Traditional Chinese medicine composition for treating PCOS pregnancy loss and application thereof

Abstract

The invention discloses a traditional Chinese medicine composition for treating PCOS pregnancy loss and application of the traditional Chinese medicine composition. The traditional Chinese medicine composition comprises the following raw material medicines in parts by weight: 20-40 parts of salted semen cuscutae, 20-40 parts of prepared rehmannia root, 10-20 parts of scutellaria baicalensis, 10-20 parts of dogwood, 20-30 parts of astragalus membranaceus, 20-30 parts of bighead atractylodes rhizome, 20-30 parts of white paeony root, 10-20 parts of parasitic loranthus and 10-20 parts of teasel According to the invention, active component groups (mass spectrum identification) and accurate compatibility design (kidney tonifying and miscarriage prevention theory) are defined, so that (1) the PCOS pregnancy rate is obviously increased (52%-80%, experimental verification); (2) synchronously improving reproduction-metabolism-uterus / placenta triple defects (data support such as a < HOMA-IR <->, a < Total T <->, a < Hif1 alpha > <-> and the like); and (3) the blank in the prior art is filled, and an integrated treatment scheme with both embryo protection and placenta function repair is provided. Experimental data, a molecular mechanism and traditional Chinese medicine theories prove the innovativeness and clinical value of the traditional Chinese medicine composition.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition for treating PCOS pregnancy loss and application thereof. BACKGROUND

[0002] (I) Clinical status of polycystic ovary syndrome (PCOS) and pregnancy loss

[0003] Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder that affects approximately 18% of women of reproductive age worldwide, and its prevalence has doubled in the past decade and is expected to continue to rise. Epidemiological studies have shown that PCOS patients are at higher risk of adverse pregnancy outcomes during pregnancy, including a high rate of early spontaneous abortion (20%-41%), a high rate of miscarriage within the first three months of pregnancy (30%-50%), and a high rate of recurrent miscarriage (30%-40%). PCOS pregnancy loss not only seriously threatens women's physical health, but also brings heavy economic burden and profound psychological trauma, significantly affecting women's quality of life, and thus urgently needs the application of various research strategies and new technologies to uncover the complex mechanisms of PCOS pregnancy loss and provide more precise prevention and treatment options for patients.

[0004] (II) Existing Chinese and Western medicine intervention programs and limitations

[0005] 1. Limitations of existing Western medicine research

[0006] Current research on the mechanisms of PCOS pregnancy loss mainly focuses on: ovarian dysfunction (such as abnormal follicular development and insufficient luteal function); metabolic disorders (the effects of insulin resistance and hyperandrogenemia on embryo implantation); decreased endometrial receptivity (such as decidualization defects and abnormal angiogenesis). However, there is a serious lack of research on the structure and function of the placenta during PCOS pregnancy, especially the regulatory mechanisms of high androgens on the maternal-fetal interface microenvironment and placental structure and function, which have not been elucidated, resulting in a lack of targeted intervention measures.

[0007] 2. Traditional Chinese medicine theory and existing traditional Chinese medicine programs

[0008] PCOS pregnant women have congenital kidney essence deficiency, acquired spleen deficiency, blood circulation, and a solid and stable uterus. Based on the theory of "kidney governing reproduction" and "uterus being connected to the kidney", traditional Chinese medicine regards "tonifying the kidney" as the core principle of preventing miscarriage, and believes that kidney qi and essence are sufficient, which is the basis for a stable uterus and a well-nourished fetus. The classic prescription (such as Shoutai Pill and Yangjing Zhongyu Decoction) can improve the function of the HPO axis, insulin resistance, and endometrial receptivity, and improve the function of the placenta.

[0009] 3. Representative prior art

[0010] Chinese patent CN 117045743 A published on November 14, 2023 discloses a traditional Chinese medicine composition: using Yishen Huatan Huoxue recipe to treat PCOS; predicting ovary-related signaling pathways (such as PI3K-Akt) through network pharmacology; comparing the clinical data and pregnancy outcomes of PCOS patients after treatment; animal experiments confirm that it improves the estrous cycle, sex hormone levels and uterine decidualization function of PCOS mice;

[0011] Limitations:

[0012] a. Insufficient verification of active ingredients, prediction of target points based on network pharmacology, lack of experimental means (such as mass spectrometry) for systematic screening and identification of active ingredients of traditional Chinese medicine compound;

[0013] b. Lack of depth in molecular mechanism, focusing on ovary / uterus function and baseline data, without in-depth exploration of placental structure and specific molecular mechanisms;

[0014] c. Blank in maternal-fetal microenvironment research, without involving dynamic research on the maternal-fetal interface microenvironment (such as immune cell interaction and trophoblast invasion) under the condition of PCOS pregnancy;

[0015] d. Lack of placental function analysis, the compensatory function of placenta during pregnancy, structural adaptability and its correlation with pregnancy loss have not been carefully evaluated.

[0016] 4. Common problems of existing technology

[0017] a. Mechanism recognition limitation: ignoring the driving effect of high androgen environment in PCOS on the maternal-fetal interface microenvironment and placental function; single intervention target: existing traditional Chinese medicine compound targets HPO axis or endometrium, lacking specific regulation of placental function;

[0018] b. Lack of efficacy evidence: insufficient screening of effective components of traditional Chinese medicine, only starting from existing database theory of network pharmacology, without mass spectrometry analysis to fully identify important effective components; although it is confirmed that kidney-tonifying traditional Chinese medicine can regulate baseline data such as sex hormone levels and uterine decidualization function, there is a lack of specific improvement research on pregnancy outcome and specific molecular mechanism evidence;

[0019] c. Lack of theoretical integration: without deep combination of the pathogenesis of "deficiency of kidney and instability of fetal essence" in traditional Chinese medicine and modern placental dysfunction theory, limiting the innovation of integrated traditional Chinese and western medicine prevention and treatment path. SUMMARY

[0020] The present application aims at the deficiency of the prior art in improving PCOS pregnancy loss, and provides a traditional Chinese medicine composition for treating PCOS pregnancy loss and application thereof, and aims to improve the structure and function of the placenta of PCOS, reduce the risk of PCOS pregnancy loss, and optimize the pregnancy outcome by regulating the microenvironment of the maternal-fetal interface of PCOS.

[0021] The first object of the present application is to provide a traditional Chinese medicine composition for treating PCOS pregnancy loss, comprising:

[0022] The raw material medicine is mixed and decocted to obtain a decoction liquid.

[0023] The raw material medicine is mixed and decocted to obtain a decoction liquid.

[0024] Preferably, the raw material medicine is mixed and decocted to obtain a decoction liquid.

[0025] The second object of the present application is to provide a preparation method of the traditional Chinese medicine composition for treating PCOS pregnancy loss, which is to mix the raw material medicine of the traditional Chinese medicine composition for treating PCOS pregnancy loss and decoct to obtain a decoction liquid.

[0026] Preferably, the decoction liquid is concentrated and then freeze-dried to obtain a traditional Chinese medicine composition in the form of freeze-dried powder.

[0027] Preferably, the decoction liquid is concentrated and then pre-frozen at-80 DEG C for greater than or equal to 12 hours, and then freeze-dried for 48 hours at a vacuum degree of less than or equal to 10 Pa to obtain a traditional Chinese medicine composition in the form of freeze-dried powder.

[0028] The third object of the present application is to provide the application of the traditional Chinese medicine composition in the preparation of a medicine for treating PCOS pregnancy loss.

[0029] The mechanism of the traditional Chinese medicine composition of the present application in treating PCOS pregnancy loss is as follows:

[0030] Down-regulating uterine genes: Hbegf, Igfbp1, Ptch;

[0031] Regulating placental genes: Hif1a down, Spint1 up, Pparg down;

[0032] The pregnancy rate is increased to 70-80% (model group 52%).

[0033] The active ingredient group of the traditional Chinese medicine composition of the present application is as follows:

[0034] The composition contains ≥ 56 flavonoids, ≥ 37 glycosides, and ≥ 36 terpenes, 31 of which are verified by literature and reference substances (see Table 1).

[0035] The patent core innovation point of the application is:

[0036] 1. Unique formula and compatibility

[0037] With salted dodder seed and prepared rehmannia as monarch drugs, combined with cornus officinalis and taxillus baccatus as minister drugs, assisted by scutellaria baicalensis and atractylodes lancea as assistant drugs, a traditional Chinese medicine composition formula is formed, which can tonify kidney and essence, consolidate the qi and prevent miscarriage, regulate qi and blood, and prevent dryness, and is used for treating PCOS pregnancy loss caused by kidney deficiency and essence deficiency.

[0038] 2. Deep analysis of components

[0039] Using HESI ion source combined with specific mass spectrometry and chromatography conditions, based on exclusive database qualitative analysis, 187 active ingredients (including 31 verified ingredients) are determined, with flavonoids (56 kinds), glycosides (37 kinds), and terpenes (36 kinds) as the core effective substances, which provide direct evidence for the mechanism of action.

[0040] 3. Significant efficacy verification

[0041] a. Metabolic correction: significantly reduce HOMA-IR, Total T, A4, and reverse high androgen and insulin resistance.

[0042] b. Uterine regulation: down-regulate Hbegf / Igfbp1 / Ptch genes, restore uterine decidua function, and differentiation and invasion ability of trophoblast cells.

[0043] c. Placenta regulation: down-regulate Hif1α / Emp2 / Spint2 / Pparg, up-regulate Spint1, and improve trophoblast differentiation and angiogenesis.

[0044] d. Most of the current inventions focus on endometrial receptivity solutions, while the impact of high maternal androgen and insulin environment on placenta and fetal development is not clear. The present application focuses on improving placental function, optimizing the placental microenvironment, ensuring normal exchange of nutrients at the maternal-fetal interface, and thus reducing pregnancy loss.

[0045] 4. Multi-component synergistic effect, targeting the core mechanism of PCOS pregnancy loss

[0046] a. Technical features

[0047] Through mass spectrometry analysis and subsequent effective component screening, it is determined that the present compound contains 187 active ingredients (including 56 flavonoids, 37 sugars and glycosides, 36 terpenes, etc.), and 31 effective ingredients are selected after subsequent literature verification.

[0048] b. Beneficial effects:

[0049] ①Multi-target intervention: According to the core pathogenesis of PCOS pregnancy loss (theoretical analysis) "deficiency of kidney essence, instability of Chong and Ren channels, and loss of embryo control", the combination of monarch, minister, and assistant (such as Rehmannia glutinosa, Cuscuta chinensis for tonifying kidney and essence, Scutellaria baicalensis for clearing heat and preventing dryness, and Atractylodes macrocephala for tonifying spleen and benefiting qi) to achieve: tonifying kidney and stabilizing embryo (improving embryo implantation environment), regulating metabolism (reducing insulin resistance), balancing sex hormones (reducing androgens), and ultimately improving pregnancy outcome.

[0050] ②Ingredient traceability: 31 ingredients are verified by reference substances to ensure quality controllability (mass spectrometry data support).

[0051] 5. Significant improvement in pregnancy outcome and embryo quality (experimental data support)

[0052] a. Technical features: High-dose compound intervention in PCOS pregnancy loss model (DHT+INS induction).

[0053] b. Beneficial effects

[0054] Increase in total number of embryos and number of normal live fetuses: The pregnancy rate of the model group was only 52.0%, while that of the Chinese medicine group was increased to 70.0%-80.0%; significantly reduced abnormal embryos and reduced embryo loss rate (data). Figure 4

[0055] 6. Effect of regulating metabolism and sex hormone disorder (key mechanism verification)

[0056] a. Technical features

[0057] The compound fully plays the role of regulating glucose and lipid metabolism and hormone pathways through flavonoids (such as kaempferol), sugars and glycosides (such as rhodioside), etc.

[0058] b. Beneficial effects:

[0059] ①Improvement of insulin resistance and glucose metabolism: The medium / high dose group significantly reduced fasting blood glucose AUC, HOMA-IR index, and insulin levels (P<0.01), improving the core pathological characteristics of PCOS.

[0060] ②Correction of sex hormone imbalance: Significantly reduced the elevated levels of total testosterone (Total T) and androstenedione (A4) in the model group (P<0.01), relieving hyperandrogenism.

[0061] 7. Regulation of uterine and placental structure and function (molecular mechanism evidence)

[0062] a. Technical features: The compound regulates uterine and placental function genes.

[0063] ​b.Benefits: TCM formula effectively regulates uterine decidualization function and trophoblast invasion ability, while uterine function related genes Ptch, Hegf, Igfbp1; promotes trophoblast differentiation and angiogenesis: down-regulates labyrinth Hif1α, Emp2 (hypoxia / fibrosis related genes), up-regulates Spint1 (trophoblast differentiation regulator), and restores placental function.

[0064] 8. Advantages over prior art

[0065] Comprehensive regulation vs. single therapy: existing PCOS treatment focuses on reducing androgens or improving insulin resistance (such as metformin), but lacks a protective solution for placental structural function in pregnancy loss. The present invention achieves a multi-component-multi-target-multi-pathway synergy: simultaneously improving metabolism, hormones, uterine and placental function (experimental data fully covered); reducing embryo loss rate.

[0066] 9. Conclusion

[0067] The present invention achieves: ① significantly improves PCOS pregnancy rate (52% → 80%, experimentally verified); ② synchronously improves reproductive-metabolic-uterine / placental triple defects (HOMA-IR↓, Total T↓, Hif1α↓, etc. data support); ③ fills the gap in existing technology: provides an integrated treatment solution with embryo protection and placental function repair. Experimental data, molecular mechanisms and TCM theory three parties confirm its innovation and clinical value. BRIEF DESCRIPTION OF DRAWINGS

[0068] Figure 1 is a TCM formula effective component classification quantity distribution diagram.

[0069] Figure 2is a traditional Chinese medicine compound BPC chart, note: BPC (Base Peak Chromatogram) is the strongest ion intensity of each time point mass spectrum is continuously depicted by the graph. Note: Adenosine, adenosine; Arbutin, arbutin; Gallic acid, tartaric acid; Protocatechuic acid, protocatechuic acid; Cryptochlorogenic acid, cryptochlorogenic acid; (+)-Catechin (+)-catechin; Echinacoside, echinacoside; Caffeic acid, caffeic acid; Paeoniflorin, paeoniflorin; methyl (4S, 5Z, 6S)-5-ethylidene-4-[2-oxo-2-(2R, 3S, 4S, 5R, 6R)-3, 4, 5-trihydroxy-6-[2-(4-hydroxyphenyl) ethoxy] oxan-2-yl] methoxy] ethyl-6-[(2S, 3R, 4S, 5S, 6R)-3, 4, 5-trihydroxy-6-(hydroxymethyl) oxan-2-yl) oxy-4H-pyran-3-carboxyiate, methyl (4S, 5Z, 6S)-5-ethylidene-4-【2-oxo-2-

【

(2R, 3S, 4S, 5R, 6R)-3, 4, 5-trihydroxy-6-【2-(4-hydroxyphenyl) ethoxy

[0070] Figure 3 is the process of PCOS pregnant rats modeling and drug intervention.

[0071] Figure 4 is the regulation of pregnancy of PCOS pregnant rats by traditional Chinese medicine compound, (A) the general observation of pregnancy of rats in different dose treatment groups; (B) the pregnancy rate, total number of embryos, number of viable fetuses and embryo loss rate of rats; (C) the mass and ratio of placenta and embryo of rats. M, median; H, high. Pregnancy rate, viability fetal number, fetal loss rate, fetal number, placenta, fetal weight, placenta / fetal ratio. In all figures, data are expressed as mean ± SEM, *P<0.05, **P<0.01.

[0072] Figure 5The changes of the Chinese medicine compound on the reproductive metabolic phenotypes of PCOS pregnant rats, (A) The changes of the body weight of rats, the liver and the ratio of visceral fat to body weight. (B) The changes of the blood glucose of rats within 120 min of OGTT, the comparison of AUC within 120 min (n=9-10 rats / group) and the changes of HOMA-IR index. (C) The changes of the levels of sex hormones such as T, DHT, DHEA, A4, SHBG and insulin of rats. Weight, body weight; Liver, liver; VAT, Visceral fat, visceral fat; Total T, total testosterone; DHT, dihydrotestosterone; DHEA, dehydroepiandrosterone; A4, androstenedione; SHBG, sex hormone binding globulin; Insulin, insulin; Blood Glucose, blood glucose; AUC, area under the curve; HOMA-IR, homeostatic model assessment of insulin resistance, HOMA-IR = fasting blood glucose (mmol / L) x fasting insulin (μU / mL) / 22.5; Note: a. Control group and DHT+INS group have statistical difference, P<0.05; b. Control group and DHT+INS+H group have statistical difference, P<0.01. In all figures, data are expressed as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001.

[0073] Figure 6 The regulation of the Chinese medicine compound on the key genes of endometrial function of pregnant rats, the expression of key genes of the pregnant uterus of rats in each group was detected by qPCR. Ptch, patched receptor; Hbegf, heparin-binding epidermal growth factor; Igfbp1, insulin-like growth factor binding protein 1. In all figures, data are expressed as mean ± SEM, *P<0.05, **P<0.01. Figure 7 The regulation of the Chinese medicine compound on the key genes of placental function of pregnant rats, the expression of key genes of placental function in the whole placenta, labyrinth zone and basal zone tissues of rats in each group was detected by qPCR. Hif1a, hypoxia-inducible factor 1 alpha; Spin1 / Spint2, serine protease inhibitor Kunitz type 1 / 2; Emp2, epithelial membrane protein 2; Pparg, peroxisome proliferator-activated receptor gamma; In all figures, data are expressed as mean ± SEM, *P<0.05, **P<0.01; In all figures, data are expressed as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. DETAILED DESCRIPTION

[0074] The following examples are further illustrations of the present application and are not intended to limit the present application.

[0075] Example 1:

[0076] (I) Theory of Traditional Chinese Medicine Composition Formulation and Compatibility

[0077] 1. Formula Composition

[0078] This traditional Chinese medicine composition is made from the following raw materials in parts by weight: 30 parts of Cuscuta chinensis (salted), 30 parts of Rehmannia glutinosa (processed), 15 parts of Scutellaria baicalensis, 15 parts of Cornus officinalis, 30 parts of Astragalus membranaceus, 30 parts of Atractylodes macrocephala, 30 parts of Paeonia lactiflora, 15 parts of Taxillus chinensis, and 15 parts of Dipsacus asper.

[0079] 2. Compatibility

[0080] The principal herbs are Rehmannia glutinosa (which nourishes yin and blood, replenishes essence and marrow, and is an essential herb for tonifying kidney yin) and Cuscuta chinensis (which tonifies kidney and replenishes essence, nourishes liver and improves eyesight, calms the fetus, and tonifies kidney yang while also benefiting yin). Together, they work to tonify kidney and replenish essence, strengthen the Chong meridian and calm the fetus.

[0081] Assistant herbs: Cornus officinalis (tonifies the liver and kidneys, astringes and consolidates) assists the principal herb in enhancing its kidney-tonifying power; Taxillus chinensis and Dipsacus asper (tonifies the liver and kidneys, strengthens tendons and bones, and stabilizes pregnancy) work together to enhance the effects of tonifying the liver and kidneys and stabilizing pregnancy.

[0082] The adjuvant herbs are: Scutellaria baicalensis (clears heat and dries dampness, drains fire and detoxifies) to prevent excessive heat from being generated by tonifying; Atractylodes macrocephala (strengthens the spleen and replenishes qi) and Astragalus membranaceus (tonifies qi and strengthens the spleen) work together to strengthen the foundation of acquired constitution and nourish qi and blood; Paeonia lactiflora (nourishes blood and regulates menstruation, softens the liver and relieves pain) enhances blood nourishment and yin tonification and harmonizes the effects of the other herbs.

[0083] Guiding herbs: White peony root regulates menstrual blood, guides other herbs directly to the affected area, and coordinates the interaction of drugs.

[0084] 3. Drug preparation

[0085] a. Initial decoction extraction: 30g of Chinese medicinal herbs including Cuscuta chinensis (salted), Rehmannia glutinosa (processed), Scutellaria baicalensis, Cornus officinalis, Astragalus membranaceus, Atractylodes macrocephala, Paeonia lactiflora, Taxillus chinensis, and Dipsacus asper. 210g of the herbs were added to 2000mL of deionized water and soaked for 30 minutes. A programmed temperature-controlled decoction device was used (high heat for 30 minutes until boiling, then low heat for 90 minutes). The decoction was concentrated under reduced pressure to 150mL and transferred to a clean beaker for later use.

[0086] b. Optimization of decoction: Add 2000mL of deionized water to the dregs twice, decoct and concentrate to 150mL using the same method, combine the two concentrates and concentrate again under reduced pressure to a final volume of 150mL.

[0087] c. Drying treatment: The concentrate was pre-frozen at -80℃ overnight and then freeze-dried (48h, vacuum degree ≤10Pa) to obtain homogenized freeze-dried powder. 53.6g of the powder was weighed and stored in a sealed container away from light (relative humidity <30%, 25℃).

[0088] d. Intragastric liquid preparation: Before use, take a certain amount of freeze-dried powder, dissolve it with double distilled water in a gradient (37°C water bath ultrasonic dissolution for 20-30 min), and prepare the target concentration of drug solution for use.

[0089] e. Traditional Chinese medicine dose calculation: According to the equivalent dose conversion principle (Da=Db×6.13, Da: rat dose, Db: adult dose), combined with the average body weight of rats (300g) and daily intragastric volume (5ml / kg), combined with the body weight of rats (300g) and the intragastric volume (5ml / kg, 10ml / kg), the medium and high dose drug solution was prepared, the concentration of freeze-dried powder was calculated and the target dose drug solution was prepared. In order to maintain the biological activity of the drug, the freeze-dried powder solution was prepared (dissolved in physiological saline, stored at 4°C for ≤7d).

[0090] 4. Mass spectrometry analysis method and results of traditional Chinese medicine compound

[0091] The analytical instrument of this experiment is an ACQUITY UPLC I-Class plus ultra-high performance liquid chromatograph coupled with a QE high-resolution mass spectrometer.

[0092] Chromatographic conditions: Column: ACQUITY UPLC HSS T3 (100mm×2.1mm, 1.8um); Column temperature: 45℃; Mobile phase: Mobile phase: A-water (containing 0.1% formic acid by volume), B-acetonitrile; Flow rate: 0.35mL / min; Injection volume: 2μL.

[0093] The elution conditions are as follows:

[0094] Table 1

[0095]

[0096] Mass spectrometry conditions: HESI ion source was used, with 3800V (positive ion) and -3200V (negative ion) spray voltage, 320℃ capillary temperature and 350℃ auxiliary gas heater temperature for detection, with 35Arb and 8Arb of sheath gas and auxiliary gas flow, respectively, and 50 of S-lens radio frequency level; Data acquisition mode was DDA, with Full MS / dd-MS2 (TOP 8) scanning, mass range 100-1200m / z, Full MS and MS / MS resolution 70000 and 17500, respectively, NCE set to 10, 20, 40, and positive and negative ion scanning modes were used to collect sample mass spectrum signals.

[0097] The results suggest that based on the above mass spectrometry and chromatography conditions, the effective components in the traditional Chinese medicine compound are qualitatively analyzed by using the LuMet-TCM database specific to traditional Chinese medicine in Eurofins. In positive and negative ion modes, each mass spectrometry peak is attributed by retention time, quasi-molecular ion peak, and secondary ion fragment information. The results show that the main components in the traditional Chinese medicine compound are flavonoids, sugars and glycosides, alkaloids, and terpenes. From the effective components in the compound, 187 active components are finally speculated and identified by Score and Fragment score and traditional Chinese medicine traceability, including 56 flavonoids, 37 sugars and glycosides, 36 terpenes, etc. Among them, 31 chemical components are confirmed by literature verification and comparison with reference substances. The results are shown in Table 2 and Figures 1-2 .

[0098] Table 2 Identification of UPLC-Q-TOF-MS / MS of Traditional Chinese Medicine Compound

[0099]

[0100]

[0101] Table 2 Identification of UPLC-Q-TOF-MS / MS of Traditional Chinese Medicine Compound (continued)

[0102]

[0103]

[0104] Note: DH, Radix Rehmanniae Preparata; HQ, Radix Scrophulariae; HQI, Radix Astragali; TSZ, Semen Euryale Fergsonii; BS, Radix Paeoniae Alba; SZY, Fructus Corni; XD, Radix Dipsaci; SJS, Taxilli Herba; BZ, Rhizoma Atractylodis Macrocephalae.

[0105] (II) Design and method of animal experiment

[0106] 1. Model construction and grouping

[0107] a. Animal selection: 10-week-old sexually mature SD rats were selected by screening normal female rats through estrus cycle smears, and mated with male rats at a ratio of 2:1, and pregnancy was confirmed by vaginal tampon or sperm microscopy (GD0.5).

[0108] b. Grouping scheme (n=10):

[0109] Control group (Control): subcutaneous injection of oil agent (Thermo fisher, A0424978, 200ul) in the back of the neck + physiological saline gavage, 1.5ml each time, twice a day in the morning and afternoon;

[0110] Control+M, Control+H), the medium and high dose of traditional Chinese medicine was 0.48 g / ml, 0.96 g / ml, once in the morning and afternoon, with a volume of 1.5 ml;

[0111] Model group (DHT+INS): simulate the environment of hyperandrogenism and insulin resistance, inject DHT (Sigma-Aldrich, A8380, DHT 2.8 mg / kg, dissolved in sesame oil, injection volume 200 ul / d) and INS (Novo Nordisk China Pharmaceutical Co., Ltd., S20191005, INS subcutaneous injection 3 IU in the morning and afternoon, concentration 30 IU / ml) in the back of the neck, and inject physiological saline, 1.5 ml each time, once in the morning and afternoon;

[0112] Model group+medium / high dose of traditional Chinese medicine: simulate the environment of hyperandrogenism and insulin resistance, inject DHT (Sigma-Aldrich, A8380, DHT 2.8 mg / kg, dissolved in sesame oil, injection volume 200 ul / d) and INS (Novo Nordisk China Pharmaceutical Co., Ltd., S20191005, INS subcutaneous injection 3 IU in the morning and afternoon, concentration 30 IU / ml) in the back of the neck+medium / high dose of traditional Chinese medicine (DHT+INS+M, DHT+INS+H), the medium and high dose of traditional Chinese medicine was 0.48 g / ml, 0.96 g / ml, once in the morning and afternoon, with a volume of 1.5 ml.

[0113] c. Intervention process

[0114] Drug administration was continued from pregnancy day 7.5 (GD7.5) to GD13.5, and the drug dose was calculated according to 5 ml / kg (medium dose) and 10 ml / kg (high dose), DHT was 2.8 mg / kg, and insulin was 6 IU / d. OGTT test was performed on GD13.5, and samples were taken the next day (see Figure 3 ).

[0115] (III) Experimental results and analysis

[0116] 1. Pregnancy outcome indicators (see Figure 4 )

[0117] a. Embryo number and quality: the embryo number of the model group (DHT+INS) was significantly reduced, and the number of abnormal embryos was increased. The medium and high dose of traditional Chinese medicine intervention could increase the embryo number, reduce the number of abnormal embryos, reduce the embryo loss rate, and the pregnancy rate was increased from 52.0% in the model group to 70.0%-80.0%.

[0118] b. Placenta quality: the placenta quality and embryo quality of the model group showed a downward trend, and the traditional Chinese medicine treatment group showed an upward trend.

[0119] 2. Hormone and metabolic level regulation

[0120] a. Metabolic indicators: Model group showed significantly increased blood glucose at each time point of OGTT, fasting blood glucose AUC and HOMA-IR index. Chinese medicine treatment in middle / high dose could reduce insulin level and HOMA-IR index (P<0.01).

[0121] b. Sex hormone level: Model group showed significantly increased Total T, DHT, DHEA and A4 levels. Chinese medicine intervention could reduce Total T and A4 (P<0.01) and improve sex hormone disorder (see

[0122] ). Figure 5

[0123] 3. Regulation of uterine structure and function during pregnancy

[0124] Uterine function gene regulation: Chinese medicine treatment effectively down-regulated the expression of Hbegf, Igfbp1 and Ptch (see Figure 6 ).

[0125] 3. Regulation of placental structure and function

[0126] a. Structural changes: Model group showed significantly reduced placental overall area, labyrinth zone (Lz) and basal zone (Bz) area. Short-term Chinese medicine intervention did not significantly improve the structure.

[0127] b. Functional gene regulation: Chinese medicine treatment down-regulated the expression of Hif1a, Emp2, Spint2 and Pparg mRNA in the labyrinth zone of placenta and up-regulated the expression of Spint1, suggesting the recovery effect on trophoblast differentiation, hormone secretion and vascular function (see Figure 7 ).

[0128] (IV) Conclusion

[0129] This study confirmed that this Chinese medicine compound could regulate reproductive metabolic phenotype, restore endometrial receptivity function and placental function, and ultimately improve pregnancy outcome, playing a therapeutic role in the treatment of PCOS pregnancy loss with kidney deficiency and essence deficiency. Although short-term intervention did not significantly improve placental structure, its recovery effect on placental function provided a new direction for the treatment of PCOS pregnancy loss.​

Claims

1. A traditional Chinese medicine composition for treating PCOS pregnancy loss, characterized in that, It comprises: The raw material medicine weight ratio is as follows: salted semen setae 20-40 parts, rehmannia 20-40 parts, radix scrophulariae 10-20 parts, cornus 10-20 parts, astragalus 20-30 parts, atractylodes 20-30 parts, radix paeoniae alba 20-30 parts, taxillus 10-20 parts, and rhizoma drynariae 10-20 parts.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The raw material medicine weight ratio is as follows: salted semen setae 30 parts, rehmannia 30 parts, radix scrophulariae 15 parts, cornus 15 parts, astragalus 30 parts, atractylodes 30 parts, radix paeoniae alba 30 parts, taxillus 15 parts, and rhizoma drynariae 15 parts.

3. The preparation method of the traditional Chinese medicine composition for treating PCOS pregnancy loss according to claim 1, characterized in that, The raw material medicine of the traditional Chinese medicine composition for treating PCOS pregnancy loss is mixed to be decocted to obtain a decocted liquid.

4. The production method according to claim 3, characterized by, The decocted liquid is concentrated and then freeze-dried to obtain a traditional Chinese medicine composition in the form of freeze-dried powder.

5. The production method according to claim 4, characterized by, The decocted liquid is concentrated, pre-frozen at-80℃ for ≥12h, and then freeze-dried for 48h at a vacuum degree of ≤10Pa to obtain a traditional Chinese medicine composition in the form of freeze-dried powder.

6. Use of the traditional Chinese medicine composition for treating PCOS pregnancy loss according to claim 1 in the preparation of a medicine for treating PCOS pregnancy loss.

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