A composition for regulating ovarian lipid metabolism and improving fertility

Abstract

This application relates to a composition for regulating ovarian lipid metabolism and improving fertility. The composition, by weight, comprises or mainly consists of the following materials: 6-9 parts ginseng, 12-20 parts yam, 3-9 parts rose, 9-15 parts kudzu root, 9-15 parts mulberry, 9-15 parts polygonatum, 3-9 parts donkey-hide gelatin, 1-5 parts cinnamon, and 6-12 parts wolfberry. The advantages of this invention are: the raw materials are derived from food and medicine homologous substances, ensuring high safety; it can improve drug-induced gonadal dysfunction, improve estrous cycle disorders, regulate drug-induced endocrine dysfunction, and promote gonadal organ development; it reduces serum testosterone levels in female animals and improves cystic follicles; it improves the fertility of drug-induced hypogonadism rats; it regulates ovarian lipid metabolism and improves metabolic ovarian dysfunction caused by APOA1 accumulation; and it reduces the expression levels of ovarian retinoid X receptor α (RXRα) and nuclear factor E2-related factor 2 (Nrf2).

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine application, specifically relating to a composition for regulating ovarian lipid metabolism and improving fertility, and can also be used as a food therapy composition or health product that is both food and medicine. Background Technology

[0002] In vertebrates, reproductive function is regulated by the neuroendocrine system comprised of the hypothalamus-pituitary-gonadal (HPG) axis. This system regulates gonadal hormone synthesis and reproductive function by modulating the release of pituitary follicle-stimulating hormone (FSH) and luteinizing hormone (LH). FSH and LH secreted by the HPG axis act as the master switch and regulatory center for the function of the female ovaries and male testes.

[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), elevated FSH levels (>25 U / L), and fluctuating decline in estrogen levels, primarily estradiol (E2). Ovarian insufficiency is a progressive process. The final stage of POI is premature ovarian failure (POF), characterized by further increases in gonadotropin levels (FSH>40 U / L) and a significant decrease in estrogen levels, accompanied by varying degrees of hypoestrogenic symptoms. This not only affects fertility and impacts the patient's normal life and health but also has a significant psychological impact. As the condition progresses, it may also lead to various serious complications such as heart disease and osteoporosis. Therefore, early intervention, management, and treatment of POI patients to prevent the condition from worsening and to avoid the occurrence of POF is of positive practical significance for the patient's physical and mental health and the promotion of family harmony.

[0004] Literature reports that the incidence of POI (Posterior Ovarian Inflammation) is 1%-4%, with a global incidence rate of 1.9%, showing an increasing trend and affecting younger individuals. The causes of POI are complex, including genetic factors, autoimmune factors, iatrogenic factors, environmental factors, and unhealthy lifestyle habits. The cause is unclear in more than half of POI cases, classifying it as idiopathic POI. Among these, iatrogenic factors, such as chemotherapy, radiotherapy, surgery, and endometrial infections causing ovarian damage, account for 50% of POI cases.

[0005] Currently, effective treatments for POI remain very limited. The primary treatment is hormone replacement therapy (HRT), which supplements with exogenous estrogen and progesterone to feedback-lower endogenous gonadotropin levels and improve FSH and E2 levels. However, HRT requires individualized treatment based on diagnosis, and the methods and dosages vary considerably. Literature reports that HRT treatment does not significantly improve pregnancy rates in POI patients, and HRT carries risks for women with a history of ovarian or breast cancer, increasing the risk of thrombosis, stroke, and other complications. Therefore, finding safe and effective drugs to improve POI, combat aging, and enhance fertility has significant social and economic implications.

[0006] Currently, there are no studies on the correlation between abnormal ovarian glucose and lipid metabolism leading to cystic ovaries and subsequently inducing premature ovarian failure, especially regarding the impact of emergency contraceptive pills on ovarian tissue metabolic function. Levonorgestrel-containing emergency contraceptive pills (ECPs) are the most widely used emergency contraceptives on the market. Literature reports that some patients with ovarian cysts have taken LNG-ECPs three times a month, or more than ten times a year. This suggests that long-term use of emergency contraceptive pills may lead to ovarian dysfunction and may even be associated with infertility. The inventors of this application found in their research that long-term, repeated (12 cycles) oral administration of levonorgestrel-containing emergency contraceptives (ECPs) to rats significantly reduced the number of primordial follicles and significantly increased the number of atresia in the rat ovaries (p<0.05). No significant improvement was observed even after three estrous cycles following discontinuation of the drug (Hu Yingyi et al., Effects of repeated administration of levonorgestrel-containing emergency contraceptives on fertility and offspring health in female rats, Chinese Journal of Reproduction and Contraception, 2024, 44(6): 562-573.). Further research revealed that LNG-ECPs can interfere with ovarian glucose and lipid metabolism, leading to the accumulation of apolipoprotein A1 (ApoA1) protein in ovarian tissue, upregulating nuclear factor E2-related factor 2 (Nrf2) and glucose and lipid metabolism-related factor retinoid X receptor alpha (RXRα), thus impairing ovarian function. ApoA1 accumulation is the target of LNG-ECPs damaging the ovaries.

[0007] Therefore, it is desired that a safe and effective product can reduce the elevated levels of the lipid metabolite ApoA1 in the ovaries caused by LNG, downregulate the expression levels of Nrf2 and RXRα, improve ovarian function, delay POI, promote follicle development, and enhance fertility. The inventors have used POI, a common form of hypogonadism, as an example to study the application of the composition in regulating the HPG axis, improving gonadal axis function, and enhancing fertility. Since the HPG axis is the central regulator of reproductive function, the effects of regulating FSH and LH are applicable not only to women but also to the regulation of male reproductive function. Summary of the Invention

[0008] This invention provides a formula for a medicinal and edible composition (such as a dietary therapy composition) that improves reproductive function decline and enhances fertility. The composition contains a certain proportion of ginseng, yam, kudzu root, mulberry, polygonatum, rose, donkey-hide gelatin, cinnamon, and wolfberry.

[0009] According to one aspect of the present invention, a composition is provided, comprising or mainly consisting of the following materials in parts by weight: 6-9 parts ginseng, 12-20 parts Chinese yam (Huai yam), 3-9 parts rose, 9-15 parts kudzu root, 9-15 parts mulberry, 9-15 parts polygonatum, 3-9 parts donkey-hide gelatin, 1-5 parts cinnamon, and 6-12 parts wolfberry.

[0010] In some embodiments, the composition comprises or mainly consists of the following materials in parts by weight: 8-9 parts ginseng, 13-16 parts yam, 9-11 parts kudzu root, 10-13 parts mulberry, 10-13 parts polygonatum, 5-7 parts rose, 5-7 parts donkey-hide gelatin, 2-4 parts cinnamon, and 9-12 parts wolfberry.

[0011] In some embodiments, the composition further comprises, by weight: 9-15 parts of Poria cocos and 6-12 parts of Angelica sinensis.

[0012] In a specific embodiment, the composition of the present invention comprises or mainly consists of the following materials in parts by weight: 9 parts ginseng, 15 parts yam, 10 parts kudzu root, 12 parts mulberry, 12 parts polygonatum, 6 parts rose, 6 parts donkey-hide gelatin, 3 parts cinnamon, and 12 parts wolfberry.

[0013] According to another aspect of the invention, a product is provided, which may be a pharmaceutical, food, or health product, comprising the composition described above or an extract prepared from the composition described above, and optionally corresponding product-acceptable excipients, such as pharmaceutical, food science, or health product-acceptable excipients.

[0014] According to the present invention, the product has one or more (including all) of the following functions: Improves pathological reproductive function damage and physiological reproductive function decline; Regulates hypothalamic-pituitary-gonadal function; It promotes the development of primordial follicles, delays the depletion of the primordial follicle pool, and reduces atretic follicles; It reduces serum testosterone levels in female animals and improves cystic follicle formation; Increase the chances of conception; It regulates ovarian lipid metabolism, downregulates ovarian ApoA1 levels, and downregulates ovarian retinoid X receptor α (RXRα) and nuclear factor E2-related factor 2 (Nrf2) expression.

[0015] The product of this invention can be in the form of solid granules, capsules or tablets, or it can be prepared as an oral liquid, soft drink or other liquid preparation or liquid beverage, or it can be prepared as candy, pastry or other forms.

[0016] In some embodiments, the extract is prepared by a method comprising the following steps: Take the ginseng, yam, kudzu root, mulberry, polygonatum, rose, and wolfberry in the formula of the above composition and decoct them in water to obtain the first decoction. Take a small amount of the first decoction and boil it together with the cinnamon in the formula until it boils. Then filter it to obtain the second decoction. Mix the second decoction with the first decoction. Dissolve the donkey-hide gelatin in rice wine in the formula and add it to the mixed decoction. Concentrate under reduced pressure to obtain the extract.

[0017] In other embodiments, the extract is prepared by a method comprising the following steps: Take the ginseng, yam, kudzu root, mulberry, polygonatum, rose, and wolfberry in the formula of the above composition, crush them, add 8-10 times the amount of water and soak for 0.5-2 hours, then decoct for 5 minutes to 1 hour to obtain the first decoction. Take a small amount of the first decoction and boil it together with the cinnamon in the formula until it boils, then filter to obtain the second decoction. Mix the second decoction with the first decoction, dissolve the donkey-hide gelatin in rice wine in the formula and add it to the mixed decoction, concentrate under reduced pressure at 80-90°C to a density of 1.10-1.15 (55-65°C) to obtain the extract.

[0018] According to another aspect of the present invention, the use of the composition in the preparation of products that improve pathological reproductive dysfunction and physiological reproductive function decline is provided. Animal experiments show that, at experimental doses, the composition of the present invention can antagonize the toxic side effects of chemotherapy drugs on the reproductive system, increase the ovarian organ coefficient, improve pathological ovarian insufficiency and ovarian function decline, increase ovarian quality, facilitate the recovery of gonadal organ function, enhance gonadal function, promote the recovery of gonadal function, and assist in improving iatrogenic ovarian dysfunction or physiological ovarian function decline.

[0019] According to another aspect of the present invention, the use of the composition in the preparation of a product for regulating HPG function is provided. Preliminary animal experiments have shown that, in cases of pathological endocrine dysfunction induced by the chemotherapy drug cyclophosphamide (CTX), the composition of the present invention, at experimental doses, can reduce the increase in FSH and LH levels caused by CTX and reduce serum testosterone (T) levels in female animals, indicating that the composition is beneficial for regulating the function of the hypothalamus-pituitary-gonadal axis, improving endocrine function, and has an ameliorative effect on endocrine dysfunction caused by iatrogenic gonadal dysfunction or physiological gonadal dysfunction and hypofunction.

[0020] According to another aspect of the present invention, the composition is provided for use in the preparation of products that promote primordial follicle development, delay primordial follicle depletion, and reduce atretic follicles. Animal experiments have shown that, at experimental doses, the composition of the present invention can antagonize the decrease in primordial follicle rate and growing follicle rate and the increase in atretic follicle rate caused by chemotherapy drugs, increase the primordial follicle rate and growing follicle rate, and reduce the atretic follicle rate.

[0021] According to another aspect of the present invention, the use of the composition in the preparation of a product that reduces serum testosterone levels in female animals and improves cystic follicles is provided.

[0022] According to another aspect of the present invention, the use of the composition in the preparation of products that improve fertility is provided. Animal experiments have shown that, compared with animal models of ovarian damage induced by chemotherapy drugs, rats treated with the composition at the experimental dose achieved a pregnancy rate of 100%, significantly higher than the 50% pregnancy rate in the model group. This indicates that the present invention has a good effect on improving fertility loss caused by iatrogenic ovarian damage.

[0023] According to another aspect of the present invention, the composition is provided for use in the preparation of products that regulate ovarian lipid metabolism, downregulate ovarian ApoA1 levels, and downregulate the expression of ovarian retinoid X receptor α (RXRα) and nuclear factor E2-related factor 2 (Nrf2). The inventors have discovered for the first time that emergency contraceptives containing levonorgestrel can interfere with ovarian lipid metabolism, leading to the accumulation of ApoA1 protein in the ovaries. In rats, ApoA1 can induce estrous cycle disorders, the appearance of cystic follicles in the ovaries, accompanied by a significant reduction in the number of primordial follicles, growing follicles, and mature follicles, a significant increase in the number of atretic follicles, and increased expression of Nrf2 and RXRα, resulting in ovarian dysfunction characterized by POI (Potentially Ovary Injury Indication). ApoA1 accumulation is a target of ovarian damage caused by LNG-ECPs. Administration of the composition of the present invention significantly reduces the protein expression of the LNG-induced ovarian lipid metabolite ApoA1, reduces the protein expression of Nrf2 and RXRα, improves ovarian function, and enhances fertility.

[0024] Unlike the numerous traditional Chinese medicinal materials used in existing literature, the composition involved in this application is a dietary therapy composition with a simple formulation, using all commonly used everyday ingredients from various parts of China. All ingredients in the formulation are listed in the food and medicine homology list published by the National Health Commission and other relevant departments, meaning they can be used simultaneously as food and traditional Chinese medicine. The properties of these ingredients have been recorded and widely applied in ancient Chinese texts, with no obvious toxic side effects and good safety. The entire formula utilizes the combined effects of common ingredients—tonifying qi and yin, strengthening the spleen and stomach, nourishing blood and securing essence, and soothing the liver and relieving depression—to improve ovarian lipid metabolism, regulate gonadal hormone levels, promote gonadal development, and increase fertility.

[0025] The formula follows the principles of traditional Chinese medicine, utilizing the combined effects of common ingredients in replenishing qi and yin, strengthening the spleen and stomach, nourishing blood and securing essence, and soothing the liver and relieving depression. The entire formula is nourishing without causing stagnation, moisturizing without being greasy, and warming without being drying. Ginseng replenishes qi, and Polygonatum replenishes qi and yin, strengthens the spleen and kidneys; these two together serve as the principal ingredients. Dioscorea strengthens the spleen and stomach, and tonifies the kidneys and astringes essence; Lycium barbarum nourishes the liver and kidneys, and benefits essence and vision; Mulberry nourishes yin and blood; Donkey-hide gelatin replenishes blood and enhances immunity, working together to strengthen the spleen and replenish qi, nourish the liver and kidneys, and simultaneously replenish qi and blood, serving as the assistant ingredients. Kudzu root generates fluids and quenches thirst, promotes the upward movement of clear yang, and unblocks meridians; Rose promotes qi circulation, soothes the liver and relieves depression, harmonizes blood and relieves pain; Poria strengthens the spleen, eliminates dampness, transforms greasy substances, and prevents tonification from hindering the stomach; these three ingredients ensure that tonification is not stagnant, prevent qi stagnation, and promote the circulation of qi and blood, serving as the adjuvant ingredients. Cinnamon, used to tonify fire and boost yang, guides the fire back to its source and directs the medicine to the kidneys, allowing the medicinal power to reach the lower burner directly, thus serving as the guiding herb. The formula combines "tonification," "transformation," and "dredging" to form a unique system of "tonification without stagnation." Ginseng, polygonatum, mulberry, wolfberry, and donkey-hide gelatin are for "tonification," yam and kudzu root are for "transformation," and rose and cinnamon are for "dredging." These substances work synergistically to improve hypothalamic-pituitary-gonadal endocrine dysfunction, regulate gonadal hormone levels, regulate ovarian ApoA1 levels, improve ovarian lipid metabolism, promote follicle and sperm development, enhance gonadal function, prevent and improve symptoms of gonadal dysfunction and reduced fertility caused by pathological (e.g., chemotherapy or emergency contraception) or physiological ovarian insufficiency or male hypogonadism, reduce ApoA1 accumulation, regulate lipid metabolism, and thus enhance fertility.

[0026] This dietary therapy formula is simple to prepare and suitable for industrial production. From the perspective of "prevention of disease," it addresses the decline in HPG axis function, providing an effective method for improving fertility in women of reproductive age and delaying and improving endocrine function in women going through menopause.

[0027] This application first establishes an early iatrogenic rat model of ovarian injury by administering the chemotherapy drugs cyclophosphamide (CTX) and levonorgestrel (LNG) to rats, and then explores the adjuvant therapeutic effect of the composition based on this model. The results showed that, compared with normal controls, the ovarian organ coefficient of the model group rats was reduced; histopathological examination revealed a decrease in the number and rate of primordial follicles in the ovaries of the model group, a significant increase in the number and rate of atretic follicles, and an increasing trend in serum FSH and LH levels, suggesting that CTX causes early ovarian injury, exhibiting characteristics of POI. After 21 days of gavage administration of the composition of this invention, the ovarian organ coefficient of female rats showed an increasing trend, the rate of primordial follicles increased, the rate of atretic follicles decreased, serum FSH, LH, and testosterone (T) levels decreased, and the pregnancy rate of rats significantly increased. This indicates that the composition of this invention can regulate hypothalamic-pituitary-gonadal function, improve endocrine status, enhance gonadal function, and increase pregnancy rate, and can be used as an adjuvant therapy to improve fertility.

[0028] Taking female animals as an example, the composition of this invention can improve iatrogenic ovarian dysfunction or physiological ovarian dysfunction, and its safety is superior to that of the control drug, estradiol benzoate. Unlike traditional Chinese medicine, since the composition of this invention does not increase E2 when regulating FSH / LH levels, it is speculated that the composition of this invention may also be suitable for men, improving male gonadal function. Acute toxicity tests showed that after a single oral administration of >10g / kg of the composition, no abnormalities were observed and no animals died after 14 days of observation.

[0029] Animal experiments showed that administration of the composition of the present invention can significantly reduce the increase in ApoA1 content in the ovary caused by LNG, downregulate the expression of oxidative factor Nrf2 and lipid metabolism-related transcription factor RXRα, improve ovarian function, delay POI, promote follicle development, and improve fertility.

[0030] In summary, the advantages of the composition in this invention are: (1) the raw materials are derived from food and drug homologous substances, and the safety is relatively high; (2) it can improve iatrogenic gonadal dysfunction, improve estrous cycle disorder, regulate HPG axis endocrine dysfunction caused by chemotherapy drugs, and promote the development of gonadal organs (follicles or testes), especially promoting the development of primordial follicles; (3) it can improve the fertility of drug-induced hypogonadism rats; (4) it can regulate ovarian lipid metabolism, reduce the expression level of ovarian ApoA1 protein induced by contraceptive drugs, and improve metabolic ovarian dysfunction caused by ApoA1 accumulation; (5) it can downregulate the expression level of ovarian glucose and lipid metabolism-related transcription factor RXRα. Attached Figure Description

[0031] Figure 1The effects of intraovarian injection of ApoA1 on estrous cycle (A), ovarian histopathology (BC), ovarian total cholesterol (D), and follicle counts at each stage (EH) in rats were shown (n=6). P <0.05, compared with the blank control group or sham surgery group; P <0.01, compared with the blank control group or sham surgery group; Figure 2 The following are shown: (A) Effects of LNG on the expression of ApoA1, the oxidative signaling molecule Nrf2, and the glucose and lipid metabolism-related transcription factor RXRα (n=3); (B) Effects of low and high doses of the composition of the present invention on the expression of ApoA1, Nrf2, and RXRα (n=3). Indicates the comparison between the two groups P <0.05; Indicates the comparison between the two groups P <0.01; Indicates the comparison between the two groups P <0.001. Detailed Implementation

[0032] The following detailed embodiments are merely illustrative in nature and are not intended to limit the invention or its uses. Furthermore, this document is not limited to the foregoing prior art or the invention itself, or to any theory described in the following detailed embodiments or examples. Unless otherwise stated, the methods, reagents, and conditions used in the examples are conventional methods, reagents, and conditions in the art.

[0033] Preparation Example Preparation of the composition This composition can be formulated into any one of granules, capsules, pills, tablets, powders, or nanoformulations. The following examples are of granules.

[0034] Granules were prepared using conventional methods, using the following qualified medicinal materials: 9g of ginseng (Shanghai Hongqiao Traditional Chinese Medicine Pieces Co., Ltd., batch number: 250609, origin: Baishan, Jilin); 15g of yam (Shanghai Yutiancheng (Shanghai) Pharmaceutical Co., Ltd., batch number: 2025050203, origin: Jiaozuo, Henan); 10g of kudzu root (Shanghai Hongqiao Traditional Chinese Medicine Pieces Co., Ltd., batch number: 250520, origin: Anqing, Anhui); 12g of mulberry (Shanghai Wanshicheng Pharmaceutical Products Co., Ltd., batch number: 20250617-1, origin: Heze, Shandong); and 12g of prepared polygonatum (Shanghai Hongqiao Traditional Chinese Medicine Pieces Co., Ltd., batch number: 250409, origin: [missing information - likely a place name]). 12g of Yunnan Wenshan, 6g of rose petals (Shanghai Dedatang Chinese Medicine Co., Ltd., Lanzhou, Gansu), and 12g of wolfberry (Shanghai Hongqiao Chinese Medicine Pieces Co., Ltd., batch number: 250410, origin: Zhongwei, Ningxia) are pulverized. The pulverized herbs are soaked in 8 times the amount of water for 45 minutes, then decocted in a micro-pressure sealed decoction machine for 18 minutes. A small amount of the decoction is taken and 3g of cinnamon (Shanghai Yanghetang Chinese Medicine Pieces Co., Ltd., batch number: 20250509; origin: Dongxing, Guangxi) is added and boiled together. The decoction is filtered and mixed with other decoctions. 6g of donkey-hide gelatin beads (Shanghai Hongqiao Chinese Medicine Pieces Co., Ltd., batch number: 250701, origin: Hunan) are dissolved in rice wine and added to the above decoction. The mixture is concentrated under reduced pressure at 85°C to a density of 1.10-1.15 (55-65°C) to obtain a clear paste. The obtained extract was spray-dried for 37 minutes to prepare granules, which were then passed through a 100-mesh sieve. A small amount of dextrin was added and mixed evenly, and the mixture was granulated through a 12-mesh sieve. The final granule moisture content was 3.24%. The resulting granules were packaged in aluminum foil bags. When using, the granules were dissolved in purified water and administered to the animals by gavage according to their body weight.

[0035] Test case The composition's effects on improving iatrogenic ovarian insufficiency (POI) and its influence on fertility, ovarian lipid metabolism, oxidative stress indicators, and glucose and lipid metabolism-related transcription factors. Rat models of poikiloma (POI) and cystic ovary syndrome (cystic ovary syndrome) were established using cyclophosphamide (CTX) and levonorgestrel (LNG), respectively. The efficacy of the composition in improving POI was evaluated by measuring rat body weight, ovarian coefficient, serum hormone levels, follicle counts at each stage, and conception rate. The effects of the composition on LNG-induced ovarian lipid metabolism accumulation, antioxidant stress capacity, and glucose and lipid metabolism-related factors were evaluated by detecting the expression levels of ApoA1, RXRα, MDA, and Nrf2.

[0036] 1.1 Animal Purchase and Feeding: In the following examples, female SD rats (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) weighed 215±15g at purchase, were 8-9 weeks old, and were 10-11 weeks old at the start of the experiment. Male SD rats weighed 315±15g at purchase, were 7-9 weeks old. SD rats were housed in rat cages at the Barrier Environment Rodent Room of Shanghai Institute of Biomedical Technology, with no more than 5 rats per cage. They had free access to water and food and were acclimatized in the animal room for 3 days before the experiment; the room temperature was 22-24℃, the relative humidity was 40-70%, and the light and darkness were 12 hours each. After arrival, the animals were acclimatized for one week, and vaginal smears were taken every morning for 5 consecutive days. Animals with irregular estrous cycles were culled.

[0037] 1.2 Construction of a rat ovarian injury model: CTX-induced ovarian insufficiency (POI) model: SD female rats with regular estrous cycles were randomly divided into a blank control group and a CTX-induced POI model group according to body weight. The blank control group consisted of normal rats, fed routinely without any treatment. The solvent control group rats were simultaneously injected intraperitoneally with an equal volume of normal saline (NS). Rats in the CTX-induced POI model group received an initial intraperitoneal injection of 75 mg / kg CTX (with normal saline as the solvent) on day 1 (D1), followed by continuous intraperitoneal (ip) injections of CTX (5 mg / kg) from day 2 to day 21 (D2-D21) for 21 consecutive days to establish the POI model. Estrous cycles were observed for 5 consecutive days from day 10 to day 14 to monitor for any disruption in the estrous cycle. On day 14 after CTX administration, the POI model group animals were further divided into: a model group, a positive control group (estradiol benzoate), a low-dose (0.85 g / kg) group of the composition, and a high-dose (1.7 g / kg) group of the composition, with at least 10 rats in each group.

[0038] LNG-induced cystic ovarian injury model in rats: SD female rats with regular estrous cycles were randomly divided into four groups (n=10 per group) according to body weight: ① blank control group; ② 0.25 mg / kg LNG group; ③ low-dose combination group; and ④ high-dose combination group. Groups ②–④ were administered 0.25 mg / kg LNG by gavage for 28 consecutive days. The blank control group was fed normally without any treatment.

[0039] ApoA1-induced rat ovarian injury model: SD female rats with regular estrous cycles were randomly divided into two groups according to body weight: a blank control group and an ApoA1 injection group. The blank control group was fed normally, while the ApoA1 injection group received 50 μL of ApoA1 solution at a concentration of 50 μg / ml in each ovary.

[0040] 1.3 Grouping and administration of CTX and LNG-induced ovarian injury rats In a CTX-induced rat ovarian injury model: Immediately after CTX injection on day 14, the positive control drug and the test drug were administered until day 35. From day 14 to day 35, the following treatments were given: ③ Model group: Intraperitoneal injection (ip) of CTX (5 mg / kg, day 14-21) + equal dose of pure water by gavage (days 14-35); ④ Positive control group: ip CTX (day 14-21) + subcutaneous estradiol benzoate (0.4 mg / kg, twice weekly, day 14-35); ⑤ Low-dose combination (hereinafter referred to as low-dose): ip CTX (5 mg / kg, day 14-21) + low-dose (0.85 g / kg, day 14-35) by gavage; ⑥ High-dose combination group (hereinafter referred to as high-dose): ip CTX (5 mg / kg, day 14-21) + High dose (1.70 g / kg, days 14-35) administered by gavage. Administer for 21 consecutive days. One hour after administering CTX, administer the test sample (positive control or low and high doses of the combination).

[0041] In the LNG-induced rat ovarian injury model: 8 days after LNG administration, rats in the low-dose and high-dose groups were simultaneously administered a low-dose (0.85 g / kg) and a high-dose (1.70 g / kg) dietary therapy composition by gavage for 21 consecutive days. The blank control group and the 0.25 mg / kg LNG group were administered the corresponding volume of 0.5% CMC-Na by gavage.

[0042] 1.4 Dissection and Tissue Collection: At the end of drug administration, the animals were anesthetized, blood was collected, and the rats were euthanized. Both ovaries and uteruses were dissected. The tissues were weighed, and the ovarian and uterine coefficients were calculated. One ovary and uterine tissue was fixed in 4% paraformaldehyde, routinely embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) to observe histological changes in the ovary and count the different types of follicles. The other ovary was stored at -80°C for protein analysis.

[0043] 1.5 Pathological observation of rat ovarian sections: Histological changes in rat ovaries were observed and recorded under a microscope. The percentage of each follicle was calculated. The total number of follicles = number of primordial follicles + number of growing follicles (primary follicles + secondary follicles) + number of mature follicles + number of atretic follicles + corpus luteum. The percentage of follicles at a certain stage = (number of follicles at that stage / total number of follicles) × 100%. 1.6 Mating: Four rats were kept in each group and mated with male rats in a 2:1 ratio (female:male) for one week after the end of drug administration. The female rats were observed to see if they became pregnant and the number of litters was counted.

[0044] 1.7 Protein Detection: Ovarian tissue was weighed and placed in a 1.5 ml EP tube. The corresponding volumes of RIPA lysis buffer, protease inhibitor, and phosphatase inhibitor were added for lysis. The tissue was cut into small fragments to ensure complete lysis. After thorough lysis, the tissue was centrifuged at 1200×g, 4℃, for 15 min, and the supernatant was collected. Protein concentration was determined according to the instructions of the Sangon Biotech BCA kit. The absorbance was measured at 562 nm, and a standard curve was plotted based on the standard concentrations to calculate the sample protein concentration. The sample was mixed with 5× loading buffer at a 1:4 ratio and denatured at 95℃ for 5-10 min. Electrophoresis was performed on a 10% separating gel at 110V. The membrane was transferred, washed, and incubated with primary antibody (RXRα, Nrf2, ApoA1, or GAPDH, purchased from CST or Abcam) overnight at 4℃. The membrane was washed three times with TBST and incubated with secondary antibody (mouse or rabbit anti-antibody) at 37℃ for 1 h. Wash the membrane with TBST, add developing solution to the PVDF membrane, react in the dark for 1 min, place it in a gel electrophoresis system, photograph the protein bands and analyze them.

[0045] 1.8 Data Statistics Experimental data are expressed as Mean ± SD. Statistical analysis was performed using GraphPad Prism (version 8.0.2, GraphPad Software, San Diego, California USA). Comparisons among multiple groups were performed using ANOVA for significance testing; post-hoc pairwise comparisons were performed using Dunnett's test. For indicators that did not meet the above criteria, comparisons among multiple groups were performed using the Kruskal-Wallis test, and post-hoc pairwise comparisons among multiple groups were performed using an uncorrected Dunn's test. P <0.05 is the standard for statistical significance.

[0046] 1.9 Results: 1.9.1. Effects of the composition on rat body weight and general condition: After 21 days of continuous gavage, compared with the blank control group, the body weight of rats in the model group was reduced ( P >0.05); Compared with the model group, there was no significant change in body weight in the positive group, the low-dose group, and the high-dose group (0.85g / kg, 1.70g / kg) of the composition. P >0.05).

[0047] 1.9.2. Effects of the composition on the organ coefficients of the uterus and ovary in rats Ovarian organ index: After CTX treatment, the ovarian weight and ovarian organ index of rats were reduced compared with the blank control group, indicating that cyclophosphamide can damage rat ovarian tissue, and the ovarian injury model was successfully established. Compared with the model group, the ovarian organ index of the positive control drug (estradiol benzoate) group increased by 19.41%, and the ovarian organ index of rats in the low-dose and high-dose (0.85g / kg, 1.70g / kg) groups increased by 8.79% and 19.05%, respectively. This shows that the composition increases ovarian organ weight in a dose-dependent manner, similar to the effect of estradiol, as shown in Table 1.

[0048] Table 1. Effects of the composition on ovarian wet weight and ovarian organ coefficient in cyclophosphamide-induced POI rats (Mean±SD)

[0049] Uterine organ coefficient: After CTX treatment, the weight of uterine organs and the uterine organ coefficient of rats were reduced compared with the blank control group. Compared with the blank group, the endometrial thickness of rats in the model group did not change significantly; compared with the model group, the endometrial thickness of the positive control group and the low-dose group increased, indicating that the composition can increase endometrial thickness, which is conducive to conception and embryo implantation. See Table 2.

[0050] Table 2. Effects of the composition on the uterine organ coefficient in cyclophosphamide-induced POI rats (Mean ± SD)

[0051] a P <0.05, compared with the blank group; b P <0.05, compared to the model group; c P <0.05, compared with the estradiol benzoate group; d P <0.05, compared with the low-dose group; e P <0.05, compared with the high-dose group 1.9.3 Effect of the composition on the count of follicles at various stages in rat ovaries Primordial follicle rate: Compared with the control group, the primordial follicle rate of rats in the model group was significantly reduced. P <0.05); Compared with the model group, the primordial follicle rate of rats in the positive group and the low-dose (0.85 g / kg) group was increased, and the primordial follicle rate of rats in the high-dose (1.70 g / kg) group was significantly increased ( P<0.05); Growing follicle rate: Compared with the blank group, the number of growing follicles in the model group rats decreased; compared with the model group, the growing follicle rate of rats in the low and high dose groups of the composition (0.85g / kg, 1.70g / kg) showed an increasing trend. Atretic follicle rate: Compared with the blank group, the atretic follicle rate of rats in the model group rats was significantly increased ( P <0.05); Compared with the model group, the rate of atretic follicles in the positive group rats did not change significantly ( P >0.05), the low and high doses of the composition (0.85 g / kg, 1.70 g / kg) of rats showed a decrease in the rate of atretic follicles, while the high dose (1.70 g / kg) group showed a significant decrease in the rate of atretic follicles. P <0.05), indicating that the composition can promote primordial follicle development, delay the depletion of the primordial follicle pool, and reduce the rate of atretic follicles. See Table 3.

[0052] Table 3. Effects of the composition on the follicle rate at each stage of the ovary in cyclophosphamide-induced POI rats (Mean ± SD)

[0053] Note: Experimental data are expressed as Mean ± SD, and statistical analysis was performed using GraphPad Prism 8.0. Comparisons among multiple groups were performed using ANOVA for significance testing; pairwise comparisons among multiple groups were performed using Tukey's test. a P <0.05, compared with the blank group; b P <0.05, compared to the model group; c P <0.05, compared with the estradiol benzoate group; d P <0.05, compared with the low-dose group; e P <0.05, compared with the high-dose group.

[0054] 1.9.4 Effects of the composition on endocrine indicators in rats: In POI diagnosis, elevated FSH levels are a more stable and critical indicator than changes in E2. In this study, compared with the blank control group, after continuous intraperitoneal injection of CTX in rats for 3 weeks, serum FSH and E2 levels were elevated, indicating hormone metabolism disorder and suggesting that cyclophosphamide had begun to damage the HPG axis, but the damage was still in its early stages. Exogenous estradiol treatment did not significantly change FSH levels. After 21 days of oral administration of the combined treatment, serum FSH levels in both the low- and high-dose dietary therapy groups were lower than those in the model group, approaching the levels of the normal control group; LH levels in the low-dose dietary therapy group were lower than those in the model group. See Table 4.

[0055] A detailed analysis of the FSH data revealed that within the FSH ≤ 7.50 mIU / ml range, the proportion of rats in the model group (42.86%, 3 / 7) was significantly higher than that in the low-dose group (85.71%, 6 / 7). Conversely, within the FSH > 7.50 mIU / ml range, the proportion of rats in the model group (57.14%, 4 / 7) decreased to 14.29% (1 / 7) in the low-dose group. (See Table 5.)

[0056] A detailed analysis of the LH data revealed that within the LH ≤ 3.0 mIU / ml range, the proportion of rats in the model group was 71.43% (5 / 7), compared to 42.86% (3 / 7) in the low-dose group. Within the LH > 3.0 mIU / ml range, the proportion of rats in the model group was 28.57% (2 / 7), compared to 14.29% (1 / 7) in the high-dose (1.70 g / kg) group. See Table 6.

[0057] The above results indicate that the composition of the present invention can reduce serum FSH and LH levels and improve hormone metabolism disorders caused by CTX.

[0058] Literature reports that cyclophosphamide (CTX) can overactivate primordial follicles, accelerate the depletion of ovarian reserve, and ultimately lead to a decrease in the number of primordial follicles, manifested by an initial increase followed by a decrease in E2 levels. In this study, the E2 level in the model group was higher than that in the blank control group, indicating that the ovaries had been stimulated by CTX and had begun to deplete follicular reserve. After administration of low and high doses of the composition, the E2 level was lower than that in the model group, suggesting that the composition may be able to antagonize the overactivation of primordial follicles induced by CTX, protect the ovaries, and prevent excessive depletion of ovarian reserve.

[0059] The inventors compared the effects of this composition and the traditional Chinese medicine formula (referring to CN1745775, the formula being: Epimedium 12 parts, Curculigo orchioides 10 parts, Leonurus japonicus 40 parts, donkey-hide gelatin 8 parts, Polygonum multiflorum 30 parts, cinnamon 22 parts, Ligustrum lucidum 40 parts, deer antler 8 parts, and ginseng 30 parts) on serum testosterone (T) levels in female rats. The results showed that the traditional Chinese medicine formula had no significant effect on serum T levels. P >0.05), while this composition reduces serum T levels in a dose-dependent manner at both low and high doses, with a significant difference at high doses ( P <0.05), which indicates that changes in the components of traditional Chinese medicine can significantly affect changes in serum hormone levels, and different compositions of traditional Chinese medicine have significantly different effects.

[0060] Table 4. Effects of the composition on serum FSH, LH, E2 and T levels in cyclophosphamide-induced POI female rats (Mean ± SD)

[0061] Note: Experimental data are expressed as Mean ± SD, and statistical analysis was performed using GraphPad Prism 8.0. Comparisons of serum FSH and E2 among multiple groups were performed using ANOVA for significance testing; pairwise comparisons among multiple groups were performed using Tukey's test; comparisons of serum LH and T data among multiple groups were performed using the Kruskal-Wallis test, and post-hoc pairwise comparisons among multiple groups were performed using Dunn's test. a P <0.05, compared with the blank group; b P <0.05, compared to the model group; c P <0.05, compared with the estradiol benzoate group; d P <0.05, compared with the low-dose group; e P <0.05, compared with the high-dose group.

[0062] Table 5. Stratified analysis of serum FSH levels

[0063] Table 6. Stratified analysis of serum LH levels

[0064] 1.9.5 Effect of the composition on the fertility of POI rats: Four rats were selected from each of the blank control group, model group, and composition groups (two rats each for low and high doses) for mating experiments. The results showed that the pregnancy rate in the blank control group was 75% (3 / 4 rats), the pregnancy rate in the model group was 50% (2 / 4 rats), and the pregnancy rate in both the low and high dose composition groups was 100% (4 / 4 rats). There was no significant change in the number of fetuses among the groups. P >0.05). This indicates that the composition can significantly improve the pregnancy rate in POI rats, as shown in Table 7.

[0065] Table 7. Effects of the composition on pregnancy rate and litter size in cyclophosphamide-induced POI rats (Mean ± SD)

[0066] Note: Experimental data are expressed as Mean ± SD, and statistical analysis was performed using GraphPad Prism 8.0. Comparisons among multiple groups were performed using ANOVA for significance testing; pairwise comparisons among multiple groups were performed using Tukey's test. P <0.05, compared to the model group.

[0067] 1.9.6 Effects of the composition on the organ coefficients of major organs in rats: Compared with the control group, the organ coefficients of the heart, liver, and lungs in the model group rats were increased ( P >0.05), indicating that CTX treatment can affect the heart, liver, and lungs of rats; compared with the model group, the estradiol benzoate group showed significantly increased organ coefficients in the liver, kidney, and lungs ( P <0.05%, indicating that estradiol exacerbated the effects of CTX on the aforementioned organs. The effects of both low- and high-dose groups of the composition on the organ coefficients of the heart, liver, spleen, lungs, and kidneys were weaker than those of the positive control group, especially the liver and kidney organ coefficients, which were significantly lower than those of the estradiol benzoate group. P The result was <0.05%, indicating that the composition was safer than the positive control drug estradiol benzoate. The results are shown in Table 8.

[0068] Table 8. Effects of the composition on changes in organ coefficients of heart, liver, and spleen in POI model rats.

[0069] Note: Experimental data are expressed as Mean ± SD, and statistical analysis was performed using GraphPad Prism 8.0. Comparisons among multiple groups were performed using ANOVA for significance testing; pairwise comparisons among multiple groups were performed using Tukey's test. a P <0.05, compared with the blank group; b P <0.05, compared to the model group; c P <0.05, compared with the positive group; d P <0.05, compared with the low-dose KJ-1 group; e P <0.05, compared with the high-dose KJ-1 group 1.9.7 Effects of the composition on ApoA1 accumulation in rat ovaries and on the expression of glucose and lipid metabolism-related transcription factor RXRα RXRα, a factor associated with glucose and lipid metabolism, is a key molecule that regulates glucose and lipid metabolism, inflammation, and various metabolic diseases. It binds to various other nuclear receptors to form heterodimers, which together regulate the expression of downstream genes.

[0070] Compared with the blank control group, after ApoA1 injection, rats experienced estrous cycle disorder, with a significant increase in interestrus and a significant decrease in proestrus. Figure 1 In rats (A), primordial follicles are lost in the ovaries, follicular development is arrested, and cystic follicles appear. Figure 1 BC), increased total cholesterol in the ovaries, and abnormal lipid metabolism ( Figure 1 In the middle D phase, the number of primordial follicles, growing follicles, and mature follicles decreased significantly, while the number of atretic follicles increased significantly. Figure 1The significant activation of ApoA1 in the ovary (EH), glucose and lipid metabolism-related factors RXRα, and oxidative factor Nrf2 indicates that ApoA1 accumulation in the ovary can impair ovarian function.

[0071] After treatment with progestin-LNG, the expression level of ApoA1 protein was significantly increased compared with the normal control group, and its downstream Nrf2 and RXRα were also upregulated synchronously; after silencing ApoA1 (siApoA1), when LNG was applied again, the expression level of ApoA1 no longer increased, and RXRα and Nrf2 were no longer activated by LNG. Figure 2 The results (A) indicate that ApoA1 is a target of LNG-ECPs that damage the ovaries. LNG damages ovarian function by activating ApoA1. The damage to ovarian function caused by ApoA1 is related to oxidative factors and glucose and lipid metabolism regulators.

[0072] Treatment with low and high doses of the composition significantly downregulated LNG-induced ApoA1 protein expression, while Nrf2 and RXRα expression were also significantly downregulated. Figure 2 (B) This indicates that the composition can alleviate LNG-induced ovarian damage, repair ovarian function, and improve fertility by downregulating the expression of LNG-induced ovarian lipid metabolism protein ApoA1, oxidative factor Nrf2, and glucose and lipid metabolism-related transcription factor RXRα.

[0073] 1.9.8 Effects of the composition on serum antioxidant markers and expression of glucose and lipid metabolism-related transcription factor RXRa in POI rats: Malondialdehyde (MDA) is an indicator of lipid peroxidation; elevated levels suggest increased oxidative stress or cell damage. Superoxide dismutase (SOD) is a core antioxidant enzyme that scavenge free radicals in the body; decreased SOD activity suggests a decline in antioxidant capacity.

[0074] The inventors also used a combination of some common medicinal and edible herbs (ginseng 9g, yam 15g, mulberry 12g, polygonatum 12g, and wolfberry 12g) as a control group to compare the effects of the composition on serum MDA, SOD, and RXRα. The results of this study showed that, compared with the solvent group, the serum MDA and RXRα levels in the model group rats were significantly increased. P <0.05), SOD level slightly decreased ( P >0.05), indicating that CTX and LNG can cause lipid peroxidation damage and abnormal lipid metabolism in POI rats. Although the serum MDA level in the control group was lower than that in the model group, no significant difference was observed. P >0.05); while the serum MDA and RXRα levels in rats in both the low- and high-dose groups of the composition decreased in a dose-dependent manner, with significant differences. P<0.05), suggesting that the composition of the present invention may improve ovarian damage by alleviating lipid peroxidation damage and reducing RXRα expression.

[0075] The above results indicate that variations in the components of traditional Chinese medicine (TCM) can significantly affect antioxidant capacity and the expression of glucose and lipid metabolism-related factors, and TCM compositions with different components exhibit significantly different efficacy. This composition has significant antioxidant effects and improves the expression of glucose and lipid metabolism factors. The results are shown in Table 9.

[0076] Table 9. Effects of the composition on oxidative parameters and retinoid X receptor α (RXRα) expression levels in POI rats (Mean ± SD)

[0077] Note: Experimental data are expressed as Mean ± SD, and statistical analysis was performed using GraphPad Prism 8.0. Comparisons among multiple groups were performed using ANOVA for significance testing; pairwise comparisons among multiple groups were performed using Tukey's test. a P <0.05, compared to the solvent group; b P <0.05, compared with the model group; c P <0.05, compared with the control group; d P <0.05, compared to the low-dose group of the composition; e P <0.05, compared to the high-dose group of the composition.

[0078] In summary, the composition of this invention can improve HPG axis function, improve endocrine levels, increase ovarian weight, increase the number of primordial follicles, reduce the rate of atretic follicles, decrease serum testosterone levels in female rats, downregulate ApoA1 accumulation in the ovaries induced by the contraceptive LNG, regulate ovarian lipid metabolism, downregulate the expression of lipid peroxide MDA, oxidative factor Nrf2, and ovarian glucose and lipid metabolism-related transcription factor RXRα, repair ovarian function, and improve fertility. Its safety profile is superior to estradiol. It can assist in improving pathological ovarian dysfunction or physiological ovarian dysfunction decline.

Claims

1. A composition, comprising or mainly consisting of the following materials in parts by weight: Ginseng 6-9 parts, yam 12-20 parts, rose 3-9 parts, kudzu root 9-15 parts, mulberry 9-15 parts, polygonatum 9-15 parts, donkey-hide gelatin 3-9 parts, cinnamon 1-5 parts, wolfberry 6-12 parts.

2. The composition according to claim 1, wherein, By weight, the composition comprises or mainly consists of the following materials: 8-9 parts ginseng, 13-16 parts yam, 9-11 parts kudzu root, 10-13 parts mulberry, 10-13 parts polygonatum, 5-7 parts rose, 5-7 parts donkey-hide gelatin, 2-4 parts cinnamon, and 9-12 parts wolfberry.

3. The composition according to claim 1 or 2, wherein, The composition further comprises, by weight, 9-15 parts of Poria cocos and 6-12 parts of Angelica sinensis.

4. The composition according to claim 1, wherein, By weight, the composition comprises or mainly consists of the following materials: 9 parts ginseng, 15 parts yam, 10 parts kudzu root, 12 parts mulberry, 12 parts polygonatum, 6 parts rose, 6 parts donkey-hide gelatin, 3 parts cinnamon, and 12 parts wolfberry.

5. A product, which is a pharmaceutical, food, or health product, comprising a composition according to any one of claims 1 to 4 or an extract prepared from a composition according to any one of claims 1 to 4, and optionally excipients acceptable to the respective product.

6. The product according to claim 5, wherein, The product has one or more of the following functions: Improves pathological reproductive function damage and physiological reproductive function decline; Regulates hypothalamic-pituitary-gonadal function; It promotes the development of primordial follicles, delays the depletion of the primordial follicle pool, and reduces atretic follicles; It reduces serum testosterone levels in female animals and improves cystic follicles; Increase the chances of conception; It regulates ovarian lipid metabolism, downregulates the content of ovarian apolipoprotein A1 (APOA1), and downregulates the expression of ovarian retinoid X receptor α (RXRα) and nuclear factor E2-related factor 2 (Nrf2).

7. The product according to claim 5 or 6, wherein, The product is a solid granule, capsule or tablet, or prepared as an oral liquid, soft drink or other liquid preparation or beverage, or prepared as a candy or pastry.

8. The product according to any one of claims 5 to 7, wherein, The extract is prepared by a method comprising the following steps: The ingredients of the above composition, namely ginseng, yam, kudzu root, mulberry, polygonatum, rose, and wolfberry, are decocted in water to obtain the first decoction. A small amount of the first decoction is boiled together with the cinnamon in the formula amount, and then filtered to obtain the second decoction. The second decoction is mixed with the first decoction. The donkey-hide gelatin in the formula amount is dissolved in rice wine and added to the mixed decoction. The mixture is then concentrated under reduced pressure to obtain the extract.

9. The product according to any one of claims 5 to 7, wherein, The extract is prepared by a method comprising the following steps: Take the ginseng, yam, kudzu root, mulberry, polygonatum, rose, and wolfberry in the prescribed amounts of the composition, pulverize them, add 8-10 times the amount of water and soak for 0.5-2 hours, then decoct for 5 minutes to 1 hour to obtain the first decoction. Take a small amount of the first decoction and boil it together with the prescribed amount of cinnamon until boiling, then filter to obtain the second decoction. Mix the second decoction with the first decoction, dissolve the prescribed amount of donkey-hide gelatin in rice wine and add it to the mixed decoction, concentrate under reduced pressure at 80-90°C to a density of 1.10-1.15 to obtain the extract.

10. Use of the composition according to any one of claims 1 to 4 in the preparation of the following products: Products that improve pathological reproductive function damage and physiological reproductive function decline; Products that regulate hypothalamic-pituitary-gonadal axis function; Products that promote the development of primordial follicles, delay the depletion of the primordial follicle pool, and reduce atresia. Products that lower serum testosterone levels in female animals and improve cystic follicles; Products that improve conception rates; Products that regulate ovarian lipid metabolism, downregulate ovarian APOA1 levels, and downregulate the expression of ovarian retinoid receptor X α (RXRα) and nuclear factor E2-related factor 2 (Nrf2).

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