Traditional Chinese medicine composition for treating perimenopausal syndrome

Abstract

The invention relates to the technical field of traditional Chinese medicine, and discloses a traditional Chinese medicine composition for treating perimenopausal syndrome, which is composed of podophyllum hexandrum, lagotis, honeysuckle fruit, bamboo shavings, pilose antler and sea-buckthorn. The composition takes podophyllum hexandrum as a monarch drug, sea-buckthorn and honeysuckle fruit as ministerial drugs to enhance the effects of promoting blood circulation and regulating menstruation, cornu cervi pantotrichum for replenishing essence and blood, warming yang and dispelling cold, and bamboo shavings and lagotis herb as adjuvant drugs for cooling blood and clearing heat, so that a three-dimensional system of promoting blood circulation, tonifying and clearing heat is formed. It is verified that the composition can relieve uterine atrophy, improve organic decline of a reproductive system, reduce whole blood viscosity and abnormally-increased leukocyte number, inhibit inflammation, accurately adjust serum E2, LH, PROG and AMH levels so as to remodel hormone balance and avoid side effects of exogenous hormones. The composition is based on the advantages of Tibetan medicines and the multi-target effect of natural medicinal materials, conforms to the modern research and development trend and is high in clinical transformation value.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a traditional Chinese medicine composition for treating perimenopausal syndrome. Background Technology

[0002] Perimenopausal syndrome (PMS) is a group of clinical symptoms caused by fluctuations in sex hormone levels during the decline of ovarian function in women. It is characterized by autonomic nervous system dysfunction, accompanied by neuropsychiatric symptoms and metabolic abnormalities. Clinical manifestations include hot flashes, sweating, menstrual irregularities, palpitations, insomnia, depression or anxiety, memory decline, and cognitive impairment. Perimenopausal is a crucial period in the transition of female reproductive aging, typically occurring between the ages of 45 and 55 and lasting 10-15 years. With the significant increase in female life expectancy (from approximately 45 years in the early 20th century to around 80 years currently), postmenopausal survival time has increased dramatically. This change may make women more susceptible to the long-term effects of PMS, such as hot flashes, osteoporosis, and increased cardiovascular and metabolic risks. PMS has a high incidence rate and affects a large population. According to reports, by 2030, the number of perimenopausal women in China will reach approximately 280 million, with a PMS incidence rate as high as 61.0% among women aged 40-65. In the clinical management of PMS, hormone replacement therapy (HRT) is a major intervention in modern medicine. While HRT can effectively alleviate PMS symptoms, long-term use may lead to various adverse reactions and complications, such as endometrial hyperplasia and breast tenderness. It can also increase the risk of endometrial cancer, breast cancer, and acute pancreatitis, thus seriously harming women's physical and mental health. Therefore, the clinical treatment of PMS urgently needs to find safer and more effective treatment options. Traditional Chinese medicine (TCM) has the advantages of good efficacy, low cost, high safety, convenient administration, and wide availability, playing an important role in the treatment of PMS. Therefore, developing drugs with good efficacy and fewer adverse reactions from TCM to replace synthetic drugs with serious adverse reactions (such as hormone preparations) has good application prospects and has become an important direction of modern pharmaceutical research. Summary of the Invention

[0003] The Liuwei Taoerqi Pill formula provided by this invention is based on Tibetan medicine theory and optimized by modern pharmacological research. It is precisely designed to target the core pathological characteristics of perimenopausal syndrome, namely "hormonal imbalance and reproductive decline", and provides a safe and effective new option for the treatment of perimenopausal syndrome.

[0004] On the one hand, the present invention provides a traditional Chinese medicine composition for treating perimenopausal syndrome, wherein the traditional Chinese medicine composition comprises, by weight, 200-300 parts of *Taoerqi*, 80-150 parts of *Honglian*, 170-260 parts of *Seabuckthorn*, 165-255 parts of *Lonicera japonica*, 75-152 parts of *Zhuru*, and 31-57 parts of *Lurong*.

[0005] Furthermore, the traditional Chinese medicine composition comprises, by weight, 250-270 parts of *Taoerqi*, 100-120 parts of *Honglian*, 200-240 parts of *Seabuckthorn*, 200-240 parts of *Lonicera japonica*, 100-120 parts of *Zhuru*, and 40-50 parts of *Lurong*. Preferably, the traditional Chinese medicine composition comprises, by weight, 264 parts of *Taoerqi*, 110 parts of *Honglian*, 220 parts of *Seabuckthorn*, 220 parts of *Lonicera japonica*, 110 parts of *Zhuru*, and 44 parts of deer antler.

[0006] Furthermore, the traditional Chinese medicine composition is prepared into a clinically acceptable formulation by adding conventional excipients according to conventional processes.

[0007] Furthermore, the dosage form of the traditional Chinese medicine composition is an aqueous solution, granules, powder, capsules, or pills.

[0008] Furthermore, the dosage of the traditional Chinese medicine composition is 7-8g / day.

[0009] On the other hand, the present invention provides a method for preparing Liuwei Taoerqi Pills, the method comprising using the above-mentioned traditional Chinese medicine composition, the preparation steps of which are as follows: Peach nut, Coptis chinensis, honeysuckle fruit, bamboo shavings and deer antler are ground together into a fine powder, sieved, and mixed medicinal powder is obtained. Sea buckthorn was boiled twice with water, then filtered and concentrated. The concentrated liquids from the two concentrations were combined to obtain an extract. Mix the powdered medicine with the extract to obtain Liuwei Taoer Qiwan; Preferably, the mass ratio of water to sea buckthorn during the decoction is 8-12:1; Preferably, the powdered medicine and the extract are mixed and then dextrin and water are added to prepare Liuwei Taoerqi Pills.

[0010] Furthermore, the relative density of the extract is 1.10-1.20.

[0011] On the other hand, the present invention provides a Liuwei Taoerqi Pill for treating perimenopausal syndrome, wherein the Liuwei Taoerqi Pill is prepared by the preparation method described above.

[0012] On the other hand, the present invention provides the application of the traditional Chinese medicine composition or the Liuwei Taoerqi Pill prepared by the above preparation method in the treatment or prevention of perimenopausal syndrome.

[0013] Furthermore, the treatment or prevention of perimenopausal syndrome includes alleviating uterine atrophy in perimenopausal women, increasing the uterine coefficient, and improving organic decline of the reproductive system.

[0014] Furthermore, the treatment or prevention of perimenopausal syndrome also includes improving blood circulation, reducing abnormally high white blood cell counts, suppressing inflammatory responses, and regulating hormone balance.

[0015] The technical solution of this invention has the following advantages: This invention, Liuwei Taoer Qiwan, is constructed based on Tibetan medicine theory and modern research, and has significant efficacy in treating "Fulong disease" (corresponding to perimenopausal syndrome, etc.) in Tibetan medicine. Its core advantages are: 1. In the formula of Liuwei Taoerqi Pill, Taoerqi is the principal ingredient, which has the functions of regulating menstruation and nourishing blood, promoting blood circulation and removing blood stasis, reducing swelling and relieving pain. It is a commonly used medicine in Tibetan medicine for treating gynecological diseases and blood disorders. The "Wish-Fulfilling Tree" states: "Aomao Sai (Taorqi) has special effects in treating blood disorders and women's diseases." The assistant ingredients are sea buckthorn and honeysuckle fruit, which enhance the blood-activating and menstruation-regulating effects of the principal ingredient; deer antler is used to benefit essence and blood, strengthen kidney yang, warm yang and dispel cold, and enhance the overall tonifying effect of the formula. The adjuvant ingredients are bamboo shavings and Coptis chinensis, which on the one hand can assist the principal ingredient in nourishing blood and regulating menstruation, and on the other hand have the effects of cooling blood, clearing heat and reducing inflammation, which are effective for gynecological inflammation. This forms a three-dimensional system of "blood activation + tonification + heat clearing", avoiding the shortcomings of traditional single medicines.

[0016] 2. The Liuwei Taoerqi Pill provided in this application can significantly alleviate uterine atrophy, increase the uterine coefficient, and improve the organic decline of the reproductive system in perimenopausal model rats.

[0017] 3. The Liuwei Taoerqi Pill provided in this application reduces whole blood viscosity (low / medium / high shear) and improves circulation at clinically equivalent doses; it also reduces white blood cell count and inhibits inflammation.

[0018] 4. The Liuwei Taoerqi Pill provided in this application precisely regulates serum E2, LH, PROG, and AMH levels, restoring hormonal balance and avoiding the side effects of exogenous hormones. This prescription has been verified through animal experiments, utilizing multi-target treatment with natural medicinal materials, preserving the advantages of Tibetan medicine, conforming to modern research and development trends, and possessing high clinical translational value. Attached Figure Description

[0019] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0020] Figure 1 This is a 100× image of vaginal cell smears from rats in the Sham and OVX groups after modeling. Figure 2 The image shows the serum E2 concentration results of rats in the Sham and OVX groups after modeling. Compared with the Sham group, the OVX group showed a significant difference (P < 0.0001, n = 6).

[0021] Figure 3 The figure shows the effects of Liuwei Taoerqi Pill on body weight and food intake in perimenopausal model rats. Compared with the Sham group, ###P<0.001; compared with the OVX group, *P<0.05, **P<0.01, ***P<0.001, n=3. Figure 4 The image shows the effects of Liuwei Taoerqi Pill on the morphology and pathology of the uterus in a perimenopausal model rat. A: Appearance of the rat uterus; B: Histopathological changes in the rat uterine tissue; C: Changes in endometrial thickness, endometrial epithelial cell thickness, and number of endometrial glands in the rat.

[0022] Figure 5 The figure shows the effect of Liuwei Taoerqi Pill on organ indices in perimenopausal model rats. Compared with the Sham group, ####P<0.0001; compared with the OVX group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n=5; Figure 6 The graph shows the effect of Liuwei Taoerqi Pill on the blood routine of perimenopausal model rats. Compared with the Sham group, ####P<0.0001; compared with the OVX group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n=5; Figure 7 The figure shows the effect of Liuwei Taoerqi Pill on hemorheology in perimenopausal model rats. Compared with the Sham group, ####P<0.0001; compared with the OVX group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n=5; Figure 8 The figure shows the effect of Liuwei Taoerqi Pill on serum hormone levels in perimenopausal model rats. Compared with the Sham group, ####P<0.0001; compared with the OVX group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n=7. Detailed Implementation The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0023] For those not specifying the specific experimental procedures or conditions in the examples, they are all operated or conducted according to the conventional experimental procedures or conditions in the art. For reagents or instruments without indicating the manufacturer, they are all conventional reagent products or instruments that can be obtained through commercial purchase.

[0024] I. Experimental Materials 1. Main Reagents Sodium carboxymethyl cellulose (CMC-Na), shape: colorless, transparent, viscous liquid, storage conditions: cool and dry, specification: 500 g / bottle, batch number: F2314831, expiration date: 2023.06.30 - 2027.06.29, production unit: Shanghai Aladdin Biochemical Technology Co., Ltd.

[0025] 2. Main Instruments HZY-C3200 balance, Fuzhou Huazhi Scientific Instruments Co., Ltd. BSA224S-CW balance, Sartorius Scientific Instruments (Beijing) Co., Ltd. L530 centrifuge, Hunan Xiangyi Laboratory Instrument Development Co., Ltd. JB-L8 paraffin embedding machine, Wuhan Junjie Electronics Co., Ltd. LEiCA RM2245 paraffin slicer, Shanghai Leica Instruments Co., Ltd. JB-L8 freezing table, Wuhan Junjie Electronics Co., Ltd. JK-6 tissue spreading machine, Wuhan Junjie Electronics Co., Ltd. 101-1B oven, Shangyu Huyue Instrument Equipment Factory, Shaoxing City BC-5100 automatic hematology analyzer, Shenzhen Mindray Bio-Medical Electronics Co., Ltd. 3. Experimental Animals (1) Basic Information Species: SD rats, grade: SPF grade, weight / age: 180 - 200 g / 8 weeks old, gender: female, quantity: 40, supply unit: Beijing Vital River Laboratory Animal Technology Co., Ltd., license number: SCXY (Beijing)

[0026] (2) Animal Facilities Facility name: Experimental animal breeding, facility address: Animal building of Tianjin University of Traditional Chinese Medicine, Beihua South Road, Jinghai District, Tianjin, experimental animal use license number: SYXK (Tianjin) 2020-0005, issuing unit: Tianjin Science and Technology Bureau.

[0027] (3) Animal Feed Feed name: SPF mouse and rat maintenance feed, sterilization method: 60Co-irradiation, Supplier: Beijing Huafukang Biotechnology Co., Ltd., Animal Feed Production License: SCXK(Beijing)2024-0003, Issued by: Beijing Municipal Science and Technology Commission.

[0028] Feed Testing: Each batch of feed has a quality certificate and a self-inspection report provided by the feed supplier. Every year, the feed supplier provides a recent third-party feed testing report.

[0029] Feed Testing Results: All test indicators meet the requirements.

[0030] (4)Animal Drinking Water Source of Drinking Water: Sterile water prepared by the experimental animal drinking water dispenser MN-LM2000J. Drinking method: Direct filling and supply in drinking water bottles, and animals drink freely.

[0031] Drinking Water Testing: The company conducts self-inspection of microbial indicators for drinking water every quarter, and sends it to a professional third-party testing agency for hygienic evaluation testing every year. [[ID=I5]]

[0032] Drinking Water Testing Results: All test indicators meet the requirements.

[0033] (5)Animal Rearing Conditions Rats: Housed in polypropylene rat cages. The cage specifications are length × width × height = 48 cm × 35 cm × 20 cm. All female rats, and at most 5 animals are housed in each cage.

[0034] Temperature: Set temperature range 20~26°C, Humidity: Set humidity range 40%-70%, Air change rate: Not less than 15 times of fresh air per hour, Lighting: 12 hours light and 12 hours dark alternating.

[0035] Rearing Conditions Testing Results: No abnormal situations affecting the experimental results occurred during the experiment.

[0036] (6)Animal Rearing Management Except for special requirements (such as fasting, water deprivation), animals eat and drink freely. The drinking water bottles and the drinking water in the bottles are replaced every day and not reused. After use, the drinking water bottles are sterilized by high pressure in a pulsating vacuum sterilizer and then used again.

[0037] The chassis of the animal rearing cages is replaced once a day, the rearing cages are replaced once a week, and the cage covers are replaced once every two weeks. All rearing cages enter the barrier environment for use after being sterilized by high pressure in a pulsating vacuum sterilizer. The animal rearing cage racks are cleaned and disinfected by wiping once a week; The animal rearing observation room is cleaned and disinfected every day, and the scope includes: the ground, countertops, desks, doors, windows and air inlets and outlets. The disinfectants used include: benzalkonium bromide disinfectant, peracetic acid solution and 84 disinfectant. The three disinfectants are used alternately, one per week, and cannot be mixed.

[0038] (7) Animal reception and inspection Laboratory animals are received jointly by laboratory personnel, animal welfare department staff, and veterinarians. Upon receipt, the transport vehicle is inspected to ensure it meets requirements and that it possesses a certificate of conformity from the animal supplier. The certificate's information is confirmed to match the species, grade, sex, and quantity of the animals requested for purchase. After the outer packaging meets requirements and is verified to be correct, the animals are moved to the quarantine area.

[0039] In the quarantine room, the animal packaging was opened, and the number of animals of each sex was verified to match the information on the animal certificate. Each animal was then examined individually (including sex, head, torso, tail, limbs, fur, mental state, and activity level). After quarantine, the animals were placed in animal cages, tagged, and placed on the acclimatization racks in the quarantine room. No abnormal animals were found.

[0040] Animals were required to undergo a 10-day acclimatization period before the experiment, during which they were observed daily. No abnormalities were found during the acclimatization period.

[0041] (8) Animal identification Individual identification: Animals are marked by tagging their ears.

[0042] Cage labeling: A cage label is hung on the animal cage. The cage label includes the project number, animal species, group, cage number, number of animals, animal number, sex, and project leader.

[0043] Labeling of the feeding and observation room: A label should be posted in the feeding and observation room, which includes the project number, project name, experimental stage, project leader, and main experimental personnel.

[0044] (9) Animal welfare The animal use methods in this experiment were approved by the Animal Experiment Ethics Committee of Tianjin University of Traditional Chinese Medicine. Approval number: TCM-LAEC2025058j1640.

[0045] 4. Data Processing In this application, all experimental data were replicated three times or more. Experimental results were plotted using GraphPad Prism 9 software and analyzed using SPSS 26.0 statistical software. Results are expressed as mean ± standard deviation. One-way ANOVA or t-tests were used to analyze sample differences. A p-value < 0.05 was considered statistically significant. The significance level was set at α = 0.05. P A value ≤0.05 indicates a statistically significant difference.

[0046] Example 1. Preparation of Liuwei Taoerqi Pills This embodiment provides the prescription for Liuwei Taoerqi Pills and the preparation method of Liuwei Taoerqi Pills.

[0047] The prescription for Liuwei Taoerqi Pills is as follows: 264g of Taoerqi, 110g of Honglian, 220g of Sea Buckthorn, 220g of Honeysuckle Fruit, 110g of Bamboo Shavings, and 44g of Deer Antler.

[0048] Preparation method of Liuwei Taoerqi Pill: (1) 264g of Peach No. 7, 110g of Coptis chinensis, 220g of Honeysuckle Fruit, 110g of Bamboo Shavings, and 44g of Deer Antler are ground into fine powder and passed through a 100-mesh sieve to obtain mixed medicinal powder. (2) Add 10 times the mass of water to 220 g of sea buckthorn, decoct for 1 hour, filter to obtain decoction A, concentrate decoction A to a relative density of 1.10~1.20 by vacuum concentration to obtain concentrated decoction A; then add 10 times the mass of water to sea buckthorn, decoct for 1 hour, filter to obtain decoction B, concentrate decoction B to a relative density of 1.10~1.20 by vacuum concentration to obtain concentrated decoction B; mix concentrated decoction A and concentrated decoction B to obtain extract.

[0049] (3) Mix the mixed powder with an appropriate amount of dextrin (=1000-mixed powder-sea buckthorn extract amount*(1-water%), where water is the water content of sea buckthorn extract), add the above extract and an appropriate amount of purified water, and make 1000g of soft material that can be formed into a ball and is not sticky. Put it into a kneading machine and knead it 2-3 times. Then transfer it to a pelleting machine (the mold is the smallest size mold for making concentrated pellets, 3mm). Adjust the speed of the strip and the rolling roller to make concentrated pellets of uniform size.

[0050] (4) Dry and polish the concentrated pills. First, spread the prepared wet pills evenly on a stainless steel tray with mesh, 2 kg / tray, and put it into a hot air circulating oven at 80℃. Dry until it makes a crisp sound when lightly bitten, about 3-4 hours. Then, put the dried pills into a pot, rotate them, moisten them with an appropriate amount of ethanol solution, and remove the hairs. Add a small amount of 250-mesh talc powder, rotate and blow air until dry, add a small amount of pharmaceutical solid paraffin, rotate until dry and shiny, and you will get Liuwei Taoer Qiwan.

[0051] Example 2. Quality control method for Liuwei Taoerqi Pills (1) Specifications of Liuwei Taoerqi Pills: The diameter of Liuwei Taoerqi Pills is 2.3 mm, and the average weight of each pill is 0.0131 g. According to the definition of micro-pills (micro-pills refer to spherical entities with a diameter of less than 2.5 mm composed of drugs and excipients), Liuwei Taoerqi Pills belong to micro-pills.

[0052] (2) Disintegration time: Refer to Section (0108) of the General Rules for Pill Preparations in the Chinese Pharmacopoeia. The method for determining the disintegration time limit is as follows: Select a basket with an appropriate mesh size (use a mesh size of about 0.42 mm for pills with a diameter of less than 2.5 mm; use a mesh size of about 1.0 mm for pills between 2.5 and 3.5 mm; use a mesh size of about 2.0 mm for pills with a diameter of more than 3.5 mm); and perform the test by adding a baffle according to the method for testing the disintegration time limit (refer to Section 0921 of the General Rules for Pill Preparations in the Chinese Pharmacopoeia). The pills should completely disintegrate and pass through the mesh within 2 hours. If there are small particles that do not pass through the mesh, but have softened and have no hard core, they can be considered as compliant. The disintegration time of Liuwei Taoerqi Pills is about 20 minutes, which is much less than the 120 minutes required by the Pharmacopoeia.

[0053] (3) Microscopic identification of the characteristics of Liuwei Taoerqi Pill: Take this product and observe it under a microscope. The bone fragments of deer antler are irregular in shape, pale yellow or pale gray, with fine longitudinal texture and dot-like pores on the surface. The unossified bone tissue is nearly colorless, with irregular edges and many irregular block protrusions, among which stripes can be faintly seen. Taoerqi: The vessels are mostly reticulate vessels or spiral vessels. Honglian: The fibrous cells are long strips or long spindles, about 120 μm long, with indistinct pits and pits.

[0054] (4) Specific qualitative detection of Coptis chinensis in Liuwei Taoerqi Pill using thin-layer chromatography: Take 0.5g of this product, grind it into a fine powder, add 10ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 2ml of methanol, and use it as the test solution. Separately, take echinacoside and verbascoside reference standards (the hallmark active ingredients of Coptis chinensis), and prepare solutions containing 0.3mg and 0.4mg per ml respectively with methanol, as reference solutions.

[0055] Thin-layer chromatography (TIC) was performed according to the standard operating procedures specified in Chapter 0502 of the Pharmacopoeia of the People's Republic of China. 5 μl of the test solution and 2 μl each of the echinacoside and verbascoside reference solutions were spotted separately onto the same polyamide TLC plate. The plate was developed using a methanol-acetic acid-water mixture (volume ratio 2:1:7). The plate was then removed, dried, and examined under ultraviolet light (365 nm). Fluorescent spots of the same color appeared at the corresponding positions in the chromatogram of the test sample and the reference samples.

[0056] (5) Drug safety testing using thin-layer chromatography: Podophyllotoxin is the main active ingredient in pepoxetine, but it is also highly toxic (potentially irritating to the human gastrointestinal tract, liver, and kidneys; excessive intake may cause adverse reactions such as nausea, vomiting, and liver and kidney damage). Its content directly affects the safety of the formulation; therefore, the following tests are performed on podophyllotoxin: Take an appropriate amount of this product, grind it into a fine powder, take 7.5g, add 10ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 10ml of methanol to prepare the test solution. Separately, take podophyllotoxin reference standard, add methanol to prepare a solution containing 2mg per ml to prepare the reference solution.

[0057] Thin-layer chromatography (TLC) was performed according to the standard operating procedures specified in Chapter 0502 of the Pharmacopoeia of the People's Republic of China. Two μL of each of the test solution and the reference solution were spotted separately onto the same silica gel GF254 TLC plate. Cyclohexane-ethyl acetate-formic acid (volume ratio 4:5:0.5) was used as the developing solvent. The plate was developed, removed, dried, and examined under ultraviolet light (254 nm). In the chromatogram of the test sample, the spot appearing at the corresponding position as in the chromatogram of the reference sample should be smaller than the spot in the reference sample. This indicates that this method can be used for the limit determination of podophyllotoxin in *Liuwei Taoerqi Wan* (a traditional Chinese medicine preparation) of *Lysimachia christinae*.

[0058] (6) Determined by high performance liquid chromatography.

[0059] Perform the procedure in accordance with the general technical requirements, operating principles and system suitability conditions for high performance liquid chromatography (HPLC) as specified in Chapter 0512 of the Pharmacopoeia of the People's Republic of China.

[0060] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase (Agilent ZORBAX Eclipse Plus 4.6 mm × 250 mm, 5 μm); acetonitrile-methanol-tetrahydrofuran (10:15:1) was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution as specified in the table below; the flow rate was 1.0 mL / min; the column temperature was 30 °C; and the detection wavelength was 331 nm. The theoretical plate number, calculated based on the echinacoside peak, should not be less than 4000. Chromatographic conditions are shown in Table 1.

[0061] Table 1

[0062] Preparation of reference solution: Take an appropriate amount of echinacoside reference standard, accurately weigh it, and add 30% ethanol to prepare a solution containing 20 μg per ml.

[0063] Preparation of the test solution: Take Liuwei Taoerqi pills, grind them into a fine powder, take about 1g, weigh accurately, place in a 100ml brown volumetric flask, accurately add 50ml of 30% ethanol, weigh, soak for 30 minutes, sonicate (power 500W, frequency 53kHz) for 30 minutes, cool, weigh again, replenish the lost weight with 30% ethanol, shake well, filter, and take the filtrate to obtain the test solution.

[0064] For the assay, accurately pipette 10 µl each of the reference solution and the test solution and inject them into the liquid chromatograph for determination.

[0065] This product contains not less than 0.4 mg of echinacoside (C33H46O20) per 1g.

[0066] Experimental Example 1. Verification of the efficacy of Liuwei Taoerqi Pills 1. Model making Forty healthy female SD rats, 8 weeks old and weighing 180-200 g, were selected and acclimatized for 10 days. After the acclimatization period, bilateral ovariectomy was performed to establish the rat model.

[0067] 2. Modeling Method SD rats were divided into a model group and a control group. The model group consisted of 32 healthy female SD rats, and the control group consisted of 8 healthy female SD rats. The modeling methods for the different groups are as follows: Model group (hereinafter referred to as OVX group): Rats were anesthetized by intraperitoneal injection of 5% sodium pentobarbital (45 mg / kg) and fixed in a prone position. The surgical site was located at the intersection of the bilateral costal arches 1.5 cm below and 1 cm lateral to the spine. After preparing the surgical area with an electric shaver, the area was disinfected triple with povidone-iodine solution from the inside out. The skin and subcutaneous fascia were longitudinally incised with sterile surgical scissors, with an incision size of about 1 cm. After blunt dissection of the muscle layer with scissors, the white fat pad was found along the incision. The fat pad was pulled out of the incision with forceps, revealing the ovary and fallopian tubes it contained. The ovarian tissue was gently pulled out using surgical instruments. After ligating the fallopian tubes with 4-0 medical sutures, the ovaries were removed with small scissors. After the operation, the fat pads and uterine horns were returned to the abdominal cavity. The muscles were first sutured with surgical sutures (4-0, absorbable), and then the outer skin was sutured with surgical sutures (3-0, non-absorbable). After each layer of suture was completed, it was disinfected with povidone-iodine solution.

[0068] Control group (hereinafter referred to as Sham group): The ovaries were not removed, only fat of the same volume next to the ovaries was removed. Other steps were the same as those in the OVX group to ensure the controllability of surgical trauma factors.

[0069] 3. Model Evaluation Vaginal smears: Experimental rats were fed according to standardized procedures. Two weeks after ovariectomy, vaginal smears were examined once daily (starting at 10:00 AM) for 10 consecutive days. Giemsa staining was used, and changes in the estrous cycle were observed under a microscope in both groups of rats. Figure 1 Compared with the Sham group, vaginal smear observation after modeling showed that the estrous cycle of rats in the OVX group was disordered, and typical estrous changes could not be observed for a long time. A large number of white blood cells were present, indicating that the bilateral ovariectomy in rats was a successful model.

[0070] Serum E2 (Estradiol) levels: At week 4 post-modeling, blood was collected from the inner canthus of the eyes and centrifuged to obtain rat serum. The E2 levels in the Sham and OVX groups were detected using a rat E2 ELISA kit. Figure 2 The results of E2 content detection in the serum of rats after modeling showed that the E2 content in the OVX group was significantly lower than that in the Shan group (P<0.0001), indicating that the modeling was successful.

[0071] 4. Experimental Grouping Model rats were randomly grouped according to their body weight, as detailed in Table 2.

[0072] Table 2 Experimental Groups and Administration Methods

[0073] Note: N =Number of animals 5. Drug preparation Six-Flavor Peach Seven Pills: Weigh an appropriate amount of the Liuwei Taoerqi Pills prepared in Example 1, grind them evenly, and then dissolve them using a 0.5% CMC-Na solution with magnetic stirring. After thorough mixing, prepare a Liuwei Taoerqi Pills suspension with final concentrations of 0.1350 g / mL and 0.0675 g / mL. Weigh using an electronic balance with an accuracy of 0.001 g, prepare under normal conditions, and prepare immediately before use.

[0074] Estradiol Valerate Tablets (Positive Test): Estradiol Valerate Tablets: Appearance: Pale yellow sugar-coated tablets, white after removing the coating; Specification: 1 mg / tablet; Storage conditions: below 30℃; Batch number: 8075A; Expiry date: 2023.02.15-2028.02.14; Manufacturer: DELPHARM Lille SAS.

[0075] Take an appropriate amount of estradiol valerate tablets and dissolve them using a magnetic stirring method with 0.5% CMC-Na solution as the solvent. After thorough mixing, prepare a 0.009 mg / mL estradiol valerate tablet suspension. Prepare under normal conditions and use immediately.

[0076] 6. Dosage setting and administration Liuwei Taoerqi Pill Group: The clinically applicable dosage of Liuwei Taoerqi Pill for adult women is 7.5 g per day. Based on an average adult female weight of 70 kg, the clinically equivalent dose for rats (calculated as 280 g) is estimated using the Meeh-Rubner formula based on body surface area, which is 0.675 g of medicinal slices / kg. Based on previous experimental results, the dosage of Liuwei Taoerqi Pill in this experiment was set as follows: the clinically equivalent dose and twice the clinically equivalent dose, namely 0.675 g of medicinal slices / kg (SHP-L group) and 1.350 g of medicinal slices / kg (SHP-H group), respectively. Administration was via gavage, with a gavage volume of 10 mL / kg.

[0077] Estradiol Valerate Tablets (Positive Drug - EV Group): The clinical daily dose is 1 mg. Based on the average weight of an adult female of 70 kg, the clinical equivalent dose in rats, calculated using the Meeh-Rubner formula based on body surface area, is 0.09 mg / kg. Administration is by gavage, with a gavage volume of 10 mL / kg.

[0078] Control group (Sham group) and model group (OVX group): 0.5% CMC-Na was administered by gavage at a volume of 10 mL / kg.

[0079] 7. Testing Indicators (1) Observation of physical signs in rats After drug administration, the daily characteristics of the rats were observed, and changes in rat body weight and food intake were recorded at intervals. The results are as follows: Figure 3 According to Tables 3-4, after drug administration intervention, compared with the OVX group, neither SHP-H nor SHP-L changed the average body weight of rats (P<0.01) nor the average food intake of rats (P<0.05). Table 3. Changes in body weight (g) of rats in each group

[0080] Table 4. Changes in food intake (g) of rats in each group

[0081] In addition, changes in the rats' fur were recorded. Rats in the Sham group were in good spirits, active, with smooth, white fur and formed, moderately loose stools. Rats in the OVX group were in poor spirits, lethargic, and had yellowish, rough, and dull fur. Drug intervention improved the rats' mental state and fur color to varying degrees.

[0082] (2) Morphological and pathological observation of rat uterine tissue Twenty-eight days after administration, rats were fasted for 12 hours following the last gavage treatment. Rats were then anesthetized, blood was collected, and the rats were dissected. The intact uterus was removed, photographed, and weighed. A portion of the uterine tissue was fixed in 4% PFA fixative for HE staining to detect pathological changes. The remaining uterus was cryopreserved and flash-frozen in liquid nitrogen. Both SHP-H and SHP-L improved uterine atrophy and pathological changes in rat uterine tissue, increasing endometrial thickness, endometrial epithelial cell thickness, and the number of endometrial glands (P<0.05). Figure 4 See Table 5.

[0083] Table 5. Changes in the endometrium of rats in each group

[0084] (3) Comparison of organ indices in rats After blood collection, the uterus, kidneys, adrenal glands, thymus, spleen, and liver of the rats were removed. Each tissue was washed with physiological saline, and then the remaining water was blotted dry with filter paper or gauze. The weight of each tissue was accurately recorded, and the organ index was calculated. The organ index calculation formula is as follows: Organ Index = Organ Mass (mg) / Body Weight (g). The results are as follows: Figure 5 See Table 6. Both SHP-H and SHP-L significantly increased the uterine index in rats (P<0.01), while SHP-H increased the kidney index and adrenal index (P<0.05).

[0085] Table 6. Changes in organ indices of rats in each group (mg / g)

[0086] (4) Detection of hematological parameters in rats After anesthesia, the rats' whiskers were trimmed to prevent hemolysis during orbital blood collection. A sterilized capillary tube was inserted into the inner canthus of the rat's eye at a 45° angle to the face to collect blood. The first drop of blood was discarded, and approximately 0.5 mL of whole blood was collected in a 1.5 mL EDTA-lined EP tube. A blood analyzer was used to detect the number of red blood cells, white blood cells, lymphocytes, and hemoglobin in the rat's whole blood. The results are shown in Table 7. Figure 6 Compared with the whole blood counts of SHP-H (7.99±0.25 red blood cells, 8.44±1.41 white blood cells, 6.76±1.2 lymphocytes, and 139±3.54) and SHP-L (8.05±0.4 red blood cells, 10.06±1.13 white blood cells, 7.98±0.77 lymphocytes, and 142.4±4.83), and OVX (7.89±0.11 red blood cells, 9.86±2.05 white blood cells, 6.62±0.89 lymphocytes, and 141.6±2.19), the red blood cell count, white blood cell count, lymphocyte count, and hemoglobin content in whole blood were not significantly affected.

[0087] Table 7. Changes in routine blood tests of rats in each group

[0088] (5) Detection of hemorheological parameters in rats Immediately after necropsy, blood was collected from the abdominal aorta using blood collection tubes containing heparin sodium, approximately 1 mL of whole blood was collected. The low-shear rate viscosity, medium-shear rate viscosity, and high-shear rate viscosity of the rat whole blood were measured using a blood rheology analyzer. η1 (1 / s) represents the low-shear rate viscosity of whole blood, η2 (30 / s) represents the medium-shear rate viscosity, and η3 (200 / s) represents the high-shear rate viscosity. The results are shown in Table 8 and... Figure 7 SHP-L reduced the low-shear rate viscosity, medium-shear rate viscosity, and high-shear rate viscosity of whole blood in rats (P<0.05).

[0089] Table 8. Changes in hemorheology of rats in each group

[0090] (6) Detection of serum hormone levels in rats Whole blood was collected using vacuum blood collection tubes. After standing at room temperature for 2 hours, the blood was centrifuged at 4000 rpm for 10 minutes, and the supernatant serum was collected and stored at -80℃. The levels of E2, PROG, AMH, and LH in rat serum were detected using an ELISA kit. Serum hormone level detection strictly followed the standardized operating procedures of the ELISA kit. The results are shown in Table 9. Figure 8 SHP-H (54.63±6.65) and SHP-L (48.24±4.37) both increased E2 levels in rat serum (P<0.05) and decreased LH levels (P<0.01). SHP-H also increased AMH and PROG levels in rat serum (P<0.05).

[0091] Table 9. Changes in serum hormone levels in rats of different groups

[0092] 7. Experimental Conclusions (1) Liuwei Taoerqi Pill can alleviate uterine tissue atrophy in perimenopausal model rats and significantly increase the uterine coefficient in perimenopausal model rats.

[0093] (2) All clinical equivalent dose groups of Liuwei Taoerqi Pills can significantly reduce whole blood viscosity (low shear, medium shear and high shear) in perimenopausal model rats.

[0094] (3) Liuwei Taoerqi Pill can regulate the serum levels of E2, LH, PROG and AMH in perimenopausal model rats.

[0095] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A traditional Chinese medicine composition for treating perimenopausal syndrome, characterized in that, The traditional Chinese medicine composition comprises, by weight, 200-300 parts of *Taoerqi*, 80-150 parts of *Honglian*, 170-260 parts of *Seabuckthorn*, 165-255 parts of *Lonicera japonica*, 75-152 parts of *Zhuru*, and 31-57 parts of *Lurong*.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The traditional Chinese medicine composition comprises, by weight, 250-270 parts of *Taoerqi*, 100-120 parts of *Honglian*, 200-240 parts of *Seabuckthorn*, 200-240 parts of *Lonicera japonica*, 100-120 parts of *Zhuru*, and 40-50 parts of *Lurong*. Preferably, the traditional Chinese medicine composition comprises, by weight, 264 parts of *Taoerqi*, 110 parts of *Honglian*, 220 parts of *Seabuckthorn*, 220 parts of *Lonicera japonica*, 110 parts of *Zhuru*, and 44 parts of deer antler.

3. The traditional Chinese medicine composition according to any one of claims 1-2, characterized in that, The traditional Chinese medicine composition is prepared into a clinically acceptable formulation by adding conventional excipients using conventional processes.

4. The traditional Chinese medicine composition according to any one of claims 1-3, characterized in that, The dosage form of the traditional Chinese medicine composition is aqueous solution, granules, powder, capsules or pills.

5. A method for preparing Liuwei Taoerqi Pills, characterized in that, The preparation method includes preparation using the traditional Chinese medicine composition according to any one of claims 1-4, and the preparation steps are as follows: Peach nut, Coptis chinensis, honeysuckle fruit, bamboo shavings and deer antler are ground together into a fine powder, sieved, and mixed medicinal powder is obtained. Sea buckthorn was boiled twice with water, then filtered and concentrated. The concentrated liquids from the two concentrations were combined to obtain an extract. Mix the powdered medicine with the extract to obtain Liuwei Taoer Qiwan; Preferably, the mass ratio of water to sea buckthorn during the decoction is 8-12:1; Preferably, the powdered medicine and the extract are mixed and then dextrin and water are added to prepare Liuwei Taoerqi Pills.

6. The preparation method according to claim 5, characterized in that, The relative density of the extract is 1.10-1.

20.

7. A Liuwei Taoerqi Pill for treating perimenopausal syndrome, characterized in that, The Liuwei Taoerqi Pills are prepared using the preparation method described in any one of claims 5-6.

8. The use of the traditional Chinese medicine composition according to any one of claims 1-5 or the Liuwei Taoerqi Pill according to claim 7 in the treatment or prevention of perimenopausal syndrome.

9. The application according to claim 8, characterized in that, The treatment or prevention of perimenopausal syndrome includes alleviating uterine atrophy in perimenopausal women, increasing the uterine coefficient, and improving the organic decline of the reproductive system.

10. The application according to claim 8, characterized in that, The treatment or prevention of perimenopausal syndrome also includes improving blood circulation, reducing abnormally high white blood cell counts, suppressing inflammatory responses, and regulating hormone balance.

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