Application of hawthorn leaf product in preparation of product for improving intestinal flora and research method

Abstract

The invention discloses application of a hawthorn leaf product in preparation of a product for improving intestinal flora and a research method, belongs to the technical field of improvement of alteration of intestinal flora, and particularly discloses application of the hawthorn leaf product in improvement of alteration of intestinal flora and the research method of the hawthorn leaf product. SPF-level male mice are selected and stored in a constant-temperature and constant-humidity illumination fixed animal room; after one week of adaptive feeding, randomly dividing the crataegus pinnatifida bunge into six groups which are respectively marked as a Control group, an HFD group, a low-dose crataegus pinnatifida bunge leaf group HL100, a high-dose crataegus pinnatifida bunge group HL500, a low-dose vitexin group Vitexin 6 and a high-dose vitexin group Vitexin 30; collecting the sample: collecting fresh mouse excrement one week before the experiment is finished; and then excrement DNA extraction and 16S rRNA sequencing are carried out to analyze intestinal flora. The invention verifies that the hawthorn leaf product can be used for preparing the product for improving the intestinal flora, and the hawthorn leaf product preferably selects hawthorn leaves or vitexin as a raw material.

Description

Technical Field

[0001] The present invention relates to the technical field of improving intestinal flora imbalance, and specifically relates to the application and research method of using hawthorn leaf products to prepare products for improving intestinal flora. Background Art

[0002] Studies have shown that hawthorn leaves and vitexin have a mitigating effect on weight gain in high-fat diet mice, which is achieved through mechanisms such as regulating lipid metabolism, suppressing appetite, and improving insulin resistance.

[0003] However, there is currently little research on the use of hawthorn leaves in improving intestinal flora imbalance. The present invention specifically provides an application of using hawthorn leaf products to prepare products for improving intestinal flora, as well as a research method for improving intestinal flora imbalance with hawthorn leaf products.

[0004] Based on this, the present invention designs an application and research method of using hawthorn leaf products to prepare products for improving intestinal flora to solve the above problems. Summary of the Invention

[0005] In view of the above-mentioned drawbacks of the prior art, the present invention provides an application and research method of using hawthorn leaf products to prepare products for improving intestinal flora.

[0006] To achieve the above objectives, the present invention is realized through the following technical solutions: An application of a hawthorn leaf product in improving intestinal flora imbalance.

[0007] Furthermore, the hawthorn leaf product is hawthorn leaf powder or hawthorn leaf extract.

[0008] Furthermore, the hawthorn leaf extract is vitexin.

[0009] To better achieve the objectives of the present invention, the present invention also provides a research method for the application of the hawthorn leaf product in improving intestinal flora imbalance, including the following steps: Step 1: Animal experiment design Select SPF-grade male mice and keep them in an animal room with constant temperature, humidity, and light; after one week of adaptive feeding, randomly divide them into six groups, denoted as: Control group, HFD group, low-dose hawthorn leaf group HL100, high-dose hawthorn group HL500, low-dose vitexin group Vitexin6, and high-dose vitexin group Vitexin30; Step 2: Sample collection: One week before the end of the experiment, collect fresh mouse feces in centrifuge tubes and store them in the refrigerator; Step 3: Extract fecal DNA and perform 16S rRNA sequencing to analyze the intestinal flora.

[0010] Further, in Step 1, the temperature in the animal house is 25±2°C; the humidity is 55±5%; the lighting is 12 h with day-night cycle; feeding is carried out for 12 weeks to establish a disease model.

[0011] Further, in Step 2, collect fresh mouse feces in a centrifuge tube and store them in a -80°C refrigerator. Each mouse has an independent space during the collection process to avoid cross-contamination of samples.

[0012] Further, in Step 1, for the Control group, a low-fat diet is used; for the HFD group, a high-fat diet is used; for the low-dose hawthorn leaf group HL100, a high-fat diet + hawthorn leaf 100 mg / kg bw / day; for the high-dose hawthorn group HL500, a high-fat diet + hawthorn leaf 500 mg / kg bw / day; for the low-dose vitexin group Vitexin6, a high-fat diet + vitexin 6 mg / kg bw / day; for the high-dose vitexin group Vitexin30, a high-fat diet + vitexin 30 mg / kg bw / day.

[0013] Further, in Step 3, identify the intestinal microbiota of the previously collected mouse feces. Extract the total genomic DNA samples from the mouse fecal samples, divide them into six groups, and store them at -20°C; use a NanoDrop ND1000 ultraviolet spectrophotometer and agarose gel electrophoresis to measure the quantity and quality of the extracted DNA. Then, use the Illumina MiSeq platform to perform 16S rRNA gene amplicon sequencing on each sample; use the forward primer 338F and the reverse primer 806R to perform PCR amplification on the V3-V4 region of the bacterial 16S rRNA gene; use Agencourt AMPure XP magnetic beads for product purification, and then elute in the elution buffer to prepare the library; use an Agilent 2100 bioanalyzer to evaluate the fragment size distribution and concentration of the library; sequence the qualified library based on the insert fragment size on the HiSeq platform; after sequencing, splice the obtained PE reads according to the overlapping relationship to obtain the original sequence, and perform filtering and quality control; based on the Uparse software, perform OTU clustering on the non-redundant sequences according to 97% similarity, and remove chimeras during this process; then, compare with the Silva128 / 16S bacterial database, annotate the optimized sequences to obtain classification information, and flatten the sample sequences according to the minimum sample sequence number before data analysis.

[0014] Further, in step three, Alpha diversity analysis and Beta diversity analysis are performed through Motherur software and QIIME software, and the differences between groups are analyzed; the R language tool is used to make a rank-abundance curve to explain the diversity, draw a pan / core species curve to judge whether the sample size is sufficient, evaluate the total species richness and the number of core species in feces, and make a Venn diagram and a community bar chart to show the composition and similarity of species; based on two distance algorithms, QIIME software is used for principal coordinate analysis, statistical analysis and non-metric multi-dimensional scaling analysis, calculate the distance between samples and obtain a distance matrix; LefSe software is used to analyze the differences in the relative abundances of species between groups; then, the Wilcoxon rank sum test is used to test the difference consistency of different species in different subgroups; finally, LDA is used to estimate the impact of these different species on the differences between groups.

[0015] The present invention verifies that hawthorn leaf products can be used to prepare products for improving intestinal flora, and the hawthorn leaf products are preferably hawthorn leaves or vitexin. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention, and those of ordinary skill in the art can obtain other drawings without creative efforts based on these drawings.

[0017] Figure 1 Effects of hawthorn leaves and vitexin on the richness and diversity of intestinal flora in HFD-induced obese mice. a. Venn diagram of OTUs in the Control group and the HFD group; b. Venn diagram of OTUs in the HFD group and the HL100 group; c. Venn diagram of OTUs in the HFD group and the Vitexin6 group; d. Sobs index; e. Simpson diversity index; f. Ace index; g. Chao index; h. Shannon diversity index. Compared with the Control group, P<0.001; compared with the HFD group, *P<0.05, **P<0.01, ***P<0.001.

[0018] Figure 2 β diversity analysis of each group. a. PCoA analysis; b. NMDS analysis; c. PCA analysis; d. PLS-DA analysis.

[0019] Figure 3Effects of hawthorn leaves and vitexin on the gut microbiota of HFD-induced obese mice. a. Relative abundances of gut microbiota at the phylum level; b. Ratio of Firmicutes to Bacteroidetes (F / B) of gut microbiota; c. Relative abundances of gut microbiota at the genus level.

[0020] Figure 4 It is the LEfSe clustering tree.

[0021] Figure 5 It is the histogram of LDA scores based on LEfSe analysis. Detailed implementation manners

[0022] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0023] Example 1: Research experiment on hawthorn leaf products for improving gut microbiota dysbiosis 1. Materials and methods 1.1 Reagents: Commercial hawthorn leaf powder, vitexin (purchased from Chengdu Profide Reference Technology Co., Ltd., batch number: JOT-10242).

[0024] 1.2 Animal experiment design: 60 specific pathogen-free (SPF)-level C57BL / 6J male mice (six weeks old, 18 - 22 g) were purchased from Zhuhai BestTest Bio-Tech Co, Ltd. and kept in an animal room with constant temperature, humidity and light (temperature 25 ± 2°C; humidity 55 ± 5%; light 12 h day-night cycle). After one week of adaptive feeding, they were randomly divided into six groups (n = 10), namely Control group (low-fat diet), HFD group (high-fat diet), low-dose hawthorn leaf group HL100 (high-fat diet + hawthorn leaf 100 mg / kg bw / day), high-dose hawthorn group HL500 (high-fat diet + hawthorn leaf 500 mg / kg bw / day), low-dose vitexin group Vitexin6 (high-fat diet + vitexin 6 mg / kg bw / day), high-dose vitexin group Vitexin30 (high-fat diet + vitexin 30 mg / kg bw / day). They were fed ad libitum, and the body weight changes of the mice were recorded weekly.

[0025] 1.3. Sample collection: One week before the end of the experiment, fresh mouse feces were collected into 1.5 mL centrifuge tubes and stored in an -80 °C refrigerator until further study. Each mouse had an independently disinfected space during feces collection to avoid sample cross-contamination.

[0026] 1.4. Fecal DNA extraction and 16S rRNA sequencing for analyzing gut microbiota: The previously collected mouse feces were sent to Shenzhen Anborelle Pharmaceutical Technology Co., Ltd. for gut microbiota identification. Total genomic DNA samples were extracted from the fecal samples of 60 mice, divided into six groups, and stored at -20 °C for further analysis. The quantity and quality of the extracted DNA were determined using a NanoDrop ND1000 ultraviolet spectrophotometer and agarose gel electrophoresis. Then, using the Illumina MiSeq platform, 16S rRNA gene amplicon sequencing was performed on each sample according to the conventional protocol. The V3-V4 region of the bacterial 16S rRNA gene was amplified by polymerase chain reaction (PCR) using the forward primer 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and the reverse primer 806R (5′-GGACTACHVGGGTWTCTAAT-3′). In addition, Agencourt AMPure XP magnetic beads were used for product purification, and then eluted in elution buffer to prepare the library. The fragment size distribution and concentration of the library were evaluated using an Agilent 2100 bioanalyzer. The qualified library based on the insert fragment size was sequenced on the HiSeq platform. After sequencing was completed, the sequenced PE reads were spliced according to the overlapping relationship to obtain the original sequences, which were then filtered and quality-controlled. Based on the Uparse software (v7.0.1090), the present invention set the classification confidence level to 70%. OTU clustering was performed on the non-redundant sequences (excluding single sequences) with 97% similarity, and chimeras were removed during this process. Then, the optimized sequences were annotated by comparison with the Silva128 / 16S bacterial database to obtain classification information, and the sample sequences were flattened according to the minimum sample sequence number before data analysis.

[0027] Alpha diversity analysis and beta diversity analysis were performed using Motherur software (version 1.30.2) and QIIME software, and the differences between groups were analyzed. The R language tool was used to create rank-abundance curves to explain diversity, and pan / core species curves were plotted to determine whether the sample size was sufficient, evaluate the total species richness and the number of core species in feces, and create Venn diagrams and community bar charts to display the species composition and similarity. Based on two distance algorithms (unweighted_unifrac and weighted_unifrac), principal coordinate analysis (PCoA), statistical analysis, and non-metric multidimensional scaling analysis (NMDS) were performed using QIIME software to calculate the distances between samples and obtain a distance matrix. LEfSe software was used to analyze the differences in the relative abundances of species between groups.

[0028] One-way analysis of variance test and Tukey-Kramer test were used to compare the abundances of gut microbiota in each group. In LEfSe analysis, non-parametric Kruskal-Wallis (KW) and rank tests were mainly used to detect the differences in the abundances of species between different taxa to obtain significantly different species. Then, the Wilcoxon rank-sum test was used to test the difference consistency of different species in different subgroups; finally, linear discriminant analysis (LDA) was used to estimate the effects of these different species on the differences between groups.

[0029] Example 2: Effects of hawthorn leaves and vitexin on the gut microbiota of mice The occurrence and development of obesity are often accompanied by reduced gut microbial diversity, gut microbiota imbalance, and dysfunction. To verify the effects of hawthorn leaves and vitexin on the gut microbiota of high-fat diet-induced obese mice, the feces of mice were detected. Venn diagrams were used to analyze the similarity and overlap of the operational taxonomic unit (OTUs) compositions between different groups. The number of OTUs in each sample was obtained, and the Venn diagram was used to display the number of OTUs common to and unique to the samples, intuitively showing the OTU overlap between samples. A total of 337 OTUs were identified in the gut microbiota of the Control group and the HFD group, of which the number of OTUs specific to the HFD group was 154 ( Figure 1 a), which was 152 less than that of the Control group. In contrast, the number of OTUs in the low-dose hawthorn leaf and vitexin groups increased by 23 and 31, respectively, compared with the HFD group ( Figure 1b, c), indicating that the intervention of vitexin and hawthorn leaves in the diet of mice can help increase the number of intestinal microbial OTUs to a certain extent and restore the intestinal environment of mice. The α-diversity index can reflect the diversity of the microbial flora in the sample, and the Chao, Ace, and Sobs indices are often used to evaluate the richness of the microbial community. It can be found in the α-diversity analysis that compared with the Control group, the Chao, Ace, and Sobs indices in the HFD group decreased significantly, indicating a significant reduction in the richness and diversity of the intestinal microbiota. After adding hawthorn leaves or vitexin, the diversity and richness of the intestinal flora were restored to a certain extent. Among them, the low-dose vitexin group had the most significant effect ( Figure 1 d-h).

[0030] The differences in microbial composition between different groups can be seen through the β-diversity index. Unweighted (only considering the presence or absence of species) UniFrac distance was used for principal component analysis (PCoA). When the distance between two samples on the graph is closer, their microbial composition is more similar. Each point in the graph represents a different sample, and the positional relationship of these points in the graph can intuitively reflect the degree of difference between samples and the similarity of community composition, etc. PCoA ( Figure 2 a) results showed that there were significant differences between the Control group and the HFD group, indicating that HFD changed the characteristics of the intestinal microbiota in mice, leading to microbiota dysbiosis. There was no overlap between the high-dose hawthorn leaf group and the HFD group, indicating that hawthorn leaves had a certain therapeutic effect on HFD-fed mice. In the NMDS analysis ( Figure 2 b), the vitexin group and the high-dose hawthorn leaf group did not overlap with the HFD group, and the vitexin group showed a trend close to the Control group. Then PCA and PLS-DA analyses were carried out to further verify the differences between the HFD group and the hawthorn leaf and vitexin groups ( Figure 2 c-d), which were similar to the previous results, showing significant differences between the intervention group and the HFD group.

[0031] In addition, the effects of hawthorn leaves and vitexin on the composition of the intestinal microbiota were also studied. The F / B ratio of the Firmicutes / Bacteroidetes in the HFD group was lower than that in the Control group. After treatment with vitexin, the F / B value increased slightly compared with the HFD group, but no significant difference was shown.

[0032] To conduct a more comprehensive analysis of the intestinal microbiota in mice, the distribution of the microbiota at the genus level was studied. It was found that compared with the Control group, HFD led to an increase in the relative abundances of Flintibacter, Acetatifactor, and Bacteroides, and a decrease in the relative abundances of Faecalibaculum, Paramuribaculum, and Sporofaciens. Compared with the HFD group, both the vitexin and hawthorn leaf groups increased the relative abundance of Acetatifactor, and the low-dose hawthorn leaf and vitexin groups slightly increased the relative abundance of Roseburia; the high-dose hawthorn leaf group slightly increased the relative abundance of Flintibacter ( Figure 3 a-c).

[0033] Figure 3 Explanation of the professional terms in a: Candidate phylum: Candidatus Saccharibacteria; Phylum Chloroflexota; Phylum Fusobacteriota; Phylum Cyanobacteriota; SR1: A phylum within the domain Bacteria, also known as Bacillota; Phylum Planctomycetota; Phylum Bacteroidota; Phylum Actinomycetota; Phylum Bacillota (formerly known as Firmicutes); Phylum Pseudomonadota; Balneolota: A phylum within the domain Bacteria; Phylum Mycoplasmatota; Calditrichota: A phylum within the domain Bacteria; Synergistota: A phylum within the domain Bacteria; Phylum Verrucomicrobiota; Phylum Campylobacterota; Phylum Deinococcota; Deferribacterota: a phylum belonging to the domain Bacteria; Figure 3 Explanation of professional terms in c: Genus Alistipes: Alistipes; Genus Flintibacter: Flintibacter; Genus Lawsonibacter: Lawsonibacter; Genus Olsenella: Olsenella; Genus Paramuribaculum: Paramuribaculum; Genus Duncaniella: Duncaniella; Genus Acetatifactor: Acetatifactor; Genus Roseburia: Roseburia; Genus Kineothrix: Kineothrix; Genus Lactobacillus: Lactobacillus; Genus Sporofaciens: Sporofaciens; Genus Anaerotignum: Anaerotignum; Genus Faecalibaculum: Faecalibaculum; Genus Mucispirillum: Mucispirillum; Genus Bacteroides: Bacteroides; Genus Vescimonas: Vescimonas; Genus Laedolimicola: Laedolimicola; LEfSe (Linear Discriminant Analysis) can discover the contribution of species with different abundances in different groups. In the LEfSe clustering tree, nodes of different colors represent the microbial communities that play important roles in the groups represented by the colors. From the inside to the outside, each circle represents species at the phylum, class, order, family, and genus levels in turn. In the LDA graph (LDA score > 2), different colors represent microbial groups with significant effects in different groups. Only when the absolute value of the LDA value is greater than 2 will it be shown in the graph, demonstrating biomarkers with statistical differences. The length of the bars in the bar graph represents the magnitude of the effects of significantly different species between different groups. In order to explore the characteristic microorganisms regulated by hawthorn leaves and vitexin, LEfSe analysis was performed ( Figure 4). The number of different species in the six groups of mice was 62. The number of differential species in the Control group was 27, and that in the HFD group was 16. At the genus level, when analyzing the six groups, the genera showing differential characteristics in the Control group were Faecalibaculum and Lawsonibacter, while the differential characteristic genus level in the HFD group was Dorea, and the differential characteristic families were Coprobacillaceae and Prevotellaceae. In the high-dose vitexin group, Acetatifactor, Duncaniella, and Romboutsia were the genera with differential characteristics, while in the low-dose hawthorn leaf group, the genera showing differential characteristics were Paramuribaculum and Streptococcus. By observing the changes in species, such as Dorea, Coprobacillaceae, and Prevotellaceae, it is speculated that the gut microbiota disorder induced by a high-fat diet may be related to these species ( Figure 5 ).

[0034] Figures 4-5 Explanation of professional terms in Acetatifactor: Acetatifactor; Lachnospiraceae: Lachnospiraceae; Duncaniella: Duncaniella; Betaproteobacteria: Betaproteobacteria; Romboutsia: Romboutsia; Peptostreptococcaceae: Peptostreptococcaceae; Ruthenibacterium: Ruthenibacterium; Muribaculum: Muribaculum; Eubacteriale: Eubacteriale; Clostridia: Clostridia; Schaalia: Schaalia; Salmonella: Salmonella; Selenomonadales: Selenomonadales; Selenomonadaceae: Selenomonadaceae; Solibaculum: Solibaculum; Genus Megamonas: Megamonas; Genus Paramuribaculum: Paramuribaculum; Family Streptococcaceae: Streptococcaceae; Genus Streptococcus: Streptococcus; Genus Dorea: Dorea; Genus Hoylesella: Hoylesella; Genus Thomasclavelia: Thomasclavelia; Family Coprobacillaceae: Coprobacillaceae; Genus Paludihabitans: Paludihabitans; Genus Ligilactobacillus: Ligilactobacillus; Family Prevotellaceae: Prevotellaceae; Class Negativicutes: Negativicutes; Order Eubacteriales: Eubacteriales; Genus Aminipila: Aminipila; Family Neisseriaceae: Neisseriaceae; Order Neisseriales: Neisseriales; Genus Neisseria: Neisseria; Family Veillonellaceae: Veillonellaceae; Order Veillonellales: Veillonellales; Genus Veillonella: Veillonella; Family Erysipelotrichaceae: Erysipelotrichaceae; Genus Faecalibaculum: Faecalibaculum; Class Erysipelotrichia: Erysipelotrichia; Order Erysipelotrichales: Erysipelotrichales; Genus Lawsonibacter: Lawsonibacter; Genus Butyricicoccus: Butyricicoccus; Genus Caproiciproducens: Caproiciproducens; Genus Calorithrix: Calorithrix; Phylum Calditrichota: Calditrichota; Genus Klebsiella: Klebsiella; Order Caryophanales: Caryophanales; Genus Paenibacillus: Paenibacillus; Family Paenibacillaceae: Paenibacillaceae; Genus Calditrichia: Calditrichia; Family Calditrichaceae: Calditrichaceae; Order Calditrichales: Calditrichales; Genus Odoribacter: Odoribacter; Family Odoribacteraceae: Odoribacteraceae; Genus Guopingia: Guopingia; Family Christensenellaceae: Christensenellaceae; Genus Neglecta: Neglecta; Genus Acutalibacter: Acutalibacter; Class Gammaproteobacteria: Gammaproteobacteria; Family Enterobacteriaceae: Enterobacteriaceae; Order Enterobacterales: Enterobacterales; Genus Fumia: Fumia; Genus Intestinimonas: Intestinimonas.

[0035] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements will not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Application of a hawthorn leaf product in improving intestinal flora imbalance.

2. The use of the hawthorn leaf product according to claim 1 in improving intestinal flora imbalance, characterized in that: The hawthorn leaf product is hawthorn leaf powder or hawthorn leaf extract.

3. The use of the hawthorn leaf product according to claim 2 in improving intestinal flora imbalance, characterized in that: The hawthorn leaf extract is vitexin.

4. A research method for using the hawthorn leaf product according to any one of claims 1 to 3 in improving intestinal flora imbalance, characterized in that: The following steps are involved: Step 1. Animal experiment design: SPF male mice were selected and kept in an animal room with constant temperature, humidity and lighting. After one week of adaptive feeding, they were randomly divided into six groups, namely: Control group, HFD group, low-dose hawthorn leaf group HL100, high-dose hawthorn group HL500, low-dose vitexin group Vitexin6, and high-dose vitexin group Vitexin30. Step 2. Sample collection: One week before the end of the experiment, collect fresh mouse feces into centrifuge tubes and store them in the refrigerator; Step 3: Fecal DNA extraction and 16S rRNA sequencing to analyze intestinal flora.

5. The method for improving intestinal flora imbalance using a hawthorn leaf product according to claim 4, characterized in that: In step 1, the temperature of the animal room was 25±2°C; the humidity was 55±5%; the light was on for 12 hours, with a day and night cycle; and the disease model was established by feeding for 12 weeks.

6. The method for improving intestinal flora imbalance using a hawthorn leaf product according to claim 4, characterized in that: In step 2, fresh mouse feces were collected into centrifuge tubes and stored in a -80°C refrigerator. Each mouse had an independent space during the collection process to avoid cross-contamination of samples.

7. The method for improving intestinal flora imbalance using a hawthorn leaf product according to claim 6, characterized in that: In step 1, the Control group was on a low-fat diet; the HFD group was on a high-fat diet; the low-dose hawthorn leaf group HL100 was on a high-fat diet plus hawthorn leaf 100 mg / kg bw / day; the high-dose hawthorn group HL500 was on a high-fat diet plus hawthorn leaf 500 mg / kg bw / day; the low-dose vitexin group Vitexin6 was on a high-fat diet plus vitexin 6 mg / kg bw / day; and the high-dose vitexin group Vitexin30 was on a high-fat diet plus vitexin 30 mg / kg bw / day.

8. The method for improving intestinal flora imbalance using a hawthorn leaf product according to claim 7, characterized in that: In step three, the previously collected mouse feces were used for intestinal microbial identification. Total genomic DNA samples were extracted from the mouse fecal samples, divided into six groups, and stored at -20°C. The quantity and quality of the extracted DNA were determined using a NanoDrop ND1000 UV spectrophotometer and agarose gel electrophoresis. Then, the 16S rRNA gene amplicon of each sample was sequenced using the Illumina MiSeq platform. The V3-V4 region of the bacterial 16S rRNA gene was PCR amplified using forward primer 338F and reverse primer 806R. The product was purified using Agencourt AMPure XP magnetic beads and then eluted in elution buffer to prepare the library. The Agilent The fragment size distribution and concentration of the library were evaluated by 2100 bioanalyzer. Qualified libraries based on insert fragment size were sequenced on the HiSeq platform. After sequencing, the PEreads obtained by sequencing were spliced ​​according to overlapping relationships to obtain the original sequence. Based on Uparse software, OTU clustering was performed on non-repetitive sequences according to 97% similarity, and chimeras were removed in the process. Then, the optimized sequences were compared with the Silva128 / 16S bacterial database, annotated to obtain classification information, and the sample sequences were flattened according to the minimum number of sample sequences before data analysis.

9. The method for improving intestinal flora imbalance using a hawthorn leaf product according to claim 8, characterized in that: In step three, Alpha diversity analysis and Beta diversity analysis were performed using Motherur software and QIIME software, and differences between groups were analyzed; Rank abundance curves were created using R language tools to explain diversity, pan / core species curves were drawn to determine whether the sample size was sufficient, the total species richness and the number of core species in feces were evaluated, and Venn diagrams and community bar charts were created to display species composition and similarity; based on two distance algorithms, QIIME software was used to perform principal coordinate analysis, statistical analysis, and non-metric multidimensional scaling analysis, calculate the distance between samples, and obtain a distance matrix; LEfSe software was used to analyze the differences in relative abundance of species between groups; Then, the Wilcoxon rank sum test was used to test the consistency of differences among different species in different subgroups; finally, LDA was used to estimate the impact of these different species on the differences between groups.

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