Use of cinnamic acid derivatives

Abstract

The present application provides use of compounds represented by formula (I) and formula (II) in the preparation of drugs for improving male reproductive ability. In the present application, different oligoasthenospermia models are constructed, and the improvement effects of the compounds represented by formula (I) and formula (II) on male reproductive ability impairment are verified by means of sperm count analysis, sperm motility analysis, mRNA expression level analysis of inflammatory and antioxidant genes, testicular histopathological damage analysis, etc. The experimental results show that the compounds represented by formula (I) and formula (II) increase the sperm count in mice with oligoasthenospermia, improve the sperm motility of the mice with oligoasthenospermia, ameliorate testicular histopathological damage in the mice, suppress the expression of inflammatory factors, and increase the expression of antioxidant genes, demonstrating great potential in the treatment of oligoasthenospermia.

Description

Application of cinnamic acid derivatives

[0001] This application claims priority to the Chinese patent application No. 202411084063.X filed on August 7, 2024 with the China National Intellectual Property Office, the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application relates to the field of medicine, in particular to the application of cinnamic acid derivatives. BACKGROUND

[0003] Healthy reproduction is an important factor for maintaining the stability of the country, society and nation, and is also an important cornerstone for the continuation of the species. However, the widespread existence of oligoasthenospermia directly causes many infertile men of childbearing age. Due to factors such as environmental pollution and life pressure, more than 50% of couples cannot have children due to male infertility. Male infertility is mainly caused by oligoasthenospermia, and low sperm count and poor sperm motility are the most important factors. The causes of oligoasthenospermia are complex and diverse, and diseases (including diabetes), pathogen infection, genetic defects, environmental pollutants and radiation exposure are important predisposing factors.

[0004] At present, there is no specific treatment drug for oligoasthenospermia, and methods such as diet therapy and traditional Chinese medicine treatment are generally used. SUMMARY

[0005] Therefore, the present application provides the application of cinnamic acid derivatives, which can significantly improve the sperm count and sperm motility of oligoasthenospermia mice.

[0006] Hydroxycinnamic acid (HCA) is a plant phenolic acid widely existing in cereals, fruits and vegetables. It has been widely studied due to its anti-inflammatory, antioxidant, antibacterial, antitumor and anti-platelet aggregation effects. However, due to the main functional group of antioxidant activity is phenolic hydroxyl group, which is easy to be oxidized, thus there are also problems of low bioavailability, low stability and rapid metabolism of pharmacokinetics. In addition, due to the dissociation effect of carboxylic acid group, it is difficult to penetrate the cell membrane, which limits its biological activity. Therefore, it is of great significance to modify HCA to increase its hydrophobicity and promote its penetration into the cell membrane, in order to prolong the drug time, improve the bioavailability and increase the biological activity.

[0007] 4-hydroxycinnamic acid methyl ester (KS-3) is an ester derivative of 4-hydroxycinnamic acid, which exists in the roots, stems and leaves of many medicinal plants. It has been confirmed that KS-3 has anti-inflammatory and antioxidant properties, but the existing technology has not disclosed its effect of preventing or treating oligoasthenospermia.

[0008] The applicant improves the bioavailability of KS-3 and the sperm count and sperm motility of oligoasthenozoospermic mice by synthesizing and administering KS-3 and esterification compounds of C2-C10 of KS-3. Based on this, the application provides the use of a compound shown in formula (I) in the preparation of a medicine for improving male reproductive capacity:

[0009] wherein R is selected from H or R1-CO-, and R1 is selected from C2-C10 alkyl.

[0010] The compound shown in formula (I) is methyl 4-hydroxycinnamate and esterification derivatives thereof. When R is H, it is methyl 4-hydroxycinnamate, and when R is R1-CO-, it is an esterification derivative of methyl 4-hydroxycinnamate.

[0011] In some specific implementations, R1 is selected from C2-C10 alkyl, including straight-chain alkyl and branched-chain alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, n-heptyl, isobutyl, nonyl, decyl, etc., and preferably n-butyl, n-pentyl, n-hexyl, and n-heptyl.

[0012] In some specific implementations, the compound shown in formula (I) is KS-3, which is methyl 4-hydroxycinnamate, and the structural formula is as follows:

[0013] The compound shown in formula (I) is methyl 4-hydroxycinnamate butyrate, and the structural formula is as follows:

[0014] The compound shown in formula (I) is methyl 4-hydroxycinnamate valerate, and the structural formula is as follows:

[0015] The compound shown in formula (I) is methyl 4-hydroxycinnamate hexanoate, and the structural formula is as follows:

[0016] The compound shown in formula (I) is methyl 4-hydroxycinnamate heptanoate, and the structural formula is as follows:

[0017] The source of the compound shown in formula (I) is not particularly limited in the application, and can be purchased directly from the market or prepared according to the method disclosed in patent CN117550978A.

[0018] Further, the application provides the use of a compound shown in formula (II) in the preparation of a medicine for improving male reproductive capacity:

[0019] wherein R is selected from H or R1-CO-, and R1 is selected from C2-C10 alkyl.

[0020] R3 is selected from hydrogen or C1-C10 alkoxy;

[0021] R2 is selected from hydrogen or C1-C10 alkyl;

[0022] R2 and R3 are not hydrogen simultaneously.

[0023] In some specific embodiments, R3 is selected from hydrogen or C1-C10 alkoxy, wherein C1-C10 alkoxy includes linear alkoxy and branched alkoxy, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy, tert-butoxy, n-pentoxy, isopentoxy, n-hexoxy, n-heptoxy, isobutoxy, nonoxy, decoxy, etc., preferably C1-C8 alkoxy, more preferably C1-C7 alkoxy, and most preferably C1-C6 alkoxy.

[0024] In some specific embodiments, R2 is selected from hydrogen or C1-C10 alkyl, wherein C1-C10 alkyl includes linear alkyl and branched alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, n-heptyl, isobutyl, nonyl, decyl, etc., preferably C1-C8 alkyl, more preferably C1-C7 alkyl, and most preferably C1-C6 alkyl.

[0025] In some specific embodiments, R is selected from H or R1-CO-, and R1 is selected from C2-C10 alkyl. In some specific embodiments, R1 is selected from C2-C10 alkyl, including linear alkyl and branched alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, n-heptyl, isobutyl, nonyl, decyl, etc., preferably n-butyl, n-pentyl, n-hexyl, n-heptyl.

[0026] Specifically, when R3 is selected from hydrogen and R2 is selected from methyl, it is a compound represented by formula (I), which will not be described herein. When R3 is selected from hydrogen and R2 is selected from alkyl other than methyl, it is a derivative of the compound represented by formula (I), which can be, for example, the following structural compound:

[0027] When R3 is selected from alkoxy and R2 is selected from hydrogen or alkyl, the compound represented by formula (II) can be the following compound:

[0028] Ferulic acid, the chemical name of which is 4-hydroxy-3-methoxycinnamic acid, has the following structural formula:

[0029] Methyl ferulate has the following structural formula:

[0030] Methyl ferulate-butyrate, the structural formula is as follows:

[0031] Methyl ferulate-valerate, the structural formula is as follows:

[0032] Methyl ferulate-hexanoate, the structural formula is as follows:

[0033] Methyl ferulate-heptanoate, the structural formula is as follows, which can be prepared by referring to the method disclosed in patent CN117550978A:

[0034] The compound provided in the application can be used for preparing a medicine for improving male reproductive capacity, specifically, for improving or ameliorating the impairment of male reproductive capacity. In some specific implementation manners, the impairment of male reproductive capacity can be caused by pollutants such as microplastics or plasticizers, diseases such as diabetes, or commonly used medicines such as sodium valerate. Experiments have proved that the above reasons can cause oligoasthenospermia in mice, and the compound provided in the application has an ameliorating effect on the impairment of male reproductive capacity caused by various reasons. In some specific implementation manners, the male can be a human, a mouse, a rat or a rabbit.

[0035] In some specific implementation manners, the improvement of male reproductive capacity includes preventing and treating oligoasthenospermia and / or protecting testicular tissue. Specifically, the prevention and treatment of oligoasthenospermia include increasing the number of sperm and / or improving the motility of sperm. Experimental results show that the compound provided in the application can significantly increase the number of sperm and improve the motility of sperm in oligoasthenospermia mice. The protection of testicular tissue includes one or more of improving tissue morphology, reducing the level of inflammatory factors and up-regulating the level of antioxidant factors. Among them, improving tissue morphology includes improving the arrangement of seminiferous tubules and improving the atrophy of spermatocytes. Among them, the inflammatory factors include interleukin 1β and / or tumor necrosis factor; and the antioxidant factor is NAD(P)H quinone dehydrogenase 1. Experimental results show that the compound provided in the application significantly down-regulates the expression of inflammatory genes IL-1β and TNF-α and up-regulates the expression of antioxidant gene NQO-1 in oligoasthenospermia mice.

[0036] The application respectively constructs a nano microplastic (PSNP) and a plasticizer (DEHP) induced Kunming mouse (KM) environmental pollution type oligoasthenospermia model, a streptozotocin (STZ) induced Kunming mouse (KM) diabetes type oligoasthenospermia model, and a valproic acid sodium (VPA) induced Kunming mouse (KM) antiepileptic drug type oligoasthenospermia model, and verifies the improvement effect of the compounds represented by formula (I) and formula (II) on the damage of male reproductive ability through sperm count analysis, sperm motility analysis, inflammation and antioxidant gene mRNA expression level analysis, testicular tissue pathological damage analysis and the like. The experimental results show that the compounds represented by formula (I) and formula (II) increase the sperm count of oligoasthenospermia mice, improve the sperm motility of oligoasthenospermia mice, improve the histopathological damage of the testis, inhibit the expression of inflammatory factors and increase the expression of antioxidant genes, and have great potential in the treatment of oligoasthenospermia. BRIEF DESCRIPTION OF DRAWINGS

[0037] Fig. 1 is the body weight statistical result of the mice in test example 1;

[0038] Fig. 2 is the sperm count statistical result of the mice in test example 1;

[0039] Fig. 3 is the sperm motility statistical result of the mice in test example 1;

[0040] Fig. 4 is the body weight statistical result of the mice in test example 2;

[0041] Fig. 5 is the sperm count statistical result of the mice in test example 2;

[0042] Fig. 6 is the sperm motility statistical result of the mice in test example 2;

[0043] Fig. 7 is the interleukin-1β (IL-1β) expression amount statistical result of the mice in test example 2;

[0044] Fig. 8 is the tumor necrosis factor (TNF-α) expression amount statistical result of the mice in test example 2;

[0045] Fig. 9 is the NAD(P)H quinone dehydrogenase 1 (NQO-1) expression amount statistical result of the mice in test example 2;

[0046] Fig. 10 is a tissue section staining diagram of the mice in test example 2;

[0047] Fig. 11 is the body weight statistical result of the mice in test example 3;

[0048] Fig. 12 is the sperm count statistical result of the mice in test example 3;

[0049] Fig. 13 is the sperm motility statistical result of the mice in test example 3;

[0050] Fig. 14 is a statistical result of the expression of interleukin-1 beta (IL-1β) in mice in Test Example 3;

[0051] Fig. 15 is a statistical result of the expression of tumor necrosis factor (TNF-α) in mice in Test Example 3;

[0052] Fig. 16 is a statistical result of the expression of NAD(P)H quinone dehydrogenase 1 (NQO-1) in mice in Test Example 3;

[0053] Fig. 17 is a staining image of a tissue section of mice in Test Example 3;

[0054] Fig. 18 is a statistical result of the body weight of mice in Test Example 4;

[0055] Fig. 19 is a statistical result of the number of sperm in mice in Test Example 4;

[0056] Fig. 20 is a statistical result of the sperm motility in mice in Test Example 4;

[0057] Fig. 21 is a statistical result of the number of sperm in mice in Test Example 5;

[0058] Fig. 23 is a statistical result of the sperm motility in mice in Test Example 5;

[0059] Fig. 23 is a statistical result of the sperm motility in mice in Test Example 5;

[0060] Fig. 24 is a statistical result of the number of sperm in mice in Test Example 6;

[0061] Fig. 25 is a statistical result of the sperm motility in mice in Test Example 6;

[0062] Fig. 26 is a statistical result of the number of sperm in mice in Test Example 7;

[0063] Fig. 27 is a statistical result of the sperm motility in mice in Test Example 7;

[0064] Fig. 28 is a result of the expression of IL6 in the testis of mice in Test Example 7;

[0065] Fig. 29 is a statistical result of the expression of HO1 in the testis of mice in Test Example 7. DETAILED DESCRIPTION

[0066] It should be understood that the expression "one or more of" includes individually each of the objects recited after the expression and various combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in connection with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0067] The use of the terms "including," "containing," or "comprising" and variations thereof, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items not specifically listed, unless otherwise specified or limited by context.

[0068] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the application remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0069] The use of any and all examples, or exemplary language herein, for example, are intended merely to better illuminate the application and do not pose a limitation on the scope of the application unless otherwise claimed. No language is such that it will be construed as indicating any non-claimed element as essential to the practice of the application.

[0070] Further, the numerical ranges and parameters setting forth the broadest scope of the application are approximations, and are only chosen to encompass the more precise values in the particular examples. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Also, as used herein, the notation "about" when

[0071] The present application provides the use of a compound of formula (I) and formula (II) in the manufacture of a medicament for improving male reproductive capacity:

[0072] In formula (I), R is selected from H or R1-CO-, R1 is selected from C2-C10 alkyl;

[0073] In formula (II), R is selected from H or R1-CO-, R1 is selected from C2-C10 alkyl;

[0074] R3 is selected from hydrogen or C1-C10 alkoxy;

[0075] R2 is selected from hydrogen or C1-C10 alkyl;

[0076] R2 and R3 are not hydrogen at the same time.

[0077] The application respectively constructs a nano microplastic (PSNPs) and plasticizer (DEHP) induced Kunming mouse (KM) environmental pollution type oligoasthenospermia model, a streptozotocin (STZ) induced Kunming mouse (KM) diabetes type oligoasthenospermia model, and a valproic acid sodium (VPA) induced Kunming mouse (KM) antiepileptic drug type oligoasthenospermia model, and verifies the improvement effect of the compounds represented by formula (I) and formula (II) on the damage of male reproductive ability through sperm count analysis, sperm motility analysis, inflammation and antioxidant gene mRNA expression level analysis, testicular tissue pathological damage analysis and the like. The experimental results show that the compounds represented by formula (I) and formula (II) increase the sperm count of oligoasthenospermia mice, improve the sperm motility of oligoasthenospermia mice, improve the histopathological damage of their testes, inhibit the expression of inflammatory factors and increase the expression of antioxidant genes, and have great potential in the treatment of oligoasthenospermia.

[0078] The application of 4-hydroxycinnamic acid methyl ester and its esterification product provided by the application is further described below in combination with examples.

[0079] In the following examples, each compound can be purchased from the market or prepared according to methods well known to those skilled in the art.

[0080] KS-3 is 4-hydroxycinnamic acid methyl ester, the structural formula of which is as follows, and can be purchased from the market:

[0081] 4-hydroxycinnamic acid methyl ester butyrate, the structural formula of which is as follows, can be prepared according to the method disclosed in patent CN117550978A:

[0082] 4-hydroxycinnamic acid methyl ester valerate, the structural formula of which is as follows, can be prepared according to the method disclosed in patent CN117550978A:

[0083] 4-hydroxycinnamic acid methyl ester hexanoate, the structural formula of which is as follows, can be prepared according to the method disclosed in patent CN117550978A:

[0084] 4-hydroxycinnamic acid methyl ester heptanoate, the structural formula of which is as follows, can be prepared according to the method disclosed in patent CN117550978A:

[0085] Resveratrol butyrate, the structural formula of which is as follows, can be prepared according to the method disclosed in patent CN117550978A:

[0086] Resveratrol valerate, the structural formula of which is as follows, can be prepared according to the method disclosed in patent CN117550978A:

[0087] Ferulic acid, chemical name 4-hydroxy-3-methoxycinnamic acid, structural formula as follows, can be purchased from the market:

[0088] Methyl ferulate, structural formula as follows, can be purchased from the market:

[0089] Methyl ferulate-butyrate, structural formula as follows, can be prepared according to the method disclosed in patent CN117550978A:

[0090] Methyl ferulate-valerate, structural formula as follows, can be prepared according to the method disclosed in patent CN117550978A:

[0091] Methyl ferulate-hexanoate, structural formula as follows, can be prepared according to the method disclosed in patent CN117550978A:

[0092] Methyl ferulate-heptanoate, structural formula as follows, can be prepared according to the method disclosed in patent CN117550978A:

[0093] 4-Coumaric acid propyl ester-valerate (CoA-L5R3), structural formula as follows, is prepared by the following method: esterifying the carboxyl group of 4-coumaric acid with alcohol to synthesize 4-coumaric acid propyl ester with propanol as solvent and concentrated sulfuric acid as catalyst; acylating the phenolic hydroxyl group of 4-coumaric acid propyl ester with valeric acid to synthesize 4-coumaric acid propyl ester-valerate with DCM as solvent, DCC and DMAP as condensing agent.

[0094] 4-Coumaric acid butyl ester-valerate (CoA-L5R4), structural formula as follows, is prepared by the following method: esterifying the carboxyl group of 4-coumaric acid with alcohol to synthesize 4-coumaric acid butyl ester with butanol as solvent and concentrated sulfuric acid as catalyst; acylating the phenolic hydroxyl group of 4-coumaric acid butyl ester with valeric acid to synthesize 4-coumaric acid propyl ester-valerate with DCM as solvent, DCC and DMAP as condensing agent.

[0095] 4-Coumaric acid valerate-valerate (CoA-L5R5), structural formula as follows, is prepared by the following method: esterifying the carboxyl group of 4-coumaric acid with alcohol to synthesize 4-coumaric acid valerate with pentanol as solvent and concentrated sulfuric acid as catalyst; acylating the phenolic hydroxyl group of 4-coumaric acid valerate with valeric acid to synthesize 4-coumaric acid valerate-valerate with DCM as solvent, DCC and DMAP as condensing agent.

[0096] In each of the following test examples, the sperm number and sperm motility evaluation method is as follows:

[0097] After the epididymal sample is collected, it is cut into small pieces and placed in a 24-well plate. 1000 μL of PBS is added, and incubation is performed at 37°C for 10 minutes. After incubation, the filter is taken, and 10 μL of the filtrate is added to a cell counting plate. A sperm analyzer is used to analyze the sperm number and sperm motility.

[0098] The steps for inflammation and antioxidant gene mRNA in testicular tissue are as follows:

[0099] qPCR is used to determine the expression of related inflammatory factors and antioxidant genes in testicular tissue, including interleukin 1β (IL-1β), tumor necrosis factor (TNF-α), and NAD(P)H quinone dehydrogenase 1 (NQO1).

[0100] The histological analysis is as follows:

[0101] The collected testicular tissue samples are fixed with freshly prepared 4% paraformaldehyde, and then stained with hematoxylin and eosin (H&E). Specifically, the testicular tissue paraffin sections are baked in an oven at 65°C for at least 1.5 hours to ensure that the paraffin on the tissue sections is fully melted. Then the sections are placed in xylene twice to remove the melted paraffin and dehydrated in gradient ethanol. The sections are stained with hematoxylin and then with eosin, and then dehydrated with ethanol. After staining, the sections are clarified twice in xylene and fixed with neutral resin. The stained sections are scanned and observed.

[0102] Test Example 1

[0103] (1) Experimental animals

[0104] KM male mice 8 weeks old, room temperature maintained at 23±1°C, humidity maintained at 50±10%. Light and dark cycle for 12 hours, clean food and water can be obtained at will for feeding.

[0105] (2) Experimental method

[0106] 60 KM mice were divided into Control group, Model group, Resveratrol butyrate + Model group, Resveratrol valerate + Model group, KS-3 butyrate + Model group, KS-3 valerate + Model group, 10 mice in each group. Except for the Control group, each mouse was given 200 μL of nano-micro plastic (PSNPs) (0.75 mg / ml) and 200 μL of plasticizer di(2-ethylhexyl) phthalate (DEHP) (60 mg / ml) by gavage daily to construct environmental pollutant type oligoasthenospermia. The Resveratrol butyrate + Model group, Resveratrol valerate + Model group, KS-3 butyrate + Model group, and KS-3 valerate + Model group were also given 200 μL of corresponding drugs (15 mM) by gavage daily. The Control group was given corn oil by gavage as a control. The KM mice were weighed at the same time every day before gavage and the data were recorded. On the 28th day, tissue samples were collected, and the epididymis was taken for sperm count and sperm motility detection.

[0107] (3) Experimental results

[0108] The data are expressed as mean ± standard error of the mean (SEM), *P < 0.05; **P < 0.01; ***P < 0.001, and the differences between the two groups were evaluated by independent sample T test. All the graphs were drawn by GraphPad Prism 8.0.

[0109] Referring to FIG. 1, FIG. 2, and FIG. 3, FIG. 1 is the weight statistics of mice in Test Example 1, FIG. 2 is the sperm number statistics of mice in Test Example 1, and FIG. 3 is the sperm motility statistics of mice in Test Example 1. As shown in FIG. 1, FIG. 2, and FIG. 3, the body weight of mice in each group did not change significantly except for the Resveratrol valerate + Model group; the sperm number of mice in each treatment group did not change significantly; the Model group significantly reduced the sperm motility of mice, indicating that the oligoasthenospermia mouse model was successfully constructed; compared with the Model group, the KS-3 butyrate + Model group, the KS-3 valerate + Model group, and the Resveratrol valerate + Model group significantly increased the sperm motility of oligoasthenospermia mice. Therefore, KS-3 butyrate, KS-3 valerate, and Resveratrol valerate can increase the sperm motility of oligoasthenospermia mice caused by PSNPs + DEHP.

[0110] Test Example 2

[0111] The difference from Test Example 1 is that the experimental method is as follows:

[0112] The 40 KM mice were divided into Control group, Model group, KS-3 valerate + Model group and KS-3 valerate group, 10 in each group. The Model group and the KS-3 valerate + Model group were each given 200 μL PSNPs (1.5 mg / ml) and 200 μL DEHP (120 mg / ml) by gavage every two days to construct the environmental pollutant type oligoasthenospermia, and the KS-3 valerate + Model group was treated by gavage with KS-3 valerate (30 mM) every two days, the Control group was given corn oil by gavage as a control, and the KS-3 valerate group was treated by gavage with KS-3 valerate (30 mM) every two days, and tissue samples were collected on the 40th day, the epididymis was taken for sperm count and sperm motility detection, and the testis was taken for histological and inflammatory and antioxidant gene mRNA analysis, see Figures 4, 5, 6, 7, 8, 9 and 10 for the results, Figure 4 is the body weight statistical result of the mice in Test Example 2, Figure 5 is the sperm number statistical result of the mice in Test Example 2, Figure 6 is the sperm motility statistical result of the mice in Test Example 2, Figure 7 is the interleukin-1β (IL-1β) expression statistical result of the mice in Test Example 2, Figure 8 is the tumor necrosis factor (TNF-α) expression statistical result of the mice in Test Example 2, Figure 9 is the NAD(P)H quinone dehydrogenase 1 (NQO-1) expression statistical result of the mice in Test Example 2, and Figure 10 is the histological section staining diagram of the mice in Test Example 2. As can be seen from Figures 4, 5 and 6, the body weights of the mice in each group did not change significantly; the Model group significantly reduced the sperm number and sperm motility of the mice, indicating that the oligoasthenospermia mouse model was successfully constructed; compared with the Model group, the KS-3 valerate + Model group and the KS-3 valerate significantly increased the sperm number and sperm motility of the oligoasthenospermia mice, so it can be seen that KS-3 valerate can improve oligoasthenospermia by increasing sperm number and sperm motility. As can be seen from Figures 7, 8 and 9, the Model group significantly up-regulated the expression of inflammatory genes IL-1β and TNF-α and down-regulated the expression of antioxidant gene NQO-1, and the mRNA expression levels of NQO1, IL-1β and TNF-α in the testis of the mice in the KS-3 valerate + Model group were significantly changed compared with those in the Model group, so it can be seen that the supplementation of KS-3 valerate can significantly reduce the inflammation in the testis of the oligoasthenospermia mice caused by PSNPs + DEHP and increase the antioxidant mRNA expression (P<0.05). As can be seen from Figure 10, the Model group caused disordered arrangement of the seminiferous tubules, and the spermatocytes were atrophic and damaged, while KS-3- valerate can effectively alleviate this condition, indicating that KS-3- valerate can improve the pathological damage of the testis of the oligoasthenospermia mice.

[0113] Test Example 3

[0114] The difference from Test Example 1 is that the experimental method is as follows:

[0115] Forty KM mice were divided into Control group, Model group, Model+KS-3 group, Model+KS-3 butyrate group, Model+KS-3 valerate group, 8 mice in each group. In addition to the Control group, on the first and second days of the experiment, each group was injected with streptozotocin (STZ) (80 mg / kg) to construct diabetic oligoasthenospermia, and the fasting blood glucose was detected as evidence of successful modeling. At the same time, the Model+KS-3 group, the Model+KS-3 butyrate group, and the Model+KS-3 valerate group were treated with KS-3 (15 mM), KS-3 butyrate (15 mM), and KS-3 valerate (15 mM), respectively, once a day. The Control group was treated with corn oil as a control, and STZ (120 mg / kg) was injected on the 17th day to extend the model time. On the 34th day, tissue samples were collected, the epididymis was taken for sperm count and sperm motility detection, and the testis was taken for histological molecular and inflammatory and antioxidant gene mRNA analysis.

[0116] The results are shown in Figures 11, 12, 13, 14, 15, 16, and 17. Figure 11 is the weight statistics of the mice in Test Example 3, Figure 12 is the sperm count statistics of the mice in Test Example 3, Figure 13 is the sperm motility statistics of the mice in Test Example 3, Figure 14 is the interleukin-1β (IL-1β) expression statistics of the mice in Test Example 3, Figure 15 is the tumor necrosis factor (TNF-α) expression statistics of the mice in Test Example 3, Figure 16 is the NAD(P)H quinone dehydrogenase 1 (NQO-1) expression statistics of the mice in Test Example 3, and Figure 17 is the histological section staining of the mice in Test Example 3.

[0117] As can be seen from FIGS. 11, 12 and 13, KS-3, KS-3 butyrate and KS-3 valerate all increased the body weight of mice; the Model group significantly reduced the sperm count and sperm motility of mice, indicating that the oligoasthenozoospermia mouse model was successfully constructed; compared with the Model group, the KS-3 butyrate + Model group and the KS-3 valerate + Model group significantly increased the sperm count and sperm motility of oligoasthenozoospermia mice, and the KS-3 + Model group significantly increased the sperm count of oligoasthenozoospermia mice, so it can be seen that KS-3, KS-3 butyrate and KS-3 valerate can improve oligoasthenozoospermia by increasing sperm count and sperm motility. As can be seen from FIGS. 14, 15 and 16, the Model group significantly up-regulated the expression of inflammatory genes IL-1β and TNF-α and down-regulated the expression of antioxidant gene NQO-1, and the mRNA expression levels of NQO1, IL-1β and TNF-α in the testes of mice in the Model + KS-3 group, the Model + KS-3 butyrate group and the Model + KS-3 valerate group were significantly changed compared with those in the Model group, so it can be seen that the supplementation of the KS-3 group, KS-3 butyrate and KS-3 valerate can significantly reduce the inflammation of the testes of STZ-induced oligoasthenozoospermia mice and increase the expression of antioxidant mRNA (P<0.05). As can be seen from FIG. 17, the Model group caused disordered arrangement of the seminiferous tubules, and the spermatocytes were atrophic and damaged, while KS-3 butyrate and KS-3 valerate can effectively alleviate this condition, indicating that KS-3 butyrate and KS-3 valerate can improve the pathological damage of the testes of oligoasthenozoospermia mice.

[0118] Test Example 4

[0119] The difference from Test Example 1 is that the experimental method is as follows:

[0120] Twenty-five KM mice were divided into a Control group, a Model group, a KS-3 + Model group, a KS-3 butyrate + Model group and a KS-3 valerate + Model group, 5 mice in each group. On the 0th day of the experiment, each group was given intragastric administration of sodium valproate (VPA) (500 mg / kg) every day except the Control group to construct drug-induced oligoasthenozoospermia, and each group was given intragastric administration of KS-3 (15 mM), KS-3 butyrate (15 mM) and KS-3 valerate (15 mM) once every two days respectively except the Control group, and the Control group was given intragastric administration of corn oil as a control. On the 35th day, tissue samples were collected, and the epididymides were taken for sperm count and sperm motility detection.

[0121] The results are shown in FIGS. 18, 19 and 20. FIG. 18 is a graph showing the body weight of mice in Test Example 4, FIG. 19 is a graph showing the sperm count of mice in Test Example 4, and FIG. 20 is a graph showing the sperm motility of mice in Test Example 4. As shown in FIGS. 18-20, KS-3, KS-3 butyrate and KS-3 valerate all increased the body weight of mice. Model group significantly reduced the sperm count and sperm motility of mice, indicating that the oligoasthenozoospermia mouse model was successfully constructed. Compared with Model group, KS-3+Model group, KS-3 butyrate+Model group and KS-3 valerate+Model group significantly increased the sperm count and sperm motility of oligoasthenozoospermia mice. Therefore, KS-3, KS-3 butyrate and KS-3 valerate can improve oligoasthenozoospermia by increasing sperm count and sperm motility.

[0122] Test Example 5

[0123] The difference from Test Example 1 is that the experimental method is as follows:

[0124] Fifty KM mice were divided into Control group, Model group, Model+KS3 valerate group, Model+KS3 hexanoate group and Model+KS3 heptanoate group, with 10 mice in each group. Except for Control group, each mouse was intragastrically administered 200 μL PSNPs (1.5 mg / ml) and 200 μL DEHP (120 mg / ml) every two days to construct environmental pollutant type oligoasthenozoospermia, and Model+KS3 valerate group, Model+KS3 hexanoate group and Model+KS3 heptanoate group were treated with KS3 valerate (15 mM), KS3 hexanoate (15 mM) and KS3 heptanoate (15 mM) respectively every two days. Control group was administered corn oil as a control, and tissue samples were collected on the 35th day for epididymal sperm count and sperm motility detection.

[0125] The results are shown in FIGS. 21, 22 and 23. FIG. 21 is a graph showing the sperm count of mice in Test Example 5, FIG. 23 is a graph showing the sperm motility of mice in Test Example 5, and FIG. 23 is a graph showing the sperm forward motility of mice in Test Example 5. As shown in FIGS. 21-23, Model group significantly reduced the sperm count, sperm motility and forward sperm motility of mice, indicating that the oligoasthenozoospermia mouse model was successfully constructed. Compared with Model group, KS-3 valerate, KS-3 hexanoate and KS-3 heptanoate significantly increased the sperm count, sperm motility and forward sperm motility of oligoasthenozoospermia mice. Therefore, KS-3 valerate, KS-3 hexanoate and KS-3 heptanoate can improve oligoasthenozoospermia by increasing sperm count, sperm motility and forward sperm motility.

[0126] Test Example 6

[0127] Forty 6-week-old KM mice were divided into Control group, Model group, Model+FA (ferulic acid) group, Model+MeFA (methyl ferulate) group, Model+MeFA-Butyrate (methyl ferulate-butyrate), Model+MeFA-Pentanoate (methyl ferulate-pentanoate), Model+MeFA-Hexanoate (methyl ferulate-hexanoate), Model+MeFA-Heptanoate group, 5 mice in each group. Except for the Control group, each mouse was intragastrically administered 200 μL PSNPs (1.5 mg / ml) and 200 μL DEHP (120 mg / ml) every other day to construct the environmental pollutant type oligoasthenospermia, and each group was intragastrically administered methyl ferulate C4-C7 ester drugs (30 mM) every other day for treatment, the Control group was administered corn oil for control, and tissue samples were collected on day 35. The epididymis was taken for sperm count and sperm motility detection. The results are shown in FIGS. 24 and 25, FIG. 24 is the sperm count statistical result of test example 6, and FIG. 25 is the sperm motility statistical result of test example 6. As can be seen from FIGS. 24 and 25, the Model group significantly reduced the sperm count and sperm motility of mice, indicating that the oligoasthenospermia mouse model was successfully constructed. Compared with the Model group, the Model+MeFA, Model+MeFA-Pentanoate, Model+MeFA-Heptanoate groups significantly increased the sperm count and sperm motility of oligoasthenospermia mice, so methyl ferulate C4-C7 esters can improve oligoasthenospermia by increasing sperm count and sperm motility.

[0128] Test Example 7

[0129] The 60 male 6-week-old KM mice were divided into Control group (n=5), Model group (n=5), Model group (n=5), Model+CoA-L5R3 group (n=10), Model+CoA-L5R4 group (n=10), Model+CoA-L5R5 group (n=10), Model+vitamin E (VE) group (n=10), Model+coenzyme Q10 (Q10) group (n=10). Except for the Control group, each was intragastrically administered 100 μL of tripterygium glycosides to induce oligoasthenospermia every day, and each was intragastrically administered 7.5 mM of CoA-L5R3, CoA-L5R4, CoA-L5R5, and positive drugs VE and Q10 every day, the Control group was intragastrically administered corn oil as a control, and the tissue sample collection was performed on the 35th day, the epididymis was taken for sperm count and sperm motility detection, and qPCR detection of testicular tissue was used for IL6 and HO1 expression detection. The results of the epididymis are shown in FIGS. 26 and 27; FIG. 26 is the statistical results of the sperm count of the mice in Test Example 7, and FIG. 27 is the statistical results of the sperm motility of the mice in Test Example 7. As shown in FIGS. 26 and 27, the Model group significantly reduced the sperm count and sperm motility of the mice, indicating that tripterygium glycosides successfully induced oligoasthenospermia in mice. Compared with the Model group, the Model+CoA-L5R4 group significantly increased the sperm count and sperm motility of the oligoasthenospermia mice and was better than the positive drugs vitamin E and coenzyme Q10 at the same molar number, so KS3-Pentanoate right ester can improve oligoasthenospermia by increasing sperm count and sperm motility.

[0130] The results of the testis are shown in FIGS. 28 and 29, FIG. 28 is the testicular IL6 expression results of the mice in Test Example 7, and FIG. 29 is the testicular HO1 expression statistical results of the mice in Test Example 7. As shown in FIGS. 28 and 29, the Model group significantly reduced the expression of the antioxidant gene HO1 and increased the expression of the inflammatory factor IL6, and all the compound treatment groups significantly reduced the IL6 increase caused by the model; only CoA-L5R4 could effectively increase the expression of the antioxidant gene HO1 and was significantly higher than the positive drugs VE and Q10 at the same molar number. Therefore, KS3-Pentanoate right ester can improve oligoasthenospermia by reducing testicular inflammation and improving antioxidant, and L5R4 is the best.

[0131] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.

Claims

1. The application of the compound shown in formula (II) in the preparation of drugs that improve male fertility: wherein R is selected from H or R1-CO-, said R1 being selected from C2-C10 alkyl; R3 is selected from hydrogen or C1-C10 alkoxy; R2 is selected from hydrogen or C1-C10 alkyl; R2 and R3 are not simultaneously hydrogen.

2. Use according to claim 1, characterized in that, R3 is selected from C1-C6 alkoxy; R2 is selected from C1-C6 alkyl.

3. Use of a compound of formula (I) for the preparation of a medicament for improving male fertility: wherein, R is selected from H or R1-CO-, said R1 being selected from C2-C10 alkyl.

4. Use according to claim 1, 2 or 3, characterized in that, The improvement of male reproductive capacity comprises prevention of oligoasthenospermia and / or protection of testicular tissue.

5. Use according to claim 4, characterized in that, The prevention of oligoasthenospermia comprises increasing sperm number and / or improving sperm motility.

6. Use according to claim 4, characterized in that, The protection of testicular tissue comprises one or more of improving tissue morphology, reducing inflammatory factor levels and upregulating antioxidant factor levels.

7. Use according to claim 6, characterized in that, The inflammatory factor comprises interleukin 1 beta and / or tumor necrosis factor; The antioxidant factor is NAD(P)H quinone dehydrogenase 1.

8. Use according to claim 1, 2 or 3, characterised in that, The improvement of male reproductive capacity comprises amelioration of male reproductive capacity impairment caused by pollutants, diseases or drugs.

9. Use according to claim 8, characterized in that, The pollutants are microplastics and plasticizers; the diseases are diabetes; the drugs are sodium valproate.

10. Use according to claim 1, 2 or 3, characterised in that, The male is a human or a mouse.

11. Use according to any one of claims 1 to 10, characterized in that, The R1 is selected from C2-C7 alkyl.

12. Use according to claim 11, characterized in that, The R is selected from H, butylcarbonyl, pentylcarbonyl, hexylcarbonyl or heptylcarbonyl.

13. Use according to claim 1, 2 or 3, characterised in that, The compounds have one of the following structures:

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