Traditional Chinese medicine composition for preventing or treating early abortion of polycystic ovarian syndrome as well as preparation method and application of traditional Chinese medicine composition

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a traditional Chinese medicine composition for preventing or treating early abortion of polycystic ovarian syndrome as well as a preparation method and application thereof. The traditional Chinese medicine composition is prepared from glossy privet fruit, morinda officinalis, radix rehmanniae preparata, astragalus membranaceus, codonopsis pilosula, Chinese yam, rhizoma atractylodis, radix ophiopogonis, rhizoma anemarrhenae and coptis chinensis, has the effects of tonifying kidney and spleen, clearing heat and drying dampness and harmonizing chong and ren channels, and is mainly used for preventing or treating early abortion of polycystic ovarian syndrome. The traditional Chinese medicine composition disclosed by the invention has a synergistic effect in the aspect of treating early abortion caused by the polycystic ovarian syndrome, systematically deals with complex endocrine metabolism disorder of a patient with the polycystic ovarian syndrome, and provides a brand new treatment strategy for miscarriage prevention. Meanwhile, the novel application of the traditional Chinese medicine composition in preparation of medicines for preventing and treating early abortion of polycystic ovarian syndrome is defined. Therefore, the traditional Chinese medicine composition has important clinical significance and wide market application prospects.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a traditional Chinese medicine composition for preventing or treating early miscarriage in polycystic ovary syndrome, its preparation method, and its uses. Background Technology

[0002] Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder in women of reproductive age, characterized by ovulation disorders, hyperandrogenemia, and insulin resistance. Due to endocrine imbalances, insulin resistance, and chronic inflammation, PCOS patients experience decreased endometrial receptivity and poor oocyte quality, resulting in a high miscarriage rate of 30%–40% in early pregnancy (the first trimester). This seriously harms women's physical and mental health; therefore, effectively reducing the incidence of early miscarriage in women with PCOS is a pressing issue that needs to be addressed.

[0003] Modern medicine has limited effectiveness in treating PCOS combined with early miscarriage, severely impacting population quality and reproductive health. The "Healthy China 2030" Plan and the "14th Five-Year Plan for National Economic and Social Development and the Long-Term Goals for 2035" place "reproductive health" in a crucial position. Therefore, research on the diagnosis and treatment of PCOS, particularly its reproductive health aspects, is a scientific issue of significant national strategic importance. Thus, research on PCOS and its associated early miscarriage is urgently needed.

[0004] Currently, clinical treatment for maintaining pregnancy in PCOS patients mainly relies on Western medicine: 1. Progesterone supplementation: such as progesterone, which is essential for maintaining early pregnancy, but some PCOS patients have progesterone resistance. 2. Metformin: used to improve insulin resistance, but its safety in early pregnancy remains controversial, and its effect on non-insulin-resistant patients is limited. 3. Low-dose aspirin: used to improve endometrial blood flow and prevent thrombosis, but its efficacy as a single drug is uncertain. It is evident that existing Western medicine treatments are mostly "replacement" or "counteracting" therapies, such as exogenous progesterone supplementation, which fail to fundamentally correct the overall endocrine and metabolic disorders in PCOS patients. Furthermore, some patients are insensitive to conventional Western medicine treatments (such as progesterone resistance), and long-term or combined use may lead to side effects such as gastrointestinal reactions and increased liver burden.

[0005] In addition, existing treatments often only target one aspect of miscarriage (such as progesterone supplementation or improved blood flow), failing to simultaneously address the multiple intertwined pathogenesis of PCOS patients, including kidney deficiency, spleen deficiency, phlegm-dampness, blood stasis, and internal heat, resulting in unsatisfactory pregnancy preservation success rates. Current research on traditional Chinese medicine mainly focuses on improving ovulation or metabolic symptoms of PCOS, while there is still a lack of compound traditional Chinese medicine preparations specifically targeting early miscarriage caused by PCOS.

[0006] Therefore, it is necessary to develop a traditional Chinese medicine composition that has a definite curative effect and few side effects, and can prevent and treat early miscarriage in polycystic ovary syndrome. Summary of the Invention

[0007] This invention aims to overcome the shortcomings of existing technologies and solve the following core technical problems: how to provide a traditional Chinese medicine composition that can fundamentally and systematically regulate the constitution of PCOS patients by tonifying the kidneys and spleen, clearing heat and drying dampness, and harmonizing the Chong and Ren meridians, thereby effectively reducing the early miscarriage rate in PCOS patients. This invention not only aims to stabilize pregnancy but also to intervene in the fundamental pathogenesis of "kidney and spleen deficiency with internal damp-heat" that leads to miscarriage in PCOS patients.

[0008] Specifically, the present invention is achieved through the following technical solutions: In a first aspect, the present invention provides a traditional Chinese medicine composition for the prevention or treatment of early miscarriage in polycystic ovary syndrome, the composition being made from the following raw materials in parts by weight: 10-40 parts of Ligustrum lucidum, 5-30 parts of Morinda officinalis, 5-30 parts of Rehmannia glutinosa, 20-50 parts of Astragalus membranaceus, 10-40 parts of Codonopsis pilosula, 20-50 parts of Dioscorea opposita, 5-30 parts of Atractylodes lancea, 5-30 parts of Ophiopogon japonicus, 10-40 parts of Anemarrhena asphodeloides, and 1-20 parts of Coptis chinensis.

[0009] Alternatively, in the above-mentioned traditional Chinese medicine composition, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: 15-30 parts of Ligustrum lucidum, 10-20 parts of Morinda officinalis, 10-20 parts of Rehmannia glutinosa, 25-35 parts of Astragalus membranaceus, 15-30 parts of Codonopsis pilosula, 25-35 parts of Dioscorea opposita, 10-20 parts of Atractylodes lancea, 10-20 parts of Ophiopogon japonicus, 15-30 parts of Anemarrhena asphodeloides, and 5-15 parts of Coptis chinensis.

[0010] As an optional method, in the above-mentioned traditional Chinese medicine composition, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: 20 parts of Ligustrum lucidum, 15 parts of Morinda officinalis, 15 parts of Rehmannia glutinosa, 30 parts of Astragalus membranaceus, 20 parts of Codonopsis pilosula, 30 parts of Dioscorea opposita, 15 parts of Atractylodes lancea, 15 parts of Ophiopogon japonicus, 20 parts of Anemarrhena asphodeloides and 10 parts of Coptis chinensis.

[0011] Alternatively, in the above-mentioned traditional Chinese medicine composition, the traditional Chinese medicine composition has the effects of tonifying the kidney and strengthening the spleen, clearing heat and drying dampness, and regulating the Chong and Ren meridians.

[0012] In a second aspect, the present invention provides a method for preparing the traditional Chinese medicine composition described in the first aspect above, the method comprising the following steps: weighing each raw material in the weight proportions described in the first aspect above, pre-treating, extracting, and concentrating.

[0013] Preferably, in the above preparation method, the traditional Chinese medicine composition of the present invention can be prepared using conventional methods known in the art.

[0014] Preferably, the extraction methods used in this invention include, but are not limited to, decoction extraction, reflux extraction, ultrasonic extraction, and supercritical extraction.

[0015] Preferably, water, commonly used organic solvents in the art, or mixtures thereof can be used to extract the treated active pharmaceutical ingredient.

[0016] Preferably, the organic solvent is methanol, ethanol, propanol, butanol, ethyl acetate, petroleum ether, etc.

[0017] Preferably, the process includes weighing each raw material, pulverizing, extracting, and concentrating.

[0018] Preferably, the extraction method is decoction, and / or the extraction solvent is water or ethanol, and / or the number of decoctions is 1-3, and / or the decoction time for each decoction is 1-3 hours.

[0019] Preferably, water extraction is used (e.g., decoction with water).

[0020] Preferably, the preparation method of the traditional Chinese medicine composition of the present invention includes the following steps: weighing each raw material according to the weight proportions described in the first aspect above, mixing them, soaking them in water for 30-60 minutes, with the amount of water being 8-12 times the total weight of the medicinal materials. First decoction for 1.5-2 hours, filtering to obtain the juice; then adding 6-8 times the amount of water to the dregs, decoct for 1-1.5 hours, and filtering. Combining the two filtrates.

[0021] In a third aspect, the present invention provides the use of the traditional Chinese medicine composition described in the first aspect above, or the traditional Chinese medicine composition prepared by the preparation method described in the second aspect above, in the preparation of a medicament for the prevention or treatment of early miscarriage in polycystic ovary syndrome.

[0022] Alternatively, in the above-described uses, the early miscarriage refers to a miscarriage occurring within the first three months of pregnancy.

[0023] Alternatively, in the above-described uses, the drug may be in solid, semi-solid, or liquid form.

[0024] Alternatively, in the above-described uses, the drug may be a decoction, powder, oral liquid, ointment, granules, tablet, or capsule.

[0025] Compared with the prior art, the present invention has the following advantages: (1) Combining disease and syndrome, with strong targeting: The Chinese medicine composition of this invention is specifically designed for early miscarriage in the specific population of PCOS. The pathogenesis of traditional Chinese medicine is closely combined with the pathology of Western medicine, and its targeting is far stronger than the general pregnancy-preserving formula.

[0026] (2) Multi-target integrated regulation: The Chinese medicine composition of the present invention regulates the function of the hypothalamus-pituitary-ovarian axis by tonifying the kidney, improves the supply of qi and blood by invigorating the spleen and replenishing qi, and improves insulin resistance and intrauterine microenvironment by clearing heat and drying dampness. It works synergistically from multiple key links to reduce the risk of miscarriage.

[0027] (3) Treating both the symptoms and the root cause, with stable therapeutic effect: The Chinese medicine composition of this invention not only "safe" the pregnant fetus, but also "regulate" the body of the unpregnant. By improving the fundamental physical condition of PCOS patients, it creates a better and more stable intrauterine environment for embryo implantation and development, which is expected to reduce the recurrence rate.

[0028] (4) High safety: The Chinese medicine composition of the present invention is derived from natural Chinese medicine, avoiding the potential side effects of Western medicine and safety concerns during pregnancy, and patients have good compliance. Attached Figure Description

[0029] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 ELISA was used to detect various indicators in mouse serum.

[0030] Figure 2 Statistics on the number of surviving mouse embryos and the rate of embryo loss.

[0031] Figure 3 HE staining of mouse decidual tissue.

[0032] Figure 4 TUNEL staining of mouse decidual tissue. Detailed Implementation

[0033] The traditional Chinese medicine composition of this invention is made from raw materials such as Ligustrum lucidum, Morinda officinalis, Rehmannia glutinosa, Astragalus membranaceus, Codonopsis pilosula, Dioscorea opposita, Atractylodes lancea, Ophiopogon japonicus, Anemarrhena asphodeloides, and Coptis chinensis. It has the effects of tonifying the kidney and spleen, clearing heat and drying dampness, and regulating the Chong and Ren meridians. It is mainly used to prevent or treat early miscarriage of polycystic ovary syndrome.

[0034] The compatibility mechanism and formulation explanation of the traditional Chinese medicine composition of this invention are as follows: Chief herbs: Morinda officinalis warms and tonifies kidney essence, Rehmannia glutinosa warms and tonifies essence and blood, and Astragalus membranaceus warms and tonifies qi. These three warming and tonifying herbs serve as the chief herbs, targeting the characteristics of PCOS caused by cold and dampness, and together tonifying qi, blood, and essence. The combined use of Morinda officinalis and Rehmannia glutinosa enhances the ability to warm and tonify kidney essence, while Astragalus membranaceus warms and tonifies the qi of the whole body to aid in the qi transformation effect.

[0035] The assistant herbs are: Codonopsis pilosula and Dioscorea opposita, which are neutral in nature and sweet in taste. When used together, they strengthen the spleen and replenish qi, thus aiding the spleen's digestive function. Atractylodes lancea is pungent and warm, which dries dampness, strengthens the spleen, and dispels wind and cold. These three herbs act as assistant herbs, providing both tonification and dispersion, targeting the pathogenesis of spleen qi deficiency and internal dampness.

[0036] Assistant drug: Glossy privet fruit regulates the thoroughfare and conception vessels, replenishes essence and nourishes blood, and helps the monarch drug to tonify the kidney and replenish essence. Phlegm-dampness is transformed from body fluid. The internal generation of phlegm-dampness involves the pathogenesis of water metabolism, and body fluid must be damaged. Anemarrhena rhizome and Ophiopogon root clear heat and nourish yin, promote the production of body fluid and relieve thirst, and restrict the pungent and dry nature of the monarch drug to prevent yin injury and dryness transformation, and can also block the pathogenesis change of qi stagnation transforming into heat.

[0037] Assistant envoy drug: Coptis chinensis is bitter and cold, clears heat, dries dampness and resolves phlegm, and has the effect of reducing the excessive fire of the thoroughfare vessel. It is the only bitter and cold drug in the formula, which can not only harmonize the formula to warm yang without generating dryness, but also harmonize the formula to nourish yin without generating dampness.

[0038] In addition, the combination of Dioscorea opposita, Coptis chinensis, Anemarrhena rhizome and Ophiopogon root can also be used to treat the condition of secondary transmission of PCOS to consumptive thirst or already developed consumptive thirst.

[0039] The whole formula is formulated in such a way that it tonifies the kidney without generating heat, strengthens the spleen without causing stagnation, clears heat without damaging yang, and removes dampness without injuring yin, and together they achieve the effects of tonifying the kidney and strengthening the spleen, clearing heat and preventing miscarriage.

[0040] The efficacy and main treatment of each medicinal flavor in the traditional Chinese medicine composition described in the present invention are as follows: "Glossy privet fruit" is the mature fruit of Ligustrum lucidum Ait. of the genus Ligustrum in the family Oleaceae, Glossy Privet which has the effects of nourishing the liver and kidney, improving eyesight and blackening hair. It tastes sweet and bitter, is cool in nature, and belongs to the liver and kidney meridians. It is mainly used to treat liver-kidney yin deficiency, dizziness and tinnitus, soreness and weakness of the waist and knees, premature whitening of hair, unclear vision, internal heat and thirst, and steaming bone fever. The role of "glossy privet fruit" in the traditional Chinese medicine composition of the present invention is to nourish the yin of the liver and kidney, clear away asthenic heat, and consolidate the thoroughfare and conception vessels.

[0041] "Morinda officinalis root" is the dried root of Morinda officinalis How of the family Rubiaceae, Indian Mulberry which is mainly produced in Guangdong and Guangxi. It tastes sweet and pungent, is slightly warm in nature, and belongs to the kidney and liver meridians. It has the effects of tonifying the kidney yang, strengthening the muscles and bones, and dispelling wind-dampness, and is mainly used to treat impotence and spermatorrhea, cold uterus and infertility, irregular menstruation, cold pain in the lower abdomen, rheumatic arthralgia, and flaccidity of muscles and bones. The role of "Morinda officinalis root" in the traditional Chinese medicine composition of the present invention is to tonify the kidney yang, strengthen the muscles and bones, warm the uterine cavity, and together with glossy privet fruit, achieve the effect of tonifying both yin and yang.

[0042] "Prepared rehmannia root" is a traditional Chinese medicine prepared by processing raw rehmannia root. Traditional medicine believes that it tastes sweet, is slightly warm in nature, and belongs to the liver and kidney meridians, and has the effects of enriching blood and nourishing yin, replenishing essence and marrow. Clinically, it is often used to treat sallow complexion, dizziness and palpitation caused by blood deficiency, and soreness and weakness of the waist and knees, night sweating and spermatorrhea caused by liver-kidney yin deficiency. Because of its greasy nature, it is likely to hinder digestion, so those with weak spleen, fullness in the middle, loose stools, and stagnant phlegm should use it with caution. The role of "prepared rehmannia root" in the traditional Chinese medicine composition of the present invention is to nourish yin and blood, replenish essence and marrow, and provide a material basis for the fetal element.

[0043] "Astragalus membranaceus" is the plant Astragalus membranaceus (Fisch.) Bunge of the genus Astragalus in the family Leguminosae, Astragalus membranaceus (Fisch.) Bge. Var. mongholicus (Bge.) Hsiao or Astragalus mongholicus Bunge, Astragalus membranaceus (Fisch.) Bge.The dried root of Astragalus membranaceus. It has a sweet taste and slightly warm nature; it enters the spleen and lung meridians. It has the effects of tonifying qi and raising yang, consolidating the exterior and stopping sweating, promoting diuresis and reducing swelling, generating fluids and nourishing blood, promoting circulation and relieving pain, promoting pus drainage and detoxification, and promoting wound healing and tissue regeneration. It is mainly used to treat qi deficiency and fatigue, poor appetite and loose stools, sinking of middle qi, chronic diarrhea and rectal prolapse, hematochezia and metrorrhagia, spontaneous sweating due to exterior deficiency, edema due to qi deficiency, internal heat and thirst, blood deficiency and chlorosis, hemiplegia, numbness and pain, carbuncles and boils that are difficult to heal, and chronic ulcers that do not heal. In the herbal composition of this invention, Astragalus membranaceus plays the role of tonifying qi and consolidating the exterior, strengthening the spleen and raising yang; sufficient qi ensures the fetus is supported.

[0044] "Codonopsis" refers to the plant Codonopsis pilosula, belonging to the Campanulaceae family. Dangshen (Codonopsis pilosula (Franch.) Nannf.) Codonopsis pilosula Dangshen Modesta (Codonopsis pilosula Nannf. var. modesta (Nannf.) L.T.Shen) or Sichuan Codonopsis Codonopsis tangshen Oliv. The dried root of the herb. The first two are mainly produced in Gansu and Sichuan; the latter is mainly produced in Sichuan, Hubei, and Shaanxi. It is neutral in nature, sweet in taste, and enters the spleen and lung meridians. It has the effects of strengthening the spleen and lungs, nourishing blood and promoting body fluids. It is mainly used to treat spleen and lung qi deficiency, poor appetite and fatigue, cough and wheezing, qi and blood deficiency, sallow complexion, palpitations and shortness of breath, thirst due to fluid depletion, and internal heat and thirst. The role of "Codonopsis pilosula" in the herbal composition of this invention is to tonify the middle energizer and replenish qi, strengthen the spleen and lungs, and enhance the qi-tonifying effect of Astragalus membranaceus.

[0045] "Yam" refers to the plant *Dioscorea opposita*, belonging to the genus *Dioscorea* of the family Dioscoreaceae. Yam The rhizome of *Dioscorea opposita*. It has a sweet taste and neutral properties; it enters the spleen, lung, and kidney meridians. It has the effects of tonifying the spleen and stomach, promoting body fluid production and benefiting the lungs, and tonifying the kidneys and astringing essence. It is mainly used for spleen deficiency with poor appetite, chronic diarrhea, lung deficiency with cough and asthma, kidney deficiency with seminal emission, leukorrhea, frequent urination, and thirst due to deficiency heat. In the herbal composition of this invention, *Dioscorea opposita* tonifies the spleen and stomach, tonifies the kidneys and astringes essence, tonifies both qi and yin, and balances the three jiaos (upper, middle, and lower burners).

[0046] "Atractylodes" refers to the plant Atractylodes lancea, belonging to the genus Atractylodes in the family Asteraceae. Honewort Or Northern Cangshu Large-headed Atractylodes Rhizome The dried rhizome of *Atractylodes lancea*. It has a pungent and bitter taste, and is warm in nature; it enters the spleen, stomach, and liver meridians. It has the effects of drying dampness and strengthening the spleen, dispelling wind and cold, and improving eyesight. It is mainly used to treat dampness obstructing the middle jiao, abdominal distension, diarrhea, edema, beriberi, rheumatic pain, colds due to wind and cold, night blindness, and blurred vision. In this invention's herbal composition, *Atractylodes lancea* functions to dry dampness and strengthen the spleen, remove phlegm and dampness from the body, improve PCOS phlegm-dampness constitution, and clear obstacles for fetal health.

[0047] "Ophiopogon japonicus" refers to the plant Ophiopogon japonicus, belonging to the Liliaceae family. Lilyturf Root The dried tuberous root of Ophiopogon japonicus. It tastes sweet and slightly bitter, is slightly cold in nature, and enters the stomach, lung, and heart meridians. It has the effects of nourishing yin and moistening the lungs, benefiting the stomach and promoting fluid production, and clearing the heart and relieving irritability. It is mainly used to treat dry cough due to lung dryness, cough due to yin deficiency, sore throat, thirst due to fluid depletion, internal heat and thirst, irritability and insomnia, and constipation due to intestinal dryness. In the herbal composition of this invention, Ophiopogon japonicus nourishes yin and promotes fluid production, clears the heart and relieves irritability, moistens the lungs and clears the stomach, and prevents the warming and drying effects of other herbs.

[0048] "Anemarrhena asphodeloides" refers to the plant Anemarrhena asphodeloides, belonging to the genus Anemarrhena in the family Liliaceae. Anemarrhena RhizomeThe dried rhizome of *Anemarrhena asphodeloides*. It tastes bitter and sweet, and is cold in nature; it enters the lung, stomach, and kidney meridians. It has the effects of clearing heat and purging fire, nourishing yin and moistening dryness. It is mainly used to treat exogenous febrile diseases, high fever and thirst, dry cough due to lung heat, steaming bone fever, internal heat and thirst, and constipation due to intestinal dryness. In the herbal composition of this invention, *Anemarrhena asphodeloides* acts to clear heat and purge fire, nourish yin and moisten dryness, targeting the yin deficiency and internal heat mechanism of PCOS.

[0049] "Huanglian" refers to the plant *Coptis chinensis*, belonging to the genus *Coptis* in the family Ranunculaceae. Chinese Goldthread Coptis triangularis Coptis Coptis deltoidea C.Y. Cheng et Hsiao Or Cloud Link Coptis teeta Wall. The dried rhizomes of the herbs. The above three are commonly known as "Wei Lian," "Ya Lian," and "Yun Lian," respectively. Wei Lian and Ya Lian are mainly produced in Sichuan and Hubei. Yun Lian is mainly produced in Yunnan. It is bitter and cold in nature; it enters the heart, spleen, stomach, liver, gallbladder, and large intestine meridians. It has the effects of clearing heat and drying dampness, purging fire and detoxifying. Internally, it can be used to treat damp-heat fullness, vomiting and acid reflux, diarrhea, jaundice, high fever and delirium, excessive heart fire, restlessness and insomnia, palpitations, hematemesis due to blood heat, red eyes, toothache, thirst, carbuncles and boils; externally, it can treat eczema, damp sores, and ear discharge. In the herbal composition of this invention, "Huang Lian" functions to clear heat and dry dampness, purge fire and detoxify. Modern research has confirmed that it can improve insulin resistance and clear damp-heat in the uterus.

[0050] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0051] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0052] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0053] Preparation Example: A traditional Chinese medicine composition for the prevention or treatment of early miscarriage in polycystic ovary syndrome (Huanglianqishan Yin, HLQSY), said traditional Chinese medicine composition is made from the following raw materials: 20 g of Ligustrum lucidum, 15 g of Morinda officinalis, 15 g of Rehmannia glutinosa, 30 g of Astragalus membranaceus, 20 g of Codonopsis pilosula, 30 g of Dioscorea opposita, 15 g of Atractylodes lancea, 15 g of Ophiopogon japonicus, 20 g of Anemarrhena asphodeloides, and 10 g of Coptis chinensis.

[0054] The preparation method of the traditional Chinese medicine composition includes the following steps: Mix the above ten medicinal materials, soak them in water for 60 minutes (the amount of water is 8 times the total weight of the medicinal materials), decoct for 1.5 hours initially, filter and collect the juice; add 6 times the amount of water to the dregs, decoct for about 1 hour, and filter again. Combine the two filtrates, vacuum seal them, and store them in a refrigerator at 4°C for later use.

[0055] Example of effect: To verify the efficacy of the traditional Chinese medicine composition of this invention, a pregnancy maintenance experiment was conducted in a PCOS model pregnant mouse.

[0056] 1. Experimental Materials and Methods Pregnant mice that successfully developed the model were randomly divided into a blank control group, a model control group, a low-dose group of the traditional Chinese medicine composition of this invention, a medium-dose group of the traditional Chinese medicine composition of this invention, and a high-dose group of the traditional Chinese medicine composition of this invention. Specific groupings included: Control, PCOS, PCOS+HLQSY-L group, PCOS+HLQSY-M group, PCOS+HLQSY-H group, and PCOS+berberine (BER) group. Berberine (BER) is the key active ingredient in the traditional Chinese medicine composition Huanglianqishan Yin (HLQSY) of this invention.

[0057] 1.1 Animal Model Construction For the PCOS model, mice were injected subcutaneously into the back of the neck with dehydroepiandrosterone (DHEA, 6 mg / 0.1 kg) dissolved in 0.1 mL of soybean oil for 21 consecutive days to establish the PCOS mouse model. The control group was administered the same volume of soybean oil to the back of the neck daily.

[0058] For the PCOS pregnancy model, the PCOS animal models and their controls were mated with age-matched male ICR mice (female:male = 2:1), and pregnancy was initially confirmed by detecting vaginal plugs; the corresponding dates were recorded as gestational days (GD). Successfully established PCOS pregnancy mouse models were randomly divided into PCOS group, PCOS+HLQSY-L group, PCOS+HLQSY-M group, PCOS+HLQSY-H group, and PCOS+BER (MCE, USA) group. The control and model groups were administered PBS by gavage. HLQSY at low, medium, and high concentrations were 1.46 g / kg / d, 2.92 g / kg / d, and 5.84 g / kg / d, respectively. The PCOS+BER group received 200 mg / kg / d BER. Treatment continued until days 8 and 14 (GD8 and GD14), at which point the mice were euthanized using intraperitoneal isoflurane (MCE, USA), and decidual tissue and blood samples were collected.

[0059] 1.2 ELISA Serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), free testosterone, and total testosterone in mice were detected using a mouse ELISA kit (ELK Biotechnology, China). The ELISA procedure was performed according to the manufacturer's instructions. The absorbance (OD) values ​​of each well were measured at 450 nm using a microplate reader, a standard curve was plotted, and the concentrations of each hormone, inflammatory cytokine, and AMH were calculated.

[0060] 1.3 Number of surviving embryos and embryo loss rate The number of surviving embryos and the embryo loss rate are assessed by observing the morphology of the uterus. A characteristic feature of a surviving embryo is the absence of blood flow within the uterus, well-developed embryos, and a large, slightly reddish size. If the uterus is "bamboo-shaped," the embryo volume is significantly reduced, the embryos within the uterus are dark brown, and embryo resorption is confirmed. Embryo loss rate = (Number of resorbed embryos / (Number of resorbed embryos + Number of surviving embryos)) × 100%.

[0061] 1.4 HE staining Clinical and mouse decidual tissues were fixed in 4% paraformaldehyde (Beyotime, China), embedded in paraffin, and prepared into 4 μm tissue sections. After dewaxing with xylene, the sections were rehydrated with graded ethanol, stained with hematoxylin (Beyotime, China) and eosin (Beyotime, China), cleared, and mounted with neutral resin (Beyotime, China). Histopathological changes were observed and photographed under a microscope (Olympus, Japan).

[0062] 1.5 TUNEL staining Apoptosis was assessed using the TUNEL assay kit (Millipore, USA) according to the manufacturer's instructions. First, tissue sections were fixed with 4% paraformaldehyde (Beyotime, China) and then treated with TUNEL reagent in the dark for 1 h. Cells were then washed with PBS. Nuclei were stained with DAPI (Beyotime, China), dehydrated, cleared, and mounted. Finally, apoptotic cells were observed under a fluorescence microscope (Olympus, Japan).

[0063] 1.6 Statistical Analysis Statistical analysis and graphing were performed using SPSS v21.0 (SPSS, USA) and GraphPad Prism 9.0 (GraphPad, USA). Data are expressed as mean ± standard deviation. The Shapiro-Wilk method was used for normality testing, independent samples t-tests were used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. P < 0.05 was considered statistically significant.

[0064] 2. Experimental Results (1) The serum levels of free testosterone, total testosterone, LH, or FSH in mice in the Control and PCOS groups were detected by ELISA. The results showed that the levels of LH, total testosterone, and free testosterone were increased in the PCOS group, while the FSH level was decreased, indicating that the PCOS model was successfully established (see [link to study]). Figure 1 ).

[0065] (2) The number of surviving embryos and the embryo loss rate were measured on days 8 and 14. Compared with the control group, the number of surviving embryos in the model group was significantly reduced and the embryo loss rate was significantly increased. With the increase of HLQSY drug concentration, the number of surviving embryos gradually increased and the embryo loss rate gradually decreased (see Figure 2 ).

[0066] (3) HE staining of decidual tissue showed that, compared with the control group, the decidual tissue lesions in the PCOS group were more severe. However, the addition of HLQSY significantly improved the decidual tissue lesions, and the treatment with high concentrations of HLQSY and BER was even more effective (see Figure 3 ).

[0067] (4) TUNEL assays were performed to detect the number of apoptotic cells at the maternal-fetal interface. The results showed that the number of apoptotic cells at the maternal-fetal interface was higher in the PCOS group than in the Control group. HLQSY inhibited apoptosis at the maternal-fetal interface in a dose-dependent manner, and BER also showed a good effect in improving apoptosis (see [link to study]). Figure 4 ).

[0068] The above results indicate that the herbal composition HLQSY of this invention significantly increases the number of surviving embryos and reduces the embryo loss rate in PCOS mice in a dose-dependent manner. Furthermore, the severity of decidual tissue lesions gradually improves, the number of apoptotic cells gradually decreases, and its key active ingredient BER also exhibits good therapeutic effects. This demonstrates that the herbal composition of this invention can effectively treat early miscarriage caused by PCOS.

[0069] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A traditional Chinese medicine composition for the prevention or treatment of early miscarriage in polycystic ovary syndrome, characterized in that: The traditional Chinese medicine composition is made from the following raw materials in parts by weight: 10-40 parts of Ligustrum lucidum, 5-30 parts of Morinda officinalis, 5-30 parts of Rehmannia glutinosa, 20-50 parts of Astragalus membranaceus, 10-40 parts of Codonopsis pilosula, 20-50 parts of Dioscorea opposita, 5-30 parts of Atractylodes lancea, 5-30 parts of Ophiopogon japonicus, 10-40 parts of Anemarrhena asphodeloides, and 1-20 parts of Coptis chinensis.

2. The traditional Chinese medicine composition according to claim 1, characterized in that: The traditional Chinese medicine composition is made from the following raw materials in parts by weight: 15-30 parts of Ligustrum lucidum, 10-20 parts of Morinda officinalis, 10-20 parts of Rehmannia glutinosa, 25-35 parts of Astragalus membranaceus, 15-30 parts of Codonopsis pilosula, 25-35 parts of Dioscorea opposita, 10-20 parts of Atractylodes lancea, 10-20 parts of Ophiopogon japonicus, 15-30 parts of Anemarrhena asphodeloides, and 5-15 parts of Coptis chinensis.

3. The traditional Chinese medicine composition according to claim 2, characterized in that: The traditional Chinese medicine composition is made from the following raw materials in parts by weight: 20 parts of Ligustrum lucidum, 15 parts of Morinda officinalis, 15 parts of Rehmannia glutinosa, 30 parts of Astragalus membranaceus, 20 parts of Codonopsis pilosula, 30 parts of Dioscorea opposita, 15 parts of Atractylodes lancea, 15 parts of Ophiopogon japonicus, 20 parts of Anemarrhena asphodeloides, and 10 parts of Coptis chinensis.

4. The traditional Chinese medicine composition according to claim 1, characterized in that: The traditional Chinese medicine composition has the effects of tonifying the kidney and spleen, clearing heat and drying dampness, and regulating the Chong and Ren meridians.

5. The method for preparing the traditional Chinese medicine composition according to any one of claims 1 to 4, characterized in that: The preparation method includes the following steps: weighing each raw material according to the weight proportions of any one of claims 1 to 4, pre-treating, extracting, and concentrating.

6. The use of the traditional Chinese medicine composition according to any one of claims 1 to 4, or the traditional Chinese medicine composition prepared by the preparation method according to claim 5, in the preparation of a medicament for the prevention or treatment of early miscarriage in polycystic ovary syndrome.

7. The use according to claim 6, characterized in that: The term "early miscarriage" refers to miscarriage within the first three months of pregnancy.

8. The use according to claim 6, characterized in that: The drug is in solid, semi-solid, or liquid form.

9. The use according to claim 8, characterized in that: The medicine is in the form of decoction, powder, oral liquid, ointment, granules, tablets or capsules.

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