Application of combination of callicarpa nudiflora particles and lactobacillus reuteri in preparation of medicine for treating ulcerative colitis

Abstract

The invention provides application of combination of callicarpa nudiflora particles and lactobacillus reuteri in preparation of a medicine for treating ulcerative colitis, and belongs to the technical field of traditional Chinese medicines. The invention finds that the combined use of callicarpa nudiflora particles and lactobacillus reuteri has a synergistic effect on improvement of enteritis symptoms of mice, the disease activity index of the mice is reduced, the colon shortening degree is relieved, the score of pathological sections is reduced, and the bile acid metabolic disorder and immune cell imbalance tend to be normal, so that the treatment purpose is achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of traditional Chinese medicine, and relates to a medicine for treating ulcerative colitis, in particular to a S. nudiflora granule and the use of the S. nudiflora granule and Lactobacillus reuteri in the preparation of a medicine for treating ulcerative colitis. BACKGROUND

[0002] Ulcerative colitis (UC) is a chronic non-specific inflammatory disease of the colon and rectum mucosa and submucosa. It is generally believed in the academic circle that ulcerative colitis is caused by the combined action of genetic susceptibility, intestinal flora disorder, immune regulation abnormality and environmental factors. Ulcerative colitis is considered to be a form of inflammatory bowel disease, which is characterized by intestinal damage and clinical symptoms such as vomiting, diarrhea, and digestive system diseases. Patients with ulcerative colitis often show intestinal microecological imbalance, intestinal mucosal barrier damage, increased intestinal epithelial barrier permeability, and intestinal inflammatory reaction. At present, chemical induction of ulcerative colitis model is a relatively mature method and has been widely used in the field of anti-inflammatory drugs. The most common methods are trinitrobenzene sulfonic acid / ethanol combined induction and dextran sulfate sodium induction. The induction methods include enema administration and free drinking water method. The existing animal models are mainly based on rats and mice, and male mice are commonly used. According to the experimental needs, chronic or acute models can be constructed by controlling the dosage. Whether the ulcerative colitis model is successfully constructed is mainly determined by detecting some classic indicators. After the model is constructed, the animal's food intake, blood stool condition, stool formation degree, mental state, body weight change, and pathological detection results of the lesion site are usually used as indicators to determine whether the model is successfully constructed. Further detection of serum cytokine expression and colon tissue-related protein expression is also performed.

[0003] S. nudiflora Callicarpa nudiflora S. nudiflora granule is a single-ingredient medicine prepared from S. nudiflora and has been listed in the 2025 edition of Chinese Pharmacopoeia. It has the functions of relieving inflammation, detoxifying, astringing, and stopping bleeding, and has been selected in the 2023 edition of the Expert Consensus on Digestive Ulcer TCM Diagnosis and Treatment. Current research has found that the components isolated from S. nudiflora include flavonoids, terpenoids, essential oils, and polyphenols, etc. The polysaccharide and flavonoid extract of S. nudiflora can improve the intestinal flora imbalance of ulcerative colitis mice, promote the generation of short-chain fatty acids, and play a protective role on the colon mucosa.

[0004] The gut microbiota, tightly connected epithelial cells, and the mucus layer covering them form a crucial selective barrier in the gut. One of the significant factors influencing intestinal barrier dysfunction is gut microbiota dysbiosis. Therefore, strengthening the mucosal barrier is considered a key mechanism in probiotic therapy for intestinal diseases. For example, certain gut commensal bacteria can restore intestinal epithelial function by promoting mucus layer formation and secreting antimicrobial factors. Adding *Lactobacillus* species can improve barrier integrity in IL-10-deficient mice and inhibit the further development of colitis. *Lactobacillus plantarum* (…) L. plantarum ZLP001 enhances the intestinal barrier by strengthening epithelial defenses and regulating the gut microbiota. (Common Bacteroides) Bacteroides vulgatus The mucus-degrading enzymes produced by probiotics may affect the intestinal mucosal barrier function. Studies have shown that gut microbiota is related to the body's inflammatory response. Probiotics protect gut health by reducing intestinal inflammation to protect the intestinal epithelial mucosal barrier, directly inhibiting the proliferation of pathogenic bacteria, and regulating ganglion cell proliferation and apoptosis (Guan Jiaqi, et al. Research progress on the role of probiotics in promoting intestinal development [J]. Food Science, 2020).

[0005] Chinese patent CN111671827B discloses the application of *Callicarpa nudiflora* extract in the preparation of a drug for relieving ulcerative colitis. The *Callicarpa nudiflora* extract, by weight, comprises 20... 50 portions of total flavonoids from naked flower purple artichoke, 15 25 samples of total phenylethanol glycosides from Callicarpa nudiflora and 1 Composed of five parts of Callicarpa nudiflora polysaccharide, it can effectively treat ulcerative colitis, but the therapeutic effect is generally limited.

[0006] Chinese patent CN112646744B discloses the application of a strain of Lactobacillus reuteri in the prevention and relief of ulcerative colitis. The provided Lactobacillus reuteri CCFM1135 can tolerate the human gastrointestinal environment and can significantly reduce the disease activity index during the course of ulcerative colitis and improve colonic mucosal damage. However, the patent also discloses another strain of Lactobacillus reuteri FCQHC8L6, which does not have the effect of treating ulcerative colitis, indicating that not all Lactobacillus reuteri have the effect of treating or relieving ulcerative colitis.

[0007] Studies by Huang Meifang et al. have found that oral mesalazine enteric-coated tablets can treat ulcerative colitis. Oral mesalazine enteric-coated tablets combined with Saccharomyces boulardii powder can further enhance the therapeutic effect. On the basis of the combination of the first two, oral administration of Callicarpa nudiflora granules can further enhance the therapeutic effect. However, this study cannot prove that Saccharomyces boulardii combined with Callicarpa nudiflora granules has a therapeutic effect without oral mesalazine enteric-coated tablets (Huang Meifang, et al. Study on the effect and mechanism of Callicarpa nudiflora granules combined with Saccharomyces boulardii powder in the treatment of mild to moderate ulcerative colitis [J]. Modern Diagnosis and Treatment, 2024). SUMMARY

[0008] The present application is directed to the problem of poor therapeutic effect of ulcerative colitis in the prior art, and provides the use of Callicarpa nudiflora granules combined with Lactobacillus reuteri in the preparation of a drug for treating ulcerative colitis.

[0009] To achieve the above object, the technical scheme adopted by the present application is as follows: On the one hand, the present application provides the use of Callicarpa nudiflora granules combined with Lactobacillus reuteri in the preparation of a drug for treating ulcerative colitis.

[0010] Preferably, the Lactobacillus reuteri is L. reuteri DSM 17938.

[0011] Preferably, the mass of the Callicarpa nudiflora granules is 6-12 mg, and the bacterial amount of the Lactobacillus reuteri is 2x10 6 -2x10 8 CFU.

[0012] In some specific embodiments, the mass of the Callicarpa nudiflora granules is 8.32 mg, and the bacterial amount of the Lactobacillus reuteri is 2x10 7 CFU.

[0013] Preferably, the use includes preparing a drug for reducing disease activity index, preparing a drug for improving colon shortening, preparing a drug for improving bile acid metabolism disorder, or preparing a drug for improving immune cell imbalance.

[0014] On the other hand, the present application provides a pharmaceutical composition comprising Callicarpa nudiflora granules and the above-mentioned Lactobacillus reuteri.

[0015] Preferably, the pharmaceutical composition comprises 6-12 mg of Callicarpa nudiflora granules and 2x10 6 -2x10 8 CFU of Lactobacillus reuteri.

[0016] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0017] Preferably, the dosage form of the pharmaceutical composition includes tablets, pills, capsules, powders, and ointments.

[0018] Preferably, the dosage form of the pharmaceutical composition is a powder.

[0019] Compared with the prior art, the present application has the following beneficial effects: The naked flower purple pearl particle and lactobacillus reuteri have a synergistic effect on the improvement of intestinal inflammation symptoms of mice, which is reflected in the decrease of disease activity index of mice, the decrease of colon shortening degree, the decrease of pathological section score, and the normality of bile acid metabolism disorder and immune cell imbalance, thereby achieving the treatment purpose. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The figure is the influence of naked flower purple pearl particle on the composition of mouse cecum content in the example (horizontal level). NC is the normal group, and LHZZG is the naked flower purple pearl particle group.

[0021] Figure 2 The figure is the influence on the DAI score of UC mice in the example. Compared with the normal group: ### represents P<0.001; compared with the model group: * represents P<0.05, ** represents P<0.01, and *** represents P<0.001.

[0022] Figure 3 The figure is the influence on the colon length of UC mice in the example.

[0023] Figure 4 The figure is the influence on the colon pathological changes of UC mice in the example.

[0024] Figure 5 The figure is the flow cytometry of Th17 and Treg in the spleen of mice in the example. A is the flow cytometry of Th17, and B is the flow cytometry of Treg. DETAILED DESCRIPTION

[0025] Unless otherwise specified, the various raw materials and reagents in the present application are purchased from commercial suppliers, and experiments are carried out according to the operating instructions. Unless otherwise specified, the instruments, equipment, devices, etc. used in the present application are conventional instruments, equipment, devices, etc. and experiments are carried out according to the operating instructions and the supporting reagents.

[0026] In order to make the purpose, technical scheme and advantages of the present application more clear and obvious, the present application is further described in detail below in combination with examples. Unless otherwise specified in the examples, the experiments are carried out according to the conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, all reagents or instruments are conventional products that can be purchased on the market. In order to better illustrate the present application, numerous specific details are given in the specific embodiments below. The specific embodiments described herein are only used to explain the present application and do not constitute any limitation on the present application.

[0027] The "drug" described in the present application includes the aforementioned naked flower purple pearl particle or naked flower purple pearl particle and lactobacillus reuteri (hereinafter referred to as active ingredient) and acceptable adjuvant. In specific embodiments, the active ingredient described in the present application is provided in an effective amount (e.g. a therapeutically effective amount) in the drug.

[0028] The "pharmaceutically acceptable excipients" described in the present application include inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants and / or oils. Excipients such as cocoa butter, colorants, coating agents, sweeteners, flavoring agents and perfuming agents can also be present in the pharmaceutical compositions described.

[0029] The "medicaments" of the present application can be prepared according to known methods, such as those set forth in the general rules for preparation in the Japanese Pharmacopoeia (16th edition), the United States Pharmacopoeia and the European Pharmacopoeia (9th edition). Depending on the dosage form.

[0030] The active ingredients, pharmaceutically acceptable excipients in the "medicaments" described in the present application will vary depending on the identity, size and / or condition of the subject being treated and further depending on the route of administration of the composition. The medicaments can comprise between 0.1% and 100% (w / w) of the active ingredient.

[0031] The "treatment" described in the present application means, unless otherwise indicated, reversing, alleviating, lessening the symptoms of the condition or disease to which the term applies or one or more symptoms of such condition or disease, inhibiting the progress of the condition or disease or one or more symptoms thereof, or preventing the condition or disease or one or more symptoms thereof. The term "treatment" as used by the application refers to the therapeutic act, as "treatment" is defined immediately above.

[0032] The "effective amount" described in the present application means an amount sufficient to elicit the desired biological response. The effective amount of the active ingredients of the present application can vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration and the age and health of the subject.

[0033] In certain embodiments, the effective amount is a therapeutically effective amount. The effective amount is the amount of the first active ingredient described in the present application in a single dose. In certain embodiments, the effective amount is the combined amount of the active ingredients described in the present application in multiple doses.

[0034] The term "therapeuticly effective amount" as used in this invention refers to an amount sufficient to provide therapeutic benefit in the treatment of a condition or sufficient to delay or minimize one or more symptoms associated with that condition. For traditional Chinese medicine compositions or extracts, a therapeutically effective amount means an amount that provides therapeutic benefit in the treatment of a condition, alone or in combination with other therapies. The term "therapeuticly effective amount" may encompass amounts that improve overall therapy, reduce or avoid symptoms, signs, or causes of a condition, and / or enhance the therapeutic efficacy of another therapeutic agent. In some embodiments, a therapeutically effective amount is an amount sufficient to treat any of the diseases or conditions described.

[0035] As used herein, the terms "subject" or "patient" are well known in the art and are used interchangeably to refer to mammals, including dogs, cats, rats, mice, monkeys, cows, horses, goats, sheep, pigs, camels, and most preferably humans. This term does not imply a specific age or sex. Therefore, adult and neonatal subjects, whether male or female, are included.

[0036] Example 1: Application of Naked Flower Purple Calligraphy Granules in Combined Treatment of Ulcerative Colitis with Lactobacillus reuteri 1. Experimental materials 1.1 Laboratory Animals Fifty-two male BALB / c mice, weighing 20±2g, were purchased from Zhejiang Vital River Laboratory Animal Co., Ltd., production license number: SYXK (Shanghai) 2019-0027. They were provided with free access to food and water. The animal experiments were approved by the Ethics Committee of Shanghai Institute of Pharmaceutical Industry (approval number 2022ZY083).

[0037] 1.2 Experimental Materials The Naked Flower Purple Callicarpa Granules (batch number 211107) were provided by Jiangxi Puzheng Pharmaceutical Co., Ltd.

[0038] Lactobacillus reuteri (DSM 17938) was purchased from Shanghai Yibei Biotechnology Co., Ltd.

[0039] 1.3 Experimental Reagents Dextran sulfate sodium (DSS) was purchased from MP Company, USA. Neomycin sulfate (lot number J0622A), streptomycin sulfate (lot number J0611A), ampicillin sodium (lot number M0517C), and vancomycin hydrochloride (lot number M0512C) were purchased from Dalian Meilun Biochemical Co., Ltd., China. Cholic acid (CA), deoxycholic acid (DCA), and lithocholic acid (LCA) were purchased from Adamas. 3-oxo-lithocholic acid (3-oxo-LCA) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. The purity of all the above reference standards was not less than 98.0%. Mouse regulatory T cell staining kit and Th17 staining kit were purchased from Hangzhou Lianke Biotechnology Co., Ltd. TNF-α (lot number BY-EM220467), IL-1β (lot number BY-EM220174), and MUC-2 (lot number BY-EM228112) ELISA kits were purchased from Shanghai Boyan Biotechnology Co., Ltd. DNA extraction kit (DP302) was purchased from Beijing Tiangen Biotech Co., Ltd. MRS (batch number HB0384-1) culture medium was purchased from Shanghai Kangxing Biotechnology Co., Ltd. Water was ultrapure water; acetonitrile and formic acid were LCMS grade (Adamas); phosphate buffered saline (PBS, batch number G4202) and tissue fixative (batch number G1101) were purchased from Wuhan Saiweier Biotechnology Co., Ltd.; tissue preservation solution (batch number 5240101367) was purchased from Shanghai Meitianni Biotechnology Co., Ltd.; and fecal occult blood test reagent (colloidal gold method) was purchased from Shanghai Sangon Biotech Co., Ltd. All other reagents were of analytical grade.

[0040] 1.4 Main Instruments Exion LC™ High Performance Liquid Chromatograph (Shimadzu Corporation, Japan); Triple Quadrupole Mass Spectrometer (AB Q-TRAP 4500, ABsciex Corporation, USA); Varioskan Flash Full-Wavelength Multifunctional Microplate Reader (Thermo Fisher Scientific, USA); Electronic Analytical Balance (MS105, Mettler Toledo Instruments Ltd., Switzerland); Ultrapure Water System (Merck Chemical Technology Ltd.); HT165 High-Speed ​​Benchtop Centrifuge (Xiangtan Xiangyi Instrument Co., Ltd.); -80 ℃ Ultra-Low Temperature Freezer (Thermo Fisher Scientific, USA); Bactron Anaerobic Workstation (Shellab Corporation, USA); Fully Automated Rapid Sample Grinder (JXFSTPRP-32L, Shanghai Jingxin Industrial Co., Ltd.).

[0041] 2. Experimental Methods 2.1 Effects of Naked Flower Purple Beauty Granules on the Intestinal Microbiota of Mice Twelve male BABL / c mice were acclimatized for one week, weighed, grouped, numbered, and randomly divided into two groups: a normal control group and a group receiving *Callicarpa nudiflora* granules (416 mg / kg). The mice were administered the medication by gavage for seven days. After the last administration, the mice were euthanized, and the cecal contents were collected and sequenced by Beijing Novogene Biotechnology Co., Ltd. The V3 and V4 sequences of 16S rDNA in the mouse intestinal bacteria were determined using high-throughput methods.

[0042] Sequencing data undergoes splicing, quality control, and homology comparison to ensure more accurate and reliable results. OmicStudio tools are used to identify representative sequences for each OTU and annotate them for the species.

[0043] 2.2 Preparation of Lactobacillus reuteri oral gavage solution Lactobacillus reuteri was cultured anaerobically overnight at 37°C in MRS medium until it reached the logarithmic growth phase. The bacteria were collected, centrifuged at 3000g for 5 minutes, and resuspended in sterile physiological saline to a final experimental concentration of 1×10⁻⁶. 9 CFU / ml.

[0044] 2.3 Grouping and Modeling Forty male BABL / c mice were used and acclimatized for one week. After weighing and numbering, they were randomly divided into five groups of eight mice each: a normal group, a model group, a group receiving *Callicarpa nudiflora* granules (416 mg / kg; considering each mouse weighs approximately 20 g, the calculated dosage per mouse is approximately 8.32 mg), and a group receiving *Lactobacillus reuteri* (1.0 × 10⁻⁶ mg / kg). 9 CFU / kg, considering that each mouse weighs approximately 20g, the calculated bacterial load per mouse is approximately 2×10⁻⁶. 7 CFU), combined treatment group (416 mg / kg of Callicarpa nudiflora granules + 1.0 × 10⁻⁶ Lactobacillus reuteri granules), 9 CFU / kg). All mice were initially fed an antibiotic regimen for 5 days. The antibiotic regimen consisted of vancomycin hydrochloride 50 mg / kg, neomycin sulfate 100 mg / kg, ampicillin sodium 100 mg / kg, and streptomycin sulfate 100 mg / kg, administered daily by gavage to deplete the intestinal flora. Three days after recovery, mice were pre-treated with 1.0 × 10⁻⁶ CFU / kg antibiotics. 9Single-cell colonization of *Lactobacillus reuteri* was performed for 7 days using CFU / kg doses in both the control and combination groups. To ensure colonization, fecal samples were randomly collected from both uncolonized and colonized mice. RT-qPCR was used to quantify *Lactobacillus reuteri*. First, bacterial genomic DNA was extracted according to the bacterial genomic DNA extraction kit instructions, and purity was determined. Primers for the *Lactobacillus reuteri* genome were designed and synthesized by Shanghai Ruimian Biotechnology Co., Ltd. The forward primer was 5'-GTGCTTGCACCTGTGGACG-3' (SEQ ID NO.1); the reverse primer was 5'-CCATTGTGGCCGATCAGTCT-3' (SEQ ID NO.2). A 20 μL reaction volume was prepared according to the RT-qPCR kit instructions. The reaction program was: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 60℃ annealing for 20 s, 72℃ extension for 30 s, 40 cycles; final extension at 72℃ for 5 min.

[0045] After confirming successful colonization of Lactobacillus reuteri, except for the normal group, the other mice were allowed to drink purified water containing 3.5% DSS freely and were administered the drug by gavage daily. The normal group and the model group were given an equal volume of sterile water by gavage. The intervention was continued for 7 days.

[0046] 2.4 Disease activity index and colon length During the experiment, the general condition of the mice was observed daily, and changes in body weight and fecal characteristics were recorded. The Disease Activity Index (DAI) was calculated according to Table 1. The DAI assessment method was based on the study by Han et al. (Han F, Zhao X, Li X, et al. Bovine lactoferricin ameliorates intestinal inflammation and mucosal barrier lesions in colitis through NF-κB / NLRP3 signaling pathways[J]. Journal of Functional Foods, 2022). After the last administration, the mice were sacrificed and colon samples were collected. The distance from the anus to the ileocecal junction was carefully measured.

[0047] Table 1 Disease Activity Index Scoring Criteria

[0048] 2.5. Colonic pathological and histological studies One cm of colon tissue was taken from each sample and fixed in 10% tissue fixative for one week. The colon tissue samples were then rinsed with water and dehydrated by graded ethanol treatment. After paraffin embedding, sectioning, baking, dewaxing, and hydration, the sections were stained with hematoxylin, differentiated with 1% hydrochloric acid ethanol, and then stained with 0.5% eosin after running water blueing. The sections were then mounted with neutral resin and placed under an optical microscope for histological observation and histopathological scoring of the colon sections.

[0049] Colonic biopsy pathological scoring was performed according to the scoring criteria reported by Shang et al. (Shang L, Yu H, Liu H, et al. Recombinant antimicrobialpeptide microcin J25 alleviates DSS-induced colitis via regulating intestinal barrier function and modifying gut microbiota[J]. Biomedicine & Pharmacotherapy, 2021). Specific scoring criteria included inflammatory cell infiltration, depth of inflammatory infiltration, degree of crypt damage, and pathological extent. Regarding inflammatory cell infiltration: 0 = no inflammatory cell infiltration; 1 = mild inflammatory cell infiltration; 2 = moderate inflammatory cell infiltration; 3 = severe inflammatory cell infiltration; 4 = extremely severe inflammatory cell infiltration. Regarding the depth of inflammatory infiltration: 0 = no inflammatory infiltration; 1 = infiltration reaching the mucosal layer; 2 = infiltration reaching the mucosal layer and submucosa; 3 = complete infiltration. Regarding the degree of crypt damage: 0 = no crypt damage; 1 = 1 / 3 crypt damage; 2 = 2 / 3 crypt damage; 3 = complete crypt damage. Regarding the pathological extent: 0 = no pathological damage; 1 = 1%~25% pathological damage; 2 = 26%~50% pathological damage; 3 = 51%~75% pathological damage; 4 = 76%~100% pathological damage.

[0050] 2.6 Detection of intestinal inflammation and intestinal barrier-related factors The remaining colon tissue was rinsed thoroughly with pre-cooled PBS, and an appropriate weight was cut. PBS was added at a 1:9 ratio, and the tissue was thoroughly ground in a homogenizer at 60 Hz for 120 s. After centrifugation at 5000 r / min for 10 min, the supernatant was collected for detection. The ELISA procedure was strictly performed according to the kit instructions.

[0051] 2.7 Detection of bile acids in feces Each fecal sample was mixed with pre-cooled methanol at a 1:50 liquid ratio and then sonicated in ice water for 30 seconds (250 W, 40 kHz). It was then centrifuged at 14,000 rpm at 4 °C for 10 minutes. 10 μL of CA-d5 (1 μg / mL) was added to each 100 μL of supernatant as an internal standard. The bile acid content in the samples was then determined.

[0052] 2.8 Flow cytometry analysis Spleen single-cell suspensions were prepared using a 100 μm cell filter. Th17 staining included cell counting, activation with PMA / iononomycin (250×) and BFA / Monensin (250×), anti-mouse CD3ε, FITC, and anti-mouse CD4, PerCP-Cy5.5 staining for 15 min, and anti-mouse IL-17A incubation with PE for 15 min. Treg staining involved incubating cells with PE-CD25 and FITC-CD4 for 15 min. Cells were then blocked with anti-mouse CD16 / CD32, purified after permeabilization for 15 min, and subsequently stained with anti-mouse Foxp3 and PE for 15 min. Detection was performed using Cytoflex (Beckman Coulter, Brea, USA), and analysis was performed using CytExpert 2.4.

[0053] 2.9 Data Statistics and Analysis Statistical analysis was performed using GraphPad Prism 9.5.1 software (GraphPad Software, CA, USA). Results are expressed as mean ± standard deviation (x̄ ± s). T-tests were used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. A p-value less than 0.05 was considered statistically significant.

[0054] 3. Results 3.1 The content of Lactobacillus spp. in the feces of mice after administration of Naked Flower Purple Callicarpa granules increased. like Figure 1 As shown, the content of Lactobacillus in the feces of mice after intervention with Callicarpa nudiflora granules was significantly increased.

[0055] 3.2. Lactobacillus reuteri can colonize the mouse intestines via oral administration. Lactobacillus reuteri successfully colonized the mouse intestine after 7 days of oral administration. The relative abundance of Lactobacillus reuteri in the uncolonized and colonized groups were 2.12±0.38 and 5.68±0.42, respectively (n=6, P<0.001).

[0056] 3.3 Effects on disease activity index and colon length in mice The results are as follows Figure 2 As shown, the DAI score of the model group mice was significantly higher than that of the normal group (P<0.001). Mice exhibited weight loss, diarrhea, and bloody stools in the later stages of the experiment. Some improvement was observed in groups treated with either *Lactobacillus reuteri* or *Callicarpa nudiflora* granules alone (P<0.05), while the group treated with *Callicarpa nudiflora* granules after colonization with *Lactobacillus reuteri* showed the most significant improvement (P<0.001).

[0057] Similar results were observed in colon length, with the colon in the model group mice being significantly shorter than that in the normal group (P<0.001). Intervention with *Lactobacillus reuteri* or *Callicarpa nudiflora* granules alone had some allergic effect, but no significant difference was observed. The combination group showed a more significant effect in reducing the shortening of colon length in mice (P<0.01). Measurement results are shown in Table 2 and... Figure 3 As shown.

[0058] Table 2 Effects on colon length in UC mice ( ±s, n=8)

[0059] Note: Compared with the normal group: ### represents P<0.001. Compared with the model group: ** represents P<0.01.

[0060] 3.4 Effects on colonic histopathology Histopathological examination results as follows Figure 4 The results showed that significant inflammatory pathological changes were observed in the colon sections of the model group mice, including a large number of inflammatory cell infiltrations in the mucosa and submucosa. While intervention with either *Lactobacillus reuteri* or *Callicarpa nudiflora* granules alone showed some improvement in colonic mucosal damage in mice, intervention with *Callicarpa nudiflora* granules after colonization of *Lactobacillus reuteri* demonstrated the most significant therapeutic effect on colonic mucosal damage. Not only was the degree of inflammatory cell infiltration significantly reduced, but these phenotypic changes were also reflected in the scoring (Table 3), confirming that *Lactobacillus reuteri* played a synergistic role in the protective effect of *Callicarpa nudiflora* granules on the intestinal mucosa.

[0061] Table 3 Comparison of colon section pathological scores ( ±s, n=6)

[0062] Note: Compared with the normal group: ### represents P<0.001. Compared with the model group: ** represents P<0.01, *** represents P<0.001.

[0063] 3.5 Effects on inflammation and intestinal barrier-related factors The results are shown in Table 4. Compared with the normal group, the expression of IL-10 in the colon tissue of the model group mice was significantly higher. The levels of IL-10 decreased significantly (P<0.001). After intervention with *Lactobacillus reuteri*, *Callicarpa nudiflora* granules, and the combination of both, IL-10 levels showed a certain degree of increase, but this effect was most significant in the combination group (P<0.001). Similarly, compared with the normal group, the expression of IL-1β in the colonic tissue of the model group mice was significantly increased (P<0.001). Intervention with *Lactobacillus reuteri* or *Callicarpa nudiflora* granules alone also had a certain inhibitory effect on the abnormally elevated expression of IL-1β, but the combination showed the most significant inhibitory effect on IL-1β in the colonic tissue of mice, demonstrating a superior anti-inflammatory effect. MUC-2 is a high-molecular-weight glycoprotein secreted by intestinal goblet cells and has a protective effect on the intestine. Compared with the normal group of mice, the expression of MUC-2 in the colon tissue of the model group mice was significantly reduced (P<0.001). Lactobacillus reuteri or Callicarpa nudiflora granules alone produced a certain recovery effect, but the combined use had a better effect (P<0.001), which confirmed that the presence of Lactobacillus reuteri has a synergistic effect on the intestinal protective effect of Callicarpa nudiflora granules.

[0064] Table 4. Effects of Naked Flower Purple Beauty Granules on the Levels of IL-10, IL-1β, and MUC-2 in Colonic Tissue of Mice with Ulcerative Colitis ( ±s, n=6)

[0065] Note: Compared with the normal group: ### represents P<0.001. Compared with the model group: * represents P<0.05, ** represents P<0.01, *** represents P<0.001.

[0066] 3.6 Effects on bile acids in feces Detection of bile acid levels revealed a disordered bile acid metabolism in the model group mice. Compared with the normal group, the levels of CA (P<0.001), DCA (P<0.05), and LCA (P<0.01) in the model group samples were significantly increased, while 3-oxo-LCA was significantly decreased (P<0.01). Although intervention with Lactobacillus reuteri or Callicarpa nudiflora granules alone had some improvement effect, the changes in bile acid content in the feces of mice in the combined treatment group were the most significant. The significant decreases in CA (P<0.01), DCA (P<0.01), and LCA (P<0.001), and the significant increase in 3-oxo-LCA (P<0.01) indicated that the disordered bile acid metabolism in the feces was corrected (Table 5). In summary, mice with ulcerative colitis exhibited bile acid metabolism disorder. The effects of administering Callicarpa nudiflora granules and Lactobacillus reuteri alone on correcting this disorder were not significant. However, after Lactobacillus reuteri colonized the mice, Callicarpa nudiflora granules effectively corrected the disorder.

[0067] Table 5 Effects on bile acid changes in UC mice ( ±s, n=6)

[0068] Note: Compared with the normal group: ### represents P<0.001. Compared with the model group: * represents P<0.05, ** represents P<0.01, *** represents P<0.001.

[0069] 3.7 Effects on the Th17 / Treg balance in mice The results are as follows Figure 5 As shown in Table 6, the proportions of Th17 cells and Treg cells in the spleen of mice treated alone (either the *Callicarpa japonica* granule group or the *Lactobacillus reuteri* group) did not change significantly compared to the model group. However, after the combined use of the two treatments, the proportion of Th17 cells in the spleen significantly decreased (P<0.001), while the proportion of Treg cells significantly increased (P<0.001). This demonstrates that *Callicarpa japonica* granules, with the participation of *Lactobacillus reuteri*, significantly improved the imbalance of immune cells in mice with enteritis, exhibiting a synergistic effect.

[0070] Table 6. Comparison of Th17 and Treg levels in the spleen of mice in each group ( ±s, n=6)

[0071] Note: Compared with the normal group: ### represents P<0.001. Compared with the model group: * represents P<0.05, *** represents P<0.001.

[0072] In summary, animal experiments conducted in this invention have shown that both *Callicarpa nudiflora* granules and *Lactobacillus reuteri* improve enteritis symptoms in mice, as evidenced by a decrease in disease activity index, reduced colonic shortening, and decreased pathological section scores. In particular, the combined use of *Callicarpa nudiflora* granules and *Lactobacillus reuteri* significantly improves enteritis symptoms in mice, exhibiting a synergistic effect. The bile acid metabolism disorder and immune cell imbalance in mice tend to normalize, thereby achieving the therapeutic goal.

[0073] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. The use of Callicarpa nudiflora granules combined with Lactobacillus reuteri in the preparation of drugs for treating ulcerative colitis.

2. The use according to claim 1, characterized in that, The Lactobacillus reuteri mentioned is L. reuteri DSM 17938.

3. The use according to claim 1, characterized in that, The mass of the *Callicarpa nudiflora* granules is 6-12 mg, and the bacterial count of *Lactobacillus reuteri* is 2 × 10⁻⁶. 6 -2×10 8 CFU.

4. The use according to claim 3, characterized in that, The mass of the *Callicarpa nudiflora* granules was 8.32 mg, and the bacterial count of *Lactobacillus reuteri* was 2 × 10⁻⁶. 7 CFU.

5. The use according to any one of claims 1-4, characterized in that, The applications include the preparation of drugs to reduce disease activity index, drugs to improve colon shortening, drugs to improve bile acid metabolism disorders, or drugs to improve immune cell imbalance.

6. A pharmaceutical composition, characterized in that, Includes granules of Callicarpa nudiflora and Lactobacillus reuteri as described in claim 2.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition comprises 6-12 mg of Callicarpa nudiflora granules and 2×10 6 -2×10 8 CFU Lactobacillus reuteri 8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

9. The pharmaceutical composition according to claim 7, characterized in that, The dosage forms of the pharmaceutical composition include tablets, pills, capsules, powders, and ointments.

10. The pharmaceutical composition according to claim 9, characterized in that, The dosage form of the pharmaceutical composition is a powder.

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