Immune active black bean seed coat galactomannan, and preparation method and application thereof

Abstract

The application discloses a black bean seed coat galactomannan with immunocompetence and a preparation method thereof; first, black bean seed coats are taken as raw materials, and crude polysaccharides are obtained through acid extraction and alcohol precipitation; then, the crude polysaccharides are separated and purified through ion exchange column and gel column chromatography to obtain purified polysaccharides. The prepared polysaccharides are homogeneous heteropolysaccharides composed of mannose, galactose, glucose and arabinose, and the molar ratio is 0.556:0.398:0.056:0.011, and the polysaccharides have a triple helix structure. In vitro experiments show that the black bean seed coat galactomannan can improve the phagocytosis of RAW264.7 cells and promote the secretion of cytokines NO, TNF-alpha and IL-6. The black bean seed coat galactomannan can be used as an immunoregulator in the fields of health food, medicine and cosmetics, provides a theoretical basis for further development of black bean seed coat resources, and improves the utilization value of black beans and by-products.

Description

Technical Field

[0001] This invention relates to the field of bioactive substance preparation technology, specifically to an immunomodulatory black soybean seed coat galactomannan, its preparation method, and its application. Background Technology

[0002] Immune regulation is one of the important defense strategies for maintaining human health. With the improvement of living standards, the demand for foods and medicines that promote immune function has greatly increased. Compared with chemically synthesized substances, consumers prefer natural products and their extracts that have a lower metabolic burden and are safer. At the same time, the medical, food, and other related industries are gradually increasing their investment in the production and research and development of such products. Polysaccharides are long-chain high-molecular-weight carbohydrates formed by monosaccharides linked by glycosidic bonds. They are widely available and, like nucleic acids and proteins, are essential substances for life activities. Studies have found that plant polysaccharides, as a low-toxicity and highly efficient biological response modifier, have a wide range of activities including antioxidant, anti-tumor, and immunomodulatory effects. In particular, their immunomodulatory capabilities have been widely used in many fields such as the food, biomedicine, and pharmaceutical industries.

[0003] Black soybeans (Glycine max (L.) Merr.) are a traditional Chinese food and medicine crop, rich in various nutrients including protein, fatty acids, pigments, isoflavones, and other bioactive substances. The seed coat, a byproduct of black soybean processing, is characterized by high yield and low production cost. Most importantly, the rich polysaccharides, dietary fiber, anthocyanins, and other active ingredients in the black soybean skin endow it with excellent medicinal activity. In traditional Chinese medicine, black soybean skin, also known as "liao bean husk," is used for detoxification, diuresis, nourishing yin and blood, and calming the liver and kidneys. Modern medical research also shows that the dietary fiber in black soybean skin has an anti-obesity effect to some extent, and the abundant polyphenols can stimulate intestinal cells to secrete GLP-1, promoting the production of NO in the aorta and thus improving vascular function. These characteristics make black soybean skin a potential raw material for the large-scale production of affordable and effective immune-boosting health products.

[0004] Acid extraction, as one of the effective methods for polysaccharide extraction, facilitates the precipitation of certain polysaccharides containing acidic groups by lowering the pH of the solution. However, the presence of a large amount of H₂ in the system also contributes to its effectiveness. + It can effectively inhibit the dissolution of acidic impurities and improve the purity of polysaccharides. Currently, the specific pharmacodynamics and material basis of the biological activity of black soybean hull polysaccharide components are still unclear, and no research reports on the immunomodulatory activity of black soybean hull polysaccharides have been found. Summary of the Invention

[0005] This invention overcomes the shortcomings of existing technologies by providing an immunomodulatory galactomannan from black soybean seed coat and its preparation method. The resulting polysaccharide exhibits uniform composition and good immunomodulatory activity, providing a new approach and theoretical basis for the full utilization of black soybean resources.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is as follows: a black soybean seed coat galactomannan with immunomodulatory activity, wherein the black soybean seed coat galactomannan contains α-configured pyranose with a peak molecular weight of 134753 Da, a weight-average molecular weight of 134194 Da, and a number-average molecular weight of 130178 Da; it has a uniform composition, consisting of mannose, galactose, glucose, and arabinose, with a molar ratio of 0.556:0.398:0.056:0.011; the main chain is connected by →4)-β-D-Manp-(1→4)-β-D-Manp-(1→), and the side chains α-D-Galp-(1→, α-D-Galp-(1→6)-α-D-Glcp-(1→ and α-L-Araf-(1→) are connected to the main chain through the O-6 bond of →4,6)-β-D-Manp-(1→), and have a triple helix structure.

[0007] A method for preparing an immunomodulatory black soybean seed coat galactomannan includes the following steps:

[0008] (1) The crude polysaccharide was obtained by hot acid extraction of black bean skin, concentration of the extract, precipitation with ethanol and freeze-drying.

[0009] (2) After separating the crude polysaccharide by DEAE-52 anion exchange cellulose column, it was dialyzed and then purified by Sephadex G-100 dextran gel column. The polysaccharide was collected by phenol-sulfuric acid method and then freeze-dried to obtain pure polysaccharide, namely black soybean seed coat galactomannan.

[0010] Furthermore, in step (1) hot acid extraction, the pH of the extract is 2.0; the mass-to-volume ratio of black bean skin sample to extract is 1g:30-50mL; the extraction time is 40-60min; and the temperature of the extract is 90-100℃.

[0011] Furthermore, in the ethanol precipitation process described in step (1), the volume ratio of anhydrous ethanol to concentrated liquid is 3-4:1, and precipitation is carried out at 4°C for 10-14 hours.

[0012] Furthermore, the DEAE-52 cellulose packing used in step (2) needs to be pretreated. The pretreatment method is as follows: first soak the packing in 0.2mol / L HCl for 2 hours, rinse the packing with deionized water until neutral, then soak the packing in 0.2mol / L NaOH for 2 hours, rinse with deionized water until neutral, and the column size is 6cm×50cm.

[0013] Furthermore, in step (2), during the separation process using the DEAE-52 cellulose column, the sample concentration was 5-10 mg / mL, the eluent was deionized water, the elution rate was 0.5 mL / min, the polysaccharide components were tracked and collected using the phenol-sulfuric acid method, and the dialysis bag pore size was 500 Da.

[0014] Furthermore, in step (2), during the Sephadex G-100 gel purification process, the column size is 2cm×60cm, the sample concentration is 3-5mg / mL, and deionized water is used for elution at a rate of 0.2mL / min.

[0015] In addition, the present invention also provides the application of the above-mentioned immunomodulatory black soybean seed coat galactomannan in the preparation of health functional foods and immunomodulatory drugs.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The black soybean seed coat galactomannan of this invention has a uniform composition and clear structure, exhibits good immunomodulatory activity, and is a safe and non-toxic natural extract. The polysaccharide in this invention can enhance the phagocytic capacity of RAW264.7 cells within a concentration range of 50-400 mg / mL, and promote the secretion of cytokines such as NO, TNF-α, and IL-6. It can be used as a raw material for health functional foods and immunomodulatory drugs.

[0018] The raw materials for preparing black soybean seed coat galactomannan of the present invention are easy to obtain, the process is simple and the cost is low, which is conducive to large-scale development and production. Attached Figure Description

[0019] Figure 1 The images show the elution profiles of the black soybean peel polysaccharide prepared in Example 1 of this invention using DEAE-52 column chromatography and Sephadex G-100 gel column chromatography.

[0020] Figure 2 The composition of the black soybean skin polysaccharide prepared in Example 1 of this invention is determined by ion chromatography.

[0021] Figure 3 Molecular weight determination diagram of HPGPC, a polysaccharide from black soybean skin prepared in Example 1 of this invention.

[0022] Figure 4 UV spectrum analysis of black soybean skin polysaccharide prepared in Example 1 of this invention.

[0023] Figure 5 FT-IR spectrum analysis of black soybean skin polysaccharide prepared in Example 1 of this invention.

[0024] Figure 6 The hydrogen spectrum of black soybean peel polysaccharide prepared in Example 1 of this invention (1 H NMR spectrum).

[0025] Figure 7 Carbon spectrum of black soybean skin polysaccharide prepared in Example 1 of this invention ( 13 (C NMR spectrum).

[0026] Figure 8 DEPT 135 NMR spectrum of black soybean skin polysaccharide prepared in Example 1 of this invention.

[0027] Figure 9 Homonuclear chemical shift correlation spectrum of black soybean peel polysaccharide prepared in Example 1 of this invention ( 1 H- 1 H COSY diagram).

[0028] Figure 10 The heteronuclear single quantum coherence spectrum (HSQC spectrum) of black soybean skin polysaccharide prepared in Example 1 of this invention.

[0029] Figure 11 Heteronuclear multicarbon correlation (HMBC) spectrum of black soybean skin polysaccharide prepared in Example 1 of this invention.

[0030] Figure 12 NOESY diagram of black soybean skin polysaccharide prepared in Example 1 of this invention.

[0031] Figure 13 Experimental analysis diagram of Congo red polysaccharide prepared in Example 1 of this invention.

[0032] Figure 14 The molecular structure diagram of the black soybean skin polysaccharide prepared in Example 1 of this invention.

[0033] Figure 15 The graph shows the determination of the DPPH free radical scavenging ability of the black soybean skin polysaccharide prepared in Example 1 of this invention.

[0034] Figure 16 The in vitro cytotoxicity analysis of black soybean peel polysaccharide prepared in Example 1 of this invention against RAW264.7 cells.

[0035] Figure 17 The effect of black soybean peel polysaccharide prepared in Example 1 of this invention on the phagocytic activity of RAW264.7 cells is shown in the figure.

[0036] Figure 18 The effect of black soybean peel polysaccharide prepared in Example 1 of this invention on NO secretion in RAW264.7 cells is shown in the figure.

[0037] Figure 19 The effect of black soybean peel polysaccharide prepared in Example 1 of this invention on TNF-α secretion in RAW264.7 cells.

[0038] Figure 20 The effect of black soybean peel polysaccharide prepared in Example 1 of this invention on IL-6 secretion in RAW264.7 cells. Detailed Implementation

[0039] The present invention will be further described below with reference to specific embodiments.

[0040] Example 1

[0041] Extraction, separation and purification of polysaccharides from black soybean skin:

[0042] 1. Take 100g of black bean seed coat, wash it clean, add hydrochloric acid solution pre-adjusted to pH 2.0 at a mass-to-volume ratio of 1:40 (g / mL), extract in a 90℃ water bath for 40 min, filter and collect the filtrate.

[0043] 2. Concentrate the filtrate to 20% of its original volume, add 4 times the volume of anhydrous ethanol for precipitation, precipitate overnight at 4°C, filter and discard the filtrate, the solid part is crude polysaccharide, collect and freeze dry after the ethanol evaporates to obtain 8.33 g of product, the crude sugar yield is 8.33%.

[0044] 3. The crude polysaccharide was dissolved in deionized water to a concentration of 10 mg / mL and separated using a pretreated DEAE-52 cellulose column (6 cm × 50 cm). Elution was performed with deionized water at a rate of 0.5 mL / min, and the polysaccharide components were tracked and collected using the phenol-sulfuric acid method. The collected eluent was then dialyzed using a 500 Da dialysis bag to remove small molecules.

[0045] 4. The washed fraction separated from DEAE-52 cellulose was dissolved again, and the supernatant was collected by stirring and centrifugation (centrifugation conditions: 3000 rpm, 10 min). Purification was performed using a Sephadex G-100 dextran gel column with deionized water at a rate of 0.2 mL / min. The eluted fraction was tracked and collected using the phenol-sulfuric acid method, concentrated, and lyophilized to obtain the polysaccharide BBHW. The elution curve is shown in Figure 1. Figure 1 As shown.

[0046] Example 2

[0047] Extraction, separation and purification of polysaccharides from black soybean skin:

[0048] 1. Take 200g of black bean seed coat, wash it clean, add hydrochloric acid solution pre-adjusted to pH 2.0 at a mass-to-volume ratio of 1:30 (g / mL), extract in a 95℃ water bath for 50 min, filter and collect the filtrate.

[0049] 2. Concentrate the filtrate to 15% of its original volume, add 4 times the volume of anhydrous ethanol for precipitation, precipitate overnight at 4°C, filter and discard the filtrate, the solid part is crude polysaccharide, collect and freeze dry after the ethanol evaporates to obtain 14.07 g of product, the crude sugar yield is 7.04%.

[0050] 3. The crude polysaccharide was dissolved in deionized water to a concentration of 10 mg / mL and separated using a pretreated DEAE-52 cellulose column (6 cm × 50 cm). Elution was performed with deionized water at a rate of 0.5 mL / min, and the polysaccharide components were tracked and collected using the phenol-sulfuric acid method. The collected eluent was then dialyzed using a 500 Da dialysis bag to remove small molecules.

[0051] 4. The water-washed fraction separated from DEAE-52 cellulose was dissolved again, and the supernatant was collected by stirring and centrifugation (centrifugation conditions: 3000 rpm, 10 min). The supernatant was purified using a Sephadex G-100 dextran gel column, eluted with deionized water at a rate of 0.2 mL / min, and the eluted fraction was tracked and collected by the phenol-sulfuric acid method, concentrated and lyophilized to obtain polysaccharide BBHW.

[0052] Example 3

[0053] 1. Take 500g of black bean seed coat, wash it clean, add HCl solution pre-adjusted to pH 2.0 at a mass-to-volume ratio of 1:35 (g / mL), extract in a 100℃ water bath for 60 min, filter and collect the filtrate.

[0054] 2. Concentrate the filtrate to 10% of its original volume, add 4 times the volume of anhydrous ethanol for precipitation, precipitate overnight at 4°C, filter and discard the filtrate, the solid part is the crude polysaccharide, collect it and freeze dry after the ethanol evaporates to obtain 36.95g of product, the crude sugar yield is 7.39%.

[0055] 3. The crude polysaccharide was dissolved in deionized water to a concentration of 10 mg / mL and separated using a pretreated DEAE-52 cellulose column (6 cm × 50 cm). Elution was performed with deionized water at a rate of 0.5 mL / min, and the polysaccharide components were tracked and collected using the phenol-sulfuric acid method. The collected eluent was then dialyzed using a 500 Da dialysis bag to remove small molecules.

[0056] 4. The water-washed fraction separated from DEAE-52 cellulose was dissolved again, and the supernatant was collected by stirring and centrifugation (centrifugation conditions: 3000 rpm, 10 min). The supernatant was purified using a Sephadex G-100 dextran gel column, eluted with deionized water at a rate of 0.2 mL / min, and the eluted fraction was tracked and collected by the phenol-sulfuric acid method, concentrated and lyophilized to obtain polysaccharide BBHW.

[0057] Example 4

[0058] Analysis of the main components and monosaccharide composition of polysaccharide BBHW in Example 1 above:

[0059] 1. Determination of total sugar content in BBHW polysaccharide: Using D-glucose as a standard, the total sugar content in the extract was determined by the sulfuric acid-phenol method. A 0.2 mg / mL glucose standard solution was prepared and mixed according to Table 1 below:

[0060] Table 1

[0061]

[0062] The obtained standard curve is: y = 15.06x - 0.0003 (R²) 2 =0.9978); 0.1 mL of BBHW sample (1 mg / mL) was diluted to 2 mL to prepare the same colorimetric system, and its absorbance at 490 nm was measured. The total sugar content was calculated to be 95.22% ± 3.11%.

[0063] 2. Determination of BBHW polysaccharide protein content: Using bovine serum albumin as a standard, the protein content in the extract was determined by the Coomassie brilliant blue method. A 0.1 mg / mL glucose standard solution was prepared and mixed according to Table 2 below:

[0064] Table 2

[0065]

[0066] The obtained standard curve is: y = 7.6729x + 0.0782(R²). 2 =0.9664); 0.1 mL of BBHW sample (1 mg / mL) was diluted to 1 mL to prepare the same colorimetric system, and its absorbance at 595 nm was measured. The total protein content was calculated to be 5.08% ± 0.44%.

[0067] 3. Determination of galacturonic acid content in polysaccharide BBHW: Using galacturonic acid as a standard, the galacturonic acid content in the extract was determined by the m-hydroxybiphenyl colorimetric method. A 0.1 mg / mL galacturonic acid standard solution was prepared and mixed according to Table 3 below:

[0068] Table 3

[0069]

[0070] The obtained standard curve is: y = 10.679x - 0.0178 (R²) 2=0.995); 0.06 mL of BBHW sample (1 mg / mL) was diluted to 6 mL to prepare the same colorimetric system, and its absorbance at 520 nm was measured. The uronic acid content was calculated to be 4.42% ± 0.55%.

[0071] 4. Determination of Monosaccharide Composition of Polysaccharide BBHW: The monosaccharide composition of polysaccharide BBHW was determined by ion chromatography. 5 mg of sample was accurately weighed and added to 2 mL of 3M trifluoroacetic acid. Hydrolysis was carried out at 120 °C for 3 h. The hydrolysate was dried under nitrogen. 5 mL of deionized water was added to fully dissolve the product, and 50 μL was diluted to 1 mL. After centrifugation at 12000 rpm for 5 min, the supernatant was collected, filtered, and analyzed by IC50. The column conditions were: Dionex Carbopac. TM PA20 (3*150mm) column, selected mobile phase: A: H2O, B: 15mM NaOH, C: 15mM NaOH & 100mM NaAc, controlled flow rate: 0.3mL / min, column temperature: 30℃, sample loading volume: 25μL. Analytical results are as follows. Figure 2 As shown, the polysaccharide BBHW is composed of mannose, galactose, glucose and arabinose, with a molar ratio of 0.556:0.398:0.056:0.011.

[0072] Example 5

[0073] Structural characterization of the polysaccharide BBHW prepared in Example 1 above

[0074] 1. Determination of molecular weight of polysaccharide BBHW: The molecular weight and purity of polysaccharide BBHW were determined by HPGPC using a BRT105-103-101 tandem gel column (8×300mm), with 0.2M NaCl solution as the mobile phase, a flow rate of 0.8mL / min, a column temperature of 40℃, and a differential detector RID-10A. The analysis was performed with an injection volume of 25μL.

[0075] The sample and standard were prepared into 2 mg / mL solutions, filtered through a 0.22 μm filter membrane, and analyzed under the chromatographic conditions described above. The equation for the lgMp-RT (peak molecular weight) correction curve was obtained as: y = -0.2025x + 10.934R 2 =0.9998; The equation for the lgMw-RT (weight-average molecular weight) correction curve is: y = -0.2004x + 10.872R 2 =0.9998; The equation for the lgMn-RT (number-average molecular weight) correction curve is: y = -0.201x + 10.876R 2 =0.9996; Test results are as follows Figure 3The peak time of the sample was 28.664 minutes. The prepared polysaccharide BBHW had uniform composition, with a peak molecular weight Mp = 134753 Da, a weight-average molecular weight Mw = 134194 Da, and a number-average molecular weight Mn = 130178 Da. Its polydispersity coefficient Mw / Mn = 1.031, indicating that the molecular weight distribution of polysaccharide BBHW is very narrow.

[0076] 2. UV spectroscopy analysis: Prepare a 4 mg / mL polysaccharide BBHW solution and scan its UV absorption spectrum in the 200-400 nm range. For example... Figure 4 The results showed that the solution did not show ultraviolet absorption at 260 nm and 280 nm, indicating that BBHW does not contain protein or nucleic acid.

[0077] 3. FT-IR Spectroscopy Analysis: After thoroughly drying the polysaccharide BBHW, weigh 1 mg of powder and grind it with 100 mg of KBr powder until homogeneous. Compress the mixture into tablets. FT-IR spectroscopy was used to analyze the polysaccharide powder at 400-4000 cm⁻¹. -1 Within the specified range, polysaccharide samples were scanned, and the results are as follows: Figure 5 As shown, at 3600-3200, 3000-2800, 1400-1200, and 1200-1000 cm -1 The region is the characteristic absorption peak of polysaccharides. At 3410 cm⁻¹ -1 There are relatively broad absorption peaks on the left and right sides, which are absorption peaks of the stretching vibration of -OH, at 1638 cm⁻¹. -1 The absorption peak at 1423 cm⁻¹ is generated by the stretching vibration of the -C=O bond. -1 and 2926cm -1 The absorption peaks at these locations are generated by the angular vibration and stretching vibration of CH in -CH2- or -CH-, respectively, in the range of 1200-1000 cm⁻¹. -1 The three absorption peaks within the range indicate that BBHW contains a pyranose sugar ring, 1027 cm⁻¹. -1 The presence of a characteristic absorption peak for glucose indicates that the BBHW molecule contains some glucose.

[0078] 4. Methylation analysis to determine the BBHW glycosidic bond type of polysaccharide: Take 3 mg of polysaccharide sample, add 1 mL of anhydrous DMSO, quickly add anhydrous alkaline solution, sonicate to completely dissolve, and then add iodomethane solution. React under magnetic stirring at 30℃ in a water bath for 60 min, and finally add 2 mL of ultrapure water to the mixture to terminate the methylation reaction.

[0079] Prepare 1 mL of 2M trifluoroacetic acid (TFA), add methylated polysaccharide, hydrolyze for 90 min, and concentrate to dryness after complete hydrolysis. Dissolve the hydrolysis product in 2 mL of deionized water, reduce with 60 mg sodium borohydride for 8 hours, neutralize the reaction system with glacial acetic acid, concentrate, and dry in a 100°C oven. Add 1 mL of acetic anhydride for acetylation and react at 100°C for 1 h. After cooling, add 3 mL of toluene, concentrate under reduced pressure, and evaporate to dryness. Repeat 5-6 times to remove excess acetic anhydride. Dissolve the acetylated product in 3 mL of CH2Cl2 and transfer to a separatory funnel. Add a small amount of distilled water for extraction, remove the upper aqueous phase, and repeat the operation 3-4 times. Dry the CH2Cl2 layer with an appropriate amount of anhydrous sodium sulfate and bring the volume to 10 mL. Analyze the sample using an Agilent GCMS 6890-5973 gas chromatography-mass spectrometry system. The results are shown in Table 4.

[0080] GC-MS conditions: An RXI-5SIL MS column (30m*0.25mm*0.25um) was used. The temperature program was as follows: initial temperature 120℃, increased to 250℃ at a rate of 3℃ / min, and held for 5 min. The injector and detector temperatures were both 250℃. High-purity helium was used as the carrier gas at a flow rate of 1 mL / min.

[0081] Table 4. Methylation analysis of polysaccharides BBHW

[0082]

[0083] 5. NMR analysis of the chemical structure of polysaccharide BBHW: 50 mg of the polysaccharide sample was repeatedly dissolved in 0.5 mL of D₂O and freeze-dried to ensure sufficient exchange of active hydrogens. Then, at room temperature (25 °C), the sample was dissolved in 0.5 mL of D₂O and analyzed using a 600 MHz NMR spectrometer. 1 H NMR spectrum, 13 C NMR spectrum, DEPT135 one-dimensional spectrum and two-dimensional spectrum.

[0084] Analysis results as follows Figure 6-12 , Figure 6 For the hydrogen spectrum ( 1 H NMR spectrum), Figure 7 Carbon spectrum ( 13 (C NMR spectrum), Figure 8 This is the DEPT 135 spectrum (DEPT 135 NMR spectrum). Figure 9 For homonuclear chemical shift correlation spectrum ( 1 H- 1 H COSY diagram), Figure 10 This is a heteronuclear single-quantum coherence spectrum (HSQC diagram). Figure 11 This is a heteronuclear multicarbon correlation spectrum (HMBC diagram). Figure 12The diagram shows the NOESY pattern. The main glycosidic bond structure of the polysaccharide BBHW isolated and purified from black soybean hulls is as follows: the main chain is linked by glycosidic bonds of →4)-β-D-Manp-(1→4)-β-D-Manp-(1→), and the side chains α-D-Galp-(1→, α-D-Galp-(1→6)-α-D-Glcp-(1→ and α-L-Araf-(1→) are linked to the main chain by O-6 bonds of →4,6)-β-D-Manp-(1→). Detailed NMR spectral analysis is shown in Table 5.

[0085] Table 5. NMR analysis of polysaccharides in BBHW

[0086]

[0087] 6. Congo Red Experiment for Helical Structure of Polysaccharide BBHW: 5 mg of polysaccharide sample was weighed and prepared into a 0.5 mg / mL solution, which was then thoroughly mixed with an equal volume of 80 μmol / L Congo red reagent. A 1 mol / L NaOH solution was gradually added to the mixture until the final NaOH concentration reached 0.5 mol / L. The solution was then scanned using a UV-Vis spectrometer to determine the maximum absorption wavelength under each NaOH concentration condition.

[0088] Analysis results as follows Figure 13 Within the NaOH concentration range of 0.1-0.3 mol / L, the maximum absorption wavelength of the Congo red solution increased after the addition of the polysaccharide sample, indicating a redshift. This suggests that the polysaccharide BBHW may have a regular triple helix structure, enabling the Congo red to bind with the polysaccharide. As the NaOH solution concentration continued to increase, the intermolecular hydrogen bonds were disrupted, and the triple helix structure gradually transformed into a random helix, resulting in a weakened redshift, manifested as a decrease in the maximum absorption wavelength of the solution.

[0089] In summary, black soybean hull polysaccharide BBHW is a homogeneous galactomannan with a triple-helical structure. Its molecular weight is 134194 Da, composed of mannose, galactose, glucose, and arabinose in a molar ratio of 0.556:0.398:0.056:0.011. NMR results show that the sugar residues in polysaccharide BBHW are in α and β configurations. The primary structural unit contains five residues: →4)-β-D-Manp-(1→, α-D-Galp-(1→, α-L-Araf-(1→, →6)-α-D-Glcp-(1→, and →4,6)-β-D-Manp-(1→), linked as follows: Figure 14 .

[0090] Example 6

[0091] Example 1: Determination of the free radical scavenging ability of black soybean skin polysaccharide BBHW DPPH

[0092] A 0.1 mmol / L solution of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) was prepared with anhydrous ethanol and mixed with polysaccharide solutions of different concentrations in a ratio (v / v = 4:1). After shaking and mixing, the mixture was reacted at room temperature in the dark for 30 min, and the absorbance was measured at 517 nm. The scavenging rate was calculated according to the following formula.

[0093]

[0094] A sample : Absorbance of polysaccharide sample solution; A control : Absorbance of control solution; A blank absorbance of blank solution

[0095] Results of the DPPH free radical scavenging ability of black soybean skin polysaccharide (BBHW) as follows: Figure 15 As shown, when the mass concentration increased from 0.5 mg / mL to 4 mg / mL, the DPPH free radical scavenging ability of the polysaccharide extract also gradually increased, with a maximum scavenging rate of 85.26% ± 2.53% at 4 mg / mL. This indicates that black soybean skin polysaccharide has a certain DPPH free radical scavenging ability.

[0096] Example 7

[0097] Identification of the in vitro immunomodulatory activity of black soybean skin polysaccharide BBHW prepared in Example 1

[0098] 1. Polysaccharide BBHW cytotoxicity assay: RAW264.7 cells were cultured to the logarithmic growth phase in 1640 medium (containing 1% penicillin and streptomycin) at 37°C and 5% CO2, and then seeded into 96-well plates (1×10⁻⁶ cells / wells). 4 Cells were cultured in wells (number of cells / well), and then treated with different concentrations of polysaccharide extracts for 24 h. 10 μL of MTT solution was added to each well, and the cells were cultured in the dark for another 4 h. The supernatant was removed and the cells were stored separately at -20℃. 100 μL of DMSO was added to each well for 10 min to dissolve the cells. The absorbance of each well was measured at OD 570 nm using a microplate reader. A control group was used for normalization, and the relative cell viability of each group was calculated. Each group was replicated in triplicate.

[0099] like Figure 16 As shown, within the concentration range of 50-400 μg / mL, there was no significant difference in cell survival rate with increasing polysaccharide concentration (P>0.05), which also reflects the low toxicity and non-toxicity characteristics of black soybean skin polysaccharide as a natural polysaccharide.

[0100] 2. Effect of polysaccharide BBHW on phagocytosis of RAW264.7 cells: The effect of BBHW on the phagocytic capacity of RAW264.7 cells was evaluated using a neutral red phagocytosis assay. Cells were seeded in 96-well plates according to the method described in section 1 above and treated with the polysaccharide. PBS and LPS (2 μg / mL) were used as blank and positive control groups, respectively, instead of polysaccharide samples. After 24 h of culture, the supernatant was discarded, and 100 μL of PBS solution containing 0.02% neutral red was added to each well. Cells were cultured for another 3 h, and the supernatant was discarded. Cells were washed with PBS, and 100 μL of lysis buffer (ethanol: glacial acetic acid = 1:1) was added to each well. Lysis was performed at room temperature for 2 h. After the neutral red in the cells was released and dissolved, the absorbance at 540 nm was measured using a microplate reader. The control group was normalized, and the relative phagocytic index of each group was calculated, with three replicates per group.

[0101] The effect of polysaccharide BBHW on phagocytosis in RAW264.7 cells, as follows: Figure 17 As shown, at a concentration of 50 μg / mL, the polysaccharide had no significant effect on the phagocytic capacity of macrophages (P>0.05). However, with increasing concentration, the polysaccharide BBHW significantly promoted the phagocytic capacity of macrophages, exhibiting a clear concentration-dependent effect.

[0102] 3. Effects of polysaccharide BBHW on NO, TNF-α and IL-6 secretion in RAW264.7 cells: Cell supernatants collected in step 1 above were used to measure the secretion of each cytokine in the supernatant using a nitric oxide (NO) assay kit and TNF-α and IL-6 ELISA kits, with 4 replicates per group.

[0103] Effects of polysaccharide BBHW on NO secretion in RAW264.7 cells, such as Figure 18 As shown, when the polysaccharide concentration reached 200 μg / mL, the amount of NO secreted by cells began to increase significantly. The overall secretion amount increased with the increase of drug concentration, and finally reached 30 μM at a concentration of 400 μg / mL. Figure 19 , 20 The results indicate that the polysaccharide BBHW significantly promoted the secretion of TNF-α and IL-6 by RAW264.7 cells in a dose-dependent manner. Furthermore, the polysaccharide promoted TNF-α secretion to a greater extent than IL-6. TNF-α helps kill or inhibit tumor cell proliferation, while also enhancing the immune response and inducing the secretion of other immune factors; IL-6 can further stimulate and activate other immune cells and is an important immune factor.

[0104] In vitro experiments showed that the polysaccharide BBHW not only possesses the ability to scavenge free radicals to a certain extent, but also enhances the phagocytic capacity of RAW264.7 cells and promotes the secretion of cytokines NO, TNF-α, and IL-6. It exhibits excellent immunomodulatory activity and has the potential for use in food and pharmaceutical development.

Claims

1. A method for preparing galactomannan from black soybean seed coat with immunomodulatory activity, characterized in that, Includes the following steps: (1) The crude polysaccharide was obtained by hot acid extraction of black bean skin, concentration of the extract, precipitation with ethanol and freeze drying. The pH of the extract was 2.0 during hot acid extraction. The mass-volume ratio of black bean skin sample to extract was 1g:30~50mL. The extraction time was 40~60 min. The temperature of the extract was 90~100℃. (2) After separating the crude polysaccharide by DEAE-52 anion exchange cellulose column, it was dialyzed, and then the dialyzed polysaccharide was purified by Sephadex G-100 dextran gel column. The pure polysaccharide, namely black soybean seed coat galactomannan, was obtained by tracking and collecting the polysaccharide using the phenol-sulfuric acid method and then freeze-drying. The black soybean seed coat galactomannan contains α-configured pyranose with a peak molecular weight of 134,753 Da, a weight-average molecular weight of 134,194 Da, and a number-average molecular weight of 130,178 Da. It has a homogeneous composition consisting of mannose, galactose, glucose, and arabinose in a molar ratio of 0.556: 0.398: 0.056: 0.

011. The main chain is linked by →4)-β-D-Manp-(1→4)-β-D-Manp-(1→), and the side chains α-D-Galp-(1→, α-D-Galp-(1→6)-α-D-Glcp-(1→ and α-L-Araf-(1→) are connected to the main chain via O-6 bonds of →4,6)-β-D-Manp-(1→), exhibiting a triple helix structure.

2. The method for preparing an immunomodulatory black soybean seed coat galactomannan according to claim 1, characterized in that, In step (1), during the ethanol precipitation process, the volume ratio of anhydrous ethanol to concentrated liquid is 3~4:1, and precipitation is carried out at 4 ℃ for 10~14 hours.

3. The method for preparing an immunomodulatory black soybean seed coat galactomannan according to claim 1, characterized in that, The DEAE-52 cellulose packing used in step (2) needs to be pretreated. The pretreatment method is as follows: first soak the packing in 0.2 mol / L HCl for 2 hours, rinse the packing with deionized water until neutral, then soak the packing in 0.2 mol / L NaOH for 2 hours, rinse with deionized water until neutral, and the column size is 6 cm × 50 cm.

4. The method for preparing an immunomodulatory black soybean seed coat galactomannan according to claim 1, characterized in that, In step (2), during the separation process using the DEAE-52 cellulose column, the sample concentration was 5~10 mg / mL, the eluent was deionized water, the elution rate was 0.5 mL / min, the polysaccharide components were tracked and collected by the phenol-sulfuric acid method, and the dialysis bag pore size was 500 Da.

5. The method for preparing an immunomodulatory black soybean seed coat galactomannan according to claim 1, characterized in that, In step (2), during the Sephadex G-100 gel purification process, the column size is 2 cm × 60 cm, the sample concentration is 3-5 mg / mL, and deionized water is used for elution at a rate of 0.2 mL / min.

6. The application of an immunomodulatory black soybean seed coat galactomannan obtained by any one of claims 1-5 in the preparation of health functional foods and immunomodulatory drugs.

Images