Preparation method and application of traditional Chinese medicine composition for treating ulcerative colitis

Abstract

The invention relates to the technical field of medicine, in particular to a preparation method and application of a traditional Chinese medicine composition for treating ulcerative colitis, and the preparation method comprises the following steps: S1, weighing coptis chinensis, bitter ginseng and donkey-hide gelatin for later use; s2, extracting coptis chinensis, bitter ginseng and colla corii asini in proportion; the mass ratio of the coptis chinensis to the bitter ginseng to the donkey-hide gelatin is 1: 1: 1; s3, filtering the extracted and combined liquid medicine, and performing low-temperature drying to obtain the traditional Chinese medicine composition; the prepared traditional Chinese medicine composition can relieve UC mouse colon shortening and body mass reduction, effectively reduce colitis symptom factor infiltration and intestinal epithelial cell apoptosis and up-regulate expression of colon tight junction protein, and it is shown that the coptis chinensis and sophora flavescens decoction has the good effects of resisting inflammation, inhibiting colon epithelial cell apoptosis and promoting intestinal mucosa repair.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a preparation method and application of a traditional Chinese medicine composition for treating ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is typically characterized by non-specific, uncontrollable, and chronic immune-mediated intestinal inflammation. The pathological changes are mainly manifested as colonic ulcer erosion, which is diffuse, superficial, and continuous. The disease has a long course and is accompanied by repeated attacks, and the recurrence rate of patients accounts for about 52.7% of the total number of cases. The onset age of the disease tends to be younger, and it can cause a series of serious complications such as intestinal obstruction, intestinal perforation, and bleeding. The etiology and pathogenesis of UC are complex. At present, clinical treatment mainly relies on aminosalicylates, glucocorticoids, immunosuppressants, and biological inhibitors. Although such treatments have a high short-term clinical remission rate, they have a long treatment cycle, unstable efficacy, high recurrence rate, and large side effects, making it difficult for patients to tolerate. Therefore, seeking effective treatment drugs with small side effects has become a key problem to be solved urgently.

[0003] Huanglian Kushen Decoction originated from "Puji Fang" Volume 212 in the Ming Dynasty. The whole prescription consists of Coptis chinensis, Sophora flavescens, and donkey-hide gelatin. In the prescription, Coptis chinensis is bitter in taste and cold in nature, and it belongs to the heart, spleen, stomach, liver, gallbladder, and large intestine meridians. It has the effects of clearing heat and drying dampness, purging fire and detoxifying, and can clear the damp-heat toxin in the intestine. It is the monarch drug. For symptoms such as diarrhea, abdominal pain, and bloody stools caused by the accumulation of damp-heat, it can fundamentally eliminate the cause and improve the intestinal state. Sophora flavescens is bitter in taste and cold in nature, and it belongs to the heart, liver, stomach, large intestine, and bladder meridians. It can clear heat and dry dampness, kill insects and promote diuresis, and assist Coptis chinensis in enhancing the power of clearing heat and drying dampness. It is the minister drug, further strengthening the clearance of intestinal damp-heat and helping to improve the intestinal environment. Donkey-hide gelatin is used as an adjuvant. It is sweet in taste and neutral in nature, and it belongs to the lung, liver, and kidney meridians. Its function of nourishing yin and replenishing blood has a special meaning in this prescription. On the one hand, it can replenish the yin and blood consumed by the patient due to long-term bloody stools or long-term retention of damp-heat. On the other hand, donkey-hide gelatin has a certain hemostatic effect and can play a hemostatic role for intestinal bleeding. However, there is still no relevant report on the improvement effect of Huanglian Kushen Decoction on ulcerative colitis. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method and application of a traditional Chinese medicine composition for treating ulcerative colitis.

[0005] The solution of the present invention is as follows:

[0006] A preparation method of a traditional Chinese medicine composition for treating ulcerative colitis, comprising the following steps:

[0007] S1. Weigh Coptis chinensis, Sophora flavescens, and donkey-hide gelatin, and set aside;

[0008] S2. Extract Coptis chinensis, Sophora flavescens, and donkey-hide gelatin in proportion; the mass ratio of Coptis chinensis, Sophora flavescens, and donkey-hide gelatin is 1:1:1;

[0009] S3. Filter the combined extracted liquid and dry it to obtain the traditional Chinese medicine composition.

[0010] As a preferred technical solution, in S2, the extraction is carried out by the water decoction method. Coptis chinensis, Sophora flavescens, and donkey-hide gelatin are decocted with 10 times the volume of water, and the decoction liquids are combined to obtain the liquid medicine.

[0011] As a preferred technical solution, after Coptis chinensis, Sophora flavescens, and donkey-hide gelatin are mixed, they are decocted with 10 times the volume of water. The obtained decoction liquid is combined and filtered, and then freeze-dried by a freeze dryer into freeze-dried powder for standby. The freeze-drying ratio is 0.307.

[0012] As a preferred technical solution, Coptis chinensis, Sophora flavescens, and donkey-hide gelatin are respectively decocted with 10 times the volume of water. The obtained decoction liquids are combined and filtered, and then freeze-dried by a freeze dryer into freeze-dried powder for standby. The freeze-drying ratios are respectively: 0.17 for Coptis chinensis, 0.21 for Sophora flavescens, and 0.86 for donkey-hide gelatin.

[0013] As a preferred technical solution, the freeze-drying ratio is the ratio of the weight of the raw material to the pure dry matter remaining after low-temperature drying of the filtrate obtained by decocting with 10 times the volume of water.

[0014] As a preferred technical solution, the drying is low-temperature drying. The low-temperature drying is freeze-drying.

[0015] The present invention also discloses the application of the traditional Chinese medicine composition in preparing a drug for treating ulcerative colitis. The drug for treating ulcerative colitis contains the traditional Chinese medicine composition prepared by the preparation method of the traditional Chinese medicine composition for treating ulcerative colitis.

[0016] As a preferred technical solution, the traditional Chinese medicine composition can inhibit the weight loss and shortening of the colon length of mice with ulcerative colitis, improve the disease activity index, inhibit the increase in the levels of pro-inflammatory factors in serum and colon tissue, increase the secretion of anti-inflammatory factors in serum 、 reverse the histopathological changes in the terminal colon tissue, increase the number of goblet cells, mucin secretion, and promote the regeneration of colon epithelium and intestinal mucosa repair. At the same time, up-regulate the protein expression of ZO-1 and Claudin-1 in colon tissue;

[0017] The dosage of the traditional Chinese medicine composition per kilogram of body weight per day ≥ 0.00896 mg;

[0018] The disease activity index includes weight change, fecal characteristics, and blood in the stool.

[0019] As a preferred technical solution, the promotion of intestinal epithelial regeneration and the enhancement of intestinal mucosal repair are that the traditional Chinese medicine composition up-regulates the expressions of CK20, KLF5, and LGR5 mRNAs in the colon tissues of ulcerative colitis mice.

[0020] Due to the adoption of the above technical solution, a preparation method and application of a traditional Chinese medicine composition for treating ulcerative colitis include the following steps: S1. Weigh Coptis chinensis, Sophora flavescens, and donkey-hide gelatin for standby; S2. Extract Coptis chinensis, Sophora flavescens, and donkey-hide gelatin in proportion; the mass ratio of Coptis chinensis, Sophora flavescens, and donkey-hide gelatin is 1:1:1; S3. Filter the combined extracted liquid and perform drying to obtain the traditional Chinese medicine composition.

[0021] Advantages of the present invention:

[0022] Coptis chinensis is bitter in taste and cold in nature, and belongs to the heart, spleen, stomach, liver, gallbladder, and large intestine meridians. It has the effects of clearing heat and drying dampness, purging fire and detoxifying, and can clear the damp-heat toxin in the intestine. It is the monarch drug. For symptoms such as diarrhea, abdominal pain, and bloody stools caused by the accumulation of damp-heat, it can fundamentally remove the cause and improve the inflammatory state of the intestine.

[0023] Sophora flavescens is bitter in taste and cold in nature, and belongs to the heart, liver, stomach, large intestine, and bladder meridians. It clears heat and dries dampness, kills parasites and promotes diuresis, assisting Coptis chinensis to enhance the power of clearing heat and drying dampness. It is the ministerial drug, further strengthening the clearance of intestinal damp-heat, helping to improve the intestinal environment and reduce the inflammatory response.

[0024] Donkey-hide gelatin is sweet in taste and neutral in nature, and belongs to the lung, liver, and kidney meridians. Its function of nourishing yin and blood has special significance in this formula. It is the adjuvant drug. On the one hand, it can tonify the yin and blood consumed by the patient due to long-term bloody stools or long-term retention of damp-heat. On the other hand, donkey-hide gelatin has a certain hemostatic effect, can play a hemostatic effect on intestinal bleeding, nourish the intestine at the same time, and promote the healing of ulcers.

[0025] The three herbs are combined and complement each other. Coptis chinensis and Sophora flavescens dispel pathogenic factors, while donkey-hide gelatin strengthens healthy qi. Together, they achieve the effects of clearing heat and drying dampness, cooling blood and hemostasis, and nourishing yin and blood. Through the use of an acute ulcerative colitis animal model induced by 3% DSS, after intragastric administration of the traditional Chinese medicine composition and each of its herbs for intervention, a pharmacodynamic comparison study was carried out. It was found that the traditional Chinese medicine composition of the present invention can relieve the shortening of the mouse colon and the weight loss, effectively reduce the infiltration of colon inflammatory factors, intestinal epithelial cell apoptosis, and up-regulate the expression of colon tight junction proteins, indicating that Coptis-Sophora decoction has good anti-inflammatory, inhibitory effects on colon epithelial cell apoptosis and intestinal mucosal repair, and the effect is significantly better than the single herbs of Coptis chinensis, Sophora flavescens, and donkey-hide gelatin, as well as other compound prescriptions containing related components.

[0026] The traditional Chinese medicine composition of the present invention can not only inhibit the elevation of the levels of pro-inflammatory factors TNF-α and IL-1β, up-regulate the level of the anti-inflammatory factor IL-10, and reduce the inflammation of the colon tissue (reduction of inflammatory cell infiltration in the colon tissue), but also increase the number of goblet cells, promote the secretion of mucin, reduce the apoptosis of epithelial cells in the colon tissue, and up-regulate the expression of tight junction proteins ZO-1, Claudin-1, intestinal epithelial cell repair factors, and intestinal stem cell proliferation regulatory factors (up-regulate the expression of LGR5, KLF5, and CK20 mRNA). Furthermore, it can promote intestinal epithelial repair, enhance the integrity of the intestinal epithelial barrier function and the maintenance of mucosal immune homeostasis, achieve the synergistic treatment of ulcerative colitis by dual mechanisms, and the effect is significantly better than that of the single-herb group. It reflects the characteristic of the integrated treatment of traditional Chinese medicine of "multiple components - multiple pathways", provides an innovative strategy for the modern transformation of traditional compound prescriptions and the clinical treatment of UC, and is expected to become a new generation of original traditional Chinese medicine drugs for anti-ulcerative colitis. Description of the Drawings

[0027] 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 use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0028] Figure 1 It is the diagram of the effects of Huanglian Kushen Decoction, Sophora flavescens, Coptis chinensis, and Colla Corii Asini on UC mice in Experiment 1 of the present invention; wherein n = 6;

[0029] Figure 2 It is the diagram of HE staining and histopathological scoring of UC mice after intervention with Huanglian Kushen Decoction, Sophora flavescens, Coptis chinensis, and Colla Corii Asini in Experiment 1 of the present invention; wherein n = 6;

[0030] Figure 3 It is the diagram of the effects of the number of goblet cells and mucin secretion in the colon of UC mice after intervention with Huanglian Kushen Decoction, Sophora flavescens, Coptis chinensis, and Colla Corii Asini in Experiment 1 of the present invention, wherein n = 6;

[0031] Figure 4 It is the diagram of the effects of apoptosis of colon epithelial cells in UC mice after intervention with Huanglian Kushen Decoction, Sophora flavescens, Coptis chinensis, and Colla Corii Asini in Experiment 1 of the present invention, wherein n = 6;

[0032] Figure 5 It is the diagram of the effects of the expression of LGR5, KLF5, and CK20 mRNA in the colon tissue of UC mice after intervention with Huanglian Kushen Decoction, Sophora flavescens, Coptis chinensis, and Colla Corii Asini in Experiment 1 of the present invention, wherein n = 6;

[0033] Figure 6 This is the graph showing the effects of Coptis and Sophora Flavescens Decoction, Sophora flavescens, Coptis chinensis, and donkey-hide gelatin on serum inflammatory factors in UC mice in Experiment 1 of the present invention. Among them n = 6;

[0034] Figure 7 This is the graph showing the effects of Coptis and Sophora Flavescens Decoction, Sophora flavescens, Coptis chinensis, and donkey-hide gelatin on inflammatory factors in the colon tissues of UC mice in Experiment 1 of the present invention. Among them n = 6;

[0035] Figure 8 This is the graph showing the effects of Coptis and Sophora Flavescens Decoction, Sophora flavescens, Coptis chinensis, and donkey-hide gelatin on the expression of ZO-1 protein in UC mice in Experiment 1 of the present invention. Among them n = 6;

[0036] Figure 9 This is the graph showing the effects of Coptis and Sophora Flavescens Decoction, Sophora flavescens, Coptis chinensis, and donkey-hide gelatin on the expression of Claudin-1 protein in UC mice in Experiment 1 of the present invention. Among them n = 6;

[0037] Figure 10 This is the graph showing the effects of Coptis and Sophora Flavescens Decoction, Compound 1, and Compound 2 on UC mice in Experiment 2 of the present invention. Among them n = 6;

[0038] Figure 11 This is the graph of HE staining and histopathological scoring of UC mice after intervention with Coptis and Sophora Flavescens Decoction, Compound 1, and Compound 2 in Experiment 2 of the present invention. Among them n = 6. Detailed implementation manners

[0039] To make up for the above deficiencies, the present invention provides a preparation method and application of a traditional Chinese medicine composition for treating ulcerative colitis to solve the problems in the above background technology.

[0040] In order to make the technical means, creative features, achieved purposes and effects of the present invention easy to understand, the present invention will be further described below in conjunction with specific embodiments.

[0041] Example 1

[0042] S1. Weigh Coptis chinensis, Sophora flavescens, and donkey-hide gelatin and set them aside;

[0043] S2. Using the water decoction method, the Coptis chinensis, Sophora flavescens, and donkey-hide gelatin are mixed and then decocted with 10 times the volume of water, and the decoction liquids are combined to obtain a medicinal liquid; the mass ratio of Coptis chinensis, Sophora flavescens, and donkey-hide gelatin is 1:1:1;

[0044] S3. After the obtained medicinal liquid is filtered, it is freeze-dried into a freeze-dried powder and set aside using a freeze-dryer, and the freeze-drying ratio is 0.307.

[0045] Example 2

[0046] S1. Weigh Coptis chinensis, Sophora flavescens, and donkey-hide gelatin, and set aside.

[0047] S2. Decoct Coptis chinensis, Sophora flavescens, and donkey-hide gelatin separately with 10 times their volume of water, and combine the obtained decoctions. The mass ratio of Coptis chinensis, Sophora flavescens, and donkey-hide gelatin is 1:1:1.

[0048] S3. Filter the combined medicinal liquid, and use a freeze dryer to freeze-dry it into freeze-dried powder for standby. The freeze-drying ratios are as follows: Coptis chinensis 0.17, Sophora flavescens 0.21, and donkey-hide gelatin 0.86.

[0049] Experiment 1:

[0050] 1 Materials

[0051] 1.1 Animals

[0052] Healthy female C57BL / 6J mice, 7 - 8 weeks old, weighing 18 - 22 g, were provided by Beijing Speywood Biotechnology Co., Ltd. The animal production license number: SCXK (Jing) 2022 - 0007. The mice were housed in an SPF-class experimental animal room, with free access to food and water, humidity 55% ± 5%, temperature (22 ± 1) °C. After 7 days of adaptive feeding, the experiment was started. This research protocol was reviewed and approved by the Animal Experiment Ethics Committee of the First Affiliated Hospital of Yunnan University of Traditional Chinese Medicine (No. DW - 2025 - 007).

[0053] 1.2 Reagents

[0054] Dextran sulfate sodium (DSS) was purchased from MP Biomedicals (product number: YD08001); AB-PAS staining kit (G1285), cytokine ELISA detection kits were purchased from Wuhan GeneMe, IFN-γ (JYM0540Mo), TNF-α (JYM0621Mo), IL-1β (JYW1136Mo), IL-10 (JYM0005Mo), IL-6 (JYM0012Mo) antibodies, and ZO-1 were purchased from abcam (ab214228), and claudin-1 was purchased from proteintech (28674 - 1 - AP); TaKaRa PrimeScript RT Master Mix, product number: (RR036A); TaKaRa SYBR Premix Ex Taq II (Tli RNaseHPlus), product number: (RR820A); Total RNA extraction kit: Tiangen Biochemical Technology Co., Ltd. (DP431); Primers were all synthesized by Shanghai Sangon.

[0055] 1.3 Preparation of the formula of Coptis and Sophora Decoction (traditional Chinese medicine composition) and freeze-dried powder of the formula drugs

[0056] Coptis chinensis, Sophora flavescens, and donkey-hide gelatin (1:1:1) were all purchased from the Chinese medicine pharmacy of Yunnan Provincial Hospital of Traditional Chinese Medicine. In the central laboratory of our hospital, a total of 384 g of crude drugs were decocted with 10 times the volume of water. After the obtained decoction was combined and filtered, it was freeze-dried into freeze-dried powder for standby using a freeze dryer, and the freeze-drying ratio was 0.307. Coptis chinensis, Sophora flavescens, and donkey-hide gelatin were weighed separately to prepare single-herb samples, with a dose of 90 g each, and they were decocted with 10 times the volume of water. After the obtained decoction was combined and filtered, it was freeze-dried into freeze-dried powder for standby using a freeze dryer. The freeze-drying ratios were as follows: Coptis chinensis 0.17, Sophora flavescens 0.21, and donkey-hide gelatin 0.86.

[0057] 1.4 Instruments

[0058] Automatic dehydrator (ASP300S), rotary microtome (RM2235), upright microscope + image system (DM2500), Leica, Germany;

[0059] 2 Methods

[0060] 2.1 Preparation and grouping of UC mouse model (ulcerative colitis mice)

[0061] 88 female C57BL / 6J mice were randomly divided into a blank group (CON), a model group (MOD), a sulfasalazine group (SASP group), a low-dose Huanglian Kushen Decoction group (HKS.L group), a high-dose Huanglian Kushen Decoction group (HKS.H group), a low-dose Coptis chinensis group (HL.L), a high-dose Coptis chinensis group (HL.H), a low-dose Sophora flavescens group (KS.L), a high-dose Sophora flavescens group (KS.H), a low-dose donkey-hide gelatin group (EJ.L), and a high-dose donkey-hide gelatin group (EJ.H), with 8 mice in each group. Except for the blank group that was given drinking water, the other groups were allowed to freely drink 3% DSS, and intragastric administration was carried out simultaneously during modeling for 7 consecutive days. During the experiment, the body weight changes, mental state, and fecal characteristics of the mice were monitored daily, and the disease activity index (DAI) score and fecal occult blood test were performed. On the 8th day after administration, the animals were sacrificed by cervical dislocation after blood collection from the eyeballs. The spleen weight of the mice was weighed, the colon length of the mice was measured, and part of the colon tissue was fixed in 4% paraformaldehyde. Subsequently, tissues such as the colon, liver, and colon contents were immediately frozen in liquid nitrogen and stored at -80 °C for subsequent index detection.

[0062] 2.2 Drug dosage and preparation

[0063] Drug dosage: The dosages of sulfasalazine, Coptis chinensis, Sophora flavescens, and donkey-hide gelatin were referred to the daily dosage for adults in the instructions and pharmacopoeia. According to the clinical drug dosage conversion method in "Pharmacological Experiment Methodology", the drug dosages for mice were obtained as follows. The Huanglian Kushen Decoction referred to the dosage in ancient medical books and combined with the drug preparation extraction rate, and the dosages for each group were as shown in Table 1 below:

[0064] Table 1 Grouping of mice and administration doses

[0065]

[0066]

[0067] Drug preparation: Weigh the drug accurately. First, add pure water with a volume of 50% of the total volume. After complete dissolution by ultrasound, add 50% pure water and mix well. Use it immediately after preparation. Administration is by gavage.

[0068] 2.3 Observation of pathological changes in colon tissue by HE staining

[0069] Take the mouse colon tissue fixed with 4% paraformaldehyde, embed it in paraffin, section it, dewax it, and then perform HE staining. Observe the pathological morphological changes of the colon tissue under a light microscope (200 times magnification), and conduct histopathological scoring according to the degree of inflammatory cell infiltration in the colon and the degree of intestinal epithelial damage. The scoring criteria are as follows: 0 point: The tissue shows normal appearance and no inflammatory reaction is observed; 1 point: Slight leukocyte infiltration is observed; 2 point: Moderate leukocyte infiltration exists; 3 point: Obvious leukocyte infiltration, accompanied by moderate fibrosis, thickening of the colon wall, moderate reduction of goblet cells, and loss of some crypt structures; 4 point: Transmural leukocyte infiltration appears, a large number of goblet cells are reduced, extensive fibrosis, and general loss of crypt structures.

[0070] 2.4 Detection of mucin in colon tissue by Alcian blue - periodic acid Schiff (AB - PAS) staining

[0071] Dewax the paraffin sections with xylene and gradient ethanol. Stain according to the instructions using Alcian blue staining solution, then wash with water, dehydrate with ethanol, and immerse in PBS staining solution in the dark. Then briefly immerse in hematoxylin differentiation solution and blue - returning solution for nuclear staining, and finally wash with absolute ethanol and perform.

[0072] 2.5 Detection of apoptosis by TUNEL method

[0073] Fix the colon tissue with 4% formaldehyde solution at room temperature overnight. Immerse it in wax, embed it, and section it. Detect apoptosis according to the instructions of the TUNEL detection kit. Observe and collect images and analyze under the microscope.

[0074] 2.6 Detection of inflammatory factors in mouse serum and colon tissue by ELISA

[0075] Collect mouse blood samples and centrifuge them at room temperature for 15 min (speed 3500 r·min -1) The serum was separated, and the serum sample was restored to a liquid state at room temperature. The levels of IFN-γ, TNF-α, IL-1β, IL-10, and IL-6 in the serum were detected according to the method described in the ELISA kit instructions. The colon tissues of each group of mice were homogenized with PBS and centrifuged at 4°C for 20 min (speed 2000 r·min -1 ), and after dilution 10-fold, the levels of IFN-γ, TNF-α, IL-1β, IL-10, and IL-6 in the colon tissues were detected according to the method described in the ELISA kit instructions.

[0076] 2.7 Detection of the protein expression of ZO-1 and Claudin-1 in colon tissues by immunohistochemical staining

[0077] The paraffin sections were dewaxed to water, repaired with 50× sodium citrate antigen repair solution, and then incubated with 3% H2O2 at room temperature for 25 min to eliminate the activity of endogenous peroxidase. Subsequently, the sections were blocked with goat serum for 40 min, and incubated overnight at 4°C with rabbit anti-mouse primary antibodies ZO-1 (1:1000) and Claudin-1 (1:1000) respectively. Then, HRP secondary antibody was selected and incubated at room temperature for 40 min. After washing, DAB chromogenic solution was added dropwise to cover the tissue, the nuclei were stained with hematoxylin, differentiated and then blued, and sealed with neutral resin. Photographs were taken under a microscope (200×).

[0078] 2.8 Detection of the mRNA expression of LGR5, KLF5, and CK20 in mouse colon tissues by RT-qPCR

[0079] The tissue was crushed using a homogenizer, and total RNA was extracted from mouse colon tissues using a Tiangen kit, and the RNA concentration was measured. It was reverse transcribed into cDNA using a TaKaRa kit. The PCR reaction conditions were: 95°C, 30 s; 95°C, 5 s; 60°C, 20 s; for a total of 40 cycles. The relative expression levels of each gene were calculated using the 2-ΔΔCT method. The primer sequences are shown in Table 2.

[0080] Table 2 Primers

[0081]

[0082] 2.9 Statistical analysis

[0083] Statistical analysis was performed using statistical software SPSS 26.0 and GraphPad Prism 8.0.1 software. The experimental results are all expressed as , and one-way ANOVA was used for comparison between groups. P < 0.05 was considered statistically significant.

[0084] 3 Results

[0085] 3.1 Effects of Huanglian Kushen Decoction, Sophora flavescens, Coptis chinensis, and Colla Corii Asini on body weight and disease activity index of UC mice

[0086] As Figure 1 shown, the body weight of mice in the MOD group decreased, accompanied by severe diarrhea, bloody stools and other symptoms. At the same time, the colon length was significantly shortened, and the disease activity index (DAI) and spleen index were significantly increased. Compared with the MOD group, the body weight of mice in the HKS.H and KS.H groups showed a trend of recovery, while the body weight of mice in the other groups still showed varying degrees of decline. It is worth noting that the HKS.H group showed significant advantages in improving colon morphology, with an increased colon length (p < 0.05), significantly better than the KS.L / H and EJ.L / H groups (p < 0.01). In terms of the disease activity index, the DAI score of the HKS.L / H group was significantly reduced (p < 0.05), and was better than that of each single herb group (p < 0.05). Among them, the DAI scores of the KS.L / H, HL.H, and EJ.L single herb groups showed abnormal rebound phenomena. The spleen index of the HKS.H group tended to be normal, while the KS.H group showed significant splenomegaly and the spleen index increased accordingly (p < 0.01). The results showed that Huanglian Kushen Decoction was significantly better than Sophora flavescens, Coptis chinensis, and Colla Corii Asini in inhibiting the decrease of body weight, shortening of colon length, and improving the disease activity index of mice.

[0087] Figure 1 CON: blank group; MOD: model group; SASP: sulfasalazine group; HKS.L: low-dose Huanglian Kushen group; HKS.H: high-dose Huanglian Kushen group; KS.L: low-dose Sophora flavescens group; KS.H: high-dose Sophora flavescens group; HL.L: low-dose Coptis chinensis group; HL.H: high-dose Coptis chinensis group; EJ.L: low-dose Colla Corii Asini group; EJ.H: high-dose Colla Corii Asini group; compared with the MOD group, * P < 0.05, ** P < 0.01, *** P < 0.001; compared with the HKS.H group, # P < 0.05, ## P < 0.01, ### P < 0.001.

[0088] 3.2 Effects of Huanglian Kushen Decoction, Sophora flavescens, Coptis chinensis, and Colla Corii Asini on histopathology of colon tissues in UC mice

[0089] HE staining was used to detect the pathological changes of the terminal colon tissues to evaluate the pathological morphological changes of the mouse colon. The results showed ( Figure 2)The colon tissues of the MOD group mice presented typical pathological features of ulcerative colitis: severe loss or disordered arrangement of glandular structures, complete disappearance of crypts, highly edematous submucosa accompanied by massive infiltration of inflammatory cells, ulcer depth invading the muscular layer, and significantly increased histopathological scores (p < 0.001). The HKS.H group showed a significant improvement effect, with complete and clear glandular arrangement, regular reconstruction of crypts, significantly reduced infiltration of inflammatory cells, and significantly lower histopathological scores compared with the MOD group (p < 0.001), and it was significantly better than other intervention groups. The HL.L / H group partially alleviated the pathological damage, with complete glandular arrangement, but there were still irregular distortions on the surface of the crypts; the KS.L group improved and reduced the infiltration of inflammatory cells, with partial recovery of crypts, and the histopathological scores showed a downward trend; the EJ.H / L group had glandular defects, partial disappearance of crypt structures, massive infiltration of inflammatory cells, and the histopathological scores tended to be those of the MOD group. The results showed that the compound Huanglian Kushen Decoction could significantly reverse the pathological changes of the terminal colon tissues, while the three herbs of Kushen, Huanglian, and Ejiao had no obvious improvement effect on the pathology of the terminal colon tissues.

[0090] Figure 2 CON: blank group; MOD: model group; SASP: sulfasalazine group; HKS.L: low-dose Huanglian Kushen group; HKS.H: high-dose Huanglian Kushen group; KS.L: low-dose Kushen group; KS.H: high-dose Kushen group; HL.L: low-dose Huanglian group; HL.H: high-dose Huanglian group; EJ.L: low-dose Ejiao group; EJ.H: high-dose Ejiao group; compared with the MOD group, * P < 0.05, ** P < 0.01, *** P < 0.001; compared with the HKS.H group, # P < 0.05, ## P < 0.01, ### P < 0.001.

[0091] 3.3 Effects of Huanglian Kushen Decoction, Kushen, Huanglian, and Ejiao on the number of goblet cells and mucin secretion in UC mice

[0092] Goblet cells are a special type of epithelial cells arranged on multiple mucosal surfaces, maintaining the intestinal barrier by secreting mucus. Moreover, goblet cells can secrete various immune molecules, such as antimicrobial proteins, chemokines, and cytokines, and participate in the regulation of innate immune functions. As seen by AB-PAS staining, the crypt structures in the colon tissues of the MOD group mice disappeared, the number of goblet cells decreased and some goblet cells showed vacuolization, with a scattered distribution, and the mucus layer was severely damaged and the secretion decreased; compared with the MOD group, each administration group could increase the number of goblet cells, and the increase in the number of goblet cells in the HKS.H group was more obvious, with a neat arrangement, increased acidic glycoprotein and neutral glycogen, and increased mucus secretion; the number of goblet cells in the EJ.H group and the HL.H group increased to some extent, but no significant improvement was seen (Figure 3 ) It shows that Huanglian Kushen Decoction significantly increases the number of goblet cells and the secretion of mucin, while Kushen, Huanglian, and Ejiao have no obvious improvement effect on the number of goblet cells and the secretion of mucin.

[0093] Figure 3 CON: Blank group; MOD: Model group; SASP: Sulfasalazine group; HKS.L: Low-dose Huanglian Kushen group; HKS.H: High-dose Huanglian Kushen group; KS.L: Low-dose Kushen group; KS.H: High-dose Kushen group; HL.L: Low-dose Huanglian group; HL.H: High-dose Huanglian group; EJ.L: Low-dose Ejiao group; EJ.H: High-dose Ejiao group.

[0094] 3.4 Effects of Huanglian Kushen Decoction, Kushen, Huanglian, and Ejiao on apoptosis of colonic epithelial cells in UC mice

[0095] Regulating apoptosis of colonic epithelial cells is a key pathological link in maintaining the integrity of the mucosal barrier. TUNEL staining quantitative analysis showed ( Figure 4 ) that the density of apoptotic positive cells in the surface layer and crypt area of the colonic mucosa in the MOD group increased (p < 0.01), and the mean fluorescence intensity increased (p < 0.001), suggesting that there was excessive programmed cell death in the pathological process of UC. After drug intervention, each administration group could significantly inhibit apoptosis of colonic epithelial cells in UC mice. The effects of the HKS.H and KS.L groups were the best, and the effect of the HKS.H group was significantly better than those of the KS.H, HL.L, and EJ.L / H groups (p < 0.05). Specifically, the number of apoptotic cells in the mucosal layer and crypts of the colonic tissue of UC mice decreased. Statistical analysis found that the density of apoptotic positive cells was lower than that in the MOD group (p < 0.001), and the mean fluorescence intensity returned to the normal level, indicating that Huanglian Kushen Decoction could significantly inhibit apoptosis of colonic epithelial cells in UC mice.

[0096] Figure 4 CON: Blank group; MOD: Model group; SASP: Sulfasalazine group; HKS.L: Low-dose Huanglian Kushen group; HKS.H: High-dose Huanglian Kushen group; KS.L: Low-dose Kushen group; KS.H: High-dose Kushen group; HL.L: Low-dose Huanglian group; HL.H: High-dose Huanglian group; EJ.L: Low-dose Ejiao group; EJ.H: High-dose Ejiao group; compared with the MOD group, * P < 0.05, ** P < 0.01, *** P < 0.001; compared with the HKS.H group, # P < 0.05, ## P < 0.01, ### P < 0.001.

[0097] 3.5 Effects of Huanglian Kushen Decoction, Kushen, Huanglian, and Ejiao on the Expression of LGR5, KLF5, and CK20 mRNA in the Colonic Tissues of UC Mice

[0098] Studies on the molecular mechanisms of intestinal homeostasis regulation have shown that LGR5, as a key receptor of the Wnt signaling pathway, regulates the proliferation activity of intestinal stem cells by mediating the nuclear translocation of β-catenin, and plays a core role in maintaining the integrity of the intestinal crypt-villus structure and mucosal barrier function. The transcription factor KLF5 regulates the differentiation process of intestinal epithelial cells by inhibiting the promoter activity of FOXA1, and its expression level is significantly positively correlated with the morphogenesis of intestinal villi. CK20, as a specific molecular marker for the mature differentiation of intestinal epithelium, the abundance of its mRNA can quantitatively reflect the differentiation status of colonic mucosal cells. In this study, RT-qPCR technology was used to detect that the expressions of CK20, KLF5, and LGR5 mRNA in the intestinal tissues of mice in the MOD group were significantly decreased (p < 0.05). After drug intervention, the HKS.H group significantly up-regulated the levels of CK20, KLF5, and LGR5 mRNA (p < 0.05), and the levels of CK20, KLF5, and LGR5 mRNA in the other drug-administered groups showed an upward trend. The results indicate that Huanglian Kushen Decoction shows significant advantages in promoting intestinal epithelial regeneration and intestinal mucosal repair.

[0099] Figure 5 CON: Blank group; MOD: Model group; SASP: Sulfasalazine group; HKS.L: Low-dose Huanglian Kushen group; HKS.H: High-dose Huanglian Kushen group; KS.L: Low-dose Kushen group; KS.H: High-dose Kushen group; HL.L: Low-dose Huanglian group; HL.H: High-dose Huanglian group; EJ.L: Low-dose Ejiao group; EJ.H: High-dose Ejiao group; compared with the MOD group, * P < 0.05, ** P < 0.01, *** P < 0.001; compared with the HKS.H group, # P < 0.05, ## P < 0.01, ### P < 0.001.

[0100] 3.6 Effects of Huanglian Kushen Decoction, Kushen, Huanglian, and Ejiao on the Levels of IFN-γ, TNF-α, IL-1β, IL-10, and IL-6 in the Serum of UC Mice

[0101] Cytokines are involved in maintaining intestinal homeostasis. The imbalance of pro-inflammatory and anti-inflammatory cytokines caused by the overreaction of the immune system plays a crucial role in the occurrence and development of UC. The changes of cytokines in the sera of mice in each experimental group were detected by ELISA. The levels of pro-inflammatory cytokines TNF-α and IFN-γ in the sera of mice in the MOD group were significantly increased (p < 0.05), the level of IL-6 showed no obvious change, and the anti-inflammatory cytokine IL-10 showed a decreasing trend. After drug intervention, the pro-inflammatory cytokines could be significantly regulated in each group: in terms of inhibiting TNF-α, HKS.L / H showed a more significant dose-dependence (p < 0.05); in terms of regulating the level of IFN-γ, the inhibitory effect of HKS.H was significantly better than that of other intervention groups (p < 0.05), while EJ.H did not reduce the level of IFN-γ; at the same time, the expression of IL-1β could be reduced in all drug-administered groups (p < 0.01), the level of IL-6 was not significantly regulated, the level of IL-10 showed an increasing trend in the groups except the KS.H group, and the increase amplitude of the level of IL-10 in the HKS.H group was the most obvious( Figure 6 ). The results showed that Coptis and Sophora flavescens Decoction and the three herbs could significantly inhibit the increase of serum pro-inflammatory factors and increase the expression level of anti-inflammatory factors, and the compound had a significantly better regulatory effect on IFN-γ, IL-1β and IL-10 than the three herbs of Sophora flavescens, Coptis chinensis and Colla Corii Asini.

[0102] Figure 6 CON: blank group; MOD: model group; SASP: sulfasalazine group; HKS.L: low-dose Coptis and Sophora flavescens group; HKS.H: high-dose Coptis and Sophora flavescens group; KS.L: low-dose Sophora flavescens group; KS.H: high-dose Sophora flavescens group; HL.L: low-dose Coptis chinensis group; HL.H: high-dose Coptis chinensis group; EJ.L: low-dose Colla Corii Asini group; EJ.H: high-dose Colla Corii Asini group; compared with the MOD group, * P < 0.05, ** P < 0.01, *** P < 0.001; compared with the HKS.H group, # P < 0.05, ## P < 0.01, ### P < 0.001.

[0103] 3.7 Effects of Coptis and Sophora flavescens Decoction, Sophora flavescens, Coptis chinensis and Colla Corii Asini on the levels of IFN-γ, TNF-α, IL-1β, IL-10 and IL-6 in the colon tissues of UC mice

[0104] Further detection of the changes in inflammatory factors in the colon tissues of mice in each group showed that the levels of IFN-γ and TNF-α in the MOD group of mice were significantly increased (p < 0.05, p < 0.01), IL-1β and IL-6 showed an upward trend, and the level of the anti-inflammatory cytokine IL-10 was significantly decreased (p < 0.05). Compared with the MOD group, HKS.L / H could simultaneously inhibit the expression of IFN-γ (p < 0.05), TNF-α (p < 0.05) and IL-1β. Among them, the inhibitory effect of the HKS.H group on IFN-γ was significantly better than that of all single-herb groups (p < 0.05), and HKS.L / H significantly up-regulated the level of IL-10 in a dose-dependent manner; the SASP group significantly inhibited TNF-α (p < 0.001) and increased the content of IL-10 (p < 0.001), but failed to significantly regulate the levels of other inflammatory factors; although KS.L could reduce TNF-α (p < 0.01), its high-dose group instead exacerbated the expression of TNF-α; HL.L and EJ.L only partially inhibited IFN-γ and TNF-α (p < 0.05), and had no obvious regulatory effect on key anti-inflammatory factors such as IL-10( Figure 7 ). It is indicated that Huanglian Kushen Decoction can inhibit the increase of pro-inflammatory factors in colon tissues, up-regulate the expression level of the anti-inflammatory factor IL-10, and the compound has a significantly better regulatory effect on IFN-γ, TNF-α and IL-10 than the three herbs of Kushen, Huanglian and Ejiao.

[0105] Figure 7 CON: blank group; MOD: model group; SASP: sulfasalazine group; HKS.L: low-dose Huanglian Kushen group; HKS.H: high-dose Huanglian Kushen group; KS.L: low-dose Kushen group; KS.H: high-dose Kushen group; HL.L: low-dose Huanglian group; HL.H: high-dose Huanglian group; EJ.L: low-dose Ejiao group; EJ.H: high-dose Ejiao group. Compared with the MOD group, * P < 0.05, ** P < 0.01, *** P < 0.001; compared with the HKS.H group, # P < 0.05, ## P < 0.01, ### P < 0.001.

[0106] 3.8 Effects of Huanglian Kushen Decoction, Kushen, Huanglian, and Ejiao on the Protein Expressions of ZO-1 and Claudin-1 in the Colon Tissues of UC Mice

[0107] Claudin is a key supporting protein for the physical barrier of the intestine. Immunohistochemical staining and analysis of the positive area ratio showed that the expression of ZO-1 and Claudin-1 in the colon tissue of mice in the MOD group was decreased. The expression of ZO-1 and Claudin-1 proteins could be up-regulated in the remaining drug administration groups, and the expression of ZO-1 and Claudin-1 proteins in the HKS.L / H group was significantly up-regulated, and the positive area ratio was better than that of the other drug administration groups( Figure 8 - 9 ), and the expression of ZO-1 and Claudin-1 proteins could also show an upward trend in the other groups. It is indicated that Coptis and Sophora Flavescens Decoction and the three herbs all significantly up-regulated the expression of ZO-1 and Claudin-1 proteins in the colon tissue, and the regulatory effect of Coptis and Sophora Flavescens Decoction on ZO-1 and Claudin-1 was significantly better than that of the three herbs.

[0108] Figure 8 CON: blank group; MOD: model group; SASP: sulfasalazine group; HKS.L: low-dose Coptis and Sophora Flavescens group; HKS.H: high-dose Coptis and Sophora Flavescens group; KS.L: low-dose Sophora Flavescens group; KS.H: high-dose Sophora Flavescens group; HL.L: low-dose Coptis group; HL.H: high-dose Coptis group; EJ.L: low-dose donkey-hide gelatin group; EJ.H: high-dose donkey-hide gelatin group. Compared with the MOD group, * P < 0.05, ** P < 0.01, *** P < 0.001; compared with the HKS.H group, # P < 0.05, ## P < 0.01, ### P < 0.01.

[0109] Figure 9 CON: blank group; MOD: model group; SASP: sulfasalazine group; HKS.L: low-dose Coptis and Sophora Flavescens group; HKS.H: high-dose Coptis and Sophora Flavescens group; KS.L: low-dose Sophora Flavescens group; KS.H: high-dose Sophora Flavescens group; HL.L: low-dose Coptis group; HL.H: high-dose Coptis group; EJ.L: low-dose donkey-hide gelatin group; EJ.H: high-dose donkey-hide gelatin group; compared with the MOD group, * P < 0.05, ** P < 0.01, *** P < 0.001; compared with the HKS.H group, # P < 0.05, ## P < 0.01, ### P < 0.01.

[0110] Conclusion

[0111] The pathogenesis of ulcerative colitis is relatively complex, and there are certain limitations in the currently marketed drugs, so the clinical demand is urgent. Its ultimate treatment goal is to induce and maintain clinical remission for a long time, and to normalize inflammatory markers and achieve mucosal healing. Huanglian Kushen Decoction originated from "Puji Fang" in the Ming Dynasty and has the effects of clearing heat and drying dampness, purging fire and detoxifying. Through the overall concept of traditional Chinese medicine of "eliminating pathogenic factors and strengthening healthy qi", it alleviates the pathological process of ulcerative colitis (UC). The results of this study found that this formula can not only inhibit pro-inflammatory factors TNF-α and IL-1β, up-regulate the level of anti-inflammatory factor IL-10, and reduce colonic tissue inflammation (decrease in inflammatory cell infiltration in colonic tissue), but also increase the number of goblet cells, promote mucin secretion, reduce apoptosis of colonic tissue epithelial cells, and up-regulate the expression of tight junction proteins ZO-1, Claudin-1, intestinal epithelial cell repair factors, and intestinal stem cell proliferation regulatory factors (up-regulate the expression of LGR5, KLF5, and CK20 mRNA), thereby promoting intestinal epithelial repair, enhancing the integrity of the intestinal epithelial barrier function and maintaining mucosal immune homeostasis, achieving dual-mechanism synergistic treatment of ulcerative colitis, and the effect is significantly better than that of the single-herb group. It reflects the integrated treatment characteristics of traditional Chinese medicine of "multiple components - multiple pathways", provides an innovative strategy for the modern transformation of traditional compound prescriptions and the clinical treatment of UC, and is expected to become a new generation of original traditional Chinese medicine for anti-ulcerative colitis.

[0112] Experiment 2:

[0113] 1.1 Preparation and grouping of UC mouse models

[0114] Sixty-four female C57BL / 6J mice were randomly divided into a blank group (CON), a model group (MOD), a low-dose Huanglian Kushen Decoction group (HKS.L group), a high-dose Huanglian Kushen Decoction group (HKS.H group), a low-dose Compound 1 group (Compound 1.L), a high-dose Compound 1 group (Compound 1.H), a low-dose Compound 2 group (Compound 2.L), and a high-dose Compound 2 group (Compound 2.H), with 8 mice in each group. Except for the blank group given drinking water, the other groups freely drank 3% DSS, and were given intragastric administration while modeling, and continuously administered for 7 days. During the experiment, the body weight changes, mental state, and fecal traits of the mice were monitored daily, and the disease activity index (DAI) score and fecal occult blood test were performed. On the 8th day after administration, the animals were sacrificed by decapitation after taking blood from the eyeballs, the spleen weight of the mice was weighed, the colon length of the mice was measured, and part of the colon tissue was fixed in 4% paraformaldehyde, and then the colon, liver and other tissues and colon contents were immediately frozen in liquid nitrogen and stored at -80 °C for subsequent index detection.

[0115] 1.2 Preparation of freeze-dried powders of Huanglian Kushen Decoction formula (traditional Chinese medicine composition), Compound 1, and Compound 2

[0116] Coptis chinensis, Sophora flavescens, and donkey-hide gelatin (1:1:1) were all purchased from the Traditional Chinese Medicine Pharmacy of Yunnan Provincial Hospital of Traditional Chinese Medicine. In the central laboratory of our hospital, a total of 384 g of crude drugs were decocted with 10 times the volume of water. After the obtained decoction was combined and filtered, it was freeze-dried into freeze-dried powder for standby using a freeze-dryer. The freeze-drying ratio was 0.307. A total of 4 doses of Compound 1, totaling 540 g, and 4 doses of Compound 2, totaling 756 g, were respectively decocted with 10 times the volume of water. After the obtained decoction was combined and filtered, it was freeze-dried into freeze-dried powder for standby using a freeze-dryer. The freeze-drying ratios were: Compound 1: 0.57, Compound 2: 0.47.

[0117] Compound 1: 10 g of chrysanthemum, 5 g of licorice, 2 g of bupleurum, 5 g of angelica, 12 g of wolfberry, 3 g of dandelion, 1 g of tangerine peel, 3 g of salvia, 5 g of ginseng, 5 g of amomum, 5 g of coptis, 3 g of purslane, 12 g of Chinese yam, 6 g of donkey-hide gelatin, 20 g of scutellaria, 18 g of agrimony, 20 g of sophora flavescens

[0118] Compound 2: 10 g of notoginseng, 10 g of plantain herb, 30 g of agrimony, 10 g of mugwort leaf, 15 g of donkey-hide gelatin, 5 g of mint, 15 g of sophora flavescens, 30 g of purslane, 8 g of aurantii fructus immaturus, 15 g of Glehnia littoralis, 15 g of folium isatidis, 5 g of forsythia, 5 g of licorice, 8 g of coptis, 8 g of platycodon grandiflorum.

[0119] 1.3 Drug dosage and preparation

[0120] Drug dosage: For Compound 1, Compound 2, and Coptis and Sophora Decoction, referring to the published patent and ancient medical books for the drug dosage and combining with the drug preparation extraction rate, the dosing doses for each group are shown in Table 3 below:

[0121] Table 3 Grouping and dosing doses of mice

[0122] Grouping Drug administration Dose (mg / kg / d) Route of drug administration CON Normal saline i.g MOD Normal saline i.g HKS.L Huanglian Kushen Decoction 0.00896 mg / kg / d i.g HKS.H Huanglian Kushen Decoction 0.02689 mg / kg / d i.g Compound 1.L Compound 1 6.4125 g / kg / d i.g Compound 1.H Compound 1 12.825 g / kg / d i.g Compound 2.L Compound 2 7.4025 g / kg / d i.g Compound 2.H Compound 2 14.805 g / kg / d i.g

[0123] Drug preparation: Weigh the drugs accurately, first add 50% of the total volume of pure water, dissolve all by ultrasonic wave, then add 50% of pure water and mix well. Prepare and use immediately, and administer by gavage.

[0124] 1.4 HE staining to observe the pathological changes of colon tissue

[0125] The paraffin-embedded, sectioned, and dewaxed mouse colon tissues fixed with 4% paraformaldehyde were subjected to HE staining. The pathological morphological changes of the colon tissues were observed under a light microscope (200×), and histopathological scores were made according to the inflammatory cell infiltration and the degree of intestinal epithelial damage. The scoring criteria were as follows: 0 point: The tissue showed normal appearance and no inflammatory reaction was observed; 1 point: Slight leukocyte infiltration was observed; 2 point: Moderate leukocyte infiltration was present; 3 point: Obvious leukocyte infiltration was accompanied by moderate fibrosis, thickening of the colon wall, moderate goblet cell reduction, and loss of some crypt structures; 4 point: Transmural leukocyte infiltration occurred, with a significant reduction in goblet cells, extensive fibrosis, and general loss of crypt structures.

[0126] 1.5 Statistical analysis

[0127] Statistical analysis was performed using statistical software SPSS 26.0 and GraphPad Prism 8.0.1. The experimental results were all expressed as . One-way analysis of variance (ANOVA) was used for comparison between groups. P < 0.05 was considered statistically significant.

[0128] 2 Results

[0129] 2.1 Effects of Huanglian Kushen Decoction, Compound 1, and Compound 2 on the body weight and disease activity index of UC mice

[0130] As Figure 9 shown, the body weight of the mice in the MOD group decreased, accompanied by severe diarrhea, bloody stools and other symptoms. At the same time, the colon length was significantly shortened, and the disease activity index (DAI) and spleen index were significantly increased. Compared with the MOD group, the mice in the HKS.L / H group showed a trend of weight recovery. The body weights of the mice in the other groups still showed varying degrees of decrease, and the body weight of the Compound 1.L group decreased most significantly. The HKS.H group showed significant advantages in improving colon morphology, with an increase in colon length (p < 0.05), which was significantly better than the other two compound groups. Among them, the colon shortening in the Compound 1.L group and the Compound 2.L group was the most significant (p < 0.01). In terms of the disease activity index, the DAI score of the HKS.L / H group was significantly reduced (p < 0.05), and it was better than the other two compound groups (p < 0.05). The spleen index of the HKS.H group tended to be normal, while the spleen indices of the other groups decreased to varying degrees. The results showed that Huanglian Kushen Decoction was significantly better than the other two compound groups in inhibiting the decrease in mouse body weight, inhibiting colon length shortening and improving the disease activity index.

[0131] Figure 10CON: Blank group; MOD: Model group; HKS.L: Low-dose Coptis and Sophora flavescens group; HKS.H: High-dose Coptis and Sophora flavescens group; Compound 1.L: Low-dose Compound 1 group; Compound 1.H: High-dose Compound 1 group; Compound 2.L: Low-dose Compound 2 group; Compound 2.H: High-dose Compound 2 group; Compared with the MOD group, * P < 0.05, ** P < 0.01, *** P < 0.001; Compared with the HKS.H group, # P < 0.05, ## P < 0.01, ### P < 0.001.

[0132] 2.2 Effects of Coptis and Sophora flavescens decoction, Compound 1, and Compound 2 on the histopathology of the colon tissues of UC mice

[0133] HE staining was used to detect the pathological changes in the terminal colon tissues of mice to evaluate the pathological morphological changes of the colon tissues of mice. The results showed ( Figure 10 ) In the MOD group, the colon tissues of mice presented typical pathological features of ulcerative colitis: severe loss or disordered arrangement of glandular structures, complete disappearance of crypts, highly edematous submucosa with massive infiltration of inflammatory cells, ulcer depth invading the muscular layer, and significantly increased histopathological scores (p < 0.001). The HKS.H group showed a significant improvement effect, with complete and clear glandular arrangement, regular reconstruction of crypts, significantly reduced infiltration of inflammatory cells, and significantly lower histopathological scores compared with the MOD group (p < 0.001), and was significantly better than other intervention groups.

[0134] The Compound 1.H group and the Compound 2.H group partially alleviated the pathological damage, with complete glandular arrangement, but there were still irregular twists on the surface of the crypts, and the histopathological scores showed a downward trend; the Compound 1.L group and the Compound 2.L group had glandular defects, partial disappearance of crypt structures, massive infiltration of inflammatory cells, and the histopathological scores tended to be those of the MOD group. The results showed that the Coptis and Sophora flavescens decoction compound could significantly reverse the histopathological changes in the terminal colon tissues, while the improvement effects of the two compound groups on the histopathology of the terminal colon tissues were not obvious.

[0135] Figure 11 CON: Blank group; MOD: Model group; HKS.L: Low-dose Coptis and Sophora flavescens group; HKS.H: High-dose Coptis and Sophora flavescens group; Compound 1.L: Low-dose Compound 1 group; Compound 1.H: High-dose Compound 1 group; Compound 2.L: Low-dose Compound 2 group; Compound 2.H: High-dose Compound 2 group; Compared with the MOD group, * P < 0.05, ** P < 0.01, *** P < 0.001; Compared with the HKS.H group, # P < 0.05, ##P < 0.01, ### P < 0.001.

[0136] The foregoing has shown and described the basic principles, principal features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and what is described in the above embodiments and the specification is only to illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will also have various changes and improvements, and all such changes and improvements fall within the scope of the present invention as claimed. The scope of the present invention as claimed is defined by the appended claims and their equivalents.

Claims

1. A preparation method of a traditional Chinese medicine composition for treating ulcerative colitis, characterized in that, It includes the following steps: S1. Weigh Coptis chinensis, Radix Sophorae Flavescentis and Colla Corii Asini, and set aside; S2. Extract Coptis chinensis, Radix Sophorae Flavescentis and Colla Corii Asini in proportion; the mass ratio of Coptis chinensis, Radix Sophorae Flavescentis and Colla Corii Asini is 1:1:1; S3. Filter the combined extracted liquid, and dry it to obtain the traditional Chinese medicine composition.

2. The preparation method of a traditional Chinese medicine composition for treating ulcerative colitis according to claim 1, characterized in that : In S2, the extraction is carried out by the water decoction method. Coptis chinensis, Radix Sophorae Flavescentis and Colla Corii Asini are decocted with 10 times the volume of water, and the decoction liquids are combined to obtain the liquid medicine.

3. The preparation method of a traditional Chinese medicine composition for treating ulcerative colitis according to claim 1, characterized in that: The drying is low-temperature drying.

4. Use of the traditional Chinese medicine composition in preparing a medicament for treating ulcerative colitis, characterized in that: The medicine for treating ulcerative colitis contains the traditional Chinese medicine composition prepared by the preparation method of the traditional Chinese medicine composition for treating ulcerative colitis described in any one of claims 1 to 3.

5. Use of the traditional Chinese medicine composition according to claim 4 in the preparation of a medicament for treating ulcerative colitis, characterized in that: The traditional Chinese medicine composition can inhibit weight loss and shortening of colon length in ulcerative colitis mice, improve the disease activity index, inhibit the increase in the levels of pro-inflammatory factors in serum and colon tissues, and increase the secretion of anti-inflammatory factors in serum. 、 It can reverse the histopathological changes in the terminal colon tissue, increase the number of goblet cells, mucus protein secretion, promote the regeneration of colon epithelium and intestinal mucosa repair, and at the same time up-regulate the protein expression of ZO-1 and Claudin-1 in colon tissues. The dosage of the traditional Chinese medicine composition per kilogram of body weight per day ≥ 0.00896 mg; The disease activity index includes body weight change, fecal character and blood in stool.

6. Use of the traditional Chinese medicine composition according to claim 5 in the preparation of a medicament for treating ulcerative colitis, characterized in that: The promotion of intestinal epithelial regeneration and the strengthening of intestinal mucosa repair are that the traditional Chinese medicine composition up-regulates the expression of CK20, KLF5 and LGR5 mRNA in the colon tissue of ulcerative colitis mice.

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