Application of prepared rehmannia root extract in preparation of medicine for treating ovarian injury

Abstract

The invention relates to the technical field of traditional Chinese medicines, in particular to application of a prepared rehmannia root extract in preparation of a medicine for treating ovarian injury. The inventor finds a single traditional Chinese medicine for treating ovarian injury based on a traditional Chinese medicine compound core medicinal material data mining model. The prepared rehmannia root is taken as a representative core medicinal material screened and output by a model, efficacy verification is carried out in a cyclophosphamide induced ovarian injury mouse model, and the result shows that the prepared rehmannia root can improve E2 and AMH levels, reduce FSH and LH levels, improve orbital cycle disorder and reduce oxidative stress level, so that the prepared rehmannia root can improve the ovarian reserve function of a mouse, and can be used for treating ovarian injury. And a certain improvement effect is achieved on ovarian injury. The invention provides a quantifiable traditional Chinese medicine compound medicinal material screening thought, realizes a closed loop of a method from data mining to efficacy verification, and provides a theoretical basis and an experimental basis for developing novel traditional Chinese medicines for treating ovarian injury.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine, in particular to the use of Radix Rehmanniae Preparata extract in the preparation of a drug for treating ovarian damage. BACKGROUND

[0002] Ovarian damage refers to the damage to the structure and function of the ovary caused by multiple factors such as chemotherapy, radiotherapy, autoimmune reaction, environmental toxins, infection or surgery, which is often manifested as a decrease in the number of follicles, a decrease in estrogen levels, an increase in gonadotropin, ovulation disorders and infertility, and may progress to premature ovarian failure and other diseases in severe cases. At present, hormone replacement therapy is often used in clinical practice to improve symptoms, but this therapy has strong efficacy dependence, significant rebound after drug withdrawal, and may increase the risk of breast cancer and cardiovascular events when used for a long time.

[0003] In recent years, traditional Chinese medicine has attracted widespread attention in regulating female endocrine and protecting ovarian function. Studies have shown that classic Chinese medicine compounds such as Siwu Decoction and Zuogui Pill can have a certain protective and repair effect on ovarian damage through mechanisms such as improving the ovarian microenvironment, antioxidant and anti-apoptosis. Therefore, intervention strategies based on traditional Chinese medicine are expected to provide a safe and effective new idea for the prevention and treatment of ovarian damage, and have become a hot research direction in this field.

[0004] However, current research on traditional Chinese medicine for treating ovarian damage mainly focuses on the effects of traditional Chinese medicine compounds as a whole or Chinese patent medicines, and lacks independent screening and efficacy verification of single traditional Chinese medicines. SUMMARY

[0005] The present application aims to at least partially solve one of the problems in the related art. To this end, one object of the present application is to provide the use of Radix Rehmanniae Preparata extract in the preparation of a drug for treating ovarian damage. The inventors found a single traditional Chinese medicine for treating ovarian damage based on a traditional Chinese medicine compound core medicinal material data mining model. More specifically, the present application uses a data mining method to deeply mine a single traditional Chinese medicine with the most potential for treating ovarian damage in traditional Chinese medicine compounds, and further verifies it in a cyclophosphamide-induced ovarian damage mouse model. It is found that Radix Rehmanniae Preparata extract can improve the estrous cycle disorder caused by cyclophosphamide in mice, improve the ovarian reserve function of mice, has a certain therapeutic effect on ovarian damage, can reduce the oxidative stress level of cyclophosphamide-induced ovarian damage mice, and can increase the number of primordial follicles, primary follicles and secondary follicles, and reduce the number of atretic follicles. The method provided by the present application for finding a single traditional Chinese medicine for treating ovarian damage based on a traditional Chinese medicine compound core medicinal material data mining model provides a new idea for mining single traditional Chinese medicines in compounds, and lays a foundation for further research and development of innovative preparations for treating ovarian damage.

[0006] To this end, the first aspect of the present application provides the use of Radix Rehmanniae Preparata extract in the preparation of a drug for treating ovarian damage.

[0007] The present application inventors construct a core medicinal material screening model based on traditional Chinese medicine compound data mining, collect a traditional Chinese medicine compound database for treating ovarian damage, extract medicinal material information involved in the compound, combine medicinal material usage frequency, four qi and five flavor properties, use clustering analysis, association rule mining and traditional Chinese medicine compound association analysis algorithm, and establish a repeatable and scalable candidate medicinal material screening framework. The model can mine a core medicinal material list highly associated with a specific disease from a large number of compounds, and is suitable for candidate drug screening for various diseases caused by ovarian damage. The present application takes prepared rehmannia as a representative core medicinal material screened by the model, and carries out efficacy verification in a cyclophosphamide-induced ovarian damage mouse model. The results show that prepared rehmannia can increase E2 and AMH levels, reduce FSH and LH levels, improve estrous cycle disorder, and reduce oxidative stress, indicating that it can improve ovarian reserve function in mice and has a certain improvement effect on ovarian damage. The present application proposes a quantifiable traditional Chinese medicine compound medicinal material screening idea, and realizes a method closed loop from "data mining" to "efficacy verification", providing a theoretical basis and experimental basis for developing new traditional Chinese medicine for treating ovarian damage.

[0008] According to the embodiment of the present application, the prepared rehmannia extract is obtained by the following extraction method:

[0009] S1: dry prepared rehmannia is crushed to obtain prepared rehmannia powder;

[0010] S2: 8-12 times the volume of water is added to the prepared rehmannia powder, and extraction treatment is carried out, the extraction treatment conditions being: temperature 80-100°C, time 1-2h;

[0011] S3: the liquid after the extraction treatment is filtered, and the obtained filtrate is the prepared rehmannia extract.

[0012] According to the embodiment of the present application, the extraction method of the prepared rehmannia extract further comprises:

[0013] S4: 8-12 times the volume of water is added to the residue obtained after the filtration treatment, and the extraction step 1-3 times of step S2 is repeated, and the obtained filtrate is combined with the filtrate obtained in step S3 to obtain the prepared rehmannia extract.

[0014] According to the embodiment of the present application, the extraction method of the prepared rehmannia extract further comprises:

[0015] S5: the filtrate obtained in step S3 or S4 is reduced pressure concentrated to obtain a prepared rehmannia water extract.

[0016] According to the embodiment of the present application, the temperature during the reduced pressure concentration is 55-70°C.

[0017] According to an embodiment of the present application, the extraction method of the Rehmannia glutinosa extract further comprises:

[0018] S6: freeze-drying the water extract of Rehmannia glutinosa obtained after the reduced-pressure concentration, to obtain a freeze-dried powder of Rehmannia glutinosa.

[0019] According to an embodiment of the present application, the administration dosage of the Rehmannia glutinosa extract is 200-600 mg / kg / d.

[0020] The second aspect of the present application provides a drug for treating ovarian damage. According to an embodiment of the present application, the drug comprises a Rehmannia glutinosa extract.

[0021] According to an embodiment of the present application, the Rehmannia glutinosa extract is obtained by the following extraction method:

[0022] S1: crushing dry Rehmannia glutinosa to obtain Rehmannia glutinosa powder;

[0023] S2: adding 8-12 times the volume of water to the Rehmannia glutinosa powder and performing extraction treatment, the extraction treatment conditions being: temperature 80-100°C, time 1-2 h;

[0024] S3: filtering the liquid after the extraction treatment, the obtained filtrate being the Rehmannia glutinosa extract.

[0025] According to an embodiment of the present application, the extraction method of the Rehmannia glutinosa extract further comprises:

[0026] S4: adding 8-12 times the volume of water to the residue obtained after the filtering treatment, repeating the extraction step 1-3 times of step S2, and combining the obtained filtrate with the filtrate obtained in step S3, to obtain the Rehmannia glutinosa extract.

[0027] According to an embodiment of the present application, the extraction method of the Rehmannia glutinosa extract further comprises:

[0028] S5: performing reduced-pressure concentration on the filtrate obtained in step S3 or S4, to obtain a water extract of Rehmannia glutinosa.

[0029] According to an embodiment of the present application, the temperature during the reduced-pressure concentration is 55-70°C.

[0030] According to an embodiment of the present application, the extraction method of the Rehmannia glutinosa extract further comprises:

[0031] S6: freeze-drying the water extract of Rehmannia glutinosa obtained after the reduced-pressure concentration, to obtain a freeze-dried powder of Rehmannia glutinosa.

[0032] According to an embodiment of the present application, the administration dosage of the Rehmannia glutinosa extract is 200-600 mg / kg / d.

[0033] The present application takes Radix Rehmanniae Preparata as a representative core medicinal material of the model screening output, and carries out efficacy verification in a cyclophosphamide-induced ovarian damage mouse model, and the results show that Radix Rehmanniae Preparata can improve the levels of E2 and AMH, reduce the levels of FSH and LH, improve estrous cycle disorder, and reduce oxidative stress level, suggesting that it can improve the ovarian reserve function of mice and has a certain improvement effect on ovarian damage. The present application proposes a quantifiable screening idea of traditional Chinese medicine compound medicinal materials, and realizes the method closed loop from 'data mining' to 'efficacy verification', providing a theoretical basis and experimental basis for developing new traditional Chinese medicine for treating ovarian damage.

[0034] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0035] The above and / or additional aspects and advantages of the present application will become apparent and be readily understood from the following description, taken in conjunction with the accompanying drawings, in which:

[0036] Figure 1 It is a four-gas statistical graph of 167 kinds of traditional Chinese medicines in embodiment 1 of the present application;

[0037] Figure 2 It is a five-flavor statistical graph of 167 kinds of traditional Chinese medicines in embodiment 1 of the present application;

[0038] Figure 3 It is a complex network graph of 167 kinds of traditional Chinese medicines in embodiment 1 of the present application;

[0039] Figure 4 It is a clustering graph of the first 27 kinds of traditional Chinese medicines in embodiment 1 of the present application;

[0040] Figure 5 It is a body weight change graph of Radix Rehmanniae Preparata treating ovarian damage in embodiment 2 of the present application;

[0041] Figure 6 It is an estrous cycle broken line graph of Radix Rehmanniae Preparata treating ovarian damage in embodiment 2 of the present application;

[0042] Figure 7 It is a hormone level detection graph of Radix Rehmanniae Preparata treating ovarian damage in embodiment 2 of the present application;

[0043] Figure 8 It is an oxidative stress level detection graph of Radix Rehmanniae Preparata treating ovarian damage in embodiment 2 of the present application;

[0044] Figure 9 It is a mouse tissue HE staining graph of Radix Rehmanniae Preparata treating ovarian damage in embodiment 2 of the present application. DETAILED DESCRIPTION

[0045] Embodiments of the present application are described in detail below. The embodiments described below are exemplary and are intended to explain the present application, but are not to be understood as limiting the present application.

[0046] It should be noted that the terms "first", "second" are used only for descriptive purposes, and should not be construed as indicating or implying relative importance or a specific number of the technical features indicated. Thus, the features defined with "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, unless otherwise stated, the meaning of "a plurality of" is two or more.

[0047] The endpoints of the ranges and any values described herein are not limited to the precise values stated. The ranges and values should be construed to be approximations that allow for significant variation. Within each range, other values are contemplated, and the endpoints of the ranges are included in the range. Other values outside the stated ranges are also contemplated.

[0048] In order to facilitate the understanding of the present application, certain technical and scientific terms are defined below. Unless otherwise apparent from context, all other technical and scientific terms used herein have the meanings typically understood by one of ordinary skill in the art to which this application belongs.

[0049] In this document, the terms "comprise" or "comprising" are open-ended, that is, they mean including, but not limited to, what is recited.

[0050] In this document, the terms "optionally", "optional" or "optional" generally mean that the event or circumstance subsequently described can or can not occur, and the description includes situations in which the event or circumstance occurs, as well as situations in which it does not occur.

[0051] According to a specific embodiment of the present application, the present application provides a drug screening method based on traditional Chinese medicine compound core medicinal material data mining model for preventing and treating ovarian damage, comprising:

[0052] 1) Search and screen data to obtain traditional Chinese medicine compounds;

[0053] 2) Unify and enter traditional Chinese medicine compounds;

[0054] 3) Analyze the use frequency, four qi and five flavors of each traditional Chinese medicine in the traditional Chinese medicine compound to obtain core single medicine;

[0055] 4) Construct a cyclophosphamide-induced ovarian damage mouse model to verify the effect of the core single medicine on ovarian damage.

[0056] According to a specific embodiment of the present application, in step 1), the CNKI full-text database and WanFang Database are searched for clinical literature on the treatment of ovary damage by traditional Chinese medicine from the date of establishment of the database to July 31, 2024, with the keywords being: "ovary damage", "traditional Chinese medicine".

[0057] According to a specific embodiment of the present application, in step 1), the specific method for screening data is to follow the inclusion criteria and exclusion criteria during the screening process:

[0058] Inclusion criteria: ① Ovary damage as the first diagnosis; ② Traditional Chinese medicine compound as the main treatment in the treatment group, with complete compound components and definite efficacy; ③ Inclusion of the same prescription once;

[0059] Exclusion criteria: ① Pure animal experiment research literature; ② No inclusion of cases or non-compliance with the diagnostic criteria for ovary damage; ③ Main research means other than traditional Chinese medicine compound;

[0060] Diagnostic criteria: ① Age <40; ② Irregular menstruation or amenorrhea for at least 4 months or more; ③ At least 2 times of serum FSH >25U / L (interval >4 weeks).

[0061] According to a specific embodiment of the present application, in step 2), the name of traditional Chinese medicine is uniformly standardized according to the 2020 edition of the People's Republic of China Pharmacopoeia, and EXCEL is used to collect the components of the compound, and a database is established to prepare for the next step of analysis.

[0062] According to a specific embodiment of the present application, in step 3), it includes:

[0063] a. Use IBM SPSS Statistics 26 software to perform frequency statistics on the components of the compound;

[0064] b. Use SPSS Modeler l8.0 software to perform association analysis on the components of the compound;

[0065] c. Use OriginPro 2024b to perform cluster analysis on the top 27 herbs by frequency, with Radix Rehmanniae as the core drug;

[0066] d. Use the Traditional Chinese Medicine Inheritance Auxiliary Platform to count the four properties and five flavors of all components in the compound.

[0067] According to a specific embodiment of the present application, in step 4), it includes:

[0068] a. Select female 8-week C57BL / 6 mice for a single injection of 100mg / kg cyclophosphamide to establish a mouse model of ovary damage;

[0069] b. Receiving the treatment of Radix Rehmanniae preparata for 28 days;

[0070] c. After the sample collection, detecting the serum hormone level, oxidative stress level and HE staining of the ovary tissue of the mice.

[0071] The core single herb Radix Rehmanniae preparata extract is screened through the above method, and the efficacy verification is carried out in the cyclophosphamide-induced ovary damage mouse model. The results show that Radix Rehmanniae preparata can improve the levels of E2 and AMH, reduce the levels of FSH and LH, improve the estrous cycle disorder, and reduce the oxidative stress level, which indicates that it can improve the ovarian reserve function of the mice and has a certain improvement effect on the ovary damage. The application provides a quantifiable screening idea of traditional Chinese medicine compound medicinal materials, and realizes the method closed loop from "data mining" to "efficacy verification", which provides a theoretical basis and experimental basis for developing a new type of traditional Chinese medicine for treating ovary damage.

[0072] According to one specific embodiment of the application, the application provides the use of Radix Rehmanniae preparata extract in the preparation of a medicine for treating ovary damage. It should be noted that there is no special restriction on the preparation method of Radix Rehmanniae preparata extract, and the water-extracted Radix Rehmanniae preparata is preferably used to obtain Radix Rehmanniae preparata extract.

[0073] According to one specific embodiment of the application, the Radix Rehmanniae preparata extract is obtained by the following extraction method:

[0074] S1: The dried Radix Rehmanniae preparata is subjected to a crushing treatment to obtain Radix Rehmanniae preparata powder;

[0075] S2: 8-12 times the volume of water is added to the Radix Rehmanniae preparata powder, and an extraction treatment is performed, and the extraction treatment conditions are: temperature 80-100℃, time 1-2h;

[0076] S3: The liquid after the extraction treatment is subjected to a filtration treatment, and the obtained filtrate is the Radix Rehmanniae preparata extract.

[0077] It should be noted that there is no special restriction on the crushing treatment conditions, and preferably, the product after the crushing treatment is passed through a 20-40 mesh sieve to obtain Radix Rehmanniae preparata powder.

[0078] According to one specific embodiment of the application, in step S2, when the extraction treatment is performed, 8 times, or 9 times, or 10 times, or 11 times, or 12 times the volume of water is added to the Radix Rehmanniae preparata powder.

[0079] According to a specific embodiment of the present application, in step S2, the extraction processing condition is that the temperature can be 80℃, 81℃, 82℃, 83℃, 84℃, 85℃, 86℃, 87℃, 88℃, 89℃, 90℃, 91℃, 92℃, 93℃, 94℃, 95℃, 96℃, 97℃, 98℃, 99℃ or 100℃, and the time can be 1.0h, 1.1h, 1.2h, 1.3h, 1.4h, 1.5h, 1.6h, 1.7h, 1.8h, 1.9h or 2.0h.

[0080] According to a specific embodiment of the present application, the extraction method of the Rehmannia glutinosa extract further comprises:

[0081] S4: adding 8-12 times volume of water into the residue obtained after the filtration processing, repeating the extraction steps 1-3 times of step S2, and combining the filtrate obtained in step S4 with the filtrate obtained in step S3, so as to obtain the Rehmannia glutinosa extract.

[0082] According to a specific embodiment of the present application, in step S4, 8 times, or 9 times, or 10 times, or 11 times, or 12 times volume of water can be added into the residue obtained after the filtration processing, the extraction steps of step S2 can be repeated 1 time, or 2 times, or 3 times, and the filtrate obtained in step S4 can be combined with the filtrate obtained in step S3, so as to obtain the Rehmannia glutinosa extract.

[0083] According to a specific embodiment of the present application, the extraction method of the Rehmannia glutinosa extract further comprises:

[0084] S5: performing vacuum concentration on the filtrate obtained in step S3 or S4, so as to obtain a Rehmannia glutinosa water extract.

[0085] It should be noted that the condition for vacuum concentration is not particularly limited, and all conditions known in the art that can perform vacuum concentration on the filtrate are encompassed in the scope of the present application. Preferably, the temperature during the vacuum concentration is 55-70℃. For example, the temperature during the vacuum concentration is 55℃, 56℃, 57℃, 58℃, 59℃, 60℃, 61℃, 62℃, 63℃, 64℃, 65℃, 66℃, 67℃, 68℃, 69℃, 70℃, etc.

[0086] According to a specific embodiment of the present application, the extraction method of the Rehmannia glutinosa extract further comprises:

[0087] S6: performing freeze-drying processing on the Rehmannia glutinosa water extract obtained after the vacuum concentration, so as to obtain a Rehmannia glutinosa freeze-dried powder.

[0088] It should be noted that the conditions for freeze-drying treatment are not particularly limited, and all conditions capable of freeze-drying treatment on the prepared Radix Rehmanniae Preparata water extract after the pressure reduction and concentration are included in the scope of the present application.

[0089] According to a specific embodiment of the present application, the administration dose of the Radix Rehmanniae Preparata extract is 200-600 mg / kg / d. For example, the administration dose for a subject can be 200 mg / kg / d, 300 mg / kg / d, 400 mg / kg / d, 500 mg / kg / d, 600 mg / kg / d, etc.

[0090] It should be noted that the natural aging of the ovary damage or ovarian function decline is due to the natural physiological degenerative changes of the body, which is an endogenous change; the cyclophosphamide-induced model provided by the present application is caused by single or multiple administrations, which causes direct damage to ovarian cells, reduction of follicles, hormone abnormalities, etc., and belongs to an acute toxicity damage model, which has the advantages of high controllability, good repeatability, and short modeling cycle. Although the present application verifies the efficacy of the Radix Rehmanniae Preparata extract on the cyclophosphamide-induced ovarian damage mouse model, it shows that the Radix Rehmanniae Preparata can increase the levels of E2 and AMH, reduce the levels of FSH and LH, improve the estrous cycle disorder, and reduce the oxidative stress level, which suggests that it can improve the ovarian reserve function of mice and has a certain improvement effect on ovarian damage, but also provides a theoretical basis and experimental basis for the development of new Chinese medicine for treating ovarian damage.

[0091] According to a specific embodiment of the present application, the present application provides a medicine for treating ovarian damage, which comprises a Radix Rehmanniae Preparata extract.

[0092] According to a specific embodiment of the present application, the Radix Rehmanniae Preparata extract is obtained by the above preparation method.

[0093] The schemes of the present disclosure will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be regarded as limiting the scope of the present disclosure. If the specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or according to the product instructions are used. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be obtained by purchase.

[0094] Example 1: Mining the most potential single Chinese medicine for treating ovarian damage from Chinese medicine compound

[0095] 1. Collection of Chinese medicine compound

[0096] The clinical literature on traditional Chinese medicine treatment of ovarian damage was collected from the China Journal Net Full-text Database (CNKI) and Wanfang Database Knowledge Service Platform (Wanfang Database) up to July 31, 2024. The search keywords were "ovarian damage" and "traditional Chinese medicine". After removing duplicates, 157 compound prescriptions and 167 traditional Chinese medicines were collected.

[0097] 2. Analysis of traditional Chinese medicine compound

[0098] The names of 167 medicinal materials were corrected, and the results are shown in Table 1. The 157 compound prescriptions were imported into IBM SPSS Statistics 26 software for frequency statistics, with a total frequency of 1867 times. The top 27 traditional Chinese medicines with a frequency of 20 or more were selected and made into a table (Table 2). The top 10 frequencies were: Radix Rehmanniae Preparata, Semen Cuscutae, Angelica sinensis, Fructus Lycii, Rhizoma Dioscoreae, Herba Epimedii, Fructus Corni, Glycyrrhiza uralensis, Radix Paeoniae Alba, and Salvia miltiorrhiza. Clustered heat maps of the top 27 traditional Chinese medicines were visualized using OriginPro 2024b software, as shown in Figure 3 , and the four properties and five flavors of the 167 medicinal materials were counted Figure 1 , 2 .

[0099] Table 1. Correction of drug names

[0100]

[0101]

[0102] Table 2. Drug frequency table

[0103]

[0104] 3. Association analysis of traditional Chinese medicine compound

[0105] The 157 compound prescriptions were imported into SPSS Modeler 18.0 software, and the Apriori association rule algorithm was used to analyze the prescriptions. The results were imported into Cytoscape 3.9.1 software for visualization, as shown in Figure 4 . The thickness of the connection between two drugs represents the level of link between the two drugs. The thicker the line, the higher the link between the two drugs, indicating that the two drugs appear in the same prescription more frequently. The size and color of each drug node icon represent the size of the link between the drug and other drugs. The darker the color, the larger the link number, indicating that the drug has a higher degree of association with other drugs, which means that this drug plays a core role in the compatibility of prescriptions. Based on the results of traditional Chinese medicine complex network analysis, Radix Rehmanniae Preparata is the most important core drug.

[0106] Example 2. Role of Radix Rehmanniae Preparata in the treatment of ovarian damage

[0107] 1. Preparation of Radix Rehmanniae Preparata extract

[0108] Take 50 g of Radix Rehmanniae Preparata, crush it through a 20-40 mesh sieve, and add 10 times the volume of purified water to the powder. Heat the mixture to 100°C and maintain the temperature for 1 hour. Filter the mixture while it is hot, and collect the filtrate. Add 10 times the volume of purified water to the residue and repeat the extraction process 3 times. Combine all the filtrates, concentrate them under reduced pressure at 55°C, and then freeze-dry the concentrate to obtain the Radix Rehmanniae Preparata water extract.

[0109] 2. Construction of a cyclophosphamide-induced ovarian injury mouse model and grouping

[0110] C57BL / 6 wild-type mice were used in the experiment. The mice were raised in an environment with a temperature of 25±2°C and a humidity of 55±5%, with a 12-hour light-dark cycle, free access to water and food. After 7 days of adaptive feeding, the experimental animals were randomly divided into 6 groups: a normal control group (NC), a cyclophosphamide model group (CTX), a low-dose Radix Rehmanniae Preparata group (RG-L), a medium-dose Radix Rehmanniae Preparata group (RG-M), a high-dose Radix Rehmanniae Preparata group (RG-H), and a coenzyme Q10 positive drug group (CoQ10). There were 10 mice in each group. Except for the normal control group, the other groups were injected intraperitoneally with 100 mg / kg of cyclophosphamide on the eighth day to establish an ovarian injury model. The control group was injected intraperitoneally with 10 mg / kg of normal saline. From one week before the injection of cyclophosphamide to the day of the mice's death, the control and model groups were given 10 mg / kg / d of normal saline by gavage, the Radix Rehmanniae Preparata groups were given 200 mg / kg / d, 400 mg / kg / d, and 600 mg / kg / d of Radix Rehmanniae Preparata by gavage, respectively, and the positive drug group was given 100 mg / kg / d of CoQ10 solution. The first day of gavage was designated as D1, and the mice were sacrificed 28 days after gavage. The serum and ovaries were collected. The weight of the ovaries and the ovarian index (ovarian index % = ovarian wet weight / mouse body weight x 100%) were recorded.

[0111] 3. Observation indicators

[0112] General condition: During the experiment, the body weight changes, hair loss, estrous cycle, etc. of the mice in each group were recorded.

[0113] Hormone index detection: The changes in E2, FSH, AMH, LH, etc. in the serum were detected using an ELISA kit.

[0114] Biochemical index detection: The levels of SOD and MDA in the serum were detected using a kit.

[0115] Ovarian pathological section observation: The HE-stained (hematoxylin-eosin-stained) sections of each group were observed under an optical microscope: the changes in the number of follicles at each stage.

[0116] Statistical analysis: expressed as mean ± standard deviation, statistical calculation was performed using T test, and P < 0.05 was considered statistically significant.

[0117] 4. Results

[0118] 1) Comparison of general conditions

[0119] On the eighth day after the modeling drug was punched in, the body weight of the CTX group, the CoQ10 group, the RG-L group, the RG-M group and the RG-H group decreased, and after continuing the drug treatment, the body weight of the mice in the CoQ10 group, the RG-L group, the RG-M group and the RG-H group began to rise, until the end of modeling, the body weight of the mice in the CTX group was generally lower than that in the other groups. Figure 5 During the modeling process, the mice in the CTX group had a hair loss phenomenon in the back of the neck, and the other groups did not have obvious hair loss phenomenon. Further observation of the estrous cycle of the mice was carried out, and the vaginal smear detection of the mice was carried out at the same time every morning in the last week of the end of modeling. As shown in the results, the RG-L group, the RG-M group and the RG-H group can improve the estrous cycle disorder of the mice caused by cyclophosphamide. Figure 6

[0120] 2) The results of hormone level detection showed that the AMH and E2 levels of the mice in the ovary damage model decreased, and the LH and FSH levels increased. After 28 days of drug intervention treatment of Radix Rehmanniae Glutinosae, it was found that the RG-L group, the RG-M group and the RG-H group could obviously increase the AMH and E2 levels in the serum of the mice, and reduce the LH and FSH levels. It is proved that Radix Rehmanniae Glutinosae can improve the ovarian reserve function of the mice, and has a certain treatment effect on the ovary damage. Figure 7

[0121] 3) The results of biochemical index detection showed that Radix Rehmanniae Glutinosae can reduce the oxidative stress level of the mice with ovary damage caused by cyclophosphamide. Figure 8

[0122] 4) The results of ovary tissue section staining showed that Radix Rehmanniae Glutinosae can increase the number of primordial follicles, primary follicles and secondary follicles, and reduce the number of atretic follicles. Figure 9

[0123] The present application can improve the development and apoptosis state of follicles in the ovary tissue of the Radix Rehmanniae Glutinosae treatment group through HE staining, increase the serum AMH level, and further support the recovery of the ovarian reserve function. In addition, the E2 level rises, the FSH and LH levels decrease, which indicates that the feedback regulation of the HPO axis is improved, the function of granulosa cells is enhanced, the hormone synthesis ability of follicles is enhanced, and it is comprehensively judged that Radix Rehmanniae Glutinosae can improve the function of follicles, and then relieve the performance of premature ovarian failure.

[0124] ​​​​In addition, the intervention of Rehmannia glutinosa in this study can significantly reduce the level of MDA in serum, improve the level of SOD, have antioxidant capacity, help to reduce the oxidative damage and apoptosis of ovarian tissue, thereby improving the environment of follicular development and promoting the synthesis of endogenous estrogen.

[0125] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", "some embodiments" or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. Furthermore, different embodiments or examples described in the present specification and the features of different embodiments or examples can be combined and combined by those skilled in the art without contradiction.

[0126] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. Use of a Radix Rehmanniae Preparata extract in the preparation of a medicament for treating ovarian damage.

2. Use according to claim 1, characterized in that, The Radix Rehmanniae Preparata extract is obtained by the following extraction method: S1: subjecting dried Radix Rehmanniae Preparata to a pulverization treatment to obtain a Radix Rehmanniae Preparata powder; S2: adding 8-12 times the volume of water to the Radix Rehmanniae Preparata powder and subjecting to an extraction treatment, the extraction treatment conditions being: temperature 80-100°C, time 1-2h; S3: subjecting the liquid after the extraction treatment to a filtration treatment, the filtrate obtained being the Radix Rehmanniae Preparata extract.

3. Use according to claim 2, characterized in that, The extraction method of the Radix Rehmanniae Preparata extract further comprises: S4: adding 8-12 times the volume of water to the drug residue obtained after the filtration treatment, repeating the extraction step 1-3 times of step S2, and combining the filtrate obtained with the filtrate obtained in step S3, so as to obtain the Radix Rehmanniae Preparata extract.

4. Use according to claim 3, characterized in that, The extraction method of the Radix Rehmanniae Preparata extract further comprises: S5: subjecting the filtrate obtained in step S3 or S4 to a reduced-pressure concentration to obtain a Radix Rehmanniae Preparata water extract; Optionally, the temperature during the reduced-pressure concentration is 55-70°C; Optionally, the extraction method of the Radix Rehmanniae Preparata extract further comprises: S6: subjecting the Radix Rehmanniae Preparata water extract obtained after the reduced-pressure concentration to a freeze-drying treatment, so as to obtain a Radix Rehmanniae Preparata freeze-dried powder.

5. Use according to claim 4, characterized in that, The administration dose of the Radix Rehmanniae Preparata extract is 200-600mg / kg / d.

6. A medicament for treating ovarian damage, characterized by, The Radix Rehmanniae Preparata extract.

7. The medicament according to claim 6, characterized in that, The Radix Rehmanniae Preparata extract is obtained by the following extraction method: S1: subjecting dried Radix Rehmanniae Preparata to a pulverization treatment to obtain a Radix Rehmanniae Preparata powder; S2: adding 8-12 times the volume of water to the Radix Rehmanniae Preparata powder and subjecting to an extraction treatment, the extraction treatment conditions being: temperature 80-100°C, time 1-2h; S3: subjecting the liquid after the extraction treatment to a filtration treatment, the filtrate obtained being the Radix Rehmanniae Preparata extract.

8. The medicament according to claim 7, characterized in that, The extraction method of the Radix Rehmanniae Preparata extract further comprises: S4: adding 8-12 times the volume of water to the drug residue obtained after the filtration treatment, repeating the extraction step 1-3 times of step S2, and combining the filtrate obtained with the filtrate obtained in step S3, so as to obtain the Radix Rehmanniae Preparata extract.

9. The medicament according to claim 8, characterized in that, The extraction method of the Radix Rehmanniae Preparata extract further comprises: S5: subjecting the filtrate obtained in step S3 or S4 to a reduced-pressure concentration to obtain a Radix Rehmanniae Preparata water extract; Optionally, the temperature during the reduced-pressure concentration is 55-70°C; Optionally, the extraction method of the Radix Rehmanniae Preparata extract further comprises: S6: subjecting the Radix Rehmanniae Preparata water extract obtained after the reduced-pressure concentration to a freeze-drying treatment, so as to obtain a Radix Rehmanniae Preparata freeze-dried powder.

10. The medicament according to claim 9, characterized in that, The administration dose of the Radix Rehmanniae Preparata extract is 200-600mg / kg / d.

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