Application of trigonelline in preparation of medicine for treating obese polycystic ovarian syndrome

Abstract

The invention discloses application of trigonelline or pharmaceutical salt thereof in preparation of a medicine for treating obesity-related polycystic ovarian syndrome. In-vivo experiments prove that the trigonelline has an obvious treatment effect on a polycystic ovarian syndrome rat model, and can be used for treating phlegm-dampness type PCOS rats, recovering the estrus cycle of the rats and reducing fasting blood-glucose and insulin resistance conditions of the rats. More importantly, the trigonelline is small in side effect, free of liver and kidney toxicity and incomparable in safety. In addition, the trigonelline plays a role through a key target GLP-1R. Therefore, the trigonelline can be used for preparing the medicine for treating the obese polycystic ovarian syndrome.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine, and particularly relates to application of trigonelline in preparation of a medicine for treating obesity type polycystic ovary syndrome. BACKGROUND

[0002] Polycystic ovary syndrome is the most common reproductive endocrine disease affecting women of childbearing age, and clinically presents with infertility, obesity, hirsutism, acne and the like. A large amount of epidemiological data shows that obesity is closely related to polycystic ovary syndrome, and about 38% to 88% of women with polycystic ovary syndrome are accompanied by obesity or overweight. Studies have shown that obesity can exacerbate hyperandrogenism and insulin resistance in patients with polycystic ovary syndrome, and increase the risk of cardiovascular diseases, type 2 diabetes and the like, which seriously affects the physical and mental health of the patients. At present, western medicine generally adopts laparoscopic ovarian puncture, drug ovulation promotion and drug anti-androgen and insulin sensitivity enhancement and the like for the treatment of obesity type polycystic ovary syndrome. However, these treatment methods are mostly phase intervention behaviors, and have the disadvantages of great surgical trauma, limited drug treatment effect, no positive prevention effect on long-term complications, great side effect, inability to long-term use, high recurrence rate after drug withdrawal and the like. Therefore, polycystic ovary syndrome is still a hot and difficult problem in the field of gynecological endocrinology, and it is of great significance to actively explore effective treatment methods and their mechanisms.

[0003] In recent years, traditional Chinese medicine has achieved good effects in the clinical treatment of obesity type polycystic ovary syndrome. According to syndrome differentiation and treatment, traditional Chinese medicine divides polycystic ovary syndrome into damp-heat of liver meridian type, phlegm-dampness and blood stasis blocking type, kidney deficiency and blood stasis type and the like. Among them, obesity patients belong to phlegm-dampness and blood stasis blocking type patients, and mainly present with oligo- or amenorrhea, little amount and pale color, and the patients are mostly obese, with greasy fur and slippery pulse or accompanied by cough, asthma and much sputum, and heavy limbs and the like. Traditional Chinese medicine believes that the basic pathogenesis of obesity type polycystic ovary syndrome is phlegm-dampness blocking as the root and blood stasis as the branch. It is stated in Jing Yue Quan Shu (Complete Works of Jing Yue) that although all five zang organs can cause phlegm, they are all related to the spleen and kidney. The spleen is in charge of water and dampness, and water and dampness can cause phlegm. Therefore, the formation of phlegm is related to the spleen, and the root of phlegm is related to the kidney. Therefore, all phlegm syndromes are related to the spleen and kidney. According to traditional Chinese medicine theory and long-term clinical practice, the patent application with the publication number CN104162094A discloses a traditional Chinese medicine composition for treating obesity type polycystic ovary syndrome, which can degrade fat through multiple pathways and multiple targets, promote the recovery of ovary function, promote the growth and development of follicles, induce ovulation, and has good effects in the clinical application in the treatment of polycystic ovary syndrome, especially obesity type polycystic ovary syndrome.

[0004] Traditional Chinese medicine compound ingredients and mechanism of action are complex, which is not conducive to popularization and application. Medlar kidney, soapweed thorn phlegm, is a commonly used drug for clinical treatment of polycystic. Trigonelline derived from medlar and soapweed thorn has the effect of regulating glucose and lipid metabolism, and has anti-inflammatory and antioxidant stress effect, and its clinical application is mainly used for diabetes and central nervous system diseases. However, there is no related research report on the preparation of polycystic ovary syndrome drugs. SUMMARY

[0005] The purpose of the present application is to provide a kind of application of trigonelline in the preparation of drug for treating obesity type polycystic ovary syndrome.

[0006] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0007] The first aspect of the present application provides a kind of application of trigonelline or its pharmaceutical salt in the preparation of drug for treating obesity type polycystic ovary syndrome.

[0008] The structural formula of the trigonelline (CAS:535-83-1) is as follows:

[0009] .

[0010] The drug for treating obesity type polycystic ovary syndrome is trigonelline or its pharmaceutical salt as the only active ingredient.

[0011] The pharmaceutical salt is acid addition salt formed by trigonelline and the following acid: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid or apricot acid.

[0012] The second aspect of the present application provides a kind of pharmaceutical preparation, which is prepared from trigonelline or its pharmaceutical salt and medically acceptable adjuvant.

[0013] The dosage form of the pharmaceutical preparation is selected from liquid preparation, tablet or capsule.

[0014] The administration mode of the pharmaceutical preparation is oral, intravenous injection or intraperitoneal injection.

[0015] The third aspect of the present application provides a kind of pharmaceutical composition, which is prepared from trigonelline or its pharmaceutical salt and other drugs for treating obesity type polycystic ovary syndrome.

[0016] The other drug for treating obesity type polycystic ovary syndrome is selected from insulin sensitizer (such as metformin), oral dydrogesterone, lipid regulating drug (such as statin), and any combination thereof.

[0017] With the above technical scheme, the present application has the following advantages and beneficial effects:

[0018] The in vivo experiment of the present application proves that trigonelline has obvious therapeutic effect on polycystic ovary syndrome rat model, can treat PCOS rats of phlegm-damp type, restore their estrous cycle, and reduce fasting blood glucose and insulin resistance. More importantly, trigonelline has small side effects, no liver and kidney toxicity, and has incomparable safety. In addition, trigonelline plays a role through the key target GLP-1R. Therefore, trigonelline can be used for preparing a drug for treating obesity type polycystic ovary syndrome.

[0019] The experimental results of the present application show that trigonelline can improve the levels of serum testosterone and luteinizing hormone of polycystic ovary syndrome model rats, thereby reversing their disturbed sexual cycle, and has significant therapeutic effect on polycystic ovary syndrome. In addition, trigonelline or its metabolites can also affect the energy metabolism center of the hypothalamus through the blood-brain barrier, affect the expression of GLP-1R, and then regulate the secretion of Kisspeptin, so as to correct the reproductive endocrine level of the model rats, and improve their disturbed sexual cycle and polycystic-like state of the ovary.

[0020] The present application finds that trigonelline can improve the endocrine level of polycystic ovary syndrome patients, improve insulin resistance, restore ovarian function, promote ovulation, and is beneficial to pregnancy, and has small side effects and good safety. Trigonelline monomer derived from plants will be a more comfortable, safer and more precise treatment choice for patients who are worried about the composition of the compound.

[0021] In summary, trigonelline can improve the levels of peripheral blood testosterone and luteinizing hormone by affecting the expression of GLP-1R and regulating the secretion of Kisspeptin, and finally achieve the effect of treating polycystic ovary syndrome. Therefore, trigonelline can be applied to prepare a drug for treating polycystic ovary syndrome, and has good prospects. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is a schematic diagram of the results of the influence of trigonelline on the sexual hormone level of rats in Example 1.

[0023] Figure 2 It is a schematic diagram of the results of HE staining of the ovarian tissue of rats in Example 1.

[0024] Figure 3 It is a schematic diagram of the results of the detection of fasting blood glucose (FBG), fasting insulin (FINS) and insulin resistance index (HOMA-IR) of rats in Example 1.

[0025] Figure 4 It is a schematic diagram of the results of the influence of trigonelline on the sexual hormone level of rats in Example 2.

[0026] Figure 5 Figure 2 is a schematic diagram of the results of HE staining of rat ovarian tissue in Example 2.

[0027] Figure 6 Figure 3 is a schematic diagram of the results of detection of fasting blood glucose, fasting insulin, and insulin antagonistic index of rats in Example 2.

[0028] Figure 7 Figure 4 is a schematic diagram of the results of detection of hypothalamic GLP-1R mRNA expression level and hypothalamic GLP-1R and Kisspeptin level of rats in Example 2.

[0029] Figure 8 Figure 5 is a schematic diagram of a rat brain positioning map. DETAILED DESCRIPTION

[0030] In order to more clearly illustrate the present application, the present application will be further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the specific description below is illustrative rather than limiting, and should not limit the protection scope of the present application.

[0031] Example 1

[0032] Sprague-Dawley rats were selected to prepare a polycystic ovary syndrome animal model, and the successful model was intervened with drugs. ELISA, RT-PCR and other methods were used for detection, and statistical software SPSS16.0 was used for analysis, in order to clarify the therapeutic effect of trigonelline on the polycystic ovary syndrome animal model.

[0033] 1. Materials

[0034] 1.1 Experimental animals

[0035] 50 SPF female Sprague-Dawley rats aged 5 weeks were provided by Beijing Vitoohua Experimental Animal Technology Co., Ltd. and were raised in the SPF experimental animal center of Shanghai Changhai Hospital. The feeding conditions were strictly controlled, including indoor temperature of 20-26℃, temperature difference not more than 4℃, and relative humidity maintained between 40%-70%, noise level maintained at not more than 60 decibels. The feeding density was controlled at not more than 4 rats per cage. The experiment was audited and approved by the experimental animal ethics committee of the medical ethics committee of Shanghai Changhai Hospital, with the ethics review number: CHEC (A.E) 2022-011, and all experimental operations conformed to the relevant regulations of animal experiment ethics.

[0036] 1.2 Main reagents

[0037] 60% fat energy high-fat diet was purchased from Jiangsu Cooperation Bioengineering Co., Ltd., letrozole chemical analysis product was purchased from Beijing Solabio Technology Co., Ltd., trigonelline was purchased from MedChemExpress company, liraglutide injection was purchased from Novo Nordisk Company, Denmark, serum testosterone (T), estradiol (E2), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) enzyme-linked immunoassay (ELISA) detection kit was purchased from Wuhan Elabscience Biotechnology Co., Ltd.

[0038] 1.3 Experimental method

[0039] 1.3.1 Establishment of polycystic ovary syndrome (i.e. obesity type polycystic ovary syndrome) animal model with phlegm-dampness syndrome

[0040] 5-week-old Sprague-Dawley rats were adaptively fed for 5 days, the blank group was given SPF level experimental mouse maintenance feed, and physiological saline 5 ml / kg was given by gavage every day, and the rats were free to eat and drink water; the model group rats were given 60% fat energy high-fat diet, and letrozole 1 mg / kg (1% carboxymethylcellulose sodium solution was prepared to suspend letrozole, the concentration dose was 1 mg / kg) was given by gavage every day, and the rats were free to eat and drink water, for a total of 28 days of modeling. After the modeling was completed, 2 rats were randomly selected from each group to identify whether the model was successful. From the 21st day of modeling, vaginal exfoliative cell detection was performed on all rats, and body weight was measured, and rats with estrous cycle disorder and body weight exceeding normal rat weight by 20% were considered to meet the criteria for phlegm-dampness type PCOS rat model, and were included in the subsequent drug intervention experiment for 28 days. Rats that did not meet the criteria were discarded.

[0041] 1.3.2 Grouping of experimental rats and drug intervention:

[0042] Normal group (Control group): physiological saline 5 ml / kg by gavage + SPF level experimental mouse maintenance feed;

[0043] Model group (PCOS group): letrozole 1 mg / kg by gavage + high-fat diet;

[0044] Traditional Chinese medicine group (TRG group): trigonelline (1.5 mg / kg) by gavage treatment + high-fat diet;

[0045] Western medicine group (Lira group): subcutaneous injection of liraglutide injection 400 μg / kg for treatment + high-fat diet.

[0046] 2. Observation index and results

[0047] 2.1 Estrous cycle of rats

[0048] The rats were subcutaneously injected with DHEA (dehydroepiandrosterone) for 21 days, and the vaginal exfoliative cytology was performed. The vaginal exfoliative cells were in a keratinized state, which was similar to the anovulatory symptoms of polycystic ovary syndrome, indicating that the modeling was successful, and the success rate of the model was 63.2% according to the statistics.

[0049] After 21 days of drug intervention, the vaginal exfoliative cytology of the rats was performed, and the changes of the sexual cycle of the rats were observed according to the histological characteristics of the vaginal smear at each stage of the sexual cycle of the rats. The recovery rate of the sexual cycle of the traditional Chinese medicine group was 75%, which had a significant difference compared with the model group without drug intervention. It is shown that trigonelline has a significant improvement effect on the sexual cycle of the polycystic ovary syndrome rat model, and its effect is not different from that of the positive control drug liraglutide.

[0050] 2.2 Morphological changes of rat ovarian tissue

[0051] After drug intervention, the bilateral ovaries of the rats were surgically removed, and the removed ovarian tissue was fixed for 48 hours. Then, the tissue was soaked in different concentrations of ethanol dehydrating agent. The tissue was soaked in xylene I for 8 minutes and then transferred to xylene II for 8 minutes. The paraffin was heated and melted, and the treated tissue was placed in the center of the embedding frame. Paraffin was dropped, and the tissue was taken out after cooling. The thickness of the section was set to 4 μm, and the glass slide was used to take out the section and placed in water at a temperature of 40°C. The glass slide with the expanded section was placed in a constant temperature oven at 60°C for drying. The section was dyed by hematoxylin-eosin staining method. The results are shown in Figure 2 Figure 2 The HE staining results of the ovarian tissue of the rats in Example 1 are shown in the schematic diagram. As can be seen from the diagram, in the normal group, the traditional Chinese medicine group and the western medicine group, different developmental stages of follicles were observed in the ovaries, and the arrangement of granulosa cells was relatively dense, accompanied by a large number of corpus luteum formation. In the model group, the ovaries showed obvious polycystic changes.

[0052] 2.3 Detection of hormone levels in rats

[0053] 2.3.1 ELISA detection of serum sex hormones in rats

[0054] After the drug intervention of the rats, the rats in the diestrus phase were taken blood from the abdominal aorta according to the vaginal exfoliative cytology. The serum sex hormones were detected by enzyme-linked immunosorbent assay (ELISA), and the results are shown in Figure 1 Figure 1 ​​Figure 1 shows the results of the effect of trigonelline on the sex hormone levels in rats in Example 1. A is the result of the level of androgen (T), B is the result of the level of luteinizing hormone (LH), C is the result of the level of follicle-stimulating hormone (FSH), and D is the result of LH / FSH. As can be seen from the figure, after the end of drug intervention, compared with the Control group of rats, the PCOS group had increased serum testosterone (T) levels (P<0.01), and the PCOS group had increased LH levels (P<0.001), indicating that the polycystic ovary syndrome animal model was successfully established; compared with the PCOS group, the TRG group and the Lira group had decreased T levels (P<0.05), and the Lira group had decreased LH / FSH (P<0.05), indicating that TRG can improve the sex hormone levels of rats and has a therapeutic effect.

[0055] 2.3.2 Results of blood glucose detection and insulin release test in rats

[0056] At the end of the experiment, the insulin release test was performed by taking blood from the orbital vein of the rats, and the fasting blood glucose of the rats was detected by a blood glucose meter. The results are shown in Figure 3 Figure 3 Figure 1 shows the results of the effect of trigonelline on the sex hormone levels in rats in Example 1. A is the result of the level of androgen (T), B is the result of the level of luteinizing hormone (LH), C is the result of the level of follicle-stimulating hormone (FSH), and D is the result of LH / FSH. As can be seen from the figure, after the end of drug intervention, compared with the Control group of rats, the PCOS group had increased serum testosterone (T) levels (P<0.01), and the PCOS group had increased LH levels (P<0.001), indicating that the polycystic ovary syndrome animal model was successfully established; compared with the PCOS group, the TRG group and the Lira group had decreased T levels (P<0.05), and the Lira group had decreased LH / FSH (P<0.05), indicating that TRG can improve the sex hormone levels of rats and has a therapeutic effect.

[0057] Example 2

[0058] Sprague-Dawley rats were selected to prepare a polycystic ovary syndrome animal model, and the hypothalamic GLP-1 / GLP-1R / kisspeptin signaling pathway was used as the starting point. Combined with stereotactic injection of adeno-associated virus into the arcuate nucleus of the hypothalamus, ELISA, qRT-PCR, and other methods were used for detection, and statistical software SPSS16.0 was used for analysis to further explore the mechanism of action of trigonelline in the treatment of phlegm-damp type polycystic ovary syndrome.

[0059] 1. Materials

[0060] 1.1 Experimental animals

[0061] ​50 SPF female Sprague-Dawley rats aged 5 weeks were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. and were raised in the SPF experimental animal center of Shanghai Changhai Hospital. The feeding conditions were strictly controlled, including indoor temperature of 20-26℃, temperature difference not more than 4℃, and relative humidity maintained between 40%-70%, noise level maintained at not more than 60 decibels. The feeding density was controlled at not more than 4 rats per cage. The experiment was audited and approved by the Experimental Animal Ethics Committee of the Medical Ethics Committee of Shanghai Changhai Hospital, ethics review number: CHEC (A.E) 2022-011, and all experimental operations complied with the relevant regulations of animal experiment ethics.

[0062] 1.2 Main reagents

[0063] 60% fat energy high-fat diet was purchased from Jiangsu Cooperation Bioengineering Co., Ltd., letrozole chemical analysis product was purchased from Beijing Solabio Technology Co., Ltd., trigonelline was purchased from MedChemExpress company, liraglutide injection was purchased from Novo Nordisk (Denmark), Depo-50 was purchased from Rhea-D France, antibody anti-GLP-1R was purchased from Abways (Shanghai) Trading Co., Ltd., antibody anti-kisspeptin was purchased from Thermo Fisher Scientific (China) Co., Ltd., serum testosterone (T), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) enzyme-linked immunoassay (ELISA) test kit was purchased from Wuhan Elabscience Biotechnology Co., Ltd.

[0064] 1.3 Experimental method

[0065] 1.3.1 r-GLP-1RR shRNA adeno-associated virus stereotactic injection method in the hypothalamic arcuate nucleus to construct GLP-1 receptor knockout rats

[0066] The rats were anesthetized with intramuscular injection of Depo-50, and after the rats were fully anesthetized, they were fixed in a prone position on a brain stereotactic injection instrument. The rat skull top hair was removed with a hair clipper to fully expose the nearby fontanel. After disinfection with iodophor, the skull surface was exposed by cutting along the midline of the skull with surgical scissors, and the skull was fully exposed by wiping the skull with a sterile cotton swab. Hemostasis was achieved by wiping the skull with a sterile cotton swab, fully exposing the skull herringbone and sagittal suture, and removing the skull membrane with a sterile cotton swab dipped in a small amount of hydrogen peroxide solution to ensure that the anterior fontanel and posterior fontanel positioning points could be clearly seen. The rat was fixed in a prone position on a brain stereotactic injection instrument. The rat skull was kept level by adjusting the rat fixation position with the brain stereotactic injection instrument fixation device. Referring to the rat brain positioning map (as shown in Figure 8 , the rat was fixed in a prone position on a brain stereotactic injection instrument. The rat skull was kept level by adjusting the rat fixation position with the brain stereotactic injection instrument fixation device. Referring to the rat brain positioning map (as shown in Figure 8The rat brain atlas schematic diagram, the box is to determine the hypothalamus specific location, the bregma position as the coordinate zero point, the bregma 2.0~4.5 mm behind the position of the arcuate nucleus of hypothalamus, 0.5 mm away from the midline, close to the median eminence, 9.6~10.0 mm below the skull surface. After making good positioning marks, use dental drill to drill on the corresponding projection position of the skull surface to penetrate the skull. Use 10 μl microsyringe to inject slowly, the injection time is 10 min, the needle retention time is 10 min, and the syringe is slowly withdrawn after the injection is completed without abnormality. After operation, the wound is sutured and erythromycin eye ointment is applied to the wound to prevent infection. Rats are caged after operation, and the adaptive feeding is ended after the wound heals for subsequent experiments.

[0067] 1.3.2 Establishment of polycystic ovary syndrome animal model with phlegm-dampness syndrome

[0068] After the stereotactic injection of the arcuate nucleus of the hypothalamus of the rat, the rats were recovered for 1 week, and then the formal modeling experiment of the polycystic syndrome rat model was started. The blank group was fed with SPF level experimental rats, and physiological saline 5 ml / kg was given by gavage every day, and the rats were free to eat and drink water; the model group rats were fed with 60% fat energy high-fat feed, and letrozole 1 mg / kg (1% carboxymethylcellulose sodium solution was prepared to suspend letrozole, the concentration dose was 1 mg / kg) was given by gavage every day, and the rats were free to eat and drink water, for a total of 28 days of modeling. Each group had 10 rats, and 2 rats were randomly selected from each group after modeling to identify whether the model was successful. From the 21st day of modeling, vaginal exfoliative cell detection was performed on all rats, and body weight was measured. Rats with menstrual cycle disorder and body weight exceeding normal rat weight by 20% were considered to be phlegm-dampness type PCOS rat models, and were included in the subsequent drug intervention experiment of rats meeting the phlegm-dampness type PCOS standard. Atypical rats were discarded.

[0069] Control con The blank group was fed with SPF level experimental rats, and physiological saline 5 ml / kg was given by gavage every day, and the rats were free to eat and drink water; PCOS con The PCOS group was fed with 60% fat energy high-fat feed, and physiological saline 5 ml / kg was given by gavage every day, and the rats were free to eat and drink water; PCOS r -GLP-1R The TRG group was fed with 60% fat energy high-fat feed, and physiological saline 5 ml / kg was given by gavage every day, and the rats were free to eat and drink water; TRG con The TRG group was fed with 60% fat energy high-fat feed, and 1.5 mg / kg of trigonelline was given by gavage every day, and the rats were free to eat and drink water; TRG r-GLP-1RGroup: 60% fat energy high-fat diet, intragastric administration of trigonelline 1.5 mg / kg per day, free drinking water; each group is intervened for 4 weeks. On the 21st day from modeling, all rats were detected by vaginal exfoliative cytology, and body weight was measured. Rats with menstrual cycle disorders and body weight exceeding normal rat weight by 20% were considered to meet the criteria for PCOS rat models of phlegm-damp type and were included in the subsequent drug intervention experiment. Those who did not meet the criteria were discarded.

[0070] 1.3.3 Grouping of experimental rats and drug intervention:

[0071] Control con Group: normal saline 5 ml / kg intragastric administration + SPF level experimental rat maintenance feed;

[0072] PCOS con Group: letrozole 1 mg / kg intragastric administration + high-fat diet;

[0073] PCOS r-GLP-1R Group: letrozole 1 mg / kg intragastric administration + high-fat diet;

[0074] PCOS con Group: normal group rats to construct PCOS model; PCOS r-GLP-1R is the construction of PCOS model of hypothalamic arcuate nucleus GLP-1R gene silencing rats.

[0075] TRG con : TRG intragastric treatment (1.5 mg / kg) + high-fat diet;

[0076] TRG r-GLP-1R : TRG intragastric treatment (1.5 mg / kg) + high-fat diet;

[0077] TRG con is the construction of PCOS model of normal group rats treated with TRG; TRG r-GLP-1R is the construction of PCOS model of hypothalamic arcuate nucleus GLP-1R gene silencing rats treated with TRG.

[0078] 2. Observation index and results

[0079] 2.1 Rat estrous cycle

[0080] Rats were subcutaneously injected with DHEA, and vaginal exfoliative cytology was performed 21 days later. The continuous keratinization of vaginal exfoliative cells was similar to the anovulatory symptoms of polycystic ovary syndrome, indicating that the modeling was successful. According to the statistics, the success rate of the model was 86.3%.

[0081] The rats were given drug intervention for 21 days, and then vaginal exfoliative cytology was performed. According to the histological characteristics of the vaginal smear of each stage of the rat sexual cycle, the changes of the rat sexual cycle were observed. con The rats in the Control group all had normal estrous cycles, and the estrous cycle lasted for 4-5 days; the rats in the PCOS group had irregular estrous cycles, and no complete estrous period was observed; the rats in the TRG con group had normal estrous cycles, and the normal rate of the estrous cycle was 73%; and the rats in the TRG r-GLP-1R group had irregular estrous cycles, and no complete estrous period was observed. con The rats in the TRG r-GLP-1R group had irregular estrous cycles, and no complete estrous period was observed. The above results show that trigonelline can improve the estrous cycle of rats with polycystic ovary syndrome by GLP-1 receptor.

[0082] 2.2 Morphological changes of rat ovarian tissue

[0083] After the drug intervention was completed, the bilateral ovaries of the rats were surgically removed. The removed ovarian tissue was fixed for 48 hours, and then immersed in different concentrations of ethanol dehydrating agent. The tissue was immersed in xylene I for 8 minutes, and then transferred to xylene II for 8 minutes. The paraffin was first heated and melted, and the treated tissue was placed in the center of the embedding frame. Paraffin was dropped, and the tissue was taken out after cooling. The thickness of the section was set to 4 pm, and the glass slide was used to take out the section and placed in water at 40°C. The glass slide carrying the expanded section was placed in a constant temperature oven at 60°C for drying. The section was dyed by hematoxylin-eosin staining method, and the results are shown in Figure 5 . Figure 5 The results of HE staining of rat ovarian tissue in Example 2 are shown in the figure. As can be seen from the figure, the Control con group, the TRG con group, the PCOS con group, the PCOS r-GLP-1R group and the TRG r-GLP-1R group showed multiple corpora lutea and follicles at different stages of development in the ovary.

[0084] 2.3 ELISA detection of serum sex hormones of rats

[0085] After the drug intervention of the rats was completed, the rats in the estrus interval were taken blood from the abdominal aorta, and the serum sex hormones were detected by enzyme-linked immunosorbent assay (ELISA). The results are shown in Figure 4 . Figure 4Figure 2 shows the results of the effect of trigonelline on the sex hormone levels of rats in Example 2. A is the results of the androgen (T) level, B is the results of the LH / FSH, C is the results of the follicle-stimulating hormone (FSH) level, D is the results of the luteinizing hormone (LH) level, and E is the results of the GLP-1 level. As can be seen from the figure, the T, LH level, LH / FSH ratio, and GLP-1 level of the PCOS group were significantly higher than those of the Control group (P<0.001, P<0.01, P<0.05). The T, LH level, LH / FSH ratio, and GLP-1 level of the TRG group were significantly lower than those of the PCOS group (P<0.001, P<0.05). The effect of trigonelline on the TRG group was significantly reversed (P<0.05), while the T, LH level, LH / FSH ratio, and GLP-1 level of the PCOS group were not significantly changed compared with the PCOS group (P>0.05). This indicates that trigonelline improves the sex hormone-related levels of rats through GLP-1R. con con con con r-GLP-1R r-GLP-1R con

[0086] 2.4 Results of blood glucose detection and insulin release test of rats

[0087] At the end of the experiment, the insulin release test was performed by taking blood from the orbital vein of the rats, and the fasting blood glucose of the rats was detected by a blood glucose meter. After the drug intervention was completed, the insulin release test was performed by taking blood from the orbital vein of the rats, the fasting blood glucose of the rats was detected by a blood glucose meter, and the insulin resistance index (fasting insulin x fasting blood glucose / 22.5, HOMA-IR) was calculated. The results are shown in Figure 3. Figure 6 Figure 6 Figure 4 shows the results of the detection of fasting blood glucose, fasting insulin, and insulin resistance index of rats in Example 2. As can be seen from the figure, the FBG, FINS, and HOMA-IR levels of the PCOS group were significantly higher than those of the Control group (P<0.0001, P<0.001). The FBG, FINS, and HOMA-IR levels of the TRG group were significantly lower than those of the PCOS group (P<0.0001, P<0.05). The effect of trigonelline on the TRG group was significantly reversed (P<0.05), while the FBG, FINS, and HOMA-IR levels of the PCOS group were not significantly changed compared with the PCOS group (P>0.05). con con con con r-GLP-1R r-GLP-1R con ​​​​​​​​​​​​​​FBG, FINS, HOMA-IR levels were not significantly changed (P>0.05). Fenugreek can reduce fasting blood glucose, fasting insulin, insulin resistance index by regulating GLP-1R, thereby playing its therapeutic effect.

[0088] 2.5 Detection of hypothalamic GLP-1R mRNA expression level and detection of hypothalamic GLP-1R and Kisspeptin levels

[0089] The results are shown in Figure 7 , Figure 7 The results of the detection of hypothalamic GLP-1R mRNA expression level and the detection of hypothalamic GLP-1R and Kisspeptin levels in rats in Example 2 are shown in the schematic diagram, wherein A is a schematic diagram of qRT-PCR, GLP-1R mRNA content compared with the internal reference β-ACTIN; B is a schematic diagram of qRT-PCR, GLP-1R mRNA content compared with the internal reference GAPDH; C is a schematic diagram of qRT-PCR, Kisspeptin mRNA content; D is a schematic diagram of Western Blot, GLP-1R and Kisspeptin protein content.

[0090] The qRT-PCR results show that, compared with the Control con group, the GLP-1R mRNA expression level of the PCOS con group decreased significantly (P<0.05), and the GLP-1R expression level of the TRG con group increased significantly (P<0.001), indicating that the mechanism of action of fenugreek is related to the increase of GLP-1R mRNA expression (P<0.05); the GLP-1R mRNA expression of the PCOS r-GLP-1R group and the TRG r-GLP-1R group was significantly lower than that of the Control con group, and the expression was extremely low (P<0.0001), indicating that r-GLP-1R virus transfection indeed affected the expression of hypothalamic GLP-1R.

[0091] The Western Blot results show that, compared with the Control con group, the GLP-1R level of the PCOS con group, the PCOS r-GLP-1R group, the TRG con group and the TRG r-GLP-1R group decreased significantly (P<0.0001); compared with the PCOS con group, the GLP-1R level of the PCOS r-GLP-1R group and the TRG r -GLP-1RGLP-1R levels in the group were significantly decreased (P<0.0001); compared with TRG con PCOS group compared with Control group r-GLP-1R PCOS group compared with Control group r-GLP-1R GLP-1R expression levels in the group were significantly decreased (P<0.0001). Compared with Control con PCOS group compared with Control group con PCOS group compared with Control group r-GLP-1R PCOS group compared with Control group con PCOS group compared with Control group r-GLP-1R Kisspeptin expression levels in the group were significantly increased (P<0.001, P<0.05); compared with PCOS con PCOS group compared with Control group r-GLP-1R PCOS group compared with Control group r-GLP-1R Kisspeptin expression levels in the group were significantly increased (P<0.001), TRG con Kisspeptin expression levels in the group were significantly decreased (P<0.0001); compared with TRG con PCOS group compared with Control group r-GLP-1R PCOS group compared with Control group r-GLP-1R Kisspeptin expression levels in the group were significantly increased (P<0.001). It is shown that trigonelline can reduce the expression level of Kisspeptin by activating GLP-1R.

[0092] The present application respectively discusses the effectiveness and mechanism of action of trigonelline in treating obesity type polycystic ovary syndrome. The experimental results show that trigonelline can improve the levels of serum testosterone, luteinizing hormone and follicle-stimulating hormone of polycystic ovary syndrome model rats, thereby reversing the disturbed sexual cycle; further research results show that trigonelline or its metabolites can affect the hypothalamic energy metabolism center by passing through the blood-brain barrier, affect GLP-1R expression, and then regulate the secretion of Kisspeptin, effectively correct the reproductive endocrine level of the model rats, and ultimately achieve the effect of treating polycystic ovary syndrome. Therefore, trigonelline can be applied to the preparation of a drug for treating obesity type polycystic ovary syndrome, and has good prospects.

[0093] The above only describes the preferred embodiments of the present application, and does not limit the present application in any form. Although the present application has been disclosed as above with the preferred embodiments, it is not intended to limit the present application. Any skilled person in the art can make some changes or modifications to the above-mentioned technical content without departing from the scope of the technical solution of the present application, and any simple modification, equivalent change and modification of the above-mentioned embodiments made according to the technical essence of the present application are still within the scope of the present application.

Claims

1. Use of trigonelline or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of obesity type polycystic ovary syndrome, characterized in that, The structural formula of the trigonelline is as follows: 。 2. Use of trigonelline or a pharmaceutically acceptable salt thereof according to claim 1 for the manufacture of a medicament for the treatment of obesity type polycystic ovary syndrome, characterized in that, The medicine for treating obesity type polycystic ovary syndrome is taken as the only active ingredient of trigonelline or its medicinal salt.

3. Use of trigonelline or a pharmaceutically acceptable salt thereof according to claim 1 for the manufacture of a medicament for the treatment of obesity type polycystic ovary syndrome, characterized in that, The medicinal salt is the acid addition salt of trigonelline and the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

4. A pharmaceutical preparation, characterized by, The medicine is made of trigonelline or its medicinal salt and medically acceptable auxiliary materials.

5. The pharmaceutical preparation according to claim 4, characterized in that, The dosage form of the medicine preparation is selected from liquid medicine, tablet or capsule.

6. The pharmaceutical preparation according to claim 4, characterized in that, The administration mode of the medicine preparation is oral, intravenous injection or intraperitoneal injection.

7. A pharmaceutical composition, characterized by, The medicine is made of trigonelline or its medicinal salt and other medicines for treating obesity type polycystic ovary syndrome.

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