Chinese wolfberry and astragalus compound polysaccharide with immunity enhancing effect as well as preparation method and application of Chinese wolfberry and astragalus compound polysaccharide

Abstract

The invention discloses a wolfberry-astragalus compound polysaccharide with an immunity enhancing effect as well as a preparation method and application thereof, and belongs to the technical field of natural active polysaccharides and immunoregulation. The wolfberry-astragalus compound polysaccharide is obtained by separately extracting and purifying wolfberry polysaccharide and astragalus polysaccharide and compounding according to a specific mass ratio, and the total sugar content is 70-80%, the uronic acid content is 10-14%, and the protein content is 15-25%. In-vitro cell experiments and in-vivo animal experiments verify that the wolfberry-astragalus compound polysaccharide can significantly promote the phagocytic function of macrophages, up-regulate the secretion of NO, TNF-alpha and IL-6, significantly promote the functions of immune cells, and improve the indexes of immune organs and the level of immune globulin, thereby enhancing the immune function of the organism. The compound is stable in preparation process, high in purity, natural in source and high in safety, can be used for preparing medicines, health foods or functional foods for enhancing immunity or improving immunocompromise, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of natural active polysaccharides and immunomodulatory technology, specifically relating to a Lycium barbarum-Astragalus membranaceus complex polysaccharide that enhances immunity, as well as the preparation method of the complex polysaccharide and its application in enhancing the body's immunity and improving low immune function. Background Technology

[0002] The immune system is a vital defense system for recognizing and eliminating pathogens and maintaining homeostasis. It is primarily composed of two major systems: cellular immunity and humoral immunity. Its functional state directly affects the body's ability to defend against infections, diseases, and external stimuli. Under the influence of various factors such as prolonged mental stress, unbalanced nutritional intake, environmental factors, infection, disease, or drug intervention, the body's immune system is prone to dysfunction. This manifests as a decrease in the number of immune cells, damage to the structure and function of immune organs, and a decline in immune response capacity, leading to reduced immunity and a significant increase in health risks.

[0003] Currently, products used to enhance immunity mainly include chemically synthesized immunomodulators and naturally derived immune enhancers. While some chemically synthesized immunomodulators are characterized by rapid onset and well-defined effects, long-term or high-dose use may cause toxic side effects, affecting normal physiological functions and limiting their application in healthy individuals and long-term interventions. Naturally derived immune enhancers, on the other hand, have attracted attention due to their higher safety profile and wider applicability. However, existing products mostly rely on single active ingredients with relatively limited target areas, making it difficult to achieve systematic and synergistic regulation of complex immune processes. Therefore, there is still room for improvement in their immune-enhancing effects.

[0004] Polysaccharides, as a class of natural macromolecules widely found in both medicinal and edible resources, possess advantages such as low toxicity, good biocompatibility, structural diversity, and multi-target regulation, showing promising research and application prospects in the field of immunomodulation. Studies have shown that polysaccharides can participate in the body's immune regulation through multiple pathways, including regulating immune cell function, promoting the secretion of immune-related factors, and improving the state of immune organs. However, current research on the immunomodulatory activity of polysaccharides largely focuses on polysaccharides from single sources, and their immunomodulatory effects are limited by the singularity of their structure and function, making it difficult to fully utilize the potential synergistic effects between polysaccharides.

[0005] Both Lycium barbarum polysaccharides and Astragalus membranaceus polysaccharides are common natural immunomodulatory polysaccharides, and studies have confirmed their respective advantages in immunomodulation. However, current research on the combined application of Lycium barbarum and Astragalus membranaceus polysaccharides is relatively limited, especially regarding the intrinsic relationship between the compounding ratio, structural stability, and immune-enhancing effects. Furthermore, some existing compound polysaccharide preparation processes employ a method of co-extraction after mixing raw materials, which can easily lead to complex polysaccharide composition and poor batch stability, thereby affecting the immunomodulatory effects and application reliability of the product.

[0006] Therefore, there is an urgent need to develop a Lycium barbarum-Astragalus complex polysaccharide with controllable preparation process, clear composition and structural characteristics, and synergistic immune-enhancing effects, in order to overcome the problems of limited immune-enhancing effects and insufficient product stability in existing technologies, thereby expanding the application scope and practical value of natural polysaccharides in the field of immune regulation. Summary of the Invention

[0007] The purpose of this invention is to provide a Lycium barbarum-Astragalus membranaceus compound polysaccharide with immune-enhancing effects. By optimizing the preparation process and compounding ratio, the purity and immunomodulatory activity of the compound polysaccharide are improved, and its application in improving low immune function is clarified. This invention solves the problems of limited immune effects of single polysaccharides, unstable preparation process of compound polysaccharides, and unclear immune synergistic effects in the prior art.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A compound polysaccharide of Lycium barbarum and Astragalus membranaceus with immune-enhancing effects is prepared by separately extracting and purifying Lycium barbarum polysaccharides (LBPs) and Astragalus membranaceus polysaccharides (APSs) and then mixing them. The total sugar content of the compound polysaccharide is 70-80%, the uronic acid content is 10-14%, and the protein content is 15-25%. The mass ratio of Lycium barbarum polysaccharides to Astragalus membranaceus polysaccharides is 1:5 to 5:1 (preferably 1:1).

[0009] The compound polysaccharide of Lycium barbarum and Astragalus membranaceus has a stable molecular weight distribution, with the molecular weight between that of single Lycium barbarum polysaccharide and single Astragalus membranaceus polysaccharide. Its monosaccharide composition includes mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose. Its microstructure exhibits the characteristic of coexistence of chain structure and irregular aggregates. With the change of the compound ratio of Lycium barbarum polysaccharide and Astragalus membranaceus polysaccharide, the microstructure can gradually change from being dominated by chain structure to being dominated by spherical aggregates and short chain structure.

[0010] The preparation method of the Lycium barbarum-Astragalus complex polysaccharide includes the following steps: (1) Separate extraction: The raw materials of wolfberry and astragalus were washed, dried and crushed respectively, and then mixed with water. They were heated and extracted, filtered and centrifuged to remove insoluble residues, and wolfberry polysaccharide extract and astragalus polysaccharide extract were obtained respectively. The temperature of the heating extraction was 80-100℃, the extraction time was 90-150min, and the mass-volume ratio of raw materials to water was 1:(20-40)g / mL. (2) Separate purification: The Lycium barbarum polysaccharide extract and Astragalus membranaceus polysaccharide extract obtained in step (1) were subjected to membrane filtration, deproteinization, decolorization and dialysis, and then dried to obtain purified Lycium barbarum polysaccharide and Astragalus membranaceus polysaccharide, respectively; among them, the membrane filtration adopted an ultrafiltration membrane with a molecular weight cutoff of 3000-10000 Da; the deproteinization treatment adopted the Sevage method; the dialysis adopted a dialysis bag with a molecular weight cutoff of 3000-4000 Da; when purifying the Lycium barbarum polysaccharide extract, a two-phase extraction step was also included after dialysis: dipotassium hydrogen phosphate and a hot mixture of choline chloride and 1,4-butanediol were added to the Lycium barbarum polysaccharide dialysis solution, vortexed and centrifuged, the lower phase liquid was collected and then dried; (3) Mixing: The wolfberry polysaccharide and astragalus polysaccharide obtained in step (2) are mixed at a mass ratio of 1:5 to 5:1, preferably at a mass ratio of 1:1, to obtain the wolfberry-astragalus complex polysaccharide.

[0011] The aforementioned Lycium barbarum and Astragalus membranaceus polysaccharide can be used to prepare drugs, health foods, or functional foods that enhance immunity or improve immunodeficiency. The immunodeficiency includes immunosuppression caused by drugs, diseases, or stress factors. The Lycium barbarum and Astragalus membranaceus polysaccharide can improve the body's immune function by promoting macrophage phagocytosis, upregulating the secretion of NO, TNF-α, and IL-6, and improving problems such as atrophy of immune organs, reduction of blood cells, and decreased immunoglobulin levels in immunosuppressed organisms.

[0012] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention uses the method of extracting and purifying Lycium barbarum polysaccharide and Astragalus membranaceus polysaccharide separately and then compounding them, which avoids the problems of complex polysaccharide composition and poor batch stability caused by mixed extraction, significantly improves the purity and immunomodulatory activity of the compound polysaccharide, and clarifies the correlation between the compounding ratio and the immune effect, thereby maximizing the immune synergistic effect. (2) The compound polysaccharide of Lycium barbarum and Astragalus membranaceus of the present invention can significantly promote the phagocytic function of RAW264.7 macrophages and the secretion of NO, TNF-α and IL-6 in vitro. The compound ratio of 1:1 (sL@A33) has the best effect, which is significantly better than the single polysaccharide treatment group, demonstrating good in vitro immunomodulatory activity. (3) The compound polysaccharide of Lycium barbarum and Astragalus membranaceus of the present invention can significantly improve immune dysfunction such as weight loss, atrophy of immune organs, reduction of blood cells and reduction of immunoglobulin levels in immunosuppressed mice in vivo, and can effectively alleviate the state of low immune function caused by various factors. (4) The compound polysaccharide of wolfberry and astragalus of the present invention is derived from natural sources, using wolfberry and astragalus, which are both food and medicine, as raw materials. It has high safety, no obvious toxic side effects, and is suitable for long-term use. The preparation process is stable and controllable, easy to industrialize, and has good application prospects. Attached Figure Description

[0013] Figure 1 Chromatogram of the single polysaccharide and the compound polysaccharide of Lycium barbarum and Astragalus membranaceus prepared in Example 1 of this invention and its molecular weight.

[0014] Figure 2 : Monosaccharide composition diagram of the single polysaccharide and the Lycium barbarum-Astragalus complex polysaccharide prepared in Example 1 of this invention.

[0015] Figure 3 The thermal decomposition curve of the single polysaccharide and the compound polysaccharide of Lycium barbarum and Astragalus membranaceus prepared in Example 1 of this invention.

[0016] Figure 4 Microscopic structures of the single polysaccharide and the Lycium barbarum-Astragalus membranaceus complex polysaccharide prepared in Example 1 of this invention. (A) LBPs; (B) APSs; (C) sL@A51; (D) sL@A42; (E) sL@A33; (F) sL@A24; (G) sL@A15.

[0017] Figure 5 The results of the effects of the single polysaccharide and the compound polysaccharide of Lycium barbarum and Astragalus membranaceus prepared in this invention on the viability of RAW264.7 macrophage cells.

[0018] Figure 6 The results of the assay of the immunomodulatory activity of the single polysaccharide and the compound polysaccharide of Lycium barbarum and Astragalus membranaceus prepared in this invention on RAW264.7 macrophages are shown in the figure. Phagocytic capacity (A); NO secretion level (B); TNF-α secretion level (C); IL-6 secretion level (D).

[0019] Figure 7 Figure 1 shows the results of the assay of the immunomodulatory activity of the preferred Lycium barbarum and Astragalus membranaceus polysaccharide sL@A33 and the control Lycium barbarum and Astragalus membranaceus polysaccharide mL@A33 on RAW264.7 macrophages. (A) Cell viability; (B) Phagocytic activity; (C) NO secretion level; (D) TNF-α secretion level; (E) IL-6 secretion level. Detailed Implementation

[0020] Example 1: Preparation of Lycium barbarum and Astragalus membranaceus compound polysaccharide Different proportions of Lycium barbarum and Astragalus membranaceus polysaccharides (sL@A series) were prepared by separate extraction methods: (1) Separate extraction and separation: The wolfberry and astragalus slices were pulverized, and 55g of each pulverized sample was weighed. They were heated at 90℃ for 120min with water at a ratio of 1:30 (w / v, dry basis). The mixture was then centrifuged (3000r / min, 10min) to remove insoluble residues. The wolfberry polysaccharide supernatant and the astragalus polysaccharide supernatant were obtained respectively.

[0021] (2) Separate purification and drying: The supernatant was concentrated to 200 mL at 50 °C and then separated by convective flow through a retardant membrane (molecular weight: 5000 Da) at a ratio of concentrate:water (1:2.5) until the retardant with a molecular weight greater than 5000 Da was 100 mL. The obtained retardant was deproteinized with Sevage reagent, and the supernatant was collected and dialyzed with flowing pure water (MWCO: 3500 Da) for 72 h. The Astragalus polysaccharide dialysate was centrifuged, filtered, concentrated to 30 mL, and freeze-dried to obtain Astragalus polysaccharide, named APSs. A hot mixture of dipotassium hydrogen phosphate and choline chloride and 1,4-butanediol was added to the Lycium barbarum polysaccharide dialysate, wherein the weight of the Lycium barbarum polysaccharide dialysate was 45%, the weight of dipotassium hydrogen phosphate was 27%, and the weight of the hot mixture of choline chloride and 1,4-butanediol was 28%. After swirling for 10 seconds, the lower phase was obtained by centrifugation. Lycium barbarum polysaccharides (LBPs) were obtained by freeze-drying.

[0022] (3) Mixing: LBPs and APSs were mixed in different mass ratios to obtain different proportions of Lycium barbarum and Astragalus membranaceus compound polysaccharides. The specific combinations and names are as follows: LBPs: APSs = 5: 1 (sL@A51); LBPs: APSs = 4: 2 (sL@A42); LBPs: APSs = 3: 3 (sL@A33); LBPs: APSs = 2: 4 (sL@A24); LBPs: APSs = 1: 5(sL@A15).

[0023] In contrast, a complex polysaccharide of Lycium barbarum and Astragalus membranaceus was prepared by mixed extraction: Weigh 55g each of pulverized wolfberry slices and astragalus slices, place them in the same beaker, add 1650mL of water (sample to water ratio of 1:30 w / v), heat and extract at 90℃ for 120 minutes, centrifuge to remove insoluble residue, concentrate the supernatant to 200mL at 50℃, dilute with 500mL of water, separate through an ultrafiltration membrane with a molecular weight cutoff of 5000 Da to a retentate of 100mL, dialyze through a dialysis bag with a molecular weight cutoff of 3500Da for 3 days, centrifuge, filter and concentrate to 30mL, freeze-dry to obtain the mixed extracted wolfberry and astragalus complex polysaccharide (named mL@A33).

[0024] Example 2: Structural Characterization of Lycium barbarum and Astragalus membranaceus Polysaccharides 1. Molecular weight distribution analysis The molecular weight of polysaccharides is closely related to their activity and is an important parameter for characterizing polysaccharides. In this study, high-performance liquid chromatography-size exclusion chromatography-laser light scattering-differential tandem detection (HPSEC-MALLS-RI) was used to measure and analyze the molecular weight distribution of polysaccharides. The results are as follows: Figure 1 As shown in Table 1, the single polysaccharides LBPs, APSs, and five proportions of Lycium barbarum and Astragalus membranaceus complex polysaccharides exhibited different numbers of chromatographic peaks, with significant differences in molecular weight and peak area ratio. Specifically, LBPs had the highest molecular weight, displaying three peaks, with values ​​of 4.801 × 10⁻⁶. 5 Da (24.7%), 3.331×10 4 Da (54.8%) and 2.890×10 4 Da (20.5%). The APSs had the lowest molecular weight and showed only one narrow peak, with a molecular weight of 7.701 × 10⁻⁶. 4 Da (100.0%). For five different proportions of Lycium barbarum and Astragalus membranaceus complex polysaccharides (sL@A51, sL@A42, sL@A33, sL@A24, and sL@A15), as the content of LBPs gradually decreased and the content of APSs correspondingly increased, the chromatographic peaks of the Lycium barbarum and Astragalus membranaceus complex polysaccharides gradually changed from three to one. The molecular weights of the five Lycium barbarum and Astragalus membranaceus complex polysaccharides also fell between those of single polysaccharides LBPs and APSs. Generally, polysaccharides with larger molecular weights are less likely to enter cells to exert their biological activity, while polysaccharides with smaller molecular weights are more likely to exert biological effects across membranes. However, polysaccharides with relatively small molecular weights have no biological activity. Changes in the molecular weight of complex polysaccharides may affect their biological activity.

[0025] 2. Monosaccharide composition analysis Monosaccharide composition analysis plays an indispensable role in the structural characterization and bioactivity research of polysaccharides. Monosaccharide composition affects the chain structure and higher-order structure of polysaccharides, which is a crucial factor influencing their efficacy. Figure 2 As shown in Table 2, mannose (Man), rhamnose (Rha), glucuronic acid (GlcA), galacturonic acid (GalA), glucose (Glc), galactose (Gal), and arabinose (Ara) were present in different molar ratios in APSs (0.87%, 1.58%, 0.28%, 1.22%, 38.61%, 18.10%, 39.34%), LBPs (2.62%, 3.16%, 1.40%, 1.55%, 22.63%, 25.43%, 43.21%), and sL@A51 (2.31%, 2.96%, 1.11%, 1.47%, 20.7 ....22%, 2.22%, 38.61%, 18.10%, 39.34%), LBPs (2.62%, 3.16%, 1.40%, 1.55%, 22.63%, 25.43%, 43.21%), and sL@A51 (2.31%, 2.96%, 1.11%, 1.47%, 20.71%, 2.96%). The percentages of APSs and LBPs were 4.76%, 46.68%, sL@A42 (1.90%, 2.78%, 0.94%, 1.44%, 30.59%, 22.38%, 39.97%), sL@A33 (1.52%, 1.97%, 0.79%, 1.40%, 34.07%, 21.85%, 38.40%), sL@A24 (1.46%, 2.25%, 0.63%, 1.24%, 37.12%, 20.48%, 36.82%), and sL@A15 (0.94%, 1.71%, 0.37%, 0.85%, 48.13%, 17.82%, 30.18%). Both APSs and LBPs were primarily composed of glucose, arabinose, and galactose. The five different proportions of Lycium barbarum and Astragalus membranaceus (LBPs) polysaccharides were mainly characterized by varying molar ratios of glucose, galactose, and arabinose. As the content of LBPs gradually decreased and the content of APSs (alpha-lipidose polysaccharides) increased accordingly, the monosaccharide composition gradually shifted from being dominated by arabinose and galactose to being dominated by arabinose and glucose. The molar ratio of arabinose in the Lycium barbarum and Astragalus membranaceus polysaccharide sL@A51 was 46.68%, which was higher than that in the monosaccharides LBPs and APSs. Similarly, the molar ratio of glucose in the Lycium barbarum and Astragalus membranaceus polysaccharide sL@A51 was 48.13%, also higher than that in the monosaccharides LBPs and APSs. These different monosaccharide compositions form the basis of the polysaccharide structure and lead to differences in biological activity.

[0026] 3. Thermal stability analysis Thermal property analysis of LBPs, APSs and five different proportions of Lycium barbarum and Astragalus membranaceus polysaccharides, as shown below Figure 3As shown, the thermal analysis curves of single polysaccharides and complex polysaccharides are similar, indicating that three main processes occurred during thermal decomposition. The first stage occurred at approximately 30-220℃, during which all seven samples experienced a slight weight loss of about 8.0%, attributed to the evaporation of adsorbed water remaining in the polysaccharide pores. The second stage occurred from 265℃ to 365℃, during which the polysaccharides underwent severe weight loss, specifically manifested as the breakage and decomposition of sugar chains. LBPs, APSs, and five different proportions of complex polysaccharides (sL@A51, sL@A42, sL@A33, sL@A24, and sL@A15) began to lose weight at 273.1°C, 271.7°C, 267.6°C, 268.8°C, 269.9°C, 270.9°C, and 271.9°C, respectively, and the weight loss ceased at 333.1°C, 341.3°C, 362.0°C, 331.69°C, 352.1°C, 347.9°C, and 345.1°C, respectively. During this stage, the weight changes of each polysaccharide were 30.34%, 56.97%, 53.85%, 36.37%, 60.19%, 55.11%, and 52.17%, respectively. In this stage of mass change, it can be seen that the mass change of single polysaccharide APSs is significantly higher than that of LBPs, while the composite polysaccharide sL@A33 shows the greatest mass loss in this stage. The third stage is from 365℃ to 800℃, and the mass loss in this stage is attributed to the thermal decomposition of the polysaccharide skeleton. The final residual amounts of LBPs, APSs, and five composite polysaccharides with different proportions (sL@A51, sL@A42, sL@A33, sL@A24, and sL@A15) are 25.26%, 25.23%, 20.37%, 25.57%, 19.76%, 24.72%, and 24.45%, respectively. The DSC curves of the seven polysaccharide samples show similar trends, all having two obvious exothermic peaks, but with slight differences in peak width. These differences are attributed to the differences and variations in the chemical composition, molecular weight, structure, and water content of the seven polysaccharide samples.

[0027] 4. Atomic force microscopy analysis Atomic force microscopy (AFM) images reveal the nanoscale structural and morphological features of LBPs, APSs, and five composite polysaccharides (sL@A51, sL@A42, sL@A33, sL@A24, and sL@A15) in different proportions. Planar and three-dimensional AFM images of the seven polysaccharide samples are shown below. Figure 4As shown, LBPs exhibit chain-like structures of varying lengths in two-dimensional images, with their molecular chains evenly distributed in water and slight aggregation forming irregular aggregates. In their three-dimensional images, these irregular aggregates appear as columnar protrusions. APSs show numerous spherical aggregates and short chain-like structures in two-dimensional images, while the irregular spherical aggregates appear as numerous sharp peak-like protrusions in their three-dimensional images. For the five complex polysaccharides (sL@A51, sL@A42, sL@A33, sL@A24, and sL@A15), as the LBP content gradually decreases and the APS content increases accordingly, their microstructure gradually changes from a chain-like structure dominated by LBPs and irregular aggregates to a spherical aggregate and short chain structure dominated by APSs. Notably, all seven polysaccharide samples exhibit varying degrees of aggregation in aqueous solution. The above aggregates may be due to the entanglement of polysaccharide molecules by intramolecular and intermolecular van der Waals forces and hydrogen bonds, forming various aggregate structures.

[0028] Example 3: Study on the immunomodulatory activity of Lycium barbarum and Astragalus membranaceus compound polysaccharides 3.1 Assay of cellular-level immunomodulation 1. Cell viability assay RAW264.7 cells are commonly used to evaluate immunomodulatory activity. The effects of LBPs, APSs, and five different proportions of compound polysaccharides (sL@A51, sL@A42, sL@A33, sL@A24, and sL@A15) on the cell viability of RAW264.7 macrophages were shown in the following results. Figure 5 As shown, within the concentration range of 60-250 μg / mL, LBPs, APSs, and five different proportions of compound polysaccharides (sL@A51, sL@A42, sL@A33, sL@A24, and sL@A15) did not significantly affect the viability of RAW264.7 cells. Therefore, concentrations of 60, 125, and 250 μg / mL were selected for subsequent studies.

[0029] 2. Neutral red phagocytic activity The phagocytic activity of macrophages can reflect the state of the innate immune response. Enhanced phagocytic activity is considered one of the most significant markers of RAW264.7 cell activation. In this study, the effects of different concentrations of LBPs, APSs, and five different proportions of complex polysaccharides (sL@A51, sL@A42, sL@A33, sL@A24, and sL@A15) on the phagocytic activity of RAW264.7 macrophages were determined by assessing the uptake of neutral red dye. Figure 6As shown in Figure A, compared with the blank control group, the Lycium barbarum and Astragalus membranaceus polysaccharide sL@A33 promoted the phagocytic activity of macrophages, and the phagocytic activity was the highest at 250 μg / mL, which was significantly higher than that of LBPs at the same concentration. P <0.01). LBPs, APSs, and five different proportions of complex polysaccharides (sL@A51, sL@A42, sL@A33, sL@A24, and sL@A15) at different concentrations, after 24 hours of treatment, had only a weak positive effect on the phagocytic activity of macrophages. P >0.05).

[0030] 3. Determination of NO content NO is an important mediator of the immune response, and its release can indirectly reflect the activation state of macrophages. Studies have shown that appropriately promoting NO production is necessary for the immune system to attack exogenous substances. This study used Griess reagent to detect the effects of LPS (positive control), different concentration gradients (60-250 μg / mL) of LBPs, APSs, and five different proportions of complex polysaccharides (sL@A51, sL@A42, sL@A33, sL@A24, and sL@A15) on NO secretion levels in macrophages after 24 h of treatment. Results are as follows: Figure 6 As shown, compared with the control group, the secretion level of NO was significantly increased under LPS stimulation. P <0.0001), reaching 11.49 μM. Compared with the control group, all polysaccharide treatment groups significantly stimulated the secretion level of NO in RAW264.7 cells (<0.0001), reaching 11.49 μM. P <0.0001), the NO content produced by RAW264.7 cells gradually increased with the increase of polysaccharide concentration from 60-250 μg / mL. Under the same concentration conditions, the Lycium barbarum and Astragalus membranaceus compound polysaccharide sL@A33 group showed the best immune activation effect, with NO secretion levels reaching 8.33, 8.78 and 9.10 μM, respectively, which were significantly higher than those of the LBPs group (5.94, 7.05 and 7.25 μM) and APSs (6.73, 6.74 and 6.91 μM) treatment groups alone. P <0.05). The above results indicate that the synergistic effect among polysaccharide components may be optimized through specific molecular ratios, with the Lycium barbarum and Astragalus membranaceus compound polysaccharide sL@A33 exhibiting the best immunomodulatory synergistic effect.

[0031] 4. Cytokine assay analysis Activated RAW264.7 cells can directly participate in the defense against pathogen invasion by releasing cytokines such as TNF-α and IL-6. To investigate the differences in immunomodulatory activity of LBPs, APSs, and five different ratios of complex polysaccharides (sL@A51, sL@A42, sL@A33, sL@A24, and sL@A15), RAW264.7 cells were pretreated with polysaccharides at different concentration gradients (60-250 μg / mL) for 24 h. The secretion levels of cytokines such as TNF-α and IL-6 in the cell culture supernatant were detected using an ELISA kit, with LPS selected as a positive control. Figure 6 As shown in C and D, all polysaccharide treatment groups significantly stimulated RAW264.7 cells to secrete TNF-α and IL-6 within the concentration range of 60-250 μg / mL. P <0.0001). At a high concentration of 250 μg / mL, the Lycium barbarum and Astragalus membranaceus polysaccharide sL@A33 group exhibited the best immune activation effect: its TNF-α secretion reached 367.00 pg / mL, which was 8.72% and 7.63% higher than the single polysaccharide treatment groups (LBPs (335.00 pg / mL) and APSs (339.03 pg / mL), respectively. P <0.0001; P <0.01). Similarly, compared with the LBPs and APSs groups, the IL-6 secretion level reached its highest when macrophages were treated with 250 μg / mL of Lycium barbarum and Astragalus membranaceus polysaccharide sL@A33, which was 5.38 and 2.07 pg / mL higher than the LBPs and APSs groups, respectively. Notably, the synergistic enhancing effect of Lycium barbarum and Astragalus membranaceus polysaccharide sL@A33 on TNF-α and IL-6 was highly consistent with the results of the previous NO secretion experiment. Among all polysaccharide treatment groups, this polysaccharide treatment group had the strongest stimulatory effect on the production of cytokines TNF-α and IL-6, thereby better enhancing immune function.

[0032] Example 4. Comparison of immunomodulatory activities between Lycium barbarum and Astragalus membranaceus compound polysaccharide sL@A33 and control compound polysaccharide mL@A33 4.1 Cell viability assay To evaluate the potential immunostimulatory activity of two Lycium barbarum and Astragalus membranaceus compound polysaccharides, their effect on the viability of RAW264.7 macrophage cells was investigated. Cell viability was measured using a CCK-8 assay after 24 h of treatment with the compound polysaccharides. Figure 7 As shown in Figure A, compared with the control group, the compound polysaccharides mL@A33 and sL@A33 did not significantly affect the viability of RAW264.7 cells at different concentrations (60, 125, and 250 μg / mL). Based on these results, concentrations of 60, 125, and 250 μg / mL were selected for subsequent experiments.

[0033] 4.2 Neutral Red Phagocytic Activity Phagocytic activity of RAW264.7 macrophages is an important means for the immune system to maintain homeostasis, and it is also the basis for immune responses and an indicator of immune function activation. The effects of Lycium barbarum and Astragalus membranaceus polysaccharides mL@A33 and sL@A33 on the phagocytic activity of RAW264.7 cells were determined by neutral red uptake assay. The effects of the two Lycium barbarum and Astragalus membranaceus polysaccharides on the phagocytic function of RAW 264.7 cells are shown in [link to study]. Figure 7 B, compared with the control group, the sL@A33 treatment group showed significantly higher phagocytic activity at 250 μg / mL ( P <0.01). This indicates that the macrophage phagocytic activity of the complex polysaccharide, extracted separately and then mixed, is enhanced. At the experimental concentration, mL@A33 had only a weak positive effect on the phagocytic ability of RAW264.7 macrophage cells ( P >0.05). The two complex polysaccharides had a weaker effect on phagocytosis than they had on NO and other cytokines.

[0034] 4.3 Determination of NO content NO is one of the active messenger cytokines secreted by macrophages, which can enhance their phagocytic and metabolic capabilities. This study used Griess reagent to detect the effects of LPS (positive control), different concentration gradients (60-250 μg / mL) of mL@A33 and sL@A33 on NO secretion levels in macrophages after 24 h of treatment. The experimental results showed that ( Figure 7 C), compared with the control group, the NO secretion level of RAW264.7 cells was significantly increased under stimulation with mL@A33 and sL@A33 at concentrations of 60-250 μg / mL. P <0.05). Furthermore, under the same concentration conditions, the sL@A33 group exhibited the best immune activation effect, with NO secretion levels reaching 6.06, 9.25, and 9.64 μM, respectively, significantly higher than the mL@A33 group ( P <0.05).

[0035] 4.4 Cytokine assay analysis Besides NO, cytokines also participate in immune responses and are important mediators in the immune system's communication network. Cytokine secretion levels are considered an important indicator of immune responses. To further elucidate the potential immunomodulatory activities of the compound polysaccharides mL@A33 and sL@A33, the effects of the two Lycium barbarum and Astragalus membranaceus compound polysaccharides on cytokine (TNF-α and IL-6) secretion were evaluated. LPS stimulation significantly increased the secretion of IL-6 and TNF-α (…). P<0.001). Compared with the control group, after treatment with two kinds of Lycium barbarum and Astragalus membranaceus compound polysaccharides at different concentration gradients (60-250 μg / mL), the secretion levels of cytokines such as TNF-α and IL-6 were significantly increased. Figure 7 D / E). At a high dose of 250 μg / mL, the TNF-α secretion level in the mL@A33 group was 1.46 times that of the control group (D / E). P <0.01), the TNF-α level in the sL@A33 group was 1.64 times that of the control group ( P <0.01)( Figure 7 D). Similarly, the IL-6 level in the mL@A33 group was 1.16 times that of the control group ( P <0.01), the IL-6 level in the sL@A33 group was 1.83 times that of the control group ( P <0.01)( Figure 7 E). These results confirm that both complex polysaccharides induced significant immune activation by upregulating the secretion of NO and cytokines in RAW264.7 cells. Natural polysaccharides extracted from plants of different sources also exhibited immunomodulatory effects by increasing the secretion of cytokines such as NO, IL-6, and TNF-α. Furthermore, compared to the same concentration of mLA33, sL@A33 was more effective in promoting the secretion of cytokines by RAW264.7 cells. Overall, sL@A33 showed better immunomodulatory activity than mL@A33, which may be due to structural differences between the two Lycium barbarum and Astragalus membranaceus complex polysaccharides caused by different extraction methods.

Claims

1. A Lycium barbarum and Astragalus membranaceus compound polysaccharide with immune-enhancing effects, characterized in that, The wolfberry-astragalus complex polysaccharide is prepared by extracting and purifying wolfberry polysaccharide and astragalus polysaccharide separately and then mixing them together; the total sugar content of the wolfberry-astragalus complex polysaccharide is 70-80%, the uronic acid content is 10-14%, and the protein content is 15-25%.

2. The Lycium barbarum and Astragalus membranaceus compound polysaccharide according to claim 1, characterized in that, The mass ratio of the mixed wolfberry polysaccharide and astragalus polysaccharide is 1:5 to 5:

1.

3. The Lycium barbarum and Astragalus membranaceus compound polysaccharide according to claim 2, characterized in that, The mass ratio of the mixed wolfberry polysaccharide and astragalus polysaccharide is 1:

1.

4. The Lycium barbarum and Astragalus membranaceus compound polysaccharide according to claim 1, characterized in that, The compound polysaccharide of Lycium barbarum and Astragalus membranaceus has a stable molecular weight distribution, with a molecular weight between that of single Lycium barbarum polysaccharide and single Astragalus membranaceus polysaccharide; its monosaccharide composition includes mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose; its microstructure exhibits the characteristics of chain structure and irregular aggregates coexisting.

5. A method for preparing the Lycium barbarum and Astragalus membranaceus complex polysaccharide according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Separate extraction: The raw materials of wolfberry and astragalus were washed, dried and crushed respectively, and then mixed with water, heated and extracted. The insoluble residues were removed by filtration and centrifugation to obtain wolfberry polysaccharide extract and astragalus polysaccharide extract respectively. (2) Purification: The Lycium barbarum polysaccharide extract and Astragalus membranaceus polysaccharide extract obtained in step (1) were subjected to membrane filtration, deproteinization, decolorization, dialysis and drying to obtain Lycium barbarum polysaccharide and Astragalus membranaceus polysaccharide respectively; (3) Mixing: Mix the wolfberry polysaccharide and astragalus polysaccharide obtained in step (2) to obtain the wolfberry-astragalus complex polysaccharide.

6. The preparation method according to claim 5, characterized in that, In step (1), the heating extraction temperature is 80-100℃, the time is 90-150 min, and the mass-volume ratio of raw material to water is 1:(20-40)g / mL.

7. The preparation method according to claim 5, characterized in that, In step (2), the membrane filtration uses an ultrafiltration membrane with a molecular weight cutoff of 3000-10000 Da; the deproteinization treatment uses the Sevage method; the dialysis uses a dialysis bag with a molecular weight cutoff of 3000-4000 Da; when purifying the Lycium barbarum polysaccharide extract, a two-phase extraction step is also included after dialysis: dipotassium hydrogen phosphate and a hot mixture of choline chloride and 1,4-butanediol are added to the Lycium barbarum polysaccharide dialysis solution, vortexed and centrifuged, the lower phase liquid is collected and then dried.

8. The use of the Lycium barbarum and Astragalus membranaceus compound polysaccharide according to any one of claims 1-4 in the preparation of products for enhancing the body's immunity.

9. The application according to claim 8, characterized in that, The product is used to improve or alleviate a state of weakened immune function.

10. The application according to claim 9, characterized in that, The aforementioned state of weakened immune function includes immunosuppression caused by drugs, diseases, or stress factors; the product is a drug, health food, or functional food; the Lycium barbarum and Astragalus membranaceus compound polysaccharide enhances the body's immune function by promoting macrophage phagocytosis and upregulating the secretion of NO, TNF-α, and IL-6.

Images