CN115779035B - Traditional Chinese medicine composition for treating or improving ovarian reserve function decline and preparation method and application thereof - Google Patents

Abstract

The application provides a traditional Chinese medicine composition for treating or improving ovarian reserve function decline, and a preparation method and application thereof, wherein the traditional Chinese medicine composition comprises the following raw materials: radix Glehniae, vinegar tortoise plastron, wine glossy privet fruit, chinese angelica, prepared rehmannia root, wine cornus, semen cuscutae, medlar, wine cistanche, fragrant solomonseal rhizome, white paeony root, vinegar nutgrass galingale rhizome and mulberry. The Chinese medicinal composition can improve DOR ovary reserve function and follicular maturation and development, can obviously improve AFC and AMH, HIF-1α, VEGF, ang-1 and Ang-2 levels in serum, and reduce FSH and FSH/LH; the increase of the number of follicles at each level of DOR mice is promoted, the ovarian atrophy is improved, the granulosa cells are orderly arranged, and the vascularity is increased; meanwhile, the expression of DOR mouse ovary SIRT1, VEGF, HIF-1 alpha, ang-1 and Ang-2 proteins is promoted, and NF-KB expression is inhibited, so that the ovary reserve function is improved.

Description

Traditional Chinese medicine composition for treating or improving ovarian reserve function decline and preparation method and application thereof

Technical Field

The application relates to the technical field of traditional Chinese medicine preparations, in particular to a traditional Chinese medicine composition for treating or improving ovarian reserve function decline, and a preparation method and application thereof.

Background

Hypoovarial stores (DOR), also known as decreased ovarian stores, refers to a decrease in the number of oocytes and/or decreased quality of ovaries in females before age 40, clinically manifested as hypomenorrhea, late menstrual period, amenorrhea, infertility, female climacteric symptoms, etc., accompanied by an increase in Follicle Stimulating Hormone (FSH), a decrease in estrogen (E2) and anti-mullerian hormone (AMH), a decrease in the number of follicles in sinuses (AFC), and DOR severely affects female fertility.

DOR pathogenesis is not clear, and the etiology is currently considered to be related to heredity, immune factors, age, enzyme deficiency, psychosocial stress, iatrogenic factors and the like. In recent years, exploration of the pathogenesis of DOR is a hotspot and difficulty in the gynaecological field. Many researchers believe that the mechanism of DOR may be closely related to oocyte mitochondrial function in the ovary, follicular granulosa cell apoptosis, level of oxidative stress in the ovary, inflammatory response, angiogenesis, etc. Therefore, the research on the pathogenesis of DOR, the improvement of clinical symptoms of DOR patients, the alleviation of accelerated occlusion of follicles, the improvement of oocyte quality and the improvement of ovarian reserve function are the problems to be solved urgently in the current research.

DOR is a serious disease affecting female reproductive health, and has no specific treatment until now because of unclear etiology. The traditional Chinese medicine treatment has unique advantages in the aspect of treating diseases with unclear etiology because of the system of treating the syndrome differentiation. Therefore, there is an urgent need to develop a drug that assists DOR patients in improving ovarian function.

Disclosure of Invention

Therefore, the application aims to provide a traditional Chinese medicine composition for treating or improving the ovarian reserve function decline, and a preparation method and application thereof.

Based on the above object, the first aspect of the present application provides a traditional Chinese medicine composition for treating or improving ovarian failure, comprising the following raw materials: radix Glehniae, vinegar tortoise plastron, wine glossy privet fruit, chinese angelica, prepared rehmannia root, wine cornus, semen cuscutae, medlar, wine cistanche, fragrant solomonseal rhizome, white paeony root, vinegar nutgrass galingale rhizome and mulberry.

Further, the traditional Chinese medicine composition comprises the following raw materials in parts by weight: 10-20g of radix glehniae, 5-20g of vinegar tortoise plastron, 10-20g of wine glossy privet fruit, 10-20g of Chinese angelica, 10-20g of prepared rehmannia root, 10-20g of wine cornus, 10-20g of semen cuscutae, 10-20g of medlar, 10-20g of wine cistanche, 10-20g of polygonatum odoratum, 6-15g of white paeony root, 6-15g of vinegar nutgrass galingale rhizome and 5-20g of mulberry.

Further, the traditional Chinese medicine composition comprises the following raw materials in parts by weight: 15g of radix glehniae, 10g of vinegar tortoise plastron, 15g of wine glossy privet fruit, 10g of Chinese angelica, 10g of prepared rehmannia root, 15g of wine cornus, 10g of semen cuscutae, 15g of medlar, 10g of wine cistanche, 15g of rhizoma polygonati officinalis, 10g of white paeony root, 10g of vinegar nutgrass galingale rhizome and 10g of mulberry.

Further, the traditional Chinese medicine composition also comprises one or more auxiliary medicines for improving or treating the concurrent symptoms.

Further, the dosage form of the traditional Chinese medicine composition comprises any one of decoction, solution, granule, suspension, capsule, powder, tablet, emulsion, dripping pill, injection, suppository, enema, aerosol, patch or drop.

Based on the same inventive concept, a second aspect of the present application provides a method for preparing the Chinese medicinal composition according to any one of the above first aspects, comprising: weighing the raw materials according to the parts by weight, mixing the raw materials, and adding water for decoction to obtain the traditional Chinese medicine composition for treating or improving the ovarian reserve function.

Further, the raw materials are mixed and then decocted with water for at least two times, and the decoction obtained by at least two times of decoction is combined to obtain the water decoction of the traditional Chinese medicine composition.

Based on the same inventive concept, the third aspect of the present application provides an application of the traditional Chinese medicine composition of any one of the first aspect or the traditional Chinese medicine composition prepared by any one of the second aspect in preparing a medicine for treating or improving the ovarian reserve function.

The traditional Chinese medicine composition (also called Harv's yin-nourishing decoction) for treating or improving the hypofunction of ovarian reserve is an empirical formula for treating DOR in Harv gynaecology, and has the characteristics of Harv's menstruation regulation according to the aspects of liver menstruation regulation, menstruation regulation and blood nourishing, such as water nourishing and fire nourishing, menstruation regulation and blood nourishing before qi regulation, menstruation regulation and blood nourishing, spleen strengthening and stomach protecting before menstruation regulation and menstruation regulation. The prescription can improve DOR ovarian function and reproductive dysfunction from the aspects of nourishing water, containing wood, nourishing yin and blood. Meanwhile, the method has low price and simple manufacturing process, can greatly reduce the treatment cost and lighten the social and economic pressures of patients.

The vinegar tortoise plastron and the prepared rehmannia root are used as the monarch, and the vinegar tortoise plastron is a blood and meat loving product, has the effects of tonifying essence and marrow, tonifying kidney and nourishing blood, nourishing yin and suppressing yang, and can tonify liver and kidney yin to dispel internal heat, suppress liver yang to calm internal wind, and is used for hectic fever and night sweat due to liver and kidney yin deficiency, dizziness, yin deficiency and yang hyperactivity and the like. The prepared rehmannia root is used for replenishing essence and marrow, replenishing blood and nourishing yin, greatly nourishing five viscera and true yin, and greatly nourishing five viscera and true water, and is used for treating blood deficiency and sallow complexion, soreness and weakness of waist and knees, palpitation and insomnia and the like due to essence and blood deficiency. The main components of the vinegar tortoise plastron are Aspartic Acid (Aspartic Acid), threonine (Threonine), methionine (Methionine), phenylalanine (Phenylalanine), leucine (leucoine) and calcium carbonate (Calcium Carbonate), so that the main chemical components are amino acids. The quality of oocytes is related to the amino acid turnover rate. Oxidative Stress (OS) can lead to endocrine dysfunction of the ovaries, reduced quality of oocytes, induction of apoptosis of Granulosa Cells (GCs), and thus induction of follicular atresia, which is a major cause of impaired ovarian function. The research shows that the amino acid can counteract OS caused by exogenous substances and protect reproductive system, so that the amino acid in the tortoise plastron can counteract OS caused by exogenous substances, thereby improving oocyte quality and ovary function. The vinegar tortoise plastron and the prepared rehmannia root are compatible, so that the DOR ovarian reserve function and the maturation and development of follicles can be better improved.

The ministerial drugs in the recipe are semen cuscutae to nourish yin and yang, and the ministerial drugs in the recipe are used for nourishing yin and yang and are used for resolving yin in yang. It is combined with cistanche deserticola to tonify kidney yang and replenish essence and blood to treat yin from yang. The wine cornus, medlar and glossy privet fruit have the effects of nourishing liver and kidney, replenishing essence and improving eyesight, and are matched with mulberry to nourish yin and enrich blood, promote the production of body fluid and moisten dryness so as to assist the functions of rehmannia glutinosa and tortoise plastron to nourish yin. Dang Gui can tonify blood and regulate menstruation, and has the actions of activating blood, promoting blood circulation, and tonifying blood without stagnation. It is combined with Sheng Rong and sang Mulberry to moisten intestines and relieve constipation and treat constipation due to deficiency of fluid from liver and kidney yin. White peony root, radix Paeoniae alba nourishes blood, astringes yin, calms liver and softens liver to assist prepared rhizome of rehmannia and Chinese angelica to replenish essence and nourish blood, assist tortoise plastron to calm liver yang. And white peony root, cornus wine and mulberry are used for tonifying kidney and storing, astringing liver yin, astringing essence and arresting sweating due to sour and astringent nature, so as to prevent the storage loss caused by liver and kidney deficiency, the blood and essence leakage and the night sweat and the body fluid.

Radix Glehniae nourishes lung and stomach yin and body fluid, and is combined with Yu Zhu to nourish yin and moisten dryness, promote salivation and quench thirst, and is used for yin deficiency and internal heat, dry mouth and dry throat, and aims at assisting Jin Pingmu and strengthening soil to make wood. The glehnia root and the fragrant solomonseal rhizome have the effects of moistening lung, nourishing stomach, promoting the production of body fluid and increasing liquid, and have definite curative effect in clinical compatibility treatment of DOR. The multiple effective components of radix Glehniae-rhizoma Polygonati Odorati can be used for treating DOR by regulating multiple protease activities to participate in cell proliferation apoptosis, hormone metabolism, inflammation, immune response, etc. The radix glehniae-polygonati officinalis medicine has remarkable effects on the aspects of antioxidation, anti-inflammatory, anti-aging, immunity improvement and the like, and the antioxidation is a key measure for delaying the decline of the ovarian function. Therefore, the glehnia root and the fragrant solomonseal rhizome can delay the decline of the ovarian function, thereby improving the DOR ovarian reserve function.

All the above medicines are ministerial medicines to nourish yin essence, yin blood and yin fluid.

Finally, the women are easy to be flung and depressed, the nutgrass galingale rhizome is used for assisting the liver to relieve depression and the blood circulation to nourish yin but not greasy. The whole formula mainly nourishes water and contains wood, nourishes yin and nourishes blood, and reflects the thought that the disease causes of the haar genre.

The traditional Chinese medicine composition is prepared under the guidance of thousands of years of traditional Chinese medicine practice theory, and it is known by a person skilled in the art that due to the complexity of traditional Chinese medicine formulas, the traditional Chinese medicine scientific formulas are an innovative process, and the effective formulas can be obtained only by taking the synergy among the traditional Chinese medicine components into consideration in the formula process, even if two formulas with very high similarity can change the drug property of the whole formula due to the difference of one traditional Chinese medicine. The application selects the specific thirteen natural plant traditional Chinese medicine components for combination and mutually synergistic cooperation, has the characteristic of multi-target point play for treating DOR, can effectively improve DOR ovarian function, promotes follicular maturation and development, has definite therapy and no obvious adverse reaction.

From the above, the traditional Chinese medicine composition for treating or improving the ovarian reserve function and the preparation method and the application thereof can improve the DOR ovarian reserve function and the maturation and development of follicles, can obviously improve the levels of AMH, HIF-1 alpha, VEGF, ang-1 and Ang-2 in human AFC and serum, and can reduce FSH and FSH/LH; the increase of the number of follicles at each level of DOR mice is promoted, the ovarian atrophy is improved, the granulosa cells are orderly arranged, and the vascularity is increased; meanwhile, the expression of DOR mouse ovary SIRT1, VEGF, HIF-1 alpha, ang-1 and Ang-2 proteins is promoted, and NF-KB expression is inhibited, so that the ovary reserve function is improved.

Drawings

In order to more clearly illustrate the technical solutions of the present application or related art, the drawings that are required to be used in the description of the embodiments or related art will be briefly described below, and it is apparent that the drawings in the following description are only embodiments of the present application, and other drawings may be obtained according to the drawings without inventive effort to those of ordinary skill in the art.

FIG. 1 is a graph of analysis results of common targets of vinegar tortoise plastron and DOR;

FIG. 2 is a PPI network diagram of a therapeutic DOR effect target of tortoise plastron with vinegar;

FIG. 3 is a network diagram of the treatment of DOR "drug-component-target-disease" with vinegar tortoise plastron;

FIG. 4A is a graph of the biological process analysis results of GO functional enrichment analysis of vinegar tortoise plastron treatment DOR;

FIG. 4B shows the results of a cellular component analysis of GO functional enrichment analysis of vinegar tortoise plastron treatment DOR;

FIG. 4C shows the molecular functional analysis results of GO functional enrichment analysis of the treatment DOR with vinegar tortoise plastron;

FIG. 5 is a graph showing the results of KEGG functional enrichment analysis of DOR treatment with vinegar tortoise plastron;

FIG. 6 is a diagram of a network of DOR component-target-pathway for treatment with tortoise plastron vinegar;

FIG. 7 is a graph of analysis results of the joint targets of radix Glehniae-rhizoma Polygonati Odorati and DOR;

FIG. 8 is a network diagram of DOR protein interactions of radix Glehniae-rhizoma Polygonati Odorati treatment;

FIG. 9 is a diagram of a DOR drug-component-target-disease interaction network for radix Glehniae-rhizoma Polygonati Odorati treatment;

FIG. 10 is a schematic diagram of key DOR targets for radix Glehniae-rhizoma Polygonati Odorati treatment;

FIG. 11 is a bar chart of an enrichment analysis of the action target of radix Glehniae-rhizoma Polygonati Odorati on DOR;

FIG. 12 is a KEGG pathway enrichment analysis bubble plot of Glehniae radix-rhizoma Polygonati Odorati treatment DOR;

FIG. 13 is a schematic diagram of cell morphology at each stage of the estrus cycle (Rayleigh staining 100X), wherein A is the estrus, B is the estrus, C is the estrus, D is the estrus; (1) the arrows are nucleated epithelial cells, (2) the arrows are keratinocytes, (3) the arrows are leukocytes;

fig. 14 is a schematic diagram of the morphology of the ovary tissue of each group of mice (HE staining 50×), in which a is a blank group, B is a model group, C is a western medicine group, D is a chinese medicine group, E is a chinese medicine control 1 group, and F is a chinese medicine control 2 group; arrow a is the original follicle, arrow b is the primary follicle, arrow c is the secondary follicle, arrow d is the glaaffer follicle, arrow e is the corpus luteum, and arrow f is the atresia follicle;

FIG. 15 is a graph showing the results of comparison of the expression of ovarian tissue proteins in mice of each group.

Detailed Description

For the purposes of promoting an understanding of the principles and advantages of the disclosure, reference will now be made in detail to the following specific examples.

It should be noted that unless otherwise defined, technical terms used in the following examples have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.

EXAMPLE 1 theoretical study of Harvey's yin-nourishing decoction

1. Network pharmacology-based analysis of mechanism of action of vinegar tortoise plastron in DOR treatment

1 study method

1.1 Vinegar tortoise plastron component and its action target screening

Taking 'GUI BAN' as a keyword, setting a Score cutoff of more than 20 minutes and a P-value of less than 0.05 by default, searching chemical components and targets of the vinegar tortoise plastron through BATMAN-TCM (http:// bionet. Ncpsb. Orc/BATMAN-TCM /), merging and de-weighting to obtain chemical components and target information of the vinegar tortoise plastron, and carrying out name standardization through a Uniprot database (https:// www.uniprot.org /).

1.2 screening of targets related to reduced ovarian reserve (diminished ovarian reserve, DOR)

Setting 'diminished ovarian reserve' as a keyword, searching target information related to DOR in an on-line human Mendelian genetic database (Online Mendelian Inheritance in Man, OMIM, https:// www.omim.org /), a DisGeNET database (https:// www.disgenet.org /) and a GeneCards database (https:// www.genecards.org /), merging and de-duplicating to obtain target information of DOR, and carrying out name standardization through a Uniprot database.

1.3 construction of the "drug-ingredient-target-disease" network

And (3) introducing the action target of the components of the vinegar tortoise plastron and the relevant target of the DOR into a Venny 2.1.0 website (https:// bioinfo gp. Cnb. Csic. Es/tools/Venny /) to obtain an intersection target, namely the action target of the vinegar tortoise plastron for treating the DOR. The tortoise plastron, DOR, chemical components of tortoise plastron and DOR action target for tortoise plastron treatment are imported into Cytoscape software (https:// Cytoscape. Org /), so as to construct a network diagram of medicine-component-target-disease.

1.4 Protein-Protein interaction (PPI) network construction

The DOR action target of the vinegar tortoise plastron treatment is imported into STRING database (https:// www.string-db. Org /), the selected species is "Homo sapiens", the condition is set as "combinedscore > 0.15", and PPI results are downloaded. And importing the result into Cytoscape software to further construct the PPI network diagram.

1.5 Gene Ontology (GO) and genome encyclopedia (Kyoto Encyclopedia of Genes and Genomes, KEGG) analysis

And performing GO function enrichment analysis and KEGG channel enrichment analysis on the DOR action target of the vinegar tortoise plastron treatment by using a DAVID database (https:// DAVID. Ncifcrf. Gov /). GO functional enrichment assays mainly include biological processes (Biological process, BP), cellular composition (Cellular component, CC), and molecular function (Molecular function, MF). Screening the first 20 results of GO enrichment analysis to prepare a bubble chart for visualization; the 20 pathways were visualized as bubble charts prior to screening KEGG enrichment analysis.

1.6 "component-target-pathway" network construction

The chemical components of the vinegar tortoise plastron, the DOR action target spot of the vinegar tortoise plastron treatment and the KEGG enrichment pathway are imported into Cytoscape software, and a component-target spot-pathway network diagram is constructed.

2 results

2.1 screening of chemical components of Vinegar tortoise plastron

6 chemical components of tortoise plastron with vinegar, including Aspartic Acid (Aspartic Acid), threonine (Threonine), methionine (Methionine), phenylalanine (Phenylalanine), leucine (Leucine) and calcium carbonate (Calcium Carbonate), were obtained by BATMAN-TCM database search. The chemical components correspond to 445 related targets, and 283 targets are obtained after merging and de-duplication.

2.2 screening of DOR action targets of vinegar tortoise plastron treatment

169 DOR related targets are obtained through OMIM database mining; the DisGeNET database is mined to obtain 43 DOR related targets; the GeneCards database is mined to obtain 1230 DOR related targets; and combining and de-duplicating to obtain 1262 DOR related targets. The screened action targets of the components of the vinegar tortoise plastron and the DOR related targets are imported into Venny 2.1 (https:// bionfogp. Cnb. Csic. Es/tools/Venny /), and 63 common targets are obtained and are used as the action targets of the vinegar tortoise plastron for treating DOR (figure 1).

2.3 "drug-composition-target-disease" network construction

The "drug-component-target-disease" interaction relationship network was obtained using Cytoscape software processing (fig. 3). The total number of nodes in the network is 71, 150 edges are formed, the node on the left side represents the chemical component of tortoise plastron, the node on the middle represents the DOR action target of tortoise plastron treatment, the connecting line between the nodes represents the corresponding relation between the nodes, the area and the color transparency of the node represent the degree value, and the greater the area and the darker the color, the more important the node. The tortoise plastron treatment DOR is based on complex synergistic effects of multiple components and multiple targets.

2.4 PPI network construction and analysis

The 63 tortoise plastron therapeutic DOR action targets are input into the STRING database, wherein no interaction relationship exists between 3 targets and other targets, so that 60 targets are finally obtained to participate in the PPI network (figure 2). Centrality is a main topology parameter for determining importance of a network node, and the larger the Degree value (deviee), the Betweenness (Betweenness), and the shortest path (routing), the more important the node in the PPI network. The median average value is 78.77, the shortest path centrality average value is 0.440, and the degree average value is 20.00 as calculated by the Network Analyzer. The total of 3 parameters of 11 targets are higher than the average value, and are key targets for treating DOR by using the vinegar tortoise plastron (Table 1). In the PPI network, the node color and size represent the target egg whiteness value, and the larger the node is, the darker the color is, and the explanatory value is larger; the more lines represent the interaction relationship between proteins, the greater the degree of association, the thicker the lines, and the higher the binding score (combined score).

TABLE 1 Vinegar tortoise plastron treatment DOR key target

2.5GO functional enrichment analysis

GO function enrichment analysis is carried out on 63 tortoise plastron treatment DOR action targets through the DAVID database, 252 BP enrichment results, 38 CC enrichment results and 55 MF enrichment results are obtained in total. The top 20 items were selected for visual presentation (fig. 4A, 4B, and 4C) ordered by P values. Among them, BP mainly involves adenylate cyclase activating adrenergic receptor signaling antibodies (adenylate cyclase-activating adrenergic receptor signaling pathway), mitochondrial electron transport, succinic acid to ubiquinone (mitochondrial electron transport, succinate to ubiquinone), arginine catabolism process (arginine catabolic process), aging (aging), tricarboxylic acid cycle (tricarboxylic acid cycle), positive regulation of cytosolic calcium ion concentration (positive regulation of cytosolic calcium ion concentration involved in phospholipase C-activating G-protein coupled signaling pathway), and the like; CC mainly involves mitochondria (mitochondron), succinate dehydrogenase (complex II) [ mitochondrial respiratory chain complex II, succinate dehydrogenase complex (ubiquinone) ], plasma membrane component (integral component of plasma membrane), lipid raft (membrane raft), mitochondrial matrix (mitochondrial matrix), perinucleosome (perikaryon), mitochondrial inner membrane (mitochondrial inner membrane), glutamatergic synapse (glutamatergic synapse), plasma membrane cavern (canela), cytoplasmic membrane (plasma membrane), mitochondrial outer membrane (mitochondrial outer membrane), etc.; MF mainly involves adenine flavin dinucleotide (flavin adenine dinucleotide binding), protein binding (protein binding), nitric oxide synthase (nitrone-oxide synthase activity), succinic acid dehydrogenase (succinate dehydrogenase) activity, norepinephrine (norepinephrine binding), epinephrine (epinephrine binding), arginine binding (arginine binding), oxidoreductase activity, acting on paired donors, binding or reducing molecular oxygen (oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, NAD (P) H as one donor, and incorporation of one atom of oxygen), amino acid binding (amino acid binding), and the like.

2.6KEGG pathway enrichment analysis

Inputting 63 tortoise plastron therapeutic DOR action targets into a DAVID database for KEGG pathway enrichment analysis, and obtaining 50 pathways through total enrichment. The top 20 lanes are selected for visualization processing (fig. 5) ordered by P values. Among them, cAMP signaling pathway (cAMP signaling pathway), cGMP-PKG signaling pathway (cGMP-PKG signaling pathway), calcium signaling pathway (Calcium signaling pathway), renin secretion (Renin secretion), amino acid biosynthesis (Biosynthesis of amino acids) and the like are closely related to tortoise plastron treatment DOR.

2.7 "component-target-pathway" network construction

The "component-target-pathway" interaction relationship network was obtained using the Cytoscape software process (fig. 6). Nodes on the left side of the network represent chemical components of the tortoise plastron, nodes on the middle part of the network represent therapeutic DOR action targets of the tortoise plastron, nodes on the right side of the network represent therapeutic DOR related paths of the tortoise plastron, and connecting lines among the nodes represent corresponding relations between the nodes. From the figure, the vinegar tortoise plastron plays a role in treating DOR through multiple components, multiple targets and multiple signal paths.

3 conclusion analysis

3.1 chemical composition analysis of Vinegar tortoise plastron

Through database mining analysis, the main chemical components of the vinegar tortoise plastron are amino acids. The quality of oocytes was found to be related to amino acid turnover. Oxidative Stress (OS) can lead to endocrine dysfunction of the ovaries, reduced quality of oocytes, induction of apoptosis of Granulosa Cells (GCs), and thus induction of follicular atresia, which is a major cause of impaired ovarian function. It was found that amino acids counteract OS caused by exogenous substances, protecting the reproductive system.

Reduced Luteinizing Hormone (LH) concentrations in follicles can lead to reduced oocyte quality. It was found that aspartic acid was able to induce LH synthesis and release in rats, improving oocyte quality. The preovulatory follicular fluid was found to contain a high concentration of threonine, which plays an important role in maintaining the osmotic balance between extracellular fluid and follicular cells. Protein kinase B (Akt) is involved in cellular processes such as cell growth, survival, proliferation and metabolism. Akt activity is regulated downstream of phosphatidylinositol-3-kinase (PI 3K) in response to various extracellular stimuli. This process is mediated by serine or threonine phosphorylation in downstream substrates. Akt, in mammalian ovaries, synergistically regulates follicular and oocyte development with other kinases. In addition, the kinase can regulate GCs apoptosis throughout folliculogenesis. In oocytes, akt is involved in controlling meiotic recovery and regulates polar body emissions and spindle tissue in metaphase II. Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates cell growth and proliferation. AKT and mTOR signaling pathways play a fundamental and coordinated role in regulating primordial follicular dormancy and maintaining female reproductive life length. In addition to primordial follicles, mTOR signaling is also important for GCs growth in sinus follicles. Amino acids such as leucine, arginine and glutamine can stimulate mammalian mTOR pathways that play a key role in primordial follicle activation. Researchers can accelerate activation of primordial follicles and alter expression of the corresponding factors by adding leucine, glutamine, arginine, or a combination thereof to the mouse ovarian tissue culture medium.

3.2 analysis of DOR action target for treatment with Vinegar tortoise plastron

The research screens 63 action targets closely related to DOR treatment of tortoise plastron, PPI network analysis discovers interaction among the targets, and key targets such as brain-derived neurotrophic factor (BDNF), tumor Necrosis Factor (TNF), prostaglandin endoperoxide synthase 2 (PTGS 2), tyrosine Hydroxylase (TH), nitric oxide synthase 3 (NOS 3), nitric oxide synthase 2 (NOS 2) and the like are obtained through screening. It was found that BDNF plays an important role in the development and regulation of ovaries, in terms of egg production, follicular recruitment and germ cell survival, and in terms of nuclear and cytoplasmic maturation of oocytes. TNF is a non-glycosylated protein of molecular weight 17kDa, which has a wide range of biological roles including not only regulation of pro-inflammatory responses, but also control of cell differentiation, tissue turnover and recombination. Previous studies have found that TNF plays multiple roles in ovarian function in a variety of species. The main function of the Corpus Luteum (CL) is to produce progesterone. Adequate progesterone is critical to determine the physiological duration of the estrus cycle and to achieve successful pregnancy. TNF has luteinizing and luteinizing effects in CL. Inflammatory processes are necessary for proper follicular development and many physiological reproductive processes. Ovulation is triggered by gonadotrophin excitations which induce expression of PTGS2 in the GCs of the preovulatory follicles. Each ovulation is accompanied by an acute inflammation driven by PTGS2 expression and a physical tear of the ovarian tissue at the site of release of the oocyte. In addition, TNF- α is also an important inflammatory factor in humans, and can induce neutrophil infiltration, causing an inflammatory response. Excessive inflammation may lead to poor oocyte quality or anovulation. Researchers find that moxibustion can up-regulate Nrf2/HO-1 signal pathway, inhibit TNF-alpha expression, and improve ovarian reserve function of mice. Nitric Oxide (NO) has a broad meaning in the regulation of ovarian function. It was found that NO has the effects of inhibiting GCs apoptosis, increasing estradiol (E2) level in follicular fluid, and increasing follicular size. NO exists in cells mainly in three subtypes: neural NOS (nNOS/NOS 1), inducible nitric oxide synthase (iNOS/NOS 2). And endothelial nitric oxide synthase (eNOS/NOS 3). NOS2 in immature follicular GCs may inhibit apoptosis of GCs, thereby preventing follicular locking. Several studies have shown that gonadotrophin increases expression and activity of NOS 3. Tyrosine Hydroxylase (TH) plays a functional role in ovarian growth, maturation and ovulation.

3.3 analysis of DOR bioinformatics for treatment with Vinegar tortoise plastron

According to GO analysis, tortoise plastron may treat DOR by modulating various biological processes such as mitochondrial-mediated granulosa apoptosis, OS, energy metabolism, amino acid biosynthesis, etc. The KEGG pathway enrichment results mainly relate to aspects of cAMP signaling pathway (cAMP signaling pathway), cGMP-PKG signaling pathway (cGMP-PKG signaling pathway), calcium signaling pathway (Calcium signaling pathway), renin secretion (Renin secretion), amino acid biosynthesis (Biosynthesis of amino acids) and the like. Pituitary gonadotrophin, follicle Stimulating Hormone (FSH) and LH are capable of promoting follicular recruitment, and sinus follicle growth, maturation and selection. Expression of Follicle Stimulating Hormone Receptor (FSHR) in GCs is a prerequisite for maintenance of monthly follicle recruitment, FSH-mediated oocyte maturation, selection and estrogen synthesis. LH modulates proliferation and anti-apoptotic signals in GCs, and is accompanied by androgen production in membranous cells, ultimately inducing dominant follicular ovulation and progesterone production. The cAMP signaling pathway, also known as the adenylate cyclase pathway, is a G protein coupled receptor-triggered signaling cascade for cellular communication. G-protein coupled receptors, FSHR and luteinizing hormone receptor (LHCGR) activate FSH and LH biological functions through the cAMP/protein kinase A (/ PKA) pathway. GCs apoptosis is the main cause of follicular occlusion. It was found that Reactive Oxygen Species (ROS) -induced OS also plays an important role in regulating the status and function of GCs. ROS are natural byproducts of intracellular aerobic metabolism in mitochondria. ROS concentrations in normal cells help to maintain physiological function, but excessive ROS accumulation can disrupt cellular homeostasis and lead to OS-induced cellular damage and mitochondrial dysfunction. During follicular development, metabolic rates are often accelerated to meet nutritional and energy requirements, which inevitably lead to excessive production ROS in amounts and further induces OS in follicles. ROS levels in follicular fluid are closely related to follicular development, occlusion and ovarian disease. Excessive ROS induced autophagy of oocytes, ultimately leading to follicular locking and premature ovarian failure. It was found that elevated cAMP levels can enhance antioxidant capacity and activation of cAMP-PKA can prevent the shift in mitochondrial permeability. Research results show that cAMP can improve the oxidation resistance of cells by mediating cAMP-PKA-Bad pathway in mitochondria, restore mitochondrial function, inhibit apoptosis and alleviate GCs oxidation injury. The cGMP-PKG signaling pathway can regulate physiological processes such as apoptosis and inflammatory responses. NO stimulates guanylate cyclase to produce cyclic guanosine monophosphate (cGMP), and in turn activates cGMP-dependent Protein Kinases (PKGs) and the like, involved in meiosis of oocytes, affecting maturation of oocytes. Maturation of oocytes is associated with changes in plasma membrane electrical properties and changes in ion channel function and distribution. Ion channels and transporters play an important role in regulating membrane potential, ca ²⁺ Play a critical role in regulating intracellular signaling pathways. Ca (Ca) ²⁺ Cell physiology is regulated by modulating the meiotic cell cycle and apoptosis of oocytes. Ca released from the intracellular storage and/or influx from the extracellular medium ²⁺ Can lead to intracellular Ca ²⁺ Levels and ROS moderately increased. Ca (Ca) ²⁺ Is triggered by increased ROS levels in the physiological range, leading to a loss of maturation-promoting factor (MPF) leading to diploid resumption of meiosis and metaphase II arrest of the oocyte. When Ca is ²⁺ Continued elevation beyond the physiological range induces excessive ROS production to cause aging of the oocyte OS. Increased Ca ²⁺ Triggering Fas ligand mediated apoptosis of the oocyte. In addition, OS also triggers mitochondrial mediated apoptosis of oocytes. It has also been found that molecular blockade of the ovarian renin-angiotensin system (OVRAS) inhibits oocyte maturation and ovulation. Follicular membrane cells are a source of large amounts of renin in human ovarian follicular fluid. Angiotensin II is an active ingredient of the renin cascade and has many potential roles in the ovary and uterus. Key roles of OVRAS in ovarian physiology and disease, OVRAS is important for follicular development/occlusion, ovulation and steroid hormone secretionInfluence. The activity of OVRAS is regulated by gonadotrophin and depends on the activation of proteolytic enzymes in the growth follicular region. Angiotensin and angiotensin receptors are widely distributed in ovarian follicles, preovulatory follicular cells and GCs, and postovulatory parietal particles-luteal cells, regulating the production of steroid hormones. In humans, caloric restriction inhibits the hypothalamic-pituitary-gonadal axis by decreasing LH secretion and by disrupting the ovulation cycle through central inhibition. Past studies have found that puberty delay and reduced fertility are both associated with caloric restriction. The reduction in available calories is a major factor in inhibiting LH release and the ovulation cycle. There are studies showing that dietary amino acid balance strongly affects the fertility of drosophila, and this finding suggests that both the calorie content and the amino acid level of the diet affect reproductive function. Malnutrition, excessive physical exercise and psychological stress can lead to reduced ovarian activity, reduced estrogen levels, and thus, the initiation of hypothalamic amenorrhea. It was found that dietary deficiency of essential amino acids rapidly resulted in cessation of the oestrus cycle in rats and that amino acid imbalance in the ovaries prevented oocyte maturation, stopped ovulation and stopped oestrus cycle. Carbon metabolism provides the necessary carbon valences and energy for amino acid synthesis.

In conclusion, network pharmacology is adopted to explore the active ingredients, action targets and signal paths of the tortoise plastron treatment DOR, and the characteristics of multiple ingredients, multiple targets and multiple paths of the tortoise plastron treatment DOR are found, so that theoretical basis and scientific basis are provided for subsequent researches.

2. Based on network pharmacology, the action mechanism of radix glehniae-polygonati officinalis medicine on treating ovarian reserve function decline is explored

1 materials and methods

1.1 DOR target information collection

DOR related target information is searched in a GeneCards database (https:// www.genecards.org /), a DisGeNET database (https:/www.disgenetorg /) and a human online Mendelian genetic database (Online Mendelian Inheritance in Man, OMIM, https:// www.omim.org /) by taking "Deminished Ovarian Reserve" DOR as a keyword. And merging and de-duplicating the disease targets retrieved from the three disease databases to obtain DOR related targets.

1.2 screening of effective active ingredients and target spots of radix glehniae and rhizoma polygonati officinalis

The chemical components of the glehnia root and the fragrant solomonseal rhizome are searched through a pharmacological database and an analysis platform (Traditional Chinese Medicine Database and Analysis Platform, TCMSP, http:// sp.nwu.edu.cn/tcmsp.php) of a traditional Chinese medicine system, and the active components are primarily screened under the conditions that the Oral Bioavailability (OB) is more than or equal to 30 percent and the drug property (DL) is more than or equal to 0.18. Retrieving the smile sequence numbers of the components obtained by screening according to the PubCHem Cid of the components in PubCHem (https:// Pubchem. Ncbi. Nih. Gov /); the person without PubCHem Cid realizes the conversion of component codes in OpenBabel software according to the 3D structure of the effective components of the traditional Chinese medicine, and obtains a component smile serial number. The active protein target of the drug action is predicted by using each component smile sequence number and selecting a species of 'Homo sapiens' through a SwisstargetPreedition database (http:// www.swisstargetprediction.ch /), and the active protein target is standardized by using a Uniprot protein database (http:// www.uniprot.org) as protein target information of the compound action.

1.3 construction of drug-ingredient-target-disease interaction network

The DOR disease target and the radix glehniae-polygonati officinalis component target are led into a Venny 2.1.0 platform (https:// bioinfo gp. Cnb. Cis/tools/Venny /), and a Wen graph is drawn to take intersection, namely the effect target of the radix glehniae-polygonati officinalis medicine on treating DOR. The interaction network diagram of the radix glehniae-polygonati officinalis drug pair and DOR 'drug-component-target-disease' is constructed by introducing radix glehniae "," radix polygonati officinalis "," DOR "," radix glehniae active ingredient "," radix polygonati officinalis active ingredient "and" radix glehniae-polygonati officinalis drug pair treatment DOR action target "into Cytoscape3.9.1 software (https:// cytoscape. Org /).

1.4 Protein-Protein interaction (PPI) network construction

And importing the combined action target points of the two into a String database (https:// String-db. Org /) to construct a PPI network model, wherein the type of the selected protein is 'Homo sapiens', the minimum interaction value is set to be 'medium confidence (0.400'), and the disconnected nodes in the network are hidden to obtain a PPI result. And visualizing the PPI result by using Cytoscape3.9.1 software, and further constructing a PPI network diagram. Further analysis was performed using the Cytohubba insert selection value (Degree) for the target of front 30.

1.5 Gene Ontology (GO) and genome encyclopedia (Kyoto Encyclopedia of Genes and Genomes, KEGG) analysis

The target of the radix glehniae-polygonati officinalis drug on treatment DOR is imported into a DAVID database (https:// DAVID. Ncifcrf. Gov /), select Identifier is selected as Official Gene symbol, gene list is checked, homosapiens is selected as a species, and GO function enrichment analysis and KEGG channel enrichment analysis are performed. GO functional enrichment assays mainly include biological processes (Biological process, BP), cellular components (Cellular component, CC), and molecular functions (Molecular function, MF). And screening GO enrichment analysis TOP10 data according to the P value ascending order, and drawing a GO enrichment analysis bar chart and a KEGG channel enrichment analysis bubble chart by adopting a microbial communication platform (http:// www.bioinformatics.com.cn /).

2 results

2.1 DOR target collection

Selecting 1230 relevant targets with score >40 from the GeneCards database; 43 targets are obtained by searching in the DisGeNET database, and 169 targets are obtained by searching in the OMIM database. And removing the repeated values to obtain 1262 DOR disease targets.

2.2 screening of effective active ingredients and target spots of radix Glehniae-rhizoma Polygonati Odorati

According to the screening conditions, 8 radix glehniae active ingredients such as quercetin, beta-sitosterol and 8 radix polygonati officinalis active ingredients such as polygonatum odoratum polysaccharide and N-cis feruloyl tyramine are finally obtained. The two are not repeated, so that 16 effective chemical components of the glehnia root-polygonatum odoratum medicine are obtained in total, and the detailed information of the compounds is shown in table 2. 331 radix glehniae active ingredient acting targets, 326 radix polygonati officinalis active ingredient acting targets, and deleting repeated values after merging to obtain 502 relevant targets.

TABLE 2 basic information of Glehniae radix-Polygonatum odoratum medicine on active ingredients

2.3 construction of "drug-ingredient-target-disease" interaction network

1262 DOR disease targets and 502 radix glehniae-polygonati officinalis drug-to-active ingredient targets are led into a Venny 2.1.0 platform to draw a Wen graph, and 162 intersection targets are finally obtained, namely, the effect targets of the radix glehniae-polygonati officinalis drug on DOR treatment. The screening results are shown by wien plot (fig. 7).

The interaction network diagram of the radix glehniae-polygonati officinalis drug pair and DOR 'drug-component-target spot-disease' is drawn by using Cytoscape3.9.1 software (figure 9), and the active components with the top five rank of the degree value are polygonatum odoratum polysaccharide, bergamot, quercetin, N-cis feruloyl tyramine and psoralen are further analyzed. The top triangle node in the network represents a "DOR" disease; the center nodes on the left side and the right side respectively represent radix glehniae and rhizoma polygonati officinalis; the plurality of nodes of the outer ring of the central nodes of the left side and the right side represent effective active ingredients of medicines, and the effective active ingredients of the polygonatum are denoted by corresponding codes; the node in the middle part represents the acting target of radix glehniae-polygonati rhizoma drug on DOR treatment; the connection lines between the nodes represent the correspondence between the nodes. The radix glehniae-polygonati officinalis drug is based on complex synergistic effect of multiple components and multiple targets for treating DOR.

2.4 PPI network construction and analysis

162 targets of radix glehniae-polygonati officinalis for treating DOR are imported into a String database, and as 2 targets and other targets have no interaction relationship, the PPI network with 160 targets participating is finally obtained. The result was imported into cytoscape3.9.1 software to construct a PPI network (fig. 8). The centrality is a main topological parameter for judging the importance of a network node, and the larger the Degree value (deviee), the Betweenness and the shortest path (Closeness) are, the more significant the importance of the node in the PPI network is. The PPI network comprises 160 nodes and 2003 edges, wherein the size and color of the nodes represent the degree value of a target point, and the larger the nodes are, the darker the color is, which indicates that the degree value of the nodes in the network is larger; the more lines represent the interaction relationship between proteins, the greater the degree of association, the thicker the lines, and the higher the binding score (combined score).

The target points with the front 30 degree values, including AKT1, TNF, EGFR, STAT, MAPK3 and the like, are calculated by a Network Analyzer and are key target points (figure 10) for treating DOR by coastal glehnia root-polygonatum, wherein the horizontal axis represents the degree value, and the vertical axis represents the key target point name.

2.5 GO functional enrichment analysis

GO enrichment analysis is performed on the key targets of the 162 glehnia littoralis-polygonatum odoratum drugs for DOR treatment by using a David data platform, TOP10 data of BP, CC and MF enrichment results are respectively screened, and are drawn into a bar graph (fig. 11). In fig. 11, the left bar represents biological processes (Biological process, BP), the middle bar represents cellular components (Cellular component, CC), the right bar represents molecular functions (Molecular function, MF), and the ordinate energy score represents Enrichment factors, the larger the value the more important.

The results show that GO analysis resulted in 543 biological processes in total, including drug response (response to drug), aging (aging), negative regulation of apoptotic processes (negative regulation of apoptotic process), positive regulation of ERK1 and ERK2 cascades (positive regulation of ERK and ERK2 cascades), positive regulation of MAP kinase activity (positive regulation of MAP kinase activity), and the like; 71 cell components including plasma membrane (perinuclear region of cytoplasm), pericytoplasmic domain (neuronal cell body), neuronal cell bodies (membrane raft), cytoplasm (cytosol), mitochondria (mitochondron), etc.; and 16 molecular functions including protein tyrosine kinase activity (protein tyrosine kinase activity), identical protein binding (identical protein binding), protein binding (protein binding), and the like.

2.6KEGG pathway enrichment analysis

Inputting the action targets of 162 radix glehniae-polygonati officinalis medicines on DOR treatment into a DAVID database for KEGG pathway enrichment analysis, and obtaining 153 pathways through total enrichment. The top 20 paths were selected and plotted as bubble graphs in ascending order of P values (FIG. 12). The color of the bubble from light to dark represents-Lg (P) value from small to large, the area of the bubble represents the gene count of the pathway, and the horizontal axis represents the gene enrichment number of the pathway. The larger the bubbles, the darker the color, representing the more pronounced the enrichment.

The KEGG enrichment pathway shows that Glehniae radix-Polygonatum odoratum drug is closely related to the pathways such as endocrine resistance (Endocrine resistance), prolactin signaling pathway (Prolactin signaling pathway), phosphatidylinositol 3-kinase/protein kinase B (phosphotidylinosol-3-kinase/pro-tein kinase B, PI 3K/Akt) signaling pathway (PI 3K-AKT signaling pathway), FOXO signaling pathway (FoxO signaling pathway), apoptosis (Apoptisis), estrogen signaling pathway (Estrogen signaling pathway), HIF-1signaling pathway (HIF-1 signaling pathway) and the like for treatment of DOR. In addition, the radix glehniae-rhizoma polygonati officinalis drug pair is also closely related to the processes of treating DOR, such as cancer, insulin signaling pathway, etc. (FIG. 12).

3. Analysis of results

The radix glehniae and the rhizoma polygonati officinalis have the effects of moistening lung, nourishing stomach, promoting fluid production and increasing fluid, and have definite curative effects in clinical compatibility and treatment of DOR. The research is based on a network pharmacology method, and explores the complex network relation of radix glehniae-polygonati officinalis medicine on multiple components, multiple targets, multiple paths and DOR.

Ovarian granulosa cells are involved as the primary functional cells in primordial follicular development, follicular maturation, ovulation, luteal formation, etc. Studies have shown that protein expression levels in granulosa cells affect the growth and development of oocytes, and that apoptosis causes follicular locking, a key process leading to reduced ovarian reserve function. Mitochondria are the source of free radical generation and are also targets for free radical attack. Oxidative stress is considered to be a major driver of ovarian failure, and free radical accumulation-induced oxidative stress can impair mitochondrial function, causing follicular apoptosis and developmental disorders, leading to reduced ovarian reserve function. Antioxidant is therefore a key measure to delay the decline of ovarian function.

3.1 analysis of chemical composition of Glehniae radix-rhizoma Polygonati Odorati

From the interaction network diagram of medicine-component-target spot-disease, the main active components of the glehnia root-polygonatum medicine for treating DOR are polygonatum polysaccharide, quercetin, psoralen and the like.

Modern pharmacological researches have shown that the main components of radix glehniae and rhizoma polygonati officinalis have remarkable effects of resisting oxidation, inflammation, aging, improving immunity and the like. The polygonatum polysaccharide can enhance the scavenging ability of free radicals by improving the serum superoxide dismutase activity of the aging mice, inhibit lipid peroxidation and reduce the content of malondialdehyde, thereby reducing the damage degree of the organism tissues of the aging mice and delaying aging. Quercetin is a dihydroflavonoid phytoestrogen, has a structure similar to that of mammal estrogen 17 beta-estradiol, can be combined with estrogen receptors to exert estrogen-like effect, promotes development of follicles at various levels to a certain extent, and improves ovarian function; meanwhile, the quercetin is also an ideal peroxide free radical scavenger and an effective lipid peroxidation inhibitor, and a large number of animal experiments prove that through up-regulating oxidative stress related genes in ovaries, the oxidative stress in ovarian tissues is obviously reduced in a dose-dependent manner, the oxidation resistance of the ovaries is improved, the follicular development and the proliferation of granulosa cells at all levels can be promoted, the estrogen secretion of the granulosa cells is increased, and thus the aging of the ovaries is delayed. Psoralen binds to the Estrogen Receptor (ER) and initiates expression of a series of genes regulated by estrogen receptor elements to exert pseudo-estrogenic activity. Other compounds such as beta-sitosterol can promote proliferation of granulosa cells, inhibit apoptosis, relieve GnRH secretion reduction by affecting estrogen receptor signaling pathway, and exert antiaging effect.

3.2 analysis of the association of the composition with the target and the pathway of the Glehniae radix-Polygonatum odoratum drug

The research results show that the coastal glehnia root-fragrant solomonseal rhizome medicine can participate in various biological processes such as cell proliferation apoptosis, hormone metabolism, inflammation, immune response and the like by regulating various protease activities for treating DOR, and key gene targets of the coastal glehnia root-fragrant solomonseal rhizome medicine comprise AKT1, TNF, EGFR, mTOR, MAPK1, ESR1, SRC, AR and the like, and are mainly enriched in PI3K/Akt signal paths, apoptosis, estrogen signal paths and the like.

The PI3K/Akt signaling pathway is one of the most important signaling pathways for cell proliferation and apoptosis, and is closely related to oocyte growth, primordial follicular development, and proliferation, differentiation, apoptosis, autophagy of granulosa cells. Abnormal expression of the downstream molecules FOX03, mTOR in the pathway can lead to excessive activation of primordial follicular cells, promote apoptosis of granulosa cells, lead to ovarian hyperplasia, follicular depletion and DOR. Protein kinase (AKT 1) is an important downstream target enzyme in the P13K signaling pathway, playing an important role in regulating ovarian growth and maturation, and regulating apoptosis. AKT 1-underexpressed females have abnormal menstrual cycle, reduced fertility, significantly reduced primordial follicle numbers, and compensatory elevation of serum Luteinizing Hormone (LH). Furthermore, studies have shown that AKT1 overactivation leads to a defect in primordial follicular development and a decline in ovarian reserve function.

Estrogens are the major sex hormones in women, maintaining female reproductive system function, and involved in gonadotrophin secretion and follicular maturation. Estrogens exert their functions by binding to the estrogen receptor alpha protein (erα) encoded by the estrogen receptor 1 gene (ESR 1) and the estrogen receptor beta protein (erβ) encoded by the estrogen receptor 2 gene (ESR 2). ESR1 is expressed mainly on follicular membranes and exerts a corresponding biological effect in combination with estrogens. SRC is a downstream protein of ESR1 in the estrogen signaling pathway and is involved in processes such as cell proliferation, differentiation and adhesion. Androgens produced by ovaries are a necessary condition for normal periodic secretion of estradiol (E2), and have important effects on follicular development of the sinuses, and can promote proliferation of granulosa cells. Androgen Receptor (AR) can up-regulate FSH receptor expression, promoting FSH secretion.

In addition, as EGFR plays an important role in maintaining ovarian function, LH induces EGFR activation, which can promote cumulus cell expansion and oocyte maturation. The MAPK signaling pathway is a basic signaling pathway regulating cell proliferation, apoptosis, differentiation and transcription, and is involved in delaying oocyte senescence. The development of ova is associated with MAPK-1 activity, and abnormally activated MAPK-1 can lead to increased apoptosis, excessive development of early micronucleus cells and reduced oocyte quality. TNF is associated with ovarian cell growth, differentiation and apoptosis, affecting the balance of follicular proliferation and apoptosis, oocyte maturation and ovulation. It has been demonstrated that TNF- α is an immune factor secreted by lymphocytes and granulosa cells, plays a central role in activating and maintaining inflammatory responses, inhibits estrogen synthesis by down-regulating expression of genes in granulosa cells, and promotes granulosa apoptosis, follicular locking by endocrine or paracrine pathways, ultimately leading to DOR. TNF signaling can also cause immune and inflammatory diseases by mediating inflammatory transcription factors and activating MAPK signaling pathways, possibly leading to DOR formation by inducing an increase in intracellular related lymphocytes and inflammatory factors.

In conclusion, according to the network pharmacological analysis result, the effective components of the radix glehniae-polygonati officinalis drug pair can participate in multi-target and multi-channel treatment DOR through regulating the activities of various proteases.

Example 2 clinical study of Harvey's yin-nourishing decoction

1 clinical data

1.1 case Source

The DOR patient in the study is 30 cases of DOR patients with liver-kidney yin deficiency meeting the nano-discharge standard, which are seen in the first affiliated gynaecology clinic of Tianjin traditional Chinese medicine university in the year 2020 from 12 months to 2022 and 3 months. And 30 normal healthy women meeting the selection criteria were included.

1.2 diagnostic criteria

1.2.1 Western diagnostic criteria

Reference is made to "practical gynaecology endocrinology" (2 nd edition), to "chinese gynaecology science" (2 nd edition), to "bolonia standard" and to the relevant literature:

(1) Patient age < 40 years;

(2) Any two of the following 3 indices are satisfied, wherein sex hormone level is examined at least 2 times or more, 4 weeks apart.

(1) 10mIU/mL < Follicle Stimulating Hormone (FSH) < 40mIU/mL;

(2) the anti-mullerian hormone (AMH) is less than or equal to 1.1ng/mL;

(3) the number of ovaries of bilateral ovaries basic sinus follicles (AFC) is less than or equal to 5.

(1) And (2) can be diagnosed.

1.2.2 dialectical criteria of TCM

Is formulated by referring to "Chinese medicine science", and "Chinese medicine New medicine clinical research guidelines" and "diagnostic therapeutic Standard of Chinese medical symptoms".

Main symptoms are as follows: (1) the menstrual flow is small; (2) advanced or misplaced menstruation; (3) infertility;

secondary symptoms: (1) soreness and weakness of waist and knees; (2) dizziness and tinnitus; (3) dysphoria with feverish sensation in the chest; (4) hot perspiration; (5) insomnia and dreaminess; (6) dryness of eyes; (7) dry mouth and throat;

tongue pulse: a red tongue with little coating and a deep, thready or thready and rapid pulse.

The main symptoms are 1 item, the secondary symptoms are 3 items or more, and the diagnosis can be realized by combining tongue pulse.

1.3 nanobars standard

1.3.1 inclusion criteria

(1) Meets DOR Western diagnosis standards;

(2) Meets the traditional Chinese medicine differentiation standard of liver-kidney yin deficiency;

(3) Women aged 19-40 years with sexual history;

(4) Patients are informed with consent and can be treated as required.

1.3.2 exclusion criteria

(1) Incorporation of other endocrinopathies, such as: thyroid dysfunction, polycystic ovary syndrome, hypercarrhytidemia, adrenocortical hyperplasia and the like;

(2) Congenital dysplasia of reproductive organs, or hypoovarianism due to damage from acquired organic diseases;

(3) Patients with serious cardiovascular and cerebrovascular diseases, liver diseases, kidney diseases, blood system diseases, malignant tumors and other medical diseases or psychiatric diseases;

(4) Women in lactation and gestation period;

(5) Hormone supplementation or other hormone-like drugs that affect the trial are administered in approximately 3 months;

(6) Severe allergic constitution, or allergic to traditional Chinese medicine.

1.3.3 healthy female selection criteria for matching

(1) Women aged 19-40 years with sexual history;

(2) Patient informed consent;

(3) Those that do not meet DOR diagnostic criteria;

(4) Meets the above exclusion criteria.

1.4 reject and drop criteria

(1) The combined administration or the administration of the medicines does not reach the prescribed treatment course;

(2) Self-stopping treatment, or automatically logging out of the investigator;

(3) For other reasons, the access is lost, and the complete data is not available.

1.5 termination criteria

(1) Allergic reaction patients appeared during the study period;

(2) Pregnant women during the study period.

2 study method

2.1 case grouping

30 DOR patients meeting the nano-row standard are selected as DOR traditional Chinese medicine groups, and 30 healthy women matched with the DOR traditional Chinese medicine groups are selected as normal control groups. Wherein, the DOR traditional Chinese medicine group adopts the front-back control of the DOR traditional Chinese medicine group, and the influence of the Harvey's yin-nourishing soup on the DOR patients with liver-kidney yin deficiency is observed.

2.2 treatment regimen

2.2.1 DOR traditional Chinese medicine group scheme:

(1) The specific medicines of the Hu-nationality nourishing soup are as follows:

radix glehniae 20g vinegar tortoise plastron 15g glossy privet fruit 15g angelica sinensis 10g prepared rehmannia root 20g wine cornus officinalis 15g mulberry 20g dodder seed 20g medlar 15g wine cistanche 10g fragrant solomonseal rhizome 15g white paeony root 10g vinegar nutgrass galingale rhizome 10g.

(2) The preparation method and the administration method of the traditional Chinese medicine decoction: the Chinese medicinal preparation is decocted in the first affiliated hospital of Tianjin Chinese medicine university, 1 dose per day, and taken 30min after breakfast and supper.

(3) Treatment course: the medicine is continuously taken for 3 menstrual cycles without stopping taking medicine during menstrual period.

2.2.2 normal control protocol:

no drug intervention.

2.3 preservation and detection of serum samples

(1) Serum collection and preservation

Elbow venous blood was collected on days 2-4 of the menstrual cycle, 9-10 hours from morning. After 30 minutes of standing, centrifugation is carried out for 10 minutes at 3000rpm, serum is separated into clean EP tubes, and the clean EP tubes are stored in a refrigerator at-80 ℃ and uniformly detected.

(2) Serum detection and calculation

All sera of this study were tested uniformly by the easy-to-grow gene technology (Tianjin) company, inc., using enzyme-linked immunosorbent assay (ELISA).

2.4 observations index

2.4.1 basic information

Age, menstrual history, wedding history, etc.

2.4.2 sex hormone markers

Serum was collected before and after treatment of the DOR herbal group to detect FSH (follicle stimulating hormone), LH (luteinizing hormone), E2 (estradiol), AMH, and FSH/LH values were calculated.

2.4.3 bilateral sinus follicle count (AFC)

AFC before and after treatment of the DOR drug group was checked and recorded. The color Doppler examination of vagina is carried out on days 2-4 of menstrual cycle, and is completed by the same doctor in the first affiliated gynaecology clinic of the hospital of Tianjin traditional Chinese medicine university.

2.4.4 angiogenesis-related index

Serum was collected from the normal control group before treatment and the DOR traditional Chinese medicine group before and after treatment to detect HIF-1α (hypoxia inducible factor-1α), VEGF (vascular endothelial growth factor), ang-1 (angiopoietin-1), ang-2 (angiopoietin-2).

2.5 statistical analysis

The study uses Microsoft Excel 2019 software to create a database and SPSS22.0 statistical software to analyze the data. (1) The comparison between the two groups before treatment is carried out, the measurement data accords with a normal inspector, the measurement data is expressed by mean ± standard deviation (x ± S), if the measurement data meets the requirement of the t-test for the variance-aligned person, and if the measurement data does not meet the requirement of the approximate t-test for the variance-aligned person; the non-conformity with the normal inspector is represented by the median and quartile distance M (P25, P75) and the rank sum inspection is adopted. (2) Comparing the measured data difference values in the groups before and after treatment, and adopting paired t test for the measured data difference values to be in accordance with a normal tester; the difference of the measured data does not conform to the normal distribution, and a rank sum test is used. Wherein P < 0.05 represents that the difference is statistically significant.

3 results of the study

The study includes 60 cases, and finally completes 57 cases, 30 DOR Chinese medicinal groups include 3 cases and 27 cases; the normal control group included 30 cases, and completed 30 cases. Adverse reactions did not occur during the treatment period of patients in DOR traditional Chinese medicine group.

3.1 comparison of two sets of baseline data

The ages of the normal control group and the DOR traditional Chinese medicine group are in conformity with normal, and the difference has no statistical significance (P is more than 0.05) through t test, so that the traditional Chinese medicine composition has comparability. (see Table 3)

TABLE 3 age comparison of DOR traditional Chinese medicine group and normal control group (x.+ -. S)

3.2 comparison of the two groups of angiogenesis indicators before treatment

Comparing the DOR traditional Chinese medicine group with the normal control group angiogenesis index (see table 4), the serum HIF-1 alpha of the DOR traditional Chinese medicine group patient is lower than that of the normal control group through t test, and the difference is statistically significant (P is less than 0.01). The difference between VEGF, ang-1 and Ang-2 in DOR traditional Chinese medicine group is lower than that in normal control group (P < 0.05).

HIF-1 a, VEGF, ang-1 and Ang-2 are pro-angiogenic factors closely related to ovarian angiogenesis, and are indexes for evaluating serum pro-angiogenesis, and the reduction of the indexes suggests that the local angiogenesis capacity of the ovaries is insufficient, the follicular blood supply is reduced, and the functions of the ovaries are damaged. The serum HIF-1 alpha, VEGF, ang-1 and Ang-2 of the DOR traditional Chinese medicine group patients are lower than those of the normal control group, which shows that the serum angiogenesis promoting index of the DOR traditional Chinese medicine group patients is lower than that of the normal control group, and shows that the ovarian local blood supply of the DOR traditional Chinese medicine group is reduced and the ovarian function is damaged.

Table 4 comparison of the angiogenesis index (x.+ -. S) of the DOR traditional Chinese medicine group with the normal control group

3.3 comparison of sex hormone levels before and after treatment with DOR traditional Chinese medicine group and AFC

The FSH and FSH/LH of DOR traditional Chinese medicine groups before and after treatment are compared (see table 5 and table 6), after rank sum test, the FSH and FSH/LH are reduced compared with those before treatment, and the difference is statistically significant (P is less than 0.01).

The LH and E2 before and after treatment were compared (see Table 6), and the difference was statistically significant by rank-sum test with LH decrease (P > 0.05) and E2 increase (P > 0.05).

The AMH and AFC were compared before and after treatment (see Table 7), and the difference was statistically significant (P < 0.05) with the elevation of AMH and AFC.

The current evaluation indexes of the ovarian function mainly comprise: FSH, E2, AMH, AFC and FSH/LH, these indices are the most immediate indices for assessing ovarian function. FSH, FSH/LH, LH were all decreased after treatment compared to pre-treatment, E2, AMH and AFC were increased compared to pre-treatment, indicating a better improvement in sex hormone levels and AFC after treatment, indicating a better improvement in ovarian function after treatment.

Table 5 FSH comparison before and after DOR traditional Chinese medicine group treatment

TABLE 6 comparison of LH, E2 and FSH/LH before and after treatment with DOR traditional Chinese medicine group

Table 7 AMH and AFC comparisons (x±s) before and after DOR traditional Chinese medicine group treatment (n=27)

3.4DOR comparison of angiogenesis indicators before and after treatment with traditional Chinese medicine group

Referring to Table 8, after paired t-test, HIF-1. Alpha., VEGF and Ang-2 were elevated earlier in DOR traditional Chinese medicine group treatment, and the differences were statistically significant (P < 0.01). Ang-1 increased earlier after DOR treatment with the traditional Chinese medicine group, and the difference was statistically significant (P < 0.05). The serum HIF-1 alpha, VEGF, ang-1 and Ang-2 of the patient are all elevated earlier after DOR traditional Chinese medicine group treatment, which shows that the angiogenesis index is improved before treatment, thereby being beneficial to the local blood supply of the ovary and further improving the ovary function.

Table 8 comparison of angiogenesis indicators before and after treatment in DOR traditional Chinese medicine group (x±s) (n=27)

EXAMPLE 3 animal Experimental study of Harvey's yin-nourishing decoction

1 Experimental materials

1.1 laboratory animals

A total of 36 SPF grade C57BL/6J healthy female mice were used in this study, including 30 SPF grade C57 female mice of 52 weeks of age and 6 SPF grade C57 female mice of 8 weeks of age. All of the above were purchased from Beijing vitamin Torilhua laboratory animal technologies Co., ltd (license number: SCXK (Beijing) 2021-0006). All mice were kept by special humans in an environment of an animal barrier system room (license number: SYXK 2021-0003) of the Probiotics gene technology (Tianjin) Limited company. The raising environment parameters are controlled to be 22+/-2 ℃ throughout the year, the relative humidity is 50+/-10%, and the artificial control of illumination and darkness are replaced for 12 hours. The feeding process is carried out according to the instructions of nursing and use of experimental animals, all mice are uniformly fed with standard feed, eat and drink water freely, and the padding is replaced every two weeks and Saturday. The experiment follows the regulations of experimental animal management, and all animals pass the ethical examination of animal experiments (ethical YSY-DWLL-2021052) of the first affiliated hospital of the university of Tianjin traditional Chinese medicine.

1.2 Experimental medicine and preparation

Harvey's yin-nourishing soup: 15g of coastal glehnia root, 20g of vinegar tortoise plastron, 15g of wine glossy privet fruit, 10g of Chinese angelica, 20g of prepared rehmannia root, 20g of wine cornus, 20g of mulberry, 20g of dodder seed, 15g of medlar, 10g of wine cistanche, 15g of fragrant solomonseal rhizome, 10g of white paeony root and 10g of vinegar nutgrass galingale rhizome.

Traditional Chinese medicine control group 1: the Hu's yin-nourishing soup is prepared by removing vinegar and tortoise plastron.

Traditional Chinese medicine control group 2: the Hu's yin-nourishing soup removes radix glehniae and rhizoma polygonati officinalis.

All the above traditional Chinese medicines are purchased from the first affiliated hospital outpatient pharmacy of Tianjin traditional Chinese medicine university. Decocting the raw materials with water, concentrating to obtain crude drug 1.69g/ml, and storing in refrigerator at 4deg.C.

Coenzyme Q10 capsule: purchased from the first affiliated hospital pharmacy at the university of Tianjin traditional Chinese medicine, specifications: 10 mg/grain. Production family: maritime pharmaceutical trust friendship pharmaceutical factory limited, national pharmaceutical standard: H19999132. taking coenzyme Q10 capsules (10 mg/granule), preparing into a suspension by double distilled water, wherein the concentration of the suspension is 0.26mg/ml, and storing in a refrigerator at 4 ℃ for standby.

2 Experimental methods

2.1 Establishment of DOR mouse model

The study selected naturally senescent mice as DOR mice model. All mice were fed adaptively for 3 days, 9 a day earlier: 00 on all mice were examined for colporrhagia and monitored continuously for 10 days. The vaginal abscission cells of the mice were observed under a light microscope, and the estrus cycle changes of the mice were recorded. Based on the modeling method, if the mice at the age of 52 weeks have estrus interstasis or disorder compared with the mice at the age of 8 weeks, the mice accord with the DOR model. 6 mice with 8 weeks of estrus cycle law and 30 mice with 52 weeks of estrus conforming to DOR model are finally screened in the experiment.

2.2 Experimental grouping

By observing daily vaginal smears, 6 mice selected at 8 weeks of age were set as a blank group and 3 mice/cage bred. The selected 30 mice which meet the DOR model and are 52 weeks old are divided into the following groups according to a random number table method: model group, western medicine group, chinese medicine control 1 group, chinese medicine control 2 group, 6, 3/cage raising each group. And (5) entering a formal experiment.

2.3 experimental dosing regimen:

(1) blank group: normal saline, and the stomach is irrigated according to the dosage of 0.3ml/20 g/d;

(2) model group: normal saline, and the stomach is irrigated according to the dosage of 0.3ml/20 g/d;

(3) western medicine group: coenzyme Q10, lavage at 0.3ml/20g/d dose;

(4) the traditional Chinese medicine group: the Hu's yin-nourishing soup is used for stomach irrigation according to the dosage of 0.3ml/20 g/d.

(5) Traditional Chinese medicine control group 1: the Hu's yin-nourishing soup is prepared by removing vinegar tortoise plastron, and the stomach is irrigated according to the dosage of 0.3ml/20 g/d.

(6) Traditional Chinese medicine control group 2: the Hu's yin-nourishing soup is prepared by removing radix glehniae and rhizoma polygonati officinalis, and the stomach is irrigated according to the dosage of 0.3ml/20 g/d.

Each group was administered 1 time daily for 6 weeks.

Referring to "pharmacological experimental methodology" and related literature, equivalent doses for mice were calculated (dose per 20g per day for mice = clinical dose for humans/70 kg x 9.1 x 0.02). The Chinese medicine group mice are daily pre-mixed with Harvey's yin-nourishing soup according to the mass of 0.507g/20g, the western medicine group mice are daily pre-mixed with coenzyme Q10 according to the mass of 0.078mg/20 g.

2.4 sample collection and processing

(1) On the day of the end of the experiment, all mice fasted with water. The next earliest 9:00 starts anesthesia and draws materials. All mice were weighed and recorded prior to anesthesia.

(2) Blood sample material selection: after the mice were anesthetized, the eyeballs were collected and left to stand at room temperature for 1 hour, centrifuged at 3000rpm for 10 minutes, and the supernatant was collected and stored in a-80 ℃ refrigerator for ELISA detection.

(3) Ovarian material selection: after blood is taken, the mice are killed after cervical dislocation, the mice are fixed on an operation table, abdominal cavities are exposed, the ovaries and the uterus on two sides are rapidly taken under the aseptic condition, redundant adipose tissues are removed as much as possible, and the mice are placed on a high-precision electronic weighing machine for weighing the weight respectively and recording. Left ovaries of each mouse were stored in 4% paraformaldehyde fixative for HE staining to observe ovary morphology. The right ovary of each mouse was stored in a cryopreservation tube. The tinfoil paper package is put in a refrigerator at the temperature of minus 80 ℃ for preservation, so as to be used for detecting the ovarian tissue protein: SIRT1 (SiR 2-associated enzyme 1,Silent mating type information regulation 2 homolog 1), NF-kB (Nuclear transcription factor kB), HIF-1α, VEGF, ang-1, ang-2.

2.5 observations index

2.5.1 general case

The general condition of all mice was observed daily, including: the mice coat, body shape, food intake, drinking water, urination and defecation, movement conditions and mental states are recorded. Mice were weighed weekly and recorded.

2.5.2 estrus cycle

(1) Day 10 before the end of the experiment, 9:00 on all mice were tested for vaginal abscission cytology. Taking a cosmetic cotton swab, soaking with a small amount of physiological saline, inserting into the vagina of a mouse at about 0.5cm, and lightly rotating for 1 turn. After taking out, the slide glass is uniformly smeared, dried and fixed by methanol. And (3) observing the morphology of the shed cells on the fixed slide after Rayleigh staining under a positive microscope, judging and recording the estrus period stage of the mice, continuously smearing and detecting for 10 days, and recording the estrus period change of the mice.

(2) The mouse vaginal abscission cells mainly include: non-nucleated keratinocytes, nucleated epithelial cells and leukocytes. Mature female mice typically have an estrus cycle of 4-5 days, comprising, in order: the estrus, estrus later and estrus interval appear in this cycle, namely regular estrus period. If the sequence is disordered or stagnates in a certain period, the cycle is irregular estrus.

2.5.3 ovarian, uterine index determination

The mice were weighed, the bilateral ovaries were removed intact, surrounding adipose tissue was peeled off as much as possible, and weighed and recorded.

Ovaries index = bilateral total wet weight of ovaries (mg)/pre-sacrifice body weight (g) ﹡%.

Uterine index = total wet uterine weight (mg)/pre-sacrifice body weight (g) ﹡%.

2.5.4 enzyme-Linked immunosorbent assay (ELISA) for detecting serotonin levels

The serum of mice in the experimental study is uniformly determined by the gene technology of easy-to-grow (Tianjin), and ELISA kit is adopted to detect FSH, LH, E2 and AMH of the serum of mice. ELISA detection was performed exactly according to the kit protocol.

2.5.5 HE staining of ovarian tissue

Ovarian tissue was left in 4% paraformaldehyde fixative for at least 24h. After paraffin embedding, slicing and HE staining, the morphology of the ovarian tissue is observed under a microscope.

2.5.6 protein immunoblotting (western blot) method for detecting protein expression in mouse ovary tissue

Taking out the frozen ovarian tissue, and measuring the expression of SIRT1, HIF-1 alpha, VEGF, ang-1, ang-2 and NF-kB proteins in the ovarian tissue by using a western blot method.

2.6 statistical analysis

The study uses Microsoft Excel 2019 software to create a database and SPSS22.0 statistical software to analyze the data. The measurement data accords with the normal and variance uniformity inspector, expressed by mean ± standard deviation (x ± S), adopts single factor analysis of variance, and adopts LSD method for comparison. The measurement data do not conform to the normal and variance alignment inspector, expressed by the median and quartile distance M (P25, P75) and adopts rank sum inspection. Wherein P < 0.05 represents that the difference is statistically significant.

3 results of experiments

3.1 general condition, ovarian, uterine index comparison

Referring to table 9, the mice of each group were normal in feeding, drinking, mental activities and bowel movements during the experiment. The model group, western medicine group and traditional Chinese medicine group mice can see the phenomenon of dehairing at a small amount of back and neck before administration. After administration, the dehairing condition of mice in the traditional Chinese medicine group is relieved. During the experiment, mice in the blank group gain more rapidly, and mice in the model group gain more slowly. At the end of the experiment, the body weight, the ovary index and the uterus index of the western medicine group and the traditional Chinese medicine group are increased compared with the model group, but the difference among the groups has no statistical significance (P is more than 0.05). The weight, the ovary index and the uterus index of the western medicine group and the Chinese medicine group are increased compared with the model group, and the increasing amplitude of the Chinese medicine group is the largest, the increasing amplitude of the Chinese medicine group is the next time of the Chinese medicine control group 1 and the Chinese medicine control group 2, and the increasing amplitude of the western medicine group is the smallest.

Table 9 comparison of body weight, ovary and uterus index (x±s) for each group of mice (n=6)

Note that: delta: p > 0.05 compared to model group.

3.2 estrus cycle comparison

The estrus cycle of mice comprises the pre-estrus, the post-estrus and the estrus interval, which are generally 4-5 days. Referring to FIG. 13, A is the form of a preestrus cell, mainly a nucleated epithelial cell ((1) arrow), accompanied by a small number of non-nucleated keratinocytes. B is estrus cell morphology, with large numbers of non-nucleated keratinocytes ((2) arrow) predominantly, with small numbers of nucleated epithelial cells. C is the morphology of the cells in the late estrus, the non-nucleated keratinocytes, the nucleated epithelial cells and the white blood cells ((3) arrow) are all visible and the ratio is comparable. D is estrus interphase cell morphology, see large numbers of leukocytes.

In the experiment, the estrus cycle of the normal group is about 4-5 days, and the estrus cycle of the model group is about 7-10 days or no cycle. Before administration, compared with blank group, the estrus cycle of mice in model group, western medicine group and Chinese medicine group is all stagnant, prolonged or disturbed, and can be judged as successful molding. After administration, estrus cycle of western medicine group and traditional Chinese medicine groups is shortened to 6-8 days or no cycle, and is slightly improved compared with mice of model group, which shows that estrus cycle is shortened after administration, and the medicine has the function of recovering ovary function.

3.3 serum FSH, LH, E2, AMH comparison

Referring to Table 10, the differences were statistically significant (P > 0.05) in the model group compared to the two FSH and LH groups in the blank group. Compared with a blank group, the model group E2 and AMH are reduced, the difference is statistically significant (P is less than 0.05), and the mice in the model group are prompted to have hypoovariance. Western groups E2 and AMH, LH, FSH were elevated compared to the model group, wherein the FSH differences were statistically significant (P < 0.05). Compared with two FSH and LH groups in the traditional Chinese medicine group and the model group, the difference has no statistical significance (P is more than 0.05). The traditional Chinese medicine group E2 and AMH are increased compared with the model group, and the difference has statistical significance (P is less than 0.05), which indicates that the serum hormone of the traditional Chinese medicine group is improved compared with the model group. Compared with the model group, the FSH of the traditional Chinese medicine 1 group is reduced, the LH, E2 and AMH are all increased, and the difference has no statistical significance (P is more than 0.05). Compared with the model group, FSH and LH in the traditional Chinese medicine group 2 are reduced, E2 and AMH are both increased, and the difference has no statistical significance (P is more than 0.05).

Compared with the model group, the traditional Chinese medicine groups E2 and AMH are increased compared with the model group, which shows that the serum hormone of the traditional Chinese medicine group is improved compared with the model group, the sex hormone level is improved before treatment, and the ovarian function is improved well after treatment.

Table 10 serum FSH, LH, E2, AMH comparison (x±s) for each group (n=6)

Note that: delta: p < 0.05 compared to the blank; * : p < 0.05 compared with the model group;

3.4 comparison of ovarian histomorphology

Referring to fig. 14, it can be seen under the mirror that the ovaries of the mice in the blank group (shown in a of fig. 14) are clear in structure, the cortical and medullary distribution is normal, the follicles at each stage are visible, the morphology and the number are normal, the arrangement of granulosa cells is dense and regular, and the blood vessels and nerves are rich. The ovaries of the mice in the model group (shown as B in FIG. 14) are obviously atrophic, the structures of cortex and medulla are not clear, the original follicles (shown as arrow a) are obviously reduced, the arrangement of granular cells is loose, the closed follicles (shown as arrow f) are increased, the vascularity is reduced, and the morphology is irregular. Compared with the model group, the western medicine group improves the number of follicles and relieves the ovarian atrophy. Compared with the model group, the number of follicles at each level of the traditional Chinese medicine group is obviously increased compared with the model group, the ovarian atrophy condition is improved, the granular cells are orderly arranged, and the vascular distribution is increased. The number of follicles of each level of the traditional Chinese medicine control group 1 and the traditional Chinese medicine control group 2 is increased compared with that of the model group, but the improvement of the number of follicles and the ovarian atrophy is inferior to that of the traditional Chinese medicine group. In conclusion, the follicular number of the traditional Chinese medicine group is most obviously increased, which indicates that the traditional Chinese medicine group obviously improves the ovarian atrophy condition and the ovarian function.

3.5 SIRT1/HIF-1 alpha/VEGF pathway-related protein expression in ovarian tissue

As shown in Table 11 and FIG. 15, the expression of SIRT1, HIF-1. Alpha., VEGF, ang-1, ang-2 was decreased in the model group compared to the blank group, and the NF-kB expression was increased compared to the blank group, and the difference was statistically significant (P < 0.05). The SIRT1, VEGF and Ang-2 of western medicine group and Chinese medicine group are raised (P < 0.05) compared with the model group, the HIF-1α and Ang-1 are raised (P < 0.01) compared with the model group, and the NF-kB is lowered (P < 0.05) compared with the model group.

Compared with the model group, the expression of SIRT1, VEGF, ang-2, HIF-1 alpha and Ang-1 in the western medicine group and each group of traditional Chinese medicines is increased, the rising amplitude of the traditional Chinese medicine group is maximum, the rising amplitude of the western medicine group is the next time, and the rising amplitude of the traditional Chinese medicine control group 1 and the traditional Chinese medicine control group 2 is minimum. The NF-kB expression of the western medicine group and each group of the traditional Chinese medicines is reduced, the reduction amplitude of the traditional Chinese medicine group is the largest, the reduction amplitude of the western medicine group is the smallest, the traditional Chinese medicine control 1 group and the traditional Chinese medicine control 2 group show that after the treatment of the traditional Chinese medicine group, the SIRT1/HIF-1 alpha/VEGF channel related protein expression of the ovarian tissue is obviously improved, the local blood supply of the ovary is facilitated, and the ovarian function is further improved.

Table 11 comparison of the expression of mouse ovarian tissue protein (x.+ -. S) for each group

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Note that: delta: p < 0.05 compared to the blank; * : p < 0.05 compared with the model group; * *: p < 0.01 compared to model group.

Example 4 specific case of Harbour Kidney-tonifying and phlegm-resolving prescription treatment

The medical records:

sun Mou female, 38 years old, married. Date of visit: 2021-3-16.

Complaints: the contraception is carried out for 1 year.

The current medical history: plain menstruation is regular, 5/27-28 days, red color, little blood clots, no dysmenorrhea, sometimes soreness of waist, and no breast distending pain. Nake, poor sleep, one time a day of stool, and shaping. Pale tongue with thin and white coating and thready pulse.

Past history of: IVF was previously failed in three hospital northern physicians due to ovum quality problems.

Auxiliary inspection: 2020.11AMH:0.79

2021.1.8M2 six items: FSH 15.86LH:7.94E2:147P:1.53

Diagnosis: ovarian failure; infertility (infertility)

Syndrome differentiation: syndrome of liver depression and kidney deficiency

The prescription comprises the following components:

the above traditional Chinese medicine failed IVF in three hospital of North medicine after 1 month. After that, the treatment with traditional Chinese medicine is repeated, and 2021.10.16 detects HCG:1174, later follow-up, has produced a male infant.

Those of ordinary skill in the art will appreciate that: the discussion of any of the embodiments above is merely exemplary and is not intended to suggest that the scope of the disclosure, including the claims, is limited to these examples; the technical features of the above embodiments or in the different embodiments may also be combined under the idea of the present disclosure, the steps may be implemented in any order, and there are many other variations of the different aspects of the embodiments of the present disclosure as described above, which are not provided in details for the sake of brevity.

The disclosed embodiments are intended to embrace all such alternatives, modifications and variances which fall within the broad scope of the appended claims. Accordingly, any omissions, modifications, equivalents, improvements, and the like, which are within the spirit and principles of the embodiments of the disclosure, are intended to be included within the scope of the disclosure.

Claims (6)

1. The traditional Chinese medicine composition for treating or improving the ovarian reserve function is characterized by comprising the following raw materials in parts by weight: 10-20g of radix glehniae, 5-20g of vinegar tortoise plastron, 10-20g of wine glossy privet fruit, 10-20g of Chinese angelica, 10-20g of prepared rehmannia root, 10-20g of wine cornus, 10-20g of semen cuscutae, 10-20g of medlar, 10-20g of wine cistanche, 10-20g of polygonatum odoratum, 6-15g of white paeony root, 6-15g of vinegar nutgrass galingale rhizome and 5-20g of mulberry.

2. The traditional Chinese medicine composition according to claim 1, wherein the traditional Chinese medicine composition is composed of the following raw materials in parts by weight: 15g of radix glehniae, 10g of vinegar tortoise plastron, 15g of wine glossy privet fruit, 10g of Chinese angelica, 10g of prepared rehmannia root, 15g of wine cornus, 10g of semen cuscutae, 15g of medlar, 10g of wine cistanche, 15g of rhizoma polygonati officinalis, 10g of white paeony root, 10g of vinegar nutgrass galingale rhizome and 10g of mulberry.

3. The traditional Chinese medicine composition according to claim 1, wherein the dosage form of the traditional Chinese medicine composition comprises any one of decoction, solution, granule, suspension, capsule, powder, tablet, emulsion, dripping pill, injection, suppository, enema, aerosol, patch or drop.

4. A method of preparing the traditional Chinese medicine composition of any one of claims 1 to 3, comprising: weighing the raw materials according to the parts by weight, mixing the raw materials, and adding water for decoction to obtain the traditional Chinese medicine composition for treating or improving the ovarian reserve function.

5. The method for preparing a Chinese medicinal composition according to claim 4, wherein the water decoction of the Chinese medicinal composition is obtained by mixing the above raw materials, decocting with water at least twice, and mixing the decoctions of the two decoctions.

6. Use of a traditional Chinese medicine composition according to any one of claims 1 to 3 or a traditional Chinese medicine composition prepared according to any one of claims 4 to 5 in the preparation of a medicament for treating or improving hypoovarianism.