Application of long-leaf menthone in regulation of biological intestinal flora and test method of long-leaf menthone

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses application of long-leaf menthone in regulation of biological intestinal flora and a test method. The long-leaf menthone disclosed by the invention can be used as an intestinal microecological preparation, is used for regulating intestinal flora of a mouse infected with the abdominal cavity, and is expected to become a new intestinal microecological preparation for treating the abdominal cavity infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of piperitone in regulating the biological intestinal flora and a testing method thereof. Background Art

[0002] Intra-abdominal infection is the second most common infectious disease in ICU and hospitalized patients, with a mortality rate as high as 20% - 30%. A large number of microorganisms inhabit the human intestine, including pathogenic microorganisms, which can lead to poor prognosis in critically ill patients. To prevent the invasion of pathogenic microorganisms, the human intestine has established four major barriers, one of which is the intestinal flora. The intestinal flora can inhibit the colonization of pathogenic microorganisms in the intestine through multiple mechanisms such as nutritional competition, secretion of antimicrobial substances, and production of metabolites (such as short-chain fatty acids, etc.), which helps prevent intestinal and systemic infections. Research shows that the function of the human intestinal flora is more important than the intestinal mucus barrier and immune barrier. After the intestinal flora is dysregulated, the function of the intestinal microbial barrier is damaged, leading to more susceptible colonization of pathogenic microorganisms in the intestine and ultimately resulting in intestinal bacterial infection.

[0003] Intestinal microecological agents can regulate the intestinal flora and are potential treatment measures for intra-abdominal infection. Probiotics (live bacterial preparations, common probiotics include Lactobacillus, Bifidobacterium, etc.), prebiotics (growth promoters for beneficial bacteria, such as oligosaccharides, fructooligosaccharides, etc.), and synbiotics (combined preparations of live bacteria and promoters) are currently the most easily available intestinal microecological agents, which can play a role in regulating the intestinal flora. However, the above-mentioned intestinal microecological agents have certain limitations in the prevention and treatment of intra-abdominal infection after being evaluated by animal experiments and clinical studies. Among them, the effect of probiotics is closely related to the number of live bacteria contained. Beneficial effects can only be produced when the number of live bacteria in the intestine reaches 1×10 6 . Probiotics have the disadvantages of short storage time and unstable effects. Active probiotics themselves contain drug-resistant genes or drug-resistant plasmids, etc. These gene information can be transferred from probiotics to intestinal pathogenic bacteria, causing drug-resistant bacterial infections in the host. In addition, since synbiotics also contain active probiotics, problems such as the transfer of drug-resistant genes need to be considered. Prebiotics have the disadvantages of short-lasting efficacy and often causing flatulence when in excess, which limits their application.

[0004] Therefore, it is a new research and development direction to focus on precisely targeting the improvement or treatment of specific diseases and enabling intestinal microecological agents to play a greater role in improving intra-abdominal infection. It is urgent to explore safe and effective intestinal microecological agents that can regulate the intestinal flora. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of piperitone in regulating the biological intestinal flora to solve the above technical problems.

[0006] One invention of the present invention relates to the application of piperitone in regulating the biological intestinal flora.

[0007] Preferably, the organism is a mouse.

[0008] Preferably, the mouse is constructed by intraperitoneal infection with drug-resistant Escherichia coli.

[0009] Preferably, the piperitone is R-(+)-piperitone.

[0010] Another aspect of the present invention relates to the application of the above-mentioned piperitone as an intestinal microecological preparation.

[0011] Another aspect of the present invention relates to an intestinal microecological preparation with R-(+)-piperitone as the only active ingredient.

[0012] Another aspect of the present invention relates to a test method for the application of biological intestinal flora, comprising the following steps:

[0013] Establish a first control group, a second control group, a third control group, and a fourth control group each including the same number of biological test subjects;

[0014] Use edible oil to perform gavage operations on the first control group and the second control group for a preset number of gavage days. After completing the gavage operations, perform injection operations with corresponding normal saline on the first control group and injection operations with corresponding multi-drug resistant Escherichia coli on the second control group;

[0015] Use R-(+)-piperitone to perform gavage operations on the third control group for a preset number of gavage days, and use cefixime to perform gavage operations on the fourth control group for a preset number of gavage days. After completing the gavage operations, perform injection operations with corresponding multi-drug resistant Escherichia coli on the third control group and the fourth control group respectively;

[0016] Perform survival observations on the first control group, the second control group, the third control group, and the fourth control group for a preset observation duration to determine the survival status corresponding to each biological test subject, wherein the survival status includes a survival state and a death state;

[0017] Collect the corresponding colon tissues of all biological test subjects in the survival state to obtain a tissue sample set including each colon tissue;

[0018] Extract DNA from each colon tissue in the tissue sample set, perform PCR amplification on each extracted DNA sample, and prepare a sequencing library based on each obtained PCR product;

[0019] Perform sequencing analysis on the sequencing library to obtain the analysis results of the corresponding biological intestinal flora.

[0020] Preferably, the first comparison group and the second comparison group are respectively subjected to gavage operations for a preset number of consecutive gavage days using edible oil. After completing the gavage operations, the first comparison group is subjected to an injection operation with corresponding normal saline, and the second comparison group is subjected to an injection operation with corresponding multi-drug resistant Escherichia coli, including:

[0021] The edible oil is used to perform a gavage operation on each biological test subject in the first comparison group and the second comparison group at 20 ml / kg for a preset number of consecutive gavage days;

[0022] After completing the gavage operation, each biological test subject in the first comparison group is subjected to an injection operation with corresponding normal saline at 10 ml / kg, and each biological test subject in the second comparison group is subjected to an injection operation with corresponding multi-drug resistant Escherichia coli at 10 ml / kg.

[0023] Preferably, the third comparison group is subjected to a gavage operation for a preset number of consecutive gavage days using R-(+)-pulegone, and the fourth comparison group is subjected to a gavage operation for a preset number of consecutive gavage days using cefixime. After completing the gavage operations, the third comparison group and the fourth comparison group are respectively subjected to an injection operation with corresponding multi-drug resistant Escherichia coli, including:

[0024] R-(+)-pulegone is used to perform a gavage operation on each biological test subject in the third comparison group at 20 ml / kg for a preset number of consecutive gavage days;

[0025] Cefixime is used to perform a gavage operation on each biological test subject in the fourth comparison group at 26 ml / kg for a preset number of consecutive gavage days;

[0026] After completing the gavage operation, each biological test subject in the third comparison group and the fourth comparison group is respectively subjected to an injection operation with corresponding multi-drug resistant Escherichia coli at 10 ml / kg.

[0027] Preferably, all biological test subjects in the corresponding survival state are collected for the corresponding colon tissues to obtain a tissue sample set including each colon tissue, including:

[0028] All biological test subjects in the corresponding survival state are anesthetized to convert all biological test subjects from the survival state to the death state, and all biological test subjects in the corresponding death state are collected for the colon tissues to obtain a tissue sample set including each colon tissue;

[0029] The tissue sample set is stored at a corresponding preset storage temperature.

[0030] Preferably, DNA extraction is performed on each colon tissue in the tissue sample set, and each obtained DNA sample is subjected to PCR amplification, and a sequencing library is prepared based on each obtained PCR product, including:

[0031] Using CTAB to perform DNA extraction based on bacterial genomes on each colon tissue in the tissue sample set to obtain each DNA sample;

[0032] Performing PCR amplification on each DNA sample using specific primers to obtain each PCR product;

[0033] Using AMpure XT beads and Qubit to purify and quantify each of the PCR products to obtain a sequencing library.

[0034] Preferably, sequencing analysis is performed on the sequencing library to obtain analysis results of the corresponding biological intestinal flora, including:

[0035] Using a sequencer to perform sequencing analysis on the sequencing library to obtain paired-end reads;

[0036] Using overlap to perform paired-end splicing on the paired-end reads to obtain ASV characteristic sequences;

[0037] Performing diversity analysis based on the ASV characteristic sequences to obtain analysis results of the corresponding biological intestinal flora.

[0038] Piperitone disclosed by the present invention can be used as an intestinal microecological preparation to regulate the intestinal flora of mice with abdominal infection, and is expected to become an effective ingredient of a new intestinal microecological preparation for the treatment of abdominal infection, providing new ideas and methods for the treatment of abdominal infection. Description of the Drawings

[0039] Figure 1 Weight curve graph and mortality rate of mice with abdominal infection of ECO K1; wherein, A: Weight curve graph of mice with abdominal infection of ECO K1; B: Mortality rate of mice with abdominal infection of ECO K1; *: p < 0.05;

[0040] Figure 2 ASV (Amplicon Sequence Variants) distribution of intestinal flora of mice with abdominal infection of ECO K1: Venn diagram based on the total sequence OTU (Operational Taxonomic Unit) of each group;

[0041] Figure 3Dilution curves and rank-abundance curves for the analysis of the alpha diversity of the intestinal microbiota of ECO K1-infected abdominal cavity mice. Among them, A: Dilution curves for the analysis of the alpha diversity of the intestinal microbiota of ECO K1-infected abdominal cavity mice; B: Rank-abundance curves for the analysis of alpha diversity.

[0042] Figure 4 Correlation analysis of the beta diversity of the intestinal microbiota of ECO K1-infected abdominal cavity mice; among them, A: Principal coordinate analysis (PCoA) of the beta diversity of the intestinal microbiota of ECO K1-infected abdominal cavity mice; B: Analysis of the similarity between groups of mouse intestinal microbiota: UPGMA hierarchical clustering analysis based on the sample distance matrix of the bray curtis algorithm.

[0043] Figure 5 Relative abundances of the top 30 intestinal microorganisms at the phylum level and genus level for each group of mice; among them, A: Relative abundances of the top 30 intestinal microorganisms at the phylum level for each group of mice; B: Relative abundances of the top 30 intestinal microorganisms at the genus level for each group of mice.

[0044] Figure 6 Composition and differences of the mouse intestinal microbiota, among them, A: Composition and differences of the mouse intestinal microbiota: barplot significance difference analysis; B: Cluster heatmap of the top 30 intestinal microorganisms at the genus level.

[0045] Figure 7 shows the composition and differences of the mouse intestinal microbiota, among them, A: Composition and differences of the mouse intestinal microbiota: Histogram of LDA score distribution; B: Composition and differences of the mouse intestinal microbiota: LEfSe cladogram. The root of the cladogram represents bacteria within the domain. The size of each node represents their relative abundance. Nodes of species with no significant differences are yellow, and nodes of species with significant differences are red.

[0046] Figure 8 Is a flowchart of the test method for the application of the intestinal microbiota of the corresponding organism. Specific embodiments

[0047] The following is a further detailed description through specific embodiments:

[0048] An embodiment of the present invention proposes the application of piperitone in regulating the intestinal microbiota of organisms, as well as a test method for the application of the intestinal microbiota of organisms. Specifically, referring to Figure 1-8 This application and test method can be specifically described as follows:

[0049] 1. Experimental method

[0050] 1.1 Animals

[0051] First, a first control group, a second control group, a third control group, and a fourth control group can be established, each including the same number of biological test subjects. Herein, the corresponding biological test subjects can specifically be mice. The first control group can be the NC group, the second control group can be the Mode1 group, the third control group can be the PU group, and the fourth control group can be the CFM group.

[0052] For example:

[0053] Forty healthy ICR mice (4 - 6 weeks old, half male and half female, body weight 18 - 22 g) were provided by the Animal Center of Xinjiang Medical University. The experimental animal use license number is: SYXK(Xin)2018 - 0003. During the entire adaptation and research period, all animals were maintained under specific specific pathogen - free (SPF) conditions with a 12 - hour light - dark cycle (room temperature 23 ± 3°C, relative humidity 40 - 70%), and were allowed to eat and drink freely. All animal studies were conducted in accordance with the protocol approved by the Animal Ethics Committee of Xinjiang Medical University.

[0054] 1.2 Reagent preparation

[0055] (1) Mixed anesthetic: Mix 250 mg of Zoletil 50 (Virbac, France) with 2.5 ml of xylazine hydrochloride (China Shengda Animal Pharmaceutical Co., Ltd., 100 mg / ml), and dilute it with 22.5 ml of sterile normal saline to a 10 mg / ml mixed solution for standby.

[0056] (2) Preparation of the concentration of R-(+)-pulegone (PU, Sigma): Take an appropriate amount of PU and prepare it into a 1 mg / ml concentration with edible oil, mix it magnetically, and filter it aseptically through a 0.22 μm filter membrane.

[0057] (3) Cefixime (CFM, positive control drug) solution: Weigh 19.5 mg of cefixime (Qilu Pharmaceutical Co., Ltd., China) aseptically, add 5 ml of normal saline to dissolve it, and then make up the volume to 15 ml. The concentration is 1.3 mg / ml, and it is stored at 4°C for standby.

[0058] 1.3 Experimental strains

[0059] Multidrug - resistant Escherichia coli (ECO K1): Select clinically screened drug - resistant strains, which are multidrug - resistant Escherichia coli producing extended - spectrum beta - lactamase (ESBL) and positive for biofilm (named ECO K1). After identification, its ESBL gene typing is CTX - M - 14 strain. Antibiotic susceptibility tests showed that ECO K1 was resistant to penicillins, cephalosporins, monobactams, fluoroquinolones, and sulfonamides.

[0060] 1.4 Establishment of a mouse model of intraperitoneal infection with multidrug-resistant Escherichia coli (ECO K1)

[0061] 1.4.1 Preparation of bacterial suspension

[0062] Resuscitate and subculture ECO K1, pick a single colony and culture it in a constant temperature shaker at 37°C and 200 rpm for 18 h. Prepare different bacterial suspensions with sterile LB liquid medium, and use a McFarland turbidimeter to make bacterial suspensions with different turbidities, and store them at 4°C for later use.

[0063] 1.4.2 Establishment of the mouse intraperitoneal infection model and setting of experimental groups

[0064] The first control group and the second control group can be continuously gavaged with edible oil for a preset number of days. After completing the gavage operation, the first control group is injected with corresponding normal saline, and the second control group is injected with corresponding multidrug-resistant Escherichia coli.

[0065] Specifically, each biological test subject in the first control group and the second control group can be continuously gavaged with edible oil at 20 ml / kg for a preset number of days; after completing the gavage operation, each biological test subject in the first control group is injected with corresponding normal saline at 10 ml / kg, and each biological test subject in the second control group is injected with corresponding multidrug-resistant Escherichia coli at 10 ml / kg.

[0066] For example:

[0067] Randomly divide the mice into 4 groups, with 10 mice in each group, half male and half female. Fast the mice for 12 h before the experiment, but do not restrict water intake.

[0068] The healthy control group (NC group) and the model group (Mode1 group) are gavaged with edible oil at 20 ml / kg. During this period, the mice are fed normally, once a day for a total of 3 days. 1 h after the edible oil gavage on the 3rd day, the NC group is intraperitoneally injected with normal saline at a dose of 10 ml / kg, and the Model group is intraperitoneally injected with ECO K1 at 3.0×10 8 cfu / ml.

[0069] The third control group is continuously gavaged with R-(+)-pulegone for a preset number of days, and the fourth control group is continuously gavaged with cefixime for a preset number of days. After completing the gavage operation, the third control group and the fourth control group are respectively injected with corresponding multidrug-resistant Escherichia coli.

[0070] Specifically, perform gavage on each biological test subject in the third comparison group with R-(+)-pulegone at a dose of 20 ml / kg for a preset number of consecutive gavage days; perform gavage on each biological test subject in the fourth comparison group with cefixime at a dose of 26 ml / kg for a preset number of consecutive gavage days; after completing the gavage operation, perform injection of corresponding multidrug-resistant Escherichia coli on each biological test subject in the third comparison group and the fourth comparison group at a dose of 10 ml / kg.

[0071] For example:

[0072] The PU group was gavaged with PU at 20 ml / kg, and the CFM group (positive drug control group) was gavaged with CFM at 26 ml / kg. During this period, the mice were fed normally, once a day for a total of 3 days. 1 hour after the liquid medicine gavage on the 3rd day, ECO was intraperitoneally injected at a dose of 10 ml / kg at 13.0 * 10 8 cfu / ml.

[0073] 1.5 Body weight and mortality of mice

[0074] Perform survival observation on the first comparison group, the second comparison group, the third comparison group, and the fourth comparison group for a preset observation duration to determine the survival status corresponding to each biological test subject, where the survival status includes a survival state and a death state.

[0075] For example:

[0076] Observe the daily body weight changes of the mice and record the death situation of the mice within 24 hours after intraperitoneal injection of the strain.

[0077] 1.6 Sample collection

[0078] Collect the corresponding colon tissues of all biological test subjects in the survival state to obtain a tissue sample set including each colon tissue.

[0079] Specifically, all biological test subjects in the survival state can be anesthetized to convert all biological test subjects from the survival state to the death state, and collect the colon tissues of all biological test subjects in the death state to obtain a tissue sample set including each colon tissue, and store the tissue sample set at a corresponding preset storage temperature.

[0080] For example:

[0081] To avoid abdominal infection caused by intraperitoneal injection, the operation was strictly carried out under sterile conditions. The mice that survived after 24 hours were anesthetized and sacrificed, and the colon tissues of the mice were collected into sterile tubes and immediately stored at -80 °C for subsequent 16S rDNA microbiome sequencing analysis.

[0082] 1.716S rDNA Microbiome Sequencing Analysis

[0083] 1.7.1 Extraction and PCR Amplification of Bacterial Genomic DNA

[0084] DNA extraction was performed on each colon tissue in the tissue sample set, and each extracted DNA sample was subjected to PCR amplification. Sequencing libraries were prepared based on the obtained PCR products.

[0085] Specifically, CTAB was used to extract DNA based on bacterial genomes from each colon tissue in the tissue sample set to obtain each DNA sample; specific primers were used to perform PCR amplification on each DNA sample to obtain each PCR product, and AMpure XT beads and Qubit were used to purify and quantify each PCR product respectively to obtain a sequencing library.

[0086] For example:

[0087] CTAB method was used to extract bacterial genomic DNA from the collected colon tissue. The obtained DNA samples were detected for DNA concentration and purity by ultraviolet spectrophotometer, and the DNA quality was detected by 2% agarose gel electrophoresis. Specific primers (341F: 5CCTACGGGNGGCWGCAG-3; 805R: 5'-GACTACHVGGGTATCTAATCC-3') were used to perform PCR amplification on the V3-V4 hypervariable region of the bacterial 16S rDNA gene.

[0088] PCR experiments were carried out in a 25 μL mixture containing 12.5 μL Phusion hot start flex 2X Master Mix, 50 ng template DNA, and 2.5 μL each of forward and reverse primers. PCR was initially denatured at 98 °C for 30 s, then cycled at 54 °C for 30 s for 27 cycles, elongated at 72 °C for 45 s, and finally extended at 72 °C for 10 min.

[0089] 1.7.2 Library Construction and Quality Assessment

[0090] AMPure XT beads (Beckman Coulter Genomics, USA) were used to purify and Qubit (Invitrogen, USA) was used to quantify the PCR products to prepare a sequencing library. Agilent 2100 Bioanalyzer (Agilent, USA) and a library quantification kit from Hlumina (Kapa Biosciences, USA) were used to evaluate the quality of the DNA library.

[0091] 1.7.316S rDNA Microbiome Sequencing Analysis

[0092] Sequencing analysis was performed on the sequencing library to obtain the analysis results of the intestinal flora of the corresponding organisms.

[0093] Specifically, the sequencing library was sequenced using a sequencer to obtain paired-end reads; the paired-end reads were paired-end assembled using overlap to obtain ASV characteristic sequences; diversity analysis was performed based on the ASV characteristic sequences to obtain the analysis results of the intestinal flora of the corresponding organisms.

[0094] For example:

[0095] The library was sequenced using a NovaSeq 6000 sequencer to obtain paired-end reads of 2×250bp. The raw data was paired-end assembled using overlap, and after quality control and chimera filtering, high-quality reads were obtained, denoised to obtain the ASV characteristic sequences and abundance table, and Alpha and Beta diversity analyses were performed based on the ASV characteristic sequences to study the richness and diversity of species in each group of samples and compare the similarity of species diversity between samples. Principal Coordinates Analysis (PCoA) was performed based on the R language, and UPGMA hierarchical clustering analysis and Anosim function were used to study the differences in community structure between groups. The SILVA and NT-16S databases were used to analyze the species with significant differences at different bacterial taxonomic levels. Potential microbial biomarkers in each group were determined by LEfSe analysis (LDA score threshold: 2.0). Finally, based on the PICRUSt2 function prediction results, differential analysis was performed using STAMP.

[0096] 1.8 Statistical Analysis

[0097] Fisher's exact test was used to compare the differences in samples without biological replicates. The Mann-Whitney U test was used to compare the differences between two groups of samples with biological replicates, and the Kruskal-Wallis test was used to compare multiple groups of samples with biological replicates. P<0.05 was considered statistically significant.

[0098] 2 Results

[0099] 2.1 Weight Changes and Mortality of Mice after PU Intervention

[0100] The weight changes of the mice are shown in Figure 1In the NC group of mice with abdominal cavity infection by ECO K1, the body weight showed an increasing trend, and the difference in body weight increase compared to the previous day was statistically significant. In the Model group, CFM group, and PU group, the body weight of the mice continued to increase from D1 to D3 (day 1 to day 3), but decreased on D4 (day 4). It is considered that the injection of bacterial solution into the abdominal cavity on D3 caused abdominal cavity infection, resulting in reduced appetite and slower metabolism in the mice, leading to weight loss.

[0101] The death situations of the mice in each group were observed. The mortality rate of the mice in the Model group was 90%. Compared with the NC group, the mortality rate increased and the difference was statistically significant (p < 0.05), indicating that the abdominal cavity infection model was relatively successful. Compared with the Model group, the mortality rates of the mice in the CFM group and PU group were significantly reduced (p < 0.05), and PU was more effective in significantly reducing the mortality rate of the mice than CFM. The results are as Figure 1 shown in

[0102] 2.2 ASV distribution of the intestinal flora of mice

[0103] As Figure 2 shown, the 16S rDNA microbiome of the colon tissue samples of the mice was analyzed by sequencing. The obtained Venn diagram of the ASV distribution of the intestinal flora showed that the NC group, Model group, CFM group, and PU group had 754, 878, 230, and 312 characteristic sequences respectively, and there were 305 characteristic sequences in total among the four groups. In the Model group, after the mice were infected with drug-resistant Escherichia coli, there were 938 characteristic sequences; after the intervention of CFM and PU, there were 663 and 831 characteristic sequences respectively. That is, the number of characteristic sequences reduced after the intervention of CFM was 168 more than that of PU, indicating that the influence of CFM on the characteristic sequences was slightly greater than that of PU. It shows that the composition of the intestinal flora of the mice in the Model group changed significantly, and CFM and PU had an impact on the flora composition.

[0104] 2.3 Alpha and Beta diversity analysis of the intestinal flora of mice

[0105] As Figure 3 shown, based on the ASV feature table, the Alpha diversity analysis of the intestinal flora was carried out through dilution curves and rank-abundance curves. As the number of sequences increased, the dilution curves of each group tended to be stable, indicating that the sequencing depth and the amount of sequencing data were reasonable and sufficient to reflect species diversity. The abundance and evenness of the intestinal flora of the mice in the Model group were significantly reduced, while CFM and PU had significant regulatory effects. In addition, Figure 3 as shown in

[0106] Principal coordinate analysis (PCoA) of Beta diversity, Figure 4A) It can be obtained that the sample regions of the NC group and the Model group were significantly separated, while the PU group and the CFM group both deviated from the Model group, forming another microbial community. That is, the composition of the intestinal microbiota of mice in the Model group changed significantly compared with that of the NC group. Severe peritoneal inflammation in mice significantly affected the distribution of the intestinal microbiota, and PU and CFM significantly regulated the distribution of the intestinal microbiota in Model group mice. In addition, the sample regions of the PU group and the NC group were close, indicating that PU could significantly regulate the intestinal microbiota of peritoneal infection mice and make it tend to the intestinal microbiota distribution of normal mice.

[0107] 2.4 Analysis of the similarity between groups of mouse intestinal microbiota

[0108] Based on the bray curtis algorithm, the distance matrix between samples was obtained, and the samples were clustered by the UPGMA method to construct a clustering tree ( Figure 4 B). It can be analyzed that the branch connection distance between the Model group and the other three groups was far, while the branch connection distances between the samples of the NC group, the CFM group, and the PU group were close. That is, the composition of the intestinal microbiota in the Model group changed greatly, while the intestinal microbiota compositions of the NC group, the CFM group, and the PU group had high similarity. Compared with the CFM group, the PU group and the NC group had higher similarity. After PU treatment, it could significantly regulate the distribution and composition of the intestinal microbiota of peritoneal infection mice and make it tend to the level of the intestinal microbiota of normal mice.

[0109] The Anosim similarity analysis of the sample distance matrix showed that R = 0.39 and p = 0.003 (p < 0.05), indicating that the differences in the distribution and composition of the intestinal microbiota between inter-group samples were significantly greater than those within-group samples, and the grouping effect was good. Further analysis showed that:

[0110] (1) There were significant differences in the distribution and composition of the intestinal microbiota between the NC group vs the Model group and the NC group vs the CFM group (p < 0.05), while there was no significant difference in the distribution and composition of the intestinal microbiota between the NC group vs the PU group (p > 0.05). It indicated that peritoneal inflammation in mice affected the distribution and composition of the intestinal microbiota, and PU significantly regulated the distribution and composition of its intestinal microbiota, while CFM failed to improve it.

[0111] (2) There was no significant difference in the distribution and composition of the intestinal microbiota between the Model group vs the CFM group (p > 0.05), and there was a significant difference between the Model group vs the PU group (p < 0.05). The above results further indicated that PU significantly regulated the community structure of the intestinal microbiota of peritoneal infection mice and made it tend to the intestinal microbiota structure of normal mice, thereby playing a potential anti-infection role, and the effect was better than that of CFM.

[0112] 2.5 Composition and difference analysis of mouse intestinal microbiota

[0113] Given that the composition and abundance of the intestinal flora in mouse colon tissue change significantly, the inter-group differential analysis of the relative abundances of the top 30 intestinal microorganisms was performed at the phylum and genus levels to explore the regulatory effect of PU on the intestinal microorganisms of mice infected with drug-resistant Escherichia coli.

[0114] At the phylum level, Campylobacter, Bacteroidetes, Firmicutes, and Proteobacteria were the main components of the relative abundances of the top 30 intestinal microorganisms at the phylum level in each group of mice ( Figure 5 A). Compared with the NC group, the abundance of Proteobacteria in the Model group increased significantly, while the abundances of Campylobacter and Firmicutes decreased. After CFM and PU intervention, this change trend was reversed. In addition, the abundance of Bacteroidetes decreased to a certain extent in the other three groups except the NC group (see Table 1).

[0115] At the genus level, Muribaculaceae_unclassified, Bacteroides, and Alloprevotella in Bacteroidetes; Helicobacter and Escherichia-Shigella in Proteobacteria were the main components of the relative abundances of the top 30 intestinal microorganisms at the genus level in the intestinal flora of each group of mice ( Figure 5 B). Compared with the NC group, the abundances of Escherichia-Shigella and Muribaculaceae_unclassified in the Model group increased significantly, showing the opposite trend in the CFM group and the PU group. The abundances of Helicobacter, Bacteroides, and Alloprevotella, which decreased in the Model group, were improved after PU intervention. The abundances of Bacteroides and Alloprevotella also decreased in the CFM group (see Table 2). Through the above analysis of the relative abundances of the TOP30 intestinal microorganisms at the phylum and genus levels in each group of mice, the results showed that PU and CFM had a regulatory effect on the relative abundances of the intestinal flora in mice, improved the distribution and structure of the intestinal flora in mice, and exerted a potential anti-infection effect. The relative abundances of the intestinal flora in the PU group and the NC group were similar at the phylum and genus levels, that is, the PU group significantly improved the relative abundances of the intestinal flora in mice with peritoneal inflammation.

[0116] After the barplot difference analysis of the bacterial community abundances at the genus level in each group, compared with the NC group, the abundances of most bacterial communities in the Model group were significantly reduced, while those in the PU group and CFM group were significantly increased. However, the abundances of g_Parabacteroides and g_Muribaculum in the CFM group were significantly reduced( Figure 6 A). According to the species relative abundance table, the top 30 bacterial communities with relative abundances at the genus level were clustered based on the abundance distribution of taxonomic units or the similarity degree among samples, and the heatmap was used to reflect the similarities and differences in the colony composition of different samples( Figure 6 B). The clustering branches showed that most of the bacterial communities in the Model group were significantly different from those in the other three groups, while the abundances of bacterial communities in the PU group and CFM group tended to be those of normal mice. Compared with the NC group, the abundances of hT002, Escherichia-Shigella, Enterococcus and other bacterial communities in the Model group were significantly increased, while the abundances of Oscillibacter, Lachnospiraceae NK44136group, Lachnospiraceae unclassified, Acinetobacter, Staphylococcus and other bacterial communities were significantly reduced. In the PU group and CFM group, the changes in the abundances of the above bacterial communities showed opposite trends, further improving the intestinal microbial homeostasis of mice with abdominal infection and playing an anti-infection role.

[0117] The linear discriminant analysis effect size (LEfSe, Figure 7A ) was used to further identify the characteristic biomarkers of intestinal microorganisms between groups, and 99 biomarkers (LDA score log10>2) were found. In addition, from the phylogenetic tree diagram from phylum to genus( Figure 7B ), it can be seen that there are significant differences in the abundances of bacterial communities at different levels, and there are differences in the effects of CFM and PU on bacterial communities.

[0118] Fifteen functional categories of gut microbiota with significant differences between groups were predicted by PICRUSt2, and nine functional pathways were significantly enriched, including replication, recombination and repair proteins, glycosyltransferases, biosynthesis of vancomycin-like antibiotics, inorganic ion transport and metabolism, cell cycle - Caulobacter, mismatch repair, epithelial cell signaling in Helicobacter pylori infection, glyoxylate and dicarboxylate metabolism, and electron transfer carriers. The functions with higher representation in the Model group were enriched in replication, recombination and repair proteins, glycosyltransferases, inorganic ion transport and metabolism, glyoxylate and dicarboxylate metabolism, and electron transfer carrier pathways. The functions with higher representation in the PU group were enriched in mismatch repair and epithelial cell signaling in Helicobacter pylori infection pathways.

[0119] Table 1: Changes in the abundance of gut microbiota at the phylum level

[0120]

[0121] Table 2: Changes in the abundance of gut microbiota at the genus level

[0122]

[0123] Taken together, the above experiments can fully demonstrate that R-(+)-pulegone can improve the gut microbial homeostasis of mice with abdominal infection and play an anti-infective role.

[0124] The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those of ordinary skill in the art can make many modifications and variations based on the concept of the present invention without creative efforts. Therefore, all technical solutions that can be obtained by those skilled in the art in the technical field of the present invention through logical analysis, reasoning, or limited experiments based on the concept of the present invention on the basis of the prior art should be within the protection scope determined by the claims.

Claims

1. Application of long-leaf menthone in regulating intestinal flora.

2. The use of long-leaf mint ketone in regulating the intestinal flora of organisms according to claim 1, characterized in that: The organism is a mouse.

3. The use of long-leaf mint ketone in regulating the intestinal flora of organisms according to claim 2, characterized in that: The mice were constructed by intraperitoneal infection with drug-resistant Escherichia coli.

4. The use of long-leaf mint ketone in regulating the intestinal flora of organisms according to claim 3, characterized in that: The longleaf menthol is R-(+)-longleaf menthol.

5. Use of the longifolia menthone according to any one of claims 1 to 4 as an intestinal microecological preparation.

6. An intestinal microecological preparation with R-(+)-menthol as the only active ingredient.

7. A test method for the application of biological intestinal flora, characterized in that: The following steps are involved: Establishing a first comparison group, a second comparison group, a third comparison group, and a fourth comparison group, respectively including the same number of biological test subjects; Performing intragastric gavage operation on the first comparison group and the second comparison group respectively for a preset number of days using edible oil, and after completing the intragastric gavage operation, performing an injection operation of corresponding physiological saline on the first comparison group, and performing an injection operation of corresponding multidrug-resistant Escherichia coli on the second comparison group; The third control group is subjected to a gavage operation for a preset number of days using R-(+)-longifolia menthone, and the fourth control group is subjected to a gavage operation for a preset number of days using cefixime, and after the gavage operation is completed, the third control group and the fourth control group are respectively subjected to an injection operation of corresponding multidrug-resistant Escherichia coli; Performing survival observation for a preset observation time on the first comparison group, the second comparison group, the third comparison group, and the fourth comparison group to determine a survival state corresponding to each biological test subject, wherein the survival state includes a living state and a dead state; Collecting corresponding colon tissues from all biological test subjects in corresponding survival states to obtain a tissue sample set including each colon tissue; Extracting DNA from each colon tissue in the tissue sample set, performing PCR amplification on each extracted DNA sample, and preparing a sequencing library based on each obtained PCR product; The sequencing library is sequenced and analyzed to obtain the analysis results of the corresponding biological intestinal flora.

8. The test method for the application of biological intestinal flora according to claim 7, characterized in that: The first comparison group and the second comparison group are respectively subjected to intragastric administration for a preset number of days using edible oil, and after the intragastric administration, the first comparison group is injected with physiological saline, and the second comparison group is injected with multidrug-resistant Escherichia coli, including: Each biological test subject in the first comparison group and the second comparison group is gavaged with edible oil at a rate of 20 ml / kg for a preset number of days; After the intragastric administration, each biological test subject in the first comparison group was injected with 10 ml / kg of normal saline, and each biological test subject in the second comparison group was injected with 10 ml / kg of multidrug-resistant Escherichia coli.

9. The test method for the application of biological intestinal flora according to claim 7, characterized in that: The third control group is subjected to a gavage operation for a preset number of days using R-(+)-longifolia menthone, and the fourth control group is subjected to a gavage operation for a preset number of days using cefixime. After the gavage operation is completed, the third control group and the fourth control group are respectively subjected to an injection operation of corresponding multidrug-resistant Escherichia coli, including: Each biological test subject in the third comparison group is gavaged with R-(+)-menthol at 20 ml / kg for a preset number of days; Using cefixime, gavage each biological test subject in the fourth comparison group at 26 ml / kg for a preset number of days; After the intragastric administration, each biological test subject in the third comparison group and the fourth comparison group was injected with the corresponding multidrug-resistant Escherichia coli at 10 ml / kg.

10. The test method for the application of biological intestinal flora according to claim 7, characterized in that: The corresponding colon tissues are collected from all the biological test subjects in the corresponding survival state to obtain a tissue sample set including each colon tissue, including: Performing anesthesia on all the biological test subjects in the corresponding living state to convert all the biological test subjects from the living state to the dead state, and collecting colon tissues from all the biological test subjects in the corresponding dead state to obtain a tissue sample set including each colon tissue; The tissue sample set is stored at a sample corresponding to a preset storage temperature.

11. The test method for the application of biological intestinal flora according to item 7, characterized in that: DNA is extracted from each colon tissue in the tissue sample set, each extracted DNA sample is amplified by PCR, and a sequencing library is prepared based on each obtained PCR product, including: Using CTAB, DNA based on bacterial genome is extracted from each colon tissue in the tissue sample set to obtain each DNA sample; Specific primers were used to perform PCR amplification on each DNA sample to obtain each PCR product; AMpure XTbeads and Qubit were used to purify and quantify each PCR product to obtain a sequencing library.

12. The test method for the application of biological intestinal flora according to claim 7, characterized in that: The sequencing library is sequenced and analyzed to obtain the analysis results of the corresponding biological intestinal flora, including: Performing sequencing analysis on the sequencing library using a sequencer to obtain double-end reads; Using overlap to perform double-end splicing on the double-end reads to obtain an ASV characteristic sequence; Diversity analysis was performed based on ASV characteristic sequences to obtain the analysis results of the corresponding biological intestinal flora.

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