Use of red ginseng polysaccharide in the preparation of a pharmaceutical composition for preventing and treating inflammatory bowel disease

Abstract

This invention discloses the application of red ginseng polysaccharide in the preparation of pharmaceutical compositions for the prevention and treatment of inflammatory bowel disease, belonging to the field of medical formulation technology. It is the first to clearly demonstrate that red ginseng polysaccharide promotes the expression of bile salt hydrolase and 7α-hydroxysteroid dehydrogenase by targeting and upregulating the relative abundance of Bacteroides acidophilus and Clostridium lysate in the intestine, thereby driving the production of 7-ketolithocholic acid in the intestine. Through the TGR5 activation mediated by 7-ketolithocholic acid, it improves intestinal damage caused by inflammatory bowel disease, providing a solid experimental basis and theoretical support for the application of red ginseng polysaccharide in the preparation of drugs or functional foods for the prevention and treatment of inflammatory bowel disease.

Description

Technical Field

[0001] This invention belongs to the field of medical preparation technology, specifically relating to the application of red ginseng polysaccharide in the preparation of pharmaceutical compositions for the prevention and treatment of inflammatory bowel disease. Background Technology

[0002] Inflammatory bowel disease (IBD) is a group of chronic, relapsing inflammatory bowel diseases, mainly including ulcerative colitis (UC) and Crohn's disease (CD). Clinically, it is characterized by abdominal pain, diarrhea, mucus and bloody stools, and damage to the intestinal structure. The disease course is prolonged and does not heal, which seriously reduces the quality of life of patients, and long-term illness significantly increases the risk of colorectal cancer.

[0003] Currently, clinical treatments for IBD mainly include aminosalicylic acids, glucocorticoids, immunosuppressants, and biologics. These drugs only relieve symptoms and cannot reverse the disease progression at its root. They also have drawbacks such as significant long-term side effects, high relapse rates, and poor or no response in some patients, making them insufficient to meet clinical treatment needs. Recent studies have confirmed that gut microbiota dysbiosis and bile acid metabolism disorders are core driving factors in the development of IBD: IBD patients have significantly reduced gut microbiota diversity, decreased abundance of beneficial bacteria, and excessive proliferation of pathogenic bacteria, leading to gut microbiota-mediated abnormal secondary bile acid metabolism, significantly downregulation of key protective bile acid levels, and ultimately, intestinal barrier disruption, immune imbalance, and chronic intestinal inflammation.

[0004] 7-Ketolithocholic acid (7-KLI) is a key secondary bile acid produced by gut microbiota metabolism. Current research confirms that it can promote intestinal stem cell self-renewal and epithelial repair, and inhibit intestinal inflammatory responses by targeting signaling pathways such as Wnt, making it an important protective metabolite for maintaining intestinal homeostasis. However, 7-KLI levels are significantly downregulated in the gut of IBD patients, and the degree of downregulation is negatively correlated with disease activity. *B. acidifacicens* and *C. scindens* are core symbiotic bacteria in the gut involved in bile acid biotransformation. Their expressed BSH and 7α-HSDH are key rate-limiting enzymes catalyzing 7-KLI production. The significantly reduced abundance of these two strains in the gut of IBD patients is a core reason for insufficient 7-KLI levels. Therefore, targeting and regulating *B. acidifacicens* and *C. scindens* to restore intestinal 7-KLI levels represents a novel target and effective strategy for the prevention and treatment of IBD.

[0005] Red ginseng is a processed product of ginseng, a plant belonging to the genus Panax in the family Araliaceae. It is a traditional Chinese medicine and food with the same origin. Red ginseng polysaccharides are one of the most abundant active ingredients in red ginseng, and current research has confirmed that they possess various biological activities, including anti-inflammatory, immunomodulatory, and gut microbiota regulation. Previous studies have shown that ginseng polysaccharides can improve colitis symptoms by regulating gut microbiota and inhibiting inflammatory pathways. However, current technology has not yet reported on red ginseng polysaccharides improving intestinal damage in IBD by targeting and regulating *B. acidifacicis* and *C. scindens* to mediate the production of 7-ketolithocholic acid. Its regulatory role and mechanism on the "gut microbiota-bile acid axis" in IBD have not yet been elucidated, greatly limiting the development and application of red ginseng polysaccharides in the prevention and treatment of IBD. Summary of the Invention

[0006] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0007] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide the application of red ginseng polysaccharide in the preparation of pharmaceutical compositions for the prevention and treatment of inflammatory bowel disease.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution: the red ginseng polysaccharide upregulates Bacteroides acidophilus in the intestine through targeted upregulation. Bacteroides acidifaciens, B. acidifaciens ) and Clostridium lysate ( Clostridium scindens, C.scindens The relative abundance of 7α-ketolithocholic acid promotes the expression of bile salt hydrolase (BSH) and 7α-hydroxysteroid dehydrogenase (7α-HSDH), thereby driving the production of 7-ketolithocholic acid in the intestine. Through the activation of TGR5 mediated by 7-ketolithocholic acid, it improves intestinal damage caused by inflammatory bowel disease (IBD). The red ginseng polysaccharide is a homogeneous polysaccharide obtained from red ginseng through water extraction, alcohol precipitation, protein removal, decolorization, and dialysis purification. It has a molecular weight of 24.3 kDa, and the extraction method includes... Red ginseng slices were pulverized to obtain red ginseng powder, which was then subjected to water extraction and alcohol precipitation to obtain crude red ginseng polysaccharide. Red ginseng crude polysaccharide was deproteinized using Sevag reagent, and the process was repeated until no obvious protein precipitate was observed. The polysaccharide solution after deproteinization was decolorized with resin, the eluent was collected, dialyzed with deionized water, and then freeze-dried after dialysis to obtain crude red ginseng polysaccharide. Crude red ginseng polysaccharide was purified using a two-step method combining DEAE-52 cellulose anion exchange chromatography and dextran gel G-100 filtration chromatography. The eluent was eluted with a NaCl gradient, and the absorbance at 490 nm was measured using the phenol-sulfuric acid method to plot the elution curve. The target polysaccharide peaks were combined, concentrated under reduced pressure, and desalted by dialysis. The resulting product was then loaded onto a pre-swollen, equilibrated Sephadex G-100 gel column and eluted with ultrapure water. The absorbance at 490 nm was measured using the phenol-sulfuric acid method to plot the elution curve, and the symmetrical single-peaked target component was collected. After freeze-drying, homogeneous pure red ginseng polysaccharide was obtained.

[0009] As a preferred embodiment of the application of the red ginseng polysaccharide described in this invention in the preparation of a pharmaceutical composition for the prevention and treatment of inflammatory bowel disease, wherein the red ginseng polysaccharide has the effect of restoring the abundance of Bacteroides acidophilus and Clostridium lysate in the intestine of an inflammatory bowel disease model to normal levels.

[0010] As a preferred embodiment of the application of the red ginseng polysaccharide described in this invention in the preparation of a pharmaceutical composition for the prevention and treatment of inflammatory bowel disease, wherein: the red ginseng polysaccharide has the effect of increasing 7-ketolithocholic acid in intestinal contents, and the content of 7-ketolithocholic acid is positively correlated with the abundance of Bacteroides acidophilus and Clostridium lysate.

[0011] As a preferred embodiment of the application of the red ginseng polysaccharide described in this invention in the preparation of a pharmaceutical composition for the prevention and treatment of inflammatory bowel disease, wherein: the improvement of intestinal damage caused by inflammatory bowel disease includes alleviating weight loss and shortening of colon length caused by inflammatory bowel disease, improving colonic tissue pathological damage, repairing the intestinal mucosal barrier, and inhibiting local intestinal inflammatory response.

[0012] As a preferred embodiment of the application of the red ginseng polysaccharide described in this invention in the preparation of a pharmaceutical composition for the prevention and treatment of inflammatory bowel disease, wherein: the repair of the intestinal mucosal barrier includes upregulating the expression of tight junction proteins ZO-1 and Occludin in colon tissue and reducing intestinal permeability.

[0013] As a preferred embodiment of the application of the red ginseng polysaccharide described in this invention in the preparation of a pharmaceutical composition for the prevention and treatment of inflammatory bowel disease, wherein: the inhibition of local intestinal inflammatory response includes downregulating the expression levels of pro-inflammatory factors TNF-α, IL-6, and IL-17 in colon tissue.

[0014] As a preferred embodiment of the application of the red ginseng polysaccharide described in this invention in the preparation of a pharmaceutical composition for the prevention and treatment of inflammatory bowel disease, wherein: the water extraction and alcohol precipitation treatment includes, wherein the mass ratio of red ginseng powder to pure water is 1:15, water extraction is performed twice, each time for 1.5 hours, the two extracts are combined, the residue is removed by suction filtration, and then concentrated by vacuum rotary evaporation to a concentrate with a solid content of 15%.

[0015] As a preferred embodiment of the application of the red ginseng polysaccharide described in this invention in the preparation of a pharmaceutical composition for the prevention and treatment of inflammatory bowel disease, wherein: the alcohol precipitation is performed by adding anhydrous ethanol to the concentrated liquid for alcohol precipitation, the final amount of ethanol used is 80% by volume, and the mixture is placed in a refrigerator at 4°C for 16 hours for alcohol precipitation.

[0016] As a preferred embodiment of the application of the red ginseng polysaccharide described in this invention in the preparation of a pharmaceutical composition for the prevention and treatment of inflammatory bowel disease, wherein: the gradient elution with NaCl solution is performed sequentially with 0, 0.1 mol / L NaCl, 0.2 mol / L NaCl and 0.3 mol / L NaCl solutions, with 250 mL of each gradient elution, a flow rate of 1 mL / min, and 10 min per tube.

[0017] As a preferred embodiment of the application of the red ginseng polysaccharide described in this invention in the preparation of a pharmaceutical composition for the prevention and treatment of inflammatory bowel disease, wherein the pharmaceutical composition is an oral preparation, including tablets, capsules, granules, powders, suspensions, solutions, and enteric-coated preparations.

[0018] Beneficial effects of this invention: This invention addresses the shortcomings of existing clinical treatments for IBD and the technological gap in research on the mechanism of action of red ginseng polysaccharide. It provides the application of red ginseng polysaccharide in the preparation of pharmaceutical compositions for the prevention and treatment of inflammatory bowel disease. For the first time, it reveals a novel mechanism of action of red ginseng polysaccharide: by upregulating the abundance of B. acidifaciens and C. scindens, promoting the production of 7-ketolithocholic acid, repairing the intestinal barrier, and inhibiting intestinal inflammation. This provides a safe, effective, widely available, and long-term usable natural active substance solution for the prevention and treatment of IBD. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. 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.

[0020] Figure 1 The present invention provides elution curves and molecular weight diagrams for the separation and purification of red ginseng polysaccharides, wherein... Figure 1 (a) Elution curve of crude red ginseng polysaccharide (RGP) on a DEAE-52 cellulose column. Figure 1 (b) shows the elution curve of RGP-I on a Sephadex G-100 gel column. Figure 1 (c) represents the molecular weight of pure red ginseng polysaccharide RGP-Ⅰ-Ⅰ.

[0021] Figure 2This is a statistical chart showing the effect of red ginseng polysaccharide of the present invention on improving intestinal damage in IBD mice. Figure 2 (a) shows the colon length of mice in different groups. Figure 2 (b) shows the IL-6 levels in different groups of mice. Figure 2 (c) shows the TNF-α content in different groups of mice. Figure 2 (d) represents the IL-17 levels in different groups of mice. Figure 2 (e) H&E staining of colon tissue from different groups of mice. Figure 2 (f) Western Blot was used to detect the expression levels of ZO-1, Occludin, Claudin-1, IL-1β and TNF-α proteins in different groups of mice.

[0022] Figure 3 PCoA analysis of gut microbiota in different groups of mice.

[0023] Figure 4 A bar chart showing the phylum level of the gut microbiota in different groups of mice.

[0024] Figure 5 Bar chart showing the species-level intestinal flora of different groups of mice.

[0025] Figure 6 (a) represents the ratio of Firmicutes relative abundance to Bacteroidetes relative abundance (F / B) in the gut microbiota of different groups of mice. Figure 6 (b) represents the relative abundance of B. acidifaciens in different groups of mice. Figure 6 (c) represents the relative abundance of C. scindens in different groups of mice.

[0026] Figure 7 This is a graph showing the detection of 7-ketolithocholic acid content in colonic contents according to the present invention. Figure 7 (a) Volcano distribution map of the model group and the high-dose red ginseng polysaccharide group treated to establish the ulcerative colitis model. Figure 7 (b) shows the 7-ketolithocholic acid content in different groups of mice.

[0027] Figure 8 For the present invention B.acidifaciens and C.scindens A statistical graph showing the improvement of intestinal damage in IBD mice by combined intervention. Figure 8 (a) shows colon photographs of mice in different groups, corresponding to groups A through F from top to bottom. Figure 8 (b) shows the 7-ketolithocholic acid content in different groups of mice. Figure 8 (c) shows the IL-6 levels in different groups of mice. Figure 8 (d) represents the IL-17 levels in different groups of mice. Figure 8 (e) represents the TNF-α content in different groups of mice. Detailed Implementation

[0028] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0029] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0030] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0031] Example 1 This embodiment provides a method for preparing red ginseng polysaccharides, specifically:

[0032] Red ginseng slices were pulverized by a pulverizer and passed through a 40-mesh sieve to obtain red ginseng powder. 1 kg of red ginseng powder was taken and 15 times the mass of pure water was added. The mixture was refluxed and extracted twice, 1.5 hours each time. The two extracts were combined and filtered with a Buchner funnel to remove the residue. The filtrate was concentrated by rotary evaporation under reduced pressure to a concentrate with a solid content of 15%.

[0033] Anhydrous ethanol was slowly added to the concentrate while stirring until the final volume fraction of ethanol reached 80%. After sealing, the mixture was placed in a refrigerator at 4°C for 16 hours to allow alcohol precipitation. The supernatant was discarded, the precipitate was collected, and the precipitate was washed three times with a small amount of anhydrous ethanol. The precipitate was then freeze-dried to obtain crude red ginseng polysaccharide. The crude red ginseng polysaccharide was reconstituted with pure water to a mass concentration of 10 mg / mL, and Sevag reagent (chloroform: n-butanol = 4:1, v / v) was added. The mixture was shaken vigorously for 30 min, allowed to stand and separate into layers, and the organic phase and denatured protein layer were discarded. The operation was repeated 6 times until no obvious protein layer precipitated. The deproteinized polysaccharide solution was decolorized by AB-8 macroporous resin, and the eluent was collected and dialyzed with deionized water for 48 h (molecular weight cutoff 3500 Da). After dialysis, the solution was freeze-dried to obtain crude red ginseng polysaccharide.

[0034] The crude red ginseng polysaccharide was purified using a two-step method combining DEAE-52 cellulose anion exchange chromatography and dextran gel G-100 filtration chromatography. Specifically, after acid-base pretreatment, DEAE-52 cellulose was wet-packed and fully equilibrated with ultrapure water. The deproteinized polysaccharide sample was dissolved and loaded onto the column, and eluted sequentially with gradients of 0, 0.1 mol / L NaCl, 0.2 mol / L NaCl, and 0.3 mol / L NaCl solutions, 250 mL for each gradient, at a flow rate of 1 mL / min. Elution was performed for 10 minutes per tube. The absorbance of the collected eluent was measured at 490 nm using the phenol-sulfuric acid method, and an elution curve was plotted. The target polysaccharide peak components were combined, concentrated under reduced pressure, and desalted by dialysis. The eluent was then loaded onto a pre-swollen and equilibrated Sephadex G-100 gel column and eluted with ultrapure water at a flow rate of 0.2 mL / min for 10 minutes per tube. The absorbance at 490 nm was measured using the phenol-sulfuric acid method, and an elution curve was plotted. The target components with symmetrical single peaks were collected and freeze-dried to obtain homogeneous pure red ginseng polysaccharide.

[0035] Figure 1 The figure shows the elution curve and molecular weight diagram of the purified red ginseng polysaccharide of this invention. The red line corresponds to the gradient of the NaCl solution. Figure 1 (a) Elution curve of crude red ginseng polysaccharide (RGP) on a DEAE-52 cellulose column. Figure 1 In (a), RGP-Ⅰ represents the RGP fraction eluted with 0 mol / L NaCl, and RGP-ⅠI represents the RGP fraction eluted with 0.1 mol / L NaCl; Figure 1 (b) shows the elution curve of RGP-I on a Sephadex G-100 gel column. Figure 1 (b) RGP-Ⅰ-I represents the first component of RGP-Ⅰ eluted with ultrapure water, and RGP-Ⅰ-II represents the second component of RGP-Ⅰ eluted with ultrapure water; Figure 1 (c) represents the molecular weight of pure red ginseng polysaccharide RGP-Ⅰ-Ⅰ. It can be seen that the molecular weight of pure red ginseng polysaccharide RGP-Ⅰ-Ⅰ obtained in this invention is 24.3 kDa.

[0036] Example 2

[0037] This embodiment is used to verify the ameliorative effect of red ginseng polysaccharide on DSS-induced IBD in mice. Specifically: 1. Laboratory animals and grouping Forty SPF-grade male C57BL / 6 mice, aged 6-8 weeks and weighing 18-22g, were randomly divided into four groups after one week of acclimatization: normal control group, model group (DSS), red ginseng polysaccharide group (DSS+RGP-II, 400mg / kg / d), and positive control group (mesalazine, 5-ASA, 40mg / kg / d), with 10 mice in each group.

[0038] 2. Establishment and administration of an ulcerative colitis (IBD) model Except for the normal control group, all other groups of mice were given free access to 3% (w / v) DSS aqueous solution for 7 consecutive days to establish an ulcerative colitis model; the normal control group was given free access to sterile pure water. At the same time as model establishment, the drugs were administered by gavage. The normal control group and the model group were given the same volume of sterile physiological saline by gavage, and each drug administration group was given the corresponding dose of drug by gavage once a day for 7 consecutive days.

[0039] During the experiment, the mice's body weight, food and water intake, fecal characteristics and blood in stool were recorded daily, and the disease activity index (DAI) was calculated. It should be noted that the Disease Activity Index (DAI) is a commonly used indicator in this field for scoring mouse models of ulcerative colitis. It combines three indicators: weight loss, stool characteristics, and rectal bleeding. The scoring criteria divide each indicator into 0-4 points, and the final average of the three indicators is taken as the DAI value, which can directly reflect the severity of intestinal inflammation and the treatment effect.

[0040] After the experiment, the mice were euthanized by cervical dislocation, colon tissue was collected, colon length was measured, and colon tissue and colon contents samples were collected and stored at -80℃ for later use.

[0041] 3. Experimental Results Figure 2 The figure shows the statistical effects of different groups on intestinal damage in IBD mice. In the figure, the control group represents the normal control group, DSS represents the model group, DSS+RGP-II represents the red ginseng polysaccharide group treated to establish an ulcerative colitis model, and DSS+5-ASA represents the positive drug control group treated to establish an ulcerative colitis model. in, Figure 2 (a) shows the colon length of mice in different groups. Figure 2 (b) shows the IL-6 levels in different groups of mice. Figure 2 (c) shows the TNF-α content in different groups of mice. Figure 2 (d) represents the IL-17 levels in different groups of mice. Figure 2 (e) H&E staining of colon tissue from different groups of mice. Figure 2 (f) Western Blot was used to detect the expression levels of ZO-1, Occludin, Claudin-1, IL-1β and TNF-α proteins in different groups of mice.

[0042] Compared with the normal control group, the model group mice showed significant weight loss, diarrhea with bloody stools, increased DAI scores, and shortened colon length (p<0.01). The colonic crypt structure was severely damaged, with extensive inflammatory cell infiltration and a reduction in goblet cells in the mucosal layer. The expression of tight junction proteins ZO-1 and Occludin in the colonic tissue was significantly downregulated, while the levels of pro-inflammatory factors TNF-α, IL-6, and IL-17 were significantly increased. Red ginseng polysaccharide could alleviate the disease symptoms and colonic pathological damage in the model mice, upregulate the expression of ZO-1 and Occludin, downregulate the levels of pro-inflammatory factors, repair the intestinal mucosal barrier, and inhibit intestinal inflammation. The high-dose group showed the most significant effect, comparable to that of the positive control drug mesalazine.

[0043] Example 3

[0044] This embodiment is used to verify the regulatory effect of red ginseng polysaccharide on the intestinal flora of mice with colitis. Specifically: 1. Experimental Methods Colonic contents samples were taken from mice in the normal control group, model group, and high-dose red ginseng polysaccharide group treated with an established ulcerative colitis model in Example 2. Total microbial DNA was extracted using the QIAamp PowerFecal ProDNA Kit. After quality control, sequencing libraries were constructed and PE150 high-throughput sequencing was performed on the Illumina NovaSeq 6000 platform. After quality filtering and host contamination removal, the raw data were used to complete species classification annotation, functional and metabolic pathway analysis, and metagenomic assembly. Statistical analysis of community diversity, intergroup differences, and correlations was carried out simultaneously.

[0045] 22. Experimental Results Figures 3-6 The results of the analysis of gut microbiota diversity and species composition in different groups are presented. Figure 3 PCoA analysis of gut microbiota in different groups of mice. Figure 4 Bar chart showing the phylum-level gut microbiota of different groups of mice. Figure 5 Bar chart showing the species-level gut microbiota of different groups of mice. Figure 6 (a) represents the ratio of Firmicutes relative abundance to Bacteroidetes relative abundance (F / B) in the gut microbiota of different groups of mice. Figure 6 (b) represents the relative abundance of B. acidifaciens in different groups of mice. Figure 6(c) shows the relative abundance of C. scindens in different groups of mice. It can be seen that, compared with the normal control group, the intestinal flora structure of the model group mice was disordered, and the relative abundance of key bile acid metabolism bacteria B. acidifaciens and C. scindens was significantly downregulated (p<0.01). Red ginseng polysaccharide can significantly reverse the above-mentioned flora abnormalities: the high-dose group can effectively restore flora homeostasis, and all dose groups can significantly upregulate the relative abundance of B. acidifaciens and C. scindens (p<0.01), achieving targeted regulation of the core flora of bile acid metabolism.

[0046] Example 4

[0047] This embodiment is used to investigate the effect of red ginseng polysaccharide on intestinal bile acid metabolism in mice with colitis. Specifically: 1. Experimental Methods Samples of cecal contents from mice in the normal control group, model group, and high-dose red ginseng polysaccharide group treated with an established ulcerative colitis model were taken from Example 2. Targeted bile acid metabolomics detection was performed using an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) platform to quantitatively analyze the changes in 7-ketolithocholic acid content and its correlation with intestinal flora.

[0048] 2. Experimental Results Figure 7 The graph shows the detection of 7-ketolithocholic acid content in colonic contents of different groups. Group A represents the normal control group, Group B represents the model group, and Group C represents the high-dose red ginseng polysaccharide group treated to establish an ulcerative colitis model. Figure 7 (a) Volcano distribution map of the model group and the high-dose red ginseng polysaccharide group treated to establish the ulcerative colitis model. Figure 7 (b) shows the 7-ketolithocholic acid content in different groups of mice.

[0049] It can be seen that, compared with the normal control group, the content of 7-ketolithocholic acid in the colon contents of the model group mice was significantly reduced (p<0.01); compared with the model group, red ginseng polysaccharide can significantly increase the content of 7-ketolithocholic acid in the colon contents (p<0.05 or p<0.01), and the high-dose group can restore the 7-ketolithocholic acid level to close to the level of the normal control group.

[0050] Example 5

[0051] This embodiment is used to verify the mechanism by which red ginseng polysaccharides improve intestinal damage in IBD, specifically: 1. B. acidifaciens, C. scindens recovery experiment Sixty specific pathogen-free (SPF) grade male C57BL / 6 mice were divided into 6 groups of 10 mice each: normal control group, model group, antibiotic + red ginseng polysaccharide (400 mg / kg / d) group (treated to establish an ulcerative colitis model), antibiotic + red ginseng polysaccharide (400 mg / kg / d) + B. acidifaciens + C. scindens (inactivated) group (treated to establish an ulcerative colitis model), antibiotic + red ginseng polysaccharide (400 mg / kg / d) + B. acidifaciens (inactivated) + C. scindens group (treated to establish an ulcerative colitis model), and antibiotic + red ginseng polysaccharide (400 mg / kg / d) + B. acidifaciens + C. scindens group (treated to establish an ulcerative colitis model). Mice were first treated with broad-spectrum antibiotics (ampicillin 1g / L, vancomycin 0.5g / L, neomycin 1g / L, metronidazole 1g / L) to clear the intestinal flora. A DSS ulcerative colitis model was established according to the method in Example 2. The mice were given the corresponding drugs by gavage. The experiment lasted for 7 days. The weight and DAI score of the mice were recorded. Samples were collected for testing after the experiment.

[0052] 2. Experimental Results Figure 8 The graph shows the statistical effect of combined intervention of B. acidifaciens and C. scindens on improving intestinal damage in IBD mice. Group A represents the normal control group, Group B represents the model group, Group C represents the group treated with antibiotics + red ginseng polysaccharide (400 mg / kg / d) after establishing an ulcerative colitis model, Group D represents the group treated with antibiotics + red ginseng polysaccharide (400 mg / kg / d) + B. acidifaciens + C. scindens (inactivated) after establishing an ulcerative colitis model, Group E represents the group treated with antibiotics + red ginseng polysaccharide (400 mg / kg / d) + B. acidifaciens (inactivated) + C. scindens after establishing an ulcerative colitis model, and Group F represents the group treated with antibiotics + red ginseng polysaccharide (400 mg / kg / d) + B. acidifaciens + C. scindens after establishing an ulcerative colitis model. Figure 8 (a) shows colon photographs of mice in different groups, corresponding to groups A through F from top to bottom. Figure 8 (b) shows the 7-ketolithocholic acid content in different groups of mice. Figure 8 (c) shows the IL-6 levels in different groups of mice. Figure 8 (d) represents the IL-17 levels in different groups of mice. Figure 8 (e) represents the TNF-α content in different groups of mice.

[0053] It can be seen that after antibiotics cleared the gut microbiota, the alleviating effects of red ginseng polysaccharide on weight loss and colon shortening in mice with colitis, as well as its intestinal barrier repair and anti-inflammatory effects, were significantly weakened, confirming that its effect in improving IBD depends on the gut microbiota. Combined intervention with B. acidifaciens and C. scindens significantly alleviated DSS-induced colitis symptoms and colonic pathological damage in mice, increased intestinal 7-ketolithocholic acid levels, mimicking the intestinal protective effect of red ginseng polysaccharide, confirming that the effect of red ginseng polysaccharide in improving IBD can be mediated by the gut microbiota.

[0054] In summary, this invention systematically verified the preventive and therapeutic effects of red ginseng polysaccharide on IBD using a mouse ulcerative colitis model induced by dextran sulfate sodium (DSS): red ginseng polysaccharide can significantly alleviate weight loss and colon length shortening in colitis mice, reduce the disease activity index (DAI), and improve pathological damage such as colonic crypt destruction and inflammatory cell infiltration; it can significantly upregulate the expression of tight junction proteins ZO-1 and Occludin in colonic tissue, repair the intestinal mucosal barrier, and reduce intestinal permeability; it can significantly downregulate the expression of pro-inflammatory factors TNF-α, IL-6, and IL-17 in colonic tissue, and inhibit local intestinal inflammatory response.

[0055] This invention, through metagenomics and targeted bile acid metabolomics sequencing, reveals the mechanism of action of red ginseng polysaccharides: red ginseng polysaccharides can significantly restore the diversity of intestinal flora in colitis mice and specifically upregulate... B.acidifaciens and C.scindens The relative abundance of 7-ketolithocholic acid was significantly increased in the colon contents of mice, and the content of 7-ketolithocholic acid was significantly positively correlated with the abundance of Bacteroides acidophilus and Clostridium lysate.

[0056] This invention further verified the mechanism of action through antibiotic gut microbiota clearance experiments and fecal microbiota transplantation (FMT) experiments: after antibiotics cleared the intestinal microbiota, the ameliorative effect of red ginseng polysaccharide on colitis was significantly weakened; replenishment B.acidifaciens With C. scindens It can significantly alleviate the symptoms of colitis in mice and upregulate the level of 7-ketolithocholic acid in the intestine; it confirms that the effect of red ginseng polysaccharide in improving IBD depends on the regulation of the "intestinal flora-7-ketolithocholic acid" axis.

[0057] This invention is the first to discover that red ginseng polysaccharides can specifically target and regulate... B.acidifaciens and C.scindens, The study revealed a novel molecular mechanism by which red ginseng polysaccharides promote the production of 7-ketolithocholic acid, a protective secondary bile acid in the intestine, thus improving the theoretical research on the bioactivity of red ginseng polysaccharides and providing a solid scientific basis for their application in the prevention and treatment of IBD.

[0058] This invention systematically verifies that red ginseng polysaccharide can comprehensively intervene in the occurrence and development of IBD from multiple dimensions, including alleviating clinical symptoms, repairing the intestinal mucosal barrier, inhibiting excessive inflammatory response, and restoring intestinal microecology and bile acid metabolism homeostasis. Its effects are clear and show significant dose dependence, providing a new active substance selection for the clinical intervention of IBD.

[0059] This invention targets the core points of IBD development and progression. B.acidifaciens-C.scindens The "-7-ketolithocholic acid" axis provides a research paradigm for the targeted regulation of the gut microbiota-bile acid axis by natural polysaccharides in the prevention and treatment of IBD, and also provides a new strategy for the precise intervention of the microecology of IBD.

[0060] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The application of red ginseng polysaccharide in the preparation of pharmaceutical compositions for the prevention and treatment of inflammatory bowel disease, characterized in that: The red ginseng polysaccharide targets and upregulates Bacteroides acidophilus in the gut (… Bacteroides acidifaciens ) and Clostridium lysate ( Clostridium scindens The relative abundance of 7-ketolithocholic acid promotes the expression of bile salt hydrolase and 7α-hydroxysteroid dehydrogenase, thereby driving the production of 7-ketolithocholic acid in the intestine. Through the TGR5 activation mediated by 7-ketolithocholic acid, it improves intestinal damage caused by inflammatory bowel disease. The extraction method for the red ginseng polysaccharide includes, Red ginseng slices were pulverized and then subjected to water extraction and alcohol precipitation to obtain crude red ginseng polysaccharide; The crude red ginseng polysaccharide was obtained by repeatedly deproteinizing with Sevag reagent until no protein layer precipitated. The resulting solution was then decolorized with resin, dialyzed with deionized water, and freeze-dried to obtain the crude red ginseng polysaccharide. Crude red ginseng polysaccharide was sequentially separated by DEAE-52 cellulose anion exchange chromatography and Sephadex G-100 gel column filtration chromatography. During the chromatography process, NaCl solution gradient elution and ultrapure water elution were used. The absorbance was detected at 490 nm using the phenol-sulfuric acid method, and the elution curve was plotted. The target single peak components were combined, concentrated, dialyzed for desalting, and freeze-dried to obtain homogeneous red ginseng polysaccharide with a molecular weight of 24.3 kDa.

2. The use of the red ginseng polysaccharide as described in claim 1 in the preparation of a pharmaceutical composition for the prevention and treatment of inflammatory bowel disease, characterized in that: The improvement of intestinal damage caused by inflammatory bowel disease includes alleviating weight loss and shortening of colon length caused by inflammatory bowel disease, improving colonic tissue pathological damage, repairing the intestinal mucosal barrier, and inhibiting local inflammatory response in the intestine.

3. The application of the red ginseng polysaccharide as described in claim 2 in the preparation of a pharmaceutical composition for the prevention and treatment of inflammatory bowel disease, characterized in that: The repair of the intestinal mucosal barrier includes upregulating the expression of tight junction proteins ZO-1 and Occludin in colon tissue and reducing intestinal permeability.

4. The application of the red ginseng polysaccharide as described in claim 2 in the preparation of a pharmaceutical composition for the prevention and treatment of inflammatory bowel disease, characterized in that: The inhibition of local intestinal inflammation includes downregulating the expression levels of pro-inflammatory factors TNF-α, IL-6, and IL-17 in colon tissue.

5. The use of the red ginseng polysaccharide as described in claim 1 in the preparation of a pharmaceutical composition for the prevention and treatment of inflammatory bowel disease, characterized in that: The water extraction and alcohol precipitation process includes water extraction in which the mass ratio of red ginseng powder to pure water is 1:15, water extraction is performed twice, each time for 1.5 hours, the two extracts are combined, the residue is removed by vacuum filtration, and then concentrated by vacuum rotary evaporation to a concentrate with a solid content of 15%.

6. The use of the red ginseng polysaccharide as described in claim 5 in the preparation of a pharmaceutical composition for the prevention and treatment of inflammatory bowel disease, characterized in that: The alcohol precipitation is carried out by adding anhydrous ethanol to the concentrated liquid, with the final amount of ethanol used reaching 80% of the system by volume, and then placing it in a refrigerator at 4°C for 16 hours for alcohol precipitation.

7. The use of the red ginseng polysaccharide as described in claim 1 in the preparation of a pharmaceutical composition for the prevention and treatment of inflammatory bowel disease, characterized in that: The gradient elution with NaCl solution consisted of sequential elution with 0, 0.1 mol / L NaCl, 0.2 mol / L NaCl, and 0.3 mol / L NaCl solutions, with 250 mL eluted per gradient at a flow rate of 1 mL / min for 10 min per tube.

8. The use of the red ginseng polysaccharide as described in claim 1 in the preparation of a pharmaceutical composition for the prevention and treatment of inflammatory bowel disease, characterized in that: The pharmaceutical composition is an oral preparation, including tablets, capsules, granules, powders, suspensions, solutions, and enteric-coated preparations.

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