Novel radix paeoniae alba polysaccharide and application thereof

Abstract

The invention discloses a novel white paeony root polysaccharide for activating an intestinal Nrf2 signal channel and application, the molecular weight of the novel white paeony root polysaccharide is 8.104 * 10 < 3 > Da, and the structural formula is shown in Figure 1. The novel radix paeoniae alba polysaccharide obtained by the invention has the effect of activating an intestinal Nrf2 signal channel, can enhance an intestinal mucosal barrier and improve intestinal injury, and can be used for preparing medicines for preventing and treating Nrf2-mediated intestinal diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, in particular to novel white peony polysaccharide and its application in preparing an intestinal Nrf2 activator and preparing medicines for preventing and treating intestinal diseases mediated by Nrf2. Background Art

[0002] Nuclear factor erythroid 2 related factor 2 (Nrf2) is a key transcription factor. Under normal circumstances, it binds to Kelch-like ECH-associated protein 1 (Keap1), is inactive and degraded by ubiquitination, and can dissociate from Keap1 after activation, activating the cell's antioxidant adaptive response to cope with various injuries. Patients with intestinal diseases have significant oxidative stress in the intestine, and activation of the Nrf2 signaling pathway can reduce intestinal oxidative stress and protect intestinal tissue by activating antioxidant genes.

[0003] White peony root is a well-known Chinese herbal medicine, which comes from the dried root of Paeonia lactiflora Pall., a plant of the Ranunculaceae family. It has the effects of nourishing blood and regulating menstruation, restraining yin and stopping sweating, softening the liver and relieving pain, and calming liver yang. In traditional Chinese medicine, it can be used to treat spontaneous sweating, night sweats, yin deficiency fever, irregular menstruation, etc. At present, the research and application of white peony root mainly focuses on small molecular compounds such as paeoniflorin, while ignoring the large molecular polysaccharides of peony, resulting in a waste of resources. Summary of the invention

[0004] Based on the technical problems existing in the background technology, the present invention proposes a new type of white peony polysaccharide and its application in the preparation of intestinal Nrf2 activators and drugs for the prevention and treatment of Nrf2-mediated intestinal diseases, and obtains a white peony polysaccharide with a new structure, which can activate the intestinal Nrf2 signaling pathway, enhance the intestinal mucosal barrier, and improve intestinal damage.

[0005] The novel white peony polysaccharide proposed in the present invention has the following molecular structure formula:

[0006]

[0007] The molecular weight of the new white peony polysaccharide is 8.104×10 3 Da.

[0008] The present invention proposes the use of the novel white peony polysaccharide in the preparation of an intestinal Nrf2 activator.

[0009] The invention provides an application of the intestinal Nrf2 activator in the preparation of medicines for preventing and treating Nrf2-mediated intestinal diseases.

[0010] Beneficial technical effects of the present invention:

[0011] The novel uniform white peony polysaccharide obtained by the present invention can significantly activate the intestinal Nrf2 signaling pathway, increase the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), reduce the level of malondialdehyde (MDA), enhance the intestinal mucosal barrier, and improve intestinal damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is the molecular structural formula of the novel white peony polysaccharide proposed in the present invention;

[0013] Figure 2 The elution curve of the crude white peony polysaccharide proposed in the present invention passing through the primary column and the secondary column;

[0014] Figure 3 This is the molecular weight distribution diagram of the novel white peony polysaccharide proposed in the present invention;

[0015] Figure 4 This is the monosaccharide composition liquid phase diagram proposed by the present invention (A is the monosaccharide standard product diagram, B is the monosaccharide composition diagram of the sample);

[0016] Figure 5 The starch iodine test analysis diagram proposed by the present invention (A is a sample-iodine complex color comparison diagram, and B is a sample-iodine complex ultraviolet absorption curve);

[0017] Figure 6 This is the infrared spectrum of the novel white peony polysaccharide proposed in the present invention;

[0018] Figure 7 This is the ultraviolet spectrum of the novel white peony polysaccharide proposed in the present invention;

[0019] Figure 8 This is a nuclear magnetic resonance spectrum (NMR) analysis diagram of the novel white peony polysaccharide proposed in the present invention (A is 1 HNMR; B is 13 CNMR spectrum; C is 1 H– 1 H-COSY; D is HSQC; E is HMBC);

[0020] Fig. 9 The novel white peony polysaccharide proposed in the present invention improves the intestinal damage of mice induced by dextran sulfate sodium (DSS) (A is a cross-sectional view of colon tissue; B is a morphological score; C is hematoxylin and eosin (H&E) staining of colon sections; D is PAS staining of colon sections; E is colon tissue pathology score; F is the number of goblet cells in each crypt of the colon);

[0021] Fig.10The novel white peony polysaccharide proposed in the present invention enhances the intestinal mucosal barrier of dextran sodium sulfate (DSS) model mice (A, B, C are protein immunoblotting (Western blot) to detect the protein expression levels of membrane integrin (Occludin) and tight junction protein (ZO-1); D and E are immunofluorescence detection of membrane integrin (Occludin) protein expression);

[0022] Fig.11 This is a graph showing that the novel white peony polysaccharide proposed in the present invention reduces intestinal oxidative stress in dextran sodium sulfate (DSS) model mice;

[0023] Fig.12 This is a diagram showing that the novel white peony polysaccharide proposed in the present invention activates the intestinal Nrf2 signaling pathway of dextran sulfate sodium (DSS) modeled mice (A is the Nrf2 protein expression level; B is the Keap-1 protein expression level). DETAILED DESCRIPTION

[0024] The present invention will be further explained below in conjunction with specific embodiments.

[0025] Example 1

[0026] The polysaccharide of white peony was extracted by water extraction and alcohol precipitation. 500g of white peony powder was weighed and placed in a 5L beaker. 85% ethanol was added and allowed to stand at room temperature for 10 hours for degreasing and decolorization. The ethanol was removed by vacuum filtration. The precipitate was collected and repeatedly added with 85% ethanol. After three times, the precipitate was dried, 5L distilled water was added, the solid-liquid ratio was 1:10, and the extraction was carried out at 85°C for 2 hours. Repeated 3 times. After the extracts were combined, they were concentrated by vacuum distillation until viscous. The rotary evaporator pressure was 60pa, the water bath temperature was 60°C, the speed was 40r per minute, and tap water was condensed. After the extract was concentrated, it was placed in a 5L beaker, 4 times the volume of anhydrous ethanol was added, and the precipitation was precipitated for 24 hours after stirring. After centrifugation, the precipitate was dried to obtain crude polysaccharide. The crude polysaccharide of white peony was redissolved in distilled water, and amylase was added. 50μL amylase was added to each 1L polysaccharide solution. After reacting at 60°C for 1 hour, it was inactivated in a boiling water bath for 10 minutes. After centrifugation at 8000 r / min for 5 minutes, the supernatant was frozen to remove protein, and then centrifuged (8000 r / min for 5 minutes) after thawing to discard the precipitate. The mixture was frozen and thawed repeatedly until no white precipitate was produced, and 51.2903 g was obtained by freeze-drying.

[0027] Weigh crude white peony polysaccharide and dissolve it in distilled water to prepare a solution with a mass concentration of 50 mg / ml. Centrifuge at a speed of 8000 r / min for 5 minutes to obtain the supernatant, pour the supernatant into a diethylaminoethyl-52 (DEAE-52) cellulose anion exchange column, and elute with distilled water at a flow rate of 5 ml / min, 1 minute per tube, 100 tubes, and determine the elution curve by the phenol-sulfuric acid method ( Figure 2The sugar-enriched eluate was dialyzed using a 3500Da dialysis bag with running water, tap water for 12 hours, and distilled water for 12 hours, then concentrated to a slightly viscous state and freeze-dried.

[0028] The freeze-dried polysaccharide obtained in the previous step was weighed and dissolved in distilled water to prepare a solution with a mass concentration of 50 mg / ml. The solution was centrifuged at a speed of 8000 r / min for 5 minutes to obtain the supernatant, which was poured into a Sephadex G-75 gel column and eluted with distilled water at a flow rate of 0.2 ml / min. Each tube was eluted for 10 minutes. 100 tubes were eluted and the elution curve was determined by the phenol-sulfuric acid method ( Figure 2 The sugar-containing eluate with a higher yield was collected, and the sample was loaded and eluted multiple times. After enrichment, it was concentrated and freeze-dried, weighing 20.67 g.

[0029] Example 2

[0030] 1) Homogeneity and molecular weight determination

[0031] High performance size exclusion chromatography-multi-angle light scattering-refractive index detector (HPSEC-MALLS-RID) was used.

[0032] Sample preparation: Weigh the white peony polysaccharide prepared in Example 1, and prepare a solution with a mass concentration of 5 mg / mL with 0.1 mol / L sodium nitrate, filter through a 0.22 μm filter membrane, and set aside.

[0033] Instrument model: Waters liquid phase system, connected in series with Waters 2414 differential detector and Brookhaven BI-MwA laser scattering detector. Experimental conditions: TSK G5000 PWxl (7.8x300 mm) and TSK 4500PWxl (7.8x300 mm) columns in series, eluted with 0.1 mol / L sodium nitrate solution, flow rate 0.5 mL / min, column temperature 40°C, injection volume 100 μL.

[0034] The data were collected, analyzed and calculated by ParSEC Chromatography software. The molecular weight curve of the sample was as follows: Figure 3 As shown in the figure, the dotted line represents the multi-angle laser light scattering signal, and the solid line represents the difference signal. After testing, the molecular weight distribution of the purified white peony polysaccharide was uniform, with a molecular weight of 8.104×10 3 Da.

[0035] 2) Monosaccharide composition

[0036] The monosaccharide composition of the novel uniform white peony polysaccharide was determined by 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization method.

[0037] Determination method: Weigh a certain amount of sample, 8 monosaccharide standards, and internal standard (glucosamine hydrochloride) to prepare a solution with a mass concentration of 6 mg / ml. Sample hydrolysis: Pipette 1 ml of sample solution into an ampoule, add 0.5 ml of 3 mol / L hydrochloric acid, seal with an alcohol burner, react in an oven at 110°C for 1 hour, and then add 3 mol / L sodium hydroxide solution until the pH value reaches 7. Derivatization: Pipette 0.4 ml of monosaccharide standard, internal standard and hydrolyzed sample into test tubes respectively, use deionized water as blank, add 0.4 ml of 0.3 mol / L sodium hydroxide solution and 0.4 ml of 0.5 mol / L PMP-methanol solution into each test tube, incubate in a 70°C water bath for 100 minutes, add 0.5 ml of 0.3 mol / L hydrochloric acid after cooling, mix well, add 2 ml of chloroform, vortex for 30 seconds, then aspirate the chloroform in the lower layer, repeat 3 times, centrifuge at 8000 r / min for 5 minutes, take the supernatant, filter through a 0.22 μm filter membrane and set aside.

[0038] Chromatographic parameters: mobile phase is acetonitrile: phosphate buffer (pH adjusted to 6.8 with 2 mol / L sodium hydroxide solution) = 82:18; chromatographic column: C18 column (250 mm × 4.6 mm, l) with octadecylsilane bonded silica as filler; mobile phase flow rate: 1 mL / min; detection wavelength: 250 nm; injection volume: 20 μL.

[0039] like Figure 4 As shown, Figure 4 The A part of the graph represents the peak order of monosaccharide standards, which are mannose (Man), internal standard, rhamnose (Rha), ribose (Rib), glucuronic acid (GlcA), galacturonic acid (GlaA), glucose (Glc), galactose (Gal), and arabinose (Ara). The red line represents the sample. Figure 4 From the B part, it can be seen that the novel uniform white peony root polysaccharide is a glucan.

[0040] 3) Starch iodine test

[0041] A 1 mL aqueous solution containing 2 mg iodine, 10 mg potassium iodide and 1 mg of novel uniform white peony polysaccharide or starch was heated to 90 ° C and diluted to 25 mL. The absorption curve of the sample-iodine complex was then measured in the range of 400 to 800 nm using a UV-visible spectrophotometer (Thermo Scientific). The dyeing principle between potassium iodide and starch is due to the redox reaction of iodine ions with hydroxyl groups in starch molecules, and the formation of a dark blue-purple iodized starch complex. The energy level spacing in the resulting complex corresponds to the absorption spectrum in the visible light region. The intensity of the resulting blue depends on the content of amylose. Waxy starch with little or no amylose will turn red.

[0042] like Figure 5 As shown, the negative reaction of the sample with iodine-potassium iodide reagent indicated that the novel uniform white peony root polysaccharide was a non-starch polysaccharide.

[0043] 4) Infrared spectroscopy analysis

[0044] Weigh 2 mg of the dried new uniform white peony polysaccharide powder, mix it thoroughly with a small amount of potassium bromide, grind it, press it into tablets, and place it on a Fourier infrared spectrometer scanner at 4000-400 cm -1 Scanning analysis was performed within a range of wavenumbers.

[0045] The analysis results are as follows Figure 6 As shown, at 3369cm -1 A broad and intense peak is shown near 2930cm, which is due to the stretching vibration of hydroxyl groups. -1 The band in the region is the characteristic absorption of CH stretching vibration. 1080-1242cm -1 The peaks at indicate the presence of COC and CO linkages.

[0046] 5) Protein and nucleic acid content determination

[0047] Weigh the dried new uniform white peony polysaccharide sample into a test tube, add deionized water to prepare a test solution with a mass concentration of 1 mg / mL, and scan it in the wavelength range of 190-400 nm on a UV-visible spectrophotometer (Thermo Scientific) at room temperature. The scanning results are as follows: Figure 7 As shown, there are no obvious absorption peaks at 260 nm and 280 nm, which means that there are minimal nucleic acids and proteins in the sample.

[0048] 6) Methylation reaction

[0049] 20 mg of the new uniform white peony polysaccharide sample was dissolved in 4 ml of anhydrous dimethyl sulfoxide (DMSO) under a nitrogen atmosphere, and 200 mg of fresh powdered sodium hydroxide was added. After ultrasonication for 30 minutes, the mixture was shaken for 30 minutes. 1 ml of methyl iodide was added, and the mixture was shaken for 60 minutes under nitrogen protection. After cooling in an ice bath, 1 ml of methyl iodide was added repeatedly, and the mixture was shaken for 60 minutes under nitrogen protection. 1 ml of water was added to stop the reaction, and 2 ml of chloroform was added to mix well and wait for stratification. The chloroform layer was aspirated, and chloroform was continued to be added to the remaining water layer to mix well. After stratification, the chloroform layer was aspirated. After the two chloroform layers were mixed, the mixture was centrifuged at a speed of 10,000 r / min for 10 minutes, the upper water layer was aspirated, and 1 g of anhydrous sodium sulfate was added to completely remove the remaining water and then spin-dried.

[0050] After the sample was dried, 2 ml of 2M trifluoroacetic acid (TFA) was added to dissolve it, and it was transferred to a screw cap tube, heated at 120 ° C for 2 hours, and then dried after cooling. 2 mL of water was added to dissolve it, and then 20 mg of sodium borodeuteride was added, incubated at room temperature for 2 hours, shaken overnight, 4 M acetic acid was added until the pH value was 6 to destroy the excess reducing agent, and then 2 ml of methanol was added, and it was dried and repeated 4 times. 2 ml of pyridine-acetic anhydride (1:1) was added, and it was transferred to a screw cap tube, heated at 120 ° C for 2 hours, and then dried after returning to room temperature, and 2 ml of dichloromethane was added to dissolve it again, and centrifuged at a speed of 10000 r per minute for 10 minutes, the supernatant was dried, and then 2 ml of dichloromethane was added to dissolve it, and then 2 ml of dichloromethane was added to dissolve it, and it was filtered through a 0.22 μm filter membrane for use.

[0051] Detection conditions: Agilent GC-MS, loaded with HP-5 capillary column (30mm×0.32mm×0.25μm), initial temperature 80℃, increased to 170℃ at a rate of 20℃ per minute, maintained for 1 minute, increased to 180℃ at a rate of 3℃ per minute, maintained for 5 minutes, increased to 280℃ at a rate of 20℃ per minute, maintained for 2 minutes. He flow rate 1ml / min, ion source temperature 200℃. The glycosidic bond analysis results of the new uniform white peony polysaccharide are shown in Table 1.

[0052] Table 1 Glycosidic bond analysis of the new uniform white peony polysaccharide

[0053]

[0054] 7) Nuclear magnetic resonance spectroscopy analysis

[0055] Weigh 50 mg of the dried sample, dissolve it in 0.5 mL of heavy water as solvent and freeze-dry it, repeat 3 times, and analyze it with 1H NMR spectrum, 13C NMR spectrum 35 and 2D NMR spectrum (1H–1H-COSY, HSQC, HMBC spectrum) on a VNMRS600 spectrometer. Use a 5 mm Z gradient field multi-nuclear formal probe and a 5 mm Z gradient field triple resonance trans probe. Dextran sodium sulfate is used as an internal reference.

[0056] The results are as follows Figure 8 As shown in Part A, 1 In the H nuclear magnetic resonance spectrum (NMR), the anomeric hydrogens with chemical shifts of 5.38, 5.35, 4.94, 4.63, 5.22, and 4.66 ppm in the novel homogeneous white peony polysaccharide were labeled as H-1 of sugar residues A, B, C, D, E, and F, respectively. The cross-peak signal of C / H in HSQC showed that the C-1 of A, B, C, D, E, and F were 102.23, 102.37, 101.18, 106.80, 94.50, and 98.37 ppm, respectively.

[0057] For residue A with the strongest signal, due to the cross peaks of H-1 / H-2, H-2 / H-3, H-3 / H-4, H-5 / H-6 and H-5 / H-6' in the COSY spectrum, the chemical shifts of H-2, H-3, H-4, H-5, H-6 and H-6' were 3.61, 3.95, 3.63, 3.82, 3.84 and 3.74 ppm, respectively. Then, based on the C / H correlation in the HSQC spectrum, the peaks around 74.3, 75.82, 79.39, 73.67 and 63.09 ppm were attributed to the C-2, C-3, C-4, C-5 and C-6 of residue A. 13 C resonance and the downfield chemical shift of C-4 at 79.39 ppm and 72.72 ppm confirmed the existence of a (1→4) bond, therefore, based on the methylation results and literature, residue A was assigned to (1→4)-α-Glcp-(1→

[0058] For residue B, the chemical shift of H-2 was 3.58 ppm due to the H-1 / H-2 correlation in the COSY spectrum, and the chemical shift of C-2 was deduced to be 73.86 ppm based on the C2 / H2 correlation in the HSQC spectrum. The chemical shifts of H-3, H-4, H-5, H-6 and C-3, C-4, C-5 and C-6 were fully assigned in the COSY and HSQC spectra, respectively. Due to the downfield shifts of C-4 (75.27 ppm) and C-6 (71.95 ppm), residue B was assigned to →4)-6-α-Glcp-(1→

[0059] For residue C, H2 / C2 (3.58 / 74.3 ppm), H3 / C3 (3.69 / 75.36 ppm), H4 / C4 (3.4 / 71.8 ppm), H5 / C5 (3.94 / 75.98 ppm), and H6 / C6 (3.92 / 63.09 ppm) deduced in the HSQC and COSY spectra were assigned to α-Glcp-(1→

[0060] For residue D, its chemical shift at 4.63 / 106.8 ppm in the HSQC spectrum indicated that it was a β-glucose residue. Based on the chemical shifts of H2 / C2, H3 / C3, H4 / C4, H5 / C5 and H6 / C6 deduced from the HSQC spectrum and COSY spectrum, residue D was assigned as →6)β-Glcp-(1→ due to the downfield chemical shift of C-6 at 72.57.

[0061] For residue E, the cross peak at 3.65 / 79.54 ppm in the HSQC spectrum indicated a downfield shift of C-4 (79.54 ppm) in residue E, and E was assigned to be α-D-Glcp-(4→

[0062] For residue F, its chemical shift at 4.66 / 98.37 ppm in the HSQC spectrum indicated that it was a β-glucose residue. The chemical shifts of H2 / C2, H3 / C3, H4 / C4, H5 / C5, and H6 / C6 were deduced from the HSQC spectrum and COSY spectrum. Considering all the chemical signals and combining with the methylation analysis, it can be inferred that the F residue is β-Glcp-(1→.

[0063] Table 2 New uniform white peony polysaccharide 1 H and 13 C chemical shift assignment

[0064]

[0065]

[0066] In the HMBC spectrum, the presence of cross peaks AH1-AC4, AH1-BC4, and BH1-AC4 indicated that the main chain of PRAP-W consisted of →4)-αGlcp-(1→4,6)-α-Glcp-(1→4), the cross peak FH1-BC4 indicated that residue F and residue B together constituted the 1-terminal of the main chain, and the cross peak CH1-BC6 indicated that residue C was linked to O-6 of residue B via a (1→6) bond. The cross peaks AH1-DC6 and DH1-EC4 indicated that residue D was linked to residue A via a (1→6) bond and to the reducing end via a (1→4)-α-Glcp.

[0067] The molecular structure of the new white peony polysaccharide is as follows Figure 1 shown.

[0068] Example 3

[0069] 1) Establishment of mouse model and grouping and drug administration

[0070] After one week of adaptive feeding, 50 7-week-old C57BL / 6 mice were randomly divided into 5 groups (10 mice in each group): blank group, model group, positive drug sulfasalazine group (100 mg / kg / d), new uniform white peony polysaccharide low-dose group (40 mg / kg / d), and new uniform white peony polysaccharide high-dose group (100 mg / kg / d), and marked.

[0071] From the 1st day to the 10th day, mice in the positive drug group, the new uniform white peony polysaccharide low-dose group, and the new uniform white peony polysaccharide high-dose group were gavaged with different concentrations of drugs once a day, and the model group and the blank group were given equal doses of normal saline, with a gavage volume of 10 μL / g. From the 4th day to the 10th day, the blank group had free access to water, and the other four groups were fed with 3% dextran sulfate sodium (w / v) aqueous solution.

[0072] After 10 days of treatment, the mice were euthanized by carbon dioxide and tested for relevant indicators. The colon was removed after the mice were dissected, rinsed with phosphate buffered saline (PBS), quickly frozen in liquid nitrogen, and stored at -80°C for later use.

[0073] 2) The novel uniform white peony polysaccharide improved the intestinal damage induced by dextran sulfate sodium (DSS) in mice

[0074] A portion of the distal colon of mice was longitudinally cut open and flattened, and the morphology of the colon section was observed and scored using a double-blind protocol.

[0075] 1 cm colon tissue of each group of mice was randomly collected and fixed in 4% paraformaldehyde (pH 7.0) for 24 hours. The fixed samples were then dehydrated, waxed, embedded, cut into 6 μm slices on a microtome, and placed on glass slides for air drying.

[0076] Hematoxylin & eosin (H&E) staining: paraffin sections were dewaxed with xylene for 10 minutes, repeated twice, and then rehydrated with ethanol from high to low concentrations, 100%-70% for 5 minutes each, soaked in distilled water for 5 minutes after rehydration, stained with hematoxylin for 10 minutes, rinsed with running water for 10 minutes to remove the remaining color, washed twice with distilled water, stained with eosin for 30 seconds, and immediately rinsed in 70% ethanol twice, each time for 10 seconds, then soaked in ethanol from low to high concentrations for dehydration, each time for 10 seconds, put in xylene for 5 minutes to make it transparent, and then sealed with neutral resin after drying. Observed and photographed under an optical microscope, and 8 different areas were selected for each section in each group to observe the structural morphology of intestinal mucosa.

[0077] Periodic acid-Schiff (PAS) staining: paraffin sections were dewaxed with xylene, rehydrated with gradient ethanol, rinsed with distilled water, stained with periodic acid for 10 minutes, rinsed with distilled water for 10 minutes, stained with Schiff's reagent at 37°C for 40 minutes, rinsed with running water for 5 minutes, stained with hematoxylin for 30 seconds, rinsed with running water for 2 minutes, dehydrated, transparent, and then dried and mounted. Observed and photographed under an optical microscope, 6 complete intestinal glands were selected for each section of each group, and the number of goblet cells per unit length on the intestinal glands was counted.

[0078] like Fig. 9 As shown in part A, the colon mucosa of mice modeled with dextran sulfate sodium (DSS) had a rough and uneven appearance, showing fine granules and accompanied by reddish-brown bleeding and mucosal shedding. The symptoms were significantly alleviated after drug intervention, and the high-dose group of the new uniform white peony polysaccharide had the mildest symptoms.

[0079] according to Fig. 9 From the results of Part B, it can be seen that the morphological scores of mice in the dextran sodium sulfate (DSS) modeling group were significantly increased, while the morphological scores of the drug-treated group were lower.

[0080] Depend on Fig. 9 From the C part, we can see that compared with the blank group, the tight junctions of the colon in the dextran sulfate sodium (DSS) modeling group mice were destroyed, the intercellular spaces became larger, the mucosal damage of the model group mice was severe, a large number of neutrophils infiltrated, and the crypts were severely deformed and atrophied, while the symptoms of the drug-treated group were milder.

[0081] like Fig. 9 As shown in part D, the model group mice lost a large number of colon goblet cells, and the goblet cell loss was less after drug intervention, proving that the new uniform white peony polysaccharide can significantly improve the symptoms of colon goblet cell loss caused by dextran sulfate sodium (DSS) modeling.

[0082] Conclusion: The new uniform white peony polysaccharide can significantly improve intestinal damage induced by dextran sulfate sodium (DSS) in mice.

[0083] 3) Enhanced the intestinal mucosal barrier of dextran sulfate sodium (DSS) model mice

[0084] Weigh 100 mg of colon tissue frozen at -80°C, chop it up thoroughly, place it in a homogenizer, add 1 mL of pre-cooled protein lysis buffer, grind it thoroughly, and homogenize it. Centrifuge the homogenate at low temperature, take the supernatant, add the sample buffer after testing the protein concentration by the kit, boil it at 100°C for 10 minutes, and store it at -80°C for use, and use it for Western blot to detect the expression of membrane integral protein (Occludin) and tight junction protein (ZO-1).

[0085] Immunofluorescence was used to detect the expression level of colon tissue membrane integrin (Occludin), apoptosis and proliferation of colon epithelial cells. Paraffin sections were dewaxed with xylene, rehydrated with gradient ethanol, and rinsed with distilled water. The tissue sections were placed in a repair box filled with ethylenediaminetetraacetic acid (EDTA) antigen repair buffer (pH 8.0) to repair antigens; then, they were blocked, and 3% bovine serum albumin (BSA) was dripped into the tissue circle to evenly cover the tissue, and blocked at room temperature for 30 minutes. The corresponding primary antibody was then dripped into each well and incubated in a 4℃ wet box overnight. The next day, the sections were washed three times with distilled water, fluorescent secondary antibodies were added, incubated at room temperature for 1 hour, and 4',6-diamidino-2-phenylindole (DAPI) staining solution was then added. After staining, the sections were sealed with anti-fluorescence quenching sealing agents. The sections were placed in a scanner to collect images. The cell nuclei stained with 4',6-diamidino-2-phenylindole (DAPI) were blue under ultraviolet excitation, and positive expression was red light labeled with the corresponding fluorescent marker.

[0086] like Fig.10 As shown in the data, the expression levels of membrane integrin (Occludin) and tight junction protein (ZO-1) in the colon of mice in the modeling group were significantly reduced, but the drug-treated group significantly improved the phenomenon of decreased expression levels of intestinal tight junction proteins, indicating that the new uniform white peony polysaccharide can improve the intestinal barrier damage caused by dextran sulfate sodium (DSS) modeling and enhance the intestinal mucosal barrier.

[0087] 4) Reduced intestinal oxidative stress in dextran sodium sulfate (DSS) model mice

[0088] Colonic tissue homogenate was prepared according to the instructions of the kit, and the protein concentration was detected by the kit. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), and the level of malondialdehyde (MDA) in the supernatant of colonic tissue of each group were determined by microplate reader.

[0089] Depend on Fig.11 It can be seen that compared with the blank group, the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) in the colon mucosal tissue of the modeling mice were reduced, and the level of malondialdehyde (MDA) was increased, while the drug-treated group could effectively prevent the excessive oxidative stress response of the intestinal mucosa of dextran sulfate sodium (DSS) modeling mice, among which the high-dose group of the new uniform white peony polysaccharide had the most significant effect, indicating that the effect of the new uniform white peony polysaccharide in improving intestinal damage may be related to reducing the level of intestinal oxidative stress.

[0090] 5) Activated the intestinal Nrf2 signaling pathway in dextran sulfate sodium (DSS) model mice

[0091] The colon tissue protein extract was prepared according to the above method, and the protein concentration was detected by a kit. The levels of Nrf2 and Keap-1 in the protein were detected by Western blot.

[0092] like Fig.12 As shown, compared with the blank group, the Nrf2 level in the modeling group was significantly downregulated, and the Keap-1 level was significantly upregulated, indicating that the antioxidant signaling pathway in the modeling group was inhibited. Compared with the model group, the situation in the drug-treated group was significantly improved, indicating that the new uniform white peony polysaccharide can activate the intestinal Nrf2 signaling pathway.

[0093] Although the embodiments of the present application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present application. The scope of the present application is defined by the appended claims and their equivalents, and all shall be included in the scope of protection of the present application.

Claims

1. A novel white peony polysaccharide, characterized in that: The molecular structure is as follows: The molecular weight of the new white peony polysaccharide is 8.104×10 3 Da.

2. Use of the novel white peony polysaccharide as claimed in claim 1 in the preparation of intestinal Nrf2 activator.

3. Use of the intestinal Nrf2 activator according to claim 2 in the preparation of drugs for preventing and treating Nrf2-mediated intestinal diseases.

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