A method for separating and purifying plant polysaccharides

Abstract

The application discloses a plant polysaccharide separation and purification method in the technical field of plant polysaccharide extraction and separation, which comprises the following steps: raw material pretreatment, extraction, magnetic affinity adsorption, magnetic separation and collection, desorption and release, secondary magnetic separation, purification and drying. The method uses magnetic nanoparticles with surface modification of affinity ligands, realizes high-efficiency selective recognition and separation of plant polysaccharide molecules based on specific reversible combination of the affinity ligands and the plant polysaccharide molecules. The method has the advantages that each step is simple in operation, the magnetic separation process is easy to realize automatic control, the ultrafiltration membrane separation technology is mature, the whole process has good continuity, is easy to enlarge to an industrial production scale, has strong separation selectivity, high purification efficiency, is green and environment-friendly, and has mild conditions, is suitable for separation and purification of various plant polysaccharides such as rhizoma polygonati, medlar, ganoderma lucidum, lentinus edodes, dendrobium officinale and radix astragali, and has good industrial application prospect.

Description

Technical Field

[0001] This invention relates to the field of plant polysaccharide extraction and separation technology, specifically a method for separating and purifying plant polysaccharides. Background Technology

[0002] Plant polysaccharides are a class of high-molecular-weight polymers composed of multiple monosaccharide molecules linked by glycosidic bonds. They are widely found in medicinal and edible plants such as Polygonatum sibiricum, Lycium barbarum, Ganoderma lucidum, Lentinus edodes, Dendrobium officinale, and Astragalus membranaceus. Numerous studies have shown that plant polysaccharides possess a variety of important biological activities, including immunomodulation, antioxidation, antitumor activity, hypoglycemia, and hypolipidemia, and have broad application prospects in functional foods, pharmaceuticals, and health products.

[0003] Currently, the extraction of plant polysaccharides mainly employs methods such as hot water extraction, acid extraction, and alkaline extraction. These traditional methods suffer from drawbacks such as high extraction temperatures, long processing times, and low extraction rates. For purification, conventional methods include ethanol precipitation, Sevag deproteinization, and column chromatography (such as ion exchange chromatography and gel size exclusion chromatography). Among these, ethanol precipitation is the most commonly used method for crude polysaccharide extraction, but it consumes large amounts of organic solvents, and the precipitation process easily co-precipitates impurities such as proteins and pigments, resulting in low purity of the crude polysaccharides. Sevag deproteinization requires repeated operations, is cumbersome, and easily leads to polysaccharide loss. While column chromatography offers good separation, the equipment is expensive, the operation is complex, and the throughput is small, making it difficult to meet the needs of large-scale industrial production.

[0004] In recent years, researchers have attempted to apply new technologies such as ultrasound-assisted extraction, microwave-assisted extraction, and enzymatic extraction to the extraction of plant polysaccharides, which have improved extraction efficiency to some extent. However, in the separation and purification stage, there is still a lack of a method that is simple to operate, has good selectivity, high efficiency, and is easy to scale up industrially.

[0005] Based on this, the present invention designs a method for separating and purifying plant polysaccharides to solve the above problems. Summary of the Invention

[0006] The purpose of this invention is to provide a method for isolating and purifying plant polysaccharides to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A method for isolating and purifying plant polysaccharides includes the following steps:

[0009] S1. Raw material pretreatment: After drying the plant raw materials, pulverize them and pass them through a 40-100 mesh sieve to obtain plant raw material powder. The plant raw material powder obtained after pulverizing and sieving has a uniform particle size, which is conducive to the full contact between the solvent and the raw materials in the subsequent extraction process and improves the dissolution efficiency of polysaccharides.

[0010] S2. Extraction: Add the plant raw material powder from S1 to the extraction solvent, stir and extract at 50-90℃ for 0.5-3 hours, centrifuge, collect the supernatant, and obtain the crude extract of plant polysaccharides.

[0011] S3. Magnetic affinity adsorption: Magnetic nanoparticles with surface-modified affinity ligands are prepared and added to the crude plant polysaccharide extract in S2. The mixture is stirred and adsorbed at 20-40℃ for 0.5-2 hours, so that the plant polysaccharide molecules selectively bind to the surface of the magnetic nanoparticles through the affinity ligands.

[0012] S4. Magnetic separation and collection: An external magnetic field is applied to the mixed system in S3 to separate the magnetic nanoparticles loaded with plant polysaccharides from the liquid phase. The supernatant is discarded to obtain the magnetic nanoparticle-polysaccharide complex.

[0013] Among them, magnetic nanoparticles have superparamagnetism and can respond quickly and achieve solid-liquid separation under the action of an external magnetic field. The separation process is simple and fast, without the need for cumbersome operations such as filtration or centrifugation.

[0014] S5. Desorption and release: Add a desorbent to the magnetic nanoparticle-polysaccharide complex in S4, and stir at 20-40℃ for 0.5-2 hours to desorb and release the plant polysaccharide from the surface of the magnetic nanoparticles.

[0015] The desorption mechanism is based on a competitive substitution mechanism. Since the concentration of the desorbent solution is much higher than the concentration of polysaccharides bound to the surface of the magnetic nanoparticles, according to the law of mass action, the competitive equilibrium shifts in the direction that favors the binding of the desorbent solution, thereby causing the plant polysaccharide molecules to dissociate from the affinity ligands of the magnetic nanoparticles and be released into the liquid phase.

[0016] S6. Secondary magnetic separation: An external magnetic field is applied to the mixed system in S5 to separate and remove magnetic nanoparticles, and the desorption liquid containing plant polysaccharides is collected.

[0017] Secondary magnetic separation separates the desorbed magnetic nanoparticles from the desorption solution containing plant polysaccharides. The magnetic nanoparticles can be washed, regenerated, and recycled, while the desorption solution contains a high concentration of purified plant polysaccharides.

[0018] S7. Purification: The desorption solution in S6 is purified by membrane separation, and the purified polysaccharide solution is collected.

[0019] Ultrafiltration membrane separation technology is a membrane separation method based on differences in molecular weight. It offers advantages such as mild operating conditions, no need for chemical reagents, high separation efficiency, and ease of continuous operation. It effectively retains plant polysaccharide molecules with molecular weights larger than the cutoff value, while allowing smaller molecule impurities to pass through the membrane pores and be removed, thereby achieving further purification and concentration of plant polysaccharides. The membrane separation purification process can be combined with dialysis or elution to further improve desalination efficiency and purity.

[0020] S8. Drying: The polysaccharide solution in S7 is dried to obtain purified plant polysaccharides;

[0021] Drying methods can include vacuum freeze-drying, spray drying, or vacuum drying. Vacuum freeze-drying can remove moisture under low temperature and low pressure conditions, maximizing the preservation of the bioactivity and structural integrity of plant polysaccharides, resulting in loose and porous polysaccharide powder with good resolubility, making it the preferred drying method. Spray drying is suitable for large-scale continuous production and has high production efficiency. Vacuum drying is simple to operate and has a lower cost, but the drying temperature is relatively high, which may have some impact on some heat-sensitive polysaccharides.

[0022] Furthermore, the extraction solvent in S2 is an eutectic solvent aqueous solution, composed of hydrogen bond donors and hydrogen bond acceptors in a molar ratio of 1:0.5 to 3. It has excellent properties such as low vapor pressure, low volatility, good thermal stability, biodegradability, simple preparation, and low cost. The hydrogen bond donors are selected from one or more of lactic acid, malic acid, citric acid, ethylene glycol, and glycerol, and the hydrogen bond acceptors are selected from one or more of choline chloride, betaine, and tetramethylammonium chloride.

[0023] Among them, the hydrogen bond donors and hydrogen bond acceptors in the eutectic solvent can form hydrogen bonds with components such as cellulose and hemicellulose in the plant cell wall, thereby disrupting the cell wall structure and promoting the dissolution of polysaccharides; at the same time, the ionic components in the eutectic solvent can interact with polysaccharide molecules, thereby improving the solubility and extraction efficiency of polysaccharides.

[0024] Furthermore, the mass concentration of the eutectic solvent aqueous solution is 20%–80%, and the mass-to-volume ratio of plant raw material powder to eutectic solvent aqueous solution is 1:5–1:30 g / mL.

[0025] Furthermore, the magnetic nanoparticles in S3 are Fe3O4 nanoparticles or Fe3O4@SiO2 core-shell structured nanoparticles, and the affinity ligands are phenylboronic acid groups or their derivatives.

[0026] Fe3O4 nanoparticles are simple to prepare, low in cost, and have strong magnetic responsiveness. The core-shell structured nanoparticles use Fe3O4 as the magnetic core and are coated with an outer SiO2 shell. The SiO2 shell not only protects the magnetic core from oxidation and corrosion and improves the chemical stability of the nanoparticles, but its surface is also rich in silanol groups, which facilitates subsequent functionalization modification. At the same time, the coating of the SiO2 shell can effectively prevent the aggregation of magnetic nanoparticles and maintain good dispersion.

[0027] Benzylboronic acid groups and their derivatives are a class of affinity ligands that can specifically and reversibly bind to compounds containing cis-vicinal diol structures. Plant polysaccharide molecules contain a large number of cis-vicinal diol structures. Under appropriate pH conditions, Benzylboronic acid groups can form stable five-membered cyclic borate ester bonds with cis-vicinal diol structures, thereby achieving specific recognition and selective binding of plant polysaccharide molecules.

[0028] This affinity binding is highly selective. The phenylboronic acid group has a strong affinity for polysaccharide molecules containing the cis-ortho-diol structure, while it hardly binds to proteins, pigments, small molecule impurities, etc. that do not contain this structure. Therefore, it can achieve highly efficient and selective separation of plant polysaccharides.

[0029] Furthermore, the desorbent in S5 is a buffer solution containing sorbitol or mannitol, with a pH of 7.0–9.0 and a concentration of sorbitol or mannitol of 100–500 mmol / L. Both sorbitol and mannitol are saccharide compounds containing cis-vicinal diol structures, and their affinity for phenylboronic acid groups is comparable to or even stronger than that of plant polysaccharide molecules, thereby causing plant polysaccharide molecules to dissociate from phenylboronic acid groups.

[0030] Furthermore, the membrane separation and purification in S7 uses an ultrafiltration membrane with a molecular weight cutoff of 10–100 kDa, which is suitable for the purification needs of most medicinal plant polysaccharides.

[0031] Furthermore, the plant materials in S1 are selected from one or more of the following: Polygonatum sibiricum, Lycium barbarum, Ganoderma lucidum, Lentinus edodes, Dendrobium officinale, and Astragalus membranaceus. All of these plants are common medicinal and edible plants rich in active polysaccharides. Polygonatum sibiricum polysaccharides have immunomodulatory and anti-fatigue effects; Lycium barbarum polysaccharides have antioxidant, liver-protecting, and vision-improving effects; Ganoderma lucidum polysaccharides have immunomodulatory and anti-tumor activities; Lentinus edodes polysaccharides have been developed into immunomodulatory drugs; Dendrobium officinale polysaccharides have moisturizing and anti-aging effects; and Astragalus membranaceus polysaccharides have immune-enhancing and antiviral effects.

[0032] Compared with the prior art, the beneficial effects of the present invention are:

[0033] 1. This invention utilizes magnetic nanoparticles with surface-modified affinity ligands to achieve efficient and selective recognition and separation of plant polysaccharide molecules based on the specific and reversible binding of affinity ligands to plant polysaccharide molecules.

[0034] 2. This invention uses magnetic separation technology to replace traditional separation operations such as filtration, centrifugation, and sedimentation. The magnetic separation process can be completed in seconds to minutes, and the magnetic nanoparticles can be recycled after simple washing and regeneration.

[0035] 3. The purification method proposed in this invention uses mild conditions, which avoids the destruction of glycosidic bonds in polysaccharide molecules by strong acids, strong alkalis or high temperatures, and is conducive to maintaining the natural structure, molecular weight and biological activity of plant polysaccharides.

[0036] 4. The method of the present invention is simple to operate in each step, the magnetic separation process is easy to automate, the ultrafiltration membrane separation technology is mature, the whole process has good continuity, and it is easy to scale up to industrial production scale. Attached Figure Description

[0037] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 This is a flowchart illustrating the method of the present invention. Detailed Implementation

[0039] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Example 1: Isolation and purification of Polygonatum polysaccharides

[0041] S1. Take dried Polygonatum rhizome, crush it with a pulverizer and pass it through a 60-mesh sieve to obtain Polygonatum powder.

[0042] S2. Mix choline chloride and lactic acid in a molar ratio of 1:2 and stir at 80°C until a homogeneous and transparent eutectic solvent is formed. Dilute the eutectic solvent with deionized water to a mass concentration of 50% to obtain an aqueous solution of the eutectic solvent.

[0043] Weigh 100g of Polygonatum powder and add 2000mL of eutectic solvent aqueous solution (mass-volume ratio 1:20g / mL). Stir and extract at 70℃ for 2 hours. After extraction, centrifuge at 8000rpm for 15 minutes and collect the supernatant to obtain crude extract of Polygonatum polysaccharide.

[0044] S3. Fe3O4 nanoparticles with a particle size of about 20 nm were prepared by coprecipitation method. The Fe3O4 nanoparticles were dispersed in an ethanol-water mixed solvent, and 3-aminophenylboronic acid was added. The mixture was stirred at room temperature for 12 hours to allow the phenylboronic acid groups to bind to the surface of the nanoparticles through the amino groups, thus obtaining magnetic nanoparticles with phenylboronic acid groups on the surface.

[0045] The prepared magnetic nanoparticles were added to the crude extract of Polygonatum polysaccharide at a ratio of 5 mg / mL, and stirred and adsorbed at 30°C for 1 hour, so that Polygonatum polysaccharide molecules could selectively bind to the surface of the magnetic nanoparticles through phenylboronic acid groups.

[0046] S4. Place the mixture from S3 next to a permanent magnet and apply an external magnetic field to rapidly enrich the magnetic nanoparticles loaded with Polygonatum polysaccharide onto the container wall. Discard the supernatant containing impurities such as proteins and pigments to obtain the magnetic nanoparticle-polysaccharide complex.

[0047] S5. Add a Tris-HCl buffer solution (containing 300 mmol / L sorbitol) with a pH of 8.0 to the magnetic nanoparticle-polysaccharide complex, and stir at 30°C for 1 hour to desorb and release Polygonatum polysaccharide from the surface of the magnetic nanoparticles.

[0048] S6. Apply an external magnetic field to the mixed system in S5 to separate and remove the magnetic nanoparticles, and collect the desorption solution containing Polygonatum polysaccharide.

[0049] S7. The desorption solution in S6 is purified by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 30 kDa. The retentate is collected to obtain the purified Polygonatum polysaccharide solution.

[0050] S8. The purified Polygonatum polysaccharide solution was freeze-dried under vacuum to obtain Polygonatum polysaccharide powder.

[0051] The extraction rate of Polygonatum polysaccharide obtained in this embodiment was 12.8%, the polysaccharide purity was 92.5%, and the protein content was 1.2%.

[0052] Example 2: Isolation and purification of Lycium barbarum polysaccharides

[0053] S1. Take dried wolfberry fruit, remove the seeds, grind it with a grinder and pass it through an 80-mesh sieve to obtain wolfberry powder.

[0054] S2. Mix betaine and citric acid in a molar ratio of 1:1.5 and stir at 70°C until a homogeneous and transparent eutectic solvent is formed. Dilute the eutectic solvent with deionized water to a mass concentration of 60% to obtain an aqueous solution of the eutectic solvent.

[0055] Weigh 100g of wolfberry powder and add 1500mL of eutectic solvent aqueous solution (mass-volume ratio 1:15g / mL). Stir and extract at 80℃ for 1.5 hours. After extraction, centrifuge at 10000rpm for 10 minutes and collect the supernatant to obtain crude extract of wolfberry polysaccharide.

[0056] S3. Fe3O4 nanoparticles were prepared by solvothermal method, and then a SiO2 shell was coated on the surface of Fe3O4 by sol-gel method to obtain Fe3O4@SiO2 core-shell structured nanoparticles.

[0057] Fe3O4@SiO2 nanoparticles were dispersed in toluene, and 4-carboxyphenylboronic acid and a coupling agent were added. The mixture was reacted under reflux for 24 hours to allow the phenylboronic acid groups to combine with the amino groups on the SiO2 surface through the carboxyl groups, thus obtaining core-shell magnetic nanoparticles with phenylboronic acid groups modified on the surface.

[0058] The prepared Fe3O4@SiO2 magnetic nanoparticles were added to the crude extract of Lycium barbarum polysaccharide at a ratio of 8 mg / mL, and the mixture was stirred and adsorbed at 25°C for 1.5 hours.

[0059] S4. Apply an external magnetic field to the mixed system in S3 to separate the magnetic nanoparticles loaded with Lycium barbarum polysaccharides from the liquid phase. Discard the supernatant to obtain the magnetic nanoparticle-polysaccharide complex.

[0060] S5. Add a phosphate buffer solution (containing 200 mmol / L mannitol) with a pH of 8.5 to the magnetic nanoparticle-polysaccharide complex and stir and desorb for 1.5 hours at 25°C.

[0061] S6. Apply an external magnetic field to the mixed system in S5 to separate and remove the magnetic nanoparticles, and collect the desorption solution containing wolfberry polysaccharides.

[0062] S7. The desorbed solution from S6 is purified by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 50 kDa at an operating pressure of 0.15 MPa, and the retentate is collected.

[0063] S8. The purified wolfberry polysaccharide solution is spray-dried at an inlet air temperature of 180℃ and an outlet air temperature of 80℃ to obtain wolfberry polysaccharide powder.

[0064] The extraction rate of Lycium barbarum polysaccharides obtained in this embodiment was 15.3%, the polysaccharide purity was 89.7%, and the protein content was 1.8%.

[0065] Example 3: Isolation and purification of Ganoderma lucidum polysaccharides

[0066] S1. Take dried Ganoderma lucidum fruiting bodies, crush them with a pulverizer and pass them through a 100-mesh sieve to obtain Ganoderma lucidum powder.

[0067] S2. Tetramethylammonium chloride and glycerol are mixed at a molar ratio of 1:1 and stirred at 90°C until a homogeneous and transparent eutectic solvent is formed. The eutectic solvent is then diluted with deionized water to a mass concentration of 40% to obtain an aqueous solution of the eutectic solvent.

[0068] Weigh 100g of Ganoderma lucidum powder and add 2500mL of eutectic solvent aqueous solution (mass-volume ratio 1:25g / mL). Stir and extract at 60℃ for 3 hours. After extraction, centrifuge at 6000rpm for 20 minutes and collect the supernatant to obtain crude extract of Ganoderma lucidum polysaccharides.

[0069] S3. Fe3O4 nanoparticles with a particle size of about 15 nm were prepared by coprecipitation method. The Fe3O4 nanoparticles were dispersed in ethanol, and 3-aminophenylboronic acid was added. The mixture was stirred at room temperature for 18 hours to obtain magnetic nanoparticles with phenylboronic acid groups on the surface.

[0070] The prepared Fe3O4 magnetic nanoparticles were added to the crude extract of Ganoderma lucidum polysaccharides at a ratio of 3 mg / mL, and the mixture was stirred and adsorbed at 35°C for 0.5 hours.

[0071] S4. Apply an external magnetic field to the mixed system in S3 to separate the magnetic nanoparticles loaded with Ganoderma lucidum polysaccharides from the liquid phase, discard the supernatant, and obtain the magnetic nanoparticle-polysaccharide complex.

[0072] S5. Add HEPES buffer solution (containing 400 mmol / L sorbitol) at pH 7.5 to the magnetic nanoparticle-polysaccharide complex and stir and desorb for 0.5 hours at 35°C.

[0073] S6. Apply an external magnetic field to the mixed system in S5 to separate and remove the magnetic nanoparticles, and collect the desorption solution containing Ganoderma lucidum polysaccharides.

[0074] S7. The desorbed solution from S6 is purified by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 100 kDa at an operating pressure of 0.25 MPa, and the retentate is collected.

[0075] S8. The purified Ganoderma lucidum polysaccharide solution was freeze-dried under vacuum to obtain Ganoderma lucidum polysaccharide powder.

[0076] The extraction rate of Ganoderma lucidum polysaccharides obtained in this embodiment was 8.6%, the polysaccharide purity was 94.2%, and the protein content was 0.9%.

[0077] Example 4: Isolation and purification of lentinan

[0078] S1. Take dried shiitake mushroom fruiting bodies, crush them with a grinder and pass them through a 40-mesh sieve to obtain shiitake mushroom powder.

[0079] S2. Mix choline chloride and ethylene glycol at a molar ratio of 1:0.5 and stir at 75°C until a homogeneous and transparent eutectic solvent is formed. Dilute the eutectic solvent with deionized water to a mass concentration of 30% to obtain an aqueous solution of the eutectic solvent.

[0080] Weigh 100g of shiitake mushroom powder and add 3000mL of eutectic solvent aqueous solution (mass-volume ratio 1:30g / mL). Stir and extract at 90℃ for 0.5 hours. After extraction, centrifuge at 12000rpm for 5 minutes and collect the supernatant to obtain crude shiitake mushroom polysaccharide extract.

[0081] S3. Fe3O4 nanoparticles were prepared by solvothermal method, and then SiO2 shell was coated by sol-gel method to obtain Fe3O4@SiO2 core-shell structured nanoparticles. Fe3O4@SiO2 nanoparticles were dispersed in toluene, 4-carboxyphenylboronic acid was added, and the reaction was carried out under reflux for 20 hours to obtain core-shell structured magnetic nanoparticles with phenylboronic acid groups on the surface.

[0082] The prepared Fe3O4@SiO2 magnetic nanoparticles were added to the crude extract of shiitake polysaccharide at a ratio of 10 mg / mL, and the mixture was stirred and adsorbed at 20°C for 2 hours.

[0083] S4. Apply an external magnetic field to the mixed system in S3 to separate the magnetic nanoparticles loaded with lentinan from the liquid phase. Discard the supernatant to obtain the magnetic nanoparticle-polysaccharide complex.

[0084] S5. Add a carbonate buffer solution (containing 500 mmol / L mannitol) with a pH of 9.0 to the magnetic nanoparticle-polysaccharide complex and stir and desorb for 2 hours at 20°C.

[0085] S6. Apply an external magnetic field to the mixed system in S5 to separate and remove the magnetic nanoparticles, and collect the desorption solution containing lentinan.

[0086] S7. The desorbed solution from S6 is purified by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa at an operating pressure of 0.1 MPa, and the retentate is collected.

[0087] S8. The purified lentinan solution is vacuum dried at 50°C to obtain lentinan powder.

[0088] The extraction rate of lentinan obtained in this embodiment was 18.5%, the polysaccharide purity was 87.4%, and the protein content was 2.1%.

[0089] Example 5: Isolation and purification of Dendrobium officinale polysaccharides

[0090] S1. Take dried Dendrobium officinale stems, crush them with a pulverizer and pass them through a 60-mesh sieve to obtain Dendrobium officinale powder.

[0091] S2. Mix betaine and malic acid at a molar ratio of 1:2.5 and stir at 85°C until a homogeneous and transparent eutectic solvent is formed. Dilute the eutectic solvent with deionized water to a mass concentration of 70% to obtain an aqueous solution of the eutectic solvent.

[0092] Weigh 100g of Dendrobium officinale powder and add 1000mL of the above eutectic solvent aqueous solution (mass-volume ratio 1:10g / mL). Stir and extract at 75℃ for 2.5 hours. After extraction, centrifuge at 9000rpm for 12 minutes and collect the supernatant to obtain the crude extract of Dendrobium officinale polysaccharides.

[0093] S3. Fe3O4 nanoparticles with a particle size of about 25 nm were prepared by coprecipitation method. The Fe3O4 nanoparticles were dispersed in an ethanol-water mixed solvent, 3-aminophenylboronic acid was added, and the reaction was stirred at room temperature for 15 hours to obtain magnetic nanoparticles with phenylboronic acid groups on the surface.

[0094] The prepared Fe3O4 magnetic nanoparticles were added to the crude extract of Dendrobium officinale polysaccharide at a ratio of 6 mg / mL, and the mixture was stirred and adsorbed at 30°C for 1 hour.

[0095] S4. Apply an external magnetic field to the mixed system in S3 to separate the magnetic nanoparticles loaded with Dendrobium officinale polysaccharide from the liquid phase, discard the supernatant, and obtain the magnetic nanoparticle-polysaccharide complex.

[0096] S5. Add a Tris-HCl buffer solution (containing 250 mmol / L sorbitol) with a pH of 8.0 to the magnetic nanoparticle-polysaccharide complex, and stir and desorb for 1 hour at 30°C.

[0097] S6. Apply an external magnetic field to the mixed system in S5 to separate and remove the magnetic nanoparticles, and collect the desorption solution containing Dendrobium officinale polysaccharide.

[0098] S7. The desorbed solution from S6 is purified by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 30 kDa at an operating pressure of 0.2 MPa, and the retentate is collected.

[0099] S8. The purified Dendrobium officinale polysaccharide solution was freeze-dried under vacuum to obtain Dendrobium officinale polysaccharide powder.

[0100] The extraction rate of Dendrobium officinale polysaccharide obtained in this embodiment was 22.1%, the polysaccharide purity was 93.8%, and the protein content was 1.1%.

[0101] Example 6: Isolation and purification of Astragalus polysaccharides

[0102] S1. Take dried Astragalus root, grind it with a pulverizer and pass it through an 80-mesh sieve to obtain Astragalus powder.

[0103] S2. Mix choline chloride with lactic acid and glycerol in a molar ratio of 1:1:1 (the total amount of hydrogen bond donors to the molar ratio of hydrogen bond acceptors is 2:1), stir at 80°C until a homogeneous and transparent eutectic solvent is formed, and dilute the eutectic solvent with deionized water to a mass concentration of 55% to obtain an aqueous solution of the eutectic solvent.

[0104] Weigh 100g of Astragalus powder and add 2000mL of eutectic solvent aqueous solution (mass-volume ratio 1:20g / mL). Stir and extract at 65℃ for 2 hours. After extraction, centrifuge at 8000rpm for 15 minutes and collect the supernatant to obtain crude extract of Astragalus polysaccharide.

[0105] S3. Fe3O4 nanoparticles were prepared by solvothermal method, and then SiO2 shell was coated by sol-gel method to obtain Fe3O4@SiO2 core-shell structured nanoparticles. Fe3O4@SiO2 nanoparticles were dispersed in toluene, 4-carboxyphenylboronic acid was added, and the reaction was carried out under reflux for 24 hours to obtain core-shell structured magnetic nanoparticles with phenylboronic acid groups on the surface.

[0106] The prepared Fe3O4@SiO2 magnetic nanoparticles were added to the crude extract of Astragalus polysaccharide at a ratio of 7 mg / mL, and the mixture was stirred and adsorbed at 28℃ for 1.2 hours.

[0107] S4. Apply an external magnetic field to the mixed system in S3 to separate the magnetic nanoparticles loaded with Astragalus polysaccharide from the liquid phase. Discard the supernatant to obtain the magnetic nanoparticle-polysaccharide complex.

[0108] S5. Add a phosphate buffer solution (containing 350 mmol / L mannitol) with a pH of 8.2 to the magnetic nanoparticle-polysaccharide complex and stir and desorb for 1.2 hours at 28°C.

[0109] S6. Apply an external magnetic field to the mixed system in S5 to separate and remove the magnetic nanoparticles, and collect the desorption solution containing astragalus polysaccharide.

[0110] S7. The desorbed solution from S6 is purified by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 50 kDa at an operating pressure of 0.18 MPa, and the retentate is collected.

[0111] S8. The purified Astragalus polysaccharide solution is spray-dried at an inlet air temperature of 170℃ and an outlet air temperature of 75℃ to obtain Astragalus polysaccharide powder.

[0112] The extraction rate of Astragalus polysaccharide obtained in this embodiment was 16.7%, the polysaccharide purity was 91.3%, and the protein content was 1.5%.

[0113] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0114] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for isolating and purifying plant polysaccharides, characterized in that, Includes the following steps: S1. Raw material pretreatment: After drying, the plant raw materials are pulverized and sieved to obtain plant raw material powder; S2. Extraction: Add the plant raw material powder from S1 to the extraction solvent, stir and extract at 50-90℃ for 0.5-3 hours, centrifuge, collect the supernatant to obtain crude plant polysaccharide extract; S3, Magnetic affinity adsorption: Magnetic nanoparticles with surface-modified affinity ligands are prepared and added to the crude plant polysaccharide extract in S2. The mixture is stirred and adsorbed at 20-40°C for 0.5-2 hours, so that the plant polysaccharide molecules are selectively bound to the surface of the magnetic nanoparticles through the affinity ligands. S4. Magnetic separation and collection: An external magnetic field is applied to the mixed system in S3 to separate the magnetic nanoparticles loaded with plant polysaccharides from the liquid phase. The supernatant is discarded to obtain the magnetic nanoparticle-polysaccharide complex. S5. Desorption and release: Add a desorbent to the magnetic nanoparticle-polysaccharide complex in S4, and stir at 20-40℃ for 0.5-2 hours to desorb and release the plant polysaccharide from the surface of the magnetic nanoparticles. S6. Secondary magnetic separation: An external magnetic field is applied to the mixed system in S5 to separate and remove magnetic nanoparticles, and the desorption liquid containing plant polysaccharides is collected. S7. Purification: The desorption solution in S6 is purified by membrane separation, and the purified polysaccharide solution is collected. S8. Drying: The polysaccharide solution in S7 is dried to obtain purified plant polysaccharides.

2. The method for isolating and purifying plant polysaccharides according to claim 1, characterized in that, The extraction solvent in S2 is an eutectic solvent aqueous solution, composed of a hydrogen bond donor and a hydrogen bond acceptor in a molar ratio of 1:0.5 to 3. The hydrogen bond donor is selected from one or more of lactic acid, malic acid, citric acid, ethylene glycol, and glycerol, and the hydrogen bond acceptor is selected from one or more of choline chloride, betaine, and tetramethylammonium chloride.

3. The method for isolating and purifying plant polysaccharides according to claim 2, characterized in that, The mass concentration of the eutectic solvent aqueous solution is 20% to 80%, and the mass-volume ratio of plant raw material powder to eutectic solvent aqueous solution is 1:5 to 1:30 g / mL.

4. The method for isolating and purifying plant polysaccharides according to claim 1, characterized in that, The magnetic nanoparticles in S3 are Fe3O4 nanoparticles or Fe3O4@SiO2 core-shell structured nanoparticles, and the affinity ligands are phenylboronic acid groups or their derivatives.

5. The method for isolating and purifying plant polysaccharides according to claim 1, characterized in that, The desorbent in S5 is a buffer solution containing sorbitol or mannitol, wherein the pH of the buffer solution is 7.0 to 9.0, and the concentration of sorbitol or mannitol is 100 to 500 mmol / L.

6. The method for isolating and purifying plant polysaccharides according to claim 1, characterized in that, The membrane separation and purification described in S7 uses an ultrafiltration membrane with a molecular weight cutoff of 10–100 kDa.

7. The method for isolating and purifying plant polysaccharides according to claim 1, characterized in that, The plant material mentioned in S1 is selected from one or more of Polygonatum sibiricum, Lycium barbarum, Ganoderma lucidum, Shiitake mushroom, Dendrobium officinale, and Astragalus membranaceus.

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