Application of yellow tea polyphenol in preparation of medicine for preventing and / or treating colitis

Abstract

The invention discloses application of yellow tea polyphenol in preparation of a medicine for preventing and / or treating colitis, and belongs to the technical field of biological medicine preparation, animal experiments prove that the yellow tea polyphenol can reduce the DAI index of DSS-induced colitis mice and relieve weight loss and colon shortening. By adopting the yellow tea polyphenol provided by the invention, inflammatory response, tissue damage and immune disorder related to colitis can be relieved; the pathological state of colon tissue is improved; therefore, the effect of treating and preventing the colitis is achieved, and the traditional Chinese medicine composition has an extremely wide application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine preparation, and particularly relates to application of yellow tea polyphenol in preparation of a medicine for preventing and / or treating colitis. BACKGROUND

[0002] Inflammatory bowel disease (IBD) is a chronic and recurrent specific gastrointestinal inflammation disease. It mainly includes ulcerative colitis (UC) and Crohn's disease (CD) types. The lesions of CD can occur in the small intestine or large intestine, but the most common is the colon and rectum. In contrast, UC only affects the colon and rectum, and inflammation is limited to the mucosa. The characteristics of UC are chronic inflammation and ulceration of the rectal and colonic mucosa, and the clinical symptoms include pain, weight loss, blood in the stool and intestinal mucosal ulceration. UC causes inflammation, colonic barrier damage, oxidative stress imbalance and intestinal microbial environment imbalance. Among them, epithelial damage is one of the larger characteristics of UC, and ulcerative colitis can cause epithelial cell ulceration and necrosis, infiltration of immune cells such as mucus-secreting goblet cells, hormone-secreting enteroendocrine cells, neutrophils, Paneth cells, and microfolds cells, destruction of the mucosal layer that breaks the physical barrier, and causes mucosal barrier permeability disorder. UC triggers inflammation and immune disorders in the colonic mucosa and submucosa, significantly increasing the risk of colorectal cancer (CRC), and UC patients have a 2-3 times higher risk of CRC than healthy people, which has become an important challenge to global public health. Therefore, the treatment goal is to control inflammation and OS to achieve mucosal healing and reduce the risk of hospitalization. Patients are usually treated with corticosteroids, aminosalicylates, antibiotics, immunosuppressive drugs and monoclonal antibodies. However, these drugs can cause a variety of adverse reactions, loss of efficacy and high cost, and many patients have difficulty accepting treatment. For these reasons, many other substances are considered to help treat inflammatory bowel disease.

[0003] It has become a research hotspot to find and utilize active factors in natural functional foods for the prevention and treatment of colitis. Huang tea is a Chinese characteristic tea, which is rich in tea polyphenols, amino acids and soluble sugars. The content of tea polyphenols detected in Huang tea is close to that in green tea, accounting for 20.44%-27.66% of the dry weight of Huang tea. In recent years, the functional research and application of tea polyphenols have been more in-depth and extensive. In addition to having antioxidant activity, studies have shown that tea polyphenols also have health benefits such as reducing blood lipids, preventing and treating cancer, inhibiting bacteria and viruses, and preventing and treating cardiovascular and cerebrovascular diseases. Among them, catechins account for about 70%-80% of the total polyphenols in tea, which plays an important role in the formation of color, aroma and taste quality of tea. Tea polyphenols have broad potential in the treatment of colitis, but there is little research on Huang tea polyphenols. Whether it has better effects on preventing and improving colitis needs to be explored through experiments, and its active mechanism needs to be clarified. SUMMARY

[0004] In view of the above problems, the purpose of the present application is to provide the application of Huang tea polyphenols in the preparation of drugs for preventing and / or treating colitis. Studies have shown that Huang tea polyphenols can significantly reduce inflammation, tissue damage, immune disorders related to colitis, regulate bacterial metabolism, and improve intestinal barrier function, and have good therapeutic effect and development value.

[0005] The present application provides the application of Huang tea polyphenols in the preparation of drugs for preventing and / or treating colitis.

[0006] As a preferred, the polyphenols include gallocatechin (GC), epicatechin gallate (ECG), epicatechin (EC), gallocatechin gallate (GCG) and epigallocatechin gallate (EGCG).

[0007] As a preferred, the colitis is ulcerative colitis.

[0008] As a preferred, Huang tea polyphenols can reduce disease activity index, reduce weight loss and shorten colon.

[0009] As a preferred, Huang tea polyphenols can relieve colitis mainly by inhibiting inflammatory factors, increasing anti-inflammatory factors, improving intestinal barrier function and regulating intestinal flora.

[0010] As a preferred, Huang tea polyphenols can reduce colonic tissue damage.

[0011] Compared with the prior art, the present application provides a detection method of Huang tea extract tea polyphenols. The present application has the following beneficial effects: 1. The Huang tea polyphenols provided by the present application can reduce the disease activity index of DSS-induced colitis mice, reduce weight loss and shorten the colon.

[0012] 2. The yellow tea polyphenol provided by the application can relieve the crypt atrophy, epithelial cell damage, goblet cell reduction and large influx of inflammatory cells caused by DSS, and significantly reduce the histological score.

[0013] 3. The yellow tea polyphenol provided by the application can relieve the colon tissue damage.

[0014] 4. Compared with the DSS group of mice, the pro-inflammatory factors IL-6, TNF-α and IL-1β in the colon of the yellow tea polyphenol intervention group of mice are reduced, the monocyte chemotactic protein-1 (MCP-1) is also reduced, the anti-inflammatory factor IL-10 is increased, and the expression of Occludin and Z0-1 genes is also up-regulated. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is a graph of the changes in the body weight (A) and the disease activity index (DAI) (B) of mice in different groups; Figure 2 is a schematic diagram of the colon length of mice in different groups; Figure 3 is an HE staining (A) and a tissue damage score graph (B) of the colon tissue of mice in different groups; Figure 4 is a staining (A) of goblet cells of mice in different groups, and a relative quantitative analysis graph (B) thereof; Figure 5 is a graph of the expression differences of the colon proteins Occuldin (A) and ZO-1 (C) of mice in different groups; and a relative quantitative analysis graph of Occuldin (B) and ZO-1 (D); Figure 6 is a graph of the cytokine expression data of the mRNA levels of TNF-α, IL-6, IL-1β, IL-10, MCP-1, Occludin and ZO-1 in the colon tissue regulated by the yellow tea polyphenol. DETAILED DESCRIPTION

[0016] It is easy to understand that, according to the technical solution of the application, those skilled in the art can propose a plurality of structure modes and implementation modes which can be replaced with each other without changing the essential spirit of the application. Therefore, the following specific embodiments and the accompanying drawings are only exemplary descriptions of the technical solution of the application, and should not be regarded as the whole or as a limitation or restriction on the technical solution of the application.

[0017] REFERENCE Figures 1-6 As shown in the accompanying drawings, the application provides a technical solution: the application of the yellow tea polyphenol in the preparation of a drug for preventing and / or treating colitis.

[0018] The polyphenols include gallocatechin (GC), epicatechin gallate (ECG), epicatechin (EC), gallocatechin gallate (GCG), and epigallocatechin gallate (EGCG).

[0019] The colitis is ulcerative colitis.

[0020] The yellow tea polyphenols can reduce the disease activity index and reduce weight loss.

[0021] The yellow tea polyphenols can relieve colitis mainly by inhibiting inflammatory factors, increasing anti-inflammatory factors, and improving intestinal barrier function.

[0022] The yellow tea polyphenols can reduce colonic tissue damage.

[0023] Example 1: Experimental grouping and process (1) Experimental animals: healthy male C57BL / 6J mice were selected and randomly divided into 6 groups, n=7 for each group, and the experiment was started after 7 days of adaptation.

[0024] (2) Model establishment: the mice were allowed to freely drink distilled water containing 2.5% DSS (w / v) for 7 days to induce acute ulcerative colitis in the mice, and the body weight change, fecal character, and bleeding of the mice were recorded during the period.

[0025] (3) Polyphenol administration: the yellow tea polyphenols were prepared into administration solutions at different dosages, the mice in the Positive Control group were administered with 100 mg / kg / d mesalazine solution by gavage, the mice in the low-dose tea polyphenol group (DSS+LTP group), the medium-dose tea polyphenol group (DSS+MTP group), and the high-dose tea polyphenol group (DSS+HTP group) were administered with 100 mg / kg / d, 200 mg / kg / d, and 250 mg / kg / d polyphenol solutions by gavage once a day for 7 consecutive days, and the mice in each group were allowed to freely drink distilled water during the period.

[0026] (4) The total experimental days were 14 days, from day 1 to day 7: the mice in the Normal Control group were allowed to freely drink distilled water, and the mice in the DSS group, the Positive Control group, the DSS+LTP group, the DSS+MTP group, and the DSS+HTP group were allowed to freely drink distilled water containing 2.5% DSS (w / v). From day 8 to day 14: the mice in the Positive Control group were administered with 100 mg / kg / d mesalazine solution by gavage, and the mice in the DSS+LTP group, the DSS+MTP group, and the DSS+HTP group were administered with 100 mg / kg / d, 200 mg / kg / d, and 250 mg / kg / d TP solutions by gavage once a day, and the mice in each group were allowed to freely drink distilled water.

[0027] (5) Sample collection and processing: Collect the colon tissue, take the entire intestinal segment from the anus to the end of the cecum, rinse the bloodstains with pre-cooled sterile PBS buffer solution, then stretch the colon naturally, lay it on white paper, and record the colon length with a ruler. Divide the colon tissue into 4 parts, 3 of which are quickly frozen with liquid nitrogen and stored in a -80°C refrigerator for subsequent RT-qPCR experiments. The other part is used for histopathological analysis, and 4% paraformaldehyde is used for fixation.

[0028] Example 2: Evaluation index and results To systematically evaluate the therapeutic effect of yellow tea polyphenols on colonitis mouse models, different dose treatment groups were set up, and disease signs, intestinal histological changes, and inflammatory response levels were observed in multiple dimensions.

[0029] (1) Changes in mouse body weight and disease activity index (DAI) The activity of the mice, mental state, fur luster, and stool state were observed daily, and the body weight of the mice was monitored. The disease activity index (Disease activity index, DAI) score = (body weight index + stool shape + bleeding condition) / 3, which can be scored according to Table 1 standard.

[0030] Table 1 Disease activity index (DAI) score standard

[0031] (2) Colon length recovery Colon length is an important objective indicator of the degree of inflammatory damage. The colon of the model group mice was significantly shortened, indicating severe tissue inflammation and edema. After intervention with yellow tea polyphenols, the colon length recovered to varying degrees, as shown in Table 2. Figure 2

[0032] (3) Improvement of colon histology (H&E staining) and tissue damage score The mouse colon tissue was fixed in 4% paraformaldehyde for 24 h, then the fixed tissue was embedded in paraffin, and 5 μm sections were cut by a microtome. The colon tissue was stained with HE, and the pathological damage degree of the colon tissue section was observed under a microscope.

[0033] Table 2 Colon tissue damage score

[0034] (4) Intestinal barrier function detection: The expression levels of cup cells, Occuldin and ZO-1 were evaluated by immunohistochemistry, and the specific steps are as follows: ​Paraffin section deparaffinization to water: the section was placed in a dehydration box in turn, and gradient dehydration treatment was carried out. The specific dehydration process was as follows: environmental protection type deparaffinization liquid I 10 min, environmental protection type deparaffinization liquid II 10 min, environmental protection type deparaffinization liquid III 10 min, anhydrous ethanol I 5 min, anhydrous ethanol II 5 min, anhydrous ethanol III 5 min, distilled water washing.

[0035] Antigen repair: the section was immersed in repair solution and repaired at high temperature. Slowly cool at room temperature, then immerse in PBS (PH 7.4) and wash on a shaker for 3 times, 5 min each time.

[0036] Blocking endogenous peroxidase: the section was immersed in 3% H2O2 for 25 min. Rinse with water, then immerse in PBS (PH 7.4) and wash on a shaker for 3 times, 5 min each time.

[0037] Serum blocking: 3% BSA covers the tissue at room temperature for 30 min.

[0038] Add primary antibody: shake off the blocking solution, add diluted primary antibody, and incubate overnight at 4°C.

[0039] Add secondary antibody: immerse the section in PBS (PH 7.4) and wash on a shaker for 3 times, 5 min each time. After shaking dry, add diluted secondary antibody, and incubate at room temperature for 50 min.

[0040] DAB color development: immerse the section in PBS (PH 7.4) and wash on a shaker for 3 times, 5 min each time. After shaking dry, add freshly prepared DAB color developing solution. Under a microscope, observe the positive as brownish yellow, and immediately use tap water to terminate the reaction.

[0041] Re-stain the nucleus: hematoxylin re-staining for 3 min, wash with tap water. Differentiate hematoxylin in differentiation solution for a few seconds, and wash again. Return blue after hematoxylin returns blue, and then rinse.

[0042] Dehydration and mounting: place in 75% alcohol, 85% alcohol, anhydrous ethanol I, anhydrous ethanol II, n-butanol, and xylene I for 5 min each time, then dry and mount with mounting medium.

[0043] Microscopy: place under a microscope for observation, and then score.

[0044] (5) Real-time quantitative PCR analysis RNA was extracted using total RNA extraction kit, and the concentration and purity of RNA were measured. The RNA was reversely transcribed into cDNA by using a reverse transcription instrument, and qPCR was performed by using specific primers (TNF-a, IL-1β, IL-6, IL-10, Occludin, ZO-1, MCP-1, etc.). The procedure was as follows: pre-denaturation at 95°C for 30 s; PCR reaction: 95°C for 10 s; 60°C for 30 s; 40 cycles of reaction. The specificity of primers was determined by observing the melting curve, and the CT value was used for analysis. The relative expression of target genes in qPCR was calculated by using the 2(-ACt) method, and the differences between groups were compared.

[0045] Result analysis: (1) The symptom relief of polyphenols in Huangcha on mice with ulcerative colitis DSS can induce severe diarrhea and hematochezia in mice, and the body weight decreases significantly, which is the characteristic of ulcerative colitis. Polyphenols in Huangcha can significantly improve these symptoms. The DAI score is based on the body weight, diarrhea and hematochezia of mice. The DAI value of the Huangcha polyphenol treatment group can be significantly reduced, and the body weight decrease of the mice with ulcerative colitis can be inhibited.

[0046] Another symptom of DSS colitis is the shortening of the colon. After the mice were sacrificed, the colon length of each experimental group was measured. Compared with the control group, the Huangcha polyphenol intervention group significantly reduced the shortening of the colon.

[0047] Histological scoring showed that the colon mucosa structure of the Normal Control group was complete and had no lesions, while the DSS group showed significant characteristics of ulcerative colitis: a large number of crypt loss, diffuse necrosis of the mucosa and submucosa, and infiltration of neutrophils and lymphocytes, with a sharp increase in damage score. After the intervention of Huangcha polyphenols, the colon mucosa integrity gradually recovered, the number of crypts increased, and the inflammatory infiltration decreased, and the scores decreased, among which the high-dose group had the most significant effect, indicating that tea polyphenols can effectively inhibit DSS-induced colitis, maintain mucosal morphology and promote tissue repair.

[0048] Goblet cells are distributed between the mucosal columnar epithelium and can secrete mucin Muc-2, which can protect the intestinal epithelium from pathogen and mechanical damage. Compared with the Normal Control group, the number of goblet cells in the colon tissue of the DSS group of mice decreased. DSS-induced ulcerative colitis significantly reduced the number of goblet cells in the colon tissue, and the epithelial mucosa thickness decreased and the mucous membrane disappeared. Compared with the DSS group, the number of goblet cells in the Huangcha polyphenol intervention group increased, and there were significant differences between the DSS+HTP group and the DSS+5-ASA group of mice. The colon tissue maintained the function of secreting mucus, and the thickness of the mucosa layer was restored.

[0049] The Occludin and ZO-1 proteins in the colon of the mice were semi-quantified by the method of immunohistochemical staining. Compared with the Normal control group, the epithelial cells of the DSS group were light in color, and the cells were arranged in disorder, indicating that the intestinal barrier was severely damaged. After treatment, the number of Occludin and ZO-1 proteins in the administration group increased, and the positive cell staining of the DSS+HTP group was deeper than that of the DSS group, and the epithelial cells were arranged in order, indicating that the yellow tea polyphenol can effectively restore the expression of tight junction proteins Occludin and ZO-1, and has a positive significance for the recovery of the intestinal barrier of the mice.

[0050] (2) Inflammatory inhibition effect of yellow tea polyphenol on ulcerative colitis mice DSS induction significantly increased the pro-inflammatory factors IL-6, TNF-α, IL-1β and chemokine MCP-1 in UC mice, and the expression of barrier proteins Occludin, ZO-1 and anti-inflammatory factor IL-10 decreased sharply, and the intestinal mucosal immune-barrier double collapse; the medium and high doses of yellow tea polyphenol can simultaneously down-regulate IL-6, TNF-α, IL-1β and MCP-1, and up-regulate IL-10, Occludin and ZO-1 to normal level, and mesalazine also showed similar but smaller improvement, indicating that yellow tea polyphenol can relieve ulcerative colitis to a certain extent.

[0051] The above provided examples are not intended to limit the scope covered by the present application, and the described steps are not intended to limit the execution order. Those skilled in the art can make obvious improvements to the present application in combination with the existing common knowledge, which also falls within the protection scope defined by the claims of the present application.

Claims

1. Application of yellow tea polyphenols in the preparation of drugs for the prevention and / or treatment of colitis.

2. The application of the yellow tea polyphenols according to claim 1 in the preparation of drugs for the prevention and / or treatment of colitis, characterized in that, Polyphenols include gallocatechin (GC), epicatechin gallate (ECG), epicatechin (EC), gallocatechin gallate (GCG), and epigallocatechin gallate (EGCG).

3. The application of the yellow tea polyphenols according to claim 1 in the preparation of drugs for the prevention and / or treatment of colitis, characterized in that, Colitis is ulcerative colitis.

4. The application of the yellow tea polyphenols according to claim 1 in the preparation of drugs for the prevention and / or treatment of colitis, characterized in that, Yellow tea polyphenols can reduce disease activity index and aid in weight loss.

5. The application of the yellow tea polyphenols according to claim 1 in the preparation of drugs for the prevention and / or treatment of colitis, characterized in that, Yellow tea polyphenols mainly alleviate colitis by inhibiting inflammatory factors, increasing anti-inflammatory factors, and improving intestinal barrier function.

6. The application of the yellow tea polyphenols according to claim 1 in the preparation of drugs for the prevention and / or treatment of colitis, characterized in that, Yellow tea polyphenols can reduce damage to colon tissue.

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