Application of kaempferol in prevention and treatment of premature ovarian insufficiency caused by chemotherapeutic drugs

Abstract

The invention relates to application of kaempferol in preparation of drugs, food or health care products for preventing or treating premature ovarian insufficiency diseases caused by chemotherapy drugs. Experiments prove that the estrus cycle disorder of mice exposed to chemotherapeutics can be improved by the administration of the kaempferol; the kaempferol administration can improve the ovarian organ coefficient of the mouse exposed by the chemotherapeutic drug; the administration of kaempferol can effectively improve mouse ovarian hormone level disorder caused by exposure of chemotherapeutic drugs; the kaempferol can increase the number of effect follicles at all levels, reduce the number of atresia follicles and increase the total number of follicles. Experiments prove that kaempferol has a potential value in early-onset ovarian insufficiency caused by chemotherapeutic drugs, and a new method and a new idea are provided for clinical treatment of POI. The method has good popularization and application values.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of kaempferol in the prevention and treatment of premature ovarian insufficiency caused by chemotherapy drugs. Background Technology

[0002] There are many causes of infertility, including female factors, male factors, and unexplained infertility. Among female factors, pelvic factors and ovulation disorders are particularly prominent causes of infertility. There are many causes of ovulation disorders in women of reproductive age, with premature ovarian insufficiency being an important one.

[0003] Premature ovarian insufficiency (POI) refers to decreased ovarian function in women before the age of 40. The main symptoms include menstrual irregularities (amenorrhea, oligomenorrhea), follicle-stimulating hormone (FSH) levels >25 U / L (intermittent >4 weeks, on two consecutive occasions), and fluctuating decline in estrogen levels. The overall incidence of POI is approximately 3.7%. Decreased ovarian function in women is a progressive process. Two other conditions associated with POI are diminished ovarian reserve (DOR) and premature ovarian failure (POF).

[0004] DOR (Decrease in Oocyte Count) refers to a decrease in the number and / or quality of oocytes in the ovary, leading to ovarian insufficiency and decreased fertility. This is accompanied by decreased anti-Müllerian hormone (AMH) levels, a reduced antral follicle count (AFC), and elevated basal FSH levels (≥10 IU / L). POF (Positive Perimenopause) refers to the onset of amenorrhea, elevated gonadotropin (Gn) levels (FSH > 40 U / L), and decreased estrogen levels in women under 40 years of age, accompanied by varying degrees of perimenopausal symptoms. It represents the final stage of POI (Positive Perimenopause).

[0005] Studies have reported that iatrogenic factors, such as the radiotherapy and chemotherapy processes for tumors, as well as environmental factors, are important causes of poikilogenic inflammation (POI). They all share the common characteristic of increasing levels of reactive oxygen species (ROS) in the body. The most common ROS include superoxide anion (O2). - ), hydrogen peroxide (H2O2) and hydroxyl radicals (OH) -At low to moderate concentrations, free radicals (ROS) exhibit beneficial effects on cellular responses and immune function. However, above moderate concentrations, these free radicals disrupt the body's antioxidant defense system, leading to oxidative stress. These ROS react with various biomolecules, namely lipids, DNA, and proteins, causing oxidative damage that results in structural damage to biomolecules, such as single-strand breaks (SSBs) or double-strand breaks (DSBs) in DNA, as well as DNA-DNA or DNA-protein crosslinks, leading to cell death. ROS can also increase apoptosis in ovarian granulosa cells by disrupting biomolecules and activating related signaling pathways.

[0006] Previously, starting from the mechanism of reducing the level of overactivated reactive oxygen species in POI patients, we conducted screening trials on some antioxidant foods and drugs reported both domestically and internationally, and obtained a relatively safe and effective drug: kaempferol. In 2024, two different research teams from China simultaneously confirmed that kaempferol plays an important role in the control of circulatory system diseases, both by reducing oxidative stress to alleviate myocardial ischemia damage (1,2) and by improving coronary atherosclerosis (3). In addition, kaempferol has also shown important potential in neurodegenerative diseases and injuries (4-6). However, previous domestic and international studies have not addressed its specific effects on ovarian function damage or failure, and whether the use of kaempferol before or after ovarian injury will have a positive effect has not yet been reported.

[0007] Chinese patent document CN104434908A discloses the application of kaempferol in drugs that inhibit organ transplant rejection. This invention is the first to disclose the pharmacological effects of kaempferol in inhibiting organ transplant rejection in recipients. Furthermore, its efficacy is best when used in short-term combination with low-dose cyclosporine, inducing long-term survival of islet grafts in recipients. This avoids the various side effects caused by long-term use of full-dose cyclosporine, reduces the economic burden of long-term anti-rejection treatment for patients, and is more conducive to a high quality of life for organ transplant patients in the long term. This invention also discovers the immunomodulatory mechanism of kaempferol: it can significantly upregulate the number of regulatory T lymphocytes in mouse recipients and downregulate the gene expression of IL-6 in islet grafts, thereby exerting anti-inflammatory and anti-rejection effects.

[0008] Chinese patent document CN106880626A discloses the application of kaempferol in the preparation of drugs for the prevention and treatment of retinal damage. Kaempferol is found in various edible plants (including fruits, vegetables, and legumes) and traditional Chinese medicines (chrysanthemum, raspberry, dodder seed, astragalus, and ginkgo leaves, etc.). By observing the protective effect of kaempferol on hydrogen peroxide (H2O2)-induced oxidative stress damage to human retinal pigment epithelial cells (ARPE-19), and by observing the changes in the expression of apoptosis-related molecules in ARPE-19 cells after H2O2 treatment, this study explored the anti-apoptotic effect of kaempferol on oxidatively damaged ARPE-19 cells and its molecular mechanism of action, providing a theoretical basis for the future use of kaempferol in the prevention and treatment of ARPE-19 cell damage-related eye diseases.

[0009] There are currently no reports regarding the use of kaempferol in early-onset ovarian insufficiency induced by chemotherapy drugs. Summary of the Invention

[0010] The purpose of this invention is to address the shortcomings of the prior art by providing the application of kaempferol in the prevention and treatment of premature ovarian insufficiency caused by chemotherapy drugs.

[0011] In a first aspect, the present invention provides the use of kaempferol in the preparation of drugs, foods or health products for the prevention or treatment of early-onset ovarian insufficiency caused by chemotherapy drugs.

[0012] As a preferred example, the chemotherapy drug is cyclophosphamide.

[0013] As a preferred example, the early-onset ovarian insufficiency caused by the chemotherapy drugs is characterized by one or more of the following: estrous cycle disorder, significantly reduced ovarian organ coefficient, disordered ovarian hormone levels, increased number of atretic follicles, or decreased total number of follicles.

[0014] As a preferred example, the kaempferol is prepared into a pharmaceutical formulation by adding pharmaceutically acceptable excipients and a carrier, wherein the excipients include at least one of fillers, disintegrants, binders, excipients, diluents, lubricants, sweeteners, and colorants.

[0015] The advantages of this invention are:

[0016] This invention demonstrates through experiments that kaempferol administration can improve estrous cycle disorders in mice exposed to chemotherapy drugs; kaempferol administration can improve the ovarian organ coefficient in mice exposed to chemotherapy drugs; kaempferol administration can effectively improve ovarian hormone level disorders in mice induced by chemotherapy drug exposure; kaempferol can increase the number of follicles at all levels of effect, reduce the number of atretic follicles, and increase the total number of follicles.

[0017] This invention experimentally demonstrates the potential value of kaempferol in chemotherapy-induced premature ovarian insufficiency (POI), and provides its application in the preparation of drugs, foods, or health products for the prevention or treatment of POI. It offers new methods and ideas for the clinical treatment of POI. This invention has significant potential for widespread application. Attached Figure Description

[0018] Figure 1 : Ovarian tissue organ coefficients of each group of mice (***) / ### (P < 0.001).

[0019] Figure 2 Kaempferol can effectively improve irradiation-induced ovarian hormone level disorders in mice. (A) Protective effect of kaempferol on anti-Müllerian hormone (AMH) in mice; (B) Protective effect of kaempferol on estrogen (E2) in mice; (C) Protective effect of kaempferol on follicle-stimulating hormone (FSH) in mice; (D) Protective effect of kaempferol on luteinizing hormone (LH) in mice. / ## P < 0.01; *** / ### (P < 0.001).

[0020] Figure 3 Follicle counts in each group of mice. (A) Primordial follicle counts in each group of mice; (B) Primary follicle counts in each group of mice; (C) Preantral follicle counts in each group of mice; (D) Antral follicle counts in each group of mice; (E) Atretic follicle counts in each group of mice; (F) Total follicle counts in each group of mice. (***) / ### (P < 0.001). Detailed Implementation

[0021] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0022] Example 1

[0023] 1. Experimental Materials:

[0024] Experimental animals: The mice used in this experiment had the genetic background of C57BL / 6J and were purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd. They were 6-8 weeks old and were housed in a constant temperature animal room with alternating day and night lighting and a temperature of 20℃.

[0025] Kaempferol: MedChemExpress

[0026] Cyclophosphamide: MedChemExpress

[0027] Mouse Estrogen (E2), Luteinizing Hormone (LH), Follicle-Stimulating Hormone (FSH), and Anti-Müllerian Hormone (AMH) ELISA Detection Reagents: Shanghai Zhuocai Biotechnology Co., Ltd.

[0028] Hematoxylin dye: Zhuhai Beso Biotechnology Co., Ltd.

[0029] Eosin dye: Zhuhai Beso Biotechnology Co., Ltd.

[0030] Giemsa Dyes: Zhuhai Beso Biotechnology Co., Ltd.

[0031] II. Experimental Methods:

[0032] (a) Experimental design:

[0033] (i) Prevention group: Female mice were divided into chemotherapy drug administration group and chemotherapy drug administration + kaempferol administration group. All female mice in the kaempferol administration group were given intraperitoneal injection of kaempferol at 20 mg / kg every 2 days for 28 days before chemotherapy drug administration, for a total of 14 times. Mice in the chemotherapy drug administration group were given the same volume of solvent intraperitoneal injection every day. On day 29 and day 31, all mice were given intraperitoneal injection of cyclophosphamide at 30 mg / kg and 120 mg / kg, respectively, for a total of 2 times.

[0034] (ii) Treatment group: Female mice were divided into chemotherapy drug administration group and chemotherapy drug administration + kaempferol administration group. On day 1 and day 3, all mice were given intraperitoneal injections of cyclophosphamide at 30 mg / kg and 120 mg / kg, respectively, for a total of 2 times. All female mice in the kaempferol administration group were given intraperitoneal injections of kaempferol at 20 mg / kg every 2 days for 28 days after the end of chemotherapy drug administration, for a total of 14 times. Mice in the chemotherapy drug administration group were given the same volume of solvent intraperitoneal injection every day.

[0035] (b) Experimental setup: Forty-eight female mice were randomly divided into a blank control group (Control), a chemotherapy drug administration group (CTX), and a chemotherapy drug administration group (CTX+KAE). 1 ) and chemotherapy drug administration + kaempferol treatment group (CTX + KAE) 2 After receiving no treatment, the subjects underwent subsequent experiments including organ coefficient, follicle count, estrous cycle and ovarian hormone level testing (3 repeated experiments).

[0036] (c) Estrogenic cycle: After all the expected number of intraperitoneal injections of cyclophosphamide or kaempferol were completed, the specific stage of the estrous cycle of the mice was determined by detecting the morphological changes of vaginal exfoliated cells at 08:00 and 20:00 every day. The 21-day complete evaluation was carried out and statistical analysis was performed.

[0037] (d) Hormone levels: At 08:00 on the second day after the end of the estrous cycle assessment experiment, mice were anesthetized, blood was collected from the eyeballs to prepare serum, and the levels of AMH, E2, FSH and LH in the mice were detected by ELISA.

[0038] (e) Organ coefficient: The weight of mice in each group was measured after anesthesia, and the bilateral ovarian tissues of the mice were dissected and weighed after blood was collected from the eyeballs. The organ coefficient was calculated and analyzed.

[0039] (f) Follicle counting: Ovarian tissue from mice was used to prepare paraffin specimens, and follicles were counted on the specimens to assess the changes in the number of follicles at each stage and the total number of follicles.

[0040] (g) Statistical analysis: GraphPad Prism 9.0 and SPSS 22.0 software were used for analysis. All data are expressed as mean ± standard deviation (Means ± SD). Unpaired t-tests were used to analyze differences among continuous data. P < 0.05 was considered statistically significant.

[0041] III. Experimental Results:

[0042] (a) Kaempferol administration improved estrous cycle disorders in mice exposed to chemotherapy drugs: In three batches of experiments, chemotherapy drug administration increased the rate of estrous cycle disorders in mice. Compared with the chemotherapy drug-only group, 72.22±12.73% of mice showed estrous cycle disorders, while the rate in the chemotherapy drug administration + kaempferol prophylactic administration group was 33.33±8.34%, which was statistically different. Furthermore, compared with the chemotherapy drug-only group, 72.22±12.73% of mice showed estrous cycle disorders, while the rate in the chemotherapy drug administration + kaempferol treatment group was 29.89±9.62%, which was also statistically different.

[0043] Table 1

[0044]

[0045] Table 1: Estrogenic cycle disorder rate in each group of mice (**) / ## (P < 0.01)

[0046] (b) Kaempferol administration reduced the ovarian organ coefficient in chemotherapy-exposed mice: Regarding the ovarian organ coefficient, compared to the control group, the ovarian organ coefficient was significantly reduced in the chemotherapy-only group. Intraperitoneal injection of kaempferol before and after chemotherapy administration reversed this trend to some extent, showing a statistically significant difference compared to the chemotherapy-only group (see [link to relevant documentation]). Figure 1 ).

[0047] (c) Kaempferol administration effectively improved ovarian hormone level disorders in mice induced by chemotherapy drug exposure: Compared with the control group, the hormone levels in mice treated with chemotherapy drugs alone were significantly disordered. Administration of kaempferol before and after chemotherapy drug exposure significantly increased anti-Müllerian hormone levels in irradiated mice. Figure 2 A) and estrogen ( Figure 2 B) levels, and can significantly reduce follicle-stimulating hormone (FSH) levels in irradiated mice. Figure 2 C) and luteinizing hormone ( Figure 2 D) Level.

[0048] (d) Effects of kaempferol on irradiated mouse follicles: Kaempferol can increase the number of follicles at all stages of effect. Figure 3 A / B / C / D), reducing the number of atretic follicles ( Figure 3 E), and increase the total number of follicles ( Figure 3 F), there is a statistical difference between the two.

[0049] References:

[0050] 1. YueZ, ZhangY, ZhangW, ZhengN, WenJ, RenL, etal. Kaempferolalleviatesmyocardialischemiainjuryby reducing oxidative stress via theHDAC3-mediatedNrf2 signaling pathway. Journal of advanced research. 2024.

[0051] 2. Zhang L, Liu

[0052] 3.ChuT,WangY,Wang S,LiJ,LiZ,WeiZ,etal.Kaempferolregulatingmacrophagefoamingand atherosclerosisthroughPiezo1-mediatedMAPK / NF-κBandNrf2 / HO-1signalingpathway.Journalofadvanced research.2024.

[0053] 4. PilottoF, SmeelePH, ScheideggerO, DiabR, SchobesbergerM, Sierra-DelgadoJA, et al.

[0054] 5. Han

[0055] 6. YuanY, ZhaiY, ChenJ, XuX, WangH. KaempferolAmelioratesOxygen-GlucoseDeprivation / Reoxygenation-InducedNeuronalFerroptosisbyActivatingNrf2 / SLC7A11 / GPX4Axis. Biomolecules. 2021; 11(7).

[0056] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. Application of kaempferol in the preparation of drugs, foods or health products for the prevention or treatment of early-onset ovarian insufficiency caused by chemotherapy drugs.

2. The application according to claim 1, characterized in that, The chemotherapy drug mentioned is cyclophosphamide.

3. The application according to claim 1 or 2, characterized in that, The manifestations of early-onset ovarian insufficiency caused by the chemotherapy drugs mentioned above include one or more of the following: estrous cycle disorder, significant reduction in ovarian organ coefficient, disordered ovarian hormone levels, increased number of atretic follicles, or decreased total number of follicles.

4. The application according to any one of claims 1-3, characterized in that, Kaempferol is prepared into a pharmaceutical formulation by adding pharmaceutically acceptable excipients and carriers, wherein the excipients include at least one of fillers, disintegrants, binders, excipients, diluents, lubricants, sweeteners, and colorants.

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