Application of pathway enrichment analysis differential metabolite in analysis of medicine for treating climacteric syndrome

Abstract

The invention provides application of a pathway enrichment analysis differential metabolite in analysis of a medicine for treating climacteric syndrome, and belongs to the technical field of biological pharmacy. And comparing and analyzing metabolites of the drug group and the placebo group before and after medication, and screening to obtain the metabolite homovanillic acid enriched in the dopamine synaptic pathway. The potential mechanism of relieving anxiety symptoms by adjusting a dopaminergic synaptic pathway through metabonomics analysis of drugs for treating climacteric syndromes lays a foundation for subsequent drug research.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and specifically relates to an application of pathway enrichment analysis of differential metabolites in drug analysis for treating menopausal syndrome. Background Art

[0002] Perimenopausal syndrome is often referred to as menopausal syndrome, which refers to a series of physical and mental symptoms caused by fluctuations or decreases in sex hormones before and after menopause in women. Perimenopausal is the stage when women transition from regular menstruation in the reproductive period to menopause, including the onset of endocrine, biological and clinical characteristics related to decreased ovarian function to one year after the last menstrual period. The symptoms of perimenopausal syndrome mainly include menstrual changes, vasomotor symptoms, and psychoneurological symptoms. It is more common in women aged 45 to 55. The clinical manifestations of perimenopausal women vary greatly due to their different mental states, living environments, etc. The treatment goal is to relieve recent symptoms, improve the quality of life of patients, and detect them early to reduce the risk of osteoporosis and cardiovascular diseases.

[0003] Traditional treatments are mainly based on psychological counseling and oral medication. However, the side effects associated with these drugs, such as dizziness, drowsiness, and constipation, often limit their use, and some patients may not respond well to currently available drug treatments. At the same time, multiple studies have shown that the use of traditional Chinese medicine can effectively and economically relieve menopausal symptoms and improve overall quality of life. However, there is little research on the treatment and relief mechanisms of traditional Chinese medicine.

[0004] Extensive research has been conducted in the prior art to explore the causes of the increased mood disorders experienced during the menopausal transition. In addition to the potential impact of VMS, research suggests that there appears to be a potential correlation between the presence of mood disorders and decreased ovarian function, as it has been observed that decreased estradiol levels lead to increased degradation and metabolic inactivation of dopamine, norepinephrine, and serotonin, along with downregulation of monoamine neurotransmitter activity. In other words, the involvement of dopamine in regulating anxiety may be related to regulating estrogen levels.

[0005] Furthermore, it has been shown that anxiety-inducing effects can be observed by inhibiting dopamine pathways. It has been found that a decrease in dopamine release in the medial prefrontal cortex (PFC) of rodents leads to an increase in anxiety-related behaviors. This finding is consistent with previous studies showing a correlation between dopamine and anxiety, as evidenced by the increased incidence of anxiety symptoms in patients with Parkinson’s disease. In this neurodegenerative disease, a decrease in dopaminergic prominence has been observed. These empirical observations support the hypothesis that anxiety may be attributed to a deficiency in dopamine levels. The role of dopamine in the pathophysiology of anxiety has been highlighted in earlier studies and has recently been re-emphasized. For example, the prefrontal cortex (PFC), hippocampus, and extended amygdala are brain regions responsible for processing emotions, including basal and stress-induced anxiety states. These regions are influenced by dopamine-rich midbrain regions that are thought to play a role in the formation of anxiety-like behaviors. Dopamine neurons in these target regions are important for emotion processing. Studies by Zweifel et al. have shown that interference with dopamine-dependent components can lead to the emergence of a generalized anxiety-like phenotype, and that activation of the mesolimbic dopamine system plays a crucial role in avoiding generalized anxiety. The dopaminergic system can also exert anxiolytic effects through its interaction with corticotropin-releasing hormone (CRH) and its receptor CRHR1.

[0006] This study starts from the mechanism of drugs for the treatment of perimenopausal syndrome in women, in order to achieve the development of safe and effective drugs for treatment. Summary of the invention

[0007] To this end, the technical problem to be solved by the present invention is to provide an application of pathway enrichment analysis of differential metabolites in the analysis of drugs for the treatment of menopausal syndrome.

[0008] The present invention provides a technical solution: application of pathway enrichment analysis of differential metabolites in drug analysis for the treatment of menopausal syndrome, by comparative analysis of metabolites of a drug group and a placebo group before and after use, screening out homovanillic acid, an enriched metabolite in the dopaminergic synaptic pathway.

[0009] Preferably, the metabolite menaquinone enriched in the vitamin digestion and absorption pathway is also obtained.

[0010] Preferably, homovanillic acid, a metabolite enriched in the tyrosine metabolic pathway, is also obtained.

[0011] Preferably, the medicine is a traditional Chinese medicine preparation with Cyperus rotundus and White Peony Root as main ingredients.

[0012] Preferably, the metabolite homovanillic acid enriched in the dopaminergic synaptic pathway is screened by the following method, comprising: S1. Experimental grouping: Participants who meet the inclusion criteria are randomly divided into a drug group and a placebo group; S2. Experimental process and sample collection: serum samples were collected from the drug group and the placebo group at three time points: before medication, during the experiment, and at the end of the experiment; S3. Metabolomics analysis, including: S3.1. After processing the serum samples, perform metabolite quantitative analysis; S3.2, identification of differential metabolites by three parameters: VIP value, FC value and p value; S3.3. Perform KEGG enrichment analysis on differential metabolites.

[0013] The present invention also provides another technical solution: application of homovanillic acid, a metabolite enriched in the dopaminergic synaptic pathway, in drug analysis for treating menopausal syndrome.

[0014] Preferably, the application includes comparative analysis of metabolites in the drug group and the placebo group before and after use, screening and identifying differential metabolites, and performing KEGG enrichment analysis, and drugs that enrich the metabolite homovanillic acid in the dopaminergic synaptic pathway have therapeutic effects.

[0015] Preferably, the analysis also includes the enrichment of the metabolite menaquinone in the vitamin digestion and absorption pathway.

[0016] Preferably, it is characterized in that the analysis also includes the enrichment of metabolite homovanillic acid in the tyrosine metabolic pathway.

[0017] The present invention also provides a technical solution: a pharmaceutical composition comprising a drug for regulating the enrichment of homovanillic acid metabolites in dopaminergic synaptic pathways.

[0018] Beneficial effects: The application of the pathway enrichment analysis of differential metabolites provided by the present invention in the analysis of drugs for the treatment of menopausal syndrome is to screen for the enriched metabolite homovanillic acid in the dopaminergic synaptic pathway by comparing and analyzing the metabolites of the drug group and the placebo group before and after use. The potential mechanism of regulating the dopaminergic synaptic pathway to regulate anxiety symptoms by metabolomics analysis of drugs for the treatment of menopausal syndrome lays the foundation for subsequent drug research. Through metabolic pathway analysis, the present invention combines metabolomics data analysis with biology, targets key differential metabolic pathways from the huge metabolic pathway database of KEGG, and provides a research direction for the in-depth development of subsequent disease treatment plans. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to make the contents of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings.

[0020] Figure 1a This is a schematic diagram of the comprehensive distribution of differential metabolites in the drug group before and after administration of Example 1 of the present invention; Figure 1b This is a schematic diagram of the comprehensive distribution of differential metabolites in the placebo group before and after medication in Example 1 of the present invention; Figure 2 This is a schematic diagram of comparative analysis of differential metabolites between two experimental groups in Example 1 of the present invention; Figure 3 This is a schematic diagram of comparative analysis of differential metabolites between the two experimental groups and before and after medication in Example 1 of the present invention; Figure 4a This is a schematic diagram of the analysis of the differential metabolite homovanillic acid in the drug group before and after administration of the present invention; Figure 4b This is a schematic diagram of the analysis of the differential metabolite homovanillic acid in the placebo group before and after the medication of the present invention; Figure 5a This is a schematic diagram of the analysis of the differential metabolite menaquinone in the drug group before and after the administration of the present invention; Figure 5b Schematic diagram of the analysis of the differential metabolite menaquinone in the placebo group before and after the medication of the present invention. DETAILED DESCRIPTION

[0021] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments. The principles and features of the present invention are described below in combination with the accompanying drawings. It should be noted that the embodiments in this application and the features in the embodiments can be combined with each other without conflict. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention.

[0022] The therapeutic drug used in this manual is Xiangshao Granules, which is produced by Sichuan Hairong Pharmaceutical Co., Ltd., China; other experimental reagents, unless otherwise specified, are commercially available products.

[0023] In the present invention, a double-blind, randomized, placebo-controlled, multicenter design was used to study the effects of drugs on perimenopausal and postmenopausal Chinese women and their mechanisms of action based on metabolomics analysis. 360 participants were randomly divided into two groups: one group took therapeutic drugs (n=180) and the other group took placebo (n=180) for 8 weeks. The main outcome measure was the Self-Rating Anxiety Scale (SAS) score, which was evaluated at baseline, 4 weeks and 8 weeks. The main focus was on the changes in SAS scores observed after the 8-week intervention period. In addition, in a subset of 96 patients, blood samples were collected at enrollment and after 8 weeks of treatment for metabolomics and transcriptomics analysis, as well as network pharmacology analysis, to study the mechanism of influence on anxiety symptoms.

[0024] In the present invention, the therapeutic drug is taken as an example of Xiangshao Granules, which is a pure herbal medicine with Cyperus rotundus and White Peony Root as the main drugs, and Bupleurum Root, Chuanxiong Rhizoma and Toosendan Fructus as auxiliary drugs. In animal experiments and randomized controlled trials, it has been observed that it has a strong safety and significant therapeutic effect in the treatment of perimenopausal symptoms, especially symptoms related to emotional symptoms. Existing studies believe that the regulation of drug symptoms of perimenopausal depression in women, the potential mechanism may involve the increase of regulating serum neurotransmitter levels and hippocampal brain-derived neurotrophic factor (BDNF), which has been identified as an antidepressant factor. Bupleurum saponin A is considered to be an active ingredient related to these mechanisms. However, the mechanism by which drugs relieve anxiety symptoms during perimenopause needs further study.

[0025] The present invention uses metabolomics to analyze the treatment mechanism, and can also use transcriptomics to analyze the treatment mechanism, as well as a combination of the two to perform mechanism analysis. "Omics" encompasses a comprehensive approach to examine the complete molecular spectrum of cells and tissues, including various disciplines such as genomics, transcriptomics, proteomics, metabolomics, epigenomics, and epitranscriptomics. Transcriptomics is the study of the transcriptome, including gene structure, expression, and regulation, while metabolomics is the downstream of genomics, transcriptomics, and proteomics, and aims to characterize a large number of metabolites in biological systems that are related to pathophysiological interventions or genetic modifications. Through the method of the present invention, the mechanism of the efficacy of traditional Chinese medicine preparations in treating perimenopausal anxiety symptoms can be explored based on the analysis of metabolites, and the potential mechanism of drug treatment can be further analyzed in combination with genomics, thereby providing a basis and ideas for future drug improvement or the development of new drugs.

[0026] 1. Screening of Experimental Participants The experimental participants in the present invention came from 18 clinical centers, and a total of 360 generally healthy perimenopausal or postmenopausal women with anxiety symptoms were recruited from December 2021 to June 2023. These participants were randomly assigned to a drug group or a placebo group. Assessments were performed at three different time intervals: baseline, 4 weeks later, and 8 weeks later. A baseline medical review was conducted using a standardized questionnaire and repeated at subsequent visits. In addition, in order to more deeply study the mechanism of the drug's effect on anxiety symptoms, blood samples were extracted from selected participants for analysis at enrollment and after 8 weeks of treatment. The study was approved by the Human Research Ethics Committee of Peking Union Medical College Hospital (HS-2958D) and registered at ClinicalTrials.gov (NCT05003336) on August 10, 2021. All participants provided written informed consent before the trial.

[0027] Participants included individuals aged 40 to 65 years with an intact uterus and at least one ovary. Eligible participants were required to be in the menopausal transition stage, characterized by a persistent difference in menstrual length of at least 7 days or amenorrhea lasting more than 60 days, or the postmenopausal stage, defined as amenorrhea lasting at least 12 consecutive months. The determination of these stages was based on the use of the 2011 Reproductive Aging Stages Workshop +10 criteria. In addition, participants had to exhibit mild to moderate anxiety symptoms, as demonstrated by a Self-Rating Anxiety Scale (SAS) score of 69 or lower and 50 or higher. In addition, participants were required to exhibit significant menopausal symptoms, as evidenced by a modified Kupperman score of 16 or higher.

[0028] Participants were excluded if they had a history of prior use of exogenous reproductive hormones, psychotropic medications, or other treatments for anxiety, or if they had used health products in the past four weeks. In addition, women with anxiety or other psychiatric disorders not related to menopause, as well as women with a Self-Rating Depression Scale (SDS) score of 70 or higher during screening, were also excluded. Women with a well-documented history of severe systemic illness, including but not limited to hypothyroidism / hyperthyroidism, unstable coronary artery disease, severe hypertension, pheochromocytoma, severe liver or kidney disease, gynecological or breast malignancies, and women with known allergies or sensitivities to study products, or women with a history of alcohol or drug abuse, were also not eligible for study participation.

[0029] 2. Experimental Grouping A randomized design was used to divide participants into the pegylated shao group and the placebo group. The randomization schedule was generated using computer software. The randomization sequence was generated using SAS software (SAS Institute, Cary, North Carolina, USA). Throughout the study, participants and investigators were unaware of the group assignment.

[0030] 3. Experimental Process In the drug group of the present invention, participants took Xiangshao granules, three times a day, 4 grams of Xiangshao granules each time, brewed with warm water, and taken within 30 minutes for 8 weeks. In the placebo group of the present invention, participants took Xiangshao granule simulators under the same scheme. Xiangshao granule simulators are mainly composed of corn dextrin, soluble starch, food coloring, flavor correctors, and 150g of clear paste extracted from every 1000g of Xiangshao granules. Compared with real drugs, the potential therapeutic effect of Xiangshao granule simulators is 9.3%. In addition, it has the same visual and taste characteristics as Xiangshao granule drugs, as well as similar packaging and labeling.

[0031] The experiment was marked as follows: "in A" refers to the entry group of the drug group, and "out A" refers to the exit group of the drug group. Similarly, "in B" refers to the entry group of the placebo group, and "out B" refers to the exit group of the placebo group. Self-administered questionnaires were used, including a general situation questionnaire and a self-rating anxiety scale (SAS) questionnaire. Anxiety symptoms were assessed using the SAS questionnaire (a total of 20 items). Each item was assigned a score from 1 to 4, with 1 indicating no or rarely, 2 indicating sometimes, 3 indicating often, and 4 indicating always. The questionnaire consisted of 15 negative statements scored in order (1-4) and 5 positive statements scored in reverse order (4-1). The total SAS score was calculated by the sum of the scores of each question. The standardized total score of the SAS was obtained by applying a multiplication factor of 1.25 to the total score and then rounding it off. Individuals with a standardized total SAS score of less than 50 were classified as having no anxiety disorder. A standardized total score of 50-59 was scored as mild, 60-69 was scored as moderate, and 70 or more was scored as severe.

[0032] Clinical outcomes were determined by analyzing the change in SAS scores from baseline scores after the 8-week treatment period. The SAS reduction rate was calculated as: (SAS score at baseline - SAS score after 8 weeks of treatment) / (SAS score at baseline - 25) x 100%. Categorical variables were used to examine clinical improvement based on the degree of change in the SAS. The classification criteria were as follows: a reduction of less than 30% was classified as "ineffective", a reduction of 30% to 50% was classified as "relief", a reduction of 50% to 80% was classified as "moderately effective", and a reduction of more than 80% was classified as "highly effective". In addition, a SAS score of less than 50 at the 8-week time point was considered an indication of symptom cure.

[0033] 4. Sample Analysis (A) Metabolic analysis using metabolite identification technology.

[0034] Serum samples were diluted with LC-MS grade water to a final concentration of 53% methanol. The samples were then centrifuged at 15,000 g and 4°C for 20 min. Finally, the supernatant was injected into the LCMS / MS system for analysis. Metabolites were quantitatively analyzed using an ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) system. The raw data files generated by UPLC-MS were processed using Compound Discoverer3.1 (CD3.1, ThermoFisher), and peak alignment, peak picking, and quantification were performed for each metabolite. Afterwards, the peak intensity was normalized to the total spectral intensity. It was then compared with the mzCloud, mzVault, and Masslist databases to obtain accurate qualitative and relative quantitative results.

[0035] Metabolites were annotated using the KEGG database (https: / / www.genome.jp / kegg / pathway.html), HMDB database (https: / / hmdb.ca / metabolites), and LIPIDMaps database (http: / / www.lipidmaps.org / ). Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed on metabolic data processing software, such as metax software. Univariate analysis (t-test) was used to calculate statistical significance (p value). Metabolites with VIP>1 and p value <0.05 and fold change22 or FCS0.5 were considered differential metabolites. Volcano plots were screened for enriched metabolites based on the log2 (fold change) and -log10 (p value) of metabolites using ggplot2 in R language.

[0036] (B) Transcriptome sequencing and analysis.

[0037] First, library preparation and sequencing were performed. RNA integrity was assessed using the RNA kit (RNA Nano 6000 Assay Kit) of the Bioanalyzer2100 system (Agilent Technologies, CA, USA). Total RNA was used as input material for RNA sample preparation. Library preparation was performed using the TruSeg PE Cluster Kit v3 (Illumina). After evaluating the quality of the library, sequencing was performed on the Illumina Novaseg platform to generate 150 bp double-end sequences.

[0038] Transcriptome data were analyzed. The raw data were processed by fastp software, that is, the sequences containing adapters, sequences containing ploy-N, and low-quality sequences were removed from the raw data to obtain high-quality data. Then, the cleaned double-end sequences were aligned with the reference genome GRCh38 / hg38 using the transcriptome alignment tool (Hisat2 v2.0.5). The number of sequences for each gene was counted using feature counting v1.5.0-p3. Then, the FPKM (Reads Per Kilobase of exon model per Million mapped reads) of each gene was calculated based on the length of each gene and the number of sequences of the gene. The data of the drug group and the placebo group were differentially expressed using the DESeq2 R (1.20.0) software package. The obtained p-values ​​were adjusted using the Benjamini and Hochberg method to control the false discovery rate. Genes with adjusted p-values ​​≤ 0.05 found by DESeq2 R (1.20.0) were classified as differentially expressed. Common gene enrichment analysis tools (such as ClusterProfiler package) were used to perform enrichment analysis of differentially expressed genes, including Gene Ontology (GO) and KEGG enrichment analysis of differentially expressed genes, in which gene length bias was corrected. GO or KEGG terms with a corrected p value of < 0.05 were considered to be significantly enriched in differentially expressed genes.

[0039] (III) Statistical analysis

[0040] Statistical analysis Continuous variables with normal distribution were expressed as mean ± standard deviation. Categorical variables were expressed as numbers and percentages. Comparisons of continuous variables between groups were assessed by analysis of variance. Depending on the sample size, Pearson chi-square test or Fisher's exact test were used to compare categorical data frequencies. For comparisons involving non-normally distributed data sets, the Wilcoxon rank sum test was used. Statistical analyses were performed using SAS software, version 9.4, and P < 0.05 was considered statistically significant.

[0041] Example 1 This example is an application of pathway enrichment analysis of differential metabolites in the analysis of drugs for treating menopausal syndrome, and the enriched metabolite homovanillic acid in the dopaminergic synaptic pathway is screened out.

[0042] The process is as follows: S1. Experimental grouping: Participants who meet the inclusion criteria are randomly divided into a drug group and a placebo group; S2. Experimental process and sample collection: serum samples were collected from the drug group and the placebo group at three time points: before medication, during the experiment, and at the end of the experiment; S3. Metabolomics analysis, including: S3.1. After processing the serum samples, perform metabolite quantitative analysis; S3.2, identification of differential metabolites by three parameters: VIP value, FC value and p value; S3.3. Perform KEGG enrichment analysis on differential metabolites.

[0043] Specific: Based on 360 participants, 180 of whom were assigned to the drug group and 180 to the placebo group. Participants in the two groups showed similar baseline characteristics, including age, menopausal status, and SAS scores. No statistically significant differences were presented in these variables. In the clinical analysis, the completion rate of the drug group was 165 / 180 (91.7%) and the completion rate of the placebo group was 162 / 180 (90.0%), of which 166 participants in the drug group and 165 participants in the placebo group completed at least 4 weeks of the study. A total of 96 patients were included in the omics study, providing comprehensive omics data for entry (in) and exit (out) samples. Each patient provided a transcriptome and a metabolome sample at two different time points before and after medication. A total of 384 omics samples were obtained, including 192 transcriptome samples and 192 metabolome samples. The entry group of the drug group was marked as "in A" and the exit group was marked as "out A". Similarly, the entry group of the placebo group was marked as "in B" and the exit group was called "outB". Through quality inspection, 4 transcriptome samples and 7 metabolome samples were found to be unqualified, and a total of 373 samples were included in the omics analysis.

[0044] After the two groups of participants were treated with medication according to the above experimental process, the changes in anxiety symptoms in both groups were significantly lower than before treatment in SAS scores (P<0.0001). The scores of the medication group were 47.7±8.78 at 4 weeks and 44.7±7.94 at 8 weeks; the scores of the placebo group were 47.8±8.27 at 4 weeks and 47.2±8.79 at 8 weeks. After 8 weeks, the SAS score of the medication group was significantly lower than that of the placebo group (P=0.0086). The proportion of participants in the medication group who were classified as "cured" increased from 58.6% at 4 weeks to 69.8% at 8 weeks. At the 4th and 8th week time points, the cure rates of the placebo group were 54.0% and 56.9%, respectively. The clinical improvement of the two groups after 8 weeks was statistically significant (P=0.0151).

[0045] Metabolites in the drug group and the placebo group before and after medication were compared and analyzed, and differential metabolites were identified by three parameters: VIP (Variable Importance in The Projection), FC (Fold Change), and p-value. VIP was calculated by the PLS-DA model, referring to the contribution of the metabolite to the group. FC was defined as the ratio of each metabolite to the mean of the quantitative values ​​of all biological replicates between groups. The p-value was calculated using a t-test, indicating that the difference was significant. The threshold was set as VIP>1.0, FC>1.2 or |log2FC]≥0.26, and p-value<0.05. The number of differential metabolites screened is shown in Table 1.

[0046] Table 1 The number of metabolites with different concentrations between the drug group and the placebo group

[0047] See also Figure 1a-1b , shows the comprehensive distribution of differential metabolites in the two groups before and after medication. The left side is the drug group and the right side is the placebo group. The horizontal axis represents the fold change and logarithmic transformation of the metabolite differences between the groups, and the vertical axis represents the significance level of these differences. Each data point in the figure corresponds to a specific metabolite, where significantly upregulated metabolites are represented in red, and significantly downregulated metabolites are represented in green. The size of the point corresponds to the VIP value. It can be seen that compared with the placebo group, the number of metabolite types in the drug group before and after medication increased significantly. See Figure 2 , shows the comparative analysis of the differential metabolites between the drug group and the placebo group, intuitively showing the common and differential metabolites between the two groups, and the relationship between the differential metabolites. It can be seen that the drug group and the placebo group have 6 common metabolites, of which the drug group has 28 differential metabolites, and the placebo group has 9 differential metabolites. Figure 3 , showing the comparative analysis of differential metabolites between the drug group and the placebo group before medication, after medication, before and after medication in the drug group, and before and after medication in the placebo group.

[0048] KEGG pathway enrichment analysis was used to determine the main biological functions performed by differential metabolites. The enrichment results come from the KEGG pathway, and the hypergeometric test technique is used to identify the pathways that enrich the differential metabolites relative to the entire identified metabolite background. In this way, the differential metabolites involved in the main biochemical metabolism and signal transduction pathways are determined. KEGG pathways that meet the p-value <0.05 threshold are considered to be significantly enriched in differential metabolites. Referring to Table 2, through KEGG pathway enrichment analysis, this example identified two statistically significant pathways, namely: dopaminergic synapses (P<0.05) and vitamin digestion and absorption (P<0.05). In addition, tyrosine metabolism (P=0.053) showed the same trend.

[0049] Table 2 KEGG pathway analysis results of differential metabolite enrichment

[0050] Among them, the dopaminergic synaptic pathway and the vitamin digestion and absorption pathway are more prominent. Figure 4a-4b and Figure 5a-Figure 5b , showing the differences in metabolites between the drug group and the placebo group before and after medication, Figure 4a-4b The metabolite homovanillic acid is shown, Figure 5a-Figure 5b The metabolite mephenone is shown. Through pathway analysis, the enriched metabolites of dopaminergic synaptic pathway, mephenone, vitamin digestion and absorption, and tyrosine pathway were obtained.

[0051] Example 2 This embodiment provides an application of homovanillic acid, a metabolite enriched in the dopaminergic synaptic pathway, in the analysis of drugs for treating menopausal syndrome.

[0052] Based on the experimental process and results of Example 1, after 8 weeks of medication, the cure rate of the drug group was 69.8%, and the cure rate of the placebo group was 56.9%, and the clinical improvement was statistically significant (P=0.0151). Figure 4a-4b and Figure 5a-Figure 5b The metabolomics analysis results showed the differences in metabolites between the drug group and the placebo group before and after medication. Figure 4a-4b As shown in the figure, the enrichment of the metabolite homovanillic acid in the dopaminergic synaptic pathway was more significant in the drug group after medication, and the same result was found in the placebo group. Figure 5a-Figure 5b As shown, the enrichment of vitamin A metabolite menaquinone in the drug group after medication was more significant, and the same result was found compared with the placebo group.

[0053] After drug treatment, the drug treatment effect was better when the dopaminergic synaptic pathway enriched the metabolite homovanillic acid more significantly. At the same time, the metabolite mephenone enriched in vitamin digestion and absorption was also more significant. Secondly, the tyrosine pathway also showed that the metabolite homovanillic acid was enriched.

[0054] Example 3 This embodiment provides a pharmaceutical composition, which contains a drug that regulates the dopaminergic synaptic pathway to enrich homovanillic acid metabolites. Thus, by regulating the dopaminergic synaptic pathway, homovanillic acid metabolites are enriched under in vivo interaction, thereby achieving the treatment of female menopausal syndrome.

[0055] It may further include drugs that regulate vitamin digestion and absorption and enrich the metabolite menaquinone.

[0056] It may further include drugs that regulate tyrosine metabolism and enrich the metabolite homovanillic acid.

[0057] In addition to the active ingredients, the drug may also include one or more pharmaceutically acceptable carriers or excipients according to the requirements of different dosage forms.

[0058] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. Application of pathway enrichment analysis of differential metabolites in drug analysis for the treatment of menopausal syndrome, characterized in that: The metabolites of the drug group and the placebo group before and after medication were compared and analyzed, and homovanillic acid, a metabolite enriched in the dopaminergic synaptic pathway, was screened out.

2. The use according to claim 1, characterized in that: The metabolite menaquinone, which is enriched in the vitamin digestion and absorption pathway, was also obtained.

3. The use according to claim 1, characterized in that: The enriched metabolite homovanillic acid in the tyrosine metabolic pathway was also obtained.

4. The use according to any one of claims 1 to 3, characterized in that: The medicine is a traditional Chinese medicine preparation with Cyperus rotundus and White Peony Root as main ingredients.

5. The use according to claim 4, characterized in that: Pathway enrichment analysis of differential metabolites was performed using the following methods, including: S1. Experimental grouping: Participants who meet the inclusion criteria are randomly divided into a drug group and a placebo group; S2. Experimental process and sample collection: serum samples were collected from the drug group and the placebo group at three time points: before medication, during the experiment, and at the end of the experiment; S3. Metabolomics analysis, including: S3.

1. After processing the serum samples, perform metabolite quantitative analysis; S3.2, identification of differential metabolites by three parameters: VIP value, FC value and p value; S3.

3. Perform KEGG enrichment analysis on differential metabolites.

6. Application of homovanillic acid, a metabolite enriched in dopaminergic synaptic pathways, in the analysis of drugs for the treatment of menopausal syndrome.

7. The use according to claim 6, characterized in that: The application includes comparative analysis of metabolites in the drug group and the placebo group before and after use, screening and identifying differential metabolites, and performing KEGG enrichment analysis. Drugs that enrich the metabolite homovanillic acid in the dopaminergic synaptic pathway are therapeutic.

8. The use according to claim 6, characterized in that: The drug analysis also includes the enrichment of the metabolite mephenazone in the vitamin digestion and absorption pathway.

9. The use according to claim 6, characterized in that: The drug analysis also includes enrichment of the metabolite homovanillic acid in the tyrosine metabolic pathway.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a drug for regulating the enrichment of homovanillic acid metabolites in dopaminergic synaptic pathways.

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