Traditional Chinese medicine Li medicine compound composition for treating premature ovarian insufficiency and application thereof

Abstract

The invention belongs to the field of medicines, and particularly relates to a traditional Chinese medicine Li medicine compound composition for treating early-onset ovarian insufficiency and application of the traditional Chinese medicine Li medicine compound composition. The traditional Chinese medicine composition can significantly improve the survival rate of SD rat granular cells, and has a significant treatment effect on POI rats. After the traditional Chinese medicine composition is clinically taken by a patient, the traditional Chinese medicine syndrome points of POI patients can be remarkably improved, the effect is quick, the cure rate is high, the curative effect is good, and the treatment effect on premature ovarian insufficiency is remarkable.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to a traditional Chinese medicine Liyao compound composition for treating premature ovarian insufficiency and an application thereof. Background Art

[0002] Premature ovarian insufficiency (POI) refers to a clinical syndrome characterized by a decline in the ovarian pool reserve due to various factors before the age of 40, leading to follicular dysfunction and consequent ovarian hypoactivity. It is characterized by menstrual irregularities (such as amenorrhea or oligomenorrhea) accompanied by high gonadotropin levels and low estrogen levels. It manifests as secondary amenorrhea and infertility, often accompanied by perimenopausal symptoms such as nighttime sweating, insomnia, and memory loss. The severity of clinical symptoms varies, with younger patients experiencing milder symptoms. The clinical diagnostic criteria are as follows: amenorrhea or oligomenorrhea for four months before the age of 40, two consecutive serum follicle-stimulating hormone (FSH) levels greater than 25 IU / L separated by >4 weeks, and low estrogen levels. Furthermore, an FSH level of 15-25 IU / L indicates a high-risk group for premature ovarian insufficiency. Since premature ovarian insufficiency will directly affect the patient's fertility, timely intervention and treatment of patients with premature ovarian insufficiency and restoration of their ovarian function are currently urgent issues that need to be addressed.

[0003] Currently, hormone replacement therapy (HRT), the primary treatment option for POI, can alleviate symptoms but carries an increased risk of thrombosis and breast cancer, and has limited impact on restoring ovarian function. In contrast, Traditional Chinese Medicine (TCM), with its holistic approach and multi-target intervention, offers unique advantages in improving treatment safety and ovarian function. However, existing TCM compositions for POI are limited to simple combinations of traditional kidney-tonifying, spleen-strengthening, and liver-soothing herbs. These compositions suffer from several significant deficiencies: first, they rely excessively on commonly available Chinese medicinal materials from mainland China, lacking innovative applications of specialized herbs; second, they fail to fully integrate the therapeutic essence of classic formulas; and third, they face bottlenecks in improving clinical efficacy, making it difficult to meet patient needs. This situation urgently requires a breakthrough solution. Based on this, we innovatively propose a treatment strategy that combines classic TCM formulas with the strengths of Li medicine and authentic Hainan herbs. Hainan's unique geographical environment and climatic conditions have nurtured specialty medicinal herbs such as gujianglong (Longjiang Dragon) and agarwood. Modern pharmacological research has confirmed that these herbs contain unique active ingredients. For example, the flavonoids in gujianglong (Longjiang Dragon) possess significant anti-inflammatory and antioxidant properties, while the sesquiterpenes in agarwood have unique regulatory functions on the neuroendocrine system. Combining these specialty herbs with traditional classical prescriptions leverages both the systemic conditioning benefits of classic formulas and the unique efficacy of these herbs, creating an innovative treatment model that combines the essence of traditional Chinese medicine with distinctive characteristics. This innovative approach, integrating the essence of traditional Chinese medicine, not only provides new breakthroughs in the treatment of POI but also opens new paths for the modernization of Traditional Chinese Medicine. Summary of the Invention

[0004] The invention provides an alkaloid compound, an extraction method and application thereof, which can be effective.

[0005] The technical solution of the present invention is achieved as follows:

[0006] Disclosed is a traditional Chinese medicine Liyao compound composition for treating premature ovarian insufficiency. Each dose comprises the following components by weight: 10-30g of Cuscuta seeds, 10-30g of Rhizoma Cynoglossi, 10-30g of Radix Dipsaci, 10-15g of Radix Rehmanniae Preparata, 10-15g of Radix Angelicae Sinensis, 10-15g of Rhizoma Chuanxiong, 10-15g of Radix Paeoniae Alba, 5-15g of Radix Bupleuri, 5-15g of Atractylodes Macrocephalae, 10-20g of Radix Pseudostellariae, 4-8g of Amomum villosum, 10-20g of Lilium Bupleurum, 10-20g of Radix Salviae Miltiorrhizae, 5-15g of Radix Scutellariae, 4-8g of Aquilariae Resinica, and 4-8g of Radix Glycyrrhizae Preparata.

[0007] Furthermore, the traditional Chinese medicine Liyao compound composition comprises the following components by weight in each dose: Cuscuta chinensis 20g, Rhizoma Cynoglossi 20g, Radix Dipsaci 20g, Rehmannia glutinosa 12g, Angelica sinensis 12g, Ligusticum chuanxiong 12g, White Peony 12g, Bupleurum 10g, Atractylodes macrocephala 10g, Pseudostellariae pseudoginseng 15g, Amomum villosum 6g, Lilium 15g, Salvia miltiorrhiza 15g, Scutellaria baicalensis 10g, Aquilaria sinensis 6g, and Radix Glycyrrhizae 6g.

[0008] The preparation method of the above-mentioned Chinese medicine composition comprises the following steps:

[0009] Take the prescribed amount of Cuscuta australis and wrap it with sterile gauze, then add the prescribed amount of Rhizoma Cyperi, Radix Dipsaci, Radix Rehmanniae Preparata, Radix Angelicae Sinensis, Rhizoma Chuanxiong, White Peony Root, Radix Bupleuri, Atractylodes Macrocephalae, Radix Pseudostellariae, Fructus Amomi, Lilium, Salvia miltiorrhizae, Radix Scutellariae, Aquilariae, and Radix Glycyrrhizae, add water and boil, filter to remove the residue to obtain the medicinal liquid.

[0010] Furthermore, decoct twice and combine the two medicinal liquids; add water to completely immerse the medicinal materials and 2 to 3 cm higher during the first decoction, and decoct for 25 to 30 minutes; add 1 / 2 to 2 / 3 of the water in the second decoction, and decoct for 20 to 30 minutes; decoct until the volume of the medicinal liquid is 1 / 3 of the volume of the added water.

[0011] A traditional Chinese medicine Liyao compound preparation for treating premature ovarian insufficiency comprises the above-mentioned traditional Chinese medicine Liyao compound composition.

[0012] The Chinese medicine preparation may further include pharmaceutically acceptable excipients, which can be selected and added by those skilled in the art according to actual preparation requirements, and the present invention does not impose any particular limitation thereto.

[0013] The Chinese medicine preparation can be granules, powders, tablets, capsules, pills or mixtures, etc. Those skilled in the art can prepare the preparation according to actual needs, and the present invention is not particularly limited thereto.

[0014] The use of the above-mentioned traditional Chinese medicine Liyao compound composition or traditional Chinese medicine preparation in the preparation of a medicament for treating premature ovarian insufficiency.

[0015] Furthermore, in the application, the Chinese medicine Liyao compound composition or Chinese medicine preparation is taken once a day, divided into two doses for warm administration.

[0016] A traditional Chinese medicine Liyao compound preparation for treating premature ovarian insufficiency contains the above traditional Chinese medicine composition.

[0017] Beneficial effects of the present invention:

[0018] The Li medicine composition described in this invention builds on classic formulas such as Shoutai Pills and Siwu Decoction, innovatively incorporating distinctive Li medicine herbs such as Guojianglong (Guojianglong) and Hainan Agarwood. Through scientific compatibility principles and rigorous preparation techniques, this innovative composition forms a comprehensive treatment plan with comprehensive theoretical support and significant clinical advantages. The development of this innovative composition not only inherits and develops traditional Chinese medicine theory but also deeply explores and enhances ethnic medicine resources, potentially providing safer and more effective treatment options for POI patients.

[0019] The prescription of the present invention comprises 16 Chinese medicinal herbs: Cuscuta seeds, Rhizoma Coptidis, Radix Dipsaci, Radix Rehmanniae Preparata, Radix Angelicae Sinensis, Rhizoma Chuanxiong, Radix Paeoniae Albae, Radix Bupleuri 10g, Rhizoma Atractylodis Macrocephalae 10g, Radix Pseudostellariae 15g, Fructus Amomi 6g, Bulbus Lilii 15g, Radix Salviae Miltiorrhizae 15g, Radix Scutellariae 10g, Radix Aquilariae 6g, and Radix Glycyrrhizae Preparata 6g. The monarch herbs are: Cuscuta seeds, Rhizoma Coptidis, Radix Dipsaci, Radix Rehmanniae Preparata; the minister herbs are: Bulbus Lilii, Radix Angelicae Sinensis, Radix Paeoniae Albae, Radix Bupleuri, Radix Pseudostellariae, and Radix Atractylodes; the adjuvant herbs are: Radix Salviae Miltiorrhizae, Rhizoma Chuanxiong, Radix Scutellariae baicalensis, Fructus Amomi, and Bulbus Lilii; and the guiding herbs are: Radix Glycyrrhizae Preparata. Cuscuta + Dipsacus root: imitates Shoutai Pills to tonify the kidney and strengthen Chong; Cuscuta balances yin and yang, Dipsacus root nourishes the liver and kidney, unclogs blood vessels, and targets the root cause of kidney deficiency; Cross-River Dragon (Li medicine): dispels rheumatism, activates blood circulation and unclogs the meridians, and synergizes with Dipsacus root to enhance the effect of unblocking joints, reflecting the idea of ​​"unblocking before replenishing"; Rehmannia root: nourishes the kidney and replenishes essence, and forms a "seeking yang in yin" combination with Cuscuta seed; Cross-River Dragon, a Li medicine, is listed as the main medicine alongside traditional kidney-tonifying medicines, highlighting the innovative feature of the "unblocking and tonifying" treatment method; the combination of main medicines embodies the functions of tonifying the kidney, replenishing essence, strengthening tendons and unblocking meridians. Lily + Bupleurum + White Peony Root: soften the liver and clear away heat, targeting the potential pathogenesis of liver depression turning into heat (lily clears the heart and calms the mind, preventing emotional disorders); Pseudostellaria baicalensis + Atractylodes macrocephala: lightly tonifies the spleen, avoiding the warm and dry nature of ginseng in the original formula of Sijunzi Decoction, more suitable for modern physiques; Angelica sinensis: links Siwu Decoction and Xiaoyao Powder, nourishes blood and activates blood circulation, and regulates liver blood together with white peony root; Features: Pseudostellaria baicalensis replaces ginseng, lily replaces Poria cocos, retaining the framework of Sijunzi Decoction and Xiaoyao Powder, but focusing more on nourishing yin and clearing heat; Layout of assistant drugs: highlights the effects of softening the liver and nourishing blood, invigorating qi and relieving depression. Salvia miltiorrhiza + Chuanxiong: Invigorates blood circulation and regulates menstruation, with Chuanxiong also promoting upward movement of the head and eyes, while Salvia miltiorrhiza cools blood and calms the mind, balancing the warming properties of Angelica sinensis. Scutellaria baicalensis: Clears heat and stabilizes pregnancy, counteracting the warming properties of Rehmannia glutinosa and Cuscuta chinensis, combined with Bupleurum chinense for a "clearing and soothing" approach. Amomum villosum (a southern medicinal herb) + Aquilaria sinensis: Invigorates the spleen and dissipates dampness, drawing Qi back to the kidneys, and addressing the greasy, nourishing effects of tonics. Key features: The southern medicinal combination of Aquilaria sinensis and Amomum villosum not only aids the spleen and stomach in digestion, but also guides Qi back to the source, enhancing the kidney-tonifying effect of the lower jiao. Adjuvant design: Highlights the effects of invigorating blood, clearing heat, invigorating the spleen, and descending Qi. The guiding herb, Roasted Licorice root, harmonizes the various herbs, mitigating the bitter and cold properties of Bupleurum chinense and Scutellaria baicalensis, and assisting Pseudostellaria chinensis and Atractylodes macrocephala in tonifying the spleen. This prescription uses Shoutai Pills (Cuscuta chinensis and Dipsacus asper) as the foundation to nourish the kidney and stabilize the fetus, Xiaoyao Powder (Bupleurum chinense, Angelica sinensis, White Peony Root, Atractylodes macrocephala) to soothe the liver and relieve depression, Rehmannia glutinosa, Angelica sinensis, Chuanxiong, and White Peony Root to nourish and regulate blood, and Pseudostellaria baicalensis, Atractylodes macrocephala, and Roasted Licorice root to invigorate Qi and strengthen the spleen. It integrates the essence of classic prescriptions, reflecting the holistic view of Traditional Chinese Medicine that "the kidney is the foundation of the innate, the spleen is the foundation of the acquired, and the liver is responsible for regulating Qi." The combination nourishes without stagnating (such as Amomum villosum and Aquilaria sinensis to regulate Qi and invigorate the spleen), warms without drying (Rehmannia glutinosa combined with Scutellaria baicalensis to control dryness and heat), and regulates Qi excessively (a light dose of Bupleurum chinense to prevent loss of Qi), with distinct layers, working together to harmonize the liver and kidney, nourish both Qi and blood, and soothe the liver and strengthen the spleen.

[0020] Correspondence table between core drugs and traditional Chinese and Western medicine mechanisms

[0021] Core drugs Chinese medicine efficacy Mechanism of action of Western medicine Cuscuta, Dipsacus asper, Rehmannia root Tonifies the kidneys and replenishes essence (main medicine) Inhibit follicle apoptosis and regulate FSH / E2 levels Bupleurum, white peony root, and Pseudostellaria baicalensis Soothing the liver and strengthening the spleen (minister medicine) Improve HPO axis function and reduce stress damage Salvia miltiorrhiza, Scutellaria baicalensis, and Rhizoma Cyperi Active blood fever (adjuvant) Antioxidant, anti-fibrosis, immune regulation Roasted Licorice Harmonizing various medicines (adjuvant medicines) Synergistic effect and reduced side effects

[0022] (1) The present invention is based on the classic Chinese medicine prescriptions Shoutai Pills and Xiaoyao Powder. Through exquisite compatibility and innovative adaptation, lily, scutellaria, and salvia miltiorrhiza are incorporated to enhance the effects of nourishing yin, clearing heat, promoting blood circulation and regulating menstruation. Local specialty medicinal materials, including Li medicine Guojianglong, southern medicine Amomum villosum, and agarwood, are introduced, making a total of 16 Chinese medicine prescriptions. This prescription inherits the core efficacy of traditional prescriptions and combines the unique advantages of local specialty medicinal materials, showing significant efficacy in improving decreased ovarian reserve (DOR). Compared with similar products, this solution not only has distinct ethnic medicine characteristics, but also has broad market prospects and development potential due to its scientific formulation and definite efficacy, providing new ideas for the modernization and innovation of traditional Chinese medicine.

[0023] (2) The present invention has unique advantages:

[0024] Fusion of ethnic medicine: Guojianglong (Li ethnic group) dredges meridians, while agarwood and Amomum villosum (Yangchun Amomum villosum authentic medicinal material) dissipate dampness, reflecting the wisdom of "adapting to local conditions" in the use of medicine;

[0025] Combining static and dynamic: Kidney-tonifying medicine (static) is combined with Rhizoma Dioscoreae, Salvia miltiorrhiza, and Aquilaria sinensis (dynamic) to prevent stagnation;

[0026] Calming design: Lily and Salvia miltiorrhiza soothe the mind and calm the nerves, in line with the theory of "heart and kidney communication";

[0027] By using classic prescriptions as a foundation, ethnic medicines for synergy, and combining dynamic and static medicines, a triple-burner conditioning system of "tonifying the kidneys and unblocking the meridians - nourishing the blood and soothing the liver - strengthening the spleen and harmonizing the stomach" is formed, which has considerable clinical reference value.

[0028] (3) The traditional Chinese medicine Li medicine compound composition of the present invention exerts its effects by regulating the hypothalamus-pituitary-ovary axis (HPO axis) through multiple targets, improving the ovarian microenvironment and follicle development, and regulating immune and metabolic functions. It has multi-target synergy: it intervenes in the three major links of HPO axis, ovarian microenvironment, and immune metabolism at the same time; it enhances the effect of local medicine: Guojianglong (Li medicine) is anti-inflammatory and agarwood (southern medicine) regulates metabolism, supplementing the deficiencies of classical prescriptions; it is highly safe: it avoids the side effects of exogenous hormone replacement therapy (HRT) (such as the risk of thrombosis). Through the above mechanism, this prescription may delay the progression of POI and partially restore ovarian function, providing a new strategy for the combined treatment of POI with traditional Chinese and Western medicine.

[0029] (4) The traditional Chinese medicine Liyao compound composition of the present invention can significantly improve the survival rate of granulosa cells in SD rats and has a significant therapeutic effect on POI rats. After clinical use, patients can significantly improve the TCM syndrome score of POI patients, with rapid effect, high cure rate, and good efficacy. It is also effective in treating premature ovarian insufficiency. DETAILED DESCRIPTION

[0030] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0031] Example 1

[0032] A traditional Chinese medicine Liyao compound composition for treating premature ovarian insufficiency. Each dose comprises the following components by weight: 20g of Cuscuta seeds, 20g of Rhizoma Cynoglossi, 20g of Radix Dipsaci, 12g of Rehmannia root, 12g of Angelica sinensis, 12g of Ligusticum chuanxiong, 12g of Paeonia lactiflora, 10g of Bupleurum chinense, 10g of Atractylodes macrocephala, 15g of Pseudostellariae pseudoginseng, 6g of Amomum villosum, 15g of Lilium bulb, 15g of Salvia miltiorrhiza, 10g of Scutellaria baicalensis, 6g of Aquilaria sinensis, and 6g of prepared Licorice root.

[0033] The preparation method of the above-mentioned traditional Chinese medicine Liyao compound composition comprises the following steps:

[0034] Take the prescribed amount of Cuscuta seeds and wrap it with gauze. Then add the prescribed amount of Rhizoma Cyperi, Radix Dipsaci, Radix Rehmanniae Preparata, Radix Angelicae Sinensis, Rhizoma Chuanxiong, Radix Paeoniae Alba, Radix Bupleuri, Atractylodes Macrocephalae, Radix Pseudostellariae, Fructus Amomi, Lilium Bupleuri, Radix Salviae Miltiorrhizae, Radix Scutellariae, Aquilariae Reticulatae, and Radix Glycyrrhizae Preparata. Boil the mixture for 25-30 minutes, filter, and collect the medicinal liquid, which should be 1 / 3 of the volume of the water added. Add water to the medicinal residue and boil it for a second time, adding 1 / 2 to 2 / 3 of the volume of the first boiling point (approximately 600-800 mL) for 20-30 minutes. Combine the two medicinal liquids (totaling approximately 500-600 mL).

[0035] Example 2

[0036] A traditional Chinese medicine Liyao compound composition for treating premature ovarian insufficiency. Each dose comprises the following components by weight: 10g of Cuscuta seed, 10g of Rhizoma Cynoglossi, 10g of Radix Dipsaci, 10g of Radix Rehmanniae Preparata, 10g of Radix Angelicae Sinensis, 10g of Rhizoma Chuanxiong, 10g of Radix Paeoniae Alba, 5g of Radix Bupleuri, 5g of Atractylodes Macrocephalae, 10g of Radix Pseudostellariae, 4g of Amomum villosum, 10g of Lilium Bupleurum, 10g of Salvia miltiorrhizae, 5g of Scutellariae Baicalensis, 4g of Aquilariae Resinica, and 4g of Radix Glycyrrhizae Preparata.

[0037] The preparation method is the same as that in Example 1.

[0038] Example 3

[0039] Disclosed is a traditional Chinese medicine Liyao compound composition for treating premature ovarian insufficiency. Each dose comprises the following components by weight: 30g of Cuscuta seeds, 30g of Rhizoma Cynoglossi, 30g of Radix Dipsaci, 15g of Rehmannia root, 15g of Angelica sinensis, 15g of Ligusticum chuanxiong, 15g of White Peony root, 15g of Bupleurum chinense, 15g of Atractylodes macrocephala, 20g of Pseudostellariae pseudoginseng, 8g of Amomum villosum, 20g of Lilium bulb, 20g of Salvia miltiorrhiza, 15g of Scutellaria baicalensis, 8g of Aquilaria sinensis, and 8g of prepared Licorice root.

[0040] The preparation method is the same as that in Example 1.

[0041] The prepared Chinese herbal medicine Liyao compound composition can be taken as a decoction in two doses, or can be prepared into various dosage forms by adding corresponding excipients for convenient administration, such as granules, powders, tablets, capsules, pills, or mixtures. Alternatively, the composition can be prepared directly using Chinese herbal medicine decoction-free (formulated) granules, depending on the dosage of the various medicinal materials in the formula.

[0042] Investigation on the effect of the traditional Chinese medicine Liyao compound composition of the present invention

[0043] 1. Clinical trials

[0044] 1.1 Research subjects

[0045] A retrospective study was conducted between August 2021 and August 2024, involving 84 patients with premature ovarian insufficiency (POI) who were treated with self-prescribed prescriptions or conventional hormone therapy at the First Affiliated Hospital of Hainan Medical University and Hainan Modern Women and Children's Hospital. This study was approved by the Ethics Committee of Hainan Medical University, and the patients and their families signed informed consent.

[0046] 1.2 Diagnostic standards of traditional Chinese medicine and Western medicine

[0047] Western medicine diagnostic criteria: formulated with reference to the "Obstetrics and Gynecology" (10th edition) and the "Expert Consensus on Clinical Diagnosis and Treatment of Premature Ovarian Insufficiency (2023 Edition)" issued by the Menopause Group of the Obstetrics and Gynecology Branch of the Chinese Medical Association: ① 18 years old < age < 40 years old; ② Oligomenorrhea or amenorrhea for more than 4 months; ③ At least two serum basal FSH levels > 25 IU / L (with an interval of more than 4 weeks).

[0048] Diagnostic criteria of Traditional Chinese Medicine: Drafted with reference to the "Tenth Edition of Traditional Chinese Medicine Gynecology", the "Guidelines for the Diagnosis and Treatment of Premature Ovarian Insufficiency with Integrated Traditional Chinese and Western Medicine (2022 Edition)" issued by the Obstetrics and Gynecology Committee of the Chinese Association of Integrated Traditional Chinese and Western Medicine, the "Guiding Principles for Clinical Research of New Chinese Medicines (Trial)", and the "Terminology for Clinical Diagnosis and Treatment of Traditional Chinese Medicine Part 2: Syndromes":

[0049] Disease name and diagnostic criteria: POI can be classified into the categories of "oligomenorrhea", "late menstruation", "amenorrhea", and "premature cessation of menstruation" in traditional Chinese medicine. According to the extension of menstrual cycle, infrequent menstruation to amenorrhea, it is diagnosed as a related disease in traditional Chinese medicine. Amenorrhea is divided into primary amenorrhea and secondary amenorrhea. Primary amenorrhea refers to females over 16 years old who have no menstruation despite the development of secondary sexual characteristics, or females over 14 years old who have no secondary sexual characteristics and menstruation. Secondary amenorrhea refers to the cessation of menstruation for 3 cycles or more than 6 months after the onset of menstruation; late menstruation refers to the menstrual cycle being extended for more than 7 days, or even 3 to 5 months, and occurring for more than 3 cycles in a row; oligomenorrhea refers to normal menstrual cycle, but the menstrual volume is significantly less than 1 / 2 of the normal menstrual volume, or less than 20 ml, or the menstrual period is less than 2 days, or even stops at a drop.

[0050] Diagnostic criteria for this syndrome: ① Late menarche, late or irregular menstruation, or even amenorrhea; ② Shortened menstrual duration; ③ Reduced menstrual volume; ④ Dark-colored, watery menstrual blood with the presence of clots. Secondary symptoms: ① Weakness in the waist and knees; ② Irritability or breast tenderness; ③ Insomnia and frequent dreams; ④ Hot flashes and night sweats. Tongue and pulse examination: Pale tongue with a thin white coating and a deep, wiry pulse.

[0051] The main symptoms must meet items ①②③, and the secondary symptoms must meet two or more criteria. The syndrome can be diagnosed based on the tongue and pulse.

[0052] 1.3 Inclusion and Exclusion Criteria

[0053] Inclusion criteria: Patients who met both the following criteria were included: ① Meet the aforementioned diagnostic criteria for both traditional Chinese medicine and Western medicine; ② Age between 18 and 40 years; ③ Treatment with the Yangyin Shugan prescription or hormone therapy alone; ④ Continuous treatment for 3–4 months, with at least one bFSH and anti-Müllerian hormone (AMH) test result within 1 day before and 1 day after treatment; and ⑤ Complete medical records.

[0054] Exclusion criteria: Patients who met any of the following conditions were excluded: ① POI caused by surgery, radiotherapy, or chemotherapy; ② Patients with gynecological reproductive organ malignancies; ③ Severe heart, liver, or kidney damage; ④ Concomitant use of other similar medications during treatment; ⑤ Severe mental disorders; ⑥ Failure to return for follow-up visits as required by the study.

[0055] 1.4 Intervention methods

[0056] The control group received sequential estrogen-progestin therapy. Patients were treated with a combination of oral estradiol tablets and estradiol dydrogesterone tablets (estradiol tablets contain 1 mg of estradiol; estradiol dydrogesterone tablets contain 1 mg of estradiol and 10 mg of dydrogesterone). One estradiol tablet was taken daily for the first 14 days, followed by one estradiol dydrogesterone tablet daily for the next 14 days. A course of treatment consisted of 28 days. After each course, the medication was discontinued for 2 days before the next course was resumed, for a total of three courses. During the medication period, patients were advised to use non-hormonal contraceptive methods, avoid grapefruit, and exercise moderately.

[0057] The Liyao compound prescription group received the same treatment as the control group, combined with a self-prescribed prescription. The prescription consisted of 20g of Cuscuta seeds, 20g of Rhizoma Cynanchifoliae, 20g of Radix Dipsaci, 12g of Rehmannia glutinosa, 12g of Radix Angelicae Sinensis, 12g of Rhizoma Chuanxiong, 12g of Radix Paeoniae Alba, 10g of Radix Bupleuri, 10g of Atractylodes macrocephala, 15g of Radix Pseudostellariae, 6g of Amomum villosum, 15g of Lilium Bupleuri, 15g of Radix Salviae Miltiorrhizae, 10g of Radix Scutellariae, 6g of Aquilariae Resinatum, and 6g of Radix Glycyrrhizae Preparata. Add approximately 1000mL of water, sufficient to completely submerge the herbs and leave them 2-3cm above the surface. Boil for 25-30 minutes until the liquid volume is reduced by 2 / 3. Filter and remove approximately 300mL of the liquid, reserving the residue. Continue decocting the residue with water, using 1 / 2-2 / 3 of the initial decoction (approximately 600-800mL) for 20-30 minutes. Decoction twice, filter, combine the two medicinal solutions (total about 500-600 mL), and take warm in two doses.

[0058] The medication should be discontinued if menstruation occurs during treatment.

[0059] Both groups received treatment for 3 consecutive months.

[0060] 1.5 Efficacy evaluation

[0061] (1) Comparison of clinical efficacy between the two groups

[0062] Total effective rate = (number of cured cases + markedly effective cases + effective cases) / total number of cases × 100%.

[0063] Recovery: After treatment, the patient's menstruation and follicle-stimulating hormone (FSH) and anti-Müllerian hormone (AMH) levels return to normal, and clinical symptoms such as sudden fever in the face and neck, and night sweats disappear.

[0064] Markedly effective: The patient's menstruation returns to normal, FSH and AMH levels are significantly improved, and clinical symptoms and signs are significantly improved.

[0065] Effective: The patient's menstruation, clinical symptoms and signs have improved, and FSH and AMH levels have no significant changes;

[0066] Ineffective: The patient is still amenorrhea, clinical symptoms and signs are not improved or even aggravated, and FSH and AMH levels have no significant changes.

[0067] (2) Comparison of TCM syndrome scores between the two groups before and after treatment

[0068] A symptom scale for kidney deficiency and liver depression was developed. Symptoms include menstrual cycle, menstrual volume, menstrual color and quality, irritability, breast engorgement, lower back pain and knee weakness, nocturia, depression, chest tightness and sighing, hot flashes and sweating, decreased libido, and insomnia. No symptoms were scored as 0; occasional symptoms as 1; mild symptoms as 2; and significant symptoms as 3, with a total score of 0-36.

[0069] 1.6 Research Results

[0070] The results of a retrospective study showed that there was no significant difference in the clinical efficacy of this product compared with conventional treatment for POI patients (P>0.05), but the cure rate of this product was higher than that of the control group, and the difference between the two was statistically significant (P<0.05). There was no significant difference in the TCM symptom scores before treatment between the two (P>0.05). After treatment, both this product and conventional treatment significantly reduced the TCM symptom scores of patients (P<0.01), but the TCM symptom scores of the TCM combination group decreased more significantly (P<0.01). This suggests that in the treatment of POI, rapid results, high cure rates, and good efficacy are the potential advantages of the TCM Liyao compound composition described in the present invention.

[0071] Table 1 Clinical efficacy test results (n=84)

[0072]

[0073] Note: △ Compared with the Chinese medicine treatment group, P>0.05(χ 2 =0.16, P = 0.75); *P < 0.05 (X 2 =4.27,P=0.04).

[0074] Table 2 Comparison of TCM syndrome scores (n=84)

[0075]

[0076]

[0077] 2. Cell experiments

[0078] 2.1 Establishment of POI cell model

[0079] Healthy female adult Sprague-Dawley rats, 7-8 weeks old, weighing 200-220 g, were fed an adaptive diet for 3 days and subcutaneously injected with 40 IU of pregnant mare serum gonadotropin (PMSG) (manufacturer: PROSPEG; catalog number: hor-272; lot number: 714PFMSG20). Forty-eight hours later, the rats were sacrificed by cervical dislocation, and the ovaries were rapidly dissected and placed in PBS. Red blood cells on the ovarian surface were rinsed with PBS, and the rinsed ovaries were placed in serum-free DMEM / F12 medium. Follicles were punctured with a 1 ml hollow needle to release the granulosa cells within the follicles. The cells were dispersed into a single cell suspension by pipetting and filtered through a 200-mesh stainless steel cell sieve. After centrifugation at 800 rpm for 6 minutes, the supernatant was discarded and the cells were collected. DMEM / F12 medium / penicillin-streptomycin / fetal bovine serum was added to the pellet at the bottom of the centrifuge tube, and the suspension was resuspended by pipetting to obtain a single cell suspension. The cells were then seeded into 6-well plates. Add DMEM / F12 / penicillin-streptomycin / fetal bovine serum culture medium to the cell culture flask, culture in a 37°C, 5% CO2 incubator for 24 hours, change the medium to remove non-adherent cells, and continue culturing. After 24 hours, add 200 μL of cyclophosphamide-containing serum to the adherent rat ovarian granulosa cells separated and cultured for 24 hours, and culture in a 37°C, 5% CO2 incubator for 48 hours.

[0080] 2.2 Preparation of drug-containing serum

[0081] The Chinese medicine decoction-free granules were prepared at the First Affiliated Hospital of Hainan Medical University. The self-prepared formula was as follows: Cuscuta chinensis 20g, Rhizoma Cynoglossi 20g, Radix Dipsaci 20g, Radix Rehmanniae Preparata 12g, Radix Angelicae Sinensis 12g, Rhizoma Chuanxiong 12g, Radix Paeoniae Alba 12g, Radix Bupleuri 10g, Rhizoma Atractylodis Macrocephalae 10g, Radix Pseudostellariae 15g, Fructus Amomi 6g, Lilium 15g, Radix Salviae Miltiorrhizae 15g, Radix Scutellariae 10g, Rhizoma Aquilariae 6g, and Radix Glycyrrhizae Preparata 6g. Each dose was diluted with 100mL of boiling water to obtain the drug stock solution. (Example 2 of the present invention)

[0082] Recipe 1: Cuscuta 20g, Dipsacus root 20g, Rehmannia root 12g, Angelica sinensis 12g, Ligusticum chuanxiong 12g, White Peony root 12g, Atractylodes macrocephala 10g. Add 49mL of boiling water to each dose to prepare the original solution.

[0083] Split formula 2: Rhizoma Cyperi 20g, Radix Bupleuri 10g, Radix Pseudostellariae 15g, Fructus Amomi 6g, Lilium 15g, Radix Salviae Miltiorrhizae 15g, Radix Scutellariae 10g, Aquilariae 6g, Radix Glycyrrhizae 6g. Add 51mL of boiling water to each dose to prepare the original solution.

[0084] The preparation method is the same as that in Example 1 of the present invention.

[0085] Forty SPF female Sprague-Dawley rats were randomly divided into five groups of 10 rats each using a random number table: a blank group, a self-prepared formula group, a modified formula 1 group, and a modified formula 2 group. The self-prepared formula, modified formula 1, and modified formula 2 groups were administered serum by gavage at 10 times the rat clinical equivalent dose, once daily in the morning and evening for 7 days; the blank control group received an equal volume of normal saline. On the day of the final dose, rats were fasted but not watered. Two hours after administration, blood was collected from the abdominal aorta under sterile conditions, centrifuged, and complement was inactivated in a water bath at 56°C for 30 minutes. The blood was sterilized by filtration through a 0.22 μm microporous filter and stored at -20°C until use. Subsequent intervention concentrations were determined based on cell proliferation curves.

[0086] 2.3 Grouping and Dosing

[0087] Granulosa cells from normal and POI SD rats were obtained, trypsinized, and then added with DMEM (containing 10% FBS) culture medium to a centrifuge tube, pipetting, and mixing. When the cell density approached 70%, the serum-free culture medium was replaced and cultured for another 24 hours to synchronize the cells in the G0 phase. Each 10-well cell culture was divided into a group, for a total of 5 groups. The groups and stimulation methods were as follows: the blank group was treated with conventional culture medium, the model group was cultured with 10% blank serum, and the self-prepared formula group, the split formula group 1, and the split formula group 2 were cultured with the corresponding 10% drug-containing serum. After 24 hours of culture, the cell survival rate of each group was measured.

[0088] 2.4 CCK-8 method survival rate detection

[0089] After 24 days of conditioned culture of granulosa cells from normal and POI SD rats, 10 μL of CCK-8 test solution was added to all wells and incubated in an incubator for 3 hours. Absorbance at 450 nm was measured using a microplate reader, and the survival rate of each group was calculated according to the formula: % survival rate = [(experimental group - blank group) / (control group - blank group)] * 100%. Granulosa cells from normal SD rats were added to the blank group, while granulosa cells from POI SD rats were added to the remaining treatment groups.

[0090] 2.5 Experimental Results

[0091] The survival rate of granular cells of SD rats in the POI model group was significantly reduced (P < 0.01). The drug-containing serum intervention of the self-prepared prescription, split prescription 1, and split prescription 2 groups significantly increased the survival rate of granular cells of SD rats (P < 0.01). Compared with the self-prepared prescription group, the survival rate of granular cells of SD rats in the split prescription 1 and split prescription 2 groups was significantly lower than that in the self-prepared prescription group (P < 0.01). This suggests that the self-prepared prescription group, split prescription 1 group, and split prescription 2 groups can significantly improve the survival rate of granular cells of POI model rats cultured in vitro, and compared with the split prescription 1 group and split prescription 2 group, the self-prepared prescription group has better efficacy. This suggests that compared with the two split prescription groups, the effect of the formula described in the present invention is significantly improved.

[0092] Table 3 Survival rate of granulosa cells in each group of SD rats (n=10, )

[0093]

[0094] Note: △ Compared with the blank group, P < 0.01; *Compared with the model group, P < 0.01; # Compared with the self-prepared prescription group, P < 0.01.

[0095] 3. Animal Experiments

[0096] 3.1 Experimental animals

[0097] Fifty eight-week-old, adult female Wistar rats weighing 250 ± 20 g (purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., quality certificate number: 430727201101514646) were selected. The animals were housed at the Animal Experiment Center of Hainan Medical University. All procedures and animal experiments were approved by the Ethics Committee of Hainan Medical University. The animals were maintained at 20–25°C and 50 ± 5% humidity with free access to food and water. All procedures and animal experiments were approved by the Ethics Committee of Hainan Medical University.

[0098] 3.2 Reagent and drug preparation

[0099] Reagents: Rat FSH, LH, E2, and AMH enzyme-linked immunosorbent assay kits (Thermo Fisher Scientific, USA); chloral hydrate, liquid nitrogen, OCT embedding medium, ethanol-acetic acid-formaldehyde fixative, 1% ammonia water, 0.7% eosin stain, 70% ethanol, 80% ethanol, 90% ethanol, xylene; physiological saline (Shenzhen Reward Life Science Co., Ltd.); cyclophosphamide for injection (Jiangsu Shengdi Pharmaceutical Co., Ltd.).

[0100] Traditional Chinese medicine decoction-free granules were prepared at the First Affiliated Hospital of Hainan Medical University. The self-prepared formula consisted of 20g of Cuscuta seeds, 20g of Rhizoma Cynoglossi, 20g of Radix Dipsaci, 12g of Radix Rehmanniae Preparata, 12g of Radix Angelicae Sinensis, 12g of Rhizoma Chuanxiong, 12g of Radix Paeoniae Alba, 10g of Radix Bupleuri, 10g of Atractylodes Macrocephalae, 15g of Radix Pseudostellariae, 6g of Amomum villosum, 15g of Lilium Bupleuri, 15g of Radix Salviae Miltiorrhizae, 10g of Radix Scutellariae, 6g of Aquilariae Reticulatae, and 6g of Radix Glycyrrhizae Preparata. Each dose was diluted with 100mL of boiling water to prepare the stock solution.

[0101] Recipe 1: Cuscuta 20g, Dipsacus root 20g, Rehmannia root 12g, Angelica sinensis 12g, Ligusticum chuanxiong 12g, White Peony root 12g, Atractylodes macrocephala 10g. Add 49mL of boiling water to each dose to prepare the original solution.

[0102] Split formula 2: Rhizoma Cynanchum 20g, Radix Bupleuri 10g, Radix Pseudostellariae 15g, Fructus Amomi 6g, Bulbus Lilii 15g, Radix Salviae Miltiorrhizae 15g, Radix Scutellariae 10g, Aquilariae Reticulatae 6g, Radix Glycyrrhizae Preparata 6g. Add 51mL of boiling water to each dose to prepare the original solution.

[0103] 3.3 Modeling

[0104] After 3 days of adaptive feeding and 10 days of observation of the estrous cycle, 40 rats were randomly injected intraperitoneally with cyclophosphamide to establish a POI rat model. The specific steps are as follows: After the rats' adaptive training, 50 mg kg-1 cyclophosphamide was injected intraperitoneally on the first day, and then the dose was reduced by 1 time and injected once a day for 1 week. During the modeling period, the rats were placed in sterile cages and free to eat and move. Vaginal smears were performed daily to observe the estrous cycle. When the estrous cycle of the modeling group rats was irregular and abnormal serum neutral hormone levels were detected, the model was successful. The remaining 10 rats served as the control group and were injected intraperitoneally with the same volume of normal saline as the modeling group every day during the entire modeling period.

[0105] 3.4 Intervention

[0106] One week after modeling, the rats with successful modeling were randomly divided into four groups: model group, self-prescription group, separated prescription group 1, and separated prescription group 2. The dosage of Chinese medicine solution for the self-prescription group, separated prescription group 1, and separated prescription group 2 was calculated according to the human-animal dosage conversion method. The blank group (normal rats) and the model group were given an equal amount of normal saline by gavage once a day for 30 consecutive days.

[0107] 3.5 Detection indicators and methods

[0108] 3.5.1 Serum hormone level detection

[0109] One week after modeling, the rats with successful modeling were randomly divided into four groups: model group, self-prescription group, separated prescription group 1, and separated prescription group 2. The self-prescription group, separated prescription group 1, and separated prescription group 2 were given an equal amount of Chinese medicine by gavage according to the human-animal drug conversion method. The blank group and the model group were given an equal amount of normal saline by gavage once a day for 30 consecutive days.

[0110] 3.5.2 Organ wet weight and organ coefficient

[0111] After anesthesia, blood was collected from the abdominal aorta of the rats. After centrifugation at 4000 rpm, the serum was stored in a -80°C ultra-low temperature refrigerator for testing. The levels of FSH, LH, E2 and AMH in the serum were then detected by enzyme-linked immunosorbent assay (ELISA).

[0112] 3.6 Experimental Results

[0113] After modeling, serum FSH and LH levels in the model group were significantly increased (P < 0.01). After intervention with Chinese medicine, FSH and LH levels were reduced in all groups (P < 0.01), with the self-prescription group showing superior effects compared with the separated prescription 1 and separated prescription 2 groups (P < 0.05, P < 0.01). After modeling, serum E2 and AMH levels, ovarian index, uterine index, number of antral follicles, and estrous cycle were significantly decreased (P < 0.01). After intervention with Chinese medicine, E2 and AMH levels, ovarian index, uterine index, number of antral follicles, and estrous cycle were increased in all groups (P < 0.01). The self-prescription group showed superior effects compared with the separated prescription 1 and separated prescription 2 groups (P < 0.05, P < 0.01).

[0114] Table 4 Serum estrogen levels of rats in each group (n=10, )

[0115]

[0116] Note: △ Compared with the blank group, P < 0.01; *Compared with the model group, P < 0.01; # Compared with the self-prepared prescription group, P < 0.05; ## Compared with the self-prepared prescription group, P < 0.01.

[0117] Table 5 Results of organ levels and estrous cycle in rats (n=10, )

[0118]

[0119] Note: △ Compared with the blank group, P < 0.01; *Compared with the model group, P < 0.01; # Compared with the self-prepared prescription group, P < 0.05; ## Compared with the self-prepared prescription group, P < 0.01.

[0120] The results of animal experiments confirmed that the self-prepared prescription group, split prescription group 1, and split prescription group 2 can all significantly treat POI rats, but the self-prepared prescription group has better efficacy than the split prescription group 1 and the split prescription group 2. The results of this experiment suggest that compared with the other split prescription experimental groups, the therapeutic effect of the prescription described in the present invention is significantly higher than that of the split prescription group.

[0121] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A traditional Chinese medicine Liyao compound composition for treating premature ovarian insufficiency, characterized in that: Each dose includes the following components by weight: Cuscuta chinensis 10-30g, Rhizoma Cynoglossi 10-30g, Dipsacus asper 10-30g, Rehmannia glutinosa 10-15g, Angelica sinensis 10-15g, Ligusticum chuanxiong 10-15g, White Peony 10-15g, Bupleurum 5-15g, Atractylodes macrocephala 5-15g, Pseudostellaria 10-20g, Amomum villosum 4-8g, Lilium 10-20g, Salvia miltiorrhiza 10-20g, Scutellaria baicalensis 5-15g, Aquilaria sinensis 4-8g, and Radix Glycyrrhizae 4-8g.

2. The Chinese medicinal compound composition according to claim 1, wherein Each dose includes the following components by weight: Cuscuta chinensis 20g, Rhizoma Cynoglossi 20g, Radix Dipsaci 20g, Rehmannia glutinosa 12g, Radix Angelicae Sinensis 12g, Rhizoma Chuanxiong 12g, White Peony 12g, Radix Bupleuri 10g, Atractylodes macrocephala 10g, Radix Pseudostellariae 15g, Fructus Amomi 6g, Lilium 15g, Radix Salviae Miltiorrhizae 15g, Radix Scutellariae 10g, Aquilariae Reticulatae 6g, and Radix Glycyrrhizae Radix 6g.

3. The method for preparing the traditional Chinese medicine Liyao compound composition according to claim 1 or 2, wherein: The following steps are involved: Take the prescribed amount of Cuscuta australis and wrap it with sterile gauze, then add the prescribed amount of Rhizoma Cyperi, Radix Dipsaci, Radix Rehmanniae Preparata, Radix Angelicae Sinensis, Rhizoma Chuanxiong, White Peony Root, Radix Bupleuri, Atractylodes Macrocephalae, Radix Pseudostellariae, Fructus Amomi, Lilium, Salvia miltiorrhizae, Radix Scutellariae, Aquilariae, and Radix Glycyrrhizae, add water and boil, filter to remove the residue to obtain the medicinal liquid.

4. The preparation method according to claim 3, wherein Decoction was performed twice, and the two medicinal solutions were combined. For the first decoction, water was added to completely immerse the medicinal materials and to be 2 to 3 cm higher, and decoction was performed for 25 to 30 minutes. The volume of water added for the second decoction was 1 / 2 to 2 / 3 of the first water, and the decoction time was 20 to 30 minutes. The volume of the medicinal solution was 1 / 3 of the volume of the added water.

5. A traditional Chinese medicine Liyao compound preparation for treating premature ovarian insufficiency, characterized in that: It contains the Chinese medicine composition according to claim 1 or 2.

6. The traditional Chinese medicine Liyao compound preparation according to claim 5, characterized in that: The traditional Chinese medicine preparation also includes pharmaceutically acceptable excipients.

7. The traditional Chinese medicine Liyao compound preparation according to claim 5, characterized in that: The Chinese medicine preparation is in the form of granules, powders, tablets, capsules, pills or mixtures.

8. Use of the traditional Chinese medicine Liyao compound composition according to claim 1 or 2 or the traditional Chinese medicine preparation according to any one of claims 5 to 7 in the preparation of a medicament for treating premature ovarian insufficiency.

9. The use according to claim 8, characterized in that The Chinese medicine Liyao compound composition or Chinese medicine preparation is taken once a day, divided into two doses for warm administration.

10. A Chinese medicine granule for treating premature ovarian insufficiency, characterized in that: A compound composition of the traditional Chinese medicine Li medicine according to claim 1 or 2.

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