Asterias amurensis glucan as well as preparation method and application thereof

Abstract

The invention discloses asterias amurensis glucan as well as a preparation method and application thereof, and belongs to the technical field of biological medicine, the asterias amurensis glucan is obtained by combining an enzymolysis method with anion exchange column chromatography and gel column chromatography purification, the main chain structure of the asterias amurensis glucan is 1, 4-linked alpha-D-glucose, and the main chain structure of the asterias amurensis glucan is 1, 4-linked alpha-D-glucose. A terminal alpha-D-glucose branch chain is arranged at the O-6 position of a main chain, and meanwhile, a small amount of mannose and galactose are contained. Moreover, it is proved for the first time that the asterias amurensis glucan can serve as an intestinal barrier protective agent, and oxidative damage, caused by H2O2, of intestinal epithelial cells is improved by up-regulating an Nrf2 signal channel and tight junction protein. Therefore, the asterias amurensis glucan prepared by the invention has a huge potential of being developed into medicines and health-care products for protecting intestinal tracts.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to Achyranthes polysaccharide and a preparation method and application thereof. Background Art

[0002] The intestine is the body's first line of defense. It can not only selectively absorb nutrients, but also resist the invasion of toxins and microorganisms in the intestine. Intestinal epithelial cell damage is a major pathological feature of various chronic diseases (such as inflammatory bowel disease, metabolic syndrome, etc.), mainly manifested as epithelial cell barrier destruction, oxidative stress damage, etc. Damaged intestinal tissue is accompanied by neutrophil and T lymphocyte infiltration, epithelial cell necrosis and ulcers. Defects in the barrier function of epithelial cells and changes in mucus secretion can lead to increased intestinal permeability; at the same time, the mucosal immune system is exposed to intestinal contents, which can lead to excessive production of proinflammatory factors, increase inflammation and oxidative stress response. Therefore, finding compounds that can resist oxidative stress and improve intestinal epithelial barrier function damage is of great significance for the treatment and relief of inflammatory bowel disease and metabolic syndrome.

[0003] Active polysaccharides from natural resources such as fungi, animals and plants are a class of low-toxic and highly effective natural antioxidants that have significant effects on protecting the intestinal barrier and resisting oxidative stress. For example, hemp seed polysaccharides can protect intestinal epithelial cells from hydrogen peroxide-induced oxidative stress. Chitosan can alleviate ulcerative colitis in mice by enhancing intestinal barrier function. Discovering new active polysaccharides with intestinal protective effects has important clinical significance for the prevention and treatment of diseases such as inflammatory bowel disease and metabolic syndrome.

[0004] Asterias are common sea star species along the coast of my country, commonly known as sea stars or star fish. Asterias amurensis is a representative sea star that is abundant in Qingdao. As a marine Chinese medicine, it has the effects of relieving pain, calming the liver and soothing the stomach. Polysaccharides are one of the main active ingredients of Chinese medicine sea stars. Literature has reported that it has significant immunomodulatory and neuroprotective effects, and can resist alcoholic fatty liver damage in mice, and has good development and application prospects. In addition, as a carnivorous animal, sea stars are very harmful to the coastal shellfish farming industry such as scallops, oysters, mussels, and abalone, causing huge economic losses to the marine aquaculture industry every year. The discovery of sea star polysaccharides can turn waste into treasure, improve the utilization rate of sea star resources, and have both economic and social benefits.

[0005] At present, there are still few reports on the extraction method and structural characteristics of Achyranthes polyspinus visceral glucan, and there are no reports on its role in protecting intestinal barrier function and anti-oxidative stress. Summary of the invention

[0006] The purpose of the present invention is to provide a kind of Achyranthes polyspinus glucan and its preparation method and application. The Achyranthes polyspinus glucan has the function of protecting the intestinal barrier.

[0007] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions:

[0008] The present invention provides a polysaccharide glucan, the main chain structure of which is 1,4-linked α-D-glucose, and there is a terminal α-D-glucose branch at the O-6 position of the main chain, and the branch also contains mannose and galactose. The structural formula of the polysaccharide glucan is specifically:

[0009] .

[0010] Furthermore, the monosaccharide composition of the Achyranthes polysaccharide glucan is glucose, mannose, galacturonic acid, glucuronic acid, arabinose, and galactose, and the molar ratios thereof are 89.2:5.0:1.5:1.6:1.5:1.2, respectively.

[0011] The present invention also provides a method for preparing the Achyranthes polysaccharide, which comprises the following steps:

[0012] (1) drying, crushing and sieving the viscera of the Polygonum multicornucopia to obtain viscera powder;

[0013] (2) After defatting the viscera powder, heat and stir in an alkaline solution overnight; after stirring, adjust the pH to 6-7 with a strong acid solution;

[0014] (3) adding papain to the solution of step (2) for enzymatic hydrolysis, removing protein with trichloroacetic acid after centrifugation, and then precipitating crude polysaccharides with anhydrous ethanol;

[0015] (4) dialyzing the crude polysaccharide and then freeze-drying it to obtain freeze-dried polysaccharide;

[0016] (5) separating and eluting the freeze-dried polysaccharide through anion exchange column chromatography, collecting the eluate to obtain a water-wash component;

[0017] (6) The water-washed fraction is purified by gel column chromatography, concentrated, and freeze-dried to obtain the polysaccharide.

[0018] Furthermore, in step (2), methanol is used for degreasing; the alkaline solution is 0.1 mol / L NaOH; the strong acid solution is concentrated hydrochloric acid; and the heating temperature is 60°C-80°C.

[0019] Furthermore, in step (3), the mass volume ratio of the amount of papain added to the volume of the solution is 1%-3%, and the enzyme activity of papain is 100,000 U / g; the amount of trichloroacetic acid used is 4%-7% of the volume of the supernatant collected after centrifugation.

[0020] Furthermore, the unit of the added amount of papain is g, and the unit of the solution volume is mL.

[0021] Furthermore, in the dialysis in step (4), the molecular weight cutoff of the dialysis bag is 3.5 kDa; and the dialysis time is 36 h-48 h.

[0022] Furthermore, in the step (5), the Aesculus truncatula dextran is eluted with 0, 0.2, and 0.5 mol / L NaCl, and the water-washed fraction is collected.

[0023] Furthermore, the purified eluent in step (6) is pure water; the flow rate of the eluent is 0.3 mL / min.

[0024] Furthermore, in the step (1), the viscera of the Polygonum multispinum are dried at 60°C-80°C and sieved with a mesh size of 40.

[0025] The present invention also provides the use of the Achyranthes polyspinus glucan in the preparation of medicines or health products for protecting the intestinal barrier.

[0026] Furthermore, the medicine or health product contains 12.5 μg / mL-200 μg / mL of Achyranthes polysaccharide.

[0027] Compared with the prior art, the beneficial effects and advantages of the present invention are:

[0028] The raw material used in the present invention is the Sea Star, which is a marine animal with a wide range of sources. It is harmful to the aquaculture industry, can be turned into treasure, and the utilization value and industrial application of starfish can be improved. The Sea Star glucan described in the present invention is extracted and purified by enzymatic hydrolysis, and combined with methylation analysis, nuclear magnetic resonance and other technologies to prove that there is 1,4-linked α-D-glucose in the Sea Star glucan, there is a terminal α-D-glucose branch at the O-6 position of the main chain, and there are a small amount of mannose and galactose structures. In addition, the present invention confirms for the first time that the Sea Star glucan can be used as an intestinal barrier protector, by upregulating the Nrf2 signaling pathway and tight junction proteins, to improve the oxidative damage of intestinal epithelial cells caused by H2O2. Therefore, the Sea Star glucan prepared by the present invention has great potential to be developed into a drug and health product for protecting the intestine. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1These are the separation diagrams of the Achyranthes polyspinus glucan SP-1; wherein A is the Q-Sepharose Fast Flow separation diagram, and B is the Sephacryl S-300 separation diagram.

[0030] Figure 2 It is a high performance gel permeation chromatogram of the Achyranthes polysaccharide SP-1.

[0031] Figure 3 This is a monosaccharide composition diagram of the Achyranthes polysaccharide glucan SP-1.

[0032] Figure 4 It is the infrared spectrum of the Achyranthes polyspinus glucan SP-1.

[0033] Figure 5A For the polysaccharide glucan SP-1 1 H spectrum.

[0034] Figure 5B For the polysaccharide glucan SP-1 13 C spectrum.

[0035] Figure 5C It is the COSY spectrum of the Achyranthes polyspinus glucan SP-1.

[0036] Figure 5D It is the HSQC spectrum of the Achyranthes polyspinus glucan SP-1.

[0037] Figure 5E It is the HMBC spectrum of the Achyranthes polyspinus glucan SP-1.

[0038] Fig. 5F It is the NOESY spectrum of the Achyranthes polyspinus glucan SP-1.

[0039] Figure 6 This is a schematic diagram of the structure of Achyranthes polysaccharide SP-1.

[0040] Figure 7 The figure shows the effect of Achyranthes polysaccharide SP-1 on the viability of Caco-2 cells; A shows the effect on the viability of Caco-2 cells, and B shows the effect on cells damaged by H2O2.

[0041] Figure 8 The RT-qPCR method was used to detect the effect of Achyranthes polysaccharide SP-1 on the genes of Nrf2 / HO-1 signaling pathway in Caco-2.

[0042] Fig. 9Western blot was used to detect the effect of Achyranthes polysaccharide SP-1 on Nrf2 / HO-1 signaling pathway proteins in Caco-2; A is the protein expression level of Nrf2, B is the protein expression level of NQO1, C is the protein expression level of HO-1, and D is the protein expression level of SOD1.

[0043] Fig.10 The protective effect of Achyranthes polysaccharide SP-1 on tight junction protein damage caused by H2O2. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods, but the scope of protection claimed by the present invention is not limited to the scope described in the examples.

[0045] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased.

[0046] Example 1

[0047] 1. Extraction and purification of Achyranthes polysaccharide SP-1

[0048] (1) Crush the viscera of the spiny sea cucumber (100 g) dried at 60°C and pass through a 40-mesh sieve for later use.

[0049] (2) The viscera powder was defatted with methanol and then stirred with 0.1 mol / L NaOH at 60 °C overnight. After stirring, the solution was cooled and the pH was adjusted to 6-7 with concentrated hydrochloric acid.

[0050] (3) Add 1% (m / v) papain (the enzymatic activity of papain is 100,000 U / g) to the solution with adjusted pH, where the unit of mass in the mass-to-volume ratio (m / v) is g and the unit of volume is mL), and stir continuously for 24 h at 47 °C. Collect the supernatant after centrifugation, add 5% (v / v) trichloroacetic acid to remove protein, and then add 3 times the volume of pre-cooled anhydrous ethanol to precipitate the crude polysaccharide.

[0051] (4) Centrifuge at 5000 rpm for 30 min, take out the precipitate and dissolve it in distilled water. The dissolved solution is dialyzed in distilled water using a dialysis bag with a molecular weight cutoff of 3.5 kDa for 36 h and then freeze-dried.

[0052] (5) The freeze-dried samples were separated by anion exchange column chromatography (Q Sepharose Fast Flow). The Aesculus polysaccharide was eluted with 0, 0.2, and 0.5 mol / L NaCl. The eluted fractions were automatically collected, and the sugar content was measured by the sulfuric acid-phenol method. The elution volume-absorbance curve was plotted.

[0053] (6) The water-washed component (i.e., the 0 mol / L NaCl elution component) was purified by gel column chromatography (Sephacryl S-300) with pure water as the eluent at a flow rate of 0.3 mL / min. The linear peak tip was collected, concentrated by a rotary evaporator, and freeze-dried to obtain Achyranthes polysaccharide SP-1.

[0054] like Figure 1 As shown in A, the QFF column chromatography chromatogram of Achyranthes polysaccharide SP-1 shows that the water washing fraction was collected to obtain the crude polysaccharide. Figure 1 As shown in B, SP-1 was purified using Sephacryl S-300 to obtain a purified polysaccharide fraction.

[0055] 2. Determination of the purity and molecular weight of SP-1 by high performance gel permeation chromatography (HPGPC)

[0056] (1) Preparation of polysaccharide samples: Accurately weigh 2.5 mg of the SP-1 prepared above, dissolve it in 500 μL of 0.2 M NH4HCO3 mobile phase, and filter it with a 0.22 μm filter membrane. Weigh dextran standards of different molecular weights (M w : 4.32, 12.6, 70.8, 126, 289 kDa) 2.5 mg each, dissolved in 500 μL 0.2 M NH4HCO3 mobile phase, and filtered with a 0.22 μm filter membrane.

[0057] (2) Chromatographic conditions: Chromatographic column: TSK G3000PW XL column; mobile phase: 0.2 M NH4HCO3; flow rate: 0.6 mL / min; column temperature: 35 ℃.

[0058] (3) Plotting the standard curve: Use the logarithm of the weight-average molecular weight of the standard as the ordinate and the retention time as the abscissa to plot the standard curve and obtain the linear regression equation. Calculate the molecular weight of the glucan in the viscera of Achyranthes polyspinus by the peak time of the sugar components.

[0059] (4) The results are as follows Figure 2 As shown, SP-1 is a single symmetrical peak, indicating that its molecular weight distribution is uniform and its purity is high. After calculation, the molecular weight of SP-1 is 12.9 kDa.

[0060] 3. Determination of monosaccharide composition of SP-1 by high performance liquid chromatography (HPLC)

[0061] (1) Weigh 5 mg of SP-1, dissolve it in 500 μL of 2 M trifluoroacetic acid, hydrolyze it at 110 °C for 6 h, and evaporate it to dryness. Take 100 μL of polysaccharide hydrolyzate or monosaccharide mixed standard solution, add equal volumes of 0.3 M NaOH solution and 0.5 M PMP methanol solution, and derivatize at 70 °C for 30 min. Then add 100 μL of 0.3 M HCl solution to neutralize the above solution. Then extract the excess PMP with chloroform, and filter the aqueous phase with a 0.22 μm filter membrane.

[0062] (2) HPLC determination of SP-1 monosaccharide composition: chromatographic column: Agilent ZORBAX C18 column; detection wavelength: 245 nm; mobile phase: phosphate buffer (pH 7.4): acetonitrile (83:17, v / v); column temperature: 35 °C; flow rate: 1.0 mL / min.

[0063] The results are as follows Figure 3 As shown, the monosaccharide composition of the Achyranthes polysaccharide glucan SP-1 is glucose, mannose, galacturonic acid, glucuronic acid, arabinose, and galactose, and the molar ratios are 89.2:5.0:1.5:1.6:1.5:1.2, respectively, indicating that SP-1 is a glucan containing a small amount of mannose, galacturonic acid, glucuronic acid, arabinose, and galactose.

[0064] 4. Infrared spectrum of Achyranthes polysaccharide SP-1

[0065] Take 2 mg of dried SP-1 sample and mix it with a small amount of dried potassium bromide powder, and press it into tablets. Use Thermo Nicolet Fourier transform infrared spectrometer to measure its infrared absorption, scanning range: 4000-500 cm -1 .

[0066] like Figure 4 As shown, 3385.25 cm -1 The signal peak at 2930.77 cm is the stretching vibration peak of -OH. -1 The signal peak at 1649.94 cm -1 The signal peak at 1200-1000 cm -1 The signal peaks at 930.24 and 846.60 cm-1 are the characteristic peaks of pyranose COC and COH. -1 The signal peak at represents the presence of α-configuration sugar residues. The results show that SP-1 is an α-configuration pyranose.

[0067] 5. Methylation analysis to determine the linkage mode of SP-1 sugar chains

[0068] Methylation analysis method: Take a small amount of SP-1 sample (2~3 mg), add 500 μL DMSO to dissolve; add 1 mg NaOH and incubate for 30 min; add 50 μL iodomethane solution to react for 1 h; add 1 mL water and 2 mL dichloromethane, vortex mix, centrifuge, and discard the aqueous phase. Repeat the water washing for 3 times; draw the lower dichloromethane phase and blow dry with nitrogen; add 100 μL 2M TFA, react at 121℃ for 90 min; evaporate to dryness at 30℃; add 50 μL 2 mol / L ammonia water, 50 μL 1 mol / L NaBD4, mix and react at room temperature for 2.5 h; add 20 μL acetic acid to terminate the reaction, blow dry with nitrogen, wash twice with 250 μL methanol, blow dry with nitrogen; add 250 μL acetic anhydride, vortex mix, react at 100℃ for 2.5 h; add 1 mL water and let stand for 10 min; add 500 μL dichloromethane, vortex mix, centrifuge, discard the water phase, and repeat the water washing for 3 times. Take the lower dichloromethane phase and detect it on the gas chromatography-mass spectrometer. The sugar residue type of A. polyspinosa glucan SP-1 was determined by comparing the methylation results with the standard spectrum.

[0069] Table 1 Methylation analysis results of SP-1

[0070] As shown in Table 1, glucose in SP-1 mainly exists in the form of →4)-Glcp-(1→, →4, 6)-Glcp-(1→ and T-Glcp-(1→. Mannose mainly exists in the form of →2)-Manp-(1→, →3)-Manp-(1→, →2,4)-Manp-(1→, →3,6)-Manp-(1→. In addition, there is a small amount of →3,4)-Galp-(1→. The existence of →4, 6)-Glcp-(1→ proves that SP-1 has a branched structure.

[0071] 6. Determination of the sugar residue linkage sequence of SP-1 by NMR spectroscopy

[0072] Two-dimensional NMR method: 30 mg of Achyranthes polysaccharide SP-1 was dissolved in 500 μL of D2O (99.9%, Sigma) and placed at room temperature for several hours to allow complete hydrogen ion exchange. The sample was then freeze-dried and the exchange was repeated three times. The analysis was performed using a JEOL-ECP 600 MHz NMR instrument (Japan). The one-dimensional H NMR spectrum was recorded at 25 °C ( 1 H-NMR、Carbon Spectroscopy( 13 C-NMR), two-dimensional spectra such as 1 H / 1H correlation spectroscopy (COSY), the relationship between hydrogen nuclei close in space 1 H / 1 HNOESY spectroscopy (NOESY), heteronuclear single quantum correlation spectroscopy (HSQC) and 1 Heteronuclear multicarbon correlation spectroscopy (HMBC) of H.

[0073] Combining the spectra and relevant literature, the carbon and hydrogen chemical shifts of different sugar residues in SP-1 were assigned. The results are shown in Table 2 and Figure 5A-5F As shown. The glucans in SP-1 are mainly →4)-α-D-Glc-(1→, T-α-D-Glc-(1→, →4,6)-α-D-Glc-(1→, which is consistent with the methylation results. Combining HMBC and NOESY spectra, it can be found that →4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→ and →4)-α-D-Glcp-(1→4)-α-6-D-Glcp-(1→) are linked, proving that the main chain is →4)-α-D-Glc-(1→, and there is a branched structure at the O-6 position; HMBC The correlation signals at 5.21 / 77.4 and 5.00 / 75.8 indicate the existence of the linkage mode of →4,6)-α-D-Glcp-(1→4)-α-1,3,4-D-Galp-(1→4)-α-1,2,4-D-Man-(1→; the correlation signals at 5.18 / 78.1 indicate the existence of the linkage mode of α-1,2-D-Man-(1→2). The correlation signals at 4.90 / 3.71 indicate the existence of the linkage mode of α-1,3-D-Man-(1→3)-α-1,3,4-D-Gal-(1→. Since T-α-D-Glcp is the only terminal sugar, all the ends are T-α-D-Glcp.

[0074] Table 2 Chemical shifts of different sugar residues in SP-1

[0075]

[0076] Note: -: Not attributed.

[0077] Therefore, if Figure 6 As shown in the figure, the main chain structure of Achyranthes polysaccharide SP-1 was determined to be 1,4-linked α-D-glucose, with a terminal α-D-glucose branch at the O-6 position of the main chain, and also some mannose and galactose structures. Its structural formula is as follows Figure 6 shown.

[0078] Example 2

[0079] 1. Polysaccharide glucan SP-1 resists oxidative stress damage of intestinal epithelial cell barrier caused by H2O2

[0080] (1) Cell culture: Human colon adenocarcinoma Caco-2 cells were cultured in MEM medium containing 20% ​​fetal bovine serum, 1% non-essential amino acids, and 1% sodium pyruvate; the culture conditions were 37°C and 5% CO2.

[0081] (2) CCK-8 method was used to detect the effect of Achyranthes polysaccharide SP-1 on the viability of Caco-2 cells: cells in the logarithmic phase were taken and 2×10 4 The cells were inoculated at a density of 10 cells / well in a 96-well plate and cultured for 24 h. The supernatant was discarded, and different concentrations of SP-1 prepared in Example 1 (12.5, 25, 50, 100, 200 μg / mL) were added to each well, with 3 replicates for each concentration, and the cells were incubated for 24 h. Subsequently, the cells were damaged with 5 mM H2O2 for 3 h. After that, 10 μL of CCK-8 solution was added to each well, and after incubation for 1 h, the OD value of each well was detected at 450 nm using an enzyme reader to calculate the cell viability of each well.

[0082] The results are as follows Figure 7 As shown in Figure A, compared with the blank group, SP-1 did not cause a decrease in cell viability in the range of 12.5-200 μg / mL, and was safe and non-toxic. Figure 7 Figure B shows that within this concentration range, SP-1 can significantly resist the decrease in cell viability caused by H2O2 and play an anti-oxidative damage role.

[0083] (3) Study on the effect of A. polyspinosa glucan SP-1 on the oxidative stress injury of intestinal epithelial cell barrier caused by H2O2: Caco-2 monolayer cells were randomly divided into a blank group, a H2O2 group, a low-dose SP-1 group, and a high-dose SP-1 group. The first two groups were added with complete culture medium, and the latter two groups were added with complete culture medium containing 25 and 50 μg / mL of A. polyspinosa glucan SP-1, respectively, for 24 h. Afterwards, the cells were washed with PBS, and 5 mM H2O2 was added to damage the cells for 3 h, except for the blank group.

[0084] RT-qPCR technology was used to detect the gene expression of Nrf2 / HO-1 signaling pathway in Caco-2 cells: The total mRNA of each group of cells was extracted using an RNA extraction kit. Reverse transcription was performed using a cDNA first-strand synthesis kit. Subsequently, a real-time fluorescence quantitative PCR system (20 μL) was prepared: 2 μL of cDNA template, 4 μL of upstream and downstream primers, 10 μL of SYBR Green qPCR mixture, and 4 μL of DEPC water. A fluorescence quantitative PCR instrument was used to amplify according to the set program (incubation stage: 95 ℃, 2 min; PCR stage: 95 ℃, 15 sec, 60 ℃, 20 sec; melting curve stage: 95 ℃, 15 sec, 60 ℃, 15 sec, 95 ℃, 15 sec. Number of cycles: 40). GAPDH was used as the internal reference gene and 2 -ΔΔCt Methods The relative expression level of the target gene was calculated.

[0085] Oxidative stress is an important cause of intestinal barrier damage in inflammatory bowel disease, metabolic syndrome, etc. The Nrf2 / HO-1 signaling pathway is the body's main anti-oxidative stress pathway. Nrf2 can activate the expression of downstream antioxidant enzymes (such as SOD1, HO-1, etc.) to resist oxidative stress.

[0086] The results are as follows Figure 8 As shown in the results, H2O2 can significantly reduce the expression of genes related to the Nrf2 / HO-1 anti-oxidative stress signaling pathway in Caco-2 cells (Nrf2, NQO1, HO-1, SOD1), and the cells are damaged by oxidation. However, adding 25 and 50 μg / mL of SP-1 can increase the expression of related genes and protect intestinal epithelial cells.

[0087] Western blot technique was used to detect the expression of Nrf2 / HO-1 signaling pathway proteins and tight junction proteins in Caco-2 cells: an appropriate amount of RIPA lysis buffer was taken, and the protease inhibitor PMSF was added 2-3 min before use, and the solution was set aside. The old cell culture medium was removed, the cells were rinsed with PBS, 100 μL of the pre-prepared lysis buffer was added to each well, and after the cells were fully lysed, the lysate was transferred to a sterile tube and centrifuged at 12000 r for 15 min in a 4 ℃ centrifuge. The supernatant was the required protein sample. The protein content in the sample was determined using the BCA protein concentration assay kit. The protein sample was diluted with SDS-PAGE loading buffer, mixed well, and denatured at 100 ℃ for 10 min. After being taken out, it was cooled to room temperature, and stored in a -20 ℃ refrigerator after aliquoting. A 5% concentrated gel was prepared, and according to the molecular weight of the target protein, an 8-12% separation gel was prepared for protein electrophoresis (60 V, 30 min; 160 V, 40 min). Subsequently, the proteins on the separation gel were transferred (300 mA) to the PVDF membrane. The transfer plate was assembled in the order of blackboard, sponge, 3 layers of transfer filter paper, gel, PVDF membrane, 3 layers of transfer filter paper, sponge, and transparent white board. No bubbles were generated between the gel and the PVDF membrane, and the transfer buffer was replenished in time to prevent the gel block from drying out. The transfer time depends on the molecular weight of the protein. The PVDF membrane was blocked in the blocking solution at room temperature for 1 h. According to the instructions for use of the antibody, the target protein band was added to the corresponding primary antibody and incubated at 4°C overnight. The membrane was washed 3 times (10 min / time) with TBST buffer at room temperature. The PVDF membrane was incubated with horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (H+L) or goat anti-rabbit IgG (H+L) secondary antibodies at room temperature for 1 h, and the membrane was washed 3 times (10 min / time) with TBST buffer. Protein development was performed on a gel imager using an ultrasensitive chemiluminescence detection kit. β-actin was used as the internal reference protein, and Image J software was used to perform relative quantitative analysis of the target protein bands.

[0088] like Fig. 9 As shown in the results, 25 and 50 μg / mL of Achyranthes polysaccharide SP-1 can significantly increase the H2O2-induced decrease in the protein expression of cellular antioxidant stress pathway proteins such as Nrf2, NQO1, HO-1, and SOD1, play an antioxidant role, and protect the intestinal barrier.

[0089] Tight junction proteins (such as ZO-1 and Occludin) play an important role in maintaining the barrier function of intestinal cells. When intestinal cells are oxidatively damaged, the expression of tight junction proteins decreases. Fig.10The results showed that 25 μg / mL and 50 μg / mL of Achyranthes polysaccharide visceral glucan SP-1 could significantly increase the expression of cell tight junction proteins (such as ZO-1 and Occludin) caused by H2O2, and had a significant protective effect on intestinal barrier function.

[0090] The above factual cases only represent the technical solutions of the present invention, rather than limiting the experiments. Although we have improved the experimental solutions, researchers in the same field can still make further improvements to the experimental solutions described above or make scientific equivalent replacements for the experimental links. These changes do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A polysaccharide from Astragalus multispinus, characterized in that: The main chain structure of the Asteraceae polyspinata glucan is 1,4-linked α-D-glucose, and there is a terminal α-D-glucose branch at the O-6 position of the main chain, and the branch also contains mannose and galactose. The structural formula of the Asteraceae polyspinata glucan is specifically as follows: 。 2. The Achyranthes polytrichum glucan according to claim 1, characterized in that The monosaccharide composition of the Achyranthes polysaccharide glucan is glucose, mannose, galacturonic acid, glucuronic acid, arabinose and galactose, and the molar ratios thereof are 89.2:5.0:1.5:1.6:1.5:1.2, respectively.

3. The method for preparing the Achyranthes polysaccharide according to claim 1 or 2, characterized in that: The preparation method comprises the following steps: (1) drying, crushing and sieving the viscera of the Polygonum multicornucopia to obtain viscera powder; (2) After defatting the viscera powder, heat and stir in an alkaline solution overnight; after stirring, adjust the pH to 6-7 with a strong acid solution; (3) adding papain to the solution of step (2) for enzymatic hydrolysis, removing protein with trichloroacetic acid after centrifugation, and then precipitating crude polysaccharides with anhydrous ethanol; (4) dialyzing the crude polysaccharide and then freeze-drying it to obtain freeze-dried polysaccharide; (5) separating and eluting the freeze-dried polysaccharide through anion exchange column chromatography, collecting the eluate to obtain a water-wash component; (6) The water-washed fraction is purified by gel column chromatography, concentrated, and freeze-dried to obtain the Achyranthes polysaccharide.

4. The preparation method according to claim 3, characterized in that: In the step (2), methanol is used for degreasing; the alkaline solution is 0.1 mol / L NaOH; the strong acid solution is concentrated hydrochloric acid; and the heating temperature is 60°C-80°C.

5. The preparation method according to claim 3, characterized in that: In the step (3), the mass volume ratio of the amount of papain added to the volume of the solution is 1%-3%, and the enzyme activity of papain is 100,000 U / g; the amount of trichloroacetic acid used is 4%-7% of the volume of the supernatant collected after centrifugation.

6. The preparation method according to claim 3, characterized in that: The molecular weight cutoff of the dialysis bag in the dialysis in step (4) is 3.5 kDa; and the dialysis time is 36 h-48 h.

7. The preparation method according to claim 3, characterized in that: In the step (5), the Aesculus truncatula dextran is eluted with 0, 0.2, and 0.5 mol / L NaCl, and the water-washed fraction is collected.

8. The preparation method according to claim 3, characterized in that: The purified eluent in step (6) is pure water; the flow rate of the eluent is 0.3 mL / min.

9. Use of the Achyranthes polysaccharide as claimed in claim 1 or 2 in the preparation of medicines or health products for protecting the intestinal barrier.

10. The use according to claim 9, characterized in that: The medicine or health product contains 12.5 μg / mL-200 μg / mL of Achyranthes polysaccharide glucan.

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