CN118593622A - A pharmaceutical composition for treating perimenopausal syndrome and its preparation method and application - Google Patents

Abstract

The present invention belongs to the technical field of pharmaceutical preparations, and specifically relates to a pharmaceutical composition for treating perimenopausal syndrome, and further discloses its preparation method and application. The pharmaceutical composition for treating perimenopausal syndrome of the present invention comprises raw materials including rhizoma anemarrhenae, cortex phellodendri, radix rehmanniae, radix astragali, angelica sinensis, epimedium, calcined dragon bone, calcined oyster shell, and stir-fried spiny jujube seeds. The pharmaceutical composition for treating perimenopausal syndrome of the present invention has indications including multiple symptoms of perimenopausal syndrome: hot flashes, sweating, sleep disorders, and mood disorders (anxiety, depression), thereby improving the quality of life of perimenopausal women as a whole.

Description

A pharmaceutical composition for treating perimenopausal syndrome and its preparation method and application

Technical Field

The present invention belongs to the technical field of pharmaceutical preparations, and specifically relates to a pharmaceutical composition for treating perimenopausal syndrome, and further discloses a preparation method and application thereof.

Background Art

Perimenopausal syndrome (PMPS) is a general term for a series of symptoms caused by abnormal fluctuations in estrogen levels in the body due to ovarian dysfunction. It mainly includes vasomotor symptoms, menstrual cycle disorders, urogenital atrophy symptoms, and psychoneurological symptoms. In particular, hot flashes and sweating are the first symptoms of medical treatment. In particular, with the decline of estrogen levels, women at this stage will also face a greatly increased risk of cognitive dysfunction, dementia, cardiovascular disease, venous thrombosis, osteoporosis, obesity, type 2 diabetes and other diseases. As a physiological stage that every woman must go through, this process is irreversible. Studies have shown that there are currently about 130 million women in my country who are in perimenopause. It is estimated that by 2030, the number of women in my country who are in perimenopause will reach 280 million. In particular, as the life expectancy of Chinese women continues to increase, women will spend at least 1/3 of their time in the postmenopausal state, which seriously affects their physical and mental health.

At present, Western medicine mainly uses menopausal hormone therapy (MHT), and the drugs are mainly estrogen and progesterone used alone or in combination, which are quick in onset and have definite effects on perimenopausal vasomotor symptoms, urogenital postmenopausal syndrome and osteoporosis. However, due to the many precautions and contraindications of Western medicine treatment, such as endometrial hyperplasia, breast tenderness or (and) swelling, endometrial cancer, and breast cancer, its clinical acceptance and application frequency are greatly limited. Therefore, how to actively prevent and treat PMPS and how to prevent and treat perimenopausal related diseases are the main bottleneck problems currently faced in clinical practice.

Traditional Chinese medicine often treats PMPS in the form of compound prescriptions. This method has the advantage of multiple targets, which can not only relieve the patient's troubling symptoms, but also most of these Chinese medicines belong to phytoestrogen Chinese medicines, which can improve the state of estrogen deficiency in the patient's body, achieving the effect of "treating both state and target at the same time". In the long-term clinical treatment of this disease, many Chinese patent medicines with reliable efficacy have been formed. At present, from the establishment of each database to June 31, 2022, a total of 33 prescriptions and 124 Chinese medicines for PMPS intervention can be retrieved in the four databases of "Chinese Pharmacopoeia 2020 Edition", "National Compilation of Chinese Patent Medicine Standards", "Ministry of Health Drug Standards for Chinese Medicine Formula Preparations", and "State Drug Administration Single-Page Standards", with a total frequency of 292 times. However, the symptoms mainly targeted by traditional prescriptions are still menstrual disorders, irritability, insomnia, hot flashes and sweating, etc., indicating that the attention of Chinese patent medicines is still on the symptoms of PMPS, while ignoring the long-term complications caused by perimenopause and menopause, such as cardiovascular and cerebrovascular diseases, osteoporosis, memory loss and Alzheimer's disease, lipid and glucose metabolism disorders and related diseases. Therefore, according to the theory of "state-target differentiation and treatment", a "state-adjusting" drug that can improve PMPS symptoms and a "targeting" drug that can regulate the low estrogen state are needed to obtain a more group-specific clinical efficacy.

Therefore, this field expects that on the basis of expanding health education to continue to consolidate the effectiveness of Chinese patent medicine in treating PMPS, it is necessary to further systematically summarize the TCM characteristics of perimenopausal and long-term menopausal complications based on the theory of "state-target differentiation and treatment", and carry out targeted research and development of Chinese patent medicines to obtain more group-specific clinical efficacy.

Summary of the invention

To this end, the technical problem to be solved by the present invention is to provide a pharmaceutical composition for treating perimenopausal syndrome, and further disclose its preparation method and application.

In order to solve the above technical problems, the pharmaceutical composition for treating perimenopausal syndrome described in the present invention is characterized in that its raw materials are composed of: 5-30 weight parts of Anemarrhena asphodeloides, 5-30 weight parts of Phellodendron chinense, 5-30 weight parts of Rehmannia glutinosa, 5-30 weight parts of Astragalus membranaceus, 5-30 weight parts of Angelica sinensis, 5-30 weight parts of Epimedium, 10-60 weight parts of calcined Dragon Bone, 10-60 weight parts of calcined Ostreae Conchae, and 10-30 weight parts of stir-fried Ziziphus jujuba seeds.

Specifically, the pharmaceutical composition for treating perimenopausal syndrome comprises the following raw materials: 8 parts by weight of Anemarrhena asphodeloides, 8 parts by weight of Phellodendron chinense, 14 parts by weight of Radix Rehmanniae, 14 parts by weight of Radix Astragali, 8 parts by weight of Radix Angelicae Sinensis, 8 parts by weight of Epimedium, 14 parts by weight of calcined Dragon Bone, 14 parts by weight of calcined Concha Ostreae, and 14 parts by weight of stir-fried Semen Ziziphi Spinosae.

The invention also discloses the use of the pharmaceutical composition for preparing medicine for treating perimenopausal syndrome.

Specifically, the perimenopausal syndrome includes at least one of the following symptoms (1)-(4):

(1) Perimenopausal hot flashes and sweating;

(2) perimenopausal sleep disorders;

(3) perimenopausal depression;

(4) Perimenopausal anxiety.

The invention also discloses a pharmaceutical preparation for treating perimenopausal syndrome, comprising the pharmaceutical composition.

Specifically, the pharmaceutical preparation for treating perimenopausal syndrome includes at least one of tablets, capsules, powders, mixtures, pills, granules, solutions, syrups, decoctions, patches, suppositories, aerosols, ointments or injections.

Specifically, the pharmaceutical preparation for treating perimenopausal syndrome further comprises clinically acceptable excipients;

Preferably, the auxiliary material includes at least one of a filler, a disintegrant, a lubricant, a suspending agent, a binder, a sweetener, a flavoring agent, a preservative or a matrix;

Preferably, the filler comprises at least one of starch, pregelatinized starch, lactose, mannitol, chitin or microcrystalline cellulose;

Preferably, the disintegrant comprises at least one of starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose or cross-linked sodium carboxymethyl cellulose;

Preferably, the lubricant comprises at least one of magnesium stearate, sodium lauryl sulfate, talc or silicon dioxide;

Preferably, the suspending agent comprises at least one of polyvinyl pyrrolidone, microcrystalline cellulose, agar or hydroxypropyl methylcellulose;

Preferably, the adhesive comprises at least one of starch slurry, polyvinyl pyrrolidone or hydroxypropyl methylcellulose;

Preferably, the sweetener comprises at least one of saccharin sodium, aspartame, sucrose, sodium cyclamate or glycyrrhetinic acid;

Preferably, the flavoring agent includes essence;

Preferably, the preservative includes at least one of parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chloroethidine acetate or eucalyptus oil;

Preferably, the matrix comprises at least one of PEG6000, PEG4000 or insect wax.

The invention also discloses a method for preparing the pharmaceutical preparation for treating perimenopausal syndrome, comprising the step of mixing selected amounts of the rhizoma anemarrhenae, cortex phellodendri, radix rehmanniae, radix astragali, angelica sinensis, epimedium, calcined dragon bone, calcined oyster shell and stir-fried spiny jujube seeds.

Specifically, the preparation method of the pharmaceutical preparation for treating perimenopausal syndrome comprises: taking selected amounts of the rhizoma anemarrhenae, cortex phellodendri, radix rehmanniae, radix astragali, angelica sinensis, epimedium, calcined dragon bone, calcined oyster shell and stir-fried spiny jujube seeds, mixing them, adding water to boil them, collecting the decoction and concentrating it into an extract, and adding conventional excipients to process the selected dosage form;

Preferably, the amount of water added is 5-15 times by weight of the total amount of the API; and/or,

Preferably, the decocting step is decocting 1-3 times; and/or,

Preferably, the decocting step lasts for 1-3 hours; and/or,

Preferably, the concentration step is to concentrate the decoction to a thick paste having a relative density of 1.25 at 60°C.

The present invention also discloses the use of the pharmaceutical composition for preparing a drug having at least one of the following effects (1) to (6):

(1) Improve perimenopausal hot flashes and sweating;

(2) Improve perimenopausal sleep disorders;

(3) Improve perimenopausal anxiety;

(4) Improve perimenopausal depression;

(5) Improve overall quality of life during perimenopause;

(6) Promote the production of endogenous estrogen.

The pharmaceutical composition for treating perimenopausal syndrome of the present invention comprises raw materials including rhizoma anemarrhenae, phellodendron, radix rehmanniae, astragalus, angelica, epimedium, calcined dragon bone, calcined oyster, and stir-fried spiny jujube seeds. In the above-mentioned composition, this prescription is designed for the core pathogenesis of perimenopausal women, namely, kidney essence deficiency, imbalance of yin and yang, and insufficient qi and blood. The kidney nourishes the original yin and yang, the kidney essence is sufficient, the heavenly essence is abundant, and women's menstruation is on time; the kidney essence is deficient, the heavenly essence is exhausted, the yin and yang are imbalanced, and the qi and blood are weak, then the menstruation is interrupted and the shape is bad, which manifests as menstrual disorders, hot flashes, night sweats, anxiety and depression, insomnia, fatigue, palpitations, dizziness, fear of cold and wind, spontaneous sweating, night sweats and many other discomforts. The treatment should focus on replenishing kidney essence, regulating yin and yang, and replenishing qi and blood. "Essence and Qi are divided into Yin and Yang, and Yin and Yang cannot be separated." This prescription uses Shengdi and Epimedium as the main ingredients. Shengdi nourishes Yin and benefits the kidney, while Epimedium warms the kidney and strengthens Yang. The two herbs are used together to balance Yin and Yang, targeting the pathological basis of insufficient kidney essence and imbalance of Yin and Yang in perimenopausal women. Zhimu and Huangbai are cold in nature and belong to the kidney. Shengdi nourishes Yin and produces fluid, clears the deficiency heat in the kidney, Huangqi replenishes Qi and consolidates the exterior, and Angelica nourishes blood and regulates menstruation, replenishes Qi and blood, and the above four herbs are the ministers, which synergistically restore the normal physiological state of perimenopausal women; calcined dragon bone and calcined oyster are astringent, calm the mind and suppress Yang, and stir-fried Ziziphus jujuba seeds are sweet, sour and moist, nourish blood and nourish the liver, calm the mind and calm the mind. The three are adjuvants, further enhancing the efficacy of this prescription on hot flashes, sweating, irritability, insomnia, and dreaminess in patients with perimenopausal syndrome.

The pharmaceutical composition for treating perimenopausal syndrome of the present invention can be clinically used to treat perimenopausal syndrome, especially with symptoms such as hot flashes, sweating, insomnia, anxiety, depression, etc., and is also suitable for early prevention of long-term symptoms of perimenopause, such as perimenopausal osteoporosis and perimenopausal obesity. For patients with severe sweating, increase the dosage of calcined dragon bone and calcined oyster; for patients with severe insomnia, increase the dosage of stir-fried spiny jujube seeds; for patients with severe hot flashes, increase the dosage of rhizoma anemarrhenae and phellodendron.

The pharmaceutical composition for treating perimenopausal syndrome of the present invention has indications including multiple symptoms of perimenopausal syndrome: hot flashes and sweating, sleep disorders, and mood disorders (anxiety, depression), and has an overall improvement effect on the symptoms of perimenopausal syndrome, thereby effectively improving the quality of life of perimenopausal women. Preliminary clinical studies have shown that after 12 weeks of intervention in patients with perimenopausal syndrome, the average daily number of hot flashes and sweating can be effectively reduced, the severity of hot flashes and sweating can be improved, and at the same time, perimenopausal insomnia, perimenopausal depression, and perimenopausal anxiety can be synergistically improved, and the quality of life of perimenopausal women can be improved in all aspects, and the long-term efficacy is stable, the clinical safety is good, and no obvious clinical adverse reactions/events are observed.

The pharmaceutical composition for treating perimenopausal syndrome of the present invention has been confirmed for the first time through animal experiments that the pharmaceutical combination can effectively control abnormal body temperature fluctuation levels caused by low estrogen, and provides a treatment plan for perimenopausal syndrome, especially hot flashes and sweating.

The basic experimental research of the pharmaceutical composition for treating perimenopausal syndrome of the present invention is as follows:

① The pharmaceutical composition of the present invention can promote the expression level of E2 in a dose-dependent manner;

② By observing and measuring the body surface temperature and core body temperature of OVX rats and the characteristics of body temperature changes in the hot flash induction model, and evaluating the intervention effect of the pharmaceutical composition of the present invention, it was found that it can effectively improve the abnormal changes in the body surface and core body temperature of OVX rats and inhibit the increase in the tail temperature of rats in the hot flash induction experiment;

③ The pharmaceutical composition of the present invention improves the E2 level without changing the uterine morphology, increasing the uterine wet weight and endometrial thickness of rats after OVX, and has good reproductive system safety.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

FIG1 is a result of the drug combination of the present invention promoting the production of endogenous estrogen in KGN cells;

Figure 2 shows the serum estradiol level in rats two weeks after ovariectomy; Note: Compared with the SHAM group, ^### P<0.001;

FIG3 is an infrared thermal imaging diagram of rat body surface temperature;

Figure 4 shows the differences in the fluctuation range of body surface temperature and heat dissipation sensitivity among rats; Note: Compared with the SHAM group, ^### P<0.001; compared with the OVX group, *P<0.05, **P<0.01, ***P<0.001;

Figure 5 (A)-(C) shows the core body temperature fluctuation of rats; Note: Compared with the SHAM group, ^### P<0.001; Compared with the OVX group, *P<0.05;

Figure 6 shows the fluctuation of body temperature in the tail of rats after CGRP induction; Note: Compared with the SHAM group, ^# P<0.05; Compared with the OVX group, * P<0.05;

Figure 7 shows the results of rat uterine morphology;

Figure 8 shows the wet weight of rat uterus; Note: Compared with the SHAM group, ^### P<0.001; Compared with the OVX group, ***P<0.001;

Figure 9 shows the results of HE staining of rat uterus morphology (×5);

Figure 10 shows the results of endometrial thickness in rats; Note: compared with the SHAM group, ^### P<0.001; compared with the OVX group, **P<0.01, ***P<0.001.

DETAILED DESCRIPTION

Example 1

The pharmaceutical composition for treating perimenopausal syndrome described in this embodiment comprises the following raw materials: 8 parts by weight of Anemarrhena Rhizoma, 8 parts by weight of Phellodendron Amurense, 14 parts by weight of Radix Rehmanniae, 14 parts by weight of Radix Astragali, 8 parts by weight of Radix Angelicae Sinensis, 8 parts by weight of Epimedium, 14 parts by weight of calcined Bone Dragon, 14 parts by weight of calcined Concha Ostreae, and 14 parts by weight of stir-fried Semen Ziziphi Spinosae.

Weigh Rhizoma Anemarrhenae, Phellodendron Amurense, Radix Rehmanniae, Radix Astragali, Radix Angelicae Sinensis, Epimedium, Calcinated Dragon Bone, Calcinated Oyster Shell and Fried Semen Ziziphi Spinosae according to the selected weight portions, mix well, and boil twice. Based on the total weight of the mixed raw materials, add 10 times the weight of water each time and boil for 1.5 hours. Combine the medicinal liquids, filter, and concentrate the filtrate to a thick paste with a relative density of 1.25 at 60°C.

Example 2

The pharmaceutical composition for treating perimenopausal syndrome described in this embodiment comprises the following raw materials: 5 parts by weight of Anemarrhena asphodeloides, 30 parts by weight of Phellodendron chinense, 5 parts by weight of Radix Rehmanniae, 30 parts by weight of Radix Astragali, 5 parts by weight of Radix Angelicae Sinensis, 30 parts by weight of Epimedium, 10 parts by weight of calcined Bone Dragon, 60 parts by weight of calcined Concha Ostreae, and 10 parts by weight of stir-fried Semen Ziziphi Spinosae.

Weigh Rhizoma Anemarrhenae, Phellodendron Amurense, Radix Rehmanniae, Radix Astragali, Radix Angelicae Sinensis, Epimedium, Calcinated Dragon Bone, Calcinated Oyster Shell and Fried Semen Ziziphi Spinosae according to the selected weight portions, mix well, and boil for 3 times. Based on the total weight of the mixed raw materials, add 5 times the weight of water each time and boil for 1 hour. Combine the medicinal liquids, filter, and concentrate the filtrate to a thick paste with a relative density of 1.25 at 60°C.

Example 3

The pharmaceutical composition for treating perimenopausal syndrome described in this embodiment comprises the following raw materials: 30 parts by weight of Anemarrhena Rhizoma, 5 parts by weight of Phellodendron Amurense, 30 parts by weight of Radix Rehmanniae, 5 parts by weight of Radix Astragali, 30 parts by weight of Radix Angelicae Sinensis, 5 parts by weight of Epimedium, 60 parts by weight of calcined Bone Dragon, 10 parts by weight of calcined Concha Ostreae, and 30 parts by weight of stir-fried Semen Ziziphi Spinosae.

Weigh Rhizoma Anemarrhenae, Phellodendron Amurense, Radix Rehmanniae, Radix Astragali, Radix Angelicae Sinensis, Epimedium, Calcinated Dragon Bone, Calcinated Oyster Shell and Fried Semen Ziziphi Spinosae according to the selected weight portions, mix well, and boil twice. Based on the total weight of the mixed raw materials, add 15 times the weight of water each time and boil for 3 hours. Combine the medicinal liquids, filter, and concentrate the filtrate to a thick paste with a relative density of 1.25 at 60°C.

Experimental example

1. Analysis and evaluation of animal research results (1) The pharmaceutical composition of the present invention can supplement endogenous estrogen in menopausal rats

KGN cells are human ovarian granulosa cells derived from human ovarian cancer tissue. Since they highly express aromatase, the addition of androgen (Testosterone, T) can be used to observe whether they have the ability to promote the production of endogenous estrogen.

Experimental materials and instruments

Thirty-two healthy female SD rats, 3 months old, were purchased from the Department of Experimental Animal Science, Peking University Health Science Center and housed at Peking University Health Science Center. Rats were raised under specific pathogen-free (SPF) conditions and fed with soybean-free feed (Beijing Keao Xieli Feed Co., Ltd.). The ambient temperature was controlled at 22-25°C, the humidity was 55±5%, and the light-dark cycle was 12/12 hours (lighting time 7:00-19:00), with free access to food and water.

Experimental methods

Experimental groups and drug administration

The mice were randomly divided into 4 groups, 8 mice in each group, and the specific groups were as follows:

①Sham group: the sham operation group, the operation method is the same as other groups, but only the same volume of fat around the ovary is removed. The mice are fed with ordinary feed and gavage with the same volume of sterile drinking water once a day;

②OVX group: also known as the model group, the surgery method was dorsal incision, bilateral ovaries were removed, and the layers were sutured; they were fed with ordinary feed and gavage with an equal volume of sterile drinking water once a day;

③ Group E2: positive control group, based on OVX, estradiol valerate tablets (trade name: Progynova) were used, converted to the human dose of 1 mg/d, and intragastrically administered at 0.21 mg/kg/d, once a day;

④ GNS group: 1 times the weight of the pharmaceutical composition finally prepared in Example 1 was taken and intragastrically administered at 1.1 mg/kg/d, once a day.

Dosage

The preventive administration method was adopted. All rats were adaptively fed for 1 week before undergoing OVX surgery. During the administration process, the rats were fed with soybean-free feed and given drug intervention to each group (administered according to the above experimental grouping information). The administration period was 8 weeks.

Experimental data detection and processing

Serum preparation

The samples were collected 24 hours after the last administration, and the subjects were fasting but not drinking water. 0.3% pentobarbital (40 mg/kg) was injected intraperitoneally for anesthesia. After fixation in the supine position, the abdominal cavity was disinfected and opened, the abdominal aorta was exposed, and 10 ml of blood was collected using a vacuum blood collection tube containing a coagulant. The blood was allowed to stand at room temperature for 2 hours, and centrifuged at 1000 g for 20 minutes. The supernatant was collected and placed in cryopreservation tubes and stored in a -80°C refrigerator.

Detection indicators and methods

Enzyme-linked immunosorbent assay (ELISA) was used to detect serum estradiol levels, and the specific operation method was strictly in accordance with the instructions of the kit.

Data analysis

SPSS27.0 software was used for statistics, and univariate statistical tests were used to compare the differences between samples in different groups. For normally distributed data, ANOVA (multiple group test) and Student's t-test (two-group test) were used for analysis; for non-normally distributed data, Kruskal-Wallis H-test (multiple group test) and Mann-Whitney Utest (two-group test) were used for analysis. P<0.05 was considered statistically significant.

Experimental Results

The results of the ability of the pharmaceutical composition to promote the production of endogenous estrogen; as shown in Figure 1, the pharmaceutical composition of the present invention can promote the expression of E2 levels, and is dose-dependent and T-dose-dependent.

(II) Effect of the drug composition on the thermoregulatory function of OVX rats

In this study, a menopausal model was established by performing OVX surgery on healthy female adult Sprague-Dawley (SD) rats. The Chinese medicine group and the positive drug group were given the drug composition and estradiol valerate (E2), respectively. The sham operation group was used as a blank control to observe the regulatory effect of the drug composition on the fluctuation of rat surface temperature and core temperature, as well as the improvement of drug-induced hot flashes, and to explore the mechanism of action of Chinese medicine.

Experimental animals

70 adult female SD rats aged 10 weeks were used. Rats were housed in the Animal Department of Peking University Health Science Center under specific pathogen free (SPF) conditions with 2-3 rats/cage. Rats were allowed to adapt to the environment in the rat room for 1 week before the experiment. All rats had unrestricted access to drinking water and soybean-free pelleted feed (Beijing Keao Xieli Feed Co., Ltd.) until the end of the experiment to eliminate the effects of phytoestrogens.

Experimental drugs

Preparation of freeze-dried powder of this pharmaceutical composition: All raw materials are boiled according to 5 pairs/portion of compound medicine, 5L of water is added to each portion for the first decoction, soaked for 30 minutes, boiled over high heat and then decocted over low heat for 90 minutes, filtered while hot with a 200-mesh sieve, and the filtrate is quickly cooled with cold water; 5L of water is added to the second decoction, boiled over high heat and then decocted over low heat for 60 minutes, filtered while hot with a 200-mesh sieve, and the filtrate is quickly cooled with cold water. The two extracts are combined for concentration, and the concentrate is centrifuged (speed 6000R/min, 15-20 minutes), the precipitate is removed, and after centrifugation, it is concentrated again to an appropriate volume, and vacuum freeze-dried to obtain freeze-dried powder of the compound medicine, which is sealed, weighed, packaged, and stored for animal experiments.

Main experimental reagents

Isoflurane, Wright-Giemsa complex staining solution, calcitonin gene-related peptide, estradiol radioimmunoassay kit (B05PZA), TRIZOL (Invitrogen), HiFiScript cDNA first-strand synthesis kit (CW2569), SYBRFAST qPCR Kit Master Mix (2×) (KK4601).

Main experimental instruments and consumables

Infrared thermal imager, small animal core temperature implant, digital-to-analog conversion module, small animal signal receiver, universal small animal anesthesia machine, animal heart perfusion system, upright optical microscope, -80℃ low-temperature refrigerator, rat brain mold, animal heart perfusion system, fully automatic γ radioimmunoassay counter, high-speed refrigerated centrifuge, Nanodrop spectrophotometer, real-time quantitative PCR instrument.

Experimental animal model

After the rats adapted to the environment for 1 week, their body weights were measured, and 13 rats were randomly selected as the sham operation group (SHAM group) according to their body weights. During the operation, 4 rats from the SHAM group were randomly selected to be implanted with body temperature implants. The remaining rats were given OVX surgery, and 12 rats were randomly selected from them to be implanted with body temperature implants.

Establishment of OVX model and implantation of intraperitoneal temperature telemetry implant

Anesthetize the rats with a small animal anesthesia machine (anesthetic is isoflurane). After the pain in the rats' limbs disappears, fix the rats in a supine position on a sterile operating table. Shave the long hair on the abdomen, and disinfect the abdominal skin with iodine and medical alcohol. After the rats are successfully anesthetized, perform surgery under a sterile environment. Make a 2-3 cm long longitudinal incision on the abdomen of the rat, and bluntly separate the muscles of the rat's abdomen. The ovary is located below the kidney. Find the milky white fat tissue surrounding the ovary, gently pull it up, confirm that it is the ovary, clamp and ligate the nearest blood vessels within 0.5-1.0 cm from the Y-shaped uterus with a thin thread, remove the ovary, and ligate the fat tissue. Treat the contralateral side in the same way. If a body temperature implant is required, place the core body temperature wireless signal transmitter implant into the left side of the abdominal cavity and adjust it to the appropriate position, and check for omissions and bleeding. Use standard 3-0 surgical thread to suture the incision sites in turn, disinfect the wound again, apply wound dressings externally, and inject penicillin intraperitoneally for anti-infection treatment for 3 days. In the SHAM group, only the fat around the ovaries was removed, and the size of the fat just had to be the same as the ovaries.

Debugging of abdominal temperature telemetry implant

Turn on the implant power through the magnet switch, use a radio to tune to 550KHz and approach the implant to confirm that the power is on. Move the rat to the telemetry core temperature signal receiver, select the corresponding implant type and number through Dataquest ART software, and judge whether the implant position is appropriate based on the signal waveform and intensity before turning off the implant.

Modeling success determination

The estrous cycle of rats was observed by vaginal exfoliated cytology. Starting from the third day after ovariectomy, the cells were stained with Wright-Giemsa complex stain for seven consecutive days to monitor vaginal exfoliated cells, and blood was collected from the eye sockets to measure serum estrogen levels. If there was no change in the estrous cycle in the vaginal smear of the rats after OVX, it was in a continuous estrus period, and the smear showed mainly white blood cells with a small amount of sticky secretions and a decrease in estrogen levels, the model was successfully established.

Experimental animal grouping

The experimental animals were randomly divided into a blank control group (SHAM group, n=13), a menopausal model group (OVX group, n=13), a low-dose treatment group given the drug combination after OVX (GNS-L group, n=9), a regular-dose treatment group given the drug combination after OVX (GNS-H group, n=13), a regular-dose group given estradiol valerate supplementary treatment after OVX (E2-L group, n=9), and a high-dose group given estradiol valerate supplementary treatment after OVX (E2-H group, n=13).

Drug configuration

According to the weight of the freeze-dried powder, the freeze-dried powder yield is about 33.5g/pack. According to the normal 60kg adult female taking 1 pack per day (about 33.5g freeze-dried powder), and the reference human-rat equivalent dose ratio (1:6.3), the high-dose group needs to take 3.5g/kg of freeze-dried powder of the pharmaceutical composition per rat per day, and the low-dose group needs to take 1.76g/kg of freeze-dried powder of the pharmaceutical composition per rat per day. E2 is calculated based on the normal 60kg adult female taking 2mg per day, and the conventional dose group needs to take 0.21mg/kg of crude drug per rat per day, and the high-dose group needs to take 0.42mg/kg of crude drug per rat per day. Taking into account the individual differences in rat weight, a gavage dose of 1ml/100g rat weight is used for liquid preparation.

Pharmacological interventions

SHAM group: soybean meal feed was removed and 1 ml/100 g body weight of sterile drinking water was gavaged once a day;

OVX group: soybean meal-free feed, 1 ml/100 g body weight sterile drinking water was gavaged once a day;

E2-L group: soybean meal-free feed, oral administration of 1 ml/100 g body weight of E2 regular dose solution, once a day;

E2-H group: soybean meal-free feed, oral administration of 1 ml/100 g body weight of E2 high-dose solution, once a day;

GNS-L group: soybean meal-free feed, 1 ml/100 g body weight of low-dose GNS solution was gavaged once a day;

GNS-H group: soybean meal-free feed, 1 ml/100 g body weight of GNS regular dose liquid was gavaged once a day;

Rats began to receive medication 2 weeks after surgery, with gavage time from 8:30 to 9:30 am every day, and medication lasted for 8 weeks.

Index detection and methods

(1) Vaginal exfoliated cytology smear: Vaginal exfoliated cells were monitored starting from the third day after OVX surgery. One person fixed the rat, and the other person inserted a cotton swab moistened with saline into the rat's vagina, gently rotated it clockwise for 2-3 times, and smeared the cotton swab with secretions from left to right on the slide. After the slide was naturally air-dried, 2-3 drops of Wright-Giemsa composite stain were added to cover the entire specimen smear. After staining for 1-2 minutes, an equal amount of 0.01M phosphate buffer (PH6.4-6.8) was added, and the slide was gently shaken, mixed thoroughly, and then stained for 3-5 minutes. Rinse with tap water for 5 minutes, and observe under a microscope after absorbing the water.

(2) Serum estradiol concentration determination: Blood (300 μl) was collected through the angular vein, incubated at 37°C for 30 minutes, and centrifuged at 3000 rpm for 15 minutes at 4°C. Serum estradiol concentration was determined in the supernatant using a commercially available estradiol radioimmunoassay kit (B05PZA) according to the manufacturer's instructions.

Temperature measurement

(1) Measurement of the body surface temperature of the neck, back, and tail of rats: In a constant temperature environment of 20°C, the rats were placed in a 20*20*40cm body temperature test box. Between 8:00 and 11:00 am on the 8th week of intervention (week 10), an infrared thermometer was placed 1 meter vertically away from the middle skin of the rat's back to measure the body surface temperature of the neck (NST), back (BST), and tail (TST) of the rats within 3 hours. The average of the body surface temperature measured three times every 5 minutes was recorded. The difference between the highest body surface temperature measured at each part within 3 hours and the lowest body surface temperature at the corresponding part was used as the evaluation value of the body surface temperature fluctuation range. The ratio of the highest value measured by TST divided by the highest value measured by the trunk, i.e., BST, at this time, was used as the evaluation index of the heat dissipation sensitivity of the rats.

(2) Measurement of rat core body temperature (CBT): The 16 rats with implants were evenly divided into SHAM, OVX, E2-H, and GNS-H groups (n = 4 in each group). The rat cages were placed on the signal receiver 2 weeks and 10 weeks after ovariectomy (one day before sampling). The 24-hour data (from 8:00 am to 8:00 am the next day) were collected continuously using the Dataquest ART receiving software, and the 24-hour average value and temperature fluctuation were analyzed.

(3) Heat dissipation induction experiment in rats: Rats were fixed in a fixator at a constant temperature of 20°C and injected with 10 μg/kg calcitonin gene-related peptide dissolved in physiological saline via the tail vein. The rats were then placed in a 20*20*40 cm body temperature test box. After the rats were calm for 5 minutes, the tail temperature of the rats was measured with an infrared thermometer. The average value of the tail temperature was recorded three times every 3 minutes for 2 hours.

Source

After 8 weeks of drug treatment, the rats with successful modeling were selected and anesthetized by intraperitoneal injection of 1% sodium pentobarbital (80 mg/kg) and fixed in supine position. (1) Blood specimens: The rat's thorax was opened at the xiphoid process of the sternum to expose the heart. A blood collection needle was connected to a negative pressure blood collection tube to collect 2 ml of blood from the apex of the heart. The blood was placed in a 37°C incubator for 30 minutes and then centrifuged at 3600 rpm for 15 minutes. After centrifugation, the upper serum was collected in an EP tube for later use. (2) Weighing of the uterus: The abdominal cavity was opened to find the uterus. The upper part was taken from the stumps of the uterine horns on both sides and the lower part was stopped at the intersection of the two uterine cavities. The uterus was removed and the surrounding tissues of the uterus were removed. The peritoneal fluid was absorbed with filter paper and weighed. (3) Fixed tissue sampling: 18 rats (n = 3 in each group) were selected after blood was drawn. The chest cavity was opened to expose the heart, the right atrium was cut open, a catheter was inserted from the left ventricle to the ascending aorta, and the heart was perfused with normal saline (0.9%, 4°C) and paraformaldehyde (4%, 4°C). After perfusion, the brain and uterus were immediately removed by craniotomy and fixed in 4% paraformaldehyde containing 0.1 mol/L PB for later use.

Statistical methods

GraphPad Prism 9.0 software was used for drawing, and SPSS25 software was used for statistical analysis. The measured data were expressed as mean ± standard error. For comparison between two groups, if the distribution was normal, the independent sample t test was selected, and if the distribution was non-normal, a non-parametric test was used. For comparison between multiple groups, if the distribution was normal and the variance was equal, one-way ANOVA was selected, otherwise Welch's correction value was used and Dunnett's T3 test was performed. P<0.05 was considered statistically significant. RT-PCR data were analyzed by independent sample t test for relative quantification.

result

General

During the experiment, the rats in the SHAM group were in general good condition, with shiny fur and good mental state, and no obvious abnormalities in food and water intake. The rats in the OVX group, E2-L group, E2-H group, GNS-L group, and GNS-H group were in general good condition, with slightly rough fur, and no obvious abnormalities in food and water intake. However, compared with the rats in the SHAM group, the rats had increased food intake and slightly decreased activity. All groups had no obvious trauma or fighting behavior.

Verify that OVX modeling is successful

Two weeks after OVX, Wright-Giemsa staining showed that in the SHAM group, vaginal detached cell smears indicated the presence of an estrous cycle, including proestrus (mainly nucleated epithelial cells, Figure 2 (A)), estrus (mainly cornified anucleated epithelial cells, Figure 2 (B)), metestrus (nucleated epithelial cells, cornified anucleated epithelial cells and leukocytes in similar proportions, Figure 2 (C)), and diestrus (mainly leukocytes, Figure 2 (D)). In all groups after OVX, the detached cells in the vaginal smears were always leukocytes and were in diestrus (Figure 2 (D)). In addition, as shown in Figure 2 (E), two weeks after ovariectomy, before drug intervention, the serum estradiol concentrations of rats in the OVX, E2-L, E2-H, GNS-L, and GNS-H groups were significantly lower than those in the SHAM group (P < 0.001), while there was no significant difference in serum E2 concentrations among the rats in the groups undergoing OVX before intervention.

Effects of this drug combination on body surface temperature of OVX rats

As shown in Figure 3, after 8 weeks of drug intervention, the surface temperature of the rats' neck, back, and tail was dynamically monitored for 3 consecutive hours using an infrared thermal imager to observe the changes in the surface temperature of various parts of the rats. Abnormal heat dissipation was observed in the tail of the rats in the OVX group and the E2-L group, which was manifested as an abnormal increase in the tail temperature when the trunk temperature was low.

As shown in Figure 4 (A)-(D), the fluctuation ranges of NST, BST and TST of rats in each group were compared. The results showed that after 8 weeks of intervention, the fluctuation range of NST in OVX group was significantly higher than that in other groups, and there was no statistical difference in the fluctuation range of NST among SHAM, E2-H and GNS-H groups (P>0.05); while there was no statistical difference in the fluctuation range of BST and TST among the groups (P>0.05). By calculating the ratio of the highest TST value of rats in each group during the observation period to the BST at this time, the sensitivity of rats to heat dissipation in the tail was reflected. It was found that the OVX group was significantly higher in heat dissipation sensitivity than the SHAM (P<0.001), E2-H (P<0.001), GNS-L (P=0.03), GNS-H (P<0.01) groups, but there was no significant statistical difference with the E2-L group (P>0.05). These results suggest that GNS intervention can significantly improve abnormal body temperature fluctuations in rats after OVX, increase the body temperature threshold for heat dissipation, and restore the rats' ability to regulate body temperature. The effect is better than that of the E2-L group, and the therapeutic effect is dose-dependent.

Effects of this drug combination on core body temperature fluctuations in OVX rats

As shown in Figure 5(A)-(C), the core temperature fluctuations of rats were monitored and recorded by telemetry core temperature implants. It was found that there was no statistically significant difference in the mean values of circadian CBT of rats in each group (P>0.05), but the rhythmic fluctuations of circadian temperature in the OVX group tended to disappear, mainly reflected in the reduction of the sharp drop in CBT caused by daytime sleep and the shortened duration. By calculating the standard deviation of rats' body temperature throughout the day to reflect the discrete degree and fluctuation of rats' CBT, it was found that the fluctuation amplitude of CBT in OVX rats was significantly lower than that in the SHAM group (P<0.01). After 8 weeks of E2 and GNS intervention, the fluctuation amplitude of CBT showed a recovery trend, and the difference was statistically significant (P<0.05).

Effects of this drug combination on heat dissipation in OVX rats

As shown in Figure 6 (A)-(B), after the tail vein injection of CGRP (10 μg/kg), the tail surface temperature of rats in each group gradually increased and reached the maximum value after 40-50 minutes. Then, the tail temperature of rats in the OVX group maintained a high value for about 50 minutes and then slowly decreased. The other groups showed different degrees of body temperature decrease after reaching the highest value. The degree of change in rat tail temperature was evaluated by calculating the area under the curve, and it was found that OVX significantly increased the CGRP-induced increase in tail skin temperature (P=0.04). Both E2 and GNS high-dose interventions can significantly inhibit the CGRP-induced increase in tail skin temperature (P=0.02), but the E2 and GNS low-dose groups have no significant inhibitory effect on this (P>0.05). It shows that E2 and GNS intervention can regulate the peripheral vasodilation activity of rats, thereby effectively improving the tail heat dissipation phenomenon caused by CGRP, and the therapeutic effect is dose-dependent.

(III) Effects of this drug combination on sex hormone levels in OVX rats and its reproductive system safety

Experimental Materials and Methods

Experimental animals

Same as Experimental Example (II).

Experimental drugs

Same as Experimental Example (II).

Main experimental reagents

Sucrose, PB buffer powder, cationic anti-slip slides, OCT frozen section embedding medium, hematoxylin staining solution, eosin staining solution, anhydrous ethanol (64-17-5), xylene (1330-20-7), neutral gum mounting medium (FP-0001), and coverslip.

Main experimental instruments and consumables

High-speed refrigerated centrifuge, fully automatic γ-radioimmunoassay counter, 1/100,000 electronic balance, frozen slicer, upright optical microscope, electric oven.

Experimental animal model

Same as Experimental Example (II).

Experimental animal grouping, drug configuration, and drug intervention

Same as Experimental Example (II).

Changes in uterine shape

After the rats were perfused and the samples were collected, the abdominal cavity was opened to check whether the ovaries were completely removed and whether there was any abdominal reimplantation. The uterus was then severed about 1 cm below the junction of the two uteri. The "Y"-shaped uterus was completely removed and the surrounding tissues were peeled off. The uterus was cleaned in saline, wiped dry and placed on a clean tissue photography backing plate to photograph the morphology of the uterus in each group.

Preparation of frozen sections

(1) Tissue dehydration

The perfusion-fixed uterine tissue in the first section was removed from the paraformaldehyde fixative and placed in a 10% sucrose solution at 4°C for 24 hours. After sinking to the bottom, the solution was replaced with 20% sucrose for 24 hours and finally placed in a 30% sucrose solution for 7 days, during which fresh sucrose was replaced every two days.

(2) Tissue embedding

Use filter paper to absorb the moisture on the surface of the dehydrated uterine tissue, cut out a 1 cm long uterine tissue block above the uterine horn and put it into an embedding box of appropriate size filled with OCT, put the embedding box into liquid nitrogen, quickly freeze the tissue block, and then place it in a -80℃ refrigerator for slicing.

(3) Frozen sections

The uterine tissue stored in the -80℃ refrigerator was taken out and transferred to a -20℃ freezing microtome to rewarm for 1 hour. The uterine tissue was cut into 20μm thick slices and spread on a cationic adsorbed slide. All slices were placed on a clean bench at room temperature for about 2 hours to dry naturally, then put into a slice box and placed in a -20℃ refrigerator.

(4) Baked slices

Before subsequent staining, the slides were removed and placed in an oven at 60°C for 50 min to prevent the tissue from falling off.

Hematoxylin and eosin (HE) staining

(1) Place the slide in distilled water for 4 minutes, then in hematoxylin staining for 8 minutes, wash with water for 3 minutes, then place in hydrochloric acid ethanol differentiation solution for 10 seconds, place in ammonia blueing solution for 10 seconds, wash with water for 30 seconds, then place in eosin staining solution for 3 minutes, wash with water for 2 minutes, and observe under an optical microscope.

(2) Place the slides in 80%, 95% ethanol I, and 95% ethanol II for 1 minute each, 100% ethanol I and 100% ethanol II for 3 minutes each, and xylene I and xylene II for 3 minutes each. Seal the slides with neutral gum, observe under a light microscope, and store them in a slice box.

Statistical methods

GraphPad Prism 9.0 software was used for drawing, SPSS25 software was used for statistical analysis, and the measured data were expressed as mean ± standard error. For comparison among multiple groups, if the distribution was normal and the variance was equal, one-way ANOVA was selected, otherwise Welch's correction value was used and Dunnett's T3 test was performed. P<0.05 was considered statistically significant.

result

Effects of GNS on uterine morphology and uterine wet weight in OVX rats

As shown in Figure 7, the uterus of the SHAM group was full and bilateral ovaries were visible, while the uterus of the OVX group was significantly thinner and linear, and the uterine horns were thinner and narrower. There was no obvious difference in the uterine morphology between the E2-L group, the GNS-L group, the GNS-H group and the OVX group, but the uterus of the E2-H group was significantly thicker than that of the OVX group, but it had not yet fully recovered to the level of the SHAM group.

In order to explain the effect of rat body weight on uterine wet weight, the uterine wet weight coefficient (uterine wet weight coefficient = uterine wet weight/rat body weight) was used to compare the changes in uterine wet weight between groups. The results are shown in Figure 8. After OVX, the rat uterine wet weight coefficient was significantly lower than that of the SHAM group (P<0.001). Supplementation with high-dose E2 could restore the rat uterine wet weight coefficient to a certain extent (P<0.001), but it had not yet recovered to the level of the SHAM group. There was no statistical difference in the uterine wet weight coefficients of the E2-L group, GNS-L group, and GNS-H group compared with the OVX group (P>0.05).

Effects of this drug combination on uterine morphology in OVX rats

As shown in Figure 9, in terms of histopathology, HE staining showed that the epithelial layer of the uterine cavity of rats in the SHAM group was thicker, mostly pseudostratified columnar epithelium and pseudostratified ciliated columnar epithelium. The superficial layer of the stroma was dense, and the stroma cells were larger. In contrast, the endometrial glands of rats in the OVX group were sparse, and the glandular epithelial cells were short columnar. After supplementing with different doses of E2, the epithelial layer of the uterine cavity showed varying degrees of thickening, mostly pseudostratified columnar epithelium and pseudostratified ciliated columnar epithelium, among which the thickening in the E2-H group was more obvious, and there were many glands of varying sizes. The uterine morphological characteristics of the GNS-L and GNS-H groups were similar to those of the OVX group.

Effect of this drug combination on endometrial thickness in OVX rats

As shown in Figure 10, the endometrial thickness of rats in each group was statistically measured and found that the endometrial thickness of the OVX group was significantly lower than that of the SHAM group (P<0.001), while the endometrial thickness of rats in the E2-L (P<0.01) and E2-H groups (P<0.001) groups increased to varying degrees, and the difference was statistically significant. After supplementing with GNS, no matter high or low doses, the endometrial thickness did not increase, indicating that GNS has better reproductive system safety than E2.

2. Analysis and evaluation based on clinical outpatient observation research results

Based on clinical outpatient observations, the clinical research scale involved in this experimental example includes the following parameters.

1. The modified Kupperman Index (KI) is a menopausal symptom evaluation scale based on the Kupperman scoring questionnaire developed in 1952. It includes the following 13 items: vasomotor contraction, paresthesia, insomnia, nervousness, depression, dizziness, fatigue, joint and muscle pain, headache, palpitations, paresthesia, sexual intercourse pain and urinary system symptoms. Each symptom is divided into 4 levels according to the Likert scale: no symptoms (0 points), mild symptoms (1 point), moderate symptoms (2 points) and severe symptoms (3 points). The modified vasomotor items are set to four weight coefficients, and the paresthesia, insomnia and nervousness items are weighed with two weight coefficients, and the remaining items are weighed with one weight coefficient. The total score can be divided into 4 groups: none (0≤score≤6 points), mild (7≤score≤15 points), moderate (16≤score≤30 points) and severe (≥31 points).

2. Pittsburgh sleep quality index (PSQI) is a sleep quality assessment scale compiled by Dr. Buysse, a psychiatrist at the University of Pittsburgh, in 1989. The scale is suitable for evaluating sleep quality in patients with sleep disorders and mental disorders, and is also suitable for evaluating sleep quality in general people. The scale consists of 9 questions, the first 4 questions are fill-in-the-blank questions, and the last 5 questions are multiple-choice questions, of which the 5th question contains 10 small questions. PSQI is used to assess the sleep quality of the subjects in the past month. Only the 18 self-assessment items commonly used for scoring are used here. The 18 items consist of 7 components, each component is scored on a scale of 0-3, and the cumulative score of each component is the total score of PSQI, with a total score range of 0-27 points. The higher the score, the worse the sleep quality.

3. The TCM syndrome score scale adopts a joint evaluation method between doctors and patients. The main evaluation aspects include: (1) symptoms related to menopausal syndrome Yin Yang Qi and blood imbalance syndrome; (2) tongue image (photographs are required for traceability); (3) pulse; (4) TCM syndrome differentiation. The tongue image, pulse, and TCM syndrome differentiation are filled in by the doctor; the symptoms related to menopausal syndrome Yin Yang Qi and blood imbalance syndrome are asked by the doctor, and the patient self-rates the symptoms according to his or her own feelings. 0 points means no such symptom, and 3 points means the most severe symptom.

At present, the modified prescription of the pharmaceutical composition of the present invention has been used in clinical practice, and an observational two-way cohort study of the prescription is being carried out in Guang'anmen Hospital of China Academy of Chinese Medical Sciences. At present, 18 women with menopausal syndrome who meet the requirements and use the modified prescription of the present invention in outpatient clinics have been preliminarily collected. One course of treatment is 12-14 doses of the medicine, two courses of treatment are 24-28 doses of the medicine, three courses of treatment are 36-42 doses of the medicine, and so on.

Treatment effect standard

Complete remission: symptoms disappear, hormone indicators return to normal, and Kupperman score decreases by more than 80%;

Markedly effective: Symptoms are relieved, hormone indicators are significantly improved and tend to normal critical values, and Kupperman score is reduced by 50%-80%;

Effective: Symptoms improved, hormone indicators slightly improved, Kupperman score reduced by 20%-50%;

Ineffective: No changes in symptoms and hormone indicators, and Kupperman score decreased by less than 20%.

Outpatient observation results

(1) Observation of clinical onset time

Preliminary observation results show that the shortest onset time for the drug of the present invention to improve menopausal symptoms is 9 days (9 doses), the average onset time is 17.23 days, and the fastest time for complete relief of symptoms is 26 days (26 doses).

(2) Clinical efficacy observation

In this experimental example, the complete remission rate, marked effectiveness rate, effectiveness rate, ineffectiveness rate and total effectiveness rate of menopausal symptoms after 2 and 4 weeks of treatment are shown in Table 1 below.

Table 1 Efficacy results of menopausal symptoms treated for 2 and 4 weeks (%)

(3) Improvement in Kupperman score

In this experimental example, the Kupperman scores after 2 weeks and 4 weeks of treatment were significantly improved compared with those before treatment (P < 0.05). The specific results are shown in Table 2-3 below.

Table 2 Improvement of Kupperman score after 2 weeks of treatment

Table 3 Improvement of Kupperman score after 4 weeks of treatment

It can be seen that according to the above evaluation of the improvement of Kupperman score, it was found that after 2 weeks of treatment, the menopausal symptoms based on Kupperman score had a marked efficacy of 16.67%, an effective rate of 41.67%, and a total effective rate of 58.34%, with significant differences before and after treatment (P < 0.05). After 4 weeks of treatment, the menopausal symptoms based on Kupperman score had a cure rate of 6.67%, a marked efficacy of 33.33%, an effective rate of 46.67%, and a total effective rate of 86.67%, with significant differences before and after treatment (P < 0.05).

(4) Improvement in the average daily frequency and severity of hot flashes and sweating

In this experimental case, the disappearance rate (frequency reduced to 0) of hot flashes and sweating after 4 weeks of treatment with KI was 21.42%, and the average daily frequency and severity of hot flashes and sweating after 2 and 4 weeks of treatment were significantly improved compared with before treatment (P < 0.05). The specific results are shown in Tables 4-5 below.

Table 4 Hot flashes and sweating in KI after 2 weeks of treatment

Table 5 Hot flashes and sweating in KI after 4 weeks of treatment

(5) Improvement in the Pittsburgh Sleep Index

In this experimental case, the patient's Pittsburgh sleep index improved significantly after 2 and 4 weeks of treatment (P < 0.05). The specific results are shown in Tables 6-7 below.

Table 6 Pittsburgh Sleep Index after 2 weeks of treatment

Table 7 Pittsburgh Sleep Index after 4 weeks of treatment

(6) Improvement of TCM syndrome scores

In this experimental case, the TCM syndrome scores of the patients improved significantly after 2 and 4 weeks of treatment (P < 0.05). The specific results are shown in Tables 8-9 below.

Table 8 TCM syndrome scores after 2 weeks of treatment

Table 9 TCM syndrome scores after 4 weeks of treatment

In summary, the present embodiment is based on the follow-up of 18 women who used the modified prescription of the pharmaceutical composition to treat menopausal syndrome in outpatient clinics. It is preliminarily proved that the shortest onset time of the drug of the present invention to improve menopausal symptoms is 9 days (9 doses), the average onset time is 17.23 days, and the fastest time for complete symptom relief is 26 days (26 doses). For the overall Kupperman score improvement, the effective rate of one course of treatment (2 weeks) is 16.67%, the effective rate is 41.67%, and the total effective rate is 58.34%. The Kupperman score has a significant difference before and after treatment (P < 0.05). The symptom relief rate of two courses of treatment (4 weeks) is 6.67%, the effective rate is 33.33%, the effective rate is 46.67%, and the total effective rate is 86.67%. The Kupperman score has a significant difference before and after treatment (P < 0.05). Regarding the improvement of the average number of hot flashes and sweating per day and the severity, the disappearance rate (number of times reduced to 0) of KI hot flashes and sweating after 4 weeks of treatment was 21.42%, and the average number of hot flashes and sweating per day and the severity were significantly improved (P < 0.05). Regarding the improvement of the Pittsburgh Sleep Index, there was a significant difference between the improvement of the Pittsburgh Sleep Index before treatment and after 2 weeks and 4 weeks of treatment (P < 0.05). Regarding the improvement of the TCM syndrome score, the TCM syndrome score was significantly improved after 2 weeks and 4 weeks of treatment (P < 0.05).

The above results prove that the drug of the present invention has a significant effect on the overall menopausal symptoms of women with perimenopausal syndrome, especially on hot flashes, sweating and sleep disorders.

3. Analysis and evaluation of the results of the 12-week interventional self-controlled clinical trial

In this experimental example, according to the clinical experimental project, the clinical research scale involved is the same as that of Experimental Example 2.

(1) Improvement in the average daily frequency of hot flashes and sweating

In this experimental example, based on the medication effects of 35 subjects, after 12 weeks of treatment with the drug of the present invention, the average frequency of hot flashes decreased from 7.47 times/day at baseline to 2.61 times/day, and the average percentage change of hot flashes was -65%. The specific results are shown in Table 10 below.

Table 1035 Daily Hot Flash Frequency Changes in Subjects (Mean±SD)

time Number of hot flashes per day GNS (N=36) Baseline Mean(SD) 7.47(3.06) Week 4 Mean(SD) 5.06(2.59) Change from baseline LsMean(Std) -2.280(0.250) 95% CI -2.778,-1.782 Week 8 Mean(SD) 4.11(3.06) Change from baseline LsMean(Std) -3.219(0.371) 95% CI -3.959,-2.479 Week 12 Mean(SD) 2.61(2.31) Change from baseline LsMean(Std) -4.489(0.344) 95% CI -5.175,-3.804 t(P) -10.65(<.0001)

(2) Improvement in the severity of hot flashes and sweating

In this experimental example, based on the medication effects of 36 subjects, the evaluation results of GNS in reducing the hot flash severity score showed that after 12 weeks of intervention, the hot flash severity score after 12 weeks of using this drug combination decreased from an average of 2 points at baseline to 1.23 points, a decrease of 0.77 points. There was a significant difference between the before and after comparisons. The specific results are shown in Table 11 below.

Table 1 Changes in severity of hot flashes among 1136 subjects (Mean±SD)

(3) Improvement in Hot Flash-Related Daily Interference Scale (HFRDIS) score

In this experimental case, based on the medication effects of 36 subjects, there was a significant difference in the HFRDIS score between the patients before and after treatment, compared with the baseline 45.19 points). At the 12th week of intervention, the HFRDIS score dropped significantly to 18.81 points, which was statistically significant (P<0.0001). The specific results are shown in Table 12 below.

Table 1 HFRDIS scale scores of 236 subjects (Mean±SD)

time HFRDIS score GNS (N=36) Baseline Mean(SD) 45.19(25.04) Week 4 Mean(SD) 25.31(18.10) 95% CI 19.18,31.43 Week 8 Mean(SD) 22.36(19.49) 95% CI 15.77,28.96 Week 12 Mean(SD) 18.81(16.23) 95% CI 0.93,1.52 t(P) -6.70(<.0001)

(4) Pittsburgh Sleep Quality Inventory (PSQI) score

In this experimental example, based on the medication effects of 36 subjects, the results of PSQI scores are shown in Table 13. The data of the 36 subjects before and after the intervention showed significant differences. After 12 weeks of intervention, the PSQI scale score was 6.86 points, which was significantly different from the baseline score of 11.03 points (P<0.0001).

Table 1: PSQI scale scores of 336 subjects (Mean±SD)

(5) Patient Health Questionnaire-9 (PHQ-9) scale score

In this experimental example, based on the medication effects of 36 subjects, the results of the PHQ-9 scale score are shown in Table 14. The data of the 36 subjects before and after the intervention showed significant differences. After 12 weeks of intervention, the average score of the PHQ-9 scale was 4.00 points, which was significantly different from the baseline PHQ-9 average score of 8.14 points (P<0.0001).

Table 1 PHQ-9 scale scores of 436 subjects (Mean±SD)

time PHQ-9 score GNS (N=36) Baseline Mean(SD) 8.14(4.79) Week 4 Mean(SD) 6.28(4.67) 95% CI 4.70,7.86 Week 8 Mean(SD) 5.06(4.06) 95% CI 3.68,6.43 Week 12 Mean(SD) 4.00(2.92) 95% CI 3.01,4.99 t(P) -5.85(<.0001)

(6) Modified Kupperman Index (KMI)

In this experimental example, based on the medication effects of 36 subjects, the results of KMI score are shown in Table 15. The data of the 36 subjects before and after the intervention showed significant differences. After 12 weeks of intervention, the KMI score was 17.37 points, which was significantly different from the baseline PHQ-9 average score of 26.42 points (P<0.0001).

Table 1 KMI scores of 536 subjects (Mean±SD)

time KMI Rating GNS (N=36) Statistics (Z-score) P-value Baseline Mean(SD) 26.14(8.31) -0.16 0.874 Week 4 Mean(SD) 20.14(7.13) -0.58 0.562 95% CI 17.73,22.55 Week 8 Mean(SD) 19.03(6.96) -0.94 0.351 95% CI 16.67,21.38 Week 12 Mean(SD) 15.33(7.18) -1.16 0.250 95% CI 12.90,17.76 t(P) -7.08(<.0001)

(7) Generalized Anxiety Disorder Scale (GAD-7) score

In terms of GAD-7 scale score results, the results are shown in Table 16. The data of 36 subjects showed significant differences before and after the intervention. After 12 weeks of intervention, the average score of the KMI scale was 3.03 points, which was significantly different from the baseline PHQ-9 average score of 6.67 points (P<0.0001).

Table 1 GAD-7 scale scores of 636 subjects (Mean±SD)

time GAD-7 score GNS (N=36) Baseline Mean(SD) 6.67(4.21) Week 4 Mean(SD) 4.25(3.71) 95% CI 5.24,8.09 Week 8 Mean(SD) 3.78(3.92) 95% CI 2.45,5.10 Week 12 Mean(SD) 3.03(2.68) 95% CI 2.12,3.93 t(P) -7.08(<.0001)

(8) TCM syndrome (yin-yang qi and blood imbalance syndrome) scoring scale

In terms of the TCM syndrome score scale, the results are shown in Table 17 below. The data of the 36 subjects showed significant differences before and after the intervention. After 12 weeks of intervention, the average score of the TCM syndrome score was 12.83 points, which was significantly different from the baseline TCM syndrome score of 7.14 points (P<0.0001).

Table 17 TCM syndrome score scale scores of the two groups of subjects (Mean±SD)

time TCM Syndrome Score GNS (N=36) Statistics (Z-score) P-value Baseline Mean(SD) 12.83(3.87) -0.35 0.725 Week 4 Mean(SD) 10.11(4.19) -0.16 0.871 95% CI 8.69,11.53 Week 8 Mean(SD) 8.53(3.65) -1.36 0.177 95% CI 7.29,9.76 Week 12 Mean(SD) 7.14(3.30) -1.18 0.244 95% CI 6.02,8.25 t(P) -7.18(<.0001)

5. Safety Evaluation

In the clinical trial of the present invention, only one subject had an adverse event during the entire follow-up process, which manifested as breast pain, and was determined to be unrelated to the test drug. In addition, there were no significant differences in the clinical blood routine, urine routine, urine NAG enzyme, liver and kidney function index abnormalities and other vital signs of all patients before and after treatment.

In summary, the pharmaceutical composition of the present invention can effectively reduce the average daily number of hot flashes and sweating, improve the severity of hot flashes and sweating, and improve the potential effects of insomnia, depression, anxiety, TCM syndrome score (yin-yang, qi and blood imbalance syndrome) and other aspects after intervening in patients with perimenopausal syndrome, and has good safety.

Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the protection scope of the invention.

Claims (10)

1. A pharmaceutical composition for treating perimenopausal syndrome, characterized in that its raw materials are composed of: 5-30 weight parts of Anemarrhena asphodeloides, 5-30 weight parts of Phellodendron chinense, 5-30 weight parts of Radix Rehmanniae, 5-30 weight parts of Astragalus membranaceus, 5-30 weight parts of Angelica sinensis, 5-30 weight parts of Epimedium, 10-60 weight parts of calcined Dragon Bone, 10-60 weight parts of calcined Ostreae Conchae, and 10-30 weight parts of stir-fried Semen Ziziphi Spinosae. 2. The pharmaceutical composition for treating perimenopausal syndrome according to claim 1, characterized in that its raw materials are composed of: 8 parts by weight of Anemarrhena Rhizoma, 8 parts by weight of Phellodendron chinense, 14 parts by weight of Radix Rehmanniae, 14 parts by weight of Radix Astragali, 8 parts by weight of Angelica Sinensis, 8 parts by weight of Epimedium, 14 parts by weight of calcined Ossobucon, 14 parts by weight of calcined Concha Ostreae, and 14 parts by weight of stir-fried Semen Ziziphi Spinosae. 3. Use of the pharmaceutical composition according to claim 1 or 2 for preparing a medicament for treating perimenopausal syndrome. 4. The use according to claim 3, characterized in that the perimenopausal syndrome includes at least one of the following symptoms (1)-(4): (1) Perimenopausal hot flashes and sweating; (2) perimenopausal sleep disorders; (3) perimenopausal depression; (4) Perimenopausal anxiety. 5. A pharmaceutical preparation for treating perimenopausal syndrome, characterized in that it comprises the pharmaceutical composition according to claim 1 or 2. 6. The pharmaceutical preparation for treating perimenopausal syndrome according to claim 5, characterized in that the pharmaceutical preparation comprises at least one of tablets, capsules, powders, mixtures, pills, granules, solutions, syrups, decoctions, patches, suppositories, aerosols, ointments or injections. 7. The pharmaceutical preparation for treating perimenopausal syndrome according to claim 5 or 6, characterized in that the pharmaceutical preparation further comprises a clinically acceptable excipient; Preferably, the auxiliary material includes at least one of a filler, a disintegrant, a lubricant, a suspending agent, a binder, a sweetener, a flavoring agent, a preservative or a matrix; Preferably, the filler comprises at least one of starch, pregelatinized starch, lactose, mannitol, chitin or microcrystalline cellulose; Preferably, the disintegrant comprises at least one of starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose or cross-linked sodium carboxymethyl cellulose; Preferably, the lubricant comprises at least one of magnesium stearate, sodium lauryl sulfate, talc or silicon dioxide; Preferably, the suspending agent comprises at least one of polyvinyl pyrrolidone, microcrystalline cellulose, agar or hydroxypropyl methylcellulose; Preferably, the adhesive comprises at least one of starch slurry, polyvinyl pyrrolidone or hydroxypropyl methylcellulose; Preferably, the sweetener comprises at least one of saccharin sodium, aspartame, sucrose, sodium cyclamate or glycyrrhetinic acid; Preferably, the flavoring agent includes essence; Preferably, the preservative includes at least one of parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chloroethidine acetate or eucalyptus oil; Preferably, the matrix comprises at least one of PEG6000, PEG4000 or insect wax. 8. A method for preparing the pharmaceutical preparation for treating perimenopausal syndrome according to any one of claims 5 to 7, characterized in that it comprises the step of mixing selected amounts of the Rhizoma Anemarrhenae, Rhizoma Rehmanniae, Radix Astragali, Radix Angelicae Sinensis, Herba Epimedii, Calcinated Ossobuxi, Calcinated Concha Ostreae and Fried Semen Ziziphi Spinosae. 9. The method for preparing the pharmaceutical preparation for treating perimenopausal syndrome according to claim 8, characterized in that it comprises: taking selected amounts of the Rhizoma Anemarrhenae, Cortex Phellodendri, Radix Rehmanniae, Radix Astragali, Radix Angelicae Sinensis, Herba Epimedii, Calcinated Ossobuxi, Calcinated Oyster and Stir-fried Semen Ziziphi Spinosae, mixing them, adding water to boil them, collecting the decoction and concentrating it into an extract, and adding conventional auxiliary materials to process the selected dosage form; Preferably, the amount of water added is 5-15 times by weight of the total amount of the API; and/or, Preferably, the decocting step is decocting 1-3 times; and/or, Preferably, the decocting step lasts for 1-3 hours; and/or, Preferably, the concentration step is to concentrate the decoction to a thick paste having a relative density of 1.25 at 60°C. 10. Use of the pharmaceutical composition according to claim 1 or 2 for preparing a drug having at least one of the following effects (1) to (6): (1) Improve perimenopausal hot flashes and sweating; (2) Improve perimenopausal sleep disorders; (3) Improve perimenopausal anxiety; (4) Improve perimenopausal depression; (5) Improve overall quality of life during perimenopause; (6) Promote the production of endogenous estrogen.