Application of chrysin-7-O-beta-glucuronic acid in gastrointestinal function improvement

Abstract

The invention discloses an application of chrysin-7-O-beta-glucuronic acid in improvement of functions of intestines and stomach. The chrysin-7-O-beta-glucuronic acid is derived from a natural food material with homology of medicine and food; the chrysin-7-O-beta-glucuronic acid can improve the intestinal microenvironment, relieve the oxidative stress and inflammatory response of intestinal mucosa, promote the integrity recovery of intestinal epithelial barrier, and cooperatively regulate the immune homeostasis of the intestinal tract, so that the chrysin-7-O-beta-glucuronic acid is helpful for recovering the normal permeability and peristalsis function of the intestinal tract, and can be used for preventing and treating intestinal diseases. The invention provides an intestinal function improvement scheme which is safer, can be applied for a long time and is easier to accept.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of drug application, and particularly relates to application of chrysin-7-O-beta-glucuronide in improvement of gastrointestinal function. BACKGROUND

[0002] Gastrointestinal dysfunction is a common health problem in modern society, including functional dyspepsia, irritable bowel syndrome, chronic gastritis, constipation, diarrhea and other symptoms, which seriously affects people's quality of life. According to statistics, the prevalence rate of gastrointestinal dysfunction in Chinese adults is as high as 20-30%, and it is showing an increasing trend year by year. Factors such as fast pace of life, irregular diet, increased stress and misuse of antibiotics can all lead to gastrointestinal dysfunction. At present, the treatment of gastrointestinal dysfunction mainly relies on drug intervention, such as prokinetic drugs, acid suppressants, probiotic preparations, etc., but these drugs often only target a single symptom, and long-term use can easily lead to drug resistance, reduced efficacy or rebound of symptoms after drug withdrawal.

[0003] Traditional Chinese medicine theory believes that gastrointestinal dysfunction is mainly related to weakness of the spleen and stomach, internal accumulation of damp heat and disorder of qi movement. The spleen and stomach are the basis of acquired constitution and the source of qi and blood, and dysfunction of the spleen and stomach will lead to poor digestion and absorption of food, thereby affecting the health of the whole body. Modern research has confirmed that damage to the intestinal mucosal barrier function and disorder of the intestinal immune function are important pathological bases of gastrointestinal dysfunction. In recent years, protecting the intestinal mucosa and reducing intestinal inflammation to improve gastrointestinal function have become a research hotspot. Natural food materials that are pharmacognosy are increasingly favored by people due to their safety, no side effects and suitability for long-term use. In view of this, the present application proposes the application of chrysin-7-O-beta-glucuronide in improvement of gastrointestinal function. SUMMARY

[0004] The purpose of the present application is to provide an application of chrysin-7-O-beta-glucuronide in improvement of gastrointestinal function, to study the effect of chrysin-7-O-beta-glucuronide on improving gastrointestinal function, and to provide a scientific basis for clinical application of chrysin-7-O-beta-glucuronide in adjuvant therapy of ulcerative colitis and intestinal epithelial cell damage.

[0005] In order to achieve the above technical effects, the present application is realized by the following technical scheme: application of chrysin-7-O-beta-glucuronide in improvement of gastrointestinal function.

[0006] Further, the application of chrysin-7-O-beta-glucuronide in ulcerative colitis.

[0007] Further, the application of chrysin-7-O-beta-glucuronide in intestinal epithelial cell damage.

[0008] Furthermore, the jujube-7-O-β-glucuronic acid is derived from natural food ingredients that are both medicinal and edible.

[0009] Furthermore, the natural food ingredient that is both medicinal and edible is one of Scutellaria baicalensis and Oroxylum indicum.

[0010] Furthermore, the jujube-7-O-β-glucuronic acid improves the intestinal microenvironment, reduces oxidative stress and inflammatory response of the intestinal mucosa, promotes the restoration of intestinal epithelial barrier integrity, and synergistically regulates intestinal immune homeostasis, thereby helping to restore normal intestinal permeability and peristaltic function and alleviate discomfort related to gastrointestinal dysfunction.

[0011] The beneficial effects of this invention are:

[0012] 1. The natural component of this invention, apigenin-7-O-β-glucuronic acid (also known as apigenin-7-O-glucuronide), belongs to the natural flavonoid polyphenols and can be extracted from Scutellaria baicalensis and Oroxylum indicum. Its structure contains a flavonoid skeleton and a glucuronic acid group, which has certain antioxidant properties. Studies suggest that this type of flavonoid-related component may play a supporting role in intestinal mucosal barrier function (such as tight junction proteins and epithelial integrity) by regulating intestinal inflammatory response and reducing oxidative stress, and under certain conditions, it may also affect intestinal immune-related signals, thereby helping to maintain intestinal microecology and mucosal homeostasis.

[0013] 2. Animal experiments have shown that the natural ingredients of this invention can effectively reduce intestinal damage in mice with ulcerative colitis and lower the level of pro-inflammatory factors.

[0014] 3. Cell experiments show that the natural ingredients of this invention can significantly improve intestinal epithelial cell damage. Attached Figure Description

[0015] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a schematic diagram illustrating the effect of the natural ingredients of this invention on the body weight changes in mice with ulcerative colitis.

[0017] Figure 2 This is a schematic diagram illustrating the effect of the natural ingredients of this invention on the DAI score of mice with ulcerative colitis.

[0018] Figure 3 This is a schematic diagram illustrating the effect of the natural components of this invention on the colon length of mice with ulcerative colitis.

[0019] Figure 4 This is a schematic diagram illustrating the effect of the natural components of this invention on the level of the pro-inflammatory cytokine IL-1β in the colon of mice with ulcerative colitis.

[0020] Figure 5 This is a schematic diagram illustrating the effect of the natural components of this invention on the level of the pro-inflammatory cytokine IL-6 in the colon of mice with ulcerative colitis;

[0021] Figure 6 This is a schematic diagram illustrating the effect of the natural components of this invention on the level of pro-inflammatory cytokine 6TNF-α in the colon of mice with ulcerative colitis.

[0022] Figure 7 The natural component of this invention promotes KLF4 expression to improve DSS-induced Caco-2 cell damage. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1

[0025] This embodiment provides the practical application of jujubenin-7-O-β-glucuronic acid in mice with ulcerative colitis. The specific steps are as follows:

[0026] 1. Modeling, grouping, and drug administration

[0027] ① Forty C57BL / 6 mice were selected and acclimatized for 7 days, then randomly divided into control group, model group, positive control group, and experimental drug group.

[0028] A. The control group was given normal food and water, while the other three groups were given DSS to establish a model. The modeling method was as follows: a 3% (w / v) sterile DSS solution was prepared using drinking water and used to replace the mice's daily drinking water for 7 days. Subsequently, the mice were given normal drinking water for 3 consecutive days.

[0029] B. Mice exhibiting weight loss, soft stools, or blood in their feces indicate successful modeling.

[0030] ② During the modeling period, mice in the positive control group were administered 100 mg / kg of sulfasalazine by gavage daily, mice in the experimental drug group were administered 25 mg / kg of the natural ingredient of this invention by gavage daily, and mice in the control group and the model group were administered the same volume of physiological saline by gavage daily.

[0031] ③During the modeling and drug administration period, the weight of mice was measured daily, and the DAI score was calculated. After the modeling and drug administration period ended, the mice were sacrificed, and colon tissue was taken to measure its length. A portion of the tissue was used to detect the levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in the colon tissue.

[0032] DAI scoring criteria: 0 points, no weight loss, normal stool consistency, no rectal bleeding; 1 point, weight loss of 1-5%, loose stool, blood in stool; 2 points, weight loss of 5-10%, loose stool, blood in stool; 3 points, weight loss of 10-20%, diarrhea, anal bleeding; 4 points, weight loss of more than 20%, diarrhea, anal bleeding.

[0033] 2. Drug Acquisition

[0034] The positive control drug sulfasalazine is commercially available; the natural ingredients of this invention are also commercially available.

[0035] 3. Experimental Results and Conclusions

[0036] like Figures 1-3 As shown, the results of the mouse body weight change rate indicated that the control group mice showed a steady increase in body weight, while the model group mice experienced a rapid decrease in body weight starting from day 4 of DSS intervention. The decrease in body weight was slowed to varying degrees after intervention with sulfasalazine and the natural ingredient of this invention. The DAI score results showed that, compared to the control group, the DAI score of the model group mice increased rapidly starting from day 4 of modeling, while the DAI score of mice after intervention with sulfasalazine and the natural ingredient of this invention was lower than that of the model group. The colon length results showed that, compared to the control group, the colon length of the model group mice was significantly shortened, while intervention with sulfasalazine and the natural ingredient of this invention alleviated the colon shortening.

[0037] Figures 1-3 In the case where n=10, compared with the control group, ##P<0.01; compared with the model group, **P<0.01.

[0038] like Figures 4-6 As shown, compared with the control group, the levels of pro-inflammatory factors IL-1β, IL-6 and TNF-α in the colon tissue of mice in the model group were significantly increased, while the levels of pro-inflammatory factors in the colon tissue of mice were reduced to varying degrees after intervention with sulfasalazine and the natural ingredients of this invention.

[0039] Figures 4-6 In the study, n=10, compared with the control group, ##P<0.01; compared with the model group, *P<0.05, **P<0.01.

[0040] The above experimental results demonstrate that the natural ingredients of this invention effectively improved weight loss, increased DAI scores, and shortened colon length in mice with ulcerative colitis, alleviated intestinal damage, and reduced the level of inflammatory factors in the colon. This suggests that the natural ingredients of this invention have the effect of improving gastrointestinal function.

[0041] Example 2

[0042] This embodiment provides the practical application of jujubenin-7-O-β-glucuronic acid in intestinal epithelial cell injury. The specific steps are as follows:

[0043] 1. Cell culture, induction and intervention

[0044] Caco-2 cells were cultured in DMEM / F12 medium containing 10% FBS and then placed in a 37 ℃, 5% CO2 cell culture incubator. Cells were passaged every 2-3 days, and cells in the logarithmic growth phase were used for experiments. The experimental groups were: Vehicle group, DSS group, and DSS + succinate-7-O-β-glucuronic acid group.

[0045] Intestinal epithelial cell injury was modeled in two groups: the DSS group and the DSS + jujubenin-7-O-β-glucuronic acid group. The modeling method involved treating Caco-2 cells with 3% DSS (the same dose as in animal experiments) for 24 hours to induce Caco-2 cell injury. During the modeling process, the DSS + jujubenin-7-O-β-glucuronic acid group was simultaneously treated with 60 μM jujubenin-7-O-β-glucuronic acid; the other two groups were cultured with an equal volume of the drug solvent.

[0046] 2. Imprinting

[0047] Total protein was extracted from cells, and protein concentration was determined using the BCA method. After heat denaturation, the protein was separated by SDS-PAGE and transferred to a PVDF membrane, which was then blocked with 5% skim milk for 1 h. The membrane was incubated overnight at 4 °C with the primary antibody for the target protein, washed, and then incubated with the secondary antibody at room temperature for 1 h. Quantitative analysis was performed using Image J after ECL development.

[0048] 3. Immunofluorescence detection

[0049] After cell treatment, cells were fixed with 4% paraformaldehyde for 15 min. Then, cells were permeabilized with 0.5% Triton X-100 at room temperature for 15 min and blocked with goat serum blocking solution for 30 min. Cells were then incubated with primary antibody at 4°C for 12 h. After washing, cells were incubated with fluorescein-labeled secondary antibody at room temperature for 1 h. Cell nuclei were stained with DAPI solution and an anti-fluorescence quencher. Images were captured using a fluorescence microscope, and positive expression regions were quantitatively analyzed using Image Pro Plus 6.0.

[0050] 4. KLF4 transcriptional activity detection

[0051] After seeding cells, KLF4 Luciferase Reporter Plasmid was transfected with Lipotamine 3000. After transfection, cells were treated with or without drugs for 24 h, and luciferase values ​​were measured according to the instructions of the dual luciferase assay kit. Renilla was co-transfected to normalize the luciferase values ​​of the KLF4 reporter gene, thereby assessing KLF4 transcriptional activity.

[0052] 5. Apoptosis level detection

[0053] Apoptosis levels were detected by flow cytometry. After drug intervention, cells were collected and incubated at room temperature in the dark for 5 min according to the instructions of the apoptosis assay kit. 10 μL of PI (20 mg / L) was added, and flow cytometry was performed immediately. Simultaneously, TUNEL staining was used to observe apoptosis. Caco-2 cells from each group were collected, fixed with 4% paraformaldehyde for 15 min, permeabilized, and then incubated with TUNEL reaction solution at 37 °C for 1 h. Finally, TUNEL staining results were observed using fluorescence microscopy, and TUNEL-positive areas were quantified using ImageJ. In addition, Caco-2 cell supernatant was collected, and LDH levels were measured according to the LDH assay kit instructions.

[0054] 6. Experimental Results and Conclusions

[0055] like Figure 7 In group A, compared with the Vehicle group, the expression levels of TJP1, OCLN, and KLF4 proteins in the intestinal epithelial cells of the DSS group were significantly decreased, while the expression levels of TJP1, OCLN, and KLF4 proteins in the intestinal epithelial cells were significantly increased after intervention with the natural ingredients of this invention.

[0056] Figure 7 In group B, compared with the Vehicle group, the fluorescence expression levels of TJP1, OCLN, and KLF4 in intestinal epithelial cells of the DSS group were significantly decreased, while the fluorescence expression levels of TJP1, OCLN, and KLF4 in intestinal epithelial cells were significantly increased after intervention with the natural ingredients of this invention.

[0057] Figure 7 In C, compared with the Vehicle group, the KLF4 transcription level in the intestinal epithelial cells of the DSS group was significantly decreased, while the KLF4 transcription level in the intestinal epithelial cells was significantly increased after intervention with the natural ingredients of this invention.

[0058] Figure 7In D, compared with the Vehicle group, the apoptosis level in the intestinal epithelial cells of the DSS group was significantly increased, while the apoptosis level in the intestinal epithelial cells was significantly decreased after intervention with the natural ingredients of this invention.

[0059] Figure 7 In E, compared with the Vehicle group, the DSS group showed a significant increase in apoptosis-positive expression in intestinal epithelial cells, while the natural ingredients of this invention significantly decreased apoptosis-positive expression in intestinal epithelial cells after intervention.

[0060] Figure 7 In F, compared with the Vehicle group, the LDH activity in the supernatant of intestinal epithelial cells in the DSS group was significantly increased, while the LDH activity in the supernatant of intestinal epithelial cells was significantly decreased after intervention with the natural ingredients of this invention.

[0061] Figure 7 In the given case, n=6, *P<0.05, **P<0.01.

[0062] In summary, the natural components of this invention effectively improve the expression level of tight junction proteins in intestinal epithelial cells, inhibit intestinal epithelial cell apoptosis, and reduce the LDH level in the cell supernatant. This suggests that the natural components of this invention improve the integrity of intestinal epithelial cells.

Claims

1. Application of jujube-7-O-β-glucuronic acid in improving gastrointestinal function.

2. The application according to claim 1, characterized in that, The application of jujube-7-O-β-glucuronic acid in ulcerative colitis.

3. The application according to claim 1, characterized in that, The application of jujube-7-O-β-glucuronic acid in intestinal epithelial cell injury.

4. The application according to claim 1, characterized in that, The eugenol-7-O-β-glucuronic acid is derived from natural food ingredients that are both medicinal and edible.

5. The application according to claim 4, characterized in that, The natural food ingredient that is both medicinal and edible is one of Scutellaria baicalensis and Oroxylum indicum.

6. The application according to claim 1, characterized in that, The jujube-7-O-β-glucuronic acid improves the intestinal microenvironment, reduces oxidative stress and inflammatory response of the intestinal mucosa, promotes the restoration of intestinal epithelial barrier integrity, and synergistically regulates intestinal immune homeostasis, thereby helping to restore normal intestinal permeability and peristaltic function and alleviate discomfort related to gastrointestinal dysfunction.

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