A traditional Chinese medicine composition for treating decreased ovarian reserve and a preparation method and application thereof

Abstract

The application discloses a traditional Chinese medicine composition for treating decreased ovarian reserve function, and the traditional Chinese medicine composition contains the following components in parts by weight: prepared rehmannia 10-20 parts, raw rehmannia 10-20 parts, north saussurea 8-15 parts, ophiopogon 10-20 parts, donkey-hide gelatin 5-10 parts, Chinese yam 8-15 parts, dendrobium 8-15 parts, turtle shell 8-15 parts, gordon euryale seed 8-15 parts, dodder seed 8-15 parts and radix morindae officinalis 8-15 parts. The traditional Chinese medicine composition can improve the follicular development disorder and sex hormone disorder of rats with cyclophosphamide-induced insufficient ovarian reserve function, and the number of atretic follicles and corpus luteum is obviously reduced, so the traditional Chinese medicine composition has a good treatment prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine, and particularly discloses a composition for treating diminished ovarian reserve and a preparation method and application thereof. BACKGROUND

[0002] Ovarian reserve refers to the ability of follicle growth, development and formation of fertilizable oocytes in the ovarian cortex, which reflects the reproductive potential of women and can be simply understood as the ability of the ovary to produce oocytes and ovulate. When the premature depletion of primordial follicles stored in the ovary, follicle growth disorder and accelerated atresia lead to a decrease in the number of follicles or a decline in the quality of oocytes, which in turn leads to a decline in reproductive potential and endocrine dysfunction, it is called diminished ovarian reserve (DOR). DOR is mainly manifested in clinical practice as oligomenorrhea, decreased menstrual flow, even amenorrhea, infertility and a series of other perimenopausal clinical symptoms. If not intervened early, it will gradually evolve into premature ovarian failure (POF), which seriously affects the reproductive health and quality of life of patients. Therefore, early intervention and prevention are urgently needed for DOR patients to protect ovarian function and improve the quality of life and reproductive function of patients.

[0003] Traditional Chinese medicine treatment mainly balances qi and blood, yin and yang, regulates the Zangfu organs and the Chong and Ren meridians, and supplements deficiency and eliminates excess. CN118615370A discloses a preparation for improving ovarian reserve function, which comprises the following components in parts by weight: red dates 20-30 parts, safflower 10-30 parts, donkey-hide gelatin 10-18 parts, angelica 10-16 parts, astragalus 8-14 parts, ligustrum 8-12 parts, pueraria 6-12 parts, medlar 6-10 parts, mulberry 4-10 parts, and longan meat 4-8 parts. The preparation has the effects of tonifying qi and nourishing blood, regulating endocrine, nourishing yin and tonifying blood, regulating menstruation, stimulating female hormones and follicle production, regulating the ovary, and delaying ovarian decline.

[0004] CN118615369A discloses a traditional Chinese medicine composition for improving age-related ovarian function decline and infertility, which comprises mulberry, pueraria, poria cocos and medlar. The composition follows the concept of homology between traditional medicine and food in traditional medicine, reasonably matches the four qi and five flavors of traditional Chinese medicine, and follows the principles of ascending, descending, floating and serving as monarch, minister, assistant and messenger, and has the effects of improving age-related ovarian reserve function decline, endocrine dysfunction, menstrual disorder and decreased fertility.

[0005] CN117752743A discloses a traditional Chinese medicine composition for maintaining ovarian follicles. Its active ingredients include the following raw materials by weight: 10-20 parts of Dendrobium, 12-25 parts of Longan pulp, 8-15 parts of Polygonatum, 5-8 parts of Chicken Follicle Gum, and 6-15 parts of Lilium. This composition, based on the Traditional Chinese Medicine (TCM) reproductive axis of "Kidney-Tiangui-Chongren-Baogong" and the ebb and flow of qi, blood, yin and yang in the internal organs, provides the theoretical basis for normal follicular growth and excretion. This corresponds to the cyclical changes and functions of the "Hypothalamus-Pituitary-Ovary" axis in modern medicine. Follicular growth depends on both the abundant kidney essence, the innate foundation, and the spleen and stomach, the acquired foundation, the generation of qi and blood, and other internal organs. This prescription mainly uses Dendrobium, Polygonatum and Lilium as the main medicines, which have the effects of nourishing the kidney and yin, invigorating qi and strengthening the spleen, moistening the lungs and calming the mind. The three are used together to nourish the congenital kidney, replenish the acquired spleen, and regulate the mind. Combined with ingredients of blood and flesh, they can strengthen the spleen and kidney, regulate Chong and Ren, increase follicular fluid, promote follicle growth, and improve egg quality, with the effect of "1+1+1>3"; the auxiliary medicines mainly include chicken foam gelatin, Ligustrum lucidum and other medicines, which help to promote blood circulation and remove blood stasis, clear away heat and detoxify, balance the endocrine system, and harmonize the body's qi and blood, and have a certain therapeutic effect on ovarian problems; the adjuvant milk thistle and mulberry can regulate the body's spleen and stomach functions and promote the absorption and metabolism of drugs.

[0006] CN114699474A discloses a Chinese medicine composition for treating decreased ovarian reserve function and its application. The Chinese medicine composition is made from the following raw materials: Rehmannia root, stir-fried Atractylodes macrocephala, Chinese yam, Chinese angelica root, stir-fried white peony root, stir-fried Ziziphus jujuba seed, moutan bark, bupleurum root, dipsaccharum officinale, Codonopsis pilosula, Glehnia littoralis, Cuscuta australis, Ligustrum lucidum fruit, and dried tangerine peel. The composition primarily tonifies the kidney and replenishes essence, but yin-tonifying herbs are often greasy and should be supplemented with herbs that warm yang and invigorate qi to achieve tonification without stagnation. Essence and blood share the same origin and mutually nourish each other, so supplementing essence with herbs that invigorate blood can double the effectiveness. Rehmannia root and stir-fried Atractylodes macrocephala are the main ingredients, nourishing yin and blood, and replenishing essence and marrow. Cuscuta australis fruit, Glehnia littoralis fruit, Glehnia littoralis, Chinese yam, dipsaccharum officinale, Codonopsis pilosula, and dried tangerine peel are the secondary ingredients, tonifying the liver and kidney, replenishing essence and marrow, invigorating qi and strengthening the spleen, and warming the kidney and supporting yang. The monarch and the minister work together to replenish both yin and yang, and replenish essence and blood. At the same time, angelica, fried white peony root, fried spinach seed, and peony bark are used as adjuvants to nourish blood, soften the liver, assist yin, clear away heat, calm the mind and soothe the nerves.

[0007] CN112618644A discloses a traditional Chinese medicine compound composition for treating decreased ovarian reserve function, comprising the following components in parts by weight: Radix Rehmanniae Preparata 25-30 parts, Angelica 10-30 parts, Radix Paeoniae Alba 10-30 parts, Fructus Corni 10-15 parts, Eucommia ulmoides 5-15 parts, Rhizoma Atractylodis Macrocephalae 10-25 parts, Poria 5-15 parts, Radix Curculignis 5-10 parts, Radix Glycyrrhizae Preparata 15-20 parts, Radix Astragali 15-20 parts, Radix Codonopsis 10-20 parts, Fructus Jujubae 5-15 parts, Colla Corii Asini 5-10 parts, Radix Morindae Officinalis 10-15 parts, Semen Lagenariae 10-15 parts, Radix Hedysari 10-30 parts, Radix Dioscoreae 10-20 parts, and Semen Euryale 10-15 parts. In the above scheme, Radix Rehmanniae Preparata, Radix Astragali, Radix Codonopsis, and Semen Euryale are used as monarch drugs to play the effects of tonifying kidney and essence, filling marrow, benefiting qi and nourishing blood, and generating fluid; Angelica, Radix Paeoniae Alba, Fructus Corni, Colla Corii Asini, Radix Morindae Officinalis, Semen Lagenariae, etc. are used as ministerial drugs to play the effects of tonifying kidney and assisting yang, and making yang channel excited; Radix Curculignis, Eucommia ulmoides, Rhizoma Atractylodis Macrocephalae, Poria, Radix Dioscoreae, etc. are used as auxiliary drugs to play the effect of assisting essence and marrow transportation; Radix Hedysari, Radix Glycyrrhizae Preparata, and Fructus Jujubae are used as ministerial drugs to make Radix Hedysari tonify kidney and strengthen waist, and guide various drugs to descend to kidney, chong and ren, and Radix Glycyrrhizae Preparata and Fructus Jujubae regulate various drugs. SUMMARY

[0008] The technical problem to be solved by the present application is to provide a composition for treating decreased ovarian reserve function based on a new treatment idea and a preparation method thereof.

[0009] The present application proposes a "pregnancy 'device'", i.e., "tian-qi-uterus" forms a "heaven-earth" component, which together forms the female reproductive "pregnancy 'device'". Tian-qi is derived from the parents' congenital kidney essence, essence into qi, in the sky, gather to form qi water, descend to the earth (uterus) as rain and dew; the uterus accommodates the chong and ren, and the yin blood and yin liquid, under the action of the yang qi (containing the fire of the gate of life) of the governor, transpiration in the upper, nourishing tian-qi, up and down, endless, constitutes the small universe of female reproduction, and becomes the female reproductive "pregnancy 'device'".

[0010] Tian-qi is the basic substance of female reproduction. It is generally believed that there are two conventional paths to nourish tian-qi: one is the postnatal spleen and stomach water and grain essence; the other is the same origin of qi and blood, i.e., the same origin of liver and kidney.

[0011] Decreased ovarian reserve function is considered as "early exhaustion of qi water". The present application proposes a new treatment idea: one, nourish yin blood and yin liquid; two, appropriately supplement the fire of the gate of life; three, according to the age characteristics of the disease, one is that people generally have great pressure, heart fire is strong, and yin liquid is easily consumed; two is that people generally eat high-calorie food such as greasy and thick-tasting food, which easily causes heat accumulation in the stomach, and makes the stomach dry and damage the fluid, so the stomach dryness is treated, and the kidney yin is nourished to retreat the virtual heat.

[0012] Based on the above treatment idea, the application provides a new traditional Chinese medicine composition for treating decreased ovarian reserve, which comprises the following components in parts by weight: 10-20 parts of prepared rehmannia, 10-20 parts of raw rehmannia, 8-15 parts of north safflower, 10-20 parts of ophiopogon, 5-10 parts of donkey-hide gelatin, 8-15 parts of yam, 8-15 parts of dendrobium, 8-15 parts of turtle shell, 8-15 parts of gordon euryale seed, 8-15 parts of semen astragali complanati, and 8-15 parts of morindae.

[0013] Preferably, the traditional Chinese medicine composition comprises the following components in parts by weight: 10-15 parts of prepared rehmannia, 10-15 parts of raw rehmannia, 8-12 parts of north safflower, 8-12 parts of ophiopogon, 5-8 parts of donkey-hide gelatin, 8-12 parts of yam, 8-12 parts of dendrobium, 8-12 parts of turtle shell, 8-12 parts of gordon euryale seed, 8-12 parts of semen astragali complanati, and 8-12 parts of morindae.

[0014] In a preferred embodiment, the traditional Chinese medicine composition comprises the following components in parts by weight: 12 parts of prepared rehmannia, 12 parts of raw rehmannia, 10 parts of north safflower, 10 parts of ophiopogon, 6 parts of donkey-hide gelatin, 10 parts of yam, 10 parts of dendrobium, 10 parts of turtle shell, 10 parts of gordon euryale seed, 10 parts of semen astragali complanati, and 10 parts of morindae.

[0015] The application further provides application of the traditional Chinese medicine composition in preparation of a medicine or preparation for treating decreased ovarian reserve.

[0016] Preferably, the preparation is any one of granules, paste, brewing agent, capsule, tablet, powder.

[0017] In an embodiment, the preparation is water decoction.

[0018] The application further provides a preparation method of the traditional Chinese medicine composition, which comprises the following steps: soaking components of the traditional Chinese medicine composition except donkey-hide gelatin in water, pouring the water and the medicinal materials into a decocting pot, decocting for the first time, boiling for the first time, then turning to small fire for decocting for the second time, filtering out medicinal juice, adding water to boil for the second time, then turning to small fire for decocting for the second time, filtering out medicinal juice, mixing the medicinal juice of the first and second decocting, filtering, and finally slowly decocting and concentrating to obtain juice, and adding melted donkey-hide gelatin.

[0019] Preferably, the components of the traditional Chinese medicine composition except donkey-hide gelatin are soaked in 5-20 times of water of the total mass of the components for 0.3-1.0 hours.

[0020] Preferably, 3-8 times of water of the total mass of the components is added for the second decocting.

[0021] Preferably, the first decocting is boiled with large fire, and then turned to small fire for 15-40 minutes; the second decocting is boiled with large fire after adding water, and then turned to small fire for 12-40 minutes.

[0022] In the prescription of the application:

[0023] Cooked Rehmannia is sweet and slightly warm, focusing on nourishing blood and yin, nourishing the kidneys and liver; Raw Rehmannia is sweet, cold and moist, good at nourishing yin and cooling blood, nourishing yin and producing body fluid. Cooked Rehmannia is good at nourishing, while Raw Rehmannia is good at clearing. The combination of the two medicines can take into account both clearing and nourishing, and can nourish yin and kidney, replenish essence and fill marrow, and can also nourish blood and produce blood, nourish yin and cool blood, and have the dual functions of nourishing yin and nourishing blood, and are both the main medicines.

[0024] The ministerial herb, donkey-hide gelatin, nourishes yin and blood, nourishing the yin of the lungs, liver, and kidneys. Chinese yam nourishes the yin of the lungs, spleen, and kidneys. Adenophora australis nourishes yin and clears the lungs, benefiting the stomach and producing fluid. Ophiopogon japonicus nourishes the yin and fluid of the lungs and stomach, while also nourishing heart yin and clearing heart heat. Together, these four herbs replenish the yin fluids of the five zang organs, prioritizing lung yin and nourishing kidney water, achieving the goal of "tonifying the lungs and invigorating the kidneys." Dendrobium officinale, with its sweet and cooling properties, enters the stomach, nourishing yin and producing fluid to treat dryness in the stomach, while also nourishing kidney yin and relieving deficiency-induced heat. Tortoise shell nourishes yin and suppresses yang, nourishing the liver and kidneys while also quenching deficiency-induced heat. Together, these herbs nourish yin and reduce heat, combining both nourishment and clearing, nourishing yin without causing greasy sensations and purging heat without damaging yin. The ministerial herb and ministerial herb work in harmony, nourishing yin and blood, replenishing the yin of the five zang organs, and prioritizing kidney yin, ensuring that the yin fluid entering the uterus contains the essence of human yin fluid.

[0025] When the heart yin is sufficient, the heart fire is cleared, the heart qi flows downward, the liver is nourished, the liver qi is regulated, and the spleen (stomach) yin is restored, the pivotal organs will benefit. All of the above work together to help the Chong and Ren channels to gather the yin blood and yin fluid in the uterus in an orderly manner.

[0026] Cuscuta chinensis is good at replenishing kidney qi, which can help the uterus seal and absorb. "The static is yin, the dynamic is yang" states in the Suwen (Suwen) chapter on Yin and Yang. Cuscuta chinensis replenishes kidney qi, while Morinda officinalis warms and nourishes kidney yang. These two herbs should be used together in yin-related medicines to prevent other yin-tonifying herbs from being static and inactive.

[0027] Morinda officinalis enters the Mingmen meridian and is adept at warming and nourishing the Mingmen fire, helping the yin blood and yin fluid in the uterus to evaporate and nourish the Tiangui. Gorgon fruit nourishes the kidneys and strengthens essence, replenishing the Ren meridian and promoting the smooth flow of yin fluid, preventing stagnation. When used in conjunction with other yin-tonifying herbs, it can nourish without stagnation and benefit without injury.

[0028] The whole formula nourishes Yin and blood, nourishes the Yin fluids of the five internal organs, replenishes kidney Qi, and warms and nourishes the fire of the gate of life. It combines tonification and purgation, clearing and replenishing, nourishing without stagnation, benefiting without harming, and achieving movement within stillness, all working together to nourish Yin and blood, warm Yang and transform Qi.

[0029] The research in this application found that the three combinations of "prepared rehmannia, raw rehmannia", "tortoise shell, dendrobium", and "cuscuta chinensis, euryale ferox, and gorgon fruit" in the above-mentioned prescription can improve the efficacy of the prescription.

[0030] Cooked Rehmannia focuses on nourishing blood and yin, while raw Rehmannia focuses on nourishing yin and blood. Cooked Rehmannia is good at nourishing, while raw Rehmannia is good at clearing. The combination of the two medicines can take into account both clearing and nourishing, and the effect of nourishing yin and blood is enhanced.

[0031] Turtle shell can nourish yin and strengthen the kidney, and can also control deficiency heat; Dendrobium is sweet and cool and enters the stomach, and can nourish yin and produce fluid to treat stomach dryness, and can also nourish kidney yin and reduce deficiency heat. The two medicines are combined to enhance the effect of nourishing yin and reducing deficiency heat.

[0032] The milkvetch seed is good at tonifying kidney-Qi, and the Ba Ji Tian is warm and tonifying the fire of the Gate of Life. The two are combined in the Yin-type medicine to prevent the static of other medicines. The Gualou Xi is good at benefiting kidney and consolidating essence, and can tonify the deficiency of the Ren Channel and dredge the Yin fluid, so that the Yin fluid moves without stagnation. The Yin-type medicine is combined with the other Yin-tonifying medicines, and the effect is better. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 The follicles of different groups of rats were counted;

[0034] Figure 2 The serum E2, FSH, LH and AMH contents of different groups of rats were compared;

[0035] Figure 3 The effect of different concentrations of cyclophosphamide on the proliferation of KGN cells;

[0036] Figure 4 The effect of the group without milkvetch seed, Ba Ji Tian and Gualou Xi, the group without Gualou Xi and the whole prescription group on the E2 secretion of KGN cells;

[0037] Figure 5 The effect of the group without Rehmannia glutinosa and Radix Rehmanniae and the whole prescription group on the E2 secretion of KGN cells;

[0038] Figure 6 The effect of the group without Dendrobium nobile and Plastrum Testudinis, the group without Dendrobium nobile and Plastrum Testudinis and Wolfberry and Dogwood, the group without Plastrum Testudinis and the whole prescription group on the E2 secretion of granulosa cells. DETAILED DESCRIPTION

[0039] The above and / or other aspects of the present application will become more apparent by describing in detail the preferred embodiments thereof with reference to the attached drawings.

[0040] Example 1 Preparation of a traditional Chinese medicine composition for treating decreased ovarian reserve function.

[0041] Formula: Radix Rehmanniae Preparata 12g, Radix Rehmanniae 12g, Radix Hedysari 10g, Radix Ophiopogonis 10g, Colla Corii Asini 6g, Rhizoma Dioscoreae 10g, Dendrobium nobile 10g, Plastrum Testudinis 10g, Gualou Xi 10g, milkvetch seed 10g, Ba Ji Tian 10g

[0042] Preparation method: the above traditional Chinese medicines except Colla Corii Asini are soaked in water for 30 min, 10 times the amount of water is added and soaked for 0.5 h. After soaking, the water and the medicinal materials are poured into a decocting pot. The first decoction is boiled with a large fire, and then the fire is turned to a small one for about 20 min. The medicinal juice is filtered out. The second decoction is added with 5 times the amount of water, boiled with a large fire, and then the fire is turned to a small one for 15 min. The medicinal juice is filtered out. The medicinal juices of the first and second decoctions are mixed, filtered, and finally slowly decocted and concentrated with a small fire to obtain the juice. Colla Corii Asini (melted) is added to prepare the traditional Chinese medicine composition.

[0043] Example 2 Effects of different concentrations of traditional Chinese medicine compositions on the number of follicles at each level and the levels of various hormones in serum of rats with ovarian reserve insufficiency model.

[0044] Experimental animals: 60 female Sprague Dawley rats, 7 weeks old, 180-200 g, purchased from Jiangxi University of Traditional Chinese Medicine (Nanchang, China) animal facilities [license number: SCXK (Jiangxi) 2023-0001]. The feeding environment of the experimental animals is temperature (23±2) ℃, relative humidity 50%-60%, 12-hour day-night rhythm, and free access to food and water.

[0045] Experimental grouping: This experiment is divided into 6 groups, namely the blank group, the model group, the traditional Chinese medicine low concentration group, the traditional Chinese medicine medium concentration group, the traditional Chinese medicine high concentration group and the dehydroepiandrosterone (DHEA) group, with 10 rats per cage. Except for the blank group, each group of mice was given intraperitoneal injection of cyclophosphamide (CTX) (50 mg / kg / d) for 7 consecutive days, and the blank group was injected with the same amount of normal saline. On the 8th day after modeling, the mice in the traditional Chinese medicine low, medium and high concentration groups were given different concentrations of traditional Chinese medicine prepared in Example 1 (5.78 g / kg, 11.55 g / kg and 23.1 g / kg) by gavage for treatment, and the blank group was given the same dose of normal saline, and the DHEA group was given DHEA solution 0.67 mg / mL by gavage, for 2 weeks. Among them, the concentration of traditional Chinese medicine is calculated based on the total weight of traditional Chinese medicine raw materials. For example, the total weight of traditional Chinese medicine raw materials in Example 1 is 110 g, and the human weight is calculated according to 60 kg. The conversion coefficient of rats and humans is 6.3, so the concentration is 110 / 60*6.3=11.55 g / kg. The above concentration is referred to as medium concentration, and the low concentration and high concentration are 1 / 2 and 2 times respectively.

[0046] Specimen collection: After the end of administration, the rats were fasted for 12 h, and the blood was collected from the heart after intraperitoneal injection of 10% chloral hydrate solution. After centrifugation at 3000 rpm / min for 10 min, the serum was collected, and the levels of estradiol (E2), follicle-stimulating hormone (FSH), luteinizing hormone (LH) and anti-Mullerian hormone (AMH) in the serum were detected by ELISA.

[0047] HE staining to observe the morphological changes of ovarian tissue: The removed mouse ovarian tissue was fixed with paraformaldehyde, dehydrated with gradient ethanol, transparentized with xylene, embedded with paraffin, and cut into 4-5 μm thick sections. According to the operation procedure, after HE staining of the ovarian tissue sections, neutral resin was used for mounting, and the morphological changes of the ovarian tissue of each group of mice were observed under a microscope and photographed. Every 5 sections were counted, and only the follicles with clear oocyte nuclei were counted. The number of follicles at each level of ovarian tissue = the sum of the number of follicles at each level of the counted sections x 5.

[0048] Statistical analysis: all data are expressed as mean ± standard deviation Statistical analysis and drawing of all data were performed by SPSS and GraphPad Prism software version 10.0. The data were tested for normality and homogeneity of variance, and if they were in line, one-way ANOVA and LSD intergroup comparison were used, and if they were not in line, Dunnett test was used, P<0.05 difference was statistically significant.

[0049] (1) The effect of traditional Chinese medicine composition on the follicle count of each group of rats.

[0050] As shown in Table 1 and Figure 1 Compared with the normal group, the number of follicles in the model group was significantly reduced (P<0.05), and the number of atretic follicles and corpus luteum was significantly increased (P<0.05). Compared with the model group, the number of follicles in the western medicine group was significantly increased (P<0.05), and the number of atretic follicles and corpus luteum was significantly reduced (P<0.05); the number of primordial follicles in the low concentration of traditional Chinese medicine group was significantly increased (P<0.05), and the number of corpus luteum was significantly reduced (P<0.05); the number of primordial follicles and primary follicles in the medium concentration of traditional Chinese medicine group was significantly increased (P<0.05), and the number of atretic follicles and corpus luteum was significantly reduced (P<0.05); the number of follicles in the high concentration of traditional Chinese medicine group was significantly increased (P<0.05), and the number of atretic follicles and corpus luteum was significantly reduced (P<0.05).

[0051] Table 1 Follicle count of each group of rats

[0052]

[0053] Note: compared with the blank group, *P<0.05; compared with the model group, # P<0.05.

[0054] (2) The effect of traditional Chinese medicine composition on the serum E2, FSH, LH and AMH levels of model rats.

[0055] As shown in Table 2 and Figure 2 Compared with the blank group, the serum E2 (estradiol) and AMH (anti-Mullerian hormone) levels of the model group were significantly reduced, and the FSH (follicle stimulating hormone) and LH (luteinizing hormone) levels were significantly increased, with statistical significance (P<0.05). Compared with the model group, the serum E2 and AMH levels of the DHEA group were significantly increased, and the LH and FSH levels were significantly reduced, with statistical significance (P<0.05). The serum E2 and AMH levels of the low, medium and high concentration of traditional Chinese medicine groups were increased, and the FSH and LH levels were reduced, and the high dose of traditional Chinese medicine group had the best improvement effect (P<0.05).

[0056] Comparison of serum E2, FSH, LH and AMH levels in each group of rats

[0057]

[0058] Note: compared with the blank group, *P<0.001; compared with the model group, # P<0.01; ## P<0.001; ### P<0.0001.

[0059] The above results show that the traditional Chinese medicine formula can improve the follicular development disorder and sex hormone disorder of rats with cyclophosphamide-induced ovarian reserve insufficiency, and the high concentration group is the best.

[0060] Example 2: Effect of different traditional Chinese medicine formulas on the secretion of estradiol by granulosa cells in ovarian reserve insufficiency.

[0061] Ovarian granulosa cells (GCs) are a key cell population that promotes the development and maturation of oocytes. The proliferation and differentiation of GCs directly affect the growth initiation, development, ovulation, corpus luteum formation, and steroid hormone secretion of follicles, as well as other ovarian function activities. Estradiol, as a marker hormone in the development of GCs and oocytes, is mainly secreted by GCs. Therefore, GCs are often used as research objects to study the effects of drugs on ovarian function changes and follicular development.

[0062] Granulosa cell culture: KGN cells were cultured in DMEM / F12 + 10% FBS + 1% double-antibody medium in an incubator set at 37°C and 5% CO2. When subculturing, the culture medium was discarded, the cells were rinsed with PBS buffer to remove residual serum, 1-2 mL of trypsin was added to digest the cells for 2-3 min, 4-6 mL of culture medium was added to stop the digestion, and the cells were blown apart using a pipette. Appropriate cell suspension was added to a new culture dish.

[0063] Establishment of granulosa cell damage model: KGN cells were seeded in a 96-well plate at a density of 3×10 5 μg / mL, 50 μg / mL, 100 μg / mL, 500 μg / mL, and 1000 μg / mL, respectively. After 24 h of culture, CCK-8 was used to detect cell viability to screen the optimal concentration of cyclophosphamide-induced granulosa cell damage model.

[0064] Preparation of each traditional Chinese medicine formula decoction:

[0065] Full group: Rehmanniae Radix Recens 12 g, Rehmanniae Radix 12 g, Radix Glehniae 10 g, Radix Ophiopogonis 10 g, Colla Corii Asini 6 g, Rhizoma Dioscoreae 10 g, Dendrobium 10 g, Plastrum Testudinis 10 g, Semen Euryale 10 g, Semen Litchi 10 g, Radix Morindae Officinalis 10 g;

[0066] Dendrobium group: Rehmanniae Radix Recens 12 g, Rehmanniae Radix 12 g, Radix Glehniae 10 g, Radix Ophiopogonis 10 g, Colla Corii Asini 6 g, Rhizoma Dioscoreae 10 g, Plastrum Testudinis 10 g, Semen Euryale 10 g, Semen Litchi 10 g, Radix Morindae Officinalis 10 g;

[0067] Dendrobium and Plastrum Testudinis group: Rehmanniae Radix Recens 12 g, Rehmanniae Radix 12 g, Radix Glehniae 10 g, Radix Ophiopogonis 10 g, Colla Corii Asini 6 g, Rhizoma Dioscoreae 10 g, Semen Euryale 10 g, Semen Litchi 10 g, Radix Morindae Officinalis 10 g;

[0068] Semen Euryale group: Rehmanniae Radix Recens 12 g, Rehmanniae Radix 12 g, Radix Glehniae 10 g, Radix Ophiopogonis 10 g, Colla Corii Asini 6 g, Rhizoma Dioscoreae 10 g, Plastrum Testudinis 10 g, Semen Litchi 10 g, Radix Morindae Officinalis 10 g;

[0069] Semen Litchi, Radix Morindae Officinalis and Semen Euryale group: Rehmanniae Radix Recens 12 g, Rehmanniae Radix 12 g, Radix Glehniae 10 g, Radix Ophiopogonis 10 g, Colla Corii Asini 6 g, Rhizoma Dioscoreae 10 g, Plastrum Testudinis 10 g;

[0070] Rehmanniae Radix Recens and Rehmanniae Radix group: Radix Glehniae 10 g, Radix Ophiopogonis 10 g, Colla Corii Asini 6 g, Rhizoma Dioscoreae 10 g, Plastrum Testudinis 10 g, Semen Euryale 10 g, Semen Litchi 10 g, Radix Morindae Officinalis 10 g;

[0071] Dendrobium, Plastrum Testudinis, Fructus Lycii and Fructus Corni group: Rehmanniae Radix Recens 12 g, Rehmanniae Radix 12 g, Radix Glehniae 10 g, Radix Ophiopogonis 10 g, Colla Corii Asini 6 g, Rhizoma Dioscoreae 10 g, Fructus Lycii 10 g, Fructus Corni 10 g, Semen Euryale 10 g, Semen Litchi 10 g, Radix Morindae Officinalis 10 g;

[0072] Plastrum Testudinis group: Rehmanniae Radix Recens 12 g, Rehmanniae Radix 12 g, Radix Glehniae 10 g, Radix Ophiopogonis 10 g, Colla Corii Asini 6 g, Rhizoma Dioscoreae 10 g, Semen Euryale 10 g, Semen Litchi 10 g, Radix Morindae Officinalis 10 g.

[0073] Each decoction piece was weighed according to the dose. All decoction pieces were purchased from the Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine. The first decoction was added with an appropriate amount of water to immerse the medicinal materials (except Colla Corii Asini), soaked for 30 min, boiled with strong fire, and decocted with weak fire for 30 min. The filtrate was filtered with clean gauze. The second decoction was prepared as above to obtain the medicinal juice with a crude drug concentration of 1 g / mL. If the formula contained Colla Corii Asini, the Colla Corii Asini was added. The mixture was stored at 4°C for standby.

[0074] Preparation of Krebs-Ringer's buffer solution: weigh sodium chloride (AR) 8.0 g, potassium chloride (AR) 0.2 g, calcium chloride (AR) 20.2 g, sodium dihydrogen phosphate dihydrate (AR) 0.05 g, sodium bicarbonate (AR) 1.0 g and magnesium chloride (AR) 0.1 g, add distilled water to 1 L, add 1.0 g of glucose, adjust the pH value to 7.39-7.41, and store at 4°C for standby.

[0075] Preparation of drug-containing intestinal absorption solution of each traditional Chinese medicine formula: the rats were sacrificed at the dislocation of the spine, the abdominal cavity was opened, and about 10±2 cm long of each intestinal segment (duodenum, jejunum, ileum and colon) was quickly taken out. The rat intestine was separated from the mesentery. After being washed with Krebs-Ringer's buffer solution, the intestinal segment was turned over so that the mucosa was on the outside and the serosa was on the inside. The end of the intestinal segment was ligated with surgical thread. After 2 mL of blank Krebs-Ringer's buffer solution was added to the small capsule, the other end of the intestinal segment was closed to form a sausage shape. The intestinal capsule was placed in a conical flask containing traditional Chinese medicine decoction and placed in a 37°C water bath for incubation. During the incubation, a mixture of O2 and CO2 was continuously introduced into the conical flask to maintain the activity of the isolated intestinal capsule. After 2 h, the liquid in the intestinal capsule was taken out, filtered through a 0.22 μm microporous filter, and stored at -80°C.

[0076] ELISA biochemical detection: six-well plates were prepared and the cells were grown to about 80% confluence. After replacing the medium with the drug, the cell culture medium in the six-well plates was collected and centrifuged to obtain the supernatant. The E2 ELISA kit was used to determine the E2 level in the supernatant according to the instructions.

[0077] (1) Effect of different concentrations of cyclophosphamide on KGN cell proliferation

[0078] We used gradient concentrations (0, 50, 100, 500 and 1000 μg / mL) of CTX to treat KGN cells, and used CCK-8 to detect cell viability to screen the optimal concentration of CTX-induced granulosa cell damage model. The experimental results showed that when the concentration of CTX was 500 and 1000 μg / mL, the level of KGN cell viability was significantly decreased (P<0.001) Figure 3 ). However, when the intervention concentration was 1000 μg / mL, the number of cells was very small, the cell deformation synapse was more severe, and the viability was very poor. Therefore, we chose 500 μg / mL of cyclophosphamide to induce granulosa cell damage model for subsequent experiments.

[0079] (2) Effect of drug-containing intestinal absorption solution of Gouteng Badixian Sishu group, Gouteng Badixian group and whole formula group on the content of estradiol in granulosa cell culture medium

[0080] The KGN cells were intervened by the drug-containing intestinal absorption liquid of the group without Cuscuta, Radix Morindae Officinalis and Semen Euryale, the group without Semen Euryale and the whole prescription group, compared with the blank group and the model group. After 24 hours, the supernatant of the KGN cell culture medium of each group was collected, and the level of E2 of each group was detected by using the ELASA kit. The results showed that compared with the blank group, the level of E2 of the granulosa cell culture medium of the model group was significantly down-regulated (P<0.001). Compared with the model group, the levels of E2 of the group without Cuscuta, Radix Morindae Officinalis and Semen Euryale, the group without Semen Euryale and the whole prescription group were significantly increased (P<0.01). Compared with the group without Cuscuta, Radix Morindae Officinalis and Semen Euryale, the level of E2 of the granulosa cell of the group without Semen Euryale was significantly increased (P<0.01) Figure 4

[0081] (3) Effects of the drug-containing intestinal absorption liquid of the group without Radix Rehmanniae and Radix Rehmanniae Preparata and the whole prescription group on the content of estradiol in the granulosa cell culture medium

[0082] The KGN cells were intervened by the drug-containing intestinal absorption liquid of the group without Radix Rehmanniae and Radix Rehmanniae Preparata and the whole prescription group, compared with the blank group and the model group. After 24 hours, the supernatant of the KGN cell culture medium of each group was collected, and the level of E2 of each group was detected by using the ELASA kit. The results showed that compared with the blank group, the level of E2 of the granulosa cell culture medium of the model group was significantly down-regulated (P<0.001). Compared with the model group, the levels of E2 of the group without Radix Rehmanniae and Radix Rehmanniae Preparata and the whole prescription group were significantly increased (P<0.001). Compared with the group without Radix Rehmanniae and Radix Rehmanniae Preparata, the level of E2 of the granulosa cell of the whole prescription group was significantly increased (P<0.001) Figure 5

[0083] (4) Effects of the drug-containing intestinal absorption liquid of the group without Dendrobium and Plastrum Testudinis, the group without Dendrobium and Plastrum Testudinis plus Lycium barbarum and Cornus officinalis, the group without Plastrum Testudinis and the whole prescription group on the content of estradiol in the granulosa cell culture medium

[0084] The KGN cells were intervened by the drug-containing intestinal absorption liquid of the group without Dendrobium and Plastrum Testudinis, the group without Dendrobium and Plastrum Testudinis plus Lycium barbarum and Cornus officinalis, the group without Plastrum Testudinis and the whole prescription group, compared with the blank group and the model group. After 24 hours, the supernatant of the KGN cell culture medium of each group was collected, and the level of E2 of each group was detected by using the ELASA kit. The results showed that compared with the blank group, the level of E2 of the granulosa cell culture medium of the model group was significantly down-regulated (P<0.001). Compared with the model group, the levels of E2 of the group without Dendrobium and Plastrum Testudinis, the group without Dendrobium and Plastrum Testudinis plus Lycium barbarum and Cornus officinalis, the group without Plastrum Testudinis and the whole prescription group were significantly increased (P<0.01). Compared with the group without Dendrobium and Plastrum Testudinis, the level of E2 in the granulosa cell culture medium of the group without Dendrobium and Plastrum Testudinis plus Lycium barbarum and Cornus officinalis was increased (P<0.05), and the level of E2 in the granulosa cell of the group without Dendrobium was significantly increased (P<0.001) Figure 6 , and the level of E2 of the whole prescription group was the highest.

[0085] ​​From the cell level, it is proved that the traditional Chinese medicine prescription can improve the estrogen level of granulosa cells induced by cyclophosphamide. Gouqi and Shanzhu are commonly used yin-tonifying herbs in gynecology, but in this prescription, the combination of Biejia and Shihu is still significantly better than Gouqi and Shanzhu. Therefore, the combination of Shihu and Biejia in the prescription has a unique role in the treatment of ovarian reserve dysfunction.

[0086] Effect of traditional Chinese medicine prescription on ovarian reserve dysfunction in Example 3.

[0087] This study included 35 patients with decreased ovarian reserve (DOR) who visited the gynecological outpatient department of Jiangxi University of Traditional Chinese Medicine Hospital and the Second Affiliated Hospital from November 2022 to May 2023. All patients met the conditions for single group observation study. The average age of the study subjects was (35.40±4.14) years, and the average age of menarche was (13.54±1.25) years.

[0088] 3.1 Diagnostic criteria

[0089] The diagnosis of DOR patients is based on the following criteria:

[0090] (1) Clinical symptoms: including menstrual disorders (such as irregular menstrual cycle, light menstrual flow) or accompanied by infertility; soreness of waist and knees; decreased sexual desire; dry throat and mouth; little and dry vaginal discharge;

[0091] (2) Laboratory tests: bilateral antral follicle count (AFC) <6; anti-Mullerian hormone (AMH) <1.1 ng / mL; and / or basal follicle-stimulating hormone (FSH) between 10 IU / L and 40 IU / L (two tests should be performed with an interval of more than 1 month, and both tests should meet the above criteria).

[0092] 3.2 Inclusion criteria

[0093] (1) Age 18 to 45 years old;

[0094] (2) Meet the diagnostic criteria for DOR;

[0095] (3) Voluntarily participate in the study and sign the informed consent form.

[0096] 3.3 Exclusion criteria

[0097] (1) Allergic or incompatible to Chinese herbal medicine;

[0098] (2) Pregnant or lactating women;

[0099] (3) More than one year of menopause;

[0100] (4) Abnormal uterine bleeding (except ovulation disorder bleeding);

[0101] (5) Patients who are taking or have taken hormone drugs in the last 3 months;

[0102] (6) Patients with endometrial heterotopia, uterine adenomyosis, submucous myoma or non-submucous myoma with a diameter greater than 4 cm;

[0103] (7) Patients with unknown nature of pelvic mass;

[0104] (8) Patients with polycystic ovary syndrome, hyperprolactinemia, hyperandrogenemia, diabetes, thyroid dysfunction or other endocrine diseases affecting ovulation;

[0105] (9) Patients receiving specialized treatment due to cardiovascular, liver, kidney, lung, biliary or hematopoietic system diseases (such as hemoglobin < 90 g / L), malignancies or mental diseases, etc.;

[0106] (10) Patients participating in other clinical trials or having participated in other clinical trials in the last month;

[0107] (11) Patients deemed unsuitable for the study by the investigator.

[0108] 3.4 Study Methods

[0109] This study adopts a single group observational design. After the completion of screening and baseline visits, all included patients receive traditional Chinese medicine treatment. The specific prescription and dosage are as follows: Radix Rehmanniae Preparata 12 g, Radix Rehmanniae 12 g, North Shashen 10 g, Ophiopogon 10 g, Colla Corii Asini 6 g, Yam 10 g, Dendrobium 10 g, Tortoise Plastrum 10 g, Gorgon 10 g, Semen Eurycoma 10 g, Ba Ji Tian 10 g. The traditional Chinese medicine decoction pieces are provided by Guangdong Yifang Pharmaceutical Co., Ltd.

[0110] Method of taking medicine: 1 dose per day, decocted with water, taken twice a day in the morning and evening, 250 mL each time, continuously taken for 3 months.

[0111] 3.5 Observation Indexes

[0112] 3.5.1 Indexes Related to Ovarian Reserve Function

[0113] Before treatment and 3 months after the end of treatment, venous blood was drawn on the 2nd-3rd day of the menstrual cycle to detect AMH, FSH, E2 and FSH / LH values, and ultrasound examination was performed to measure AFC.

[0114] 3.5.2 Clinical Symptoms

[0115] Before treatment and 3 months after the end of treatment, the patient's menstrual cycle, menstrual volume, soreness of waist and knees, decreased libido, dry throat and mouth, and 6 items of vaginal discharge volume, dryness and dryness were evaluated, and the total score was 4 grades, each counted as 0, 2, 4, 6 points, and the scoring criteria were as follows:

[0116] Table 3 Scoring of Clinical Symptoms

[0117]

[0118] 3.5.3 Safety indicators

[0119] Adverse reactions were recorded during treatment. Blood routine and liver function tests were performed before treatment and 3 months after treatment.

[0120] 3.6 Statistical analysis

[0121] Statistical analysis was performed using SPSS 27.0 software. Continuous variables were expressed as mean ± standard deviation if they met the normal distribution. Paired t-test was used for comparison of indicators before and after treatment. Non-normal distribution data were analyzed by Wilcoxon signed rank test. P<0.05 was considered statistically significant. 4 Results

[0122] 4.1 Ovarian reserve function-related indicators

[0123] The ovarian reserve function-related indicators before and after treatment were analyzed by paired t-test. The results showed that:

[0124] · After treatment, FSH, E2 and FSH / LH values were significantly lower than before treatment, with statistically significant differences (P<0.05);

[0125] · After treatment, AMH and AFC increased compared with before treatment, with statistically significant differences (P<0.05) (see Table 4).

[0126] Table 4 Changes in ovarian reserve function-related indicators before and after treatment

[0127] n=35

[0128] Note: Compared with before treatment, # P>0.05, *P<0.05.

[0129] 4.2 TCM-related symptom scores

[0130] The TCM-related symptom scores before and after treatment were analyzed by Wilcoxon signed rank test. The results showed that the clinical symptoms of patients improved significantly after treatment. Menstrual cycle, menstrual volume, soreness of waist and knees, decreased libido, dry throat and mouth, and total score were lower than before treatment, with statistically significant differences (P<0.05) (see Table 5). The TCM-related symptom score table is shown in Table 3.

[0131] Table 5 Changes in TCM-related symptom scores before and after treatment

[0132] n=35 ​

[0133]

[0134] 5.3 Adverse reactions

[0135] The patients were subjected to safety examination before and after treatment, and no obvious abnormality was found, and no adverse reactions and safety events occurred during treatment.

[0136] 5. Conclusion

[0137] The traditional Chinese medicine treatment has a significant improvement effect on the clinical symptoms of DOR patients, can reduce the FSH, FSH / LH and E2 values, increase the AFC quantity and AMH value, and thus improve the ovarian reserve function to a certain extent.

[0138] In summary, the traditional Chinese medicine composition of the present application can improve the estrogen level of cyclophosphamide-induced granulosa cells with insufficient ovarian reserve function, and the combination of "dendrobium, turtle shell", "cuscuta, radix morindae officinalis, gordon euryale seed" and "prepared rehmannia, raw rehmannia" has a good synergistic effect in the treatment of insufficient ovarian reserve function.

[0139] The present application provides a new composition for treating decreased ovarian reserve function, and there are many methods and approaches to realize the technical solution, and the above description is only the preferred embodiment of the present application, it should be pointed out that for ordinary skilled in the art, without departing from the principles of the present application, can make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application. The components not explicitly described in the embodiment can be realized by using the existing technology.

Claims

1. A traditional Chinese medicine composition for treating decreased ovarian reserve, characterized in that, The traditional Chinese medicine composition is made from raw materials in the following weight proportions: 10-20 parts of prepared rehmannia, 10-20 parts of raw rehmannia, 8-15 parts of north adenosma, 10-20 parts of ophiopogon, 5-10 parts of donkey-hide gelatin, 8-15 parts of Chinese yam, 8-15 parts of dendrobium, 8-15 parts of turtle shell, 8-15 parts of gordon euryale seed, 8-15 parts of semen astragali complanati, and 8-15 parts of morindae.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The traditional Chinese medicine composition is made from raw materials in the following weight proportions: 10-15 parts of prepared rehmannia, 10-15 parts of raw rehmannia, 8-12 parts of north adenosma, 8-12 parts of ophiopogon, 5-8 parts of donkey-hide gelatin, 8-12 parts of Chinese yam, 8-12 parts of dendrobium, 8-12 parts of turtle shell, 8-12 parts of gordon euryale seed, 8-12 parts of semen astragali complanati, and 8-12 parts of morindae.

3. The traditional Chinese medicine composition according to claim 1, characterized in that, The traditional Chinese medicine composition is made from raw materials in the following weight proportions: 12 parts of prepared rehmannia, 12 parts of raw rehmannia, 10 parts of north adenosma, 10 parts of ophiopogon, 6 parts of donkey-hide gelatin, 10 parts of Chinese yam, 10 parts of dendrobium, 10 parts of turtle shell, 10 parts of gordon euryale seed, 10 parts of semen astragali complanati, and 10 parts of morindae.

4. Use of the traditional Chinese medicine composition of any one of claims 1-3 in the preparation of a medicament for treating decreased ovarian reserve.

5. Use according to claim 4, characterized in that, The dosage form of the medicament is any one of granules, paste, decoction, capsules, tablets, and powder.

6. The method for preparing the traditional Chinese medicine composition according to any one of claims 1-3, characterized in that, The method comprises the following steps: soaking the raw materials of the traditional Chinese medicine composition except donkey-hide gelatin in water, pouring the water and the medicinal materials into a decocting pot after soaking, decocting for the first time, boiling with a large fire, then decocting with a small fire, and filtering out medicinal juice; decocting for the second time by adding water, boiling with a large fire, then decocting with a small fire, and filtering out medicinal juice, mixing the medicinal juice of the first and second decocting, filtering, finally slowly decocting and concentrating to obtain juice, and adding melted donkey-hide gelatin.

7. The production method according to claim 6, characterized by, The raw materials of the traditional Chinese medicine composition except donkey-hide gelatin are soaked in water in an amount of 5-20 times the total weight of the raw materials for 0.3-1.0 hours.

8. The preparation method according to claim 6, characterized in that Water in an amount of 3-8 times the total weight of the raw materials is added for the second decocting.

9. The preparation method according to claim 6, characterized in that The first decocting is boiled with a large fire, then decocted with a small fire for 15-40 minutes; the second decocting is boiled with a large fire after adding water, then decocted with a small fire for 12-40 minutes.

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