Traditional Chinese medicine composition for treating inflammatory bowel disease as well as preparation method and application thereof

Abstract

The invention discloses a traditional Chinese medicine composition for treating inflammatory bowel disease as well as a preparation method and application thereof, and belongs to the technical field of medicines. The traditional Chinese medicine composition is prepared from the following components in parts by mass: 15-17 parts of radix pulsatillae, 10-12 parts of cortex fraxini, 10-12 parts of wine-processed rhizoma coptidis, 30-32 parts of cortex phellodendri, 15-17 parts of radix paeoniae alba, 10-12 parts of radix angelicae sinensis, 10-12 parts of radix aucklandiae and 10-12 parts of liquorice root. The traditional Chinese medicine composition provided by the invention is prepared from natural medicines, has low side effects, and provides a new choice for rehabilitation treatment and prevention of patients with inflammatory bowel diseases.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and more specifically to a Chinese medicine composition for treating inflammatory bowel disease, and a preparation method and application thereof. Background Art

[0002] Inflammatory bowel disease is a group of chronic and recurrent diseases characterized by intestinal inflammation, mainly including ulcerative colitis and Crohn's disease. These diseases not only seriously affect the digestive tract, causing symptoms such as abdominal pain, diarrhea, and mucus, pus and blood in the stool, but may also involve inflammatory lesions of extraintestinal organs such as joints and internal organs. The causes of inflammatory bowel disease involve genetics, immune response, environmental factors and intestinal microbial imbalance, but the specific mechanism is not yet fully understood. In recent years, the increase in the incidence of inflammatory bowel disease is closely related to changes in lifestyle and environmental factors.

[0003] Currently, the drugs used to treat inflammatory bowel disease include 5-aminosalicylic acid drugs, corticosteroids, immunosuppressants and biological agents. 5-aminosalicylic acid drugs are used to treat mild to moderate inflammatory bowel disease, and corticosteroids are used to treat moderate to severe inflammatory bowel disease, mainly for rapid control of inflammation and relief of symptoms. Immunosuppressants such as azathioprine and 6-mercaptopurine are mainly used to maintain remission and reduce the use of steroids, and are secondarily used for patients with severe illness or who are ineffective with other treatments. Biological agents targeting specific immune response pathways, such as anti-TNF-α antibodies such as infliximab and adalimumab, are suitable for patients with moderate to severe inflammatory bowel disease. However, long-term use of existing drugs has potential side effects. Summary of the invention

[0004] In view of the above problems, the present invention provides a Chinese medicine composition for treating inflammatory bowel disease, and a preparation method and application thereof. The Chinese medicine composition provided by the present invention is composed of natural medicines and has low side effects, providing a new option for the rehabilitation treatment and prevention of patients with inflammatory bowel disease.

[0005] The first object of the present invention is to provide a traditional Chinese medicine composition for treating inflammatory bowel disease, which is prepared from the following components in parts by mass: 15 to 17 parts of Pulsatilla scutellariae, 10 to 12 parts of Qinpi, 10 to 12 parts of Coptis chinensis, 30 to 32 parts of Phellodendron amurense, 15 to 17 parts of White Peony Root, 10 to 12 parts of Angelica sinensis, 10 to 12 parts of Costusroot Root and 10 to 12 parts of Licorice.

[0006] For example, Pulsatillae is 15, 16 or 17 parts, Cortex Fraxini is 10, 11 or 12 parts, Rhizoma Coptidis is 10, 11 or 12 parts, Cortex Phellodendri is 30, 31 or 32 parts, Radix Paeoniae Alba is 15, 16 or 17 parts, Radix Angelicae Sinensis is 10, 11 or 12 parts, Radix Aucklandiae is 10, 11 or 12 parts, and Radix Glycyrrhizae is 10, 11 or 12 parts. However, it is not limited to the listed values, and other values ​​not listed in the above numerical range are also applicable, so they will not be repeated here.

[0007] In a preferred embodiment of the present invention, it is prepared from the following components in parts by mass: 15 parts of Pulsatilla striata, 10 parts of Qinpi, 10 parts of Coptis chinensis, 30 parts of Phellodendron amurense, 15 parts of White Peony Root, 10 parts of Angelica sinensis, 10 parts of Aucklandia lappa and 10 parts of Licorice.

[0008] The second object of the present invention is to provide a method for preparing the above-mentioned Chinese medicine composition for treating inflammatory bowel disease, comprising the following steps:

[0009] A mixture is obtained by mixing Pulsatilla striata, Cortex qinpi, Rhizoma coptidis, Cortex phellodendri, Radix paeoniae alba, Radix angelicae sinensis, Radix aucklandiae and Radix glycyrrhizae, and then soaking the mixture in water and decocting it for 3 to 4 times with a slow fire. After the decocting is completed, the decoction is combined, filtered and concentrated, and the pH is adjusted to 5 to 6, and the mixture is subjected to post-treatment after standing and centrifugation. For example, the pH is adjusted to 5, 5.2, 5.4, 5.6, 5.8 or 6, etc., but it is not limited to the listed values. Other values ​​not listed in the above numerical range are also applicable, and they will not be repeated here.

[0010] The present invention adjusts the pH value to 5-6, which can help the traditional Chinese medicine composition maintain the best stability and activity of the medicinal components, and at the same time, standing still is more conducive to dissolving and releasing the medicinal components into the solution.

[0011] In a preferred embodiment of the present invention, the ratio of the mixture to water is 110 g to 123 g: 1000 mL. For example, the ratio of the mixture to water is 110 g: 1000 mL, 113 g: 1000 mL, 116 g: 1000 mL, 119 g: 1000 mL or 123 g: 1000 mL, etc., but is not limited to the listed values. Other values ​​not listed in the above numerical range are also applicable, and will not be repeated here.

[0012] In a preferred embodiment of the present invention, the soaking time is 1.5h to 2h. For example, the soaking time is 1.5h, 100min, 110min or 2h, etc., but it is not limited to the listed values. Other values ​​not listed in the above numerical range are also applicable, and they will not be repeated here.

[0013] In a preferred embodiment of the present invention, the decoction time is 30 minutes to 40 minutes. For example, the decoction time is 30 minutes, 32 minutes, 34 minutes, 36 minutes, 38 minutes or 40 minutes, etc., but it is not limited to the listed values. Other values ​​not listed in the above numerical range are also applicable, and they will not be repeated here.

[0014] In a preferred embodiment of the present invention, the standing time is 12 hours to 14 hours. For example, the standing time is 12 hours, 12.5 hours, 13 hours, 13.5 hours, 14 hours, etc., but it is not limited to the listed values. Other values ​​not listed in the above value range are also applicable, and they will not be repeated here.

[0015] The third object of the present invention is to provide the use of the above-mentioned traditional Chinese medicine composition for treating inflammatory bowel disease in the preparation of traditional Chinese medicine preparations for treating inflammatory bowel disease.

[0016] In a preferred embodiment of the present invention, the Chinese medicine composition is combined with pharmaceutically acceptable excipients to prepare a Chinese medicine preparation.

[0017] In a preferred embodiment of the present invention, the dosage form of the Chinese medicine preparation is decoction, granules, mixture, tablets, capsules, pills, powders, granules, syrups, microcapsules or pastes.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The traditional Chinese medicine composition prepared by the present invention consists of 15 to 17 parts of Pulsatilla scutellariae, 10 to 12 parts of Qinpi, 10 to 12 parts of Coptis chinensis, 30 to 32 parts of Phellodendron chinense, 15 to 17 parts of White Peony Root, 10 to 12 parts of Angelica sinensis, 10 to 12 parts of Costusroot lonicera and 10 to 12 parts of Licorice. As a natural medicine combination preparation, the traditional Chinese medicine composition of the present application has lower side effects on the human body than traditional medicines and is suitable for long-term use.

[0020] The Chinese medicine composition provided by the present invention has the effects of clearing away heat and detoxicating, cooling blood and stopping dysentery according to the theory of traditional Chinese medicine. Among them, Phellodendron chinense is bitter and cold in nature and is good at detoxifying and treating sores. At the same time, it is good at clearing the dampness and heat in the lower jiao, and is good at clearing the dampness and heat in the lower jiao. Combined with the characteristics of intestinal local administration, Phellodendron chinense is used as the monarch medicine, which can not only enhance the effect of clearing the dampness and heat in the lower jiao, but also attract and retain various medicines in the lower jiao to prolong the local drug effect. Pulsatilla striata is bitter and cold and enters the blood, Coptis chinensis is bitter and cold, and it dries dampness and thickens the intestines, and Cortex qin is bitter and astringent and stops dysentery. At the same time, the three medicines all have excellent detoxification and dysentery stopping effects, and they are used as ministers to assist Phellodendron chinense to enhance its effect of clearing heat and stopping dysentery; White peony root is bitter, sour and slightly cold in nature and flavor, and the "Ben Jing" says that it: it is mainly used for abdominal pain caused by evil qi, removes blood stasis, breaks up hard masses, treats cold and heat hernia, relieves pain, promotes urination, and invigorates qi. Recurrent inflammatory bowel disease is prone to postnatal nutritional ulceration, which leads to spleen weakness and liver abuse, making abdominal pain and diarrhea worse. White peony root is bitter to dry dampness and sour to astringe, and is good at calming the liver and relieving pain. Therefore, it is used as a ministerial drug for clinical pathogenesis and patient symptoms to strengthen the efficacy of this prescription in a targeted manner. With the help of angelica and costus root to promote blood circulation and promote qi circulation, the coordination of qi and blood and clearing away heat and drying dampness can be carried out simultaneously, so that the stool pus will heal itself when the blood circulation is reached, and the heaviness in the back will be removed by regulating qi. Licorice is used to detoxify and reconcile various medicines, and the patient's intestinal discomfort caused by drug stimulation such as excessive heat clearing is alleviated, thereby reducing damage to the body. The Chinese medicine composition thus formed is suitable for treating various inflammatory bowel diseases of the left colon and rectum caused by intestinal dampness and heat, including inflammatory bowel diseases such as ulcerative colitis and Crohn's disease. By the mode of rectal administration, the drug is directly acted on the local part of the left colon and rectum, and its effect of clearing away heat and detoxifying, cooling blood and stopping dysentery can be more directly exerted, thereby alleviating the symptoms of inflammatory bowel disease. The Chinese medicine composition of the present invention adopts the principle of compatibility of monarch, minister, assistant and guiding ingredients, and will not cause problems such as osteoporosis and immunosuppression caused by corticosteroids, or infection and liver damage caused by immunosuppressants, thereby reducing the risk of side effects that may be caused by the Chinese medicine composition to a certain extent.

[0021] The Chinese medicine composition prepared by the present invention can achieve the following effects in the treatment of inflammatory bowel disease: (1) reduce inflammation and abdominal pain, promote intestinal mucosal healing, and reduce the inflammatory response of the colon and rectum by clearing away heat and detoxifying, thereby relieving a series of inflammation-related clinical manifestations such as abdominal pain in patients. (2) By reducing the inflammatory response, reducing the scope of inflammation and the depth of the lesion, it can exert its good effects of cooling blood, stopping bleeding and stopping diarrhea. (3) By reducing inflammation and repairing the intestinal mucosa, the frequency of diarrhea can be reduced to a certain extent. (4) By relieving the symptoms of abdominal pain, diarrhea, and mucus and bloody stools, the quality of life of patients can be improved, and the living and economic burden of patients can be reduced.

[0022] The Chinese medicine composition prepared by the present invention comprises a variety of effective ingredients, such as phytosterols including β-sitosterol and stigmasterol, phellodendron lactone, etc., which can play a role in multiple pathways and multiple targets, and is more effective for inflammatory bowel disease with complex pathology. The Chinese medicine composition prepared by the present invention can be used for supplementary treatment when mesalazine is intolerant, and can also be used in combination with mesalazine for treatment.

[0023] The present invention directly acts on the inflammation site through rectal administration, which may enhance the therapeutic effect and reduce systemic side effects. In addition to the rectal administration, if the lesion site of inflammatory bowel disease is high, the drug cannot reach the ulcer surface, and the treatment effect may be unsatisfactory; and frequent enema may cause serious complications such as colon edema. The Chinese medicine composition of the present invention can also be used in a targeted manner for different patient conditions, and it is convenient for patients to choose the dosage form of granules, mixtures, tablets, capsules, pills, powders, powders, syrups, microcapsules or ointments according to their own conditions.

[0024] The traditional Chinese medicine composition prepared by the present invention not only targets local inflammation, but also improves the efficacy and recurrence prevention and control capabilities by adjusting the overall immune response and internal environment of the human body.

[0025] Compared with expensive biological preparations, the Chinese medicine composition provided by the present invention has a relatively low cost and is easier to popularize. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The longitudinal sections of the colon of rats in each group were unfolded, among which A is the blank group; B is the model group; C is the Chinese medicine group; and D is the mesalazine enema group.

[0027] Figure 2 The colon tissue pathological sections and HE staining images of rats in each group, among which A is the blank group; B is the model group; C is the Chinese medicine group; and D is the mesalazine enema group.

[0028] Figure 3 The flow cytometric plots of the percentage of Th17 cells in each group of rats, among which A is the blank group; B is the model group; C is the Chinese medicine group; and D is the mesalazine enema group.

[0029] Figure 4 The flow cytometric graphs of the percentage of Treg cells in each group of rats, among which A is the blank group; B is the model group; C is the Chinese medicine group; and D is the mesalazine enema group. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] It should be noted that the Chinese herbal medicines used in the present invention all meet the quality requirements of the Chinese Pharmacopoeia for medicinal materials. Pulsatilla chinensis is the dried root of the Ranunculaceae plant Pulsatilla chinensis, and the Latin name of the Ranunculaceae plant Pulsatilla chinensis is Pulsatilla chinensis (Bunge) Regel. Coptis chinensis is the dried rhizome of the Ranunculaceae plant Coptis chinensis, and the Latin name of the Ranunculaceae plant Coptis chinensis Franch. Phellodendron chinense is the dried bark of the Rutaceae plant Phellodendron chinense Schneid. The Latin name of the Rutaceae plant Phellodendron chinense Schneid. Qinpi is the dried branch bark or dry bark of the Oleaceae plant Fraxinus rhynchophylla Hance, and the Latin name of the Oleaceae plant Fraxinus rhynchophylla Hance is Fraxinus rhynchophylla Hance. White peony root is the dried root of Paeonia lactiflora Pall., a plant of the Ranunculaceae family. The Latin name of the Paeonia lactiflora Pall. is Paeonialactiflora Pall. Angelica sinensis is the dried root of Angelica sinensis, a plant of the Umbelliferae family. The Latin name of Angelica sinensis, a plant of the Umbelliferae family, is Angelica sinensis (Oliv.) Diels. Costus root is the dried root of Costus root, a plant of the Asteraceae family. The Latin name of Costus root, a plant of the Asteraceae family, is Aucklandia lappa Decne. Licorice root is the dried root and rhizome of Glycyrrhiza uralensis, a plant of the Umbelliferae family. The Latin name of Glycyrrhiza uralensis Fisch. is Glycyrrhiza uralensis Fisch.

[0032] It should be noted that helper T cells 17 were recorded as Th17 cells, regulatory T cells were recorded as Treg cells, interleukin 6 was recorded as IL-6, interleukin-17A was recorded as IL-17A, tumor necrosis factor-α was recorded as TNF-α, interleukin-10 was recorded as IL-10, transforming growth factor-β was recorded as TGF-β, 2,4,6-trinitrobenzenesulfonic acid was recorded as TNBS, and normal saline was recorded as NS.

[0033] It is understandable that those skilled in the art know that the temperature of the slow fire is 80°C to 120°C.

[0034] Example 1

[0035] This embodiment provides a traditional Chinese medicine composition for treating inflammatory bowel disease, which is prepared from 15g of Pulsatilla striata, 10g of Qinpi, 10g of Coptis chinensis, 30g of Phellodendron amurense, 15g of White Peony Root, 10g of Angelica sinensis, 10g of Aucklandia lappa and 10g of Licorice.

[0036] According to the above formula, each component is weighed and mixed to obtain a mixture. 1000 ml of clean water is added to 110 g of the mixture and soaked for 1.5 hours. The mixture is simmered for 3 times, each time for 0.5 hours. The decoctions are combined and filtered. The filtrate obtained is concentrated to 100 ml, the pH is adjusted to 5, and the mixture is allowed to stand for 12 hours for filtration. The filtered filtrate is centrifuged, and the filtrate obtained by centrifugation is added with purified water to 100 ml, mixed, packaged, and sterilized.

[0037] Example 2

[0038] This embodiment provides a traditional Chinese medicine composition for treating inflammatory bowel disease, which is prepared from 16g of Pulsatilla striata, 12g of Qinpi, 11g of Coptis chinensis, 32g of Phellodendron amurense, 17g of White Peony Root, 11g of Angelica sinensis, 12g of Aucklandia lappa and 12g of Licorice.

[0039] According to the above formula, each component is weighed and mixed to obtain a mixture. 1000 ml of clean water is added to 123 g of the mixture and soaked for 2 hours. The mixture is simmered for 4 times, each time for 40 min. The decoctions are combined and filtered. The filtrate obtained is concentrated to 100 ml, the pH is adjusted to 5.5, and the mixture is allowed to stand for 13 hours for filtration. The filtered filtrate is centrifuged, and the filtrate obtained by centrifugation is added with purified water to 100 ml, mixed, packaged, and sterilized.

[0040] Example 3

[0041] This embodiment provides a traditional Chinese medicine composition for treating inflammatory bowel disease, which is prepared from 17 g / portion of Pulsatilla striata, 11 g / portion of Qinpi, 12 g / portion of Coptis chinensis, 31 g / portion of Phellodendron amurense, 16 g / portion of White Peony Root, 12 g / portion of Angelica sinensis, 11 g / portion of Aucklandia lappa and 11 g / portion of Licorice.

[0042] According to the above formula, each component is weighed and mixed to obtain a mixture. 1000 ml of clean water is added to 121 g of the mixture and soaked for 100 min. The mixture is simmered for 3 times, each time for 35 min. The decoction is combined and filtered. The filtrate obtained is concentrated to 100 ml, the pH is adjusted to 6, and the mixture is allowed to stand for 14 hours for filtration. The filtrate after filtration is centrifuged, and the filtrate obtained by centrifugation is added with purified water to 100 ml, mixed, packaged, and sterilized.

[0043] Comparative Example 1

[0044] Mesalazine enema solution, specification is 60g:4g, batch number: H20150127, produced by Swiss Vivo Pharmaceuticals.

[0045] According to the Methodology of Chinese Medicine Pharmacology Research, the dosage of enteritis enema solution for 60 kg adult is 110 g, and 6.17 times of the dosage for 60 kg adult is 678.70 g, which means the dosage of Chinese medicine for 60 kg rat should be 678.70 g, which is 11.31 g / kg. The standard dosage of Chinese medicine for rat is 11.31 g / kg / d, which is the standard dosage for rat enema. Similarly, the dosage of mesalamine enema solution is 0.41 g / kg / d.

[0046] The Chinese medicine group was given 2 ml enema of the Chinese medicine composition, the mesalazine enema group was given 2 ml enema of mesalazine enema solution, and the model group and the blank group were given 2 ml enema of normal saline.

[0047] It should be noted that the above drugs are all stored in a refrigerator at 4°C for future use. The experiment of the present invention has passed the animal ethics review of Southwest Medical University, and the ethics number of the experiment is: swmu20230087.

[0048] 1. Experimental groups and dosage

[0049] 28 clean-grade Sprague-Dawley male rats, weighing 220g to 240g, were randomly divided into two groups using a random number table method, namely a blank group of 6 rats and a model group of 22 rats. After all rats were fasted but not watered for 24 hours, the blank group rats were enemaed with 1.1mL 0.9% NS, and the remaining rats were enemaed with 1.1mL 5% TNBS / ethanol solution. After the modeling was completed, 18 rats were randomly selected from the modeling group and divided into 3 groups, with 6 rats in each group. Combined with the 6 rats in the blank group, a total of 4 groups were obtained, namely the blank group, model group, Chinese medicine group, and mesalazine enema group, with 6 rats in each group. The treatment methods of each group are as follows:

[0050] A Blank group: 0.9% saline enema 2 mL daily for 10 consecutive days.

[0051] Model group B: 0.9% saline enema 2 mL daily for 10 consecutive days.

[0052] C Chinese medicine group: 2 mL of the Chinese medicine composition was administered by enema every day for 10 consecutive days. It should be noted that the Chinese medicine group refers to the Chinese medicine composition prepared in Example 1.

[0053] D Mesalazine enema group: 2 mL of mesalazine enema solution was given daily for 10 consecutive days.

[0054] 2. Indicators and testing methods

[0055] Starting from the first day of modeling, the rats in each group were weighed at a fixed time every day, and the stool characteristics, fecal occult blood test results or bloody stools were recorded. Fecal occult blood test uses a fecal occult blood qualitative test kit, namely the o-tolidine method: collect fresh feces and test in time. Use a sterilized bamboo stick to pick up a small amount of feces, about 20 mg, and apply it on white sterilized cotton. Use a 1mL empty needle to extract the reagent O-tolidine Solution, add 2 drops of about 0.1mL to different parts of the feces, and then add 2 drops of oxidant, about 0.1ml, immediately start timing and observe the color change, and the interpretation is completed within 2 minutes.

[0056] The interpretation is as follows:

[0057] a. If no color develops within 2 minutes after adding the reagent, the occult blood test is negative and is recorded as -.

[0058] b. 10 seconds after adding the reagent, the color changes from light green to green and is interpreted as +.

[0059] c. After adding the reagent, the color initially appears light green, then gradually turns into obvious blue-brown, and is judged as ++.

[0060] d. After adding the reagent, the color immediately turns blue-brown and gradually turns dark brown, and is judged as +++.

[0061] e. After adding the reagent, the color immediately turns blue-black-brown and is judged as ++++.

[0062] The disease activity index scores of mice are shown in Table 1. It should be noted that the disease activity index score is the sum of the weight loss score, stool characteristics score and occult blood score.

[0063] Table 1 Disease activity index scores of mice

[0064] Weight loss (%) Stool characteristics Occult blood in stool / visible blood in stool score 0～1％ normal Normal / occult blood (-) 0 1～5％ Loose but formed Gross blood in stool / occult blood (+) 1 6～10％ Loose Gross blood in stool / occult blood (++) 2 11～15％ Loose stools Gross blood in stool / occult blood (+++) 3 >15％ Severe loose stools Gross blood in stool / occult blood (++++) 4

[0065] 3. Experimental Animal Handling and Sample Collection

[0066] Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate. After anesthesia was successful, the rat's abdominal cavity was quickly opened, and a blood collection needle was inserted into the rat's abdominal aorta to collect blood. About 6mL to 8mL of blood was collected and stored in a biochemical tube, and then naturally coagulated and separated. Then, it was centrifuged at 3000 rpm, and the serum was extracted and stored in a 2mL centrifuge tube and stored in a -80℃ refrigerator. After blood collection, the rats were killed by cervical dislocation, and the intestine from the anus to the end of the cecum was freed. The intestine was cleaned with ice saline and unfolded, and the length of the colon of each rat was measured with a vernier caliper and recorded. After the measurement was completed, the intestine was cut open along the longitudinal axis, the intestinal contents were washed with ice saline, wiped dry, and laid flat on the operating table. The gross morphology of the colon tissue and the damage of the colon mucosa were observed with the naked eye, and the degree of colon mucosal damage was improved, as shown in Table 2.

[0067] Table 2 Scoring criteria for colon mucosal injury

[0068]

[0069] After the colon mucosal injury degree was scored, 5 cm of the diseased rat colon tissue was cut proximally from 2 cm away from the anus of each rat colon and divided into 3 sections on average. The proximal intestinal section was fixed and preserved with a 4% mass concentration of paraformaldehyde solution. The volume of formaldehyde was 4 to 5 times that of the diseased rat colon tissue, so that the formaldehyde immersed the diseased rat colon tissue to prepare colon pathological tissue sections, hematoxylin-eosin staining and colon mucosal histological injury scores under a microscope, as shown in Table 3. The distal intestinal section was cleaned with ice phosphate buffer solution and stored in phosphate buffer solution at 4°C. It was ground and sieved in time to prepare a single cell suspension of colon tissue for flow cytometric detection of the proportion of Th17 cells and Treg cells.

[0070] Table 3 Colon mucosal histological injury scores

[0071]

[0072] 4. Experimental methods and statistical analysis

[0073] 4.1. Flow cytometric detection of the proportion of Th17 cells and Treg in colon tissue of rats in each group

[0074] Fresh rat colon tissue was placed in a glass culture dish with 10 mL phosphate buffer solution at 4°C to fully clean blood and contaminants, and then the colon tissue was cut into small pieces of about 1 mm with scissors, and then washed with PBS at 4°C. After washing, the cell suspension was gently ground with a grinder and sieved with a 200-mesh nylon mesh, and then centrifuged and red-broken repeatedly until the single-cell suspension was clarified, and then centrifuged again to collect the cell precipitate, and the collected cells were set aside.

[0075] The staining steps were based on the staining method in Section 2.11 of Wang Qinsha’s master’s thesis titled “A Study on the Mechanism of Invigorating Qi and Strengthening Spleen to Enhance the Efficacy of Chemotherapy in Mice with Spleen Deficiency Colorectal Cancer by Regulating Treg and Th17 Cells”. The expression of Th17 cells and Treg cells was detected by flow cytometry.

[0076] 4.2. Determination of serum cytokine levels in rats of each group

[0077] The serum of each group of rats collected in the experiment was tested by sandwich enzyme-linked immunosorbent assay. The operation steps of the kit were followed one by one. After the operation was completed, the absorbance value was measured at a wavelength of 450nm using an enzyme marker. According to the linear regression equation of the standard curve and the dilution multiple, the actual concentration of cytokines in the sample was calculated.

[0078] The statistical method used was the Social Science Statistical Package Software Version 29, and one-way analysis of variance was used for inter-group comparison. When the variance test was equal, the least significant difference test was used; when the variance was unequal, the Tamhani test was used. P < 0.05 was statistically significant, P < 0.01 was statistically significant, P < 0.001 was extremely statistically significant, and P > 0.05 was not statistically significant. Group A was the blank group, Group B was the model group, Group C was the Chinese medicine group, and Group D was the mesalazine enema group. It should be noted that the sample size was 6, and the results were based on To express.

[0079] Compared with group A, group C has *P<0.05, **P<0.01, and ***P<0.001;

[0080] Compared with group B, group C, # means P < 0.05, ## means P < 0.01, and ### means P < 0.001;

[0081] Compared with group D, & in group C was P<0.05, && was P<0.01, and &&& was P<0.001.

[0082] 5. Experimental results

[0083] 5.1. Three days after modeling, all groups of rats were uniformly given enema intervention at a fixed time for 10 consecutive days. The blank group and the model group were given 2 ml of normal saline enema, the Chinese medicine group was given 2 ml of Chinese medicine composition enema solution for enema, and the mesalazine enema group was given 2 ml of mesalazine solution for enema. During the intervention period, the weight and fecal characteristics of the rats were recorded every day, the fecal occult blood of the rats was qualitatively detected, and the disease activity index was scored. The specific scores are shown in Table 4.

[0084] Table 4 Disease activity index scores of rats in each group before and after continuous intervention for 10 days

[0085] Grouping Disease activity index score before intervention Disease activity index score after intervention A blank group 0.72±0.46 0.42±0.50 Model group B 7.33±1.37*** 5.35±1.46*** C Chinese medicine group 7.72±1.02*** 4.58±1.43***# D Mesalazine enema group 8.11±0.47*** 4.65±1.34***#

[0086] Compared with the blank group, the DAI scores of the model group, the Chinese medicine group and the mesalazine group were significantly increased, and the P values ​​were all <0.001, which was extremely statistically significant. Compared with the model group, the scores of the Chinese medicine group and the mesalazine enema group decreased, which was statistically significant, P<0.05. Compared with the mesalazine enema group, the DAI score of the Chinese medicine group decreased, which was not statistically significant, P>0.05. The above results show that after treatment with Chinese medicine composition enema and mesalazine enema, the disease activity index score of UC model rats decreased, and the Chinese medicine group can effectively relieve the symptoms of ulcerative colitis such as diarrhea and bloody stool caused by TNBS.

[0087] 5.2. The colon tissue of the rats was taken out and laid flat on the operating table. The colon length of each rat was measured and recorded. The analysis showed that compared with the blank group, the colon length of the rats in the model group, the Chinese medicine group and the mesalazine enema group was significantly shortened, P<0.001, and the difference was extremely statistically significant. Compared with the model group, the colon length of the rats in the Chinese medicine group and the mesalazine enema group increased, and the difference was statistically significant, P<0.05. The score of the degree of colon mucosal injury of rats in each group is shown in Table 5.

[0088] Compared with the blank group, the colon mucosal injury scores of the model group, compound pulsatilla enema group and mesalazine enema group increased, and the difference was extremely statistically significant, P<0.001. Compared with the model group, the colon mucosal injury scores of the compound pulsatilla enema group and mesalazine enema group decreased, and the difference was statistically significant, P<0.05. The results of the colon mucosal histological injury scores of rats in each group are shown in Table 5. Compared with the blank group, the colon mucosal histological injury score of the model group was significantly increased, and the difference was extremely statistically significant, P<0.001. Compared with the model group, the compound pulsatilla enema group and mesalazine enema group could reduce the colon mucosal histological injury score, and the difference was extremely statistically significant, P<0.001.

[0089] Table 5 Colon length, colon mucosal injury score, colon mucosal histological injury score of rats in each group

[0090] Grouping Colon length (cm) Colon mucosal injury score Colon mucosal histological injury score A blank group 20.08±1.79 0.50±0.55 1.00±0.89 Model group B <![CDATA[14.83±1.25 *** ]]> <![CDATA[4.83±0.75 *** ]]> <![CDATA[5.33±0.52 *** ]]> C Chinese medicine group <![CDATA[16.78±1.80 ***# ]]> <![CDATA[3.33±1.21 ***# ]]> <![CDATA[2.33±0.52 **### ]]> D Mesalazine enema group <![CDATA[16.60±0.45 ***# ]]> <![CDATA[3.50±1.05 ***# ]]> <![CDATA[2.50±0.55 **### ]]>

[0091] The colon was cut open along the longitudinal axis, and the contents of the colon were flushed clean with ice saline, then spread out on the operating table for observation. Figure 1 , Figure 1 As can be seen from A, the colon of the blank group rats had no adhesion to the surrounding abdominal tissues, the entire colon was unobstructed, the colon was longer and more elastic, the intestinal mucosa was normal without congestion, edema, erosion or ulcer, the intestinal wall was thin and ruddy, and there was no hyperplasia, hypertrophy or stenosis. Figure 1 As can be seen from B, in the model group, rats had severe adhesions around the colon and the peritoneal tissues, the colon length was significantly shortened, the intestinal wall was dark red in color, hyperplastic, hypertrophic and inelastic, the intestinal cavity was obviously narrowed, intestinal contents were retained in the proximal colon, the colon mucosa was congested, swollen, eroded, and multiple ulcer surfaces were visible. Figure 1 As can be seen from C, there was no adhesion between the colon tissue and the surrounding abdominal tissue in the Chinese medicine group, the intestinal wall was light red and slightly thickened, the colon length was longer than that in the model group, the colon elasticity was good, there was no stenosis of the intestinal cavity, no obvious ulcer surface was found in the intestinal mucosa, and there was no retention of contents in the proximal colon. Figure 1As can be seen from D, there was no adhesion between the colon tissue and the surrounding abdominal tissue in the mesalazine enema group rats, the intestinal wall was light red in color, the intestinal wall was slightly thickened, the colon length was longer than that in the model group, the colon elasticity was good, there was no stenosis of the intestinal cavity, no obvious ulcer surface was found in the intestinal mucosa, and no retention of proximal colon contents was found.

[0092] 5.3. Pathological section observation revealed that Figure 2 As can be seen from A, there was no inflammatory cell infiltration in the colon mucosa of the blank group rats, the epithelial tissue was intact, the structure was clear, the glands were arranged regularly, and the goblet cells were abundant. Figure 2 As can be seen from B in the figure, in the model group, inflammatory cells diffusely infiltrated deep into the muscular layer to the serosa, the colonic crypt cells decreased or even disappeared, the intestinal mucosal epithelial cells necrotic and sloughed off, the glands decreased or even some glands disappeared and could not be seen, the intestinal mucosa could be seen as crater-like ulcers or intestinal wall thickening, a large amount of fibrous tissue was repaired, and even the mucosal layer, submucosa, lamina propria, and muscular layer disappeared, and only fibrous tissue repair and proliferation and scar formation were seen in the intestinal wall. Figure 2 As can be seen from Figures C and D, the infiltration of inflammatory cells in the colon tissue of rats in the Chinese medicine group and mesalazine enema group was less than that in the model group, the glands were arranged irregularly, and the glandular ducts had irregular morphology.

[0093] 5.4. Pass Figure 3 The single cell suspension flow cytometry test of mid-colon tissue showed that the proportion of Th17 cells in each group was as follows: Figure 3 Q1-UL is the upper left quadrant in the figure, Q1-UR is the upper right quadrant, and the upper right quadrant is CD4 + IL-17A + The cells expressing positive expression, i.e., Th17 cells, Q1-LL is the lower left quadrant, and Q1-LR is the lower right quadrant.

[0094] Figure 3 A is the blank group, B is the model group, C is the Chinese medicine group, and D is the mesalazine enema group. Compared with the blank group, the proportion of Th17 cells in the colon tissue of rats in the model group, Chinese medicine group, and mesalazine enema group was significantly increased, with significant differences. Compared with the model group, the proportion of Th17 cells in the colon tissue of rats in the Chinese medicine group and mesalazine enema group was significantly reduced, with extremely significant differences, P<0.001. Compared with the mesalazine enema group, the proportion of Th17 cells in the colon tissue of rats in the Chinese medicine group was obviously not significantly different, and was not statistically significant.

[0095] The proportion of Treg cells in each group is as follows Figure 4 As shown, it should be noted that Figure 4 Q1-UL is the upper left quadrant in the figure, Q1-UR is the upper right quadrant, and is CD25 + Foxp3 +The cells expressing positive expression, namely Treg cells, Q1-LL is the lower left quadrant, and Q1-LR is the lower right quadrant.

[0096] Figure 4 A in the figure is the blank group, B is the model group, C is the Chinese medicine group, and D is the mesalazine enema group. Compared with the blank group, the proportion of Treg cells in the colon tissue of rats in the model group and the mesalazine enema group was significantly reduced, with significant differences (P<0.01). However, there was no statistically significant difference between the blank group and the Chinese medicine group (P>0.05). Compared with the model group, the proportion of Treg cells in the colon tissue of rats in the Chinese medicine group and the mesalazine enema group was not statistically significant; compared with the mesalazine enema group, the proportion of Treg cells in the colon tissue of rats in the Chinese medicine group was significantly increased, with statistical significance (P<0.01).

[0097] Analysis of the Th17 / Treg ratio in Table 6 shows that compared with the blank group, the Th17 / Treg ratio in the colon tissue of rats in the model group, the Chinese medicine group and the mesalazine enema group was significantly increased, and the difference was statistically significant. Among them, the increase in the model group was the most significant, P<0.001. Compared with the model group, the Th17 / Treg ratio in the colon tissue of rats in the Chinese medicine group and the mesalazine enema group was significantly reduced, and the difference was statistically significant, P<0.01; compared with the mesalazine enema group, the Th17 / Treg ratio in the colon tissue of rats in the Chinese medicine group was reduced, and the difference was statistically significant, P<0.05.

[0098] Table 6 The percentage of Th17 cells and Treg cells and the Th17 / Treg ratio in each group

[0099] Group Th17 cells (%) Treg cells (%) Th17 / Treg ratio A blank group 0.20±0.10 11.32±3.57 0.02±0.01 Model group B 1.39±0.18*** 6.54±1.10** 0.22±0.04*** C Chinese medicine group 0.70±0.32**### 9.25±1.71&& 0.07±0.02*###& D Mesalazine enema group 0.63±0.26**### 4.91±1.86*** 0.13±0.02***##

[0100] According to the experimental test results in Table 7, compared with the blank group, the concentrations of proinflammatory cytokines IL-6, IL-17A and TNF-α in the serum of rats in the model group, Chinese medicine group and mesalazine enema group were all increased. Among them, the increase trend in the model group was extremely significant, P<0.001. Compared with the model group, the concentrations of proinflammatory cytokines IL-6, IL-17A and TNF-α in the Chinese medicine group and mesalazine enema group were reduced. Among them, the Chinese medicine group and mesalazine enema group had a significant decrease in IL-6, and the difference was statistically significant, P<0.01; the decrease in IL-17A was extremely significant, and the difference was extremely statistically significant, P<0.001; in TNF-α, the decrease in the mesalazine enema group was statistically significant, P<0.05, and the decrease trend in the Chinese medicine group was significantly lower than that in the mesalazine enema group, and the difference was statistically significant, P<0.01. There was no statistically significant difference between the Chinese medicine group and the mesalazine enema group, P>0.05.

[0101] Table 7 Changes in the concentration of pro-inflammatory cytokines in the serum of rats in each group

[0102] Group IL-6 (pg / mL) IL-17A (pg / mL) TNF-α(pg / mL) A blank group 26.90±2.15 13.52±1.27 27.31±3.29 Model group B 45.98±8.30*** 21.57±1.87*** 40.56±5.04*** C Chinese medicine group 36.89±4.44**## 17.49±1.21***### 32.63±2.59*## D Mesalazine enema group 37.11±6.22**## 18.69±0.73***### 36.16±2.77**#

[0103] According to the experimental test results in Table 8, compared with the blank group, the concentrations of IL-10 and TGF-β in the model group were reduced. Among them, the model group decreased in IL-10, and the difference was statistically significant, P<0.05; the model group decreased in TGF-β, and the difference was statistically significant, P<0.01. Compared with the model group, IL-10 increased in the Chinese medicine group and the mesalamine enema group, and the difference was statistically significant, P<0.05; while TGF-β increased significantly in the Chinese medicine group and the mesalamine enema group, and the difference was statistically significant, P<0.001. In the concentration of TGF-β, the concentration levels of the Chinese medicine group and the mesalamine enema group were higher than those of the blank group. There was no statistically significant difference between the Chinese medicine group and the mesalamine enema group in the above indicators, P>0.05.

[0104] Table 8 Changes in the concentration of anti-inflammatory cytokines in the serum of rats in each group

[0105] Group IL-10 (pg / mL) TGF-β(pg / mL) A blank group 6.69±1.42 40.16±1.60 Model group B 5.23±0.63* 34.31±1.80** C Chinese medicine group 6.41±0.88# 45.52±3.70**### D Mesalazine enema group 6.37±0.25# 45.64±3.41**###

[0106] In addition, the Chinese medicinal compositions prepared in Example 2 and Example 3 were scored for the disease activity index, as shown in Table 9. It can be seen that after treatment with the Chinese medicinal compositions prepared in Example 2 and Example 3, the disease activity index scores of rats decreased, indicating that the Chinese medicinal compositions prepared in Example 2 and Example 3 can effectively alleviate diarrhea and bloody stools caused by TNBS in ulcerative colitis.

[0107] Table 9 Disease Activity Index Scores of Important Compositions in Examples 2 and 3

[0108] Grouping Disease activity index score before intervention Disease activity index score after intervention Example 2 7.68±0.95 4.92±1.23 Example 3 7.53±0.61 4.84±1.56

[0109] In summary, the Chinese medicine composition prepared by the present invention can, in the ulcerative colitis model rats, enable the relevant cytokines to participate in and down-regulate the proliferation and differentiation of local Th17 cells and Treg cells in the colon through local enema, thereby affecting the expression of Th17-related cytokines IL-6, IL-17A, TNF-α and Treg-related cytokines IL-10, TGF-β in peripheral serum, regulating the balance of Th17 / Treg, and achieving the purpose of alleviating and treating intestinal inflammation in ulcerative colitis model rats. The Chinese medicine composition can not only regulate the local inflammation of the colon of ulcerative colitis model rats, but also achieve systemic regulation of inflammation by regulating the secretion of relevant cytokines.

[0110] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0111] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A Chinese medicine composition for treating inflammatory bowel disease, characterized in that: The invention is prepared from the following components in parts by weight: 15-17 parts of Pulsatilla scutellariae, 10-12 parts of Qinpi, 10-12 parts of Coptis chinensis, 30-32 parts of Phellodendron amurense, 15-17 parts of White Peony Root, 10-12 parts of Angelica sinensis, 10-12 parts of Costusroot Root and 10-12 parts of Licorice.

2. A Chinese medicine composition for treating inflammatory bowel disease according to claim 1, characterized in that: The invention is prepared from the following components in parts by mass: 15 parts of Pulsatilla striata, 10 parts of Qinpi, 10 parts of Coptis chinensis, 30 parts of Phellodendron amurense, 15 parts of White Peony Root, 10 parts of Angelica sinensis, 10 parts of Aucklandia lappa and 10 parts of Licorice.

3. A method for preparing the Chinese medicine composition for treating inflammatory bowel disease according to claim 1 or 2, characterized in that: The following steps are involved: The mixture is obtained by mixing Pulsatilla striata, Qinpi, Rhizoma Coptidis, Phellodendron amurense, White Peony Root, Angelica Sinensis, Aucklandia lappa and Licorice, and water is added to the mixture to soak it, and then decocted with a slow fire for 3 to 4 times. After the decocting is completed, the decoctions are combined, filtered and concentrated, the pH value is adjusted to 5 to 6, and the mixture is allowed to stand and centrifuged, and then post-processed to obtain the mixture.

4. The method for preparing a Chinese medicine composition for treating inflammatory bowel disease according to claim 3, characterized in that: The ratio of the mixture to water is 110 g to 123 g: 1000 mL.

5. The method for preparing a Chinese medicine composition for treating inflammatory bowel disease according to claim 3, characterized in that: The soaking time is 1.5h to 2h.

6. The method for preparing a Chinese medicine composition for treating inflammatory bowel disease according to claim 3, characterized in that: The decoction time is 30min to 40min.

7. The method for preparing a Chinese medicine composition for treating inflammatory bowel disease according to claim 3, characterized in that: The standing time is 12h to 14h.

8. Use of the Chinese medicine composition for treating inflammatory bowel disease according to claim 1 in preparing a Chinese medicine preparation for treating inflammatory bowel disease.

9. Use of the Chinese medicinal composition for treating inflammatory bowel disease according to claim 8 in preparing a medicament for treating inflammatory bowel disease, characterized in that: The Chinese medicine composition is prepared with pharmaceutically acceptable auxiliary materials to obtain the Chinese medicine preparation.

10. Use of the Chinese medicine composition for treating inflammatory bowel disease according to claim 8 in preparing a Chinese medicine preparation for treating inflammatory bowel disease, characterized in that: The dosage forms of Chinese medicine preparations are decoctions, granules, mixtures, tablets, capsules, pills, powders, granules, syrups, microcapsules or pastes.

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