Application of dandelion polysaccharide in preparation of medicine for improving barrier function of inflammatory bowel disease

Abstract

The invention discloses application of dandelion polysaccharide in preparation of a medicine for improving the barrier function of inflammatory bowel disease, and relates to the field of traditional Chinese medicine. 107 action targets of dandelion for improving dextran sodium sulfate to influence ulcerative colitis are determined through a network pharmacology strategy; molecular docking results prove that dandelion has good binding force with potential molecular targets. Then, an in-vivo exposure experiment is carried out, and it is verified that the dandelion polysaccharide can effectively improve UC mouse symptoms. Meanwhile, glycogen in the tissue is detected through an alcian blue-periodate-Schiff staining experiment, and it is verified that the dandelion polysaccharide can effectively improve intestinal barrier damage caused by the inflammatory bowel disease. The invention provides application of dandelion polysaccharide in drugs, food or health care products for preventing and treating intestinal barrier damage caused by inflammatory bowel diseases.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine, and particularly to the application of dandelion polysaccharide in the preparation of a drug for improving the barrier function of inflammatory bowel disease. Background Art

[0002] Dandelion (Taraxacum mongolicum Hand.-Mazz) is a perennial herbaceous plant with white latex in the plant. It is one of the materials that are both medicine and food announced by the International Health and Family Planning Commission. It is distributed almost everywhere in China except in the southern region. Its medicinal value has long been recorded in various medical skills. It is mild in nature, sweet and slightly bitter in taste, and can clear heat and detoxify, disperse swelling and dissipate nodules, and has the laudatory name of "natural antibiotic". The chemical components of dandelion mainly include polysaccharides, flavonoids, triterpenoids, sterols, coumarins and other compounds. Among them, polysaccharides, as the main component, have various functions such as anti-tumor, anti-virus, anti-aging, anti-radiation, and hypoglycemic.

[0003] Inflammatory bowel disease (IBD) is a group of chronic non-specific intestinal inflammatory diseases. In recent years, the damage of the intestinal membrane barrier function has been considered as a key factor in the pathogenesis of IBD.

[0004] Previous studies and reports mainly focused on the anti-tumor research of the mixed extracts of dandelion with various active ingredients and crude polysaccharides, etc., and did not explore its effect on the inflammatory bowel disease barrier. Summary of the Invention

[0005] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art, and provide the application of dandelion polysaccharide in the preparation of a drug for improving the barrier function of inflammatory bowel disease.

[0006] The technical solution of the present invention is as follows:

[0007] The application of dandelion polysaccharide in the preparation of a drug for improving the barrier function of inflammatory bowel disease, wherein the dandelion polysaccharide is the active ingredient in the drug;

[0008] The screening method of the active ingredient includes the following steps:

[0009] S1: Screen the active ingredients of dandelion from the traditional Chinese medicine comprehensive database;

[0010] S2: Then draw a Venn diagram of the component-disease intersection targets according to the active ingredients and UC disease targets to obtain the intersection targets;

[0011] S3: Construct a PPI network for the intersection targets, screen out the key hub genes, and obtain the core targets for dandelion to treat UC;

[0012] S4: Then prepare dandelion polysaccharide, verify the binding ability between dandelion polysaccharide and the core targets, and prove that dandelion polysaccharide can effectively improve the symptoms of UC mice.

[0013] Furthermore, the preparation method of the dandelion polysaccharide comprises the following steps:

[0014] S1: Take dandelion decoction pieces, crush and sieve them to obtain dandelion powder. First, extract the dandelion powder with petroleum ether, then add absolute ethanol and extract it for defatting in a water bath, filter by suction. The precipitate is dried until there is no alcohol smell to obtain defatted dandelion powder;

[0015] S2: Take the defatted dandelion powder, add water, extract it by reflux, filter, take the filtrate and concentrate it under reduced pressure, precipitate it with alcohol, filter by suction after standing, and redissolve the precipitate in water to obtain a crude dandelion polysaccharide solution;

[0016] S3: Remove the protein in the crude dandelion polysaccharide solution with a mixed solvent of chloroform and n-butanol, take the supernatant, concentrate it, and freeze-dry it to obtain dandelion polysaccharide.

[0017] Furthermore, in step S1, the addition amount of petroleum ether is 4-6 times the mass of the dandelion powder, and the petroleum ether is extracted at room temperature for 20-30 h; add absolute ethanol and extract it on a water bath at 90-100 °C for 2-3 h.

[0018] Furthermore, in step S2, the amount of water added is 18-24 times the mass of the defatted dandelion powder, and the extraction is carried out by reflux for 2-4 times, 1-3 h each time.

[0019] Furthermore, in step S3, the volume ratio of chloroform to n-butanol is 4:1.

[0020] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0021] The beneficial effects of the present invention are as follows: In the present invention, first, through a network pharmacology strategy, it is clear that 107 action targets of dandelion in improving dextran sulfate sodium-induced ulcerative colitis are identified; the molecular docking results confirm that dandelion has good binding ability with potential molecular targets. Subsequently, through in vivo exposure experiments, it is verified that dandelion polysaccharide can effectively improve the symptoms of UC mice. At the same time, we detected glycogen in tissues through Alcian blue-periodic acid-Schiff staining (AB-PAS) experiments and verified that dandelion polysaccharide can effectively improve the intestinal barrier damage caused by inflammatory bowel disease. The present invention provides an application of dandelion polysaccharide in drugs, foods or health products for preventing and treating intestinal barrier damage caused by inflammatory bowel disease. Description of the Drawings

[0022] Figure 1 It is a Venn diagram of the intersection targets of dandelion-UC;

[0023] Figure 2 It is the visualization result of the PPI network diagram;

[0024] Figure 3 They are the top ten core targets and their Degree values;

[0025] Figure 4 It is the result of GO functional analysis;

[0026] Figure 5 It is the result of KEGG pathway enrichment analysis;

[0027] Figure 6 It is the ingredient-core target-pathway network;

[0028] Figure 7 It is the molecular docking diagram of Quercetin with the top ten targets;

[0029] Figure 8 It is the docking diagram of Scopoletin with the top ten targets;

[0030] Figure 9 It is the effects of dandelion polysaccharide on the change rate of mouse body weight, DAI score and colon length (n = 6). Among them, A: the change rate of mouse body weight; B: DAI score; C: the colon length of mice (compared with the control group: # indicates P < 0.0001; compared with the DSS group: * indicates p < 0.05, *** indicates p < 0.001, **** indicates p < 0.0001);

[0031] Figure 10 It is the effects of dandelion polysaccharide on the expressions of inflammatory cytokines IL-1β, IL-6, and TNF-α in mouse colon tissues (n = 8). In the figure, compared with the control group: # indicates P < 0.0001; compared with the DSS group: * indicates p < 0.05, *** indicates p < 0.001, **** indicates p < 0.0001;

[0032] Figure 11 It is the HE staining result;

[0033] Figure 12 It is the AB-PAS staining result. Detailed implementation manners

[0034] The embodiments of the present invention will be described in detail below. The following described embodiments are exemplary and are only used to explain the present invention, and should not be construed as a limitation to the present invention. For those techniques or conditions not specified in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. Those reagents or instruments not specified by the manufacturer can be obtained as conventional products through commercial purchase.

[0035] The technical solution of the present invention will be further described below with specific experiments.

[0036] Reagents used in the following examples:

[0037] Dandelion; Sulfasalazine; Chloroform; n-Butanol; D-Anhydrous Glucose (Lot No. 110833-201908) was purchased from the National Institutes for Food and Drug Control; Concentrated sulfuric acid; Phenol; Coomassie Brilliant Blue G-250; Absolute ethanol; Petroleum ether (60°C - 90°C); 4% (g / ml) Paraformaldehyde Universal Tissue Fixative; Xylene; n-Butanol; HE Staining Kit; Interleukin (IL)-1β, IL-6, and Tumor Necrosis Factor α (TNF-α) ELISA Kits

[0038] Instruments used in the following examples:

[0039] Chinese herbal medicine grinder; Electric heating mantle; Vacuum concentration instrument; Freeze dryer; Multifunctional microplate reader; BAS124S Electronic Balance (Sartorius Scientific Instruments Beijing Co., Ltd.); Ultraviolet spectrophotometer; RM2125 Rotary Microtome (Leica Microsystems GmbH, Germany); Automatic Slide Stainer - VS200

[0040] Animals used in the following examples:

[0041] 60 male C57BL / 6J mice, 6 - 8 weeks old, 18 - 21 g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd., and the source institution license number is SCXK(Liao)2020 - 0001. The animals were housed in the Institute of Traditional Chinese Medicine Health Industry, China Academy of Chinese Medical Sciences, and the experimental animal license number is SYXK(Gan)2023 - 0008. All animal experiments were approved by the Animal Welfare and Ethics Committee of the Institute of Traditional Chinese Medicine Health Industry, China Academy of Chinese Medical Sciences (Approval No.: 2023001).

[0042] The test method is as follows:

[0043] 1. Collection of Components of Dandelion against UC and Target Screening

[0044] Screen the active ingredients of dandelion from the Traditional Chinese Medicine Comprehensive Database (Herb; http: / / herb.ac.cn / Detail / ?v=HERB004401&label=Herb). Then, obtain the chemical structures of the selected bioactive compounds from PubChem and import them into the Swiss Target Prediction database (http: / / www.Swisstargetprediction.cn / ) to predict the drug targets. Combine the GeneCards database (https: / / www.genecards.org / ), OMIM database (https: / / www.omim.org), and DisGeNET database (https: / / disgenet.org). Enter the keyword "Ulcerative Colitis" to search for the targets related to this disease and remove duplicate targets. Finally, obtain the target genes of the disease. Use VENNY2.1 (https: / / bioinfogp.cnb.csic.es / tools / venny / ) to input the action targets of the drug and the targets of the disease to create a Venn diagram. The intersection gives the common targets of the two, which are considered the potential targets for dandelion to intervene in UC infection.

[0045] 2. PPI network construction

[0046] Input the intersection targets of the drug and the disease into the String database (https: / / cn.string-db.org / ), select the species "Homo sapiens" to obtain the protein-protein interaction relationship data, and export it as a TSV type file. Use Cytoscape3.9.1 to visualize the protein-protein interaction relationship data, and use the "cytoHubba" plugin in the application of Chaixiong polysaccharide in the preparation of drugs for improving inflammatory bowel disease, combined with the DC, BC, and CC algorithms to screen the core targets.

[0047] 3. Functional and pathway analysis of dandelion targets for treating UC

[0048] Search for enriched genes in the DAVID database (https: / / david.ncifcrf.gov / ), use Microbial Informatics (http: / / www.bioinformatics.com.cn / static / others / jvenn / example.html) to generate a bubble chart for GO and KEGG enrichment analysis. Finally, use the Cytoscape3.9.1 software to construct a network relationship diagram of components-action targets-pathways.

[0049] 4. Molecular docking verification

[0050] Dock the top ten core target proteins obtained from the PPI network data with the components. Use AutodockVina software for molecular docking. Take the drug as the ligand and the protein as the receptor. Select the all-atom docking method and output the top 10 conformations ranked by the docking score. Use Pymol software to visualize the docking results.

[0051] 5. Preparation of Polysaccharide

[0052] Crush the dandelion decoction pieces and pass through a No. 3 sieve. Extract the powder with 5 times the amount of petroleum ether (boiling range of 60 °C - 90 °C) at room temperature for 24 h, filter out the petroleum ether (boiling range of 60 °C - 90 °C), add absolute ethanol and extract and degrease in a water bath at 90 - 100 °C for 2 h, filter by suction. Dry the precipitate in a blast dryer at 50 °C until there is no alcohol smell to obtain the degreased powder. Take the dandelion degreased powder, add 20 times the amount of water, reflux and extract 2 times, each time for 2 h. Filter while it is hot, combine the dandelion filtrates, concentrate under reduced pressure to a crude drug content of 0.25 g / mL, use 80% (v / v) alcohol content as the end point of alcohol precipitation, let it stand overnight at 4 °C and then filter by suction. Dissolve the precipitate in water to obtain the crude dandelion polysaccharide solution. Remove the protein in the crude polysaccharide solution by the Sevag method (chloroform - n-butanol = 4:1), take the supernatant, concentrate, and freeze-dry for 72 h to obtain dandelion polysaccharide.

[0053] 6. Modeling and Administration

[0054] After 7 days of adaptive feeding, except for the normal group, the experimental groups were all fed with 3.0% (g / ml) DSS solution instead of drinking water. Divide the mice into 6 groups (n = 10 / group), randomly divided into normal (Ctrl) group, model (DSS) group, sulfasalazine (SASP, 100 mg / kg) group, low-dose dandelion polysaccharide (PGY-L, 50 mg / kg) group, medium-dose dandelion polysaccharide (PGY-M, 100 mg / kg) group, and high-dose dandelion polysaccharide (PGY-H, 200 mg / kg) group. Each group of animals received treatment from day 1 to day 7. On the 8th day, after fasting for 12 h without water deprivation, collect blood by eye socket puncture, centrifuge at 4 °C for 10 min, and store the supernatant at -80 °C. Then, sacrifice the mice by cervical dislocation. Freeze the distal colon specimens in liquid nitrogen or immediately fix them in 4% paraformaldehyde solution for further analysis.

[0055] 7. Evaluation of Mouse Body Weight and DAI Score

[0056] Observe and record the body weight, fecal traits, and blood in feces of the mice during the entire experiment period, and perform DAI scoring according to the scoring criteria in Table 1.

[0057] DAI = body weight loss rate + fecal traits + blood in feces;

[0058] Table 1 DAI Scoring Criteria

[0059]

[0060] 8.ELISA detection of mouse serum inflammatory factor levels

[0061] After the last administration, mice were fasted but not watered for 12 h, their eyeballs were removed and blood was collected. The blood was placed in a centrifuge tube and centrifuged at 3000 r / min. -1 Centrifuge for 10 minutes, take the supernatant, and store at -80°C for later use. Detect TNF-α, IL-6, and IL-1β according to the kit instructions.

[0062] 9. Colon Histopathological Observation

[0063] Mice were killed by cervical dislocation, colon tissue was quickly isolated, colon length was recorded, and part of the colon was fixed with 4% paraformaldehyde. After dehydration, embedding, sectioning, HE staining, and AB-PAS staining, the slides were mounted and the pathological changes of colon tissue were observed under a microscope.

[0064] Here are the results:

[0065] 1. Screening of active ingredients

[0066] Based on the herb database, 11 active ingredients from dandelion were screened based on the parameters xlogp≤5, hydrogen donor≤5, and hydrogen acceptor≤10.

[0067] Table 1 Ingredient screening based on herb database

[0068]

[0069]

[0070] 2. Intersection targets of dandelion for the treatment of UC

[0071] After screening, 194 targets of dandelion active ingredients and 3751 UC disease targets were obtained. A Venn diagram of component-disease intersection targets was drawn, and 107 intersection targets were obtained. Figure 1 .

[0072] 3. PPI network construction and key hub gene screening

[0073] The above 107 potential anti-inflammatory targets were input into the String platform to obtain the PPI network, which was then imported into Cytoscape 3.9.1 software for network visualization. Figure 2 , Figure 2 The network contains 107 nodes and 227 edges. The cytoHubba plug-in is then used to analyze the PPI network and select core targets based on the degree value. Figure 3 ,Depend on Figure 3It can be seen that the top ten targets are SRC(24), PIK3R1(21), PIK3CA(20), PIK3CB(19), STAT3(18), PTK2(16), AKT1(13), ESR1(13), CYP2C9(9), and CYP2C19(9), indicating that the above proteins may be important targets for dandelion in the treatment of UC.

[0074] 4. Functional and Pathway Analysis of Targets for Dandelion in the Treatment of UC

[0075] GO and KEGG enrichment analyses were performed on 107 intersection targets. The results of GO functional analysis showed ( Figure 4 ) that the intersection targets were mainly involved in processes such as phosphorylation, transmembrane receptor protein tyrosine kinase signaling pathway, and positive regulation of kinase activity in the biological process (BP); were mainly related to receptor complex, cell plasma membrane, cytoplasm, etc. in the cellular component (CC); and were mainly related to transmembrane receptor protein tyrosine kinase activity, protein tyrosine kinase activity, ATP binding, etc. in the molecular function (MF).

[0076] The results of KEGG pathway enrichment analysis showed ( Figure 5 ) that the potential targets of dandelion against UC were mainly involved in signaling pathways such as Pathways in cancer, PI3K-Akt signaling pathway, and Focal adhesion. According to the component-core target-pathway network ( Figure 6 ), important targets such as SRC(24), PIK3R1(21), PIK3CA(20), and PIK3CB(19) were involved in the regulation of these pathways.

[0077] 5. Results of Molecular Docking

[0078] In this study, two components, quercetin and scopoletin, were selected for molecular docking with the top ten targets, and the results are shown in Table 2. Among them, the binding affinity lower than 0 kcal·mol -1 indicated that the ligand molecule could spontaneously bind to the receptor protein, while lower than -5.00 kcal·mol -1It indicates that the ligand molecule has good binding affinity. The best docking results are quercetin with AKT1 (-7.6 kcal / mol). Quercetin has good binding affinity with SRC, PIK3R1, PIK3CA, STAT3, PTK2, AKT1, ESR1, CYP2C9, and CYP2C19. Scopolamine has good binding affinity with PIK3CB, PTK2, ESR1, CYP2C9, and CYP2C19. The docking results of PTK2, ESR1, CYP2C9, and CYP2C19 with quercetin and scopolamine are both good, which may play an important role in the treatment of UC by dandelion.

[0079] Table 2 Molecular docking results

[0080]

[0081]

[0082] 6. Effects of dandelion polysaccharide on the body weight change rate, DAI score, and colon length of mice

[0083] During the experiment, the mice in the Ctrl group were in a stable state, with shiny hair and a steadily increasing body weight. The mice in the model group were listless and had severe bloody stools. After the administration of sulfasalazine and dandelion polysaccharide, the condition of UC mice improved. As Figure 9 shown, compared with the Ctrl group, the body weight of the mice in the DSS group decreased significantly, and the DAI score increased significantly (P < 0.0001). After the administration of dandelion polysaccharide, compared with the DSS group, the body weight change rate and DAI score of the PGY group also showed a similar trend, with a significant increase in the body weight of the mice and a significant decrease in the DAI score. Compared with the Ctrl group, the colon length of the mice treated with DSS was significantly shortened (P < 0.0001). After the administration of dandelion polysaccharide, the colon length recovered significantly (P < 0.0001).

[0084] 7. Detection results of the levels of inflammatory factors in the serum of mice

[0085] The severity of colon inflammation in UC mice is directly related to the content of inflammatory factors. The results are as Figure 10 shown. The inflammatory factors IL-1β, IL-6, and TNF-α in the colon tissues of DSS-induced mice were significantly higher than those in the normal group (p < 0.0001). In contrast, the intervention with dandelion polysaccharide could effectively reduce the levels of IL-1β, IL-6, and TNF-α (p < 0.0001), and the intervention efficiency showed a concentration-dependent manner.

[0086] 3.8 Effects of dandelion polysaccharide on the pathological changes of the colon in UC mice

[0087] As Figure 11The HE staining results showed that the colon structure of the normal group mice was intact; after DSS modeling, the colon crypts of the mice disappeared, a large number of inflammatory cells infiltrated, and the colon tissue was severely damaged; compared with the DSS group, the damage of the colon villus morphology and crypts in the SASP group and different dose groups of dandelion polysaccharide had obvious recovery, the infiltration of inflammatory cells decreased, and the treatment group significantly alleviated the pathological state of UC mice.

[0088] To further explore the effect of dandelion polysaccharide on the colonic mucus barrier in the UC model, we observed the colon tissue by AB-PAS staining method. Figure 12 The results showed that compared with the normal group, the mucin in the colon tissue of the model group mice was significantly reduced and the mucus barrier was damaged. After administration of dandelion polysaccharide, the level of mucus protein in the colon tissue of the mice increased significantly, and the damage of the colon mucus barrier was repaired. The medium and high doses of dandelion polysaccharide showed a good effect on repairing the colon mucus barrier.

[0089] In summary, in the present invention, first, through the network pharmacology strategy, 107 action targets for dandelion to improve dextran sulfate sodium-induced ulcerative colitis were identified; the molecular docking results confirmed that dandelion had good binding ability with potential molecular targets. Subsequently, through in vivo exposure experiments, it was verified that dandelion polysaccharide could effectively improve the symptoms of UC mice. At the same time, in the present invention, by detecting glycogen in tissues through Alcian blue-periodic acid-Schiff staining (AB-PAS) experiments, it was verified that dandelion polysaccharide could effectively improve the intestinal barrier damage caused by inflammatory bowel disease. Therefore, the present invention provides an application of dandelion polysaccharide in drugs, foods or health products for preventing and treating intestinal barrier damage caused by inflammatory bowel disease.

[0090] The above-described embodiments only represent the preferred embodiments of the present invention, and the description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the present invention patent. It should be noted that for those of ordinary skill in the art, according to the technical solutions and concepts described above, other various corresponding changes and deformations can also be made, and all these changes and deformations should fall within the protection scope of the claims of the present invention.

Claims

1. Use of dandelion polysaccharide in the preparation of a drug for improving the barrier function of inflammatory bowel disease, characterized in that, The dandelion polysaccharide is the active ingredient in the drug; The screening method of the active ingredient comprises the following steps: S1: Screen the active ingredients of dandelion from the traditional Chinese medicine comprehensive database; S2: Then draw a Venn diagram of the component-disease intersection targets based on the active ingredients and UC disease targets to obtain the intersection targets; S3: Construct a PPI network with the intersection targets, screen out the key hub genes, and obtain the core targets for dandelion to treat UC; S4: Then prepare dandelion polysaccharide, verify that dandelion polysaccharide has binding ability with the core targets, and prove that dandelion polysaccharide can effectively improve the symptoms of UC mice.

2. The application according to claim 1, characterized in that, The preparation method of the dandelion polysaccharide comprises the following steps: S1: Take dandelion decoction pieces, crush and sieve them to obtain dandelion powder. First, extract the dandelion powder with petroleum ether, then add absolute ethanol and extract it for defatting in a water bath, filter by suction. The precipitate is dried until there is no alcohol smell to obtain defatted dandelion powder; S2: Take the defatted dandelion powder, add water, reflux and extract, filter, take the filtrate, concentrate it under reduced pressure, precipitate with alcohol, stand still and then filter by suction. The precipitate is dissolved in water to obtain a crude dandelion polysaccharide solution; S3: Remove the protein in the crude dandelion polysaccharide solution with a mixed solvent of chloroform and n-butanol, take the supernatant, concentrate it, and freeze-dry it to obtain dandelion polysaccharide.

3. The application according to claim 1, wherein In step S1, the addition amount of petroleum ether is 4-6 times the mass of the dandelion powder, and the petroleum ether is extracted at room temperature for 20-30 h; then add absolute ethanol and extract it on a water bath at 90-100 °C for 2-3 h.

4. The application according to claim 1, characterized in that, In step S2, the amount of water added is 18-24 times the mass of the defatted dandelion powder, and the reflux extraction is carried out 2-4 times, each time for 1-3 h.

5. The application according to claim 1, wherein In step S3, the volume ratio of chloroform to n-butanol is 3-5:

1.

6. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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