Cassava polysaccharide as well as preparation method and application thereof

Abstract

The invention provides cassava polysaccharide as well as a preparation method and application thereof, and belongs to the technical field of polysaccharide extraction. The preparation method of the cassava polysaccharide comprises the following steps: grinding cassava tuberous roots, mixing the ground cassava tuberous roots with water, carrying out enzymolysis by adopting medium-temperature alpha-amylase under an ultrasonic condition, carrying out enzyme deactivation, centrifuging, taking supernate, carrying out alcohol precipitation, centrifuging, taking precipitate, redissolving the precipitate, dialyzing, and freeze-drying, so as to obtain the cassava polysaccharide. The prepared cassava polysaccharide can effectively improve the immunocompetence of cells and mice, improve the pathological state of colon tissue and promote tissue repair and regeneration. The preparation method of the cassava polysaccharide is simple, convenient and easy to implement, green, environmentally friendly and suitable for large-scale application and popularization.

Description

Technical Field

[0001] This invention relates to the field of polysaccharide preparation technology, and in particular to a cassava polysaccharide, its preparation method, and its application. Background Technology

[0002] Natural polysaccharides, due to their unique biological activities, show broad application prospects in immunomodulation, antioxidant activity, and antitumor effects. Among these biological activities, their immunomodulatory properties have particular clinical significance, as a strong and well-regulated immune system is the main defense mechanism against various pathological conditions. Immunodeficiency is closely associated with increased susceptibility to bacterial, viral, and fungal infections, as well as an increased risk of malignant tumors. Unlike synthetic immunomodulators, plant-derived polysaccharides have the advantage of regulating immune function through multiple pathways and hierarchical mechanisms, with minimal side effects.

[0003] Cassava (Manihot esculenta Crantz) is an important agricultural product in tropical regions and a staple crop rich in starch and non-starch polysaccharides. This abundant polysaccharide content, coupled with its widespread cultivation and economic importance, makes cassava a promising source of bioactive polysaccharides for therapeutic applications. Previous studies have identified various polysaccharides in cassava with significant bioactivity. For example, Charles isolated a heteropolysaccharide composed of multiple monosaccharides from cassava roots, exhibiting specific molecular weights and hemicellulose-derived components [Charles, AL, Huang, TC, & Chang, YH (2008). Structural analysis and characterization of a mucopolysaccharide isolated from roots of cassava (Manihot esculenta Crantz L.). FOOD HYDROCOLLOIDS, 22(1), 184-191.]. Furthermore, cassava polysaccharide (CP) possesses various biological functions, including enhancing exercise endurance in animal models and demonstrating antioxidant and hepatoprotective effects. The combination of high safety, abundant availability, and low production costs makes CP particularly attractive for developing immunomodulatory functional materials.

[0004] Polysaccharide extraction methods mainly include hot water extraction, enzymatic extraction, and microwave extraction. Hot water extraction is simple but has a low yield. Microwave extraction is simple, efficient, safe, and highly selective. However, this method consumes more electricity, and the relatively high microwave power can easily lead to scorching, damaging the polysaccharide structure. Enzymatic extraction often uses amylase, cellulase, and other enzymes, which facilitate the dissolution of polysaccharides from the cell wall and result in a high polysaccharide extraction rate. Summary of the Invention

[0005] In view of this, the present invention provides a cassava polysaccharide, its preparation method and application, to solve the problem of low polysaccharide activity in various enzymatic hydrolysis preparations.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing cassava polysaccharides, comprising the following steps:

[0008] (1) Grind the cassava tubers to obtain cassava pulp;

[0009] (2) Mix cassava pulp with water, add medium-temperature α-amylase, and sonicate at 60-70℃ for 200-250 min to obtain enzymatic hydrolysate;

[0010] (3) After the enzyme is inactivated by the enzyme hydrolysate, centrifuge and collect the supernatant. Concentrate the supernatant and precipitate it with alcohol. Collect the precipitate.

[0011] (4) After dissolving the precipitate, dialyze it, concentrate the dialysate, freeze dry it, and obtain cassava polysaccharide.

[0012] Preferably, the amount of mesophilic α-amylase added is 4500–5500 U / g, based on the weight of the cassava tuber.

[0013] Preferably, the mass-to-volume ratio of the cassava pulp to water is 1g:2-3mL.

[0014] Preferably, the power of the ultrasonic treatment is 200-400W.

[0015] Preferably, the supernatant is concentrated to 1 / 5 to 1 / 3 of its original volume and then anhydrous ethanol is added for alcohol precipitation until the volume fraction of ethanol in the system is 75 to 85%. After alcohol precipitation, the mixture is allowed to stand for 2 to 12 hours.

[0016] Preferably, the concentration in steps (3) and (4) is rotary evaporation concentration, with a temperature of 45-55°C and a rotation speed of 180-220 rpm.

[0017] Preferably, the dialysis uses a dialysis bag with a molecular weight cutoff of 3.5 kDa, and the dialysis time is 48–72 hours.

[0018] Preferably, the dialysate is concentrated to 1 / 10 to 1 / 8 of its original volume and then freeze-dried at a temperature of -10°C to -5°C and a vacuum of 8 to 15 Pa.

[0019] The present invention also provides cassava polysaccharides obtained according to the preparation method described above.

[0020] This invention provides the application of the cassava polysaccharide in at least one of the following:

[0021] (1) Application in the preparation of drugs that enhance immunity;

[0022] (2) Application in the preparation of drugs that promote the repair and regeneration of damaged intestines.

[0023] By adopting the above technical solution, the present invention has the following beneficial effects: The preparation of cassava polysaccharide of the present invention includes the following steps: cassava tubers are ground, mixed with water, enzymatically hydrolyzed using medium-temperature α-amylase under ultrasonic conditions, the enzyme is inactivated, the supernatant is collected by centrifugation, precipitated with alcohol, centrifuged again, the precipitate is collected, the precipitate is reconstituted and dialyzed, and then freeze-dried to obtain the final product. The cassava polysaccharide prepared by the present invention can effectively improve the immune capacity of macrophages and mice, and can also improve the pathological state of colon tissue, promoting tissue repair and regeneration. The preparation method of cassava polysaccharide of the present invention is simple, easy to implement, green and environmentally friendly, and suitable for widespread application. Attached Figure Description

[0024] Figure 1 The elution curve of cassava polysaccharide on a DEAE-Sephacel column is shown.

[0025] Figure 2 Elution curves of CP1-CP5 from cassava polysaccharides on a dextran gel G-200 column.

[0026] Figure 3 The UV-Vis spectra of CP1-CP5 are shown.

[0027] Figure 4 The Fourier transform infrared spectra of CP1–CP5 are shown.

[0028] Figure 5 The cell viability of RAW264.7 macrophages in each treatment group in Example 3.

[0029] Figure 6 This refers to the phagocytic activity of RAW264.7 macrophages in each treatment group in Example 3.

[0030] Figure 7 The NO concentration of RAW264.7 macrophages in each treatment group in Example 3.

[0031] Figure 8 The secretion level of TNF-α in RAW264.7 macrophages in each treatment group in Example 3.

[0032] Figure 9 The level of IL-6 secreted by RAW264.7 macrophages in each treatment group in Example 3.

[0033] Figure 10 The level of IL-10 secreted by RAW264.7 macrophages in each treatment group in Example 3.

[0034] Figure 11 This is a bar chart showing the changes in body weight of mice in each treatment group during the spleen cell proliferation assay.

[0035] Figure 12 This is a bar chart showing the food intake of mice in each treatment group during the spleen cell proliferation assay.

[0036] Figure 13 This is a bar chart showing the water intake of mice in each treatment group during the spleen cell proliferation assay.

[0037] Figure 14 This is a bar chart showing the spleen index of mice in each treatment group during the spleen cell proliferation assay.

[0038] Figure 15 This is a bar chart showing the thymus index of mice in each treatment group during the spleen cell proliferation assay.

[0039] Figure 16 This study shows the proliferation of spleen T lymphocytes in mice under different treatment groups in the ConA-induced model during a spleen cell proliferation assay.

[0040] Figure 17 This study shows the proliferation of spleen T lymphocytes in mice under different treatment groups in the LPS-induced model during a spleen cell proliferation assay.

[0041] Figure 18 The value represents the IgA expression level of mice in each treatment group in Example 4.

[0042] Figure 19 The value represents the IgM expression level of mice in each treatment group in Example 4.

[0043] Figure 20 The IgG expression levels of mice in each treatment group in Example 4.

[0044] Figure 21 The expression level of TNF-α in mice in each treatment group in Example 4.

[0045] Figure 22 The expression level of IL-2 in mice in each treatment group in Example 4.

[0046] Figure 23 The expression level of IL-4 in mice in each treatment group in Example 4.

[0047] Figure 24 The expression level of IL-6 in mice in each treatment group in Example 4.

[0048] Figure 25 The expression level of IL-10 in mice in each treatment group in Example 4.

[0049] Figure 26The protein expression level of ZO-1 in the intestinal tissue of mice in each treatment group in the cyclophosphamide (Cy) induced model.

[0050] Figure 27 The protein expression level of occludin in the intestinal tissue of mice in each treatment group in the cyclophosphamide (Cy) induced model.

[0051] Figure 28 The protein expression level of Claudin-1 in the intestinal tissue of mice in each treatment group in the cyclophosphamide (Cy) induced model.

[0052] Figure 29 The results of H&E staining of intestinal tissue from mice in each treatment group in the cyclophosphamide (Cy) induced model are shown. Detailed Implementation

[0053] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0054] The mesophilic α-amylase of this invention was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0055] Example 1

[0056] Take 100g of cassava tubers and grind them to obtain cassava pulp. Mix the cassava pulp with 300mL of water, add 5000U / g of mesophilic α-amylase (based on the mass of cassava), and sonicate at 65℃ for 200min to obtain the enzymatic hydrolysate. The sonication power is 300W. Inactivate the enzyme in the hydrolysate by boiling it in a water bath for 5min. After centrifugation at 4000g for 20min, collect the supernatant. Rotate the supernatant at 50℃ and 200rpm to 1 / 3 of its original volume, add anhydrous ethanol to a concentration of 80% for alcohol precipitation, let it stand at room temperature for 12h, and collect the precipitate by centrifugation at 5000g for 15min. Dissolve the precipitate completely, centrifuge at 5000g for 20min to obtain the supernatant, and place the supernatant in a dialysis bag with a molecular weight cutoff of 3.5kDa for 72h to obtain the dialysate. The dialysate was rotary evaporated to 1 / 10 of its original volume and then freeze-dried at -10℃ and 8Pa to obtain crude cassava polysaccharide (CPCR).

[0057] To purify the polysaccharides, cPCR was further fractionated using sequential chromatography (MC99-3). First, anion exchange chromatography was performed on a DEAE cellulose DE-52 column, followed by molecular size fractionation on a Sephadex G-200 gel filtration column. Elution curves were monitored using the phenol-sulfuric acid method, with continuous detection at 490 nm. Based on the elution curves, fractions containing polysaccharides were combined, concentrated under reduced pressure, and lyophilized to obtain purified polysaccharide fractions, named CP1, CP2, CP3, CP4, and CP5.

[0058] Example 2. Structural characteristics of polysaccharide components

[0059] The molecular weight of the purified fractions was determined by high-performance gel permeation chromatography (HPGPC) according to the method established by Wang et al. [Wang, J., Ge, B., Li, Z., Guan, F., & Li, F. (2016). Structural analysis and immunoregulation activity comparison of five polysaccharides from Angelica sinensis. CARBOHYDRATE POLYMERS, 140, 6-12.]. Monosaccharide composition analysis was performed using gas chromatography-mass spectrometry (GC-MS) (ICS 5000+, Thermo). Purity was assessed using UV-Vis spectroscopy (TU-1810APC, Persee) to detect potential protein and nucleic acid contamination. Simultaneously, Fourier transform infrared spectroscopy (FT-IR) analysis (Nicolet iZ-10, Thermo) was performed according to the procedure described by Mahepali et al. [Mahepali Mahabati, Adila Aipire, Pengfei Yuan, Xiaoying Liu, Shanshan Cai, Alimu Aimaier, Dilinigeeer Ziyayiding, Mayila Yasheng, Dilinazi Abudujilile, & Li, J. (2020). Comparison of structural characteristics and immunoregulatory activities of polysaccharides from four natural plants. FOOD AND AGRICULTURAL IMMUNOLOGY, 31(1), 545-562.] to characterize functional groups and structural features.

[0060] The elution curves of polysaccharides isolated from cassava roots are as follows: Figures 1-4 As shown. Chromatographic purification by cPCR yielded five well-separated fractions ( Figure 1 The components were named CP1, CP2, CP3, CP4, and CP5, respectively. Each component (CP1–CP5) showed a single elution peak, indicating that they were homogeneous. Figure 2 The yield distribution shown indicates the order of CP yields: CP1>CP2>CP3>CP4>CP5.

[0061] like Figure 3As shown, the purified CPs components (CP1-CP5) were characterized by UV-Vis spectroscopy. The results showed that none of the components exhibited significant absorption peaks at 260 nm or 280 nm, indicating that nucleic acids and proteins were absent from the CP1-CP5 components. This demonstrates the high purity of the obtained purified polysaccharide components, providing a reliable basis for Fourier transform infrared spectroscopy detection.

[0062] Fourier transform infrared spectroscopy confirmed the characteristic functional groups of cassava polysaccharides (CP1–CP5). Figure 4 The results showed that the infrared spectra of the five CP samples were basically similar, but with slight differences in intensity. All fractions exhibited broad OH stretching vibrations (3237-3275 cm⁻¹). -1 ) and CH elastic belt (2928-2955cm) -1 This is a typical characteristic of polysaccharides. (At 1200-900 cm⁻¹) -1 Within the range (the "fingerprint region" of the polysaccharide), multiple characteristic peaks were observed. The peak value was 1148 cm⁻¹. -1 (C–O–C / OH) and 1014–1030 cm -1 (Pyranose ring vibration) further confirmed the glycosidic bond, and the characteristic absorption at 832 cm⁻¹ -1 836cm -1 This indicates the presence of an α-configuration. Studies have shown that polysaccharides containing an α-configuration pyran ring possess significant immunomodulatory activity.

[0063] The molecular weight and monosaccharide composition of the cassava polysaccharide prepared by this invention are shown in Table 1.

[0064] Table 1 Monosaccharide composition and molecular weight

[0065]

[0066] Table 1 shows significant differences among CP1-CP5. CP1 and CP5 are neutral polysaccharides mainly composed of arabinose and glucose, while CP2-CP4 are acidic polysaccharides (36.9-62.4 kDa) containing uronic acid and heterogeneous monosaccharides. Among these samples, CP1 has the highest glucose content (97.0%) and the lowest molecular weight (3.0 kDa), while CP2 has the highest mannose content (5.6%) and the largest molecular weight (62.4 kDa; 20.8 times larger than CP1). Notably, polysaccharides rich in mannose, glucose, and galactose have shown potent anti-inflammatory effects. These structural changes, including monosaccharide composition, molecular weight, and glycosidic bond type, may significantly affect their biological activity and immunomodulatory potential.

[0067] Example 3. Evaluation of in vitro immunomodulatory activity

[0068] 1. Cell Culture and Processing

[0069] RAW264.7 mouse cells were cultured at 37°C and 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin / streptomycin. After 48 h of culture, the cells were sputtered at 1 × 10⁻⁶ cells / mL. 5 Cells were seeded at a density of 100 cells / mL in 96-well plates and allowed to adhere for 24 h before treatment. Lipopolysaccharide (LPS, 1.0 μg / mL) was used as a positive control, while cassava polysaccharides (CPCR, CP1, CP2, CP3, CP4, and CP5) at final concentrations of 62.5, 250.0, and 1000.0 μg / mL were used as negative controls. After 24 h of treatment, cell viability and phagocytic capacity were measured in each treatment group.

[0070] 2. Cell viability

[0071] Cell viability in each group was assessed using the Cell Count Kit-8 (CCK-8) assay. After 24 hours of treatment, the original cell supernatant was aspirated from each well, and CCK-8 solution was added to each well. The wells were then incubated at 37°C for 2 hours. Absorbance (OD) was measured at 450 nm using a microplate reader (Multiskan FC, Thermo). Cell viability was calculated using the following formula.

[0072] Cell viability = A2 / A1 × 100%

[0073] In the formula: A1 is the absorbance of the normal control group; A2 is the absorbance of the sample group or positive group.

[0074] The results are as follows Figure 5 As shown, the results indicate that cell viability remained above 100% under the influence of lipopolysaccharide (LPS) and different concentrations of CP. This demonstrates that no observable cytotoxicity was observed within the tested concentration range, thus validating the suitability of CP for subsequent experiments.

[0075] 3. Assessment of macrophage phagocytic capacity in RAW264.7

[0076] The phagocytic activity of RAW264.7 macrophages was assessed using a neutral red uptake assay. After treatment, cells were incubated with neutral red solution for 2 hours, followed by extraction of the internalized dye with acidified ethanol. Absorbance (OD) was measured at 540 nm. Cellular phagocytic capacity was calculated using the following formula, and the results are shown below. Figure 6 As shown.

[0077] Phagocytosis rate / % = A2- / A1 × 100%

[0078] In the formula: A1 is the absorbance of the normal control group; A2 is the absorbance of the sample group or positive group.

[0079] The phagocytic capacity of macrophages is a core mechanism of immune defense, and its enhancement is a key parameter for assessing immunostimulatory activity. Results showed that, compared with the control group, all concentrations of CP (62.6, 250, and 1000.0 μg / mL) significantly enhanced the phagocytic capacity of RAW264.7 cells. CP4 at a concentration of 1000 μg / mL exhibited the strongest phagocytic pro-activation effect, with a phagocytic rate of 116.0% (p<0.001), demonstrating superior efficacy compared to the LPS positive control (p<0.01).

[0080] 4. Measurement of nitric oxide production

[0081] The concentration of nitric oxide (NO) in the culture supernatant was determined using the Griess method with a NO ELISA kit (Shanghai Enzyme-Link Biotechnology Co., Ltd.). After 24 hours of treatment, the cell culture supernatant was collected and mixed with Griess reagent according to the kit instructions. The absorbance was measured at 540 nm, and the NO concentration was calculated using a sodium nitrite standard curve. The results are shown below. Figure 7 As shown.

[0082] Nitric oxide (NO) is a key immunomodulatory molecule that plays a crucial role in macrophage-mediated immune responses, including antitumor, antiviral, and lymphocyte proliferative activities, and serves as an indirect indicator of macrophage activation status. The results showed that, compared to the control group, all three concentrations of CPs significantly promoted NO production in RAW264.7 cells (p<0.001). Furthermore, CPs exhibited a concentration-dependent increase in NO production (except for CP1). This indicates that CPs can effectively activate the immune response function of macrophages.

[0083] 5. Cytokine secretion analysis

[0084] The secretion levels of pro-inflammatory and anti-inflammatory cytokines, including TNF-α, IL-6, and IL-10, in the culture supernatant were quantitatively detected using a tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10) ELISA kit (Shanghai Enzyme-Link Biotechnology Co., Ltd.) according to the manufacturer's instructions. Triple copies of the samples were analyzed. Results are as follows: Figures 8-10 As shown.

[0085] Cytokines play a crucial role in the proliferation, differentiation, and functional regulation of immune cells, with TNF-α, IL-6, and IL-10 playing key regulatory roles in immune responses. The results showed that, across all three concentration ranges, the CPs components did not significantly enhance TNF-α secretion. Figure 8) However, except for CP1, the TNF-α secretion level was positively correlated with the polysaccharide concentration. Compared with the control group, CPCR and CP1 significantly promoted the secretion of IL-6 (p<0.05, Figure 9 ). In addition, CPCR, CP1, CP2, and CP4 significantly increased the secretion of IL-10 (p<0.05, Figure 10 ). These results indicate that CPCR and CPs not only promote the proliferation and phagocytic activity of RAW264.7 cells but also stimulate the secretion of immunomodulatory factors such as NO, IL-6, and IL-10. This demonstrates their potential to maintain immune balance.

[0086] Considering that CP1 and CP2 have higher yields, different molecular weights and monosaccharide composition characteristics, and excellent in vitro immunomodulatory activities, the present invention selects CPCR, CP1, and CP2 for subsequent animal experiments to further clarify their immunomodulatory mechanisms in vivo.

[0087] Example 4. Evaluation of Immunomodulatory Activity in Animals

[0088] 1. Experimental Animals and Housing Conditions

[0089] SPF-grade male BALB / c mice (6 weeks old, body weight: 24.5±1.2 g) were provided by Taikang Medical Testing Services Co., Ltd. (Animal Use License No.: SYXK (Ji) 2021-006). A total of 48 mice were housed in an SPF-grade barrier environment with controlled environmental conditions (temperature: 20±2°C, humidity: 55±10%, 12 h light / dark cycle), and had free access to standard rodent food and water. All experimental procedures strictly adhered to Chinese regulations on animal welfare and ethics.

[0090] 2. Experimental Design and Treatment Regimen

[0091] After 7 days of adaptive feeding, the mice were randomly divided into six experimental groups (8 mice per group):

[0092] (1) Normal control group (NC): Oral gavage with distilled water;

[0093] (2) Model control group (MC): Cyclophosphamide (Cy) + oral gavage with distilled water;

[0094] (3) Positive control (PC): The cyclophosphamide (Cy) treatment group received levamisole (40 mg / kg body weight);

[0095] (4) CPCR group: The cyclophosphamide (Cy) treatment group received CPCR (350 mg / kg body weight);

[0096] (5) CP1 group: Cyclophosphamide (Cy) treatment group received CP1 (350 mg / kg body weight);

[0097] (6) CP2 group: Cyclophosphamide (Cy) treatment group received CP2 (350 mg / kg body weight).

[0098] The protocol for day 21 consisted of three phases: (1) Days 8-10: Immunosuppression model was established by inducing cyclophosphamide (Cy). Except for the NC group, all groups received intraperitoneal injections of cyclophosphamide (Cy, 80 mg / kg body weight) dissolved in sterile saline daily to establish the immunosuppression model; the NC group received an equal volume of sterile saline injection. (2) Days 11-20: Treatment phase. The NC and MC groups were orally administered 200 μL of distilled water daily, while the treatment groups were administered their respective polysaccharide solutions or levamisole (total volume 200 μL). Body weight and food intake were monitored and recorded every 3 days throughout the experiment. Mouse body weight and food and water intake were recorded as follows: Figures 11-13 As shown. (3) Day 21 (endpoint): Blood and tissue collection: Peripheral blood was collected under anesthesia via the retroorbital plexus prior to euthanasia for cervical dislocation. The weight of the primary and secondary immune organs (spleen and thymus) was immediately weighed to calculate the organ index. Cecal contents were rapidly frozen in liquid nitrogen and stored at -80°C for subsequent microbiome analysis. As described by Xie et al. [Xie, H., Fang, J., Farag, MA, Li, Z., Sun, P., & Shao, P. (2022). Dendrobium officinaleafpolysaccharides regulation of immune response and gut microbiotacomposition in cyclophosphamide-treated mice. Food Chemistry: X, 13, 100235.], the organ index was calculated as organ weight (mg) per 100g body weight.

[0099] Figures 11-13 The results showed that Cy induction significantly reduced the body weight of mice (p<0.01 compared to NC), while intervention with CPCR and CPs restored body weight, especially for CP2, where body weight almost returned to pre-treatment levels. Figure 11 There were no significant differences in food intake or water consumption among the groups. Figure 12 , Figure 13 This suggests that the weight-improving effects of CPCRs and CPs may stem from their specific immunomodulatory mechanisms, rather than just nutritional supplementation.

[0100] Regarding immune organ indices, Cy treatment reduced spleen and thymus indices in mice. Figure 14 , Figure 15 Following CPCR and CPs intervention, spleen indices showed highly significant improvement (p<0.001 compared to MC), while thymus indices did not change significantly. These results suggest that CPs may exert an immunomodulatory effect by enhancing spleen immune function. In summary, CPs significantly improved Cy-induced weight loss and spleen atrophy, demonstrating their potential to reverse drug-induced immunosuppression.

[0101] 3. Spleen cell proliferation test

[0102] After sacrificing mice and weighing the spleens, spleen cells were harvested for spleen cell proliferation assays. Under aseptic conditions, mouse spleens were harvested and prepared as a single-cell suspension via mechanical disruption and filtration. The cell suspension was adjusted to 2 × 10⁶ cells / mL using complete culture medium. 5 Cells / well. To specifically induce the proliferation of T lymphocytes and B lymphocytes, the experimental groups were stimulated for 12 h with concanavalin A (ConA, 1 μg / mL) and LPS (0.1 μg / mL), respectively. After stimulation, the supernatant was removed, and each well was replenished with culture medium containing 10% CCK-8 solution. After incubation at 37°C in a 5% CO2 atmosphere for 4 h, the optical density at 450 nm was measured using a microplate reader to assess cell proliferation. The results are shown below. Figure 16 , 17 As shown.

[0103] The thymus and spleen, as important central and peripheral immune organs, are key sites for the proliferation, differentiation, and maturation of immune cells. Their morphological changes can directly reflect the body's immune status through organ indices. Lymphocyte proliferation is a key biological indicator for assessing immune responses, playing a crucial role in both cellular and humoral immunity. B lymphocytes mediate humoral immune responses by secreting specific antibodies, while T lymphocytes play a central regulatory role in cellular immunity. Figure 16 and Figure 17 The characteristics of ConA-induced and LPS-induced samples were displayed. The results showed that the survival rates of T cells and B cells in the MC group were significantly lower than those in the NC group (p<0.001), confirming the successful establishment of the immunosuppressive model. Compared with the MC group, all treatment groups exhibited different immunomodulatory effects. Among them, CPCR showed the most significant promoting effect on T and B lymphocyte proliferation (p<0.05), while CP1 and CP2 also showed significant immunomodulatory activity, with CP2 being more effective than CP1. This indicates that CP2 can enhance immune function by promoting lymphocyte proliferation.

[0104] 3. Quantitative analysis of serum immunoglobulins and cytokines

[0105] Blood samples were coagulated at room temperature for 30 min, then centrifuged at 3000×g for 15 min to obtain serum. The concentrations of immunoglobulins (IgA, IgM, and IgG) and cytokines (IL-2, IL-4, IL-6, IL-10, and TNF-α) in serum samples were quantitatively analyzed using a commercially available ELISA kit. All analyses were performed strictly according to the manufacturer's instructions, with each sample analyzed in triplicate to ensure data reliability and accuracy. The expression levels of immunoglobulins (IgA, IgM, IgG) and cytokines (TNF-α, IL-2, IL-4, IL-6, IL-10) in mouse serum are shown below. Figures 18-25 As shown.

[0106] Immunoglobulins play a crucial role in immune protection. IgG molecules are essential for helping the body fight off invading pathogens. IgM is the first antibody produced in the primary antibody response and is mainly produced by B cells. IgA plays an important role in immune system protection due to its significant distribution across the systemic and mucosal immune systems. The results showed no significant differences in IgA and IgM levels among the groups. Figure 18 , Figure 19 ). Figure 20 The results showed that CP2 significantly increased serum IgG levels compared with the MC and PC groups (p<0.01), indicating that CP2 can improve the decrease in immunoglobulin levels induced by cyclophosphamide (Cy).

[0107] Regarding cytokine regulation, macrophages, as key effector cells in immune regulation, play a crucial role in the immune response by releasing inflammatory cytokines and maintaining the homeostasis of the cytokine network. The results showed no significant difference in TNF-α levels. Figure 21 This is consistent with the results of macrophage studies. Figure 8 Compared with the NC group, CP2 treatment significantly promoted the secretion of IL-2, IL-4, and IL-10 in mouse serum (p<0.05). Compared with the MC group, CP2 treatment significantly reversed the Cy-induced decrease in IL-2, IL-4, IL-6, and IL-10 levels (p<0.05 or 0.001). Figures 22-25 These results indicate that CP2 has a significant therapeutic effect on Cy-induced immunosuppression in mice. Its mechanism may involve dual regulation: on the one hand, promoting the secretion of immunoglobulin IgG, and on the other hand, upregulating the expression levels of anti-inflammatory cytokines (IL-2, IL-4, IL-6, and IL-10), thereby exerting its immunomodulatory function.

[0108] 4. Western Blot analysis of intestinal tight junction proteins

[0109] To assess changes in intestinal barrier integrity, the protein expression levels of ZO-1, Occludin, and Claudin-1 in colonic tissue were determined by Western blot analysis, following the method described by Li et al. [Li,Y.,Zheng,J.,Wang,Y.,Yang,H.,Cao,L.,Gan,S.,Ma,J.,&Liu,H.(2023).Immuno-stimulatory activity of Astragalus polysaccharides in cyclophosphamide-induced immunosuppressed mice by regulating gut microbiota.INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES,242(Pt 2),124789.]. Methods: Colonic tissue samples were homogenized in protein extraction buffer, and the total protein concentration was determined using the BCA assay. Equal amounts of protein were separated by SDS-PAGE and transferred to a PVDF membrane. After blocking and incubation with primary and secondary antibodies, protein bands were observed using enhanced chemiluminescence. The results are shown below. Figures 26-28 As shown.

[0110] The results showed that the MC group significantly reduced the expression levels of ZO-1, Occludin, and Claudin-1 in the small intestine of mice (p<0.001), which was consistent with the phenotype of Cy-induced intestinal mucosal barrier damage and increased intestinal permeability. Intervention with cPCR, CP1, and CP2 significantly upregulated ZO-1 in the intestine of immunosuppressed mice. Figure 26 Occludin Figure 27 ) and Claudin-1 ( Figure 28 This suggests that they may play a potential role in protecting intestinal mucosal integrity by regulating tight junction protein expression.

[0111] 5. Histopathological examination

[0112] Colon tissue samples from each group of mice were fixed in 10% neutral buffered formalin, processed according to standard histological procedures, and embedded in paraffin. Sections (5 μm thick) were prepared and morphologically evaluated by staining with hematoxylin and eosin (H&E). Pathological evaluation was performed according to the method of Xie et al. [Xie, H., Fang, J., Farag, MA, Li, Z., Sun, P., & Shao, P. (2022). Dendrobium officinale leaf polysaccharides regulation of immune response and gut microbiota composition in cyclophosphamide-treated mice. Food Chemistry: X, 13, 100235.], and the results are as follows. Figure 29 As shown.

[0113] Figure 29 The results showed that the MC group exhibited significant pathological changes, characterized by loose tissue and mucosal damage. The histological features of the colonic tissue in the PC, CPCR, CP1, and CP2 groups were similar to those in the NC group, showing intact epithelial structure, regularly arranged intestinal villi, and compact tissue structure. These results indicate that intervention with CPCR, CP1, and CP2 significantly improved the pathological state of the colonic tissue and promoted tissue repair and regeneration.

[0114] As can be seen from the above embodiments, the present invention provides a cassava polysaccharide, its preparation method and application. The cassava polysaccharide prepared by the present invention can effectively improve the immune capacity of macrophages and mice.

[0115] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing cassava polysaccharides, characterized in that, Includes the following steps: (1) Grind the cassava tubers to obtain cassava pulp; (2) Mix cassava pulp with water, add medium-temperature α-amylase, and sonicate at 60-70℃ for 200-250 min to obtain enzymatic hydrolysate; (3) After the enzyme is inactivated by the enzyme hydrolysate, centrifuge and collect the supernatant. Concentrate the supernatant and precipitate it with alcohol. Centrifuge and collect the precipitate. (4) After dissolving the precipitate, dialyze it, concentrate the dialysate, freeze dry it, and obtain cassava polysaccharide.

2. The method according to claim 1, characterized in that, The amount of mesophilic α-amylase added, based on the weight of the cassava tuber, is 4500–5500 U / g.

3. The method according to claim 1, characterized in that, The mass-to-volume ratio of the cassava pulp to water is 1g:2-3mL.

4. The method according to claim 1, characterized in that, The power of the ultrasonic treatment is 200-400W.

5. The method according to claim 1, characterized in that, After concentrating the supernatant to 1 / 5 to 1 / 3 of its original volume, anhydrous ethanol is added for alcohol precipitation until the volume fraction of ethanol in the system is 75 to 85%. After alcohol precipitation, the mixture is allowed to stand for 2 to 12 hours.

6. The method according to claim 1, characterized in that, The concentration described in steps (3) and (4) is rotary evaporation concentration, with a temperature of 45-55°C and a rotation speed of 180-220 rpm.

7. The method according to claim 1, characterized in that, The dialysis was performed using a dialysis bag with a molecular weight cutoff of 3.5 kDa, and the dialysis time was 48–72 hours.

8. The method according to claim 1, characterized in that, The dialysate is concentrated to 1 / 10 to 1 / 8 of its original volume and then freeze-dried at a temperature of -10°C to -5°C and a vacuum of 8 to 15 Pa.

9. The cassava polysaccharide prepared by any one of claims 1 to 8.

10. The use of the cassava polysaccharide according to claim 9 in at least one of the following: (1) Application in the preparation of drugs that enhance immunity; (2) Application in the preparation of drugs that promote the repair and regeneration of damaged intestines.

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