A method for extracting polysaccharides from *Nostoc commune* with immunomodulatory activity

Abstract

This invention provides a method for extracting polysaccharides from *Nostoc commune* with immunomodulatory activity. Belonging to the field of functional food technology, the method includes the following steps: pulverizing dried *Nostoc commune* into powder; using the powder obtained in step S1 as raw material, extracting with 60% ethanol under ultrasonic assistance to obtain a crude polysaccharide extract; removing the ethanol from the crude polysaccharide extract in step S2 using a vacuum rotary evaporator, precipitating the polysaccharide from a 90% ethanol solution, and then dialyzing and freeze-drying it to obtain purified crude polysaccharide; eluting the purified crude polysaccharide using a Sephadex G-75 column, collecting the eluent in separate tubes and plotting elution curves, collecting the eluent from the tube with the peak on the elution curve and freeze-drying it to obtain a low molecular weight polysaccharide, which is NCEP.

Description

Technical Field

[0001] This invention belongs to the field of functional food technology, specifically relating to a method for extracting polysaccharides from *Nostoc commune* that have immunomodulatory activity. Background Technology

[0002] Immune regulation refers to the interactions between immune cells and molecules within the immune system, as well as with other systems such as the neuroendocrine system, to ensure that the immune response is maintained at an optimal level. Therefore, immune regulation relies on the immune system. Currently, drugs that enhance human immune function often cause numerous side effects while regulating immune activity. Therefore, finding a natural, non-toxic immunomodulator is a key research focus, and polysaccharides have been found to possess excellent immunomodulatory effects.

[0003] Nostoc commune, also known as lichen, is a combination of fungi and algae. It typically grows in dark, damp places, is dark in color, and somewhat resembles softened black fungus. Nostoc commune is rich in protein, various vitamins, and minerals such as phosphorus, zinc, and calcium. Currently, there are no reports on the immune activity of Nostoc commune in research, thus indicating its potential for research and development. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for extracting NCEP from *Nostoc commune* (a type of plant) with immunomodulatory activity. The NCEP extracted by this invention exhibits significant scavenging activity against DPPH and hydroxyl radicals, and also demonstrates significant in vitro immunomodulatory function.

[0005] The specific technical solution is as follows:

[0006] A method for extracting polysaccharides from *Nostoc commune* with immunomodulatory activity includes the following steps:

[0007] S1: Crush the dried lichen into powder;

[0008] S2: The powder obtained in step S1 is used as raw material and extracted with 60% ethanol under ultrasonic assistance to obtain crude polysaccharide extract of Nostoc commune.

[0009] S3: After removing the ethanol from the crude polysaccharide extract of Nostoc commune in step S2 using a vacuum rotary evaporator, the polysaccharide is precipitated from the 90% ethanol solution and then dialyzed and freeze-dried to obtain purified Nostoc commune crude polysaccharide.

[0010] S4: The purified crude polysaccharide of Nostoc commune was eluted using a Sephadex G-75 column. The eluent was collected in separate tubes and the elution curve was plotted. The eluent from the tube with the peak on the elution curve was collected and freeze-dried to obtain a low molecular weight polysaccharide, which is NCEP.

[0011] NCEP is a homogeneous polysaccharide with a molecular weight of 20.466 kDa. The monosaccharide composition of NCEP consists of glucose (Glc), xylose (Xyl), galactose (Gal), glucuronic acid (Glc-UA), mannose (Man), rhamnose (Rha), fucose (Fuc), and arabinose (Ara).

[0012] In addition, the method for extracting polysaccharides from *Nostoc commune* with immunomodulatory activity provided by the present invention may also have the following additional technical features:

[0013] In the above technical solution, in step S2, the specific method of extraction with 60% ethanol under ultrasonic assistance is as follows: using an ultrasonic instrument, the ratio of raw material to 60% ethanol is 1:10-1:30 g / ml, the extraction temperature is 30℃-70℃, the ultrasonic time is 40-200 minutes, and the ultrasonic power is 180-600W.

[0014] In the above technical solution, in step S4, the conditions for using a Sephadex G-75 column to elute the purified crude polysaccharide of *Gnaphalium affine* are as follows: the diameter and length of the column are 1.6 cm × 60 cm, the elution rate is 1 mL / min, and 2 mL / tube.

[0015] The method for extracting polysaccharides from *Nostoc commune* with immunomodulatory activity according to the present invention has the following advantages compared with the prior art:

[0016] 1. The crude polysaccharide of *Nostoc commune* was purified by ultrasonic-assisted extraction with 60% ethanol, followed by precipitation with 90% ethanol solution, dialysis, and freeze-drying. The purified polysaccharide was then eluted using a Sephadex G-75 column to obtain NCEP. The NCEP extracted using this method showed significant scavenging activity against DPPH and hydroxyl radicals, as well as significant in vitro immunomodulatory function.

[0017] 2. This invention uses common lichen as raw material, ensuring the green and safe nature of the raw material, improving the utilization rate of lichen, broadening the application scope of lichen, and providing a basis for the in-depth development of lichen series products. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of an experiment of the present invention to explore the extraction of polysaccharides from *Nostoc commune* using a single-factor method, considering factors such as liquid ratio, extraction parameters, time, temperature, and ultrasonic power.

[0019] Figure 2 This is a schematic diagram illustrating the optimization of optimal process parameters for extracting polysaccharides from *Gnaphalium affine* using response surface methodology, according to the present invention.

[0020] Figure 3 This is a schematic diagram illustrating the structural analysis of a polysaccharide according to the present invention;

[0021] Figure 4 and Figure 5 This is a schematic diagram illustrating the in vitro immunomodulatory activity (cell viability, cell ROS and NO content, cell phagocytic capacity, and cellular inflammatory factors IL-6, TNF-α, and IL-1β) of a low molecular weight polysaccharide from *Nostoc commune* according to the present invention.

[0022] Figure 6 This is a schematic diagram of sample clustering heat from the present invention;

[0023] Figure 7 This is a bar chart of differentially expressed genes in the samples of this invention;

[0024] Figure 8 Venn diagram for statistical analysis of differentially expressed genes in samples of this invention;

[0025] Figure 9 This is a bar chart of GO analysis of the samples from this invention;

[0026] Figure 10 Bubble diagram of KEGG signal pathway enrichment analysis for samples in this invention; Detailed Implementation

[0027] The following are specific implementation cases and appendices. Figure 1-10 The present invention will be further described, but the present invention is not limited to these embodiments.

[0028] Example 1:

[0029] A method for extracting polysaccharides from *Nostoc commune* with immunomodulatory activity includes the following steps:

[0030] S1: Crush the dried lichen into powder;

[0031] S2: The powder obtained in step S1 is used as raw material and extracted with 60% ethanol under ultrasonic assistance to obtain crude polysaccharide extract of Nostoc commune.

[0032] S3: After removing the ethanol from the crude polysaccharide extract of Nostoc commune in step S2 using a vacuum rotary evaporator, the polysaccharide is precipitated from the 90% ethanol solution and then dialyzed and freeze-dried to obtain purified Nostoc commune crude polysaccharide.

[0033] S4: The purified crude polysaccharide of Nostoc commune was eluted using a Sephadex G-75 column. The eluent was collected in separate tubes and the elution curve was plotted. The eluent at the peak of the elution curve was collected and freeze-dried to obtain a low molecular weight polysaccharide, which is NCEP.

[0034] NCEP is a homogeneous polysaccharide with a molecular weight of 20.466 kDa. The monosaccharide composition of NCEP consists of glucose (Glc), xylose (Xyl), galactose (Gal), glucuronic acid (Glc-UA), mannose (Man), rhamnose (Rha), fucose (Fuc), and arabinose (Ara).

[0035] In an embodiment of the present invention, in step S2, the specific method of extraction with 60% ethanol under ultrasonic assistance is as follows: using an ultrasonic instrument, the ratio of raw material to 60% ethanol is 1:10-1:30 g / ml, the extraction temperature is 30℃-70℃, the ultrasonic time is 40-200 minutes, and the ultrasonic power is 180-600W.

[0036] In an embodiment of the present invention, in step S4, the conditions for eluting the purified crude polysaccharide of *Gnaphalium affine* using a Sephadex G-75 column are as follows: the column diameter and length are 1.6 cm × 60 cm, the elution rate is 1 mL / min, and 2 mL / tube.

[0037] Example 2:

[0038] 1. Optimization of extraction parameters for Nostoc commune polysaccharide: Based on single-factor experiments, the effects of four factors—ultrasound time, temperature, ultrasonic power, and material-liquid ratio—on the yield of Nostoc commune polysaccharide were studied using response surface methodology. The yield was evaluated using Box-Behnken design and named A, B, C, and D, respectively, as shown in Table 1.

[0039] Table 1 Variables and levels of Box–Behnken design.

[0040]

[0041] Table 1

[0042] 2. Single-factor experimental analysis, preliminary single-factor studies such as... Figure 1 As shown, the model was constructed by detecting the effects of the ratio of material to liquid, extraction parameters time, temperature, and ultrasonic power on the extraction rate of crude polysaccharide extract from *Gnaphalium affine*.

[0043] The yield of crude polysaccharide extract from *Nostoc commune* first increased and then decreased with temperature (30-70℃). When the temperature exceeded 70℃, the yield decreased significantly (e.g., Figure 1 (As shown in A in the figure). The reason for the decrease in NCEP yield is that high temperature causes oxidation and thermal decomposition of polysaccharides; therefore, 70℃ is the optimal temperature.

[0044] according to Figure 1As shown, at a ratio of 1:20 (g / mL), the yield significantly increased to 3.47 mg / g. Under constant temperature, solid-liquid ratio, and time of 70℃, 1:40, and 20 min, respectively, the extraction rate of crude polysaccharide extract from *Nostoc commune* increased with increasing power, but decreased over time, reaching a maximum of 6.734 mg / g at 540 W.

[0045] 3. Optimization of extraction process parameters: Based on single-factor data, response surface methodology was conducted to further optimize the extraction conditions of the crude polysaccharide extract from *Nostoc commune*. A total of 29 groups were randomly selected, as shown in Table 2.

[0046] The quadratic polynomial function representing the NCEP content is as follows:

[0047] Y = 8.02 - 0.1050A + 0.31B + 0.1333C - 0.55D - 0.355AB + 0.5975AC - 0.5475AD - 0.4875BC + 0.1325BD + 0.275CD - 0.8287A² - 0.6787B² - 0.6512C² - 0.1612D², where Y represents the NCEP content (mg / g), and A, B, C, and D are the values ​​of extraction temperature, material-to-liquid ratio, extraction time, and ultrasonic power, respectively.

[0048] Table 2 Three factors three levels of BBD for extraction yield of NCEP and the predicted.

[0049]

[0050] Table 2

[0051] Table 3 illustrates the statistical significance of each model coefficient through response surface methodology. The model's deterministic modulus (R² = 0.9404) shows a sufficient model fit, with almost perfect agreement between expected and actual values. Furthermore, the model can explain most of the variation in the extraction of crude polysaccharide from *Nostoc commune* due to the adjusted coefficient of determination (0.8809). The F-value is sufficiently high (14.83), and the P-value is significantly low (P < 0.0001), indicating that the model and related variables are significant. The P-value (0.0859) and F-value (4.31) are above 0.051, indicating a good fit. The results show that the order of P-values ​​for the four autonomous conditions is B > C > A > D, indicating that the ratio of raw material to liquid (B) has the greatest impact on the extraction effect.

[0052] Table 3 Analysis of variance for the results of BBD.

[0053]

[0054] Table 3

[0055] The effects of various factors on NCEP extraction rate first increase and then decrease, based on three-dimensional response surface plots and contour plots (such as...). Figure 2 As shown in the figure, the surface is convex in the middle and concave around the edges. The results indicate that the surface has a highest point, suggesting optimal conditions. Therefore, under the conditions of 54℃, 597.7W, 141.98min, and a solid-liquid ratio of 1:22.98, the predicted yield (8.335 mg / g) was obtained. A verification experiment was conducted under improved conditions (i.e., a solid-liquid ratio of 1:23, 54℃, 142min, 600W), achieving an extraction rate of 8.27 ± 0.08 mg / g (n=3), close to the predicted yield. Suitable temperature can improve the solubility of polysaccharides and promote their mass transfer, while excessively high temperatures can lead to oxidation and thermal degradation. Therefore, ultrasound-assisted extraction has significant benefits for the development of *Nostoc commune*.

[0056] Specifically, as shown in Table 4, the polysaccharide content, protein content, and uronic acid content of NCEP were 83.84%±0.27, 1.25%±0.05, and 6.35%±0.29, respectively, indicating that NCEP is an acidic polysaccharide.

[0057] Table 4 The physicochemical properties of NCE

[0058]

[0059] Table 4

[0060] Example 3:

[0061] 1. In the existing technology, the structural characterization of NCEP and the analysis of its 113 monosaccharides were performed using a practical high-performance anion exchange chromatography instrument, high-performance ion exchange chromatography, and pulsed amperometric detector. The molecular weight of NCEP was analyzed using high-performance gel permeation chromatography-evaporative light scattering detector. The conditions were as follows: Agilent 1260 Infinity ELSD, TSK Gel G-3000PWXL column (7.8 × 300 nm). NCEP sample (10 mg) was mixed with KBr powder (1 g), and... The particles were scraped into 1 mm translucent particles within the range and analyzed by Fourier transform infrared spectroscopy (Equinx 55, Brucher, Germany).

[0062] like Figure 3As shown, the NCEP extracted in this application exhibits a single symmetrical peak of polysaccharide, indicating that NCEP is a homogeneous polysaccharide. Furthermore, the molecular weight of NCEP is 20.466 kDa, significantly different from the 501.267 kDa and 245 kDa of polysaccharides extracted in existing technologies. Therefore, the NCEP extracted in this application is a homogeneous polysaccharide.

[0063] The monosaccharide composition of NCEP consists of glucose (Glc), xylose (Xyl), galactose (Gal), glucuronic acid (Glc-UA), mannose (Man), rhamnose (Rha), fucose (Fuc), and arabinose (Ara). Polysaccharides rich in glucose and xylose are speculated to possess potential immunomodulatory activities, such as... Figure 3 As shown, at 3420 cm -1 A broad and strong peak appeared at this point, which is the result of OH tensile vibration. 2940cm -1 The weak absorption band at this point indicates that it is attributed to the stretching vibration of CH. The asymmetric stretching band of COO– observed in NCEP is caused by this. and The absorption band indicates that NCEP contains acidic uronic acid. NCEP exhibits characteristic absorption peaks of polysaccharides. The absorption peak at [location] is generated by the bending vibrations of COC and COH caused by the pyranose ring, indicating that NCEP is a pyranose-type polysaccharide. NCEP also lists the 800 cm⁻¹ [value]. -1 and 900 cm -1 The absorption band at the location indicates the presence of α and β configurations of pyranose polysaccharides.

[0064] 2. In vitro antioxidant activity of NCEP, DPPH and hydroxyl radicals are widely used to evaluate the total antioxidant activity of compounds.

[0065] like Figure 4-5 As shown, NCEP exhibits dose-dependent DPPH and hydroxyl radical scavenging activity; NCEP achieves a DPPH scavenging rate of 51.8% at 5.0 mg / mL, and the trend of NCEP scavenging hydroxyl radicals is the same. Therefore, NCEP extracted with 60% ethanol using ultrasound assistance has a strong antioxidant capacity against DPPH and hydroxyl radicals, which is consistent with the high antioxidant activity of low molecular weight polysaccharides.

[0066] Specifically, for the NCEP assay of DPPH and hydroxyl radical scavenging activity, ascorbic acid (Vc) was selected as a positive control.

[0067] 3. In vitro immunomodulatory activity of NCEP: This invention focuses on the role of NCEP in the viability of mouse mononuclear macrophage leukemia cells (RAW264.7) and the production of ROS, NO and inflammatory factors.

[0068] like Figure 4-5 As shown, compared with the blank control group (Control), NCEP at concentrations of 12.5-200 µg / mL significantly improved cell viability (p<0.05). The cell viability of RAW264.7 cells treated with 100 µg / mL NCEP reached 137.43±2.17%, indicating that NCEP is non-toxic to RAW264.7 cells and has potential macrophage activation capacity.

[0069] NCEP affects the immune processes of macrophages. Compared with the control group, NCEP dose-dependently increased NO production (p<0.05). NCEP at 12.5 µg / mL induced NO production close to that LPS (p>0.05), while NCEP at 50 µg / mL was significantly higher than LPS (p<0.05). Simultaneously, NCEP treatment also significantly enhanced phagocytic activity (p<0.05). Furthermore, notably, when the NCEP concentration exceeded 50 µg / mL, the phagocytic capacity of NCEP was significantly superior to that of LPS.

[0070] As a low-molecular-weight soluble polysaccharide, IL-1β stimulates macrophages and pro-inflammatory cytokines, and further responds to inflammatory signals by triggering the production of the inflammatory cytokine IL-6. Figure 4-5 As shown, NCEP induced the release of IL-6, TNF-α, and IL-1β from RAW264.7 cells in a dose-dependent manner. TNF-α plays a crucial role in the host defense system, inducing numerous immunomodulatory responses and promoting the secretion of chemokines and inflammatory factors at the site of infection, thereby contributing to the clearance of pathogens from the site of infection. IL-6 promotes phagocytosis and adhesion and is an important bioactive cytokine for macrophage immune function. It can be stimulated by large amounts of TNF-α, thereby regulating T cell activation, B cell differentiation, and lymphocyte typing, exerting a non-specific immunomodulatory effect. In particular, NCEP (50 µg / mL) was significantly more effective than LPS in promoting IL-6 secretion (P>0.05), and NCEP induced a more effective level of IL-1β than LPS.

[0071] Therefore, it can be seen that the immunomodulatory mechanism by which NCEP enhances immunity is through promoting the production of ROS and upregulating the secretion of interleukin-6 (IL-6), tumor necrosis factor (TNF-α), and interleukin-1β (IL-1β).

[0072] ROS is a metabolic byproduct of oxidation during aerobic cell growth and an important signaling molecule involved in maintaining normal cellular physiological functions. Appropriate ROS production is beneficial for enhancing the immunomodulatory activity of RAW264.7 macrophages. NCEP significantly increased intracellular ROS production levels in a dose-dependent manner. Furthermore, 50 μg / mL NCEP showed similar levels of ROS release as LPS. These data indicate that NCEP has a significant immunomodulatory effect by promoting the production of TNF-α, IL-6, IL-1β, and ROS.

[0073] 4.16S RNA transcriptome sequencing results revealed differentially expressed genes (DEGs) in the transcriptome, which can specifically demonstrate the biological function of drugs in this process, facilitating the exploration of the mechanisms behind gene changes. Transcriptome sequencing technology has advantages such as high throughput, high accuracy, and high sensitivity. To explore the potential immunomodulatory mechanism of NCEP on RAW264.7 cells, 16S RNA transcriptome sequencing experiments were performed. Statistical analysis was performed on six samples, yielding a total of 37.65 Gb of clean data. The effective data for different samples ranged from 5.72 to 6.82 Gb, with Q30 bases in the sample index ranging from 93.37% to 94.42% and an average GC percentage of 48.78%. The three samples showed good reproducibility within the same group, with a clear separation between the NCEP group and the control group, indicating a large number of differentially expressed genes compared to the control group.

[0074] Experimental error was controlled using DESeq2. p < 0.05 and |log2-fold change| ≥ 2 were considered criteria for filtering DEGs. A total of 1093 DEGs were identified between the Control and NCEP groups, of which 567 were upregulated and 526 were downregulated. A total of 43 targets, including 26 BPs, 2 CCs, and 15 MFs, obtained through GO enrichment were used for GO enrichment analysis (p < 0.05), and bar charts were plotted. NCEPs exert antioxidant activity and are involved in various BPs, CCs, and MFs. For example, they are involved in biological processes related to stimulus responses, immune system processes, viral processes, circadian rhythm processes, detoxification, and biomineralization.

[0075] like Figure 6-10As shown, KEGG enrichment analysis revealed 316 pathways enriched by NCEP (p < 0.05), and the bubble plot shows the gene proportions of the top 20 KEGG signaling pathways. The pathways enriched by NCEP mainly included cytokine-cytokine receptor interactions, cancer pathways, IL-17 signaling pathway, TNF pathway, Rap1 signaling pathway, C-type lectin receptor signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway, lipid and atherosclerosis pathways, etc. Therefore, the interaction between NCEP and immunomodulatory activity enriched BPs, CCs, MFs, and KEGG pathways, indicating that NCEP can counteract the immunomodulatory response of RAW264.7 cells through multiple pathways.

[0076] Specifically, for CCK-8 assay, RAW 264.7 macrophages were used to measure cell proliferation using the CCK-8 assay. RAW264.7 macrophages (1×10⁻⁶ cells) were used. 4 Cells were cultured in 96 wells for 24 h (1 cell / well), then co-cultured for 24 h with either LPS (1 μg / mL) or fresh medium containing NCEP (0, 1.56, 3.125, 6.25, 12.5, 25, 50, 100, 200, and 400 μg / mL), followed by continuous incubation at 37°C with 10 μL of CCK-8 for 1.5 h. Cells were analyzed at 490 nm using an Infinite 200 PRO microplate reader (Tecan, Switzerland). Relative cell viability is expressed as a percentage of the Control group. Each treatment was performed in triplicate.

[0077] Phagocytic capacity was assessed using a neutral red assay on RAW264.7 cells. RAW264.7 cells (1×10⁻⁶) 5 Cells (1 cell / mL) were seeded overnight in 96-well plates and incubated with different concentrations of NCEP or LPS (1 μg / mL). 20 μL of neutral red was added to the cells for 2 hours, followed by washing six times with PBS. Cell lysate was added, and the plates were gently agitated for 10 minutes after removing the supernatant. Absorbance was recorded at 540 nm using an Infinite 200 PRO microplate reader (Tecan, Switzerland).

[0078] Measurements of NO, cytokine, and ROS production. RAW264.7 cells (1×10⁻⁶) were used. 5Cells were incubated for 24 hours in 24-well plates with different concentrations of NCEP and LPS (1 μg / mL) as described above. After 24 hours, the concentrations of NO and cytokines (IL-1β, TNF-α, and IL-6) were determined according to the manufacturer's protocol. DCFH-DA was used to measure intracellular ROS production. Cells were incubated with DCFH-DA (10 μM) in the dark at 37°C for 30 min. Staining images were captured using a fluorescence microscope (Zeiss AXIO, Germany).

[0079] Transcriptome sequencing was performed. Total RNA was extracted from normal RAW264.7 cells and NCEP-treated (50 µg / mL) RAW266.7 cells using Trizol reagent (Beijing Teronsen Biotechnology Co., Ltd.). RNA library sequencing was performed by GeneDenovo Biotechnology Co. (Guangzhou, China) using an Illumina Novaseq 6000. Omicsmart (http: / / www.omicsmart.com) was used for bioinformatics analysis. Genes with a false discovery rate (FDR) < 0.05 and a log2-fold change ≥ 2 were considered differentially expressed genes (DEGs).

[0080] In the description of this invention, the term "a plurality of" refers to two or more. Unless otherwise explicitly defined, the terms "upper," "lower," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the invention. The terms "connection," "installation," "fixing," etc., should be interpreted broadly. For example, "connection" can be a fixed connection, a detachable connection, or an integral connection; it can be a direct connection or an indirect connection through an intermediate medium. For those skilled in the art, the specific meaning of the above terms in this invention can be understood according to the specific circumstances.

[0081] In the description of this invention, the terms "one embodiment," "some embodiments," "specific embodiment," etc., refer to a specific feature, structure, material, or characteristic described in connection with that embodiment or example, which is included in at least one embodiment or example of the invention. In this invention, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0082] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for extracting polysaccharides from *Nostoc commune* with immunomodulatory activity, characterized in that, Includes the following steps: S1: Crush the dried lichen into powder; S2: The powder obtained in step S1 is used as raw material and extracted with 60% ethanol under ultrasonic assistance to obtain crude polysaccharide extract of Nostoc commune. S3: After removing the ethanol from the crude polysaccharide extract of Nostoc commune in step S2 using a vacuum rotary evaporator, the polysaccharide is precipitated from the 90% ethanol solution and then dialyzed and freeze-dried to obtain purified Nostoc commune crude polysaccharide. S4: The purified crude polysaccharide of Nostoc commune was eluted using a Sephadex G-75 column. The eluent was collected in separate tubes and the elution curve was plotted. The eluent at the peak of the elution curve was collected and freeze-dried to obtain a low molecular weight polysaccharide, which is NCEP. The NCEP is a uniform polysaccharide with a molecular weight of 20.466 kDa, and its monosaccharide composition consists of glucose (Glc), xylose (Xyl), galactose (Gal), glucuronic acid (Glc-UA), mannose (Man), rhamnose (Rha), fucose (Fuc), and arabinose (Ara).

2. The method for extracting *Nostoc commune* polysaccharides with immunomodulatory activity according to claim 1, characterized in that, In step S2, the specific method of extraction with 60% ethanol under ultrasonic assistance is as follows: using an ultrasonic instrument, the raw material and 60% ethanol are mixed at a ratio of 1:10-1:30 g / ml, the extraction temperature is 30℃-70℃, the ultrasonic time is 40-200 minutes, and the ultrasonic power is 180-600W.

3. The method for extracting *Nostoc commune* polysaccharides with immunomodulatory activity according to claim 1, characterized in that, In step S4, the Sephadex G-75 column was used to elute the purified crude polysaccharide of Nostoc commune under the following conditions: the column diameter and length were 1.6 cm × 60 cm, the elution rate was 1 mL / min, and the elution rate was 2 mL / tube.

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