Application of patchouli alcohol as flora regulator

Abstract

The invention discloses an application of patchouli alcohol as a flora regulator. According to the application of patchouli alcohol provided by the invention in preparation of medicines, foods, drinks or health-care products for promoting growth and / or proliferation of bacteria, the bacteria are selected from one or two of bacteria of the phylum firmicules and bacteria of the phylum actinobacteria, and the application of patchouli alcohol in preparation of medicines, foods, drinks or health-care products is also provided, wherein the bacteria are selected from one or two of bacteria of the phylum firmicules and bacteria of the phylum actinobacteria. The patchouli alcohol performs targeted regulation on intestinal flora by improving the abundance of intestinal probiotics and reducing the abundance of harmful bacteria, and provides a new thought and direction for development of drugs for targeted regulation of the intestinal flora in future.

Description

TECHNICAL FIELD

[0001] The present application relates to the use of amygdalol as a microbiota modulator. BACKGROUND

[0002] Inflammatory Bowel Disease (IBD) is a complex group of gastrointestinal diseases, mainly including Crohn's disease (CD) and ulcerative colitis (UC). These diseases are characterized by bloody diarrhea, abdominal pain, systemic infection and even death. The exact pathogenesis of inflammatory bowel disease has not been fully elucidated, and intestinal flora imbalance is considered to be a factor. This imbalance can lead to mucosal dysfunction and immune dysregulation, further triggering intestinal inflammation. The balance of intestinal microbiota is crucial for maintaining gastrointestinal health, and any imbalance can lead to disease. Some harmful flora, such as Helicobacte, Bacteroides and Parasutterella, are considered biomarkers of IBD. These bacteria not only overgrow in the intestine, but also can destroy the intestinal barrier, triggering infection and inflammation. In contrast, probiotics are a class of microorganisms that are beneficial to the host, can inhibit the proliferation of pathogenic bacteria, enhance the function of the intestinal barrier, and relieve intestinal inflammation. Therefore, treatment targeting intestinal flora modulation is of great significance for the research and treatment of IBD.

[0003] Currently, intestinal flora modulators are mainly probiotics, such as Bifidobacterium, Dubosiella, Romboutsia and Turicibacter, which have the potential to replace or assist in the treatment of IBD. In addition, some natural compounds have also been found to have intestinal flora modulating function. For example, the invention patent application with publication number CN115869330A mentions that specnuzhenin can significantly change the abundance of Butyrivibrio and other bacteria in the intestinal flora of aging model mice, thereby having an intestinal flora modulating effect.

[0004] Prunol, a sesquiterpene compound and one of the main active ingredients of patchouli, possesses various pharmacological effects. According to patent application CN106511323A, prunol shows good efficacy in treating enteritis. It can reduce myeloperoxidase activity to alleviate inflammatory responses, promote the expression of tight junction proteins and mucins, and protect the intestinal epithelial mucosal barrier. Despite its good efficacy in treating enteritis, prunol has low oral bioavailability. Currently, there are no reports on the regulation of gut microbiota by prunol. Summary of the Invention

[0005] The technical problem this invention aims to solve is to provide a novel intestinal flora regulator to promote the growth of beneficial bacteria and / or inhibit the growth of harmful bacteria. To this end, this invention provides an application of baicalein as a flora regulator. This invention is the first to discover that baicalein can significantly affect the composition of the intestinal flora. Specifically, baicalein can significantly increase the absolute abundance and / or relative abundance of beneficial bacteria, including *Dubosiella*, *Romboutsia*, *Turicibacter*, *Clostridia UCG-014*, and *Eubacterium nodatum* from the phylum Firmicutes, as well as *Bifidobacterium* from the phylum Actinobacteria. Simultaneously, baicalein significantly reduced the absolute and / or relative abundance of harmful bacteria in enteritis, including Bacteroides, Alistipes, and Parabacteroides bacteria (Bacteroidetes); Parasutterella and Escherichia Shigella bacteria (Proteobacteria); and Helicobacter spp. (Campilobacterota), significantly improving inflammation and repairing the intestinal barrier in mice with enteritis. Baicalein, by increasing the abundance of beneficial gut bacteria and reducing the abundance of harmful bacteria, targets and regulates the gut microbiota, providing new ideas and directions for future drug development that targets and regulates the gut microbiota.

[0006] This invention provides the application of baicalein in the preparation of pharmaceuticals, food products, or health products that promote bacterial growth and / or proliferation, wherein the bacteria are selected from one or both of Firmicutes and Actinobacteriota bacteria.

[0007] In one embodiment, the Firmicutes bacteria are selected from one, two, three, four, or five species of the genera *Dubosiella*, *Romboutsia*, *Turicibacter*, *Clostridia* UCG-014, and *Eubacterium nodatum*; preferably, they are selected from the genus *Dubosiella*.

[0008] In one embodiment, the Actinobacteriota bacteria are selected from the Bifidobacterium genus; preferably, the Bifidobacterium genus bacteria are selected from one or both of Bifidobacterium pseudolongum and Bifidobacterium animalis; more preferably, they are selected from Bifidobacterium pseudolongum.

[0009] In one embodiment, the bacteria are selected from bacteria of the phylum Actinobacteriota.

[0010] In one embodiment, the bacteria are bacteria from the gut microbiota.

[0011] In one embodiment, the intestine is the human intestine or the mouse intestine.

[0012] In one embodiment, the drug, food, or health product may also include pharmaceutical excipients.

[0013] In one embodiment, the sole active ingredient of the drug, food, or health product is baicalein.

[0014] The single dose of the baicaleol can be administered according to the subject's weight, and the non-limiting example range can be 0.05 mg / kg to 50 mg / kg, for example 1 mg / kg to 10 mg / kg, for example 4 mg / kg.

[0015] In one embodiment, the drug, food, or health product is administered orally.

[0016] The dosage form of the drug, food, or health product is a conventional dosage form for oral administration in the art, such as oral liquid, tablets, or granules, for example, oral liquid.

[0017] In one embodiment, the mass concentration of baicalein in the oral liquid is (0.1-10) mg / mL, for example (0.1-1) mg / mL, or even 0.4 mg / mL.

[0018] The frequency of application of the medicine, food, or health product is the conventional frequency of application in the art, such as once a day, twice a day, or three times a day; for example, once a day.

[0019] In one embodiment, the drug, food, or health product is used to treat and / or prevent diseases associated with a decrease in the number of one or both of the Firmicutes and Actinobacteriota bacteria, such as colitis.

[0020] In one embodiment, the colitis is colitis associated with decreased expression levels of the Ocln or Cdh1 gene.

[0021] This invention provides the application of baicalein in the preparation of drugs, food products or health products that inhibit the growth and / or proliferation of bacteria; wherein the bacteria are selected from one, two or three of the phyla Bacteroidetes, phyla Proteobacteria and phyla Campilobacterota.

[0022] In one embodiment, the Bacteroidetes bacteria are selected from one, two, or three of the genera Bacteroides, Alistipes, and Parabacteroides; preferably, they are selected from one or two of the genera Bacteroides and Parabacteroides.

[0023] In one embodiment, the Bacteroides bacteria are selected from one or both of Bacteroides stercorirosoris and Bacteroides sartorii.

[0024] In one embodiment, the Parabacteroides bacteria are selected from Parabacteroides goldsteinii.

[0025] In one embodiment, the Proteobacteria are selected from one or both of the genera Parasutterella and Escherichia Shigella; preferably, they are selected from the genus Parasutterella.

[0026] In one embodiment, the Parasutterella bacteria are selected from Burkholderiales bacterium.

[0027] In one embodiment, the Campilobacterota bacteria are selected from the Helicobacter genus.

[0028] In one embodiment, the bacteria are selected from one or both of the phyla Bacteroidetes and phyla Proteobacteria.

[0029] In one embodiment, the bacteria are bacteria from the gut microbiota.

[0030] In one embodiment, the intestine is the human intestine or the mouse intestine.

[0031] In one embodiment, the drug, food, or health product may also include pharmaceutical excipients.

[0032] In one embodiment, the sole active ingredient of the drug, food, or health product is baicalein.

[0033] The single dose of the baicaleol can be administered according to the subject's weight, and the non-limiting example range can be 0.05 mg / kg to 50 mg / kg, for example 1 mg / kg to 10 mg / kg, for example 4 mg / kg.

[0034] In one embodiment, the drug, food, or health product is administered orally.

[0035] The dosage form of the drug, food, or health product is a conventional dosage form for oral administration in the art, such as oral liquid, tablets, or granules, for example, oral liquid.

[0036] In one embodiment, the drug, food, or health product is an oral liquid. Preferably, the mass concentration of baicalein in the oral liquid is (0.1-10) mg / mL, for example (0.1-1) mg / mL, or even 0.4 mg / mL.

[0037] The frequency of application of the medicine, food, or health product is the conventional frequency of application in the art, such as once a day, twice a day, or three times a day; for example, once a day.

[0038] In one embodiment, the drug, food, or health product is used to treat and / or prevent diseases associated with a decrease in the number of one or both of Firmicutes and Actinobacteriota bacteria, such as colitis.

[0039] In one embodiment, the colitis is colitis associated with decreased expression levels of the Ocln or Cdh1 gene.

[0040] This invention provides the application of baicalein in the preparation of drugs, food products or health products for the prevention and / or treatment of intestinal flora imbalance.

[0041] In one embodiment, the gut microbiota comprises the following bacteria: Firmicutes, Actinobacteriota, Bacteroidetes, Proteobacteria, and Campilobacterota.

[0042] In one embodiment, the drug, food, or health product is used to increase the absolute and / or relative abundance of one or both of the Firmicutes and Actinobacteriota bacteria in the gut microbiota.

[0043] In one embodiment, the drug, food, or health product is used to reduce the absolute and / or relative abundance of one, two, or three of the bacteria selected from the Bacteroidetes, Proteobacteria, and Campilobacterota phyla in the gut microbiota.

[0044] In one embodiment, the drug, food, or health product is used to increase the absolute and / or relative abundance of one or two of the Firmicutes and Actinobacteriota bacteria in the gut microbiota, and to decrease the absolute and / or relative abundance of one, two, or three of the Bacteroidetes, Proteobacteria, and Campilobacterota bacteria in the gut microbiota.

[0045] In one embodiment, the drug, food, or health product is used to prevent and / or treat diseases related to gut microbiota dysbiosis.

[0046] In one implementation, the disease associated with intestinal flora imbalance is colitis.

[0047] In one embodiment, the intestine is the human intestine or the mouse intestine.

[0048] In one embodiment, the drug, food, or health product may also include pharmaceutical excipients.

[0049] In one embodiment, the sole active ingredient of the drug, food, or health product is baicalein.

[0050] The single dose of the baicaleol can be administered according to the subject's weight, and the non-limiting example range can be 0.05 mg / kg to 50 mg / kg, for example 1 mg / kg to 10 mg / kg, for example 4 mg / kg.

[0051] In one embodiment, the drug, food, or health product is administered orally.

[0052] The dosage form of the drug, food, or health product is a conventional dosage form for oral administration in the art, such as oral liquid, tablets, or granules, for example, oral liquid.

[0053] In one embodiment, the drug, food, or health product is an oral liquid. Preferably, the mass concentration of baicalein in the oral liquid is (0.1-10) mg / mL, for example (0.1-1) mg / mL, or even 0.4 mg / mL.

[0054] The frequency of application of the medicine, food, or health product is the conventional frequency of application in the art, such as once a day, twice a day, or three times a day; for example, once a day.

[0055] In one embodiment, the drug, food, or health product increases the relative abundance of Bifidobacterium pseudolongum in the gut microbiota by more than 207%.

[0056] In one embodiment, the drug, food, or health product increases the relative abundance of Bifidobacterium animalis in the gut microbiota by more than 486%.

[0057] In one embodiment, the drug, food, or health product reduces the relative abundance of Bacteroides stercorirosoris in the gut microbiota by more than 67%.

[0058] In one embodiment, the drug, food, or health product reduces the relative abundance of the Bacteroides sartorii bacteria in the gut microbiota by more than 65%.

[0059] In one embodiment, the drug, food, or health product reduces the relative abundance of Parabacteroides goldsteinii in the gut microbiota by more than 53%.

[0060] In one embodiment, the drug, food, or health product reduces the relative abundance of Burkholderiales bacterium in the gut microbiota by more than 79%.

[0061] In this invention, the percentage increase or decrease in relative abundance is obtained by dividing the difference in relative abundance of bacteria before and after application of the drug, food, or health product by the ratio of the relative abundance value of bacteria before application of the drug, food, or health product, multiplied by 100%.

[0062] In this invention, relative abundance of bacteria = absolute abundance of a certain bacterium in the intestinal flora sample / sum of absolute abundance of all bacteria detected in the sample.

[0063] In this invention, the absolute abundance of bacteria is defined as the number of sequencing sequences of the bacteria. The number of bacteria in a sample is directly proportional to the number of sequencing sequences of the bacteria.

[0064] This invention provides the use of baicalein in the preparation of medicaments for the prevention and / or treatment of diseases related to intestinal flora imbalance.

[0065] In one implementation, the disease associated with intestinal flora imbalance is colitis.

[0066] In one embodiment, the drug further includes pharmaceutical excipients.

[0067] In one embodiment, the sole active ingredient of the drug is baicalein.

[0068] The single dose of the baicaleol can be administered according to the subject's weight, and the non-limiting example range can be 0.05 mg / kg to 50 mg / kg, for example 1 mg / kg to 10 mg / kg, for example 4 mg / kg.

[0069] In one implementation scheme, the drug is administered orally.

[0070] The dosage form of the drug is a conventional dosage form for oral administration in the art, such as oral liquid, tablet or granule, or, for example, oral liquid.

[0071] In one embodiment, the drug is an oral liquid, preferably, the mass concentration of baicalein in the oral liquid is (0.1-10) mg / mL, for example (0.1-1) mg / mL, or even 0.4 mg / mL.

[0072] The frequency of administration of the drug is the conventional frequency of administration in the art, such as once a day, twice a day, or three times a day; for example, once a day.

[0073] In one embodiment, the drug is used to treat and / or prevent diseases associated with a decrease in the number of one or both of Firmicutes and Actinobacteriota bacteria, such as colitis.

[0074] In one embodiment, the colitis is colitis associated with decreased expression levels of the Ocln or Cdh1 gene.

[0075] This invention provides the use of baicalein in the preparation of drugs for the prevention and / or treatment of diseases associated with reduced expression levels of the Ocln or Cdh1 genes.

[0076] In one implementation, the disease associated with decreased expression levels of the Ocln or Cdh1 gene is colitis.

[0077] In one embodiment, the drug further includes pharmaceutical excipients.

[0078] In one embodiment, the sole active ingredient of the drug is baicalein.

[0079] The single dose of the baicaleol can be administered according to the subject's weight, and the non-limiting example range can be 0.05 mg / kg to 50 mg / kg, for example 1 mg / kg to 10 mg / kg, for example 4 mg / kg.

[0080] In one implementation scheme, the drug is administered orally.

[0081] The dosage form of the drug is a conventional dosage form for oral administration in the art, such as oral liquid, tablet or granule, or, for example, oral liquid.

[0082] In one embodiment, the drug is an oral liquid, preferably, the mass concentration of baicalein in the oral liquid is (0.1-10) mg / mL, for example (0.1-1) mg / mL, or even 0.4 mg / mL.

[0083] The frequency of administration of the drug is the conventional frequency of administration in the art, such as once a day, twice a day, or three times a day; for example, once a day.

[0084] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0085] The reagents and raw materials used in this invention are all commercially available.

[0086] The positive and progressive effects of this invention are as follows:

[0087] This invention is the first to discover that baicalein can significantly affect the composition of the gut microbiota. Specifically, baicalein can significantly increase the relative abundance of beneficial bacteria, including Dubosiella, Romboutsia, Turicibacter, Clostridia UCG-014, and Eubacterium nodatum groups from the Firmicutes phylum, as well as Bifidobacterium from the Actinobacteria phylum. Simultaneously, baicalein significantly reduced the relative abundance of harmful bacteria in enteritis, including Bacteroides, Alistipes, and Parabacteroides bacteria (Bacteroidetes); Parasutterella and Escherichia Shigella bacteria (Proteobacteria); and Helicobacter bacteria (Campilobacterota), significantly improving inflammation and repairing the intestinal barrier in mice with enteritis. Baicalein's targeted regulation of the gut microbiota by increasing the relative abundance of beneficial bacteria and decreasing the relative abundance of harmful bacteria not only further confirms its important role in the treatment of enteritis but also provides new ideas and directions for future drug development. Attached Figure Description

[0088] Figure 1 This is a graph showing the trend of body weight changes in mice in the control group (Control), model group (DSS+Veh), and baicalein treatment group (DSS+PA).

[0089] Figure 2This is a graph showing the trend of fecal looseness in mice in the normal group, model group, and baicalein treatment group.

[0090] Figure 3 The graph shows the trend of fecal occult blood scores in mice in the normal group, model group, and baicalein treatment group.

[0091] Figure 4 This is a graph showing the trend of disease activity index changes in mice in the normal group, model group, and baicalein treatment group.

[0092] Figure 5 The graph shows the colon length test results of mice in the normal group, model group, and baicalein treatment group.

[0093] Figure 6 The graph shows the spleen weight coefficient test results of mice in the normal group, model group, and baicalein treatment group.

[0094] Figure 7 The graph shows the colon pathology scores of mice in the normal group, model group, and baicalein treatment group.

[0095] Figure 8 Microscopic images of the colon structure of mice in the normal group, model group, and baicalein treatment group.

[0096] Figure 9 The graph shows the results of FABP2 content detection in the serum of mice in the normal group, model group, and baicalein treatment group.

[0097] Figure 10 The image shows the results of Tjp-1 gene level detection in the colon tissues of mice in the normal group, model group, and baicalein treatment group.

[0098] Figure 11 The image shows the results of Ocln gene level detection in the colon tissues of mice in the normal group, model group, and baicalein treatment group.

[0099] Figure 12 The image shows the results of Cdh1 gene level detection in the colon tissues of mice in the normal group, model group, and baicalein treatment group.

[0100] Figure 13 The images show the colonic mucus barrier function analysis of mice in the normal group, model group, and baicalein treatment group; ZO-1 / DAPI is the result of ZO-1 protein immunofluorescence labeling; Zoom is a magnified image of the protein immunofluorescence labeling result; and PAS is the result of periodic acid Schiff (PAS) staining of mouse colon.

[0101] Figure 14 This is a graph showing the PCA analysis results based on OUT abundance.

[0102] Figure 15The relative abundance values ​​of Firmicutes in the intestines of mice in the normal group, model group, and baicalein treatment group are given.

[0103] Figure 16 The relative abundance values ​​of Bacteroidota in the intestines of mice in the normal group, model group, and baicalein treatment group are given.

[0104] Figure 17 The relative abundance ratio of Firmicutes to Bacteroidetes in the intestines of mice in the normal group, model group, and baicalein treatment group.

[0105] Figure 18 The relative abundance values ​​of Actinobacteria in the intestines of mice in the normal group, model group, and baicalein treatment group are given.

[0106] Figure 19 The relative abundance values ​​of Proteobacteria in the intestines of mice in the normal group, model group, and baicalein treatment group are given.

[0107] Figure 20 The relative abundance values ​​of Campilobacterota in the intestines of mice in the normal group, model group, and baicalein treatment group are given.

[0108] Figure 21 This is a graph showing the results of the LDA Effect Size analysis.

[0109] Figure 22 Correlation analysis of gut microbiota and enteritis indicators significantly regulated by baicalein. Detailed Implementation

[0110] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0111] Instruments and Materials

[0112] VersaMax microplate reader (Molecular Devices, USA); 5810 high-speed centrifuge (ThermoFisher, USA); Sartorius BSA223S electronic analytical balance (Sartorius, Germany); Eppendorf thermostat (Germany); Bio-Red CFX384 Touch real-time PCR instrument (USA); Zeiss LSM 900 laser scanning confocal microscope (CLSM, Germany).

[0113] Sodium dextran sulfate (DSS, molecular weight 36-50kDa, MP Biochemicals), baicalein (98% purity, Chengdu Desite Biotechnology Co., Ltd.), occult blood test kit (C027-1-1, Nanjing Jiancheng Bioengineering Institute), TNF-α (Invitrogen, Thermo Fisher Scientific), IL-1β (Invitrogen, Thermo Fisher Scientific), IL-6 ELISA kit (Invitrogen, Thermo Fisher Scientific), intestinal fatty acid-binding protein 2 (FABP2) detection kit (SEA559Mu, Wuhan Yunclone Technology Co., Ltd.), BCA protein quantification / concentration assay kit (MA0082-3, Dalian Meilun Biotechnology Co., Ltd.); primers (Sangon Biotech (Shanghai) Co., Ltd.), primer sequence: Tjp-1: F:5'-GCCGCTAAGAGCACAGCAA-3 (SEQ ID NO: 1) and R: 5'-GCCCTCCTTTTAACACATCAGA-3' (SEQ ID NO: 2), Ocln: F: 5'-TTGAAAGTCCACCTCCTTACAGA-3' (SEQ ID NO: 3) and R: 5'-CCGGATAAAAAGAGTACGCTGG-3' (SEQ ID NO: 4), Cdh1: F: 5'-CAGGTCTCCTCATGGCTTTGC-3' (SEQ ID NO:5) and R:5'-CTTCCGAAAAGAAGGCTGTCC-3' (SEQ ID NO:6), Gapdh-F:5'-TTGATGGCAACAATCTCCAC-3' (SEQ ID NO:7) and R:5'-CGTCCCGTAGACAAAATGGT-3' (SEQ ID NO:8), HiScript III RT SuperMix for qPCR (#R323, Vazyme), antibody ZO-1 (61-7300, Invitrogen); Anti-Rabbit IgG (H+L) and F(ab')2 Fragment (#4412, Cell Signaling Technology); DAPI (C1006, Shanghai Beyotime Biotechnology Co., Ltd.).

[0114] Relative abundance of bacteria = absolute abundance of the bacteria in the gut microbiota sample / sum of absolute abundance of all bacteria detected in the sample.

[0115] Absolute abundance = the number of sequencing sequences of bacteria. The number of bacteria in a sample is directly proportional to the number of sequencing sequences of bacteria.

[0116] Effect of Pogostol on Alleviating Intestinal Inflammation and Intestinal Barrier Injury in Example 1

[0117] 1.1 Test method

[0118] (1) Animals and grouping: 8-week-old C57BL / 6J male mice (weight 18 - 22 g), purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd. with SCXK (Su) 2020 - 0009, and raised in the standard SPF barrier environment of Shanghai Institute of Materia Medica, Chinese Academy of Sciences, maintaining a constant temperature (22 °C), constant humidity (55%), 12-hour day-night alternation, and free access to food and water. After 1 week of adaptive feeding, 24 mice were evenly divided into a normal group (Control), a model group (DSS + Veh), and a pogostol treatment group (DSS + PA). The three groups of mice were fed the same standard diet and had free access to water. The experiment was approved by the Animal Experiment Ethics Committee of Shanghai Institute of Materia Medica, Chinese Academy of Sciences (IACUC: 2022 - 06 - XC - 60).

[0119] (2) Administration method: After 1 week of adaptive feeding, the mice in the DSS + PA group were given pogostol solution daily (dose: 4 mg / kg per day). Pogostol was dissolved in an aqueous solution of CMCNa with a mass concentration of 0.5% before use to make the mass concentration of pogostol 0.4 mg / mL; the normal group and the model group were given an equal dose of 0.5% CMCNa, and gavaged once a day for 8 consecutive days.

[0120] (3) Model establishment: Refer to the internationally recognized method of inducing chronic ulcerative colitis with DSS. During the 1st - 6th day of drug administration, the drinking water of the mice in the model group and the pogostol treatment group was changed to a 2% DSS aqueous solution. After continuous free drinking for 6 days, the drinking water was changed back to normal drinking water for 2 days to induce acute ulcerative colitis.

[0121] During the experiment, the body weight, fecal status, and occult blood index of the mice were recorded daily; after the experiment, the mice were dissected, blood was collected from the eyeballs, the spleen weight was measured, the colon length was measured, and the lower end of the colon was fixed with 4% formalin fixative to preserve the colon tissue samples.

[0122] (4) Determination of key indicators and experimental methods:

[0123] A. Histopathological analysis

[0124] Collect the colon tissue of the mice to prepare tissue sections, observe the hematoxylin-eosin (HE) stained sections under an optical microscope, and calculate the total histopathological score according to the sum of the scores of three indicators: the degree of epithelial barrier damage, the degree of mucosal damage, and the degree of inflammatory cell infiltration.

[0125] B. Determination of inflammatory factors in tissues

[0126] The levels of TNF-α, IL-1β, and IL-6 in colon tissue were quantified using a kit following the manufacturer's instructions. Absorbance values ​​were then read at 450 nm and 570 nm using a microplate reader. Extracted protein concentrations were quantified using a BCA protein quantification kit, and the levels of cellular inflammatory factors were calculated and expressed in pg / mg.

[0127] C. Intestinal wall permeability test

[0128] The serum FABP2 level was quantified using the kit according to the manufacturer's instructions. Finally, the absorbance was read at 450 nm using a microplate reader, and the level of cellular inflammatory factors was calculated and expressed in pg / mg.

[0129] D. Detection of gene expression of tight junction proteins and adhesion junction proteins in intestinal epithelium

[0130] Gene-specific primers were designed, using mouse Gapdh as an internal reference gene. The specific steps of real-time quantitative PCR were performed according to the manufacturer's instructions, and relative quantification was calculated using 2-ΔΔCt.

[0131] E. Analysis of colonic mucus barrier function

[0132] The effect of glycyrrhizic acid on the mucus barrier function of mouse colon was studied using periodic acid-Schiff (PAS) staining. First, paraffin sections of colonic tissue were dewaxed in xylene, rehydrated with ethanol, and then immersed in Alcian Blue (pH 2.5) for 10 min, followed by rinsing twice with distilled water. Staining was then performed with periodic acid reagent and Schiff's reagent for 5 min each, followed by rinsing twice with distilled water. After dehydration with ethanol, the sections were cleared in xylene, mounted, and examined under a microscope.

[0133] 1.2 Test Results:

[0134] (1) Baiqiu Lichun relieves the symptoms of DSS-induced colitis.

[0135] Weight loss, diarrhea, and bloody stools are clinical symptoms of colitis. The Disease Activity Index (DAI) is an important indicator for evaluating the severity of colitis models by comprehensively considering parameters such as weight loss, stool looseness, and bloody stools in mice. The higher the score, the more severe the colitis.

[0136] During the experiment, the mouse body weight, fecal condition, and occult blood index were measured as follows: Figure 1 , Figure 2 and Figure 3 As shown. Disease Activity Index (DAI) is as follows. Figure 4 As shown.

[0137] Depend on Figures 1-4It can be seen that during the DSS modeling period, mice experienced severe weight loss, bloody stools, and loose feces. Administration of baicalein improved the weight loss, loose stools, and bloody stools in mice, and significantly reduced the DAI score (P<0.001).

[0138] (2) Colon length

[0139] Colon length is another important reference indicator for assessing the severity of colitis, such as... Figure 5 As shown, compared with the normal group, the colon length of mice in the model group was significantly shortened (P<0.05). The colon length of mice in the baicalein-treated group was significantly increased (P<0.05), indicating that baicalein can significantly improve the DSS-induced shortening of colon length in mice.

[0140] (3) Spleen coefficient

[0141] The spleen is an important peripheral immune organ, producing specific antibodies and other active substances. Splenomegaly occurs due to the expansion of inflammatory cells. Figure 6 As shown, compared with the normal group, the spleen coefficient of mice in the model group was significantly increased (P<0.001); compared with the model group, the spleen coefficient of mice in the baicalein-treated group was significantly decreased (P<0.05), indicating that baicalein can alleviate the problem of splenomegaly caused by colitis.

[0142] (4) Histopathological analysis

[0143] like Figure 7 As shown, compared with the normal group, the histological damage index of mice in the model group was significantly increased (P<0.005); the histological damage index of mice in the baicalein-treated group was significantly decreased compared with the model group (P<0.05).

[0144] like Figure 8 As shown, under an optical microscope, the colonic structure of the normal mice was clear, with intact crypt structures and no glandular damage. In the model group mice, localized ulceration, necrosis and sloughing of the colonic mucosal epithelium, and inflammatory cell infiltration were observed, along with severe edema in the mucosa and submucosa. In the group treated with baicalein, colonic inflammatory cell infiltration was reduced, the structure of the intrinsic glands improved, and congestion and edema were alleviated.

[0145] The above results indicate that baicalein can effectively alleviate DSS-induced colitis in mice.

[0146] Figures 1-7 In the control group, #P<0.05, ##P<0.01, and ###P<0.001; compared with the DSS+Veh group, *P<0.05, **P<0.01, and ***P<0.001.

[0147] (5) Inflammation of the colon tissue

[0148] The results of the detection of TNF-α, IL-1β and IL-6 in colon tissue are shown in Table 1.

[0149] Table 1 shows the detection results of pro-inflammatory factors TNF-α, IL-1β, and IL-6 in the colon tissue of mice in the three experimental groups.

[0150]

[0151] Data are presented as mean ± SEM. Compared to the control group, ## P<0.01, ### P<0.001; compared with the DSS+Veh group,

[0152] * P<0.05, ** P<0.01, *** P<0.001 (Control group represents normal group, DSS+Veh group represents model group, DSS+PA group represents baiqiuli alcohol treatment group).

[0153] The above results indicate that DSS-induced colitis increases the expression of inflammatory factors in the intestinal environment, while baicalein treatment can significantly inhibit the expression of inflammatory factors TNF-α, IL-1β and IL-6, indicating that baicalein has anti-inflammatory capabilities.

[0154] (6) Intestinal barrier

[0155] The content of FABP2 in serum is as follows Figure 9 As shown, the results indicated that intestinal permeability in mice was reduced after intervention with baicalein (P<0.05). This suggests that baicalein promotes the repair of the intestinal epithelial barrier, thereby preventing further damage to the intestine by harmful substances and alleviating the occurrence of colitis.

[0156] Further investigation was conducted to examine the effect of baicalein on the intestinal barrier in mice, and the results were as follows: Figures 10-12 As shown, baicalein significantly increased the level of ZO-1 mRNA (i.e., Tjp-1 gene) in the mouse colon (P<0.0001), and showed a trend of increasing the expression of Ocln and Cdh1 genes.

[0157] Figures 9-12 In the control group, #P<0.05, ##P<0.01, and ###P<0.001; compared with the DSS+Veh group, *P<0.05, **P<0.01, and ***P<0.001.

[0158] Immunofluorescence labeling of ZO-1 protein yielded the following results: Figure 13As shown, the ZO-1 protein on the periphery of colonic epithelial cells in the normal group exhibited a continuous and tight ring structure, while in the model group, ZO-1 was almost completely destroyed, irregularly dispersed on the cell periphery, and showed dim fluorescence staining. Compared with the model group, the ZO-1 protein in the baicalein-treated group was only locally destroyed, and the degree of damage was significantly reduced compared with the model group. This indicates that baicalein enhances the expression of intestinal tight junction proteins and adhesion junction proteins, promotes the repair of the intestinal epithelial barrier, and thus prevents harmful substances from further damaging the intestinal barrier, alleviating the occurrence of colitis.

[0159] The physiological structure composed of a mucus layer secreted by intestinal epithelial cells plays a vital role in maintaining the intestinal barrier function and preventing intestinal bacteria and other pathogenic antigens from entering the bloodstream and other tissues and organs. For example... Figure 13 As shown, DSS treatment significantly reduced the number of intestinal mucins; while baicalein significantly increased intestinal mucins and restored the mucosal barrier.

[0160] Example 2: Prunol improves DSS-induced gut microbiota dysbiosis in mice.

[0161] 2.1 16S rRNA sequencing analysis

[0162] After mouse dissection, the cecum was removed, and its contents were collected for 16S rRNA sequencing. Microbiome DNA was extracted using a fecal DNA isolation kit (Qiagen, Düsseldorf, Germany). The V3+V4 regions of intestinal bacterial 16S rDNA were amplified using primers 341F (5'-CCTAYGGGRBGCASCAG-3' (SEQ ID NO:9)) and 806R (5'-GGACTACNNGGGTATCTAAT-3' (SEQ ID NO:10)). The amplified products were separated using a 2.0% agarose gel and purified using a QIAquick PCR purification kit (Qiagen, Valencia, USA). Bacterial DNA was sequenced on an Illumina HiSeq PE 150 platform (Beijing Novogene Co., Ltd.), and bioinformatics analysis was performed using the Lianchuan BioCloud Platform (https: / / www.omicstudio.cn / tool).

[0163] 2.2 Test Results:

[0164] (1) Principal component analysis (PCA) based on OUT abundance

[0165] Principal component analysis (PCA) was performed on the gut microbiota of mice under different treatments, and the results are as follows: Figure 14 As shown, by Figure 14The analysis revealed that the model group and the normal group clustered separately, indicating that enteritis caused changes in the composition of the gut microbiota. Compared with the model group, the gut microbiota composition of the baicalein treatment group was closer to that of the normal group, suggesting that baicalein can improve the overall gut microbiota structure in DSS-induced enteritis mice.

[0166] (2) Differences in gut microbiota composition

[0167] Firmicutes and Bacteroides are the two dominant phyla in the gut microbiota, accounting for approximately 90% of the total flora. Changes in their relative abundance and ratios suggest certain differences in the gut microbiota composition. The abundance of different phyla in the gut microbiota was analyzed (relative abundance greater than 0.1%), and the results are shown below. Figures 15-20 See Tables 2-1 and 2-2.

[0168] Table 2-1 Relative abundance of gut microbiota in experimental group mice

[0169]

[0170] Data are expressed as mean ± SEM. Compared with the control group, #P<0.05, ##P<0.01, ###P<0.001; compared with the DSS+Veh group, *P<0.05, **P<0.01 and ***P<0.001 (DSS+Veh represents the model group, DSS+PA represents the baiqiuli alcohol treatment group).

[0171] Table 2-2 Absolute abundance of gut microbiota in experimental group mice

[0172]

[0173] Data are expressed as mean ± SEM. Compared with the control group, #P<0.05, ##P<0.01, and ###P<0.001; compared with the DSS+Veh group, *P<0.05, **P<0.01, and ***P<0.001 (Control represents the normal group, DSS+Veh represents the model group, and DSS+PA represents the baiqiuli alcohol treatment group).

[0174] Depend on Figure 15 As can be seen from Table 2-1 above, compared with the model group, mice treated with DSS showed a decrease in the relative abundance of Firmicutes. Figure 16 It can be seen that the Bacteroidetes phylum in mice treated with DSS showed an upward trend, from Figure 17It can be seen that the Firmicutes / Bacteroidetes ratio was significantly reduced in DSS-treated mice. Conversely, the baicalein treatment group significantly upregulated the abundance of Firmicutes and significantly downregulated the abundance of Bacteroidetes, while increasing the Firmicutes / Bacteroidetes ratio.

[0175] In addition, by Figure 18 , Figure 19 and Figure 20 It can be seen that the treatment with baicalein significantly increased the number of Actinobacteriota bacteria in the intestine, and showed a trend of decreasing the number of Proteobacteria and Campilobacterota bacteria.

[0176] LEfSe (Linear Discriminant Analysis Effect Size), also known as LDAE effect size analysis, can compare different groups to identify biomarkers with significant differences in abundance between groups.

[0177] LDA analysis results are as follows Figure 21 As shown in the figure and Tables 3-1 and 3-2.

[0178] Table 3-1 Relative abundance of gut microbiota in experimental group mice

[0179]

[0180] Data are expressed as mean ± SEM. Compared with the control group, #P<0.05, ##P<0.01, ###P<0.001; compared with the DSS+Veh group, *P<0.05, **P<0.01 and ***P<0.001 (DSS+Veh represents the model group, DSS+PA represents the baiqiuli alcohol treatment group).

[0181] Table 3-2 Absolute abundance of gut microbiota in experimental group mice

[0182]

[0183]

[0184] Data are expressed as mean ± SEM. Compared with the Control group, #P<0.05, ##P<0.01, and ###P<0.001; compared with the DSS+Veh group, *P<0.05, **P<0.01, and ***P<0.001 (Control represents the normal group, DSS+Veh represents the model group, and DSS+PA represents the baiqiuli alcohol treatment group).

[0185] The above results indicate that Dubosiella, Romboutsia, and Turicibacter from the Firmicutes phylum, as well as Bifidobacterium from the Actinobacteria phylum, were significantly enriched in the baiqiuli alcohol treatment group. These bacterial groups are considered to be unique to the baiqiuli alcohol treatment group.

[0186] In contrast, the genera *Bacteroides* (Bacteroidetes) and *Helicobacter* and *Parasutterella* (Proteobacteria) were significantly enriched in the enteritis group, indicating that these three genera are unique bacterial groups in mice with enteritis.

[0187] Further analysis of the regulatory effects of baicalein on specific bacterial species was conducted, and the results are shown in Tables 4-1 and 4-2 (relative abundance greater than 0.1%). Correspondingly, the results showed that baicalein significantly increased the levels of two beneficial bacteria, *Bifidobacterium pseudolongum* and *Bifidobacterium animalis*; and significantly decreased the levels of harmful bacteria, *Escherichia coli* (*Bacteroides sartorii*) and *Bacteroides stercorirosoris*, as well as *Burkholderiales bacterium*, a species belonging to the genus *Parasutterella*.

[0188] Table 4-1 Relative abundance of gut microbiota in experimental group mice

[0189]

[0190] Data are expressed as mean ± SEM. Compared with the control group, #P<0.05, ##P<0.01, ###P<0.001; compared with the DSS+Veh group, *P<0.05, **P<0.01 and ***P<0.001 (DSS+Veh represents the model group, DSS+PA represents the baiqiuli alcohol treatment group).

[0191] Table 4-2 Absolute abundance of gut microbiota in experimental group mice

[0192]

[0193]

[0194] Data are expressed as mean ± SEM. Compared with the control group, #P<0.05, ##P<0.01, ###P<0.001; compared with the DSS+Veh group, *P<0.05, **P<0.01 and ***P<0.001 (DSS+Veh represents the model group, DSS+PA represents the baiqiuli alcohol treatment group).

[0195] (3) Correlation analysis of the gut microbiota significantly regulated by baicalein and enteritis indicators

[0196] The data in the table showing the relative abundance of gut bacteria in the experimental group mice indicate that the abundance of probiotics in the baicalein-treated group was significantly higher than that in the model group.

[0197] Specifically, the relative abundance of probiotics such as *Dubosiella*, *Bifidobacterium*, *Romboutsia*, and *Turicibacter* was significantly increased in the baicalein-treated group. For example... Figure 22 As shown, *P<0.05, **P<0.01, ***P<0.001, where P values ​​represent the significance of the correlation results. These probiotics were negatively correlated with enteritis indicators such as stool softness, DAI, colonic histology score, TNF-α, and IL-6, indicating that baicalein can improve enteritis-related indicators by increasing these beneficial bacteria. Conversely, the abundance of harmful bacteria in the baicalein-treated group was significantly lower than that in the model group. Specifically, the relative abundance of harmful bacteria such as *Bacteroides*, *Helicobacter*, and *Parasutterella* was significantly reduced in the baicalein-treated group. These harmful bacteria were significantly positively correlated with enteritis indicators such as DAI and TNF-α, indicating that baicalein can improve enteritis indicators by inhibiting harmful bacteria.

[0198] In summary, this study found that baicalein has a significant effect on improving DSS-induced gut microbiota dysbiosis in mice. Specifically, baicalein significantly increased the abundance of three beneficial genera of Firmicutes (Dubosiella, Romboutsia, and Turicibacter) and two beneficial species of Bifidobacterium (Bifidobacterium pseudolongum and Bifidobacterium animalis) in the gut of patients. On the other hand, it significantly decreased the abundance of two harmful bacteria (Bacteroides sartorii and Bacteroides stercorirosoris) and one species of Burkholderiales bacterium in the Bacteroides genus. This indicates that baicalein has the ability to regulate gut microbiota balance. Therefore, baicalein, as a natural product that targets and regulates gut microbiota, has broad application prospects in the development of drugs for the treatment of enteritis.

Claims

1. The use of a baicalein in the preparation of a drug, food or health product that promotes bacterial growth and / or proliferation, wherein the bacteria are selected from one or both of Firmicutes and Actinobacteriota bacteria.

2. The application as described in claim 1, characterized in that, It meets one or more of the following conditions: (1) The Firmicutes bacteria are selected from one, two, three, four or five of the following: Dubosiella, Romboutsia, Turicibacter, Clostridia UCG-014 and Eubacterium nodatum group. (2) The Actinobacteriota bacteria are selected from the Bifidobacterium genus; (3) The bacteria are selected from the phylum Actinobacteriota. (4) The bacteria are bacteria in the gut microbiota; for example, the gut microbiota is the human gut or mouse gut; (5) The drugs, food and beverages or health products mentioned above also include pharmaceutical excipients; (6) The sole active ingredient of the drug, food, or health product is bacitrullol; (7) The single dose of the baicaleol is 0.05 mg / kg to 50 mg / kg; for example, 1 mg / kg to 10 mg / kg, or even 4 mg / kg; (8) The route of administration for the drug, food, or health product is oral administration; (9) The dosage form of the drug, food or health product is an oral liquid, tablet or granule; for example, an oral liquid; the mass concentration of baicalein in the oral liquid can be (0.1-10) mg / mL; for example (0.1-1) mg / mL, for example 0.4 mg / mL; (10) The frequency of application of the medicine, food, or health product is once a day, twice a day, or three times a day; for example, once a day. (11) The medicine, food or health product is used to treat and / or prevent diseases associated with a decrease in the number of one or both of Firmicutes and Actinobacteriota bacteria, such as colitis, for example, colitis associated with a decrease in the expression level of the Ocln or Cdh1 gene.

3. The application as described in claim 2, characterized in that, It meets one or two of the following conditions: (1) The Firmicutes bacteria mentioned are selected from Dubosiella bacteria; (2) The Bifidobacterium bacteria mentioned are selected from one or both of Bifidobacterium pseudolongum and Bifidobacterium animalis.

4. The use of a baicalein in the preparation of a drug, food product or health product that inhibits the growth and / or proliferation of bacteria; wherein the bacteria are selected from one, two or three of the phyla Bacteroideta, Proteobacteria and Campilobacterota.

5. The application as described in claim 4, characterized in that, It meets one or more of the following conditions: (1) The Bacteroidetes bacteria are selected from one, two or three of the Bacteroides, Alistipes and Parabacteroides genera; preferably, they are selected from one or two of the Bacteroides and Parabacteroides genera. (2) The Proteobacteria are selected from one or two of the genus Parasutterella and the genus Escherichia Shigella; preferably, they are selected from the genus Parasutterella. (3) The Campilobacterota bacteria are selected from the Helicobacter genus; (4) The bacteria are bacteria in the gut microbiota; for example, the gut microbiota is the human gut or mouse gut; (5) The drugs, food and beverages or health products mentioned above also include pharmaceutical excipients; (6) The sole active ingredient of the drug, food, or health product is bacitrullol; (7) The single dose of the baicaleol is 0.05 mg / kg to 50 mg / kg; for example, 1 mg / kg to 10 mg / kg, or even 4 mg / kg; (8) The route of administration for the drug, food, or health product is oral administration; (9) The dosage form of the drug, food or health product is an oral liquid, tablet or granule; for example, an oral liquid; the mass concentration of baicalein in the oral liquid can be (0.1-10) mg / mL; for example (0.1-1) mg / mL, for example 0.4 mg / mL; (10) The frequency of application of the medicine, food, or health product is once a day, twice a day, or three times a day; for example, once a day. (11) The medicine, food or health product is used to treat and / or prevent diseases associated with an increase in the number of one, two or three of the Bacteroidetes, Proteobacteria and Campilobacterota bacteria, such as colitis, or colitis associated with a decrease in the expression level of the Ocln or Cdh1 gene.

6. The application as described in claim 5, characterized in that, It meets one or more of the following conditions: (1) The Bacteroides bacteria are selected from one or both of Bacteroides stercorirosoris and Bacteroides sartorii; (2) The Parabacteroides bacteria mentioned are selected from Parabacteroides goldsteinii; (3) The Parasutterella bacteria mentioned are selected from Burkholderialesbacterium.

7. The use of a baicalein in the preparation of drugs, food products or health products for the prevention and / or treatment of intestinal flora imbalance.

8. The application as described in claim 7, characterized in that, It meets one or two of the following conditions: (1) The intestinal flora comprises the following bacteria: Firmicutes bacteria as described in any one of claims 1-3, Actinobacteriota bacteria as described in any one of claims 1-3, Bacteroidetes bacteria as described in any one of claims 4-6, Proteobacteria bacteria as described in any one of claims 4-6, and Campilobacterota bacteria as described in any one of claims 4-6; (2) The drug, food or health product is used to increase the absolute abundance and / or relative abundance of one or two of the Firmicutes and Actinobacteriota bacteria in the intestinal flora; (3) The drug, food or health product is used to reduce the absolute abundance and / or relative abundance of one, two or three of the bacteria selected from the Bacteroidota, Proteobacteria and Campilobacterota phyla in the intestinal flora; (4) The medicine, food or health product is used for the prevention and / or treatment of diseases related to intestinal flora imbalance, such as colitis; (5) The drugs, food and beverages or health products mentioned above also include pharmaceutical excipients; (6) The sole active ingredient of the drug, food, or health product is bacitrullol; (7) The single dose of the baicaleol is 0.05 mg / kg to 50 mg / kg; for example, 1 mg / kg to 10 mg / kg, or even 4 mg / kg; (8) The route of administration for the drug, food, or health product is oral administration; (9) The dosage form of the drug, food or health product is an oral liquid, tablet or granule; for example, an oral liquid; the mass concentration of baicalein in the oral liquid can be (0.1-10) mg / mL; for example (0.1-1) mg / mL, for example 0.4 mg / mL; (10) The frequency of application of the medicine, food, or health product is once a day, twice a day, or three times a day; for example, once a day. Preferably, the drug, food, or health product is used to increase the absolute abundance and / or relative abundance of one or two of the Firmicutes and Actinobacteriota bacteria in the gut microbiota, and to decrease the absolute abundance and / or relative abundance of one, two, or three of the Bacteroidetes, Proteobacteria, and Campilobacterota bacteria in the gut microbiota; The absolute abundance is the number of sequencing sequences of bacteria, and the relative abundance is the absolute abundance of bacteria in the gut microbiota divided by the sum of the absolute abundance of all bacteria detected in the sample.

9. The use of baciolin in the preparation of a medicament for the prevention and / or treatment of diseases related to intestinal flora imbalance; preferably, the disease related to intestinal flora imbalance is colitis; more preferably, the sole active ingredient of the medicament is baciolin.

10. The use of baicalein in the preparation of a medicament for the prevention and / or treatment of diseases associated with decreased expression levels of the Ocln or Cdh1 gene; preferably, the disease associated with decreased expression levels of the Ocln or Cdh1 gene is colitis; more preferably, the sole active ingredient of the medicament is baicalein.

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