Traditional Chinese medicine composition for treating ulcerative colitis

Abstract

The invention discloses a traditional Chinese medicine composition for treating ulcerative colitis, and belongs to the technical field of traditional Chinese medicines. The traditional Chinese medicine composition consists of the following components in parts by mass: 5-15 parts of codonopsis pilosula, 2-6 parts of coptis chinensis, 5-15 parts of parched white peony root, 5-15 parts of poria cocos, 5-15 parts of parched white atractylodes rhizome, 3-9 parts of scutellaria baicalensis, 3-9 parts of elecampane, 5-15 parts of angelica sinensis, 5-15 parts of kudzu vine root, 5-15 parts of smoked plum, 5-15 parts of purslane and 2-6 parts of honey-fried licorice root. The traditional Chinese medicine composition is designed for ulcerative colitis large intestine damp-heat and spleen qi weakness core pathogenesis, cold and heat are administered at the same time, reinforcing and reducing are conducted at the same time, qi and blood are regulated at the same time, both symptoms and root causes are considered, the traditional Chinese medicine composition can be used for treatment in the attack stage and has a good curative effect on prevention of relapse in the remission stage, and the safety of patients is good after long-term administration.

Description

Technical Field

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

[0002] Ulcerative colitis (UC) is a chronic nonspecific inflammatory disease of the large intestine with unknown etiology, characterized by continuous and diffuse inflammatory changes in the colorectal mucosa. The lesions mainly involve the mucosa and submucosa, and belong to the category of inflammatory bowel disease (IBD) in Western medicine. UC patients often present with persistent or recurrent diarrhea, bloody stools with mucus, abdominal pain, tenesmus, and varying degrees of extraintestinal manifestations. UC has the characteristics of recurrent and difficult-to-cure disease, which seriously affects the quality of life of patients. In addition, long-term chronic intestinal inflammation is a high-risk factor for colorectal cancer.

[0003] Modern medicine mainly formulates treatment plans for ulcerative colitis based on the severity of disease activity, the scope of lesions involved, and the type of disease (frequency of relapse, previous response to therapeutic drugs, extraintestinal manifestations, etc.). During treatment, the treatment plan should be adjusted at any time according to the patient's response to treatment and tolerance to drugs. Treatment goals are divided into short-term, medium-term, and long-term goals. Clinical response is a short-term goal, which is mainly aimed at quickly achieving improvement in clinical symptoms. The clinical response of adult UC patients refers to a decrease of at least 50% in the rectal bleeding score and stool frequency in the patient reported outcome 2 (PRO2); the medium-term treatment goals include clinical remission and biomarker compliance. Clinical remission of adult UC patients refers to a rectal bleeding score of 0 and stool frequency of 0 in PRO2, or a partial Mayo score of <3 points and no single score >1 point. Biomarker compliance includes C-reactive protein (CRP) below the upper limit of normal and fecal calprotectin (FC) decreased to 100-250ug / g. FC is more sensitive than CRP in reflecting endoscopic activity. As a non-invasive disease activity marker in UC patients, it can be used to evaluate treatment response and relapse. When endoscopy is not feasible or mucosal healing cannot be evaluated, FC can be used as an alternative to endoscopy. Endoscopic mucosal healing is the preferred long-term treatment goal for IBD. The STRIDE-II consensus recommends that if this goal has not been achieved, a change in treatment should be considered. Endoscopic mucosal healing can be evaluated by sigmoidoscopy or colonoscopy. Endoscopic mucosal healing in UC patients refers to a Mayo endoscopic score = 0, or a UC endoscopic severity index ≤ 1. Although histological remission is not currently a formal treatment goal, it can be used as an auxiliary indicator of endoscopic remission in UC, representing a deeper level of healing.

[0004] Among the current treatment options for UC patients in my country, traditional options based on oral 5-aminosalicylic acid, glucocorticoids, and immunosuppressants account for more than 80.0%, and the use rate of biological agents is about 12.0%. Aminosalicylic acid preparations are the main drugs for the treatment of mild UC, including traditional sulfasalazine (SASP) and various other types of 5-aminosalicylic acid (5-ASA) preparations. The efficacy of SASP is similar to that of other 5-ASA preparations, but adverse reactions are much more common than those of 5-ASA preparations. There is currently a lack of evidence showing that the efficacy of different types of 5-ASA preparations is different. For patients with poor symptom control after adequate aminosalicylic acid treatment (generally 24 weeks), especially those with extensive lesions, hormone treatment is used instead. After achieving symptom relief, the dose is gradually reduced until the drug is stopped. Rapid reduction can lead to early recurrence. For patients who are ineffective or intolerant to hormones, aminosalicylic acid preparations are often used in combination with thiopurine drugs in clinical practice, but aminosalicylic acid preparations can increase the bone marrow suppression toxicity of thiopurine drugs, so special attention should be paid. Refractory UC is often treated with a combination of biological agents such as infliximab, adalimumab, vedolizumab, etc. For patients with massive bleeding, perforation, cancer, or a high degree of suspicion of cancer, surgical treatment is used.

[0005] As can be seen from the above, the commonly used drugs for the treatment of UC in clinical practice include aminosalicylic acid preparations, corticosteroids, immunosuppressants, biological agents, small molecule drugs, etc. Although they have made certain progress in inducing remission, their medication courses and side effects cause great physical, mental and financial pressure on patients. Summary of the invention

[0006] The purpose of the present invention is to provide a traditional Chinese medicine composition for treating ulcerative colitis, which has good therapeutic effect on restoring the pathological morphology of intestinal mucosal tissue of patients with ulcerative colitis, improving clinical symptoms such as abdominal pain, diarrhea, mucus, pus and blood in stool, fatigue, poor appetite, etc. of patients with ulcerative colitis, and no adverse reactions occur.

[0007] The present invention first provides a traditional Chinese medicine composition, which is composed of the following components in parts by mass: 5-15 parts of Codonopsis pilosula, 2-6 parts of Coptis chinensis, 5-15 parts of stir-fried white peony root, 5-15 parts of Poria cocos, 5-15 parts of stir-fried Atractylodes macrocephala, 3-9 parts of Scutellaria baicalensis, 3-9 parts of Costus root, 5-15 parts of Angelica sinensis, 5-15 parts of Pueraria root, 5-15 parts of Prunus mume, 5-15 parts of Portulaca oleracea, and 2-6 parts of roasted Licorice root.

[0008] Specifically, the Chinese medicine composition is composed of the following components in parts by mass: 7.5 parts of Codonopsis pilosula, 3 parts of Coptis chinensis, 6 parts of stir-fried white peony root, 7.5 parts of Poria cocos, 6 parts of stir-fried Atractylodes macrocephala, 4.5 parts of Scutellaria baicalensis, 4.5 parts of Costus root, 6 parts of Angelica sinensis, 6 parts of Pueraria root, 7.5 parts of Prunus mume, 7.5 parts of Portulaca oleracea, and 3 parts of roasted Licorice root.

[0009] The Chinese medicine composition is a water decoction.

[0010] Specifically, the preparation method of the traditional Chinese medicine composition comprises the following steps: mixing the components, decocting with water, concentrating the medicinal liquid to obtain a thick paste; and then drying.

[0011] The Chinese medicine composition of the present invention is based on the core pathogenesis of ulcerative colitis: the disease is located in the large intestine, which is related to the spleen and liver; the disease is characterized by dampness and heat as the symptoms and qi deficiency as the root cause. The core syndromes targeted are dampness and heat in the large intestine and spleen deficiency; it embodies the core treatment method of "regulating qi and blood to consolidate the intestine" for the treatment of ulcerative colitis.

[0012] The Codonopsis pilosula in the formula is sweet in taste and neutral in nature, and its functions are to tonify the spleen, replenish qi and produce blood. "Bencao Congxin" calls it "tonifies the middle and replenishes qi, and harmonizes the spleen and stomach"; Coptis chinensis is bitter in taste and cold in nature, and its functions are to clear away heat, dry dampness and detoxify, and "Compendium of Materia Medica" calls it an important medicine for treating dysentery; the two medicines are used together to invigorate the spleen, dry dampness and stop diarrhea, and they are both the main medicines. White peony root tastes bitter and sour, and is slightly cold in nature. It can soften the liver and nourish the blood. After being fried, the effect of softening the liver and relieving pain is even stronger. "Medical Origin" calls it "calming the spleen meridian, treating abdominal pain, stopping diarrhea, and harmonizing blood"; Poria cocos tastes sweet and light, and is neutral in nature. It can promote diuresis, eliminate dampness, and strengthen the spleen. "Compendium of Materia Medica" calls it "an important medicine for promoting diuresis and removing dampness, and the spleen will be healthy when the water is gone"; Atractylodes macrocephala tastes sweet and bitter, and is warm in nature. It can tonify qi and strengthen the spleen, dry dampness and promote diuresis. After being fried, the effect of tonifying qi and strengthening the spleen is even stronger. "Depei Materia Medica" calls it "stops diarrhea and dysentery"; Scutellaria baicalensis tastes bitter and is cold in nature. It can clear away heat, dry dampness, and detoxify. "Shennong's Materia Medica" calls it "the main medicine for intestinal diarrhea and diarrhea". They are all minister drugs. Muxiang is pungent and pungent, warm in nature, and has the function of strengthening the spleen, promoting qi and relieving pain. "Depei Materia Medica" says it "stops heaviness after diarrhea"; Angelica sinensis is sweet and pungent in taste, warm in nature, and has the function of promoting blood circulation and relieving pain, "Mingyi Bielu" says it "warms the middle and stops pain"; Pueraria root is sweet and pungent in taste, cool in nature, and has the function of raising yang and stopping diarrhea, "Bencao Zhengyi" says it "is the best at raising the clear yang qi of the spleen and stomach"; Plum is sour and astringent in taste, neutral in nature, and has the function of astringing the intestines, "Mingyi Bielu" says it "stops diarrhea"; Purslane is sour and cold in nature, and has the function of cooling blood and stopping bleeding and stopping diarrhea, "Xinxiu Materia Medica" says it "treats malnutrition and dysentery", and they are all adjuvants. Among them, Muxiang, Angelica sinensis and stir-fried white peony root regulate qi to promote blood circulation, Pueraria root and Codonopsis pilosula, Poria cocos, stir-fried Atractylodes macrocephala strengthen the spleen and raise clearness, remove dampness and stop diarrhea, Plum and stir-fried white peony root soften the liver and suppress wood, and Purslane clears heat, cools blood and stops bleeding. Licorice is sweet and neutral in nature. It can nourish the spleen and replenish qi, relieve urgency and stop pain. Roasting it can enhance the function of nourishing the middle and relieving urgency. The "Compendium of Materia Medica" says that it "enters qi with qi medicine and enters blood with blood medicine", and is a guiding drug. The whole prescription treats both cold and hot, tonifies and purges, regulates qi and blood, and takes into account both the symptoms and the root causes. Together, it achieves the functions of "clearing dampness and heat, regulating qi and blood, and consolidating the intestines to stop diarrhea".

[0013] Second, the present invention also provides the use of the above-mentioned Chinese medicine composition in the preparation of a product for preventing and / or treating ulcerative colitis;

[0014] Specifically, the product is a medicine or a pharmaceutical preparation.

[0015] The Chinese medicine composition of the present invention is designed to target the core pathogenesis of ulcerative colitis, namely, damp-heat in the large intestine and weak spleen. It treats both cold and heat, tonifies and purges, harmonizes qi and blood, and takes both the symptoms and the root causes into consideration. It can not only be used for the treatment of the attack period, but also has a good therapeutic effect for preventing recurrence in the remission period. It is safe for patients to take it for a long time, and has research and development value. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Survival curve of mice during drug administration;

[0017] Figure 2 This is the characterization and observation of mice 7 days after administration;

[0018] Figure 3 Comparison of the body weight of mice after 7 days of administration; Note: Compared with the normal control group, ## P<0.01; compared with the model control group, * P < 0.05;

[0019] Figure 4 Statistics of DAI scores of mice after 7 days of drug administration; Note: Compared with the normal control group, ## P<0.01; compared with the model control group, ** P < 0.01;

[0020] Figure 5 This is the heat map of inflammation-related cytokines in colon tissue 7 days after administration;

[0021] Figure 6 This is a representative picture of the gross morphology of the colon 7 days after administration;

[0022] Figure 7 The colon length of mice was counted after 7 days of drug administration; Note: Compared with the normal control group, # P < 0.05;

[0023] Figure 8 Representative images of colon HE staining 7 days after administration (40×, 200×);

[0024] Fig. 9 Thickness of colon mucosa layer after 7 days of drug administration; Note: Compared with the normal control group, ## P<0.01; compared with the model control group, * P<0.05, ** P < 0.01;

[0025] Fig.10 The total score of colon tissue pathology after 7 days of administration; Note: Compared with the normal control group, ## P<0.01; compared with the model control group, * P<0.05, ** P < 0.01;

[0026] Fig.11 Representative images of the colon under transmission electron microscopy 7 days after administration (A: 15000×, 40000×; B: 5000×, 15000×); Note: Fig.11 A in the figure shows the observation of intestinal epithelial cells and intercellular junctions; B shows the observation of goblet cells; Mv: microvilli, TJ: tight junction, IJ: intermediate junction, De: desmosome, M: mitochondria, RER: rough endoplasmic reticulum, Go: Golgi apparatus, MP: mucin granules. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but not for limiting the scope of the present invention.

[0028] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0029] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0030] Example 1

[0031] The composition of the Chinese medicine composition is as follows: Codonopsis pilosula 7.5g, Coptis chinensis 3g, stir-fried white peony root 6g, Poria 7.5g, stir-fried Atractylodes macrocephala 6g, Scutellaria baicalensis 4.5g, Costusroot 4.5g, Angelica sinensis 6g, Pueraria lobata 6g, Prunus mume 7.5g, Portulaca oleracea 7.5g, and roasted Licorice root 3g.

[0032] Example 2

[0033] 1. Experimental plan

[0034] 1 Materials and Instruments

[0035] 1.1 Animals

[0036] SPF grade C57BL / 6J mice, male, 6-8 weeks old, weighing 19-21g, ordered from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., experimental animal license number: SCXK (Beijing) 2021-0006, animals were raised in the animal laboratory of Beijing University of Chinese Medicine (barrier environment), experimental unit use license number: SYXK (Beijing) 2023-0011, room temperature 22-26℃, relative humidity 40%-70%, light cycle light and dark time 12h each, good purification and ventilation system, and standard feed was given during the breeding period. Approved by the Experimental Animal Ethics Review Committee of Beijing University of Chinese Medicine, ethics review number: BUCM-2023121104-4199.

[0037] 1.2 Drugs

[0038] Codonopsis pilosula (batch number 2203036), Coptis chinensis (batch number 2211038), stir-fried white peony root (batch number 2210073), Poria cocos (batch number 2212001), stir-fried Atractylodes macrocephala (batch number 2210008), Scutellaria baicalensis (batch number 2106015), Costusroot rhizome (batch number 2203055), Angelica sinensis (batch number 2210050), Pueraria root (batch number 2211029), Prunus mume (batch number 2209084), Portulaca oleracea (batch number 2208015), and Glycyrrhiza uralensis (batch number 2301041) were purchased from Beijing Shuangqiao Yanjing Chinese Medicine Piece Factory; Guchang Zhixie Pills (concentrated pills), batch number 212450, Xi'an Afang Palace Pharmaceutical Co., Ltd., China.

[0039] 1.3 Reagents and Supplies

[0040] Dextran sulfate sodium (DSS, enteritis modeling grade, molecular weight: 36000-50000), product number 160110, batch number YC12017, MP Biomedicals, USA;

[0041] Fecal occult blood qualitative detection kit (o-tolidine method), catalog number TC0511, Beijing Regen Biotechnology Co., Ltd., China;

[0042] Physiological saline, catalog number G4702-500ML; general tissue fixative, catalog number G1101-500ML; electron microscope fixative, catalog number G1102-100ML; environmentally friendly dewaxing solution, catalog number G112; PBS buffer, catalog number G4202-500ML; grinding beads (steel), catalog number G0103-200G; Wuhan Saiweier Biotechnology Co., Ltd., China;

[0043] Xylene, Product No. 10023418; Anhydrous ethanol, Product No. 1000926; Neutral gum, Product No. 10004160; Acetone, Product No. 10000418; Sinopharm Chemical Reagent Co., Ltd., China;

[0044] Hematoxylin stain, Catalog No. BA-4041; Eosin stain, Catalog No. BA-4024; Zhuhai Beso Biotechnology Co., Ltd., China;

[0045] 812 embedding medium, product number 90529-77-4, SPI, USA;

[0046] Osmium acid, product number 18456, Ted Pella Inc, USA;

[0047] Protease and phosphatase inhibitor cocktail, cat. no. P002, Suzhou New Saimei Biotechnology Co., Ltd., China;

[0048] BCA protein quantification / concentration assay kit, catalog number MA0082-3, Dalian Meilun Biotechnology Co., Ltd., China;

[0049] LEGENDplex TM MUCytokine Release Syndrome Panel (13-plex) w / FP, Catalog No. 741023, BioLegend, USA;

[0050] LEGENDplex TM Mouse Cytokine Panel 2 (13-plex) w / FP V02, Catalog No. 741334, BioLegend, USA.

[0051] 1.4 Instruments and Equipment

[0052] ZKXFB-2 vacuum drying oven, Shanghai Yiheng Scientific Instrument Co., Ltd., China;

[0053] GFL-230 electric heating blast drying oven, Tianjin Laboterui Instrument Equipment Co., Ltd., China;

[0054] Digital constant temperature water bath, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory, China;

[0055] IKA Plate (RCT digital) magnetic stirrer, IKA, Germany;

[0056] VP-15L oil-free diaphragm vacuum pump, Qun'an Scientific Instrument Co., Ltd., China;

[0057] IMS-130 fully automatic snowflake ice machine, Changshu Xueke Electric Appliance Co., Ltd., China;

[0058] ME204 1 / 10,000 electronic balance, METTLER-TOLEDO, Switzerland;

[0059] XS105 1 / 100,000 electronic balance, METTLER-TOLEDO, Switzerland;

[0060] HistoCore Arcadia fully automatic tissue embedding machine, Leica, Germany;

[0061] HistoCore MULTICUT-semiautomatic rotary microtome, Leica, Germany;

[0062] KD-P biological tissue slide machine, Kedi Instrument Equipment Co., Ltd., Jinhua City, Zhejiang Province, China;

[0063] ASP300S fully automatic vacuum dehydrator, Leica, Germany;

[0064] ST5020 stainer / coverslipper workstation, Leica, Germany;

[0065] BX43 upright biological microscope, OLYMPUS, Japan;

[0066] Leica UC7 ultramicrotome, Leica, Germany;

[0067] Ultra 45° diamond microtome knife, Daitome, Switzerland;

[0068] HT7700 transmission electron microscope, HITACHI, Japan;

[0069] MDF-U3386S-80℃ low temperature refrigerator, SANYO, Japan;

[0070] MM400 high-throughput tissue grinder, Retsch, Germany;

[0071] Medifuge X1R refrigerated centrifuge, Thermo Fisher Scientific Inc, USA;

[0072] EPOCH2TC microplate reader, BioTek Instruments Inc, USA;

[0073] VORTEX-5 vortex mixer, Haimen Qilin Bell Instrument Manufacturing Co., Ltd., China;

[0074] LX-200 mini centrifuge, Haimen Qilin Bell Instrument Manufacturing Co., Ltd., China;

[0075] BD LSRFortessa TM Cell analyzer, BD, USA;

[0076] WELVAC200 Microplate Vacuum Manifold, Rockco Instrument Co., Ltd., China;

[0077] Rocker 300 Oil Free Vacuum Pump, Rocker Instrument Co., Ltd., China;

[0078] MS 3digital orbital shaker, IKA, Germany.

[0079] 2 Experimental methods

[0080] 2.1 Drug preparation and dosage

[0081] Embodiment 1 Chinese medicine composition, total crude drug amount 69.00g, extraction process is to add 10 times of water (mass), boil and then simmer for 1.5 hours, collect and filter the medicinal liquid and then add 10 times of water, boil and then simmer for 1.5 hours, decocted 2 times in total, merge the two medicinal liquids for concentration, put the concentrated thick paste into a vacuum drying oven at 70°C and dry for 48h, powder it after fully drying, mix it, weigh it, and place it in a desiccator for preservation, and the paste yield is 42.00%.

[0082] The dosage for mice is calculated based on the body surface area conversion method between humans and animals as follows:

[0083] The dosage of crude drugs in each group of the Chinese medicine composition is: 5.75g crude drug / kg for the low-dose group, 11.50g crude drug / kg (clinically equivalent dose) for the medium-dose group, and 23.00g crude drug / kg for the high-dose group, prepared with pure water.

[0084] Chinese medicine positive control drug Guchang Zhixie Pills: The clinical daily dosage for humans is 12.00g, which is converted to 2.00g / kg for mice. The pills are crushed in a mortar and then prepared with pure water.

[0085] Among them, the specification of Guchang Zhixie Pills: 9 pills weighing 1 gram each, 216 pills / bottle / box; manufacturer: Xi'an Afang Palace Pharmaceutical Co., Ltd.; functions and indications: harmonize the liver and spleen, astringe the intestines and relieve pain. It is used for patients with liver and spleen disharmony, diarrhea and abdominal pain, and chronic nonspecific ulcerative colitis with the above symptoms. Effective ingredients: black plum, coptis root, dried ginger, costus root, poppy shell, and corydalis. Usage and dosage: oral. 4g (36 pills) at a time, 3 times a day.

[0086] Preparation of DSS solution: Weigh the corresponding DSS powder and add it to the animal's drinking water. Use a magnetic stirrer to stir until it is fully dissolved. Filter it through a vacuum filtration device equipped with a 0.22μm organic microporous filter membrane before use. Prepare it before use.

[0087] 2.2 Animal grouping and modeling

[0088] The mice were adaptively raised for 12 days until they were stable, and 9 mice injured in fighting were eliminated, and the remaining 97 mice were included in subsequent experiments. On the first day of modeling, the mice were weighed and numbered, and randomly divided into a normal group and a modeling group according to their weight. There were 12 mice in the normal group and 85 mice in the modeling group. The normal group drank animal drinking water, and the modeling group mice freely drank animal drinking water containing 3.0wt% DSS for 4 days to establish the UC model.

[0089] During the modeling period, the overall manifestations, fecal characteristics and bloody stools of the mice were observed, and their bodies were weighed at the same time every day to monitor changes in food and water intake. Specifically, the initial water and food weight was recorded on the first day of modeling. The remaining food and water weight in each cage was weighed at the same time every day from the second day to the fourth day of modeling. The weight of the previous day was subtracted and then divided by the number of mice in the cage. The obtained value was included in the statistics as the average daily food and water intake of a single mouse in the cage.

[0090] 2.3 Animal grouping and drug intervention

[0091] After 4 consecutive days of modeling, the DSS drinking water was stopped for the modeling group. All animals were allowed to drink the drinking water freely. The disease activity index (DAI) of mice was comprehensively evaluated according to the changes in body weight, stool characteristics, and blood in stool. The mice were divided into segments according to the DAI score, and randomly grouped according to body weight in each segment. The modeling group was divided into model control group (Model), Guchang Zhixie Pill group (GCZX), low-dose group (LSZX-L), medium-dose group (LSZX-M), and high-dose group (LSZX-H), with 17 mice in each group, and 12 mice in the normal group were used as the normal control group (Control). The mice in the normal and model control groups were given drinking water by gavage, and the other groups were given corresponding drugs by gavage, with a gavage volume of 15.00 mL / kg, once a day, for 7 consecutive days.

[0092] 2.4 Sample collection

[0093] After 7 days of continuous administration, samples were collected. The collection steps were as follows: one eyeball was removed with forceps, blood was dripped into a 1.5 mL centrifuge tube, and after standing at room temperature for 4 hours, the tube was centrifuged at 3000 r / min for 10 minutes, serum was drawn, and stored in a -80 ° C refrigerator. After blood collection, the mice were killed by cervical dislocation, the abdominal cavity was exposed after alcohol disinfection, the colon was gently peeled off, placed on an ice plate, the fat tissue on the surface of the colon was removed, and the colon was placed on a grid paper for photographing and measuring the length of the colon. Feces were taken from the lower part of the colon using sterile instruments and placed in a cryopreservation tube for rapid freezing with liquid nitrogen and then transferred to a -80 ° C refrigerator for storage. The feces in the intestine were removed with pre-cooled saline, and the colon was divided into 3 parts. 1.5 cm was taken from the middle section and placed in tissue fixative for fixation, and 3 mm was taken from the middle and lower sections and placed in electron microscope fixative. The remaining colon tissue was placed in a cryopreservation tube for rapid freezing with liquid nitrogen and then transferred to a -80 ° C refrigerator for storage.

[0094] 2.5 Observation indicators, methods and results

[0095] 2.5.1 Survival

[0096] In view of the characteristics of this experimental model, the survival status of mice was observed daily, and their survival status was described by survival curve and survival rate; the results are shown in Figure 1 and Table 1.

[0097] Table 1 Statistics of mouse survival rate after 7 days of administration

[0098] Group n(first day of administration) n(7 days after administration) Survival rate (%) Normal control group 12 12 100.00 Model control group 17 7 <![CDATA[41.18 ## ]]> Positive drug intestinal solidification and antidiarrhea group 17 8 47.06 Low dose group 17 11 64.71 Medium dose group 17 11 64.71 High dose group 17 8 47.06

[0099] Note: Compared with the normal control group, ## P<0.01.

[0100] The results showed (see Table 1 and Figure 1 ), the survival rates of mice in the normal control group and the model control group were 100% and 41.18%, respectively. Compared with the normal control group, the survival rate of mice in the model control group was significantly reduced (P<0.01). The survival rates of mice in the Guchang Zhixie Pills group, LSZX-L group, LSZX-M group, and LSZX-H group were 47.06%, 64.71%, 64.71%, and 47.06%, respectively. Compared with the model control group, the Guchang Zhixie Pills and the administration group of the present invention can reduce the number of mouse deaths.

[0101] 2.5.2 Characterization Observation

[0102] Observe and describe the spirit, eyesight, posture, activity status, hair color and luster, nails, skin around the lips and anus, and defecation of the mice during the entire process of modeling and drug administration.

[0103] The mice in the normal control group were in good mental state, with sharp eyes, normal body shape, flexibility and activity, black, shiny and smooth hair, ruddy skin around the lips, and normal skin around the anus. Compared with the normal control group, the mice in the model control group were in poor mental state, with dull eyes, hunched backs, reduced activity and slow movements, messy and dull hair, thin and light-colored nails, and weak stretching, pale lips, dark purple skin around the anus, swelling and moisture, hair loss, mucus and blood in the stool, and large and foul-smelling stools.

[0104] Compared with the model control group, most mice in each drug-treated group showed improved spirits, bright eyes, normal posture, flexibility, smooth black hair, clean paws, full and rosy pads, rosy lips, and improved perianal mucus and bloody stools, swelling, and hair loss. The improvement in mouse hair was most obvious in the drug-treated group of the present invention. In addition, close observation during the drug-treated period revealed that the bloody stools in the naked eyes of individual mice in the drug-treated group of the present invention disappeared after one day of drug administration, and individual mice whose weight loss exceeded 20% of their initial weight were still alive and in good spirits. Figure 2 .

[0105] 2.5.3 Stool characteristics and blood in stool

[0106] Observe the stool characteristics and blood in the stool every day. If there is no visible blood in the stool, use an occult blood test kit for detection. The interpretation standards for fecal occult blood test results are as follows: ① Occult blood negative: no color within 2 minutes after adding the reagent; ② Occult blood +: the liquid gradually changes from light green to green 10 seconds after adding the reagent; ③ Occult blood ++: after adding the reagent, it initially appears light green and gradually becomes obvious blue-brown; ④ Occult blood +++: it immediately turns blue-brown after adding the reagent and gradually turns dark brown; ⑤ Occult blood ++++: it immediately turns blue-black-brown after adding the reagent.

[0107] The stool of the normal control group was brown-yellow rice-like, and the fecal occult blood was negative; compared with the normal control group, the model control group had mainly loose stools, and the occult blood was +++~++++.

[0108] Compared with the model control group, the Guchang Zhixie Pills group showed normal stool, soft stool, loose stool, mainly occult blood +, occasionally occult blood ++, and the stool characteristics and blood in the stool were significantly improved; the low, medium and high dose groups of the present invention showed normal stool, soft stool, occult blood negative or occult blood +, and the stool condition was significantly improved. The low dose group occasionally had loose stools, and the high dose group occasionally had occult blood ++, see Table 2 and Table 3.

[0109] Table 2 Statistics of fecal characteristics of mice after 7 days of administration

[0110] Group n Normal stool (%) Soft stool (%) Loose stool (%) Normal control group 12 12(100.00) 0(0.00) 0(0.00) Model control group 7 0(0.00) 0(0.00) <![CDATA[7(100.00) ## ]]> Positive drug intestinal solidification and antidiarrhea group 8 3(37.50) 2(25.00) <![CDATA[3(37.50) * ]]> Low dose group 11 4(36.36) 4(36.36) <![CDATA[3(27.27) ** ]]> Medium dose group 11 5(45.45) 6(54.55) <![CDATA[0(0.00) ** ]]> High dose group 8 7(87.50) 1(12.50) <![CDATA[0(0.00) ** ]]>

[0111] Note: Compared with the normal control group, ## P<0.01; compared with the model control group, * P<0.05, ** P<0.01; compared with the normal control group, the model control group Z=4.243, P=0.000; compared with the model control group, the positive drug intestinal solidifying and antidiarrhea group Z=-2.427, P=0.040, the low-dose group Z=-2.819, P=0.008, the medium-dose group Z=-3.706, P=0.000, and the high-dose group Z=-3.706, P=0.000.

[0112] Table 3 Statistics of bloody stools in mice 7 days after administration

[0113] Group n Negative(%) +(％) ++(％) +++(％) ++++(％) Normal control group 12 11(91.67) 0(0.00) 1(8.33) 0(0.00) 0(0.00) Model control group 7 0(0.00) 0(0.00) 0(0.00) 5(71.43) <![CDATA[2(28.57) ## ]]> Positive drug intestinal solidification and antidiarrhea group 8 1(12.50) 5(62.50) 2(25.00) 0(0.00) <![CDATA[0(0.00) ** ]]> Low dose group 11 5(45.45) 6(54.55) 0(0.00) 0(0.00) <![CDATA[0(0.00) ** ]]> Medium dose group 11 3(27.27) 6(54.55) 2(18.18) 0(0.00) <![CDATA[0(0.00) ** <!-- 7 -->]]> High dose group 8 5(62.50) 2(25.00) 1(12.50) 0(0.00) <![CDATA[0(0.00) ** ]]>

[0114] Note: Compared with the normal control group, ## P<0.01; compared with the model control group, ** P<0.01; compared with the normal control group, the model control group Z=3.997, P=0.000; compared with the model control group, the positive drug intestinal solidifying and antidiarrhea group Z=-3.369, P=0.000, the low-dose group Z=-3.632, P=0.000, the medium-dose group Z=-3.602, P=0.000, and the high-dose group Z=-3.369, P=0.000.

[0115] 2.5.4 Weight

[0116] The mice were weighed and recorded at the same time every day; the results are shown in Table 4 and Figure 3 From Table 4 and Figure 3 It can be seen that compared with the normal control group, the body weight of mice in the model control group was significantly reduced (P<0.01). Compared with the model control group, the body weight of mice in the Guchang Zhixie Pill group was significantly increased (P<0.05), the body weight of mice in the low-dose group was significantly increased (P<0.05), and the body weight of mice in the medium and high-dose groups showed an increasing trend.

[0117] Table 4 Statistics of mouse body weight before and after administration

[0118]

[0119] Note: Compared with the normal control group, ## P<0.01; compared with the model control group, * P<0.05

[0120] 2.5.5 Disease Activity Index Score

[0121] DAI = (weight loss% score + stool characteristics score + blood in stool score) / 3. The scoring standards are shown in Table 5.

[0122] Table 5 DAI scoring criteria

[0123] Weight loss% Stool characteristics Blood in stool Scoring 0 normal Occult blood negative 0 1-5％ Occult blood+ 1 6-10％ Soft stools Hidden Blood++ 2 11-15％ Occult blood+++ 3 >15％ Loose stools Gross blood in stool or occult blood++++ 4

[0124] The results are shown in Table 6 and Figure 4 It can be seen that compared with the normal control group, the DAI score of the model control group was significantly increased (P<0.01). Compared with the model control group, the DAI score of the Guchang Zhixie Pill group was significantly reduced (P<0.01), and the DAI scores of mice in the low, medium and high dose groups of the Chinese medicine of the present invention were significantly reduced (P<0.01).

[0125] Table 6 Statistics of DAI scores of mice after 7 days of administration

[0126] Group n DAI Rating Normal control group 12 0.06±0.19 Model control group 7 <![CDATA[3.33±0.67 ## <!-- 8 -->]]> Positive drug intestinal solidification and antidiarrhea group 8 <![CDATA[1.75±1.03 ** ]]> Low dose group 11 <![CDATA[1.33±1.00 ** ]]> Medium dose group 11 <![CDATA[1.43±0.45 ** ]]> High dose group 8 <![CDATA[0.75±0.61 ** ]]>

[0127] Note: Compared with the normal control group, ## P<0.01; compared with the model control group, ** P<0.01.

[0128] 2.5.6 Detection of inflammatory cytokines in colon tissue

[0129] Multiplex microsphere flow immunofluorescence luminescence method was used to detect inflammatory related cytokines:

[0130] Interleukin-1alpha (IL-1α), Interleukin-1beta (IL-1β), Interleukin-3 (IL-3), Interleukin-6 (IL-6), Interleukin-10 (IL-10), Interleukin-11 (IL-11), Interleukin-23 (IL-23), Interleukin-33 (IL-33), Interleukin-12p40 (IL-12p40), Interleukin-12p70 (IL-12p70), CC Motif chemokine ligand 2 (CC Motif chemokine ligand 2, CCL2), CC Motif chemokine ligand 3 (CCL3), CC Motif chemokine ligand 4 (CCL4), CXC Motif chemokine ligand 9 (CXCL9), CXC Motif chemokine ligand 10 (CXCL10), tumor necrosis factor-alpha (TNF-α), interferon-alpha (IFN-α), and vascular endothelial growth factor (VEGF).

[0131] The specific steps are as follows:

[0132] (1) Sample preparation: Prepare a mixture of protease and phosphatase inhibitors with PBS solution at a ratio of 1:100, take 300 μL and add it to a 2 mL centrifuge tube. At the same time, weigh 33.00 mg of colon tissue and add it to the tube. Cut the tissue on ice and add 6 2 mm steel balls. Grind it with a tissue grinder at 30 Hz for 1 min each time, with an interval of 30 s, for 5 to 6 times. After sufficient grinding, remove the steel balls and centrifuge the homogenate at 12,000 g, 4°C for 10 min. Transfer the supernatant to a new tube for testing.

[0133] (2) Protein concentration determination: Prepare protein standard solution according to the instructions, and use PBS solution to prepare standard curve solution; prepare working solution, calculate the amount of working solution, add 200 μL working solution to each well, mix reagent A and reagent B in the BCA kit at a ratio of 50:1, and place at room temperature; dilute the colon tissue homogenate supernatant with PBS at a certain ratio (15-20 times dilution depending on the situation); add standard and sample to 96-well plate, 20 μL per well, add 200 μL BCA working solution to each well, shake for 30 seconds to mix, and incubate at 37°C for 30 minutes; read the absorbance at 562 nm with an enzyme reader. Draw a standard curve and linear formula based on the actual concentration of the standard and the detection value, and calculate the sample detection protein concentration based on the absorbance value of the sample to be tested.

[0134] (3) Reagent preparation: Return the reagents to room temperature (20-25°C). Add 250 μL Assay Buffer to the vial of standard lyophilized powder, shake well and let stand for 10 min. Transfer an appropriate amount to a 1.5 ml centrifuge tube as the highest standard C7, and prepare the remaining standard solutions according to the instructions; dilute 25 mL 20× Wash Buffer with 475 mL deionized water to prepare 1× Wash Buffer solution.

[0135] (4) Add 100 μL of 1× Wash Buffer to each well and allow the plate to wet for 1 min at room temperature. Use a microplate vacuum filtration device to remove excess liquid and use a clean paper towel to absorb the wash buffer from the bottom of the plate.

[0136] (5) Add 25 μL of Assay Buffer to all wells; add 25 μL of standard to each standard well and 25 μL of each sample to the sample wells, and mix by pipetting.

[0137] (6) Vortex the mixed beads for 1 min to mix them evenly, then add 25 μL of mixed beads to each well. Seal the plate with a sealing film. Do not apply positive pressure when sealing the plate. Cover the plate with tin foil to avoid light, fix it on an orbital shaker, and incubate it at room temperature for 2 h at 500 rpm.

[0138] (7) After the incubation, use a microplate vacuum filtration device to absorb excess liquid, add 200 μL of 1× Wash Buffer to each well and wash for 1 min, filter the liquid, add 200 μL of 1× Wash Buffer again and wash for 1 min, and filter the liquid.

[0139] (8) Add 25 μL of detection antibodies to each well, seal the plate, and incubate at room temperature for 1 h at 500 rpm.

[0140] (9) Without filtering the liquid, add 25 μL SA-PE directly to each well, seal the plate, and incubate at room temperature for 30 min at 500 rpm. Then filter and wash twice with Wash Buffer as in step 7.

[0141] (10) Add 100 μL of 1× Wash Buffer to each well to resuspend the beads. Incubate at room temperature for 5 min at 500 rpm. Transfer the beads to the flow cytometer after pipetting several times. Repeat the above steps once. A total of 200 μL of liquid is obtained in each well for testing. Pipette 10 times before testing. TM The test was performed on a cell analyzer, and after data acquisition was completed, LEGENDplexTM data analysis software was used for data analysis.

[0142] The statistical results of cytokines related to colon tissue inflammation showed that compared with the normal control group, the levels of IL-1β, IL-3, IL-6, IL-11, TNF-α, CCL2, CCL3, CCL4, CXCL9, CXCL10, and VEGF in the model control group were significantly increased (P<0.05 or P<0.01), and IL-33 showed an increasing trend; IL-12p40, IL-12p70, IL-10, IL-23, and IFN-α were significantly decreased (P<0.01).

[0143] Compared with the model control group, the results of each drug-treated group are as follows:

[0144] In the Guchang Zhixie Pills group, IL-1β, IL-6, IL-11, and VEGF were significantly decreased (P<0.05 or P<0.01), and IL-3, TNF-α, CCL2, CCL3, CCL4, CXCL9, and CXCL10 showed a decreasing trend; IL-1α, IL-33, and IL-12p70 were significantly increased (P<0.05 or P<0.01), and IL-10, IL-23, and IFN-α showed an increasing trend.

[0145] In the low-dose group, IL-6, IL-11, CXCL10, and CCL2 were significantly decreased (P<0.05 or P<0.01), and IL-1β, IL-3, CCL3, CCL4, CXCL9, TNF-α, and VEGF showed a decreasing trend; IL-1α, IL-10, IL-23, IL-33, IL-12p70, and IFN-α were significantly increased (P<0.05 or P<0.01), and IL-12p40 showed an increasing trend.

[0146] In the medium-dose group, IL-6, IL-11, TNF-α, CXCL10, CCL4, IL-3, CCL2, CCL3, and CXCL9 were significantly decreased (P<0.05 or P<0.01), and IL-1β and VEGF showed a decreasing trend; IL-12p70, IL-23, IFN-α, and IL-10 were significantly increased (P<0.05 or P<0.01), and IL-12p40, IL-33, and IL-1α showed an increasing trend.

[0147] In the high-dose group, IL-1β, IL-3, IL-6, IL-11, TNF-α, CCL2, CCL3, CCL4, CXCL9, CXCL10, and VEGF were significantly decreased (P<0.01); IFN-α, IL-1α, IL-12p40, and IL-10 were significantly increased (P<0.05 or P<0.01), and IL-12p70, IL-23, and IL-33 showed an increasing trend (see Table 7, Table 8, Table 9, Table 10, Table 11, Table 12, Figure 5 .

[0148] Table 7 Statistics of cytokines related to inflammation in colon tissue (IL-1β, IL-3, IL-6)

[0149] Group n IL-1β (ng / mg) IL-3 (ng / mg) IL-6 (ng / mg) Normal control group 12 25.68±12.11 3.31±1.13 2.81±1.44 Model control group 7 <![CDATA[115.94±34.77 ## ]]> <![CDATA[11.20±8.60 ## ]]> <![CDATA[11.71±3.90 ## ]]> Positive drug intestinal solidification and antidiarrhea group 8 <![CDATA[62.56±25.90 ** ]]> 5.46±2.90 <![CDATA[5.59±2.60 ** ]]> Low dose group 11 100.84±78.45 8.92±10.49 <![CDATA[6.05±2.03 ** ]]> Medium dose group 11 78.71±46.94 <![CDATA[4.11±1.64 * ]]> <![CDATA[5.24±1.89 ** ]]> High dose group 8 <![CDATA[37.28±20.38 ** ]]> <![CDATA[3.78±1.74 ** ]]> <![CDATA[4.60±1.15 ** ]]>

[0150] Note: Compared with the normal control group, ## P<0.01; compared with the model control group, * P<0.05, ** P<0.01.

[0151] Table 8 Statistics of cytokines related to colonic tissue inflammation (IL-11, IL-33, TNF-α)

[0152] Group n IL-11 (ng / mg) IL-33 (ng / mg) TNF-α(ng / mg) Normal control group 12 20.32±7.89 179.28±64.05 8.21±2.16 Model control group 7 <![CDATA[49.94±12.62 ## ]]> 234.20±84.51 <![CDATA[19.09±7.13 ## <!-- 10 -->]]> Positive drug intestinal solidification and antidiarrhea group 8 <![CDATA[31.85±13.11 * ]]> <![CDATA[417.11±133.66 ** ]]> 14.36±4.79 Low dose group 11 <![CDATA[29.85±12.77 ** ]]> <![CDATA[415.59±93.37 ** ]]> 18.78±6.43 Medium dose group 11 <![CDATA[31.44±13.32 ** ]]> 279.29±100.99 <![CDATA[12.55±3.96 * ]]> High dose group 8 <![CDATA[27.56±11.81 ** ]]> 308.16±69.14 <![CDATA[8.53±3.03 ** ]]>

[0153] Note: Compared with the normal control group, ## P<0.01; compared with the model control group, * P<0.05, ** P<0.01.

[0154] Table 9 Statistics of cytokines related to colonic tissue inflammation (CCL2, CCL3, CCL4)

[0155] Group n CCL2 (ng / mg) CCL3 (ng / mg) CCL4 (ng / mg) Normal control group 12 26.90±7.09 72.24±29.80 105.82±47.65 Model control group 7 <![CDATA[84.83±29.79 ## ]]> <![CDATA[107.99±18.30 # ]]> <![CDATA[162.43±21.65 ## ]]> Positive drug intestinal solidification and antidiarrhea group 8 56.51±31.31 84.87±31.53 102.47±51.19 Low dose group 11 <![CDATA[54.37±12.04 * ]]> 112.97±36.11 148.78±60.50 Medium dose group 11 <![CDATA[46.82±16.01 * ]]> <![CDATA[76.50±31.58 * ]]> <![CDATA[104.20±44.65 ** ]]> High dose group 8 <![CDATA[35.96±16.71 ** ]]> <![CDATA[51.05±26.77 ** ]]> <![CDATA[67.40±35.36 ** ]]>

[0156] Note: Compared with the normal control group, # P<0.05, ## P<0.01; compared with the model control group, * P<0.05, **P<0.01.

[0157] Table 10 Statistics of cytokines related to colonic tissue inflammation (CXCL9, CXCL10, VEGF)

[0158]

[0159]

[0160] Note: Compared with the normal control group, # P<0.05, ## P<0.01; compared with the model control group, * P<0.05, ** P<0.01.

[0161] Table 11 Statistics of cytokines related to colonic tissue inflammation (IL-1α, IL-12p40, IL-12p70)

[0162] Group n IL-1α (ng / mg) IL-12p40 (ng / mg) IL-12p70 (ng / mg) Normal control group 12 0.76±0.31 3.65±1.01 36.40±9.07 Model control group 7 0.76±0.35 <![CDATA[2.17±0.35 ## ]]> <![CDATA[15.93±4.10 ## ]]> Positive drug intestinal solidification and antidiarrhea group 8 <![CDATA[2.53±0.72 ** ]]> 2.11±0.16 <![CDATA[31.62±17.97 * ]]> Low dose group 11 <![CDATA[1.80±0.64 ** ]]> 2.49±0.20 <![CDATA[27.31±10.07 * ]]> Medium dose group 11 1.10±0.40 2.27±0.18 <![CDATA[25.34±3.87 ** ]]> High dose group 8 <![CDATA[1.65±0.55 * ]]> <![CDATA[2.53±0.72 * ]]> 25.84±12.05

[0163] Note: Compared with the normal control group, ## P<0.01; compared with the model control group, * P<0.05, ** P<0.01.

[0164] Table 12 Statistics of cytokines related to colonic tissue inflammation (IL-10, IL-23, IFN-α)

[0165] Group n IL-10 (ng / mg) IL-23 (ng / mg) IFN-α (ng / mg) Normal control group 12 4.85±2.42 95.36±39.24 25.22±11.43 Model control group 7 <![CDATA[1.89±1.87 ## ]]> <![CDATA[55.34±14.57 ## ]]> <![CDATA[12.40±3.95 ## ]]> Positive drug intestinal solidification and antidiarrhea group 8 4.66±3.54 58.81±17.82 15.93±6.10 Low dose group 11 <![CDATA[9.90±4.73 ** ]]> <![CDATA[102.35±24.00 ** ]]> <![CDATA[33.82±4.25 ** ]]> Medium dose group 11 <![CDATA[5.39±2.20 * ]]> <![CDATA[102.92±21.79 ** ]]> <![CDATA[35.90±12.23 ** ]]> High dose group 8 <![CDATA[6.09±2.67 * ]]> 100.21±45.71 <![CDATA[35.39±8.67 ** ]]>

[0166] Note: Compared with the normal control group, ## P<0.01; compared with the model control group, * P<0.05, ** P<0.01.

[0167] 2.5.7 Observation of the gross morphology of the colon

[0168] The colon's gross length, color, damage, congestion, intestinal wall edema, adhesion, etc. were observed; the colon length from the ileocecal part to the middle of the rectum end 1 cm away from the anus was measured, and quantitative statistical analysis was performed.

[0169] The gross morphological observation of the colon showed that the colon of the mice in the normal control group was light red, the feces in the intestine were formed into particles, and the colon was relaxed. Compared with the normal control group, the colon of the mice in the model control group was grayish white, the feces in the intestine were not formed, the intestinal wall was thinner, there was inflation in the intestine, the colon length was significantly shortened (P<0.05), and the colon was severely damaged. Figure 6 .

[0170] Compared with the model control group, the colon of each drug-treated group regained its blood color, the stool was formed, the relaxation force was restored, and the colon damage was alleviated to varying degrees. Among them, the colon length of the medium and high dose groups of the present invention showed an increasing trend, as shown in Table 13. Figure 7 .

[0171] Table 13 Statistics of mouse colon length after 7 days of administration

[0172]

[0173]

[0174] Note: Compared with the normal control group, # P<0.05.

[0175] 2.5.8 Colonic histopathological examination

[0176] Hematoxylin-eosin (HE) staining was used for histopathological examination.

[0177] (1) Fixation and trimming: The colon tissue was fixed in 4% paraformaldehyde for 48 h, then trimmed into intestinal loops 4 to 6 mm high and placed in an embedding box.

[0178] (2) Dehydration and transparency: Rinse with running water for 30 minutes to remove the residual fixative, then place the tissue in a fully automatic vacuum dehydrator and dehydrate the tissue using 75% ethanol, 85% ethanol, 95% ethanol, and 100% ethanol in sequence. After dehydration, place the tissue in xylene to make it transparent.

[0179] (3) Wax embedding: Immerse the tissue in melted paraffin. Embed the tissue after the paraffin has completely penetrated the tissue. Pour melted paraffin into the metal mold, place the tissue, cover the embedding box, and inject paraffin again. Place the mold on a cold table and remove the wax block from the mold after it solidifies.

[0180] (4) Slice preparation: Use a tissue slicer to cut the tissue wax block into thin slices with a thickness of 4 μm. Place the slices in 45°C warm water for development and then remove the slices. Place the slices in a 70°C forced air drying oven for 2 h.

[0181] (5) Dewaxing: After dewaxing with xylene, the tissue sections were placed in 100% ethanol, 95% ethanol, and 75% ethanol for 5 min each, and then rinsed with pure water for 2 min.

[0182] (6) Staining: Immerse in hematoxylin staining jar for 6 min, wash with water, differentiate in 1% hydrochloric acid ethanol for a few seconds, wash with water, place in 1% ammonia solution to blue for 30 s, wash with water, and place in eosin staining solution for 20 s.

[0183] (7) Dehydration, transparency, and sealing: The dehydration and transparency steps are the same as (2), and the slides are sealed with neutral gum and air-dried.

[0184] (8) Observe and collect images under an optical microscope.

[0185] HE staining images of colon tissue showed that the colon mucosal surface of the normal control group was continuous, the crypts were arranged regularly, the goblet cells were normal in morphology, the colon tissue structure was intact, and no inflammatory cell infiltration was observed; compared with the normal control group, the colon mucosal surface of the model control group was discontinuous, the crypts were atrophied, deformed, branched, irregularly arranged, the base was thickened, the crypts were lost, the number of goblet cells decreased, the integrity of the tissue structure was significantly damaged, and the inflammatory cell infiltration and mucosal thickening were obvious; compared with the model control group, each drug-treated group showed different degrees of improvement in tissue pathological morphology and inflammatory infiltration, as shown in Figure 2. Figure 8 .

[0186] 2.5.9 Colonic histopathological scoring

[0187] The HE staining images of colon tissue were analyzed, and the histopathological evaluation items were determined according to the relevant guidelines for ulcerative colitis. The integrity of tissue structure, inflammatory infiltration, and thickness of the colon mucosal layer were observed. The following scoring criteria were formulated based on the performance of the normal control group and model control group mice in this experiment. The sum of each item was used as the total score of colon tissue pathology for statistical analysis (see Table 14).

[0188] The thickness of the colon mucosal layer includes the length from the mucosal epithelium, the mucosal lamina propria to the muscularis mucosa. The measurement method for quantitative analysis is as follows: each intestinal loop is divided into 5 regions, and the thickness of the mucosal layer at the middle of each region is measured to represent the average thickness of the region. The average thickness of the 5 regions is taken as the representative value of the mucosal layer thickness of the sample and included in the statistics.

[0189] Table 14 Pathological scoring criteria

[0190] Degree of structural damage The degree of inflammatory infiltration Mucosal layer thickness (μm) Scoring Normal epithelial cell morphology No inflammatory infiltration <250 0 Small area of ​​goblet cell loss Small infiltration at the base of the crypt 251-280 1 Extensive goblet cell loss Extensive infiltration of the crypt base 281-320 2 Small area of ​​crypt loss Invasion reaches the muscularis mucosa 321-350 3 Extensive loss of crypts Invasion reaches the submucosal layer >350 4

[0191] The results of colon tissue pathological structure showed that the colon epithelium in the normal control group had normal morphology; compared with the normal control group, the colon tissue crypts in the model control group were lost in large or small areas, and the degree of structural damage was more serious.

[0192] Compared with the model control group, the loss of goblet cells and a small area of ​​crypts was the main feature in the Guchang Zhixie Pills group, the loss of goblet cells and a small area of ​​crypts was the main feature in the low- and high-dose groups, with occasional crypt loss, and the loss of goblet cells and a small area of ​​crypts was the main feature in the medium-dose group of the present invention. The degree of damage to the colon tissue structure was alleviated in each medication group, as shown in Table 15.

[0193] Table 15 Colon histopathological score statistics after 7 days of administration (degree of structural damage)

[0194]

[0195] Note: Compared with the normal control group, ## P<0.01; compared with the model control group, *P<0.05; compared with the normal control group, the model control group Z=3.964, P=0.000; compared with the model control group, the positive drug intestinal solidifying and antidiarrhea group Z=-1.152, P=0.281, the low-dose group Z=-1.636, P=0.126, the medium-dose group Z=-1.956, P=0.069, and the high-dose group Z=-2.414, P=0.021.

[0196] The results of colon tissue pathological inflammatory infiltration showed that there was no obvious inflammatory infiltration in the colon tissue of the normal control group; compared with the normal control group, the inflammatory infiltration in the model control group reached deep into the submucosal layer, and the degree of inflammatory infiltration was significantly aggravated.

[0197] Compared with the model control group, the inflammatory infiltration in the Guchang Zhixie Pills group was infiltrated from the crypt base, the mucosal muscular layer to the submucosal layer to varying degrees, and the degree of inflammatory infiltration tended to be alleviated. The low, medium and high dose groups were mainly infiltrated to varying degrees at the crypt base, and part of the low dose group infiltrated to the submucosal layer, and a small number of the medium and high dose groups infiltrated to the mucosal muscular layer. The degree of inflammatory infiltration of the colon tissue in each dose group of the present invention was significantly alleviated, as shown in Table 16.

[0198] Table 16 Colon histopathological score statistics (inflammatory infiltration degree) after 7 days of administration

[0199]

[0200] Note: Compared with the normal control group, ## P<0.01; compared with the model control group, **P<0.01; compared with the normal control group, the model control group Z=3.997, P=0.000; compared with the model control group, the positive drug intestinal solidifying and antidiarrhea group Z=-2.103, P=0.054, the low-dose group Z=-2.010, P=0.069, the medium-dose group Z=-3.465, P=0.000, and the high-dose group Z=-3.117, P=0.001.

[0201] The statistical results of colon mucosal thickness showed that compared with the normal control group, the mucosal layer of the model control group was significantly thickened (P<0.01). Compared with the model control group, the mucosal layer thickness of the Guchang Zhixie Pill group was significantly reduced (P<0.05), and the mucosal thickness of the low, medium and high dose groups of the present invention was reduced, among which the low and high dose groups could significantly reduce the mucosal layer thickness (P<0.05 or P<0.01), see Table 17, Fig. 9 .

[0202] Table 17 Statistics of colon mucosal thickness after 7 days of administration

[0203] Group n Colon mucosal thickness (μm) Normal control group 12 207.75±34.79 Model control group 7 <![CDATA[378.57±84.60 ## ]]> Positive drug intestinal solidification and antidiarrhea group 8 <![CDATA[288.13±56.49 * ]]> Low dose group 11 <![CDATA[290.00±90.89 * ]]> Medium dose group 11 304.55±108.93 High dose group 8 <![CDATA[242.00±49.90 ** ]]>

[0204] Note: Compared with the normal control group, ## P<0.01; compared with the model control group, * P<0.05, ** P<0.01.

[0205] The total pathological score of colon tissue showed that compared with the normal control group, the total pathological score of the model control group was significantly increased (P<0.01). Compared with the model control group, the total pathological score of the Guchang Zhixie Pill group was significantly reduced (P<0.05). The total pathological scores of the low, medium and high dose groups of the present invention were reduced, among which the medium and high dose groups could significantly reduce the total pathological scores (P<0.01), as shown in Table 18. Fig.10 .

[0206] Table 18 Total colon pathology scores after 7 days of administration

[0207] Group n Total pathological score Normal control group 12 0.17±0.39 Model control group 7 <![CDATA[9.86±1.86 ## ]]> Positive drug intestinal solidification and antidiarrhea group 8 <![CDATA[6.88±2.42 * ]]> Low dose group 11 6.18±4.51 Medium dose group 11 <![CDATA[6.00±2.37 ** ]]> High dose group 8 <![CDATA[4.38±2.82 ** ]]>

[0208] Note: Compared with the normal control group, ## P<0.01; compared with the model control group, * P<0.05, ** P<0.01.

[0209] 2.5.10 Transmission electron microscopy observation of colon

[0210] (1) Fixation and trimming: When fixing tissue, reduce external force and fix the tissue in pre-cooled electron microscopy fixative in a 4°C refrigerator. Rinse with 0.1M phosphate buffer PB (PH7.4) three times, 15 minutes each time.

[0211] (2) Post-fixation: Fix with 1% osmium acid prepared in 0.1 M phosphate buffer PB (PH 7.4) at room temperature (20°C) for 2 h, and then rinse with 0.1 M phosphate buffer PB (PH 7.4) three times, each time for 15 min.

[0212] (3) Dehydration: The tissue was dehydrated in ascending order by placing it in ethanol of different concentrations for 20 min each time, and then in 100% acetone twice for 15 min each time.

[0213] (4) Infiltration, embedding, and polymerization: Use acetone and 812 embedding agent for infiltration. Insert the sample into the embedding plate and place it in a 37°C oven overnight. Place the embedded plate in a 60°C oven for polymerization for 48 hours, and remove the resin block for later use.

[0214] (5) Sectioning: Cut the resin block into 60-80 nm ultrathin sections using an ultrathin microtome and scoop out the sections using a 150-mesh copper mesh.

[0215] (6) Staining: Place the copper mesh sections in a 2% uranyl acetate saturated ethanol solution for staining for 8 min (protected from light); wash three times with 70% ethanol; wash three times with ultrapure water; stain for 8 min with a 2.6% lead citrate solution away from carbon dioxide; wash three times with ultrapure water, dry slightly with filter paper, place the copper mesh sections in a copper mesh box, and dry overnight at room temperature.

[0216] (7) Observe, collect images and analyze them under a transmission electron microscope.

[0217] Transmission electron microscopy of the colon showed that the apical microvilli of the intestinal epithelial cells of mice in the normal control group were of moderate length and thickness and were arranged neatly. Clear tight junction complexes composed of tight junctions, intermediate junctions, and desmosomes were visible between cells. The morphology and structure of intracellular organelles were normal, and the cell morphology and structure were intact. The mucin granules in the goblet cells were of moderate size, evenly and densely distributed, and the morphology and structure of organelles were normal, and the cell morphology and structure were intact.

[0218] Compared with the normal control group, the apical microvilli of intestinal epithelial cells of mice in the model control group were of different lengths, broken, fallen off, and arranged irregularly. Tight junctions were visible between cells, the intermediate junction structure was fuzzy, the connection was loose, the desmosome fibers were reduced or disappeared, the tension microfilaments of the tight junction complex were reduced, the intercellular space was significantly widened, the density of mitochondrial membrane and matrix was deeper, the cristae were expanded, the damage was serious, the rough endoplasmic reticulum was expanded, the surface ribosomes were degranulated, the Golgi apparatus was proliferated and hypertrophied, autophagic lysosomes were partially present, and the cell morphology and structure were obviously destroyed; the colonic goblet cells had pyknosis, condensed cytoplasm, condensed nuclear chromatin, significantly reduced number of mucin granules, irregular shape, scattered distribution, obvious swelling of mitochondria, fuzzy and damaged mitochondrial membrane structure, more matrix overflow, broken and disappeared cristae, showing obvious apoptotic changes, and obvious destruction of cell morphology and structure.

[0219] Compared with the model control group, the microvilli at the top of the intestinal epithelial cells of the mice in each drug-treated group were arranged more neatly, with uniform length and thickness, and occasional breaks. The tight junctions between cells and the intermediate junction structure were clear and tightly connected. The desmosome filaments increased, the tension filaments of the tight junction complex increased, the intercellular space was narrowed, the tight junction complex structure was restored to be complete and clear, the density of the mitochondrial membrane and matrix was uniform and moderate, the damage was reduced, the ribosomes attached to the surface of the rough endoplasmic reticulum, the damage to the Golgi apparatus was reduced, no obvious autophagy lysosomes were observed, and the cell morphology and structure returned to normal; the size of the goblet cell body was restored, the cytoplasm density was uniform and moderate, the cell nucleus returned to normal, the number of mucin granules increased significantly, and gathered in the cup, the swelling of the mitochondria was reduced, the mitochondrial membrane and cristae structure were complete and clear, the matrix was relatively uniform, the apoptosis phenomenon was not obvious or the degree of apoptosis was reduced, and the cell morphology and structure returned to normal. Among them, the morphology and structure of goblet cells and intestinal epithelial cells in the Guchang Zhixie Pill group and the low, medium and high doses of the present invention were significantly improved in all aspects, and the high dose group had the most obvious improvement and was close to the submicroscopic structure of the colon tissue in the normal control group, as shown in Figure 2. Fig.11 .

[0220] 2.6 Statistical methods

[0221] SPSS 20.0 was used for statistics, and the data were expressed as mean ± standard deviation. If the data conform to normal distribution, the Student's t test was used. If the data do not conform to normal distribution, nonparametric tests were used. The Mann-Whitney test was used for rank data. P<0.05 was considered statistically significant.

Claims

1. A Chinese medicine composition, which is composed of the following components in parts by weight: 5-15 parts of Codonopsis pilosula, 2-6 parts of Coptis chinensis, 5-15 parts of stir-fried white peony root, 5-15 parts of Poria cocos, 5-15 parts of stir-fried Atractylodes macrocephala, 3-9 parts of Scutellaria baicalensis, 3-9 parts of costusroot, 5-15 parts of Angelica sinensis, 5-15 parts of Pueraria root, 5-15 parts of Prunus mume, 5-15 parts of Portulaca oleracea, and 2-6 parts of roasted Licorice root.

2. The Chinese medicine composition according to claim 1, characterized in that: The Chinese medicine composition consists of the following components in parts by mass: 7.5 parts of Codonopsis pilosula, 3 parts of Coptis chinensis, 6 parts of stir-fried white peony root, 7.5 parts of Poria cocos, 6 parts of stir-fried Atractylodes macrocephala, 4.5 parts of Scutellaria baicalensis, 4.5 parts of Costusroot rhizome, 6 parts of Angelica sinensis, 6 parts of Pueraria root, 7.5 parts of Prunus mume, 7.5 parts of Portulaca oleracea, and 3 parts of roasted Licorice root.

3. The Chinese medicine composition according to claim 1 or 2, characterized in that: The Chinese medicine composition is a water decoction.

4. Use of the Chinese medicine composition according to any one of claims 1 to 3 in the preparation of a product for preventing and / or treating ulcerative colitis.

5. The use according to claim 4, characterized in that: The product is a drug or a drug preparation.

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