A soluble acid polysaccharide from pinto beans, and a method of preparing and using the same

Abstract

The application provides a soluble acid polysaccharide of Phaseolus vulgaris, and also provides a preparation method and application of the soluble acid polysaccharide of Phaseolus vulgaris. The application also provides application of the soluble acid polysaccharide of Phaseolus vulgaris in preparation of a medicine for protecting the spleen and liver, having the ability of recovering the colon barrier, regulating intestinal flora, or a health-care food for enhancing the immunity of the body. The soluble acid polysaccharide of Phaseolus vulgaris can be extracted by hot water extraction or microwave-assisted low eutectic solvent extraction, especially, the polysaccharide extracted by the microwave-assisted low eutectic solvent extraction has better drug efficacy, is safer and more controllable, and provides a new selection for clinic.

Description

Technical Field

[0001] This invention relates to a soluble acidic polysaccharide from kidney beans, its preparation method, and its uses. Background Technology

[0002] Kidney bean (Phaseolus uulgaris Linn.sp.) is an annual herbaceous plant belonging to the genus Pseudolaricis in the legume family. Its scientific name is common bean, a common edible legume, and also a traditional Chinese medicine. Kidney bean seeds are flat and round, milky white on the outside and dark brown on the inside, rich in nutrients such as protein, fat, carbohydrates, vitamins, and minerals. Literature reports the extraction and isolation of ferritin from kidney beans (Tian Tongtong, et al., Isolation and purification of ferritin from kidney beans, Journal of the Chinese Cereals and Oils Association, September 2015, Vol. 30, No. 9). Application number 201310133400.5, invention title: A method for extracting kidney bean starch and simultaneously producing kidney bean protein powder and dietary fiber powder, relates to a method for extracting kidney bean starch and simultaneously producing kidney bean protein powder and dietary fiber powder. Zheng Hongjun, et al., Research Progress on Functional Components, Bioactivity and Product Development of White Kidney Bean, Chinese Cereals and Oils Journal, December 2022, Vol. 37, No. 12, published that white kidney beans contain abundant glycoproteins, polysaccharides, polyphenols, and other components, with specific bioactivities such as antioxidant, hypoglycemic, and weight loss effects. To date, scholars both domestically and internationally have conducted relatively in-depth research on white kidney beans, and nearly one hundred compounds have been isolated from them, among which the amylase inhibitory activity of white kidney bean glycoproteins has attracted the most attention. However, research reports on polysaccharides in kidney beans are relatively few.

[0003] Polysaccharides are chain polymers that can be classified into three types: neutral, acidic, and positively charged. Acidic polysaccharides, containing acidic groups such as sulfate and uronic acid, are widely found in natural plants, animals, and microorganisms. Acidic polysaccharides possess various biological activities, including antioxidant, immunomodulatory, antitumor, hypoglycemic, and hypolipidemic effects. Compared to neutral polysaccharides, acidic polysaccharides exhibit better biological activity due to their structural characteristics, including uronic acid content, molecular weight, and monosaccharide composition. Yan et al. found that acidic polysaccharides containing a large amount of Glc and a small amount of GlcA have stronger antioxidant activity than neutral polysaccharides. Zhang et al. found that acidic polysaccharides showed better immunomodulatory activity than neutral polysaccharides, and their biological activity was positively correlated with uronic acid content, the percentage of rhamnose, arabinose, and galactose, and the degree of branching. Currently, no relevant literature reports on soluble acidic polysaccharides from kidney beans. Summary of the Invention

[0004] The technical solution of this invention is to provide a soluble acidic polysaccharide from kidney beans; another technical solution of this invention is to provide a method for preparing the soluble acidic polysaccharide from kidney beans and its uses.

[0005] This invention provides a soluble acidic polysaccharide from kidney bean (Phaseolus vulgaris Linn.), which is extracted and prepared from the seeds of kidney bean (Phaseolus vulgaris Linn.), a plant belonging to the genus Fabaceae. The polysaccharide content is 88.1-92.4 mg / 100 mg, the total uronic acid content is 19.6-24.8 mg / 100 mg, the bound phenol content is 1.4-3.0 mg GAE / 100 mg, and the degree of esterification is 15.0-18.5%.

[0006] Monosaccharides containing the following molar ratios:

[0007] Rhamnose 1.00, mannose 0.11-0.85, glucuronic acid 0.41-0.50, galacturonic acid 3.87-5.22, glucose 0.31-2.36, galactose 2.93-3.53, xylose 4.16-4.19, arabinose 8.83-13.08.

[0008] More preferably, the total polysaccharide content is 91.38±1.00 mg / 100 mg, the total uronic acid content is 24.51±0.27 mg / 100 mg, and the degree of esterification is 17.20%±1.28%.

[0009] Monosaccharides containing the following molar ratios:

[0010] Rhamnose 1.00, mannose 0.11, glucuronic acid 0.41, galacturonic acid 5.22, glucose 2.36, galactose 2.93, xylose 4.19, arabinose 13.08.

[0011] The soluble acidic polysaccharide from kidney beans of this invention is prepared by hot water extraction or microwave-assisted eutectic solvent extraction.

[0012] This invention also provides a method for preparing the aforementioned soluble acidic polysaccharide from kidney beans, characterized by comprising the following steps:

[0013] a. Take dried and pulverized kidney beans, grind them into powder, add 80%-95% ethanol to remove the alcohol-soluble components, and obtain a precipitate;

[0014] b. Add water to the precipitate, stir, and then heat in a water bath to extract. Concentrate the extract to obtain a concentrated solution.

[0015] c. Add α-amylase and saccharifying enzyme to the concentrate to remove starch, then add trypsin to remove protein. After inactivation, add 95% ethanol for precipitation, centrifuge, and obtain the precipitate.

[0016] d. The precipitate prepared in step c is re-dissolved in water, dialyzed to remove small molecules, and dried to obtain the soluble acidic polysaccharide SKBP-H from kidney beans.

[0017] This invention also provides a method for preparing the aforementioned soluble acidic polysaccharide from kidney beans, characterized by comprising the following steps:

[0018] a. Take dried and pulverized kidney beans, grind them into powder, add 80%-95% ethanol to remove the alcohol-soluble components, and obtain a precipitate;

[0019] b. Add the prepared DES (choline chloride: ethylene glycol = 1:3) to the precipitate and stir well. Extract using microwave, concentrate the extract to obtain the concentrated solution. The extraction parameters are as follows:

[0020] DES moisture content: 15-75%; extraction power: 440-840W; extraction time: 6-30min;

[0021] c. Add α-amylase and saccharifying enzyme to the concentrate to remove starch, then add trypsin to remove protein. After inactivation, add 95% ethanol for precipitation, centrifuge, and obtain the precipitate.

[0022] d. The precipitate prepared in step c is re-dissolved in water, dialyzed to remove small molecules, and dried to obtain the soluble acidic polysaccharide SKBP-DM from kidney beans.

[0023] The extraction parameters in step b are as follows: extraction time is 20-30 min, extraction power is 700-800 W, and DES water content is 30-45%.

[0024] The extraction parameters in step b are as follows: extraction time is 26.2 ± 1.2 min, extraction power is 760.6 ± 10.4 W, and DES water content is 41.5% ± 1.8%.

[0025] The present invention also provides the use of the aforementioned soluble acidic polysaccharide from kidney beans in the preparation of health foods or medicines with antioxidant effects.

[0026] The present invention also provides the use of the aforementioned soluble acidic polysaccharide from kidney beans in the preparation of health products or pharmaceuticals that stimulate immune activity.

[0027] The drug mentioned above increases the levels of CAT, SOD, and GSH in the liver, reduces the levels of MDA and ALT, enhances antioxidant capacity, and improves oxidative stress caused by immunodeficiency.

[0028] The present invention also provides the use of the aforementioned soluble acidic polysaccharide from kidney beans in the preparation of drugs that protect the spleen, improve intestinal inflammation, restore colonic barrier function, regulate intestinal flora, or health foods that help enhance the body's immunity.

[0029] The soluble acidic polysaccharides from kidney beans of this invention can be extracted using hot water extraction or microwave-assisted eutectic solvent extraction. In particular, the polysaccharides extracted using microwave-assisted eutectic solvent extraction have better efficacy and are safer and more controllable, providing a new option for clinical use. Attached Figure Description

[0030] Figure 1 Flowchart of the preparation method of soluble acidic polysaccharide from kidney bean according to the present invention;

[0031] Figure 2 Effects of extraction parameters on the yield of acidic polysaccharides extracted from kidney beans (where A, B, and C represent the effects of water content of DES solution, microwave power, and extraction time on the polysaccharide yield extracted by microwave-assisted DES extraction, respectively; D, E, and F represent the interaction effects of microwave power, extraction time, and water content of DES solution on the polysaccharide yield extracted by microwave-assisted DES extraction).

[0032] Figure 3 In vitro antioxidant activity of SKBP-H and SKBP-DM (wherein, (A) ABTS free radical scavenging activity, (B) nitric oxide free radical scavenging activity, (C) DPPH free radical scavenging activity, (D) reducing power);

[0033] Figure 4 In vitro immunomodulatory effects of SKBP-H and SKBP-DM (including their effects on (A) proliferation, (B) IL-6 secretion, (C) NO secretion and (D) TNF-α secretion of RAW 264.7 cells);

[0034] Figure 5 High-performance size exclusion chromatograms of SKBP-H and SKBP-DM (where (A) is the high-performance liquid chromatogram of monosaccharide composition (B), infrared spectrum (C) and 1H NMR spectrum (D));

[0035] Figure 6 Effects of SKBP-H and SKBP-DM on body weight and immune organ indices in CTX-induced immunosuppressed mice (where (A): changes in body weight, (B): changes in thymus index and spleen index);

[0036] Figure 7 Hematoxylin-eosin (H&E) tissue sections of mouse liver (from left to right: normal group, model group, positive group, SKBP-H group, SKBP-DM group; the figure shows representative H&E stained liver tissue sections, scale bar, 50 μm);

[0037] Figure 8Antioxidant markers in mouse liver (where A: CAT catalase, B: SOD superoxide dismutase, C: GSH glutathione peroxidase, D: MDA malondialdehyde, E: ALT alanine aminotransferase).

[0038] Figure 9 Hematoxylin-eosin (H&E) tissue sections of mouse spleen (from left to right: normal group, model group, positive group, SKBP-H group, SKBP-DM group; the figure shows representative H&E stained spleen tissue sections, scale bar, 100 μm);

[0039] Figure 10 Serum cytokine and immunoglobulin levels in mice (where A: IL-6, interleukin-6; B: TNF-α, tumor necrosis factor-α; C: INF-γ, interferon-γ; D: Ig-G, immunoglobulin G).

[0040] E: Ig-A immunoglobulin A);

[0041] Figure 11 Images and scores of hematoxylin-eosin (H&E) stained tissue sections and immunofluorescence staining of mouse colon (where A: a representative H&E stained colon tissue section, scale bar, 200 μm; B: a representative image of ZO-1 and Occuldin immunofluorescence staining).

[0042] Figure 12 Production of SCFAs in mouse cecal contents (where A: acetic acid, B: propionic acid, C: butyric acid, D: total acid);

[0043] Figure 13 PCoA analysis of gut microbiota in mice of each group;

[0044] Figure 14 The structure of the mouse gut microbiota (where A: bar chart of gut microbiota composition at the phylum level, B: bar chart of gut microbiota composition at the genus level, C: microbiota with significant differences among the top 10 phyla, and D: microbiota with significant differences among the top 30 genera).

[0045] Figure 15 Linear discriminant analysis (LDA) scores of species richness differences between the model group and the normal group, SKBP-H group, and SKBP-DM group, respectively;

[0046] Figure 16 The correlation between monosaccharide type and key gut microbiota and short-chain fatty acids. The color of the circle indicates the R-value of the Spearman correlation coefficient (orange and green indicate positive or negative correlation, respectively);

[0047] Figure 17Correlation between immune-related indicators and key genera and taxa of the gut microbiota. The color of the circle represents the R value of the Spearman correlation coefficient (orange and green indicate positive or negative correlation, respectively). Detailed Implementation

[0048] Example 1: Preparation method of soluble acidic polysaccharide from kidney bean according to the present invention

[0049] Hot water extraction method: Kidney bean raw material was dried at 60℃ for 24 hours, then pulverized using a grinder and passed through an 80-mesh sieve. The kidney bean powder was mixed with 80% ethanol (1:10, w / v) and ultrasonically cleaned to remove alcohol-soluble components. Ultrapure water (1:30, w / v) was added to the precipitate and stirred until homogeneous. Extraction was then performed in a water bath for 2 hours. The extract was concentrated to approximately 1 / 3 of its original volume using a rotary evaporator. High-temperature α-amylase (10 U / mL) and saccharifying enzyme (5 U / mL) were then added to remove starch, followed by trypsin (5 U / mL) to remove protein. The mixture was inactivated at 95℃ for 30 minutes. Precipitation was then performed overnight at 4℃ with four volumes of 95% ethanol (v / v). The precipitate was then centrifuged, redissolved in ultrapure water, and dialysis was used to remove small molecules (molecular weight cutoff, 3.5 kDa). Finally, after freeze-drying, water-soluble acidic polysaccharide from kidney beans (SKBP-H) was obtained.

[0050] Example 2: Preparation method of soluble acidic polysaccharide from kidney bean according to the present invention

[0051] The kidney bean raw material was pretreated according to the method in Example 1 to remove alcohol-soluble components. 42% DES solvent (volume fraction) was added to the sample at a ratio of 1:30 and stirred thoroughly. The mixture was then transferred to a laboratory microwave oven (MKX-H1C1A, Qingdao Microwave Innovation Technology Co., Ltd., Qingdao, China) and extracted at 761W for 26 min. After centrifugation at 4000×g for 15 min, the supernatant was collected, and this process was repeated twice. The supernatants from the two extractions were mixed and concentrated to approximately one-third of their original volume using a rotary evaporator. Then, 10 U / mL of heat-stable α-amylase was added, and the mixture was reacted at 80°C for 8 hours. After the extract cooled to room temperature, 10 U / mL of saccharifying enzyme was added, and the mixture was reacted at 59°C for 12 hours to remove starch. Finally, the mixture was heated to 95°C and incubated for 30 min to inactivate the enzyme. Next, 5 U / mL of trypsin was added, and the mixture was reacted at 40°C for 8 hours to remove protein. Finally, the mixture was heated to 95°C and incubated for 30 min to inactivate the enzyme. After the extract was cooled to room temperature, it was thoroughly mixed with four times its volume of 95% ethanol and allowed to stand overnight at 4°C. Next, the mixture was centrifuged at 5000×g for 10 minutes, the supernatant was removed, and the precipitate was collected. The precipitate was then dissolved in ultrapure water and freeze-dried under vacuum for 48 hours to obtain kidney bean soluble acidic polysaccharide (SKBP-DM), which was stored at 4°C for later use (see process flow diagram). Figure 1 ).

[0052] Example 3: Condition screening test of the preparation method of the soluble acidic polysaccharide from kidney bean of the present invention.

[0053] Microwave-assisted eutectic solvent extraction: Prepared DES (choline chloride: ethylene glycol = 1:3) was added to the precipitate and stirred until homogeneous. Extraction was then performed using a laboratory microwave oven. Other conditions were consistent with the hot water extraction method. Then, a single-factor experimental design was used to optimize the microwave-assisted eutectic solvent extraction method; the conditions investigated are shown in Table 1.

[0054] In summary, we first optimized the extraction microwave power and DES water content, and then optimized the extraction time.

[0055] Table 1 Single-factor experimental design table

[0056]

[0057] Secondly, a three-factor response surface methodology was used to further optimize the effects of various extraction parameters on the extraction rate of soluble polysaccharides from kidney beans. Independent variables included extraction time (X1, 18, 24, 30 min), extraction power (X2, 640, 740, 840 W), and DES moisture content (X3, 30, 45, 60%). Seventeen experiments were designed in this study, and the response surface factor codes are shown in Table 2. The experimental data were then analyzed using Design-Expert. The obtained data were fitted using a second-order polynomial model, as follows:

[0058] Table 2. Response surface methodology experimental design and results

[0059]

[0060]

[0061] Microwave-assisted extraction (MAE) can be used to obtain acidic polysaccharides with various biological characteristics, offering advantages such as economic feasibility, time-saving, environmental friendliness, high extraction efficiency, low solvent consumption, and low energy consumption. This invention uses DES as the extraction solvent and assists microwave technology in the extraction of SKBP-DM. Increasing microwave power can effectively accelerate the transfer of intracellular substances, but excessively high microwave power may lead to polysaccharide degradation. Figure 2 It can be seen that the extraction rate of SKBP-DM increases as the extraction time increases from 18 min to 24 min. This is because acidic polysaccharides gradually dissolve from the sample with increasing microwave time. However, after 24 min, the extraction rate gradually decreases. This may be because excessively long extraction times can lead to the degradation of acidic polysaccharides by microwaves. Increasing microwave power can effectively accelerate the transfer of intracellular substances, but excessively high microwave power may cause the degradation of acidic polysaccharides. Therefore, when the microwave power increases from 640 W to 740 W, the extraction rate of soluble polysaccharides gradually increases, while when the microwave power is increased to 840 W, the extraction rate of acidic polysaccharides decreases. Too low a water content in DES will result in excessively high solution viscosity, making it difficult to extract acidic polysaccharides by penetrating plant cells, while too high a water content may hinder the interference between DES and acidic polysaccharides, leading to a decrease in the extraction rate. Therefore, the optimal conditions finally optimized are 45%.

[0062] Based on the single-factor structure, we further employed a Box-Behnken experimental design to optimize the extraction rate of SKBP-DM. The final BBD experimental data are shown in Table 3. Furthermore, a second-order polynomial equation was generated as follows:

[0063] Y=-34.12+0.22X1+1.15X2+0.05X3-0.002X1X2+0.00002X1X3+0.0002X2X3–0.0022X1 2 –0.024X2 2–0.00004X3 2

[0064] Where Y represents the extraction rate; X1, X2 and X3 are the DES water content (%), extraction time (min) and microwave power (W), respectively.

[0065] Table 3. Analysis of variance of the regression model for microwave-assisted eutectic solvent extraction (MDE)

[0066]

[0067]

[0068] Note: X1: DES moisture content (%), v / v; X2: extraction time (min); X3: microwave power (W); MDE, R 2 =0.9896,R 2 adj =0.9762, coefficient of variation (CV) =2.59%, and adeq.precision =22.688.

[0069] *Significant difference (p<0.05), **Highly significant difference (p<0.01).

[0070] As shown in Table 2, one-way ANOVA was used to confirm the significance of the extracted parameters for yield and the forecast model. The model fit was highly significant, with a high F-value (22.69) and a very low P-value (<0.0001). Furthermore, the values ​​of the lack-of-fit term, coefficient of determination, and corrected coefficient of determination demonstrate a high degree of fit. Additionally, the values ​​of the coefficient of variation and moderate precision also demonstrate good repeatability and reliability of the fitted model.

[0071] Furthermore, the linear coefficients (X1, X2, X3), interaction coefficients (X1X2, X2X3), and quadratic coefficients (X1, X2, X3) of this fitted model equation are also present. 2 X2 2 X3 2 The interaction coefficients (X1X3) were significant when the p-value was below 0.05, but not significant when the p-value was above 0.05. These results indicate that all selected extraction parameters can significantly affect the extraction yield of SKBP-DM. Indeed, the three-dimensional response surface plots also reveal significant interactions between extraction time and extraction power, extraction power and DES water content, and extraction time and DES water content. All these data suggest that extraction time, microwave power, and DES water content are important parameters affecting SKBP-DM extraction.

[0072] Based on the analysis of the experimental results, the optimal extraction conditions were determined as follows: extraction time of 26.22 min, extraction power of 760.57 W, and DES water content of 41.65%. Verification experiments were conducted under these conditions, and the actual extraction rate was 5.14% ± 0.17%, very close to the predicted value of 5.25%. This invention optimizes the microwave-assisted DES extraction method to achieve efficient extraction of acidic polysaccharides from kidney beans, aiming to achieve more significant results in terms of extraction efficiency and bioactivity. The results show that under optimal conditions, the efficiency of extracting acidic polysaccharides from kidney beans using microwave-assisted DES reached 5.14% ± 0.17% (n = 3), which is 268% higher than the traditional water extraction method. Microwave-assisted DES extraction not only significantly improves the extraction rate but also shortens the extraction time to only 26 minutes, saving energy consumption and demonstrating environmental friendliness. Furthermore, the polysaccharide samples prepared using microwave technology exhibit superior bioactivity compared to other extraction techniques. This indicates that microwave-assisted eutectic solvent extraction (MDE) can be used as an efficient method for preparing acidic polysaccharides from kidney beans in the food industry.

[0073] Example 4: Physicochemical properties and in vitro bioactivity of the soluble acidic polysaccharide from kidney bean of the present invention.

[0074] Based on the above response surface methodology optimization results, soluble polysaccharides were extracted from kidney beans, and their physicochemical properties and antioxidant and immunomodulatory activities were determined.

[0075] Total sugars, combined phenols, total uronic acids, and total protein in soluble acidic polysaccharides from kidney beans were determined using colorimetric methods. The molecular weight and dispersibility of SKBP-H and SKBP-DM were determined using gel size exclusion chromatography with a multi-angle laser light scattering detector-differential refractive index detector (Wyatt Technology Co., Santa Barbara, CA, USA). The monosaccharide composition in the samples was determined using high-performance liquid chromatography (L-20A, Shimadzu, Japan) combined with pre-column derivatization of 1-phenyl-3-methyl-5-pyrazolone (PMP). Functional groups and esterification degrees in the samples were analyzed by FT-IR spectroscopy (PerkinElmer, Waltham, MA, USA). Glycosidic bonds in the dietary polysaccharides were analyzed by nuclear magnetic resonance spectroscopy (Bruker, Rheinstetten, Germany).

[0076] The antioxidant activity of kidney bean extracts obtained by two different methods was investigated using four indicators: ABTS, DPPH, NO radical scavenging rate, and total reducing power. Experiments were conducted with different concentrations of SKBP-H and SKBP-DM, and their IC50 values ​​were calculated. 50Values ​​were used. VC was used as a positive control. The experimental model was established by culturing RAW 264.7 macrophages in RPMI-1640 medium. Subsequently, the effects of SKBP-H and SKBP-DM on macrophage proliferation were determined by the MTT assay. The NO content released by macrophages was detected by Griess reagent. Finally, the secretion of tumor necrosis factor-α (TNF-α) and leukocyte-6 (IL-6) by macrophages was measured by an ELISA kit.

[0077]

[0078]

[0079] SKBP-H and SKBP-DM represent dietary polysaccharides from kidney beans extracted by hot water and microwave-assisted eutectic solvent extraction, respectively; the superscript (ab) between SKBP-H and SKBP-DM indicates that the difference between them is significant (p<0.05).

[0080] Experimental results showed that the total sugar content of SKBP-H and SKBP-DM was 88.60 mg / 100 mg and 91.38 mg / 100 mg, respectively, and the uronic acid content was 19.63 mg / 100 mg and 24.51 mg / 100 mg, respectively, indicating that the samples extracted using our method were mainly composed of soluble polysaccharides. Numerous studies have shown that the uronic acid content of polysaccharides is closely related to their biological activity; higher uronic acid content often indicates higher biological activity.

[0081] like Figure 3 As shown, the antioxidant activity of SKBP-DM was significantly higher than that of SKBP-H. The IC50 values ​​of SKBP-DM for scavenging ABTS, DPPH, and NO free radicals were 0.915 mg / mL, 1.511 mg / mL, and 0.697 mg / mL, respectively, which were much lower than those of SKBP-H (ABTS, 2.161 mg / mL; DPPH, 4.529 mg / mL; NO, 1.087 mg / mL). Furthermore, at a concentration of 2.5 mg / mL, the FRAP (receivable at 700 nm) concentrations of SKBP-H and SKBP-DM were 0.333 ± 0.007 and 0.947 ± 0.005, respectively, further indicating that the in vitro antioxidant activity of SKBP-DM was significantly higher than that of SKBP-H.

[0082] like Figure 4As shown, the results of cellular immunomodulatory activity experiments revealed that SKBP-H and SKBP-DM at doses between 100 and 400 μg / ml were non-toxic to RAW 264.7 macrophages. More importantly, the data showed that both SKBP-H and SKBP-DM made significant contributions to the secretion of NO, IL-6, and TNF-α in a dose-dependent manner. SKBP-DM exhibited a stronger immunomodulatory effect on RAW 264.7 macrophages than SKBP-H. Therefore, the observed difference in immunomodulatory effects between SKBP-H and SKBP-DM may be due to their structural differences. The high uronic acid content and low molecular weight of SKBP-DM may also be factors contributing to its stronger immunomodulatory effect.

[0083] Example 5: Structural characterization of the soluble acidic polysaccharide from kidney bean of the present invention.

[0084] Molecular weight is also correlated with the bioactivity of polysaccharides. Two distinct fractions were found in the soluble polysaccharides extracted from kidney beans using different methods: fraction 1 and fraction 2, with extraction times of approximately 16–20 min and 20–22 min, respectively, but fraction 2 was predominant. Figure 5 The SKBP-DM component shown in Figure A has a molecular weight of 3.18 × 10⁻⁶. 4 Da) is lower than SKBP-H (3.60×10) 4 The molecular weight of Da) is determined by microwaves. Microwaves degrade high molecular weight polysaccharides, thus reducing the molecular weight of samples prepared by microwave-assisted DES extraction. This demonstrates that different extraction methods affect the molecular weight of the sample. Furthermore, samples with lower molecular weights generally exhibit better activity.

[0085] To further understand the chemical structures of SKBP-H and SKBP-DM, their monosaccharide composition, functional groups, and glycosidic bonds were investigated using HPLC, FT-IR, and NMR. Figure 5The monosaccharide compositions of SKBP-H and SKBP-DM are mostly composed of Rha, GalA, Glc, Gal, Xyl, and Ara, but their molar ratios differ. It can be seen that the GalA molar ratio of SKBP-DM is significantly higher than that of SKBP-H, indicating a marked increase in uronic acid content, consistent with colorimetric results. This further demonstrates that the microwave-assisted DES method can extract more acidic polysaccharides compared to traditional water extraction. The ratio of HG and RG I pectin domains in pectin polysaccharides can generally be reflected by the GalA / Rha ratio (MR1 ratio). The MR1 values ​​of SKBP-H and SKBP-DM are 3.87 and 5.22, respectively, indicating that SKBP-DM is richer in HG than SKBP-H. Furthermore, Ara and Gal can also be produced from arabinogalactan (AG), indicating that both SKBP-H and SKBP-DM contain pectin polysaccharides. In addition, abundant hemicellulose has been found in the polysaccharides of many legumes. Xyl, Glc, and Man are typical monosaccharides of hemicellulose, such as arabinoxylan, glucomannan, galactomannan, and xyloglucan. Based on the molar ratio of Ara, Xyl, and Glc, it can be found that arabinolan and arabinoxylan are likely present in all SKBPs, while xyloglucan may be present in SKBP-DM.

[0086] The results are as follows Figure 5 Typical signals of pectin polysaccharides were found in SKBPs prepared by different extraction methods, namely 3443.1, 2922.3, 1741.8, 1636.5, 1410.1, 1240.8, 1103.8, and 1021.3 cm⁻¹. -1 This indicates that they contain very similar functional groups. The vibrational stretching of the OH and CH bonds produces the prominent 3443 cm⁻¹. -1 and 2922cm -1 The reason for the absorption band. The C=O stretching vibration of the esterified group at 1741 cm⁻¹. -1 Represented by the absorption band at 1621 cm⁻¹, the C=O asymmetric extension of COO⁻ extends to 1621 cm⁻¹. -1 The absorption peak at 1743 cm⁻¹ is representative. In summary, these results indicate the presence of uronic acid in SKBPs and the presence of acidic polysaccharides in kidney bean dietary polysaccharides. Furthermore, based on the 1743 cm⁻¹... -1 and 1636cm -1 The signal intensity was analyzed, and the esterification degrees of SKBP-H and SKBP-DM were calculated to be 15.8% and 17.2%, respectively, indicating that MDE can increase the esterification degree of SKBPs. Furthermore, the immunomodulatory effect of acidic polysaccharides was positively correlated with the degree of esterification. Results are as follows... Figure 5D, SKBP-H, and SKBP-DM all exhibited several similar characteristic signals in their NMR spectra. Specifically, the signal at 5.23 ppm (H-1) indicates the presence of 1,2,4-α-L-Rha; the signal at 5.17 ppm (H-1) indicates the presence of T-α-L-Araf; the signal at 5.07 ppm (H-1) is due to the presence of 1,5-α-L-Araf; the signal at 4.54 ppm (H-1) indicates the presence of 1,4-β-Xyl; the signal at 4.47 ppm is due to the presence of 1,3,6-β-D-Gal / 1,4-β-D-Gul; 3.81 ppm is attributed to GalA-OCH3; the signal at 2.08 ppm indicates the presence of O-acetyl; and the signal at 1.24 ppm indicates the presence of 1,2,4-α-L-Rha.

[0087] In general, based on the composition of monosaccharides 1 H NMR signals and monosaccharide composition, all of which contain fructose polysaccharides (RG I and HG domains) and hemicellulose (e.g., arabinoxylan, xyloglucan-arabinolan).

[0088] The following efficacy tests demonstrate the beneficial effects of the present invention.

[0089] Experimental Example 1: In vivo immunomodulatory activity of soluble acidic polysaccharides from kidney beans according to the present invention.

[0090] I. Modeling Method:

[0091] Balb / c mice were housed in a nearly thermally stable room (24°C) under a 12-hour light-dark cycle, with free access to fresh water and standard laboratory pelleted feed. After one week of acclimatization, all mice were randomly assigned to five groups (n=10 per group): normal control (NC), cyclophosphamide model group (MC), levamisole positive group (PC), SKBP-H group, and SKBP-DM group. Mice in the MC, SKBP-H, SKBP-DM, and PC groups were intraperitoneally injected with 80 mg / kg body weight (BW) / day of cyclophosphamide for three consecutive days, while mice in the NC group were given saline in the same manner. Subsequently, mice in the NC and MC groups were gavaged with 0.2 mL of sterile water, while mice in the PC, SKBP-H, and SKBP-DM groups were gavaged with SKBP-H (150 mg / kg BW / day), SKBP-DM (150 mg / kg BW / day), and LH (40 mg / kg BW / day), respectively, for 14 days. The weight of the mice was recorded every two days.

[0092] II. Experimental Results

[0093] 1. The effect of the soluble acidic polysaccharide from kidney bean on body weight and immune organ index of the present invention.

[0094] Body weight is an indicator of growth status in mice, while the spleen and thymus are major immune organs. In a cyclophosphamide-induced mouse immunodeficiency model, these indicators can be used to assess changes in growth status and immune function. During model establishment, we recorded daily changes in body weight in different groups of mice. The results are as follows: Figure 6 A showed that, starting on day 2 after cyclophosphamide injection, mice receiving the injection exhibited sustained weight loss, indicating successful model establishment. Mice were administered kidney bean soluble polysaccharides (150 mg / day BW) prepared using two different extraction techniques via gavage for 14 days. The results showed that, compared to the model group, both the kidney bean soluble polysaccharides prepared using different extraction methods and the positive control group significantly slowed the weight loss in mice. Results are as follows... Figure 6 B showed that the spleen and thymus organ indices of mice significantly decreased after cyclophosphamide injection. Compared with the normal group, the thymus and spleen indices in the model group were significantly reduced, at 1.36 mg / g and 0.64 mg / g, respectively, indicating that cyclophosphamide caused severe damage to the immune organs of mice. In both the SKBP-H and SKBP-DM groups, the thymus and spleen organ indices showed a significant increasing trend. The effect was more pronounced in the SKBP-DM sample.

[0095] 2. The effect of the soluble acidic polysaccharide from kidney bean of this invention on the liver.

[0096] The liver plays a vital role in the human immune system. It is not only an important organ of the digestive system but also possesses powerful immune functions. These functions include clearing bacteria, viruses, and other harmful substances from the bloodstream, while also participating in regulating the body's immune response. When the body's immunity is low, it may lead to increased inflammation and oxidative stress, resulting in the production of large amounts of free radicals. Free radicals are highly reactive molecules that damage biomolecules such as cell membranes, proteins, and nucleic acids, leading to cell damage and even cell death. Furthermore, excess free radicals in the body can weaken the body's antioxidant capacity, causing damage to cells and tissues.

[0097] To further evaluate the impact of SKBPs on the submicroscopic structure of the liver, this invention used HE staining to observe the histological morphology of the liver. The results are as follows: Figure 7 The results showed that, compared with the normal group, the model group had disordered hepatic cord arrangement, sinusoidal occlusion, and inflammatory cell infiltration. However, intervention with SKBPs improved these pathological changes. SKBP-DM showed a more significant improvement compared with SKBP-H, and its recovery level was closer to the normal state.

[0098] 3. The effect of the soluble acidic polysaccharide from kidney bean on liver oxidative stress in this invention.

[0099] Cyclophosphamide (CTX) can damage the liver and antioxidant enzymes, induce oxidative stress in the body, and produce large amounts of reactive oxygen species (ROS), thereby causing oxidative damage to immune cells and further reducing the body's immune function. Based on the observation of liver pathological damage, this invention further tested its antioxidant capacity.

[0100] The results of the antioxidant capacity of mice are as follows Figure 8 Compared with the model group, the levels of CAT, SOD, and GSH in the SKBP-DM group were significantly increased (P < 0.05), while the levels in the SKBP-H group were slightly increased compared with the model group, but not significantly. The levels of MDA and ALT in the SKBP-H and SKBP-DM groups were significantly lower than those in the model group (P < 0.05).

[0101] Kapok acidic polysaccharides significantly increased the levels of CAT, SOD, and GSH in the liver of immunosuppressed mice, decreased the levels of MDA and ALT, improved the antioxidant capacity of immunosuppressed mice, and alleviated oxidative stress induced by immunodeficiency.

[0102] 4: The effect of the soluble acidic polysaccharide from kidney bean of the present invention on the spleen

[0103] The spleen is one of the largest lymphatic organs in the human body and an important component of the peripheral immune system. It plays a crucial role in the body's immune system, including the proliferation, differentiation, and activation of immune cells. The spleen contains abundant lymphocytes, macrophages, dendritic cells, and other immune cells, which work together to provide vital support for antibody and cell-mediated immunity. When the body is infected or invaded by foreign substances, the spleen can respond rapidly, initiating an immune response, producing specific antibodies or activating T cells to help the body eliminate pathogens or abnormal cells.

[0104] like Figure 9 The pathological changes in spleen tissue stained with HE showed that the boundary between the red and white pulp in the normal group was clear, while the boundary between the white and red pulp in the model group was blurred and the area of ​​the white pulp was reduced. After the intervention of SKBP-H and SKBP-DM, the boundary became clear and the area of ​​the white pulp increased. The results indicate that SKBP-H and SKBP-DM can effectively improve the damage to the spleen caused by CTX.

[0105] 5. The immunomodulatory effect of the soluble acidic polysaccharide from kidney beans in this invention.

[0106] Cytokines play crucial regulatory roles in cell growth, differentiation, and intercellular interactions, and significantly influence immune inflammatory responses. Therefore, we investigated the effects of *Kidney Bean* acidic polysaccharide on the production of immune-related cytokines, including TNF-α, IFN-γ, and IL-6, in mouse serum. The results are as follows: Figure 10AB studies showed that acidic polysaccharides from kidney beans affected the levels of cytokine secretion in mouse serum. The results indicated that, compared to the normal group, the levels of TNF-α, INF-γ, and IL-6 in the model group were significantly reduced (P<0.05). After gavage administration of SKBP-H and SKBP-DM, the serum levels of immune-related cytokines TNF-α, INF-γ, and IL-6 significantly recovered (P<0.05), indicating that intervention with acidic polysaccharides from kidney beans can improve the decrease in immune function induced by cyclophosphamide treatment.

[0107] To further investigate immune status, we measured the levels of Igs in mouse serum. The results are as follows: Figure 10 As shown in the DC results, compared with the normal group, the serum IgG and IgA levels in the model group mice were significantly reduced. Conversely, in the SKBP-H and SKBP-DM groups, both IgG and IgA levels showed varying degrees of increase. This indicates that kidney bean acidic polysaccharides can improve the secretion of IgG and IgA in mice.

[0108] 6. The effect of the soluble acidic polysaccharide from kidney bean on intestinal barrier damage in this invention.

[0109] The gut is a vital organ for digestion and absorption, as well as an important site of the immune response. The intestinal mucosal immune system participates in regulating immune responses, maintaining intestinal homeostasis, and defending against external pathogens by expressing various pro-inflammatory and anti-inflammatory cytokines. Many studies have reported that cyclophosphamide treatment-induced intestinal mucosal damage can lead to apoptosis of intestinal crypt cells, resulting in decreased villus height and crypt depth. This experiment used HE staining to observe stained sections of colonic tissue under a microscope. The results are as follows: Figure 11 Compared to the normal group, the model group showed a trend of shortened villus length and significant inflammatory cell infiltration. Compared to the model group, the SKBP-H and SKBP-DM groups showed increased villus length and reduced inflammatory cell infiltration. This suggests that kidney bean acidic polysaccharides may have a positive effect on improving intestinal inflammation.

[0110] Tight junction proteins are important intercellular junction protein complexes located on the apical sidewall membranes of intestinal epithelial cells. They include occludin, ZO proteins (mainly ZO-1 and ZO-2), Claudins, and adhesion molecules. These protein complexes interact to form intercellular junctions, constituting "tight junctions," which help maintain the physiological barrier function of the intestinal mucosa. Therefore, we used immunofluorescence staining to detect the expression of two major tight junction proteins (ZO-1 and occludin) in colonic epithelial cells. The results are as follows: Figure 11In the model group (B), the fluorescence intensity and expression levels of ZO-1 and occludin were significantly lower than those in the control group. Supplementation with SKBP-H and SKBP-DM significantly improved the reduction in ZO-1 and occludin expression. By increasing the expression of tight junction proteins and promoting intestinal epithelial barrier formation, bacterial translocation, intestinal inflammation, and metabolic endotoxemia were significantly reduced. Compared to SKBP-H, SKBP-DM showed better ability to restore the colonic barrier.

[0111] 7. The promoting effect of the soluble acidic polysaccharide from kidney beans of this invention on short-chain fatty acids in cecal contents.

[0112] Polysaccharides produce large amounts of short-chain fatty acids (SCFAs) through fermentation by the gut microbiota. These SCFAs mainly include acetic acid, propionic acid, and butyric acid, which play important roles in regulating local and systemic immune responses and maintaining intestinal homeostasis. The effects of SKBP-H and SKBP-DM on the content of SCFAs in the gut are as follows: Figure 12 As shown, compared with the normal group, the levels of acetic acid, propionic acid, butyric acid, and total acid in the model group were significantly reduced (P<0.05). Treatment with SKBP-H and SKBP-DM alleviated this downward trend and increased the levels of SCFAs to varying degrees. SKBP-DM promoted the secretion of acetic acid, propionic acid, butyric acid, and total acid better than SKBP-H. These results indicate that *Kalocarpus anthelmintica* acidic polysaccharides may restore SCFA secretion levels by improving the metabolic activity of intestinal microorganisms in mice, thereby maintaining normal intestinal immune function.

[0113] 8. The effect of the soluble acidic polysaccharide from kidney bean of the present invention on the composition of intestinal microbiota.

[0114] The gut microbiota is a complex micro-ecosystem in the human body, comprising a large number of bacteria, fungi, viruses, and other microorganisms that coexist with the human body in the gut and play a vital role in human health. Polysaccharides, as prebiotics, can be utilized by the gut microbiota, playing a regulatory role and thus influencing immune function. This study explored the differences or similarities in species community composition among different groups using β-diversity analysis. Principal coordinate analysis (PCoA) based on the Bray-Curtis distance matrix was employed. The more similar the sample composition, the more similar the PCoA plot (…). Figure 13The closer the distances in the samples, the more similar and less different they are in terms of species composition. The results showed that CTX treatment altered the overall structure of the gut microbiota. As shown in the figure, most individuals in the model group clustered independently and were separated from those in the normal group. However, individuals in the SKBP-H and SKBP-DM groups clustered together, intersecting with those in the normal group and moving away from those in the model group. It can be seen that the overall microbial composition of the SKBP-H and SKBP-DM groups is significantly different from that of the model group, but similar to and closer to that of the normal group.

[0115] 9. The effect of the soluble acidic polysaccharide from kidney bean of the present invention on the composition of intestinal microbiota.

[0116] Significant differences were observed when analyzing the relative abundance of gut microbiota at the phylum level in mice. Figure 14 Results showed that Bacteroidota and Firmicutes were the two most dominant phyla at the phylum level, accounting for nearly 90% of the bacterial population. This was followed by Patescibacteria and Desulfobacterota. The results are as follows... Figure 14 As shown in Figure C, compared with the control group mice, the relative abundance of Bacteroidota was decreased in the model group mice, while the relative abundance of Firmicutes, Patescibacteria, and Desulfobacterota was increased. Compared with the model group mice, SKBP-H and SKBP-DM both had varying degrees of regulatory effects on the gut microbiota, increasing the relative abundance of Bacteroidota and decreasing the relative abundance of Patescibacteria and Desulfobacterota. Firmicutes and Bacteroidetes are the two most common bacterial phyla in the human gut.

[0117] The relative abundance distribution of gut microbiota at the genus level in each group of mice is as follows: Figure 14 As shown in B. The research results indicate that ( Figure 14(D) Compared with the control group, CTX treatment significantly increased the relative abundance of Candidatus_Saccharimonas, [Eubacterium]_siraeum_group, and Desulfovibrio, while decreasing the relative abundance of Bacteroides. SKBP-H and SKBP-DM, to varying degrees, increased the relative abundance of Bacteroides and decreased the relative abundance of Candidatus_Saccharimonas, [Eubacterium]_siraeum_group, and Desulfovibrio. Additionally, SKBP-DM significantly increased the relative abundance of Ligilactobacillus.

[0118] 10. The effect of the soluble acidic polysaccharide from kidney bean of the present invention on the composition of intestinal microbiota.

[0119] Linear discriminant analysis effect size (LEfSe) is a commonly used bioinformatics tool for identifying statistically significant and biologically meaningful biomarkers in high-dimensional data. LEfSe combines the features of linear discriminant analysis (LDA) and nonparametric tests, enabling it to identify features that demonstrate significant differences between different groups and calculate their effect size within each group.

[0120] exist Figure 15 In the LDA effect value bar chart, the genera showing significant differences between pairwise comparisons were displayed, while also identifying more genera with significant differences between groups. By observing these bar charts, it is clear which genera have a significant impact on distinguishing different groups, thus providing intuitive information for understanding the differences in microbial composition. Figure 15 The results show that, compared with the model group, the genus Muribaculaum was enriched and the genus Candidatus_Saccharimonas disappeared after SKBP-H treatment; and the genus Bacteroides and Prevotellaceae_UCG_001 were enriched and the genus Candidatus_Saccharimonas disappeared after SKBP-DM treatment.

[0121] 11. Correlation analysis of the monosaccharide structure of soluble acidic polysaccharide from kidney beans, intestinal flora, and immune function indicators in this invention.

[0122] Based on the monosaccharide composition results, we initially found that SKBP-DM had significantly higher levels of arabinose, glucose, and galacturonic acid compared to SKBP-H. Therefore, we compared their differences in gut microbiota utilization by analyzing the correlations between eight monosaccharide types in SKBP-H and SKBP-DM and their corresponding key phyla, genera, and SCFAs generated by gut microbiota regulation. The results are as follows: Figure 16 As shown, at the genus level, arabinose, glucose, galacturonic acid, and *Ligilactobacillus* are positively correlated. The gut microbiota can convert difficult-to-break-down polysaccharides into short-chain fatty acids during digestion, which play a crucial role in immunity, inflammation, and metabolism. Simultaneously, *Ligilactobacillus* shows a positive correlation with the concentrations of acetic acid, propionic acid, and butyric acid. These results indicate that, to some extent, these bacteria promote the production of short-chain fatty acids, which in turn provide nutritional support and maintain the proliferation of beneficial bacteria. Therefore, this mutually reinforcing relationship helps form a virtuous cycle, positively impacting gut health.

[0123] Polysaccharides can be utilized by the gut microbiota as an energy source, promoting the growth of beneficial bacteria and maintaining gut microbiota balance. The gut microbiota influences immune system development and function, regulating immune responses and inflammatory responses. Therefore, we performed intergroup correlation analysis on four phyla and five genera that previously identified significant differences between the model group and the normal group, as well as between SKBP-H and SKBP-DM, and the previously measured immune-related parameters. Based on the results of the correlation analysis, we assessed the association between the microbiota and immune-related parameters, further exploring the potential relationship between microbial composition and immune status. Figure 17As shown, at the phylum level, Bacteroidota was positively correlated with thymus index, serum cytokines (TNF-α, INF-γ, and IL-6), immunoglobulins (Ig-A, Ig-G), catalase, and acetate-propionic acid. Patescibacteria and Desulfobacterota were positively correlated to varying degrees with parameters related to immune organs (thymus and spleen index), serum cytokines (TNF-α, INF-γ, and IL-6), immunoglobulins (Ig-A, Ig-G), oxidative stress markers (CAT, GSH, and SOD), colonic tight junction protein expression (ZO-1, Occuldin), and cecal contents (acetic acid, propionic acid, butyric acid, and total acid). At the genus level, Bacteroides also showed varying degrees of positive correlation with parameters related to immune organs (thymus and spleen indices), serum cytokines (TNF-α, INF-γ, and IL-6), immunoglobulins (Ig-A, Ig-G), oxidative stress markers (CAT, GSH, and SOD), colonic tight junction protein expression (ZO-1, Occuldin), and cecal contents (acetic acid, propionic acid, butyric acid, and total acid). Meanwhile, potential pathogenic bacteria such as Candidatus_Saccharimonas, Desulfovibrio, and [Eubacterium]_siraeum_group showed varying degrees of negative correlation with these parameters.

[0124] Bioinformatics analysis was conducted to study the characteristics of the gut microbiota in fecal samples, revealing that SKBP-H and SKBP-DM can influence the composition and structure of the microbiota. Bacteroidota and Firmicutes are the two most dominant phyla, accounting for nearly 90% of the bacterial population. Bacteroidota and Firmicutes are microbial communities rich in glycoside hydrolases, which aid in the digestion of polysaccharides in the gastrointestinal tract that cannot be directly digested by the body. Desulfobacterota, or sulfate-reducing bacteria, produces hydrogen sulfide as a toxic byproduct; excessive amounts may be detrimental to intestinal epithelial cells. Current understanding of Patescibacteria is still limited. Studies have shown that Patescibacteria levels are high in alcoholic liver disease and aging models, potentially associated with these diseases. The results showed that CTX treatment significantly promoted the overgrowth of Patescibacteria, Desulfobacterota (phylum level), and Candidatus_Saccharimonas, [Eubacterium]_siraeum_group, and Desulfovibrio (genus level), which are generally associated with gut microbiota dysbiosis and barrier damage. However, interventions with SKBP-H and SKBP-DM significantly reduced the relative abundance of these microbiota. SKBP-DM, on the other hand, significantly increased the relative abundance of Ligilactobacillus. Correlation analysis revealed differences in the monosaccharide structure of crude polysaccharides from kidney beans prepared using different extraction techniques in terms of microbiota regulation. At the genus level, galacturonic acid, arabinose, and glucose were positively correlated with Ligilactobacillus. Ligilactobacillus is a lactic acid bacterium with antibacterial, anti-inflammatory, immunomodulatory, and gut microbiota-regulating activities, and is therefore considered a beneficial microbiota. These microbiota can convert indigestible polysaccharides into short-chain fatty acids such as acetic acid, propionic acid, and butyric acid, which are crucial for immunity, inflammation, and metabolism. Ligilactobacillus concentrations were positively correlated with those of acetic acid, propionic acid, and butyric acid. These findings suggest that these bacteria promote the production of short-chain fatty acids to some extent, creating a virtuous cycle that helps maintain gut health. The increased number of Ligilactobacillus, due to the action of arabinose, glucose, and galacturonic acid, further promotes the production of short-chain fatty acids such as acetic acid, propionic acid, and butyric acid. SKBP-DM exhibited better immunomodulatory effects in vivo than SKBP-H.

Claims

1. A soluble acidic polysaccharide from kidney beans, characterized in that: It is extracted and prepared from the seeds of Phaseolus vulgaris Linn., a plant belonging to the genus Phaseolus of the legume family. The total polysaccharide content is 88.1-92.4 mg / 100 mg, the total uronic acid content is 19.6-24.8 mg / 100 mg, the bound phenol content is 1.4-3.0 mg GAE / 100 mg, and the degree of esterification is 15.0-18.5%. Monosaccharides containing the following molar ratios: Rhamnose 1.00, mannose 0.11-0.85, glucuronic acid 0.41-0.50, galacturonic acid 3.87-5.22, glucose 0.31-2.36, galactose 2.93-3.53, xylose 4.16-4.19, arabinose 8.83-13.

08.

2. The soluble acidic polysaccharide from kidney beans according to claim 1, characterized in that: The total polysaccharide content was 91.38 ± 1.00 mg / 100 mg, the total uronic acid content was 24.51 ± 0.27 mg / 100 mg, the conjugated phenol content was 2.83 ± 0.17 mg GAE / 100 mg, and the degree of esterification was 17.20% ± 1.28%. Monosaccharides containing the following molar ratios: Rhamnose 1.00, mannose 0.11, glucuronic acid 0.41, galacturonic acid 5.22, glucose 2.36, galactose 2.93, xylose 4.19, arabinose 13.

08.

3. The soluble acidic polysaccharide from kidney beans according to claim 1 or 2, characterized in that: It is prepared by hot water extraction or microwave-assisted eutectic solvent extraction.

4. A method for preparing the soluble acidic polysaccharide from kidney beans according to any one of claims 1-3, characterized in that: It includes the following steps: a. Take dried and pulverized kidney beans, grind them into powder, add 80%-95% ethanol to remove the alcohol-soluble components, and obtain a precipitate; b. Add water to the precipitate, stir, and then heat in a water bath to extract. Concentrate the extract to obtain a concentrated solution. c. Add α-amylase and saccharifying enzyme to the concentrate to remove starch, then add trypsin to remove protein. After inactivation, add 95% ethanol for precipitation, centrifuge, and obtain the precipitate. d. The precipitate prepared in step c is reconstituted with water, dialyzed to remove small molecules, and dried to obtain the soluble acidic polysaccharide SKBP-H from kidney beans.

5. A method for preparing the soluble acidic polysaccharide from kidney beans according to any one of claims 1-3, characterized in that: It includes the following steps: a. Take dried and pulverized kidney beans, grind them into powder, add 80%-95% ethanol to remove the alcohol-soluble components, and obtain a precipitate; b. Add the prepared choline chloride:ethylene glycol = 1:3 DES to the precipitate, stir well, and extract by microwave. Concentrate the extract to obtain the concentrated solution. The extraction parameters are: DES moisture content: 15-75%; extraction power: 440-840 W; extraction time: 6-30 min; c. Add α-amylase and saccharifying enzyme to the concentrate to remove starch, then add trypsin to remove protein. After inactivation, add 95% ethanol for precipitation, centrifuge, and obtain the precipitate. d. The precipitate prepared in step c is re-dissolved in water, dialyzed to remove small molecules, and dried to obtain the soluble acidic polysaccharide SKBP-DM from kidney beans.

6. The method for preparing the soluble acidic polysaccharide from kidney beans according to claim 5, characterized in that: The extraction parameters in step b are: extraction time of 20-30 min, extraction power of 700-800 W, and DES water content of 30-45%.

7. The method for preparing the soluble acidic polysaccharide from kidney beans according to claim 6, characterized in that: The extraction parameters in step b are: extraction time of 26.2 ± 1.2 min, extraction power of 760.6 ± 10.4 W, and DES water content of 41.5% ± 1.8%.

8. The use of the soluble acidic polysaccharide from kidney beans according to any one of claims 1-3 in the preparation of health foods or pharmaceuticals with antioxidant effects.

9. The use of the soluble acidic polysaccharide from kidney beans according to any one of claims 1-3 in the preparation of health products or pharmaceuticals that stimulate immune activity.

10. The use according to claim 9, characterized in that: The drug described is designed to increase the levels of CAT, SOD, and GSH in the liver, decrease the levels of MDA and ALT, enhance antioxidant capacity, and improve oxidative stress caused by immunodeficiency.

11. The use of the soluble acidic polysaccharide from kidney beans according to claim 1 or 2 in the preparation of drugs that protect the spleen and liver, restore the colonic barrier, regulate intestinal flora, or health foods that help enhance the body's immunity.

Images