A method for significantly improving the anti-inflammatory effect of peanut meal polysaccharides

Abstract

The present invention belongs to the field of peanut meal processing technology, and in particular to a method that can significantly improve the anti-inflammatory effect of peanut meal polysaccharides. The method that can significantly improve the anti-inflammatory effect of peanut meal polysaccharides provided by the present application comprises the following steps: extracting peanut meal polysaccharides; degrading them using Vc and H2O2, and conducting experiments on the anti-inflammatory activities of IL-6, TNF-α and IL-4, respectively, to obtain the optimal degradation time. The beneficial effect of the present application is that peanut meal polysaccharides are subjected to degradation treatment, which solves the technical problem that peanut meal polysaccharides are difficult to be directly utilized by the human body. The results show that peanut meal polysaccharides degraded for 1.25 h have the strongest anti-inflammatory activity, which can reduce IL-6 levels by 37.82%; peanut meal polysaccharides degraded for 2.5 h have the greatest effect on the IL-4 expression level of RAW 264.7 cells, and have the best effect on inhibiting IL-4 release, which can reduce IL-4 levels by 39.68%, and significantly enhance anti-inflammatory activity.

Description

Technical Field

[0001] The invention belongs to the technical field of peanut meal processing, and particularly relates to a method capable of significantly improving the anti-inflammatory effect of peanut meal polysaccharides. Background Art

[0002] Peanut meal polysaccharides have antibacterial, lipid-lowering, immunomodulatory, and anti-tumor effects. However, there is currently little literature on the specific anti-inflammatory effects of peanut meal polysaccharides. Some literature reports that peanut polysaccharides have anti-inflammatory effects, but detailed experimental research on their specific anti-inflammatory activity has not been disclosed.

[0003] If peanut meal is used as raw material, polysaccharides are extracted, and their anti-inflammatory activity is deeply studied, and corresponding products are further developed, it will have positive significance for improving the comprehensive utilization rate of peanut by-products and increasing the added value of peanut meal.

[0004] Polysaccharides have good biological activity, but their macromolecular structure is difficult to be directly utilized by the human body. Similarly, peanut polysaccharides also have the above-mentioned problems. Therefore, how to moderately degrade polysaccharides to improve solubility is also a difficult technical problem to solve.

[0005] Currently, research on the functions of peanut meal polysaccharides is still lacking. The complex structure of polysaccharides also makes it difficult to further study their structure-activity relationship and functional mechanism, which limits the industrialization of peanut meal polysaccharides. Therefore, the technical problems to be solved in this application are to analyze the chemical structure of peanut polysaccharides, moderately degrade the polysaccharides to improve their solubility, and obtain the relationship between degradation time and anti-inflammatory effect, so as to achieve effective utilization of polysaccharides by biological organisms. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides a method that can significantly improve the anti-inflammatory effect of peanut meal polysaccharides. The method uses peanut cake after oil extraction as raw material, extracts it with hot water to obtain peanut meal polysaccharides, and then degrades it to improve its solubility, so as to achieve its high-value utilization.

[0007] The method provided by the present invention for significantly improving the anti-inflammatory effect of peanut meal polysaccharides comprises the following steps:

[0008] (1) Extraction of peanut meal polysaccharides:

[0009] Peanut meal was taken and peanut meal polysaccharide was extracted by hot water extraction method;

[0010] (2) Degradation of peanut meal polysaccharides:

[0011] Take the peanut polysaccharide obtained in (1), add pure water to dissolve it, add Lactobacillus powder at 35-40°C, the amount of which is 0.5-1% of the weight of the peanut polysaccharide, ferment for 1-3 hours, then add Vc and H2O2 solution, and finally add pure water to make the H2O2 concentration 75mM and the Vc concentration 20mM, mix well, and let it stand;

[0012] (3) Manganese dioxide is added to the mixed solution in (2) to terminate the reaction, centrifuged, and the precipitate is separated to obtain the supernatant. Anhydrous ethanol is then added to precipitate the solution. After the precipitation is completed, centrifuged, the supernatant is separated, and the precipitate is spread on a clean culture dish, dried, ground, and the dry powder is collected to obtain peanut meal polysaccharide with enhanced anti-inflammatory effect.

[0013] Preferably, in (2), 2.50 g of peanut meal polysaccharide is accurately weighed and placed in a 500 mL wire-top reagent bottle. An appropriate amount of pure water is added and shaken to dissolve. Then, Vc and H2O2 solutions are added, and the pure water is added to 250 mL so that the H2O2 concentration in the solution is 75 mM and the Vc concentration is 20 mM. The mixture is mixed and the reagent bottle is placed in a water bath shaker at 25-30°C for 1-2.5 h.

[0014] Preferably, in (3), manganese dioxide is added to the solution mixed in (2) at a ratio of 0.05 g / mL to terminate the reaction, and the mixture is centrifuged at 10,000 rpm for 15 min to separate the precipitate and obtain the supernatant. Anhydrous ethanol is added to the supernatant at a volume ratio of 1:4 to alcohol-liquid ratio, and the mixture is placed at 4°C for alcohol precipitation for 6 to 12 hours. After the alcohol precipitation is completed, the mixture is centrifuged at 10,000 rpm for 15 min to separate the supernatant. The precipitate is spread on a clean culture dish, placed in an oven, dried at 55°C, ground, and the dry powder is collected and sealed and stored at 4°C in the dark.

[0015] Preferably, in (2), after the peanut meal polysaccharide is degraded for 1.25 to 2.5 hours, the final product obtained contains: 1.287 to 1.329% mannose, 5.626 to 5.744% ribose, 7.819 to 9.121% rhamnose, 10.738% to 11.148% glucose, 19.567 to 21.356% galactose, 1.282 to 1.397% xylose, 17.485 to 18.689% arabinose and 6.991 to 7.536% fucose.

[0016] (3), for inhibiting the secretion of IL-6 or the release of TNF-α, the degradation time is controlled to be 1.25 h; for inhibiting the secretion of IL-4, the degradation time is controlled to be 2.5 h.

[0017] The beneficial effects of the present invention are:

[0018] (1) In this application, peanut meal polysaccharides were degraded to solve the technical problem that peanut meal polysaccharides are difficult to be directly utilized by the human body. The results showed that peanut meal polysaccharides degraded for 1.25 hours had the strongest anti-inflammatory activity and could reduce the IL-6 level by 37.82%; peanut meal polysaccharides degraded for 2.5 hours had the greatest effect on the IL-4 expression level of RAW 264.7 cells and had the best effect on inhibiting IL-4 release, which could reduce the IL-4 level by 39.68%, and the anti-inflammatory activity was significantly enhanced;

[0019] (2) For the inhibition of cellular IL-6 secretion, cellular IL-4 secretion, and inhibition of TNF-α secretion, the present application obtained the corresponding degradation time through experiments. The degradation time shows that the longer the degradation time is, the better the anti-inflammatory effect on cells is not necessarily the case;

[0020] (3) The peanut meal polysaccharide extraction process employed in this application utilizes hot water extraction, which has the advantage of minimizing the cleavage of glycosidic bonds and the destruction of the polysaccharide structure in the sample. Furthermore, the mild experimental conditions and low equipment requirements provide favorable conditions for subsequent degradation. Furthermore, the degradation process of the peanut meal polysaccharide utilizes a combination of microbial degradation, VC, and H2O2 degradation, further increasing the degradation rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Attachment Figure 1 This is the GC spectrum of the undegraded derivative of peanut meal polysaccharide;

[0022] Attachment Figure 2 This is the GC spectrum of the derivatives after 1.25h degradation of peanut meal polysaccharide;

[0023] Attachment Figure 3 This is the GC spectrum of the derivatives of peanut meal polysaccharide degradation for 2.5 hours;

[0024] Attachment Figure 4 The infrared spectra of peanut meal polysaccharides at different degradation times;

[0025] Attachment Figure 5 Effects of peanut meal polysaccharides on IL-6 expression in RAW 264.7 cells;

[0026] Attachment Figure 6 Effects of peanut meal polysaccharides on IL-4 expression in RAW 264.7 cells;

[0027] Attachment Figure 7 This is the effect of peanut meal polysaccharides on the expression level of TNF-α in RAW 264.7 cells. DETAILED DESCRIPTION

[0028] In order to enable those skilled in the art to better understand the present invention, the present invention will be further explained in conjunction with specific embodiments.

[0029] Example 1 Extraction of peanut meal polysaccharides and determination of yield and purity

[0030] 1.1 Extraction of peanut meal polysaccharides

[0031] In the process of extracting peanut meal polysaccharides and determining their yield and purity, the main reagents used include:

[0032] Table 1 Main reagents and materials

[0033] Reagents or materials Specifications or parameters Manufacturer <![CDATA[Iodine (I2)]]> analytically pure Tianjin Guangcheng Chemical Reagent Co., Ltd. Potassium iodide (KI) analytically pure Sinopharm Chemical Reagent Co., Ltd. High-temperature α-amylase Food grade Beijing Solebow Technology Co., Ltd. D-anhydrous glucose 99％ Shanghai MacLean Biochemical Technology Co., Ltd. 3,5-Dinitrosalicylic acid (DNS) analytically pure Shanghai Yuanye Biotechnology Co., Ltd. Anhydrous ethanol analytically pure Tianjin Funing Fine Chemical Co., Ltd. sulfuric acid analytically pure Sinopharm Chemical Reagent Co., Ltd. phenol analytically pure Sinopharm Chemical Reagent Co., Ltd. MSC80003 ultrafiltration membrane Molecular weight cut-off 3kDa Shanghai Mosu Scientific Instruments Co., Ltd.

[0034] The main instruments used are: electronic scale, digital overhead electronic stirrer, vertical pressure steam sterilizer, low-temperature refrigerated centrifuge, heat-collecting constant temperature heating magnetic stirrer, etc. The above equipment are common instruments available on the market and will not be described in detail here.

[0035] Place 100g of peanut meal in a beaker and add pure water at a solid-liquid ratio of 1:10. Stir for 20 minutes to ensure uniform dispersion. Seal with kraft paper and place in an autoclave (121°C, 30 minutes). After extraction, cool to room temperature. Seal the precipitate by centrifugation (5000 rpm, 15 minutes). Collect the supernatant, adjust its pH to 6.0, and measure the volume. Heat in a 90°C water bath and add 10 U / mL of high-temperature α-amylase until the supernatant does not change color when exposed to iodine reagent (2% KI and 1% I2) and the reducing sugar content no longer increases. Samples were collected at 20, 30, and 40 minutes. After enzymatic hydrolysis, separate the precipitate by centrifugation (9000 rpm, 10 minutes). Collect the supernatant, remove impurities through ultrafiltration using a 3k ultrafiltration membrane, and rotary evaporation (60°C) to obtain approximately 200mL of polysaccharide concentrate. Add anhydrous ethanol at a ratio of 1:4 and allow to precipitate overnight at 4°C. After the alcohol precipitation is completed, the supernatant is separated by centrifugation (4000 rpm, 10 min), the precipitate is spread on a clean culture dish, placed in an oven at 40-50°C for drying, ground, and the dry powder is collected and stored in a sealed container at 4°C away from light.

[0036] 1.2 Determination of the yield and purity of peanut polysaccharides

[0037] The yield and purity were determined as follows:

[0038]

[0039] The purity was determined by the phenol-sulfuric acid method. Reference: Research on steam explosion-assisted extraction of water shield polysaccharides and determination of their polysaccharide content by phenol-sulfuric acid method [J]. Grain and Oils, 2022, 35(05): 116-120, Author: Jin Jin, Liu Yanqi, Qin Lingxiang.

[0040] Calculation formula:

[0041] In Example 1, a total of 100 g of peanut meal was used to extract 5.62 g of peanut meal polysaccharide. According to the above formula, the yield of peanut meal polysaccharide was 5.62%.

[0042] After calculation, the purity is 48.83%.

[0043] Example 2 Degradation of peanut meal polysaccharides

[0044] The peanut meal polysaccharide prepared in Example 1.1 was degraded. The specific degradation method is as follows:

[0045] 2.1 Degradation of peanut meal polysaccharides

[0046] Accurately weigh 2.50g of peanut meal polysaccharide and place it in a 500mL wire-top reagent bottle. Add an appropriate amount of pure water and shake to dissolve. Heat the peanut meal polysaccharide aqueous solution to 35-40°C. Add Lactobacillus powder at an amount of approximately 0.8% of the peanut polysaccharide weight. Ferment for 2 hours. Add an appropriate amount of Vitamin C and H2O2 solution to the solution, and add pure water to 250mL to achieve an H2O2 concentration of 75mM and a Vitamin C concentration of 20mM. Mix thoroughly. Place the reagent bottle in a water bath shaker at 25°C for 1.25 hours. Repeat the above steps for another 2.5 hours.

[0047] After treatment, add manganese dioxide (MnO2) at a ratio of 0.05 g / mL to terminate the reaction. Separate the precipitate (MnO2) by centrifugation (10,000 rpm, 15 min) and obtain the supernatant. Add anhydrous ethanol at a ratio of 1:4 and allow to precipitate overnight at 4°C. After precipitation, separate the supernatant by centrifugation (10,000 rpm, 15 min). Spread the precipitate onto a clean Petri dish, dry it in an oven at 55°C, grind it, collect the dry powder, and store it in a sealed container at 4°C in the dark.

[0048] 2.2 Degradation rate of peanut meal polysaccharides

[0049] Place dried peanut meal polysaccharide powder (undegraded, degraded for 1.25 hours, and degraded for 2.5 hours) in a clean beaker. Add an appropriate amount of pure water and stir to dissolve, creating solutions of varying concentrations. Remove the Ubbelohde viscometer and inject the first set of sample solutions into ball B along tube 2. Place the instrument vertically in a 25°C constant-temperature water bath, with the bath liquid level above ball C. Allow the instrument to stand for 15 minutes. Connect a clamped latex tube to tube opening 1. Use an ear bulb to pump air at tube opening 3, slowly raising the solution level to the center of ball C. Open tube openings 1 and 3, respectively, allowing the sample solution to fall naturally. Start timing at mark m1 and stop at mark m2.

[0050] Repeat the measurement twice. The difference between the two measurements should not exceed 0.1 seconds. The average value is taken as the outflow time of the sample solution. Using pure water as the control group, repeat the above steps and measure the data of the remaining two groups.

[0051] Calculate intrinsic viscosity:

[0052] Where:

[0053] η sp (Specific viscosity) = η r -1

[0054] t n : Sample measurement time

[0055] t0: Pure water measurement time

[0056] C: sample concentration

[0057] The outflow time of pure water was 1:39:59 as measured by an Ubbelohde viscometer.

[0058] Table 2 Viscosity of polysaccharide solutions at different degradation times

[0059] Solution grouping Undegraded group Degradation 1.25h group Degradation 2.5h group time 1:59:49 1:51:10 1:45:94 <![CDATA[η r ]]> 1.200 1.116 1.064 <![CDATA[η sp ]]> 0.200 0.116 0.064 [η] 0.5719 0.4253 0.3212

[0060] According to the above formula, the viscosity of peanut meal polysaccharide solution at different degradation times is shown in Table 6-2. The degradation rate of peanut meal polysaccharide is 25.63% after 1.25 hours of degradation and 43.84% after 2.5 hours of degradation.

[0061] 2.3 Analysis of monosaccharide composition before and after peanut meal degradation

[0062] The monosaccharide composition was determined as follows:

[0063] The monosaccharide composition of the peanut meal polysaccharide sample was determined using ion exchange chromatography. Chromatographic conditions were as follows: Monosaccharide composition analysis was performed using a Dionex ICS-3000 ion chromatograph equipped with a 3×150 mm Xtimate C18 column (4.6×200 mm, 5 μm), with an injection volume of 20 μL. The mobile phase consisted of ultrapure water, 0.02 mol / L sodium hydroxide (NaOH) solution, and 0.5 mol / L sodium acetate (NaAc) solution. A gradient elution method was used, with the elution program shown in Table 4-3, a flow rate of 1.0 mL / min, and a column temperature of 30°C.

[0064] Control solution: Accurately weigh appropriate amounts of mannose (Man), ribose (Rib), rhamnose (Rha), glucuronic acid (GlcA), galacturonic acid (GalA), N-acetyl-glucosamine (GlcNAc), glucose (Glc), N-acetyl-galactosamine (GalNAc), galactose (Gal), xylose (Xyl), arabinose (Ara), and fucose (Rha), and dissolve and dilute with water to a mixed control solution containing 50 μg of each per 1 mL.

[0065] Hydrolysis of the sample: Accurately weigh an appropriate amount of sample into a 10 mL ampoule. Add 3.0 mL of 2 mol / L trifluoroacetic acid (TFA) to the ampoule. Aerate the ampoule with nitrogen, seal the tube, and incubate at 120°C for 4 h. After the acid hydrolysis is complete, pipette 1.0 mL of the sample and add an appropriate amount of methanol. Repeatedly evaporate the sample with nitrogen to remove any residual acid. Finally, reconstitute the sample with 1.0 mL of water and filter through a 0.22 μm microporous filter before use.

[0066] Table 3 Monosaccharide composition analysis ion exchange chromatography elution procedure

[0067] Elution time / min 0 15 15.01 30 Ultrapure water (%) 90 90 70 70 0.02mol / L NaOH (%) 10 10 10 10 0.5mol / L NaAc(%) 0 0 20 20

[0068] (1) Attachment Figure 1 Table 4 is the GC spectrum analysis results of the undegraded derivatives of peanut meal polysaccharides:

[0069] Table 4 GC spectrum analysis results of undegraded derivatives of peanut meal polysaccharides

[0070]

[0071]

[0072] Combine Figure 1As shown in Table 4, the elution order of the eight standard monosaccharide derivatives on the capillary column is as follows: ribose and rhamnose have similar elution times, while galactose, xylose, and arabinose are relatively close; all monosaccharides achieve good baseline separation. As shown in Table 6-3, the main monosaccharide components of the undegraded peanut meal polysaccharide are mannose (0.860%), ribose (5.632%), rhamnose (1.605%), glucose (55.156%), galactose (5.970%), xylose (0.697%), arabinose (8.629%), and fucose (4.716%), with glucose having the highest content.

[0073] (2) Analysis of monosaccharide composition after 1.25 h degradation

[0074] Attachment Figure 2 This is the monosaccharide analysis of the peanut meal polysaccharide in Example 1 after 1.25 hours of degradation using the method in Example 2. Table 5 shows the GC spectrum analysis results of the derivatives of peanut meal polysaccharide degradation for 1.25 hours:

[0075] Table 5 GC spectrum analysis results of peanut meal polysaccharide degradation 1.25h derivatives

[0076] Peak order Monosaccharide name <![CDATA[Retention time (T R / min)]]> Composition percentage (%) 1 Mannose (Man) 14.079 1.287 2 Ribose 18.041 5.626 3 Rhamnose (Rha) 19.169 9.121 4 Glucose (Glc) 29.820 10.738 5 Galactose (Gal) 34.073 21.356 6 Xylose (Xyl) 35.775 1.282 7 Arabinose (Ara) 37.154 18.689 8 Fucose (Rha) 41.978 6.991

[0077] Combine Figure 2 As shown in Table 5, the elution order of the eight standard monosaccharide derivatives on the capillary column is similar; ribose and rhamnose have similar elution times, while galactose, xylose, and arabinose are relatively close. All monosaccharides achieve good baseline separation. Table 6-4 shows that the main monosaccharide composition of peanut meal polysaccharides after 1.25 h of degradation is mannose (1.287%), ribose (5.626%), rhamnose (9.121%), glucose (10.738%), galactose (21.356%), xylose (1.282%), arabinose (18.689%), and fucose (6.991%), with galactose having the highest content.

[0078] (3) Analysis of monosaccharide composition after 2.5 h degradation

[0079] Attachment Figure 3 This is the monosaccharide analysis of the peanut meal polysaccharide in Example 1 after 1.25 hours of degradation using the method in Example 2. Table 6 shows the GC spectrum analysis results of the derivatives of peanut meal polysaccharide degradation for 1.25 hours:

[0080] Table 6 GC spectrum analysis results of peanut meal polysaccharide degradation 2.5h derivatives

[0081] Peak order Monosaccharide name <![CDATA[Retention time (T R / min)]]> Composition percentage (%) 1 Mannose (Man) 14.085 1.329 2 Ribose 18.069 5.744 3 Rhamnose (Rha) 19.178 7.819 4 Glucose (Glc) 29.860 11.148 5 Galactose (Gal) 34.100 19.567 6 Xylose (Xyl) 35.797 1.397 7 Arabinose (Ara) 37.190 17.485 8 Fucose (Rha) 42.000 7.536

[0082] Combine Figure 3As shown in Table 6, the elution order of the eight standard monosaccharide derivatives on the capillary column is similar; ribose and rhamnose have similar elution times, while galactose, xylose, and arabinose are relatively close. All monosaccharides achieve good baseline separation. Table 6-4 shows that the main monosaccharide composition of peanut meal polysaccharides after 1.25 h of degradation is mannose (1.329%), ribose (5.744%), rhamnose (7.819%), glucose (11.148%), galactose (19.567%), xylose (1.397%), arabinose (17.485%), and fucose (7.536%), with galactose having the highest content.

[0083] 2.4 Analysis of monosaccharide and uronic acid composition at different degradation times

[0084] Table 7 Percentage of monosaccharide and uronic acid composition at different degradation times

[0085]

[0086] As shown in Table 7, the glucose content of peanut meal polysaccharides decreased significantly during the degradation process, while the galactose and arabinose contents increased; the glucuronic acid content decreased, while the galacturonic acid content increased.

[0087] 2.5 Infrared spectroscopic determination results of peanut meal polysaccharides at different degradation times

[0088] Infrared spectrum scanning

[0089] The peanut meal polysaccharide sample and potassium bromide (KBr) were dried in an oven at 105°C for 4 hours, and then placed in a desiccator to cool for 2 hours. 4 mg of sample and 80 mg of KBr were accurately weighed and ground thoroughly in an agate mortar until mixed evenly. The ground sample and KBr mixture were transferred to a tableting mold and subjected to a pressure of 20 MPa for 20 seconds. The pressed potassium bromide tablets were dried for 30 minutes and scanned using a Fourier transform infrared spectrometer with a scanning range of 4000-400 cm -1 , with a resolution of 4cm -1 .

[0090] according to Figure 4 It can be seen that the three peanut meal polysaccharides all have characteristic peaks of carbohydrate compounds. -1 The broad peak at 2937.8~2962.9cm is the absorption peak caused by the stretching vibration of a large number of -OH and OH in the polysaccharide molecules. -1 and 1400.5~1416.4cm -1 The absorption peaks are caused by CH stretching vibration and bending vibration, which are characteristic absorption peaks of carbohydrate compounds.

[0091] Figure 4 In the middle, between 1651.7 and 1663.2 cm -1 The absorption peak at 1238.3~1245.1cm is caused by the C=O stretching vibration of the -COOH functional group. -1 The CO stretching vibration absorption peak is at 1048.7~1082.9cm -1 The absorption peak is the COC absorption peak of the ether bond in the pyranose ring.

[0092] The undegraded group was at 1539.8 cm -1 There is an absorption peak at 795.1cm, while the absorption peaks of the two groups of degradation are less obvious. This is caused by the vibration of the benzene ring skeleton and is one of the most important peaks for determining the presence of benzene rings in polysaccharides. -1 There is a small peak at both ends, which indicates that the degradation process leads to the displacement of the benzene ring, resulting in substitution at the ortho-meta-position. Both groups of degraded polysaccharides have an absorption peak at 1315.9 cm-1, which is the characteristic absorption peak of -CH3. It can be inferred that the degradation process causes the glycosidic bond to break, thereby generating more -CH3.

[0093] In summary, except for the substitution of the benzene ring at 1539.8 cm-1 by the ortho-meta position, the other major functional groups did not change significantly, but there were changes in quantity, indicating that the degradation process led to the cleavage of the glycosidic bond and the generation of more free groups.

[0094] 2.6 Verification of the anti-inflammatory effect of peanut meal polysaccharides

[0095] 2.6.1(1) Reagent preparation

[0096] Cell culture medium: Sterilize the wire-top reagent bottle in advance, take out 20 mL of fetal bovine serum (FBS) from the sterile biological cabinet and thaw it. Filter it with a sterilized 0.22 μm filter membrane, adjust the volume to 200 mL with DMEM high-glucose culture medium, add 2 mL of double-antibody (penicillin-streptomycin 100X), and place it into the treated wire-top reagent bottle. Store at 4°C until use.

[0097] High-glucose DMEM complete medium: DMEM high-glucose medium + 10% FBS.

[0098] Preparation of LPS solution: Accurately weigh 1.0 mg of LPS and dissolve it in 4 mL of high-glucose DMEM by ultrasonication to prepare a 0.25 mg / mL solution. Aliquot and store at -20°C.

[0099] Cell freezing solution: Pipette 3 mL FBS and 1.2 mL DMSO into a 15 mL centrifuge tube, and add high-glucose DMEM to 15 mL (72% DMEM high-glucose medium + 20% FBS + 8% DMSO). Prepare it before use.

[0100] (2) Cell culture

[0101] Cell recovery: Take the RAW 264.7 cell line out of the -80°C freezer, quickly thaw it in a water bath (37°C), and then transfer it to a clean bench. Pipette 5mL of complete culture medium into a 15mL sterile centrifuge tube, and then transfer the dissolved cell suspension into the centrifuge tube. Take 1mL of complete culture medium from the centrifuge tube and gently blow the cell freezing tube, wash it once, wash the remaining attached cells and transfer them to the centrifuge tube, discard the supernatant by centrifugation (1000rpm, 5min), replace with new complete culture medium, and gently blow to mix so that the cells are suspended and evenly distributed. Mark it and place it in an incubator (37°C, 5% CO2) for culture.

[0102] Cell culture medium replacement: Culture cells according to the above conditions and observe their condition before replacing the culture medium, usually every 1-2 days. Before replacing the culture medium, wash the culture medium twice with PBS buffer. Select cells with good growth status for the experiment.

[0103] Cell passaging: Observe cell growth under a microscope. Disinfect and sterilize the test bench, discard the original culture medium, and wash the RAW 264.7 cells at approximately 80% confluency with PBS buffer. Digest the adherent cells with an appropriate amount of trypsin. Transfer the cell suspension to a 15 mL centrifuge tube containing 3 mL of complete culture medium. Centrifuge (1000 rpm, 5 min), discard the supernatant, add 3 mL of complete culture medium, gently pipette to mix, and incubate as described above.

[0104] Cell freezing: Remove serum from the -80°C freezer and DMSO from the -4°C freezer, then thaw in a water bath (37°C). Select RAW 264.7 cells in the logarithmic phase, discard the original culture medium, and wash once with 5 mL of PBS buffer. Gently pipette to remove adherent cells. Centrifuge (1000 rpm, 5 min) and discard the supernatant. Add cell freezing solution, pipette repeatedly to evenly distribute, and transfer 1 mL per tube into cryovials. Label the tubes, place in a programmable cryovial box, and transfer to a -80°C freezer.

[0105] (3) Preparation and sterilization of polysaccharide solution

[0106] The concentration of peanut meal polysaccharide in the cell culture was 300 μg / mL. Due to the low solubility of the polysaccharide, a 10X stock solution could not be prepared. Therefore, a 2X stock solution was prepared using full culture medium, resulting in a polysaccharide solution with a concentration of 600 μg / mL.

[0107] Preparation of sample solution: In a sterile clean bench, take about 20 mL of complete culture medium and place it in a sterile centrifuge tube. Accurately weigh 0.0030 g of undegraded, degraded for 1.25 h, and degraded for 2.5 h polysaccharide powder, place them in 10 mL centrifuge tubes and add 5 mL of sterile culture medium. After vortexing to dissolve, prepare a solution with a concentration of 600 μg / mL.

[0108] Sterilization: After the centrifuge tube is completely sterilized with a large amount of alcohol spray, it is placed on a centrifuge tube rack in the clean bench. (Note: At this time, the tube mouth, the inside of the tube and the lid, as well as the liquid in the tube are still sterile, so be careful to place them separately from sterile instruments to avoid contamination.) Take 3 sterile centrifuge tubes and place them on another centrifuge tube rack in the clean bench and open the lid. After tearing open the packaging of the 0.22um sterile filter, use tweezers to grab the cylindrical tube part of the filter and place it on a sterile centrifuge tube. Use one filter for each sample to avoid cross contamination. Use a sterile syringe with a needle to draw up the prepared polysaccharide solution, take the needle cap and cover the needle, remove the needle with force, insert the syringe into the cylindrical tube of the sterile filter, press down the syringe, and transfer the filtrate to a new sterile centrifuge tube. After the filtration is completed, discard the syringe, filter and original centrifuge tube to avoid contamination. The sterilized polysaccharide solution is briefly placed at 4°C for later use.

[0109] (4) Cell treatment

[0110] A blank control group (RAW 264.7 cells + PBS), an LPS model group (RAW 264.7 cells + LPS), and an experimental group (i.e., peanut meal polysaccharide + LPS group at different degradation time periods) were set up.

[0111] Observe the cells under a microscope and select RAW 264.7 cells in the logarithmic growth phase and in good condition for the experiment. Take 500 μL of polysaccharide solution and add it to 500 μL of cell culture medium and mix well. Digest the adherent cells, resuspend them in fetal bovine serum medium, and inoculate them in the above medium. After inoculation, incubate the cells in an incubator (37°C, 5% CO2) for 24 hours. Discard the original medium and divide the cells into groups according to the experiment. Each group is treated with LPS at a final concentration of 1 μg / mL for 24 hours. After the treatment, separate the precipitate by centrifugation (1000 rpm, 5 minutes). The supernatant is collected into a sterile centrifuge tube, labeled, and stored at 4°C for short-term use and -20°C for long-term use.

[0112] (5) Determination of cytokines IL-6, IL-4, and TNF-α by ELISA

[0113] ① Reagent preparation: Remove the kit from the refrigerator in advance and equilibrate it at room temperature for 20 minutes. All liquid components must be shaken thoroughly before use.

[0114] Dilution of 20X Wash Buffer: Add purified water to dilute 1:20.

[0115] ② Reagent Procedure: Remove the desired strips from the aluminum foil bag after equilibration at room temperature for 20 minutes. Seal the remaining strips in a ziplock bag and return them to 4°C. Set up standard and sample wells and add 50 μL of standard at different concentrations to the standard wells. First, add 10 μL of the sample to be tested to the sample wells, followed by 40 μL of sample diluent; leave blank wells untouched. Add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well, except the blank wells. Seal the wells with plate sealing film and incubate in a constant temperature incubator (37°C) for 1 hour.

[0116] After incubation, discard the liquid, pat dry on absorbent paper, and fill each well with diluted wash solution. Let stand for 1 minute, then discard the wash solution and pat dry on absorbent paper. Repeat this process five times. After washing, add 50 μL each of substrates A and B to each well and incubate in a thermostat (37°C) in the dark for 15 minutes. After incubation, add 50 μL of stop solution to each well and measure the absorbance of each well at 450 nm using a microplate reader within 15 minutes.

[0117] ③Draw a standard curve

[0118] With the concentration of the standard as the horizontal axis and the corresponding OD value as the vertical axis, a linear regression curve of the standard was drawn, and the concentration value of each sample was calculated according to the curve equation.

[0119] ④Statistical analysis

[0120] The experimental data are expressed as mean ± standard deviation, and the results were repeated three times. SPSS software was used for data analysis, with P < 0.05 being considered significant and P < 0.01 being extremely significant. Graphs were drawn using Graphpad Prism 9.5.

[0121] 2.6.2(1) Effects of polysaccharides on IL-6 expression in RAW 264.7 cells

[0122] according to Figure 5It can be seen that the difference between the control group and the LPS group was extremely significant. Compared with the control group, the IL-6 level in the LPS group was significantly increased, which shows that the inflammatory model was successfully established, that is, after LPS induction, RAW 264.7 cells showed significant inflammation; there was no significant difference between the LPS group and the undegraded group. Compared with the LPS group, the IL-6 level in the undegraded group did not show a significant decrease, indicating that the peanut meal polysaccharide in the undegraded group could not effectively inhibit the secretion of IL-6 in the cells; there was a significant difference between the LPS group and the 1.25h degradation group. Compared with the LPS group, the IL-6 level in the 1.25h degradation group was significantly reduced, indicating that the peanut meal polysaccharide degraded for 1.25h can significantly inhibit the secretion of IL-6 in the cells; there was a significant difference between the LPS group and the 2.5h degradation group. Compared with the LPS group, the IL-6 level in the 2.5h degradation group was reduced to a certain extent, indicating that the peanut meal polysaccharide degraded for 2.5h can inhibit the secretion of IL-6 in the cells, but the inhibitory effect was not as obvious as that of the 1.25h degradation group.

[0123] In summary, the three peanut meal polysaccharides all showed an inhibitory effect on the secretion of IL-6 by RAW 264.7 cells induced by LPS, showing a certain anti-inflammatory activity. Among them, the peanut meal polysaccharide degraded for 1.25 h had the strongest anti-inflammatory activity, which could reduce the IL-6 level by 37.82% (compared with the LPS group). (2) Effect of polysaccharides on the expression level of IL-4 in RAW 264.7 cells

[0124] according to Figure 6 It can be seen that there is a significant difference between the control group and the LPS group. Compared with the control group, the IL-4 level in the LPS group was significantly increased, which shows that the inflammatory model was successfully established. That is, after LPS induction, RAW 264.7 cells showed significant inflammation; there was no significant difference between the LPS group and the 1.25h degradation group, but compared with the LPS group, the IL-4 level of the 1.25h degradation group was reduced to a certain extent, indicating that the peanut meal polysaccharide degraded for 1.25h can inhibit the secretion of cellular IL-4, but the inhibitory effect is not obvious; the difference between the LPS group and the 2.5h degradation group was extremely significant, compared with the LPS group, the IL-4 level of the 2.5h degradation group was significantly reduced, indicating that the peanut meal polysaccharide degraded for 2.5h can significantly inhibit the secretion of cellular IL-4; there was no significant difference between the LPS group and the non-degraded group, compared with the LPS group, the IL-4 level of the non-degraded group was slightly increased, which may be an error, but both groups of degraded polysaccharides had a certain inhibitory effect on the secretion of IL-4 by cells. It is speculated that the peanut meal polysaccharide in the non-degraded group can also inhibit the secretion of IL-4 in cells, but the effect is not obvious.

[0125] In summary, the three peanut meal polysaccharides all showed an inhibitory effect on the secretion of IL-4 by RAW 264.7 cells induced by LPS, and exhibited a certain anti-inflammatory activity. Among them, the peanut meal polysaccharide degraded for 2.5 hours had the strongest anti-inflammatory activity, which could reduce the IL-4 level by 39.68% (compared with the LPS group). (3) Effect of polysaccharides on the expression level of TNF-α in RAW 264.7 cells

[0126] according to Figure 7 It can be seen that the difference between the control group and the LPS group was extremely significant. Compared with the control group, the TNF-α level in the LPS group was significantly increased, which indicated that the inflammatory model was successfully established, that is, after LPS induction, RAW 264.7 cells showed significant inflammation; there was a significant difference between the LPS group and the undegraded group. Compared with the LPS group, the TNF-α level in the undegraded group decreased to a certain extent, indicating that the peanut meal polysaccharide in the undegraded group could inhibit the secretion of TNF-α in the cells; there was a significant difference between the LPS group and the 1.25h degradation group. Compared with the LPS group, the TNF-α level in the 1.25h degradation group was significantly reduced, indicating that the peanut meal polysaccharide degraded for 1.25h could significantly inhibit the secretion of TNF-α in the cells; there was a significant difference between the LPS group and the 2.5h degradation group. Compared with the LPS group, the TNF-α level in the 2.5h degradation group was reduced to a certain extent, indicating that the peanut meal polysaccharide degraded for 2.5h could inhibit the secretion of TNF-α in the cells, and the inhibitory effect was better than that of the undegraded group.

[0127] In summary, all three peanut meal polysaccharides inhibited LPS-induced TNF-α secretion in RAW 264.7 cells, demonstrating certain anti-inflammatory activity. Among them, the peanut meal polysaccharide that was degraded for 1.25 hours had the strongest anti-inflammatory activity, reducing TNF-α levels by 37.32% (compared to the LPS group). As shown in the figure, after LPS stimulation of RAW 264.7 cells, the levels of IL-6, IL-4, and TNF-α significantly increased from 13.12, 5.20, and 132.89 pg / mL to 17.85, 12.60, and 168.75 pg / mL, respectively. Compared with the LPS group, peanut meal polysaccharides could extremely or significantly inhibit the secretion of these three cytokines. Among them, peanut meal polysaccharides degraded for 1.25 h had the greatest effect on the expression levels of IL-6 and TNF-α in RAW 264.7 cells, and had the best effect in inhibiting the release of IL-6 and TNF-α. Compared with the LPS group, the levels of IL-6 and TNF-α could be reduced by 37.82% and 37.32%, respectively; peanut meal polysaccharides degraded for 2.5 h had the greatest effect on the expression level of IL-4 in RAW 264.7 cells, and had the best effect in inhibiting the release of IL-4, which could reduce the IL-4 level by 39.68%.

[0128] As shown in the figure, after LPS stimulation of RAW 264.7 cells, IL-6, IL-4, and TNF-α levels significantly increased from 13.12, 5.20, and 132.89 pg / mL to 17.85, 12.60, and 168.75 pg / mL, respectively. Compared with the LPS group, peanut meal polysaccharides significantly or extremely significantly inhibited the secretion of these three cytokines. Peanut meal polysaccharides degraded for 1.25 hours had the greatest effect on IL-6 and TNF-α expression in RAW 264.7 cells, and showed the best inhibitory effect on IL-6 and TNF-α release, reducing IL-6 and TNF-α levels by 37.82% and 37.32%, respectively, compared to the LPS group. Peanut meal polysaccharides degraded for 2.5 hours had the greatest effect on IL-4 expression in RAW 264.7 cells, and showed the best inhibitory effect on IL-4 release, reducing IL-4 levels by 39.68%.

[0129] Through the analysis of peanut meal polysaccharides, it was found that peanut meal polysaccharides contain relatively high proportions of monosaccharides such as arabinose, glucose, xylose, mannose and galactose. These monosaccharides give peanut meal polysaccharides better anti-inflammatory activity.

[0130] According to the experiments of this application, it can be inferred that the higher the content of mannose and xylose, the higher the anti-inflammatory activity of peanut meal polysaccharides on IL-4; the higher the content of galactose and arabinose, the higher the anti-inflammatory activity of peanut meal polysaccharides on IL-6 and TNF-α.

[0131] Therefore, this application solves the problem of low solubility of peanut meal polysaccharides and difficulty in effective utilization by degrading peanut meal polysaccharides, and conducts a large number of experiments on the anti-inflammatory activity of IL-6, TNF-α and IL-4 to obtain the optimal degradation time.

Claims

1. A method for significantly improving the anti-inflammatory effect of peanut meal polysaccharides, characterized in that: The following steps are involved: (1) Extraction of peanut meal polysaccharides: Peanut meal was taken and peanut meal polysaccharide was extracted by hot water extraction method; (2) Degradation of peanut meal polysaccharides: Take 2.50 g of peanut meal polysaccharide obtained in (1), place it in a wire-top reagent bottle, add pure water to dissolve it, add Lactobacillus powder at 35-40℃, the amount of which is 0.5-1% of the weight of peanut polysaccharide, ferment for 1-3 hours, then add Vc and H2O2 solution, and finally add pure water to make the H2O2 concentration 75 mM and the Vc concentration 20 mM, mix well, place the reagent bottle in a water bath shaker at 25-30℃ for 1-2.5 hours, and let it stand; (3) Manganese dioxide is added to the mixed solution in (2) to terminate the reaction, centrifuged, and the precipitate is separated to obtain the supernatant. Anhydrous ethanol is then added to precipitate the solution. After the precipitation is completed, centrifuged, the supernatant is separated, and the precipitate is spread on a clean culture dish, dried, ground, and the dry powder is collected to obtain peanut meal polysaccharide with enhanced anti-inflammatory effect.

2. A method for significantly improving the anti-inflammatory effect of peanut meal polysaccharides according to claim 1, characterized in that: (3) Add manganese dioxide at a ratio of 0.05 g / mL to the solution mixed in (2) to terminate the reaction, centrifuge at 10,000 rpm for 15 min, separate the precipitate, obtain the supernatant, add anhydrous ethanol to the supernatant at a volume ratio of 1:4, place it at 4°C for alcohol precipitation for 6 to 12 hours, centrifuge at 10,000 rpm for 15 min, separate the supernatant, spread the precipitate on a clean culture dish, place it in an oven, dry it at 55°C, grind it, collect the dry powder, and store it in a sealed container at 4°C in the dark.

3. A method for significantly improving the anti-inflammatory effect of peanut meal polysaccharides according to claim 1, characterized in that: (2) After the peanut meal polysaccharide was degraded for 1.25~2.5h, the final product obtained contained 1.287~1.329% mannose, 5.626~5.744% ribose, 7.819~9.121% rhamnose, 10.738%~11.148% glucose, 19.567~21.356% galactose, 1.282~1.397% xylose, 17.485~18.689% arabinose and 6.991~7.536% fucose.

4. A method for significantly improving the anti-inflammatory effect of peanut meal polysaccharides according to claim 1, characterized in that: (2) To inhibit the secretion of IL-6 or the release of TNF-α, the degradation time is controlled to be 1.25h.

5. A method for significantly improving the anti-inflammatory effect of peanut meal polysaccharides according to claim 1, characterized in that: In (2), to inhibit the secretion of IL-4 in cells, the degradation time is controlled to be 2.5h.

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