Application of lotus seed alkaloid in the preparation of drugs for treating polycystic ovary syndrome

Abstract

This invention discloses the application of lotus seed alkaloids in the preparation of drugs for treating polycystic ovary syndrome (PCOS), belonging to the field of pharmaceutical technology. This invention discovers that lotus seed alkaloids have therapeutic effects on PCOS and reveals its therapeutic mechanism. This invention provides the application of lotus seed alkaloids in the preparation of drugs for treating PCOS, demonstrating the potential to overcome existing treatment bottlenecks. This invention provides a novel pathway for developing PCOS treatment drugs that avoid other recurrence risks and also have long-term metabolic regulatory effects.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of lotus seed alkaloid in the preparation of drugs for treating polycystic ovary syndrome. Background Technology

[0002] Polycystic ovary syndrome (PCOS) is one of the most common endocrine and metabolic disorders in women of reproductive age. It is characterized by infrequent ovulation / anovulation, hyperandrogenemia, insulin resistance, and polycystic ovarian changes, constituting a complex endocrine disorder syndrome. For women with PCOS, persistent anovulation leading to infertility is the most prominent clinical problem. Although the molecular mechanisms of ovulation disorders in PCOS are not fully understood, the resulting infertility remains a challenge in clinical treatment.

[0003] Interventions for patients with fertility needs include lifestyle modifications, menstrual cycle regulation, relief of androgen symptoms, insulin sensitizers, ovulation induction therapy, ovarian drilling, and assisted reproductive technologies. However, due to the unknown etiology of PCOS, existing treatments have significant limitations: ovulation induction therapy easily induces ovarian hyperstimulation syndrome (OHSS); assisted reproductive technologies result in decreased egg quality, lower fertilization and embryo implantation rates, and increased risk of miscarriage; long-term drug treatment may exacerbate metabolic disorders, and its efficacy is unstable. For example, current first-line clinical drugs such as oral contraceptives (which regulate menstrual cycles and lower androgen levels) and the insulin sensitizer metformin both have limitations. Oral contraceptives do not improve or may even worsen metabolic abnormalities, and metformin has limited effects on improving reproductive outcomes. The fundamental reason is that existing therapies mostly target single symptoms or pathways and fail to simultaneously address the common pathological aspects of PCOS reproductive disorders and metabolic disturbances.

[0004] Therefore, given the safety deficiencies and limited efficacy of existing PCOS treatments, there is an urgent need to explore new intervention strategies based on natural drug therapies, elucidate the molecular pathways that regulate ovulation disorders and metabolic disturbances, and provide a scientific basis for developing safe and effective new PCOS treatments.

[0005] The signaling pathway consisting of the energy metabolism sensor adenosine monophosphate activated protein kinase (AMPK) and its downstream target silencing information regulator 1 (SIRT1) plays a central role in regulating systemic energy homeostasis, insulin sensitivity, and local ovarian function. Decreased activity of this pathway is closely related to the development and progression of PCOS, but to date, no drugs or candidate compounds that specifically activate this pathway to treat PCOS have entered clinical development or been approved for marketing.

[0006] Lotus seed alkaloid is a dibenzylisoquinoline alkaloid derived from the lotus seed heart, a traditional Chinese medicine. Its known pharmacological activities are concentrated in the cardiovascular system (such as antiarrhythmic activity). Current technology has not reported any association between lotus seed alkaloid and the AMPK / SIRT1 signaling pathway, nor is there any information suggesting its potential use in the treatment of polycystic ovary syndrome (PCOS). Summary of the Invention

[0007] In response to the clinical challenges of high incidence of ovarian hyperstimulation syndrome (OHSS) in existing ovulation induction treatments, poor oocyte quality in assisted reproductive technologies, and the inability of traditional drugs to simultaneously improve metabolic disorders, the present invention aims to provide the application of lotus seed alkaloid in the preparation of drugs for the treatment of polycystic ovary syndrome (PCOS), thus providing a safe and effective new clinical treatment strategy for PCOS patients.

[0008] The objective of this invention is achieved through the following technical solution: This invention provides the use of limonin or its analogues / derivatives or pharmaceutically acceptable salts thereof in the preparation of pharmaceuticals or reagents. In this use, limonin or its analogues / derivatives are used as the sole active ingredient of the pharmaceutical or reagent. The use includes one or more of the following: Application in the preparation of drugs for the prevention and / or treatment of polycystic ovary syndrome; Its application in the preparation of drugs for treating hyperandrogenemia; Its application in the preparation of drugs that promote normal follicle development; Its application in the preparation of drugs that promote the recovery of ovarian ovulation; Its application in the preparation of drugs that improve insulin resistance; Its application in the preparation of drugs for treating hyperinsulinemia; Application in the preparation of reagents that inhibit abnormal autophagy; Application in the preparation of ATG9B inhibitors; Application in the preparation of AMPK / SIRT1 signaling pathway activators.

[0009] The present invention also provides the use of compositions comprising limonene or its analogues / derivatives or pharmaceutically acceptable salts thereof in the preparation of medicaments for the prevention and / or treatment of polycystic ovary syndrome (PCOS). In this application, the compositions comprising limonene or its analogues / derivatives may further comprise other active ingredients having therapeutic effects on PCOS, such as insulin sensitizers and / or ovulation-inducing drugs. The insulin sensitizer is preferably metformin or a pharmaceutically acceptable salt thereof.

[0010] In the above applications, the lotusine analogues / derivatives preferably include isolivineine, methyl lotusine, pro-lotus leaf alkaloid, lotusine quaternary ammonium alkaloid, etc.

[0011] In the above applications, pharmaceutically acceptable salts of the lotusine or its analogues / derivatives preferably include perchlorate, hydrochloride, phosphate, methanesulfonate, citrate, etc.

[0012] In the above applications, the polycystic ovary syndrome is preferably obese polycystic ovary syndrome.

[0013] In the above applications, the drug, in addition to containing the active ingredient, may further contain a pharmaceutically acceptable carrier. The dosage forms of the drug for treating polycystic ovary syndrome include oral dosage forms (such as tablets, enteric-coated tablets, sustained-release / controlled-release tablets, capsules, etc.), injectable dosage forms (such as powder for injection, injection / solution, etc.), transdermal dosage forms (such as patches, etc.), and vaginal dosage forms (such as vaginal rings, vaginal gels / creams, etc.).

[0014] In the above applications, the daily dose of the drug containing nepeline or its analogues / derivatives or pharmaceutically acceptable salts is 40-80 mg / kg.

[0015] The present invention also provides a pharmaceutical composition for treating polycystic ovary syndrome, comprising a therapeutically effective amount of limonene or its analogues / derivatives or pharmaceutically acceptable salts thereof, and one or more pharmaceutically acceptable carriers.

[0016] Furthermore, the pharmaceutical composition also comprises at least one other active ingredient having therapeutic effects on polycystic ovary syndrome, said other active ingredient being selected from insulin sensitizers and / or ovulation-inducing drugs. Preferably, the insulin sensitizer is metformin or a pharmaceutically acceptable salt thereof.

[0017] This invention uses the natural active ingredient lotus seed alkaloid as its core, and can achieve the following clinical goals: Breaking through the safety bottleneck of ovulation induction treatment: Through the natural anti-inflammatory and anti-autophagy properties of lotus seed alkaloid, it reduces the risk of ovarian hyperstimulation caused by ovulation induction drugs, reduces serious complications such as ascites and thrombosis, and at the same time protects oocytes from excessive autophagy damage, and improves the proportion of mature follicles and embryo quality.

[0018] The present invention has the following advantages and beneficial effects: This invention utilizes limonene for the treatment of PCOS, demonstrating its potential to overcome existing treatment limitations. Compared to clomiphene, a commonly used ovulation-inducing drug (reported in literature that the incidence of OHSS in mouse models is >35%), the C57BL / 6J mouse model treated with limonene did not exhibit symptoms of ovarian hyperstimulation syndrome. Its core advantage stems from the synergistic regulation of multiple targets and pathological processes: by regulating the activity of autophagy proteins in ovarian tissue, it effectively blocks the dysfunction of follicular granulosa cells caused by abnormal autophagy; it significantly improves insulin resistance (e.g., reducing fasting insulin and HOMA-IR index); it reverses hyperandrogenemia (reducing serum testosterone levels from 4 ng / mL to 1.5 ng / mL); and it effectively controls the weight gain of the model mice. This multi-mechanism of action, encompassing autophagy, metabolism, and endocrine function, overcomes the limitation of current clinical treatments that often require combination therapy—in animal experiments, limonene alone can simultaneously improve the above key phenotypes and restore ovarian ovulation function.

[0019] In terms of translational applications, lotus seed extract exhibits significant cost and safety advantages: it utilizes lotus seed heart processing byproducts for raw material extraction, resulting in a natural composition; and it achieves effective blood drug concentrations in mice without the need for an outer shell to encapsulate the drug. This invention confirms that lotus seed extract, through a synergistic pathway of anti-autophagy, metabolism regulation, and androgen reduction, provides a novel pathway for developing PCOS treatments that avoid other relapse risks and also possess long-term metabolic regulatory effects. Attached Figure Description

[0020] Figure 1 Phenotypic changes in mice in the control group, polycystic ovary syndrome (PCOS) model group, and limonene treatment group. (A) Schematic diagram of animal experimental procedure. (B, C, D) Estrogenic cycle pattern and percentage of mice with normal estrus. P: Pre-ovulation; E: Ovulation period; M: Post-ovulation; D: Interovulatory period. (E) Morphology of mouse ovaries and follicles observed using HE staining; black arrows point to cystic follicles. (F) Mouse body weight measurement (n=6). (G) Comparison of serum testosterone levels in mice of different groups (n=6). (H, I) Quantitative analysis of LC3B, SQSTM1, and Beclin1 protein levels by Western blot (n=6). (J, K) Immunohistochemical analysis of SQSTM1 expression in the four groups (n=6). (L, M) Immunohistochemical analysis of LC3B expression in the four groups (n=6). Data are presented as mean ± standard deviation. One-way ANOVA. There was no significant difference in ns.

[0021] Figure 2(A) Chemical structure of limonin. (B) Viability of KGN cells cultured for 24 hours in DMEM / F12 medium containing 0.2% FBS with or without limonin (0-200 μM). (C) Viability of KGN cells cultured for 24 hours in DMEM / F12 medium containing 10% FBS with or without limonin (0-200 μM) (n=6). (D, E) Viability of quiescent KGN cells co-incubated with DHT (500 nM) and 40 μM or 80 μM limonin for 24 or 48 hours, and assessed by CCK-8 assay (n=6). (F) Effect of limonin on mitochondrial membrane potential of DHT-treated KGN cells measured by JC-1 fluorescent probe. (G) Quantitative data of red / green fluorescence intensity ratio in different treatment groups. (H, I) Autophagy was assessed using ImageJ software by quantitatively analyzing the colocalization of SQSTEM1 (green light) and LC3B (red light) (n=3). (J, K) Western blot and density analysis were performed on the expression of LC3B, SQSTEM1, and Beclin1 (n=3). Data are presented as mean ± standard deviation. One-way ANOVA was used. There was no significant difference in ns.

[0022] Figure 3ATG9B mediates the anti-autophagy effect of limonin. (A) Heatmap showing the expression of autophagy-related genes, oxidative stress molecules, and inflammatory cytokines in differentially expressed genes. (B) Quantitative analysis of ATG9B mRNA levels in women with polycystic ovary syndrome (PCOS) and healthy controls (n=6). (C, D) Quantitative analysis of ATG9B protein levels in women with PCOS and healthy controls (n=6). (E) 3D / 2D binding results of ATG9B to limonin. (F, G) Western blot and density analysis of ATG9B expression (n=3). (H, I) Immunofluorescence staining to show the effect of limonin on ATG9B expression in DHT-treated KGN cells (n=6). (J) Quantitative analysis of ATG9B mRNA levels in KGN cells infected with LV-Vector or LV-ATG9B (n=3). (K, L) Representative blot bands and relative expression levels of ATG9B in KGN cells infected with LV-Vector or LV-ATG9B were detected (n=3). (M, N) Western blot analysis was performed to detect the protein levels of LC3B, SQSTM1, and Beclin1 (n=3). (O, P) Quantitative analysis of the colocalization of SQSTEM1 (green light) and LC3B (red light) was performed using Image J software to assess autophagy (n=3). (Q) The effect of limonene on the mitochondrial membrane potential of DHT-treated KGN cells was measured using the JC-1 fluorescent probe. (R) Quantitative data of the red / green fluorescence intensity ratio in different treatment groups (n=3). (S, T) Western blot analysis was performed to detect the quantitative levels of LC3B, SQSTM1, and Beclin1 (n=3). Data are presented as mean ± standard deviation. Student's t-test was used. One-way ANOVA was used. There was no significant difference in ns.

[0023] Figure 4The interaction between ATG9B and AKT during the action of limonin on KGN cells. (A) 3D / 2D binding results of ATG9B and AKT. (B) Binding results of ATG9B and AKT at different angles. (C, D) Western blot and density analysis of the effect of limonin on the levels of ATG9B / AKT / mTOR pathway-related proteins in KGN cells (n=3). (E, F) Western blot and density analysis of the effect of limonin on the levels of ATG9B / AKT / mTOR pathway-related proteins in four groups of mice (n=6). (G, H) Immunohistochemical analysis of ATG9B expression in four groups (n=6). (I, J) Relative expression level of ATG9B in KGN cells transfected with NC siRNA or ATG9B siRNA (n=3). (K, L) Western blot analysis of phosphorylation activation levels of ATG9B, AKT, and mTOR in KGN cells transfected with NC siRNA or ATG9B siRNA after DHT treatment followed by 24-hour treatment with 80 μM limonene (n=3). (M, N) Relative expression level of AKT in KGN cells transfected with NC siRNA or AKT siRNA (n=3). (O, P) Western blot analysis of phosphorylation activation levels of ATG9B and AKT in KGN cells after DHT treatment followed by transfection with NC siRNA or AKT siRNA and then 24-hour treatment with 80 μM limonene (n=3). Data are presented as mean ± standard deviation. One-way ANOVA was used. There was no significant difference in ns.

[0024] Figure 5: The effect of limonin on improving glucose tolerance and anti-autophagy in PCOS rats. (A, B) Western blot and density analysis of the effect of limonin on the level of SIRT1 / AMPK pathway-related proteins in rat ovaries (n=6). (C) Line graph of blood glucose level changes in rats at different time points in the GTT experiment. (D) Line graph of blood glucose level changes in rats at different time points in the ITT experiment. (E) Fasting blood glucose test results in four groups of rats. (F) Fasting insulin test results in four groups of rats. (G) HOMA-IR calculation results in four groups of rats. (H) Effects of limonin, metformin and their mixed tablets on the autophagy phenotype in PCOS rats (n=6). CON: Control group; PCOS: Polycystic ovary syndrome model group; PCOS + Liensinine Tablets: Polycystic ovary syndrome model group + Liensinine tablets treatment group; PCOS + Metformin: Polycystic ovary syndrome model group + Metformin treatment group; PCOS + Liensinine and Metformin Compound Capsules: Polycystic ovary syndrome model group + Liensinine and Metformin compound capsules treatment group. Data are presented as mean ± standard deviation. One-way ANOVA was used. There was no significant difference in ns. Detailed Implementation

[0025] To better understand the purpose, technical solution, and advantages of this application, the present invention will be further described and illustrated below in conjunction with embodiments and accompanying drawings.

[0026] Experimental Example 1 The procedure for establishing PCOS modeling and administering lotusine to 3-week-old female C57BL / 6J mice weighing 9-16g is as follows: Figure 1 As shown in Figure A, 24 experimental animals were randomly divided into 4 groups (n=6 per group): control group, dehydroepiandrosterone (DHEA) model group (PCOS), low-concentration administration group (PLL), and high-concentration administration group (PHL). Mice in the DHEA model group, low-concentration administration group, and high-concentration administration group received subcutaneous injections of DHEA (dosage: 6 mg / 100 g body weight, dissolved in corn oil, product code: #HY-14650MedChemExpress); mice in the control group received an equal volume of corn oil; mice in the low-concentration administration group and high-concentration administration group were simultaneously treated with liensinsine (HY-N0484 MedChemExpress) by gavage (dosage: 40 mg / kg / d and 80 mg / kg / d, respectively, dissolved in 0.9% NaCl solution); mice in the control group and DHEA model group were treated with an equal volume of 0.9% NaCl solution by gavage. All mice were treated continuously for 21 days according to the above protocol. On day 22, mice were sacrificed, and samples were collected for the following tests.

[0027] Ovarian samples were immediately washed with pre-cooled PBS after collection and fixed in 4% paraformaldehyde. After fixation at 4°C for 12 hours, they were embedded in paraffin and 5 μm thick serial sections were prepared. Hematoxylin-eosin (H&E) staining was performed on 6 ovarian samples from each group, and 3 equally spaced sections (20 μm spacing) from each sample were selected for staining analysis. Serum samples from mice were collected to measure serum sex hormone levels. Proteins were extracted from other ovarian tissues to detect the target sites of limonin and autophagy-related indicators in the ovary.

[0028] Monitoring of estrous cycles starting from day 10 revealed that DHEA-induced polycystic ovary syndrome (PCOS) mice exhibited estrous cycle disturbances, characterized by prolonged interestrous periods, while control mice showed normal estrous cycles. Treatment with limonene significantly improved this disturbance. Figure 1 BD). Histological examination of ovarian tissue (HE staining) revealed that PCOS mice exhibited cortical thickening, decreased number of corpora lutea, increased number of cystic follicles, and a significant reduction in primordial and primary follicles. These pathological changes were partially reversed under low-dose liansinine treatment and almost returned to normal under high-dose liansinine treatment. Figure 1 E). Furthermore, DHEA-induced weight gain in mice was significant, while high-dose treatment with limonene inhibited this increase (E). Figure 1 F. Serum androgen (testosterone) levels were significantly elevated in the PCOS group, and treatment with limonene effectively reduced these levels (F). Figure 1 G). In summary, these results indicate that limonene can alleviate estrous cycle disorders, hormonal imbalances, and poor follicular development in PCOS mice. To explore its underlying mechanism, the expression levels of ovarian autophagy markers (SQSTM1, Beclin1, and LC3B) were assessed by immunohistochemistry and Western blotting. Polycystic ovary syndrome (PCOS) mice showed decreased expression of SQSTM1 (1:5000, Proteintech, 18420-1-AP), an increased LC3-II / LC3-I (1:2000, Abcam, ab192890) ratio, and increased Beclin1 (1:1000, Proteintech, 11306-1-AP) levels, indicating excessive activation of autophagy. Limonene treatment restored the expression of these proteins to normal levels, with high-dose limonene showing more significant effects on SQSTM1 and Beclin1. Figure 1 H, I). Immunohistochemical results were consistent with Western blot data (H, I). Figure 1 These findings suggest that limonin can alleviate abnormal autophagy activation in the ovaries of PCOS mice.

[0029] Experiment Example 2 To assess the effects of limonin on KGN cells and determine its optimal concentration, cytotoxicity was evaluated using a CCK-8 assay. KGN cells were cultured at 3 × 10⁶ cells per well. 3 KGN cells were seeded at a density of 10 μL at the bottom of 96-well plates and treated with different concentrations of lysimachia alkaloids for 24 hours. The cytotoxicity of lysimachia alkaloids was assessed using the CCK-8 assay: 10 µL of CCK-8 reagent was added to each well, and after culturing for 1.5 hours, absorbance was measured at 450 nm using a microplate reader (PerkinElmer, USA). KGN cells were placed in culture medium (DMEM / F12 medium containing 0.2% / 10% fetal bovine serum) containing different concentrations of lysimachia alkaloids. Low concentrations of lysimachia alkaloids showed minimal cytotoxicity, but cell viability decreased at 160 μM. Figure 2 (B, C). Therefore, 80 μM was set as the safety upper limit, and 40 μM (a trend with potential benefits) was selected for further research.

[0030] A dihydrotestosterone (DHT, 500 nM)-induced pathological model of human ovarian granulosa cells (KGNs) was established to simulate the hyperandrogen state in the ovarian microenvironment of PCOS patients. To investigate the effects of limonene on DHT-treated KGNs and the therapeutic effect of limonene, four groups were set up: a control group, a DHT group, a DHT + 40 µM limonene group, and a DHT + 80 µM limonene group. DHT and limonene were diluted once with dimethyl sulfoxide (DMSO) and then diluted a second time with DMEM / F12 medium without fetal bovine serum to achieve the final drug concentrations. Cell viability was assessed using a CCK-8 assay 24 or 48 hours after treatment with DHT alone or in combination with limonene. In the androgen-induced polycystic ovary syndrome model, 40 / 80 µM limonene reduced cell damage at both 24 and 48 hours, with the 80 µM limonene showing stronger protective effects at 24 hours. Figure 2Therefore, 24 hours was used as the drug action time in all the following experiments. The mechanism of action was analyzed through the following systematic experiments: Mitochondrial membrane potential was quantitatively analyzed using flow cytometry. Mitochondrial membrane potential was detected using the JC-1 fluorescent probe: Cells treated with limonene were resuspended in buffer and co-incubated with the JC-1 probe in the dark. Fluorescence intensity changes were analyzed by flow cytometry. The ratio of red fluorescence (high membrane potential) of J-mers formed under normal mitochondrial membrane potential conditions to green fluorescence (low membrane potential) of monomers formed when the membrane potential decreased was used to clarify the antagonistic effect of limonene on DHT-induced mitochondrial membrane potential decrease. Autophagy level assessment focused on key proteins. Western blotting experiments were used to detect the protein levels of SQSTEM1, Beclin1, and LC3B. Limonene increased the protein level of SQSTEM1 and decreased the levels of Beclin1 and LC3II / I, revealing the antagonistic ability of limonene against abnormal autophagy. Dihydrotestosterone induces mitochondrial depolarization (decreased red / green fluorescence ratio), while limonene can alleviate this phenomenon. Figure 2 F, G). Immunofluorescence co-staining showed that limonin increased the fluorescence intensity of SQSTM1 and decreased the fluorescence intensity of LC3B (F, G). Figure 2 H, I), which is consistent with the results of Western blotting (H, I), which is consistent with the results of Western blotting. Figure 2 J and K indicate that autophagy activity is regulated.

[0031] Experimental Example 3 This study investigated the molecular mechanism of limonin in treating PCOS using gene silencing and pharmacological blocking. Small interfering RNA (sRNA) was designed to target and knock down candidate signaling molecules, blocking the interaction between limonin and key targets, revealing the effects of limonin treatment on PCOS apoptosis and oxidative stress phenotypes in cell lines. Simultaneously, siRNA and molecular inhibitors were used to intervene at the limonin target sites to elucidate the specific molecular mechanism by which limonin treats the PCOS phenotype.

[0032] After KGN cells were cultured to 70%–90% confluence, ATG9B siRNA, AKT siRNA, and negative control siRNA were transfected into them using lipo2000 (Thermo Fisher Scientific, catalog number: 11668030) to silence the corresponding genes. The sequences of each siRNA are shown in the table below.

[0033]

[0034] Cells were collected for Western blot analysis 48 hours after transfection. In the ATG9B overexpression experiment, KGN cells were transduced using a lentiviral vector carrying the ATG9B gene (LV-ATG9B) or an empty vector (LV-NC) control (Jintuosi Gene). After infection, cells were cultured for another 72 hours and selected with puromycin to establish a KGN cell line stably expressing the ATG9B gene.

[0035] KGN cells were co-stained with SQSTEM1 and LC3B, and stained with ATG9B alone. After fixation with 4% paraformaldehyde, KGN cells were blocked with 10% goat serum for 30 minutes to block non-specific binding. The samples were then incubated overnight at 4°C with primary antibodies SQSTEM1 (1:50) and LC3 (1:50). After thorough washing with PBST, the cells were incubated for 1 hour at room temperature in the dark with secondary antibodies—Cy3-labeled goat anti-rabbit IgG and FITC-labeled goat anti-mouse IgG. Cell nuclei were stained with DAPI, and images were acquired using an Olympus BX6 fluorescence microscope.

[0036] To investigate autophagy-related genes in polycystic ovary syndrome (PCOS), we analyzed the transcriptome of granulosa cells from the GEO dataset (GSE216609) and identified differentially expressed autophagy-related genes in PCOS patients. Figure 3 A). ATG9B was selected for further study, and its mRNA and protein levels in PCOS granulosa cells were found to be significantly upregulated compared to healthy control women. Figure 3 BD). Molecular docking analysis showed that limonin and ATG9B have a strong binding affinity (BD). Figure 3 E). In dihydrotestosterone-induced KGN cells, treatment with limonene reduced ATG9B expression (E). Figure 3 FI). Functional studies showed that ATG9B overexpression altered autophagy, as evidenced by changes in the LC3-II / LC3-I ratio. Figure 3 JN). Furthermore, ATG9B overexpression attenuated the therapeutic effect of limonene (80 μM) on autophagy recovery, including partial failure to restore mitochondrial membrane potential under high-dose limonene treatment and altered the expression of SQSTM1 and LC3B (JN). Figure 3 These results suggest that ATG9B may mediate the therapeutic effect of limonin on abnormal autophagy in PCOS.

[0037] To investigate the molecular pathway by which lotus seedine regulates ATG9B, we performed molecular docking analysis. The results showed a strong binding interaction between ATG9B and AKT (binding free energy: -39.2 kcal / mol; docking score: -279.74; confidence score: 0.9305). Figure 4 A, 4B). Given that mTOR is a classic downstream effector of AKT-regulated autophagy, we examined the expression and phosphorylation levels of related proteins in the ovarian tissues of mice in different groups. Treatment with limonene significantly promoted the phosphorylation of AKT and mTOR in a dose-dependent manner, while simultaneously reducing ATG9B expression (…). Figure 4EH). Consistent results were obtained in KGN cells treated with DHT alone or in combination with limonene (EH). Figure 4 C, 4D), suggesting that lotusine may inhibit abnormal autophagy in polycystic ovary syndrome through the ATG9B / AKT / mTOR pathway.

[0038] To verify the functional relationship between ATG9B and AKT, we interfered with gene expression in KGN cells by transfecting ATG9B siRNA. In the test sequence, the knockdown efficiency of siRNA-1 exceeded 50%, and therefore it was selected for subsequent experiments. Figure 4 I, J). Under co-treatment with dihydrotestosterone and limonene (80 μM), knockdown of ATG9B enhanced the promoting effect of limonene on AKT phosphorylation. Figure 4 K, L). Conversely, knocking down AKT did not alter the inhibitory effect of limonene on ATG9B expression (K, L). Figure 4 These results suggest that ATG9B may act as an upstream target of AKT in the mechanism of pursinolide treatment for polycystic ovary syndrome, co-regulating autophagy activity.

[0039] Experiment Example 4 To investigate the target mechanism of limonin in a polycystic ovary syndrome (PCOS) animal model, we analyzed ovarian tissue from PCOS rats and examined the expression of key proteins in autophagy-related pathways. The results showed that limonin treatment significantly upregulated SIRT1 expression in ovarian tissue and promoted AMPK phosphorylation. Figure 5 (AB). These results suggest that limonene may exert its therapeutic effect by regulating the SIRT1 / AMPK signaling axis.

[0040] To investigate how lotus seed alkaloids improve insulin resistance in PCOS model rats, we conducted glucose tolerance (GTT) and insulin tolerance (ITT) tests on four groups of rats. The results of glucose level comparisons at each specified time point for each group are shown below. Figure 5 On the day of mouse sacrifice, serum was collected, and fasting blood glucose and fasting insulin levels were measured. The results showed that treatment with limonene significantly reduced blood glucose levels in rats, while also decreasing serum insulin levels and improving glucose tolerance impairment observed during DHEA modeling. Figure 5 CG).

[0041] To elucidate the effects of limonene on the SIRT1 / AMPK axis and its combination with other drugs, we treated PCOS model rats by gavage with limonene (60 mg / d / kg), metformin (200 mg / d / kg), and limonene-metformin (dose as before). Compared with the model group, except for the metformin monotherapy group, all other treatment groups showed varying degrees of improvement in the excessive autophagy state of ovarian tissue. Figure 5H). It is worth noting that the combined use of limonin and metformin showed a synergistic enhancing effect in anti-autophagy, and its effect was significantly better than that of the single-drug group (H). Figure 5 The above results indicate that lysimachia may exert its effects through SIRT1 / AMPK. Furthermore, the combined use of lysimachia and metformin has a synergistic effect in regulating and inhibiting excessive autophagy in ovarian tissue, jointly contributing to the improvement of the PCOS phenotype.

[0042] Example 1: Preparation of lotus seed alkaloid tablets Prescription: 20g of lotus seed alkaloid, 60g of microcrystalline cellulose (filler), 15g of lactose (filler), 4g of croscarmellose sodium (disintegrant), and 1g of magnesium stearate (lubricant), to make 1000 tablets.

[0043] Preparation: Mix lotusine with microcrystalline cellulose, lactose, and croscarmellose sodium in a mixer until thoroughly homogeneous. Add an appropriate amount of purified water as a binder to form a soft mass, which is then granulated through a 20-mesh sieve. Dry the wet granules at 55-60℃ until the moisture content meets the requirements (usually <3.0%), and then granulate using an 18-mesh sieve. Add magnesium stearate, mix thoroughly, and compress into tablets using a tableting machine to obtain oral tablets containing 20mg of lotusine per tablet.

[0044] Example 2: Preparation of compound capsules containing lotus seed alkaloid and metformin Prescription: 50g of lotus seed alkaloid, 500g of metformin hydrochloride, 150g of pregelatinized starch (filler and disintegrant), and 5g of magnesium stearate (lubricant), to make 1000 capsules.

[0045] Preparation: Separately pass lotusine, metformin hydrochloride, and pregelatinized starch through an 80-mesh sieve. Mix thoroughly in a three-dimensional motion mixer using an equal-volume incremental mixing method until homogeneous. Add magnesium stearate and continue mixing for 5-10 minutes. Fill the mixed powder into No. 1 gelatin empty capsules using a fully automated capsule filling machine to obtain a compound capsule containing 50mg of lotusine and 500mg of metformin hydrochloride per capsule. This formulation combines AMPK pathway regulation with classic insulin sensitization, producing a synergistic therapeutic effect.

[0046] The above embodiments are only used to help illustrate the present invention. The implementation of the present invention is not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered as equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. The use of lotusine or its analogues / derivatives or pharmaceutically acceptable salts thereof in the preparation of drugs or reagents, characterized in that: The application includes one or more of the following: Application in the preparation of drugs for the prevention and / or treatment of polycystic ovary syndrome; Its application in the preparation of drugs for treating hyperandrogenemia; Application in the preparation of drugs that promote normal follicle development; Its application in the preparation of drugs that promote the recovery of ovarian ovulation; Its application in the preparation of drugs that improve insulin resistance; Its application in the preparation of drugs for treating hyperinsulinemia; Application in the preparation of reagents that inhibit abnormal autophagy; Application in the preparation of ATG9B inhibitors; Application in the preparation of AMPK / SIRT1 signaling pathway activators.

2. The use of compositions comprising limonin or its analogues / derivatives or pharmaceutically acceptable salts thereof in the preparation of medicaments for the prevention and / or treatment of polycystic ovary syndrome.

3. The application according to claim 1 or 2, characterized in that: The aforementioned lotusine analogues / derivatives include isolivineine, methyl lotusine, pro-lotus leaf alkaloid, and lotusine quaternary ammonium alkaloid.

4. The application according to claim 1 or 2, characterized in that: The pharmaceutically acceptable salts of the lotusine or its analogues / derivatives include perchlorate, hydrochloride, phosphate, methanesulfonate, and citrate.

5. The application according to claim 1 or 2, characterized in that: The polycystic ovary syndrome mentioned is obese polycystic ovary syndrome.

6. The application according to claim 2, characterized in that: The composition also contains other active ingredients that have therapeutic effects on polycystic ovary syndrome.

7. The application according to claim 6, characterized in that: The active ingredient that has the effect of treating polycystic ovary syndrome is selected from one or more of insulin sensitizers and ovulation-inducing drugs.

8. A pharmaceutical composition for treating polycystic ovary syndrome, characterized in that: It contains lotusine or its analogues / derivatives or pharmaceutically acceptable salts thereof.

9. The pharmaceutical composition according to claim 8, characterized in that: It also contains one or more pharmaceutically acceptable carriers.

10. The pharmaceutical composition according to claim 8, characterized in that: It also contains at least one other active ingredient that has therapeutic effects on polycystic ovary syndrome, wherein the active ingredient that has therapeutic effects on polycystic ovary syndrome is selected from one or more insulin sensitizers and ovulation-inducing drugs.

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