A kind of Wogan polysaccharide and its extraction method and application

Abstract

The present invention extracts and separates Wogan polysaccharide from Wogan peel polysaccharide to obtain Wogan polysaccharide, which has a molecular weight of 50-70kDa. It has the effects of promoting NK-92MI cell proliferation, upregulating gene expression and improving cytotoxicity, and can be used to prepare drugs that promote NK-92MI cell proliferation, upregulate gene expression and improve cytotoxicity. The Wogan polysaccharide prepared by the present invention also has a certain inhibitory effect on Calu-1 lung cancer cells and can be used to prepare drugs that inhibit Calu-1 lung cancer cells, greatly improving the utilization value of Wogan peel.

Description

Technical Field

[0001] The present invention relates to the technical field of food resource utilization, and in particular to a polysaccharide from citrus aurantium and an extraction method and application thereof. Background Art

[0002] Citrus fruits are among the world's most popular fruits, with global sales of tangerines alone exceeding 29 million tons annually. With the development of the citrus processing industry, an increasing amount of peels is produced. However, much of this peel is simply discarded, placing significant pressure on the environment and wasting resources. Addressing this issue warrants greater attention and research.

[0003] Citrus peel is one of the main raw materials for producing polysaccharides such as pectin. Due to its diverse properties, it has been widely used in the food, pharmaceutical, and cosmetic industries. Extensive research has been conducted on the extraction and separation methods, structural characterization, rheological properties, antioxidant activity, and immune function of citrus polysaccharides from different varieties. For example, patent application number CN201611238361.5, entitled "A Method for Extracting and Purifying Polysaccharides from Citrus Peel," primarily involves the following steps for polysaccharide extraction and purification: 1) Preparation of crude citrus peel polysaccharide; 2) Purification of the crude citrus peel polysaccharide: A) Decolorization with activated carbon, and B) Deproteinization with the Sevag method. The resulting polysaccharide is claimed to exhibit moderate antioxidant activity, high purity, and low energy consumption. High-purity polysaccharides have significant application value in medicine, health supplements, and food. However, little research has been conducted on polysaccharides from Wogan. Polysaccharides with specific structures can enhance animal immunity and offer significant advantages and potential for the development of functional health supplements. For example, some polysaccharides can enhance the body's killing effect on invading viruses by activating natural killer (NK) cells. When NK cells are activated, they release many killing mediators, such as perforin and granzyme B. First, perforin forms pores on the target cell membrane, and then granzyme B enters through the pores, activating related proteins and causing them to die. The main purpose of this invention is to study whether the polysaccharides in Wogan peel have the characteristics of activating NK cells, and to identify and protect their structure. On this basis, the dose-effect relationship between polysaccharides and NK cell activity is further explored to provide reference data for improving the high-value utilization and product development of Wogan peel. Summary of the Invention

[0004] The purpose of the present invention is to provide a polysaccharide from Wogan orange and an extraction method and application thereof.

[0005] A Wogan polysaccharide is separated from Wogan peel and has a molecular weight of 50-70 kDa.

[0006] Furthermore, the Wogan polysaccharide is prepared by the following method:

[0007] S1: Wash and dry the peel of the mandarin orange, crush it, extract it with distilled water, and collect the supernatant;

[0008] S2: The supernatant obtained in S1 is concentrated to obtain a viscous liquid, anhydrous ethanol is added to the viscous liquid for alcohol precipitation, and the precipitate collected is the crude polysaccharide of Wogan peel;

[0009] S3: The crude polysaccharide from the peel of the mandarin orange is dissolved in distilled water to prepare a crude polysaccharide solution from the peel of the mandarin orange. The crude polysaccharide solution from the peel of the mandarin orange is passed through a MinimatePall ultrafiltration system. After passing through ultrafiltration membranes of 1000, 500, 300, 100, 70, 50, 30, 10 and 5 kDa, the 50-70 kDa polysaccharide obtained by ultrafiltration is concentrated, and then precipitated with alcohol. The precipitate is freeze-dried to obtain the said mandarin orange polysaccharide.

[0010] Another object of the present invention is to protect the use of the above-mentioned Wogan polysaccharide in the preparation of drugs for promoting NK-92MI cell proliferation, upregulating gene expression and improving cytotoxicity.

[0011] Another object of the present invention is to protect the above-mentioned drug for promoting NK-92MI cell proliferation, upregulating gene expression and improving cytotoxicity, wherein the drug comprises the above-mentioned Wogan polysaccharide.

[0012] Another purpose of the present invention is to protect the use of the above-mentioned Wogan polysaccharide in the preparation of drugs for inhibiting Calu-1 lung cancer cells.

[0013] Another object of the present invention is to protect the above-mentioned drug for inhibiting Calu-1 lung cancer cells, which contains the above-mentioned Wogan polysaccharide.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention extracts and separates polysaccharides of different molecular weights from the peel of the mandarin orange. Among them, the polysaccharides of 50-70 kDa have the effects of promoting NK-92MI cell proliferation, upregulating gene expression, and improving cytotoxicity, and can be used to prepare drugs that promote NK-92MI cell proliferation, upregulate gene expression, and improve cytotoxicity. The prepared mandarin orange polysaccharide has a certain inhibitory effect on Calu-1 lung cancer cells and can be used to prepare drugs that inhibit Calu-1 lung cancer cells, thereby improving the utilization value of the mandarin orange peel. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a graph showing the effects of different molecular weight citrus polysaccharides (OP1-OP10) on the proliferation of NK-92MI cells in Example 2;

[0017] Figure 2This is a graph showing the effects of different molecular weight citrus polysaccharides (OP1-OP10) on perforin expression in NK-92MI cells in Example 2;

[0018] Figure 3 This is a graph showing the effects of different molecular weight citrus polysaccharides (OP1-OP10) on the expression of granzyme B in NK-92MI cells in Example 2;

[0019] Figure 4 This is a graph showing the effects of different molecular weight citrus polysaccharides (OP1-OP10) on the expression of IFN-γ in NK-92MI cells in Example 2;

[0020] Figure 5 This is a graph showing the effects of different molecular weight citrus polysaccharides (OP1-OP10) on NK-92MI cell cytotoxicity in Example 2;

[0021] Figure 6 This is the monosaccharide composition diagram of OP5 in Example 2;

[0022] Figure 7 is the infrared analysis spectrum of OP5 in Example 2;

[0023] Figure 8 is the hydrogen spectrum of OP5 in Example 2;

[0024] Figure 9 This is the carbon spectrum of OP5 in Example 2. DETAILED DESCRIPTION

[0025] The present invention will be further described below in conjunction with examples and tests.

[0026] Example 1

[0027] A Wogan polysaccharide is separated from Wogan peel and has a molecular weight of 50-70 kDa.

[0028] Example 2

[0029] The Wogan polysaccharide of Example 1 was prepared by the following method:

[0030] S1: The peel of the mandarin orange was rinsed with distilled water and then dried in an oven at 40°C. The dried peel of the mandarin orange was crushed into powder using a grinder. The powder (100 g) was extracted with distilled water (1900 g) at 100°C for 3 h, and the supernatant was collected;

[0031] S2: The supernatant obtained in S1 was concentrated using a rotary evaporator to obtain a viscous liquid. The viscous liquid (100 ml) was added to anhydrous ethanol (900 ml) and stirred for 2 minutes, and then allowed to stand at 4°C for 24 hours. The precipitate was collected and named as crude polysaccharide from Wogan peel (OP).

[0032] S3: The crude polysaccharide from the peel of the mandarin orange was dissolved in distilled water at 100°C to prepare a crude polysaccharide solution from the peel of the mandarin orange. The crude polysaccharide solution from the peel of the mandarin orange was passed through a MinimatePall ultrafiltration system having ultrafiltration membranes of 1000, 500, 300, 100, 70, 50, 30, 10, and 5 kDa. After the crude polysaccharide solution from the peel of the mandarin orange passed through the MinimatePall ultrafiltration system, 10 polysaccharide solutions with different molecular weights were obtained. The 10 polysaccharide solutions with different molecular weights were concentrated and then precipitated with alcohol. All the precipitates were then freeze-dried to prepare mandarin orange polysaccharides with different molecular weights.

[0033] The 10 polysaccharide fractions of different molecular weights obtained in this example are shown in Table 1 below. The 10 polysaccharides with different molecular weights are named (OP1-OP10). Among them, OP10 has the highest proportion, accounting for 48.41%, followed by OP8 (10.25%), OP6 (9.64%), and OP4 (8.82%). The other proportions are less than 7%.

[0034] Among them, the 50-70 kDa (OP5) Wogan polysaccharide is the Wogan polysaccharide in Example 1.

[0035] Table 1

[0036]

[0037]

[0038] The above 10 polysaccharides with different molecular weights were subjected to the following experiment:

[0039] (1) Cell culture

[0040] 1. NK-92MI cells (natural killer cells)

[0041] Culture conditions: MEMα+0.2mM Inositol+0.1mMβ-mercaptoethanol+0.02mM Folic Acid+12.5% ​​HS+12.5% ​​FBS+1% P / S, 5% CO2, 37°C.

[0042] 2. Calu-1 cells (lung cancer cells)

[0043] Culture conditions: McCoy's 5A + 10% FBS + 1% P / S, 5% CO2, 37°C.

[0044] (II) NK-92MI cell activity assay

[0045] 1. Effects of different molecular weight Wogan polysaccharides (OP1-OP10) on NK-92MI cell proliferation

[0046] NK-92MI cells were seeded in 96-well plates. Four hours after passage, solutions of 62.5, 125, 250, 500, and 1000 μg / mL of Wogan polysaccharide of varying molecular weights were added to the sample group, while an equal volume of PBS was added to the control group. After 16 hours of co-culture, the proliferation rate of NK-92MI cells was measured using the CCK8 assay.

[0047] The results can be found in Figure 1 .Depend on Figure 1 As can be seen, OP1-OP10 significantly promoted the proliferation of NK-92MI cells. At certain concentrations, the proliferation rate of NK-92MI cells even exceeded 150%. For example, OP1 at 500 μg / mL and OP9 at 250 μg / mL increased the proliferation rates of NK-92MI cells to 156.45% and 160.40%, respectively. Overall, the effects of each OP on NK-92MI cell proliferation initially increased and then decreased with increasing concentration. Within the concentration range of 250 μg / mL to 500 μg / mL, the promoting effect of each OP was highest. Therefore, in subsequent experiments, this range was narrowed to identify the optimal concentration.

[0048] 2. Effects of different molecular weight Wogan polysaccharides (OP1-OP10) on NK-92MI gene expression

[0049] Total RNA from treated NK-92MI cells was extracted using TRIzol reagent. cDNA was constructed using HiScript II QRT SuperMix for qPCR (+gDNAwiper). ChamQ Universal SYBR qPCR Master Mix was used for quantitative real-time PCR (qRT-PCR) amplification. Fluorescence quantitative PCR conditions were as follows: pre-denaturation at 95°C for 3 minutes. 40 cycles of 95°C for 15 seconds, 57°C for 15 seconds, and 72°C for 20 seconds were performed. Target gene expression of perforin (PFP), granzyme B (GZMB), and interferon-γ (IFN-γ) in NK-92MI cells was determined by the fold change [2(-ΔΔCt)] method. For qRT-PCR analysis, GAPDH was selected as an internal reference gene.

[0050]

[0051]

[0052] The experimental results can be found in Figure 2-Figure 4 As shown by Figure 2It can be seen that different OPs have significantly different effects on perforin expression in NK-92MI cells. At the experimental concentrations, perforin expression in NK-92MI cells was downregulated by four Wogan polysaccharides (OP7, OP8, OP9, and OP10), while it was significantly upregulated by three Wogan polysaccharides (OP2, OP4, and OP5). Between 150 μg / mL and 250 μg / mL, the upregulation level was relatively high. Figure 3 It can be seen that at the same concentration, OP2 and OP5 can significantly increase the expression of granzyme B in NK-92MI cells. In the range of 250-350 μg / mL, the increase is large, increasing by 1.29-1.50 times (OP2) and 1.32-1.41 times (OP5). Figure 4 It can be seen that six types of Wogan polysaccharides (from OP1 to OP6) enhanced the expression of IFN-γ in NK-92MI cells. Except for OP3, all Wogan polysaccharides achieved the greatest upregulation level within the concentration range of 150μg / mL to 250μg / mL. Compared with the control group, OP5 increased the expression level of IFN-γ the most, reaching 5.39-7.40 times.

[0053] 3. Effects of different molecular weight Wogan polysaccharides (OP1-OP10) on NK-92MI cytotoxicity

[0054] NK-92MI cells were seeded into 6-well plates and divided into sample and control groups after 4 hours of passage. The sample groups were treated with 250 μg / mL of OP1-OP10 polysaccharide solutions of varying molecular weights, while the control groups were treated with the same volume of PBS (250 μg / mL). Both groups were cultured for 16 hours.

[0055] On the other hand, Calu-1 cells cultured in 96-well plates were divided into a sample group, a control group, and a blank group. Each group had 8 replicates. After the Calu-1 cells (target cells) adhered, the two groups of NK-92MI cells (effector cells) treated above were collected and added accordingly at an effective target ratio of 10:1 to culture together. After 4 hours of co-culture, the effect of Wogan polysaccharide on the cytotoxicity of NK-92MI cells was evaluated by calculating the viability of Calu-1 cells using the CCK-8 assay.

[0056] The cytotoxicity of NK-92MI cells treated with OP1-OP10 changed. Figure 5 As shown by Figure 5 As can be seen, the cytotoxicity of NK-92MI cells co-cultured with OP7, OP8, OP9, and OP10 was reduced to varying degrees. Although the other six Wogan polysaccharides can enhance the cytotoxicity of NK-92MI cells, only OP5 reached a significant level. Therefore, the structure of OP5 will be identified to analyze its structure-activity relationship.

[0057] (III) Structural identification of OP5

[0058] (1) Identification method:

[0059] 1. Monosaccharide composition detection

[0060] OP5 was hydrolyzed. Mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose were dissolved in distilled water as a mixed standard solution. The monosaccharide composition of OP5 was determined by liquid chromatography-mass spectrometry. The column temperature was 30°C. The flow rate was 1.0 mL / min. The detection wavelength was 250 nm. The injection volume was 20 μL. The mobile phase was a mixture of 0.05 M potassium dihydrogen phosphate and acetonitrile (volume ratio:83:17).

[0061] 2. Infrared detection

[0062] The polysaccharide was ground and pressed, mixed with KBr and analyzed by infrared spectroscopy in the range of 400 cm-1 to 4000 cm-1.

[0063] 3. Nuclear magnetic resonance imaging

[0064] OP5 was detected by nuclear magnetic resonance instrument 13 C NMR (150 MHz) and 1 H NMR (600 MHz) spectrum. Deuterium oxide (D2O) was used as the solvent.

[0065] (2) Results and analysis

[0066] 1. Monosaccharide composition

[0067] The monosaccharide composition of OP5 is as follows Figure 6 ( Figure 6 Comparison of the HPLC chromatogram with that of a standard product revealed that OP5 is composed of 10 monosaccharides. The proportions are 7.67% mannose, 0.27% ribose, 2.39% rhamnose, 0.42% glucuronic acid, 22.35% galacturonic acid, 15.95% glucose, 16.72% galactose, 1.30% xylose, 31.52% arabinose, and 1.41% fucose. The high galacturonic acid content indicates that it is an acidic polysaccharide.

[0068] 2. Infrared analysis

[0069] Infrared analysis of OP5 Figure 7 As shown. Figure 7It can be seen that the strong absorption peak at 3416 cm-1 is caused by the stretching vibration of the OH bond. The absorption peak at 2936 cm-1 is attributed to the stretching vibration of the C-H bond. The two absorption peaks at 1751 cm-1 and 1609 cm-1 should be the symmetric and asymmetric stretching vibration peaks of the carboxyl (-COOH) C=O bond, respectively, which indicates the presence of uronic acid in OP5. The absorption peak at 1443 cm-1 is due to the bending vibration of the C-H bond. The absorption peaks at 1234 cm-1 and 1105 cm-1 may be caused by the stretching vibration of the C-O bond. A strong absorption peak was observed at 1018 cm-1, indicating the presence of stretching vibrations of the pyran ring and the C-O-C bond in OP5. The absorption peak at 633 cm-1 may be caused by the out-of-plane bending vibration of the OH bond.

[0070] 3. Nuclear Magnetic Resonance

[0071] The hydrogen spectrum of OP5 is as follows Figure 8 As shown, the carbon spectrum is as Figure 9 As shown. Figure 8 It can be seen that in the anomeric proton region (δ4.3-5.9), there is an obvious signal at δ4.70 and it is less than 5.0, indicating that the sugar ring is mainly in β configuration. The signal at δ1.26 should be attributed to the proton of the methyl group in rhamnose. Figure 9 As can be seen in the characteristic signal region of uronic acid (δ170-190), a clear signal at δ170.66 indicates that OP5 contains uronic acid. This is consistent with the results of monosaccharide composition detection. In the anomeric carbon region (δ90-112), many signals appear at low field (δ102-112) and several signals appear at high field (δ90-102), indicating that the sugar ring is mainly in β configuration, but α configuration still exists. Two signals at δ82.33 and δ83.88 indicate that there may be a furanose structure in OP5. In addition, four signals (δ81.25, 80.66, 79.01 and 76.53) located in the δ76 to δ85 region and three signals (δ74.42, 74.12 and 71.11) located in the δ70 to δ75 region indicate that the C2, C3 and C4 carbons of pyranose have been partially substituted. Signals at δ69.65, δ69.18, and δ67.53 suggest that some pyranose residues have C6 substitutions. Four signals (δ63.86, 63.34, 61.07, and 60.73) appear between δ60 and δ64, indicating that some pyranose residues have no C6 substitutions. The signal at δ52.86 should be attributed to the carbon of the N-substituent in N-Ac (CH3CON-). The presence of the signal at δ23.85 suggests that it may be caused by the carbon of the methyl group in N-Ac. The signal at δ19.89 may be caused by the carbon of the methyl group in O-Ac (CH3COO-).

[0072] The immune efficacy of polysaccharides stems from their structure. Many factors influence their activity, including molecular weight, monosaccharide composition, solubility, side chains, and glycosidic bonds. Numerous studies have shown that polysaccharides cannot directly attack target cells such as tumor and cancer cells. Instead, they exert their biological functions through systemic immune-enhancing or immunomodulatory effects. Specifically, polysaccharides must first bind to receptors on effector cells before generating a host response. Therefore, the molecular weight of the polysaccharide is particularly important, as only an appropriate molecular weight facilitates receptor binding. In the present invention, OP5, with a molecular weight of 50-70 kDa, exhibited the highest biological activity among all OPs. This is likely because its molecular weight is neither too large nor too small, allowing it to readily bind to receptors. It then stimulates NK-92MI cells to secrete perforin, granzyme B, and IFN-γ, which kill Calu-1 cells. Monosaccharide composition is another factor influencing NK-92MI cell activity. It is well known that the complement receptor type 3 (CR3) of NK-92MI cells is a target receptor for polysaccharide recognition, which can be more easily recognized and bound by polysaccharides containing special monosaccharide components such as glucose, mannose and N-acetyl-d-glucosamine. In OP5, a large amount of glucose (15.95%) and some mannose (7.67%) were detected. This may be one of the reasons supporting its enhancement of NK-92MI cell activity. In addition, the high proportion of arabinose (31.52%) may help to improve the immune activity of OP5. Good water solubility is a necessary basis for polysaccharides to exert their effects, and the uronic acid content will affect the water solubility of polysaccharides. There is a large amount of galacturonic acid (22.35%) with highly hydrophilic carboxyl groups in OP5, which is very beneficial for its rapid dissolution in water and subsequent immune effects.

[0073] Based on the above experiments and analysis, among OP1-OP10 in this example, OP5 showed the best combined effect in promoting NK-92MI cell proliferation, upregulating gene expression, and enhancing cytotoxicity. OP5 has a molecular weight that is neither too large nor too small, ranging between 50 and 70 kDa. It is composed of arabinose (31.52%), galacturonic acid (22.35%), galactose (16.72%), glucose (15.95%), mannose (7.67%), rhamnose (2.39%), fucose (1.41%), xylose (1.30%), glucuronic acid (0.42%), and ribose (0.27%). OP5's moderate molecular weight and high content of arabinose, galacturonic acid, glucose, and mannose may account for its immunogenicity.

[0074] Example 3

[0075] The use of the Wogan polysaccharide of Example 1 in the preparation of drugs for promoting NK-92MI cell proliferation, upregulating gene expression and improving cytotoxicity.

[0076] Example 4

[0077] A drug for promoting NK-92MI cell proliferation, upregulating gene expression and improving cytotoxicity, comprising the polysaccharide of Wogan as described in Example 1.

[0078] Example 5

[0079] Use of the Wogan polysaccharide of Example 1 in the preparation of a drug for inhibiting Calu-1 lung cancer cells.

[0080] Example 6

[0081] A drug for inhibiting Calu-1 lung cancer cells, comprising the polysaccharide from Example 1.

[0082] The above description is a detailed description of the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. Any equivalent changes or modifications completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. A use of Wogan polysaccharide in the preparation of a drug for promoting NK-92MI cell proliferation, upregulating gene expression and increasing cytotoxicity, characterized in that: The Wogan polysaccharide is prepared by the following method: S1: Wash and dry the peel of the mandarin orange, crush it, extract it with distilled water, and collect the supernatant; S2: The supernatant obtained in S1 is concentrated to obtain a viscous liquid, anhydrous ethanol is added to the viscous liquid for alcohol precipitation, and the precipitate collected is the crude polysaccharide of Wogan peel; S3: The crude polysaccharide from the peel of the mandarin orange is dissolved in distilled water to prepare a crude polysaccharide solution from the peel of the mandarin orange. The crude polysaccharide solution from the peel of the mandarin orange is passed through a MinimatePall ultrafiltration system. After passing through ultrafiltration membranes of 1000, 500, 300, 100, 70, 50, 30, 10 and 5 kDa, the 50-70 kDa polysaccharide obtained by ultrafiltration is concentrated, and then precipitated with alcohol. The precipitate is freeze-dried to obtain the said mandarin orange polysaccharide.

2. A drug for promoting NK-92MI cell proliferation, upregulating gene expression and enhancing cytotoxicity, characterized in that: It contains Wogan polysaccharide prepared by the following method: S1: Wash and dry the peel of the mandarin orange, crush it, extract it with distilled water, and collect the supernatant; S2: The supernatant obtained in S1 is concentrated to obtain a viscous liquid, anhydrous ethanol is added to the viscous liquid for alcohol precipitation, and the precipitate collected is the crude polysaccharide of Wogan peel; S3: The crude polysaccharide from the peel of the mandarin orange is dissolved in distilled water to prepare a crude polysaccharide solution from the peel of the mandarin orange. The crude polysaccharide solution from the peel of the mandarin orange is passed through a MinimatePall ultrafiltration system. After passing through ultrafiltration membranes of 1000, 500, 300, 100, 70, 50, 30, 10 and 5 kDa, the 50-70 kDa polysaccharide obtained by ultrafiltration is concentrated, and then precipitated with alcohol. The precipitate is freeze-dried to obtain the said mandarin orange polysaccharide.

3. A use of Wogan polysaccharide in the preparation of a drug for inhibiting Calu-1 lung cancer cells, characterized in that: The Wogan polysaccharide is prepared by the following method: S1: Wash and dry the peel of the mandarin orange, crush it, extract it with distilled water, and collect the supernatant; S2: The supernatant obtained in S1 is concentrated to obtain a viscous liquid, anhydrous ethanol is added to the viscous liquid for alcohol precipitation, and the precipitate collected is the crude polysaccharide of Wogan peel; S3: The crude polysaccharide from the peel of the mandarin orange is dissolved in distilled water to prepare a crude polysaccharide solution from the peel of the mandarin orange. The crude polysaccharide solution from the peel of the mandarin orange is passed through a MinimatePall ultrafiltration system. After passing through ultrafiltration membranes of 1000, 500, 300, 100, 70, 50, 30, 10 and 5 kDa, the 50-70 kDa polysaccharide obtained by ultrafiltration is concentrated, and then precipitated with alcohol. The precipitate is freeze-dried to obtain the said mandarin orange polysaccharide.

4. A drug for inhibiting Calu-1 lung cancer cells, characterized in that: It contains Wogan polysaccharide prepared by the following method: S1: Wash and dry the peel of the mandarin orange, crush it, extract it with distilled water, and collect the supernatant; S2: The supernatant obtained in S1 is concentrated to obtain a viscous liquid, anhydrous ethanol is added to the viscous liquid for alcohol precipitation, and the precipitate collected is the crude polysaccharide of Wogan peel; S3: The crude polysaccharide from the peel of the mandarin orange is dissolved in distilled water to prepare a crude polysaccharide solution from the peel of the mandarin orange. The crude polysaccharide solution from the peel of the mandarin orange is passed through a MinimatePall ultrafiltration system. After passing through ultrafiltration membranes of 1000, 500, 300, 100, 70, 50, 30, 10 and 5 kDa, the 50-70 kDa polysaccharide obtained by ultrafiltration is concentrated, and then precipitated with alcohol. The precipitate is freeze-dried to obtain the said mandarin orange polysaccharide.

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