Soluble soybean polysaccharide, preparation method and application of soluble soybean polysaccharide in preparation of anti-inflammatory products

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to soluble soybean polysaccharide, a preparation method and application of the soluble soybean polysaccharide in preparation of anti-inflammatory products. The soluble soybean polysaccharide provided by the invention is obtained by taking bean dregs as a raw material, extracting by adopting ultrasonic-assisted ionic liquid 1-octyl-3-methylimidazolium bromide, deproteinizing, carrying out alcohol precipitation and further purifying by adopting gel chromatography, and the obtained soluble soybean polysaccharide at least contains neutral soybean polysaccharide and acidic soybean polysaccharide. The soluble soybean polysaccharide has the beneficial effects that the soluble soybean polysaccharide obtained by the method has a good effect in the aspect of relieving LPS-induced inflammatory injury of the liver and intestinal tracts.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a soluble soybean polysaccharide, its preparation method, and its application in the preparation of anti-inflammatory products. Background Technology

[0002] Natural plant polysaccharides are a class of biological macromolecules with effects such as immune regulation, anti-tumor, anti-radiation, anti-inflammation, anti-fatigue, and anti-aging. In recent years, they have become the preferred raw material for medical and health care products due to their high safety and accessibility.

[0003] Soybeans are rich in nutrients and are an agricultural resource with great development value. They are often used to make soybean oil, tofu and other soy products. However, a large amount of soybean residue and other by-products are generated during the processing of various soybean products. Because of their rough texture and difficulty in storage, soybean residue is currently mainly used to make protein feed for poultry and livestock, with extremely low utilization rate.

[0004] Studies have shown that soybean residue contains more than 30% soluble polysaccharides (SSPS) (based on soybean residue), making it a potentially valuable natural resource.

[0005] If an extraction and purification process can be developed to make fuller use of the soluble polysaccharides in soybean residue, it will greatly promote the development of the soybean industry. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a soluble soybean polysaccharide, its preparation method, and its application in the preparation of anti-inflammatory products.

[0007] The first aspect of the present invention is to provide an application of soluble soybean polysaccharides, specifically the application of soluble soybean polysaccharides in the preparation of anti-inflammatory products.

[0008] Preferably, the inflammation includes, but is not limited to, enteritis and gastritis.

[0009] Preferably, the soluble soybean polysaccharide is obtained by using soybean residue as raw material, extracting it with ultrasonic-assisted ionic liquid, and then purifying it through deproteinization, alcohol precipitation, and gel chromatography. It is an imidazole ionic liquid.

[0010] As a further preferred embodiment, the ionic liquid is selected from at least one of 1-octyl-3-methylimidazolium bromide, 1-ethyl-3-methylimidazolium bromide, and 1-butyl-3-methylimidazolium bromide.

[0011] Preferably, when using ionic liquids to extract soybean polysaccharides, the concentration of the ionic liquid is 0.1~2 M.

[0012] Preferably, the gel chromatography is selected from any one of: DEAE-Sepharose Fast Flow, DEAE-Sephacel, DEAE-Cellulose, and DEAE-Sepharose FF.

[0013] As a further preferred embodiment, the soluble soybean polysaccharide comprises at least neutral soybean polysaccharide and acidic soybean polysaccharide. The neutral soybean polysaccharide has a number-average molecular weight (Mn) of 518 Da and a weight-average molecular weight (Mw) of 532 Da. The acidic soybean polysaccharide includes peaks 1, 2, and 3, wherein peak 1 has a number-average molecular weight of 17082 Da and a weight-average molecular weight of 34508 Da, peak 2 has a number-average molecular weight of 1298 Da and a weight-average molecular weight of 1402 Da, and peak 3 has a number-average molecular weight of 455 Da and a weight-average molecular weight of 473 Da.

[0014] The inventors discovered that the anti-inflammatory activity of plant polysaccharides is closely related to their chemical structure, such as monosaccharide composition, molecular weight, and chain conformation. Furthermore, the chain conformation of polysaccharides also shows a trend of change with molecular weight, which has been confirmed to be closely related to changes in anti-inflammatory effects.

[0015] Based on this, the inventors used soybean residue, a large amount of waste generated during the production and processing of soybean products, as raw material. They used ionic liquids to extract soluble polysaccharides from it. The crude soybean polysaccharides obtained were then purified by gel chromatography after protein removal and alcohol precipitation. Finally, they obtained refined soluble soybean polysaccharides with specific molecular weights and specific monosaccharide compositions.

[0016] A second aspect of the present invention is to provide a method for preparing the above-mentioned soluble soybean polysaccharide, the method specifically comprising the following steps: Extraction of crude polysaccharides from S1 soybean Soybean residue was used as raw material, and ultrasonic-assisted ionic liquid was used for extraction. The mass-volume ratio of soybean residue to ionic liquid was 1 g: 10~30 mL, the extraction temperature was 50~60℃, the ultrasonic time was 10~30 min, and after extraction, the supernatant was obtained by centrifugation, which is the crude soybean polysaccharide. Purification of S2 soybean crude polysaccharide First, the protein in the crude soybean polysaccharide obtained in S1 was removed by the Sevag method. Then, crude soluble soybean polysaccharide was obtained by ethanol precipitation. Finally, the crude soluble soybean polysaccharide was purified by DEAE-Sepharose FF gel chromatography to obtain soluble soybean polysaccharide.

[0017] In the above preparation method, preferably, in S2, when deproteinizing using the Sevag method, the volume ratio of the Sevag reagent to the crude soybean polysaccharide is 1:3~5.

[0018] Preferably, in S2, when purifying crude soluble soybean polysaccharides using DEAE-Sepharose FF gel chromatography, gradient elution is performed using NaCl solution as the eluent, with the concentration of NaCl solution being 0~0.5 mol / L and the flow rate of the eluent being 0.5~2.0 mL / min.

[0019] Furthermore, the application of the soluble soybean polysaccharide provided by this invention in the preparation of products for alleviating intestinal flora imbalance is also a key technical content protected by this invention.

[0020] The beneficial effects of this invention are as follows: (1) A process for reusing soybean residue was developed. Ionic liquid was used to treat the soluble polysaccharide in soybean residue, and after purification by specific gel chromatography, a refined soluble soybean polysaccharide with a specific molecular weight was obtained. (2) The soluble soybean polysaccharide provided by the present invention includes at least soybean neutral polysaccharide and soybean acidic polysaccharide, etc. Several different types of soluble soybean polysaccharides have shown good effects in regulating intestinal flora and inhibiting inflammatory response. Attached Figure Description

[0021] Figure 1 Elution curve of DEAE-Sepharose FF ion chromatography column in Example 2; Figure 2 The retention time and molecular weight of the neutral soybean polysaccharide and acidic soybean polysaccharide obtained in Example 2 are shown. Figure 3 This is the HPLC chromatogram of neutral soybean polysaccharide from Example 2; Figure 4 Here is the HPLC chromatogram of acidic soybean polysaccharide from Example 2; Figure 5 This is an H&E staining image of mouse liver from Example 3; Figure 6 This is a diagram illustrating the effect of soybean polysaccharides on F4 / 80 expression in the liver tissue of mice with LPS-induced acute inflammation, as shown in Example 3. Figure 7 This is a diagram illustrating the effect of soybean polysaccharide on LPS-induced acute inflammation cytokines in mouse livers (Example 3). Figure 8 Figure 3 shows the effect of soybean polysaccharide on liver transaminase in mice with LPS-induced acute inflammation. Figure 9 This is a diagram illustrating the effect of soybean polysaccharide on the expression levels of TLR4 / NF-κB pathway-related mRNAs in the liver of LPS-induced acute inflammation mice, as shown in Example 3. Figure 10 This is a diagram illustrating the activation of the TLR4 / NF-κB pathway in the liver of mice with LPS-induced acute inflammation by soybean polysaccharides, as shown in Example 3. Figure 11 Figure 3 shows the effect of soybean polysaccharide on the expression of TLR4 / NF-κB pathway-related proteins in the liver of LPS-induced acute inflammation mice. Figure 12 Figure 4 shows the effect of soybean polysaccharide on lipopolysaccharide-induced acute inflammatory intestinal pathological damage in mice. Figure 13 These are histopathological images of the colon tissue of mice in each group in Example 4; Figure 14 The image shows the staining of tight junction proteins in the colon tissue of mice in each group in Example 4. Figure 15 This is a diagram illustrating the effect of soybean polysaccharide on the expression of tight junction proteins in the colon tissue of mice with LPS-induced acute inflammation, as shown in Example 4. Figure 16 Example 5 shows the α-diversity of gut microbiota in each group of mice; Figure 17 This is a diagram illustrating the effect of soybean polysaccharides on the composition of the intestinal microbiota in mice, as shown in Example 5. Figure 18 This is a graph showing the differences in gut microbial species composition among the mouse groups in Example 5; Figure 19 This is a diagram showing the intestinal microbial marker species of mice in each group in Example 5. Detailed Implementation

[0022] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments. Example

[0023] A method for preparing soluble soybean polysaccharide includes the following steps: Extraction of crude polysaccharides from S1 soybean Soybean residue was used as raw material, and ultrasonic-assisted ionic liquid extraction was performed. The ionic liquid was 1-octyl-3-methylimidazolium bromide with a concentration of 0.15 M. The mass-to-volume ratio of soybean residue to ionic liquid was 1:20. The extraction temperature was 60℃ and the ultrasonic time was 30 min. After extraction, the residue was centrifuged at 3000 r / min for 30 min to obtain the supernatant, which is crude soybean polysaccharide. Purification of S2 soybean crude polysaccharide First, the crude soybean polysaccharide obtained in S1 was concentrated to 1 / 4 of its original volume under a vacuum of 0.09 MPa and a temperature of 60°C. Then, the protein in the crude soybean polysaccharide was removed by the Sevag method, and crude soluble soybean polysaccharide was obtained by ethanol precipitation. Finally, the crude soluble soybean polysaccharide was purified by DEAE-Sepharose FF ion chromatography column to obtain refined soluble soybean polysaccharide.

[0024] When removing protein using the Sevag method, the concentrated soybean polysaccharide solution is thoroughly mixed with the Sevag reagent and shaken for 30 min, centrifuged for 1 min, and the intermediate protein layer and the lower organic phase are discarded. This step is repeated 2 to 3 times. The Sevag reagent used is chloroform: n-butanol (v:v) = 4:1, and the volume ratio of polysaccharide solution to Sevag reagent is 4:1.

[0025] The specific steps for precipitation using ethanol are as follows: Anhydrous ethanol was added to the protein-free solution until the ethanol concentration reached 95%. The ethanol was added using a slow-speed stirring method, and the mixture was stirred in one direction with a magnetic stirrer. The mixture was then allowed to stand at 4°C for 24 h, and then centrifuged at 3000 r / min for 30 min. The supernatant was removed, and the precipitate was dried at 40°C to obtain crude soluble soybean polysaccharide. Example

[0026] 2.1 Determination of molecular weight of refined soybean polysaccharides The crude soluble soybean polysaccharide obtained in Example 1 was purified using a DEAE-Sepharose FF ion chromatography column. The specific steps are as follows: (1) DEAE-Sepharose FF (Φ6.15×60 cm) pretreatment: The new gum was repeatedly rinsed with distilled water during vacuum filtration until it was neutral; (2) Elution and purification: Take 0.1 g of the dried crude polysaccharide sample, dissolve it in distilled water to prepare a solution with a concentration of 40 mg / mL, filter it through a 0.45 μm filter membrane and load it onto the sample. Use 0, 0.1, 0.2, 0.3, 0.4 and 0.5 mol / L NaCl solutions as eluents for gradient elution. The flow rate of the eluent is 1 mL / min. Collect the eluent in one tube for every 10 mL. Elute until no polysaccharide is detected in the eluent. Concentrate the collected elution peaks of each gradient by rotary evaporation, remove salt by ultrafiltration, and freeze dry to obtain the soluble soybean polysaccharide (hereinafter referred to as refined soybean polysaccharide). When purifying the sample using a DEAE-Sepharose FF ion chromatography column, the soluble soybean crude polysaccharide sample obtained in Example 1 was eluted with gradients of 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L NaCl solutions, respectively. The phenol-sulfuric acid method was used for colorimetric detection, and the absorbance was measured at a wavelength of 490 nm. Elution curves were plotted against the number of elution tubes, as shown in the figure. Figure 1 As shown.

[0027] Figure 1 The results show that after gradient elution, the refined soybean polysaccharide produced two relatively obvious elution peaks in the distilled water and 0.2 M NaCl elution portions, indicating that this portion of polysaccharide is the most important polysaccharide contained in soybean residue. For convenience, these two components are named neutral soybean polysaccharide and acidic soybean polysaccharide, respectively, in this invention.

[0028] Furthermore, Figure 2 In the figure, ab represents the retention time and molecular weight of neutral soybean polysaccharides, and cd represents the retention time and molecular weight of acidic soybean polysaccharides.

[0029] Among them, ab shows that the neutral soybean polysaccharide has an Mn of 518 Da, an Mw of 532 Da, a peak molecular weight (Mp) of 581 Da, and a Z-average molecular weight (Mz) of 543 Da.

[0030] Peaks 1, 2, and 3 shown in c are three acidic polysaccharide elution peaks. Combined with d, we can know that: peak 1 has Mn of 17082 Da, Mw of 34508 Da, Mp of 20317 Da, and Mz of 75115 Da; peak 2 has Mn of 1298 Da, Mw of 1402 Da, Mp of 1563 Da, and Mz of 1517 Da; peak 3 has Mn of 455 Da, Mw of 473 Da, Mp of 468 Da, and Mz of 490 Da.

[0031] 2.2 Determination of Monosaccharide Composition of Refined Soybean Polysaccharides The monosaccharide composition of refined soybean polysaccharides was determined by high-performance liquid chromatography (HPLC), and the specific operation is as follows: Control solution: Accurately weigh appropriate amounts of mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, N-acetyl-glucosamine, glucose, N-acetyl-galactosamine, galactose, xylose, arabinose, and fucose reference standards, dissolve and dilute with water to a mixed control solution containing 50 µg of each in 1 mL.

[0032] Derivatization procedure: Accurately pipette 250 μL of the mixed control solution into a 5 mL EP tube, add 250 µL of 0.6 mol / L NaOH and 500 µL of 0.4 mol / L 1-phenyl-3-methyl-5-pyrazolone (PMP)-methanol, react at 70 °C for 1 h, cool in cold water for 10 min, add 500 µL of 0.3 mol / L HCl to neutralize, then add 1 mL of chloroform and vortex for 1 min, centrifuge at 3000 r / min for 10 min, carefully collect the supernatant, extract 3 times, and use the supernatant for HPLC.

[0033] Hydrolysis of the sample to be tested: Accurately weigh the sample into a 10 mL ampoule, add 3.0 mL of 2 mol / L trifluoroacetic acid (TFA) into the 10 mL ampoule, fill with nitrogen, seal the ampoule, and acid hydrolyze at 120℃ for 4 h. Remove the ampoule, add methanol and nitrogen to evaporate the TFA, and redissolve in 3.0 mL of water.

[0034] Sample solution derivatization: Accurately pipette 250 µL of sample solution (i.e., the neutral soybean polysaccharide and acidic soybean polysaccharide obtained after separation) into a 5 mL EP tube, add 250 µL of 0.6 mol / L NaOH and 500 µL of 0.4 mol / L PMP-methanol, react at 70℃ for 1 h, and cool in cold water for 10 min; add 500 µL of 0.3 mol / L HCl to neutralize, then add 1 mL of chloroform, vortex for 1 min, centrifuge at 3000 r / min for 10 min, carefully collect the supernatant, extract 3 times, and collect the supernatant to obtain the sample solution.

[0035] HPLC chromatograms of neutral soybean polysaccharides and acidic soybean polysaccharides are shown in the appendix. Figure 3-4 The monosaccharide composition and content of each are shown in Table 1-2 below.

[0036] Table 1. Monosaccharide composition of neutral soybean polysaccharides Peak Retention time / min Compound name area high area / % 1 14.021 Mannose 234098 12965 10.829 2 18.353 Ribose 18631 613 0.862 3 19.035 Rhamnose 3891 168 0.180 4 22.641 Glucuronic acid 22271 634 1.030 5 26.077 Galacturonic acid 11714 257 0.542 6 30.153 glucose 910914 25002 42.136 7 34.558 Galactose 396864 9693 18.358 8 36.274 Xylose 17257 440 0.798 9 37.663 Arabic sugar 527255 11778 24.389 10 43.211 Fucose 18938 309 0.876 total 2161833 61861 100.000

[0037] Table 2 Monosaccharide composition of acidic soybean polysaccharides Peak Retention time / min Compound name area high area% 1 13.988 Mannose 188729 9802 4.584 2 18.149 Ribose 42169 1637 1.024 3 19.024 Rhamnose 193800 8153 4.707 4 22.804 Glucuronic acid 42872 1131 1.041 5 26.058 Galacturonic acid 770150 23897 18.705 6 30.107 glucose 191460 5231 4.650 7 34.472 Galactose 730116 17990 17.733 8 36.147 Xylose 585931 13811 14.231 9 37.567 Arabic sugar 932696 21113 22.653 10 43.007 Fucose 439369 8587 10.671 total 4117293 111353 100.000

[0038] The results above show that the refined soybean polysaccharides obtained by ionic liquid extraction and purification are composed of monosaccharides such as glucose, arabinose, galactose, mannose, glucuronic acid, fucose, ribose, xylose, galacturonic acid, and rhamnose. Among them, glucose is the most abundant, accounting for 42.36%, followed by arabinose, galactose, and mannose, accounting for 24.39%, 18.36%, and 10.83%, respectively. The remaining monosaccharides account for a very small proportion.

[0039] In addition, acidic soybean polysaccharides contain monosaccharides such as arabinose, galacturonic acid, galactose, xylose, fucose, rhamnose, glucose, mannose, glucuronic acid, and ribose. Among them, arabinose is the most abundant, accounting for 22.65%; followed by galacturonic acid, accounting for 18.71%. Galactose and xylose are also abundant, accounting for 17.73% and 14.23% respectively. The remaining monosaccharides account for a small proportion.

[0040] Example 3 Anti-inflammatory activity experiment of refined soybean polysaccharides 3.1 Experimental Procedure Animal model establishment and treatment: After 7 days of adaptive feeding, all mice were randomly divided into 6 groups (n=10 / group): control group, lipopolysaccharide group, crude soybean polysaccharide group, neutral soybean polysaccharide group, acidic soybean polysaccharide group, and rhamnogalacturonan group. Except for the control group and LPS group, which were administered distilled water by gavage, the other groups were administered a solution containing 200 mg / kg of the corresponding polysaccharide by gavage once daily for 7 consecutive days. On the eighth day, except for the control group, the other groups were injected intraperitoneally with 10 mg / kg LPS solution to establish the model. All mice were sacrificed 6 hours later.

[0041] Sample Collection: On the eighth day of the experiment, 6 hours after LPS treatment, the mice were observed to confirm successful modeling. Peripheral blood samples (1-1.5 mL per mouse) were collected from the inner canthus of mice with enucleated eyes. Mice were euthanized by cervical dislocation, dissected, and the liver was collected. The liver was divided into two parts; one part was used to prepare paraffin sections, and the remaining part was wrapped in aluminum foil and stored at -80°C. The small intestine and colon tissues were immediately fixed in 4% paraformaldehyde solution for morphological observation. The colon contents were collected and rapidly frozen in liquid nitrogen, then stored at -80°C for further analysis.

[0042] 3.2 Experimental Results and Discussion Figure 5 Mouse livers were stained with hematoxylin and eosin (H&E) at 200×, with a scale bar of 100 µm. The figure shows the histopathological changes in the liver tissue of each group of mice. Among them, Control group, LPS group, Neutral group, Acidic group, Crude group, and Rhamno group are all represented by rhamnose galacturonic acid group.

[0043] In the control group, the liver lobule structure was normal, the hepatic cords and hepatocytes were regularly arranged, the hepatocyte structure was intact, there was no swelling, the size and staining were uniform, the boundaries were orderly and clear, and no inflammatory cell infiltration or necrosis was observed. After LPS stimulation, the hepatic cord structure of the mice was unclear, the hepatocyte boundaries were indistinct, granular degeneration of hepatocytes was widely observed, the cytoplasm was loose and lightly stained, and eosinophilic granules were visible (blue arrows). A small number of hepatocytes showed watery degeneration, cell swelling, and loose and lightly stained cytoplasm (yellow arrows), indicating that the model was successfully established.

[0044] The results showed that the edema, inflammatory infiltration and cell necrosis of mouse hepatocytes were significantly reduced after intervention with crude soybean polysaccharide and rhamnose. In the neutral soybean polysaccharide group and the acidic soybean polysaccharide group, some degeneration of the normal structure of hepatocytes was observed, and a small number of inflammatory cells showed slight morphological changes.

[0045] Figure 6 This figure shows the effect of soybean polysaccharides on the expression of F4 / 80 cells in the liver tissue of mice with LPS-induced acute inflammation. F4 / 80 cells can effectively indicate the degree of liver damage.

[0046] During inflammation, macrophages in the liver recruit neutrophils and monocytes to the liver. Monocytes further differentiate into M1 macrophages (pro-inflammatory macrophages). The polarization of liver macrophages can be assessed by detecting the expression of F4 / 80. Figure 6 The results showed that crude soybean polysaccharides, as well as acidic and neutral soybean polysaccharides, could alleviate LPS-induced liver inflammation and macrophage activation and polarization.

[0047] The effects of soybean polysaccharides on lipopolysaccharide-induced acute inflammatory mouse liver inflammatory cytokines are as follows: Figure 7 As shown, Figure 7 The secretion levels of tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), interleukin 1β (IL-1β), and interleukin 10 (IL-10) in mouse liver tissue were shown.

[0048] The results showed that the secretion of pro-inflammatory factors TNF-α, IL-6, and IL-1β in the liver of mice after LPS stimulation was significantly higher than that in the control group, indicating that the model was successfully established. After intervention with neutral soybean polysaccharide, acidic soybean polysaccharide, crude soybean polysaccharide, and rhamnose galacturonic acid, the levels of TNF-α, IL-6, and IL-1β decreased significantly (P < 0.05), while the secretion level of the anti-inflammatory factor IL-10 increased significantly. It can be seen that the intervention of different types of soybean polysaccharides can effectively regulate the level of liver inflammation response in LPS-stimulated mice and have a protective effect on the liver. However, compared with each other, neutral soybean polysaccharide and acidic soybean polysaccharide have better regulatory effects.

[0049] The effects of soybean polysaccharides on liver transaminases in mice with lipopolysaccharide-induced acute inflammation, specifically the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in mouse liver tissue, are shown in [the table below]. Figure 8 As shown.

[0050] AST and ALT are two important enzymes found in the liver, and elevated levels of these enzymes reflect the degree of liver cell damage. Figure 8 The results showed that LPS stimulation significantly increased AST and ALT levels in the liver tissue of mice compared to the control group (P < 0.05). However, mice treated with different types of soybean polysaccharides showed a significant decreasing trend in AST and ALT levels (P < 0.05). This indicates that refined soybean polysaccharides can alleviate liver damage by reducing AST and ALT levels in the liver of inflamed mice.

[0051] The effects of soybean polysaccharides on the expression levels of TLR4 / NF-κB pathway-related mRNAs in the liver of lipopolysaccharide-induced acute inflammation mice are shown in the following results. Figure 9 .

[0052] This invention uses qRT-PCR to detect the expression of TLR4 / NF-κB pathway-related mRNAs in the livers of mice in each group. The results show that after LPS stimulation, the expression level of TLR4 mRNA in mice increased significantly, which in turn led to an increase in the expression levels of downstream MyD88, IKK, IκB and NF-κB mRNAs. Soybean polysaccharide treatment significantly inhibited the upward trend of related mRNA expression (P < 0.05). Furthermore, the neutral soybean polysaccharide group and the acidic soybean polysaccharide group showed better effects than the crude soybean polysaccharide group.

[0053] The effects of soybean polysaccharides on the expression of TLR4 / NF-κB pathway-related proteins in the liver of lipopolysaccharide-induced acute inflammation mice are shown in the following results. Figure 10-11As shown. To further explore the possible mechanism by which soybean polysaccharides inhibit the inflammatory response in mice, this invention used Western blot to detect the expression of NF-κB pathway-related proteins in the liver of mice in each group. Compared with the control group, the relative expression levels of p-NF-κB-p65 and p-IκBα in the liver of mice in the LPS group were significantly increased (P < 0.05), indicating that the liver NF-κB pathway was activated after LPS induction, and the intervention of soybean polysaccharides could inhibit the phosphorylation of NF-κB-p65 and IκBα.

[0054] In addition, the expression level of IL-1β in the model group was significantly increased compared with that in the control group, and the intervention of soybean polysaccharide could significantly inhibit the increase of IL-1β expression.

[0055] The above results indicate that soluble soybean polysaccharides can inhibit the activation of the NF-κB signaling pathway, and their protective effect against LPS-induced inflammatory liver damage in mice may be achieved by inhibiting the activation of the NF-κB signaling pathway.

[0056] Example 4 Experiment on the regulation of intestinal inflammation by soybean polysaccharides in mice The effects of soybean polysaccharides on lipopolysaccharide-induced acute inflammatory intestinal pathological damage in mice are shown in the following results. Figure 12 As shown in the figure, the pathological changes in the small intestine tissue of mice in each group are illustrated.

[0057] The image shows intestinal villi distributed on the surface of the small intestine, with a single layer of columnar epithelium and normal morphology and structure. Goblet cells are distributed between the epithelial cells. The epithelium at the base of the villi invaginates to form Lee's crypts. A double layer of smooth muscle cells separates the intestinal crypts from the submucosa. The submucosa is composed of connective tissue. The remaining part of the intestinal wall includes a muscular layer composed of smooth muscle cells and a serosa layer. No obvious inflammatory changes were observed. After LPS stimulation, intestinal epithelial cells were damaged and sloughed off (red arrows), and eosinophil infiltration in the lamina propria increased (black arrows). After intervention with different types of soybean polysaccharides, the intestinal villi were repaired, the condition of the epithelial absorptive cells improved, the number of crypts increased, and they were arranged regularly. No obvious inflammatory changes were observed.

[0058] Histopathological changes in the colon tissue of mice in each group are as follows: Figure 13 As shown, the colonic tissue of the control group mice was in a normal physiological state, without inflammation or damage. The colon of mice stimulated with LPS showed a significant reduction in some epithelial cells (brown arrows) and goblet cells (yellow arrows), disappearance of crypts, extensive inflammatory infiltration of mononuclear cells in the mucosa and submucosa, and mucosal edema in the intestinal mucosa and muscularis propria. Different types of soybean polysaccharide intervention groups significantly reduced LPS-induced pathological damage, goblet cells largely returned to normal, edema was significantly improved, and no inflammatory cell infiltration was observed, although a small amount of inflammatory response remained.

[0059] To observe the effects of soybean polysaccharides on the intestinal barrier in LPS-induced acute inflammation mice, this invention employed immunohistochemistry to study the effects of soybean polysaccharide intervention on tJs and intestinal barrier integrity. The expression levels of TJ proteins (ZO-1, occludin, and claudin-1) were analyzed in colon tissue sections. The results are as follows: Figure 14-15 As shown.

[0060] ZO-1 staining was more intense at the apical junctions of colonic mucosal epithelial cells. Conversely, Occludin and claudin-1 proteins stained more intensely on the lateral membrane of the crypt basement membrane. In the LPS group colonic tissue, their protein distribution was uneven, and their expression levels were significantly reduced. The absorbance values ​​of immunohistochemical images were measured using the Image-pro Plus 6.0 image analysis system, and the percentage of protein expression in the colon was expressed as staining intensity (IOD) values. The results showed that compared with the blank control group, the expression levels of ZO-1, Occludin, and Claudin-1 in the colonic tissue of the LPS group were significantly reduced (P < 0.01). Compared with the LPS group, the expression levels of ZO-1, Occludin, and Claudin-1 in different types of soybean polysaccharide intervention groups were significantly increased.

[0061] Example 5 An investigation into the role of soybean polysaccharides in regulating gut microbiota and improving inflammation. Dietary fiber can be broken down and fermented by colonic microorganisms. Soluble soybean polysaccharides are a type of dietary fiber and may therefore affect the composition of gut microbiota. This invention further explores the effects of refined soybean polysaccharides on the gut microbiota of mice with LPS-induced acute inflammation by analyzing the composition and changes of gut microbiota in each group of mice. Five colonic contents samples were randomly selected from each group for 16S rRNA high-throughput sequencing analysis.

[0062] (1) Analysis of α-diversity of mouse gut microbiota This invention analyzes the α diversity of mouse gut microbiota using Specaccum species accumulation curves, species abundance rank curves, and α diversity indices.

[0063] Specaccum species accumulation curve as shown Figure 16 As shown in (A), the curve flattens out as the sample size increases, indicating that the number of species in this environment does not increase significantly with the increase in sample size, suggesting that the sampling is sufficient. The species abundance ranking curve is shown below. Figure 16 As shown in (B), the control group had the highest number of ASVs, followed by the crude soybean polysaccharide group and the acidic soybean polysaccharide group, then the neutral soybean polysaccharide group and the rhamnose galacturonic acid group, with the LPS group having the lowest. The downward trend of the curve indicates a uniform sample distribution. The α-diversity index is shown below. Figure 16As shown in (C), there were no significant differences in the Chao 1 index, ACE index, Simpson index, and Shannon index among the groups (P>0.05). This means that the intervention of LPS-induced stimulation, neutral soybean polysaccharide, acidic soybean polysaccharide, crude soybean polysaccharide, and rhamnose galacturonic acid did not affect the overall richness and community diversity of the intestinal bacteria in mice.

[0064] (2) Analysis of β-diversity of mouse gut microbiota This invention evaluated the effects of soluble soybean polysaccharides on the composition of the gut microbiota in mice. Principal coordinate analysis (PCA) results are as follows: Figure 17 As shown in (A), the results of non-metric multidimensional scaling (NMDS) are as follows: Figure 17 As shown in (B) (the results are highly reliable when stress < 0.2).

[0065] The results showed that the gut microbiota of the control group and the LPS group were separated but not significantly, indicating that the composition of the gut microbiota in mice with LPS-induced acute inflammation was changed to some extent. The gut microbiota composition of the neutral soybean polysaccharide group was similar to that of the control group, but it was significantly separated from the gut microbiota of the LPS group, indicating that neutral soybean polysaccharide can regulate the composition of the gut microbiota of mice to some extent.

[0066] (3) Analysis of the differences in species composition of mouse gut microbiota This invention analyzed the differences in the composition of the gut microbiota among different groups of mice.

[0067] First, the number of common and unique species among the groups was observed. Figure 18 The petal diagram (A) shows that the number of ASVs shared by all groups is 265. Figure 18 (B) represents the species abundance at the phylum and genus levels for each group. Figure 18(D) is a box plot of differential species. At the phylum level, the gut microbiota of mice in each group mainly consisted of Firmicutes, Bacteroidota, Deferribacterota, Desulfobacterota, and Actinobacteriota, with Firmicutes and Bacteroidota being the dominant phyla in each group. Compared with the control group, the relative abundance of Firmicutes and Actinobacteriota was increased and the relative abundance of Bacteroidota was decreased in the LPS group. In contrast, the increasing trend of relative abundance of Firmicutes and Actinobacteriota and the decreasing trend of relative abundance of Bacteroidota were slowed in the neutral soybean polysaccharide group, crude soybean polysaccharide group, and rhamnosine galacturonic acid group. At the genus level, compared with the control group, the abundance of Bacteroides, Muribaculaceae, [Eubacterium]-coprostanoligenes-group, Erysipelatoclostridium, and Parabacteroides decreased in the LPS group, while the abundance of Dubosiella, Faecalibaculum, Coriobacteriaceae-UGG-002, Blautia, and Mucispirillum increased significantly. Compared with the LPS group, the abundance of Bacteroides, Erysipelatoclostridium, and Parabacteroides was significantly increased in the neutral soybean polysaccharide group, while the abundance of Dubosiella, Faecalibaculum, Coriobacteriaceae-UGG-002, Desulfovibrio, Bifidobacterium, and Mucispirillum was significantly decreased. In the crude soybean polysaccharide group, the abundance of Bacteroides, Muribaculaceae, and Erysipelatoclostridium showed an increasing trend, while the abundance of Dubosiella, Faecalibaculum, Coriobacteriaceae-UGG-002, Desulfovibrio, Bifidobacterium, and Mucispirillum showed a decreasing trend.

[0068] To further compare the differences in species composition among the samples and to demonstrate the species abundance distribution trends of each sample, Figure 18 The middle (C) is a heatmap of genus-level composition based on relative abundance. The results show that the neutral soybean polysaccharide group and the control group have stronger clustering effects, followed by the crude soybean polysaccharide group.

[0069] (4) Analysis of mouse gut microbial marker species To identify the characteristic gut microbiota of each group of mice, this invention performed linear discriminant effect clustering analysis and scoring on the colonic contents microbiota. The LEfSe analysis of the gut microbiota of each group of mice is as follows: Figure 19 As shown in (A), linear discriminant analysis (LDA) is used to estimate the impact of each species' abundance on the differential effect, thereby identifying the species that produce significant differences in sample abundance. The linear discriminant effect clustering analysis is as follows: Figure 19 As shown in Figure (B), the most significant differences in species among the groups at the phylum level were observed in Bacteroidota, Actinobacteriota, and Firmicutes. At the family level, the abundance of Bifidobacteriaceae was significantly increased in the LPS group, while the abundance of Marinifilaceae and Muribaculaceae was significantly increased in the control group. The abundance of Tannerllaceae, Enterococcaceae, and Bacteroidaceae was significantly increased in the neutral soybean polysaccharide group, and the abundance of Erysipelotrichaceae, Lactobacillaceae, Clostridiaceae, and Bacillaceae was significantly increased in the acidic soybean polysaccharide group. These results indicate that refined soybean polysaccharides can alter the composition and structure of the gut microbiota in LPS-induced inflammatory mice.

Claims

1. Application of soluble soybean polysaccharides in the preparation of anti-inflammatory products.

2. The application as described in claim 1, characterized in that, The inflammation mentioned includes, but is not limited to, enteritis and gastritis.

3. The application as described in claim 1, characterized in that, The soluble soybean polysaccharide is obtained by using soybean residue as raw material, extracting it with ultrasonic-assisted ionic liquid, and then purifying it by deproteinization, alcohol precipitation, and gel chromatography; wherein the ionic liquid is an imidazole ionic liquid.

4. The application as described in claim 3, characterized in that, The ionic liquid is selected from at least one of 1-octyl-3-methylimidazolium bromide, 1-ethyl-3-methylimidazolium bromide, and 1-butyl-3-methylimidazolium bromide.

5. The application as described in claim 3, characterized in that, When using ionic liquids to extract soybean polysaccharides, the concentration of the ionic liquid is 0.1~2 M.

6. The application as described in claim 3, characterized in that, The gel chromatography is selected from any one of DEAE-Sepharose FastFlow, DEAE-Sephacel, DEAE-Cellulose, and DEAE-Sepharose FF.

7. The application as described in any one of claims 1-6, characterized in that, The soluble soybean polysaccharide comprises at least neutral soybean polysaccharide and acidic soybean polysaccharide. The neutral soybean polysaccharide has a number-average molecular weight of 518 Da and a weight-average molecular weight of 532 Da. The acidic soybean polysaccharide includes peaks 1, 2, and 3, wherein peak 1 has a number-average molecular weight of 17082 Da and a weight-average molecular weight of 34508 Da, peak 2 has a number-average molecular weight of 1298 Da and a weight-average molecular weight of 1402 Da, and peak 3 has a number-average molecular weight of 455 Da and a weight-average molecular weight of 473 Da.

8. A method for preparing the soluble soybean polysaccharide according to any one of claims 1-7, characterized in that, The steps include the following: Extraction of crude polysaccharides from S1 soybean Soybean residue was used as raw material, and ultrasonic-assisted ionic liquid was used for extraction. The mass-volume ratio of soybean residue to ionic liquid was 1 g: 10~30 mL, the extraction temperature was 50~60℃, the ultrasonic time was 10~30 min, and after extraction, the supernatant was obtained by centrifugation, which is the crude soybean polysaccharide. Purification of S2 soybean crude polysaccharide First, the protein in the crude soybean polysaccharide obtained in S1 was removed by the Sevag method. Then, crude soluble soybean polysaccharide was obtained by ethanol precipitation. Finally, the crude soluble soybean polysaccharide was purified by DEAE-Sepharose Fast Flow gel chromatography to obtain soluble soybean polysaccharide.

9. The preparation method according to claim 8, characterized in that, In S2, during the Sevag deproteinization process, the volume ratio of Sevag reagent to crude soybean polysaccharide is 1:3~5; for the purification process, NaCl solution is used as the eluent for gradient elution, with a NaCl solution concentration of 0~0.5 mol / L and an eluent flow rate of 0.5~2.0 mL / min.

10. The use of soluble soybean polysaccharides prepared by the preparation method according to any one of claims 8-9 in the preparation of products for alleviating intestinal flora imbalance.

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