CN114805621A

CN114805621A - Polysaccharide with anti-tumor activity and application and preparation method thereof

Info

Publication number: CN114805621A
Application number: CN202210274247.7A
Authority: CN
Inventors: 吴鸿; 班俊峰; 周芳; 黄海兰; 袁彩虹
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2020-04-28
Filing date: 2020-04-28
Publication date: 2022-07-29
Anticipated expiration: 2040-04-28
Also published as: CN111363060B; CN114805621B; CN111363060A

Abstract

This application is a divisional application of 2020103543999. The application belongs to the technical field of medicines, and discloses polysaccharide with anti-tumor activity, and an application and a preparation method thereof. The polysaccharide has a structure shown in formula (I);

Description

Polysaccharide with anti-tumor activity and application and preparation method thereof

The application is a divisional application of Chinese application with the application number of 2020103543999, the application date of 2020, 4 months and 28 days, and the invention name of 'polysaccharide with anti-tumor activity and application and preparation method thereof'.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to polysaccharide with anti-tumor activity and an application and a preparation method thereof.

Background

Polysaccharides are a class of compounds consisting of a number of identical or different monosaccharides linked by alpha-or beta-glycosidic bonds. Polysaccharides derived from natural plants belong to a very important class of biological macromolecules in plants, and are also basic substances for effectively maintaining and ensuring normal running of living activities of organisms. The related plant polysaccharide reported at home and abroad has pharmacological effects of immunoregulation, anti-tumor, antivirus, antioxidation, blood pressure reduction, antithrombotic, blood sugar reduction, protection of immunological liver injury, radiation resistance and the like, and provides a good theoretical basis for better development and utilization of the plant polysaccharide in related fields of medicines, foods and the like.

As a natural immunomodulator, the plant polysaccharide has multi-target immunoregulation activity, and plays a positive role in promoting the growth of immune organs of a body, the activation of immune cells and the release of immune factors. To a certain extent, the purity of polysaccharides determines their biological activity, but the main structures of polysaccharides derived from plants are very complex and diverse, and regarding the relationship between the primary structure of polysaccharides and their biological activity, factors such as monosaccharide composition, connection mode, glycosidic bond type, branching degree, substituent species and substitution degree of polysaccharides all have different degrees of influence on the immunological activity of polysaccharides. Therefore, it is a technical problem to those skilled in the art to find polysaccharides which have stable structure-activity, effective part content (not total sugar content) of more than 50% of the total extract, and anti-tumor activity.

Disclosure of Invention

The invention aims to provide polysaccharide with anti-tumor activity and an application and a preparation method thereof. The invention extracts a new polysaccharide from echinacea plant raw materials, the polysaccharide has stable structure effect and anti-tumor activity, and can be used for preparing anti-tumor drugs and the like.

Another object of the present invention is to provide.

The invention is realized by the following steps:

in a first aspect, the present invention provides a polysaccharide having anti-tumor activity, which has a structure represented by the following formula (I):

wherein R is H or methyl.

The invention provides a novel polysaccharide molecule which has a structure shown as the formula (I), wherein R is H or methyl. The structural unit of the polysaccharide is arabinogalactan taking alpha- (1 → 5) -L-arabinogalactan as a skeleton structure. The side chain structure comprises alpha-L-Araf- (1 → → 6) -beta-D-Galp- (1 → and → 4) -alpha-D-GalpA- (wherein 1 → only consists of Galp, Galp A and Araf, and the molar ratio is 2.7: 1: 1), the research of the invention shows that the polysaccharide has antitumor activity, and in addition, the polysaccharide is derived from echinacea, can promote the activation of macrophages and secrete proinflammatory cytokines, not only can improve the phagocytosis capability of the macrophages, but also can promote the macrophages to phagocytose the echinacea polysaccharide, continuously exert the immune stimulation effect, and has wide application; in addition, the conformation and the property of the polysaccharide are relatively stable, which is beneficial to the exertion of biological effect.

Further, in some embodiments of the invention, the polysaccharide has a molecular weight of from 20kD to 50 kD.

Further, in some embodiments of the invention, the polysaccharide has a molecular weight of 38 kD.

In a second aspect, the present invention provides the use of a polysaccharide as described above in the manufacture of a medicament having one or more of the following functions:

immunoregulation, anti-tumor, antivirus, antioxidation, blood pressure lowering, antithrombotic, blood sugar lowering, immune liver injury protecting and radiation resisting.

Further, in some embodiments of the invention, the tumor is selected from liver cancer, breast cancer, colorectal cancer, or lung cancer.

In a third aspect, the present invention provides a medicament having one or more of the following functions: immunoregulation, anti-tumor, antivirus, antioxidation, blood pressure lowering, antithrombotic, blood sugar lowering, immune liver injury protecting and radiation resisting.

The medicament contains the polysaccharide as described above as an active ingredient.

Further, in some embodiments of the invention, the medicament further comprises a pharmaceutically acceptable excipient.

In a fourth aspect, the present invention provides a process for preparing a polysaccharide as described above, comprising: extracting the polysaccharide from echinacea purpurea raw material;

further, in some embodiments of the present invention, extracting the polysaccharide from echinacea purpurea raw material comprises the steps of:

(1): crushing the echinacea raw material to obtain echinacea micropowder;

(2): degreasing the echinacea purpurea micro powder;

(3): polysaccharide extraction is carried out on the degreased echinacea purpurea micro powder to obtain a crude polysaccharide extracting solution;

(4): removing protein in the crude polysaccharide extracting solution to obtain crude polysaccharide;

(5): removing inorganic salts of the crude polysaccharide to obtain a primarily purified crude polysaccharide;

(6): and purifying the primarily purified crude polysaccharide by adopting an ion chromatography column to obtain a pure polysaccharide product.

The term "Crude polysaccharides" refers to the product obtained by extracting a medicinal material or plant with water or precipitating it with ethanol, and is sometimes referred to in the art as "total polysaccharides". As known to those skilled in the art, if the crude drug contains polysaccharide, the precipitate obtained by water extraction and ethanol precipitation of the crude drug must contain polysaccharide, but may contain other non-sugar components. It will be understood by those skilled in the art that total polysaccharides do not refer to the total polysaccharides of the medicinal material or the plant, since it is difficult to obtain the total polysaccharides of the plant regardless of the extraction.

Further, in some embodiments of the present invention, the particle size of the echinacea purpurea micropowder is 0.1-10 μm.

Further, in some embodiments of the present invention, in the echinacea micropowder, the weight ratio of particles having a particle size of 0.1 μm or less is not more than 30%; the weight ratio of particles with the particle diameter of less than or equal to 1 mu m and less than or equal to 5 mu m is more than 65 percent.

Further, in some embodiments of the invention, the echinacea material is comminuted by jet milling.

Further, in some embodiments of the invention, when the echinacea raw material is pulverized by adopting an air jet pulverization method, the air pressure is controlled to be 0.35-0.85 Mpa, and the air flow is controlled to be 10-30 m ³ Min, and the feeding amount is 1.2-3 kg/h.

The particle size of the powder is closely related to the activity and polysaccharide extraction rate. In the embodiment of the invention, the material meeting the particle size requirement is prepared preferably by using a jet milling mode, the compressed air pressure and the speed of injecting into a milling cavity are closely related in the jet milling, the higher the air pressure is, the material is repeatedly collided, rubbed and sheared at the intersection point of air flow, and the crushed particle size distribution is more concentrated. In the actual operation process, control reasonable air pressure make the material can spray into crushing chamber with stable speed can, should avoid the too high pressure to lead to material and high-pressure air current intersection time too short. The method of comminution is therefore preferably employedPulverizing with air flow, controlling air pressure at 0.35-0.85 Mpa and air flow at 10-30 m ³ Min, and the feeding amount is 1.2-3 kg/h. By controlling such parameters, the activity of the polysaccharide can be maintained and the extraction rate can be improved.

Further, in some embodiments of the invention, the echinacea material is extracted from a combination of one or more of the leaves, roots, flowers and stems of an echinacea plant.

The medicinal part has high polysaccharide content, and the medicinal part is used as extraction raw material to improve polysaccharide yield.

Further, in some embodiments of the invention, the echinacea material is extracted from the echinacea plant at full bloom.

Further, in some embodiments of the invention, the echinacea plant is a 1-perennial plant;

further, in some embodiments of the invention, the echinacea plant is selected from the group consisting of echinacea purpurea (e.purpurea), echinacea angustifolia (e.angustifolia), and echinacea pallida (e.pallida).

Further, in some embodiments of the present invention, the degreasing treatment comprises step (a) or step (B):

step (A): soaking the echinacea purpurea micro powder in ethanol to degrease;

step (B): mixing the echinacea purpurea micro powder with petroleum ether, and carrying out hot reflux treatment to degrease;

further, in some embodiments of the invention, in the step (A), the mass-to-volume ratio of the echinacea purpurea micropowder to the ethanol is 1 (10-50), and more preferably 1: 30; the concentration of the ethanol is 55-95%, and the preferable concentration is 95%;

further, in some embodiments of the present invention, in the step (a), the time for soaking is 3 to 8 hours, and more preferably 3 hours;

further, in some embodiments of the present invention, in the step (B), the mass-to-volume ratio of the echinacea purpurea micropowder to the petroleum ether is 1 (30-50), and preferably 1: 30; the boiling range of the petroleum ether is 30-120 ℃, and the further optimization is 60-90 ℃;

further, in some embodiments of the present invention, in the step (B), the time of the thermal reflux treatment is 1 to 3 hours, and more preferably 2 hours.

Further, in some embodiments of the invention, the polysaccharide extraction comprises: mixing the echinacea purpurea micro powder with water for hot reflux extraction, then carrying out solid-liquid separation, taking a liquid component and concentrating to obtain the crude polysaccharide extracting solution;

further, in some embodiments of the invention, the time of the hot reflux extraction is 2 h;

further, in some embodiments of the present invention, the solid-liquid separation is performed by centrifugation;

further, in some embodiments of the present invention, the rotation speed of the centrifugation is 500 to 1000rpm, and the time of the centrifugation is 10 to 20 min.

Further, in some embodiments of the present invention, the protein in the crude polysaccharide extract is removed by water extraction and alcohol precipitation or sevage reagent method;

further, in some embodiments of the invention, removing the inorganic salts of the crude polysaccharide comprises: mixing the crude polysaccharide with an organic solvent for dialysis to remove inorganic salts;

further, in some embodiments of the present invention, the organic solvent is selected from one or more of ethanol, ethyl acetate, chloroform or n-butanol;

further, in some embodiments of the present invention, the organic solvent is ethanol, and the concentration of ethanol is 30% to 80%, and is further preferably 60%;

further, in some embodiments of the invention, the dialysis is performed with a molecular cut-off of 1.5-9 kD.

Further, in some embodiments of the invention, the ion chromatography column is selected from one or more of a macroporous cation exchange resin, an anion exchange resin, or a combination of DEAE-Sepharose Fast Flow columns; (ii) a

Further, in the present inventionIn some embodiments, the flow rate of the primary purified crude polysaccharide is controlled to be 0.8 to 1.2 mL-min when the ion chromatography column is used for purifying the primary purified crude polysaccharide ^-1 And eluting with an elution medium having a continuous pH gradient;

further, in some embodiments of the invention, the elution medium is one or more of a NaCl solution or a PBS buffer;

further, in some embodiments of the invention, the concentration of the NaCl solution is 0.05-5M;

further, in some embodiments of the present invention, after elution, the same fractions are combined according to the results of sugar color reaction and uv detection to obtain the pure polysaccharide.

The polysaccharide extracted by the preparation method provided by the invention has high purity and uniform components, maintains the antitumor bioactivity, and can be used as a medicinal raw material to prepare antitumor medicines and the like.

In another aspect, the present invention also provides an extract having anti-tumor activity, which is extracted by the method as described in any one of the above.

Further, in some embodiments of the invention, the extract contains a component of formula a and/or formula B as follows:

r in formula A, formula (I) is H, R in formula B, formula (I) is methyl (CH) ₃ )。

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a structural formula of EPDS-2.

FIG. 2 is a high performance liquid gel permeation chromatogram of EPDS-2.

FIG. 3 is EPDS-2 ¹ H-NMR spectrum.

FIG. 4 is EPDS-2 ¹³ C-NMR spectrum.

FIG. 5 is a tumor profile 12 days after gastric administration of EPDS-2.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The method for preparing the polysaccharide with the antitumor activity provided by the embodiment is as follows:

(1) root of 1-perennial Echinacea angustifolia (E. angustifolia) picked in the flowering stage was taken as a raw material for extracting polysaccharide, and it was air-dried at a low temperature of 40 ℃ for 8 hours.

(2) Crushing: micronizing the raw materials by adopting jet milling, controlling the air pressure to be 0.55-0.65 Mpa and the air flow to be 15-20 m ³ And (4) feeding the materials at a rate of 1.5-1.8 kg/h per min to obtain the micro powder. The particle size distribution of the micropowder is as follows: the weight ratio of the particles with the particle diameter less than or equal to 0.1 mu m is not more than 30 percent; the weight ratio of particles with the particle diameter of less than or equal to 1 mu m and less than or equal to 5 mu m is more than 65 percent.

(3) Degreasing: mixing the micro powder with petroleum ether according to the mass-to-volume ratio of 1:30, heating and refluxing for 1-3 h, and volatilizing the micro powder until no organic solvent exists after the heating and refluxing are finished.

(4) Extraction: mixing the degreased micro powder with 15-23 times of water, heating, refluxing and extracting for 1-3 times, wherein each time lasts for 1-3 hours; after extraction, centrifugation is performed: 1000-3000 rpm, 5-10 min; collecting supernatant, and concentrating under reduced pressure to about 1/3 to obtain crude polysaccharide extract.

(5) Protein removal: removing protein by adopting a sevage reagent method;

mixing the crude polysaccharide extracting solution and a sevage reagent according to the volume ratio of 1: 1 to remove protein, adding ethanol to adjust the alcohol concentration to 60%, removing supernatant, and collecting precipitate to obtain crude polysaccharide.

(6) Removing inorganic salts: mixing the crude polysaccharide with 60% ethanol, dialyzing in a dialysis bag with molecular cut-off of 3500D to remove inorganic salts, collecting precipitate, adding appropriate amount of deionized water, ultrasonic re-dissolving, and freeze drying to obtain primarily purified crude polysaccharide.

(7) And (3) chromatographic purification: dissolving the crude polysaccharide with water to obtain water solution, filtering with 0.45 μm filter membrane, and purifying with DEAE-Sepharose Fast Flow column

The flow rate is 1 mL/min ^-1 And (3) merging the same fractions according to the absorbance and activity results by using 0.1-0.5M NaCl elution medium to obtain the EPDS-2.

Example 2

(1) aerial parts (flowers, leaves and stems) of Dendranthema indicum (E.pallida) were collected as a raw material for extracting polysaccharides, and dried in the shade at room temperature.

(2) Crushing: the raw materials are crushed by a universal crusher until all the raw materials can pass through a 65-mesh sieve, but the powder which can pass through a 80-mesh sieve is not less than 95 percent of the total amount.

(4) Extraction: mixing the degreased micro powder with 15-23 times of water, heating and refluxing for extraction for 1-3 times, wherein each time lasts for 1-3 hours; after extraction, centrifugation is performed: 1000-3000 rpm, 5-10 min; collecting supernatant, and concentrating under reduced pressure to about 1/5 to obtain crude polysaccharide extract.

(5) Protein removal: removing protein by water extraction and alcohol precipitation;

mixing the crude polysaccharide extract with absolute ethyl alcohol according to a volume ratio of 1: 1 to remove protein, adding ethanol to adjust the alcohol concentration to 30 percent, removing supernatant fluid, and taking precipitate to obtain crude polysaccharide.

(6) Removing inorganic salts: mixing the crude polysaccharide with 60% ethanol, dialyzing in a dialysis bag with molecular cut-off of 8000D to remove inorganic salts, collecting precipitate, adding appropriate amount of deionized water, ultrasonic re-dissolving, and freeze drying to obtain primarily purified crude polysaccharide.

(7) And (3) chromatographic purification: dissolving the crude polysaccharide with water to obtain water solution with certain concentration, filtering with 0.45 μm filter membrane, and purifying with DEAE-Sepharose Fast Flow column

The flow rate is 1 mL/min ^-1 And (3) taking 0.1-0.5M NaCl as an elution medium, and merging the same fractions according to the absorbance and activity results to obtain the pure polysaccharide product of the EPDS-2.

Example 3

(1) Taking 1-years Echinacea purpurea root extracted from 9 months as raw material for extracting polysaccharide, and drying in shade at room temperature.

(2) Crushing: the raw materials are crushed by a universal crusher to be completely capable of passing through a 65-mesh sieve, but the powder capable of passing through a 80-mesh sieve is not less than 95% of the total amount.

Effect example 1

The purity and molecular weight of the pure polysaccharide prepared in example 1 were measured by high performance liquid gel permeation chromatography:

chromatographic conditions are as follows: KH with 0.02mol/L mobile phase ₂ PO ₄ The buffer solution, gel column TSK G-5000PWXL column (7.8 × 300mm) and TSK G-3000PWXL column (7.8 × 300mm) were used in series, the flow rate was 0.6mL/min, differential detector, column temperature was 35 ℃. Dissolving Echinacea purpurea polysaccharide with appropriate amount of mobile phase, and analyzing by sample injection, wherein the polysaccharide sample has a peak-out time of 20-25min and relatively similar relative molecular weight. EPDS-2 is a single symmetrical peak (see FIG. 2).

To be provided with ¹ H-NMR and ¹³ further analysis by C-NMR showed (see FIGS. 3 and 4), EPDS-2 is a homogeneous polysaccharide having a molecular weight of about 37.9kD and consisting essentially of arabinose and galactose in a ratio of 3 (1-4).

EPDS-2 is identified by high performance gel permeation chromatography and proved to be a single component (the structural formula is shown in figure 1, R is H or methyl).

Effect experiment example 2

Verification of antitumor Activity of EPDS-2

The liver cancer H22 cell strain is selected to model Kunming mice, and the animal modeling method is as follows:

culturing H22 tumor in vitro, and adjusting cell concentration to 1.5 × 10 with sterile physiological saline ⁷ Preparing cell suspension, injecting the cell suspension into abdominal cavity of mouse by syringe, observing that abdominal bulge of first generation mouse is obvious (about 7-8 days after inoculation), passaging, sterilizing abdominal cavity of mouse, directly extracting ascites cell with needle, adjusting to 1.5 × 10 ⁷ After one/mL, passage was repeated. Passage of tumor strain in mouse ascites for 3 times, extracting ascites from mouse with tumor to obtain the final product with concentration of 1.5 × 10 ⁷ 0.2mL of the cell suspension was inoculated subcutaneously into the right upper axilla of mice after 1 week of adaptive feeding. After 24h of inoculation, subcutaneous tumor-bearing mouse models are randomly grouped according to experimental requirements, and 12 mice in each group.

The dosing regimen was as follows:

control group (Model): the normal saline with the same dosage is used for gastric perfusion administration;

polysaccharide group (Experimental): 20mg/kg of the aqueous polysaccharide solution prepared in example 1 was administered by gavage;

positive Control group (Control): 40mg/kg cyclophosphamide, intraperitoneal injection;

the administration was continued for 12d following the above dose and route, fasted overnight after the last administration, and the tumor mass was completely detached and weighed, as calculated below.

Tumor inhibition (%) (average tumor weight in model group-average tumor weight in experimental group)/average tumor weight in model group × 100.

The results are shown in FIG. 5 and Table 1 below.

TABLE 1 Effect of intragastric administration of polysaccharides on tumor growth in mice

As can be seen from the results of table 1 and fig. 5, the tumor weight and volume of the polysaccharide group and the positive control group are both significantly reduced compared to the control group, and the difference is statistically significant. The polysaccharide EPDS-2 (shown in figure 1) provided by the embodiment of the invention has antitumor activity, and can be used for preparing antitumor drugs and drugs for immunoregulation, antivirus, antioxidation, blood pressure reduction, antithrombotic, blood sugar reduction, immune liver injury protection and radiation resistance.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A polysaccharide having the structure shown in formula (I):

wherein R is H or methyl.

2. The polysaccharide of claim 1, wherein:

the molecular weight of the polysaccharide is 20 kD-50 kD;

preferably, the polysaccharide has a molecular weight of 38 kD.

3. Use of a polysaccharide according to claim 1 or 2 for the preparation of a medicament,

preferably, the medicament has one or more of the following functions:

immunoregulation, anti-tumor, anti-virus, anti-oxidation, blood pressure lowering, antithrombotic, blood sugar lowering, immune liver injury protecting and anti-radiation;

preferably, the tumor is selected from liver cancer, breast cancer, colorectal cancer or lung cancer.

4. A medicament, characterized by: the medicament contains the polysaccharide according to claim 1 or 2 as an active ingredient; preferably, the medicament has one or more of the following functions: immunomodulating, anti-tumor, antiviral, antioxidant, blood pressure lowering, antithrombotic, blood glucose lowering, immune liver injury protecting, and radioprotective.

5. The method for producing a polysaccharide according to claim 1 or 2, wherein the polysaccharide is obtained by extraction from an echinacea purpurea raw material.

6. The method of claim 5, wherein:

the polysaccharide extracted from the echinacea raw material comprises the following steps:

(1): crushing the echinacea raw material to obtain echinacea micropowder;

(2): carrying out degreasing treatment on the echinacea purpurea micro powder;

7. The method of claim 6, wherein: the particle size of the echinacea purpurea micro powder in the step (1) is 0.1-10 mu m;

preferably, in the echinacea purpurea micro powder, the weight ratio of particles with the particle diameter less than or equal to 0.1 mu m is not more than 30 percent; the weight ratio of particles with the particle size of less than or equal to 5 mu m and less than or equal to 1 mu m is more than 65 percent;

preferably, the echinacea raw material is crushed by adopting a jet milling mode;

preferably, when the echinacea raw material is crushed by adopting an air flow crushing mode, the air pressure is controlled to be 0.35-0.85 Mpa, and the air flow is controlled to be 10-30 m ³ Min, the feeding amount is 1.2-3 kg/h;

preferably, the echinacea raw material is extracted from a combination of one or more of leaves, roots, flowers and stems of an echinacea plant;

preferably, said echinacea material is harvested from said echinacea plant at full bloom;

preferably, the echinacea plant is a 1-perennial plant;

preferably, the echinacea plant is selected from echinacea, echinacea angustifolia and echinacea pallida.

8. The method of claim 7, wherein:

the degreasing treatment in the step (2) comprises a step (A) or a step (B):

step (A): soaking the echinacea purpurea micro powder in ethanol to degrease;

preferably, in the step (A), the mass-to-volume ratio of the echinacea purpurea micropowder to the ethanol is 1 (10-50), and more preferably 1: 30; the concentration of the ethanol is 55-95%, and the preferable concentration is 95%;

preferably, in the step (A), the soaking time is 3-8 h, and further preferably 3 h;

preferably, in the step (B), the mass-to-volume ratio of the echinacea purpurea micropowder to the petroleum ether is 1 (30-50), and more preferably 1: 30; the boiling range of the petroleum ether is 30-120 ℃, and the more preferable boiling range is 60-90 ℃;

preferably, in the step (B), the time of the thermal reflux treatment is 1 to 3 hours, and more preferably 2 hours.

9. The method for producing according to claim 8, characterized in that:

the polysaccharide extraction in the step (3) comprises the following steps: mixing the echinacea purpurea micro powder with water for hot reflux extraction, then carrying out solid-liquid separation, taking a liquid component and concentrating to obtain the crude polysaccharide extracting solution;

preferably, the time of hot reflux extraction is 2-3 h;

preferably, solid-liquid separation is carried out in a centrifugal mode;

preferably, the rotation speed of the centrifugation is 500-1000 rpm, and the time of the centrifugation is 10-20 min.

10. The method for producing according to claim 9, characterized in that:

removing protein in the crude polysaccharide extracting solution by adopting a water extraction and alcohol precipitation method or a Sevage reagent method;

preferably, the removing of the inorganic salts of the crude polysaccharide comprises: mixing the crude polysaccharide with an organic solvent for dialysis to remove inorganic salts;

preferably, the organic solvent is selected from one or more of ethanol, ethyl acetate, chloroform or n-butanol;

preferably, the organic solvent is ethanol, and the concentration of the ethanol is 30-80%, and more preferably 60%;

preferably, the molecular interception amount during dialysis is 1.5-9 KD;

preferably, the ion chromatography column is selected from one or more of a macroporous cation exchange resin, an anion exchange resin or a combination of DEAE-Sepharose Fast Flow columns;

preferably, when the ion chromatographic column is used for purifying the primarily purified crude polysaccharide, the flow rate is controlled to be 0.8-1.2 mL-min ^-1 And eluting with an elution medium having a continuous pH gradient;

preferably, the elution medium is one or more of a NaCl solution or a PBS buffer;

preferably, the concentration of the NaCl solution is 0.05-5M;

preferably, after elution, the same fractions are combined according to the results of sugar color reaction and ultraviolet detection to obtain the pure polysaccharide product.