shaker with TBST solution.

[0153] (8) Exposure

[0154] Prepare developer A:B (1:1) in an EP tube under light - proof conditions, evenly coat it on the membrane, image it using a UVP gel imager, and determine the gray value using the image.j image analysis software. The signal level is normalized with the gray value of the β - actin antibody.

[0155] 2.12. Statistical analysis

[0156] The experimental results are expressed as mean ± standard deviation (SD), and statistical analysis of the data is performed using SPSS 22.0 software; when P < 0.05, it indicates that the difference is statistically significant. Use GraphPad Prism 8 and Origin 2021 software to draw charts.

[0157] 3. Experimental results

[0158] 3.1. Establishment of a spermatogenesis disorder mouse model induced by HFD

[0159] To evaluate the damage of HFD to the fertility of male mice, fertility assessment was performed at 8 weeks, 10 weeks, and 12 weeks after feeding with a high - fat diet. By comparing the fertility rates of the Control group and the HFD group and the number of offspring (F1), the fertility of the mice was evaluated. As Figure 3As shown, after feeding a high-fat diet for 8 weeks, the fertility rate in the HFD group decreased by 13.3% compared with the Control group, and the number of offspring (F1) decreased by 27.5%; after feeding a high-fat diet for 10 weeks, the fertility rate in the HFD group decreased by 46.7% compared with the Control group, and the number of offspring (F1) decreased by 61.25%; after feeding a high-fat diet for 12 weeks, the fertility rate in the HFD group decreased by 66.7% compared with the Control group, and the number of offspring (F1) decreased by 72.2%. Considering the modeling duration and effect, the modeling time for inducing spermatogenic dysfunction in mice by HFD was selected as 10 weeks of feeding HFD, and the body weight of mice in the HFD group was 20% higher than that in the Control group.

[0160] 3.2. Effects of LB-PK extract on physiological and biochemical indexes of mice with spermatogenic disorder induced by HFD

[0161] Long-term HFD can cause dyslipidemia, leading to increased total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL-C), and decreased high-density lipoprotein (HDL-C). As Figure 4 shown, compared with the blank group, TC, TG, LDL-C, and HDL-C in the HFD group increased by 3.36 times, 2.02 times, 2.17 times, and 1.65 times, respectively. Compared with the HFD group, after 5 weeks of drug administration, TC, TG, LDL-C, and HDL-C in each drug administration group decreased by at least 19.08%, 11.92%, 32.52%, and 10.71%, respectively. The results indicate that LB-PK extract can improve the dyslipidemia caused by HFD.

[0162] 3.3. Effects of LB-PK extract on oral glucose tolerance and insulin tolerance of mice with spermatogenic disorder induced by HFD

[0163] Long-term HFD not only causes dyslipidemia but also leads to abnormal blood glucose and insulin resistance. By measuring the blood glucose of the Control group, HFD group, and the medium-dose of each LB-PK drug administration group through OGTT and ITT experiments, the results are as Figure 5 shown. In the OGTT test, the blood glucose of mice in the HFD group increased by 1.55 times compared with the Control group, and the medium-dose of each LB-PK drug administration group decreased by 16.1%, 18.1%, and 22.3% respectively compared with the blood glucose of mice in the HFD group. In the ITT test, the blood glucose of mice in the HFD group was 1.94 times higher than that of the Control group, and the medium-dose of each LB-PK drug administration group decreased by 33.1%, 29.3%, and 34.1% respectively compared with the blood glucose of mice in the HFD group. It shows that LB-PK can effectively improve the abnormal blood glucose and insulin resistance caused by HFD.

[0164] 3.4. Effects of LB-PK extract on the fertility of mice with spermatogenic disorder induced by HFD

[0165] In order to evaluate the restorative effect of LB-PK extract on the fertility impairment of male mice caused by high fat diet, the fertility rate and the number of offspring (F1) were compared among the Control group, HFD group, and LB-PK administration groups. Figure 6 As shown. Compared with the Control group, the fertility rate of the HFD group decreased by 83.33%, and the number of offspring (F1) decreased by 67.50%. Compared with HFD, except for the LB-PKD and LB-PKE low-dose groups, which showed a slight improvement in fertility and the number of offspring (F1), the LB-PK extract groups increased by 1.5 times and more than 84.62%, respectively. This shows that LB-PK extract can effectively reverse the decline in mouse fertility caused by HFD.

[0166] 3.5 Effects of LB-PK extract on body weight, testicular and epididymal fat hypertrophy index in HFD-induced spermatogenic mice

[0167] Long-term HFD feeding can lead to fat accumulation and weight gain, and the accumulation of epididymal fat can increase inflammation in the reproductive environment of mice and cause spermatogenesis dysfunction. Figure 7 and Figure 8 As shown in the results, the LB-PK administration group showed significant inhibitory effects on the body weight, testicular index and epididymal fat hypertrophy index of HFD-induced obese spermatogenic mice. Compared with the blank group, the body weight change rate and epididymal fat weight of mice at the end of HFD intervention were significantly increased, with significant differences (P < 0.01). After administration of LB-PK extract, it showed a significant inhibitory effect on the increase of body weight of mice. Compared with the blank group, the body weight of HFD increased by 1.33 times; compared with HFD, each LB-PK administration group inhibited the increase of body weight by more than 17.99%. Compared with the blank group, the testicular weight of HFD increased by 14.86%, and each LB-PK administration group inhibited the increase of testicular weight by more than 10%. Compared with the blank group, the epididymal fat weight of HFD increased by 5.62 times; compared with HFD, the epididymal fat weight of each LB-PK administration group decreased by more than 46.63%. The results showed that LB-PK could reverse the decrease in testicular index of HFD-induced obese spermatogenic mice and effectively inhibit the increase in epididymal fat hypertrophy index of HFD-induced mice, indicating that LB-PK can significantly inhibit the weight gain and fat accumulation of mice caused by HFD.

[0168] 3.6 Effects of LB-PK extract on sex hormones in HFD-induced spermatogenesis-impaired mice

[0169] T and FSH are key gonadal hormones that regulate the development and function of reproductive organs of organisms, and are also important indicators for evaluating the status of the reproductive system. Long-term HFD feeding will lead to decreased T and FSH levels and increased E2 levels. Fig. 9As shown, compared with the blank group, testosterone T in the testes of HFD decreased by 42.76%, FSH decreased by 70.65%, and E2 increased by 2.68 times. Compared with HFD, T in each administration group increased by more than 31.04%; FSH in each administration group increased by more than 92.52%; E2 in each administration group decreased by at least 25.16%. After 5 weeks of administration, each administration group could effectively reverse the decrease in T and FSH and the increase in E2 caused by HFD in mice. The results showed that LB-PK extract could improve the sex hormone disorder caused by HFD.

[0170] 3.7 Effects of LB-PK Extract on Sperm Parameters in Mice with Spermatogenesis Disorder Induced by HFD

[0171] Long-term feeding of HFD will cause the sperm parameters of mice to deteriorate. As Fig.10 shown, after 10 weeks of modeling with a high-fat diet, compared with the blank group, the sperm motility of HFD decreased by 20.08%, the sperm count decreased by 23.66%, the abnormal sperm increased, and abnormal or headless sperm appeared (as shown in the red box). Compared with HFD, the sperm abnormalities of mice in each administration group were improved to a certain extent. Except for slightly improved in HFD+DL, HFD+EL, and HFD+EM, the sperm motility of mice in each administration group increased by more than 4.83%, while that of HFD+PH increased by 21.26%, indicating that HFD+PH could significantly improve the decrease in sperm motility caused by HFD in mice; compared with HFD, except for slightly improved in HFD+DL and HFD+EL, the sperm count of mice in the remaining administration groups increased by more than 6.62%. It shows that LB-PK extract can effectively reverse the decrease in sperm motility and count and sperm abnormalities caused by HFD in mice.

[0172] 3.8 Effects of LB-PK Extract on Testicular Histology in Mice with Spermatogenesis Disorder Induced by HFD

[0173] The testis is an important place for sperm production. Long-term feeding of HFD will cause significant changes in the morphology and function of the testes in mice, as Fig.11 shown. After 10 weeks of modeling with a high-fat diet, normal seminiferous tubules with clear lumens and spermatogenesis were observed in the blank group, which contained various germ cells. As seen, a large number of germline cells were arranged in a normal cell type, including spermatogonia, primary spermatocytes, secondary spermatocytes, round sperm, attached sperm, and sperm released into the lumen. Compared with the blank group, the testes of HFD showed deterioration and atrophy of the seminiferous tubules ( Fig.13 the boxed part in (b) in Fig.13 (b)), and there were some degenerative changes such as vacuoles ( the red star in (b) in (b)), the arrangement of spermatogenic cells was disordered, and the number of spermatogenic cells decreased significantly; compared with HFD, after 5 weeks of administration, each administration group could effectively reverse the histological damage of the testes in mice caused by HFD.

[0174] 3.9 Effects of LB-PK Extract on Testicular Lipid Deposition in HFD-Induced Spermatogenesis Disorder Mice

[0175] The testis is an important site for sperm production. Long-term feeding of HFD can lead to an increase in testicular lipids in mice, affecting the testicular microenvironment and thus the sperm production in mice. As Fig.12 and Fig.13 shown, compared with the blank group, there were many lipid droplets of different sizes and lipid accumulations in the interstitial cells of the testis in the HFD group; after 5 weeks of drug administration, each drug administration group could effectively reverse the damage of many lipid droplets of different sizes and lipid accumulations in the interstitial cells of the testis in mice caused by HFD.

[0176] 3.10 Effects of LB-PK Extract on Sperm Ultrastructure in HFD-Induced Spermatogenesis Disorder Mice

[0177] Long-term feeding of HFD can lead to changes in mouse sperm. The flagellar mitochondria of mouse sperm mainly provide energy for sperm movement, while HFD can cause damage to the flagellar mitochondria of mouse sperm, thus affecting sperm motility and the fertility of mice. As Fig.14 shown, clear mitochondrial cristae structures could be seen in the flagellar mitochondria of sperm in the blank group, with normal morphology, while in the HFD group, the mitochondrial matrix was swollen (blue box), the matrix became thinner, the electron density decreased, and even became vacuolated. The cristae became shorter due to the extension of the inner membrane, and the mitochondrial cristae structure was unclear and significantly reduced. After 5 weeks of drug administration, the mitochondrial matrix of sperm flagella increased, the electron density increased, the vacuolation decreased or disappeared, and the mitochondrial cristae structure became clear again. This indicates that LB-PK extract can effectively improve the damage of sperm flagella in mice caused by HFD.

[0178] 3.11 Effects of LB-PK Extract on Proteins in the Testis of HFD-Induced Spermatogenesis Disorder Mice

[0179] Long-term high-fat diet can lead to disorders in testosterone synthesis, increased inflammation, and endoplasmic reticulum stress (ERS) in the testis of mice, thus causing disorders in spermatogenic function in mice. Detect the key proteins for testosterone synthesis CYP17A1, 3β-HSD, STAR in each group, testicular inflammatory factors IL-1β, IL-6, TNF-α, and PERK-CHOP related pathway proteins, as Fig.15 and Fig.16As shown, the expressions of CYP17A1, 3β-HSD, and STAR proteins were all downregulated in the HFD group, with significant differences (P<0.001). After LB-PK intervention, the downregulation of CYP17A1, 3β-HSD, and STAR proteins related to testosterone synthesis in mice caused by HFD was reversed; the expressions of IL-1β, IL-6, and TNF-α proteins were all upregulated in the HFD group, with significant differences (P<0.001). After LB-PK intervention, the expressions of IL-1β, IL-6, and TNF-α related proteins in testicular inflammation of mice caused by HFD were reversed; the expressions of CHOP and P-PERK proteins were all upregulated in the HFD group, with significant differences (P<0.001). It shows that after the intervention of LB-PK extract, the testicular ERS caused by HFD can be reversed, and then to a certain extent, the testosterone synthesis disorder caused by HFD and the expression of testicular inflammatory factor-related proteins can be improved.

[0180] In summary, through the fertility assessment of mice at different modeling times, it was determined that the modeling time for the impairment of spermatogenic function induced by HFD in mice was 10 weeks of feeding HFD, and the body weight of mice in the HFD group was 20% higher than that in the Control group. Long-term feeding of HFD can lead to abnormal blood glucose and insulin resistance in mice, which in turn affects mouse spermatogenesis. By measuring the blood glucose of mice at different time periods through the oral glucose tolerance test and insulin tolerance test, it was shown that the LB-PK extract can improve the abnormal blood glucose and insulin resistance caused by HFD in mice and effectively reverse the abnormal blood glucose caused by HFD in mice. Long-term feeding of HFD not only leads to abnormal blood lipid levels in mice, causing a significant increase in serum TG, TC, HDL-C, and LDL-C in mice, but the LB-PK extract can effectively reverse the abnormal blood lipid levels caused by HFD in mice. Long-term feeding of HFD also leads to a significant decrease in serum sex hormones T and FSH and a significant increase in E2 in mice. The LB-PK extract can effectively reverse the decrease in T and FSH and the increase in E2 caused by high fat, improve the sex hormone disorder caused by HFD, and thus regulate the gonadal axis to regulate mouse spermatogenesis. Long-term feeding of HFD can lead to obesity in mice. Obesity can affect mouse spermatogenic function through different mechanisms, thereby affecting mouse fertility. Through the statistics of the body weight, testis weight, testis index, and epididymal fat hypertrophy index of mice in each group after 5 weeks of drug administration and the detection of sperm parameters of mice, it was shown that LB-PK can significantly inhibit the weight gain and fat accumulation of mice with spermatogenic disorders induced by HFD, and thus effectively reverse the decrease in sperm motility and quantity and the sperm deformity caused by HFD in mice. At the same time, by evaluating the fertility of mice in each group after 5 weeks of drug administration, it was found that the LB-PK extract can effectively reverse the decrease in mouse fertility caused by HFD. Through testicular pathological section observation, it was found that the seminiferous tubules of the testis in HFD mice deteriorated and atrophied, the spermatogenic cells were disorderly arranged, and the number of spermatogenic cells decreased significantly. Many lipid droplets and lipid accumulations of different sizes appeared in the interstitial cells of the testis. After administration of the LB-PK extract, the seminiferous tubules and the arrangement of spermatogenic cells in the testis of mice with spermatogenic disorders induced by HFD could be restored, and the lipid droplets and lipid accumulations in the interstitial cells decreased, indicating that LB-PK can reverse the testicular damage and lipid accumulation caused by HFD. Through the electron microscope observation of mouse sperm, it was found that HFD can cause damage to the mitochondria of the sperm flagella in mice, and the LB-PK extract can restore the mitochondria of the sperm flagella of mice with spermatogenic disorders induced by HFD to a certain extent. It is speculated that LB-PK may improve the fertility of mice by improving the sperm mitochondrial structure. Long-term feeding of HFD can lead to endoplasmic reticulum stress, an increase in inflammatory factors, and a decrease in T in the testis of mice. By detecting the expression of related proteins, it was found that the mechanism by which the LB-PK extract improves spermatogenic dysfunction may be related to reversing the testicular ERS caused by HFD, and then to a certain extent improving the testosterone synthesis disorder caused by HFD and downregulating the expression of related proteins of testicular inflammatory factors.

[0181] It can be seen that the LB-PK extract can effectively improve obesity caused by HFD and a series of metabolic problems caused thereby, that is, the LB-PK extract can effectively inhibit obesity caused by HFD and reverse the abnormal blood glucose and blood lipids caused by HFD, and improve the disorder of sex hormones, the decrease of sperm parameters, the abnormal mitochondria of sperm flagella and the fertility of mice caused by HFD. It may restore the normal regulatory function of the hypothalamic-pituitary-testicular axis by reversing problems such as abnormal blood glucose, blood lipids and sex hormone disorders caused by HFD; the mechanism by which the LB-PK extract improves spermatogenic dysfunction can improve ERS caused by HFD, the testosterone-related synthesis pathway and reduce the protein expression level of inflammatory factors in the testes of mice to restore sperm production, thereby improving the reproductive dysfunction caused by HFD.

[0182] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of the wolfberry-polygonatum extract in the preparation of a drug for improving spermatogenic dysfunction, characterized in that, The Lycium barbarum - Polygonatum sibiricum extract includes one or more of the water extract of Lycium barbarum - Polygonatum sibiricum, the ethanol extract of Lycium barbarum - Polygonatum sibiricum, and the crude polysaccharide of Lycium barbarum - Polygonatum sibiricum.

2. The application according to claim 1, wherein The preparation method of the water extract of Lycium barbarum - Polygonatum sibiricum includes the steps of mixing Lycium barbarum and Polygonatum sibiricum, decocting and concentrating to obtain the water extract of Lycium barbarum - Polygonatum sibiricum. The preparation method of the ethanol extract of Lycium barbarum - Polygonatum sibiricum includes the steps of mixing Lycium barbarum and Polygonatum sibiricum, performing ethanol extraction, concentrating and drying to obtain the ethanol extract of Lycium barbarum - Polygonatum sibiricum. The preparation method of the crude polysaccharide of Lycium barbarum - Polygonatum sibiricum includes the steps of performing water extraction, concentrating, precipitating with ethanol, filtering and drying on the remaining residue obtained from preparing the ethanol extract of Lycium barbarum - Polygonatum sibiricum to obtain the crude polysaccharide of Lycium barbarum - Polygonatum sibiricum.

3. The application according to claim 2, wherein When preparing the water extract of Lycium barbarum - Polygonatum sibiricum, the mass ratio of Lycium barbarum to Polygonatum sibiricum is 1:1; the number of decocting times is 3 times, the decocting time for each time is 1 h, and the solvent is water; when performing the first decocting, the dosage of the solvent is 8 times the total mass of Lycium barbarum and Polygonatum sibiricum; when performing the second decocting, the dosage of the solvent is 6 times the total mass of Lycium barbarum and Polygonatum sibiricum; when performing the third decocting, the dosage of the solvent is 4 times the total mass of Lycium barbarum and Polygonatum sibiricum.

4. The application according to claim 2, characterized in that When preparing the ethanol extract of Lycium barbarum - Polygonatum sibiricum, the mass ratio of Lycium barbarum to Polygonatum sibiricum is 1:1; the number of ethanol extraction times is 3 times, and the extracting agent is an ethanol solution with a volume percentage content of 80%; when performing the first ethanol extraction, the dosage of the extracting agent is 8 times the total mass of Lycium barbarum and Polygonatum sibiricum, and the extraction time is 60 min; when performing the second ethanol extraction, the dosage of the extracting agent is 6 times the total mass of Lycium barbarum and Polygonatum sibiricum, and the extraction time is 40 min; when performing the third ethanol extraction, the dosage of the extracting agent is 4 times the total mass of Lycium barbarum and Polygonatum sibiricum, and the extraction time is 30 min.

5. The application according to claim 2, characterized in that, When preparing the crude polysaccharide of Lycium barbarum - Polygonatum sibiricum, the number of water extraction times is 3 times, the water extraction time for each time is 2 h, and the extracting agent is water; when performing the first water extraction, the dosage of the extracting agent is 8 times the mass of the remaining residue; when performing the second water extraction, the dosage of the extracting agent is 6 times the mass of the remaining residue; when performing the third water extraction, the dosage of the extracting agent is 4 times the mass of the remaining residue.

6. The application according to claim 1, wherein The spermatogenic dysfunction includes spermatogenic dysfunction caused by a high - fat diet.

7. The application according to claim 1, characterized in that, The drug achieves the effect of improving spermatogenic dysfunction by improving blood glucose abnormality and insulin resistance, improving sex hormone disorder, improving sperm motility and quantity, and reducing the sperm malformation rate.

8. A drug for improving spermatogenic dysfunction, characterized in that, The drug includes the Lycium barbarum - Polygonatum sibiricum extract; the Lycium barbarum - Polygonatum sibiricum extract includes one or more of the water extract of Lycium barbarum - Polygonatum sibiricum, the ethanol extract of Lycium barbarum - Polygonatum sibiricum, and the crude polysaccharide of Lycium barbarum - Polygonatum sibiricum.

9. The drug according to claim 8, characterized in that, The preparation method of the water extract of Lycium barbarum - Polygonatum sibiricum includes the steps of mixing Lycium barbarum and Polygonatum sibiricum, decocting and concentrating to obtain the water extract of Lycium barbarum - Polygonatum sibiricum. The preparation method of the ethanol extract of Lycium barbarum - Polygonatum sibiricum includes the steps of mixing Lycium barbarum and Polygonatum sibiricum, performing ethanol extraction, concentrating and drying to obtain the ethanol extract of Lycium barbarum - Polygonatum sibiricum. The preparation method of the wolfberry-polygonatum crude polysaccharide comprises the steps of performing water extraction, concentration, precipitation with ethanol, filtration and drying on the remaining residue of the preparation of the wolfberry-polygonatum ethanol extract to obtain the wolfberry-polygonatum crude polysaccharide.

10. The medicament according to claim 8, characterized in that, The drug comprises pharmaceutically acceptable excipients.

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