Preparation method and application of schizophyllum commune polysaccharide nanoparticles with effects of improving intestinal inflammation and repairing mucosa

Abstract

The invention discloses a preparation method and application of schizophyllum commune polysaccharide nanoparticles with the effects of improving intestinal inflammation and repairing mucosa. The nanoparticles are formed by self-assembling schizophyllum commune polysaccharide and a vegetable protein nano core. The schizophyllum commune polysaccharide nanoparticles show excellent intestinal inflammation resistance and mucosal barrier injury repair functions, all the used materials are natural biological macromolecules, and the schizophyllum commune polysaccharide nanoparticles have good development and utilization prospects and can be widely applied to the fields of health-care products and biological medicine products.

Description

Technical Field

[0001] The invention relates to a Schizophyllum polysaccharide nanoparticle having the functions of improving intestinal inflammation and repairing mucosa, and a preparation method and application thereof, belonging to the technical field of functional health food and biomedicine. Background Art

[0002] Ulcerative colitis (UC) is an inflammatory disease of the intestine, characterized by ulcers, which mainly occur in the mucosal layer of the colon. It is characterized by chronicity, non-specificity, and diffuseness. Reports show that the number of people suffering from UC worldwide will reach 5 million in 2023, showing an increasing trend year by year, especially in Asia. It is predicted that it will increase fourfold by 2035, which should be of great concern. It is generally believed that genetic factors, environmental factors, psychological factors and bad eating habits can lead to the occurrence of UC. Its pathogenesis is related to the destruction of the intestinal mechanical barrier, decreased immunity and imbalance of intestinal flora. The early occurrence of UC is mainly caused by the destruction of the intestinal mechanical barrier due to damage to intestinal epithelial cells. When the protective structure of the intestinal epithelium is damaged, a large amount of antigens and other substances directly pass through the epithelial barrier, activating nuclear factor κB (NF-κB) and its inflammatory signaling pathway, increasing the production of proinflammatory cytokines such as TNF-α, IL-1β, IL-6, etc., leading to the aggregation of inflammatory cells and intensified inflammatory response, inducing a certain degree of local intestinal cell apoptosis, causing intestinal mucosal barrier damage, and triggering ulcers.

[0003] At present, the drugs used in clinical treatment of UC can be divided into two categories: Western medicine and traditional Chinese medicine. Common drugs for Western medicine treatment include aminosalicylic acid, steroid drugs, TNF monoclonal antibodies, immunosuppressants, biological agents, probiotics, etc.; common drugs for traditional Chinese medicine treatment include Xianglian Pills, Shenling Baizhu Powder, Wumei Pills, etc. In addition, some natural products such as flavonoids, terpenes, alkaloids and polysaccharides have also been proven to have good colitis treatment effects with few side effects, and have received widespread attention in recent years.

[0004] Schizophyllum, also known as white ginseng fungus, is not only rich in nutritional value, but also rich in polysaccharides and other active ingredients. Schizophyllum fruiting body polysaccharides have good anti-colitis effects, but as a type of macromolecule, their utilization efficiency in the body is limited after ingestion. Studies have shown that polysaccharide macromolecules are converted into small molecules such as short-chain fatty acids under the action of intestinal flora, so the timeliness of polysaccharide molecules directly exerting anti-inflammatory effects is limited. Summary of the invention

[0005] In order to overcome the problems existing in the above-mentioned prior art, the present invention provides a method for preparing Schizophyllum polysaccharide nanoparticles with the effects of improving intestinal inflammation and repairing mucosal barrier damage, aiming to make it have better effects of improving intestinal inflammation and repairing mucosal barrier damage than Schizophyllum fruiting body polysaccharide.

[0006] In order to achieve the purpose, the present invention adopts the following technical scheme:

[0007] The present invention provides a method for preparing Schizophyllum polysaccharide nanoparticles having the effects of improving intestinal inflammation and repairing mucosa, wherein the Schizophyllum polysaccharide nanoparticles are self-assembled from Schizophyllum polysaccharide and plant protein nanocores, and specifically comprises the following steps:

[0008] (1) preparing schizophyllan polysaccharide with deionized water to a 5-10 mg / mL polysaccharide stock solution;

[0009] (2) dissolving the plant protein at a concentration of 1 to 5 g / mL in an ethanol solution having a volume concentration of 65% to 85%, and stirring the mixture at a speed of 500 to 800 rpm to obtain a plant protein ethanol mixed solution;

[0010] (3) taking the plant protein ethanol mixed solution, adding deionized water under stirring at 500-800 rpm, and then continuing to stir at room temperature for 30-120 min to obtain a plant protein nanocore suspension; wherein the volume ratio of the plant protein ethanol mixed solution to the added deionized water is 1:3-5;

[0011] (4) After removing ethanol from the plant protein nanocore suspension obtained in step (3) by rotary evaporation at a heating temperature of 55 to 65° C., the polysaccharide mother solution prepared in step (1) is added thereto to make the concentration of schizophyllan polysaccharide in the system 0.1 to 1.0 mg / mL, and then the pH is adjusted to 2.0 to 5.0 by adding 0.1 to 0.5 M hydrochloric acid solution, and continuously stirred at 500 to 800 rpm for 30 to 120 min to obtain a dispersion of schizophyllan polysaccharide nanoparticles.

[0012] Furthermore, the Schizophyllum polysaccharide is a Schizophyllum fruiting body polysaccharide, and the specific preparation method can be found in patent CN117343210A.

[0013] Furthermore, the plant protein is at least one of zein and wheat prolamin.

[0014] The particle size of the Schizophyllum polysaccharide nanoparticles prepared by the present invention is in the range of 15 to 150 nm, and can be used to prepare an oral preparation for improving intestinal inflammation and / or repairing intestinal mucosa.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] 1. The present invention selects natural plant alcohol-soluble protein as raw material, adopts anti-solvent precipitation method to prepare plant protein nano-core suspension, and then mixes it with schizophyllan polysaccharide solution, and controls the parameters such as pH adjustment of the solution to form uniform and stable schizophyllan polysaccharide nanoparticles. The alcohol-soluble protein in the present invention is a common edible cereal protein, which has good biocompatibility and low price. The ethanol solution used in the anti-solvent precipitation method can be recycled by rotary evaporation, without other organic solvents, and the preparation process is convenient and green. At the same time, compared with schizophyllan polysaccharide used alone, the schizophyllan polysaccharide nanoparticles prepared by the present invention have more excellent effects on improving intestinal inflammation and repairing mucosal barrier damage, and the therapeutic effect of ulcerative colitis is better. At present, there is no report on schizophyllan polysaccharide nanoparticles in improving intestinal inflammation and repairing mucosal barrier damage, which has important development and utilization prospects.

[0017] 2. The Schizophyllum polysaccharide nanoparticles prepared by the present invention have a diameter range of 15 to 150 nm and are suitable for preparing oral preparations for improving or treating chronic intestinal inflammation caused by UC.

[0018] 3. The Schizophyllum polysaccharide nanoparticles prepared by the present invention have excellent effects on improving intestinal inflammation and mucosal repair: in vitro monolayer membrane model experiments show that it has a good alleviating effect on DSS-induced intestinal mechanical barrier damage, and effectively restores TEER values ​​and inflammatory factors IL-6 and TNF-α levels. At the same time, in vivo experiments have shown that it has a significant improvement effect on DSS-induced UC mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The particle size, PDI and Zeta charge characterization diagram of the Schizophyllum polysaccharide nanoparticles prepared in Example 1.

[0020] Figure 2 These are SEM images of the Schizophyllum polysaccharide nanoparticles prepared in Example 1, wherein (A), (B), and (C) correspond to the plant protein nanocore, SCP nanoparticles, and SCP-1 nanoparticles, respectively.

[0021] Figure 3 This is a test of the toxicity of Schizophyllum polysaccharide nanoparticles to Caco-2 cells.

[0022] Figure 4 The TEER value changes for the Caco-2 monolayer model (see Figure 4 (A) in the figure), standard curve of fluorescein sodium (see Figure 4 (B)) and absorption rate (see Figure 4 (C) in the.

[0023] Figure 5This is the effect of Schizophyllum polysaccharide nanoparticles on the TEER value of Caco-2 monolayer membrane under 2.5% DSS treatment.

[0024] Figure 6 The effect of Schizophyllum polysaccharide nanoparticles on the inflammatory factor IL-6 (see Figure 6 (A)) and TNF-α (see Figure 6 The influence of (B)).

[0025] Figure 7 The body weight of UC mice after SCP and SCP nanoparticle treatment (see Figure 7 (A)), colon length (see Figure 7 (B)), Disease Activity Index (DAI) (see Figure 7 (C)) and spleen weight (see Figure 7 (D) changes in the.

[0026] Figure 8 H&E staining images and histological scores, muscle layer thickness, crypt depth and number of mouse colon tissue (see Figure 8 (A)) and AB-PAS staining images and goblet cell numbers and positive areas (see Figure 8 (B) in the figure.

[0027] Fig. 9 is the inflammatory factor IL-6 in mouse serum (see Fig. 9 (A)), TNF-α (see Fig. 9 (B)), IL-1β (see Fig. 9 (C)), IL-10 content (see Fig. 9 (D)), IL-13 (see Fig. 9 (E)), IL-22 (see Fig. 9 (F)) determination.

[0028] Fig.10 Western blotting was used to determine the expression of Occludin and Claudin-1 proteins in colon tissue (see Fig.10 (A)) and quantitative analysis (see Fig.10 (B), (C)). DETAILED DESCRIPTION

[0029] The following examples may enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way.

[0030] The Schizophyllum polysaccharides used in the following examples are the crude Schizophyllum fruiting body polysaccharide (named SCP) prepared in Example 1 of patent CN 117343210A and the purified Schizophyllum fruiting body polysaccharide (named SCP-1).

[0031] Example 1

[0032] In this example, the following steps were used to prepare Schizophyllum polysaccharide nanoparticles:

[0033] (1) Prepare schizophyllan polysaccharide (SCP or SCP-1) with deionized water to a 5 mg / mL polysaccharide stock solution.

[0034] (2) Dissolve zein at a concentration of 2 g / mL in an ethanol solution with a volume concentration of 80%, and stir the mixture at a speed of 600 rpm to obtain a plant protein ethanol mixed solution.

[0035] (3) taking the plant protein ethanol mixed solution, adding deionized water under stirring at 600 rpm, and then continuing to stir at room temperature for 60 minutes to obtain a plant protein nanocore suspension; wherein the volume ratio of the plant protein ethanol mixed solution to the added deionized water is 1:4.

[0036] (4) After removing ethanol from the plant protein nanocore suspension obtained in step (3) by rotary evaporation at a heating temperature of 55° C., the polysaccharide mother solution prepared in step (1) was added thereto to make the concentration of schizophyllan in the system 0.2 mg / mL, and then the pH was adjusted to 4.0 by adding 0.1 M hydrochloric acid solution, and the mixture was continuously stirred at room temperature for 60 min at 600 rpm to obtain a dispersion of schizophyllan polysaccharide nanoparticles (the nanoparticles prepared using SCP and SCP-1 as raw materials were named SCP nanoparticles and SCP-1 nanoparticles, respectively).

[0037] The particle size, PDI and Zeta potential of the nanoparticles obtained in this example were characterized by a Malvern nanometer laser particle size meter. The results are as follows: Figure 1 As shown, it can be seen that the average particle size of the plant protein nanocores prepared by the antisolvent precipitation method is 55.36±4.87nm, the PDI value is 0.14, and the absolute value of the Zeta charge is 22.70mV. The average particle size of the SCP nanoparticles is 58.92±0.85nm, the PDI value is 0.19, and the absolute value of the Zeta charge is 14.67mV. The average particle size of the SCP-1 nanoparticles is 95.92±11.23nm, the PDI value is 0.17, and the absolute value of the Zeta charge is 18.60mV. Compared with the plant protein nanocores, the particle size of the schizophyllum polysaccharide nanoparticles is increased to a certain extent, but the particle size is less than 100nm, and the particle size is relatively uniform, so the schizophyllum polysaccharide nanoparticles can be selected for subsequent experiments.

[0038] Figure 2 These are SEM images of plant protein nanocores, SCP nanoparticles, and SCP-1 nanoparticles. It can be seen that the particle sizes of SCP nanoparticles and SCP-1 nanoparticles are uniform.

[0039] Example 2

[0040] In this example, the following steps were used to prepare Schizophyllum polysaccharide nanoparticles:

[0041] (1) Prepare schizophyllan polysaccharide (SCP or SCP-1) with deionized water to a 5 mg / mL polysaccharide stock solution.

[0042] (2) Dissolve wheat alcohol-soluble protein at a concentration of 2 g / mL in an ethanol solution with a volume concentration of 65%, and stir evenly at a speed of 500 rpm to obtain a plant protein ethanol mixed solution.

[0043] (3) taking the plant protein ethanol mixed solution, adding deionized water under stirring at 500 rpm, and then continuing to stir at room temperature for 30 minutes to obtain a plant protein nanocore suspension; wherein the volume ratio of the plant protein ethanol mixed solution to the added deionized water is 1:4.

[0044] (4) After removing ethanol from the plant protein nanocore suspension obtained in step (3) by rotary evaporation at a heating temperature of 55° C., the polysaccharide mother solution prepared in step (1) was added thereto to make the concentration of schizophyllan in the system 0.2 mg / mL, and then the pH was adjusted to 2.0 by adding 0.1 M hydrochloric acid solution, and the mixture was continuously stirred at room temperature for 120 min at 500 rpm to obtain a dispersion of schizophyllan polysaccharide nanoparticles (the nanoparticles prepared using SCP and SCP-1 as raw materials were named SCP nanoparticles and SCP-1 nanoparticles, respectively).

[0045] The particle size, PDI and Zeta potential of the nanoparticles obtained in this example were characterized by a Malvern nanometer laser particle size meter. The results showed that the average particle size of the plant protein nanocores prepared by the antisolvent precipitation method was 32.79 ± 0.62 nm, the PDI value was 0.19, and the absolute value of the Zeta charge was 23.57 mV. The average particle size of the SCP nanoparticles was 63.82 ± 4.76 nm, the PDI value was 0.27, and the absolute value of the Zeta charge was 17.63 mV. The average particle size of the SCP-1 nanoparticles was 105.07 ± 2.58 nm, the PDI value was 0.11, and the absolute value of the Zeta charge was 26.00 mV.

[0046] Example 3

[0047] In this example, the following steps were used to prepare Schizophyllum polysaccharide nanoparticles:

[0048] (1) Prepare schizophyllan polysaccharide (SCP or SCP-1) with deionized water to a 5 mg / mL polysaccharide stock solution.

[0049] (2) Dissolve wheat alcohol-soluble protein at a concentration of 2 g / mL in an ethanol solution with a volume concentration of 75%, and stir evenly at a speed of 800 rpm to obtain a plant protein ethanol mixed solution.

[0050] (3) taking the plant protein ethanol mixed solution, adding deionized water under stirring at 800 rpm, and then continuing to stir at room temperature for 30 minutes to obtain a plant protein nanocore suspension; wherein the volume ratio of the plant protein ethanol mixed solution to the added deionized water is 1:4.

[0051] (4) After removing ethanol from the plant protein nanocore suspension obtained in step (3) by rotary evaporation at a heating temperature of 55° C., the polysaccharide mother solution prepared in step (1) was added thereto to make the concentration of schizophyllan in the system 0.2 mg / mL, and then the pH was adjusted to 5.0 by adding 0.1 M hydrochloric acid solution, and the mixture was continuously stirred at room temperature for 30 min at 800 rpm to obtain a dispersion of schizophyllan polysaccharide nanoparticles (the nanoparticles prepared using SCP and SCP-1 as raw materials were named SCP nanoparticles and SCP-1 nanoparticles, respectively).

[0052] The particle size, PDI and Zeta potential of the nanoparticles obtained in this example were characterized by a Malvern nanometer laser particle size meter. The results showed that the average particle size of the plant protein nanocores prepared by the antisolvent precipitation method was 29.48 ± 0.89 nm, the PDI value was 0.24, and the absolute value of the Zeta charge was 25.8 mV. The average particle size of the SCP nanoparticles was 58.92 ± 0.86 nm, the PDI value was 0.19, and the absolute value of the Zeta charge was 28.10 mV. The average particle size of the SCP-1 nanoparticles was 118.80 ± 6.90 nm, the PDI value was 0.18, and the absolute value of the Zeta charge was 32.80 mV.

[0053] Example 4: In vitro monolayer film experiment of Schizophyllum polysaccharide nanoparticles

[0054] 1. Cytotoxicity assay

[0055] Caco-2 cells with good growth status were seeded in 96-well plates (1×10 4cells / well), cultured in a CO2 incubator at 37°C, and the MTT method was used to detect the effect of schizophyllan polysaccharide nanoparticles (the following experiments all used the samples prepared in Example 1) on cell survival rate. The blank culture medium was used as the control group (CTRL), and schizophyllan polysaccharide or schizophyllan polysaccharide nanoparticles (50 μg / mL) were added to the experimental group. After incubation for 12 hours, the cell supernatant was removed and washed twice with PBS. 100 μL MTT solution (0.5 mg / mL) was added to each well, and the culture was continued for 4 hours. After discarding the supernatant, 100 μL DMSO was added. After shaking on a shaker for 10 minutes under light-proof conditions, the absorbance at 570 nm was measured using an enzyme reader. The results of the toxic effects of schizophyllan polysaccharide and schizophyllan polysaccharide nanoparticles on Caco-2 cells are shown in the figure. Figure 3 As shown, Schizophyllan polysaccharide and Schizophyllan polysaccharide nanoparticles had no toxic effects on Caco-2 cells.

[0056] 2. Establishment of in vitro monolayer membrane model

[0057] 0.5 mL of Caco-2 cells with good growth status were inoculated into 12-well Transwell plates (1×10 5 / mL) was placed on the AP side of the culture chamber, and 1.5mL of complete culture medium was added to the BL side. The culture medium was changed every 48h in the first week, and then every day. The transmembrane resistance (TEER) was detected every 3 days using a cell transmembrane resistance meter in the first 2 weeks, and the TEER value was measured every day thereafter. The results are shown in Figure (A). On the 22nd day, 0.5mL of sodium fluorescein (20μg / mL) was added to the AP side, and 1.5mL of Hanks solution was added to the BL side. 200μL of the culture medium on the BL side was taken at 30, 60, 90, and 120min, respectively, to test the absorbance of the sodium fluorescein solution. Figure 4 As shown in (B), the standard curve of fluorescein sodium is y=0.03149x+0.5102(R 2 =0.996). Figure 4 As shown in (C), after adding sodium fluorescein and culturing for 120 min, the absorbance was only 0.79%, while the TEER values ​​of each well of the Transwell plate were greater than 550 Ω·cm 2 , proving that the integrity of the monolayer membrane is good and meets the requirements of transmembrane transport experiments.

[0058] The in vitro monolayer model was established in the same manner as above. On the 22nd day, 0.5 mL of 2.5% dextran sulfate sodium DSS solution was applied to the AP side for 24 h. Then, Schizophyllan or Schizophyllan nanoparticles (50 μg / mL) were applied to the AP side and cultured for 120 min. Figure 5 As shown in the figure, the TEER value of the monolayer membrane recovered to a certain extent, among which the SCP-1 nanoparticle group recovered the best.

[0059] 3. Cytokine determination

[0060] Under the condition that 2.5% DSS acted on the cell monolayer membrane for 24 hours, the cell monolayer was treated with Schizophyllum polysaccharide or Schizophyllum polysaccharide nanoparticles for 120 minutes, and the changes in IL-6 and TNF-α concentrations were detected by ELISA. According to the instructions of the kit, the samples and different concentrations of standards were added to the corresponding wells at 50μL / well, and 50μL of diluent was added to the blank group, followed by 100μL of enzyme labeling solution, covered with a sealing film and incubated at 37°C for 1h. After the incubation, the ELISA plate was taken out, the liquid was discarded, 1mL of washing solution was added to each well and allowed to stand for 30s, the washing solution was shaken off, and repeated 5 times. 50μL of biotinylated detection antibody solution and 50μL of enzyme conjugate working solution were added to each well. After incubation at 37°C in the dark for 15 minutes, 50μL of stop solution was added to each well, and the OD value of each well in the plate was measured at a wavelength of 450nm. The results are shown as follows. Figure 6 As shown. Compared with the blank group (CTRL) without DSS treatment, the concentrations of proinflammatory factors IL-6 and TNF-α increased after 2.5% DSS treatment. After administration of Schizophyllum polysaccharide or Schizophyllum polysaccharide nanoparticles, the concentrations of IL-6 and TNF-α decreased, among which the IL-6 concentration in the SCP nanoparticle group was only 34.33pg / mL, and the IL-6 concentration in the SCP-1 nanoparticle group was only 34.52pg / mL, which was significantly different from the DSS group.

[0061] Example 5: Effects of SCP nanoparticles on intestinal inflammation and mucosal barrier in UC mice

[0062] The UC mouse model was induced by 2.5% DSS to study the effects of SCP nanoparticles on intestinal inflammation and mucosal barrier in UC mice. 50 C57BL / 6 mice were divided into 5 groups (control group CTRL, DSS model group, mesalazine group, SCP group, SCP nanoparticle group). After one week of adaptive feeding of mice, the experiment began. The control group drank distilled water freely, the DSS model group and the experimental group drank 2.5% DSS freely, and the experimental group was gavaged with positive drug Mesalazine (100 mg / kg) or Schizophyllum polysaccharide (SCP, 100 mg / kg), Schizophyllum polysaccharide nanoparticles (SCP nanoparticles, 10 mg / kg).

[0063] 1. Determination of colitis-related indicators

[0064] During the experiment, the model establishment of mice was observed every day, and their coat color, activity, weight change, stool consistency and blood in stool were observed and recorded. On the 8th day, the mice were fasted for 12 hours, and the eyeballs were removed to collect blood. After the mice were killed, the required tissues were taken, and the colon length and spleen weight were measured. The disease activity index (DAI) was scored according to the standards in Table 1. The results are shown in Table 1. Figure 7 As shown, compared with the DSS group, the mice in the SCP nanoparticle group significantly alleviated the symptoms of decreased spleen weight and shortened colon, and the DAI score was improved, indicating that SCP nanoparticles play a certain role in alleviating the symptoms of UC mice.

[0065] Table 1

[0066]

[0067] 2. Alleviating effect of SCP nanoparticles on colon tissue damage in UC mice

[0068] The colon tissue was embedded in paraffin, and then sliced ​​and stained with H&E and AB-PAS. The tissue was observed and photographed under an inverted microscope, and the mucin and goblet cells in the tissue were evaluated according to Table 2. Figure 8 As shown in the results, the inflammatory state of the colon of UC mice treated with SCP nanoparticles and Mesalazine was alleviated at the histological level, the crypt structure was intact, the depth was uniform, the number was increased, the thickness of the crypt base was reduced, the infiltration of lymphocytes and plasma cells at the base was reduced, and the number of goblet cells was significantly increased; the H&E staining sections of colon tissue were scored based on the crypt structure, inflammatory infiltration and goblet cell number, and it was found that the scores were significantly reduced after treatment with SCP nanoparticles and Mesalazine. The number of goblet cells and mucin area in the Mesalazine and SCP nanoparticle groups were significantly higher than those in the DSS group (p<0.05). The results showed that SCP nanoparticle treatment can prevent the deterioration of UC by inhibiting mucus destruction and goblet cell depletion.

[0069] Table 2

[0070]

[0071]

[0072] 3. Effect of SCP nanoparticles on the levels of inflammatory factors in UC mice

[0073] ELISA was used to detect changes in the concentrations of IL-6, IL-1β, TNF-α, IL-22, IL-13, and IL-10 in mouse serum. According to the instructions of the kit, the samples and standards of different concentrations were added to the corresponding wells at 50 μL / well, and 50 μL of diluent was added to the blank group. Then 100 μL of enzyme labeling solution was added, and the plate was covered with a sealing film and incubated at 37°C for 1 hour. After the incubation, the ELISA plate was taken out, the liquid was discarded, 1 mL of washing solution was added to each well and allowed to stand for 30 seconds, the washing solution was shaken off, and repeated 5 times. 50 μL of biotinylated detection antibody solution and 50 μL of enzyme conjugate working solution were added to each well. After incubation at 37°C in the dark for 15 minutes, 50 μL of stop solution was added to each well, and the OD value of each well in the plate was measured at a wavelength of 450 nm. The results are as follows. Fig. 9 As shown, compared with the CTRL group, DSS promoted the release of pro-inflammatory factors and inhibited the release of anti-inflammatory factors, while the SCP nanoparticles and the positive control Mesalazine group effectively reversed this phenomenon, indicating that SCP nanoparticles have a certain anti-inflammatory effect.

[0074] 4. Western Blotting Analysis

[0075] Colon tissue was obtained, tissue protein was obtained by RIPA lysis buffer and grinding, protein was quantified using BCA kit, protein homogenate was mixed with loading buffer (5×), and electrophoresis samples were prepared. After electrophoresis, wet transfer, blocking and antibody incubation, the samples were observed by chemiluminescent gel imaging system. The results are shown in Fig.10 As shown in the figure, compared with the CTRL group, the expression of Occludin and Claudin-1 proteins in the DSS group was significantly reduced, and the intestinal mucosal barrier of mice was severely damaged. However, the SCP nanoparticles and the positive control Mesalazine group effectively increased the expression of tight junction proteins and repaired the intestinal mucosal barrier of mice with ulcerative colitis.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. All modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing Schizophyllum polysaccharide nanoparticles having the effects of improving intestinal inflammation and repairing mucosa, characterized in that: The schizophyllan nanoparticles are self-assembled from schizophyllan polysaccharide and plant protein nanocores.

2. The preparation method according to claim 1, characterized in that: The steps include: (1) preparing a polysaccharide mother solution by using deionized water to prepare a polysaccharide mother solution; (2) dissolving the plant protein at a concentration of 1 to 5 g / mL in an ethanol solution having a volume concentration of 65% to 85%, and stirring the mixture at a speed of 500 to 800 rpm to obtain a plant protein ethanol mixed solution; (3) taking the plant protein ethanol mixed solution, adding deionized water under stirring at 500-800 rpm, and then continuing to stir at room temperature for 30-120 min to obtain a plant protein nanocore suspension; wherein the volume ratio of the plant protein ethanol mixed solution to the added deionized water is 1:3-5; (4) After removing ethanol from the plant protein nanocore suspension obtained in step (3) by rotary evaporation, the polysaccharide mother solution prepared in step (1) is added thereto to make the concentration of Schizophyllum polysaccharide in the system be 0.1-1.0 mg / mL, and then the pH is adjusted to 2.0-5.0, and the mixture is continuously stirred at 500-800 rpm for 30-120 min to obtain a dispersion of Schizophyllum polysaccharide nanoparticles.

3. The preparation method according to claim 1 or 2, characterized in that: The Schizophyllum polysaccharide is a Schizophyllum fruiting body polysaccharide.

4. The preparation method according to claim 2, characterized in that: In step (1), the concentration of the polysaccharide mother solution is 5 to 10 mg / mL.

5. The preparation method according to claim 2, characterized in that: The plant protein is at least one of zein and wheat prolamin.

6. The preparation method according to claim 2, characterized in that: In step (4), the heating temperature for removing ethanol by rotary evaporation is 55 to 65°C.

7. The preparation method according to claim 2, characterized in that: In step (4), the pH is adjusted by adding 0.1-0.5 M hydrochloric acid solution.

8. Schizophyllan nanoparticles prepared by the preparation method according to any one of claims 1 to 7.

9. A use of the Schizophyllan polysaccharide nanoparticles according to claim 8, characterized in that: Used to prepare oral preparations for improving intestinal inflammation and / or repairing intestinal mucosa.

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