Meridian fixing pill for preventing and treating ovarian function decline as well as preparation method and application of meridian fixing pill

Abstract

The invention belongs to the technical field of traditional Chinese medicine research and development, and particularly relates to meridian fixing pills for preventing and treating ovarian function decline and a preparation method and application thereof. The meridian fixing pill for preventing and treating ovarian function decline is prepared from the following raw materials in parts by mass: 600 to 1000 parts of semen cuscutae, 700 to 1200 parts of radix paeoniae alba, 600 to 900 parts of radix angelicae sinensis, 300 to 450 parts of radix rehmanniae praeparata, 350 to 450 parts of rhizoma dioscoreae, 200 to 300 parts of poria cocos, 100 to 180 parts of schizonepeta spike and 30 to 50 parts of radix bupleuri. The preparation process of the meridian fixing pill is good in repeatability, and assessment indexes such as the yield and the paeoniflorin content of the meridian fixing pill have good stability. In addition, a female rat ovarian function decline model is induced through cyclophosphamide, and it is proved that the meridian fixing pill has a good ovarian function decline treatment effect.

Description

Technical Field

[0001] The invention belongs to the technical field of research and development of traditional Chinese medicines, and particularly relates to a Dingjing pill for preventing and treating ovarian dysfunction, and a preparation method and application thereof. Background Art

[0002] Ovarian failure refers to decreased ovarian function, premature ovarian insufficiency, and premature ovarian failure in women before the age of 40 due to various factors. The ovaries are crucial reproductive organs and are susceptible to environmental influences, such as illness, harsh environments, and improper surgery. The fast pace of life, high work pressure, and mental stress in modern society often lead to liver depression, qi stagnation, and kidney deficiency in women, leading to morbidity. The incidence of ovarian failure is increasing year by year and is becoming younger.

[0003] As a crucial female reproductive organ, the ovaries have both reproductive and endocrine functions. They serve as a reservoir for follicles, regulating their maturation and cyclical ovulation, enabling the growth and development of oocytes. The endocrine function of the ovaries refers to the secretion of sex hormones such as estrogen and progesterone, as well as various growth factors, under the control of the hypothalamic-pituitary-ovarian axis. These hormones regulate the normal development of follicles and reproductive organs. Normal ovarian function is the primary factor influencing ovarian reproductive capacity.

[0004] Ovarian failure is primarily due to decreased estrogen secretion, and hormone replacement therapy is the mainstay of treatment. Clinically, cyclical estrogen and progestin replacement, either sequentially or in combination, is commonly used to treat a range of conditions associated with ovarian failure. Estrogen alone can improve osteoporosis and atrophic vaginitis caused by ovarian failure, but this approach is only suitable for women who have undergone a hysterectomy and do not require endometrial protection. Therefore, progestin supplementation is particularly important for women with an intact uterus to counteract estrogen-induced endometrial overgrowth. Clinically, progestin alone can be used during the menopausal transition to address menstrual problems associated with ovarian failure. For patients with cancer undergoing radiotherapy or chemotherapy, ovarian tissue cryopreservation can be used to preserve ovarian function and fertility. Ovarian tissue cryopreservation involves cryopreserving a portion of ovarian tissue prior to radiotherapy or chemotherapy using cryobiological principles. While this method has been used clinically, it has certain limitations. Therefore, hormone replacement therapy is currently the most widely used treatment. While hormone therapy has some efficacy during treatment, ovarian function fluctuates significantly after discontinuation, and there are adverse reactions associated with an increased risk of cancer and thrombotic conditions.

[0005] Traditional Chinese Medicine (TCM) theory considers the human body as a whole. Ovarian dysfunction is rooted in kidney deficiency, with liver qi stagnation as a key factor, leading to an imbalance of yin and yang within the five internal organs. Therefore, TCM treatment for ovarian dysfunction prioritizes nourishing the kidneys, while also focusing on soothing the liver, strengthening the spleen, and calming the mind. However, the application of these principles varies from person to person and requires a detailed analysis of each individual's situation. TCM treatment for ovarian dysfunction primarily focuses on nourishing kidney yin. The article "Irregular Menstruation" clearly states that only when kidney qi, kidney essence, and the sea of blood are abundant, and kidney yin and yang are balanced, can the secretion of heavenly essence and the Chong and Ren meridians flow freely, allowing essence and blood to enter the uterus and transform into menstruation, and menstruation to occur regularly. Therefore, kidney tonification is a key principle in regulating menstruation. Tang Wenjie used Guishen Pills to treat premature ovarian failure due to kidney yin deficiency. Results showed that Guishen Pills improved hormone levels, enhanced ovarian function, and restored regular menstruation. Traditional Chinese Medicine states that the liver and kidney share a common origin. While tonifying the kidneys, it's also important to soothe the liver and regulate qi. Fu Qingzhu's Gynecology argues that women are born with the liver, and regulating menstruation focuses on soothing the liver and regulating qi, regulating qi and blood. He also emphasizes the use of liver-tonifying and depression-relieving methods. Blood is fundamental to women, and the liver, as the organ that stores blood, is responsible for dispersing and relieving depression. Suwen: On Pain states, "Anger causes qi to rise." If women experience emotional frustration, liver qi stagnation, loss of pliability, and stagnation of qi and blood result. Therefore, relieving qi stagnation is the primary approach. Because the liver stores blood, soothing and soothing the liver shouldn't be the primary approach. Instead, liver-tonifying and qi-regulating herbs should be supplemented with liver-tonifying and qi-regulating herbs. Professor Wu Keming believes that the high work and lifestyle pressures of modern life can make women more susceptible to qi stagnation, exacerbating liver depression. Therefore, in clinical practice, herbs that tonify the liver and kidneys, such as Cuscuta chinensis, white peony root, and Chinese angelica root, are often used to treat premature ovarian failure. Most of the traditional Chinese medicines used in clinical treatment of ovarian dysfunction enter the kidney, liver, and spleen meridians. The spleen is the foundation of acquired constitution and essential for regulating menstruation. The key is to tonify the spleen and stomach to nourish the source of blood. Therefore, tonifying the spleen is also an important approach to treating premature ovarian failure. Li Danhong analyzed the thinking and medication patterns in the clinical treatment of premature ovarian failure and found that the most commonly used medications enter the kidney, liver, and spleen meridians, focusing on nourishing kidney yin and harmonizing the liver and spleen. Furthermore, among the many clinical symptoms associated with ovarian failure, emotional symptoms are particularly prominent. These manifestations are all driven by the mind. The heart is the monarch organ, housing the spirit and governing all mental, emotional, conscious, and mental activities. Therefore, the appropriate use of medications to tonify the mind has a theoretical basis for treating ovarian failure.

[0006] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the Invention

[0007] The purpose of the present invention is to provide a Dingjing pill for preventing and treating ovarian dysfunction and a preparation method and application thereof, so as to solve the problems existing in the background art of using hormone drugs to treat ovarian dysfunction.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] Disclosed is a Dingjing pill for preventing and treating ovarian dysfunction. The Dingjing pill is prepared from the following raw materials, measured by weight: 600-1000 parts of Cuscuta seeds, 700-1200 parts of white peony root, 600-900 parts of Chinese angelica, 300-450 parts of Rehmannia root, 350-450 parts of Chinese yam, 200-300 parts of Poria, 100-180 parts of Schizonepeta spicate, and 30-50 parts of Bupleurum.

[0010] Furthermore, the Dingjing Pills are prepared from the following raw materials in parts by mass: 810 parts of Cuscuta seeds, 810 parts of white peony root, 810 parts of Chinese angelica, 405 parts of Rehmannia root, 405 parts of Chinese yam, 243 parts of Poria, 162 parts of Schizonepeta spicate and 40.5 parts of Bupleurum.

[0011] The present invention also provides a preparation method of the Dingjing Pills, comprising the following steps:

[0012] S1 take the fixed by pills 1 / 10 of the amount of each raw material by mass, drying, crushing, sieving to obtain crude drug powder;

[0013] S2. Unsieved crude drug powder was placed in a gauze bag and mixed with 9 / 10 parts by mass of the remaining raw materials, soaked in water and refluxed for 2 times, the filtrates were combined, concentrated, and spray-dried to obtain a dry powder;

[0014] S3. The dry powder and crude drug powder are mixed evenly, wet granulated, pelletized, pelletized, dried, and packaged to obtain the Dingjing pills.

[0015] Furthermore, in step S1, the product is dried in an oven at 65° C., crushed, and passed through a 100-mesh sieve.

[0016] Furthermore, in step S2, 6 times the mass of water is added, soaked for 30 minutes, refluxed for 1.5 hours, and the filtrate is collected; 6 times the mass of water is added to the residue, and refluxed for 1.5 hours again; and the filtrates of the two extractions are combined.

[0017] Furthermore, in step S2, the extract is concentrated to a relative density of 1:1.13-1.15 at 80°C.

[0018] Furthermore, in step S3, the dry powder and crude drug powder are evenly mixed, passed through a 100-mesh sieve, and wet granulated with 80% ethanol.

[0019] The present invention also provides the use of the Dingjing Pills in preparing a medicine for treating ovarian dysfunction.

[0020] Cuscuta seed is pungent and sweet in flavor, neutral in nature, and enters the liver, kidney, and spleen meridians. It has the effects of nourishing the liver and kidneys, consolidating essence and reducing urination, improving eyesight, and stopping diarrhea. The Shennong Bencao Jing states that it "repairs severe injuries, replenishes deficiencies, and strengthens Qi and strength." Cuscuta seed is a traditional liver and kidney tonic. Modern pharmacological research has shown that it contains a variety of active ingredients, including flavonoids, phenolic acids, polysaccharides, alkaloids, and amino acids. Experiments have shown that Cuscuta seed can significantly increase the secretion of sex hormones such as luteinizing hormone, follicle-stimulating estrogen, and estradiol, demonstrating its potential to repair ovarian tissue damage. Furthermore, through combination with other herbs, it has become a classic prescription frequently used in the treatment of ovarian dysfunction.

[0021] White peony root (Bai peony) tastes bitter and sour, is slightly cold in nature, and enters the liver and spleen meridians. It has the effects of nourishing blood and regulating menstruation, restraining yin and stopping sweating, softening the liver and relieving pain, and calming liver yang. White peony root has a long history of medicinal use, and total glucosides of paeony are the key active ingredient in white peony extract. Modern research has found that white peony has analgesic, immune-modulating, liver-protective, antidepressant, and cardiovascular protective effects. Dai Yixian et al. investigated the role of total glucosides of paeony in regulating the phosphatidylinositol 3-kinase (PI3K) / protein kinase B (Akt) signaling pathway in improving primary dysmenorrhea (PDM) in rats. The results showed that total glucosides of paeony improve PDM in rats by inhibiting the PI3K / Akt signaling pathway.

[0022] Angelica sinensis is sweet and pungent in flavor and warm in nature. It enters the liver, heart, and spleen meridians, and has benefits such as nourishing and activating blood circulation, regulating menstruation and relieving pain, and moistening the intestines and promoting bowel movements. Angelica sinensis was first mentioned in the Shennong Bencao Jingjuan (Collection of Materia Medica) and is known as the "holy medicine for blood diseases." Modern pharmacology confirms that angelica sinensis has antioxidant, anti-inflammatory, liver and kidney protective, and immunomodulatory effects. Chen Xiubao et al. investigated the effects of angelica sinensis polysaccharides on Th17 / Treg cells in the bone marrow of mice with aplastic anemia. The results showed that compared with the model group, the peripheral blood red blood cell, white blood cell, and platelet counts, as well as hemoglobin concentrations, were significantly increased in the treated group, while the Th17 / Treg cell ratio was significantly decreased.

[0023] Rehmannia root (Shengdihuang) is sweet and slightly warm in nature. It enters the liver and kidney meridians, and has the effects of nourishing blood and yin, replenishing essence and filling marrow. The Jingyue Complete Works considers this herb to be the purest and most potent herb in the essence and blood, and a key remedy for nourishing yin and tonifying the kidneys. Zhang Xinyou et al. investigated the effects of Rehmannia root on mice with cyclophosphamide-induced ovarian insufficiency. The results showed that E2 levels were significantly increased and FSH levels were significantly decreased in the Rehmannia root-treated group. Furthermore, compared with the model group, Treg cell expression in the plasma of mice in all Rehmannia root groups was elevated, demonstrating that Rehmannia root can improve ovarian insufficiency.

[0024] Chinese yam is sweet and neutral in nature, entering the spleen, lung, and kidney meridians. It nourishes the spleen and stomach, promotes fluid production and benefits the lungs, and tonifies the kidneys and astringes semen. The Shennong Herbal Classic states that it "tonifies deficiency, strengthens energy, and, with long-term consumption improving hearing and vision, lightens the body, and prolongs life." Chinese yam is a traditional dual-use medicine and food. Modern pharmacological experiments have shown that Chinese yam contains multiple active ingredients, with Chinese yam polysaccharides being the main active ingredient. These polysaccharides have antioxidant, anti-aging, blood pressure-lowering, immune-modulating, and anti-tumor properties, making them useful for aging and other ailments.

[0025] Poria cocos has a sweet, mild flavor and a neutral nature. It enters the heart, lung, spleen, and kidney meridians, promoting diuresis, strengthening the spleen, and calming the mind. Poria cocos was first recorded in the Shennong Bencao Jing (Shen Nong's Classic of Materia Medica), with the Yunnan variety being the most effective. Poria cocos contains a variety of chemical components, including polysaccharides, diterpenes, triterpenes, and sterols, with Poria cocos polysaccharide as the primary active ingredient. Its modern pharmacological effects include anti-tumor, immune regulation, kidney protection, cardioprotection, and liver protection. Qu Zhun et al. have found that Poria cocos polysaccharide can reduce inflammation, regulate hormone levels, and improve ovarian function in rats by activating the SIRT1 / AMPK signaling pathway.

[0026] Charcoaled Schizonepeta spicata is pungent and astringent, with a slightly warm nature. It enters the lung and liver meridians and has astringent and hemostatic properties. Schizonepeta spicata, first mentioned in Shennong's Classic of Materia Medica, is known as "false soy sauce." Its active parts are its leaves and spikes, and its volatile oils, such as menthone and pulegone, are among its key active ingredients. Wen Taoqun et al. investigated the anti-inflammatory activity of Schizonepeta volatile oils and pulegone in LPS-intoxicated mice, demonstrating their ability to inhibit the infiltration of inflammatory factors. Furthermore, the charcoaled active parts, when prepared, exhibit hemostatic properties and are used for bleeding symptoms such as hematochezia and metrorrhagia.

[0027] Bupleurum has a pungent and bitter taste and a slightly cold nature. It enters the liver, gallbladder, and lung meridians, and has the effects of dispersing fever, relieving liver depression, and elevating yang energy. Bupleurum is known as "the one that harmonizes the Qi mechanism and mediates its movement." The body's Qi mechanism relies on the spleen and stomach to rise and fall, and the liver and gallbladder to release Qi. Bupleurum has the effect of regulating liver Qi. Adding Bupleurum to prescriptions can balance the body's yin and yang, Qi and blood, and internal organs. Modern pharmacological research has shown that Bupleurum has anti-inflammatory, heat-clearing, antidepressant, hepatoprotective, and immunomodulatory effects. Chen Yuchan et al. have shown that Bupleurum polysaccharides have a significant protective effect against acute liver injury in mice.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The preparation process of the Dingjing Pills of the present invention has good repeatability, and the evaluation indicators such as the yield and the paeoniflorin content have good stability.

[0030] (2) The present invention uses cyclophosphamide to induce an ovarian dysfunction model in female rats, and uses low, medium and high doses of Dingjing Pills for experimental research to confirm that the weight changes of rats in the medium and high dose groups are the most obvious, the Th17 / Treg results decrease, and it has a significant effect on alleviating anxiety caused by the new environment, promoting the exploratory behavior and spontaneous actions of rats, improving the physiological functions of the ovaries and uterus, increasing the weight of reproductive organs, E2, AMH, IL-22, IL-21 and IL10 levels, and reducing FSH, LH and TGF-β levels; This shows that Dingjing Pills has a good effect in treating ovarian dysfunction. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a line graph of water absorption;

[0032] Figure 2 This is a flow chart of the preparation process of Dingjing Pills of the present invention;

[0033] Figure 3 This is the chromatogram of the specificity test of paeoniflorin;

[0034] Figure 4 This is the test chromatogram for the specificity of the test product;

[0035] Figure 5 The chromatogram of the specificity test of the negative preparation of Paeonia lactiflora is missing;

[0036] Figure 6 is the linear relationship diagram of paeoniflorin;

[0037] Figure 7 This is the result of thin layer identification of Cuscuta australis in the preparation;

[0038] Figure 8 This is the TLC identification result of white peony root in the preparation;

[0039] Figure 9 This is the TLC identification result of Angelica sinensis in the preparation;

[0040] Figure 10 This is the TLC identification result of Rehmannia glutinosa in the preparation;

[0041] Figure 11 This is the TLC identification result of yam in the preparation;

[0042] Figure 12 This is the TLC identification result of Poria cocos in the preparation;

[0043] Figure 13 This is the TLC identification result of Schizonepeta spicata charcoal in the preparation;

[0044] Figure 14 This is the TLC identification result of Bupleurum chinense in the preparation;

[0045] Figure 15The open field test was used to detect the anxiety behavior of rats;

[0046] Figure 16 Flow cytometry was used to detect the results of rat Th17 / Treg;

[0047] Figure 17 This is a graph showing the weight change trend of rats;

[0048] Figure 18 is the rat ovary and uterus index;

[0049] Figure 19 The results of biochemical index determination of rats;

[0050] Figure 20 The results of inflammatory factor determination in rats;

[0051] Figure 21 This is the HE staining result of rat ovary (50×);

[0052] Figure 22 This is the HE staining result of rat uterus (100×);

[0053] Description of main reference numerals:

[0054] Figure 7 In the figure, 1.2.3 are test samples, 4 is hyperoside reference, 5 is Cuscuta australis reference, and 6 is negative control; developing solvent: methanol-glacial acetic acid-water (4:1:5); inspection condition: 365 nm;

[0055] Figure 8 In the table, 1.2.3 are test samples, 4 is the reference substance for paeoniflorin, and 5 is the negative control; developing solvent: chloroform-ethyl acetate-methanol-formic acid (40:5:10:0.2); inspection conditions: daylight;

[0056] Figure 9 In the figure, 1.2.3 are test samples, 4 is the paeoniflorin reference substance, and 5 is the negative control; developing solvent: n-hexane-ethyl acetate (4:1); inspection condition: 365 nm;

[0057] Figure 10 In the test sample, 1.2.3 is the test sample, 4 is the reference sample, and 5 is the negative control; the developing solvent is ethyl acetate-methanol-formic acid (16:0.5:2); the inspection condition is daylight;

[0058] Figure 11 In the figure, 1.2.3 are test samples, 4 is the Chinese yam control material, and 5 is the negative control; developing solvent: ethyl acetate-methanol-concentrated ammonia test solution (9:1:0.5); inspection condition: 365 nm;

[0059] Figure 12In the table, 1.2.3 are test samples, 4 is the control herbal medicine Poria cocos, and 5 is the negative control; developing solvent: toluene-ethyl acetate-formic acid (20:5:0.5); inspection conditions: daylight;

[0060] Figure 13 In the table, 1.2.3 are test samples, 4 is pulegone reference substance, and 5 is negative control; developing solvent: petroleum ether (60-90°C)-ethyl acetate (37:3); inspection conditions: daylight;

[0061] Figure 14 In the figure, 1.2.3 are test samples, 4 is saikosaponin a reference substance, and 5 is a negative control; developing solvent: ethyl acetate-ethanol-water (8:2:1); inspection conditions: daylight;

[0062] Figure 15 Middle, A. Movement distance, B. Activity time in the central area, C. Still time. Compared with the Control group, ###P < 0.001; compared with the Model group, *P < 0.05, **P < 0.01, ***P < 0.001;

[0063] Figure 16 Compared with the Control group, ##P<0.01; compared with the Model group, **P<0.01, ***P<0.001;

[0064] Figure 18 Middle, A. Ovarian index, B. Uterine index, compared with the Contral group, #P < 0.05;

[0065] Figure 19 A. FSH content, B. E2 content, C. E2 content, D. AMH content, compared with the Contral group, #P < 0.05, ##P < 0.01; compared with the Model group, *P < 0.05, **P < 0.01;

[0066] Figure 20 Figure A. TGF-β content, Figure B. IL-22 content, Figure C. IL-21 content, and Figure D. IL-10 content. Compared with the Control group, #P < 0.05, ###P < 0.001; compared with the Model group, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. DETAILED DESCRIPTION

[0067] The following is a clear and complete description of the technical solution of the present invention. Obviously, the embodiments described are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts are within the scope of protection of the present invention.

[0068] Example 1: Study on the preparation process of Dingjing Pills

[0069] 1.1 Experimental reagents and standards

[0070] The main experimental reagents and standards are shown in Table 1.1.

[0071] Table 1.1 Experimental reagents and standards

[0072]

[0073]

[0074] 1.2 Experimental instruments and manufacturers

[0075] The main experimental instruments and manufacturers are shown in Table 1.2.

[0076] 1.2 Experimental instruments and manufacturers

[0077]

[0078] 1.3 Extraction process research

[0079] 1.3.1 Crushing process and powder yield

[0080] According to preliminary experimental results, the aqueous extract of the Dingjing Decoction formula requires the addition of 10-15% of medicinal excipients to form pills. Based on this, this study proposed a 27-fold magnification of the formula for pills: "Dodder Seed 810g, White Peony 810g, Angelica 810g, Rehmannia Glutinosa 405g, Chinese Yam 405g, Poria 243g, Schizonepeta Spike 162g, Bupleurum 40.5g." This amount, 1 / 10 of the labeled amount of each decoction piece, or 368.6g in total, was oven-dried at 65°C, pulverized, and passed through a 100-mesh sieve to investigate the powder yield. The results are shown in Table 1.3.

[0081] Table 1.3 Results of investigation on powder yield of medicinal materials (passing through 100 mesh sieve)

[0082]

[0083] As shown in Table 1.3, the powder recovery rate of eight Chinese medicinal materials (1 / 10 of the amount) such as Cuscuta australis and white peony root is stable, the scoring rate is high, and the loss is small, and the specific production is feasible.

[0084] 1.3.2 Investigation of water absorption of prescription medicinal materials

[0085] Weigh 136.5g of the eight Chinese medicinal materials in the original prescription of Dingjing Decoction, namely 30g of Cuscuta australis, 30g of white peony root, 30g of Chinese angelica root, 15g of Rehmannia root, 15g of Chinese yam, 9g of Poria cocos, 6g of carbonized Schizonepeta tenuifolia, and 1.5g of Bupleurum root. Place three parallel portions (labeled as samples A, B, and C) in a beaker, and then add 10 times the amount of water (i.e., 1365g) to the beaker. Record the volume of the remaining solution in the beaker every 10 minutes for the first hour and every 20 minutes for the second hour. Calculate the water absorption rate according to the following formula. The results are shown in the figure. Figure 1 and Table 1.4.

[0086] Water absorption rate (%) = (wet weight of medicinal material - dry weight of medicinal material) / dry weight of medicinal material × 100%

[0087] Table 1.4 Water absorption of medicinal materials

[0088]

[0089] Depend on Figure 1 As shown in Table 1.4, after the medicinal materials are soaked for 120 minutes, the water absorption basically tends to be stable after 100 minutes; this shows that the total amount of the original prescription of Dingjing Decoction is 136.5g, and 1365g of water, which is 10 times the total amount of the medicinal materials, is added. The water absorption rate after soaking for 120 minutes is 194.14%, indicating that the water absorption of the medicinal materials in the Dingjing Decoction prescription is moderate.

[0090] 1.3.3 Investigation of wetting agent concentration

[0091] This pill is made by mixing 9 / 10 of the dry powder of the water extract of the prescribed medicinal material and 1 / 10 of the fine powder of the prescribed medicinal material. Preliminary experiments confirmed that the viscosity of the mixture was poor. To improve the pilling process, we explored the possibility of adding a wetting agent to increase viscosity and facilitate pill formation. Based on the conventional choice of ethanol as a wetting agent, we conducted the following studies:

[0092] About 50 g of a uniform mixture of 9 / 10 dry powder of the water extract prepared in the preliminary experiment and 1 / 10 fine powder of the medicinal materials used to make the pills in the Dingjing Tang kg pills prescription was taken and investigated using different concentrations of ethanol as a wetting agent. The results are shown in Table 2.5.

[0093] Table 1.5 Results of investigation on ethanol concentration

[0094]

[0095] As shown in Table 1.5, using 80% ethanol as a wetting agent for making soft materials can achieve satisfactory molding and pelletizing effects.

[0096] 1.3.4 Orthogonal experiment on the preparation process of Dingjing Pills

[0097] The factors that affect water extraction (decoction) are mainly the following: soaking time, extraction time, extraction times and water addition amount. 4 ) design and conduct the study, details are shown in Table 1.6.

[0098] Table 1.6 Experimental factor level table

[0099]

[0100] 1.3.4.1 Investigation of the extraction rate of dry powder (dry paste)

[0101] Weigh the various Chinese medicinal materials in the proposed kilogram pill prescription, and use L9(3 4 ) experimental method was used to perform reflux extraction of 9 / 10 kg of Chinese medicinal materials in the pill formula. The extract was filtered, the filtrate was combined, concentrated to a relative density of 1:1.15-1.18 (80°C), and spray-dried to obtain the extract powder (dry paste powder) of the orthogonal sample. The paste yield was calculated; the dry paste yield was one of the evaluation indicators. The paste yield calculation formula is as follows:

[0102]

[0103] 1.3.4.2 Assessment of Paeoniflorin Content

[0104] Weigh L9(3 4 ) method, accurately weighed, placed in a 10mL volumetric flask, added with methanol to the scale, ultrasonicated (100HZ) for 30min, placed at room temperature, topped up with methanol, shaken, and filtered through a 0.45μm microporous filter to obtain the test solution. The concentration of the reference solution and liquid phase detection were carried out in accordance with the method specified for paeoniflorin under the item of white peony root (page 108) in the 2020 edition of the Chinese Pharmacopoeia. The formula for calculating the paeoniflorin content in the test sample is as follows:

[0105]

[0106] 1.3.4.3 Scoring Weight

[0107] Based on the evaluation indicators of "dry powder (paste) rate and paeoniflorin content", the paeoniflorin content indicator is more important, so its scoring weight is set as 20% for dry paste rate, that is, the minimum value of dry paste rate is 0 points and the maximum value is full marks; paeoniflorin content accounts for 80% of the weight, the minimum paeoniflorin content is 0 points and the maximum is full marks. The total score is calculated, and the data is processed through the orthogonal experimental table to evaluate the optimal preparation process. The scoring formula is as follows:

[0108] Comprehensive score = A + B

[0109]

[0110] 1.3.4.4 Specific tests

[0111] Based on the kilogram prescription quantity determined in the preliminary experiment, this orthogonal experimental study was designed. Based on this data, the preparation process and parameters of the kilogram pill prescription of Dingjing Pills were determined, providing a basis for the subsequent adaptability pilot test of this preparation.

[0112] According to the results of the preliminary experiment of kilogram pills, the prescription dosage of materials was formulated: 810g of Cuscuta seeds, 810g of white peony root, 810g of Chinese angelica, 405g of Rehmannia root, 405g of Chinese yam, 243g of Poria, 162g of Schizonepeta spicate, and 40.5g of Bupleurum; 1 / 10 of the amount of each medicinal piece marked in the prescription, i.e. a total of 368.6g, was taken, oven-dried at 65°C, crushed, and passed through a 100-mesh sieve (crude drug powder) for later use; the portion above the sieve was put into a gauze bag and the remaining 9 / 10 of the medicinal materials in the prescription were extracted with water reflux according to an orthogonal design, the filtrates were combined, concentrated to a relative density of 1:1.13-1.15 (80°C), spray-dried to obtain the extract dry powder, the dry powder was mixed with the crude drug powder, granulated with 80% ethanol, and pilled.

[0113] 1.3.4.5 Results of Dry Paste Rate Investigation

[0114] The results are shown in Tables 1.7 and 1.8.

[0115] Table 1.7 Orthogonal experiment dry paste rate results (n = 3)

[0116]

[0117] Table 1.8 Variance analysis (cream yield %)

[0118]

[0119]

[0120] It can be seen from Tables 1.7 and 1.8 that the factors affecting the dry paste rate are C>A>B>D through range analysis; variance analysis was performed in combination with the soaking time, and the results showed that each factor had no significant effect on the dry paste amount. In order to save time and improve efficiency, the optimal extraction process was determined to be A1B1C2D1, that is, adding 6 times the amount of water to extract twice, the first soaking was 15 minutes, and each extraction was 0.5 hours.

[0121] 1.3.4.6 Paeoniflorin content is the result of investigation

[0122] According to the quality detection method of peony root using paeoniflorin as described in the 2020 edition of the Chinese Pharmacopoeia, the content of paeoniflorin was determined, and the analysis results are shown in Tables 1.9 and 1.10.

[0123] Table 1.9 Results of orthogonal test for paeoniflorin (n=3, unit: mg / g)

[0124]

[0125] Table 1.10 Analysis of variance

[0126]

[0127] 1.3.4.7 Evaluation Based on Weighted Indicators

[0128] The dry paste rate and paeoniflorin content were calculated by weight and analyzed using the orthogonal test assistant. The results are shown in Tables 1.11 and 1.12.

[0129] Table 1.11 Orthogonal experiment weight index scoring results

[0130]

[0131] Table 1.12 Analysis of variance

[0132]

[0133]

[0134] As shown in Tables 1.11 and 1.12, the dry paste rate and paeoniflorin content were weighted and analyzed using the Orthogonal Test Assistant. Range analysis revealed that the influencing factors were B > C > D > A, with A3B2C3D3 being the optimal factor. A variance analysis was then performed using soaking time as the error column. The results showed no significant difference in water addition, so the optimal extraction process was determined to be A1B3C2D2: adding six times the amount of water and performing two extractions, with the first soaking time lasting 30 minutes and each extraction lasting 1.5 hours.

[0135] 1.3.4.8 Orthogonal test yield and other results

[0136] The results are shown in Table 1.13.

[0137] Table 1.13 Orthogonal test yield and other parameters

[0138]

[0139] It can be seen from Table 1.13 that there are obvious differences in the finished product rates of the 9 batches of Dingjing Pills in the orthogonal experiment and the paeoniflorin content in the finished products. In comparison, the finished product rates obtained by the preparation processes of Experiment 2 and Experiment 3 are closest to the theoretical predicted values (1000g pills), and the paeoniflorin content therein is also higher; this shows that the preparation process conditions of Dingjing Pills determined by the orthogonal experiment, namely, adding 6 times the amount of water to extract twice, soaking for 30 minutes, and extracting for 1.5 hours each time, have certain operability.

[0140] 1.3.4.9 Conclusions of the Dingjing Pills Orthogonal Test

[0141] I. The prescription of Dingjing Pills (1 kilogram pill) is clarified: Cuscuta seeds 810g, white peony root 810g, Chinese angelica 810g, Rehmannia root 405g, Chinese yam 405g, Poria 243g, Schizonepeta spicate 162g, and Bupleurum 40.5g.

[0142] II. Preparation process of Dingjing pills (see process diagram Figure 2 ): Take 1 / 10 of the labeled amount of 8 Chinese herbal medicine slices respectively, mix them evenly, dry them, grind them into fine powder, and pass them through a 100-mesh sieve for later use; put the part above the sieve into a gauze bag and add 6 times the amount of water with the remaining 9 / 10 of the medicinal slices in the prescription, soak them for 30 minutes, reflux extraction for 1.5 hours, and collect the filtrate; add 6 times the amount of water to the residue, continue reflux extraction for 1.5 hours; combine the secondary extraction filtrates, concentrate them to a relative density of 1:1.13-1.15 (80°C) extract, spray dry them to obtain dry powder, mix the dry powder and fine powder evenly, wet granulate them with 80% ethanol, make pills, make pills, dry them, and package them.

[0143] 1.4 Methodological Research

[0144] 1.4.1 Preparation of reference solution

[0145] Weigh 6.23 mg of paeoniflorin into a 10 mL volumetric flask. Add methanol to the mark to completely dissolve. Shake well and set aside. Add 5 mL of the reference substance to a 10 mL volumetric flask and dilute to the mark with methanol. Shake well. Repeat this dilution method five times and store in a refrigerator at 4°C until needed. The concentrations are 0.623, 0.311, 0.156, 0.078, 0.039, and 0.019 mg / mL, respectively. Filter through a 0.45 μm filter membrane and use the filtrate as the reference solution.

[0146] 1.4.2 Preparation of test solution

[0147] Weigh about 0.1 g of the dry paste powder obtained in "Experiment 2 under 1.3.3.5", accurately weigh it, place it in a 10 mL volumetric flask, accurately add 10 mL of methanol, ultrasonicate (100 Hz) for 30 min, let it cool to room temperature after the ultrasonication, add appropriate amount of methanol to make up the scale, shake well, and filter through a 0.45 μm microporous filter membrane to obtain the test solution.

[0148] 1.4.3 Chromatographic conditions

[0149] According to the content determination method under "Paeoniflorin" in the 2020 edition of the Chinese Pharmacopoeia, the chromatographic column PhenomenexLuna C 18 A column (5 μm, 4.6 mm × 25 cm) was used, and the mobile phase consisted of acetonitrile-0.1% phosphoric acid (14:86); the detection wavelength was 230 nm, the flow rate was 1 mL / min, and the injection volume was 10 μL. The theoretical plate number, calculated based on the paeoniflorin peak, should be no less than 2000.

[0150] 1.4.4 Specificity test

[0151] According to the method under "1.4.3", the paeoniflorin in the paeoniflorin reference substance, the test substance and the negative sample solution lacking paeoniflorin were determined respectively. Figure 3-5 .

[0152] Depend on Figure 3-5 It can be seen that the chromatographic peaks in the test sample can be well separated, the negative control has no interference, and the selected chromatographic conditions and methods meet the specificity requirements.

[0153] 1.4.5 Linear Relationship Investigation

[0154] Inject the reference solution under "1.4.1", set the concentration of the reference solution as the horizontal axis and the peak area as the vertical axis, and draw the standard curve. Figure 6 and Table 1.14.

[0155] Table 1.14 Investigation of linear relationship of paeoniflorin

[0156]

[0157] Depend on Figure 6 As can be seen from Table 1.14, the regression equation of paeoniflorin is: y=12365196.9914+23642.7463, r=0.9998, and paeoniflorin shows a good linear relationship in the range of 0.623-0.01946875 mg / ml.

[0158] 1.4.6 Precision test

[0159] Take the peony prepared in "1.4.1" and inject it repeatedly 6 times. Measure the content and calculate the RSD. The results are shown in Table 1.15.

[0160] Table 1.15 Paeoniflorin precision test results (n=6)

[0161]

[0162] As shown in Table 1.15, the peak area RSD is 1.23% and the retention time RSD is 0.125%, indicating that the instrument has good precision.

[0163] 1.4.7 Stability test

[0164] Accurately weigh the test sample powder prepared in "Experiment 2 under 1.3.3.5" and prepare the test sample solution according to the method under "2.4.2". Inject the sample at 0, 2, 4, 8, 12, 24, and 36 hours after preparation, determine the content, and calculate the RSD. The results are shown in Table 1.16.

[0165] Table 1.16 Paeoniflorin experimental results

[0166]

[0167] From 1.16, we can see that the RSD values of the peak area and content of each component of the test sample within 36 hours are calculated. The RSD values of the peak area and content are 1.5%, which indicates that the test sample is stable and reliable within 36 hours.

[0168] 1.4.8 Repeatability test

[0169] Accurately weigh 6 portions of the test sample powder prepared under "1.3.3.5" and prepare the test sample solution according to the method under "1.3.4.2". Inject the sample for detection, record the peak area 6 times and calculate the RSD. The results are shown in Table 1.17.

[0170] Table 1.17 Repeatability test results

[0171]

[0172] As shown in Table 1.17, the RSD value is 1.33%, indicating that the method has good repeatability and can be used for content determination in samples.

[0173] 1.4.9 Sample recovery test

[0174] Take 6 portions of the test sample prepared in "Experiment 3 under 1.3.3.5", each sample is about 0.1g, accurately weighed, and a certain amount of paeoniflorin reference substance is accurately added according to three gradients of high, medium and low (80%, 100% and 120% of the known content respectively). According to the method under "2.4.3", 10μL is injected and the average recovery rate of each component is calculated. The results are shown in Table 1.18.

[0175] Table 1.18 Test results of sample recovery

[0176]

[0177] As can be seen from Table 1.18, the component recovery rate is between 80-120%, indicating that the sample recovery rate of the test solution is good.

[0178] 2.5 Dingjing Pills kg pill formula 5 times magnification verification

[0179] 2.5.1 Experimental medicinal materials and suppliers

[0180] The experimental medicinal materials, batch numbers and suppliers are shown in Table 2.19.

[0181] Table 1.19 Experimental medicinal materials and suppliers

[0182]

[0183]

[0184] 2.5.2 Process Scale-up Verification Plan

[0185] In order to verify the stability and repeatability of the preparation process of Dingjing Pills (main conditions are A3B2C3D1) determined by orthogonal experiment, a 5-fold kilogram pill prescription scale-up process verification study was carried out according to the Dingjing Pills (kilogram pill) prescription and preparation process determined by orthogonal experiment.

[0186] Prescription: Cuscuta 4050g, White Peony 4050g, Angelica 4050g, Rehmannia Glutinosa 2025g, Chinese Yam 2025g, Poria 1215g, Schizonepeta Spike Charcoal 810g, Bupleurum 202.5g.

[0187] Preparation process: Take 1 / 10 of the labeled amount of each herbal medicine, mix, dry, and crush, and pass through a 100-mesh sieve to obtain a fine powder (crude herbal powder) for later use. Wrap the sieved portion with gauze and, along with 9 / 10 of the herbal medicine, add 6 times the amount of water and extract twice, soaking for 30 minutes initially and then refluxing for 1.5 hours each time. Filter, combine the extracts, and concentrate to a relative density of 1:1.13-1.15 (80°C). Spray dry the resulting aqueous extract (dry powder). Mix this dry powder with the crude herbal powder, pass through a 100-mesh sieve, and soften with 80% ethanol. Form into pellets, dry, and package. Prepare three replicates. Results are shown in Table 1.20.

[0188] Table 2. 203 batches of Dingjing pills 5 times the kilogram pill process scale-up verification results

[0189]

[0190]

[0191] Since the finished pills are sensitive to temperature and humidity, the temperature and humidity should not be too high when studying the drying temperature and drying time of the pellets. First, use circulating air at a low temperature of 30°C to dry them for 24 hours, then raise the temperature to 45°C and dry them for another 24 hours. The moisture content is tested and they are packaged after passing the test.

[0192] Example 2 Quality Standard and Preliminary Stability Study of Dingjing Pills

[0193] 2.1 Experimental Reagents

[0194] The main experimental reagents and manufacturers are shown in Table 2.1.

[0195] Table 2.1 Experimental reagents and manufacturers

[0196]

[0197]

[0198] 2.2 Experimental instruments

[0199] The main experimental instruments and manufacturers are shown in Table 2.2.

[0200] Table 2.2 Experimental instruments and manufacturers

[0201]

[0202] 3.3 Experimental methods

[0203] 3.3.1 Characteristic Identification

[0204] The properties, odor, color, etc. of three batches of 5-times-kilogram pellet preparations were observed to determine the properties of this product.

[0205] 3.3.2 Thin layer identification

[0206] Referring to the 2020 edition of the "Chinese Pharmacopoeia" and the thin layer identification methods in the literature, a thin layer identification study was carried out on Cuscuta chinensis, white peony root, Chinese angelica, Rehmannia glutinosa, etc. in the prescription.

[0207] 3.3.2.1 Thin layer chromatography identification of Cuscuta australis in preparations

[0208] Take 3g of the powder of this product, add 50mL of methanol, heat and reflux for 30min, filter, and concentrate the filtrate to 2mL as the test solution. Take another 0.5g of Cuscuta seed reference medicinal material and prepare a reference medicinal material solution in the same way. Take the hyperoside reference material and add methanol to prepare a solution containing 1mg per 1mL as the reference solution. Take 3g of negative control powder and prepare a negative control solution in the same way. According to the thin layer chromatography method (General Rule 0502), take 2μL of each of the above four solutions and spot them on the same polyamide film. Use methanol-glacial acetic acid-water (4:1:5) as the developing agent, develop, take out, dry, spray with aluminum chloride test solution, and inspect under ultraviolet light (365nm). The results are shown in the figure. Figure 7 .

[0209] Depend on Figure 7 It can be seen that the three batches of test samples, hyperoside reference substance, and Cuscuta australis control medicinal materials showed spots of the same color at the same position, and there was no obvious spot effect at the position of the negative control.

[0210] 3.3.2.2 Thin layer chromatography identification of white peony root in preparations

[0211] Take 3g of the powder of this product, add 40mL of ethanol, ultrasonicate for 5min, filter, evaporate the filtrate to dryness, dissolve the residue in 1ml of ethanol, and use it as the test solution. Take another reference substance of paeoniflorin, add ethanol to make a solution containing 1mg per 1ml, and use it as the reference solution. Take 3g of negative control powder and prepare a negative control solution in the same way. According to the thin layer chromatography method (General Rule 0502), take 10μL of each of the above four solutions and spot them on the same silica gel G thin layer plate, use chloroform-ethyl acetate-methanol-formic acid (40:5:10:0.2) as the developing agent, develop, take out, dry, spray with 5% vanillin sulfuric acid solution, and heat until the spots are clearly colored. The results are shown in the figure. Figure 8 .

[0212] Depend on Figure 8 It can be seen that the three batches of test samples and the paeoniflorin control substance showed spots of the same color at the same position, and there was no obvious spot effect at the position of the negative control.

[0213] 3.3.2.3 Thin layer chromatography identification of Angelica sinensis in preparations

[0214] Take 3g of this product powder, add 25mL of ether, ultrasonically treat for 10min, filter, evaporate the filtrate to dryness, add 1mL of ethanol to the residue to dissolve it, and use it as the test solution. Take 0.5g of Angelica sinensis control medicinal material and prepare a control medicinal material solution in the same way. Take another 3g of Angelica sinensis negative control powder and prepare a negative control solution in the same way. According to the thin layer chromatography method (General Rule 0502), take 10μL of each of the above solutions and spot them on the same silica gel G thin layer plate, use n-hexane-ethyl acetate (4:1) as the developing solvent, develop, take out, dry, and examine under ultraviolet light (365nm). The results are shown in Figure 2. Figure 9 .

[0215] Depend on Figure 9 It can be seen that the three batches of test samples and the Angelica sinensis control medicinal materials showed spots of the same color at the same position, and there was no obvious spot effect at the position of the negative control.

[0216] 3.3.2.4 Thin layer chromatography identification of Rehmannia root in preparations

[0217] Take 3g of the powder of this product, add 50ml of 80% methanol, ultrasonically treat for 30min, filter, evaporate the filtrate to dryness, dissolve the residue in 5ml of water, shake and extract 4 times with 10ml of n-butanol saturated with water, combine the n-butanol solution, evaporate to dryness, dissolve the residue in 2ml of methanol, and use it as the test solution. Take another standard of verbascoside, add methanol to make a solution containing 1mg per 1ml. Take 3g of negative control powder and make a negative control solution in the same way. Test according to the thin layer chromatography method (General Rule 0502), take 5μL of each of the three solutions mentioned above, spot them on the same silica gel G thin layer plate, use ethyl acetate-methanol-formic acid (16:0.5:2) as the developing solvent, develop, take out, dry, and soak with 0.1% 2,2-diphenyl-1-picrylhydrazyl anhydrous ethanol solution. The results are shown in the figure. Figure 10 .

[0218] Depend on Figure 10 It can be seen that no spots of the same color as those of the verbascoside reference substance appeared in the test sample.

[0219] 3.3.2.5 Thin layer chromatography identification of Chinese yam in preparations

[0220] Take 3g of the powder of this product, add 30mL of ethanol, ultrasonically extract for 30min, filter, evaporate the filtrate to dryness, dissolve the residue in 1ml of ethanol, and use it as the test solution. Take 4g of Chinese yam control medicinal material and prepare the control medicinal material solution in the same way. Take another 3g of Chinese yam negative control powder and prepare the negative control solution in the same way. According to the thin layer chromatography method (General Rule 0502), take 5μL of each of the above three solutions and spot them on the same silica gel G thin layer plate, use ethyl acetate-methanol-concentrated ammonia test solution (9:1:0.5) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and inspect under ultraviolet light (365nm). The results are shown. Figure 11 .

[0221] Depend on Figure 11 It can be seen that spots of the same color appeared in some parts of the three batches of test samples and the yam control medicinal materials, some spots were lighter, and there was no obvious spot effect at the position of the negative control.

[0222] 3.3.2.6 Thin layer chromatography identification of Poria cocos in preparations

[0223] Take 3g of the powder of this product, add 50ml of ether, ultrasonically treat for 10min, filter, evaporate the filtrate to dryness, and dissolve the residue in 1ml of methanol as the test solution. Take 0.5g of Poria cocos control medicinal material and prepare the control medicinal material solution in the same way. Take another 3g of Poria cocos negative control powder and prepare the negative control solution in the same way. According to the thin layer chromatography method (General Rule 0502), take 5μL of each of the above three solutions and spot them on the same silica gel G thin layer plate, use toluene-ethyl acetate-formic acid (20:5:0.5) as the developing agent, develop, take out, dry, spray with 2% vanillin sulfuric acid solution-ethanol (4:1) mixed solution, heat at 105℃ until the spots are clearly colored, the results are shown. Figure 12 .

[0224] Depend on Figure 12 It can be seen that the three batches of test samples and the Poria control medicinal material appeared the same spots at the same position, but in the negative control, some ingredients appeared spots of the same color at the same position.

[0225] 3.3.2.7 Thin-layer chromatography identification of Schizonepeta spicata charcoal in preparations

[0226] Take 3g of the powder of this product, add 10ml of methanol, seal, ultrasonically treat for 15min, filter, and use the filtrate as the test solution. Take pulegone reference substance, add petroleum ether (60-90℃) to make a solution containing 4mg per 1ml, as the reference solution. Take another 3g of Schizonepeta spicata charcoal negative control powder, and prepare a negative control solution in the same way. According to the thin layer chromatography method (General Rule 0502), take 5μL of each of the three solutions mentioned above, spot them on the same silica gel G thin layer plate, use petroleum ether (60-90℃)-ethyl acetate (37:3) as the developing agent, develop, take out, dry, spray with 1% vanillin sulfuric acid solution, heat until the spots are clearly colored, the results are shown. Figure 13 .

[0227] Depend on Figure 13 It can be seen that the three batches of test products and the pulegone reference product did not have the same spots at the same position.

[0228] 3.3.2.8 Thin-layer chromatography identification of Bupleurum in preparations

[0229] Take 3g of the powder of this product, add 20mL of methanol, ultrasonically treat for 10min, filter, and concentrate to 5mL as the test solution. Take the reference substance of saikosaponin a, add methanol to make a mixed solution containing 0.5mg of each in 1mL, as the reference solution. Take another 3g of the negative control powder of bupleurum, and prepare the negative control solution in the same way. According to the thin layer chromatography method (General Rule 0502), take 5μL of each of the three solutions mentioned above, spot them on the same silica gel G thin layer plate, use ethyl acetate-ethanol-water (8:2:1) as the developing agent, develop, take out, dry, spray with 2% p-dimethylaminobenzaldehyde in 40% sulfuric acid solution, heat at 60℃ until the spots are clear, and inspect under sunlight and ultraviolet light (365nm), respectively. The results are shown in Table 1. Figure 14 .

[0230] Depend on Figure 14 It can be seen that the three batches of test samples and the saikosaponin a reference substance did not have the same spots at the same position.

[0231] 3.3.3 Various inspections

[0232] 3.3.3.1 Moisture Test of Dingjing Pills

[0233] According to the "Determination of Moisture (General Rule 0832)" in Part IV of the 2020 edition of the Chinese Pharmacopoeia, the moisture content of Dingjing Pills was determined, and it is stipulated that the moisture content of this pill shall not exceed 9.0%. The results are shown in Table 1.3.

[0234] Table 1.3 Moisture test results

[0235]

[0236] 3.3.3.2 Weight Differences of Dingjing Pills

[0237] According to the "Pills (General Rule 0108)" in Part IV of the 2020 edition of the "Chinese Pharmacopoeia", 10 pills are taken as 1 portion, and a total of 10 portions of test sample are taken. The weight is weighed separately and then compared with the labeled weight of each portion. No more than 2 portions shall exceed the weight difference limit. The results are shown in Table 1.4.

[0238] Table 3.4 Weight difference results

[0239]

[0240] 3.3.3.3 Differences in the dosage of Dingjing Pills

[0241] According to the "Pills (General Rule 0108)" in Part IV of the 2020 edition of the "Chinese Pharmacopoeia", the filling quantity is indicated by weight. According to the minimum filling quantity inspection method (General Rule 0942), 10 bags of test sample were taken, and the weight of each bag was weighed separately. The filling quantity of each bag was compared with the labeled filling quantity. No more than 2 bags exceeded the filling quantity difference limit. The results are shown in Table 1.5.

[0242] Table 1.5 Results of differences in loading volume

[0243]

[0244]

[0245] 3.3.3.4 Dissolution Time of Dingjing Pills

[0246] According to the "Pills (General Rule 0108)" section of Part IV of the 2020 edition of the Chinese Pharmacopoeia, six test sample pellets were placed in a hanging basket with a mesh of appropriate aperture. Disintegration Time Test Method (General Rule 0108) for pills was followed, with baffles added. Water-based pellets should dissolve completely within 1 hour; concentrated water-based pellets, concentrated honey-based pellets, concentrated water-honey-based pellets, and paste-based pellets should dissolve completely within 2 hours. The results are shown in Table 1.6.

[0247] Table 1.6 Dissolution time results

[0248]

[0249] 3.3.4 Content detection

[0250] Prepare the sample according to the preparation method in "1.4.2" and use the linear calculation in "1.4.2" to calculate the content of paeoniflorin. The results are shown in Table 1.7.

[0251] Table 1.7 Content results of Dingjing Pills

[0252]

[0253]

[0254] 3.3.5 Accelerated and room temperature stability studies of Dingjing Pills

[0255] Three batches of Dingjing Pills, packaged and magnified 5 times, were placed in a drug stability test chamber (temperature 45°C ± 5°C; humidity 70% ± 5%) and stored at room temperature. Samples were taken at 0, 1, 2, and 3 months, respectively. The pills were observed for shape, moisture content, TLC, content, dissolution time, and other specified parameters according to the quality standards of this product. The results are shown in Tables 1.8 and 1.9.

[0256] Table 1.8 Stability results of three batches of Dingjing pills at room temperature

[0257]

[0258] Table 1.9 Accelerated stability results of three batches of Dingjing Pills

[0259]

[0260]

[0261] It can be seen from Tables 1.8 and 1.9 that all the test indicators of accelerated and room temperature stability meet the requirements of the proposed quality standards for Dingjing Pills.

[0262] Example 3 Pharmacodynamic Evaluation of Dingjing Pills on Ovarian Function Decline

[0263] 3.1 Introduction

[0264] The classical and recognized cyclophosphamide-induced ovarian dysfunction model was used to evaluate the efficacy of Dingjing Pills in treating ovarian dysfunction and provide data support for its possible clinical application.

[0265] 3.2 Experimental Materials

[0266] 3.2.1 Experimental animals

[0267] Seventy-two female Sprague-Dawley rats, 2-3 months old, weighing 180-200 g, were purchased from the Experimental Animal Research Center of the Air Force Medical University under license number SCXK(Military)2017-0020. They were housed with free access to water and food, and their bedding was changed three times per week.

[0268] 3.2.2 Test drugs

[0269] The test drug used in this experiment was Dingjing Pills, which were verified to be qualified by 5-fold magnification according to the kilogram pill prescription, with batch number 20240828-1. When administering, a sufficient amount of Dingjing Pills was weighed and moistened with an appropriate amount of sodium carboxymethylcellulose aqueous solution. After grinding until completely suspended, the pills were transferred to a graduated volumetric flask, made up to volume with sodium carboxymethylcellulose aqueous solution, and then transferred to a 50ml beaker. A magnetic stirrer was added and the pills were placed on a magnetic stirrer tray for continuous stirring until the animals were dosed to ensure the accuracy of the dosage for all animals.

[0270] 3.2.3 Experimental reagents

[0271] See Table 3.1.

[0272] Table 3.1 Experimental reagents and manufacturers

[0273]

[0274] 3.2.4 Experimental instruments

[0275] The main experimental instruments and manufacturers are shown in Table 3.2.

[0276] Table 3.2 Experimental instruments and manufacturers

[0277]

[0278]

[0279] 3.2.5 Preparation of experimental drugs

[0280] The positive drug group was given Progynova estradiol valerate tablets, which were fully crushed and dissolved in a sodium carboxymethylcellulose aqueous solution. The specific dosage is shown in Table 3.3.

[0281] The test drug groups were given different doses of Dingjing Pills, prepared according to the test drug method in 3.2.2. Specific doses are shown in Table 3.3.

[0282] Table 4.3 Configuration of therapeutic drugs

[0283]

[0284] 3.3 Experimental methods

[0285] 3.3.1 Establishment of ovarian failure rat model

[0286] This study used 2-3 month old female Sprague-Dawley rats. Cyclophosphamide was injected intraperitoneally to induce a model of ovarian failure. Rats with disrupted estrous cycles were selected for this study. A normal estrous cycle consists of a complete 5-day period: proestrus, estrus, metestrus, and diestrus. Prior to drug administration, all rats underwent estrous cycle testing. The specific procedure was to perform vaginal exfoliative cell smears on the rats daily between 7:00 and 9:00 AM. A disposable cotton swab soaked in saline was inserted approximately 1 cm into the rat's vagina. The swab was gently rotated clockwise three times before being smeared onto a glass slide. One to two drops of Lu's basic methylene blue stain were then applied, diluted with PBS, and allowed to stand for 3-5 minutes to fix. The stain was then slowly rinsed with pure water. Cell morphology was observed under a microscope for five consecutive days. If the vaginal exfoliative cells showed irregular cycles, this indicated an established model of ovarian failure.

[0287] 3.3.2 Animal grouping and drug administration

[0288] Seventy-two 2-3 month old female Sprague-Dawley rats were housed in the animal laboratory of the Institute of Pharmacology, Air Force Medical University, at an ambient temperature of 22 ± 2°C, a humidity of 60 ± 5%, and a 12-hour light cycle. The rats were acclimated for one week. During this period, the rats were numbered and weighed. Sixty female Sprague-Dawley rats, after vaginal smear examination, were randomly divided into five groups of 12 animals each: model control, positive control, low-dose drug group (DL), medium-dose drug group (ZL), and high-dose drug group (GL). Twelve normal female rats served as a blank control group.

[0289] The dosage was determined by consulting the recommended dosage of eight Chinese medicinal herbs in the 2020 edition of the Pharmacopoeia of the People's Republic of China, including Cuscuta australis, Paeonia lactiflora, Angelica sinensis, Rehmannia glutinosa, Chinese yam, Poria cocos, Schizonepeta tenuifolia charcoal, and Bupleurum chinense. The dosage of each test drug was calculated and determined based on the amount of raw herbs and decoction pieces per gram of dry powder of the prepared test drug, as well as the dosage table of the ratio of human and animal body surface area. The dose of the positive control group of estradiol valerate tablets was 0.1 mg / kg (converted from the clinical dose for adults), the dose of the low-dose Dingjing Pills group was 0.635 g / kg, the dose of the medium-dose Dingjing Pills group was 1.27 g / kg, and the dose of the high-dose Dingjing Pills group was 2.54 g / kg. The blank control group and the model control group were both given an equal volume of CMC-Na, and different test drugs were administered by gavage using equal solvents. The administration volume was 1 mL / 100 g, and the drug was administered once a day. The test was conducted after 4 consecutive weeks of administration.

[0290] 3.3.3 General behavioral observations

[0291] Observe the rats daily to see whether they are easily irritated, difficult to catch, prone to panicking and fleeing, as well as their mental state and activity level.

[0292] Results: The young rats were docile, not easily startled or jumped when being grasped; while the old rats in the control group were startled and easily jumped when being grasped, and were alert to external sounds and prone to making noises; after drug administration, the rats showed less nervousness, grasped docilely, were not easily startled or angered.

[0293] 3.3.4 Open field test

[0294] After 4 weeks of drug administration, the rats were subjected to an open field test. The open field test is a behavioral experimental method that tests the spontaneous movement and exploratory behavior of rats and reflects the anxiety level of rats in a new environment. The experimental environment was kept quiet and clean. The open field test was carried out in a black wooden box of 80×80×40 cm, with black sides and bottom. Before the experiment, the camera field of view was adjusted to the center of the box to ensure that the field of view completely covered the entire interior of the box and that the lighting in the box was uniform. The rats were placed in the same corner from the outside of the box and their free movement was observed for 15 minutes. The movement trajectory of the rats was photographed using path tracking software. After the end, the rats were put back in the cage, the feces and urine left by the rats were cleaned, and the open field was sprayed with 75% alcohol to reduce the influence of the previous rat on the behavior of other rats. The next rat was placed in according to the steps. The activity time, movement time, stationary time and movement distance in the central area of the rats were analyzed, and the results are shown in the figure. Figure 15 .

[0295] Depend on Figure 15After drug administration, an open field test was performed to investigate the effects of the drug on anxiety in rats. The results below show that compared with the blank control group, the model control group showed a significant increase in central area activity time and movement distance (P < 0.01), and a significant increase in immobility time (P < 0.01). Compared with the model control group, the positive drug control group showed a significant increase in the ratio of movement distance to central area activity time, while its immobility time decreased compared with the model group. Compared with the model control group, each drug-treated group showed an increase in movement distance and central area activity time, but a decrease in immobility time. The low-dose group showed a longer movement distance than the medium- and high-dose groups, but the central area activity time increased more significantly in the medium- and high-dose groups than in the low-dose group. There was no significant difference in immobility time between the low- and high-dose groups compared with the model control group, while the high-dose group showed a significant decrease in immobility time compared with the model control group. These open field results indicate that rats experiencing ovarian failure exhibit anxiety, with decreased spontaneous movement and exploratory behavior. Dingjing Pills can reduce anxiety in rats and enhance their spontaneous movement and exploratory behavior.

[0296] 3.3.5 Th17 / Treg ratio in rat peripheral blood

[0297] After the rat mining experiment, blood was collected through the eye socket, and 1 ml of rat blood sample was collected. 15 ml sterile centrifuge tubes were taken and 5 ml of separation solution was added to each tube; 1 ml of sample diluent was added to each blood sample, pipetted and mixed, and then slowly added to the corresponding separation solution tube. The blood sample was carefully added to the separation solution interface and centrifuged at room temperature for 30 minutes. The third transparent separation solution layer from top to bottom was taken, and the lymphocyte layer was transferred to a new centrifuge tube. 10 ml of washing solution was added, the cells were mixed, centrifuged for 10 minutes, the supernatant was discarded, and the washing was repeated twice. 2.5 ml of PBS was added to resuspend the lymphocytes, pipetted and mixed, and 200 ul was taken for flow cytometry counting; the remaining 2 ml of cells were evenly divided into two flow tubes for IL17 and FOXP3 staining.

[0298] 3.3.5.1 Th17 Detection

[0299] Th17 surface antibody labeling: centrifuge the above 1ml separated cells for 5 minutes and discard the supernatant; take another 48ml of 1640 culture medium and place it in a 50ml centrifuge tube, add 96ul CellActivation Cocktail (with BrefeldinA); add 1ml of the prepared culture medium to each sample of the discarded supernatant, resuspend in a 48-well plate, culture for 6 hours, pipette the cells evenly, place them in labeled 1.5ml centrifuge tubes, and centrifuge for 5 minutes; take 4.9ml FACS buffer and add 245ul CD4-APC antibody, pipette and mix; discard the supernatant of the centrifuged cells, add 105ul CD4 antibody to each tube, mix and pipette evenly, stand at 4℃ in the dark for 30 minutes, add 1ml FACS buffer and centrifuge for 5 minutes, add 50ul MediumA to the centrifuged cells, stand in the dark at room temperature for 20 minutes, wash with 1ml FACS buffer, vortex, centrifuge for 5 minutes, and discard the supernatant; prepare MediumB: 2450ul Medium B + 245ul IL17, at room temperature in the dark, add 50ul Medium B to the centrifuged cells and incubate at room temperature in the dark for 30min. Wash the cells with 1ml FACS buffer, centrifuge for 5min, discard the supernatant, add 500ul FACS buffer, and detect on a flow cytometer.

[0300] 3.3.5.2 Treg Detection

[0301] Take 1ml of separated cells and add them to a newly labeled 15ml centrifuge tube, centrifuge for 5 minutes, discard the supernatant, resuspend with FACS buffer and centrifuge for 5 minutes; at the same time, prepare CD4 antibody, take 4.9ml FACS buffer and add 245ul CD4-APC antibody, mix by pipetting; discard the supernatant of the centrifuged cells, add 105ul CD4 antibody mixture to each tube, pipette evenly, stand at 4℃ in the dark for 30 minutes, add 1ml FACS buffer, and centrifuge for 5 minutes; during this period, dilute 4×Fix concentrate (1 part) with Fix diluent (3 parts) to prepare fresh transcription factor Fix working solution, prepare 50ml; filter 300ml of ultrapure water with 0.22μm filter membrane, and mix True-Nuclear 1×Perm buffer was diluted at a ratio of 1:9 to prepare 350ml; 1×Fix concentrate was added to each tube of the centrifuged cells, vortexed and incubated at room temperature in the dark for 60min, 2×Perm buffer was added to each tube, centrifuged at room temperature for 5min, and the supernatant was discarded. After repeating this operation, 4.9ml of 1×Perm buffer was added to each tube, followed by 245μl of FOXP3 antibody, incubated at room temperature in the dark for 30min, 2ml of 1×Perm buffer was added to each tube, centrifuged at room temperature for 5min, and the supernatant was discarded; 2ml of FACS buffer was added to each tube, centrifuged at room temperature for 5min, and the supernatant was discarded. The cells were resuspended with 500μl FACS buffer and detected by flow cytometer.

[0302] See the results Figure 16 .

[0303] Depend on Figure 16 It can be seen that the abnormal Th17 / Treg ratio in the body is one of the main factors that induce ovarian dysfunction. When the body is in an inflammatory state, the Th17 / Treg ratio is abnormally unbalanced, and lymphocytes invade the ovarian tissue, leading to the decline of ovarian function. Therefore, in cell differentiation, Th17 cell differentiation is inhibited and Treg cell differentiation is promoted, so that the two are interconnected and mutually restricted, thereby achieving the purpose of inhibiting ovarian function decline. This experiment detected the Th17 / Treg ratio in the peripheral blood of rats. The Th17 / Treg cell ratio results are shown in the figure below. The results showed that compared with the blank control group, the Th17Treg cell ratio in the model control group was significantly increased, and there was a significant difference (P < 0.01); compared with the model control group, the Th17 / Treg cell ratio in the positive drug administration group was significantly decreased, and there was a significant difference (P < 0.01); compared with the model group, the Th17 / Treg cell ratio in the low-dose, medium-dose, and high-dose drug administration groups was significantly decreased (P < 0.001). In summary, all three dosages can inhibit the Th17 / Treg cell ratio, and there is little difference among the three groups.

[0304] 3.3.6 Collection and storage of blood samples

[0305] During the entire administration period, the rats were weighed once every 10 days. After the administration, the rats in each group were fasted for 12 hours, but were not allowed to drink water. The rats were anesthetized by intraperitoneal injection of 10% chloral hydrate, and blood was collected from the abdominal aorta. About 10 mL of blood was obtained, and after the blood was allowed to stand for 1 hour to coagulate, the serum was separated by centrifugation (4°C, 3500rPm, 20min), the upper serum was aspirated and packaged, and stored in a -80°C refrigerator. Among them, the weight effect results are shown in Figure 17 .

[0306] Depend on Figure 17 It can be seen that there was no significant difference in the weight of the rats in the groups before the experiment. During the normal feeding period, the weight of the rats in the blank control group continued to increase, the weight of the rats in the model control group continued to rise, and the weight of the rats in the positive drug control group and the individual dose groups first decreased and then increased. Ultimately, the average weight of the rats in the three drug groups was higher than that of the rats in the model control group, among which the weight change in the high-dose group was the most significant. The results show that after rats enter ovarian decline, the estrogen level in the body gradually decreases, leading to weight loss. Judging from the weight changes after drug administration, Dingjing Pills can improve the gradual weight loss trend of rats with ovarian decline, and the inhibitory effect of the high-dose group is more significant.

[0307] 3.3.7 Collection and preservation of tissue samples

[0308] After blood collection, the rats were dissected and the heart, liver, spleen, lungs, kidneys, brain, uterus, and ovaries were quickly removed on ice. The tissues were washed in pre-chilled saline to remove blood and impurities. The tissues were blotted dry with filter paper, weighed, and the data recorded. The uterus and one ovary were fixed in 4% paraformaldehyde and stored at 4°C for HE staining. The other ovary was placed in a 2 mL tissue cryovial, frozen in liquid nitrogen, and stored at -80°C. All other tissues were stored in 4% paraformaldehyde at room temperature.

[0309] 3.3.8 Calculation of organ coefficient

[0310] The ovaries and uteri obtained after autopsy of the rats were weighed and the organ index was calculated.

[0311] Organ index = organ weight (mg) / body weight (g) × 100%.

[0312] See the results Figure 18 .

[0313] Depend on Figure 18It can be seen that compared with the blank control group, the ovarian coefficient and uterine coefficient of the rats in the model control group were significantly reduced (P < 0.05); compared with the model control group, the ovarian coefficient and uterine coefficient of the positive drug group increased to varying degrees, but there was no significant difference; the ovarian coefficient and uterine coefficient of the low, medium, and high dose groups also increased to varying degrees, but there was no significant difference; this shows that after rats enter ovarian dysfunction, their ovarian and uterine gonadal organs will atrophy, and the drug group can increase the organ coefficient of the gonadal organs of rats with ovarian dysfunction and improve the atrophy of the gonadal organs. Among them, the ovarian coefficient of the high dose group increased significantly, and there was no significant difference in the uterine coefficient among the three drug groups.

[0314] 3.3.9 Determination of serum biochemical indicators

[0315] Enzyme-linked immunosorbent assay (ELISA) kits were used to measure the levels of estradiol (E2), follicle-stimulating hormone (FSH), anti-Müllerian hormone (AMH), and luteinizing hormone (LH) in rat serum. Before testing, the kits were equilibrated at room temperature for 30 minutes and the serum samples were thawed at room temperature. Specific procedures were performed according to the kit instructions.

[0316] See the results Figure 19 .

[0317] Depend on Figure 19It can be seen that compared with the model control group, the serum FSH content of the model control group increased (P < 0.05), the LH content increased significantly (P < 0.001), the E2 content decreased significantly (P < 0.01), and the AMH content decreased (P < 0.05). The results are consistent with the characteristics of sex hormones in rats with ovarian dysfunction. Compared with the model control group, the FSH and LH contents of the positive drug group decreased significantly, and the E2 and AMH contents increased significantly (P < 0.01); compared with the model control group, the FSH contents in the low-dose, medium-dose, and high-dose groups decreased significantly (P < 0.01), and the differences among the three groups were not significant; compared with the model group, the LH contents in the low-dose, medium-dose, and high-dose groups decreased, but there was a significant difference between the medium-dose and high-dose groups (P < 0.05), while there was no significant difference in the low-dose group; compared with the model group, the low-dose, medium-dose, and high-dose groups decreased significantly. The E2 content in the three groups increased to varying degrees, but there was no significant difference. Compared with the model group, the AMH content in the low-dose, medium-dose, and high-dose groups increased to varying degrees, among which the low-dose group had no significant difference, the medium-dose group had a significant difference (P < 0.05), and the high-dose group also had a significant difference (P < 0.05). By comparing the various drug-administered groups, the medium-dose and high-dose drug-administered groups had better improvements in FSH and LH hormone levels, the high-dose drug-administered group had better improvements in AMH content, and there was no significant difference in the improvement of E2 content among the three drug-administered groups. Taking into account the above hormone content changes, it was determined that Dingjing Pills can improve the serum hormone levels of rats with ovarian dysfunction, and the high-dose drug-administered group has better improvements in various hormones in rats than the other groups.

[0318] 3.3.10 Determination of related inflammatory indicators in rats

[0319] The amount of transforming growth factor-β (TGF-β), interleukin-22 (IL-22), interleukin-21 (IL-21), and interleukin-10 (IL-10) in rat ovaries was detected using an enzyme-linked immunosorbent assay kit. The kit was equilibrated at room temperature for 30 minutes before testing, and the serum sample was thawed at room temperature. The specific operation requirements were carried out according to the kit instructions. The results are shown in the table. Figure 20 .

[0320] Depend on Figure 20It can be seen that the results of the detection of factors related to inflammation in the rat ovary are as shown in the figure below. Compared with the model control group, the blank control group showed a significant increase in the content of TGF-β in the model group (P < 0.05), a significant decrease in the content of IL-22 (P < 0.001), and a significant decrease in the content of IL-21 and IL-10 (P < 0.05). Compared with the model control group, the content of TGF-β in the positive drug administration group decreased but not significantly, the content of IL-22 increased significantly (P < 0.01), and the content of IL-21 and IL-10 increased significantly (P < 0.05); compared with the model group, the content of TGF-β in the low-dose, medium-dose, and high-dose groups increased but there was no significant difference; compared with the model group, the content of IL-22 in the low-dose, medium-dose, and high-dose groups increased significantly (P < 0.001 ... significantly (P < 0.001); compared with the model group, the content of TGF-β in the low-dose, medium-dose, and high-dose groups increased significantly (P < 0.001). The IL-21 content in the low-dose and medium-dose groups increased without significant difference, and the high-dose group increased significantly (P < 0.01). Compared with the model group, the IL-10 content in the low-dose and medium-dose groups increased significantly (P < 0.05), and the high-dose group also showed significant difference (P < 0.01). In summary, it can be seen that the high-dose group had the best improvement effect on the following four indicators, indicating that Dingjing Pills can effectively improve the anti-inflammatory ability of rats with ovarian dysfunction, among which the high-dose group had better anti-inflammatory effect.

[0321] 3.3.11 HE staining of rat ovary / uterus

[0322] Pathological sections of the ovary and uterus were prepared using the paraffin embedding method and observed after hematoxylin-eosin (HE) staining. The steps are as follows: Place one ovary and uterus in a 4% paraformaldehyde solution for fixation overnight, place the tissue in an embedding frame, dehydrate through a gradient of alcohol from low to high, then place the ovary and uterus in xylene to replace the alcohol and make it transparent. Place the embedding frame in low, mixed, and high melting point wax melted in a water bath in advance to replace the xylene and embed in paraffin. Fix the paraffin on a microtome, slice continuously, and cut into 5μm thin slices. Place the slices in an oven for 2 hours, dewax them in xylene, soak them in a gradient of alcohol from high to low, and rinse with running water. Stain them with hematoxylin, rinse with running water, then immerse them in hydrochloric acid ethanol, rinse with running water, and transfer the slices to 0.2% ammonia water. Stain them with eosin, dehydrate them with anhydrous ethanol, and make them transparent by immersing them in xylene. Finally, the slices were placed in a fume hood to dry, and then dripped with gum, covered with a cover glass, and observed and photographed under a light microscope. Figure 21 and Figure 22 .

[0323] The ovary is the female gonadal organ, and the follicles in the ovary can reflect the female's fertility. Pathological sections can intuitively reflect the changes in ovarian morphology and physiological functions. The oocytes in the ovary can be divided into primordial follicles, primary follicles, secondary follicles, mature follicles and atretic follicles. Primordial follicles are located in the shallow part of the ovarian cortex, with an oogonia in the center, surrounded by a layer of flat follicular cells on the periphery; primary follicles are mostly cubic, with a zona pellucida between the egg cell and the follicular cells, and the follicles are surrounded by a layer of connective tissue composed of the follicle membrane; secondary follicles have follicular cavities, and some follicular cavities can form cumulus ovale; the follicular cavities of mature follicles are large, the cumulus ovale is obvious, and the endometrial cells are close to the granulosa layer of the follicle; the structure of the atretic follicle is fuzzy, the zona pellucida shrinks, degenerates and disappears, and the granulosa cells are loose. Figure 21 Microscopic observation of rat ovaries by HE staining revealed that the ovaries of the blank control group were mature, with a higher number of primordial follicles, primary follicles, secondary follicles, and mature follicles. Compared with the blank control group, the number of follicles at all levels in the ovaries of the model control group was significantly reduced, and the oocytes were irregular in shape, with obvious pathological changes such as nuclear condensation or oocyte atrophy and disappearance, and zona pellucida collapse. Compared with the model group, the ovarian morphology of the positive drug control group and all drug-treated groups improved to varying degrees, with an increase in the number of primordial follicles and follicles at all levels. This indicates that Dingjing Pills can improve ovarian histology, promote the development of follicular cells, and improve ovarian physiological function. The number of primordial follicles, primary follicles, and secondary follicles in the medium-dose and high-dose groups was higher than that in the low-dose group, indicating a significant improvement.

[0324] Depend on Figure 22 HE staining of the rat uterus revealed that the blank control group had a thick endometrium and a higher number of glands. Compared with the blank control group, the model control group showed significant uterine atrophy, with reduced endometrial thickness and a significantly reduced number of uterine glands, with uneven distribution. Compared with the model control group, the positive drug control group and all treatment groups showed varying degrees of improvement in uterine morphology, with increased endometrial thickness and gland number. This suggests that Dingjing Pills can improve uterine morphology and physiological function. The medium-dose group showed significant endometrial thickening and a higher number of glands.

[0325] In summary, after successful model establishment, estradiol valerate was used as a positive agent. The model group received continuous intraperitoneal injections of cyclophosphamide, while the treatment groups received low, medium, and high doses of cyclophosphamide, respectively. After four weeks of continuous administration, Dingjing Pills reduced anxiety in rats, improved spontaneous locomotion and exploratory behavior, and effectively improved the gradually declining weight of rats with ovarian failure. Flow cytometry results showed a significant decrease in the Th17 / Treg cell ratio. HE staining analysis showed that Dingjing Pills improved the physiological function of the ovaries and uterus in rats with ovarian failure. Compared with the model group, the levels of estradiol (E2), anti-Mullerian hormone (AMH), interleukin-22 (IL-22), interleukin-21 (IL-21), and interleukin-10 (IL-10) were all increased, while the levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and transforming growth factor-β (TGF-β) were all decreased. These results indicate that Dingjing Pills at medium and high doses have a significant therapeutic effect on ovarian failure.

[0326] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A Dingjing pill for preventing and treating ovarian dysfunction, characterized in that: The Dingjing Pills are prepared from the following raw materials in parts by mass: 600-1000 parts of Cuscuta seeds, 700-1200 parts of white peony root, 600-900 parts of angelica root, 300-450 parts of Rehmannia root, 350-450 parts of Chinese yam, 200-300 parts of Poria cocos, 100-180 parts of Schizonepeta spicate and 30-50 parts of Bupleurum root.

2. The Dingjing Pill for preventing and treating ovarian dysfunction according to claim 1, characterized in that: The Dingjing Pills are prepared from the following raw materials in parts by mass: 810 parts of Cuscuta seeds, 810 parts of white peony root, 810 parts of Chinese angelica root, 405 parts of Rehmannia root, 405 parts of Chinese yam, 243 parts of Poria, 162 parts of Schizonepeta spicate and 40.5 parts of Bupleurum.

3. The preparation method of Dingjing Pills according to claim 1, characterized in that: The following steps are involved: S1 take the fixed by pills 1 / 10 of the amount of each raw material by mass, drying, crushing, sieving to obtain crude drug powder; S2. Unsieved crude drug powder was placed in a gauze bag and mixed with 9 / 10 parts by mass of the remaining raw materials, soaked in water and refluxed for 2 times, the filtrates were combined, concentrated, and spray-dried to obtain a dry powder; S3. The dry powder and crude drug powder are mixed evenly, wet granulated, pelletized, pelletized, dried, and packaged to obtain the Dingjing pills.

4. The preparation method according to claim 3, characterized in that In step S1, the product is dried in an oven at 65° C., crushed, and passed through a 100-mesh sieve.

5. The preparation method according to claim 3, characterized in that In step S2, add 6 times the weight of water, soak for 30 minutes, reflux extraction for 1.5 hours, and collect the filtrate; add 6 times the weight of water to the medicinal residue, reflux extraction for 1.5 hours again; and combine the filtrates of the two extractions.

6. The preparation method according to claim 3, characterized in that In step S2, the extract is concentrated to a relative density of 1:1.13-1.15 at 80°C.

7. The preparation method according to claim 3, characterized in that In step S3, the dry powder and crude drug powder are evenly mixed, passed through a 100-mesh sieve, and wet granulated with 80% ethanol.

8. Use of the Dingjing Pills according to claim 1 in preparing a medicine for treating ovarian dysfunction.

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