Use of a pleurotus ostreatus polysaccharide in the preparation of a medicine for regulating intestinal barrier function and a health food

Abstract

This invention provides the application of *Pleurotus oosporum* polysaccharide in the preparation of drugs and health foods that regulate intestinal barrier function or protect against intestinal damage-related diseases. Its protective effect against a mouse model of intestinal damage induced by sodium dextran sulfate was investigated. Results showed that *Pleurotus oosporum* polysaccharide significantly improved symptoms such as weight loss, loose stools, and colonic atrophy caused by intestinal damage in mice, reduced the disease activity index, inhibited colonic epithelial destruction, inflammatory cell infiltration, and goblet cell necrosis, and enhanced intestinal mucosal barrier function. As a fungus, *Pleurotus oosporum* has high safety and is a potential source of active ingredients against intestinal damage, showing promising development prospects in intestinal barrier protection.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical and health food technology, specifically relating to the application of a polysaccharide from Pleurotus oosporum in the preparation of drugs and health foods that regulate or improve intestinal barrier function or damage-related diseases. Background Technology

[0002] The gut plays a crucial interfacial role between the body and its external environment. On one hand, it is responsible for absorbing essential dietary nutrients; on the other hand, it must defend against various toxins and microorganisms ingested orally. Therefore, maintaining the integrity of the intestinal barrier is a key indicator of gut health. The intestinal barrier is a multi-layered complex structure composed of normal gut microbiota, a mucus layer, soluble antimicrobial molecules, an intestinal epithelial cell layer, and the intestinal immune system. Among these, the intestinal mucus barrier, formed by goblet cells secreting mucus, is the first line of defense against microorganisms coming into contact with the intestinal epithelium and plays a vital role in maintaining intestinal barrier function. As the largest barrier tissue in the body, the integrity of the intestinal barrier is indispensable for maintaining normal physiological functions. Once its integrity is compromised, intestinal microorganisms and endotoxins can cross the damaged intestinal mucosal barrier and enter the bloodstream, causing bacterial and endotoxin translocation, leading to enterogenic infections, and in severe cases, even developing into systemic inflammatory response syndrome or multiple organ failure. Numerous studies have confirmed that intestinal barrier damage is closely related to the occurrence, development, and metastasis of various diseases through pathways such as the brain-gut axis and liver-gut axis. These include gastrointestinal diseases such as inflammatory bowel disease and colon cancer, as well as diabetes, non-alcoholic fatty liver disease, obesity, and even hyperglycemia and stroke. Therefore, protecting the intestinal barrier and maintaining homeostasis can help reduce the incidence of these diseases. Currently, developing drugs and health foods that can protect intestinal barrier function has become a hot research topic.

[0003] Current methods for improving intestinal barrier function mainly include probiotic therapy and drug therapy. Probiotic therapy involves fecal microbiota transplantation or the supplementation of probiotic preparations. Through complex interactions with the host and resident flora, it maintains the balance of the microecology, thereby protecting normal bodily functions. However, probiotic therapy also has certain side effects, such as causing discomfort like gas, bloating, constipation, and headaches. Furthermore, long-term use of synthetic probiotic products may gradually cause the intestines to lose their ability to reproduce beneficial bacteria, eventually leading to dependence. On the other hand, most chemical drugs currently used to improve intestinal barrier function have poor efficacy and are often accompanied by strong side effects. In contrast, natural products are receiving increasing attention due to their mild effects and high safety. Given the current lack of effective natural functional foods for protecting the intestinal barrier and promoting intestinal health, there is an urgent need to develop related products.

[0004] Pleurotus oosporum ( Pleurotus placentodesPleurotus oosporum is a new edible fungus variety successfully cultivated artificially in recent years by the Institute of Microbiology, Chinese Academy of Sciences. Unlike commercially available edible fungi such as king oyster mushroom and wood ear mushroom, the unique value of Pleurotus oosporum lies not only in its superior nutritional content—its protein (30.10 g / 100g), polysaccharide (7.45 g / 100g), and amino acid (19.24 g / 100g) content are significantly higher than those of common cultivated strains—but also in the qualitative difference in its functional components. Animal experiments and edible trials have confirmed its safety for consumption, and heavy metal testing results also meet national standards. This lays a crucial foundation for its transformation from a common "food ingredient" to a functional "matrix," making it an ideal resource for developing precision nutrition products. Research shows that polysaccharides, as bioactive macromolecules, have immune-enhancing, antiviral, and antioxidant effects, possessing enormous potential in the health food market; however, research on the efficacy of Pleurotus oosporum polysaccharides is relatively limited. This invention studies the protective effect of oospore polysaccharide against intestinal barrier damage, thereby potentially obtaining pharmaceutical compositions or functional products that protect the intestinal barrier and / or improve intestinal barrier damage. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide an application of Pleurotus oosporum polysaccharide in the preparation of drugs and health foods that regulate intestinal barrier function. In other words, this invention provides a new use for Pleurotus oosporum polysaccharide. As a natural fungal polysaccharide, Pleurotus oosporum polysaccharide can inhibit intestinal damage, repair and enhance the intestinal barrier, and improve intestinal diseases caused by impaired intestinal barrier function. Furthermore, this Pleurotus oosporum polysaccharide is inexpensive, naturally sourced, and has significant effects.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: Application of a polysaccharide from Pleurotus oosporum in the preparation of drugs that regulate intestinal barrier function, protect against intestinal damage, or are used in health foods.

[0007] Specifically, in the above-mentioned applications, the symptoms of intestinal damage include at least one of the following: weight loss, loose stools, colonic atrophy, destruction of the colonic epithelial mucosa, inflammatory cell infiltration, and goblet cell necrosis.

[0008] Furthermore, the use of the oospore pleuroplastin polysaccharide in the preparation of drugs or health foods for the prevention, relief or treatment of intestinal barrier damage.

[0009] Furthermore, the oospore polysaccharide is used in the preparation of drugs or health foods for promoting intestinal damage repair.

[0010] Furthermore, in the above applications, the preferred dosage of the oospore polysaccharide is 100-200 mg / kg / d, for example, it can be 100 mg / kg / d, 150 mg / kg / d, 200 mg / kg / d, etc.

[0011] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: Pleurotus oosporum polysaccharide, a naturally derived fungal polysaccharide, has been shown in this invention to effectively protect against intestinal damage and enhance the intestinal barrier. Therefore, this invention reveals that Pleurotus oosporum polysaccharide can act on the intestine, exhibiting a good protective effect against DSS-induced intestinal damage in mice. It effectively improves symptoms such as weight loss, loose stools, and colonic atrophy caused by intestinal damage, reduces the disease activity index, inhibits colonic damage, inflammatory cell infiltration, and goblet cell necrosis, promotes the expression of tight junction proteins ZO-1, Claudin-1, and Occludin, and enhances intestinal mucosal barrier function. As a fungus, Pleurotus oosporum has high safety and is a potential source of active ingredients against intestinal damage, showing promising development prospects in intestinal barrier protection. Attached Figure Description

[0012] Figure 1 The effects of oospore polysaccharide on body weight and disease index were investigated, where A represents the change in mouse body weight and B represents the disease activity index.

[0013] Figure 2 The effect of Pleurotus oosporum polysaccharide on colon length is shown in the figure. A: mouse colon tissue photograph; B: mouse colon length; BC: blank group; MC: model group; PC: positive control group; HD: high-dose Pleurotus oosporum polysaccharide group; LD: low-dose Pleurotus oosporum polysaccharide group.

[0014] Figure 3 The effect of oospore polysaccharide on colonic injury, where A: H&E staining results; B: AB-PAS staining results.

[0015] Figure 4 The effects of Pleurotus oosporioides polysaccharide on the expression of tight junction proteins in the small intestine are shown in the figures. A: Expression results of ZO-1, Occludin, and Claudin-1 in the colon; B: Quantitative results of ZO-1 expression; C: Quantitative results of Occludin expression; D: Quantitative results of Claudin-1 expression. 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.

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

[0018] In the following experiments, the polysaccharide used was the purified polysaccharide PPp-W, which can be prepared by referring to the following steps: After pulverizing 1350 g of Pleurotus oospore fruiting bodies, 70% ethanol was added at a material-to-liquid ratio of 1 g:5 ml (w / v) for extraction. Extraction was performed twice at room temperature, for 5 days and 2 days respectively. The extracts were filtered, combined, and the ethanol was recovered under reduced pressure. The residue was evaporated until no alcohol odor remained, and then extracted twice with 8 times its volume of distilled water by gentle boiling, 4 hours each time. The filtrates were filtered, combined, and concentrated under reduced pressure to a density of approximately 1.10 g / cm³ (25℃). Subsequently, 95% ethanol was slowly added to the concentrate to a final concentration of 70%. After standing at room temperature for 12 hours, the precipitate was collected by centrifugation at 4500 rpm for 6 minutes. After redissolving the precipitate, the Sevage method was used to remove proteins (chloroform-n-butanol = 4:1, polysaccharide solution to organic phase volume ratio 3:1). The polysaccharide solution after removing denatured proteins was concentrated under reduced pressure to remove reagents, then precipitated with ethanol, redissolved, and freeze-dried to finally obtain crude Pleurotus oospore polysaccharide. After dissolving the crude polysaccharide from *Pleurotus oosporum* (after protein removal), the solution was centrifuged at 5000 rpm for 6 min to remove insoluble matter. The supernatant was filtered through a 0.22 μm microporous membrane and then separated by DEAE-52 cellulose column chromatography (60 × 2.5 cm). Eluent was added with distilled water, and the polysaccharide content was determined using the phenol-sulfuric acid method. The absorbance was measured at 490 nm, and a polysaccharide elution curve was plotted. Polysaccharide fractions corresponding to the same elution peak were combined and sequentially concentrated, dialyzed (molecular weight cutoff 8000-14000), and freeze-dried. Further purification of the separated polysaccharides was achieved using Sephadex G-100 column chromatography (100 × 1.50 cm). Using pure water as the eluent, the solution was processed according to the same method as described above for DEAE-52 cellulose column chromatography, ultimately yielding a high-purity polysaccharide fraction, named PPP-W.

[0019] The aforementioned Pleurotus oosporum polysaccharide PPp-W is a homogeneous polysaccharide with a molecular weight of approximately 27.4 kDa. It is mainly composed of mannose (17.56%), glucose (6.37%), galactose (44.89%), and fucose (1.22%), and also contains a certain amount of 3-O-Me-galactose. PPp-W is irregularly coiled and sheet-like, possessing a triple helix structure and containing branches, →6)-α-D-Gal p -(1→,→6)-3-O-Me-α-D-Gal p -(1→ and →2, 6)-α-D-Galp-(1→ is the main chain, β -D-Manp-(1→ and β -L-Fuc p -(1→ non-reduced ends replace the O-2 and O-6 portions, in addition, a small amount α -1, 3-Connections - Glcp are connected to the main chain structure. See the following literature for details.

[0020] [1] Zhenhua Yin, Hao Zhang, Juanjuan Zhang, et al., Digestioncharacteristics in vitro of polysaccharide from Pleurotus placentodes and its positive effects on microbiota[J]. Journal of Functional Foods, 2025, 135:107106. doi: 10.1016 / j.jff.2025.107106.

[0021] [2] Zhenhua Yin, Xiaopeng Liu, JinmeiWang, et al., Structural characterization and anticoagulant activity of a 3-O-methylated heteroglycan from fruiting bodies of Pleurotus placentodes[J]. Frontiers in Chemistry, 2022, 10: 825127. doi: 10.3389 / fchem.2022.825127.

[0022] Application trials.

[0023] 1. Animal experimental grouping and establishment of mouse model of intestinal injury.

[0024] Male C57BL / 6 mice aged 6–8 weeks (weighing 22 ± 1 g) were fed standard food and water freely under constant temperature (23 ± 2℃) and humidity (60–70%) conditions with a 12-hour light / dark cycle. After one week of acclimatization, they were randomly divided into 5 groups (n=10): normal group (BC), model group (MC), positive control group (mesalazine enteric-coated tablets, PC), and high- and low-dose Pleurotus ostreatus polysaccharide groups (200, 100 mg / kg, HD and LD). The BC group had free access to water, while the MC, PC, HD, and LD groups had free access to 3% sodium dextran sulfate (DSS) and were simultaneously administered the corresponding drugs by gavage for 7 consecutive days. On days 8–9, all mice were switched back to normal drinking water, DSS modeling was discontinued, and the corresponding drugs were administered by gavage again. After the last administration, the mice were fasted but allowed free access to water for 12 hours, blood was collected from their eyeballs, and they were euthanized.

[0025] 2. Effects of Pleurotus oosporum polysaccharide on mouse body weight and disease index.

[0026] During the experiment, the mice were weighed at the same time every day, and the shape of their feces and the presence of blood in their stool were observed. The Disease Activity Index (DAI) was calculated based on changes in body weight (0-5 points), fecal viscosity (0-4 points), and fecal occult blood (0-5 points). The occurrence of ulcerative colitis was assessed by using the DAI score.

[0027] 3. Effect of Pleurotus ostreatus polysaccharide on colon length in mice.

[0028] Mice were euthanized by blood collection from the eyeballs, colon tissue was collected, colon length was measured, and the differences in colon length among the groups of mice were compared.

[0029] 4. Effects of Pleurotus ostreatus polysaccharide on colonic injury in mice.

[0030] Colon tissue was collected from all mice. Part of the colon tissue was cut off and fixed in 4% paraformaldehyde, while the remainder was stored at -80°C for later use.

[0031] Mouse colon tissue was fixed in 4% paraformaldehyde for more than 24 hours, followed by dehydration and paraffin infiltration. The paraffin-infiltrated tissue was 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 images were acquired under a white light scanner.

[0032] Continue to fix, embed, and section the colon tissue using the above method, then stain with AB-PAS, mount and dry the slides, and acquire images under a white light scanner to observe inflammatory cell infiltration, mucus swelling and destruction, and epidermal cell damage.

[0033] 5. Western blot was used to determine the expression of tight junction proteins in intestinal tissue.

[0034] Colon tissue was washed 2-3 times with pre-cooled PBS at 4°C to remove blood contamination. After being cut into small pieces, the tissue was placed in a homogenization tube, and two 4mm homogenization beads and 10 times the volume of lysis buffer were added for homogenization. The homogenate was then placed on ice for 30 min to lyse, followed by centrifugation at 12000 rpm for 10 min at 4°C. The supernatant was collected as the total protein solution. A portion of the undenatured protein solution was used to determine the protein concentration using a BCA kit. The remaining protein solution was denatured in boiling water for 15 min at a 4:1 ratio with 5× reducing loading buffer and stored at -20°C for later use.

[0035] SDS-PAGE electrophoresis was performed. After electrophoresis, the PVDF membrane was activated with anhydrous methanol, and proteins were transferred onto the membrane. After transfer, the membrane was blocked with 5% milk at room temperature for 30 min; then, an appropriate amount of TBST-diluted primary antibody was added. β -Actin, ZO-1, Occludin, and Claudin-1), incubated overnight at 4°C. Wash the membrane three times with TBST, then incubate with the corresponding secondary antibody dilutions (HRP-goat anti-rabbit and HRP-goat anti-mouse) on a shaker at room temperature for 30 min, washing three times. Finally, react with the ultrasensitive ECL chemiluminescence reagent, detect protein bands using an SCG-W3000 PLUS chemiluminescence imager, and calculate grayscale values ​​using ImageJ software.

[0036] 6. Statistical analysis methods.

[0037] 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.

[0038] 7. Results.

[0039] 1) Effects of Pleurotus oosporum polysaccharides on body weight and disease activity index: such as Figure 1 As shown, compared with the BC group, the MC group mice exhibited a significant decrease in body weight and an increase in DAI scores. In DSS-induced colitis mice, administration of different doses of Pleurotus oosporus polysaccharide alleviated the degree of body weight loss and the increase in DAI scores, and improved fecal viscosity and fecal occult blood. Therefore, Pleurotus oosporus polysaccharide can alleviate the above-mentioned symptoms of ulcerative colitis.

[0040] 2) The effect of Pleurotus ostreatus polysaccharide on colon length: such as Figure 2 As shown, compared with the BC group, the colon length of mice in the MC group was significantly reduced ( p<0.001); Compared with the MC group, the shortening of the mouse colon was significantly improved after administration of different doses of Pleurotus ostreatus polysaccharide. p The improvement was more pronounced in the HD group (<0.01 or 0.05). This indicates that the effect of Pleurotus ostreatus polysaccharide on improving colonic shortening in colitis mice exhibits a dose-response relationship.

[0041] 3) Effects of Pleurotus ostreatus polysaccharide on colonic injury: such as Figure 3 As shown, H&E staining histological analysis revealed extensive epithelial mucosal destruction and inflammatory cell infiltration in the MC group, while AB-PAS staining further confirmed significant goblet cell loss in the MC group. These pathological changes were alleviated after administration of different doses of Pleurotus oosporum polysaccharide, with the HD group showing relatively significant improvement. This indicates that Pleurotus oosporum polysaccharide can effectively reduce DSS damage to colonic tissue.

[0042] 4) The effect of Pleurotus oosporum polysaccharides on intestinal barrier integrity: such as Figure 4 As shown, the expression of the small intestinal tight junction proteins ZO-1 (Occludin) and Claudin-1 was detected by Western blot. Compared with the BC group, the expression levels of ZO-1, Occludin, and Claudin-1 were all decreased in the colon group of DSS-induced colitis mice, and treatment with Pleurotus oosporum polysaccharide reversed this condition. The results indicate that Pleurotus oosporum polysaccharide can protect the intestinal barrier from inflammation, which is crucial for the integrity of the intestinal barrier.

[0043] The above results indicate that Pleurotus oosporioides can enhance the intestinal mucosal barrier function, inhibit colonic mucosal edema, and suppress the occurrence and development of DSS-induced intestinal damage.

[0044] Compared to existing technologies, the differences of this invention are as follows: 1) This invention is the first to discover that Pleurotus oosporum polysaccharide has the function of protecting the intestinal barrier, which can greatly expand the scope of application of Pleurotus oosporum. 2) Experimental model: This patent uses a mouse intestinal injury model induced by sodium dextran sulfate (DSS) to examine indicators such as mouse body weight changes, colon length, degree of colon tissue damage, and tight junction proteins, to evaluate the protective effect of Pleurotus oosporum polysaccharide on the intestinal barrier. This efficacy is the first study on Pleurotus oosporum polysaccharide, which is different from other literature.

[0045] 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 polysaccharide from Pleurotus oosporum in the preparation of drugs and health foods that regulate intestinal barrier function or protect against intestinal damage.

2. Use according to claim 1, wherein The symptoms of intestinal damage include at least one of the following: weight loss, loose stools, colonic atrophy, destruction of the colonic epithelial mucosa, inflammatory cell infiltration, and goblet cell necrosis.

3. Use according to claim 1, characterized in that, The application of the polysaccharide from Pleurotus ostreatus in the preparation of drugs and health foods for the prevention, relief or treatment of intestinal barrier damage.

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

5. Use according to claim 1, characterized in that, The dosage of the polysaccharide from Pleurotus ostreatus is 100-200 mg / kg / day.

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