A method for separating and purifying a glycuronan

Abstract

This invention provides a method for separating and purifying uronic acid polysaccharides, belonging to the field of polysaccharide separation and purification technology. This invention utilizes the electrostatic adsorption of sodium ions in an aqueous sodium chloride solution around the carboxyl anions of uronic acid units, forming a charge shielding layer. Chloride ions in the aqueous sodium chloride solution are also adsorbed around the dextran gel via electrostatic interaction, effectively shielding the electrostatic attraction between the uronic acid units and the dextran gel, reducing the non-specific adsorption of uronic acid units and the dextran gel, making the uronic acid polysaccharides easy to separate and with high purity. Furthermore, sodium and chloride ions in the aqueous sodium chloride solution are strongly hydrated ions, preferentially binding to the hydroxyl groups on the dextran gel and the carboxyl groups on the uronic acid units, occupying hydrogen bond sites, disrupting the existing hydrogen bonds between the uronic acid units and the dextran gel, preventing the formation of new hydrogen bonds, reducing the adsorption of uronic acid units in the dextran gel, making the uronic acid polysaccharides easy to separate and with high purity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of polysaccharide separation and purification, and particularly relates to a separation and purification method of uronic acid-containing polysaccharide. BACKGROUND

[0002] Polysaccharide is a high-molecular carbohydrate formed by 10 or more monosaccharide molecules through glycosidic bond dehydration condensation, and the monosaccharide composition includes glucose, galactose, arabinose, mannose, rhamnose and the like. Among them, the uronic acid-containing polysaccharide is a special polysaccharide rich in uronic acid (such as glucuronic acid and galacturonic acid), has good water solubility and biocompatibility, and is easy to interact with proteins, enzymes, cytokines and the like in the organism; in terms of pharmacological activity, the uronic acid-containing polysaccharide has been proved to have multiple biological functions such as immunomodulation, antioxidant, hypoglycemic, hypolipidemic, anticoagulation and antitumor, and has extremely high application value in the fields of food, health care products, medicine and the like.

[0003] At present, the separation of polysaccharide mainly includes polysaccharide extraction from biological materials such as plants, microorganisms or insects, alcohol precipitation, protein removal, decolorization, preliminary separation and purification of polysaccharide by anion exchange resin to obtain polysaccharide crude material, and then final separation and purification by dextran gel column chromatography. In the dextran gel column chromatography, pure water is generally used as an eluent to separate and purify polysaccharide, and small molecule impurities such as monosaccharide, disaccharide and oligosaccharide in the polysaccharide crude material are removed. However, the carboxyl groups contained in the uronic acid-containing polysaccharide are easy to be non-specifically adsorbed on the dextran gel, and hydrogen bonds are formed with the hydroxyl groups in the dextran gel, so that the uronic acid-containing polysaccharide cannot be separated from the dextran gel, which leads to the omission of the step of dextran gel column chromatography during the extraction and purification of the uronic acid-containing polysaccharide, and further purification of the uronic acid-containing polysaccharide cannot be realized. SUMMARY

[0004] The present application aims to provide a separation and purification method of uronic acid-containing polysaccharide, which has good separation effect and high purity of the uronic acid-containing polysaccharide.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions: The present application provides a separation and purification method of uronic acid-containing polysaccharide, comprising: subjecting the uronic acid-containing polysaccharide crude material to dextran gel column chromatography to obtain the uronic acid-containing polysaccharide; The conditions of the dextran gel column chromatography include: eluent: sodium chloride aqueous solution; dextran gel column: Sephadex G-100 dextran gel column; elution flow rate: 3-5 s / drop; Sample loading amount: The ratio of the mass of the uronic acid polysaccharide crude material to the volume of the dextran gel column is 1 mg: 990~1100 mL.

[0006] Preferably, the mass of uronic acid in the crude uronic acid polysaccharide is 30-50% of the uronic acid polysaccharide.

[0007] Preferably, the glycouronic acid polysaccharide includes beak-tailed rostellum polysaccharide.

[0008] Preferably, the concentration of sodium chloride in the sodium chloride aqueous solution is 0.05~0.15 mol / L.

[0009] Preferably, the crude uronic acid polysaccharide is obtained by sequentially extracting, deproteinizing, decolorizing, and crudely separating and purifying biological materials containing uronic acid polysaccharides.

[0010] Preferably, the extraction of uronic acid polysaccharides includes: mixing the biomaterial containing uronic acid polysaccharides with water and then extracting it, followed by alcohol precipitation of the resulting extract to obtain the uronic acid polysaccharide raw material.

[0011] Preferably, the protein removal includes: mixing the uronic acid polysaccharide raw material obtained from the extraction of the uronic acid polysaccharide with Sevag reagent, performing protein denaturation, and then centrifuging to remove the precipitate.

[0012] Preferably, the decolorization includes: decolorizing and adsorbing the product after protein removal using a macroporous resin to obtain decolorized glycouronic acid polysaccharide.

[0013] Preferably, the crude separation and purification includes: subjecting the decolorized uronic acid polysaccharide obtained by decolorization to anion exchange column chromatography to obtain crude uronic acid polysaccharide.

[0014] Preferably, the conditions for the anion exchange column chromatography include: Eluent: pure water and sodium chloride aqueous solution; Anion exchange column: DEAE-agarose gel FF pre-packed column; Elution flow rate: 1~3 mL / min; Sample loading amount: The ratio of the mass of the decolorized uronic acid polysaccharide to the volume of the DEAE-agarose gel FF pre-packed column is 1 mg: 75~90 mL.

[0015] This invention provides a method for separating and purifying uronic acid polysaccharides, comprising: subjecting crude uronic acid polysaccharide material to dextran gel column chromatography to obtain uronic acid polysaccharides; the conditions for the dextran gel column chromatography include: eluent: sodium chloride aqueous solution; dextran gel column: Sephadex G-100 dextran gel column; elution flow rate: 3~5s / drop; sample loading: the ratio of the mass of the crude uronic acid polysaccharide material to the volume of the dextran gel column is 1mg:990~1100mL. This invention utilizes sodium ions in an aqueous sodium chloride solution to adsorb around the carboxyl anions of uronic acid units via electrostatic interaction, forming a charge shielding layer. Simultaneously, chloride ions in the aqueous sodium chloride solution adsorb around the dextran gel via electrostatic interaction, effectively shielding the electrostatic attraction between the uronic acid units and the dextran gel, reducing the non-specific adsorption of uronic acid units in the dextran gel. This results in easy separation of uronic acid-containing polysaccharides with high purity. Furthermore, sodium and chloride ions in the aqueous sodium chloride solution are strongly hydrated ions, preferentially binding to the hydroxyl groups on the dextran gel and the carboxyl groups on the uronic acid units, occupying hydrogen bond sites. This disrupts existing hydrogen bonds between the uronic acid units and the dextran gel and prevents the formation of new hydrogen bonds, thereby reducing the adsorption of uronic acid units in the dextran gel. This further facilitates the separation of uronic acid-containing polysaccharides with high purity. The results of the examples show that the separation and purification method provided by the present invention can separate and purify the crude polysaccharide of *Lycopus lucidus*, and the total content of neutral monosaccharide units in the obtained polysaccharide is 43.35%, and the content of uronic acid units is 45.65%. Attached Figure Description

[0016] Figure 1 This is the elution curve of anion exchange column chromatography in Example 1 of the present invention; Figure 2 This is the elution curve of dextran gel column chromatography in Example 1 of the present invention; Figure 3 This is an external image of the uronic acid polysaccharide contained in *Begonia beakata* according to Embodiment 1 of the present invention; Figure 4 This is a signal intensity-time graph of uronic acid polysaccharide from the beak-tailed lutea in Example 1 of the present invention; Figure 5 This is a molecular weight-time graph of the uronic acid polysaccharide contained in *Begonia beakata* in Example 1 of the present invention; Figure 6 This is a molecular conformation diagram of the uronic acid polysaccharide from *Begonia beakina* in Example 1 of the present invention; Figure 7 For glucose standard curve; Figure 8 This is a standard curve of uronic acid. Figure 9 This is a standard curve of bovine serum albumin. Figure 10 This is a chromatogram of the monosaccharide composition analysis of a mixed standard of glycouronic acid polysaccharide and monosaccharide from *Begonia beakina* in Example 1 of the present invention; Figure 11 This is the ultraviolet spectrum of the uronic acid polysaccharide of *Ligustrum beakum* in Example 1 of the present invention; Figure 12 The infrared spectrum of the uronic acid polysaccharide of the beak-tailed lutea in Example 1 of the present invention; Figure 13 This is a UV-Vis spectral shift diagram of the complexation of uronic acid polysaccharide with Congo red and Congo red in Example 1 of the present invention. Figure 14 This is a SEM image of the uronic acid polysaccharide of the beak-tailed lutea in Example 1 of the present invention; Figure 15 This is the elution curve of dextran gel column chromatography in Comparative Example 1 of this invention. Detailed Implementation

[0017] This invention provides a method for separating and purifying polysaccharides containing uronic acid, comprising: The crude polysaccharide containing uronic acid was subjected to dextran gel column chromatography to obtain the polysaccharide containing uronic acid. The conditions for the dextran gel column chromatography include: Eluent: Sodium chloride aqueous solution; Dextran gel columns: Sephadex G-100 dextran gel columns; Elution flow rate: 3~5s / drop; Sample loading amount: The ratio of the mass of the uronic acid polysaccharide crude material to the volume of the dextran gel column is 1 mg: 990~1100 mL.

[0018] In one embodiment of the present invention, the mass of uronic acid units in the crude uronic acid polysaccharide is 30-50% of the total uronic acid polysaccharide. The separation and purification method provided by the present invention has good extraction and separation effect and high purity for uronic acid polysaccharides in biomaterials with high uronic acid unit content.

[0019] In one embodiment of the present invention, the crude uronic acid polysaccharide can be obtained from biological materials containing uronic acid polysaccharides through sequential extraction of uronic acid polysaccharides, removal of proteins, decolorization, and crude separation and purification.

[0020] In one embodiment of the present invention, the biomaterial containing uronic acid polysaccharides can be a plant containing uronic acid polysaccharides, a microorganism containing uronic acid polysaccharides, or an insect containing uronic acid polysaccharides. In an embodiment of the present invention, the insect containing uronic acid polysaccharides can be a beetle. The method provided by the present invention has good extraction and separation effects on uronic acid polysaccharides from biomaterials with high uronic acid unit content.

[0021] As one embodiment of the present invention, the extraction of uronic acid polysaccharides may include: Biological materials containing uronic acid polysaccharides are mixed with water for extraction, and the resulting extract is then subjected to alcohol precipitation to obtain the uronic acid polysaccharide raw material. In this invention, water is used for the extraction of uronic acid polysaccharides. This is because polysaccharides containing uronic acid units typically have carboxyl groups, exhibiting strong polarity and good water solubility. Water, as a strongly polar solvent, can fully dissolve and extract these acidic polysaccharides, ensuring a high extraction rate.

[0022] In one embodiment of the present invention, the biomaterial containing uronic acid polysaccharides can be pretreated before use. The present invention does not have any particular limitation on the pretreatment of the biomaterial containing uronic acid polysaccharides; any pretreatment method well-known in the art can be used to degrease and depigment the biomaterial containing uronic acid polysaccharides. In one embodiment of the present invention, the pretreatment of the biomaterial containing uronic acid polysaccharides can be as follows: the biomaterial containing uronic acid polysaccharides is sequentially subjected to anhydrous ethanol impregnation, drying, pulverization, sieving, petroleum ether defatting, and ethanol defatting and depigmentation.

[0023] In one embodiment of the present invention, the mass ratio of the uronic acid polysaccharide-containing biomaterial to the volume ratio of water can be 10-20 g / mL. In another embodiment of the present invention, the mixing of the uronic acid polysaccharide-containing biomaterial and water is carried out under stirring. The present invention does not impose any particular limitation on the stirring; any stirring method well-known in the art can be used to mix the uronic acid polysaccharide-containing biomaterial and water evenly.

[0024] In one embodiment of the present invention, the extraction can be performed three times, the temperature of each extraction can be independently set to 55-60°C, and the extraction time of each extraction can be independently set to 2-2.5 hours. In another embodiment of the present invention, after each extraction, solid-liquid separation is performed to obtain a filtrate. After the final extraction, the filtrates obtained from each extraction are combined to obtain an extract. The present invention does not impose any particular limitation on the solid-liquid separation; any solid-liquid separation method well-known in the art can be used to obtain the filtrate.

[0025] In one embodiment of the present invention, the extract can be concentrated and then precipitated with alcohol.

[0026] In one embodiment of the present invention, the concentration factor can be 3 to 5 times.

[0027] In one embodiment of the present invention, the alcohol precipitation is performed using an aqueous ethanol solution, wherein the volume concentration of ethanol in the aqueous ethanol solution can be 90-95%; the temperature of the alcohol precipitation can be 4-10°C; and the alcohol precipitation time can be 12-24 hours.

[0028] As one embodiment of the present invention, after the alcohol precipitation is completed, the obtained product can be subjected to solid-liquid separation, and then the obtained solid is washed, dialyzed with pure water, concentrated and freeze-dried in sequence to obtain uronic acid polysaccharide raw material.

[0029] In one embodiment of the present invention, the solid-liquid separation can be centrifugation, the centrifugation speed can be 3000~6000 r / min, and the centrifugation time can be 5~10 min. In another embodiment of the present invention, the washing can be sequential washing with petroleum ether and anhydrous ethanol, and the number of washings can be 3~5 times independently. In another embodiment of the present invention, the pure water dialysis time can be 24~48 h, and the pure water dialysis solution is changed every 8 h. In another embodiment of the present invention, the molecular weight cutoff for pure water dialysis can be 1000~3500 Da. The present invention does not have special limitations on the concentration and freeze-drying operations; the aqueous solution obtained from alcohol-water dialysis can be concentrated to the desired volume and the water removed using methods well known in the art.

[0030] After the extraction of uronic acid polysaccharides is completed, the present invention removes proteins from the uronic acid polysaccharide raw material obtained by the extraction of uronic acid polysaccharides.

[0031] In one embodiment of the present invention, the uronic acid polysaccharide raw material can be thawed and then subjected to solid-liquid separation before use. The present invention does not impose any particular limitation on the thawing and solid-liquid separation; any thawing and solid-liquid separation method well-known in the art can be used to remove insoluble proteins from the uronic acid polysaccharide raw material.

[0032] In one embodiment of the present invention, the protein removal may include: mixing the uronic acid polysaccharide raw material extracted from the uronic acid polysaccharide with Sevag reagent to denature the protein, followed by centrifugation to remove the precipitate. In the present invention, the use of Sevag reagent for protein denaturation can disrupt the hydrogen bonds, hydrophobic interactions, and spatial conformation of the protein, causing irreversible denaturation and changing the protein from a soluble state to an insoluble state.

[0033] In one embodiment of the present invention, the volume ratio of the uronic acid polysaccharide raw material after extraction to Sevag reagent can be 1:4-5. In another embodiment, the mixing of the uronic acid polysaccharide raw material and Sevag reagent can be carried out under stirring. The present invention does not specifically limit the stirring; any stirring method well-known in the art can be used to mix the uronic acid polysaccharide raw material and Sevag reagent evenly. In one embodiment, the number of protein denaturations can be 8-10, and the time for each protein denaturation can be independently 10-15 min. In another embodiment, after each protein denaturation, centrifugation is performed to obtain a supernatant; after the final protein denaturation, the supernatants obtained from centrifugation after each protein denaturation are combined. The present invention does not specifically limit the centrifugation; any centrifugation method well-known in the art can be used to obtain the supernatant.

[0034] After protein removal, the present invention decolorizes the product obtained by protein removal to obtain decolorized uronic acid polysaccharides. In one embodiment of the present invention, the decolorization may include: decolorizing the protein-removed product using a macroporous resin. In this invention, the use of a macroporous resin for decolorization utilizes the porous structure and hydrophobic properties of the macroporous resin to selectively adsorb pigments, polyphenols, and lipid-soluble impurities in the uronic acid polysaccharides, causing the uronic acid polysaccharide solution to lighten in color and become clear and transparent. In one embodiment of the present invention, the macroporous resin may be macroporous resin D101. In one embodiment of the present invention, the eluent for decolorization may be pure water, the number of decolorization cycles may be 3-5, and the time for each decolorization cycle may be independently 8-12 hours. In one embodiment of the present invention, the effluent collected after each decolorization cycle is combined. In one embodiment of the present invention, the combined effluent is then sequentially concentrated and freeze-dried. The present invention does not have specific limitations on the concentration and freeze-drying; concentration and freeze-drying methods well-known in the art can be used to remove the eluent.

[0035] After decolorization, the present invention performs crude separation and purification on the decolorized uronic acid polysaccharide obtained by decolorization to obtain crude uronic acid polysaccharide.

[0036] In one embodiment of the present invention, the crude separation and purification may include: subjecting the decolorized uronic acid polysaccharide to anion exchange column chromatography. In this invention, the crude separation and purification of the decolorized uronic acid polysaccharide using anion exchange column chromatography utilizes the carboxyl groups and other anionic groups contained in the uronic acid polysaccharide to bind with the anion exchange resin. Different polysaccharides have different charge densities and structures, resulting in varying binding strengths. Eluting allows for the effective separation of different polysaccharide components. Furthermore, it efficiently removes neutral impurities; for example, neutral sugars, pigments, some proteins, and small molecule impurities in the uronic acid polysaccharide raw material do not bind with the resin and can be removed before elution, significantly improving the polysaccharide purity.

[0037] In one embodiment of the present invention, the decolorized uronic acid polysaccharide can be sequentially dissolved in pure water, centrifuged to obtain a supernatant, and filtered through a filter membrane before use. The present invention does not have any particular limitations on the pure water dissolution, centrifugation, and filtration processes; any well-known dissolution method can be used to dissolve the decolorized uronic acid polysaccharide raw material, a well-known centrifugation method can be used to obtain a supernatant, and a well-known filter membrane method can be used to further remove impurities from the supernatant.

[0038] As one embodiment of the present invention, the conditions for the anion exchange column chromatography may include: Eluent: pure water and sodium chloride aqueous solution; Anion exchange column: DEAE-agarose gel FF pre-packed column; Elution flow rate: 1~3 mL / min; Sample loading amount: The ratio of the mass of the uronic acid polysaccharide raw material to the volume of the DEAE-agarose gel FF pre-packed column is 1 mg: 75~90 mL.

[0039] In one embodiment of the present invention, the eluent can be pure water or an aqueous sodium chloride solution. The present invention, by limiting the eluent to an aqueous sodium chloride solution, avoids damaging the structure of the uronic acid polysaccharide, and by allowing the chloride ions in the aqueous sodium chloride solution to compete with the uronic acid polysaccharide molecules for binding to the anion exchange sites on the resin, thereby displacing and eluting the uronic acid polysaccharide adsorbed on the resin. In another embodiment of the present invention, the elution method can be gradient elution, which can be performed using pure water, 0.1 mol / L aqueous sodium chloride solution, 0.2 mol / L aqueous sodium chloride solution, and 0.3 mol / L aqueous sodium chloride solution; the elution flow rate can be 1~3 mL / min, and the elution time for each gradient can be independently 55~175 min. The present invention, by limiting gradient elution and gradually increasing the concentration of the aqueous sodium chloride solution, can sequentially elute polysaccharide components with different charges and structures, achieving fractional purification.

[0040] In one embodiment of the present invention, the anion exchange column can be a DEAE-agarose gel FF pre-packed column, and the specifications of the anion exchange column can be a 2.6cm × 60cm protein chromatography column. The present invention ensures improved adsorption and separation efficiency of uronic acid polysaccharides by limiting the type and specifications of the anion exchange column. In one embodiment of the present invention, the mass ratio of the uronic acid polysaccharide raw material to the volume of the DEAE-agarose gel FF pre-packed column can be 1mg:75~90mL. The present invention ensures that the sample loading amount is within the dynamic adsorption capacity range of the resin by limiting the sample loading amount, avoiding excessive uronic acid units leading to breakthrough loss, ensuring sufficient adsorption of uronic acid polysaccharides; ensuring more complete separation of impurities and uronic acid polysaccharides, reducing tailing and overlap, and improving separation degree and purity of uronic acid polysaccharides. In one embodiment of the present invention, the eluent from the anion exchange column chromatography can be tested using the phenol-sulfuric acid method. Based on the elution curve, the eluent corresponding to the desired polysaccharide peak is collected and sequentially concentrated, dialyzed with deionized water, and freeze-dried to obtain crude uronic acid polysaccharide. In another embodiment of the present invention, the molecular weight cutoff for the deionized water dialysate can be 1000-3500 Da. The present invention does not impose any particular limitations on the concentration and freeze-drying processes; any concentration and freeze-drying methods well-known in the art can be used to remove water. The present invention also does not impose any particular limitations on the deionized water dialysate; any deionized water dialysate methods well-known in the art can be used to remove sodium chloride and obtain crude uronic acid polysaccharide of the desired molecular weight.

[0041] After anion exchange column chromatography is completed, the present invention performs dextran gel column chromatography on the crude uronic acid polysaccharide obtained by the anion exchange column chromatography to obtain uronic acid polysaccharide.

[0042] In one embodiment of the present invention, the crude uronic acid polysaccharide can be dissolved in pure water before loading. The present invention does not have a specific limitation on the amount of pure water used; any amount of pure water known in the art can be used to dissolve the crude uronic acid polysaccharide.

[0043] In this invention, dextran gel column chromatography utilizes the molecular sieve effect to separate uronic acid polysaccharides according to molecular weight, achieving desalting, removal of small molecule impurities, and polysaccharide fractionation, thereby improving the purity and homogeneity of uronic acid polysaccharides. As one embodiment of this invention, the dextran gel column can be a Sephadex G-100 dextran gel column, and the specifications of the dextran gel column can be a 2cm × 1.6m glass chromatography column. This invention, by limiting the type and specifications of the dextran gel column, ensures the effective separation of uronic acid polysaccharides according to molecular weight, guarantees stable separation results, improves the purity of uronic acid polysaccharides, and removes impurities such as oligosaccharides, inorganic salts, and pigments. As one embodiment of this invention, the dextran gel in the dextran gel column can be activated before packing the column. As one embodiment of this invention, the activation method can be: placing the dextran gel in boiling water, and the activation time can be 2-3 hours. In one embodiment of the present invention, the dextran gel column is equilibrated with pure water and then with a sodium chloride aqueous solution before elution. The equilibration times for pure water and sodium chloride aqueous solution can be independently set to 8-12 hours, and the concentration of sodium chloride in the sodium chloride aqueous solution can be 0.05-0.1 mol / L. In another embodiment, the sodium chloride aqueous solution equilibration can be achieved by eluting the dextran gel column with a sodium chloride aqueous solution for 1-3 hours. By limiting the sodium chloride aqueous solution equilibration, the present invention utilizes the electrostatic adsorption of chloride ions in the sodium chloride aqueous solution around the dextran gel, which is beneficial for effectively shielding the electrostatic attraction between uronic acid units and the dextran gel during subsequent elution, reducing the non-specific adsorption of uronic acid units in the dextran gel. In another embodiment, the eluent can be a sodium chloride aqueous solution with a sodium chloride concentration of 0.05-0.1 mol / L. This invention utilizes sodium chloride aqueous solution as the eluent. Sodium ions in the sodium chloride aqueous solution are electrostatically adsorbed around the carboxyl anions of uronic acid units, forming a charge shielding layer. This facilitates the separation of uronic acid polysaccharides and results in high purity of the separated uronic acid polysaccharides. Furthermore, sodium and chloride ions in the sodium chloride aqueous solution are strongly hydrated ions, preferentially binding to hydroxyl groups on the dextran gel and carboxyl groups on the uronic acid units, occupying hydrogen bond sites. This disrupts existing hydrogen bonds between the uronic acid units and the dextran gel and prevents the formation of new hydrogen bonds, thereby reducing the adsorption of uronic acid units in the dextran gel. This further facilitates the separation of uronic acid polysaccharides and results in high purity of the separated uronic acid polysaccharides. In one embodiment of this invention, the elution can be isocratic elution, and the elution flow rate can be 3-5 s / drop. In another embodiment of this invention, the mass ratio of the crude uronic acid polysaccharide to the volume of the dextran gel column is 1 mg: 990-1100 mL.This invention ensures that the crude uronic acid polysaccharide sample is not overloaded and diffused by limiting the sample loading amount, thus ensuring that the crude uronic acid polysaccharide sample is effectively separated according to molecular weight and improving separation resolution and purity.

[0044] In one embodiment of the present invention, the eluent from the dextran gel column chromatography can be tested using the phenol-sulfuric acid method. Based on the elution curve, the eluent corresponding to the desired polysaccharide peak is collected, and then subjected to a first concentration, deionized water dialysis, a second concentration, and freeze-drying sequentially to obtain a uronic acid-containing polysaccharide. In one embodiment of the present invention, the molecular weight cutoff for the deionized water dialysis can be 1000-3500 Da. The present invention does not have any particular limitations on the first concentration, second concentration, and freeze-drying; any concentration and freeze-drying method well-known in the art can be used to remove water. The present invention also does not have any particular limitations on the deionized water dialysis; any deionized water dialysis method well-known in the art can be used to remove sodium chloride and obtain the uronic acid-containing polysaccharide of the desired molecular weight.

[0045] This invention utilizes sodium ions in an aqueous sodium chloride solution to adsorb around the carboxyl anions of uronic acid units via electrostatic interaction, forming a charge shielding layer. Simultaneously, chloride ions in the aqueous sodium chloride solution adsorb around the dextran gel via electrostatic interaction, effectively shielding the electrostatic attraction between the uronic acid units and the dextran gel, reducing the non-specific adsorption of uronic acid units in the dextran gel. This results in easy separation of uronic acid-containing polysaccharides with high purity. Furthermore, sodium and chloride ions in the aqueous sodium chloride solution are strongly hydrated ions, preferentially binding to the hydroxyl groups on the dextran gel and the carboxyl groups on the uronic acid units, occupying hydrogen bond sites. This disrupts existing hydrogen bonds between the uronic acid units and the dextran gel and prevents the formation of new hydrogen bonds, thereby reducing the adsorption of uronic acid units in the dextran gel. This further facilitates the separation of uronic acid-containing polysaccharides with high purity.

[0046] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0047] Example 1 After soaking in anhydrous ethanol for 24 hours, the *Echinopsis lanceolata* was successively dried, pulverized, and passed through a 40-mesh sieve. It was then mixed with petroleum ether and stirred at 60°C for 2 hours to degrease with petroleum ether. After filtration, the *Echinopsis lanceolata* was dried at 55°C for 8 hours. Next, it was mixed with 95% ethanol (volume fraction) and stirred at 60°C for 2 hours to degrease and remove pigments with ethanol. After filtration, the *Echinopsis lanceolata* was dried at 55°C for 8 hours to obtain pretreated *Echinopsis lanceolata*. The petroleum ether degreasing was performed 5 times; the ethanol degreasing and pigment removal was performed 5 times; the mass ratio of *Echinopsis lanceolata* to petroleum ether was 1 g: 20 mL; and the mass ratio of *Echinopsis lanceolata* to ethanol was 1 g: 20 mL. Pretreated *Ligustrum lucidum* was mixed with pure water at a mass-to-volume ratio of 1 g: 20 mL and stirred at 60 °C for 2 h to extract uronic acid polysaccharides. This process was repeated 4 times. The filtrates were then combined and concentrated to 1 / 5 of the filtrate volume using a rotary evaporator. The filtrate was then mixed with 95% ethanol at a volume ratio of 1:4 and placed at 4 °C for 24 h. The mixture was then centrifuged at 4000 rpm for 10 min and washed 3 times each with petroleum ether and anhydrous ethanol. After being dissolved in ultrapure water, the mixture was dialyzed using a 1000 Da dialysis bag for 24 h (with the ultrapure water replaced every 8 h). The mixture was then concentrated by rotary evaporation and freeze-dried at -20 °C to obtain the uronic acid polysaccharide raw material. After thawing the frozen uronic acid polysaccharide raw material at room temperature, filter to obtain the filtrate. Then, mix it with Sevag reagent (volume ratio: chloroform: n-butanol = 4:1) at a volume ratio of 4:1, stir for 15 minutes, centrifuge, and collect the supernatant. Repeat this operation 10 times. Then, concentrate the supernatant, mix it with macroporous resin D101 overnight, filter, and obtain a depigmented filtrate. Wash off the adsorbed polysaccharides with distilled water, concentrate the filtrate to 1 / 5 of its original volume, repeat the above operation 5 times, and then concentrate and freeze-dry. Before use, macroporous resin D101 should be soaked in 95% ethanol for 24 hours, and then rinsed with distilled water until there is no alcohol odor. Dissolve 100 mg of the above-mentioned freeze-dried uronic acid polysaccharide raw material in 3 mL of deionized water, centrifuge at 2000 r / min for 5 min, and filter the supernatant through a 0.45 μm filter membrane into a DEAE-agarose gel FF pre-packed column (2.6 cm × 60 cm) for column chromatography. Perform gradient elution using pure water, 0.1 mol / L, 0.2 mol / L, and 0.3 mol / L sodium chloride aqueous solutions as eluents. The time was 175 min for all samples; the flow rate was 1 mL / min; 7 mL of eluent was collected from each tube; the absorbance of uronic acid polysaccharides was measured at 490 nm using a UV detector via the phenol-sulfuric acid method, and the absorbance of proteins was measured at 280 nm. Elution curves were plotted with the number of collection tubes on the x-axis and the absorbance of the collected solution on the y-axis. The elution peaks corresponding to elution with pure water, 0.1 mol / L, 0.2 mol / L, and 0.3 mol / L sodium chloride aqueous solutions were named BRPS-0, BRPS-1, BRPS-2, and BRPS-3, respectively. Figure 1 As shown; according to the elution curve, the eluents corresponding to the elution peaks obtained by using 0.2 mol / L sodium chloride aqueous solution were combined, concentrated at 60°C using a rotary evaporator, dialyzed with deionized water using a 1000 Da dialysis bag (molecular weight cutoff 1000 Da) for 48 h, and then freeze-dried to obtain crude uronic acid polysaccharide containing uronic acid. Sephadex G-100 was boiled in water for 2 hours and then packed into a Sephadex G-100 (2cm × 1.6m) glass column. The column was equilibrated with pure water for 12 hours, followed by equilibration with 0.1mol / L sodium chloride for 12 hours. 20mg of crude uronic acid polysaccharide was dissolved in 2mL of pure water and added to a Sephadex G-100 dextran gel column for column chromatography. After static equilibration, elution was initiated using 0.1mol / L sodium chloride as the eluent. The eluent flow rate was controlled at 5s / drop. 3mL of eluent was collected from each tube using an automated fraction collector. The absorbance of the uronic acid polysaccharide was measured at 490nm using a UV detector via the phenol-sulfuric acid method. An elution curve was plotted with the number of collection tubes on the x-axis and the absorbance of the collected solution on the y-axis. Figure 2 As shown, based on the elution curve, a single elution peak was obtained. The eluents corresponding to the single elution peaks in tubes 16-20 were combined, concentrated by rotary evaporation, dialyzed with deionized water for 48 hours, and finally the dialysate was rotary evaporated at 60°C and freeze-dried to obtain the polysaccharide containing uronic acid from the beak-tailed pine, denoted as BRPS-2-0. The mass ratio of the uronic acid polysaccharide crude material to the volume of pure water is 10 mg: 1 mL; the loading amount of the uronic acid polysaccharide crude material is: the mass ratio of the uronic acid polysaccharide crude material to the volume of the dextran gel column is 2 mg: 50 mL; the mass of the uronic acid units in the uronic acid polysaccharide crude material is 13.16% of the uronic acid polysaccharide.

[0048] (1) The appearance of the polysaccharide containing uronic acid from Blaps rynchopetera Fabricius prepared in Example 1 was photographed using a mobile phone, and the results are as Figure 3 shown. As can be seen from the figure, the appearance of the polysaccharide containing uronic acid from Blaps rynchopetera Fabricius prepared in Example 1 is light yellowish-white and loose.

[0049] (2) Determination of the molecular weight of the polysaccharide containing uronic acid from Blaps rynchopetera Fabricius prepared in Example 1 The polysaccharide containing uronic acid from Blaps rynchopetera Fabricius prepared in Example 1 was dissolved in 0.1 mol / L aqueous NaNO3 solution (added with NaN3, the mass of NaN3 being 0.02% of the mass of the aqueous NaNO3 solution) to obtain a mixed solution. The concentration of the polysaccharide containing uronic acid from Blaps rynchopetera Fabricius in the mixed solution was 1 mg / mL. After filtration through a filter with a pore size of 0.45 μm, a chromatographic system of gel chromatography - differential - multi-angle laser light scattering system was used, with a liquid phase system U3000 (Thermo, USA), a differential detector Optilab T-rEX (Wyatt technology, CA, USA), a laser light scattering detector DAWN HELEOS Ⅱ (Wyatt technology, CA, USA), and gel exclusion chromatography columns Ohpak SB-805 HQ (300×8 mm) and Ohpak SB-803 HQ (300×8 mm) connected in series for detection; the column temperature was controlled at 45 °C, the injection volume was 100 μL, mobile phase A: 0.1 mol / L aqueous NaNO3 solution (added with NaN3, the mass of NaN3 being 0.02% of the mass of the aqueous NaNO3 solution), the flow rate was 0.6 mL / min, and the elution gradient: isocratic, 75 min. The differential signal (RI), light scattering (LS) signal, and molecular conformation diagram obtained are as Figures 4-6 shown. Figure 4 is a signal intensity - time diagram. In the spectrum, the red curve is the light scattering (LS) signal, and the blue curve is the differential signal (RI). Both show a single symmetric peak, indicating that the polysaccharide containing uronic acid from Blaps rynchopetera Fabricius has high purity and a relatively narrow molecular mass distribution (smaller dispersion coefficient); Figure 5 is a molecular weight - time diagram. In the spectrum, the red curve is the light scattering (LS) signal, the blue curve is the differential signal (RI), and the black curve is the traditional calibration curve. From the red curve and the blue curve, Mp can be read as 4.45 kDa. After calculation, Mn = 4.55 kDa, Mz = 4.86 kDa, the weight average number Mw = 4.69 kDa, and Mw / Mn = 1.03, further indicating a relatively narrow molecular mass distribution. Figure 6 The fitting slope in is 0.52 ± 0.21, and the central value is very close to 0.5, indicating that in the current solution, the molecule as a whole presents a random coil (θ solvent) conformation, the chain segments are in an ideal random coil state, without obvious swelling or curling.

[0050] (3) Physicochemical property determination (total content of neutral monosaccharide units, protein content, and uronic acid unit content) 1. Construction of the glucose standard curve 1.1 Solution Preparation Preparation of glucose standard solution: Accurately weigh 10 mg of glucose that has been kept constant in weight, place it in a 100 mL volumetric flask, add ultrapure water to make up to the mark, shake well, and the result is a 0.1 mg / mL glucose standard stock solution.

[0051] Preparation of 6% phenol solution: Weigh 6g of solid phenol, place it in a 100mL brown volumetric flask, add ultrapure water to the mark, shake well, protect from light, and store in a refrigerator for later use.

[0052] Preparation of sample solution of uronic acid polysaccharide from *Echinopsis lanceolata* prepared in Example 1: Weigh 1.7 mg of uronic acid polysaccharide from *Echinopsis lanceolata* prepared in Example 1, make up to 1 mL, shake well, and obtain a sample solution of 1.7 mg / mL.

[0053] 1.2 Plotting the Standard Curve Accurately pipette glucose standard solution to prepare control stock solutions with concentrations of 0.00, 6.25, 12.5, 25, 50, 100, and 200 μg / mL. Accurately pipette 100 μL of each concentration of control stock solution into a 15 mL test tube, add 100 μL of 6% phenol solution, then add 500 μL of concentrated sulfuric acid solution, vortex to mix, and incubate in an 80°C water bath for 15 min. After cooling to room temperature, pipette 200 μL into each well of a 96-well plate and measure the absorbance at 490 nm using a microplate reader. Perform three parallel operations and plot a glucose standard curve. Plot the standard curve with glucose concentration on the x-axis and absorbance on the y-axis. Figure 7 As shown; the regression equation is y = 0.0058x + 0.1140, and the correlation coefficient R0 is [value missing]. 2 =0.9999.

[0054] 1.3 Determination of Sample Solution Take 100 μL of the 1.7 mg / mL uronic acid polysaccharide sample solution of *Echinopsis lanceolata* prepared in Example 1 using a pipette, add 100 μL of 6% phenol solution, then add 500 μL of concentrated sulfuric acid solution, vortex to mix, and place in an 80°C water bath for 15 min. After cooling to room temperature, pipette 200 μL into each well of a 96-well plate and cool to room temperature. Measure the absorbance at 490 nm using a microplate reader. Perform the operation in parallel for 3 groups. Substitute the results into the regression equation to obtain the glucose concentration. Glucose content (g / 100g) = (Measured glucose concentration × Sample volume / Sample mass) × 100%; 2. Construction of the uronic acid standard curve 2.1 Solution Preparation Preparation of sodium tetraborate-sulfuric acid solution: Take 0.125 g of sodium tetraborate, add sulfuric acid to make up to 100 mL in a volumetric flask, and shake well to obtain the solution; Preparation of m-hydroxybiphenyl solution: Take 0.5g of sodium hydroxide and dilute to 100mL in a volumetric flask (to obtain a 0.5% sodium hydroxide solution). Then take 30mg of m-hydroxybiphenyl and dissolve it in the 0.5% sodium hydroxide solution. Dilute to 100mL in a volumetric flask with the 0.5% sodium hydroxide solution and store at 4℃ protected from light. Preparation of galacturonic acid solution: Take 10 mg of galacturonic acid, place it in a 100 ml volumetric flask, add ultrapure water to the mark, and shake well. This is the 0.1 mg / mL galacturonic acid standard stock solution.

[0055] 2.2 Plotting the Standard Curve Accurately pipette galacturonic acid solution to prepare control stock solutions with concentrations of 0, 3.125, 6.25, 12.5, 25, 50, and 100 μg / mL. Accurately pipette 200 μL of each concentration of control stock solution into 15 mL test tubes. Add 1 mL of sodium tetraborate-sulfuric acid solution to each tube in an ice-water bath, vortex to mix, and incubate at 80°C for 8 min. Remove the tubes and cool to room temperature in an ice-water bath. Add 20 μL of m-hydroxybiphenyl solution, vortex to mix, and incubate for 10 min. Pipette 200 μL of each tube into a 96-well plate and measure the absorbance at 520 nm using a microplate reader. Perform this experiment in triplicate and plot a galacturonic acid standard curve. Plot the standard curve with galacturonic acid concentration on the x-axis and absorbance on the y-axis as shown below. Figure 8 As shown, the regression equation is y = 0.0018x + 0.0490, and the correlation coefficient R0 is obtained. 2 =0.9943.

[0056] 2.3 Determination of Sample Solution The uronic acid polysaccharide sample solution of *Echinopsis lanceolata* prepared in Example 1 was diluted to 0.05 mg / mL. 200 μL of each sample was taken and 1 mL of sodium tetraborate-sulfuric acid solution was added to the sample in an ice-water bath. The sample was vortexed and mixed. The sample was then placed in an 80°C water bath for 8 min and then removed and cooled to room temperature in an ice-water bath. 20 μL of m-hydroxybiphenyl solution was added and the sample was vortexed and mixed. The sample was reacted for 10 min. 200 μL of each sample was pipetted into a 96-well plate and the absorbance was measured at 520 nm using a microplate reader. The experiment was repeated in three parallel groups. The uronic acid concentration was obtained by substituting the results into the regression equation. Glucuronic acid unit content (g / 100g) = (measured glutaronic acid concentration × sample volume / sample mass) × 100%; 3. Construction of protein standard curve (coomassie brilliant blue method) 3.1 Bovine serum albumin stock solution Mix bovine serum albumin standard with water and dissolve thoroughly to prepare a 0.2 mg / mL bovine serum albumin stock solution.

[0057] 3.2 Preparation of Coomassie Brilliant Blue Solution Dissolve 100 mg of Coomassie Brilliant Blue G-250 in 5 mL of 95% ethanol, add 10 mL of 85% phosphoric acid, and dilute to 100 mL with pure water. Filter with filter paper and set aside.

[0058] 3.3 Determination of Standard Curve for Bovine Serum Protein Concentration Bovine serum albumin (BSA) stock solution was diluted with water to concentrations of 0, 6.25, 12.5, 25, 50, 100, and 200 μg / mL, respectively. 4.0 mL of Coomassie Brilliant Blue solution was added, vortexed, and allowed to stand for 10 min. 200 μL was pipetted into each well of a 96-well plate, and the absorbance of the BSA concentration was measured at 595 nm using a microplate reader. This was performed in triplicate, and a BSA standard curve was plotted. The standard curve was plotted with BSA concentration on the x-axis and absorbance on the y-axis as shown in the figure. Figure 9 As shown, the regression equation is y = 0.0025x + 0.7653, R0 2 =0.996; 3.4 Determination of Sample Solution Take 20 μL of the 1.7 mg / mL uronic acid polysaccharide sample solution of *Echinopsis lanceolata* prepared in Example 1 using a pipette, add 200 μL of BCA working solution, incubate at 37°C for 30 min, and measure the absorbance of the protein concentration at 562 nm using an ELISA reader.

[0059] Protein content (g / 100g) = (measured protein concentration × sample volume / sample mass) × 100%; In summary, by detecting the neutral monosaccharide units, uronic acid units, and proteins in the uronic acid polysaccharide of *Euphorbia tirucalli* prepared in Example 1, it was found that the total content of neutral monosaccharide units in the uronic acid polysaccharide of *Euphorbia tirucalli* prepared in Example 1 was 43.35%, the content of uronic acid units was 45.65%, and the protein content was not detected.

[0060] (3) Monosaccharide composition analysis Accurately weigh fucose (Fuc), rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), xylose (Xyl), mannose (Man), fructose (Fru), ribose (Rib), galacturonic acid (Gal-UA), guluronic acid (Gul-UA), glucuronic acid (Glc-UA), and mannuronic acid (Man-UA), and dissolve them in water to prepare a 10 mg / mL monosaccharide standard stock solution. Then, mix the monosaccharide standard stock solutions separately to prepare a series of mixed monosaccharide standard solutions with different concentration gradients (referred to as monosaccharide mixed standards), as shown in Table 1.

[0061] Table 1. Monosaccharide mixed standard concentration gradient

[0062] Note: To ensure a goodness of fit of the standard curve ≥ 0.99, the concentration range of each monosaccharide will be adjusted according to the actual response value of each monosaccharide during the calculation.

[0063] Take a clean chromatographic bottle, weigh the uronic acid polysaccharide sample of *Symplocos buergeriana* prepared in Example 1, add 1 ml of 2 mol / L trifluoroacetic acid (TFA) solution, heat at 121°C for 2 hours, purge with nitrogen, and dry. Wash with 99.99% methanol (v / v), dry again, repeat methanol washing 3 times, dissolve in sterile water, and analyze the uronic acid polysaccharide sample and the monosaccharide unit components in the monosaccharide mixed standard using a Thermo ICS 5000+ ion chromatography system (ICS5000+, Thermo Fisher Scientific, USA) with an electrochemical detector. Use a Dionex™ CarboPac™ PA20 (150 × 3.0 mm, 10 μm) liquid chromatography column; injection volume: 5 μL; mobile phase A (H2O), mobile phase B (0.1 M NaOH), mobile phase C (0.1 M NaOH, 0.2 M NaOH, 0.2 M NaOH). NaAc), flow rate 0.5 ml / min; column temperature 30℃; elution gradient: A phase:B phase:C phase volume ratio 95:5:0, elution 0 min; A phase:B phase:C phase volume ratio 85:5:10, elution 26 min; A phase:B phase:C phase volume ratio 85:5:10, elution 42 min; A phase:B phase:C phase volume ratio 60:0:40, elution 42.1 min; A phase:B phase:C phase volume ratio 60:40:0, elution 52 min; A phase:B phase:C phase volume ratio 95:5:0, elution 52.1 min; A phase:B phase:C phase volume ratio 95:5:0, elution 60 min. This yielded the monosaccharide composition analysis chromatogram of the mixed standard of uronic acid polysaccharide and monosaccharide from *Ligustrum lucidum* in Example 1, as shown below. Figure 10As shown in the figure, the monosaccharide units in the uronic acid polysaccharide of *Ligustrum lucidum* in Example 1 consist of arabinose, rhamnose, galacturonic acid, galactose, glucuronic acid, fucose, mannose, glucose, and xylose units; their proportions are calculated and shown in Table 2.

[0064] Table 2. Monosaccharide unit composition of uronic acid polysaccharide in Example 1, *Begonia vesca*.

[0065] As shown in Table 2, the molar percentages of arabinose, rhamnose, galacturonic acid, galactose, glucuronic acid, fucose, mannose, glucose, and xylose units in the polysaccharide of *Ligustrum lucidum* prepared in Example 1 were 24.6%, 24.3%, 20.16%, 10.19%, 8.94%, 7.81%, 1.65%, 1.61%, and 0.60%, respectively.

[0066] (4) Ultraviolet spectroscopy analysis 2 mg of the uronic acid polysaccharide from *Lysimachia clethroides* prepared in Example 1 was dissolved in 4 mL of distilled water to prepare a solution with a concentration of 0.5 mg / mL. The solution was then detected using a UV-Vis spectrophotometer at room temperature within the wavelength range of 190–500 nm. The UV spectrum of the uronic acid polysaccharide from *Lysimachia clethroides* in Example 1 was obtained, as shown below. Figure 11 As shown in the figure, the uronic acid polysaccharide from *Ligustrum lucidum* in Example 1 showed no significant absorption peaks at 260 nm and 280 nm, indicating that it does not contain protein or nucleic acid impurities and has high purity.

[0067] (5) Fourier transform infrared spectroscopy analysis The infrared absorption spectrum of the uronic acid polysaccharide from *Euphorbia tirucalli* in Example 1 was determined using the KBr tablet method: 2 mg of the dried uronic acid polysaccharide sample from *Euphorbia tirucalli* in Example 1 and 500 mg of pre-dried spectrally pure KBr powder were weighed and placed together in a dry agate mortar and ground thoroughly. The mixture was then pressed into tablets using a tablet press. Using KBr as a control, the spectrum was scanned from 500 to 4000 cm⁻¹ using a Fourier transform infrared spectroscopy (FTIR) spectrometer. -1 The range was measured, and the infrared spectrum of the uronic acid polysaccharide in *Ligustrum beakum* from Example 1 was obtained as follows: Figure 12 As shown in the figure. From the figure, we can see that: 1) at 3423cm... -1 The broad and strong absorption peak at 2934 cm⁻¹ is attributed to the stretching vibration of OH; 2) at 2934 cm⁻¹ -1 The absorption peak at 1726 cm⁻¹ is attributed to the stretching vibration of CH; 3) the absorption peak at 1726 cm⁻¹ is attributed to the stretching vibration of CH; -1The weak absorption peak at 1611 cm⁻¹ is attributed to the stretching vibration of C=O in the carboxyl group, indicating the presence of a certain amount of uronic acid units. This is consistent with the results of the aforementioned chemical method for determining the content of uronic acid units and the composition of monosaccharide units; 4) At 1611 cm⁻¹ -1 The absorption peak at 1421 cm⁻¹ is attributed to adsorbed water, which may be due to the hygroscopic nature of polysaccharides, resulting in a strong absorption peak at this location. -1 The weaker absorption peak at 1247 cm⁻¹ is attributed to the stretching vibration of CH; 6) at 1247 cm⁻¹ -1 The absorption peaks at 1054 cm⁻¹ are attributed to the stretching and deformation vibrations of CO and OH; 7) at 1054 cm⁻¹ -1 The strong absorption peak at 918 cm⁻¹ is attributed to the stretching vibration of COC on the pyranose ring; 8) at 918 cm⁻¹ -1 The presence of absorption peaks in the vicinity indicates that the polysaccharide component contains β-configured glycosidic bonds.

[0068] (6) Congo Red Experiment 11.144 mg of Congo red was weighed and diluted to 200 mL to prepare an 80 μmol / L Congo red solution. A 1 mol / L sodium hydroxide solution was prepared. Six solutions were prepared according to Table 3, containing uronic acid polysaccharide from *Symplocos buergeriana* (from Example 1), Congo red solution, water, and sodium hydroxide. The solutions were allowed to stand for 10 min, with distilled water as a blank. A spectrophotometer was used to scan in the 400–700 nm wavelength range to determine the maximum absorption wavelength of *Symplocos buergeriana* polysaccharide under different concentrations of sodium hydroxide. The maximum absorption wavelength of Congo red under different concentrations of sodium hydroxide was measured using the same method without the addition of *Symplocos buergeriana* polysaccharide as a control. The results are as follows: Figure 13 As shown in the figure, the maximum absorption wavelength of the complex formed by the uronic acid polysaccharide from *Spatholobus suberectus* in Example 1 and Congo red increases slowly with increasing NaOH concentration, and then decreases slowly, indicating that the uronic acid polysaccharide from *Spatholobus suberectus* in Example 1 has a triple helix structure. All experimental data, unless otherwise specified, are the average of three parallel determinations and were processed and analyzed using OriginPro 2016 (OriginLab Inc., USA) software.

[0069] Table 3. Sample Configuration Table for Congo Red Experiment

[0070] (7) Scanning electron microscopy analysis First, double-sided tape was applied to the stage. The raw material containing uronic acid polysaccharide, the decolorized uronic acid polysaccharide, and the uronic acid polysaccharide from *Ligustrum lucidum* (Example 1) were dispersed on the double-sided tape to form a uniform thin layer. This layer was then transferred to a vacuum sputtering process. The morphology of the uronic acid polysaccharide from *Ligustrum lucidum* was scanned under a magnifying glass. The results are shown below. Figure 14As shown in the figures: The first image in the first row is a morphological image of the uronic acid polysaccharide raw material magnified 200 times, showing a rough, porous, and lamellar structure. The fourth image is a morphological image magnified 20,000 times, showing irregular lamellar edges, numerous internal cracks or pores, and fine granular material attached to or filling the pores in some areas. The overall structure is relatively loose, and the bonding force between the lamellars is weak. The first image in the second row is a morphological image of the decolorized uronic acid polysaccharide magnified 200 times, showing a honeycomb or porous blocky structure. The fourth image is a morphological image magnified 20,000 times. The morphological images show relatively smooth pore walls and a significantly reduced surface granular texture. The first image in the third row is a morphological image of the uronic acid polysaccharide from Example 1, magnified 200 times, showing that it is fragmented into layers with a finer surface texture. The third image is a morphological image magnified 5000 times, showing fine texture or tiny protrusions, a reduced number of pores but more uniform size, and fibrous structures in some areas. The fourth image is a morphological image magnified 20000 times, showing fine texture or tiny protrusions, a reduced number of pores but more uniform size, and fibrous structures in some areas.

[0071] Comparative Example 1 The difference between this comparative example and Example 1 is that the eluent for the Sephadex G-100 dextran gel column chromatography is pure water, and the dextran gel column is equilibrated with pure water only before elution. The rest is the same as in Example 1. The absorbance of the uronic acid-containing polysaccharide was measured at 490 nm using a UV detector via the phenol-sulfuric acid method. An elution curve was plotted with the number of collection tubes on the x-axis and the absorbance of the collected solution on the y-axis. Figure 15 As shown.

[0072] according to Figure 15 It can be seen that the elution peaks in the figure are three peaks and are asymmetrical. Therefore, it is evident that using pure water as the eluent cannot yield polysaccharides containing uronic acid.

[0073] In summary, the separation and purification method provided by this invention can separate and purify crude uronic acid polysaccharides with high uronic acid unit content, good separation effect, and high purity of the obtained uronic acid polysaccharides.

[0074] 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 separating and purifying polysaccharides containing uronic acid, comprising: The crude polysaccharide containing uronic acid was subjected to dextran gel column chromatography to obtain the polysaccharide containing uronic acid. The conditions for the dextran gel column chromatography include: Eluent: Sodium chloride aqueous solution; Dextran gel columns: Sephadex G-100 dextran gel columns; Elution flow rate: 3~5s / drop; Sample loading amount: The ratio of the mass of the uronic acid polysaccharide crude material to the volume of the dextran gel column is 1 mg: 990~1100 mL.

2. The separation and purification method according to claim 1, characterized in that, The mass of uronic acid units in the crude uronic acid polysaccharide is 30-50% of the total mass of the uronic acid polysaccharide.

3. The separation and purification method according to claim 1 or 2, characterized in that, The glycouronic acid polysaccharide includes beak-tailed rosary polysaccharide.

4. The separation and purification method according to claim 1, characterized in that, The concentration of sodium chloride in the sodium chloride aqueous solution is 0.05~0.15 mol / L.

5. The separation and purification method according to claim 1, characterized in that, The crude uronic acid polysaccharide was obtained by sequentially extracting, deproteinizing, decolorizing, and crudely separating and purifying biological materials containing uronic acid polysaccharides.

6. The separation and purification method according to claim 5, characterized in that, The extraction of uronic acid polysaccharides includes: mixing biological materials containing uronic acid polysaccharides with water and then extracting them, followed by alcohol precipitation of the resulting extract to obtain uronic acid polysaccharide raw materials.

7. The separation and purification method according to claim 5, characterized in that, The protein removal process includes: mixing the uronic acid polysaccharide raw material obtained from the extraction of the uronic acid polysaccharide with Sevag reagent, performing protein denaturation, and then centrifuging to remove the precipitate.

8. The separation and purification method according to claim 5, characterized in that, The decolorization process includes: adsorbing the protein-free product onto a macroporous resin to obtain a decolorized glycouronic acid polysaccharide.

9. The separation and purification method according to claim 5, characterized in that, The crude separation and purification includes: subjecting the decolorized uronic acid polysaccharide obtained by decolorization to anion exchange column chromatography to obtain crude uronic acid polysaccharide.

10. The separation and purification method according to claim 9, characterized in that, The conditions for the anion exchange column chromatography include: Eluent: pure water and sodium chloride aqueous solution; Anion exchange column: DEAE-agarose gel FF pre-packed column; Elution flow rate: 1~3 mL / min; Sample loading amount: The ratio of the mass of the decolorized uronic acid polysaccharide to the volume of the DEAE-agarose gel FF pre-packed column is 1 mg: 75~90 mL.

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