Traditional Chinese medicine composition for treating decrease of ovarian reserve function as well as preparation method and application of traditional Chinese medicine composition

Abstract

The invention discloses a traditional Chinese medicine composition for treating ovarian reserve function decline. The traditional Chinese medicine composition is prepared from the following components in parts by weight: 10-20 parts of radix rehmanniae preparata, 10-20 parts of radix rehmanniae, 8-15 parts of radix glehniae, 10-20 parts of radix ophiopogonis, 5-10 parts of colla corii asini, 8-15 parts of rhizoma dioscoreae, 8-15 parts of herba dendrobii, 8-15 parts of tortoise plastron, 8-15 parts of semen euryales, 8-15 parts of semen cuscutae and 8-15 parts of radix morindae officinalis. The traditional Chinese medicine composition can improve follicle development disorder and sex hormone disorder of rats with ovarian reserve function insufficiency induced by cyclophosphamide, the number of atresia follicles and corpus luteum is obviously reduced, and the traditional Chinese medicine composition has a good treatment prospect.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicines, and specifically designs a composition for treating decreased ovarian reserve function, and a preparation method and application thereof. Background Art

[0002] Ovarian reserve refers to the ability of ovarian cortical follicles to grow, develop, and form fertilizable oocytes. It reflects a woman's reproductive potential and can be simply understood as the ability of the ovaries to produce eggs and ovulate. When the primordial follicles in the ovaries are exhausted prematurely, follicular growth is impaired, and atresia is accelerated, resulting in a decrease in the number of follicles or a decrease in the quality of oocytes, which in turn leads to a decrease in reproductive potential and endocrine dysfunction, it is called diminished ovarian reserve (DOR). The clinical manifestations of DOR are mainly a series of perimenopausal clinical symptoms such as oligomenorrhea, decreased menstrual volume, and even amenorrhea and infertility. If not intervened early, it will gradually develop into premature ovarian failure (POF), which seriously affects the patient's reproductive health and quality of life. Therefore, early intervention and prevention are urgently needed for DOR patients to protect ovarian function and improve the patient's quality of life and reproductive function.

[0003] Traditional Chinese medicine treatment mainly balances qi, blood, yin and yang, regulates the internal organs, and replenishes deficiency and eliminates excess. CN118615370A discloses a preparation for improving ovarian reserve function, comprising the following components by weight: 20-30 parts of red dates, 10-30 parts of safflower, 10-18 parts of donkey-hide gelatin, 10-16 parts of angelica, 8-14 parts of astragalus, 8-12 parts of Ligustrum lucidum, 6-12 parts of kudzu root, 6-10 parts of wolfberry, 4-10 parts of mulberry seeds, and 4-8 parts of longan pulp. The preparation has the effects of replenishing qi and blood, regulating endocrine, nourishing yin and blood, regulating menstruation, stimulating the production of estrogen and follicles, regulating ovaries, and delaying ovarian decline.

[0004] CN118615369A discloses a Chinese medicine composition for improving age-related ovarian dysfunction and infertility. The ingredients of the Chinese medicine composition include mulberry, kudzu root, poria and wolfberry. The composition follows the concept of traditional medicine that Chinese medicine and food are of the same origin, rationally combines the four properties and five flavors of Chinese medicine, as well as the principles of ascending, descending, sinking and floating, and monarch, minister, assistant and guiding. It has the effect of improving age-related ovarian reserve dysfunction, endocrine dysfunction, menstrual disorder and decreased fertility.

[0005] CN117752743A discloses a Chinese medicine composition for maintaining follicles, and 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 foam gelatin, and 6-15 parts of lily. It is based on the reproductive axis of "kidney-Tiangui-Chongren-Baogong" in traditional Chinese medicine and the activities and transformation of the growth and decline of qi, blood, yin and yang of the internal organs, which is the theoretical basis for the normal growth and discharge of follicles, and corresponds to the periodic changes and effects of the axis of "hypothalamus-pituitary-ovary" in modern medicine. The growth of follicles depends on the abundance of kidney essence, which is the innate foundation, and is closely related to the spleen and stomach, which is the acquired foundation, to generate qi and blood, and other internal organs. The prescription mainly uses Dendrobium officinale, Polygonatum sibiricum 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 the ingredients of blood and flesh, they can strengthen the spleen and kidney, regulate the Chong and Ren meridians, increase follicular fluid, promote follicle growth, and improve egg quality, and have the effect of "1+1+1>3"; the assistant 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 adjuvants 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 reduced ovarian reserve function and its application. The Chinese medicine composition is made of the following raw materials: Radix Rehmanniae Preparata, stir-fried Atractylodes macrocephala, Chinese yam, Chinese angelica, stir-fried white peony root, stir-fried Semen Ziziphi Spinosae, Cortex Moutan, Radix Bupleuri, Radix Dipsaci, Codonopsis pilosula, Radix Adenophorae, Semen Cuscutae, Fructus Ligustri Lucidi, and Pericarpium Citri Reticulatae. It is mainly used to tonify the kidney and replenish essence, but the medicines for tonifying yin are mostly nourishing and greasy, and should be supplemented with products for warming yang and replenishing qi to replenish without retention. Essence and blood are of the same origin and nourish each other. The effect of supplementing essence with products for replenishing blood and nourishing blood can be doubled. It uses Radix Rehmanniae Preparata and stir-fried Atractylodes macrocephala as the main medicines, nourishing yin and blood, replenishing essence and filling marrow, and using Semen Cuscutae, Fructus Ligustri Lucidi, Radix Adenophorae, Chinese yam, Radix Dipsaci, Codonopsis pilosula, and Pericarpium Citri Reticulatae as the ministerial medicines, tonifying the liver and kidney, replenishing essence and marrow, replenishing qi and strengthening spleen, and warming the kidney and assisting yang. The monarch and 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 Chinese medicinal compound composition for treating decreased ovarian reserve function, comprising the following components in parts by weight: 25-30 parts of Rehmannia root, 10-30 parts of Angelica sinensis, 10-30 parts of White Peony Root, 10-15 parts of Cornus Fruit, 5-15 parts of Eucommia ulmoides, 10-25 parts of Atractylodes macrocephala, 5-15 parts of Poria cocos, 5-10 parts of Dipsacus asper, 15-20 parts of Radix Glycyrrhizae Preparata, 15-20 parts of Astragalus membranaceus, 10-20 parts of Codonopsis pilosula, 5-15 parts of Jujube, 5-10 parts of Donkey-hide Gelatin, 10-15 parts of Morinda officinalis, 10-15 parts of Cuscuta australis, 10-30 parts of Achyranthes bidentata, 10-20 parts of Dioscorea opposita and 10-15 parts of Euryale ferox. In the above scheme, Rehmannia root, Astragalus membranaceus, Codonopsis pilosula and Euryale ferox are used as the main medicines in this prescription, which play the effects of tonifying the kidney, replenishing essence and filling marrow, replenishing qi, nourishing blood and promoting body fluid; Angelica sinensis, White Peony Root, Cornus Officinalis, Donkey-hide Gelatin, Morinda Officinalis, Cuscuta Australis, etc. are all ministerial medicines, which have the effects of tonifying the kidney and assisting yang, and promoting yang; Dipsacus asper, Eucommia ulmoides, Atractylodes macrocephala, Poria cocos, Chinese yam, etc. are all adjuvant medicines, which have the effect of assisting the circulation of essence and marrow; Chinese Cyathula Bidentatae, Roasted Licorice Root and Jujube are all guiding medicines. Chinese Cyathula Bidentatae nourishes the kidney and strengthens the waist, and guides all the medicines downward to the kidney, Chong, Ren and uterus, while Roasted Licorice Root and Jujube harmonize all the medicines. Summary of the invention

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

[0009] This application proposes a "vessel for nurturing", that is, "Tian Gui-Bao Gong" forms a "heaven-earth" component, which together form the "vessel for nurturing" of female reproduction. Tian Gui comes from the innate kidney essence of parents, which turns into Qi, resides in the sky, gathers to form Gui Shui, and descends to the earth (Bao Gong) as rain and dew; Bao Gong receives the Yin blood and Yin fluid of Chong and Ren, and under the action of Yang Qi (including the fire of Mingmen) of Du, they evaporate upwards, nourish Tian Gui, and go back and forth, endlessly, forming a small universe of female reproduction, and becoming the "vessel for nurturing" of female reproduction.

[0010] Tiangui is the basic substance of female reproduction. It is generally believed that there are two conventional ways to replenish Tiangui: one is the acquired essence of spleen and stomach water and grain; the other is that Yi and Gui have the same origin, that is, essence and blood have the same origin (liver and kidney have the same origin).

[0011] The decline of ovarian reserve function is considered as "early exhaustion of Guishui". This application proposes a new treatment idea: 1. Nourish Yin blood and Yin fluid; 2. Properly supplement the fire of the gate of life; 3. According to the characteristics of the times of the disease, first, people are generally under great pressure, and their heart fire is strong, which is easy to consume (heart) Yin fluid; second, they generally overeat high-calorie foods such as greasy and thick food, which is easy to accumulate heat in the stomach and intestines, causing stomach dryness and damage to body fluids. Therefore, it is supplemented with treating stomach dryness, nourishing kidney yin and reducing deficiency heat.

[0012] Based on the above treatment ideas, the present application proposes a new Chinese medicine composition for treating decreased ovarian reserve function, which comprises the following components in parts by weight: 10-20 parts of cooked rehmannia, 10-20 parts of raw rehmannia, 8-15 parts of northern adenophora, 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 tortoise shell, 8-15 parts of euryale, 8-15 parts of dodder seed and 8-15 parts of Morinda officinalis.

[0013] Preferably, the traditional Chinese medicine composition comprises the following components in parts by weight: 10-15 parts of cooked rehmannia, 10-15 parts of raw rehmannia, 8-12 parts of northern adenophora, 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 tortoise shell, 8-12 parts of euryale, 8-12 parts of dodder seed and 8-12 parts of Morinda officinalis.

[0014] In a preferred embodiment, the Chinese medicine composition comprises the following components in parts by weight: 12 parts of cooked rehmannia, 12 parts of raw rehmannia, 10 parts of northern adenophora, 10 parts of ophiopogon, 6 parts of donkey-hide gelatin, 10 parts of yam, 10 parts of dendrobium, 10 parts of tortoise shell, 10 parts of Euryale ferox, 10 parts of dodder seed and 10 parts of Morinda officinalis.

[0015] The present application further proposes the use of the above-mentioned Chinese medicine composition in the preparation of a medicine or preparation for treating decreased ovarian reserve function.

[0016] Wherein, the preparation is any one of granules, pastes, infusions, capsules, tablets and powders.

[0017] In one embodiment, the preparation is a decoction.

[0018] The present application further proposes a method for preparing the above-mentioned Chinese medicine composition, comprising the following steps: soaking the components of the Chinese medicine composition except donkey-hide gelatin in water, then pouring the water and medicinal materials into a decocting pot, performing a first decoction, boiling over high heat, then simmering over low heat, and filtering out the medicinal juice; performing a second decoction, adding water and boiling over high heat, then simmering over low heat, filtering out the medicinal juice, mixing the medicinal juices of the first decoction and the second decoction, filtering, and finally decocting over low heat to concentrate the juice, and adding the melted donkey-hide gelatin.

[0019] Preferably, the components other than donkey-hide gelatin in the traditional Chinese medicine composition are added with water 5-20 times the mass of the total components and soaked for 0.3 h to 1.0 h.

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

[0021] Preferably, the first frying is brought to a boil over high heat, then reduced to low heat and simmered for 15-40 minutes; the second frying is brought to a boil over high heat, then reduced to low heat and simmered for 12-40 minutes.

[0022] In the prescription of this application:

[0023] Cooked rehmannia is sweet and slightly warm, and focuses on nourishing blood and yin, nourishing the kidney and liver; raw rehmannia is sweet, cold and moist, and is good at nourishing yin and cooling blood, nourishing yin and producing body fluid. Cooked rehmannia is good at tonifying, while raw rehmannia is good at clearing. The combination of the two medicines can take into account both clearing and tonifying, 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, so they are both the main medicines.

[0024] The ministerial drug, donkey-hide gelatin, nourishes yin and blood, nourishes the yin of the lungs, liver and kidneys; Chinese yam nourishes the yin of the lungs, spleen and kidneys; Northern Adenophora nourishes yin and clears the lungs, benefits the stomach and produces fluid; Ophiopogon nourishes the yin and fluid of the lungs and stomach, and can also nourish the yin of the heart and clear the heat of the heart. The four drugs are used together to replenish the yin fluid of the five internal organs, focusing on replenishing the yin of the lungs and nourishing the water of the kidneys, and playing the role of "replenishing the lungs and invigorating the kidneys". Dendrobium is sweet and cool and enters the stomach. It can nourish yin and produce fluid to treat dry stomach, and nourish kidney yin and reduce deficiency heat. Turtle shell nourishes yin and suppresses yang, nourishes the liver and kidneys, and can control deficiency fire. The combination of these drugs replenishes yin and reduces fire, replenishes and clears, nourishes yin without nourishing greasiness, and reduces fire without damaging yin. The monarch and ministers work together to nourish yin and blood, replenish the yin of the five internal organs, and focus on nourishing kidney yin, so 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 clear, 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. The above work together to help the Chong and Ren channels to orderly gather the yin blood and yin fluid in the uterus.

[0026] Cuscuta chinensis is good at replenishing kidney qi, which can help the uterus seal and absorb. "Suwen·Yin Yang Bie Lun" says: "The static is Yin, and the moving is Yang." Cuscuta chinensis replenishes kidney qi, and Morinda officinalis warms and replenishes kidney yang. The two are combined in yin medicines to prevent other yin-replenishing medicines from being static and not moving.

[0027] Morinda officinalis enters the Mingmen, is good at warming and replenishing the fire of the Mingmen, and helps the Yin blood and Yin fluid in the uterus to evaporate and nourish the Tiangui. Gorgon fruit can nourish the kidney and consolidate the essence, can replenish the deficiency of the Ren meridian, and can also clear the Yin fluid, so that the Yin fluid can flow smoothly without stagnation. When used together with other Yin-tonifying medicines, it can replenish without stagnation, and benefit without injury.

[0028] The whole formula nourishes yin and blood, nourishes the yin fluid of the five internal organs, replenishes kidney qi, and warms and replenishes the fire of the gate of life. It is both tonifying and purgative, and combines clearing and tonifying. It nourishes without stagnation, benefits without injury, and moves in stillness, playing the role of nourishing yin and blood, warming yang and transforming qi.

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

[0030] Cooked Rehmannia focuses on tonifying blood and nourishing yin, while raw Rehmannia focuses on nourishing yin and blood. Cooked Rehmannia is special in tonifying, while raw Rehmannia is good at clearing. The two medicines are combined to take into account both clearing and tonifying, and the effect of nourishing yin and blood is enhanced.

[0031] Turtle shell can nourish yin and tonify the kidney, and can also control false 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 false heat. The two medicines are used together, and the effect of nourishing yin and reducing false heat is enhanced.

[0032] Cuscuta is good at replenishing kidney qi, while Morinda officinalis warms and replenishes the fire of the gate of life. The combination of the two in yin medicines can prevent other medicines from being static and not moving; Gorgon fruit can nourish the kidney and consolidate essence, replenish the deficiency of the Ren meridian, and promote the flow of yin fluid, so that yin fluid can flow without stagnation. When used together with other yin-replenishing medicines, it replenishes without stagnation. Yin medicines can be static and dynamic with Cuscuta and Morinda officinalis, and can be dynamic and orderly with Gorgon fruit. This combination is more effective. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Count the follicles at all levels for rats in different groups;

[0034] Figure 2 Comparison of serum E2, FSH, LH and AMH levels in rats of each group;

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

[0036] Figure 4 To investigate the effects of the group without Cuscuta australis and Euryale ferox, the group without Euryale ferox and the whole formula on the secretion of E2 in KGN cells;

[0037] Figure 5 The effects of the raw and cooked rehmannia group and the whole prescription group on the secretion of E2 in KGN cells;

[0038] Figure 6 To study the effects of the tortoise shell group without Dendrobium officinale, the tortoise shell group without Dendrobium officinale and plus Lycium barbarum and Cornus officinalis, the tortoise shell group without Dendrobium officinale and the whole prescription group on the E2 secretion content of granulosa cells. DETAILED DESCRIPTION

[0039] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

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

[0041] Formula: 12g cooked rehmannia root, 12g raw rehmannia root, 10g northern adenophora root, 10g ophiopogon root, 6g donkey hide gelatin, 10g yam, 10g dendrobium, 10g tortoise shell, 10g Euryale ferox, 10g dodder seed, 10g Morinda officinalis root

[0042] Preparation method: Soak the above Chinese medicines except donkey-hide gelatin in water for 30 minutes, add 10 times water and soak for 0.5 hours, and pour the water and medicinal materials into the decoction pot after soaking. For the first decoction, boil over high heat, then turn to low heat and simmer for about 20 minutes, and filter out the juice. For the second decoction, add 5 times water, boil over high heat, turn to low heat and simmer for 15 minutes, filter out the juice, mix the juice of the first decoction and the second decoction, filter, and finally slowly simmer over low heat to concentrate the juice, add donkey-hide gelatin (melted), and prepare a Chinese medicine composition.

[0043] Example 2 Effects of different concentrations of Chinese herbal compositions on the number of follicles at various levels and serum hormones in rats with insufficient ovarian reserve function.

[0044] Experimental animals: Sixty female Sprague Dawley rats, 7 weeks old, 180-200 g, were purchased from the animal facility of Jiangxi University of Traditional Chinese Medicine (Nanchang, China) [License No.: SCXK(Gan)2023-0001]. The feeding environment of the experimental animals was (23±2)℃, relative humidity 50%-60%, 12-h circadian rhythm, and free access to food and water.

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

[0046] Specimen collection: After the administration, the animals were fasted for 12 hours, and 10% chloral hydrate solution was injected intraperitoneally into the rats for anesthesia and blood was collected from the heart. After the whole blood was collected, it was centrifuged at 3000 rpm / min for 10 minutes, and the upper serum was aspirated. The levels of estradiol (E2), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and anti-Müllerian hormone (AMH) in the serum were detected by ELISA.

[0047] HE staining to observe morphological changes in ovarian tissue: The removed mouse ovarian tissue was fixed with paraformaldehyde, dehydrated with gradient ethanol, transparentized with xylene, immersed in wax, and routinely embedded in tissue, and cut into 4-5 μm thick slices. According to the operating procedures, after HE staining of the ovarian tissue slices, neutral gum was used to seal the slices, and the morphological changes of the ovarian tissue of each group of mice were observed under a microscope, and photographed and recorded. Follicles were counted every 5 slices, and only follicles with clearly visible 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 slices × 5.

[0048] Statistical analysis: All data are expressed as mean ± standard deviation All data were statistically analyzed and plotted using SPSS and GraphPad Prism software version 10.0. Data were tested for normality and homogeneity of variance. One-way ANOVA and LSD were used for comparison between groups if they met the criteria. Dunnett's test was used for unequal variance. P < 0.05 was considered statistically significant.

[0049] (1) Effects of the Chinese medicine composition on the follicle counts at various levels in each group of rats.

[0050] As shown in Table 1 and Figure 1 As shown in the figure, compared with the normal group, the number of follicles at all levels in the model group was significantly reduced (P<0.05), and the number of atretic follicles and corpora lutea was significantly increased (P<0.05). Compared with the model group, the number of follicles at all levels in the western medicine group was significantly increased (P<0.05), and the number of atretic follicles and corpora lutea was significantly decreased (P<0.05); the number of primordial follicles and primary follicles in the low-concentration Chinese medicine group was significantly increased (P<0.05), and the number of atretic follicles and corpora lutea was significantly decreased (P<0.05); the number of primordial follicles and primary follicles in the medium-concentration Chinese medicine group was significantly increased (P<0.05), and the number of atretic follicles and corpora lutea was significantly decreased (P<0.05); the number of follicles at all levels in the high-concentration Chinese medicine group was significantly increased (P<0.05), and the number of atretic follicles and corpora lutea was significantly decreased (P<0.05).

[0051] Table 1 Follicle counts at each level in 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) Effects of Chinese medicine prescriptions on the levels of serum E2, FSH, LH, and AMH in model rats.

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

[0056] Table 2 Comparison of serum E2, FSH, LH and AMH levels in rats of each group

[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 indicate that the Chinese medicine formula can improve follicular development disorder and sex hormone disorder in rats with ovarian reserve deficiency induced by cyclophosphamide, and the high-concentration group is the best.

[0060] Example 2 Effects of different Chinese medicine prescriptions on the level of estradiol secreted by granulosa cells in patients with insufficient ovarian reserve function.

[0061] Ovarian granulosa cells (GCs) are a key cell group that promotes the development and maturation of oocytes. The proliferation and differentiation of GCs directly affect ovarian functional activities such as the growth initiation, development, ovulation, luteinization, and steroid hormone secretion of follicles. Estradiol is a landmark hormone in the development of GCs and oocytes, and its secretion is mainly responsible for GCs. Therefore, GCs are often used as research objects and play an important role in exploring the effects of drugs on changes in ovarian function and follicular development.

[0062] Granule 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. During subculturing, the 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, and then 4-6 mL of culture medium was added to terminate the digestion. The cells were blown off with a pipette, and the appropriate cell suspension was added to a new culture dish.

[0063] Establishment of granulosa cell injury model: KGN cells were cultured at 3×10 5 The cells were inoculated at a density of 100 cells / well in a 96-well plate and incubated overnight to allow them to adhere to the wall. Cyclophosphamide culture medium containing final concentrations of 0 μg / mL, 50 μg / mL, 100 μg / mL, 500 μg / mL and 1000 μg / mL were added respectively. The cell viability was detected using CCK-8 for 24 hours to screen the optimal concentration of cyclophosphamide-induced granulosa cell injury model.

[0064] Preparation of each Chinese medicine decoction:

[0065] Whole prescription: cooked rehmannia 12g, raw rehmannia 12g, northern adenophora 10g, ophiopogon 10g, donkey-hide gelatin 6g, yam 10g, dendrobium 10g, tortoise shell 10g, euryale ferox 10g, dodder seed 10g, Morinda officinalis 10g;

[0066] Dendrobium-free group: cooked rehmannia 12g, raw rehmannia 12g, northern adenophora 10g, ophiopogon 10g, donkey-hide gelatin 6g, yam 10g, tortoise shell 10g, Euryale ferox 10g, dodder seed 10g, Morinda officinalis 10g;

[0067] Dendrobium and tortoise shell group: cooked rehmannia 12g, raw rehmannia 12g, northern adenophora 10g, ophiopogon 10g, donkey-hide gelatin 6g, yam 10g, Euryale ferox 10g, dodder seed 10g, Morinda officinalis 10g;

[0068] Group without Euryale ferox: 12g cooked rehmannia root, 12g raw rehmannia root, 10g Adenophora chinensis, 10g Radix Ophiopogonis, 6g donkey hide gelatin, 10g Chinese yam, 10g Dendrobium officinale, 10g tortoise shell, 10g Cuscuta chinensis, 10g Morinda officinalis.

[0069] Group without dodder seeds, Morinda officinalis and Euryale ferox: cooked rehmannia 12g, raw rehmannia 12g, northern adenophora 10g, ophiopogon 10g, donkey hide gelatin 6g, yam 10g, dendrobium 10g, tortoise shell 10g;

[0070] The group without raw and cooked rehmannia root: Adenophora 10g, Ophiopogon japonicus 10g, donkey-hide gelatin 6g, Dioscorea opposita 10g, Dendrobium candidum 10g, Plastrum testudinis 10g, Euryale ferox 10g, Cuscuta chinensis 10g, Morinda officinalis 10g;

[0071] The group without dendrobium and tortoise shell plus wolfberry and cornus officinalis: cooked rehmannia 12g, raw rehmannia 12g, northern adenophora 10g, ophiopogon 10g, donkey-hide gelatin 6g, yam 10g, wolfberry 10g, cornus officinalis 10g, Euryale ferox 10g, dodder seed 10g, Morinda officinalis 10g;

[0072] The turtle shell-removed group: cooked rehmannia 12g, raw rehmannia 12g, northern adenophora 10g, ophiopogon 10g, donkey-hide gelatin 6g, yam 10g, dendrobium 10g, Euryale ferox 10g, dodder seed 10g, and Morinda officinalis 10g.

[0073] Weigh each decoction piece according to the composition dosage, and all decoction pieces were purchased from the Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine. For the first decoction, add an appropriate amount of water to submerge the medicinal materials (except donkey-hide gelatin), soak for 30 minutes, boil over high heat, simmer over low heat for 30 minutes, and filter the juice with clean gauze. The second decoction method is as above to obtain a Chinese medicine formula juice with a crude drug concentration of 1g / mL. If the formula contains donkey-hide gelatin, add melted donkey-hide gelatin and store at 4℃ for later use.

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

[0075] Preparation of intestinal absorption solution containing Chinese medicine formula: rats were killed by spinal dislocation, their abdominal cavities were opened, and intestinal segments (duodenum, jejunum, ileum and colon) of about 10±2cm in length were quickly taken out, and the rat intestine and mesentery were peeled off. After being rinsed with Krebs-Ringer's buffer, the intestinal segment was turned over so that the mucosal layer was outside and the serosal layer was inside. One end of the intestinal segment was ligated with surgical thread. After adding 2mL of blank Krebs-Ringer's buffer to the small sac, the other end of the ligated intestinal segment was sealed into a sausage shape. The intestinal sac was placed in a conical bottle of Chinese medicine decoction and kept warm in a 37℃ water bath. During this period, a mixed gas of O2 and CO2 was continuously introduced into the conical bottle to maintain the activity of the isolated intestinal sac. After 2h, the liquid in the intestinal sac was taken out, filtered with a 0.22μm microporous filter membrane, and stored at -80℃.

[0076] ELISA biochemical detection: Spread six-well plates and wait until the cell confluence reaches about 80%. After changing the medium, add drugs, collect the cell culture medium in the six-well plates, centrifuge and aspirate the supernatant. Use the E2 ELISA kit to measure the level of E2 in the culture medium supernatant according to the instructions.

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

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

[0079] (2) Effects of the drug-containing intestinal absorption solution of the group without dodder seeds, Morinda officinalis and Euryale ferox, the group without Euryale ferox and the whole formula on the estradiol content in the granulosa cell culture medium

[0080] KGN cells were intervened with the drug-containing intestinal absorption solution of the dodder, Morinda officinalis and Euryale ferox group, the Euryale ferox group and the whole formula group, and 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 ELASA kit was used to detect the level of E2 in each group. The results showed that compared with the blank group, the E2 level in the granular cell culture medium of the model group was significantly downregulated (P < 0.001). Compared with the model group, the E2 levels of the dodder, Morinda officinalis and Euryale ferox group, the Euryale ferox group and the whole formula group were significantly increased (P < 0.01). Compared with the dodder, Morinda officinalis and Euryale ferox group, the E2 content of the granular cells of the Euryale ferox group was significantly increased (P < 0.01) ( Figure 4 ), meanwhile, the E2 content in granulosa cells of the whole formula group was significantly higher than that of the group without Gorgon fruit.

[0081] (3) Effects of drug-containing intestinal absorption fluid from the group without raw rehmannia and the whole prescription group on the estradiol content in the granulosa cell culture medium

[0082] The drug-containing intestinal absorption solution of the group without raw rehmannia and cooked rehmannia and the whole prescription group was used to intervene in KGN cells and 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 ELASA kit was used to detect the level of E2 in each group. The results showed that compared with the blank group, the E2 level of the granular cell culture medium of the model group was significantly downregulated (P < 0.001). Compared with the model group, the E2 levels of the group without raw rehmannia and cooked rehmannia and the whole prescription group were significantly increased (P < 0.001). Compared with the group without raw rehmannia and cooked rehmannia, the E2 content of granular cells in the whole prescription group was significantly increased (P < 0.001) ( Figure 5 ).

[0083] (4) Effects of drug-containing intestinal absorption fluid on estradiol content in granulosa cell culture medium in the group with no dendrobium, no dendrobium, and no dendrobium plus wolfberry and cornus officinalis, no tortoise shell, and whole formula

[0084] KGN cells were intervened with the drug-containing intestinal absorption solution in the group without dendrobium and tortoise shell, the group without dendrobium and tortoise shell plus wolfberry and cornus officinalis, the group without tortoise shell and the whole prescription group, and 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 in each group was detected using the ELASA kit. The results showed that compared with the blank group, the level of E2 in the granular cell culture medium of the model group was significantly downregulated (P < 0.001). Compared with the model group, the E2 levels in the group without dendrobium and tortoise shell, the group without dendrobium and tortoise shell plus wolfberry and cornus officinalis, the group without tortoise shell and the whole prescription group were significantly increased (P < 0.01). Compared with the group without dendrobium and tortoise shell, the E2 content in the granular cell culture medium of the group without dendrobium and tortoise shell plus wolfberry and cornus officinalis increased (P < 0.05), and the E2 content in the granular cells of the group without dendrobium increased significantly (P < 0.001) ( Figure 6 ), meanwhile, the E2 content in the whole prescription group increased the most.

[0085] At the cellular level, it was demonstrated that the Chinese medicine formula can improve the estrogen level of granulosa cells in ovarian reserve deficiency induced by cyclophosphamide. Lycium barbarum and Cornus officinalis are commonly used gynecological yin-tonifying drugs, but in this formula, the combination of tortoise shell and dendrobium is still significantly better than Lycium barbarum and Cornus officinalis. Therefore, the combination of dendrobium and tortoise shell in the formula has a unique role in the treatment of ovarian reserve deficiency.

[0086] Example 3 Effect of Chinese medicine prescription on insufficient ovarian reserve function.

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

[0088] 3.1 Diagnostic criteria

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

[0090] (1) Clinical symptoms: including menstrual disorders (such as irregular menstrual cycle, scanty menstruation) or infertility; soreness of waist and knees; decreased libido; dry throat and mouth; scanty 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 (the interval between the two tests must be greater than 1 month, and both test results must meet the above standards).

[0092] 3.2 Inclusion criteria

[0093] (1) Aged 18 to 45;

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

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

[0096] 3.3 Exclusion criteria

[0097] (1) Those who are allergic to or incompatible with Chinese herbal medicines;

[0098] (2) Pregnant or lactating women;

[0099] (3) postmenopausal for more than one year;

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

[0101] (5) Those who are currently taking or have taken hormone drugs in the past 3 months;

[0102] (6) Patients with endometriosis, adenomyosis, submucosal fibroids, or non-submucosal fibroids with a diameter greater than 4 cm;

[0103] (7) Pelvic mass of unknown nature;

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

[0105] (9) Patients receiving specialist treatment for cardiovascular, liver, kidney, lung, biliary or hematopoietic system diseases (such as hemoglobin <90 g / L), malignant tumors or mental illness;

[0106] (10) Participating in other clinical trials or have participated in other clinical trials in the past month;

[0107] (11) Patients deemed by the researcher to be unsuitable for study participation.

[0108] 3.4 Research Methods

[0109] This study adopted a single-group observational design. After screening and baseline visits, all included patients received Chinese medicine treatment, with the specific prescriptions and dosages as follows: 12g of cooked rehmannia, 12g of raw rehmannia, 10g of northern adenophora, 10g of ophiopogon, 6g of donkey-hide gelatin, 10g of yam, 10g of dendrobium, 10g of tortoise shell, 10g of Euryale ferox, 10g of dodder seed, and 10g of Morinda officinalis. Chinese herbal medicine pieces were provided by Guangdong Yifang Pharmaceutical Co., Ltd.

[0110] Dosage: 1 dose per day, decoct in water and take warm twice a day, morning and evening, 250 mL each time, for 3 consecutive months.

[0111] 3.5 Observation indicators

[0112] 3.5.1 Ovarian reserve function related indicators

[0113] Before treatment and 3 months after the end of treatment, venous blood was drawn on the 2nd to 3rd day of the menstrual cycle to test 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 patients were evaluated for the scores of menstrual cycle, scanty menstruation, soreness of waist and knees, loss of libido, dry throat and mouth, scanty and dry vaginal discharge, and the total score. The scores were divided into 4 levels, each with 0, 2, 4, and 6 points. The scoring standard was:

[0116] Table 3 Clinical symptom scores

[0117]

[0118] 3.5.3 Safety indicators

[0119] The adverse reactions of the patients were recorded during the treatment. Blood routine and liver function tests were performed before treatment and 3 months after the end of treatment.

[0120] 3.6 Statistical analysis

[0121] SPSS 27.0 software was used for statistical analysis. Continuous variables were expressed as mean ± standard deviation if they were normally distributed. The paired t-test was used to compare the indicators before and after treatment; the Wilcoxon signed-rank test was used for non-normal distribution data; P < 0.05 was considered statistically significant.

[0122] 4 Results

[0123] 4.1 Ovarian reserve function related indicators

[0124] The paired t-test analysis of ovarian reserve function indicators before and after treatment showed that:

[0125] After treatment, the FSH, E2 and FSH / LH values ​​were significantly lower than those before treatment, and the difference was statistically significant (P < 0.05);

[0126] After treatment, AMH and AFC increased compared with those before treatment, and the difference was statistically significant (P < 0.05) (see Table 4).

[0127] Table 4 Changes of ovarian reserve function related indicators before and after treatment ( n=35)

[0128]

[0129] Note: Compared with before treatment, # P>0.05, *P<0.05.

[0130] 4.2 TCM-related symptom scores

[0131] The Wilcoxon signed rank test showed that the clinical symptoms of patients after treatment were significantly improved, and the scores of menstrual cycle, scanty menstruation, soreness of waist and knees, decreased libido, dry throat and mouth, scanty and dry vaginal discharge, and total score were all lower than before treatment, with statistically significant differences (P < 0.05) (see Table 5). The score table of TCM related symptoms is shown in Table 3.

[0132] Table 5 Changes in TCM-related symptom scores before and after treatment ( n=35)

[0133]

[0134] 5.3 Adverse Reactions

[0135] The included patients underwent safety examinations before and after treatment, and no obvious abnormalities were found. No adverse reactions or safety events occurred during the treatment.

[0136] 5 Conclusion

[0137] Traditional Chinese medicine treatment has a significant effect on improving the clinical symptoms of DOR patients, reducing FSH, FSH / LH and E2 values, increasing AFC quantity and AMH value, thereby improving ovarian reserve function to a certain extent.

[0138] In summary, the Chinese medicine formula of the present application can improve the estrogen level of granulosa cells in ovarian reserve deficiency induced by cyclophosphamide, and the combination of "Dendrobium, Tortoise Shell", "Cuscuta, Morinda, Euryale" and "Prepared Rehmannia, Raw Rehmannia" in this formula has a good synergistic effect in the treatment of ovarian reserve deficiency.

[0139] The present invention provides a new formula for treating decreased ovarian reserve function. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be considered as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A Chinese medicine composition for treating decreased ovarian reserve function, characterized in that: The traditional Chinese medicine composition comprises the following components in parts by weight: 10-20 parts of cooked rehmannia, 10-20 parts of raw rehmannia, 8-15 parts of northern adenophora, 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 tortoise shell, 8-15 parts of euryale, 8-15 parts of dodder seeds and 8-15 parts of Morinda officinalis.

2. The Chinese medicine composition according to claim 1, characterized in that: The traditional Chinese medicine composition comprises the following components in parts by weight: 10-15 parts of cooked rehmannia, 10-15 parts of raw rehmannia, 8-12 parts of northern adenophora, 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 tortoise shell, 8-12 parts of euryale, 8-12 parts of dodder seeds and 8-12 parts of Morinda officinalis.

3. The Chinese medicine composition according to claim 1, characterized in that The Chinese medicine composition comprises the following components in parts by weight: 12 parts of cooked rehmannia, 12 parts of raw rehmannia, 10 parts of northern adenophora, 10 parts of ophiopogon, 6 parts of donkey-hide gelatin, 10 parts of yam, 10 parts of dendrobium, 10 parts of tortoise shell, 10 parts of euryale, 10 parts of dodder seeds and 10 parts of Morinda officinalis.

4. Use of the Chinese medicine composition according to any one of claims 1 to 3 in the preparation of a medicine or preparation for treating decreased ovarian reserve function.

5. The use according to claim 4, characterized in that: The preparation is any one of granules, pastes, infusions, capsules, tablets and powders.

6. The use according to claim 5, characterized in that: The preparation is a water decoction.

7. The method for preparing the Chinese medicine composition according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: soaking the components of the traditional Chinese medicine composition except donkey-hide gelatin in water, then pouring the water and the medicinal materials into a medicine decocting pot, performing a first decoction, boiling over high heat, then simmering over low heat, and filtering out the medicinal juice; performing a second decoction, adding water, boiling over high heat, then simmering over low heat, filtering out the medicinal juice, mixing the medicinal juices of the first decoction and the second decoction, filtering, and finally decocting over low heat to concentrate the juice, and adding the melted donkey-hide gelatin.

8. The preparation method according to claim 7, characterized in that: Add 5-20 times the mass of water to the components other than donkey-hide gelatin in the traditional Chinese medicine composition and soak for 0.3h-1.0h.

9. The preparation method according to claim 7, characterized in that: Add 3 to 8 times the mass of the total ingredients of water for a second decoction.

10. The preparation method according to claim 7, characterized in that: For the first decoction, bring to a boil over high heat, then reduce to low heat and simmer for 15-40 minutes. For the second decoction, add water and bring to a boil over high heat, then reduce to low heat and simmer for 12-40 minutes.

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