Application of polyphenol composition in preparation of medicine for treating inflammatory bowel disease and medicine

Abstract

The invention belongs to the technical field of chemical drugs, and particularly relates to application of a polyphenol composition in preparation of drugs for treating inflammatory bowel diseases and the drugs. The polyphenol composition contains isorhamnetin, kaempferol, quercetin, ferulic acid and caffeic acid, and has an obvious anti-inflammatory effect on the inflammatory bowel disease; when the composition is used as a medicine for treating the inflammatory bowel disease, the treatment cost can be reduced, and the composition is effective in treating the inflammatory bowel disease and small in toxic and side effects after being taken for a long time.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical medicines, in particular to a polyphenol composition and application of the polyphenol composition in preparing a medicine for treating inflammatory bowel disease. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic nonspecific intestinal inflammatory disease, mainly including ulcerative colitis and Crohn's disease. Clinical manifestations include persistent or recurrent diarrhea, bloody stools with mucus, abdominal pain, and tenesmus, which seriously affect the quality of life of patients. Existing treatments mostly rely on antibiotics and immunosuppressants, but there are problems such as large toxic side effects and easy recurrence. Therefore, finding safe and effective natural medicines has become a research hotspot.

[0003] Modern research shows that polyphenol compounds exhibit significant effects in anti-oxidation and anti-inflammatory aspects, providing the possibility for the development of new anti-IBD drugs.

[0004] The present invention screens and separates polyphenols contained in natural herbal plants and finds that a specific polyphenol composition has a better effect on inflammatory bowel disease. Summary of the invention

[0005] The present invention aims to provide a use of a polyphenol composition in preparing a drug for treating inflammatory bowel disease and the drug, so as to solve the problems of large toxic and side effects and easy recurrence in existing treatment methods.

[0006] The technical solution of the present invention is as follows: A use of a polyphenol composition in preparing a drug for treating inflammatory bowel disease (IBD), wherein the polyphenol composition is a composition of isorhamnetin, kaempferol, quercetin, ferulic acid, and caffeic acid.

[0007] The use of the polyphenol composition in preparing a drug for treating inflammatory bowel disease is characterized in that it is composed of the following mass ratio: 1-50 parts of isorhamnetin, 1-50 parts of kaempferol, 1-50 parts of quercetin, 1-50 parts of ferulic acid, and 1-50 parts of caffeic acid.

[0008] The use of the polyphenol composition in preparing a drug for treating inflammatory bowel disease is characterized in that it is composed of the following mass ratio: 1 to 10 parts of isorhamnetin, 1 to 10 parts of kaempferol, 1 to 10 parts of quercetin, 1 to 10 parts of ferulic acid, and 1 to 10 parts of caffeic acid.

[0009] The use of the polyphenol composition in preparing a drug for treating inflammatory bowel disease is characterized in that it is composed of the following mass ratio: 1 part of isorhamnetin, 1 part of kaempferol, 1 part of quercetin, 1 part of ferulic acid, and 1 part of caffeic acid.

[0010] The polyphenol composition inhibits inflammatory bowel disease (IBD) by reducing the secretion of at least one of the inflammatory factors IL-1β or TNF-α in intestinal epithelial cells.

[0011] The polyphenol composition inhibits inflammatory bowel disease (IBD) by reducing ulcer bleeding.

[0012] The polyphenol composition is dissolved in water (containing 5% DMSO) and has a total concentration of 0.01-30 mg / ml, preferably 0.5-15 mg / ml.

[0013] A drug for treating inflammatory bowel disease (IBD), characterized in that it contains an effective dose of a polyphenol composition.

[0014] The present invention has found that the polyphenol composition has a better therapeutic effect on IBD.

[0015] Beneficial effects of the present invention: (1) The polyphenol composition provided by the present invention has significant antioxidant, anti-inflammatory and anti-ulcer effects, and can effectively alleviate the clinical symptoms of patients with inflammatory bowel disease (IBD), reduce inflammatory responses, and promote intestinal mucosal repair.

[0016] (2) Compared with existing treatment methods, the polyphenol composition provided by the present invention has the advantages of less toxic and side effects, high safety, and low recurrence, and is suitable for long-term treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The morphology of RAW264.7 cells after treatment with LPS and various polyphenols.

[0018] Bar=100μm.

[0019] Figure 2 The levels of inflammatory factors in the cell supernatant after the cells were treated with LPS and each monomer.

[0020] Panel A represents the NO level, and panel B represents the TNF-α level.

[0021] The horizontal axis represents different compounds at different concentrations, and the vertical axis represents the concentration of inflammatory factors.

[0022] Compared with the LPS group, P**<0.05, P*<0.01 (n=3).

[0023] Figure 3 The therapeutic effects of each group of compounds on IBD mice.

[0024] Panels AG are weight change ratio, stool characteristics score, DAI score, duration of red blood cells in stool, duration of white blood cells in stool, stool characteristics picture, and perianal bleeding.

[0025] Group 1, control group; Group 2, model group; Group 3, isorhamnetin, kaempferol, quercetin, ferulic acid, caffeic acid; Group 4, 5-ASA.

[0026] Compared with group 2, P**<0.05, P*<0.01 (n=8).

[0027] Figure 4 The anatomical conditions of mice in each group.

[0028] Panels AE represent colon images, colon length, tissue score, colon IL-1β level, and colon tissue section HE staining. Group 1, control group; Group 2, model group; Group 3, isorhamnetin, kaempferol, quercetin, ferulic acid, caffeic acid; Group 4, 5-ASA.

[0029] P*<0.01 (n=8) compared with Group 2. Bar=200μm. DETAILED DESCRIPTION

[0030] Purpose: To explore the therapeutic effect of polyphenol composition on dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD) model mice, the content of active ingredient compounds and their therapeutic effect.

[0031] Experimental methods: First, the polyphenol composition was used to treat a cellular inflammation model to observe the effect of the compound monomer on inflammation. In order to explore the effect of these polyphenol compositions in animals, the polyphenol composition monomers were combined into different combinations of agents and used to treat DSS-induced IBD model mice. The degree of inflammation relief was analyzed by observing the symptoms, tissue changes, and inflammatory factor levels of the mice.

[0032] Experimental results: Our study proved that all nine polyphenol monomers have the effect of relieving LPS-induced cell inflammation, which is manifested by maintaining cell morphology, preventing polarization and reducing the level of inflammatory factors. The polyphenol combination has the effect of treating IBD model mice, including analgesia, hemostasis, anti-inflammatory and protection of tissue damage. The combination of multiple polyphenols is more effective in relieving IBD symptoms, and the combination of isorhamnetin, kaempferol, quercetin, ferulic acid and caffeic acid has the most obvious effect.

[0033] Experimental conclusion: The combination of isorhamnetin, kaempferol, quercetin, ferulic acid and caffeic acid can treat DSS-induced IBD in mice, and the polyphenol monomers therein have different degrees of anti-inflammatory effects. It is suggested that the combination of isorhamnetin, kaempferol, quercetin, ferulic acid and caffeic acid may be used as an alternative drug for the treatment of IBD and has a good application value.

[0034] 1. Materials and Methods 1.1 Materials Dextran sulfate sodium (DSS) (Cat. No. MB5535, MeilunBio, China), 5-aminosalicylic acid (5-ASA) (Cat. No. A8231485, Macklin, China), RAW264.7 cell culture medium (Cat. No. CM-0191, Procell, China), lipopolysaccharide (LPS) (Cat. No. L2880, Sigma, USA), nitric oxide detection kit (Cat. No. S0021S, Beyotime, China), mouse TNF-α, IL-1β enzyme-linked immunosorbent assay (ELISA) kit (Cat. No. 1217202, 1210122, Dakewe, China).

[0035] Caffeic acid (CAS: 331-39-5, Item No.: C108308), ferulic acid (CAS: 1135-24-6, Item No.: F103702), gallic acid (CAS: 149-91-7, Item No.: G131992), hesperidin (CAS: 520-26-3, Item No.: H105438), isoquercetin (CAS: 482-35-9, Item No.: No.: Q109589), isorhamnetin (CAS: 480-19-3, SKU: I109591), kaempferol (CAS: 520-18-3, SKU: K107144), protocatechuic acid (CAS: 99-50-3, SKU: P104383), and quercetin (CAS: 117-39-5, SKU: Q111273) were all from Aladdin (China).

[0036] 1.2 Methods 1.2.1 Cell culture RAW264.7 cells were placed in a T25 culture flask and cultured in 5 ml complete medium (DMEM high glucose medium containing 10% fetal bovine serum and 1% penicillin + streptomycin). The cell culture incubator environment was 37°C and 5% CO2.

[0037] Recovery: Take out the frozen cells and thaw them quickly in a 37℃ water bath, add 4ml of complete culture medium, mix well, centrifuge (800rpm, 5min), remove the supernatant, add complete culture medium to resuspend and transfer to T25 cell culture flask for culture.

[0038] Passaging: After discarding the culture medium, rinse the cells with PBS. Add 2 ml of complete culture medium and gently blow off the cells. Centrifuge the cell suspension (800 rpm, 5 min) to remove the supernatant, add culture medium to resuspend and evenly distribute to two culture bottles.

[0039] Cryopreservation: Collect the cells in the culture flask and centrifuge (800rpm, 5min), discard the supernatant, add 1ml serum-free cryopreservation solution to resuspend, then freeze in a gradient cryopreservation box, take out after one day and store in liquid nitrogen.

[0040] 2.2.6 Monomeric polyphenols in the treatment of cellular inflammation models Cells in the logarithmic growth phase were inoculated into 96-well plates, and the number of cells in each well was kept constant. After waiting for adhesion, polyphenol compound solutions (dissolved in PBS) were added to each well to make the final concentrations of 2, 5, 10, and 15 μg / ml. After culturing for 2 hours, LPS (dissolved in PBS) was added to make the final concentration of 2 μg / ml. For the positive control group (LPS group), only LPS was added; the negative control group (CTRL group) was not treated. After culturing for 24 hours, the cell morphology was observed and the supernatant was taken to detect the NO and TNF-α content. The experiment was repeated three times.

[0041] 1.2.2 Animal husbandry All experimental animals used were 6-8 weeks old, 18-22g SPF grade balb / c female mice. They were cultured under experimental conditions of 22-24℃, 50-60% humidity, 12h light and 12h dark alternation. All mice were free to eat and drink freely. The experiment began after one week of acclimatization.

[0042] 1.2.3 Polyphenol combination therapy for IBD In order to determine the efficacy of polyphenol components in animals, we combined different polyphenols to treat DSS-induced colitis mice. 56 mice were randomly assigned to 7 groups: Group 1, control group; Group 2, model group; Group 3, isorhamnetin, kaempferol, quercetin, ferulic acid, caffeic acid; Group 4, 5-ASA. The above 4 groups of polyphenol compositions were all mixed according to the weight of each monomer component at 1 mass part.

[0043] The total amount of polyphenols administered was 300 mg / kg / d, and the preparation of the drug was as follows: Weigh 60 mg of isorhamnetin, kaempferol, quercetin, ferulic acid, and caffeic acid respectively and dissolve them in 10 ml of water (containing 5% DMSO).

[0044] The CTRL group was given pure water, and the other groups were given 3% DSS aqueous solution and replaced with pure water after one week. The mice were free to eat and drink. At the beginning of the experiment, the corresponding drugs were given to the mice in groups 3 and 4 for gavage treatment every day for 10 days. The CTRL and MODEL groups were given equal amounts of pure water for gavage. During the experiment, the weight of each mouse was recorded every day. Feces were collected and observed under a microscope using saline smears, and the fecal characteristics, the presence of red blood cells (occult blood) and white blood cells under the microscope, and the presence of macroscopic bloody stools were recorded. After the treatment, the mice were anesthetized with isoflurane and dissected, and the spleen index and colon length were recorded. The colon tissue of the mice was taken and added with an equal mass of PBS solution, ground into a homogenate, and stored at -80°C. The colon of each group of mice was embedded in paraffin and sectioned, and then the sections were dewaxed and HE stained, and the colon morphology was observed under a microscope.

[0045] The disease activity index (DAI) score and histological score refer to Tables 1 and 2. Spleen index = spleen weight (mg) / body weight (g).

[0046] Table 1. DAI scoring criteria

[0047] Table 2. Organization scoring criteria

[0048] 1.2.4 ELISA test ELISA kits were used to detect the levels of inflammatory factors in mouse colon tissue and cell supernatant. After the specimens (colon homogenate and cell supernatant) were diluted to appropriate concentrations, 100 μl of the specimen was mixed with 50 μl of antibody and incubated at 37°C for 90 min in the antibody-coated plate, the plate was washed, 100 μl of HRP was added and incubated for 30 min, and the plate was washed again. Finally, the color developer TMB was added and incubated for 10 min to terminate the reaction, and the OD value was detected at a wavelength of 450 nm. Standard curves were prepared using inflammatory factor standards with concentrations of 500, 250, 125, 62.5, 31.3 and 15.6 pg / ml to calculate the concentration of inflammatory factors.

[0049] 2.2.13 Statistical methods Data were analyzed using GraphPad Prism 9.0 software (GraphPad Software, USA) and SPSS 26.0 software (IBM SPSS Statistics, USA). Quantitative data are expressed as " ± sx". One-way analysis of variance (ANOVA) was used for comparison among groups, and the least significant difference (LSD) test was used for pairwise comparison. All statistical tests were two-sided, and P < 0.05 was considered statistically significant.

[0050] 2. Results 2.1 Monomeric compounds alleviate inflammation in RAW264.7 cells After treating cells with polyphenols and LPS, the cell morphology was observed under a microscope. Figure 1 As shown, the cells in the CTRL group were normal round in morphology and moderate in size ( Figure 1 K). The cells in the LPS group were polygonal, with significantly larger volumes and visible antennae ( Figure 1 J). The cells in each treatment group were normal or slightly enlarged in size and round in shape, with few or no cells showing antennae ( Figure 1 A-1I).

[0051] After treatment, the cell supernatant was stored at -80°C. The supernatant was taken to detect the NO content, and the inflammatory factor TNF-α content was detected by ELISA. Figure 2 As shown in the figure, compared with the CTRL group, the NO and TNF-α contents in the LPS group were significantly increased (P < 0.01), proving that this model is effective. At a concentration of 2 μg / ml, the NO production in the cell supernatant treated with quercetin was lower than that in the LPS group (P < 0.01). Although the NO production in the isorhamnetin, kaempferol and protocatechuic acid groups was lower than that in the LPS group, the difference was not significant (P > 0.05), and the NO in other treatment groups did not decrease. At a concentration of 5 μg / ml, the NO production in the gallic acid group and the isorhamnetin group (P < 0.05), the kaempferol group and the quercetin group (P < 0.01) decreased, while the magnitude of the NO decrease in other treatment groups was not significantly different (P > 0.05). At concentrations of 2 μg / ml and 5 μg / ml, the TNF-α level in each treatment group did not decrease significantly. At 10μg / ml and 15μg / ml concentrations, the production of NO (P<0.01) and TNF-α (except isoquercetin, the rest are P<0.01; 10μg / ml isoquercetin group P<0.05) in each treatment group was significantly reduced, and the higher the drug concentration, the greater the reduction. The above results show that all nine compounds show different degrees of dose-dependent anti-inflammatory effects.

[0052] 2.2 Polyphenol combination therapy for IBD During the ten-day treatment, we observed that the symptoms of mice in each treatment group were alleviated compared with those in group 2. Figure 3As shown, the changes in weight, stool characteristics and DAI scores in each group were different from those in group 2 (P<0.01). We can observe that bloody stools in group 2 lasted longer, and red blood cells in the stool were observed under the microscope. The bleeding in group 3 was significantly improved (P<0.01). As for the duration of white blood cells in the stool, group 3 had the best relief effect (P<0.01).

[0053] After treatment, we dissected the mice and removed the colon to measure the length of the colon. After adding PBS solution to the colon and grinding and diluting it, we measured the IL-1β level in the colon homogenate. Figure 4 As shown, the colon length of group 2 was significantly shortened and IL-1β was increased. Compared with group 2, the data of each treatment group were relieved to near normal levels (P<0.01). Subsequently, a section of colon was taken to observe the morphology after HE staining under the microscope, and the tissue score was performed accordingly. The microscopic morphology of the colon in group 2 showed significant colitis characteristics, and the tissue score was increased. After treatment, the microscopic morphology of the colon was improved and the tissue score was reduced (P<0.01), with group 3 having the better effect. In summary, the compounds in group 3 (isorhamnetin, kaempferol, quercetin, ferulic acid, and caffeic acid) have the best therapeutic effect on IBD.

Claims

1. Use of a polyphenol composition in preparing a drug for treating inflammatory bowel disease, wherein the polyphenol composition is a composition of isorhamnetin, kaempferol, quercetin, ferulic acid, and caffeic acid.

2. Use of a polyphenol composition according to claim 1 in preparing a drug for treating inflammatory bowel disease, characterized in that The invention is composed of the following mass ratios: 1-50 parts of isorhamnetin, 1-50 parts of kaempferol, 1-50 parts of quercetin, 1-50 parts of ferulic acid and 1-50 parts of caffeic acid.

3. Use of a polyphenol composition according to claim 1 in preparing a drug for treating inflammatory bowel disease, characterized in that The invention is composed of the following mass ratios: 1-10 parts of isorhamnetin, 1-10 parts of kaempferol, 1-10 parts of quercetin, 1-10 parts of ferulic acid and 1-10 parts of caffeic acid.

4. Use of a polyphenol composition according to claim 1 in preparing a drug for treating inflammatory bowel disease, characterized in that The composition is composed of the following mass ratio: 1 part of isorhamnetin, 1 part of kaempferol, 1 part of quercetin, 1 part of ferulic acid and 1 part of caffeic acid.

5. Use of a polyphenol composition according to claim 1 in preparing a drug for treating inflammatory bowel disease, characterized in that: The polyphenol composition treats symptomatic enteropathy by reducing the secretion of at least one of the inflammatory factors IL-1β or TNF-α in intestinal epithelial cells.

6. Use of a polyphenol composition according to claim 1 in preparing a drug for treating inflammatory bowel disease, characterized in that: The polyphenol composition treats inflammatory bowel disease by means of hemostasis, analgesia, anti-oxidation, anti-inflammation and the like.

7. Use of a polyphenol composition according to claim 1 in preparing a drug for treating inflammatory bowel disease, characterized in that The concentration of the polyphenol composition is 0.01-100 mg / ml.

8. Use of a polyphenol composition according to claim 1 in preparing a drug for treating inflammatory bowel disease, characterized in that The concentration of the polyphenol composition is 0.5-15 mg / ml.

9. A drug for treating inflammatory bowel disease, characterized in that: The polyphenol composition according to any one of claims 1 to 8 is contained in an effective dose.

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