Preparation method and application of high-stability synergistic compound medicament based on phaseolus calcaratus

Abstract

The invention relates to the field of compound medicaments, in particular to a preparation method and application of a high-stability synergistic compound medicament based on phaseolus calcaratus, and the preparation method comprises the following steps: adding modified phaseolus calcaratus polysaccharide, modified astragalus polysaccharide and mannitol into a mixing machine, stirring and mixing for 40-50 minutes at the relative humidity of less than or equal to 45%, adding deionized water, heating to 60-70 DEG C, increasing the rotating speed, stirring for 30-60 minutes, and cooling to room temperature to obtain the high-stability synergistic compound medicament based on phaseolus calcaratus. According to the preparation method, the phaseolus calcaratus polysaccharide is subjected to sulfation modification, the astragalus polysaccharide is subjected to acetylation modification, the phaseolus calcaratus polysaccharide and the astragalus polysaccharide are scientifically compounded with the mannitol, and finally the phaseolus calcaratus polysaccharide and the astragalus polysaccharide are formed through an accurately controlled vacuum freeze-drying process. The obtained medicament has remarkably enhanced immunoregulation activity, excellent physical and chemical stability and a definite synergistic treatment effect, and has a wide clinical application prospect in the aspect of preparing immunoregulation, anti-inflammatory and tumor adjuvant therapy medicines.

Description

Technical Field

[0001] This invention relates to the field of compound pharmaceutical preparations, and more particularly to a method for preparing a highly stable synergistic compound pharmaceutical preparation based on adzuki bean and its application. Background Technology

[0002] Plant-derived polysaccharides, as an important resource in the field of biopharmaceutical manufacturing, have attracted much attention due to their immunomodulatory, antioxidant, and low-toxicity properties. Among them, adzuki beans, as a traditional medicinal and edible plant, have shown clear potential in regulating the body's immunity and inhibiting inflammation, and have broad application prospects in the market of chronic disease management and adjuvant cancer treatment drugs.

[0003] In existing technologies, the extraction of red bean polysaccharides mostly adopts the traditional water extraction and alcohol precipitation process. Although this method is simple to operate, it has problems such as limited extraction efficiency, excessively wide molecular weight distribution of polysaccharide components, and incomplete removal of impurities, resulting in low activity of the final product and unstable quality between batches. In addition, in order to improve the stability of polysaccharides, conventional technologies often rely on adding a large amount of excipients or physical encapsulation through simple spray drying. These methods fail to fundamentally change the characteristics of polysaccharide molecules being hygroscopic and easily degraded, and still face the risk of potency decline during long-term storage.

[0004] Furthermore, existing technologies mostly focus on physically mixing adzuki bean polysaccharides with other components, lacking in-depth research and precise design on the synergistic effects between components, and do not involve enhancing the function of core active components through chemical modification. As a result, the synergistic effect of the prepared compound drugs is not obvious, and the overall performance has not achieved a breakthrough.

[0005] Therefore, based on the relevant technologies mentioned above, there is an urgent need to develop a method for preparing a highly stable synergistic compound drug based on adzuki beans and its application. Summary of the Invention

[0006] In view of this, the purpose of this invention is to propose a method for preparing a highly stable synergistic compound drug based on adzuki bean and its application, so as to solve the problems of limited activity, poor stability and insufficient synergy of adzuki bean in the prior art.

[0007] A method for preparing a highly stable synergistic compound drug based on adzuki bean, the preparation method being as follows: Modified adzuki bean polysaccharide, modified astragalus polysaccharide, and mannitol were added to a mixer. The relative humidity was ≤45%, the speed was 10-20 rpm, and the mixture was stirred for 40-50 min. Deionized water was added, the temperature was raised to 60-70℃, the speed was increased to 150-250 rpm, and the mixture was stirred for 50-70 min. Activated carbon was added, the speed was reduced to 80-100 rpm, and the mixture was stirred for 30-40 min. The mixture was filtered through 0.45 μm polyethersulfone and then freeze-dried under vacuum to obtain the compound preparation.

[0008] Two functionally enhanced modified polysaccharides were compounded with mannitol in a precise mass ratio and shaped using vacuum freeze-drying technology. This process not only solved the stability problem of polysaccharide drugs being hygroscopic and easily degraded, but also ensured that the product had a full appearance and rapid reconstitution, thanks to mannitol as an excellent freeze-drying protectant. Meanwhile, modified adzuki bean polysaccharide can quickly activate the innate immune system and exert a strong immune initiation and antioxidant effect, while modified astragalus polysaccharide can regulate and enhance specific immune function. Through the combination of the two, they not only complement each other in different stages and pathways of immune regulation, but also produce a synergistic effect, achieving comprehensive conditioning at multiple levels and on multiple targets for complex pathological states such as low immune function, chronic inflammation and adjuvant treatment of tumors.

[0009] Preferably, the mass ratio of the modified adzuki bean polysaccharide, modified astragalus polysaccharide, mannitol, deionized water and activated carbon is 1:1.9-2.1:1.7-1.8:45.1-45.3:0.04-0.06.

[0010] Preferably, the preparation steps of the modified adzuki bean polysaccharide are as follows: Step A1: Add adzuki beans to phosphate buffer, heat to 40-60℃, stir at 200-300 rpm for 15-25 min, add cellulase and pectinase, reduce the stirring speed to 100-150 rpm, add 1 mol / L sodium hydroxide solution, adjust the pH to 4.5-4.8, react for 2-3 h, after the reaction is complete, heat to 85-95℃, increase the stirring speed to 200-300 rpm, continue for 50-70 min, separate and purify, dry, and obtain adzuki bean polysaccharide powder; Step A2: Under a nitrogen atmosphere, add adzuki bean polysaccharide powder to a chlorosulfonic acid and pyridine complex reagent, cool to 0-5℃, stir at 200-300 rpm for 50-70 min, heat to 50-70℃, increase the stirring speed to 350-450 rpm, and react for 2-4 h. After the reaction is complete, cool to 2-8℃, add saturated sodium hydroxide solution, adjust the pH to 6.8-7.2, purify, and dry to obtain modified adzuki bean polysaccharide.

[0011] By using cellulase and pectinase, cell walls can be selectively disrupted, greatly improving the extraction efficiency and purity of polysaccharides. Subsequently, ultrafiltration is used to precisely enrich polysaccharides within the target molecular weight range and remove small molecule impurities, fundamentally solving the problems of high impurity content, unclear active ingredients, and unstable batch quality in traditional water extraction methods. This provides a high-purity raw material basis for subsequent modification. In addition, sulfation significantly enhances the electronegativity and antioxidant activity of adzuki bean polysaccharides and endows them with stronger immunostimulatory capabilities.

[0012] Preferably, the mass ratio of red beans, phosphate buffer, cellulase and pectinase in step A1 is 1:11-13:0.024-0.026:0.024-0.026.

[0013] Preferably, the ratio of adzuki bean polysaccharide powder to composite reagent in step A2 is 1g:6.4-6.6mL.

[0014] Preferably, the volume ratio of chlorosulfonic acid to pyridine in step A2 is 1:4.

[0015] Preferably, the preparation steps of the modified Astragalus polysaccharide are as follows: Step B1: Add Astragalus membranaceus slices to deionized water, heat to 90-110℃, reflux for 1-2 hours. After the reaction is complete, filter, concentrate, add 95% ethanol, let stand, wash and dry to obtain Astragalus membranaceus polysaccharide powder. Step B2: Under a nitrogen atmosphere, add Astragalus polysaccharide powder to anhydrous pyridine, heat to 50-70℃, stir at 150-250 rpm for 2-3 hours, add acetic anhydride, cool to 0-5℃, increase the stirring speed to 300-400 rpm, stir for 60-70 minutes, then heat to 60-70℃ and react for 4-6 hours. After the reaction is complete, cool to 20-30℃, wash the precipitate, and dry to obtain modified Astragalus polysaccharide.

[0016] Introducing acetyl groups with acetic anhydride effectively improves the water and lipid solubility of Astragalus polysaccharides. This not only enhances their bioavailability but also exposes new active sites, making their immunomodulatory function more gentle and lasting.

[0017] Preferably, the mass ratio of Astragalus membranaceus slices to deionized water in step B1 is 1:20-24.

[0018] Preferably, the mass ratio of Astragalus polysaccharide powder to acetic anhydride in step B2 is 1:2.6-2.8.

[0019] Application of a highly stable synergistic compound drug based on adzuki bean, wherein the highly stable synergistic compound drug is used to prepare drugs for immunomodulation, anti-inflammation and adjuvant tumor treatment.

[0020] The beneficial effects of this invention are: This invention provides a method for preparing a highly stable synergistic compound drug based on adzuki bean and its application. The invention involves sulfation modification of adzuki bean polysaccharide and acetylation modification of astragalus polysaccharide, followed by scientific compounding of the two with mannitol at a specific mass ratio. Finally, the mixture is shaped using a precisely controlled vacuum freeze-drying process. Compared with existing technologies, this invention fundamentally solves the problems of limited activity, poor stability, and insufficient synergistic effect of natural polysaccharides. The resulting drug exhibits significantly enhanced immunomodulatory activity, excellent physicochemical stability, and a clear synergistic therapeutic effect, showing broad clinical application prospects in the preparation of immunomodulatory, anti-inflammatory, and tumor adjuvant therapy drugs. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only for this invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a bar chart showing the polysaccharide content on day 0 and day 30 of a highly stable synergistic compound agent based on adzuki bean according to the present invention. Figure 2 Line graph showing the moisture absorption weight gain rate and polysaccharide decrease rate of a highly stable synergistic compound agent based on adzuki bean after 30 days; Figure 3 This is a bar chart showing the stimulation index of macrophages and lymphocytes in a highly stable synergistic compound drug based on adzuki bean according to the present invention. Figure 4 This is a bar chart illustrating the enhanced activity of macrophages and lymphocytes by a highly stable synergistic compound drug based on adzuki beans according to the present invention. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments.

[0024] Example 1: Preparation steps of a modified adzuki bean polysaccharide S1: Add 100g of red beans to 1100g of phosphate buffer, heat to 40℃, stir at 300rpm for 15min, add 2.4g of cellulase and 2.4g of pectinase, reduce the stirring speed to 150rpm, add 1mol / L sodium hydroxide solution, adjust the pH to 4.5-4.8, react for 2h, the reaction is complete, heat to 95℃, increase the stirring speed to 200rpm, continue for 70min, separate and purify, dry, and obtain red bean polysaccharide powder; S2: Under a nitrogen atmosphere, 10g of red bean polysaccharide powder was added to 12.8mL of chlorosulfonic acid and 51.2mL of pyridine complex reagent. The mixture was cooled to 0℃, stirred at 300rpm for 50min, heated to 70℃, and stirred at 350rpm for 4h. After the reaction was complete, the mixture was cooled to 2℃, saturated sodium hydroxide solution was added, and the pH was adjusted to 6.8-7.2. The mixture was then purified and dried to obtain modified red bean polysaccharide.

[0025] Example 2: Preparation steps of a modified adzuki bean polysaccharide S1: Add 100g of red beans to 1200g of phosphate buffer, heat to 50℃, stir at 250rpm for 20min, add 2.5g of cellulase and 2.5g of pectinase, reduce the stirring speed to 130rpm, add 1mol / L sodium hydroxide solution, adjust the pH to 4.5-4.8, react for 2.5h, the reaction is complete, heat to 90℃, increase the stirring speed to 250rpm, continue for 60min, separate and purify, dry, and obtain red bean polysaccharide powder; S2: Under a nitrogen atmosphere, 10g of adzuki bean polysaccharide powder was added to 13mL of chlorosulfonic acid and 52mL of pyridine complex reagent. The mixture was cooled to 3℃, stirred at 250rpm for 60min, heated to 60℃, and stirred at 400rpm for 3h. After the reaction was complete, the mixture was cooled to 5℃, saturated sodium hydroxide solution was added, and the pH was adjusted to 6.8-7.2. The mixture was then purified and dried to obtain modified adzuki bean polysaccharide.

[0026] Example 3: Preparation steps of a modified adzuki bean polysaccharide S1: Add 100g of red beans to 1300g of phosphate buffer, heat to 60℃, stir at 200rpm for 25min, add 2.6g of cellulase and 2.6g of pectinase, reduce the stirring speed to 100rpm, add 1mol / L sodium hydroxide solution, adjust the pH to 4.5-4.8, react for 3h, the reaction is complete, heat to 85℃, increase the stirring speed to 300rpm, continue for 50min, separate and purify, dry, and obtain red bean polysaccharide powder; S2: Under a nitrogen atmosphere, 10g of red bean polysaccharide powder was added to 13.2mL of chlorosulfonic acid and 52.8mL of pyridine complex reagent. The mixture was cooled to 5℃, stirred at 300rpm for 70min, heated to 50℃, and stirred at 450rpm for 2h. After the reaction was complete, the mixture was cooled to 8℃, saturated sodium hydroxide solution was added, and the pH was adjusted to 6.8-7.2. The mixture was then purified and dried to obtain modified red bean polysaccharide.

[0027] Example 4: Preparation steps of a modified Astragalus polysaccharide S1: Add 100g of Astragalus membranaceus slices to 2000g of deionized water, heat to 90℃, reflux for 2h, filter, concentrate, add 95% ethanol, let stand, wash and dry to obtain Astragalus membranaceus polysaccharide powder. S2: Under a nitrogen atmosphere, 10g of Astragalus polysaccharide powder was added to 100mL of anhydrous pyridine, heated to 50℃, stirred at 250rpm for 2h, 26g of acetic anhydride was added, the temperature was lowered to 5℃, the stirring speed was increased to 300rpm for 70min, and then the temperature was raised to 60℃ for 6h. After the reaction was completed, the temperature was lowered to 20℃, the precipitate was washed, dried, and the modified Astragalus polysaccharide was obtained.

[0028] Example 5: Preparation steps of a modified Astragalus polysaccharide S1: Add 100g of Astragalus membranaceus slices to 2200g of deionized water, heat to 100℃, reflux for 1.5h, filter, concentrate, add 95% ethanol, let stand, wash and dry to obtain Astragalus membranaceus polysaccharide powder. S2: Under a nitrogen atmosphere, 10g of Astragalus polysaccharide powder was added to 100mL of anhydrous pyridine, heated to 60℃, stirred at 200rpm for 2.5h, 27g of acetic anhydride was added, the temperature was lowered to 3℃, the stirring speed was increased to 350rpm for 65min, the temperature was raised to 65℃ again, and the reaction was carried out for 5h. After the reaction was completed, the temperature was lowered to 25℃, the precipitate was washed, dried, and the modified Astragalus polysaccharide was obtained.

[0029] Example 6: Preparation steps of a modified Astragalus polysaccharide S1: Add 100g of Astragalus membranaceus slices to 2400g of deionized water, heat to 110℃, reflux for 1h, filter, concentrate, add 95% ethanol, let stand, wash and dry to obtain Astragalus membranaceus polysaccharide powder. S2: Under a nitrogen atmosphere, 10g of Astragalus polysaccharide powder was added to 100mL of anhydrous pyridine, heated to 70℃, stirred at 150rpm for 3h, 28g of acetic anhydride was added, cooled to 0℃, stirred at 400rpm for 60min, then heated to 70℃ and reacted for 4h. After the reaction was completed, the temperature was lowered to 30℃, the precipitate was washed, dried, and the modified Astragalus polysaccharide was obtained.

[0030] Example 7: A method for preparing a highly stable synergistic compound drug based on adzuki bean 10g of modified adzuki bean polysaccharide (Example 1), 19g of modified astragalus polysaccharide (Example 4) and 17g of mannitol were added to a mixer. The relative humidity was ≤45%, the speed was 10 rpm, and the mixture was stirred for 50 min. 451g of deionized water was added, the temperature was raised to 60℃, the speed was increased to 250 rpm, and the mixture was stirred for 50 min. 0.4g of activated carbon was added, the speed was reduced to 80 rpm, and the mixture was stirred for 40 min. The mixture was filtered through 0.45μm polyethersulfone and then freeze-dried under vacuum to obtain the compound preparation.

[0031] Example 8: A method for preparing a highly stable synergistic compound drug based on adzuki bean 10g of modified adzuki bean polysaccharide (Example 2), 20g of modified astragalus polysaccharide (Example 5), and 17.5g of mannitol were added to a mixer. The relative humidity was ≤45%, the speed was 15 rpm, and the mixture was stirred for 45 min. 452g of deionized water was added, the temperature was raised to 65℃, the speed was increased to 200 rpm, and the mixture was stirred for 60 min. 0.5g of activated carbon was added, the speed was reduced to 90 rpm, and the mixture was stirred for 35 min. The mixture was filtered through 0.45μm polyethersulfone and then freeze-dried under vacuum to obtain the compound preparation.

[0032] Example 9: A method for preparing a highly stable synergistic compound drug based on adzuki bean 10g of modified adzuki bean polysaccharide (Example 3), 21g of modified astragalus polysaccharide (Example 6), and 18g of mannitol were added to a mixer. The relative humidity was ≤45%, the speed was 20 rpm, and the mixture was stirred for 40 min. 453g of deionized water was added, the temperature was raised to 70℃, the speed was increased to 150 rpm, and the mixture was stirred for 70 min. 0.6g of activated carbon was added, the speed was reduced to 80 rpm, and the mixture was stirred for 40 min. The mixture was filtered through 0.45μm polyethersulfone and then freeze-dried under vacuum to obtain the compound preparation.

[0033] Example 10: A method for preparing a highly stable synergistic compound drug based on adzuki bean 10g of modified adzuki bean polysaccharide (Example 1), 19g of modified astragalus polysaccharide (Example 4), 15g of mannitol, 3g of curcumin phospholipid complex, and 2g of grape seed proanthocyanidins were added to a mixer. The relative humidity was ≤45%, the speed was 10 rpm, and the mixture was stirred for 50 min. 451g of deionized water was added, the temperature was raised to 60℃, the speed was increased to 250 rpm, and the mixture was stirred for 50 min. 0.4g of activated carbon was added, the speed was reduced to 80 rpm, and the mixture was stirred for 40 min. The mixture was filtered through 0.45μm polyethersulfone and then freeze-dried under vacuum to obtain the compound preparation.

[0034] Comparative Example 1: Compared with Example 7, this comparative example only replaces "modified adzuki bean polysaccharide" with "adzuki bean polysaccharide". All other steps and parameters are the same, and will not be repeated here. The final compound preparation is obtained.

[0035] Comparative Example 2 Compared with Example 7, this comparative example only changed the mass ratio of modified red adzuki bean polysaccharide, modified astragalus polysaccharide, and mannitol from "1:1.9:1.7" to "1:1:1". All other steps and parameters are the same, and will not be repeated here. The final compound preparation is obtained.

[0036] Comparative Example 3 This comparative example differs from Example 7 only in that "vacuum freeze drying" is replaced with "spray drying". All other steps and parameters are the same, and will not be repeated here. The final compound preparation is obtained.

[0037] Comparative Example 4: Compared with Example 7, this comparative example only replaces "modified Astragalus polysaccharide" with "Astragalus polysaccharide". All other steps and parameters are the same, and will not be repeated here. The final compound medicine is obtained.

[0038] Comparative Example 5 Compared with Example 7, this comparative example only replaces the rotation speed "10 rpm" with "50 rpm" in the preparation of the compound drug. All other steps and parameters are the same, and will not be repeated in this comparative example. The final compound drug is obtained.

[0039] Comparative Example 6 Compared with Example 7, this comparative example only changed the rotation speed of "350 rpm" in the S2 process of preparing modified red bean polysaccharide to "200 rpm". All other steps and parameters are the same, and will not be repeated here. The final compound preparation was obtained.

[0040] Performance testing: Stability test Determination of moisture absorption and weight gain rate The test was conducted using the BSD-TH10 constant temperature and humidity chamber, in accordance with the testing standards of the Chinese Pharmacopoeia (2020 edition). 1. Take several weighing bottles, put them in an oven, heat them to 105℃, dry them to constant weight, cool them to room temperature, and record the weight W0. 2. Take 1.0g (W1) of each of the compound agents of Examples 7-10 and Comparative Examples 1-6, put them into weighing bottles, place them in a constant temperature and humidity test chamber, raise the temperature to 40℃±2℃, relative humidity 75%±5%, and place them for 30 days. On day 0, day 10, day 20 and day 30, samples are taken. When taking samples, cool for 30 minutes, weigh them, and record the weight as W2. 3. Calculation formula: W1: Initial weight of sample (g), W2: Total weight of weighing bottle and sample (g), W0: Weight of weighing bottle (g).

[0041] Polysaccharide content determination The testing standards were in accordance with the Chinese Pharmacopoeia (2020 edition), and a SHIMADZU UV-2700 constant ultraviolet-visible spectrophotometer was used. 1. Take 10 mg of each of the compound reagents from Examples 7-10 and Comparative Examples 1-6 in the above experiments, add deionized water, and make up to 50 mL to obtain the sample solution; 2. Take 0.5 mL of sample solution, put it into a test tube, add 0.5 mL of 5% phenol solution, shake well, add 2.5 mL of concentrated sulfuric acid, shake well, heat to 40℃, react for 30 min, cool to room temperature, and measure the absorbance at a wavelength of 490 nm. 3. Calculate polysaccharide content: C: Concentration (μg / mL), V: Volume of test solution (mL), D: Dilution factor, W: Sample weight (g). 4. Calculate the polysaccharide content decrease rate: .

[0042] Table 1. Stability test results of the examples and comparative examples Immunoactivity test Referring to the 2023 edition of the "Technical Specifications for Inspection and Evaluation of Health Foods", mouse mononuclear macrophages RAW264.7 and mouse spleen lymphocytes were used. 1. Macrophage phagocytic activity test a: RAW264.7 cells were placed in RPMI-1640 containing 10% fetal bovine serum. Place the culture medium in a CO2 incubator, heat to 37°C, and then subculture. b: Take cells in the logarithmic growth phase, digest them with trypsin, resuspend and adjust... Cell density up to 2×10 5 Cells / mL, seeded in 96-well plates, 100 μL per well. L, incubate for 24 hours; c: Blank group: Replace with 100 μL of maintenance medium containing 1% fetal bovine serum; Positive control group: 100 μL of maintenance medium containing lipopolysaccharide was added; Experimental group: The compound agents from Examples 7-10 and Comparative Examples 1-6 were respectively used with... RPMI-1640 complete medium containing 10% fetal bovine serum was prepared in 50, 100, and 200 mL solutions. A sample solution of μg / mL was incubated for 24 hours. d: Aspirate the culture medium from each well, wash the cells twice with phosphate buffer, and add... Add 100 μL of 0.1% neutral red solution and continue culturing for 2 hours. Incubation is then complete. Wash cells three times with phosphate buffer, then add 100 μL of cell lysis solution. After incubation for 2 hours, the absorbance (OD) was measured using a microplate reader at a wavelength of 540 nm. value).

[0043] 2. Lymphocyte proliferation assay a: Mouse spleen was ground into a single-cell suspension and passed through a 200-mesh sieve. Centrifuge at 1500 rpm for 5 min, resuspend the precipitate in red blood cell lysis buffer, and lyse. 3-5 min, place in RPMI-1640 complete culture medium containing 10% phosphate buffer solution Cells were resuspended in culture medium, counted, and density adjusted to 5 × 10⁶. 6 cells / mL, seeded in 96-well plates, 100 μL per well; b: Blank group: Add 100 μL of complete culture medium; Positive control group: 100 μL of complete culture medium containing concanavalin A was added; Experimental group: Take the compound agents from Examples 7-10 and Comparative Examples 1-6 respectively, and use them up. Prepare sample solutions of 50, 100, and 200 μg / mL using complete culture medium, and add... Concanavalin A solution was placed in a CO2 incubator and heated to 37°C for incubation. 72h; c: Add 20 μL of MTT solution 4 hours before the end of the culture, and continue culture for another 4 hours. Centrifuge, add 150 μL of dimethyl sulfoxide, vortex for 10 min, and then use a microplate reader to read the microplate. Absorbance was measured at a wavelength of 570 nm. 3. Stimulus Index Calculation Formula: Table 2. Results of immunological activity tests for the examples and comparative examples. Data Analysis: As can be seen from Tables 1-2, the highly stable synergistic compound preparations obtained by the present invention have superior stability and stronger immunomodulatory activity. In contrast, Comparative Example 1, due to the failure to replace the modified adzuki bean polysaccharide with adzuki bean polysaccharide, experienced a significant decline in performance. This was because the hydrophilic groups such as hydroxyl groups on the surface of the natural polysaccharide were not effectively replaced by hydrophobic sulfate groups, making it more susceptible to moisture absorption and degradation. At the same time, the electronegativity of the molecular structure was not enhanced, affecting its binding ability to immune cell receptors. Furthermore, the unmodified molecular conformation could not effectively activate key immune signaling pathways, resulting in insufficient immunostimulatory activity. Comparative Example 2 showed that the synergistic effect was weakened because the modified red adzuki bean polysaccharide, modified astragalus polysaccharide and mannitol deviated from the optimal compound ratio. The reason is that the 1:1 ratio destroyed the electrostatic interaction and hydrogen bond network between the two modified polysaccharide molecules, which affected the stability of the system. At the same time, the imbalance of the ratio caused the activation of innate immunity and specific immunity to be asynchronous, and the effective immune cascade reaction could not be formed. In addition, the specific mass ratio is the optimal ratio that can maximize the synergistic effect after being verified by a large number of experiments. Comparative Example 3 suffered a severe decrease in stability due to the use of spray drying. This is because the sample is heated instantaneously during spray drying, which can only form a loose microsphere structure, rather than the rigid porous framework formed by freeze drying. At the same time, high temperature will trigger Maillard reaction, which accelerates the decomposition and browning of polysaccharides. In addition, spray-dried products have a large specific surface area and loose structure, making them more prone to moisture absorption and clumping, and cannot provide a long-term stable protective environment. Comparative Example 4 showed that the function of the modified Astragalus polysaccharide was limited because the intermolecular and intramolecular hydrogen bond network of the natural polysaccharide was dense, which reduced its water solubility and bioavailability. At the same time, the unexposed active sites could not effectively bind to specific receptors on the surface of immune cells. In addition, the absence of acetyl groups caused it to lose the key structural basis for enhancing immune regulation by regulating the Toll-like receptor pathway. Comparative Example 5 showed a decrease in stability and immunomodulatory activity due to the replacement of the rotation speed from 10 rpm to 50 rpm during the preparation of the compound drug. This was because the excessive shear force destroyed the uniform dispersion system and potential interaction forces between mannitol and modified polysaccharide molecules, resulting in a non-uniform microstructure of the product. At the same time, static electricity may be generated, causing powder agglomeration, which affects resolubility and long-term stability. In addition, the severe mechanical action may cause physical damage to some polysaccharide chains, slightly reducing its biological activity. Comparative Example 6 showed significantly inferior product performance compared to the Example due to the reduced stirring speed during the sulfation reaction. This was because the excessively low stirring speed could not ensure sufficient mass transfer in the high-viscosity reaction system, resulting in uneven contact between the chlorosulfonic acid-pyridine composite reagent and the polysaccharide particles. Furthermore, the coexistence of local overheating and local under-reaction led to a low degree of sulfation modification and uneven distribution. In addition, the activity of polysaccharide molecule fragments that were not effectively modified was not enhanced, thereby reducing the overall immune-enhancing effect of the product. Example 10, by introducing curcumin phospholipid complex and grape seed proanthocyanidins, achieved functional expansion while maintaining excellent performance. Although its hygroscopicity was slightly higher than that of the core example, it was still within a controllable range, and its immune activity was significantly enhanced. This is because curcumin and proanthocyanidins themselves have strong antioxidant activity, which can synergistically protect the polysaccharide structure from oxidative degradation. At the same time, these active ingredients can produce an immune-enhancing effect with polysaccharides by regulating signaling pathways such as MAPK and Nrf2. In addition, the phospholipid complex technology improves the bioavailability of curcumin, enabling it to more effectively exert its anti-inflammatory and immunomodulatory functions, and achieving an overall effect of synergistic enhancement of multiple components.

[0044] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of the invention is limited to these examples; within the framework of the invention, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.

[0045] This invention is intended to cover all such substitutions, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A method for preparing a highly stable synergistic compound drug based on adzuki bean, characterized in that, The preparation method is as follows: Modified adzuki bean polysaccharide, modified astragalus polysaccharide, and mannitol were added to a mixer. The relative humidity was ≤45%, the speed was 10-20 rpm, and the mixture was stirred for 40-50 min. Deionized water was added, the temperature was raised to 60-70℃, the speed was increased to 150-250 rpm, and the mixture was stirred for 50-70 min. Activated carbon was added, the speed was reduced to 80-100 rpm, and the mixture was stirred for 30-40 min. The mixture was filtered through 0.45 μm polyethersulfone and then freeze-dried under vacuum to obtain the compound preparation.

2. The method for preparing a highly stable synergistic compound drug based on adzuki bean according to claim 1, characterized in that, The mass ratio of the modified adzuki bean polysaccharide, modified astragalus polysaccharide, mannitol, deionized water and activated carbon is 1:1.9-2.1:1.7-1.8:45.1-45.3:0.04-0.

06.

3. The method for preparing a highly stable synergistic compound drug based on adzuki bean according to claim 1, characterized in that, The preparation steps of the modified adzuki bean polysaccharide are as follows: Step A1: Add adzuki beans to phosphate buffer, heat to 40-60℃, stir at 200-300 rpm for 15-25 min, add cellulase and pectinase, reduce the stirring speed to 100-150 rpm, add 1 mol / L sodium hydroxide solution, adjust the pH to 4.5-4.8, react for 2-3 h, after the reaction is complete, heat to 85-95℃, increase the stirring speed to 200-300 rpm, continue for 50-70 min, separate and purify, dry, and obtain adzuki bean polysaccharide powder; Step A2: Under a nitrogen atmosphere, add adzuki bean polysaccharide powder to a chlorosulfonic acid and pyridine complex reagent, cool to 0-5℃, stir at 200-300 rpm for 50-70 min, heat to 50-70℃, increase the stirring speed to 350-450 rpm, and react for 2-4 h. After the reaction is complete, cool to 2-8℃, add saturated sodium hydroxide solution, adjust the pH to 6.8-7.2, purify, and dry to obtain modified adzuki bean polysaccharide.

4. The method for preparing a highly stable synergistic compound drug based on adzuki bean according to claim 3, characterized in that, The mass ratio of red beans, phosphate buffer, cellulase and pectinase in step A1 is 1:11-13:0.024-0.026:0.024-0.

026.

5. The method for preparing a highly stable synergistic compound drug based on adzuki bean according to claim 3, characterized in that, The ratio of red bean polysaccharide powder to the compound reagent in step A2 is 1g: 6.4-6.6mL.

6. The method for preparing a highly stable synergistic compound drug based on adzuki bean according to claim 3, characterized in that, The volume ratio of chlorosulfonic acid to pyridine in step A2 is 1:

4.

7. The method for preparing a highly stable synergistic compound drug based on adzuki bean according to claim 1, characterized in that, The preparation steps of the modified Astragalus polysaccharide are as follows: Step B1: Add Astragalus membranaceus slices to deionized water, heat to 90-110℃, reflux for 1-2 hours. After the reaction is complete, filter, concentrate, add 95% ethanol, let stand, wash and dry to obtain Astragalus membranaceus polysaccharide powder. Step B2: Under a nitrogen atmosphere, add Astragalus polysaccharide powder to anhydrous pyridine, heat to 50-70℃, stir at 150-250 rpm for 2-3 hours, add acetic anhydride, cool to 0-5℃, increase the stirring speed to 300-400 rpm, stir for 60-70 minutes, then heat to 60-70℃ and react for 4-6 hours. After the reaction is complete, cool to 20-30℃, wash the precipitate, and dry to obtain modified Astragalus polysaccharide.

8. The method for preparing a highly stable synergistic compound drug based on adzuki bean according to claim 7, characterized in that, The mass ratio of Astragalus membranaceus slices to deionized water in step B1 is 1:20-24.

9. The method for preparing a highly stable synergistic compound drug based on adzuki bean according to claim 7, characterized in that, The mass ratio of Astragalus polysaccharide powder to acetic anhydride in step B2 is 1:2.6-2.

8.

10. The application of a highly stable synergistic compound drug based on adzuki bean according to any one of claims 1-9, characterized in that, The highly stable synergistic compound is used to prepare drugs for immunomodulation, anti-inflammation, and adjuvant tumor treatment.

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