Citron, chicory, elsholtzia and jujube are used for preparing drugs or preparations for promoting defecation, simulating prebiotics and inhibiting harmful bacteria

Abstract

The present application relates to the field of probiotics and prebiotics, and in particular to the use of Citrus aurantium, Chichorium intybus, Elsholtzia stauntoni and Hamamelis virginiana as prebiotic mimics (pseudobiotics) that are both medicinal and edible. We first demonstrated that the water extracts of these medicinal and edible herbs significantly improved the constipation symptoms of model mice, improved the pathological damage of the colon of constipated mice, significantly increased the expression of proteins related to intestinal peristalsis and maintenance of the intestinal barrier, promoted the colonization of beneficial bacteria in the intestines of constipated mice, and exhibited effects similar to prebiotics. Therefore, any one of Citrus aurantium, Chichorium intybus, Elsholtzia stauntoni and Hamamelis virginiana or a combination thereof, or a mixture prepared with other substances, can be used to treat constipation in mice, and the mechanism of action may be related to promoting intestinal peristalsis, enhancing the function of the intestinal barrier, and increasing the abundance of beneficial bacteria in the intestines.

Description

Technical Field

[0001] This invention relates to the field of probiotics and prebiotics, specifically referring to the use of prebiotic mimics (biogenic agents) such as citron, chicory, euphorbia, and jujube seed in loperamide-induced constipation model mice to promote the colonization of beneficial intestinal bacteria and inhibit the colonization of harmful intestinal bacteria, thereby improving constipation symptoms in the model mice. Background Technology

[0002] Probiotics are live microorganisms that can colonize the human body, help alter the composition of the gut microbiota in specific sites, and are beneficial to the host. Probiotics promote health by regulating the host's mucosal and systemic immune functions, or by balancing the gut microbiota, thereby promoting nutrient absorption and maintaining intestinal health. These microorganisms can be single strains or well-defined combinations of bacteria. Probiotics are closely related to human health, and a wide variety of probiotic species have been identified.

[0003] The growth and colonization of probiotics depend on nutritional and environmental factors, making this field a focus of widespread attention in the biomedical community. Glenn Gibson, hailed as the "father of prebiotics," introduced the concept of prebiotics in 1995. Prebiotics are organic substances that cannot be digested and absorbed by the host but can selectively promote the metabolism and proliferation of beneficial bacteria in the body, thereby improving the host's health. Besides traditional prebiotics, a few researchers have attempted to use other substances, such as traditional Chinese medicine, to promote the growth and metabolism of these beneficial bacteria, but related research reports are limited.

[0004] China's experience in using traditional medicine has made an indelible contribution to the progress of global civilization. Based on extensive safety studies and long-term practical use of traditional Chinese medicine, the Chinese government has compiled a list of traditional Chinese medicines that can be used as food. However, to date, no scholar has systematically evaluated the effects of these medicinal and edible herbs on the proliferation and growth of representative probiotics.

[0005] We conducted a systematic search of traditional Chinese medicine (TCM) varieties that can be used as food (TCMs that are both food and medicine), excluding those TCMs that have been reported to affect fungal growth, and finally screening out TCMs that are both food and medicine whose effects on fungal growth have not yet been investigated. Next, we evaluated the disease-improving effects of these TCMs on loperamide-induced constipation model mice, as well as their effects on the growth and colonization of beneficial and harmful bacteria in the mouse gut.

[0006] The tested medicinal and edible herbs, including citron, chicory, elsholtzia, and raisin tree fruit, showed a promoting effect on the proliferation and growth of representative probiotics, and therefore can be considered as prebiotic mimics. The inventors of this application, Wu Junhua, Jiang Chunping, and Academician Gu Xiaosong, have named these substances capable of mimicking the functions of prebiotics "prebiomimetics." The inventors of this application hope to make original contributions to the research and development of probiotic-related products worldwide.

[0007] Citron (scientific name: *Citrus medica* L.), also known as Chinese citron, is an evergreen shrub or small tree belonging to the genus *Citrus* in the family Rutaceae. It has short petioles, elliptical leaves with a rounded apex and serrated margins; small, pale yellow flowers with a smooth, hairless surface; a thick, long style and a large stigma; and an elliptical fruit with a pale yellow, rough, and difficult-to-peel rind, and a white or slightly pale yellow inner peel.

[0008] Chicory is the dried aerial parts or root of *Cicada oleracea* or *Chicoryca indicum*, both belonging to the Asteraceae family. The stem surface is nearly smooth; the cauline leaves are few, oblong-lanceolate; the capitula are few, clustered; the bracts are shorter on the outside and longer on the inside, glabrous or sparsely pubescent at the apex; the achenes are scale-like, with short pappus.

[0009] Elsholtzia ciliata is the dried aerial part of Elsholtzia ciliata or Elsholtzia jiangensis, both belonging to the Lamiaceae family. The base is purplish-red, the upper part yellowish-green or pale yellow, and the entire plant is densely covered with white downy hairs. The stem is square-columnar, with a sub-rounded base and distinct nodes; it is brittle and easily broken. The leaves are opposite, mostly wrinkled or deciduous; when flattened, the leaf blade is oblong-ovate or lanceolate, dark green or yellowish-green, with 3-5 sparse, shallow serrations along the margin.

[0010] The seeds of the Japanese raisin tree are flat and round, slightly raised on the back, with a relatively flat flag surface, about 5 mm in diameter and 1-1.5 mm thick. The surface is reddish-brown, dark brown, or greenish-brown, glossy, with a hard seed coat, white endosperm, and pale yellow, thick cotyledons, all rich in oil.

[0011] To date, there have been no reports on the effects of the four medicinal and edible herbs disclosed in this invention—citron, chicory, elsholtzia, and jujube seed—on bacterial growth and mouse constipation, which are effective in improving constipation in mice. Furthermore, no research has been found on the effects of these four medicinal and edible herbs on promoting the accumulation of beneficial intestinal bacteria and inhibiting harmful bacteria. Summary of the Invention

[0012] In this invention, a mouse constipation model was established using loperamide gavage. Subsequently, the mice were administered four medicinal and edible herbs (hereinafter referred to as medicinal and edible herbal biostimulants): citron, chicory, elsholtzia, and jujube seed. Fecal parameters, such as fecal quantity, fecal water content, and intestinal propulsion rate, were observed. The results showed that the four medicinal and edible herbal biostimulants significantly alleviated constipation symptoms in the model mice by increasing fecal quantity, fecal water content, and improving intestinal propulsion rate, especially by promoting intestinal peristalsis.

[0013] Meanwhile, we evaluated the histological changes in the colon of mice, and found that the colonic mucosa of the model group mice was significantly damaged, accompanied by the loss of goblet cells and changes in crypt structure. The ZO-1, Occludin, and Claudin-1 proteins, which maintain the integrity and function of the intestinal barrier, were also impaired. Intervention with four kinds of herbal remedies containing biosynthetic components rescued these phenomena and increased the expression level of intestinal tight junction proteins. This may help improve the increased intestinal permeability and barrier function damage induced by lopidine, thereby restoring the defecation function of mice.

[0014] Meanwhile, we also observed the effects of gavage administration of citron, chicory, elsholtzia, and jujube seed on the gut microbiota of mice in a mouse model. 16S rRNA analysis identified newly emerging or increased beneficial bacteria in the mouse gut after treatment with citron, chicory, elsholtzia, and jujube seed; simultaneously, we also identified harmful bacteria that disappeared or decreased in number in the mouse gut after treatment with these same herbs.

[0015] The details of this invention are as follows:

[0016] The effect and mechanism of prebiotic mimics (biogenic prebiotics) of food and medicine in improving constipation symptoms in loperamide-induced model mice. The prebiotic mimics (biogenic prebiotics) of food and medicine refer to organic substances that cannot be digested and absorbed by the host, but can selectively promote the metabolism and proliferation of beneficial bacteria in the body, thereby improving the health of the host.

[0017] The effect and mechanism of prebiotic mimics (biogenic analogs) of food and medicine in improving constipation symptoms in loperamide-induced model mice. The prebiotic mimics (biogenic analogs) of food and medicine refer to any one or a combination of citron, chicory, euphorbia, or jujube.

[0018] The effect and mechanism of prebiotic mimics (biogenic analogs) of food and medicine in improving constipation symptoms in loperamide-induced model mice are as follows: when applied, one, two, three or four prebiotic mimics (biogenic analogs) of food and medicine or their extracts are mixed with other substances to form a complex, which is used to improve constipation symptoms in loperamide-induced model mice.

[0019] The effect and mechanism of prebiotic mimics (biogenic analogs) of food and medicine in improving constipation symptoms in loperamide-induced model mice. The loperamide-induced constipation model mice refer to mice that exhibit the expected disease symptoms after subcutaneous injection of loperamide hydrochloride (Lop), such as reduced activity, reduced water content of fecal particles, hard fecal texture, and reduced fecal particle size.

[0020] The effects and mechanisms of prebiotic mimics (biogenic compounds) of food and medicine in improving constipation symptoms in loperamide-induced model mice are as follows: the effects and mechanisms of improving constipation symptoms in mice are to improve the pathological tissue damage in the colon of constipated mice, significantly increase the expression of proteins related to promoting intestinal peristalsis and maintaining the intestinal barrier, and promote the colonization of beneficial bacteria in the intestines of constipated mice.

[0021] Food-medicine homologous prebiotics (biogenic analogs) promoted the colonization of beneficial intestinal bacteria and inhibited the colonization of harmful intestinal bacteria in mice with loperamide-induced constipation. Attached Figure Description

[0022] Figure 1 Summary of fecal index results for mice after different sample treatments: The fecal index of each sample is displayed in a bar chart, which visually shows the time of first black stool excretion, number of black stools excreted at 6 hours, fecal dry weight, fecal water content, and small intestinal propulsion rate for different samples. Caption: The horizontal axis represents the sample name, and the vertical axis represents the values ​​of different fecal indices; different colors represent different groups.

[0023] Figure 2 Summary of HE results: HE results visually demonstrate the effects of different treatments on the morphology of mouse colon tissue.

[0024] Figure 3 Summary of Immunohistochemical Results Comparison of Mouse Colon Tissue After Different Sample Treatments: Immunohistochemical and quantitative analysis results visually demonstrate the effects of different treatments on the expression levels of c-Kit and SCF in mouse colon tissue.

[0025] Figure 4 Summary of Western blot analysis results of colon tissue proteins after different sample treatments: The effects of different treatments on the expression levels of ZO-1, Occludin, and Claudin-1 proteins in mouse colon tissues were visually demonstrated through the results of Western blot analysis and statistical analysis of band gray values.

[0026] Figure 5 Summary of α-diversity index results of mouse fecal gut microbiota after different sample treatments: The α-diversity index of each sample is displayed in a bar chart, visually showing the magnitude of the α-diversity index among different samples. Caption: The horizontal axis represents the sample name, and the vertical axis represents the Shannoneven index at the ASV level; different colors represent different groups.

[0027] Figure 6Summary of the results of the exponential between-group difference test for the intestinal flora of mouse feces after different sample treatments: This figure shows the significant differences in the indices between the selected different groups and the changes in the indices with the experimental research. A P-value < 0.05 below the main title indicates that there are significant differences in the indices between the groups. Figure note: This figure shows the significant difference situation between the selected two groups of samples, and marks the two groups with significant differences (marked as * for 0.01 < P ≤ 0.05, marked as ** for 0.001 < P ≤ 0.01, and marked as *** for P ≤ 0.001). The abscissa is the group name, and the ordinate is the index value of each group.

[0028] Figure 7 Summary of the results of the dilution curve analysis of the intestinal flora of mouse feces after different sample treatments: The dilution curve is used to illustrate whether the sequencing data volume of the sample is sufficient. When the curve tends to be flat at the end, it indicates that the sequencing data volume is reasonable. Figure note: Abscissa, the randomly selected sequencing data volume; ordinate, the observed diversity index (such as the Shannon index).

[0029] Figure 8 Summary of the results of the PCA analysis of the intestinal flora of mouse feces after different sample treatments: A three-dimensional visual scatter plot is used to show the similarity and difference degree of the communities of the control group and the treatment group. The clustering and dispersion degree of the sample communities are reflected by the distance size between samples (default Euclidean distance algorithm). This analysis result is applied with statistical analyses such as ANOSIM / adonis / PERMAVONA to present the community difference changes between the control group and the treatment group. Figure note: The abscissa and ordinate represent two selected principal coordinate components, and the percentage represents the contribution value of the principal coordinate components to the sample composition difference. The scales of the abscissa and ordinate axes are relative distances and have no practical significance. Points of different colors or shapes represent samples of different groups. The closer the two sample points are, the more similar the species compositions of the two samples are; the chart tool can be selected to display the information of the third axis and draw a 3D-PCA graph.

[0030] Figure 9Summary of PCoA analysis results of mouse fecal gut microbiota after different sample treatments: A two-dimensional scatter plot was used to display the similarity and differences between the control and treatment groups. The distance between samples (different distance algorithms can be applied; Bray-curtis, unweighted UniFrac, weighted UniFrac, and Jaccard are commonly used in this paper) reflects the clustering degree of the sample communities. The statistical analysis results were performed using ANOSIM / adonis / PERMAVONA to present the significant differences in community composition between the control and treatment groups. Figure caption: The horizontal and vertical axes represent two selected principal components; the percentage represents the contribution of the principal components to the differences in sample composition. The scales of the horizontal and vertical axes are relative distances and have no practical significance. Points of different colors or shapes represent samples from different groups; the closer the points of two samples are, the more similar the species composition of the two samples. The chart tool can optionally display information on a third axis to create a 3D-PCA plot.

[0031] Figure 10 Summary of gut microbiota typing analysis results of mouse feces after different sample treatments: Microbiota typing plots typically use scatter plots to display the microbial composition of different samples or treatment groups to compare differences between them. The dominant bacterial community structure of different samples reflects the microbiota typing of a specific clinical sample. Caption: The upper right corner shows different sample groups; different colors represent different typings; the circled area represents the range of confidence intervals.

[0032] Figure 11 Summary of Venn plot analysis results of mouse fecal gut microbiota after different sample treatments: Venn focuses on analyzing and displaying the number of species unique to and shared by different groups, facilitating the understanding of changes in the presence or absence of species under different research conditions. This application can be used to select biomarkers for research based on unique or shared species. Caption: Different colors represent different groups (or samples). Overlapping areas indicate species shared by multiple groups (or samples), while non-overlapping areas indicate species unique to that group (or sample). Numbers represent the corresponding number of species. On the interactive page of the cloud platform, clicking on a number in the Venn plot will display the corresponding species information (e.g., ASV level) in an information box.

[0033] Figure 12Summary of Bar plot analysis results of mouse fecal gut microbiota after different sample treatments: The community Bar plot shows the composition of the top N most abundant species in all samples and the proportion of each species, with other low-abundance species grouped as "Others". This figure mainly shows the changes in the composition of dominant species in different samples / groups. Caption: The x-axis / y-axis represents the sample name, and the y-axis / x-axis represents the proportion of the species in that sample. Different colored bars represent different species, and the length of the bar represents the proportion of that species.

[0034] Figure 13 Summary of Heatmap Analysis Results of Mouse Fecal Intestinal Microbiota after Different Sample Treatments: The heatmap visually displays the distribution of the top dominant species in different samples / groups across all samples, exploring the species variation trends in the control and treatment groups. Caption: The horizontal axis represents the sample name, and the vertical axis represents the species name. A color gradient is used to represent the proportion of each species; the right side of the figure shows the numerical values ​​represented by the color gradient.

[0035] Figure 14 Summary of the bar chart results of the comparative analysis of mouse fecal gut microbiota after different sample treatments: The bar chart shows the differences in the average relative abundance of the same species between different groups, and marks whether the differences are significant, intuitively demonstrating the differences in the average relative abundance of the same species between different groups. Caption: The horizontal axis represents the species name at different taxonomic levels, and the vertical axis represents the percentage abundance of a certain species in the sample. Different colors represent different groups. The rightmost value is the p-value, *0.01. <P≤0.05,**0.001<P≤0.01,***P≤0.001。

[0036] Figure 15 Summary of fecal index results for mice after different sample treatments: The fecal index of each sample is displayed in a bar chart, which visually shows the time of first black stool excretion, number of black stools excreted at 6 hours, fecal dry weight, fecal water content, and small intestinal propulsion rate for different samples. Caption: The horizontal axis represents the sample name, and the vertical axis represents the values ​​of different fecal indices; different colors represent different groups.

[0037] Figure 16 Summary of HE results: HE results visually demonstrate the effects of different treatments on the morphology of mouse colon tissue.

[0038] Figure 17 Summary of Immunohistochemical Results Comparison of Mouse Colon Tissue After Different Sample Treatments: Immunohistochemical and quantitative analysis results visually demonstrate the effects of different treatments on the expression levels of c-Kit and SCF in mouse colon tissue.

[0039] Figure 18Summary of the results of Western blot analysis of colon tissue proteins after different sample treatments: Through the immunoblot results and statistical analysis of band gray values, it is intuitively shown the effects of different treatment methods on the protein expression levels of ZO-1, Occludin, and Claudin-1 in the colon tissues of mice.

[0040] Figure 19 Summary of the α-diversity index results of the intestinal flora in the feces of mice after different sample treatments: The bar chart shows the α-diversity index of each sample, intuitively demonstrating the size of the α-diversity index of different samples. Figure note: The horizontal axis represents the sample name, and the vertical axis represents the value of the Shannon evenness index at the ASV level. Different colors represent different groups.

[0041] Figure 20 Summary of the results of the index between-group difference test of the intestinal flora in the feces of mice after different sample treatments: This figure shows the significant differences in the index between the selected different groups and the changes in the index with the experimental study. The P value < 0.05 below the main title indicates that there are significant differences in the index between the groups. Figure note: This figure shows the significant difference situation between the selected two groups of samples, and the two groups with significant differences are marked (marked as * for 0.01 < P ≤ 0.05, marked as ** for 0.001 < P ≤ 0.01, marked as *** for P ≤ 0.001). The horizontal axis represents the group name, and the vertical axis represents the index size of each group.

[0042] Figure 21 Summary of the results of the rarefaction curve analysis of the intestinal flora in the feces of mice after different sample treatments: The rarefaction curve is used to illustrate whether the sequencing data volume of the sample is sufficient. When the curve tends to be flat at the end, it indicates that the sequencing data volume is reasonable. Figure note: The horizontal axis is the randomly selected sequencing data volume; the vertical axis is the observed diversity index (such as the Shannon index).

[0043] Figure 22 Summary of the results of the PCA analysis of the intestinal flora in the feces of mice after different sample treatments: The three-dimensional visual scatter plot is used to show the similarity and difference degrees of the communities of the control group and the treatment group. The distance between samples (default Euclidean distance algorithm) reflects the aggregation and dispersion degree of the sample communities. The results of this analysis are applied with statistical analysis ANOSIM / adonis / PERMAVONA to present the community differences between the control group and the treatment group. Figure note: The horizontal and vertical axes represent two selected principal coordinate components, and the percentage represents the contribution value of the principal coordinate components to the sample composition difference. The scales of the horizontal and vertical axes are relative distances and have no practical significance. Different colored or shaped points represent samples of different groups. The closer the two sample points are, the more similar the species composition of the two samples; the chart tool can be selected to display the information of the third axis and draw a 3D-PCA graph.

[0044] Figure 23Summary of PCoA analysis results of mouse fecal gut microbiota after different sample treatments: A two-dimensional scatter plot was used to display the similarity and differences between the control and treatment groups. The distance between samples (different distance algorithms can be applied; Bray-curtis, unweighted UniFrac, weighted UniFrac, and Jaccard are commonly used in this paper) reflects the clustering degree of the sample communities. The statistical analysis results were performed using ANOSIM / adonis / PERMAVONA to present the significant differences in community composition between the control and treatment groups. Figure caption: The horizontal and vertical axes represent two selected principal components; the percentage represents the contribution of the principal components to the differences in sample composition. The scales of the horizontal and vertical axes are relative distances and have no practical significance. Points of different colors or shapes represent samples from different groups; the closer the points of two samples are, the more similar the species composition of the two samples. The chart tool can optionally display information on a third axis to create a 3D-PCA plot.

[0045] Figure 24 Summary of gut microbiota typing analysis results of mouse feces after different sample treatments: Microbiota typing plots typically use scatter plots to display the microbial composition of different samples or treatment groups to compare differences between them. The dominant bacterial community structure of different samples reflects the microbiota typing of a specific clinical sample. Caption: The upper right corner shows different sample groups; different colors represent different typings; the circled area represents the range of confidence intervals.

[0046] Figure 25 Summary of Venn plot analysis results of mouse fecal gut microbiota after different sample treatments: Venn focuses on analyzing and displaying the number of species unique to and shared by different groups, facilitating the understanding of changes in the presence or absence of species under different research conditions. This application can be used to select biomarkers for research based on unique or shared species. Caption: Different colors represent different groups (or samples). Overlapping areas indicate species shared by multiple groups (or samples), while non-overlapping areas indicate species unique to that group (or sample). Numbers represent the corresponding number of species. On the interactive page of the cloud platform, clicking on a number in the Venn plot will display the corresponding species information (e.g., ASV level) in an information box.

[0047] Figure 26Summary of Bar plot analysis results of mouse fecal gut microbiota after different sample treatments: The community Bar plot shows the composition of the top N most abundant species in all samples and the proportion of each species, with other low-abundance species grouped as "Others". This figure mainly shows the changes in the composition of dominant species in different samples / groups. Caption: The x-axis / y-axis represents the sample name, and the y-axis / x-axis represents the proportion of the species in that sample. Different colored bars represent different species, and the length of the bar represents the proportion of that species.

[0048] Figure 27 Summary of Heatmap Analysis Results of Mouse Fecal Intestinal Microbiota after Different Sample Treatments: The heatmap visually displays the distribution of the top dominant species in different samples / groups across all samples, exploring the species variation trends in the control and treatment groups. Caption: The horizontal axis represents the sample name, and the vertical axis represents the species name. A color gradient is used to represent the proportion of each species; the right side of the figure shows the numerical values ​​represented by the color gradient.

[0049] Figure 28 Summary of the bar chart results of the comparative analysis of mouse fecal gut microbiota after different sample treatments: The bar chart shows the differences in the average relative abundance of the same species between different groups, and marks whether the differences are significant, intuitively demonstrating the differences in the average relative abundance of the same species between different groups. Caption: The horizontal axis represents the species name at different taxonomic levels, and the vertical axis represents the percentage abundance of a certain species in the sample. Different colors represent different groups. The rightmost value is the p-value, *0.01. <P≤0.05,**0.001<P≤0.01,***P≤0.001。

[0050] Figure 29 Summary of fecal index results for mice after different sample treatments: The fecal index of each sample is displayed in a bar chart, which visually shows the time of first black stool excretion, number of black stools excreted at 6 hours, fecal dry weight, fecal water content, and small intestinal propulsion rate for different samples. Caption: The horizontal axis represents the sample name, and the vertical axis represents the values ​​of different fecal indices; different colors represent different groups.

[0051] Figure 30 Summary of HE results: HE results visually demonstrate the effects of different treatments on the morphology of mouse colon tissue.

[0052] Figure 31 Summary of Immunohistochemical Results Comparison of Mouse Colon Tissue After Different Sample Treatments: Immunohistochemical and quantitative analysis results visually demonstrate the effects of different treatments on the expression levels of c-Kit and SCF in mouse colon tissue.

[0053] Figure 32Summary of the results of Western blot analysis of colon tissue proteins after different sample treatments: Through the statistical analysis of the Western blot results and band gray values, it is直观地显示出 different treatment methods have on the protein expression levels of ZO-1, Occludin, and Claudin-1 in the colon tissues of mice.

[0054] Figure 33 Summary of the α-diversity index results of the intestinal flora in the feces of mice after different sample treatments: The bar chart shows the α-diversity index of each sample,直观展示了 the size of the α-diversity index of different samples. Figure note: The abscissa represents the sample name, the ordinate represents the value of the Shannon even index at the ASV level, and different colors represent different groups.

[0055] Figure 34 Summary of the results of the index between-group difference test of the intestinal flora in the feces of mice after different sample treatments: This figure shows the significant differences in the index between the selected different groups and the changes in the index with the experimental research. The P value below the main title < 0.05 indicates that there are significant differences in the index between the groups. Figure note: This figure shows the significant difference situation between the selected two groups of samples, and the two groups with significant differences are marked (0.01 < P ≤ 0.05 is marked as *, 0.001 < P ≤ 0.01 is marked as **, P ≤ 0.001 is marked as ***). The abscissa represents the group name, and the ordinate represents the size of the index of each group.

[0056] Figure 35 Summary of the results of the dilution curve analysis of the intestinal flora in the feces of mice after different sample treatments: The dilution curve is used to说明样本的测序数据量是否足够. When the curve tends to be flat at the end, it indicates that the sequencing data volume is reasonable. Figure note: The abscissa is the randomly selected sequencing data volume; the ordinate is the observed diversity index (such as the Shannon index).

[0057] Figure 36 Summary of the results of the PCA analysis of the intestinal flora in the feces of mice after different sample treatments: A three-dimensional visual scatter plot is used to展示对照组和处理组的群落的相似度和差异度. The degree of aggregation and dispersion of the sample communities is reflected by the distance between samples (default Euclidean distance algorithm). The statistical analysis results of this analysis, such as ANOSIM / adonis / PERMAVONA, are applied to呈现对照组和处理组群落差异性变化. Figure note: The abscissa and ordinate represent two selected principal coordinate components, and the percentage represents the contribution value of the principal coordinate components to the difference in sample composition. The scales of the abscissa and ordinate axes are relative distances and have no practical significance. Different colored or shaped points represent samples of different groups. The closer the two sample points are, the more similar the species composition of the two samples is; the chart tool can be used to select to display the information of the third axis and draw a 3D-PCA graph.

[0058] Figure 37 It should be noted that the part "直观地显示出" and "直观展示了" in the translation are directly translated according to the meaning, but in a more professional context, more accurate and appropriate expressions may be used. Also, for some terms like "说明样本的测序数据量是否足够", a more accurate and standard expression could be further refined according to specific professional knowledge.Summary of PCoA analysis results of mouse fecal gut microbiota after different sample treatments: A two-dimensional scatter plot was used to display the similarity and differences between the control and treatment groups. The distance between samples (different distance algorithms can be applied; Bray-curtis, unweighted UniFrac, weighted UniFrac, and Jaccard are commonly used in this paper) reflects the clustering degree of the sample communities. The statistical analysis results were performed using ANOSIM / adonis / PERMAVONA to present the significant differences in community composition between the control and treatment groups. Figure caption: The horizontal and vertical axes represent two selected principal components; the percentage represents the contribution of the principal components to the differences in sample composition. The scales of the horizontal and vertical axes are relative distances and have no practical significance. Points of different colors or shapes represent samples from different groups; the closer the points of two samples are, the more similar the species composition of the two samples. The chart tool can optionally display information on a third axis to create a 3D-PCA plot.

[0059] Figure 38 Summary of gut microbiota typing analysis results of mouse feces after different sample treatments: Microbiota typing plots typically use scatter plots to display the microbial composition of different samples or treatment groups to compare differences between them. The dominant bacterial community structure of different samples reflects the microbiota typing of a specific clinical sample. Caption: The upper right corner shows different sample groups; different colors represent different typings; the circled area represents the range of confidence intervals.

[0060] Figure 39 Summary of Venn plot analysis results of mouse fecal gut microbiota after different sample treatments: Venn focuses on analyzing and displaying the number of species unique to and shared by different groups, facilitating the understanding of changes in the presence or absence of species under different research conditions. This application can be used to select biomarkers for research based on unique or shared species. Caption: Different colors represent different groups (or samples). Overlapping areas indicate species shared by multiple groups (or samples), while non-overlapping areas indicate species unique to that group (or sample). Numbers represent the corresponding number of species. On the interactive page of the cloud platform, clicking on a number in the Venn plot will display the corresponding species information (e.g., ASV level) in an information box.

[0061] Figure 40Summary of Bar plot analysis results of mouse fecal gut microbiota after different sample treatments: The community Bar plot shows the composition of the top N most abundant species in all samples and the proportion of each species, with other low-abundance species grouped as "Others". This figure mainly shows the changes in the composition of dominant species in different samples / groups. Caption: The x-axis / y-axis represents the sample name, and the y-axis / x-axis represents the proportion of the species in that sample. Different colored bars represent different species, and the length of the bar represents the proportion of that species.

[0062] Figure 41 Summary of Heatmap Analysis Results of Mouse Fecal Intestinal Microbiota after Different Sample Treatments: The heatmap visually displays the distribution of the top dominant species in different samples / groups across all samples, exploring the species variation trends in the control and treatment groups. Caption: The horizontal axis represents the sample name, and the vertical axis represents the species name. A color gradient is used to represent the proportion of each species; the right side of the figure shows the numerical values ​​represented by the color gradient.

[0063] Figure 42 Summary of the bar chart results of the comparative analysis of mouse fecal gut microbiota after different sample treatments: The bar chart shows the differences in the average relative abundance of the same species between different groups, and marks whether the differences are significant, intuitively demonstrating the differences in the average relative abundance of the same species between different groups. Caption: The horizontal axis represents the species name at different taxonomic levels, and the vertical axis represents the percentage abundance of a certain species in the sample. Different colors represent different groups. The rightmost value is the p-value, *0.01. <P≤0.05,**0.001<P≤0.01,***P≤0.001。

[0064] Figure 43 Summary of fecal index results for mice after different sample treatments: The fecal index of each sample is displayed in a bar chart, which visually shows the time of first black stool excretion, number of black stools excreted at 6 hours, fecal dry weight, fecal water content, and small intestinal propulsion rate for different samples. Caption: The horizontal axis represents the sample name, and the vertical axis represents the values ​​of different fecal indices; different colors represent different groups.

[0065] Figure 44 Summary of HE results: HE results visually demonstrate the effects of different treatments on the morphology of mouse colon tissue.

[0066] Figure 45 Summary of Immunohistochemical Results Comparison of Mouse Colon Tissue After Different Sample Treatments: Immunohistochemical and quantitative analysis results visually demonstrate the effects of different treatments on the expression levels of c-Kit and SCF in mouse colon tissue.

[0067] Figure 46Summary of the results of Western blot analysis of colon tissue proteins after different sample treatments: Through the statistical analysis of the Western blot results and band gray values, the effects of the protein expression levels of ZO-1, Occludin, and Claudin-1 in the colon tissues of mice with different treatment methods were visually shown.

[0068] Figure 47 Summary of the α-diversity index results of the intestinal flora in the feces of mice after different sample treatments: The bar chart was used to display the α-diversity index of each sample, visually showing the sizes of the α-diversity indices of different samples. Figure note: The abscissa represents the sample name, the ordinate represents the value of the Shannon evenness index at the ASV level, and different colors represent different groups.

[0069] Figure 48 Summary of the results of the index between-group difference test of the intestinal flora in the feces of mice after different sample treatments: This figure shows the significant differences in the indices between the selected different groups and the changes in the indices with the experimental study. The P value < 0.05 below the main title indicates that there are significant differences in the indices between the groups. Figure note: This figure shows the significant difference situation between the two selected groups of samples, and the two groups with significant differences are marked (0.01 < P ≤ 0.05 is marked as *, 0.001 < P ≤ 0.01 is marked as **, P ≤ 0.001 is marked as ***). The abscissa represents the group name, and the ordinate represents the size of the index of each group.

[0070] Figure 49 Summary of the results of the rarefaction curve analysis of the intestinal flora in the feces of mice after different sample treatments: The rarefaction curve is used to illustrate whether the sequencing data volume of the sample is sufficient. When the curve tends to be flat at the end, it indicates that the sequencing data volume is reasonable. Figure note: Abscissa, randomly extracted sequencing data volume; ordinate, observed diversity index (such as Shannon index).

[0071] Figure 50 Summary of the results of the PCA analysis of the intestinal flora in the feces of mice after different sample treatments: A three-dimensional visualized scatter plot was used to display the similarity and difference degrees of the communities of the control group and the treatment group. The degree of aggregation and dispersion of the sample communities was reflected by the distance size between samples (default Euclidean distance algorithm). The results of this analysis were applied with statistical analyses such as ANOSIM / adonis / PERMAVONA to present the community differences between the control group and the treatment group. Figure note: The abscissa and ordinate represent two selected principal coordinate components, and the percentage represents the contribution value of the principal coordinate components to the sample composition difference. The scales of the abscissa and ordinate axes are relative distances and have no practical significance. Different colored or shaped points represent samples of different groups. The closer the two sample points are, the more similar the species compositions of the two samples are; the chart tool can be selected to display the information of the third axis and draw a 3D-PCA graph.

[0072] Figure 51Summary of PCoA analysis results of mouse fecal gut microbiota after different sample treatments: A two-dimensional scatter plot was used to display the similarity and differences between the control and treatment groups. The distance between samples (different distance algorithms can be applied; Bray-curtis, unweighted UniFrac, weighted UniFrac, and Jaccard are commonly used in this paper) reflects the clustering degree of the sample communities. The statistical analysis results were performed using ANOSIM / adonis / PERMAVONA to present the significant differences in community composition between the control and treatment groups. Figure caption: The horizontal and vertical axes represent two selected principal components; the percentage represents the contribution of the principal components to the differences in sample composition. The scales of the horizontal and vertical axes are relative distances and have no practical significance. Points of different colors or shapes represent samples from different groups; the closer the points of two samples are, the more similar the species composition of the two samples. The chart tool can optionally display information on a third axis to create a 3D-PCA plot.

[0073] Figure 52 Summary of gut microbiota typing analysis results of mouse feces after different sample treatments: Microbiota typing plots typically use scatter plots to display the microbial composition of different samples or treatment groups to compare differences between them. The dominant bacterial community structure of different samples reflects the microbiota typing of a specific clinical sample. Caption: The upper right corner shows different sample groups; different colors represent different typings; the circled area represents the range of confidence intervals.

[0074] Figure 53 Summary of Venn plot analysis results of mouse fecal gut microbiota after different sample treatments: Venn focuses on analyzing and displaying the number of species unique to and shared by different groups, facilitating the understanding of changes in the presence or absence of species under different research conditions. This application can be used to select biomarkers for research based on unique or shared species. Caption: Different colors represent different groups (or samples). Overlapping areas indicate species shared by multiple groups (or samples), while non-overlapping areas indicate species unique to that group (or sample). Numbers represent the corresponding number of species. On the interactive page of the cloud platform, clicking on a number in the Venn plot will display the corresponding species information (e.g., ASV level) in an information box.

[0075] Figure 54Summary of Bar plot analysis results of mouse fecal gut microbiota after different sample treatments: The community Bar plot shows the composition of the top N most abundant species in all samples and the proportion of each species, with other low-abundance species grouped as "Others". This figure mainly shows the changes in the composition of dominant species in different samples / groups. Caption: The x-axis / y-axis represents the sample name, and the y-axis / x-axis represents the proportion of the species in that sample. Different colored bars represent different species, and the length of the bar represents the proportion of that species.

[0076] Figure 55 Summary of Heatmap Analysis Results of Mouse Fecal Intestinal Microbiota after Different Sample Treatments: The heatmap visually displays the distribution of the top dominant species in different samples / groups across all samples, exploring the species variation trends in the control and treatment groups. Caption: The horizontal axis represents the sample name, and the vertical axis represents the species name. A color gradient is used to represent the proportion of each species; the right side of the figure shows the numerical values ​​represented by the color gradient.

[0077] Figure 56 Summary of the bar chart results of the comparative analysis of mouse fecal gut microbiota after different sample treatments: The bar chart shows the differences in the average relative abundance of the same species between different groups, and marks whether the differences are significant, intuitively demonstrating the differences in the average relative abundance of the same species between different groups. Caption: The horizontal axis represents the species name at different taxonomic levels, and the vertical axis represents the percentage abundance of a certain species in the sample. Different colors represent different groups. The rightmost value is the p-value, *0.01. <P≤0.05,**0.001<P≤0.01,***P≤0.001。 Detailed Implementation

[0078] Materials and methods involved in Examples 1-4

[0079] Part 1 Experimental Materials

[0080] (1) Female ICR mice, weighing 20-25 g, were purchased from the Comparative Medicine Center of Yangzhou University, Laboratory Animal Production License No.: SCXK(Su)2022-0009, Laboratory Animal Use License No.: SYXK(Su)2019-0056. They were housed in isolation cages with free access to food and water, and the laboratory temperature was maintained at (24±)°C.

[0081] 1) Temperature, relative humidity 40%~80%, and start the experiment after 3 days of acclimatization.

[0082] (2) The Chinese medicinal herbs Citron and Chicory were purchased from Bozhou Jingwan Chinese Medicine Pieces Factory; the Chinese herbs Elsholtzia and Hovenia dulcis were purchased from Bozhou Haocao Chinese Medicinal Herbs Sales Co., Ltd.

[0083] Part Two: Experimental Methods

[0084] (1) According to the traditional decoction method, 50 g of citron, chicory, elsholtzia ciliata and Japanese raisin tree fruit were weighed, 10 times the amount of distilled water was added, soaked for 30 min, decocted for 60 min, the decoction was filtered out with gauze, 8 times the amount of distilled water was added and decocted for 40 min, the decoction was filtered out with gauze, the decoctions were combined, heated and concentrated to 25 ml, the corresponding crude drug concentrations of citron water extract, chicory water extract, elsholtzia ciliata water extract and Japanese raisin tree fruit water extract were 2 g / ml, and placed in centrifuge tubes for later use.

[0085] (2) ICR mice were randomly divided into a blank control group, a constipation group, a citron group, a chicory group, a sesame group, and a Japanese raisin tree group. Mice in the constipation group were given 4 mg / kg of [a specific antibiotic]. -1 Loperamide hydrochloride (Lop) was administered subcutaneously twice daily for four consecutive days, followed by a three-day fixation period at 8 mg / kg. -1 Lop was administered subcutaneously for 4 days.

[0086] The successful establishment of the constipation model was indicated by the following symptoms in mice: decreased activity, reduced water content in fecal particles, hardened fecal texture, and reduced fecal particle size. After constipation induction, mice in each of the traditional Chinese medicine treatment groups were administered 8 g / kg of citron water extract, chicory water extract, elsholtzia water extract, and jujube fruit water extract, respectively, via gavage. -1 Meanwhile, the control group and the model group were given an equal volume of physiological saline by gavage once a day for 7 consecutive days. All mice were provided with standard laboratory food, water, and clean cages.

[0087] (3) Determination of constipation indicators: The general physiological condition and fecal condition of mice in each group were observed daily. Fresh feces were collected from each group of mice within 3 hours on days 7 and 14 of the experiment. The number of feces was recorded, and the mice were weighed (wet weight = total weight M1 - empty plate M0). The feces were dried continuously in an 80℃ dryer for 6 hours and weighed again (dry weight = total weight M2 - empty plate M0). The water content (W) of the mouse feces was calculated and statistically analyzed to verify whether the model was successful and whether citron had a therapeutic effect. After the last administration, the mice were fasted but allowed to drink water for 16 hours. Each group of mice was housed in a single cage and administered 0.2 mL of 10% activated charcoal solution by gavage. The timing was started after the gavage ended.

[0088] Record the time of the first black feces excretion in mice and the number of black feces excreted within 6 hours, and perform statistical analysis.

[0089] Mouse fecal water content (W) = (M1-M2) / (M1-M0) × 100%

[0090] (4) Measurement of small intestinal propulsion rate: After constipation index determination, mice were fasted for 16 hours but allowed free water. Each group of mice was given 0.2 mL of 10% activated charcoal solution by gavage. Timing started after gavage and the mice were sacrificed immediately after 20 minutes. The entire intestine from the pylorus to the cecum was removed by laparotomy, slowly straightened and positioned, and photographed on white paper. The total length of the intestine (L1) and the propulsion distance of 10% activated charcoal solution from the pylorus to the cecum (L2) were then measured. The small intestinal propulsion rate (R) was calculated and statistically analyzed.

[0091] Small intestinal propulsion rate (R) = L2 / L1 × 100%

[0092] (5) Collection of fecal and colonic tissues: On day 14 of the experiment, fecal tissues were collected from each mouse, collected in Eppendorf tubes, flash-frozen in liquid nitrogen, and stored at -80°C for further analysis. On day 16 of the experiment, mice were euthanized by cervical dislocation, and the colonic tissues were separated by laparotomy and divided into two parts. One part was minced, lysed, and ground to prepare for the extraction of total tissue protein; the other part was fixed in 4% paraformaldehyde for further pathological experiments.

[0093] (6) After paraffin embedding of mouse colon tissue for H&E staining, it was cut into 5μm thick paraffin sections. The sections were placed in environmentally friendly dewaxing solution, anhydrous ethanol, and 75% alcohol for dewaxing and washing. After repeated water treatment, they were stained with 1% hematoxylin and eosin (H&E) at 25±2℃ and mounted with neutral resin.

[0094] Finally, morphological changes in colon tissue were observed and images were acquired under an optical microscope.

[0095] (7) Immunohistochemistry: Paraffin sections of mouse colon were placed in a drying oven and baked at 65°C for 30 minutes.

[0096] After a few minutes, the sections were dewaxed and dehydrated, then washed. To improve the specificity and stability of immunohistochemical staining, 2000 ml of citrate retrieval solution at pH 6.0 was prepared for antigen retrieval, thereby improving the antigen-antibody binding efficiency. Goat serum was then added for blocking. The goat serum was discarded, and c-Kit antibody (1:300) and SCF antibody (1:300) were added, followed by incubation for 1 hour. The sections were removed and rinsed with PBS-T. Excess liquid was discarded, and HRP-labeled goat anti-rabbit IgG antibody was added, followed by incubation for 30 minutes, and then rinsed with PBS-T. The sections were developed with DAB solution for 5 minutes and counterstained with hematoxylin for 1 minute. After mounting with neutral resin, the sections were observed under an optical microscope.

[0097] (8) Western blot analysis: Mouse colon tissue was collected, lysis buffer was added, and the tissue was homogenized on ice. After centrifugation, the supernatant was collected, and protein quantification and concentration were adjusted. 20 μg of protein sample was taken from each well, denatured by boiling, and then subjected to SDS-PAGE electrophoresis. The membrane was then immediately transferred using wet transfer, blocked with 5% skim milk powder for 1 h, washed with TBS-T, and subsequently, primary antibodies (ZO-1 1:5000, Occludin 1:2000, Claudin-1) were added according to the protein molecular weight.

[0098] Incubate overnight with β-actin (1:1000) and internal control (β-actin 1:60000). The next day, wash the membrane with TBS-T, add HRP-labeled goat anti-rabbit IgG secondary antibody, and incubate for 1 hour. After washing the membrane with TBS-T, develop the protein.

[0099] White stripe.

[0100] (9) 16S rRNA gene sequencing was performed using mouse fecal tissue sampled and analyzed using Illumina's Miseq PE300 microarray.

[0101] The platform uses primer 338F_806R for paired-end sequencing, amplifying and sequencing the V3-V4 regions.

[0102] The original code was processed using the FastP software (https: / / github.com / OpenGene / fastp, version 0.20.0).

[0103] Quality control of the initial sequencing sequences was performed using FLASH.

[0104] The software (http: / / www.cbcb.umd.edu / software / flash, version 1.2.7) is used for assembly. Samples are distinguished based on barcodes and primers at both ends of the sequence, and the sequence orientation is adjusted. Microorganisms

[0105] Classification metagenomic sequencing and 16S rRNA gene sequencing were performed by Shanghai Meiji Biomedical Technology Co., Ltd.

[0106] The work was completed by the company (Contract No.: MJ20240612154). All data analysis was performed on the Meiji Bio Cloud Platform.

[0107] This can be done at (https: / / cloud.majorbio.com).

[0108] Part Three: Experimental Results and Conclusions

[0109] Example 1: Effect of citron administration via gavage on defecation in a mouse model of constipation.

[0110] I. Fecal Index

[0111] like Figure 1 As shown, compared with the model group, the citron group mice showed significant differences in all fecal parameters. The time to the first black stool was significantly shortened (P < 0.001), the number of black stool particles excreted within 6 hours increased (P < 0.01), fecal dry weight and water content both increased (P < 0.001, P < 0.01), and intestinal propulsion rate also increased significantly (P < 0.001). These findings demonstrate that citron water extract can improve defecation in constipated model mice. Note: Compared with the control group, ##P < 0.01; compared with the model group, **P < 0.01; ***P < 0.001.

[0112] II. HE staining

[0113] like Figure 2 As shown, compared with the model group, the intestinal pathological morphology of mice in the citron group was improved, such as intact and neatly arranged mucosal epithelial cells, increased goblet cells, and normalized crypt structure. This demonstrates that citron water extract can repair colonic pathological damage in constipated mice. Note: →. Goblet cells; Nest structure.

[0114] III. Immunohistochemistry

[0115] like Figure 3 As shown, compared with the model group, the number of c-Kit and SCF positive cells in the citron group mice was significantly increased and densely distributed (P < 0.01 or P < 0.05). This demonstrates that citron water extract may activate the c-Kit / SCF pathway, thereby coordinating the contraction of intestinal smooth muscle and promoting intestinal peristalsis. Note: *P < 0.05, **P < 0.01.

[0116] IV. Western blot analysis

[0117] like Figure 4 As shown, compared with the model group, the expression levels of tight junction proteins ZO-1, Occludin, and Claudin-1 in the colon of mice in the citron group were significantly increased (P < 0.05 or P < 0.01). This indicates that citron water extract can repair intestinal barrier damage in Lop-induced constipation mice. Note: *P < 0.05, **P < 0.01, ***P < 0.001.

[0118] V. 16S RNA gene sequencing

[0119] (I) Alpha Diversity

[0120] 1. Alpha diversity index:

[0121] like Figure 5As shown, compared with the control group and the model group, the citron treatment group showed increased diversity index and α-diversity. These results indicate that citron can improve the species richness and diversity of the gut microbiota in constipated mice, which is beneficial for building a healthy gut microbiota.

[0122] 2. Test for differences between index groups:

[0123] like Figure 6 As shown, there were no significant differences in the α diversity index among the control group, model group, and citron group.

[0124] 3. Dilution curve analysis:

[0125] Depend on Figure 7 The dilution curves show that as the amount of randomly sampled sequencing data increases, the curves tend to flatten out. This indicates that the amount of sequencing data in the three groups is gradually reasonable, and more data will only produce a small number of new species (ASVs).

[0126] (II) β-diversity:

[0127] 1. PCA / PCoA Analysis:

[0128] like Figure 8 As shown, PCA analysis of the gut microbiota in mouse feces after different sample treatments revealed that the three groups of samples were similar, with P > 0.05. These results indicate that the three groups exhibited similar gut microbiome composition.

[0129] like Figure 9 As shown, PCoA analysis based on the relative abundance of ASV levels between groups was used to examine the community structure of the gut microbiota. It was found that after citron treatment, the gut microbiota community composition in mouse feces was significantly separated from that of the control group and the model group (P < 0.05), with PC1 and PC2 contributing 28.83% and 19.91%, respectively. These results indicate that citron treatment can alter the gut microbiota community structure in the feces of constipated mice.

[0130] 2. Sample microbial typing analysis:

[0131] like Figure 10 As shown, intestinal type analysis at the genus level in mouse fecal gut microbiota revealed that the dominant bacterial species in the control and citron groups was *g__norank_f__Muribaculaceae*, while the dominant bacterial species in the model group was *g__Lactobacillus*. The ellipses represent the 0.95 confidence interval (CI) range within each intestinal type group. Due to the small sample size, larger confidence intervals could not be formed. These results indicate that three different bacterial species emerged in the mouse fecal gut microbiota after the three different sample treatments.

[0132] (III) Community Composition Analysis

[0133] 1. Venn diagram of species:

[0134] like Figure 11 As shown, to analyze the number of unique and shared bacterial flora in mouse fecal intestinal flora compared to the control and model groups after citron treatment intervention, a species-level Venn diagram analysis was performed. The results showed that the control group had 14 unique genera, the model group had 13 unique genera, and the citron group had 10 unique genera. The control and model groups shared 5 genera, the control and citron groups shared 7 genera, and the model and citron groups shared 7 genera. The total number of genera shared across the three groups was 102. These results indicate that citron treatment altered the microbial composition.

[0135] Table 1. Intestinal bacteria that disappeared from the model group's specific flora after citron treatment.

[0136]

[0137] like Figure 11 As shown in Table 1, 13 bacteria disappeared from the model group after mice were treated with citron; these were bacteria specific to the model group. Among them, *Coriobacteriales* are commonly found in periodontal / pulp infections. *Paeniclostridium* is involved in intestinal and tissue toxicity infections. *Macrococcus* is an opportunistic pathogen associated with infections in humans and animals. *Acetatifactor* inhibits prostate cell apoptosis. *Faecalitalea* is a pathogenic and gas-producing microorganism that promotes inflammation. The amplification of *Erysipelotrichales* is associated with NASH.

[0138] These results indicate that citron treatment can reduce the presence of harmful bacteria, resulting in various effects such as: inhibiting periodontal / pulp infections, treating Parkinson's disease, reducing intestinal and tissue viral infections, reducing inflammatory responses, inhibiting prostate hyperplasia, and potentially treating NASH.

[0139] Table 2 shows the newly emerging gut microbiota after citron treatment compared to the combined control and model groups.

[0140]

[0141] like Figure 11As shown in Table 2, after mice were treated with citron, 10 new bacteria appeared, which were unique to the citron group. Among them, *Bacteroidales* maintained intestinal barrier integrity. *Lactobacillaceae* helped improve intestinal function, promote food digestion and absorption, and prevent and treat diarrhea. *Rikenellaceae* was a protective bacterium against DSS-induced colitis and a producer of SCFAs, beneficial to metabolic health. *Marinifilaceae* had a potential protective effect against food allergies. These results indicate that citron treatment can increase the number of beneficial bacteria, thus achieving various effects such as maintaining intestinal barrier integrity, improving intestinal function, promoting food digestion and absorption, preventing and treating diarrhea, and providing potential protection against food allergies.

[0142] 2. Community composition Bar diagram:

[0143] like Figure 12 As shown, the dominant bacterial species in each group was *Bacteroides muribacaceae*, and the content of this species increased to the control level after citron treatment. This study indicates that this bacterium has the effect of improving host energy metabolism and intestinal barrier integrity. Therefore, the above results suggest that citron treatment is beneficial for building a healthy gut microbiota.

[0144] 3. Community Heatmap:

[0145] like Figure 13 As shown, heatmap analysis was performed on the gut microbiota of mouse feces, and the relative abundance of the 50 most important gut microbiota genera was displayed.

[0146] (IV) Species Difference Analysis – Comparative Analysis of Two Groups

[0147] 1. Two sets of comparative bar charts:

[0148] Table 3. Fungi species with significantly increased average relative abundance in the Citron group compared to the model group.

[0149]

[0150]

[0151] Depend on Figure 14 The results showed that, compared with the model group, the relative abundance of gut microbiota in the feces of mice treated with citron was significantly altered, with significant enrichment of beneficial bacteria such as Odoribacter, Bacteroides_caecimuris, Clostridia_vadinBB60_group, and Roseburia. These results indicate that citron treatment can change the gut microbiota community structure in mouse feces, promoting the formation of a healthy gut microbiota.

[0152] Table 4 shows that, compared with the model group, the abundance of bacteria in the gut increased more than fivefold after citron treatment.

[0153]

[0154] Table 4 shows that, compared to the model group, the gut microbiota in mice treated with citron increased by 5 times or more, and these microbiota were mostly beneficial bacteria. Literature reports that *Turicibacter* is a positive regulator of host lipid biology. *Akkermansia* has beneficial effects on improving obesity and type 2 diabetes. *Odoribacter* is negatively correlated with the severity of non-alcoholic fatty liver disease and inflammatory bowel disease. *Rs-E47_termite_group* has lipid-lowering and anti-obesity effects. *Rikenella* is negatively correlated with urinary protein levels. Increased abundance of *Clostridia_vadinBB60_group* is associated with weight control, injury prevention, and reduced inflammatory responses.

[0155] These results show that citron treatment can enrich corresponding beneficial bacteria, thereby achieving various effects such as inhibiting obesity, protecting the cardiovascular system, reducing inflammation, and improving colitis.

[0156] Table 5 shows the number of bacteria in the gut that decreased to less than 1 / 5 after citron treatment compared to the model group.

[0157]

[0158] Table 5 shows that, compared to the model group, the gut microbiota in mice treated with citron, which decreased to less than 1 / 5, consisted mostly of harmful bacteria. Literature reports that *Streptococcus* is a cause of bacteremia and infective endocarditis. *Faecalibaculum* can induce depressive-like behaviors in mice via the vagus nerve. *Blautia* is a microbiota associated with inflammatory bowel disease. The *Rikenellaceae_RC9_gut_group* is positively correlated with HFD-induced "harmful indicators." *Clostridium_sensu_stricto_1* is associated with a high risk of Alzheimer's disease. *Gastranaerophilales* is a risk factor for prostatitis.

[0159] These results indicate that citron treatment can reduce the presence of harmful bacteria, thus treating or preventing bacteremia, infective endocarditis, HFD, Alzheimer's disease, prostatitis, and other conditions.

[0160] Example 2: Effect of chicory gavage administration on defecation in a mouse model of constipation.

[0161] I. Fecal Index

[0162] like Figure 15 As shown, compared with the model group, the chicory group mice showed significant differences in all fecal parameters. The time to first black stool was significantly shortened (P < 0.01), the number of black stool particles excreted within 6 hours increased (P < 0.05), fecal dry weight and water content both increased (P < 0.01), and intestinal propulsion rate also significantly increased (P < 0.01). These findings demonstrate that chicory water extract can improve defecation in constipated model mice. Note: Compared with the control group, ##P < 0.01; compared with the model group, *P < 0.05; **P < 0.01.

[0163] II. HE staining

[0164] like Figure 16 As shown, compared with the model group, the intestinal pathological morphology of mice in the chicory group was improved, such as intact and neatly arranged mucosal epithelial cells, increased goblet cells, and normalized crypt structure. This demonstrates that chicory water extract can repair colonic pathological damage in constipated mice. Note: →. Goblet cells; Nest structure.

[0165] III. Immunohistochemistry

[0166] like Figure 17 As shown, compared with the model group, the number of c-Kit and SCF positive cells in the chicory group mice was significantly increased and densely distributed (P < 0.05). This demonstrates that chicory aqueous extract may activate the c-Kit / SCF pathway, thereby coordinating intestinal smooth muscle contraction and promoting intestinal peristalsis. Note: *P < 0.05, **P < 0.01.

[0167] IV. Western blot analysis

[0168] like Figure 18 As shown, compared with the model group, the expression levels of tight junction proteins ZO-1, Occludin, and Claudin-1 in the colon of chicory-treated mice were significantly increased (P < 0.05 or P < 0.001). This indicates that chicory aqueous extract can repair intestinal barrier damage in Lop-induced constipation mice. Note: *P < 0.05, **P < 0.01, ***P < 0.001.

[0169] V. 16S RNA gene sequencing

[0170] (I) Alpha Diversity

[0171] 1. Alpha diversity index:

[0172] like Figure 19As shown, compared with the control group and the model group, the chicory treatment group showed increased diversity index and α-diversity. These results indicate that chicory can improve the species richness and diversity of the gut microbiota in constipated mice, which is beneficial for building a healthy gut microbiota.

[0173] 2. Test for differences between index groups:

[0174] like Figure 20 As shown, there were no significant differences in the α-diversity index among the control group, model group, and chicory group.

[0175] 3. Dilution curve analysis:

[0176] Depend on Figure 21 The dilution curves show that as the amount of randomly sampled sequencing data increases, the curves tend to flatten out. This indicates that the amount of sequencing data in the three groups is gradually reasonable, and more data will only produce a small number of new species (ASVs).

[0177] (II) β-diversity:

[0178] 1. PCA / PCoA Analysis:

[0179] like Figure 22 As shown, PCA analysis of the gut microbiota in mouse feces after different sample treatments revealed that the three groups of samples were similar, with P > 0.05. These results indicate that the three groups exhibited similar gut microbiome composition.

[0180] like Figure 23 As shown, PCoA analysis based on the relative abundance of ASV levels between groups was used to examine the community structure of the gut microbiota. It was found that after chicory treatment, the gut microbiota community composition in mouse feces was significantly separated from that of the control and model groups (P < 0.05), with PC1 and PC2 contributing 27.04% and 20.14%, respectively. These results indicate that chicory treatment can alter the gut microbiota community structure in the feces of constipated mice.

[0181] 3. Sample microbial typing analysis:

[0182] like Figure 24 As shown, intestinal type analysis at the genus level in mouse fecal gut microbiota revealed that the dominant bacterial species in the chicory and model groups was *g__Lactobacillus*, while the dominant bacterial species in the control group was *g__norank_f__Muribaculaceae*. The ellipses represent the 0.95 confidence interval (CI) range within each intestinal type group. Due to the small sample size, larger confidence intervals could not be formed. These results indicate that three different bacterial species emerged in the mouse fecal gut microbiota after the three different sample treatments.

[0183] (III) Community Composition Analysis

[0184] 1. Venn diagram of species:

[0185] like Figure 25 As shown, to analyze the number of unique and shared bacterial flora in mouse feces after chicory treatment intervention compared to the control and model groups, Venn diagram analysis at the species and genus level was performed. The results showed that the control group had 13 unique genera, the model group had 14 unique genera, and the chicory group had 14 unique genera. The control and model groups shared 3 genera, the control and chicory groups shared 8 genera, and the model and chicory groups shared 6 genera. The total number of genera shared across the three groups was 105. These results indicate that chicory treatment altered the microbial composition.

[0186] Table 6. Intestinal bacteria that disappeared from the model group's specific flora after chicory treatment.

[0187]

[0188]

[0189] like Figure 25 As shown in Table 6, after chicory treatment, 14 bacteria disappeared from the model group, indicating they were specific to the model group. Among these, *Coriobacteriales* are commonly found in periodontal / pulp infections. *Mycoplasma* is a mycoplasma that induces inflammation. *Paeniclostridium* is involved in intestinal and tissue toxicity infections. *Macrococcus* is an opportunistic pathogen associated with infections in humans and animals. *Faecalitalea* is a pathogenic and gas-producing microorganism that promotes inflammation. The amplification of *Erysipelotrichales* is associated with NASH.

[0190] These results indicate that chicory treatment can reduce the presence of harmful bacteria, thereby inhibiting periodontal / pulp infections, reducing intestinal and tissue viral infections, lowering inflammatory responses, and potentially treating NASH, among other benefits.

[0191] Table 7 shows the newly emerging gut microbiota after chicory treatment compared to the combined control and model groups.

[0192]

[0193] like Figure 25As shown in Table 7, 14 new bacteria emerged in mice after chicory treatment, all of which are specific to the chicory group. Among them, *Bacteroidales* maintains intestinal barrier integrity. *Lactobacillaceae* helps improve intestinal function, promotes food digestion and absorption, and prevents and treats diarrhea. *Rikenellaceae* is a protective bacterium against DSS-induced colitis and is also a producer of SCFAs, beneficial to metabolic health. *Christensenellaceae* has physiological functions such as protein catabolism and prebiotic fiber production. *Marinifilaceae* has potential protective effects against food allergies. *Butyricicoccaceae* produces SCFAs, maintaining metabolic health.

[0194] These results indicate that chicory treatment can increase the number of beneficial bacteria, resulting in various benefits such as maintaining intestinal barrier integrity, improving intestinal function, promoting food digestion and absorption, preventing and treating diarrhea, producing SCFAs, maintaining metabolic health, and providing potential protection against food allergies.

[0195] 2. Community composition Bar diagram:

[0196] like Figure 26 As shown, the dominant bacterial species in each group was *Bacteroides muribacaceae*, and the content of this species increased to the control level after chicory treatment. This study indicates that this bacterium has the effect of improving host energy metabolism and intestinal barrier integrity. Therefore, the above results suggest that chicory treatment is beneficial for building a healthy gut microbiota.

[0197] 3. Community Heatmap:

[0198] like Figure 27 As shown, heatmap analysis was performed on the gut microbiota of mouse feces, and the relative abundance of the 50 most important gut microbiota genera was displayed.

[0199] (IV) Species Difference Analysis – Comparative Analysis of Two Groups

[0200] 1. Two sets of comparative bar charts:

[0201] Table 8. Species with significantly increased average relative abundance in the chicory group compared to the model group.

[0202]

[0203] Depend on Figure 28The results showed that, compared with the model group, the relative abundance of gut microbiota in the feces of mice treated with chicory was significantly altered, with significant enrichment of beneficial bacteria such as Bacteroides_caecimuris, Peptococcaceae, Clostridia_vadinBB60_group, and Harryflintia. These results indicate that chicory treatment can change the gut microbiota community structure in mouse feces, promoting the formation of a healthy gut microbiota.

[0204] Table 9. Species with significantly decreased average relative abundance in the chicory group compared to the model group.

[0205]

[0206]

[0207] Table 10 shows the bacterial abundance in the gut that increased more than fivefold after chicory treatment compared to the model group.

[0208]

[0209] Table 10 shows that, compared to the model group, the intestinal flora in mice treated with chicory increased by 5 times or more, and these were mostly beneficial bacteria. Literature reports that butyrate produced by Roseburia inhibits bacterial translocation. ASF356 and Lachnospiraceae_NK4A136_group both have anti-obesity effects. Rikenella showed a negative correlation with urinary protein levels. Increased abundance of Clostridia_vadinBB60_group was associated with weight control, injury prevention, and reduced inflammatory responses. Intestinimonas, a butyrate-producing bacterium, plays an important role in maintaining intestinal barrier function and inhibiting inflammation. Coridextribacter showed a positive correlation with the antioxidant capacity and glucose metabolism of sow skeletal muscle.

[0210] These results show that chicory treatment can enrich corresponding beneficial bacteria, thereby achieving various effects such as inhibiting obesity, protecting the cardiovascular system, reducing inflammation, and improving colitis.

[0211] Table 11 shows the bacteria whose abundance in the gut decreased to below 1 / 5 after chicory treatment compared to the model group.

[0212]

[0213] Table 11 shows that, compared to the model group, the gut microbiota in mice treated with chicory decreased to less than 1 / 5, and was mostly composed of harmful bacteria. Literature reports that Romboutsia induces acute myeloid leukemia, leading to bone marrow necrosis. The Rikenellaceae_RC9_gut_group was positively correlated with the "harmful indicator" induced by HFD. Clostridium_sensu_stricto_1 was associated with a high risk of Alzheimer's disease. Lachnospiraceae is a potential harmful bacterium associated with obesity.

[0214] These results indicate that chicory treatment can reduce the presence of harmful bacteria, thus playing a role in treating or preventing bone marrow necrosis caused by acute myeloid leukemia, HFD, Alzheimer's disease, prostatitis, and inhibiting obesity.

[0215] Example 3: Effect of oral administration of Elsholtzia ciliata on defecation in a mouse model of constipation

[0216] I. Fecal Index

[0217] like Figure 29 As shown, compared with the model group, the mice in the Elsholtzia ciliata group showed significant differences in all fecal parameters. The time to the first black stool was significantly shortened (P < 0.01), the number of black stool particles excreted within 6 hours increased (P < 0.01), and the fecal dry weight and water content both increased (P < 0.01, P < 0.05), and the intestinal propulsion rate also increased significantly (P < 0.001). These findings demonstrate that the aqueous extract of Elsholtzia ciliata can improve the defecation status of constipated model mice. Note: Compared with the control group, ##P < 0.01; compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001.

[0218] II. HE staining

[0219] like Figure 30 As shown, compared with the model group, the intestinal pathological morphology of mice in the Elsholtzia group was improved, such as intact and neatly arranged mucosal epithelial cells, increased goblet cells, and normalized crypt structure. This demonstrates that the aqueous extract of Elsholtzia can repair colonic pathological damage in constipated mice. Note: →. Goblet cells; Nest structure.

[0220] III. Immunohistochemistry

[0221] like Figure 31 As shown, compared with the model group, the number of c-Kit and SCF positive cells in the Elsholtzia ciliata group mice was significantly increased and densely distributed (P < 0.05 or P < 0.01). This demonstrates that the aqueous extract of Elsholtzia ciliata may activate the c-Kit / SCF pathway, thereby coordinating the contraction of intestinal smooth muscle and promoting intestinal peristalsis. Note: *P < 0.05, **P < 0.01.

[0222] IV. Western blot analysis

[0223] like Figure 32 As shown, compared with the model group, the expression levels of tight junction proteins ZO-1, Occludin, and Claudin-1 in the colon of mice in the Elsholtzia ciliata group were significantly increased (P < 0.05 or P < 0.01). This indicates that the aqueous extract of Elsholtzia ciliata can repair intestinal barrier damage in Lop-induced constipation mice. Note: *P < 0.05, **P < 0.01, ***P < 0.001.

[0224] V. 16S RNA gene sequencing

[0225] (I) Alpha Diversity

[0226] 1. Alpha diversity index:

[0227] like Figure 33 As shown, compared with the control group and the model group, the diversity index and α-diversity were increased in the Elsholtzia ciliata treatment group. These results indicate that Elsholtzia ciliata can improve the species richness and diversity of the gut microbiota in constipated mice, which is beneficial for building a healthy gut microbiota.

[0228] 2. Test for differences between index groups:

[0229] like Figure 34 As shown, there were no significant differences in the α-diversity index among the control group, model group, and Elsholtzia ciliata group.

[0230] 3. Dilution curve analysis:

[0231] Depend on Figure 35 The dilution curves show that as the amount of randomly sampled sequencing data increases, the curves tend to flatten out. This indicates that the amount of sequencing data in the three groups is gradually reasonable, and more data will only produce a small number of new species (ASVs).

[0232] (II) β-diversity:

[0233] 1. PCA / PCoA Analysis:

[0234] like Figure 36 As shown, PCA analysis of the gut microbiota in mouse feces after different sample treatments revealed that the three groups showed similar patterns, with P > 0.05. This indicates that the three groups exhibited similar gut microbiome composition. Figure 37As shown, PCoA analysis based on the relative abundance of ASV levels between groups was used to examine the community structure of the gut microbiota. It was found that after treatment with *Elsholtzia ciliata*, the composition of the gut microbiota in mouse feces was significantly different from that of the control group and the model group (P < 0.05), with PC1 and PC2 contributing 28.70% and 21.02%, respectively. These results indicate that *Elsholtzia ciliata* treatment can alter the gut microbiota community structure in mouse feces.

[0235] 3. Sample microbial typing analysis:

[0236] like Figure 38 As shown, intestinal type analysis at the genus level of mouse fecal gut microbiota revealed that the dominant bacterial species in the control and model groups was *Lactobacillus*, while the dominant bacterial species in the *Elsholtzia ciliata* group was *Muribaculaceae*. The ellipses represent the 0.95 confidence interval (CI) range within each intestinal type group. Due to the small sample size, larger confidence intervals could not be formed. These results indicate that three different bacterial species emerged in the mouse fecal gut microbiota after the three different sample treatments.

[0237] (III) Community Composition Analysis

[0238] 1. Venn diagram of species:

[0239] like Figure 39 As shown, to analyze the number of unique and shared bacterial communities in mouse fecal intestinal flora compared to the control and model groups after Elsholtzia ciliata treatment intervention, Venn diagram analysis at the species and genus level was performed. The results showed that the control group had 5 unique genera, the model group had 13 unique genera, and the Elsholtzia ciliata group had 5 unique genera. The control and model groups shared 6 genera, the control and Elsholtzia ciliata groups shared 16 genera, and the model and Elsholtzia ciliata groups shared 7 genera. The total number of genera shared across the three groups was 102. These results indicate that the microbial composition changed after Elsholtzia ciliata treatment.

[0240] Table 12. Intestinal bacteria that disappeared from the model group's specific flora after treatment with Elsholtzia ciliata.

[0241]

[0242] like Figure 39As shown in Table 12, after treatment with *Elsholtzia ciliata*, 13 bacteria disappeared from the model group, indicating they were specific to the model group. Among these, *Coriobacteriales* are commonly found in periodontal / pulp infections. *Mycoplasma* is a mycoplasma that induces inflammation. *Paeniclostridium* is involved in intestinal and tissue toxicity infections. *Macrococcus* is an opportunistic pathogen associated with infections in humans and animals. *Acetatifactor* inhibits prostate cell apoptosis. *Faecalitalea* is a pathogenic and gas-producing microorganism that promotes inflammation. The amplification of *Erysipelotrichales* is associated with NASH.

[0243] These results indicate that Elsholtzia splendens treatment can reduce the presence of harmful bacteria and thus have the following effects: inhibiting periodontal / pulp infection, treating Parkinson's disease, reducing intestinal and tissue viral infections, reducing inflammatory response, inhibiting prostatic hyperplasia, and potentially treating NASH, among other benefits.

[0244] Table 13 shows the newly emerging gut microbiota after Elsholtzia ciliata treatment, compared to the combined groups of the control and model groups.

[0245]

[0246] like Figure 39 As shown in Table 13, after mice were treated with Elsholtzia ciliata, five new bacteria appeared, which are unique to the Elsholtzia ciliata group. Among them, Psychrobacter showed antibacterial activity. Lactobacillaceae can help improve intestinal function, promote food digestion and absorption, and prevent and treat diarrhea.

[0247] These results indicate that processing with Elsholtzia ciliata can increase the number of beneficial bacteria, resulting in various effects such as antibacterial activity, improved intestinal function, enhanced digestion and absorption of food, and prevention and treatment of diarrhea.

[0248] 2. Community composition Bar diagram:

[0249] like Figure 40 As shown, the dominant bacterial species in each group was *Bacteroides muribacteri*, and the content of this species increased to the control level after *Elsholtzia ciliata* treatment. This study indicates that this bacterium has the effect of improving host energy metabolism and intestinal barrier integrity. Therefore, the above results suggest that *Elsholtzia ciliata* treatment is beneficial for building a healthy gut microbiota.

[0250] 3. Community Heatmap:

[0251] like Figure 41As shown, heatmap analysis was performed on the gut microbiota of mouse feces, and the relative abundance of the 50 most important gut microbiota genera was displayed.

[0252] (IV) Species Difference Analysis – Comparative Analysis of Two Groups

[0253] 1. Two sets of comparative bar charts:

[0254] Table 14. Fungal species with significantly increased average relative abundance in the Elsholtzia group compared to the model group.

[0255]

[0256] Depend on Figure 42 The results showed that, compared with the model group, the relative abundance of gut microbiota in the feces of mice treated with Elsholtzia ciliata was significantly altered, with significant enrichment of beneficial bacteria such as Turicibacter and Clostridia_vadinBB60_group. These results indicate that Elsholtzia ciliata treatment can change the community structure of gut microbiota in mouse feces, which is beneficial to the formation of a healthy gut microbiota.

[0257] Table 15. Fungal species whose average relative abundance significantly decreased in the Elsholtzia group compared to the model group.

[0258]

[0259] Table 16 shows the bacterial abundance in the gut increased more than fivefold after Elsholtzia ciliata treatment compared to the model group.

[0260]

[0261] Table 16 shows that, compared to the model group, the gut microbiota in mice treated with *Elsholtzia ciliata* increased by 5 times or more, and these microbiota were mostly beneficial bacteria. Literature reports that *Akkermansia* has beneficial effects on improving obesity and type 2 diabetes. *Alistipes* has protective effects against liver fibrosis, cancer immunotherapy, and cardiovascular disease. ASF356, Christensenellaceae_R-7_group, and Lachnospiraceae_NK4A136_group all have anti-obesity effects. Increased abundance of *Clostridia_vadinBB60_group* is associated with weight control, injury prevention, and reduced inflammatory responses. *Eubacterium_siraeum_group* can improve colitis.

[0262] These results show that processing Elsholtzia ciliata can enrich the corresponding beneficial bacteria, thereby achieving various effects such as inhibiting obesity, protecting the cardiovascular system, reducing inflammation, and improving colitis.

[0263] Table 17 shows the bacteria whose abundance in the gut decreased to below 1 / 5 after Elsholtzia ciliata treatment compared to the model group.

[0264]

[0265] Example 4: Effect of oral administration of Hovenia dulcis seeds on defecation in a mouse model of constipation

[0266] I. Fecal Index

[0267] like Figure 43 As shown, compared with the model group, the mice in the Hovenia dulcis group showed significant differences in all fecal parameters. The time to the first black stool was significantly shortened (P < 0.01), the number of black stool particles excreted within 6 hours increased (P < 0.001), and the fecal dry weight and water content both increased (P < 0.001, P < 0.01), and the intestinal propulsion rate also increased significantly (P < 0.01). These findings demonstrate that the aqueous extract of Hovenia dulcis can improve the defecation status of constipated mice. Note: Compared with the control group, ##P < 0.01; compared with the model group, **P < 0.01; ***P < 0.001.

[0268] II. HE staining

[0269] like Figure 44 As shown, compared with the model group, the intestinal pathological morphological damage in the Hovenia dulcis group mice was improved, such as intact and neatly arranged mucosal epithelial cells, increased goblet cells, and normalized crypt structure. This demonstrates that the aqueous extract of Hovenia dulcis can repair colonic pathological damage in constipated mice. Note: →. Goblet cells; Nest structure.

[0270] III. Immunohistochemistry

[0271] like Figure 45 As shown, compared with the model group, the number of c-Kit and SCF positive cells in the Hovenia dulcis group mice was significantly increased and densely distributed (P < 0.05 or P < 0.01). This demonstrates that the aqueous extract of Hovenia dulcis may activate the c-Kit / SCF pathway, thereby coordinating the contraction of intestinal smooth muscle and promoting intestinal peristalsis. Note: *P < 0.05, **P < 0.01.

[0272] IV. Western blot analysis

[0273] like Figure 46 As shown, compared with the model group, the expression levels of tight junction proteins ZO-1, Occludin, and Claudin-1 in the colon of mice in the Hovenia dulcis group were significantly increased (P < 0.01). This indicates that the aqueous extract of Hovenia dulcis can repair intestinal barrier damage in Lop-induced constipation mice. Note: **P < 0.01, ***P < 0.001.

[0274] V. 16S RNA gene sequencing

[0275] (I) Alpha Diversity

[0276] 1. Alpha diversity index:

[0277] like Figure 47 As shown, compared with the control group and the model group, the diversity index and α-diversity were increased in the Hovenia dulcis treatment group. These results indicate that Hovenia dulcis can improve the species richness and diversity of the gut microbiota in constipated mice, which is beneficial for building a healthy gut microbiota.

[0278] 4. Test for differences between index groups:

[0279] like Figure 48 As shown, there were no significant differences in the α diversity index among the control group, model group, and Hovenia dulcis group.

[0280] 3. Dilution curve analysis:

[0281] Depend on Figure 49 The dilution curves show that as the amount of randomly sampled sequencing data increases, the curves tend to flatten out. This indicates that the amount of sequencing data in the three groups is gradually reasonable, and more data will only produce a small number of new species (ASVs).

[0282] (II) β-diversity:

[0283] 1. PCA / PCoA Analysis:

[0284] like Figure 50 As shown, PCA analysis of the gut microbiota in mouse feces after different sample treatments revealed that the three groups of samples were similar, with P > 0.05. These results indicate that the three groups exhibited similar gut microbiome composition.

[0285] like Figure 51 As shown, PCoA analysis based on the relative abundance of ASV levels between groups was used to examine the community structure of the gut microbiota. It was found that after treatment with Hovenia dulcis seeds, the gut microbiota community composition in mouse feces was significantly separated from the control group and the model group (P < 0.05), with PC1 and PC2 contributing 28.36% and 17.45%, respectively. These results indicate that Hovenia dulcis seed treatment can alter the gut microbiota community structure in the feces of constipated mice.

[0286] 5. Sample microbial typing analysis:

[0287] like Figure 52As shown, intestinal type analysis at the genus level of mouse fecal gut microbiota revealed that the dominant bacteria in the control group was *g__norank_f__Muribaculaceae*, while the dominant bacteria in the *Hovenia dulcis* group was *g__Lactobacillus*. The ellipses represent the 0.95 confidence interval (CI) range within each intestinal type group. Due to the small sample size, larger confidence intervals could not be formed. These results indicate that three different bacterial types emerged in the mouse fecal gut microbiota after the three different sample treatments.

[0288] (III) Community Composition Analysis

[0289] 1. Venn diagram of species:

[0290] like Figure 53 As shown, to analyze the number of unique and shared bacterial communities in mouse fecal intestinal flora compared to the control and model groups after treatment with Hovenia dulcis seeds, a species-level Venn diagram analysis was performed. The results showed that the control group had 14 unique genera, the model group had 17 unique genera, and the Hovenia dulcis seed group had 10 unique genera. The control and model groups shared 7 genera, the control and Elsholtzia ciliata groups shared 7 genera, and the model and Elsholtzia ciliata groups shared 3 genera. The total number of genera shared across the three groups was 101. These results indicate that the microbial composition changed after treatment with Hovenia dulcis seeds.

[0291] Table 18. Intestinal bacteria that disappeared from the model group's specific flora after treatment with Hovenia dulcis seeds.

[0292]

[0293] like Figure 53As shown in Table 18, after treatment with Hovenia dulcis seeds, 17 bacteria disappeared from the model group, indicating they were specific to the model group. Among these, Enterococcus can cause various serious infections, including urinary tract infections, bacteremia, intra-abdominal infections, and endocarditis. Coriobacteriales are associated with periodontal / pulp infections. Peptostreptococcales-Tissierellales promote inflammatory responses. Mycoplasma is a mycoplasma that induces inflammation. Paeniclostridium is involved in intestinal and tissue toxic infections. Macrococcus is an opportunistic pathogen that can cause infections in humans and animals. Acetatifactor inhibits prostate cell apoptosis. Coriobacteriales_Incertae_Sedis easily causes mild cognitive impairment in obese individuals. Faecalitalea is a pathogenic and gas-producing microorganism that promotes inflammation. Amplification of Erysipelotrichales is associated with NASH.

[0294] These results indicate that processing with Hovenia dulcis seeds can reduce the presence of harmful bacteria and thus have the following effects: inhibiting various serious infections, including urinary tract infections, bacteremia, intra-abdominal infections, and endocarditis; treating Parkinson's disease; reducing intestinal and tissue viral infections; lowering inflammatory responses; inhibiting prostatic hyperplasia; and potentially treating NASH, among other benefits.

[0295] Table 19 shows the newly emerging gut microbiota after treatment with Hovenia dulcis seeds, compared to the combined groups of the control and model groups.

[0296]

[0297] like Figure 53 As shown in Table 19, after mice were treated with Hovenia dulcis, 10 new bacteria appeared, which are unique to the Hovenia dulcis group. Among them, Eubacterium ruminantium group can improve type 2 diabetes. Olsenella enhances anti-tumor immunity. Bacteroidales helps maintain intestinal barrier integrity. Marinifilaceae has a potential protective effect against food allergies.

[0298] These results indicate that processing with Hovenia dulcis seeds can increase the number of beneficial bacteria, thereby improving type 2 diabetes, enhancing anti-tumor immunity, maintaining intestinal barrier integrity, and potentially combating food allergies, among other benefits.

[0299] 2. Community composition Bar diagram:

[0300] like Figure 54 As shown, the main bacterial species in each group was *Bacteroides muribacicae*, and the content of this species increased to the control level after treatment with *Hovenia dulcis* seeds. This study indicates that this bacterium has the effect of improving host energy metabolism and intestinal barrier integrity. Therefore, the above results suggest that treatment with *Hovenia dulcis* seeds is beneficial for building a healthy gut microbiota.

[0301] 4. Community Heatmap:

[0302] like Figure 55 As shown, heatmap analysis was performed on the gut microbiota of mouse feces, and the relative abundance of the 50 most important gut microbiota genera was displayed.

[0303] (IV) Species Difference Analysis – Comparative Analysis of Two Groups

[0304] 1. Two sets of comparative bar charts:

[0305] Table 20 shows the species with significantly increased average relative abundance in the Hovenia dulcis group compared to the model group.

[0306]

[0307] Depend on Figure 56 The results showed that, compared with the model group, the relative abundance of gut microbiota in the feces of mice treated with Hovenia dulcis seeds was significantly altered, with significant enrichment of beneficial bacteria such as Muribauculaceae, Odoribacter, Roseburia, Rikenellaceae, Rikenella, Clostridia_vadinBB60_group, Peptococcaceae, and Eubacterium_siraeum_group. These results indicate that Hovenia dulcis seed treatment can change the gut microbiota community structure in mouse feces, promoting the formation of a healthy gut microbiota.

[0308] Table 21. Species with significantly decreased average relative abundance in the Hovenia dulcis group compared to the model group.

[0309]

[0310]

[0311] Table 22 shows that, compared with the model group, the abundance of bacteria in the intestines of the Japanese raisin tree increased by more than 5 times after treatment with Japanese raisin tree seeds.

[0312]

[0313] Depend on Figure 56It was found that, compared to the model group, the intestinal flora in mice treated with Hovenia dulcis seeds increased by 5 times or more, and was mostly composed of beneficial bacteria. Literature reports that Odoribacter was negatively correlated with the severity of non-alcoholic fatty liver disease and inflammatory bowel disease. Roseburia inhibited bacterial translocation. Rikenella was negatively correlated with urinary protein levels. Clostridia_vadinBB60_group was associated with weight control, damage prevention, and reduced inflammatory response. Eubacterium_siraeum_group improved colitis. Bacteroides_caecimuris treated liver injury. These results indicate that Hovenia dulcis seed treatment can enrich corresponding beneficial bacteria, thereby achieving multiple effects such as inhibiting bacterial translocation, reducing urinary protein, controlling weight, preventing damage and reducing inflammatory response, improving colitis, and treating liver injury.

[0314] Table 23 shows the number of bacteria in the gut whose abundance decreased to less than 1 / 5 after treatment with Hovenia dulcis seeds, compared to the model group.

[0315]

[0316]

[0317] In summary, these four herbal biosynthetic agents significantly impacted the gut microbiota, not only promoting the growth and colonization of beneficial bacteria but also effectively inhibiting or reducing the number of harmful bacteria. Furthermore, these four herbal biosynthetic agents improved colonic pathological tissue damage in constipated mice and significantly increased the expression of proteins related to promoting intestinal motility and maintaining the intestinal barrier. Therefore, these four herbal biosynthetic agents can improve related disease symptoms and their treatment by regulating the gut microbiota and maintaining the intestinal barrier. Thus, any one or a combination of these four herbal biosynthetic agents has the potential to be used in the preparation of drugs or formulations that promote bowel movements, mimic prebiotics, and inhibit harmful bacteria, thereby enriching beneficial gut bacteria and inhibiting harmful bacteria, and thus improving related disease symptoms and their treatment.

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Claims

1. The application of prebiotic mimics (biogenic compounds) of the same origin as food and medicine in the preparation of drugs or preparations that promote bowel movements, mimic prebiotics and inhibit harmful bacteria, wherein the prebiotic mimics (biogenic compounds) of the same origin as food and medicine refer to any one or a combination of citron, chicory, sesame, or Japanese raisin tree fruit.

2. The use of the prebiotic mimicry (biomimetic) of the medicinal and edible homology class as described in claim 1 in the preparation of drugs or formulations that promote bowel movements, mimic prebiotics, and inhibit harmful bacteria, characterized in that: When applied, one, two, three or four prebiotic mimics (biogenic analogs) or their extracts from food and medicine are mixed with other substances to form a complex.

3. The application of the prebiotic mimicry (biogenic prebiotic) of the medicinal and edible homology class as described in claim 1 in the preparation of drugs or preparations that promote bowel movements, mimic prebiotics, and inhibit harmful bacteria, wherein promoting bowel movements, mimicking prebiotics, and inhibiting harmful bacteria refers to: improving the pathological tissue damage of the colon in constipated mice, significantly increasing the expression of proteins related to promoting intestinal peristalsis and maintaining the intestinal barrier, promoting the colonization of beneficial bacteria in the intestine, and inhibiting the number of harmful bacteria.

4. The application of the prebiotic mimicry (biogenic prebiotic) of the medicinal and edible homology class as described in claim 2 in the preparation of drugs or preparations that promote bowel movements, mimic prebiotics, and inhibit harmful bacteria, wherein promoting bowel movements, mimicking prebiotics, and inhibiting harmful bacteria refer to: improving the pathological tissue damage of the colon in constipated mice, significantly increasing the expression of proteins related to promoting intestinal peristalsis and maintaining the intestinal barrier, promoting the colonization of beneficial bacteria in the intestine, and inhibiting the number of harmful bacteria.

5. The use of the prebiotic mimicry (biomimetic) of the medicinal and edible homology class as described in any one of claims 1-4 in the preparation of drugs or formulations that promote bowel movements, mimic prebiotics, and inhibit harmful bacteria, characterized in that: The prebiotic mimic (biogenetic) of the food-medicine homology class is citron.

6. The use of the prebiotic mimicry (biomimetic) of the medicinal and edible homology class as described in claim 5 in the preparation of drugs or formulations that promote bowel movements, mimic prebiotics, and inhibit harmful bacteria, characterized in that: The term "simulated prebiotic" refers to a prebiotic that promotes the growth of beneficial bacteria in the gut.

7. The use of the prebiotic mimicry (biogenic prebiotic) of the medicinal and edible homology class as described in claim 6 in the preparation of drugs or formulations that promote bowel movements, mimic prebiotics, and inhibit harmful bacteria, characterized in that the beneficial intestinal bacteria include: Bacteroidales, Lactobacillaceae, Rikenellaceae, Marinifilaceae, Odoribacter, Bacteroides_caecimuris, Clostridia_vadinBB60_group, Roseburia, Turicibacter, Akkermansia, Rs-E47_termite_group, Rikenella, Clostridia_vadinBB60_group.

8. The use of the prebiotic mimicry (biomimetic) of the medicinal and edible homology class as described in claim 5 in the preparation of drugs or formulations that promote bowel movements, mimic prebiotics, and inhibit harmful bacteria, characterized in that: The harmful intestinal bacteria include: Coriobacteriales, Paeniclostridium, Macrococcus, Acetatifactor, Faecalitalea, Erysipelotrichales, Streptococcus, Faecalibaculum, Blautia, Rikenellaceae_RC9_gut_group, Clostridium_sensu_stricto_1, and Gastranaerophilales.

9. The use of the prebiotic mimicry (biomimetic) of the food-medicine homology as described in claim 5 in the preparation of drugs or formulations that promote bowel movements, mimic prebiotics, and inhibit harmful bacteria, characterized in that: When applied, citron or its extract, a prebiotic mimic from food and medicine, is mixed with other substances to form a complex for preparing drugs or preparations that promote bowel movements, mimic prebiotics, and inhibit harmful bacteria.

10. Citron, a prebiotic mimic of food and medicine, is used in the preparation of drugs or preparations, which have the following effects: inhibiting periodontal / pulp infection, treating Parkinson's disease, reducing intestinal and tissue viral infections, reducing inflammatory response, inhibiting prostatic hyperplasia, potentially treating NASH, maintaining intestinal barrier integrity, helping to improve intestinal function, promoting food digestion and absorption, preventing and treating diarrhea, having potential protective effects against food allergies, inhibiting obesity, protecting the cardiovascular system, reducing inflammation, improving colitis, and treating or preventing bacteremia, infective endocarditis, HFD, Alzheimer's disease, prostatitis, etc.

11. The use of the prebiotic mimicry (biomimetic) of the medicinal and edible homology class as described in any one of claims 1-4 in the preparation of drugs or formulations that promote bowel movements, mimic prebiotics, and inhibit harmful bacteria, characterized in that: The prebiotic mimic (biogenetic) of the food-medicine homology category is chicory.

12. The use of the prebiotic mimicry (biomimetic) of the food-medicine homology as described in claim 5 in the preparation of drugs or formulations that promote bowel movements, mimic prebiotics, and inhibit harmful bacteria, characterized in that: The term "simulated prebiotic" refers to a prebiotic that promotes the growth of beneficial bacteria in the gut.

13. The use of the prebiotic mimicry (probiotic analogue) of the food-medicine homology class as described in claim 6 in the preparation of drugs or formulations that promote bowel movements, mimic prebiotics, and inhibit harmful bacteria, characterized in that the beneficial intestinal bacteria include: Bacteroidales, Lactobacillaceae, Rikenellaceae, Christensenellaceae, Marinifilaceae, Butyricicoccaceae, Bacteroides_caecimuris, Peptococcac eae, Clostridia_vadinBB60_group, Harryflintia, Roseburia, Rikenella, ASF356, Lachnospiraceae_NK4A136_group, Intestinimonas, Colidextribacter.

14. The use of the prebiotic mimicry (biomimetic) of the food-medicine homology as described in claim 5 in the preparation of drugs or formulations that promote bowel movements, mimic prebiotics, and inhibit harmful bacteria, characterized in that: The harmful intestinal bacteria include: Coriobacteriales, Mycoplasma, Paeniclostridium, Macrococcus, Faecalitalea, Erysipelotrichales, Romboutsia, Rikenellaceae_RC9_gut_group, Clostridium_sensu_stricto_1, and Lachnospiraceae.

15. The use of the prebiotic mimicry (biogenic prebiotic) of the medicinal and edible homology class as described in claim 5 in the preparation of drugs or formulations that promote bowel movements, mimic prebiotics, and inhibit harmful bacteria, characterized in that: When applied, chicory or its extract, a prebiotic mimic of food and medicine, is mixed with other substances to form a complex for preparing drugs or preparations that promote bowel movements, mimic prebiotics, and inhibit harmful bacteria.

16. Chicory, a prebiotic mimic of food and medicine, is used in the preparation of drugs or formulations that have the following effects: inhibiting periodontal / pulp infection, reducing intestinal and tissue viral infections, reducing inflammatory response, potential treatment of NASH, maintaining intestinal barrier integrity, helping to improve intestinal function, promoting food digestion and absorption, preventing and treating diarrhea, producing SCFA, maintaining metabolic health, having potential protective effects against food allergies, inhibiting obesity, protecting the cardiovascular system, reducing inflammation, improving colitis, treating or preventing bone marrow necrosis caused by acute myeloid leukemia, HFD, Alzheimer's disease, prostatitis, and inhibiting obesity.

17. The use of the prebiotic mimicry (biomimetic) of the medicinal and edible homology class as described in any one of claims 1-4 in the preparation of drugs or formulations that promote bowel movements, mimic prebiotics, and inhibit harmful bacteria, characterized in that: The prebiotic mimic (biogenetic) of the food-medicine homology category is Elsholtzia ciliata.

18. The use of the prebiotic mimicry (biomimetic) of the food-medicine homology as described in claim 5 in the preparation of drugs or formulations that promote bowel movements, mimic prebiotics, and inhibit harmful bacteria, characterized in that: The term "simulated prebiotic" refers to a prebiotic that promotes the growth of beneficial bacteria in the gut.

19. The use of the prebiotic mimicry (biogenic prebiotic) of the medicinal and edible homology class as described in claim 6 in the preparation of drugs or formulations that promote bowel movements, mimic prebiotics, and inhibit harmful bacteria, characterized in that the beneficial intestinal bacteria include: Psychrobacter, Lactobacillaceae, Turicibacter, Clostridia_vadinBB60_group, Akkermansia, Alistipes, ASF356, Christensenellaceae_R-7_group、Lachnospiraceae_NK4A136_group、 Clostridia_vadinBB60_group, Eubacterium_siraeum_group.

20. The use of the prebiotic mimicry (biomimetic) of the medicinal and edible homology class as described in claim 5 in the preparation of drugs or formulations that promote bowel movements, mimic prebiotics, and inhibit harmful bacteria, characterized in that: The harmful intestinal bacteria include: Coriobacteriales, Mycoplasma, Paeniclostridium, Macrococcus, Acetatifactor, Faecalitalea, and Erysipelotrichales.

21. The use of the prebiotic mimicry (biomimetic) of the medicinal and edible homology class as described in claim 5 in the preparation of drugs or formulations that promote bowel movements, mimic prebiotics, and inhibit harmful bacteria, characterized in that: When applied, the medicinal and edible prebiotic mimic (biogenic) Elsholtzia ciliata or its extract is mixed with other substances to form a complex for the preparation of drugs or preparations that promote bowel movements, mimic prebiotics, and inhibit harmful bacteria.

22. Elsholtzia ciliata, a prebiotic mimic of food and medicine, is used in the preparation of drugs or preparations, which have the following effects: inhibiting periodontal / pulp infection, treating Parkinson's disease, reducing intestinal and tissue viral infections, reducing inflammatory response, inhibiting prostatic hyperplasia, potential treatment of NASH, antibacterial, helping to improve intestinal function, promoting food digestion and absorption, preventing and treating diarrhea, and other different effects, inhibiting obesity, protecting the cardiovascular system, reducing inflammation, improving colitis, and other different effects.

23. The use of the prebiotic mimicry (biomimetic) of the medicinal and edible homology class as described in any one of claims 1-4 in the preparation of drugs or formulations that promote bowel movements, mimic prebiotics, and inhibit harmful bacteria, characterized in that: The prebiotic mimic (biogenetic) of the food-medicine homology category is Hovenia dulcis.

24. The use of the prebiotic mimicry (biomimetic) of the medicinal and edible homology class as described in claim 5 in the preparation of drugs or formulations that promote bowel movements, mimic prebiotics, and inhibit harmful bacteria, characterized in that: The term "simulated prebiotic" refers to a prebiotic that promotes the growth of beneficial bacteria in the gut.

25. The use of the prebiotic mimicry (biogenic prebiotic) of the medicinal and edible homology class as described in claim 6 in the preparation of drugs or formulations that promote bowel movements, mimic prebiotics, and inhibit harmful bacteria, characterized in that the beneficial intestinal bacteria include: Eubacterium_ruminantium_group, Olsenella, Bacteroidales, Marinifilaceae, Muribaculaceae, Odoribacter, Roseburia, Rikenellaceae, Rikenella, Clostridia_vadinBB60_group, Peptococcaceae, Eubacterium_siraeum_group, Bacteroides_caecimuris.

26. The use of the prebiotic mimicry (biomimetic) of the food-medicine homology as described in claim 5 in the preparation of drugs or formulations that promote bowel movements, mimic prebiotics, and inhibit harmful bacteria, characterized in that the harmful intestinal bacteria include: Coriobacteriales, Peptostreptococales-Tissierellales, Paeniclostridium, Mycoplasma, Macrococcus, Acetatifactor, Coriobacteriales_Incertae_Sedis, Faecalitalea, Erysipelotrichales.

27. The use of the prebiotic mimicry (biomimetic) of the medicinal and edible homology class as described in claim 5 in the preparation of drugs or formulations that promote bowel movements, mimic prebiotics, and inhibit harmful bacteria, characterized in that: When applied, the prebiotic mimic (biogenic) Hovenia dulcis fruit or its extract, which is a food and medicine homology, is mixed with other substances to form a complex for the preparation of drugs or preparations that promote bowel movements, mimic prebiotics, and inhibit harmful bacteria.

28. Hovenia dulcis, a prebiotic mimic (biogenic agent) of medicinal and edible origin, is used in the preparation of drugs or preparations, which have the following effects: inhibiting various serious infections including urinary tract infections, bacteremia, intra-abdominal infections and endocarditis, treating Parkinson's disease, reducing intestinal and tissue viral infections, reducing inflammatory response, inhibiting prostatic hyperplasia, potentially treating NASH, improving type 2 diabetes, enhancing anti-tumor immunity, maintaining intestinal barrier integrity, potentially combating food allergies, inhibiting bacterial translocation, reducing proteinuria, controlling weight, preventing damage and reducing inflammatory response, improving colitis, and treating liver damage.

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