Traditional Chinese medicine composition as well as preparation method and application thereof

Abstract

The invention relates to the technical field of traditional Chinese medicine compositions, in particular to a traditional Chinese medicine composition and a preparation method and application thereof. Specifically, the traditional Chinese medicine composition is prepared from radix salviae miltiorrhizae, rhizoma coptidis, rhizoma anemarrhenae, radix ophiopogonis, nacre mother of pearl and semen ziziphi spinosae. The traditional Chinese medicine composition provided by the invention is simple in formula, does not contain precious, thin, toxic and endangered medicinal materials, and in a naturally aged female climacteric mouse model, the traditional Chinese medicine composition formula can obviously increase the level of E2 in plasma, lower the levels of FSH and LH and improve the pathological characteristics of uterus tissues. Test results show that the traditional Chinese medicine composition provided by the invention has a certain treatment effect on patients with perimenopausal syndromes.

Description

[0001] This application claims the priority of the Chinese patent application filed with the China Patent Office on December 13, 2023, with application number 202311719738.9 and invention name “A Chinese medicine composition, its preparation method and application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to the technical field of Chinese medicine compositions, and in particular to a Chinese medicine composition and a preparation method and application thereof. Background Art

[0003] Perimenopausal syndrome (MPS), also known as menopausal syndrome, is a condition in which women experience a decrease in estrogen (such as E2) levels in their bodies due to the disappearance or decline of ovarian function before and after menopause, which in turn stimulates the pituitary gland to release FSH and LH into the blood, causing imbalance in the hypothalamus-pituitary-gonadal axis and symptoms of neurological and endocrine disorders. Clinical manifestations include menstrual disorders, hot flashes, sweating, insomnia, fatigue, irritability, and changes in mental and psychological states. This disease is relatively common in gynecology, mostly occurring in women aged 45 to 55, 3 to 5 years before and after menopause, and can last for more than 10 years in severe cases.

[0004] There is no record of the disease name "perimenopausal syndrome" in ancient Chinese medical books, but looking at its clinical manifestations and the law of occurrence and development, it should belong to the categories of "visceral agitation", "depression", "lily disease", "pre-menopausal syndrome" and so on in Chinese medicine. Chinese medicine believes that the occurrence of this disease is due to the gradual weakening of kidney essence, exhaustion of Tiangui, Chongren deficiency or insufficient Guishui, imbalance of yin and yang, liver and kidney yin deficiency, hyperactivity of liver yang, disorder of qi and blood in viscera, and the occurrence of a variety of symptoms.

[0005] At present, Western medicine mainly uses hormone replacement therapy for perimenopausal syndrome. Although it has a positive effect, it has many contraindications, adverse reactions and side effects, which limit its application to a certain extent. Among the many Chinese patent medicines for perimenopausal syndrome in China, most contain toxic or cold medicines, which can easily cause indigestion or liver and kidney toxicity reactions and are not suitable for long-term use. Therefore, it is of great application value to develop innovative Chinese medicines with simple prescription composition and without precious, toxic and endangered medicinal materials. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a Chinese medicine composition and a preparation method and application thereof, which can be used to prevent, treat or alleviate perimenopausal syndrome.

[0007] To achieve the above object, the present invention provides a traditional Chinese medicine composition, comprising: salvia miltiorrhiza, coptis chinensis, anemarrhena asphodeloides, ophiopogon japonicus, pearl mother-of-pearl and spiny jujube seed.

[0008] As a preferred embodiment, by weight parts, the above traditional Chinese medicine composition comprises:

[0009] 10 - 30 parts of Salvia miltiorrhiza, 2 - 10 parts of Coptis chinensis, 2 - 10 parts of Anemarrhena asphodeloides, 5 - 20 parts of Ophiopogon japonicus, 10 - 50 parts of Concha Margaritifera Usta, 2 - 10 parts of Semen Ziziphi Spinosae.

[0010] As a preferred embodiment, by weight parts, the above traditional Chinese medicine composition comprises:

[0011] 15 parts of Salvia miltiorrhiza, 5 parts of Coptis chinensis, 5 parts of Anemarrhena asphodeloides, 10 parts of Ophiopogon japonicus, 25 parts of Concha Margaritifera Usta, 5 parts of Semen Ziziphi Spinosae.

[0012] The traditional Chinese medicine composition provided by the present invention is prepared by the method of nourishing yin and blood, clearing heat and tranquilizing mind. In the formula, the monarch drug Salvia miltiorrhiza is bitter in taste and slightly cold in nature, and belongs to the heart and liver meridians. Its functions are promoting blood circulation to remove stasis, clearing the heart and relieving vexation, taking into account nourishing blood and promoting blood circulation, which hits the basic pathogenesis of perimenopausal syndrome as deficiency of essence and blood. Coptis chinensis and Ophiopogon japonicus are used as ministerial drugs together. Coptis chinensis is bitter in taste and cold in nature, and belongs to the heart, spleen, stomach, liver, gallbladder and large intestine meridians. Its functions are clearing heat and drying dampness, purging fire and detoxifying. Ophiopogon japonicus is sweet in taste, slightly bitter and slightly cold in nature, and belongs to the heart, lung and stomach meridians. Its functions are nourishing yin, promoting the production of body fluid, clearing heat and relieving vexation. Anemarrhena asphodeloides and Semen Ziziphi Spinosae (fried) are used as assistant drugs together. Anemarrhena asphodeloides is bitter and cold in nature, and belongs to the lung, stomach and kidney meridians. Its functions are clearing heat and purging fire, nourishing yin and moistening dryness. Semen Ziziphi Spinosae (fried) is sweet, sour in taste and neutral in nature, and belongs to the liver, gallbladder and heart meridians. It nourishes the heart and tonifies the liver, soothes the nerves, arrests sweating and promotes the production of body fluid, and is used for restlessness and insomnia, palpitation and dreaminess, excessive sweating due to weakness of the body, and thirst due to consumption of body fluid. The envoy drug Concha Margaritifera Usta is salty and cold in nature, and belongs to the liver and heart meridians. Its efficacy is suppressing the hyperactive liver and subsiding yang, calming the nerves and relieving fright.

[0013] Through the scientific ratio among the raw materials, the components of the present invention can coordinate and promote each other, playing a role of synergistic enhancement, and having good effects of nourishing yin and blood, clearing heat and tranquilizing mind.

[0014] On the other hand, the present invention provides a preparation method of the above traditional Chinese medicine composition, comprising the following steps:

[0015] Take each traditional Chinese medicine raw material according to weight parts, extract with an extraction solvent, and the obtained extract is concentrated and dried under reduced pressure to obtain a traditional Chinese medicine extract.

[0016] As a preferred embodiment, the above extraction solvent is selected from water, aqueous ethanol or ethanol. More preferably, it is water.

[0017] As a preferred embodiment, the addition amount of the above extraction solvent is 8 - 14 times that of the traditional Chinese medicine raw materials, more preferably 12 times that of the traditional Chinese medicine raw materials; the number of extraction times is preferably 1 - 3 times, more preferably 2 times; the extraction time for each time is preferably 0.5 - 2 hours, more preferably 1 hour.

[0018] As a preferred embodiment, the above extraction method is decoction method, reflux extraction method or ultrasonic extraction method. More preferably, it is reflux extraction method.

[0019] As a preferred embodiment, the above extraction is specifically as follows:

[0020] Take nacre, reflux extract with 8 - 14 times water for 1 - 3 times, each time for 0.5 - 3 hours, then add salvia miltiorrhiza, coptis chinensis, anemarrhena asphodeloides, ophiopogon japonicus, and ziziphus jujuba var. spinosa for reflux extraction, each time for 0.5 - 3 hours, filter, add 8 - 14 times water to the medicinal residues again for reflux extraction, each time for 0.5 - 3 hours, combine the filtrates, concentrate and dry under reduced pressure to obtain the traditional Chinese medicine extract.

[0021] The above traditional Chinese medicine composition can be prepared into a traditional Chinese medicine preparation by mixing with a pharmaceutically acceptable carrier.

[0022] Based on this, the present invention provides the application of the above traditional Chinese medicine composition in the preparation of a drug for preventing, treating or alleviating perimenopausal syndrome.

[0023] The present invention also provides a drug for preventing, treating or alleviating perimenopausal syndrome, comprising the above traditional Chinese medicine composition and a pharmaceutically acceptable adjuvant.

[0024] As a preferred embodiment, the dosage form of the drug is oral liquid, granule, capsule, tablet, pill, dripping pill or sustained release preparation, etc.

[0025] The present invention has no special limitation on the type of the above adjuvant, and those skilled in the art can adjust it according to the dosage form.

[0026] When the traditional Chinese medicine composition provided by the present invention is made into an oral liquid, the extracts of each raw material are dissolved in distilled water, filtered, and syrup is added to adjust the pH value to make the oral liquid.

[0027] When the traditional Chinese medicine composition provided by the present invention is made into granules, the extracts of each raw material are mixed evenly with lactose and magnesium stearate, granulated, and dried to make granules.

[0028] When the traditional Chinese medicine composition provided by the present invention is made into tablets, each raw material is mixed evenly with lactose and magnesium stearate, granulated, and then pressed into tablets.

[0029] Other dosage forms described in the present invention can be prepared by conventional methods in the pharmacopoeia.

[0030] Compared with the prior art, the present invention provides a traditional Chinese medicine composition, comprising: salvia miltiorrhiza, coptis chinensis, anemarrhena asphodeloides, ophiopogon japonicus, nacre and ziziphus jujuba var. spinosa.

[0031] The traditional Chinese medicine composition provided by the present invention has a simple formula, does not contain precious, toxic or endangered medicinal materials. In the natural aging female menopausal mouse model, this traditional Chinese medicine composition formula can significantly increase the level of E2 in plasma, and down - regulate the levels of FSH and LH, and improve the pathological characteristics of uterine tissue. The test results show that the traditional Chinese medicine composition provided by the present invention has a certain therapeutic effect on perimenopausal syndrome patients. Description of the Drawings

[0032] Figure 1 This is the morphological diagram of the uterine tissue of ovariectomized female menopausal mice in Example 1 of the present invention;

[0033] Figure 2 This is the morphological variation diagram of the uterine tissue of each group of naturally aging mice in Example 5 of the present invention. Detailed Embodiments

[0034] In order to further illustrate the present invention, the traditional Chinese medicine composition provided by the present invention, its preparation method and application will be described in detail below in conjunction with embodiments.

[0035] The following parts are all parts by weight.

[0036] Preparation and Screening of Traditional Chinese Medicine Composition Granules in Example 1

[0037] I. Sample Preparation

[0038] 1. Preparation of Samples in Process 1 Group

[0039] Take 25 parts of nacre, reflux extract with 14 times water for 30 minutes, then add 15 parts of salvia miltiorrhiza, 5 parts of coptis chinensis, 5 parts of anemarrhena asphodeloides, 10 parts of ophiopogon japonicus, and 5 parts of ziziphus jujuba var. spinosa, reflux extract for 1 hour, filter, add 12 times water to the medicinal residues and reflux extract for 1 hour again, combine the two filtrates, concentrate and dry under reduced pressure. After obtaining the traditional Chinese medicine extract, mix it evenly with lactose and magnesium stearate, granulate, and dry to make granules.

[0040] 2. Preparation of Samples in Process 2 Group

[0041] Take 25 parts of nacre, reflux extract with 14 times water for 30 minutes, then add 15 parts of salvia miltiorrhiza, 5 parts of coptis chinensis, 5 parts of anemarrhena asphodeloides, 10 parts of ophiopogon japonicus, and 5 parts of ziziphus jujuba var. spinosa, reflux extract for 1 hour, filter, add 12 times water to the medicinal residues and reflux extract for 1 hour again, combine the two filtrates, concentrate under reduced pressure to a relative density of about 1.08 of the extract, add ethanol to make the alcohol content reach 70%, stand for 12 hours, separate and take the supernatant of alcohol precipitation, concentrate and dry under reduced pressure. After obtaining the traditional Chinese medicine extract, mix it evenly with lactose and magnesium stearate, granulate, and dry to make granules.

[0042] II. Effects of Two Process Routes on the Ovariectomized Female Menopausal Mouse Model

[0043] 1. Experimental Materials

[0044] 1.1 Test Drugs: Samples in Process 1 Group, containing 3.25 g of crude drug per gram of granule. Samples in Process 2 Group, containing 5.34 g of crude drug per gram of granule.

[0045] 1.2 Animals: Balb / c mice, female, body weight 20 - 22 g, 80 mice. Purchased from Shanghai Slake Laboratory Animal Co., Ltd. (Animal License No.: SCXK(Shanghai)2017 - 0012; Certificate No.: 20170012012742). After being approved by the laboratory animal ethics (Ethics No.: PZSHUTCM210528007), they were raised in a SPF - level environment.

[0046] 1.3 Reagents: Zoletil 50 (Virbac, France, MFGD201911), Ethylenediaminetetraacetic acid (Sangon Biotech, 2906B858), Follicle - stimulating hormone (Hengyuan Biotech, HB967 - Mu), Luteinizing hormone (Hengyuan Biotech, HB970 - Mu), Estradiol (Hengyuan Biotech, HB975 - Mu), Progesterone (Hengyuan Biotech, HB494 - Mu), 10% Neutral formaldehyde solution (Sinopharm Chemical Reagent Co., Ltd., 20180429), Hematoxylin (Zhuhai Baiso Biotechnology Co., Ltd., BA4041), Eosin (Zhuhai Baiso Biotechnology Co., Ltd., BA4099).

[0047] 2. Dose Design

[0048] The daily dose of the samples prepared by the two processes was calculated based on 65 g of crude drug equivalent per oral liquid. Assuming the mouse body weight was 20 g and the equivalent dose ratio calculated according to the body surface area conversion between humans and mice was 0.0026, the dose of the sample for every 10 g of mouse body weight was:

[0049] Samples of Process 1 group: 65×30.74%×0.0026÷20×10 = 0.026 g / 10 g·day

[0050] Samples of Process 2 group: 65×18.74%×0.0026÷20×10 = 0.016 g / 10 g·day

[0051] 3. Experimental methods:

[0052] SPF-grade Balb / c mice were surgically ovariectomized bilaterally; 10 mice were used as sham-operated controls. Starting from the 3rd day after surgery, vaginal smears were observed in the ovariectomized mice for 5 consecutive days to screen for successfully modeled mice with successful surgery and disrupted estrous cycles. On the 15th day after surgery, the successfully ovariectomized mice were randomly divided into a model group, a drug administration group (process 1), and a drug administration group (process 2), with 9 - 10 mice in each group. After grouping, the general condition of the mice was observed, and body weight was measured daily. Except for the sham-operated control group, the mice in each group were placed in a tube (50 mL centrifuge tube) for 2 hours of immobilization treatment every day for 10 consecutive days. After grouping, intragastric administration was carried out once a day for 8 weeks. On the 53rd day of drug administration, after adapting to the activity apparatus for 5 minutes, the number of activities within 5 minutes was measured. Twenty-four hours after the last drug administration, the experimental mice in each group were sacrificed, and the organ coefficients, plasma hormone levels were detected, and the pathological morphology of the uterine tissue was observed. Measurement data were expressed as mean ± standard deviation (X+S), and all data were statistically analyzed by one-way analysis of variance. A p value less than 0.05 was considered to have statistical significance (*p<0.05, **p<0.01, ***p<0.001 for comparison between the model control group and other groups respectively).

[0053] 4. Experimental results:

[0054] The body weight monitoring of the mice is shown in Table 1. The overall body weight growth trend of the experimental animals in each group was as follows. The body weight of the experimental animals in the normal control group tended to be stable after the 28th day (10 weeks old) of the experiment. The body weights of the other groups showed an upward trend. During the period from the 15th to the 25th day of the experiment, due to the immobilization intervention, the body weights of the experimental animals in each group showed a certain degree of fluctuation, and the activity of individual experimental animals decreased, but there were no other obvious abnormal signs. The process 1 group controlled the body weight of the ovariectomized mice to a certain extent, showing good results.

[0055] Table 1. Results of body weight monitoring of ovariectomized female menopausal mice

[0056]

[0057] The results of the spontaneous activity experiment of the mice are shown in Table 2. Ovariectomy and stress intervention led to a significant increase in the spontaneous activity of the model control mice compared with the normal control mice. After 8 weeks of drug treatment, each drug intervention group could reduce the spontaneous activity of the mice to a certain extent. Among them, the process 1 group had the most obvious effect, showing statistical significance compared with the model control group, and the spontaneous activity frequency was the same as that of the normal control group.

[0058] Table 2. Detection results of spontaneous activity of each group of ovariectomized female menopausal mice

[0059]

[0060] The results of the Less index and fat coefficient in each group of the organ coefficient are shown in Table 3. The process 1 group showed a good reducing effect in the detection of the Less index and fat coefficient, and there were also statistical differences. The process 2 group decreased to a certain extent in both indicators, but there was no statistical difference compared with the model group.

[0061] Table 3 Detection results of the Less index and fat coefficient in each group of ovariectomized female climacteric mice

[0062]

[0063] The results of the plasma hormone level determination are shown in Table 4. The estradiol (E2) level of the mice in the model group was significantly decreased, and the follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels were significantly increased. Both the process 1 group and the process 2 group of the test samples had a tendency to increase the E2 level in the plasma of mice. For FSH and LH, the process 1 group of the test drug could significantly reduce their levels more than the process 2 group. It shows that the sample of the process 1 group has a certain pharmacological effect on regulating the endocrine level during menopause.

[0064] Table 4. Detection results of plasma hormone levels in each group of ovariectomized female climacteric mice

[0065]

[0066]

[0067] The pathological morphological results of the uterine tissues of each group of mice are as shown in the appendix Figure 1 The pathological manifestation of the uterine tissue in the model group was atrophy of the entire endometrium, with a smooth surface, no wavy undulations, a decrease in the number of endometrial glands, and obvious atrophy of the smooth muscle. The process 1 group improved the decline of the endometrium, smooth muscle and glands to a certain extent, and the endometrial surface was slightly smoother.

[0068] Experimental conclusion:

[0069] Based on the above experimental results of the pharmacological effect evaluation, for the climacteric mouse model caused by ovariectomy and stress, the sample of the process 1 showed better pharmacological effects in each index than the sample of the process 2 group, and most of the pharmacological indexes had statistical differences compared with the model group. The sample of the process 1 group has a certain therapeutic effect on patients with perimenopausal syndrome and has great development value.

[0070] Example 2 Preparation of the oral liquid of the traditional Chinese medicine composition

[0071] Take 30 parts of Salvia miltiorrhiza, 10 parts of Coptis chinensis, 10 parts of Anemarrhena asphodeloides, 20 parts of Ophiopogon japonicus, 50 parts of Concha Margaritifera Usta, and 10 parts of Ziziphus jujuba var. spinosa.

[0072] Preparation method:

[0073] (1) First, take 50 parts by mass of nacre and decoct it with 10 times the amount of water for 60 minutes. Then add 30 parts of salvia miltiorrhiza, 10 parts of coptis chinensis, 10 parts of anemarrhena asphodeloides, 20 parts of ophiopogon japonicus, and 10 parts of wild jujube seed, and decoct for 3 times, 2 hours each time. Filter, combine the filtrates, and concentrate and dry under reduced pressure to obtain a traditional Chinese medicine extract.

[0074] (2) Take the traditional Chinese medicine extract prepared in step (1), add distilled water, add syrup, adjust the pH value, and fill into bottles to obtain an oral liquid.

[0075] Preparation of the traditional Chinese medicine composition tablet in Example 3

[0076] Take 10 parts of salvia miltiorrhiza, 2 parts of coptis chinensis, 2 parts of anemarrhena asphodeloides, 5 parts of ophiopogon japonicus, 10 parts of nacre, and 2 parts of wild jujube seed.

[0077] Preparation method:

[0078] (1) First, take 10 parts by mass of nacre and ultrasonically extract it with 8 times the amount of 50% ethanol solution for 20 minutes. Then add 10 parts of salvia miltiorrhiza, 2 parts of coptis chinensis, 2 parts of anemarrhena asphodeloides, 5 parts of ophiopogon japonicus, and 2 parts of wild jujube seed, and ultrasonically extract for 0.5 hour. Filter, and concentrate and dry the filtrate under reduced pressure to obtain a traditional Chinese medicine extract.

[0079] (2) Take the traditional Chinese medicine extract prepared in step (1), mix it evenly with lactose and magnesium stearate, granulate, and then press into tablets.

[0080] Preparation of the traditional Chinese medicine composition granule in Example 4

[0081] Take 15 parts of salvia miltiorrhiza, 5 parts of coptis chinensis, 5 parts of anemarrhena asphodeloides, 10 parts of ophiopogon japonicus, 25 parts of nacre, and 5 parts of wild jujube seed.

[0082] Preparation method:

[0083] (1) First, take 25 parts by mass of nacre and reflux extract it with 14 times the amount of water for 30 minutes. Then add 15 parts of salvia miltiorrhiza, 5 parts of coptis chinensis, 5 parts of anemarrhena asphodeloides, 10 parts of ophiopogon japonicus, and 5 parts of wild jujube seed, and reflux extract for 1 hour. Filter, add 12 times the amount of water to the residue and reflux extract for 1 hour, combine the two filtrates, and concentrate and dry under reduced pressure to obtain a traditional Chinese medicine extract.

[0084] (2) Take the traditional Chinese medicine extract prepared in step (1), mix it evenly with lactose and magnesium stearate, granulate, and dry to obtain granules.

[0085] Effect of the composition of the present invention on the natural aging female climacteric mouse model in Example 5

[0086] 1. Experimental materials

[0087] 1.1 Test drug: The intermediate obtained by preparing the composition of the present invention according to the method of Example 4

[0088] 1.2 Animals: 35 SPF-grade 18-month-old female Balb / c mice. After being approved by the Institutional Animal Care and Use Committee (ethical approval number: PZSHUTCM210528007), they were housed in an SPF-grade environment (animal license number: SCXK(Shanghai)2018-0006; certificate number: 20180006024966).

[0089] 1.3 Reagents: Zoletil 50 (Virbac, France, MFGD201911), Ethylenediaminetetraacetic acid (Sangon Biotech, 2906B858), Follicle-stimulating hormone (Hengyuan Biotech, HB967-Mu), Luteinizing hormone (Hengyuan Biotech, HB970-Mu), Estradiol (Hengyuan Biotech, HB975-Mu), Progesterone (Hengyuan Biotech, HB494-Mu), 10% Neutral formaldehyde solution (Sinopharm Chemical Reagent Co., Ltd., 20180429), Hematoxylin (Zhuhai Baiso Biotechnology Co., Ltd., BA4041), Eosin (Zhuhai Baiso Biotechnology Co., Ltd., BA4099).

[0090] 2. Dose Design

[0091] The sample of the composition of the present invention was calculated based on the crude drug content in the oral liquid equivalent to 65 g per day. Assuming the adult body weight is 60 kg, it is 1.08 g crude drug / kg / d. The equivalent dose for mice was converted to 12 g crude drug / kg / d. During the experiment, mice were divided into three dose groups: 24 g crude drug / kg (high dose), 12 g crude drug / kg (medium dose), and 6.0 g crude drug / kg (low dose) (equivalent to 2 times, equal times, and 1 / 2 times the human clinical dose respectively), and were gavaged at a volume of 10 ml / kg once a day.

[0092] 3. Experimental Method:

[0093] Vaginal smear examinations were performed on 18-month-old SPF-grade Balb / c female mice. Mice with irregular estrous cycles for 5 consecutive days were considered as perimenopausal old mice. The experimental animals were divided into a normal (young) control group of 10 mice, a model (old) control group of 7 mice, a low-dose group of the composition of the present invention of 6 mice, a medium-dose group of the composition of the present invention of 6 mice, and a high-dose group of the composition of the present invention of 6 mice. The experimental animals in the composition groups of the present invention were gavaged with the corresponding samples once a day; the experimental animals in the young mouse control group and the old mouse control group were gavaged with an equal volume of sterilized pure water once a day. The general condition of the mice was observed, and body weight was measured daily. At 24 h after gavage on the 42nd day after administration (the 43rd day), the mice in each group were sacrificed to detect organ coefficients, plasma hormone levels, and observe the pathological morphology of uterine tissues. Measurement data were expressed as mean ± standard deviation (X+S), and all data were statistically analyzed by one-way ANOVA. A p value less than 0.05 was considered statistically significant (the model control group was compared with other groups respectively using *

[0094] p < 0.05, **p < 0.01, ***p < 0.001 (indicated).

[0095] 4. Experimental results:

[0096] The body weights of the mice were monitored as shown in Table 5. The body weight of the young mouse control group was 22.82 g, and that of the old mouse control group was 24.54 g. Compared with the female old control group of mice, the body weight of the young mice in the composition group of the present invention did not increase significantly; compared with the female old control group of mice, the body weight of the mice in the composition group of the present invention also did not increase significantly.

[0097] Table 5. Detection results of body weights of each group of naturally aging menopausal mice

[0098]

[0099] The results of the Less index and fat coefficient of each group are shown in Table 6. The Less index of the group administered with the composition of the present invention did not change significantly compared with the old control group, and the fat coefficient showed a downward trend but there was no statistical difference.

[0100] Table 6. Detection results of the Less index and fat coefficient of each group of naturally aging menopausal mice

[0101]

[0102] The results of the determination of plasma hormone levels are shown in Table 7. After female mice entered menopause, ovarian function declined, manifested as a decrease in estrogen production level, such as estradiol (E2). Due to the feedback regulation mechanism of the gonadal axis, the basal levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) secreted by the hypothalamus, which promote ovarian function, instead increased during menopause, the ovarian pregnancy reserve ability decreased, and ovarian function failed. The E2 level of the old control group of mice decreased significantly, and the FSH and LH levels increased significantly. The sample of the composition group of the present invention could significantly increase the level of E2 in plasma and down-regulate the levels of FSH and LH. It shows that the sample of the composition group of the present invention has a certain pharmacological effect on regulating the endocrine level during menopause.

[0103] Table 7. Detection results of plasma hormone levels of each group of naturally aging menopausal mice

[0104]

[0105] 5. Histopathological study of uterine tissue

[0106] The results of the histopathological observation of the uterine tissue of each group of mice are shown in the appendix Figure 2As shown, in old female mice, due to ovarian function failure, estrogen levels gradually decline, leading to reproductive system decline, uterine tissue atrophy. The pathological manifestation of the uterine tissue is atrophy of the entire endometrium, with a smooth surface, no wavy undulations, a decrease in the number of endometrial glands, and obvious atrophy of the smooth muscle. The composition group of the present invention can improve the decline of the endometrium, smooth muscle, and glands to a certain extent. Compared with the old control group of mice, the endometrial thickness of the mice in the composition group of the present invention has increased, the inner diameter of the uterus has widened, and the number of glands in the uterus has also increased slightly.

[0107] 6. Experimental conclusions:

[0108] Based on the above experimental results of pharmacodynamic evaluation, for the natural aging menopausal mouse model, the composition of the present invention can relieve the signs similar to menopause in the model to a certain extent, such as plasma hormone levels and pathological characteristics of uterine tissue. It further demonstrates that the composition of the present invention can be used to treat perimenopausal syndrome.

[0109] Example 6 Effect of the composition of the present invention on the ovariectomized female menopausal mouse model

[0110] 1. Experimental materials

[0111] 1.1 Test drug: The intermediate obtained by preparing the composition of the present invention according to the method of Example 4

[0112] 1.2 Animals: Balb / c mice, female, body weight 20 - 22 g, 80 mice. Purchased from Shanghai Slack Experimental Animal Co., Ltd. (Animal License No.: SCXK (Shanghai) 2017 - 0012; Certificate No.: 20170012012742), after being approved by the experimental animal ethics (Ethics No.: PZSHUTCM210528007), they were raised in an SPF - level environment

[0113] 1.3 Reagents: Zoletil 50 (Vetoquinol, France, MFGD201911), Ethylenediaminetetraacetic acid (Sangon Biotech, 2906B858), Follicle - stimulating hormone (Hengyuan Biotech, HB967 - Mu), Luteinizing hormone (Hengyuan Biotech, HB970 - Mu), Estradiol (Hengyuan Biotech, HB975 - Mu), Progesterone (Hengyuan Biotech, HB494 - Mu), 10% Neutral formaldehyde solution (Sinopharm Chemical Reagent Co., Ltd., 20180429), Hematoxylin (Zhuhai Baiso Biotechnology Co., Ltd., BA4041), Eosin (Zhuhai Baiso Biotechnology Co., Ltd., BA4099)

[0114] 2. Dose design

[0115] The sample of the composition of the present invention is calculated based on 65g of crude drug equivalent to oral liquid per day, assuming that the adult body weight is 60kg, that is, 1.08g of crude drug / kg / d. The equivalent dose for mice is 12g of crude drug / kg / d. During the test, mice were given three dose groups of 24g of crude drug / kg (high dose), 12g of crude drug / kg (medium dose), and 6.0g of crude drug / kg (low dose) (equivalent to 2 times, equal times, and 1 / 2 times the clinical dose of humans, respectively), and gavage was performed at 10mL / kg, once a day, and the positive drug: estradiol tablets / estradiol dydrogesterone tablets composite package (fenmoston) was calculated as one tablet (containing progesterone tablets) orally per day, 12mg. Assuming the weight of the mouse is 20g, the equivalent dose ratio between the body surface area of ​​humans and mice is 0.0026, then the positive dose of Western medicine is: 12mg×0.0026÷20×10=0.0156mg / 10g·day, and the positive drug of Chinese patent medicine: Gengnian'an Capsule is calculated by daily oral administration of 2.7g (0.3g×3 capsules×3 times). Assuming the weight of the mouse is 20g, the equivalent dose ratio between the body surface area of ​​humans and mice is 0.0026, then the positive dose of Chinese patent medicine is: 2.7g×0.0026÷20×10=0.00351g / 10g·day

[0116] 3 Experimental methods

[0117] SPF grade Balb / c mice were surgically removed from both sides of the ovaries; 10 mice were used as sham operation controls. Vaginal smear observations were performed on castrated mice starting from the third day after surgery for 5 consecutive days to screen for successful surgery and successful modeling mice with estrous cycle disorders. On the 15th day after surgery, the successfully castrated mice were randomly divided into 10 model control groups, 10 positive drug (fenmoton) control groups, 10 positive drug (menopause) control groups, 10 low-dose groups of the present invention's composition, 10 medium-dose groups of the present invention's composition, and 10 low-dose groups of the present invention's composition. The experimental animals in the present invention's composition group were given a corresponding dose of sample once a day by gastric gavage; the experimental animals in the sham operation control group and the model control group were given an equal volume of sterilized pure water by gastric gavage once a day; the experimental animals in the positive drug control group were given a corresponding volume of positive drugs by gastric gavage once a day, and the general condition of the mice was observed, and body weight was tested daily. At 7-9 am on the 53rd day after administration (the 68th day of the experiment), the number of activities within 5 minutes was measured after 5 minutes of adaptation to the autonomous activity instrument. The temperature changes of the experimental mice in each group were detected by infrared thermal imaging. The animals were anesthetized 24 hours after the last administration, weighed, and the body length (length from nose to anus) was accurately measured to calculate the Less index; the abdominal cavity was opened along the midline of the abdomen, and the fat around the uterus, thymus, and spleen were taken out and weighed. The wet weight was calculated to calculate the fat coefficient, thymus coefficient, and spleen coefficient. -1Anesthetize the mice by intraperitoneal injection, take blood from the eyeballs, anticoagulate with ethylenediaminetetraacetic acid (EDTA), centrifuge at 3500 rpm for 15 min, take the plasma, and measure the contents of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), and progesterone (P) in the plasma of mice in each group. Fix the mouse uterus in 10% neutral formaldehyde solution, embed it in paraffin, section it, perform hematoxylin-eosin (HE) staining, and observe the pathological changes of the uterine tissue of mice in each group under a light microscope.

[0118] Measurement data are expressed as mean ± standard deviation (X+S). All data are statistically analyzed by one-way analysis of variance. A p-value less than 0.05 is considered to have statistical significance (*p<0.05, **p<0.01, ***p<0.001 for comparison between the model control group and other groups respectively).

[0119] 4 Experimental conclusions

[0120] 4.1 Body weight detection

[0121] Observe each group of experimental mice throughout the experiment. For the overall adult body weight growth trend of each group of experimental animals, the body weight of the experimental animals in the normal control group tended to be stable after the 27th day (10 weeks old) of the experiment, and the body weight of the Femoston group decreased compared with other groups. The body weights of other groups showed an upward trend. During the experiment, due to the braking intervention, the body weights of each group of experimental animals showed a certain degree of fluctuation, and the activity of individual experimental animals decreased, but there were no other obvious abnormal signs. However, the composition group of the present invention controlled the body weight of the castrated mice to a certain extent and had an ideal comprehensive pharmacological effect.

[0122] Table 8 Monitoring results of body weight of castrated female menopausal mice

[0123]

[0124]

[0125] 4.2 Spontaneous activity of mice

[0126] Due to ovariectomy, the estrogen level drops sharply and combined with braking stress treatment, resulting in an increase in the restlessness and activity of the model mice. Record the number of spontaneous activities of each group of mice within a certain period of time through a mouse spontaneous activity meter. The experimental results show that ovariectomy and stress intervention lead to a significant increase in the spontaneous activity of the model control mice compared with the normal control mice. Each drug intervention group can reduce the spontaneous activity of mice to a certain extent after 8 weeks of drug treatment. Among them, the high-dose group of the composition group of the present invention has the most obvious effect and has statistical significance compared with the model control group.

[0127] Table 9 Detection results of spontaneous activities of each group of castrated female menopausal mice

[0128]

[0129] 4.3 Body temperature changes

[0130] Hot flashes and night sweats often occur in menopausal women, so we used an infrared thermal imager to observe the changes in body temperature of each group of experimental mice. The body temperature of each group of mice in Table 10 was around 30°C, and no obvious changes in body temperature were observed in the castrated mouse model.

[0131] Table 10 Body temperature change test results of castrated female menopausal mice in each group

[0132]

[0133] 4.4 Organ coefficient

[0134] Less index is a commonly used indicator for evaluating the obesity and fat accumulation of mice at the macro level, while the fat coefficient further evaluates the accumulation of mouse fat tissue by separating adipose tissue and organ fat and calculating the ratio to body mass. The Less index and fat coefficient of the drug intervention group both decreased, among which the degree of decrease in the fentermone control group was the most obvious, and both had statistical differences. The decrease in the Less index of the menopause control group was more obvious, with statistical differences. The combination group of the present invention showed a good reduction effect in the detection of the Less index and the fat coefficient, and there were also statistical differences. See Table 11.

[0135] Table 11 Results of Less index and fat coefficient test in each group of castrated female menopausal mice

[0136]

[0137] Both the thymus and spleen are immune organs, and their quality is usually related to the overall immune function of the body. Under castration and stress intervention modeling conditions, the thymus index and spleen index of the model group mice decreased, and the spleen index decreased compared with the normal group. The drug intervention increased the thymus index to varying degrees, among which the effect of positive western medicine and Chinese patent medicine was more obvious, but there was no statistical difference in the data of each group. See Table 12.

[0138] Table 12 Results of thymus index and spleen index test in each group of castrated female menopausal mice

[0139]

[0140] 4.5 Measurement of plasma hormone levels

[0141] After female animals enter menopause, ovarian function declines, manifested as a decrease in estrogen production levels, such as estradiol (E2). Due to the feedback regulation mechanism of the gonadal axis, the basal levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) secreted by the hypothalamus, which promote ovarian function, instead increase during menopause. Ovariectomized female mice can simulate the failure of ovarian secretory function during menopause. The E2 level in the model mice is significantly decreased, while the FSH and LH levels are significantly increased. The positive control drug Femoston, as an E2 supplement, can significantly increase the E2 level in the plasma of mice and down-regulate the FSH and LH levels. The traditional Chinese medicine positive control drug Gengnian'an can also significantly increase the E2 level in the plasma and down-regulate the FSH and LH levels. The composition group of the present invention can increase the E2 level in the plasma of ovariectomized mice, and for FSH and LH, the composition group of the present invention can significantly reduce their levels. See Table 13.

[0142] Table 13 Determination results of plasma hormone levels in each group of ovariectomized female menopausal mice

[0143]

[0144] 4.6 Histopathological observation of uterine tissue

[0145] Due to the sharp decline in estrogen levels, the reproductive system of ovariectomized female mice declines, and the uterine tissue atrophies. The pathological manifestation of the uterine tissue is atrophy of the entire endometrium, with a smooth surface, no wavy undulations, a decrease in the number of endometrial glands, and obvious atrophy of the smooth muscle. After Femoston intervention, the uterine tissue is closer to the normal tissue morphology, manifested as obvious thickening of the endometrium, the morphological characteristics of endometrial hyperplasia with wavy appearance can be observed on the endometrial surface, an increase in the number of glands and regular arrangement, and the atrophy of the smooth muscle is also slowed down. The Gengnian'an control group and the composition of the present invention improved the decline of the endometrium, smooth muscle and glands to a certain extent, and the endometrial surface was slightly smoother.

[0146] The composition group of the present invention alleviates the signs similar to menopause in the model to a certain extent, such as weight gain, increased spontaneous activity, plasma hormone levels, histopathological characteristics of uterine tissue, fat coefficient, organ index, etc.

[0147] The description of the above embodiments is only used to help understand the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art of this technology, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A Chinese medicine composition, characterized in that: include: Danshen, Coptis chinensis, Anemarrhena asphodeloides, Ophiopogon japonicus, Mother of Pearl and Ziziphus jujuba seed.

2. The Chinese medicine composition according to claim 1, characterized in that: In parts by weight, it includes: 10-30 parts of Salvia miltiorrhiza, 2-10 parts of Coptis chinensis, 2-10 parts of Anemarrhena asphodeloides, 5-20 parts of Ophiopogon japonicus, 10-50 parts of Mother of Pearl, and 2-10 parts of Semen Ziziphi Spinosae.

3. The Chinese medicine composition according to claim 1, characterized in that In parts by weight, it includes: 15 parts of Salvia miltiorrhiza, 5 parts of Coptidis Rhizoma, 5 parts of Anemarrhena Rhizoma, 10 parts of Ophiopogon japonicus, 25 parts of Mother of Pearl, 5 parts of Semen Ziziphi Spinosae.

4. The method for preparing the Chinese medicine composition according to any one of claims 1 to 3, characterized in that: The following steps are involved: The Chinese medicinal materials are taken according to their weight proportions, and are extracted with an extraction solvent. The obtained extract is concentrated and dried under reduced pressure to obtain a Chinese medicinal extract.

5. The preparation method according to claim 4, characterized in that: The extraction solvent is selected from water, aqueous ethanol or ethanol; The extraction method is decoction, reflux extraction or ultrasonic extraction.

6. The preparation method according to claim 4, characterized in that: The extraction is specifically as follows: Take mother of pearl, extract it with 8-14 times water for 1-3 times, each time for 0.5-3 hours, then add salvia miltiorrhiza, coptis chinensis, anemarrhena asphodeloides, ophiopogon japonicus and spiny jujube seeds for reflux extraction, each time for 0.5-3 hours, filter, add 8-14 times water to the residue for reflux extraction again, each time for 0.5-3 hours, combine the filtrate, concentrate and dry under reduced pressure to obtain the Chinese medicine extract.

7. Use of the Chinese medicine composition according to any one of claims 1 to 3 in the preparation of a medicament for preventing, treating or alleviating perimenopausal syndrome.

8. A drug for preventing, treating or alleviating perimenopausal syndrome, characterized in that: The invention comprises the Chinese medicine composition according to any one of claims 1 to 3 and a pharmaceutically acceptable adjuvant.

9. The drug for preventing, treating or alleviating perimenopausal syndrome according to claim 8, characterized in that: The dosage form of the medicine is oral liquid, granules, capsules, tablets, pills, dripping pills or sustained-release preparations.

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