New application of radix bupleuri and cassia twig decoction

Abstract

The invention discloses a new application of a radix bupleuri and cassia twig decoction. The radix bupleuri and cassia twig decoction has a potential effect in the aspect of regulating imbalance of intestinal microflora. A new theoretical basis and a research direction are provided for application of the radix bupleuri and cassia twig decoction in regulation of flora, and a scientific basis is provided for application of the radix bupleuri and cassia twig decoction in intestinal health maintenance.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and particularly relates to a new use of Chaihu Guizhi Decoction. Background Art

[0002] The gut microbiota is a large and complex community of symbiotic microorganisms in the human body, and they play a crucial role in maintaining human health. Not only the health of the digestive tract, but these microorganisms also profoundly affect the overall health status of the host by producing metabolites, regulating the immune system, and interacting with other in vivo systems. In particular, recent studies have revealed that the gut microbiota is closely related to various inflammatory diseases, such as inflammatory bowel disease, non-alcoholic fatty liver disease, and some metabolic diseases. The occurrence and development of these inflammatory diseases are closely related to gut microbiota imbalance, overactivation of immune responses, and abnormalities in metabolites. Bile salt hydrolase (BSH) is a key enzyme in the gut microbiota, which directly participates in the reprogramming of host bile acid metabolism by catalyzing the hydrolysis of conjugated bile acids, and thus affects lipid homeostasis, immune regulation, and inflammatory responses. In recent years, the regulatory role of BSH and its related bacterial species in infectious and inflammatory diseases has attracted much attention, especially showing potential in intervention strategies for drug-resistant bacterial infections, intestinal inflammation, and immune-related diseases.

[0003] In the field of infectious diseases, BSH-active strains (such as Lactobacillus and Bifidobacterium) can inhibit the colonization and virulence expression of pathogenic bacteria (such as carbapenem-resistant Acinetobacter baumannii and Klebsiella pneumoniae) by regulating the bile acid metabolic profile. For example, the free bile acids produced by BSH hydrolysis can reduce the risk of drug-resistant bacterial infections by disrupting the membrane stability of pathogenic bacteria or interfering with their quorum sensing system. In addition, the antimicrobial peptides secreted by specific probiotics (such as Lactobacillus reuteri and Bifidobacterium longum) act synergistically with BSH to alleviate inflammatory responses such as bacterial pneumonia and urinary tract infections, and enhance the efficacy of antibiotics (such as polymyxin B sulfate). In the regulation of chronic inflammation, BSH-related bacterial species affect the host inflammatory pathway through the "microbiota-bile acid-immune" axis. For example, secondary bile acids (such as deoxycholic acid) mediated by BSH can activate the farnesoid X receptor (FXR) and G protein-coupled bile acid receptor (TGR5), and inhibit pro-inflammatory signaling pathways such as NF-κB, thereby reducing the low-grade inflammation associated with inflammatory bowel disease (IBD) and metabolic syndrome. Studies have also found that next-generation probiotic candidate bacterial species such as Roseburia spp. Roseburia ), and Christensenella minuta further strengthen the anti-inflammatory effect by secreting short-chain fatty acids (SCFAs) and regulating the Th17 / Treg balance.

[0004] Therefore, exploring the regulation of Bupleurum and Cinnamon Twig Decoction on the intestinal flora and deeply understanding how the intestinal flora regulates bile acid metabolism have important scientific significance. This research direction not only helps to reveal the mechanism of the interaction between microorganisms and the host, but also provides new ideas for treating diseases by regulating the intestinal flora or bile acid metabolism in the future. Summary of the Invention

[0005] The object of the present invention is to provide the application of Bupleurum and Cinnamon Twig Decoction in the preparation of drugs for regulating the intestinal flora.

[0006] Further, regulating the intestinal flora is to regulate the abundance and content of the intestinal flora.

[0007] Furthermore, the intestinal flora is the intestinal flora related to the BSH function involved in bile acid metabolism.

[0008] Furthermore, the content of the intestinal flora is Bacteroides fragilis CAG:47 , Bacteroides sp. OM05-10AA , Bacteroides finegoldii CAG:203 , Bacteroides sp. 4 3 47FAA and Clostridium hathewayi CAG:224 the content of the bacterial species.

[0009] Beneficial Effects Bupleurum and Cinnamon Twig Decoction has a potential effect in regulating the imbalance of the intestinal microbial community. This provides a new theoretical basis and research direction for the application of Bupleurum and Cinnamon Twig Decoction in regulating the flora, and provides a scientific basis for its application in maintaining intestinal health. Brief Description of the Drawings

[0010] Figure 1 is the relative abundance of BSH-related bacterial species; Figure 2 is the bar chart of the relative abundance of the intestinal flora at the phylum level; Figure 3 is the bar chart of the relative abundance of the intestinal flora at the genus level; Figure 4 is the bar chart of the relative abundance of the intestinal flora at the species level; Figure 5 is the Zscore heat map at the phylum level; Figure 6 is the Zscore heat map at the genus level; Figure 7 is the Zscore heat map at the species level; Figure 8 is the differential analysis of BSH-related bacterial species; A is the content of the bacterial species Bacteroides fragilis CAG:47 in each group, B is the content of the bacterial species Bacteroides sp. OM05-10AA in each group, C is the content of the bacterial species Bacteroides finegoldii CAG:203 in each group, D is the content of the bacterial speciesBacteroides sp. 4 3 47FAA Content in each group, where E is the bacterial strain Clostridium hathewayi CAG:224 Content in each group; Figure 9 It is a heatmap of Spearman correlation analysis between serum and intestinal UDCA and BSH-related bacterial strains. Specific implementation manners

[0011] To enable those skilled in the art to better understand the technical solution of the present invention, the following further details the technical solution of the present invention in conjunction with specific implementation manners.

[0012] Example 1 1 Experimental materials 1.1 Experimental reagents The basic information of the reagents used in this experiment is listed in Table 1.

[0013] Preparation method of Chaihu Guizhi Decoction: 47.5 g of the drug in 1 prescription amount of Chaihu Guizhi Granules (purchased from Shandong Mingren Frida Pharmaceutical Co., Ltd.) was made into 24 g of granules, packed into 8 g per bag, and each bag was dissolved in 17.09 mL of water to obtain a gavage solution with a concentration of 0.468 g / mL.

[0014] Table 1 Main experimental reagents

[0015] 1.2 Experimental instruments The basic information of the instruments used in this experiment is listed in Table 2.

[0016] Table 2 Main experimental instruments

[0017] 2 Experimental methods 2.1 Animal grouping, model establishment and drug administration After three days of adaptive feeding of Balb / c mice, 24 BALB / c mice (weighing 18±2 g) were randomly divided into 3 groups using SPSS 24.0 software, namely the control group, the model group, and the Chaihu Guizhi Decoction group (intervention group), with 8 mice in each group. Under isoflurane anesthesia, on day 0, the control group was instilled with sterile normal saline into the nasal cavity, and the remaining groups were instilled with 0.1 TCID50 HIN1 / PR8, 20 μL / mouse. On day 3, the control group was instilled with sterile normal saline into the nasal cavity, and the remaining groups were instilled with freshly cultured Staphylococcus aureus 1x10 6CFU, 20 μL per mouse. On the 4th day, the Chaihu Guizhi Decoction group (intervention group) was intragastrically administered with 9.36 g / kg (calculated as granules). The body weight, rectal temperature, water intake and food intake of the mice were recorded daily, and the mental state of the mice was observed. When the body weight of the model mice decreased, each administration group was intragastrically given the corresponding drug, and the administration volume was calculated as 0.15 mL / 10 g. The normal group and the model group were given an equal amount of normal saline. After 5 days of administration, the mice were sacrificed and the samples were taken for subsequent experiments.

[0018] 2.2 Collection and preservation of mouse intestinal content samples After the experiment, 8 mice were randomly selected from each group. The mice were anesthetized by intraperitoneal injection of 3.5% chloral hydrate (10 mL / kg). The intestinal contents were collected within 4 cm above the anus and placed in a cryopreservation tube, then quickly frozen in liquid nitrogen for 10 min, and then placed in a -80 °C refrigerator after being taken out.

[0019] 2.3 DNA extraction and preservation in mouse intestinal contents The mouse intestinal contents were placed in a 2 mL EP tube pre-cooled on ice, and InhibitEx Buffer was added for grinding and mixing. Then, the sample was heated and incubated, and the supernatant was taken after centrifugation. After mixing with proteinase K and Buffer AL, it was heated again. Subsequently, absolute ethanol was added and mixed evenly, and the lysate was added to the QIAamp spin column multiple times for adsorption and washing. Finally, DNA was eluted with Buffer ATE and incubated at 37 °C, and the DNA sample was obtained after centrifugation. The concentration and purity of the sample were detected by NanoDrop, and the integrity of DNA was detected by 1% agarose gel electrophoresis. The extracted DNA sample was finally stored in an environment of -20 °C.

[0020] 2.4 DNA sample detection Library construction and sequencing require obtaining a sufficient amount of high-quality nucleic acids. The main methods for detecting DNA samples are agarose gel electrophoresis (AGE) for analyzing the purity and integrity of DNA and Qubit for accurately quantifying the DNA concentration.

[0021] 2.5 Library construction and library inspection Take 1 μ μg of genomic DNA from the sample, randomly fragmented into fragments with a length of about 350 bp using a Covaris ultrasonic crusher, and then the library was constructed. The entire library preparation was completed through steps such as end repair, adding A-tail, adding sequencing adapters, purification, and PCR amplification. After the library construction was completed, first use AATI to detect the integrity of the library fragments and the insert fragment size. After meeting the expectations, use the O-PCR method to accurately quantify the effective concentration of the library (library effective concentration > 3 nM) to ensure the library quality.

[0022] 2.6 Sequencing on the machine After the library inspection is qualified, different libraries are pooled according to the requirements of the effective concentration and the target output data volume, and then paired-end sequencing is carried out.

[0023] 2.7 Information analysis process (1)Data quality control: In order to obtain effective data (CleanData), the raw data must be quality controlled and filtered first. If the sample is contaminated, the Bowtie2 software is used to filter the sample.

[0024] (2)Metagenome assembly: Starting from the Clean Data after quality control of each sample, Metagenome assembly is carried out; the software for assembly analysis is the MEGAHIT assembly software, and the Bowtie2 software is used to align the ScafigsCleanData after assembly of each sample.

[0025] (3)Gene prediction: The MetaGeneMark method is used for gene prediction, and the abundance information in each sample is obtained by integrating the CleanData of each sample.

[0026] (4)Species annotation: The gene catalogue is aligned with the MicroNR library to obtain the species annotation information of each gene, and the samples are aligned with the gene abundances in the database to obtain the species abundance table at different taxonomic levels.

[0027] (5)Annotation of common functional databases: Starting from the gene catalogue, functional annotation and abundance analysis of metabolic pathways (KEGG), clusters of orthologous genes (eggNOG), and carbohydrate-active enzymes (CAZy) are carried out.

[0028] (6)Based on the species abundance table and the functional abundance table, abundance clustering analysis can be carried out, mainly including PCA and PLS-DA analysis, LEfSe analysis, and Zscore heat map analysis.

[0029] 3 Experimental results 3.1 Relative abundances of BSH-related bacterial species Through metagenomic analysis, the relative abundances of bile salt hydrolase (BSH)-related bacterial species were compared between groups. See Figure 1 . The abundance of BSH bacterial species in the CGD group was significantly higher than that in the control group and the model group ( p <0.05).

[0030] 3.2 Analysis of the relative abundances of intestinal microbiota species 3.2.1 Relative abundances of species at the phylum level At the phylum level, the relative abundances of the microbial community are asFigure 2 As shown, the microbial compositions of the control group, model group, and Chaihu Guizhi Decoction (CGD) group were compared. According to the results in the figure, it can be observed that the two phyla, Bacillota and Bacteroidota, mainly occupied a significant proportion in each group, and the changes in their relative abundances showed differences among groups.

[0031] In the control group, the relative abundance of Firmicutes was 84%, while that of Bacteroidota was 15%. In the model group, the abundance of Firmicutes decreased to 70%, while the abundance of Bacteroidota increased to 24%, and the relative abundance of Verrucomicrobia increased to 4%, showing significant changes in the microbial community in the model group. Compared with the model group, the abundance of Firmicutes in the CGD group further decreased to 63%, while the abundance of Bacteroidota increased to 36%, and the relative abundance of Verrucomicrobia decreased.

[0032] 3.2.2 Relative Abundance of Species at the Genus Level At the genus level, the relative abundances of the microbial community structures in each group, as Figure 3 shown, further revealed the differences in the bacterial genus compositions among the control group, model group, and Chaihu Guizhi Decoction (CGD) group. Compared with the control group, the relative abundances of genera such as Bacteroides Bacteroides, Oscillibacter Oscillibacter, Alistipes Alistipes, Akkermansia Akkermansia, Parabacteroides and Parabacteroides Eubacterium in the model group increased, Prevotella Prevotella, Lepagella , Hungatella , Dorea , Limosilactobacillus and Bacteroides , Prevotella , Lepagella and Hungatella decreased; compared with the model group, the relative abundances of genera such as Eubacterium , Oscillibacter , Alistipes , Akkermansia , Parabacteroides , Muribaculum and Acutalibacter in the CGD group increased,

[0033] 3.2.3 Relative Abundance of Species at the Species Level At the species level, the relative abundances of the microbial community structures in each group, as Figure 4 shown, at the species level, Oscillospiraceae bacterium , Muribaculaceae bacterium ,Oscillibacter sp. , Clostridia bacterium , Akkermansia muciniphila and Alistipes sp. etc., the relative abundances of such strains increased, Lachnospiraceae bacterium , Eubacterium sp. and Hungatella sp. etc., the relative abundances of such strains decreased; in the CGD group, Lepagella muris , Hungatella sp. , Bacteroides acidifaciens and bacterium D16-54 etc., the relative abundances of such strains increased, Oscillospiraceae bacterium , Eubacterium sp. , Muribaculaceae bacterium and Akkermansia muciniphila etc., the relative abundances of such strains decreased.

[0034] 3.3 Zscore Heatmap Analysis 3.3.1 Zscore Heatmap Analysis at the Phylum Level Figure 5 shows the Z-score standardized heatmap based on the microbial community abundance data at the phylum level, used to compare the differences in microbial abundances between different groups. In the control group, phyla such as Firmicutes, Candidatus Saccharibacteria, Deinococcota, etc. showed relatively high abundances, especially Firmicutes, which was significantly enriched in the control group, suggesting its stable biological function in a healthy intestinal environment. Phyla such as Actinomycetota and Uroviricota showed relatively low abundances in the control group. In the model group, the abundances of phyla such as Verrucomicrobiota, Deferribacterota, Fusobacteriota, etc. were significantly higher than those in the control group and the CGD group. At the same time, various phyla such as Spirochaetota and Fibrobacterota also showed relatively high abundances in the model group. In the CGD group, the abundances of phyla such as Bacteroidota, Pseudomonadota, and Bacteroides were significantly higher than those in other groups. Phyla such as Candidatus Melainabacteria and Cyanobacteriota also showed relatively high abundances in the CGD group.

[0035] 3.3.2 Zscore Heatmap Analysis at the Genus Level Figure 6 shows the Z-score standardized heatmap based on the microbial community abundance data at the genus level. In the control group, the significantly enriched genera included Eubacterium , Ligilactobacillus , Limosilactobacillus , Ruminococcus ,Lactobacillus , Anaerotruncus and Roseburia etc. These genera showed higher abundances in the control group. In the model group, Oscillibacter , Alistipes , Prevotella , Parabacteroides , Hungatella , Dorea and Escherichia etc. genera showed higher abundances. The increase of these genera may be related to the intestinal microbiota dysbiosis and the formation of pathological states. In the CGD group, significantly enriched microbial genera included Bacteroides , Lepagella , Clostridium , Bacteroides acidifaciens , Parabacteroides and Mucispirillum , and these genera showed higher abundances in the CGD group.

[0036] 3.3.3 Zscore Heatmap Analysis at the Species Level Figure 7 showed the Z - score normalized heatmap based on the species - level microbial community abundance data. In the control group, significantly enriched species included Eubacterium sp. , Limosilactobacillus reuteri , Ruminococcus sp., Eubacterium plexicaudatum and Clostridiales bacterium . These species had higher abundances in the control group. In the model group, Oscillospiraceae bacterium , Alistipes sp. , Lawsonibacter sp. , Akkermansia muciniphila and Prevotella sp. MGM2 etc. species showed higher abundances. In the CGD group, significantly enriched species included Bacteroides acidifaciens , Muribaculaceae bacterium , Lepagella muris and Flintibacter muris etc.

[0037] 4 Discussion In this study, the differences in the intestinal microbiota composition among the control group, the model group, and the CGD group were systematically evaluated, and the potential role of CGD in regulating the imbalanced microbiota was investigated. The experimental results showed that the model group exhibited significant microbiota imbalance, while CGD treatment significantly changed the composition of the intestinal microbiota, which may provide a certain regulatory mechanism for the microbial balance in the disease state.

[0038] In the control group, the significant enrichment of Firmicutes and other probiotic taxa such as Lactobacillus, Eubacterium, Ruminococcus, etc. indicates their important role in maintaining intestinal health. These genera of bacteria can produce short-chain fatty acids, especially butyrate, which is crucial for the nutritional supply and immune support of intestinal epithelial cells. For example, Limosilactobacillus reuteri and Lactobacillus taiwanensis , play an important role in maintaining the intestinal microecological balance and barrier function. Limosilactobacillus reuteri Can stimulate the immune system, enhance the host's resistance to pathogens, and inhibit the growth of harmful bacteria by producing lactic acid, maintaining the balance of the intestinal flora. Lactobacillus taiwanensis Can enhance the intestinal epithelial barrier and play a certain role in alleviating intestinal inflammation caused by dextran sodium sulfate (DSS). Ruminococcus and Eubacterium As representatives of butyrate-producing bacteria, play a key role in the metabolic activities of the healthy intestine. They ferment indigestible carbohydrates such as dietary fiber to produce short-chain fatty acids (SCFAs), especially butyrate. Butyric acid is the main energy source of colonic epithelial cells, accounting for more than 70% of their energy requirements, and helps to maintain the integrity and function of the intestinal epithelium.

[0039] In contrast, dysbiosis related to intestinal inflammation and metabolic disorders was observed in the model group. In the model group Akkermansia muciniphila Was significantly enriched. It is a bacterial species closely related to the metabolism of the intestinal mucus layer and is generally considered beneficial to intestinal health. However, its over-enrichment may be related to the weakening of the intestinal barrier function and the inflammatory response. A. muciniphila The over-colonization of may lead to excessive consumption of the mucus layer, weaken the intestinal barrier function, and then trigger an inflammatory response. Therefore, although A. muciniphila Helps to maintain intestinal homeostasis at an appropriate level, its over-enrichment may have a negative impact on intestinal health. In addition, Alistipes and Prevotella The enrichment of also suggests the possible presence of inflammation and metabolic disorders in the model group. Prevotella Colonization can lead to metabolic changes in the microbiota, thereby reducing the production of IL-18, which exacerbates intestinal inflammation and potential systemic autoimmunity. Alistipes The abundance in colorectal cancer patients is significantly increased and is positively correlated with the inflammatory factor signal, indicating that it may have a pro-inflammatory role in the occurrence of colorectal cancer.

[0040] CGD treatment also had a significant impact on the composition of the intestinal microbial community. In the CGD group Bacteroidota andPseudomonadota The significant enrichment may indicate that CGD intervention has a certain selective effect on the microbial community. It is worth noting that Bacteroides fragilis The significant enrichment in the CGD group is related to its important role in maintaining host metabolic balance and regulating immune responses. Polysaccharide A is a capsular carbohydrate derived from Bacteroides fragilis and has the ability to balance pro-inflammatory and anti-inflammatory responses. In addition, Hungatella and Lawsonibacter The enrichment also indicates that CGD treatment may act through changes in the intestinal microenvironment. Hungatella hathewayi is a highly efficient glycosaminoglycan-degrading bacterium that can utilize glycosaminoglycans in the intestine to provide nutritional support for the host.

[0041] Generally speaking, different experimental treatments had significant effects on the composition and structure of intestinal microbiota at the phylum, genus, and species levels. The control group showed significant enrichment of probiotics, representing a healthy intestinal microbial structure; specific microbiota related to inflammation and dysbiosis were enriched in the model group; while the changes in the CGD group indicated its potential in regulating intestinal microbiota dysbiosis. These results provide important references for our understanding of the mechanism of action of Chaihu Guizhi Decoction in regulating the intestinal flora, and also point out the potential roles of specific microorganisms under different intestinal states.

[0042] 5 Summary This chapter revealed the potential role of Chaihu Guizhi Decoction in regulating intestinal microbiota dysbiosis by evaluating the intestinal microbiota composition of different experimental groups. The results showed that Chaihu Guizhi Decoction intervention could significantly change the imbalanced microbial community in the model group, restore the enrichment of probiotics, and inhibit the proliferation of specific potential pathogenic bacteria. In summary, Chaihu Guizhi Decoction provides a scientific basis for its application in maintaining intestinal health.

[0043] Example 2 Explore the effects of Chaihu Guizhi Decoction on BSH-related flora, that is, its specific metabolism in mice, in order to lay a scientific foundation for clarifying the relationship between Chaihu Guizhi Decoction and the intestinal flora.

[0044] 1 Experimental Materials 1.1 Experimental Reagents The basic information of the reagents used in this experiment is listed in Table 3.

[0045] Table 3 Main Experimental Reagents

[0046] 1.2 Experimental Instruments The basic information of the instruments used in this experiment is listed in Table 4.

[0047] Table 4 Main Experimental Instruments

[0048] 2 Experimental methods 2.1 Animal grouping, model establishment and drug administration After three days of adaptive feeding of Balb / c mice, 24 BALB / c mice (weighing 18 ± 2 g) were randomly divided into 3 groups using SPSS 24.0 software, namely the control group, the model group, and the Chaihu Guizhi Decoction group (intervention group), with 8 mice in each group. Under isoflurane anesthesia, on day 0, the control group was instilled with sterile normal saline into the nasal cavity, and the remaining groups were instilled with 0.1 TCID50 HIN1 / PR8, 20 μL / mouse. On day 3, the control group was instilled with sterile normal saline into the nasal cavity, and the remaining groups were instilled with freshly cultured Staphylococcus aureus 1x10 6 CFU, 20 μL / mouse. On day 4, the Chaihu Guizhi Decoction group (intervention group) was given intragastric administration of 9.36 g / kg (calculated as granules). The body weight, rectal temperature, water intake, and food intake of the mice were recorded daily, and the mental state of the mice was observed. When the body weight of the model mice decreased, each drug administration group was given the corresponding drug by intragastric gavage. The administration volume was calculated as 0.15 mL / 10 g, and the normal group and the model group were given an equal amount of normal saline. After 5 days of drug administration, the mice were sacrificed and the samples were taken for subsequent experiments.

[0049] 2.2 Bile acid detection 2.2.1 Preparation of standard quantitative curve and internal standard The standard chemicals of bile acids and their isotopes were accurately weighed and dissolved in methanol to prepare a mother liquor of 5.0 mM for storage. A certain volume of the mother liquor was precisely pipetted and added to the biological matrix without cholate (urine matrix without bile acids was used for fecal samples) to prepare a series of quantitative standard curves with concentrations of 1500, 500, 250, 50, 10, 2.5, and 1 nM, a total of seven concentrations. Among them, the concentrations of 15 internal standards, GCA-d4, TCA-d4, CA-d4, TCDCA-d9, GCDCA-d4, GUDCA-d4, TUDCA-d4, UDCA-d4, GDCA-d4, DCA-d4, GLCA-d4, LCA-d4, were 150 nM; the concentrations of CDCA-d4, TLCA-d4, β -CA-d5 were 25 nM.

[0050] 2.2.2 Pretreatment of intestinal content samples The fecal samples were taken out from the ultra-low temperature freezer and slowly thawed on ice. About 10 mg was weighed into a 1.5 mL centrifuge tube, and 15 µ L of water and homogenization beads were added for thorough homogenization, and then 180 containing internal standards was added µL of ACN / MeOH = 80 / 20 (volume ratio) solvent was placed on a thermostatic shaker, and the vibration speed was set to 1450 rpm, the temperature was 10 °C, and the time was 15 min. Then the static sample was centrifuged on a centrifuge at a speed of 13500 rpm, a temperature of 4 °C, and a time of 20 min. After centrifugation, 34 µ L was taken and placed in a clean 96-well plate, and then each sample was transferred to the 96 wells of the kit, and 86 µ L of mobile phase (mobile phase A:B = 2:1 volume ratio) was added for dilution, and the aluminum film was sealed with a heat sealer. The diluted solution was placed on a thermostatic shaker, with a vibration speed of 650 rpm, a temperature of 10 °C, and a time of 5 min. Then it was put into a centrifuge, with a centrifugal force of 4000 xg, a temperature of 4 °C, and a time of 30 min. After centrifugation, it was transferred to the liquid chromatography injection chamber and waited for injection, with an injection volume of 5 µ L.

[0051] 2.2.3 Serum sample pretreatment The serum sample was taken out from the ultra-low temperature refrigerator and slowly thawed on ice. After the sample was completely dissolved, 20 µ L was precisely pipetted into a 1.5 mL centrifuge tube, and 180 µ L of ACN / MeOH = 80 / 20 (volume ratio) solvent containing internal standard was added, and it was placed on a thermostatic shaker, with the vibration speed set to 1450 rpm, the temperature was 10 °C, and the time was 15 min. Then the static sample was centrifuged on a centrifuge at a speed of 13500 rpm, a temperature of 4 °C, and a time of 20 min. After centrifugation, 170 µ L was taken and placed in a clean 96-well plate, and then each sample was transferred to the 96 wells of the kit and dried in a freeze dryer. 60 µ L of mobile phase (mobile phase A:B = 2:1 volume ratio) was added for reconstitution, and the aluminum film was sealed with a heat sealer. It was placed on a thermostatic shaker, with a vibration speed of 650 rpm, a temperature of 10 °C, and a time of 5 min. Then it was put into a centrifuge, with a centrifugal force of 4000 xg, a temperature of 4 °C, and a time of 30 min. After centrifugation, it was transferred to the liquid chromatography injection chamber and waited for injection, with an injection volume of 5 µ L.

[0052] 2.2.4 Liquid chromatography - mass spectrometry conditions Detection was carried out using an ultra-high performance liquid chromatography - tandem mass spectrometry instrument (Acquity UPLC-Xevo TQS) from Waters Corporation, USA. The specific setting parameters are as follows.

[0053] In this study, an ACQUITY UPLC Cortecs C18 chromatographic column was used, with a guard column specification of 1.6 µ μm, a size of 2.1×5 mm, and an analytical column specification of 1.6µ M, with a size of 2.1×100 mm. Mobile phase A is water containing formic acid (pH = 3.25); mobile phase B is a mixed solution of acetonitrile / methanol (80:20). The column temperature is controlled at 30 °C, the injector temperature is set at 10 °C, and the injection volume each time is 5 µ L, and the flow rate of the mobile phase is 0.4 mL / min. The gradient elution conditions are set as follows: 0 - 1 min (5% B), 1 - 3 min (5 - 30% B), 3 - 15 min (30 - 100% B), 15 - 16 min (100 - 5% B), 16 - 17 min (5% B).

[0054] For mass spectrometry analysis, the electrospray ionization source negative ion mode (ESI-) is used, and the capillary voltage is set at 2.0 kV. The ion source temperature is 150 °C, and the desolvation temperature is 550 °C. The desolvation gas flow rate is 1000 L / Hr.

[0055] 2.2.5 Data processing The TargetLynx software of MassLynx from Waters Corporation in the United States is used for data extraction, integration, establishment of standard quantitative curves, quality control review, and output of the final reported concentration.

[0056] 3 Experimental results 3.1 Differential analysis of BSH-related strains Figure 8 In it, A is the content of the strain Bacteroides fragilis CAG:47 in each group, B is the content of the strain Bacteroides sp. OM05-10AA in each group, C is the content of the strain Bacteroides finegoldii CAG:203 in each group, D is the content of the strain Bacteroides sp. 4 3 47FAA in each group, E is the content of the strain Clostridium hathewayi CAG:224 in each group. The results of the differential analysis show that CGD significantly increased Bacteroides fragilis CAG:47 , Bacteroides sp. OM05-10AA , Bacteroides finegoldii CAG:203 , Bacteroides sp. 4347FAA and Clostridium hathewayi CAG:224 the content of the strain ( P <0.05).

[0057] 3.2 Spearman correlation analysis of UDCA and BSH-related strains Spearman correlation analysis was performed on UDCA, TUDCA, and UDCA / TUDCA in serum and intestine with BSH-related strains. Among them, strains significantly regulated by CGD or the top five strains in terms of content were selected for Spearman correlation analysis with bile acids. As Figure 9 , the analysis results show that the UDCA / TUDCA ratio in serum and intestine is related to the total amount of BSH-related strains, Bacteroidesfragilis CAG:47 , Bacteroides sp. OM05-10AA , Bacteroides finegoldii CAG:203 , Bacteroides sp. 4347FAA and Clostridium hathewayi CAG:224 showed a significant positive correlation ( P <0.05), and a significant negative correlation with Bifidobacterium dentium ( P <0.05). Moreover, the content of UDCA in the intestine was significantly positively correlated with Bacteroides fragilis CAG:47 , Bacteroides sp. OM05-10AA , Bacteroides finegoldii CAG:203 , Bacteroides sp. 4347FAA and Clostridium hathewayi CAG:224 showed a significant positive correlation ( P <0.05).

[0058] 4 Discussion In this study, the regulatory effect of Chaihu Guizhi Decoction on bile acid metabolism-related flora was systematically evaluated. At the same time, by combining the changes in the intestinal flora metabolite bile acids, the regulatory effect of Chaihu Guizhi Decoction on BSH-related flora was further demonstrated. According to the previous experimental data, CGD could significantly increase the abundance of BSH-related strains in the intestine. BSH is a key enzyme for intestinal microorganisms to participate in bile acid deconjugation, mainly including Bacteroides and Bifidobacterium. The research results showed that the change in the UDCA / TUDCA ratio was significantly correlated with the strains related to BSH activity. Among them, CGD significantly increased the Bacteroides fragilis CAG:47 , Bacteroides sp. OM05-10AA , Bacteroides finegoldii CAG:203 , Bacteroides sp. 4347FAA and Clostridium hathewayi CAG:224 content of strains, indicating that CGD could promote the hydrolysis of bile acids by regulating the abundance of BSH activity-related flora (such as Bacteroides and Bifidobacterium). Bacteroides fragilis ( Bacteroides fragilis ) contains BSH genes and enzyme activities, and can restore intestinal flora dysregulation and abnormal bile acid metabolism by inhibiting the FXR-NLRP3 signaling pathway, and reduce intestinal injury.

[0059] 5 Summary This invention first reveals the medicinal value of Chaihu Guizhi Decoction as a microecological regulator in improving intestinal flora dysregulation. Through metagenomic analysis, it is confirmed that this compound prescription can specifically up-regulate the abundance of BSH activity flora (such as Bacteroides fragilis CAG:47 ), and promote the generation of secondary bile acid metabolites. This discovery breaks through the application scope of traditional Chinese medicine prescriptions, and innovatively constructs a three-level regulatory network of "components-flora-metabolites", providing a technical solution within the scope of patent protection for the development of intervention programs for digestive system diseases based on intestinal flora reconstruction.

Claims

1. An application of Chaihu Guizhi Decoction in the preparation of a medicine for regulating intestinal flora.

2. The use of Chaihu Guizhi Decoction according to claim 1 in the preparation of a drug for regulating intestinal flora, characterized in that: Regulating the intestinal flora means regulating the abundance and content of intestinal flora.

3. The use of Chaihu Guizhi Decoction according to claim 2 in the preparation of a drug for regulating intestinal flora, characterized in that: The intestinal flora is the intestinal flora that participates in BSH-related functions of bile acid metabolism.

4. The use of Chaihu Guizhi Decoction according to claim 3 in the preparation of a drug for regulating intestinal flora, characterized in that: The intestinal flora content is Bacteroides fragilis CAG:47 , Bacteroides sp. OM05-10AA , Bacteroides finegoldii CAG:203 , Bacteroides sp. 4 3 47FAA and Clostridium hathewayi CAG:224 The content of bacterial species.

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