Pholiota nameko polysaccharide with effect of improving ulcerative colitis as well as preparation method and application of pholiota nameko polysaccharide
By isolating and purifying the structurally defined polysaccharide component PNP-1a from Nameko mushroom, the problem of unstable efficacy of existing drugs has been solved, achieving effective improvement in ulcerative colitis and enhancing treatment compliance and safety.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-02-28
- Publication Date
- 2026-03-27
AI Technical Summary
Existing drugs for treating ulcerative colitis have unstable efficacy, significant side effects, and are prone to drug resistance, leading to decreased treatment adherence. The structure and mechanism of action of Pleurotus ostreatus polysaccharide have not been fully studied, limiting its high-value development and application.
PNP-1a, a well-defined and homogeneous polysaccharide component, was isolated and purified from the fruiting body of *Pleurotus ostreatus*. Its biological functions in preventing and improving ulcerative colitis were extracted and verified using a specific preparation method, including upregulating tight junction protein expression, reducing pro-inflammatory cytokines, and enhancing antioxidant activity.
It effectively improves the symptoms of ulcerative colitis, maintains the integrity of the colonic mucosal barrier, reduces the expression of pro-inflammatory cytokines, enhances antioxidant capacity, and achieves effective relief of ulcerative colitis.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of natural product development, and in particular to a Pholiota nameko polysaccharide with the effect of improving ulcerative colitis, and a preparation method and application thereof. BACKGROUND
[0002] Ulcerative colitis (UC) is a non-specific, chronic and recurrent intestinal inflammatory bowel disease, which is a major subtype of inflammatory bowel disease (IBD). The main pathological changes are in the colonic mucosa and submucosa. The clinical manifestations are persistent or recurrent abdominal pain and diarrhea, with mucus or pus in the stool, and severe cases can develop into toxic megacolon, colorectal cancer and other complications, which seriously affect the quality of life of patients. Although the etiology of UC has not been fully elucidated, more and more researchers believe that it is related to genetics, environment, immunity and infection. At present, the clinical treatment mainly relies on aminosalicylic acid preparations (mesalazine, sulfasalazine), glucocorticoids (prednisone, budesonide), immunosuppressants (azathioprine, cyclosporine) and biological agents (infliximab, adalimumab), etc. These drugs can short-term relieve symptoms, but have problems such as unstable efficacy, obvious side effects, drug resistance and high recurrence rate after drug withdrawal, which leads to a decrease in treatment compliance. Therefore, exploring new natural active substances from natural resources that are safe, efficient and can be used for long-term intervention has become an important direction for UC management.
[0003] Edible and medicinal fungi are an important treasure trove of bioactive molecules. Fungal polysaccharides have attracted much attention due to their wide range of immunomodulatory, anti-tumor, antioxidant and anti-inflammatory activities. Pholiota nameko (Berk.) Quél. Pholiota nameko ) belongs to the genus Pholiota of the family Strophariaceae, and is a widely cultivated edible fungus in China. It not only has a delicious flavor and rich nutrition, but also contains active ingredients such as protein, terpenoids, polyphenols and polysaccharides, and has immunomodulatory, antioxidant, antitumor, hypolipidemic and other biological activities. Fungal polysaccharides, as natural active macromolecules, can exert anti-inflammatory activity by enhancing intestinal mucosal barrier, regulating intestinal flora and inhibiting NF-κB signaling pathway, which has been reported in Ganoderma lucidum, Lentinula edodes and Grifola frondosa. However, the monosaccharide composition, glycosidic bond type, branching degree, molecular weight and higher structure of polysaccharides from different sources differ significantly, and there is a strict structure-activity relationship between their biological activities and structures. However, the precise structure of Pholiota nameko polysaccharide and its dose-effect relationship and mechanism in UC models are almost blank, which greatly limits the high-value development and application of Pholiota nameko polysaccharide.
[0004] In view of the unmet clinical needs of UC and the bottleneck of high-value utilization of Pholiota nameko, there is an urgent need in the art to isolate and purify an active polysaccharide component with a clear structure and uniformity from Pholiota nameko, and to systematically evaluate its specific improvement effect and molecular mechanism on ulcerative colitis, so as to provide a solid scientific basis and technical support for the development of functional food or medicine based on Pholiota nameko polysaccharide for preventing and / or improving ulcerative colitis. SUMMARY
[0005] The present application aims to overcome the deficiencies of the prior art, and provides a Pholiota nameko polysaccharide with an improvement effect on ulcerative colitis and a preparation method and application thereof. A new polysaccharide component PNP-1a with a clear structure and uniformity is isolated and purified from Pholiota nameko fruiting bodies, and the accurate molecular structure characteristics of the polysaccharide component are elucidated, including its molecular weight, monosaccharide composition and glycosidic bond connection mode; an effective preparation method of the Pholiota nameko polysaccharide is provided; the biological function and application value of the Pholiota nameko polysaccharide in preventing and / or improving ulcerative colitis and related diseases are verified, and a scientific basis is provided for the development of functional food or medicine based on Pholiota nameko polysaccharide for preventing and / or improving ulcerative colitis.
[0006] To achieve the above-mentioned object, the present application provides the following specific schemes: In a first aspect, the present application provides a Pholiota nameko polysaccharide PNP-1a with an improvement effect on ulcerative colitis, wherein the Pholiota nameko polysaccharide PNP-1a comprises galactose, glucose, xylose, mannose, galacturonic acid, guluronic acid and glucuronic acid with a molar percentage of 11.70%, 32.34%, 9.16%, 33.71%, 4.05%, 1.31% and 7.73%; and the molecular weight ranges from 62kDa to 236kDa.
[0007] As a specific embodiment of the present application, the main chain of the Pholiota nameko polysaccharide PNP-1a comprises →3)-α-D-Manp-(1→ and →3)-α-D-Glcp-(1→, and the branch comprises β-D-Xylp-(1→3)-α-D-Manp-(1→ connected to O-2 of sugar residue →2,6)-β-D-Galp-(1→, or β-D-Xylp-(1→ connected to O-6 position of sugar residue →3,6)-β-D-Glcp-(1→.
[0008] As a specific embodiment of the present application, the Pholiota nameko polysaccharide PNP-1a has the structure of formula I: Formula I.
[0009] In a second aspect, a preparation method of the above-mentioned polysaccharide PNP-1a is provided, which comprises the following steps: extracting the fruiting bodies of Pholiota nameko with hot water, precipitating with ethanol, subjecting to DEAE seplife FF anion exchange column chromatography, and subjecting to Sephadex G-100 gel column purification and dialysis to obtain the polysaccharide PNP-1a.
[0010] As a specific embodiment of the present application, the preparation method specifically comprises the following steps: (1) drying and crushing the fruiting bodies of Pholiota nameko, and then sieving; adding water according to a solid-liquid ratio of 1:20-40 (g / mL), and then subjecting to water bath extraction; centrifuging to obtain supernatant and concentrating; adding ethanol to the concentrated solution, and then standing overnight; filtering to collect precipitate, and thus obtaining a crude polysaccharide extract; (2) adding a chloroform-n-butanol mixed solution to the crude polysaccharide extract of step (1), mixing, and then collecting the upper aqueous phase; dialyzing, and then freeze-drying to obtain a crude polysaccharide PNP; (3) dissolving the crude polysaccharide PNP of step (2) in water, and then subjecting to DEAE seplife FF anion exchange column chromatography; sequentially using pure water and NaCl solutions with different concentrations to gradient elute at a flow rate of 4 mL / min; concentrating the eluate, and then dialyzing to remove salt, and thus obtaining the polysaccharide PNP-1; (4) dissolving the polysaccharide PNP-1 of step (3) in water, and then subjecting to Sephadex G-100 gel filtration and elution; concentrating the eluate, and then dialyzing to remove salt, and freeze-drying to obtain the polysaccharide PNP-1a.
[0011] As a specific embodiment of the present application, the water bath extraction in step (1) is performed at 80-95°C for 3-5 h; and 2-4 times the volume of 90%-98% ethanol is added to the concentrated solution.
[0012] As a specific embodiment of the present application, the volume ratio of chloroform to n-butanol in the chloroform-n-butanol mixed solution in step (2) is 5:1.
[0013] As a specific embodiment of the present application, the NaCl solutions with different concentrations in step (3) include 0.1 mol / L NaCl solution, 0.2 mol / L NaCl solution and 0.3 mol / L NaCl solution.
[0014] In a third aspect, the above-mentioned polysaccharide PNP-1a or the polysaccharide PNP-1a prepared by the above-mentioned preparation method is provided for use in the preparation of a functional food for preventing and / or improving ulcerative colitis.
[0015] In a fourth aspect, the above-mentioned polysaccharide PNP-1a or the polysaccharide PNP-1a prepared by the above-mentioned preparation method is provided for use in the preparation of a drug for preventing and / or improving ulcerative colitis.
[0016] As a specific embodiment of the present application, the Pholiota nameko polysaccharide PNP-1a up-regulates the expression of tight junction proteins Occludin, ZO-1 and Claudin-1, and maintains the integrity of the colon mucosal barrier.
[0017] As a specific embodiment of the present application, the Pholiota nameko polysaccharide PNP-1a can reduce the expression levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, IL-18 and IFN-γ, and can enhance the activities of SOD, GSH-Px and CAT, and reduce the levels of iNOS and NO.
[0018] As a specific embodiment of the present application, the Pholiota nameko polysaccharide PNP-1a can be used in combination with mesalazine, and the mass ratio of the two is 1:2-4; when used in combination, the dosage of mesalazine is reduced by 30-50%, and the gastrointestinal irritation side effects caused by mesalazine can be reduced.
[0019] The Pholiota nameko polysaccharide PNP-1a exerts an effect of improving ulcerative colitis through one or more of the following mechanisms, specifically including: Repairing intestinal barrier: effectively relieving the clinical symptoms of colitis, such as increasing the length of the colon, reducing the disease activity index (DAI) score; up-regulating the tight junction proteins (such as Occludin and ZO-1) in the colon tissue, improving the microstructure of the colon, and maintaining the integrity of the colon mucosal barrier; Anti-inflammatory: reducing the expression levels of pro-inflammatory cytokines (such as TNF-α, IL-1β, IL-6 and IL-18), and reducing intestinal inflammation.
[0020] Antioxidant: enhancing the activities of SOD, GSH-Px and CAT, and reducing the levels of iNOS and NO.
[0021] Advantages of the present application 1. The present application purifies a new polysaccharide component PNP-1a from Pholiota nameko fruiting bodies, which has a clear and uniform structure.
[0022] 2. The present application provides an effective preparation method of the Pholiota nameko polysaccharide PNP-1a, which provides technical support for the extraction and preparation thereof, and has high safety.
[0023] 3. The present application verifies that the Pholiota nameko polysaccharide PNP-1a can effectively improve the symptoms related to colitis, effectively relieve the clinical symptoms of colitis, maintain the integrity of the colon mucosal barrier, reduce the expression of pro-inflammatory cytokines, reduce intestinal inflammatory response, enhance antioxidant efficacy, and achieve the effect of relieving ulcerative colitis.
[0024] 4. The Pholiota nameko polysaccharide prepared by the method of the present application has anti-colitis activity, which will greatly promote the development of Pholiota nameko related industries and has a broad application prospect. Attached Figure Description
[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other embodiments obtained by those skilled in the art without creative effort are all within the scope of protection of the present invention.
[0026] Figure 1 Purification process and preliminary structural characterization of PNP-1a; where: A is the elution curve of DEAE anion exchange column; B is the elution curve of Sephadex G-100 gel filtration; C is the ultraviolet spectrum; D is the composition of standard and PNP-1a monosaccharide; E is the infrared spectrum. Figure 2 PNP-1a nuclear magnetic resonance spectrum; where A is 1 H spectrum; B is... 13 C spectrum; C is 1 H, 1 H-COSY spectrum; D is HMBC spectrum; E is HSQC spectrum; F is NOESY spectrum.
[0027] Figure 3 Effects of PNP-1a on body weight, colon length, and histopathology in DSS mice; where: A represents changes in body weight; B represents DAI score; C represents changes in colon length; D represents HE staining of colon tissue; Note: Data are expressed as mean ± standard deviation. # P<0.05, ## P<0.01, ### P<0.001 compared with the Control group; *P<0.05, **P<0.01, ***P<0.001 compared with the DSS group; Figure 4 The effect of PNP-1a on the improvement of tight junction proteins and ultrastructure of colon tissue in DSS mice; where: A is the expression of Claudin-1, Occludin, and ZO-1 proteins; B is the relative quantitative expression of Claudin-1, Occludin, and ZO-1 proteins; C is the electron micrograph of the ultrastructure of colon tissue; Note: Data are expressed as mean ± standard deviation. # P<0.05, ## P<0.01, ### P<0.001 compared with the Control group; *P<0.05, **P<0.01, ***P<0.001 compared with the DSS group; Figure 5The effects of PNP-1a on reducing inflammatory symptoms and enhancing antioxidant activity in DSS mice; where A represents the inflammatory marker TNF-α; B represents the inflammatory marker IL-1β; C represents the inflammatory marker IL-6; D represents the inflammatory marker IFN-γ; E represents the inflammatory marker IL-18; F represents the antioxidant marker SOD; G represents the antioxidant marker GSH-Px; H represents the antioxidant marker CAT; I represents the antioxidant marker NO; J represents the antioxidant marker iNOS; Note: Data are expressed as mean ± standard deviation. # P<0.05, ## P<0.01, ### P<0.001 compared with the Control group; *P<0.05, **P<0.01, ***P<0.001 compared with the DSS group. Detailed Implementation
[0028] Unless otherwise specified, the methods involved in this application are all conventional methods in the art; unless otherwise specified, the experimental materials and reagents involved in this application are all commercially available. Based on the embodiments of this invention, unless otherwise defined, all scientific and technical terms used in this invention have the meaning known to those skilled in the art. The technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.
[0029] Unless otherwise specified, the following embodiments are all conventional methods.
[0030] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0031] Mesalazine (purity ≥99%, purchased from Sigma); the modeling agent was sodium dextran sulfate (DSS), purchased from MPBiomedical, with a molecular weight of 36,000-50,000 Da. The DSS solution involved in the following examples was prepared as follows: 3g of DSS powder was accurately weighed and dissolved in 100mL of sterile, high-purity deionized water to prepare a 3% DSS aqueous solution. All other reagents used in this invention are analytical grade or higher.
[0032] To facilitate understanding of the present invention, a more comprehensive description will be provided below, along with embodiments, but these are not intended to limit the scope of the invention. Detailed examples of specific implementations of the present invention are provided below: Example 1: Preparation, isolation and purification of PNP-1a, a polysaccharide from Mushroom Nameko. 1. Preparation of crude polysaccharide PNP from Mushroom Nameko The fruiting bodies of *Pleurotus ostreatus* were dried, pulverized, and passed through a 60-mesh sieve. The fruiting body powder was extracted at a material-to-liquid ratio of 1:30 (g / mL) in a 90℃ water bath for 3 hours. After three extractions, the extracts were combined, centrifuged (3000 r / min, 15 min), and the supernatant was collected. The supernatant was then concentrated to an appropriate volume using a rotary evaporator. Three volumes of 95% ethanol (v / v) were added to the concentrate, and the mixture was allowed to stand overnight at 4℃. After centrifugation and filtration, the precipitate was collected, which was the crude polysaccharide extract of *Pleurotus ostreatus*. Three volumes of chloroform-n-butanol (v:v=5:1) mixture were added to the aqueous solution of the above crude polysaccharide extract, and the mixture was thoroughly mixed. The upper aqueous phase was collected and dialyzed through a 3500 Da dialysis bag for 48 hours to remove small molecule components. The polysaccharide solution was then freeze-dried to obtain the crude polysaccharide PNP of *Pleurotus ostreatus*.
[0033] 2. Isolation and purification of PNP polysaccharide from Mushroom Nameko. Dissolve an appropriate amount of the crude polysaccharide sample in pure water to prepare a polysaccharide stock solution with a concentration of approximately 10 mg / mL. Centrifuge at 10,000g for 10 min. Collect the supernatant and pass it through a DEAE Seplife FF anion exchange column at a flow rate of 4 mL / min. Elute sequentially with pure water, 0.1 mol / L, 0.2 mol / L, and 0.3 mol / L NaCl solutions, collecting 15 mL tubes per eluent. Collect all eluents and determine the polysaccharide content of the eluents using the phenol-sulfuric acid method. Plot a curve, as shown below. Figure 1 As shown in Figure A. The eluents from each collection tube corresponding to the same elution peak were combined, concentrated to 1 / 5 of the original volume by rotary evaporation, dialyzed for 48-72 h to remove salt, and the polysaccharide content after ion purification was identified by the phenol-sulfuric acid method. The eluents from tubes 6-23 were collected to obtain polysaccharide PNP-1.
[0034] The above-mentioned polysaccharide PNP-1 aqueous solution was subjected to Sephadex G-100 gel filtration at a flow rate of 1 mL / min, with 2 mL collected per tube. All eluents were collected, and the polysaccharide content was determined using the phenol-sulfuric acid method. Figure 1 As shown in Figure B. The eluents from the collection tubes corresponding to the same elution peak were combined, concentrated to 1 / 5 of the original volume by rotary evaporation, and dialyzed for 48-72 h to remove salt. The eluents from tubes 7-22 were collected to obtain polysaccharide PNP-1a. The obtained polysaccharide fraction was lyophilized and stored.
[0035] Example 2 Structural characterization of PNP-1a, a polysaccharide from Mushroom Nameko. 1. Purity determination: Weigh an appropriate amount of PNP-1a and dissolve it in 1 mL of sterile water. Vortex to mix thoroughly until completely dissolved. Centrifuge and collect the supernatant. Scan the supernatant using a UV-Vis spectrophotometer in the 200-600 nm wavelength range. The scan spectrum shows no obvious absorption peaks at 260 nm and 280 nm, indicating that PNP-1a does not contain nucleic acids or proteins (see...). Figure 1 (C)
[0036] 2. Monosaccharide composition: Take a clean chromatographic vial, weigh an appropriate amount of PNP-1a, add 1 mL of 2 mol / L TFA acid solution, and heat at 121℃ for 2 h. Purge with nitrogen and dry. Wash with 99.99% methanol, then dry again, repeating the methanol washing 2-3 times. Dissolve in sterile water, transfer to a chromatographic vial, and perform analysis. Results are shown (see...). Figure 1 PNP-1a (D) is composed of galactose, glucose, xylose, mannose, galacturonic acid, guluronic acid and glucuronic acid in molar percentages of 11.70%, 32.34%, 9.16%, 33.71%, 4.05%, 1.31% and 7.73%, respectively.
[0037] 3. Molecular weight determination: PNP-1a was dissolved in a 0.1 mol / L NaNO3 aqueous solution (containing 0.02% NaN3, w / w) to a final concentration of 1 mg / mL. The solution was then filtered through a 0.45 μm filter before analysis. The results showed that the molecular weight of PNP-1a ranged from 62 kDa to 236 kDa.
[0038] 4. Fourier Transform Infrared Spectroscopy (FTIR): The KBr pellet method was used for determination. 1 mg of polysaccharide sample was thoroughly ground with 100-200 mg of KBr, compressed into a pellet, and then scanned using a Nicolet 8700 infrared spectrometer (4000-400 cm⁻¹). -1 ).like Figure 1 The image in section E shows the typical Fourier transform infrared spectrum of PNP-1a. 2927 cm⁻¹ -1 (3000-2800cm) -1 The weak absorption peak at () is a characteristic peak of the CH stretching vibration in the sugar ring. (1023 cm⁻¹) -1 1080cm -1 and 1154cm -1 The three sets of stretching peaks at 849 cm⁻¹ indicate that PNP-1a belongs to the pyranose type polysaccharide, while the peaks at 849 cm⁻¹... -1 and 932cm -1 The absorption peak at that location suggests the presence of α- and β-glycosidic bonds in PNP-1a.
[0039] 5. Methylation Analysis: Weigh 2-3 mg of the sample to be tested and dissolve it in 500 μL of DMSO. Perform methylation, hydrolysis, reduction, and acetylation treatments on the polysaccharide sequentially to obtain the methylated polysaccharide sample, ready for GC-MS analysis. The analytical instrument was an Agilent 7890A-5977B GC-MS system with an autosampler model G4567A. The injection volume was 1 μL, the split ratio was 10:1, and the carrier gas was high-purity helium. An electron impact ionization (EI) source was used, and the analyte was detected in full scan mode, with a mass scan range (m / z) of 50-350. The bonding mode of PNP-1a is shown in Table 1.
[0040] Table 1. Results of PNP-1a bond structure analysis 6. Nuclear Magnetic Resonance Analysis: Dissolve an appropriate amount of purified polysaccharide thoroughly in D2O to prepare a polysaccharide solution with a concentration greater than or equal to 40 mg / mL. Transfer the dissolved solution to an NMR tube, adding 0.5 mL. Place the NMR tube in a nuclear magnetic resonance spectrometer (Bruker 600MHz) to scan one-dimensionally. 1 H spectrum, 13 C-spectrum, two-dimensional COSY, HSQC, HMBC, and NOESY spectra. Results are shown below. Figure 2 The sugar residue signals of PNP-1a were assigned by combining AF and one-dimensional and two-dimensional NMR spectra, as shown in Table 2.
[0041] Table 2 Sugar residues 1 H and 13 Chemical shift of C / indicates that it was not recognized.
[0042] Based on the combined analysis of one-dimensional and two-dimensional NMR information, it is inferred that the PNP-1a backbone is mainly composed of →3)-α-D-Man p -(1→and→3)-α-D-Glc p -(1→ Formed, the side chain is composed of β-D-Xyl p -(1→3)-α-D-Man p -(1→linked to sugar residues→2,6)-β-D-Gal p -(1→O-2, or β-D-Xyl) p -(1→linked to sugar residues→3,6)-β-D-Glc p -(1→) O The -6 position constitutes the structure. Therefore, the possible structure of the PNP-1a polysaccharide chain is speculated to be as shown in Formula I: Formula I.
[0043] Example 3: The effect of PNP-1a polysaccharide from Mushroom Nameko on the symptoms of ulcerative colitis 1. Mouse Experimental Design: Male Kunming mice (8 weeks old) were purchased from Taibang Biotechnology Co., Ltd. and housed under standard conditions (22±2℃, 50-55% humidity, 12h light / 12h dark cycle). A UC mouse model was established using DSS (Digital Substances Supplement). After one week of acclimatization, the mice were randomly divided into 5 groups: Control group, DSS group, DSS+HPNP-1a group (PNP-1a 400mg / kg bw), DSS+LPNP-1a group (PNP-1a 200mg / kg bw), and DSS+Mesa group (mesalazine 100mg / kg bw). Control group: No medication was administered via gavage from days 1 to 14; DSS group: No medication was administered via gavage from days 1 to 7, and mice were allowed free access to a 3% DSS aqueous solution daily from days 8 to 14; DSS+HPNP-1a and DSS+LPNP-1a groups: 400 mg / kg and 200 mg / kg of the *Pleurotus ostreatus* polysaccharide from Example 1 were administered via gavage daily from days 1 to 14, and mice were allowed free access to a 3% DSS aqueous solution daily from days 8 to 14; DSS+Mesa group: 100 mg / kg of mesalazine was administered via gavage daily from days 1 to 14, and mice were allowed free access to a 3% DSS aqueous solution daily from days 8 to 14. On day 14, the mice were observed for activity and sacrificed 1 hour after the last administration. From days 8 to 14, the following indicators were recorded at fixed times daily: weight change, fecal characteristics (formed, loose, watery), and fecal blood.
[0044] 2. Indicator Detection: Weight changes, fecal characteristics, and fecal occult blood levels were recorded for all mice to assess the Disease Activity Index (DAI). Fresh colonic tissue was dissected, cleaned, and its length was measured (from the ileocecal junction to the anal verge, i.e., from the rectum to the cecum). A portion of the colonic tissue was then fixed for histopathological observation with H&E staining and ultrastructural analysis under transmission electron microscopy. The levels of TNF-α, IL-1β, IL-6, IL-18, and IFN-γ in mouse serum and colon were measured according to the commercial kit instructions. The activities of SOD, GSH-Px, and CAT, as well as the levels of NO and iNOS, were also determined.
[0045] 3. Results Analysis: For example... Figure 3 As shown in Figures A and B, mice in the Control group showed no symptoms of UC, while the DAI in the DSS group was significantly higher than that in the Control group. PNP-1a, especially HPNP-1a, significantly reduced DAI. Figure 3The results from the C-cell assay indicate that PNP-1a can inhibit colonic shortening, and all of the above results suggest that PNP-1a can effectively improve the symptoms of DAI and colonic shortening in UC mice. Figure 3 China D and Figure 4 C, H&E, and TEM staining results showed that PNP-1a improved colonic structural damage. Compared with the Control group, the DSS group mice had severe damage to the colonic mucosa, loss of colonic epithelial mucosa integrity, loss or absence of goblet cells and crypts, severe villus loss, extensive inflammatory cell infiltration, and shortened or even absent tight junctions. In contrast, the HPNP-1a and LPNP-1a groups significantly improved colonic tissue lesions in UC mice, with more orderly gland arrangement, indicating that PNP-1a can effectively protect the colonic tissue structure of UC mice, reduce inflammatory cell infiltration, and maintain the integrity of the intestinal barrier. Figure 4 The results in sections A and B of the study showed that the expression level of tight junction protein in the colon of mice in the DSS group was significantly reduced, while the expression level of tight junction protein was significantly increased after PNP-1a intervention, indicating that PNP-1a can effectively protect the intestinal barrier of mouse colon tissue and maintain high expression of tight junction protein. Furthermore, Figure 5 The results of the study showed that the levels of inflammatory factors in the serum and colon tissue of mice in the DSS group were significantly increased, but PNP-1a intervention significantly reduced its expression. Figure 5 The results from the Chinese FJ study showed that PNP-1a intervention significantly improved the antioxidant level of colonic tissue. In summary, PNP-1a, a polysaccharide from *Schefflera arvense*, can improve ulcerative colitis by repairing intestinal barrier integrity, upregulating the expression of tight junction proteins, reducing the expression of pro-inflammatory cytokines, alleviating intestinal inflammation, and enhancing antioxidant capacity.
[0046] Example 4: Synergistic effect experiment of PNP-1a combined with mesalazine in the intervention of ulcerative colitis 1. Mouse experimental design: Male Kunming mice (8 weeks old) were purchased from Taibang Biotechnology Co., Ltd. and were acclimatized for 1 week under standard conditions (22±2℃, 50-55% humidity, 12h light / 12h dark cycle). Students were randomly divided into 6 groups: Control group: free access to distilled water throughout the treatment, and daily gavage with an equal volume of normal saline; DSS group: daily gavage with an equal volume of normal saline from days 1 to 7, and free access to 3% DSS aqueous solution from days 8 to 14; Mesalazine alone group (Mesa group): daily gavage with mesalazine 300 mg / kg bw from days 1 to 14, and free access to 3% DSS aqueous solution from days 8 to 14; Low-ratio combination group (PNP-1a + Mesa-L group): daily gavage with PNP-1a 50 mg / kg bw + mesalazine 250 mg / kg bw from days 1 to 14, and free access to 3% DSS aqueous solution from days 8 to 14; Medium-ratio combination group (PNP-1a + Mesa-M group): daily gavage with PNP-1a 100 mg / kg bw + mesalazine 200 mg / kg bw from days 1 to 14, and free access to 3% DSS aqueous solution from days 8 to 14. DSS aqueous solution; combined high-ratio group (PNP-1a+Mesa-H group): PNP-1a 150mg / kg bw + mesalazine 150mg / kg bw were administered by gavage daily from day 1 to 14, and 3% DSS aqueous solution was allowed free drinking from day 8 to 14. UC mouse model was established by allowing free drinking of 3% DSS aqueous solution from day 8 to 14, lasting for 7 days; the drug was administered by gavage at fixed times daily for 14 consecutive days.
[0047] 2. Indicator detection: Same as in Example 3.
[0048] 3. Results Analysis: Comparison of DAI scores and reduction rates: Mice in the control group showed no UC-related symptoms throughout the entire process, and their DAI scores remained at 0. Mice in the DSS group began to show significant weight loss, watery stools, and bloody stools from day 10. The average DAI score on day 14 was 2.8 ± 0.3. In the mesalazine-only group, the mean DAI score on day 14 was 1.26 ± 0.2, with a DAI score reduction rate of 55%. The average DAI score on day 14 in the combined low-ratio group (PNP-1a 50 mg / kg + mesalazine 250 mg / kg) was 1.05 ± 0.18, with a DAI score reduction rate of 62.5%. The average DAI score on day 14 of the combined medium ratio group (PNP-1a 100mg / kg + mesalazine 200mg / kg) was 0.62±0.15, and the DAI score reduction rate reached 78%, which was significantly higher than that of the mesalazine alone group (P<0.01). The average DAI score on day 14 of the combined high-ratio group (PNP-1a 150mg / kg + mesalazine 150mg / kg) was 0.75±0.16, and the DAI score reduction rate was 73.2%.
[0049] Comparison of the incidence of gastrointestinal irritation: The incidence of gastrointestinal irritation in mice in the mesalazine-only group was 28% (2 out of 10 mice experienced vomiting and 3 experienced exacerbated diarrhea). The incidence of gastrointestinal irritation in the combined low-ratio group was 15% (1 out of 10 animals experienced vomiting and 1 experienced worsened diarrhea). The incidence of gastrointestinal irritation in the combined medium-ratio group decreased to 8% (only 1 out of 10 animals experienced mild diarrhea exacerbation), which was significantly lower than that in the mesalazine alone group (P<0.05). The incidence of gastrointestinal irritation in the combined high-ratio group was 10% (1 out of 10 animals experienced mild vomiting). No obvious gastrointestinal irritation symptoms were observed in the Control group and the DSS group (diarrhea in the DSS group was a symptom of the disease itself, not caused by drug stimulation).
[0050] Comparison of colon histopathological scores: The control group had a colon tissue pathology score of 0, with intact mucosal structure and no inflammatory cell infiltration. The DSS group had a colonic tissue pathology score of 3.7±0.4, with mucosal ulcers extending to the submucosa and extensive inflammatory cell infiltration. The histopathological score of the colon tissue in the mesalazine-only group was 1.8 ± 0.3; The histopathological score of the colon tissue in the combined treatment group was 0.8±0.2, which was significantly lower than that in the mesalazine-only group (P<0.01). The colonic mucosa showed only mild congestion and edema, a small amount of inflammatory cell infiltration, and the integrity of the mucosa was basically restored.
[0051] Comparison of inflammatory factor expression levels: The expression levels of TNF-α and IL-6 in the colon tissue of the DSS group were 186.5±15.2 pg / mg and 128.3±12.5 pg / mg, respectively. The expression levels of TNF-α and IL-6 in the mesalazine-only group were 102.3±10.5 pg / mg and 70.5±8.6 pg / mg, respectively. The expression levels of TNF-α and IL-6 in the combined group were 58.6±8.3 pg / mg and 42.8±6.5 pg / mg, respectively, which were significantly lower than those in the mesalazine alone group (P<0.01), indicating a more significant inhibitory effect on inflammatory factors.
[0052] When PNP-1a polysaccharide from Mushroom Nameko is used in combination with mesalazine, it has a significant synergistic effect on UC. The optimal ratio is "PNP-1a 100mg / kg + mesalazine 200mg / kg", which reduced the DAI score by 78%, significantly higher than the 55% reduction in the mesalazine alone group.
[0053] Combination therapy can reduce the dosage of mesalazine (by 33.3% compared to the monotherapy group) while ensuring enhanced anti-inflammatory effects, and can significantly reduce the incidence of gastrointestinal irritation caused by mesalazine, from 28% to 8%.
[0054] The above description of the embodiments is only for the purpose of helping to understand the method and core idea of the present invention, and is not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. A Pholiota nameko polysaccharide PNP-1a having an effect of improving ulcerative colitis, characterized in that, The Pleurotus subpellis polysaccharide PNP-1a comprises 11.70%, 32.34%, 9.16%, 33.71%, 4.05%, 1.31% and 7.73% of galactose, glucose, xylose, mannose, galacturonic acid, guluronic acid and glucuronic acid in terms of molar percentage; and the molecular weight ranges from 62 kDa to 236 kDa.
2. The Pleurotus obliquus polysaccharide PNP-1a of claim 1, characterized in that, The main chain of the Pleurotus subpellis polysaccharide PNP-1a comprises →3)-α-D-Manp-(1→ and →3)-α-D-Glcp-(1→, and the branch comprises β-D-Xylp-(1→3)-α-D-Manp-(1→ connected to O-2 of sugar residue →2,6)-β-D-Galp-(1→, or β-D-Xylp-(1→ connected to O-6 position of sugar residue →3,6)-β-D-Glcp-(1→.
3. The Pleurotus obliquus polysaccharide PNP-1a of claim 1 or 2, characterized in that, The Pleurotus subpellis polysaccharide PNP-1a has the structure of formula I: Formula I.
4. A method for preparing the Pleurotus passecol polysaccharide PNP-1a according to any one of claims 1 to 3, characterized in that, The Pleurotus subpellis polysaccharide PNP-1a is obtained by hot water extraction, ethanol precipitation, DEAE seplife FF anion exchange column chromatography, Sephadex G-100 gel column purification and dialysis.
5. The preparation method according to claim 4, characterized in that, Specifically comprising the following steps: (1) After the Pleurotus subpellis fruiting body is dried and crushed, it is sieved, water is added at a solid-liquid ratio of 1:20-40 (g / mL), water bath extraction is performed, the supernatant is taken after centrifugation and concentrated, ethanol is added to the concentrated solution, and the solution is left overnight, the precipitate is collected after filtration, and a crude polysaccharide extract is obtained; (2) A chloroform-n-butanol mixed solution is added to the crude polysaccharide extract of step (1), the upper aqueous phase is collected after mixing, dialysis and freeze-drying to obtain a crude polysaccharide PNP; (3) The crude polysaccharide PNP of step (2) is dissolved in water, the supernatant is taken and passed through a DEAE seplife FF anion exchange column, and the eluate is concentrated, dialyzed to remove salt and freeze-dried to obtain a polysaccharide PNP-1; (4) The polysaccharide PNP-1 of step (3) is dissolved in water, filtered through a Sephadex G-100 gel, eluted, dialyzed to remove salt after concentration of the eluate, and freeze-dried to obtain the Pleurotus subpellis polysaccharide PNP-1a.
6. The production method according to claim 5, wherein In step (1), the water bath extraction conditions are 80-95°C water bath extraction for 3-5h; and 2-4 times the volume of 90%-98% ethanol is added to the concentrated solution; And / or, in step (2), the volume ratio of chloroform to n-butanol in the chloroform-n-butanol mixed solution is 5:1; And / or, in step (3), the NaCl solutions of different concentrations include 0.1 mol / L NaCl solution, 0.2 mol / L NaCl solution and 0.3 mol / L NaCl solution.
7. Use of the Pleurotus subpellis polysaccharide PNP-1a of any one of claims 1-3 or the Pleurotus subpellis polysaccharide PNP-1a prepared by the preparation method of any one of claims 4-6 in the preparation of a functional food for preventing and / or improving ulcerative colitis.
8. The use of the Pleurotus subadultus polysaccharide PNP-1a of any one of claims 1-3 or the Pleurotus subadultus polysaccharide PNP-1a prepared by the preparation method of claim 4 or 5 in the preparation of a drug for preventing and / or improving ulcerative colitis.
9. Use according to claim 7 or 8, characterized in that, The Pleurotus subadultus polysaccharide PNP-1a up-regulates the expression of tight junction proteins Occludin, ZO-1 and Claudin-1, and maintains the integrity of the colonic mucosal barrier; The Pleurotus subadultus polysaccharide PNP-1a can reduce the expression levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, IL-18 and IFN-γ, and can enhance the activities of SOD, GSH-Px and CAT, and reduce the levels of iNOS and NO.
10. Use according to claim 7 or 8, characterized in that, The Pleurotus subadultus polysaccharide PNP-1a can be used in combination with mesalazine, and the mass ratio of the two is 1:2-4.