Preparation method of transdermal hydrogel for improving polycystic ovarian syndrome

Abstract

The invention discloses a preparation method of transdermal hydrogel for improving polycystic ovarian syndrome, and relates to the technical field of pharmaceutical preparations and delivery systems. Comprising the following steps: preparing a traditional Chinese medicine compound kidney-tonifying and brewing-regulating decoction which consists of astragalus membranaceus, rhizoma atractylodis, poria cocos, herba epimedii, eucommia ulmoides, semen cuscutae, radix curcumae, rhizoma cyperi, tangerine seeds, curcuma zedoary, safflower carthamus, ligusticum wallichii, rhizoma anemarrhenae, spina gleditsiae and fried mustard seeds; dissolving chitosan in the decoction to prepare a chitosan solution, and adjusting the pH value; dissolving sodium alginate in the decoction to prepare a sodium alginate solution; mixing the chitosan solution with a sodium alginate solution, continuously stirring, and forming hydrogel through electrostatic crosslinking; adjusting the pH value of the hydrogel, and storing in a dark place. The hydrogel prepared by the invention has excellent physical and chemical characteristics, a scanning electron microscope shows that the drug-loaded hydrogel has a structure with smaller pore diameter and more compact and ordered structure, and the structure is beneficial to improving the water absorbability and the drug loading rate.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical preparations and delivery systems, in particular to a manufacturing method of a transdermal hydrogel for improving polycystic ovary syndrome. BACKGROUND

[0002] Polycystic ovary syndrome (PCOS) is a gynecological endocrine disease characterized by hyperandrogenism, anovulation and polycystic-like changes in the ovary, which leads to reproductive dysfunction in women of childbearing age. The global prevalence of PCOS among women of childbearing age is 5-20%, among which 74% of affected women suffer from infertility, 54% from amenorrhea, and 41% from obesity, and are prone to a series of diseases such as insulin resistance (IR), diabetes, cardiovascular disease and psychological disorders.

[0003] However, the etiology of PCOS is complex, and there is currently no ideal PCOS drug treatment that can completely reverse its underlying hormonal disorders and treat all clinical manifestations. Western medicine treatments (such as oral contraceptives, hormone preparations, and surgery) have limitations, serious adverse reactions (such as oral contraceptive-related thrombosis risk), and recurrence of symptoms after discontinuation. In recent years, the multi-component and multi-target characteristics of traditional Chinese medicine have achieved good results in the clinical treatment of PCOS. Traditional Chinese medicine is the treasure of the Chinese nation, emphasizing the concept of holism. In traditional Chinese medicine theory, kidney deficiency is the root, and phlegm-dampness, liver stagnation and blood stasis are the main pathogenesis of PCOS. The kidney governs reproduction, and kidney deficiency is considered to be the core pathogenesis of PCOS. Spleen deficiency and phlegm-dampness can cause obesity, insulin resistance and metabolic disorders, further exacerbating the symptoms of PCOS. Liver stagnation and blood stasis are important pathological links of PCOS, and blood stasis can lead to changes in ovarian morphology (such as polycystic-like changes), further affecting reproductive function.

[0004] In traditional oral administration, traditional Chinese medicine decoction pieces need to go through a complex process of soaking and decoction, which is time-consuming and tedious, and patients have poor compliance and must be absorbed through the intestinal tract and metabolized by the liver, which is affected by the first-pass effect, resulting in reduced bioavailability. As a biocompatible and controllable material, hydrogel has become a new type of drug carrier and delivery system in recent years and is widely used in transdermal drug delivery systems (TDDS). The combination of traditional Chinese medicine and hydrogel effectively solves a series of problems faced by traditional Chinese medicine in the application of TTDS, such as low solubility, low absorption rate and instability. As a biocompatible material, hydrogel as an efficient drug delivery system has shown great potential in the treatment of PCOS, not only having excellent biocompatibility, but also effectively supporting the survival and function of stem cells.

[0005] Therefore, the present application aims to construct a transdermal drug delivery system based on hydrogel to explore the therapeutic effect and mechanism of polycystic ovary syndrome. SUMMARY

[0006] The purpose of this invention is to address the shortcomings of existing technologies by proposing a method for manufacturing a transdermal hydrogel that improves polycystic ovary syndrome.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A method for manufacturing a transdermal hydrogel for improving polycystic ovary syndrome includes the following steps: S1: Prepare a decoction of the traditional Chinese medicine compound Yishen Tiaochong Decoction, which consists of Astragalus membranaceus, Atractylodes lancea, Poria cocos, Epimedium brevicornu, Eucommia ulmoides, Cuscuta chinensis, Curcuma longa, Cyperus rotundus, Citrus reticulata seed, Curcuma zedoaria, Carthamus tinctorius, Ligusticum chuanxiong, Anemarrhena asphodeloides, Gleditsia sinensis thorns and stir-fried mustard seed; S2: Dissolve chitosan in the decoction to prepare a chitosan solution, and adjust the pH. S3: Dissolve sodium alginate in the decoction to prepare a sodium alginate solution; S4: Mix the chitosan solution with the sodium alginate solution and stir continuously to form a hydrogel through electrostatic cross-linking; S5: Adjust the pH of the hydrogel and store it away from light.

[0008] Preferred: The composition of the traditional Chinese medicine compound Yishen Tiaochong Decoction by weight is as follows: Astragalus membranaceus 15g, Atractylodes lancea 20g, Poria cocos 20g, Epimedium brevicornu 20g, Eucommia ulmoides 15g, Cuscuta chinensis 30g, Curcuma longa 15g, Cyperus rotundus 15g, Citrus reticulata seed 20g, Curcuma zedoaria 15g, Carthamus tinctorius 10g, Ligusticum chuanxiong 12g, Anemarrhena asphodeloides 20g, Gleditsia sinensis thorn 15g, and stir-fried mustard seed 10g.

[0009] Preferably, in step S2, the chitosan solution concentration is 2% w / v and the pH is adjusted to 5.0; in step S3, the sodium alginate solution concentration is 2% w / v.

[0010] Preferably: in step S4, the chitosan solution and sodium alginate solution are mixed at a volume ratio of 1:1 and stirred for 20 minutes; in step S5, the pH of the hydrogel is adjusted to 6.0 and stored at 4°C in the dark.

[0011] Preferably, the degree of deacetylation of the chitosan is not less than 85%.

[0012] Preferably, the concentration of the decoction of Yishen Tiaochong Decoction is 2.25g / ml of raw herbs.

[0013] Preferably, the hydrogel achieves a cumulative drug release rate of over 90% within 6 hours in a PBS release medium at 37°C.

[0014] A transdermal hydrogel, prepared by the above-described manufacturing method, wherein the hydrogel is loaded with active ingredients of Yishen Tiaochong Decoction, for improving metabolic disorders in polycystic ovary syndrome.

[0015] Preferably, the hydrogel has a pore size range of 10-50 μm, which allows it to adhere to the skin surface.

[0016] Preferably, the metabolic disorder includes at least one of insulin resistance, lipid metabolism abnormalities, and sex hormone level disorders.

[0017] The beneficial effects of this invention are as follows: 1. The hydrogel prepared by this invention has excellent physicochemical properties. Scanning electron microscopy shows that the drug-loaded hydrogel exhibits a smaller pore size and a more dense and ordered structure, which is beneficial to improving water absorption and drug loading rate.

[0018] 2. The hydrogel of this invention exhibits excellent adhesion properties, and can firmly adhere to various surfaces such as skin to ensure the continuous action of the drug; in vitro release experiments have confirmed that the hydrogel achieves almost complete drug release within 6 hours, and has good drug diffusion ability.

[0019] 3. The hydrogel of the present invention exhibits excellent biocompatibility; through CCK-8 cell proliferation assay and live / dead staining verification, both the blank hydrogel and the drug-loaded hydrogel can significantly promote the proliferation of HACAT and KGN cells, with high cell survival rate and very few dead cells, proving its safety and reliability.

[0020] 4. Animal experiments of this invention show that YSTC-HY hydrogel can significantly reverse the weight gain of PCOS model rats (P<0.001), reduce abnormal ovarian index, and improve glucose and lipid metabolism disorders; specifically, it significantly reduces fasting insulin, HOMA-IR index, serum triglycerides, total cholesterol and low-density lipoprotein cholesterol levels.

[0021] 5. The hydrogel treatment of the present invention can effectively regulate sex hormone levels, significantly reduce serum testosterone, luteinizing hormone and LH / FSH ratio, while increasing estradiol, follicle-stimulating hormone and progesterone levels, and restore endocrine balance; the hydrogel treatment can restore regular estrous cycles in rats, significantly reduce the number of cystic follicles in the ovary, increase the corpus luteum and developing follicles, and significantly alleviate the degree of ovarian fibrosis. Attached Figure Description

[0022] Figure 1 The structure and characterization of the hydrogel of the present invention are shown in the figures (wherein: (A) SEM image of the hydrogel; (B) pore diameter statistics; (C) hydrogel adhesion properties; (D) in vitro release experiment (VIRT)). Figure 2This is a schematic diagram illustrating the toxicity and biocompatibility of the hydrogel of the present invention (wherein: (A) CCK-8 values ​​of HACAT and KGN cells cultured in blank hydrogel and YSCT-HY at 24h and 48h; (B) (Calcein-AM / PI) live and dead staining of HACAT and KGN cells; (C) Quantitative analysis of live and dead staining; (n=3)). Figure 3 A schematic diagram of the chemical composition of YSTC-HY identified by UPLC-MS in this invention (wherein: (A) representative total ion chromatograms (TIC) in positive ion and (B) negative ion modes); Figure 4 This diagram illustrates the effects of YSCT-HY on body weight and ovarian index in PCOS rats (wherein: (A) rat body weight change curve during modeling (day 1 to day 21); (B) rat body weight change curve during treatment (day 22 to day 42); (C) rat body weight at the end of modeling; (D) rat body weight at the end of treatment; (E) wet weight of rat ovaries; (F) food intake of rats during modeling; (G) food intake of rats at the end of treatment; (H) rat ovarian index; (n=7) all values ​​represent (Mean±SEM); ∗P<0.05, ∗P<0.01, ***P<0.001). Figure 5 This is a schematic diagram illustrating the effect of YSCT-HY of the present invention on the estrous cycle of PCOS rats (wherein: (A) Giemsa staining of vaginal cell smears from rats at different time points in each group; (B) line graph of estrous cycle over 7 consecutive days in different treatment groups; (C) statistics on the regularity rate of estrous cycles (red arrow: nucleated epithelial cells, green arrow: keratinized epithelial cells, yellow arrow: leukocytes); n=7, scale bar=200μm); Figure 6 This is a schematic diagram illustrating the effect of YSCT-HY on glucose and lipid metabolism in PCOS rats (wherein: (A) serum fasting insulin; (B) serum fasting blood glucose; (C) HOMA-IR; (D) serum triglycerides; (E) serum total cholesterol; (F) serum high-density lipoprotein cholesterol; (G) serum low-density lipoprotein cholesterol; (n=5) all values ​​represent (Mean±SEM); ∗P<0.05, ∗P<0.01, ***P<0.001); Figure 7 This is a schematic diagram illustrating the effect of YSCT-HY of the present invention on serum hormone levels in PCOS rats (wherein: (A) testosterone; (B) estradiol; (C) progesterone; (D) luteinizing hormone (LH); (E) follicle-stimulating hormone (FSH); (F) LH / FSH; (n=5) all values ​​represent (Mean±SEM); ∗P<0.05, ∗P<0.01, ***P<0.001); Figure 8This is a schematic diagram illustrating the effect of YSCT-HY treatment of the present invention on the pathological morphology of the ovaries in PCOS rats (wherein: (A) appearance; (B) HE staining; (C) ovarian vesicle count; (D) Masson staining; (E) quantitative analysis of fibrosis area by Masson staining; scale bar = 200 μm; n = 5; all values ​​are expressed as (Mean ± SEM); ∗P<0.05, ∗P<0.01, ***P<0.001). Figure 9 This is an information diagram illustrating the identification of the main compounds in YSTC-HY by UPLC / MS according to the present invention. Detailed Implementation

[0023] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0024] Example 1: A study on the improvement of metabolic disorders in polycystic ovary syndrome by a transdermal hydrogel 1. Materials and Methods 1.1. Preparation and Identification of Traditional Chinese Medicine Compound Prescriptions: The Kidney-Nourishing and Cholecystitis-Regulating Decoction consists of 15 Chinese herbs: Astragalus membranaceus (15g), Atractylodes lancea (20g), Poria cocos (20g), Epimedium brevicornu (20g), Eucommia ulmoides (15g), Cuscuta chinensis (30g), Curcuma longa (15g), Cyperus rotundus (15g), Citrus reticulata (20g), Curcuma zedoaria (15g), Carthamus tinctorius (10g), Ligusticum chuanxiong (12g), Anemarrhena asphodeloides (20g), Gleditsia sinensis (15g), and stir-fried Sinapis alba (10g). These herbs were purchased from the University City Hospital Affiliated to Chongqing Medical University, and all ingredients meet the standards of the 2020 edition of the Pharmacopoeia of the People's Republic of China. All herbs in the prescription are soaked in pure water and decocted twice in an automatic Chinese medicine decoction pot. The two decoctions are combined, and the residue is filtered through gauze. The drug dosage is selected based on the optimal dosage determined in previous clinical trials by the research team. This project has received support from the Chongqing Municipal Health Commission and is currently in the clinical trial stage. The daily dose of Yishen Tiaochong Decoction for adults (70kg) is 252g of raw herbs. Based on the equivalent dose conversion of body surface area for adults and rats, the drug was concentrated to a solution of 2.25g / ml of raw herbs and stored in a -20 degree refrigerator for subsequent experiments.

[0025] 1.2. Hydrogel preparation and performance testing: The Yishen Tiaochong Decoction-loaded hydrogel (YSTC-HY) was prepared via a polyelectrolyte complex reaction between chitosan (CS, deacetylation degree ≥85%) and sodium alginate (SA). In short, a 2% (w / v) chitosan solution was first prepared by dissolving chitosan in a filtered concentrated Yishen Tiaochong Decoction decoction, and the pH was adjusted to approximately 5.0 with glacial acetic acid. Similarly, a 2% (w / v) sodium alginate solution was prepared using the same Yishen Tiaochong Decoction decoction as a solvent. The two solutions were mixed at a 1:1 volume ratio under continuous mechanical stirring for 20 minutes, forming a homogeneous hydrogel through electrostatic cross-linking. The final hydrogel had a polymer concentration of 2% (w / v), a pH adjusted to 6.0 for skin compatibility, and was stored in the dark at 4°C until further use. For the blank control hydrogel, deionized water was used instead of the Yishen Tiaochong Decoction decoction, with all other procedures remaining the same. The microstructure of the hydrogel was characterized by scanning electron microscopy (SEM; Hitachi S-650, Tokyo, Japan), and its adhesion properties were evaluated.

[0026] 1.3. Drug release in vitro In vitro drug release studies were conducted using a TD-12ATPLUS transdermal diffusion instrument (Shenzhen Huarong Analytical Instruments Co., Ltd.). YSTC-HY was placed on an artificial membrane and fixed in the test area of ​​a Franz diffusion cell. PBS was used as the release medium, and the temperature was set at 37°C to simulate body temperature. At different time points, 10 mL samples were collected, and the same volume of fresh PBS was immediately added to maintain a constant total volume. LC-MS was used to analyze the samples at each time point to determine the drug concentration, and the drug release amount was calculated using a standard curve.

[0027] 1.4. LC-MS Identification of the Chemical Composition of YSTC Hydrogel LC-MS / MS analysis was performed using a UHPLC-QExactive HF-X system (Thermo Fisher Scientific, USA) equipped with an HSST3 column (100 mm × 2.1 mm, 1.8 μm). Samples were separated using solvent A (95% water, 5% acetonitrile, 0.1% formic acid) and solvent B (47.5% acetonitrile, 47.5% isopropanol, 5% water, 0.1% formic acid) at a flow rate of 0.40 mL / min at 40 °C. Mass spectra were acquired in both positive and negative ion modes, with m / z ranging from 70 to 1050. Key parameters included a sheath gas flow rate of 50 psi, an auxiliary gas flow rate of 13 psi, spray voltages of +3500 V and −3500 V, and an ion transfer tube temperature of 325 °C. Step collision energies of 20, 40, and 60 V were used, with resolutions of MS 160,000 and MS 27,500, respectively, in DDA mode.

[0028] Metabolites were annotated using the ProgenesisQI, HMDB, and Metlin databases, as well as Majorbio's internal databases. Data processing and statistical analysis were performed on the MajorbioCloud platform (cloud.majorbio.com).

[0029] 1.5. Cell Culture HATAC cells (TCH-C388) were purchased from Haixing Biotechnology Co., Ltd., and KGN cells (LC-0603) were purchased from Pronosei Biotechnology Co., Ltd. The cells were cultured in DMEM / F2 medium (Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin and streptomycin (Gibco, USA), and the cells were cultured under standard conditions of 37°C, 5% CO2 and 95% humidity.

[0030] 1.6. Cytotoxicity and Biocompatibility Cells were digested with trypsin-EDTA at approximately 80% confluence and seeded into 96-well plates (1 × 10^3 cells / well). When cell confluence reached 50–60%, cells were assigned to control, blank hydrogel, or YSTC-HY hydrogel groups, treated with standard culture medium, blank hydrogel extract, or YSTC-HY extract, respectively. Cell viability was assessed at 24 and 48 hours using a CCK-8 assay kit, and cytotoxicity and proliferation were assessed by measuring absorbance at 450 nm.

[0031] For live / dead staining, cells were seeded into 24-well plates (1 × 10^5 cells / well). When cell confluence reached 50-60%, the same three treatments were applied. After 24 hours, cells were washed with PBS, stained with Calcein-AM (2 μM) and PI (8 μM) at 37°C for 20 minutes, washed again, and imaged under a fluorescence microscope.

[0032] 1.7. Animal Model Establishment and Treatment Thirty 6-week-old female Sprague-Dawley rats (initial weight: 160-200 g) were obtained from the Experimental Animal Center of Chongqing Medical University. The animals were housed in a controlled environment (temperature 21±1℃, relative humidity 55±5%) with a 12-hour light / dark cycle (light from 08:00 to 20:00, darkness from 20:00 to 08:00). Food and water were provided without restriction. After a 3-day acclimatization period in a specific pathogen-free (SPF) animal facility, the formal experiments began. All experimental procedures were approved by the Animal Ethics Committee of Chongqing Medical University (IACUC-CQMU-2024-10066). The rats were randomly assigned to two groups: a control group (n=10) and a model group (n=20). The control group rats were administered 0.5% sodium carboxymethyl cellulose (CMC, 10 mL / kg / day) orally by gavage along with a standard diet. The model group rats were given letrozole suspension (Jiangsu Hengrui, 1 mg / kg / day) orally by gavage and combined with a high-fat diet (45% of the energy came from fat, Jiangsu Xietong Biotechnology, batch number 20240612F) for 21 consecutive days to induce a polycystic ovary syndrome (PCOS) model (Zuo et al., 2023).

[0033] At the end of the modeling process, blood samples were collected from the orbital venous plexus to determine serum testosterone (T) and estradiol (E2) levels. In addition, three rats from each group (control group and model group) were randomly selected for ovarian histopathological examination using hematoxylin-eosin (H&E) staining. Significant differences in hormone levels and ovarian morphology between the two groups confirmed the successful establishment of the model.

[0034] After successfully establishing the PCOS model, PCOS-induced rats were randomly divided into two groups: a model group (n=7) receiving no further treatment, and a YSTC-HY treatment group (n=7) receiving topical application of YSTC-HY hydrogel to the ovarian projection area daily. The control group (n=7) remained untreated during this period. The treatment lasted for 21 days.

[0035] 1.8. Evaluation of therapeutic efficacy indicators 1.8.1 Body weight, food intake, and ovarian index of rats During treatment, rat body weight and food intake were recorded every three days. On the day before the end of the experiment, rats were fasted for 12 hours but provided with free access to water. Subsequently, blood samples were collected from the orbital venous plexus under 3% isophorene inhalation anesthesia. Samples were allowed to stand at room temperature for 30 minutes, followed by centrifugation at 3000×g for 15 minutes at 4°C to obtain serum, which was stored at −80°C until analysis. After isophorene overdose anesthesia and cervical dislocation euthanasia, bilateral ovaries were collected, rinsed with physiological saline, air-dried, and weighed. The ovarian index (%) was calculated as follows: Ovarian index (%) = bilateral ovarian weight (mg) / body weight (g) × 100%. One ovary was fixed in 4% paraformaldehyde for morphological analysis, while the other ovary was rapidly frozen in liquid nitrogen and stored at −80°C for subsequent experiments.

[0036] 1.8.2. Vaginal smear estrous cycle monitoring Starting on day 15 of modeling and treatment, vaginal cell smears were collected from rats every morning at 9:00 AM to monitor the estrous cycle. Vaginal cells were collected by irrigation with physiological saline and then stained with Giemsa (Beyotime, C0133). Changes in vaginal epithelial cells were observed under a microscope (Olympus, Tokyo, Japan) to determine the estrous cycle, which was divided into proestrus, estrus, metestrus, and diaestrus based on cytological characteristics.

[0037] 1.8.3. Serum Biochemical Indicators and Insulin Resistance Measurement Serum triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and fasting blood glucose (FBG) levels in rats were measured using a kit (Nanjing Jiancheng Biotechnology Institute, China). Fasting insulin levels (FINS) and insulin resistance levels (HOMA-IR) in rats were detected using an enzyme-linked immunosorbent assay (ELISA) kit (Quanzhou Jiubang Biotechnology Co., Ltd.). The results were calculated using the following formula: [Fasting insulin (mU / L) × Fasting blood glucose (mmol / L)] / 22.5.

[0038] 1.8.4. Enzyme-linked immunosorbent assay (ELISA) for serum sex hormone levels The levels of sex hormones in rat serum, including testosterone (T), estradiol (E2), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and progesterone (PROG), were detected using an enzyme-linked immunosorbent assay kit (Quanzhou Jiubang Biotechnology Co., Ltd.), and the LH / FSH ratio was calculated.

[0039] 1.8.5. Ovarian Morphological Analysis Ovarian tissue was fixed in 4% paraformaldehyde for 24 hours, then dehydrated using a gradient of alcohols, cleared in xylene, embedded in paraffin, and sectioned into 5 μm thick sections using a manual rotary microtome (Leica RM2235, Wetzlar, Germany). After drying, the sections were dewaxed in xylene, rehydrated with a gradient of alcohols, stained with hematoxylin and eosin (Hubei Bios, BP0211), and then mounted with neutral gel. Microscopic observation (Olympus, Japan) and imaging were performed. In addition, Masson staining (Wuhan, China, Xavier, G1006) was performed to investigate fibrotic changes in rat ovarian tissue. ImageJ software was used to analyze and quantify the pathological images to calculate the positive rate of fibrosis (i.e., the percentage of positive area to total area).

[0040] Research Results 2.1. Preparation process and physicochemical characterization of hydrogels The microstructure and morphology of the YSCT hydrogel were observed using scanning electron microscopy (SEM). Figure 1 As shown in Figure A, compared to the blank hydrogel, the YSTC hydrogel exhibits a smaller pore size and a more dense and ordered structure, indicating that the hydrogel has strong water absorption and drug loading capacity. Furthermore, the YSTC hydrogel also demonstrates excellent adhesion properties, adhering to surfaces such as glass, rubber, skin, steel pipes, and plastics. Figure 1 C). The release of YSTC-HY in PBS was studied in vitro. The results are shown in the figure. The drug in YSTC-HY showed rapid and almost complete release behavior within 6 hours, with a cumulative release rate of nearly 100%, indicating that the hydrogel formulation has good drug diffusion ability and release efficiency, and is suitable for further in vitro and in vivo pharmacodynamic studies.

[0041] 2.2. Biocompatibility of Hydrogels For hydrogels, biocompatibility is fundamental for their use as implant materials, drug carriers, or tissue engineering scaffolds. Therefore, in this invention, HACAT and KGN cells were co-incubated with blank hydrogels and drug-loaded hydrogel extracts for 24 h and 48 h, respectively. CCK-8 proliferation assays showed that, compared to the control group, both the blank hydrogel group and the drug-loaded hydrogel group significantly promoted the proliferation of HACAT and KGN cells at 24 and 48 h (OD450nm value, P<0.05), with no significant cytotoxicity. Figure 2 A). Further validation by Calcein-AM / PI staining results showed that the survival rate of cells in both hydrogel treatment groups was >95%, with very few dead cells, indicating that the blank hydrogel and its drug-treated hydrogel have good biocompatibility. Figure 2 BC).

[0042] 2.3. LC-MS identification of the chemical composition of YSTC hydrogel: The components of YSTC-HY were analyzed by LC-MS, and representative total ion chromatograms (TIC) of YSTC-HY in positive and negative ion modes were collected, as shown in Figures (3A-B). Figure 3 A represents the positive ion mode. Figure 3 B negative ion mode. Specific confirmation was achieved through comparison with mass spectrometry (MS) information and MS fragment ions of the reference standard. As a result, 35 major compounds and their sources were identified (…). Figure 9 ).

[0043] like Figure 9 The major compounds in YSTC-HY were identified by UPLC / MS.

[0044] 2.4. YSTC-HY regulates body weight and ovarian index in PCOS rats The effects of letrozole (1 mg / kg) combined with a high-fat diet on rats were studied. It was found that three weeks after modeling, the body weight of the model group increased significantly. Figure 4 A) (P<0.001), which is consistent with the characteristics of weight gain in PCOS rats. After modeling, PCOS rats received topical YSTC-HY treatment for three weeks. At the end of the experiment, the body weight of the model group was still significantly higher than that of the control group, while YSTC-HY treatment reversed the weight gain in the rats. Figure 4 B) (P<0.001), the analysis results of ovarian wet weight and ovarian index showed that YSTC-HY intervention could reduce ovarian weight and significantly increase ovarian index in letrozole-induced PCOS rats (B) (P<0.001). Figure 4 (P<0.001) There was no significant difference in food intake between the modeling period and the treatment period.

[0045] 2.5. YSTC-HY improves letrozole-induced estrous cycle disorder in PCOS rats. To assess ovarian function, Giemsa staining was performed on vaginal cell smears from rats in each group for 5 days prior to the end of drug administration. Figure 5 A. The control group rats exhibited regular estrous cycles. Proestrus was dominated by nucleated epithelial cells; estrus was primarily composed of keratinized epithelial cells; mesestrus consisted of a mixture of nucleated epithelium, keratinized epithelium, and leukocytes; and interestrus was almost entirely composed of leukocytes. In contrast, the model group rats showed a significantly prolonged interestrus period. Figure 5 In AB rats treated with YSTC-HY, 85.7% recovered and maintained normal estrous cycles. Figure 5 Treatment with C,YSTC-HY can restore abnormal estrous cycles in PCOS rats.

[0046] 2.6. YSTC-HY improves letrozole-induced abnormal glucose and lipid metabolism in PCOS rats. like Figure 6Fasting insulin (FINS) and fasting blood glucose (FBG) levels in rats were measured, and the HOMA-IR index, serum TG, TC, HDL-C, and LDL-C levels were calculated using the formula. The results showed that compared with the control group, the model group rats had significantly increased fasting insulin, HOMA-IR, serum TG, TC, and LDL-C, and significantly decreased HDL-C. Compared with the model group, the YSTC-HY group rats had significantly decreased serum TG, TC, and LDL-C levels (P<0.05), but slightly increased HDL-C levels, although the difference was not significant (P=0.08). There were no significant differences in serum fasting blood glucose levels among the groups (P>0.05). This indicates that YSTC-HY can improve abnormal glucose and lipid metabolism in PCOS rats to a certain extent.

[0047] 2.7. YSTC-HY improves serum sex hormone disorders in letrozole-induced PCOS rats. like Figure 7 The sex hormone levels of rats were examined. Compared with the control group, the model group showed significantly elevated serum testosterone (T), luteinizing hormone (LH), and LH / FSH ratios. These levels decreased after YSTC-HY intervention. Conversely, the model group showed significantly decreased serum estradiol (E2), follicle-stimulating hormone (FSH), and serum progesterone (PROG) levels compared to the control group. These hormone levels rebounded after YSTC-HY intervention. These results indicate that YSTC-HY can improve serum sex hormone levels in PCOS rats.

[0048] 2.8. YSTC-HY ameliorate letrozole-induced ovarian pathological morphological damage in PCOS rats. Subsequently, morphological analysis of rat ovarian tissue was performed using hematoxylin and eosin (H&E) staining. The results showed that follicles at various developmental stages and mature follicles were visible in the ovaries of control rats, with numerous corpora lutea and few cystic dilated follicles. In contrast, letrozole-induced PCOS model rats exhibited significant histological abnormalities, with a marked increase in the number of cystic follicles and a decrease in the number of corpora lutea, presenting typical polycystic ovary pathological features. After YSTC-HY intervention, the number of cystic dilated follicles in the ovaries of PCOS rats significantly decreased, while the number of corpora lutea and developing follicles increased. Figure 8 B). The number of cystic follicles in the ovaries of rats in each group was statistically analyzed (see [link]). Figure 8 C). The results showed that the number of cystic follicles in the model group was significantly higher than that in the control group, and this change was significantly improved after high intervention. In addition, Masson's trichrome staining results showed that the degree of ovarian fibrosis in the model group rats was significantly higher than that in the control group, and the ovarian fibrosis in rats was significantly alleviated after YSTC-HY intervention. Figure 8DE). This result indicates that YSTC-HY intervention can significantly alleviate ovarian dysfunction in PCOS rats.

[0049] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for manufacturing a transdermal hydrogel for improving polycystic ovary syndrome, characterized by, The method comprises the following steps: S1: preparing a Chinese medicine compound Yishen Tiaochong decoction, which is composed of Huangqi, Cangzhu, Fuling, Yinyanghuo, Duzhong, Tusizi, Yujin, Xiangfu, Juren, E'zhu, Honghua, Chuanqiong, Zhimu, Zaokeshu and Chaojiezi; S2: dissolving chitosan in the decoction to prepare a chitosan solution and adjusting pH; S3: dissolving sodium alginate in the decoction to prepare a sodium alginate solution; S4: mixing the chitosan solution and the sodium alginate solution, continuously stirring, and forming a hydrogel through electrostatic cross-linking; S5: adjusting the pH of the hydrogel and storing it in the dark.

2. The method for manufacturing a transdermal hydrogel for improving polycystic ovary syndrome according to claim 1, characterized in that, The Yishen Tiaochong decoction comprises, by weight, Huangqi 15 g, Cangzhu 20 g, Fuling 20 g, Yinyanghuo 20 g, Duzhong 15 g, Tusizi 30 g, Yujin 15 g, Xiangfu 15 g, Juren 20 g, E'zhu 15 g, Honghua 10 g, Chuanqiong 12 g, Zhimu 20 g, Zaokeshu 15 g and Chaojiezi 10 g.

3. The method for manufacturing a transdermal hydrogel for improving polycystic ovary syndrome according to claim 1, characterized in that, In S2, the concentration of the chitosan solution is 2% w / v, and the pH is adjusted to 5.0; in S3, the concentration of the sodium alginate solution is 2% w / v.

4. The method for manufacturing a transdermal hydrogel for improving polycystic ovary syndrome according to claim 1, characterized in that, In S4, the chitosan solution and the sodium alginate solution are mixed at a volume ratio of 1:1 and stirred for 20 minutes; in S5, the pH of the hydrogel is adjusted to 6.0, and the hydrogel is stored in the dark at 4°C.

5. The method for manufacturing a transdermal hydrogel for improving polycystic ovary syndrome according to claim 1, characterized in that, The degree of deacetylation of the chitosan is not less than 85%.

6. The method for manufacturing a transdermal hydrogel for improving polycystic ovary syndrome according to claim 1, characterized in that, The concentration of the Yishen Tiaochong decoction is 2.25 g of crude drug per milliliter.

7. The method for manufacturing a transdermal hydrogel for improving polycystic ovary syndrome according to claim 1, characterized in that, The cumulative drug release rate of the hydrogel in a PBS release medium at 37°C reaches more than 90% within 6 hours.

8. A transdermal hydrogel prepared by the method of any one of claims 1-7, characterized by: The hydrogel is loaded with active ingredients of the Yishen Tiaochong decoction and is used for improving metabolic disorders of polycystic ovary syndrome.

9. A method for manufacturing a transdermal hydrogel for improving polycystic ovary syndrome according to claim 8, characterized in that, The pore size of the hydrogel ranges from 10 μm to 50 μm, and the hydrogel can adhere to the surface of the skin.

10. A method for manufacturing a transdermal hydrogel for improving polycystic ovary syndrome according to claim 8, characterized in that, The metabolic disorders include at least one of insulin resistance, abnormal lipid metabolism and disorder of sex hormone levels.

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