Application of fructus psoraleae polysaccharide in preparation of medicine for regulating intestinal barrier function

Abstract

The invention provides an application of fructus psoraleae polysaccharide in preparation of a medicine or health food for regulating an intestinal barrier function. Specifically, the fructus psoraleae polysaccharide acts on an intestinal tract. In the intestinal tract action process, the fructus psoraleae polysaccharide effectively improves damaged intestinal tract barrier repair; the number of CD4 + and CD8 + T cells in intestinal tracts is increased, and the immune barrier of the intestinal tracts is improved; according to the present invention, the expression of tight junction proteins Claudin-1, Occludin and ZO-1 can be promoted, the intestinal mechanical barrier can be repaired and enhanced, and the polypeptide can be used for preventing, relieving or treating intestinal barrier damage, has excellent performance in the regulation of the intestinal barrier function, and has good application prospects.

Description

Technical Field

[0001] This invention belongs to the field of health care drugs or food technology, specifically relating to the application of psoralen polysaccharide in the preparation of drugs that regulate intestinal barrier function. Background Technology

[0002] With the fast pace and increasing pressure of modern life, the prevalence of unhealthy lifestyles has led to a growing prominence of intestinal dysfunction, resulting in a surge in health problems such as obesity, constipation, indigestion, and inflammatory bowel disease, posing a significant public health challenge. Against this backdrop, developing novel and effective solutions for preventing or improving intestinal-related diseases has become one of the core issues urgently needing to be addressed in the current health industry and scientific research field.

[0003] The gut is the largest digestive and immune organ in the human body, and its health directly affects the overall health of the body. Gut health mainly depends on two core elements: a stable gut microbiota balance and intact intestinal barrier function. As the largest barrier tissue, the intestinal barrier's functional integrity depends on mechanical barriers (tight junctions between epithelial cells), chemical barriers (mucus layer), and immune barriers. The integrity of the intestinal barrier plays an indispensable role in maintaining normal physiological functions. When the integrity of the intestinal barrier is damaged, causing barrier injury, microorganisms and endotoxins in the gut can cross the intestinal mucosal barrier, enter the bloodstream, cause bacterial and endotoxin translocation, promote enterogenic infections, and even develop into systemic inflammatory response syndrome or multiple organ failure.

[0004] Currently, the main methods for regulating intestinal barrier function include the use of prebiotics, probiotics, and synbiotics. However, existing technologies have some limitations. For example, probiotics are strain-specific in their effects, and their colonization ability varies among individuals, leading to unstable efficacy. Prebiotics (such as oligosaccharides and inulin), while selectively promoting the growth of beneficial bacteria, have relatively singular targets and limited ability to regulate complex gut microbiota imbalances. Excessive intake may also cause gastrointestinal discomfort such as bloating and gas production. Chemical drugs, such as those used to treat diarrhea or inflammation, often focus on symptom control and may cause side effects. Given the complexity of living environments and the trend towards diversified dietary structures, the pressures and challenges faced by the intestinal system vary greatly among individuals, and the specific mechanisms of intestinal barrier damage also differ significantly. This undoubtedly poses an obstacle to the widespread application of probiotic therapy. Natural active polysaccharides are receiving increasing attention due to their mildness and safety, but there is currently a lack of natural functional foods that protect the intestinal barrier, making the development of related products urgent.

[0005] Psoralea corylifolia ( Psoralea corylifoliaPsoralea corylifolia (L.) is a traditional Chinese medicine with the effects of warming the kidneys and tonifying yang, regulating qi and relieving asthma, and warming the spleen and stopping diarrhea. Modern pharmacological studies have shown that Psoralea corylifolia contains various active ingredients such as coumarins, flavonoids, monoterpenoid phenols, and volatile oils, exhibiting antioxidant, antitumor, antibacterial, and estrogen-like activities. However, research on the active ingredients of Psoralea corylifolia has largely focused on small molecule compounds, such as psoralen and isopsoralen. Research on the large molecule in Psoralea corylifolia—psoralea polysaccharides—is relatively limited and insufficient. Existing literature has not addressed the direct effects of psoralea polysaccharides on the intestine, an important immune organ, particularly their role in regulating intestinal barrier function.

[0006] Various intestinal diseases caused by impaired intestinal barrier function have become a significant problem affecting public health and quality of life. Currently, the number of interventions available on the market to regulate the intestinal barrier (such as specific probiotics and synbiotics) is relatively limited, and they generally suffer from high costs and insufficient stability of efficacy. Based on this, this application was developed. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide an application of psoralen polysaccharide in the preparation of drugs that regulate intestinal barrier function. In other words, this invention provides a new use for psoralen polysaccharide. As a natural plant polysaccharide, psoralen polysaccharide can increase the number of intestinal immune cells, repair and strengthen the intestinal barrier, and improve intestinal diseases caused by impaired intestinal barrier function. Furthermore, this psoralen polysaccharide is inexpensive, naturally sourced, and has significant effects.

[0008] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: Application of a psoralen polysaccharide in the preparation of drugs or health foods that regulate intestinal barrier function.

[0009] Specifically, the above-mentioned application refers to the use of psoralen polysaccharide in the preparation of drugs or health foods that enhance intestinal barrier function.

[0010] Furthermore, the above-mentioned applications refer to the use of psoralen polysaccharide in the preparation of drugs or health foods for the prevention, relief or treatment of intestinal barrier damage.

[0011] Furthermore, the above-mentioned applications refer to the use of psoralen polysaccharide in the preparation of drugs or health foods for promoting intestinal damage repair.

[0012] Furthermore, the above-mentioned applications refer to the use of psoralen polysaccharide in the preparation of drugs or health foods for improving damaged intestinal immune cells.

[0013] Furthermore, the above application refers to the use of psoralen polysaccharide at a dosage of 75~300 mg / kg / day.

[0014] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: As a naturally sourced plant polysaccharide, psoralen polysaccharide has been shown in this invention to effectively increase the number of T cells and repair and enhance the intestinal barrier. Therefore, this invention reveals that psoralen polysaccharide can act on the intestine, effectively improving the repair of damaged intestinal barriers and increasing CD4 levels in the intestine. + and CD8 + It increases the number of T cells and enhances the intestinal immune barrier; it can promote the expression of tight junction proteins Claudin-1, Occludin, and ZO-1, and repair and strengthen the intestinal mechanical barrier. Attached Figure Description

[0015] Figure 1 For pathological observation of H&E-stained intestinal tissue; Figure 2 CD4 in small intestinal tissue + and CD 8 + Number of T cells; A: CD8 + Representative immunohistochemical staining of T cells; B: CD4 + Representative immunohistochemical staining of T cells; C: CD8 + and CD 4 + T cell count; Figure 3 Expression of small intestinal tight junction proteins (Occludin, Olaudin 1, and ZO-1). Detailed Implementation

[0016] To make the technical objectives, technical solutions, and beneficial effects of the present invention clearer, the technical solutions of the present invention will be further described below in conjunction with specific embodiments. However, the embodiments are intended to explain the present invention and should not be construed as limiting the present invention, that is, the scope of protection of the present invention is not limited thereto.

[0017] For any specific techniques or conditions not specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual shall apply. Materials or equipment whose manufacturers are not specified are all conventional products that can be purchased.

[0018] In the following experiments, the psoralen polysaccharide used was deproteinized crude psoralen polysaccharide, which can be prepared according to the following steps: Psoralea corylifolia was pulverized and defatted three times with petroleum ether, each time for 3 days. The residue was evaporated to dryness, and then extracted three times with 70% ethanol solution, each time for 3 days. The residue was evaporated until no alcohol odor was detected. A certain amount of distilled water was added (the solid-liquid ratio was approximately 1 g: 20 mL), and the mixture was extracted three times at 85 ± 2℃, each time for 4 hours. The extracts were combined, filtered, and concentrated to 1 / 4 of the original volume. Anhydrous ethanol was added to adjust the ethanol volume fraction to 70%, and the mixture was allowed to stand. The mixture was then centrifuged at 4000 r / min for 8 min, and the precipitate was collected and freeze-dried to obtain crude psoralea corylifolia polysaccharide. The protein in the crude polysaccharide was removed using the Sevage method, repeated four times. The mixture was then freeze-dried to obtain deproteinized psoralea corylifolia polysaccharide (PPs).

[0019] The deproteinized psoralen polysaccharide (PPs) mainly comprises four components: PCp-I, PCp-II, PPs-1-1, and PPs-2-1, with molecular weights of 2.721 × 10⁻⁶ and 2.721 × 10⁻⁶, respectively. 4 2.850×10 4 3.98×10 6 and 4.47×10 6 The monosaccharide composition mainly includes rhamnose, arabinose, xylose, mannose, glucose, and galactose, but their proportions vary. The process for subsequent separation and purification of the deproteinized psoralen polysaccharide PPs to obtain components PCp-I, PCp-II, PPs-1-1, and PPs-2-1 can be found in the following literature: [1] Zhenhua Yin, Wei Zhang, Juanjuan Zhang, et al., Two NovelPolysaccharides in Psoralea corylifolia L and anti-A549 Lung Cancer CellsActivity In Vitro[J]. Molecules, 2019, 24, 3733; doi:10.3390 / molecules24203733; [2] Yin Zhenhua, Zhang Juanjuan, Chen Lin, et al. Isolation, purification, structural identification and effects of psoralen polysaccharide on the activity of RAW264.7 macrophages [J]. Food Science, 2019, 40(24): 27-32.

[0020] The following describes the relevant activity tests conducted on the deproteinized psoralen polysaccharides (PPs) obtained during preparation.

[0021] 1. Animal experiments:

[0022] Balb / c mice (male, 20±2g, 6-8 weeks old) were housed for one week to acclimatize to the experimental environment. During gavage, the mice had free access to water and food. They were randomly divided into 5 groups of 12 mice each, based on body weight. The blank control group (BC) and the CXT model group (MC) were administered physiological saline by gavage at 0.1 mL / 10 g. The positive control group (PC) was administered levamisole hydrochloride solution (LH, 10 mg / kg, 0.1 mL / 10 g) by gavage. The high (HD, PPsH) and low (LD, PPsL) dose groups of psoralen polysaccharide were administered 300 mg / kg and 75 mg / kg, respectively, by gavage at 0.1 mL / 10 g, once daily for 14 days. On days 15, 16, and 17 after administration, mice in the BC group were injected intraperitoneally with saline, while mice in the other groups were injected intraperitoneally with cyclophosphamide (CTX) at a dose of 70 mg / kg / d to establish a mouse model of intestinal barrier damage. After the last administration, the contents of the small intestine and cecum were collected one day later (all tissues were stored at -80°C for later use).

[0023] 2. Preparation of paraffin sections and HE staining

[0024] Mouse spleen and small intestine were collected and fixed with 4% paraformaldehyde for 24 h, followed by dehydration and paraffin infiltration. The paraffin-infiltrated tissues were embedded in an embedding machine, and the trimmed paraffin blocks were sectioned on a paraffin microtome to obtain paraffin sections 3-4 μm thick. The sections were dewaxed to water, stained with hematoxylin and eosin (HE), mounted, dried, and the images were acquired and analyzed under a white light scanner, with photographs taken at 200x magnification.

[0025] 3. Small intestinal tissue CD4 + CD8 + Measurement of T cell count CD4 levels in small intestinal tissue were measured using immunohistochemical staining (IHC). + CD8 + T cell count. Embedded paraffin tissue sections were dewaxed and rehydrated for antigen retrieval. Endogenous peroxidase was blocked in 3% hydrogen peroxide solution. Serum blocking with 3% bovine serum albumin (BSA) was performed in the histochemistry zone. The blocking solution was discarded, and the tissue was incubated overnight at 4°C with CD4 or CD8 primary antibody. After washing three times with PBS, the tissue was covered with HRP-labeled goat anti-rabbit secondary antibody corresponding to the primary antibody species. 3,3′-diaminobenzidine (DAB) staining was performed, and the cell nuclei were counterstained with hematoxylin. The tissue was then dehydrated and mounted. Finally, the results were interpreted under a white light microscope.

[0026] 4. Western blot determination of tight junction protein expression in intestinal tissue Small intestinal tissue blocks were washed 2-3 times with pre-cooled PBS at 4°C to remove blood contamination. The tissue was cut into small pieces and placed in a homogenization tube. Two 4 mm homogenization beads and 10 times the tissue volume of lysis buffer were added for homogenization. After homogenization, total lysis buffer (RIPA lysis buffer: 50× Cocktail protease inhibitor: benzyl sulfonyl chloride: phosphorylated protease inhibitor = 100: 2: 1: 1) was added and the mixture was incubated on ice for 30 min. The mixture was then centrifuged at 12000 rpm at 4°C for 10 min, and the supernatant was collected as the total protein solution. The undenatured protein solution was used to determine the protein concentration according to the BCA protein assay kit instructions. The remaining protein solution was denatured in boiling water for 15 min at a ratio of 4:1 with 5× reducing protein loading buffer and stored at -20°C for later use.

[0027] Electrophoresis was performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The PVDF membrane was soaked in anhydrous ethanol for 2 min before use. After electrophoresis, the proteins on the gel block were transferred to the PVDF membrane. After the transfer, the PVDF membrane was blocked with 5% milk at room temperature for 30 min. After blocking, the membrane was placed in a solution diluted 5000, 3000, 3000, and 3000 times with TBST, respectively. β The membrane was incubated overnight at 4°C with primary antibody solutions of Actin, ZO-1, Claudin 1, and Occludin. After washing the membrane three times with TBST, it was incubated on a shaker for 30 min with the corresponding HRP-goat anti-rabbit secondary antibody dilution. After incubation, the membrane was washed three times. Finally, a chemiluminescent reaction was performed using a high-sensitivity ECL chemiluminescence reagent. Protein bands were detected using an SCG-W3000 PLUS chemiluminescence imager, and gray values ​​were calculated using ImageJ.

[0028] 5. Statistical Analysis Methods Statistical analysis was performed using GraphPad Prism 8.0 software. Statistical differences were determined using one-way ANOVA. Experimental results are expressed as mean ± standard deviation. P A value <0.05 is considered statistically significant.

[0029] 6. Test Results

[0030] 1) Effects of psoralen polysaccharides on intestinal morphology:

[0031] To investigate the effects of PPs on intestinal morphology, histopathological analysis of small intestinal tissue was performed, and the results are as follows: Figure 1H&E staining revealed that the intestinal mucosa of mice in the BC group was intact with clear crypt structures. In the MC group, the intestinal integrity was disrupted, and the crypt structures showed deformation. Both high (PPsH) and low (PPsL) doses of psoralen polysaccharides (PPs) effectively improved CTX-induced intestinal mucosal damage, indicating that PPs can improve intestinal barrier repair.

[0032] 2) Effects of psoralen polysaccharides on immune cells: Immunohistochemistry was used to investigate CD4 in small intestinal tissue. + T and CD8 + The number of T cells, the results are as follows Figure 2 Compared to group BC, CD4 + and CD8 + The number of T cells was significantly reduced in the MC group. P <0.001), while in PPs, CD 4 + and CD 8 + The number of T cells increased significantly, especially in the PPsL group. P <0.001). This indicates that PPs can regulate CD4+ in the small intestine. + and CD8 + The number of T cells regulates and assists in the immune response and enhances the intestinal immune regulation capacity.

[0033] 3) Effects of psoralen polysaccharides on the intestinal barrier: The expression of tight junction proteins Claudin-1, Occludin, and ZO-1 in the small intestine was detected by Western blot, and the results are shown in Figure 3. Compared with the BC group, CTX caused damage to the intestinal mucosa of mice, resulting in a decrease in the expression of tight junction proteins such as Claudin-1, Occludin, and ZO-1. Administration of PPs significantly increased the expression of tight junction proteins Claudin-1, Occludin, and ZO-1. P <0.001). Further explanation is that PPs can repair and enhance the intestinal barrier, improve the connections between intestinal epithelial cells, and reduce intestinal permeability.

[0034] In summary, the psoralen polysaccharide described in this invention can effectively improve the repair of damaged intestinal barriers, enhance intestinal immune regulation, and repair and strengthen the intestinal barrier.

[0035] It should be understood that these embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of the present invention. Furthermore, it should be understood that after reading the technical content of the present invention, those skilled in the art can make various alterations or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The application of a psoralen polysaccharide in the preparation of drugs or health foods that regulate intestinal barrier function.

2. The application as described in claim 1, characterized in that, The psoralen polysaccharide is used in the preparation of drugs or health foods that enhance intestinal barrier function.

3. The application as described in claim 1, characterized in that, The application of the psoralen polysaccharide in the preparation of drugs or health foods for the prevention, relief or treatment of intestinal barrier damage.

4. The application according to claim 1, characterized in that, The application of psoralen polysaccharide in the preparation of drugs or health foods for promoting intestinal damage repair.

5. The application according to claim 1, characterized in that, The application of psoralen polysaccharide in the preparation of drugs or health foods for enhancing the immune cells of damaged intestinal cells.

6. The application according to claim 1, characterized in that, The dosage of the psoralen polysaccharide is 75~300 mg / kg / day.

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