Tremella fuciformis acidic polysaccharide, preparation method thereof and application thereof in improving ulcerative colitis

Abstract

The invention discloses a Tremella acidic polysaccharide, a preparation method thereof and an application thereof in improving ulcerative colitis, and belongs to the field of natural product development. The Tremella acidic polysaccharide comprises mannose, xylose, glucuronic acid, glucose and rhamnose, and the molar percentages are 59.2%, 23.2%, 13.9%, 1.6%, 1.7% and 0.4% respectively; the glycosidic bond connection mode is: 1,2,3-mannose, 1,3-mannose, T-xylose, 1,3-xylose, 1,4-glucuronic acid and T-mannose. The Tremella acidic polysaccharide is obtained by a purification method of ultrasonic extraction combined with hot water extraction, ethanol precipitation and ion chromatography purification combined with gel chromatography; experiments have shown that the Tremella acidic polysaccharide can effectively improve the symptoms of ulcerative colitis, laying a foundation for the development and research of ulcerative colitis drugs.

Description

Technical Field

[0001] The invention relates to the field of natural product development, and in particular to Tremella fuciformis acidic polysaccharide, a preparation method thereof and application thereof in improving ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is a nonspecific, recurrent, chronic intestinal inflammatory disease. Although the pathogenesis of UC is still unclear, pathological studies have shown that UC patients often present with intestinal inflammation, intestinal microbial disorders, and impaired intestinal barrier function. Currently, the main therapeutic drugs are aminosalicylic acids, corticosteroids, and immunosuppressants, but they are not effective for UC patients, and some patients have increased physical burden due to adverse reactions. Therefore, it is of great significance to develop potential drugs that are non-toxic and can improve the damaging effects of UC.

[0003] Tremella aurantialbau belongs to the phylum Basidiomycetes, the order Tremellaceae, the genus Tremellaceae, and is parasitic on the fungus Stereum hirsutum. It is also called yellow fungus because of its golden color. It is a high-quality edible fungus with high nutritional and medicinal value and is an important natural drug resource. At present, there is no report on the efficacy of Tremella auriculariae polysaccharide in improving ulcerative colitis. Therefore, it is of great significance to develop and utilize its medicinal value. Summary of the invention

[0004] The purpose of the present invention is to provide a Tremella fuciformis acidic polysaccharide and a preparation method thereof and application in improving ulcerative colitis, so as to solve the problems existing in the above-mentioned prior art. Tremella fuciformis acidic polysaccharide is extracted and purified from Tremella fuciformis for the first time. Experiments have shown that the Tremella fuciformis acidic polysaccharide can improve the symptoms of ulcerative colitis, effectively reduce the intestinal damage of ulcerative colitis, improve microbial disorders, enhance the intestinal barrier function and reduce the level of inflammation.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention also provides a Tremella acidic polysaccharide, which comprises mannose, xylose, glucuronic acid, glucose and rhamnose, wherein the molar percentages of the mannose, xylose, glucuronic acid, glucose and rhamnose are 59.2%, 23.2%, 13.9%, 1.6%, 1.7% and 0.4% respectively.

[0007] Preferably, the connection mode of the glycosidic bonds of the Tremella fuciformis acidic polysaccharide is: 1,2,3-mannose, 1,3-mannose, T-xylose, 1,3-xylose, 1,4-glucuronic acid and T-mannose.

[0008] The present invention also provides a method for preparing the Tremella fuciformis acidic polysaccharide, comprising the following steps: extracting Tremella fuciformis fruiting body dry product by combining ultrasonic method and water extraction method, extracting Tremella fuciformis crude polysaccharide through alcohol precipitation treatment, and then purifying the Tremella fuciformis crude polysaccharide by ion chromatography and gel chromatography to obtain Tremella fuciformis acidic polysaccharide.

[0009] Preferably, the preparation method specifically comprises the following steps:

[0010] (1) Grinding Tremella fuciformis fruiting bodies into powder, mixing with deionized water, and ultrasonically treating;

[0011] (2) extracting the solution after ultrasonic treatment with water at 75° C., filtering and concentrating the water extract to 1 / 5-1 / 3 of the original volume, separating the solution with anhydrous ethanol, and collecting the precipitate by centrifugation to obtain a crude extract of Tremella fuciformis polysaccharide;

[0012] (3) The Tremella fuciformis polysaccharide crude extract is subjected to a sevag method to remove protein components in the crude polysaccharide, followed by dialysis and freeze-drying to obtain a Tremella fuciformis polysaccharide freeze-dried product;

[0013] (4) Using ion chromatography to separate and purify the Tremella fuciformis polysaccharide lyophilized material to obtain Tremella fuciformis polysaccharide components TA1 and TA 2, and then using gel chromatography to further separate and purify Tremella fuciformis polysaccharide component TA 2 to obtain Tremella fuciformis acidic polysaccharide TA 2-1.

[0014] Preferably, in step (1), the Tremella fuciformis fruiting body powder is 100 mesh, and the mass volume ratio of the Tremella fuciformis polysaccharide fruiting body powder to deionized water is 1:2.

[0015] Preferably, in step (1), the ultrasonic conditions are: 500W ultrasound for 10 minutes.

[0016] Preferably, in step (2), the water extraction time is 3 hours.

[0017] Preferably, in step (3), the volume ratio of the Tremella fuciformis polysaccharide crude extract to the sevag reagent is 1:2.

[0018] Preferably, in step (4), the conditions for separation and purification by ion chromatography are: gradient elution with 0-1M NaCl solution at an elution rate of 0.8mL / min; the conditions for separation and purification by gel chromatography are: the eluent is 0.15M sodium chloride solution at an elution rate of 0.5mL / min.

[0019] The present invention also provides the use of Tremella fuciformis acidic polysaccharide in preparing a product for improving ulcerative colitis. More preferably, the product is a medicine, but not limited thereto.

[0020] The present invention discloses the following technical effects:

[0021] The present invention obtains polysaccharide TA 2-1 with novel structure and multiple biological activities from natural Tremella fuciformis for the first time. At the same time, through in vivo experiments on mice, it is found that the polysaccharide can improve the symptoms of ulcerative colitis, effectively reduce the intestinal damage of ulcerative colitis, reduce microbial disorders, improve intestinal barrier function and reduce inflammation levels. The present invention provides a research basis for the subsequent development and utilization of Tremella fuciformis acidic polysaccharides, and has important economic value and market value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 The elution curves of Tremella fuciformis polysaccharide of the present invention are shown in FIG. 1 ; (A) DEAE-agarose gel-FF chromatography elution curve, (B) Hiload 16 / 600 Superdex 200 gel chromatography elution curve;

[0024] Figure 2 This is the monosaccharide composition analysis of Tremella fuciformis acidic polysaccharide TA 2-1 of the present invention;

[0025] Figure 3 is the molecular weight distribution curve of Tremella acidic polysaccharide TA2-1 of the present invention;

[0026] Figure 4 FT-IR spectrum of Tremella fuciformis acidic polysaccharide TA 2-1 of the present invention;

[0027] Figure 5 This is the total ion current spectrum of TA 2-1 methylation analysis;

[0028] Figure 6 The nuclear magnetic resonance spectrum analysis of Tremella fuciformis acidic polysaccharide TA 2-1;

[0029] Figure 7 The effect of Tremella fuciformis acidic polysaccharide TA 2-1 on inhibiting UC damage in vivo; (A) DSS administration induced mouse UC model and schematic diagram of TA 2-1 administration. DAI score (B), body weight (C), survival curve (D) and colon length (E, F) of mice in each group; data are expressed as mean ± standard deviation (n = 6) #p < 0.05, ##p < 0.01, ###p < 0.001 vs control group, *p < 0.05, **p < 0.01, **p < 0.001 vs DSS-induced UC group;

[0030] Figure 8 The HE results of the effect of Tremella fuciformis acidic polysaccharide component TA 2-1 of the present invention on the intestinal pathology of UC mice (A) and its effect on the expression level of ferroptosis-related proteins in intestinal epithelial cells of UC mice (B);

[0031] Fig. 9 The present invention adopts immunofluorescence staining method to detect the effect of Tremella fuciformis acidic polysaccharide TA2-1 on the expression of tight protein molecules claudin-1 (A) and ZO-1 (B) in intestinal epithelial cells of UC mice;

[0032] Fig.10 The present invention shows the effect of Tremella fuciformis acidic polysaccharide TA2-1 on the levels of inflammatory cytokines IL-6 (A), TNF-α (B), IFN-γ (C) and MCP-1 (D) in the peripheral blood of UC mice;

[0033] Fig.11 The effect of Tremella fuciformis acidic polysaccharide TA2-1 of the present invention on the intestinal microbial composition of UC mice: (A) Venn diagram; (B) principal component analysis;

[0034] Fig.12 The heat map analysis results of the effect of Tremella fuciformis acidic polysaccharide TA 2-1 of the present invention on the intestinal microbial composition of UC mice;

[0035] Fig.13 This is the effect of Tremella fuciformis acidic polysaccharide TA 2-1 of the present invention on the relative abundance of intestinal microbial communities at the phylum level. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] Example 1 Extraction, preparation and purification of Tremella fuciformis acidic polysaccharide

[0042] The dried Tremella fuciformis was ground into powder by a high-speed pulverizer, passed through a 100-mesh sieve, and mixed with deionized water twice the volume of the powder, and subjected to ultrasonic extraction for 10 minutes at a power of 500 W in an ultrasonic instrument;

[0043] The sonicated Tremella powder solution was placed under 75°C for high-temperature water extraction. Since the Tremella was continuously expanded in the water, 75°C deionized water was continuously added during the water extraction process to keep the liquid surface covering the Tremella. After 3 hours of extraction, the expanded Tremella was removed by filtering with multi-layer gauze, and the aqueous solution was concentrated at 75°C to concentrate the Tremella extract to 1 / 5-1 / 3 of the original volume. Anhydrous ethanol was then added to make the final ethanol concentration of 75%, and the mixture was left to stand overnight at room temperature, centrifuged at 1000rpm for 10 minutes, and the precipitate was collected and dried on a sterile plate to obtain a Tremella crude extract.

[0044] The Sevag method was used to remove protein components from the crude extract of Tremella fuciformis. Deionized water was used to dissolve the dried crude extract of Tremella fuciformis, and sevag reagent (n-butanol: chloroform = 1:3, v / v) was added at a volume ratio of 1:2, and the mixture was shaken at 120 rpm for 40 minutes on a horizontal shaker to mix, and the denatured protein was removed by centrifugation. The operation was repeated once more to further remove the residual protein components; after dialysis to remove small molecules, the extract was concentrated using a rotary evaporator, and the concentrate was freeze-dried to obtain the Tremella fuciformis crude polysaccharide freeze-dried product.

[0045] The lyophilized Tremella fuciformis crude polysaccharide was dissolved in deionized water, and after passing through the membrane, a DEAE-agarose gel-FF chromatography column was used for the first step of purification, and a 0-1M NaCl solution was used for gradient elution (0.8mL / min), and 3mL was collected in each tube. The polysaccharide component content of each tube of the eluate was determined by the phenol-sulfuric acid method, and a curve graph was drawn to obtain Tremella fuciformis polysaccharide components TA 1 and TA2. TA 2 was purified in the second step using a Hiload 16 / 600Superdex 200 gel chromatography column, eluted with 0.15M / NaCl (0.5mL / min), and the polysaccharide portion was collected, and 3mL was collected in each tube. The polysaccharide component content of each tube of the eluate was determined by the phenol-sulfuric acid method, and a curve graph was drawn to obtain Tremella fuciformis acidic polysaccharide component TA2-1, which was dialyzed and freeze-dried to obtain freeze-dried Tremella fuciformis acidic polysaccharide TA 2-1 (see Figure 1 )

[0046] Example 2 Analysis of Monosaccharide Composition of Tremella Acidic Polysaccharide TA2-1

[0047] (1) Complete acid hydrolysis

[0048] Weigh about 2 mg of Tremella fuciformis acidic polysaccharide component TA2-1, add 1 mL of anhydrous methanol solution containing 1 M hydrochloric acid, seal the tube with N2, hydrolyze at 80 ° C for 16 hours, blow dry with an air pump, add 1 mL of 2 M trifluoroacetic acid, hydrolyze at 120 ° C for 1 hour, add a small amount of ethanol, dry in a water bath at 60 ° C, repeat 3 to 5 times to completely evaporate the trifluoroacetic acid.

[0049] (2) PMP derivatization

[0050] Add 0.5 mL of PMP reagent and 0.5 mL of 0.3 M NaOH solution to the dried sample obtained after complete acid hydrolysis. After the sample is fully dissolved, take 0.2 mL of it into a small centrifuge tube and place it in a 70°C water bath for 30 minutes. After centrifugation at 10,000 rpm for 5 minutes, add 0.1 mL of 0.3 M hydrochloric acid solution and 0.1 mL of distilled water and mix thoroughly. Add 1 mL of dichloromethane, mix well, extract the remaining PMP reagent, remove the dichloromethane layer, retain the water layer, and repeat three times. After filtering with a 0.22 μm filter membrane, perform HPLC detection:

[0051] A Shimadzu HPLC system (LC-10ATvp pump and SPD-10AVD UV detector) and a Compass C18 column (4.6×150 mm) were used. The mobile phase was PBS (0.1 M, pH 7.0)-acetonitrile 81:19 (v / v), the flow rate was 1.0 mL / min, the injection volume was 10 μL, and the detection wavelength was 245 nm.

[0052] The results are as follows Figure 2As shown, the Tremella fuciformis acidic polysaccharide component TA2-1 is composed of mannose (Man), xylose (Xyl) and glucuronic acid (GlcA), and also contains a small amount of glucose (Glc), fucose (Fuc) and rhamnose (Rha), with molar percentages of 59.2%, 23.2%, 13.9%, 1.6%, 1.7% and 0.4%, respectively.

[0053] Example 3 Detection of molecular weight of Tremella fuciformis acidic polysaccharide TA2-1

[0054] Weigh 1-2 mg Tremella acidic polysaccharide TA2-1, dissolve in dH2O, the sample concentration is 10 mg / mL, filter through a 0.22 μm aqueous phase filter membrane, and detect using HPSEC-MALLS-RI high performance liquid phase system. Agilent 1260 InfinityII high performance liquid phase system was used, the chromatographic column was Shodex OHpak SB-805HQ (8mm×300mm), the detector was an interferometric differential refractometer (OPTILAB T-rEx) and a multi-angle laser light scattering instrument (DAWN HELEOS II). The mobile phase was 0.2M NaCl, containing 0.02% NaN3, the elution flow rate was 0.6mL / min, and the column temperature was 35°C. The molecular weight distribution curve of TA2-1 is shown in Figure 3 The test showed that the weight average molecular weight (Mw) of Tremella fuciformis acidic polysaccharide TA2-1 was 1.27×10 5 Da, number average molecular weight (Mn) is 5.03×10 4 Da, polydispersity index (Mw / Mn) is 2.5.

[0055] Example 4 FT-IR analysis of Tremella fuciformis acidic polysaccharide TA 2-1

[0056] Tremella acidic polysaccharide TA 2-1 was analyzed by infrared spectroscopy. Figure 4 The sample showed obvious polysaccharide characteristic absorption peaks at 3600-3200 cm –1 The -OH stretching vibration absorption peak is broad and strong, at 3000~2800cm -1 The narrow and weak CH stretching vibration absorption peak is at 1647 cm -1 The bending vibration absorption peak of OH is shown at 1403 cm -1 Weak CH deformation vibration peaks were detected around 1071cm -1 There is an obvious pyran ring absorption peak at 855~810cm -1 The CH angle vibration of the α-anomer diastereomer of pyranose is shown at .

[0057] Example 5 Methylation Analysis of Tremella Acidic Polysaccharide TA 2-1

[0058] (1) Acidic sugar reduction: Dissolve 5 mg of sample in 1 mL of H2O, add 200 μL of 0.2 M morpholineethanesulfonic acid (MES) and 400 μL of 500 mg / mL carbodiimide reagent, and react at 25-30°C for 3 h. After the reaction, add 1 mL of 4 M imidazole-HCl and 600 μL of 70 mg / mL NaBD4, react at 4°C overnight, slowly add 500 μL of glacial acetic acid in an ice bath to terminate the reaction, and dialyze the reaction solution to dryness.

[0059] (2) Dissolution of sugar samples: Weigh 5 mg of dried sample and dissolve it in 0.5 mL of DMSO (dehydrated with 4A molecular sieves), fill with N2 and seal, stir magnetically until the sugar sample is fully dissolved. Add 0.5 mL of NaOH-DMSO suspension to the dissolved sugar sample, fill with N2 and seal, stir magnetically for 2 min, and mix well. Slowly add 1 mL of iodomethane in an ice bath, seal, protect from light, stir magnetically for 30 min, add 2 mL of distilled water to stop the reaction, dialyze with running water for 24 h, dialyze with distilled water for 24 h, concentrate to a small volume and freeze-dry.

[0060] (3) Repeat the above steps for the second methylation. After terminating the methylation reaction, add an equal volume of dichloromethane and stir to extract for 30 minutes. Let stand to separate the layers. Take the dichloromethane layer (lower layer) and repeat the extraction three times. Combine the extracts, add 5-7 mL of water and stir to extract the organic phase for 20 minutes. Discard the aqueous phase and repeat three times. Dry the organic phase with an air pump and add 1 mL of distilled water to freeze-dry.

[0061] (4) The methylated sample is subjected to infrared spectroscopy. If the IR spectrum is at 3400 cm -1 There is no absorption peak at 1000cm -1 If there is a high absorption peak around , it proves that the polysaccharide is completely methylated.

[0062] Hydrolysis of methylated polysaccharides: Add 1 mL of mixed acid (HCOOH:H2O:TFA=3:2:1) to the above-mentioned dried methylated sugar sample, seal with N2, and hydrolyze at 100°C for 6 hours. After the hydrolysis, repeatedly add anhydrous ethanol to evaporate the mixed acid until pH = 7 (temperature below 40°C).

[0063] (5) Reduction: Add 1 mL of 3 mg / mL NaBH4 solution and stir at room temperature for 12 h. Add about 100 μL of 50% glacial acetic acid to neutralize until neutral. Add an appropriate amount of strong acid cation exchange resin, stir magnetically for 20 min, filter (remove the resin), repeatedly add methanol to the filtrate, and evaporate the boric acid until neutral (temperature below 40°C).

[0064] (6) Acetylation: Add 0.5 mL of acetic anhydride and anhydrous pyridine, seal with N2, and react at 100°C for 2 h. After the reaction is completed, quickly add 1 mL of distilled water in an ice bath to terminate the reaction, cover the bottle cap tightly, and cool in an ice bath for 5 min. Add 2 mL of dichloromethane and 2 mL of distilled water, and back-extract the organic phase 3 times. Remove the aqueous phase, blow dry the organic phase, dissolve it in 1 mL of chromatographically pure dichloromethane, filter it, and perform GC-MS analysis.

[0065] (7) GC-MS program: The chromatographic column model was Agilent DB-35ms, the injection port temperature was 300°C, the auxiliary heater temperature was 280°C, and the heating program was as follows: initial temperature 140°C, hold for 2 min, increase to 170°C at 5°C / min, hold for 3 min, increase to 180°C at 1°C / min, hold for 5 min, increase to 220°C at 3°C / min, hold for 1 min, increase to 295°C at 20°C / min, hold for 3 min.

[0066] The total ion current of TA 2-1 methylation analysis is as follows Figure 5 The results showed that the main glycosidic bond connection modes of TA 2-1 were 1,2,3-mannose, 1,3-mannose, T-xylose, 1,3-xylose, 1,4-glucuronic acid and T-mannose (Table 1).

[0067] Table 1 Glycosidic bond connection mode of TA 2-1

[0068]

[0069] Example 6 Nuclear Magnetic Resonance Spectrum Analysis of Tremella Acidic Polysaccharide TA 2-1

[0070] 20.0 mg of the dried sample was weighed and dissolved in 0.5 mL of D2O (99.8%), and 1D NMR spectra (1H NMR, 13C NMR) and 2D NMR spectra (HSQC, HMBC) were measured at 20° C. using a Bruker Avance 600 MHz nuclear magnetic resonance spectrometer. The detection frequency of 1H NMR was 600 MHz, and the detection frequency of 13C NMR was 150 MHz.

[0071] In the 13C-NMR spectrum, the chemical shifts of 102.32 ppm and 102.42 ppm are respectively assigned to the anomeric carbon of α-1,3-D-Manp and β-1,3-D-Xylp; the chemical shift of 99.28 ppm is assigned to the anomeric carbon of α-1,2,3-D-Manp; the signal peak of 173.15 ppm is the C-6 signal peak of t-β-D-GlcAp; the signal peak of 19.26 ppm located in the high field is assigned to the anomeric carbon signal peak of the methyl part of the acetyl group, and the acetyl group may be located at O-2 of α-1,3-D-Manp (see Figure 6 ).

[0072] The following test examples will further verify and analyze the medical uses of Tremella fuciformis acidic polysaccharide TA 2-1 obtained by the present invention.

[0073] Test Example 1 Tremella Acidic Polysaccharide TA 2-1 Improves Symptoms of Ulcerative Colitis

[0074] 6-8 week old male C57BL / 6 mice were purchased and the UC mouse model was established by oral administration of dextran sulfate sodium salt (DSS) aqueous solution ( Figure 7 A). The mice were divided into four groups, namely, negative control group, model group, low-dose TA2-1 (20 mg / kg) + model group and high-dose TA2-1 (60 mg / kg) + model group. After oral administration of 2.5% DSS aqueous solution, the four groups of mice were gavaged with PBS, PBS, 20 mg / kg TA2-1 and 60 mg / kg TA2-1 aqueous solution, respectively, with a gavage volume of 100 μL, and the gavage was continued for 36 days.

[0075] The weight, disease activity index (DAI), colon length, and survival of mice were recorded. The results showed that compared with the model group, the weight, DAI, survival days, and colon length of mice in the TA 2-1 group were significantly improved, and the improvement in the high-dose TA2-1 group was higher than that in the low-dose group ( Figure 7 (in BF).

[0076] Experimental Example 2 Tremella fuciformis acidic polysaccharide TA 2-1 effectively reduces intestinal epithelial cell ferroptosis and reduces intestinal epithelial barrier function damage in UC mice

[0077] The colon tissue of mice was analyzed by hematoxylin-eosin (HE) staining. The results showed that the DSS-induced mice, i.e., the model group, had significant intestinal damage ( Figure 8 In the middle (A), damage to the villus tip, separation of the lamina propria, epithelial damage, complete disruption of the crypt-villus axis, and increased inflammatory cell infiltration were observed in the colon tissue. After TA 2-1 intervention, these phenomena were significantly alleviated in a dose-dependent manner. HE staining results showed that oral administration of TA2-1 significantly improved the pathological characteristics of the colon tissue of UC mice.

[0078] Immunohistochemistry (IHC) results showed that TA 2-1 significantly upregulated the expression of GPX4 and FTH, and decreased the expression level of ACSL4 in intestinal epithelial cells of colon tissue ( Figure 8(B). ACSL4 is an important isoenzyme in polyunsaturated fatty acid (PUFA) metabolism and is positively correlated with ferroptosis levels. In contrast, GPX4 utilizes glutathione (GSH) to detoxify lipid peroxidation and plays an important role in inhibiting ferroptosis. In addition, the results showed that TA 2-1 increased the expression of FTH, which is responsible for iron storage in the cytoplasm, resulting in a decrease in the level of free iron involved in ferroptosis. The above results indicate that TA 2-1 can effectively inhibit ferroptosis in intestinal epithelial cells.

[0079] Tight junctions (TJs) are responsible for building epithelial barriers, including transmembrane and membrane proteins and signaling molecules. TJ proteins include more than 40 different proteins, the main ones of which are claudin, zonula occludens 1 and 2 (ZO1 / 2), occludens, and F-actin. The inventors performed immunostaining of claudin-1 and ZO-1 in intestinal tissues to further evaluate the effect of TA 2-1 on tight junctions between intestinal epithelial cells. Immunofluorescence staining showed that the levels of claudin-1 and ZO-1 proteins in DSS-induced mice were significantly reduced compared with the control group ( Fig. 9 (A and B). Compared with the DSS group, TA2-1 (20 mg / kg and 60 mg / kg) intervention significantly inhibited the decrease in claudin-1 and ZO-1 expression levels.

[0080] The above data show that TA2-1 can effectively reduce intestinal epithelial cell ferroptosis and increase the level of tight junction proteins, indicating that TA2-1 can effectively reduce the damage of intestinal barrier function in UC.

[0081] Experimental Example 3 Tremella acidic polysaccharide TA2-1 significantly reduces the level of inflammatory factors in the peripheral blood of UC mice

[0082] After TA2-1 was intragastrically administered, peripheral blood serum was collected from each group of mice, and the levels of inflammatory cytokines (IL-6, TNF-α, IFNγ, and MCP-1) were determined by CBA (cytometric beadarray) to further clarify the effect of TA2-1 on the inflammatory level of UC mice. The results showed that compared with the control group, the levels of IL-6, TNF-α, IFNγ, and MCP-1 in the DSS model group were significantly increased ( Fig.10 ). Intragastric administration of TA 2-1 reduced the expression levels of peripheral inflammatory factors in UC mice, especially the high-dose TA 2-1 (60 mg / kg) group significantly reduced the levels of IL-6, TNF-α, IFNγ and MCP-1. The above results show that Tremella acidic polysaccharide TA 2-1 can significantly inhibit the inflammatory response of UC in the DSS-induced UC mouse model.

[0083] Experimental Example 4 Tremella acidic polysaccharide TA2-1 effectively improves the intestinal flora composition of mice with ulcerative colitis

[0084] Feces of mice from different groups (n=3) were collected, including the control group, DSS-administered group, and TA 2-1-treated DSS group (20 mg / kg and 60 mg / kg). Total genomic DNA was extracted from the cecal contents, and the V3-V4 region of the bacteria was amplified and sequenced using the Illumina NovaSeq 6000 system (Shanghai Painuosen Biotechnology Co., Ltd.).

[0085] Venn diagram analysis showed that there were 1958, 1942, 2396 and 2396 effective OUTs in the feces of the control group, model group, low-dose TA2-1 group and high-dose TA 2-1 group, respectively. Fig.11 The principal component analysis showed that the distance between the control group and the model group increased significantly, and TA 2-1 administration by oral gavage could effectively regulate the distance between the DSS group and the treatment group ( Fig.11 (middle B).

[0086] At the phylum level, the mouse intestinal microorganisms were mainly Firmicutes, Bacteroidetes, and Verrucomicrobia ( Fig.13 ). Compared with the control group, the beneficial Firmicutes in the microbiota of mice in the DSS group decreased, while the abundance of Bacteroidetes increased. The abundance ratio of Firmicutes / Bacteroidetes in the intestinal microorganisms of UC patients decreased. Compared with the DSS group, TA 2-1 was able to increase the abundance ratio of Firmicutes / Bacteroidetes, which indicates that TA 2-1 can significantly improve the composition of intestinal microorganisms in UC mice after oral administration.

[0087] Differences in intestinal microbial populations at the genus level Fig.12As shown: Compared with the control group, the populations of Bacteroides and Ruminococcus increased significantly, and the abundance of Akkermansia, Adlercreutzia, and Lactobacillus decreased significantly. In the TA2-1 administration group, the abundance levels of Bacteroides and Ruminococcus decreased, and the abundance of Akkermansia, Adlercreutzia, and Lactobacillus increased significantly. The level of Bacteroides increased in the intestinal microbiota of UC mice, and excessive Bacteroides is considered to be harmful to the intestinal immune system. Ruminococcus is enriched in UC patients, and its overgrowth is associated with the presence of bacterial biofilms, and endoscopic mucosal biofilms are common in patients with gastrointestinal diseases. Functional analysis of the composition of the microbiota in samples from UC patients showed that before the onset of UC, elastase activity increased, which was negatively correlated with the abundance of related bacterial groups including Adlercreutzia and Akkermansia. In addition, Kluyveromyces adlerii is a genus that metabolizes isoflavones, phenolic compounds known for their antimicrobial and anti-inflammatory functions, and its reduced abundance in UC patients may promote the inflammatory state. Bifidobacterium and Lactobacillus have received considerable attention as common probiotics, while the abundance of Bifidobacterium and Lactobacillus is reduced in UC patients clinically.

[0088] The above results indicate that compared with the DSS model group, Tremella fuciformis acidic polysaccharide TA2-1 can effectively affect the intestinal microbial composition of ulcerative colitis mice and may help alleviate the dysfunction of intestinal flora in UC.

[0089] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A Tremella acidic polysaccharide, characterized in that: It includes mannose, xylose, glucuronic acid, glucose, fucose and rhamnose, wherein the molar percentages of mannose, xylose, glucuronic acid, glucose, fucose and rhamnose are 59.2%, 23.2%, 13.9%, 1.6%, 1.7% and 0.4% respectively; The connection mode of Tremella acidic polysaccharide is: 1,2,3-mannose, 1,3-mannose, T-xylose, 1,3-xylose, 1,4-glucuronic acid and T-mannose; The weight average molecular weight of the Tremella acidic polysaccharide is 1.27×10 5 Da.

2. A method for preparing Tremella fuciformis acidic polysaccharide according to claim 1, characterized in that: The following steps are involved: The dry product of Tremella fuciformis fruiting body is extracted by combining ultrasonic method and water extraction method, and Tremella fuciformis crude polysaccharide is extracted through alcohol precipitation treatment, and then the Tremella fuciformis crude polysaccharide is purified by ion chromatography and gel chromatography to obtain Tremella fuciformis acidic polysaccharide.

3. The preparation method according to claim 2, characterized in that: The preparation method specifically comprises the following steps: (1) Grind the Tremella fuciformis fruiting bodies into powder, mix with deionized water, and perform ultrasonic treatment; (2) The solution after ultrasonic treatment is then placed at 75°C for water extraction, and then filtered and concentrated to make the water extract concentrated to 1 / 5-1 / 3 of the original volume, and then separated with anhydrous ethanol, and the precipitate is collected by centrifugation to obtain a crude extract of Tremella fuciformis polysaccharide; (3) The crude extract of Tremella fuciformis polysaccharide is subjected to sevag method to remove protein components in the crude polysaccharide, and then dialyzed and freeze-dried to obtain Tremella fuciformis polysaccharide freeze-dried product; (4) Using ion chromatography to separate and purify the Tremella fuciformis polysaccharide lyophilized material to obtain Tremella fuciformis polysaccharide components TA 1 and TA 2, and further using gel chromatography to separate and purify Tremella fuciformis polysaccharide component TA 2 to obtain Tremella fuciformis acidic polysaccharide TA 2-1.

4. The preparation method according to claim 3, characterized in that: In step (1), the Tremella fuciformis fruiting body powder is 100 mesh.

5. The preparation method according to claim 3, characterized in that: In step (1), the ultrasonic condition is: 500W ultrasonic for 10 minutes.

6. The preparation method according to claim 3, characterized in that: In step (2), the water extraction time is 3 hours.

7. The preparation method according to claim 3, characterized in that: In step (3), the volume ratio of the Tremella fuciformis polysaccharide crude extract to the sevag reagent is 1:

2.

8. The preparation method according to claim 3, characterized in that: In step (4), the conditions for separation and purification by ion chromatography are: gradient elution with 0-1 M NaCl solution at an elution rate of 0.8 mL / min; the conditions for separation and purification by gel chromatography are: the eluent is 0.15 M sodium chloride solution at an elution rate of 0.5 mL / min.

9. Use of Tremella fuciformis acidic polysaccharide according to claim 1 in preparing a product for improving ulcerative colitis.

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