A cascade separation and purification method for isolating astragalus polysaccharides from astragalus.

Abstract

This invention belongs to the field of separation and purification technology, and relates to a cascade separation and purification method for separating astragalus polysaccharides from astragalus. The method includes: first, gradient filtration of the coarse filtrate of astragalus root slices through multi-stage microfiltration membranes, collecting the clarified liquid; second, separation and concentration of the clarified liquid using an ultrafiltration membrane, collecting the membrane retentate to obtain a first concentrated liquid; third, addition of papain to the first concentrated liquid for enzymatic protein desorption, followed by centrifugation and collection of the first supernatant; fourth, addition of Sevage reagent to the first supernatant for vortex extraction, followed by centrifugation and collection of the second supernatant; fifth, treatment of the second supernatant using an ultrafiltration membrane, simultaneously washing, desalting, and concentrating to obtain a second concentrated liquid; and sixth, addition of ethanol to the second concentrated liquid for alcohol precipitation, collecting the precipitate, redissolving, and drying to obtain astragalus polysaccharides. This invention achieves the separation and purification of astragalus polysaccharides through a combination of multi-stage membrane separation, enzymatic protein desorption, extraction, washing, desalting, and alcohol precipitation, featuring good impurity removal, wide process conditions, and ease of industrialization.

Description

Technical Field

[0001] This invention relates to the field of separation and purification technology, specifically to a cascade separation and purification method for separating astragalus polysaccharides from astragalus. Background Technology

[0002] Astragalus polysaccharide (APS) is one of the main active components of Astragalus membranaceus, possessing immunomodulatory and antioxidant bioactivities, and has certain application value in traditional Chinese medicine preparations and related products. Studies have shown that the bioactivity of Astragalus polysaccharide is closely related to its molecular weight and distribution. Astragalus polysaccharides with low molecular weight and uniform molecular weight distribution generally have better water solubility, bioavailability, and pharmacological activity. Therefore, obtaining Astragalus polysaccharide products with suitable molecular weight and good uniformity has important application value.

[0003] Currently, the extraction of Astragalus polysaccharides typically employs a water extraction and alcohol precipitation method. This involves first extracting the Astragalus raw material with water, followed by concentration and alcohol precipitation of the extract. However, the Astragalus polysaccharides obtained through traditional direct extraction methods are naturally distributed, with molecular weights typically extremely large and wide-ranging, making it impossible to obtain specific low-molecular-weight polysaccharide components, resulting in poor product uniformity. To obtain low-molecular-weight Astragalus polysaccharides, existing technologies often employ acid, alkali, or conventional enzymatic hydrolysis methods for degradation. However, the bond breaking in these degradation methods is random. Although the average molecular weight is reduced, the product becomes a hodgepodge containing monosaccharides, oligosaccharides, and various structurally incomplete polysaccharide fragments. This not only disrupts the product's uniformity but may also damage the active structure of the Astragalus polysaccharides, leading to a decrease in their biological activity.

[0004] In addition to polysaccharides, Astragalus aqueous extract also contains impurities such as proteins, inorganic salts, pigments, small-molecule sugars, and suspended particles. Direct concentration and alcohol precipitation can easily lead to co-precipitation of polysaccharides and impurities, affecting the purity of the obtained Astragalus polysaccharides. To remove proteins from the aqueous extract, current technologies typically employ enzymatic or Sevage methods. Enzymatic deproteinization operates under relatively mild conditions, but when used alone, it may result in insufficient protein removal. The Sevage method can remove some proteins, but it usually requires multiple extractions, consumes large amounts of organic reagents, and may cause polysaccharide loss. Therefore, using only one deproteinization method makes it difficult to simultaneously achieve effective deproteinization and high polysaccharide yield.

[0005] Furthermore, astragalus water extract contains a certain amount of suspended particles and colloidal substances. When directly treated with small-pore microfiltration or ultrafiltration membranes, problems such as decreased filtration speed and membrane fouling are likely to occur. Pre-filtration with ordinary filter paper has limited effect on removing fine particles and colloidal substances and may still affect subsequent microfiltration and ultrafiltration processes. Summary of the Invention

[0006] To address the technical problems in existing technologies, such as the extremely large molecular weight, wide distribution, and poor uniformity of Astragalus polysaccharide direct extraction products, the random bond breaking and easy destruction of polysaccharide structure during acid-base or conventional enzymatic hydrolysis to prepare low molecular weight polysaccharides, and the difficulty in efficiently removing impurities such as proteins, small molecules, and inorganic salts from aqueous extracts, and the difficulty in achieving both purity and yield during purification, this invention provides a cascade separation and purification method for separating Astragalus polysaccharides from Astragalus. Through steps such as fractional microfiltration, ultrafiltration concentration, combined deproteinization, ultrafiltration washing and desalting, and alcohol precipitation, the separation and purification effect of Astragalus polysaccharides is improved.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A cascade separation and purification method for isolating astragalus polysaccharides from astragalus, comprising the following steps: (1) The coarse filtrate obtained by water extraction and solid-liquid separation of Astragalus membranaceus slices is sequentially filtered through multiple microfiltration membranes with progressively smaller pore sizes, and the clear liquid is collected. (2) The clarified liquid is concentrated by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 5~10 kDa, and the membrane retentate is collected to obtain the first concentrate; (3) Papain was added to the first concentrate to enzymatically decompose the protein. After inactivation and centrifugation, the first supernatant was collected. Sevage reagent was added to the first supernatant for shaking extraction. After centrifugation, the second supernatant was collected. (4) The second supernatant was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 5-10 kDa, and ultrapure water was dynamically added during the ultrafiltration process for washing, desalting and concentration to obtain the second concentrate. (5) Add ethanol to the second concentrate for alcohol precipitation, separate and collect the precipitate, redissolve the precipitate and dry it to obtain Astragalus polysaccharide.

[0008] Further, in step (1), the Astragalus membranaceus slices are crushed and ultrasonically degreased with ethanol or an aqueous solution of ethanol with a volume fraction of 70% to 95%.

[0009] Further, collect the defatted solids, and add ultrapure water in 1 to 3 portions based on the initial mass of the Astragalus membranaceus slices. The ratio of the mass of the Astragalus membranaceus slices to the cumulative volume of ultrapure water added is 1 g: (10 to 20) mL. Extract in a water bath at 70 to 90 ℃ for 60 to 90 min each time.

[0010] Further, in step (1), the coarse filtrate is subjected to gradient cascade filtration through microfiltration membranes with pore sizes of 10-20 μm, 3-5 μm, and 0.2-0.65 μm. Preferably, the coarse filtrate is subjected to gradient cascade filtration through microfiltration membranes with pore sizes of 10 μm, 5 μm, and 0.45 μm. Preferably, the microfiltration membrane is an MCE membrane.

[0011] Further, in step (2), the transmembrane pressure of the ultrafiltration concentration is 0.1~0.3 MPa, and the clarified liquid is concentrated to 1 / 10~1 / 4 of the initial volume to obtain the first concentrated liquid.

[0012] Further, in step (3), the added papain is 1% to 3% of the mass of the first concentrate; the enzymatic hydrolysis of the protein is carried out at 55 to 60 °C for 3 to 4 h, and after the enzymatic hydrolysis is completed, the temperature is raised to 70 to 90 °C and held for 10 to 15 min to inactivate the papain, and then the first supernatant is collected by centrifugation.

[0013] Further, in step (3), the Sevage reagent is prepared by mixing chloroform and n-butanol in a volume ratio of 4:1; the volume ratio of the first supernatant to the Sevage reagent is (4~5):1, and the mixture is extracted by shaking at 200 r / min for 30~50 min.

[0014] Further, in step (4), the second supernatant is subjected to ultrafiltration, and ultrapure water is added simultaneously in equal volume. The cumulative volume of ultrapure water added is 5 to 10 times the initial volume of the second supernatant.

[0015] Further, in step (5), anhydrous ethanol of 2 to 4 times its volume is added to the second concentrate, and the mixture is allowed to stand at 4 °C for 10 to 18 h for alcohol precipitation. The precipitate is then collected by centrifugation.

[0016] Furthermore, the drying process is vacuum freeze drying.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects: (1) This invention uses membrane separation methods such as gradient microfiltration, ultrafiltration concentration, and ultrafiltration washing to perform gentle physical fractionation and purification of Astragalus polysaccharides. It does not require degradation methods such as acid, alkali, or conventional enzymatic hydrolysis that are prone to random bond breaking, and can obtain Astragalus polysaccharide products with low molecular weight, narrow distribution, and good uniformity. The weight average molecular weight Mw is 5.073 × 10⁻⁶. 4 g / mol, peak molecular weight Mp is 2.812 × 10⁻⁶ g / mol. 4 With a concentration of g / mol and a dispersion coefficient Mw / Mn of 1.519, the mass fraction of the main molecular weight components can reach 99.1%, thus avoiding the destruction of polysaccharide chain integrity and biological activity during the degradation process.

[0018] (2) In this invention, the coarse filtrate is sequentially filtered through a multi-stage microfiltration membrane with progressively smaller pore sizes. This process can gradually trap and remove impurities such as suspended particles and colloids, significantly reducing the filtration load on the small-pore-size microfiltration membrane and subsequent ultrafiltration membrane, delaying membrane fouling, maintaining a high filtration flux, and facilitating the stable operation and continuous operation of the separation process.

[0019] (3) In this invention, papain is first used to enzymatically deproteinize the first concentrate, and then Sevage reagent is used to shake and extract the first supernatant to remove protein. The two deproteinization methods complement each other and can remove the protein in the Astragalus aqueous extract stepwise and thoroughly. The protein content in the obtained product is lower than the detection limit of the BCA protein quantification method. At the same time, the enzymatic hydrolysis can remove most of the protein in advance, reduce the amount of Sevage reagent used, and avoid the large amount of organic reagent and polysaccharide loss caused by multiple extractions in the single Sevage method. It takes into account both the deproteinization effect and the polysaccharide yield.

[0020] (4) In the ultrafiltration process of this invention, an equal volume of ultrapure water is added simultaneously for washing, which can simultaneously achieve desalination and concentration, effectively removing impurities such as small molecule sugars, inorganic salts and pigments, reducing the co-precipitation of impurities and astragalus polysaccharides during alcohol precipitation, and improving product purity. The total polysaccharide content of the obtained astragalus polysaccharide product can reach 98.8%~99.3%, and no protein was detected. The product is a pure white powder with good appearance quality.

[0021] (5) The process conditions of each step of the present invention are mild, the operating parameters are wide and easy to control, which is convenient for industrial production. When the raw material input is increased from 100 g to 1400 g, the purity of the obtained Astragalus polysaccharide product can still be stably maintained at more than 99%, which shows that the process of the present invention has good stability and good prospects for industrial application. Attached Figure Description

[0022] Figure 1 This is a process flow diagram of the Astragalus polysaccharide cascade separation and purification method of the present invention; Figure 2 This is a multi-angle laser scattering detection image of the Astragalus polysaccharide obtained in Example 1; Figure 3 These are electron micrographs of Astragalus polysaccharide obtained in Example 1 at different magnifications. Detailed Implementation

[0023] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are implemented based on the technical solution of the present invention, providing detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0024] Example 1: This embodiment provides a cascade separation and purification method for isolating astragalus polysaccharides from astragalus, such as... Figure 1 As shown, the specific steps are as follows: (1) Degreasing treatment: After pulverizing the Astragalus membranaceus slices, weigh 100 g of the pulverized slices, add 2000 mL of anhydrous ethanol, and treat with ultrasound at 300 W for 20 min. Filter and collect the solid. Add another 2000 mL of anhydrous ethanol to the solid, treat with ultrasound at 300 W for 10 min, filter and collect the solid. Dry the solid at room temperature to obtain defatted Astragalus membranaceus powder for later use.

[0025] (2) Water extraction and preliminary solid-liquid separation: Add 1200 mL of ultrapure water to the defatted Astragalus powder obtained in step (1), extract in a water bath at 85 ℃ for 90 min, squeeze and filter and collect the first extract; add 800 mL of ultrapure water to the obtained filter residue, extract in a water bath at 85 ℃ for 60 min, squeeze and filter and collect the second extract.

[0026] A total of 2000 mL of ultrapure water was added during the two extractions. The ratio of the initial mass of Astragalus membranaceus slices to the cumulative volume of ultrapure water added was 1 g: 20 mL.

[0027] The two extracts were combined, filtered through a 150-mesh filter, and centrifuged at 4000 r / min for 20 min. The supernatant was collected, yielding approximately 1600 mL of coarse filtrate.

[0028] (3) Gradient microfiltration: The coarse filtrate obtained in step (2) was passed through microfiltration membranes with pore sizes of 10 μm, 5 μm and 0.45 μm in sequence, and the clarified liquid was collected.

[0029] (4) First ultrafiltration concentration: An ultrafiltration membrane with a molecular weight cutoff of 10 kDa was used to concentrate the clarified solution obtained in step (3) under ultrafiltration conditions of 0.25 MPa transmembrane pressure and room temperature. Approximately 1600 mL of the clarified solution was concentrated to approximately 300 mL, and the membrane retentate was collected to obtain the first concentrate.

[0030] (5) Enzymatic decomposition of proteins: Papain was added to the first concentrate obtained in step (4), and the mass of the added papain was 1.5% of the mass of the first concentrate. The mixture was then enzymatically hydrolyzed at 55 °C for 3 h.

[0031] After enzymatic hydrolysis, the temperature was raised to 85 °C and held for 10 min to inactivate papain; then centrifuged at 10000 r / min for 10 min and the first supernatant was collected.

[0032] (6) Sevage extraction method for protein removal: Add Sevage reagent to the first supernatant obtained in step (5) at a volume ratio of 4:1, and extract by shaking at 200 r / min for 30 min.

[0033] After extraction, the layers were separated by centrifugation, and the upper aqueous phase was collected to obtain a second supernatant. The Sevage reagent was prepared by mixing chloroform and n-butanol in a volume ratio of 4:1.

[0034] (7) Ultrafiltration washing, desalination, and concentration: The second supernatant obtained in step (6) was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and ultrapure water was added simultaneously in equal volumes. When the cumulative volume of ultrafiltration permeate reached 5 times the initial volume of the second supernatant, the addition of ultrapure water was stopped, and ultrafiltration concentration was continued until the membrane filtration volume was approximately 200 mL, thus obtaining the second concentrate.

[0035] (8) Alcohol precipitation and freeze drying: Add 800 mL of anhydrous ethanol to the approximately 200 mL of the second concentrate obtained in step (7), mix well, and let it stand at 4 °C for 14 h for alcohol precipitation.

[0036] Then, centrifuge at 9000 r / min for 10 min, collect the precipitate, and add an appropriate amount of ultrapure water to completely dissolve the precipitate.

[0037] The resulting reconstituted solution was pre-frozen at -80 °C for 6 h, and then freeze-dried under vacuum for 36 h at a condenser temperature not higher than -55 °C and a vacuum degree not higher than 15 Pa to obtain 425 mg of Astragalus polysaccharide powder.

[0038] Example 2: This embodiment separates Astragalus polysaccharides according to the method described in Example 1, except that the amount of Astragalus slices crushed is 200 g, the amount of ethanol and ultrapure water is adjusted proportionally to the amount of Astragalus slices crushed, and the amounts of papain and Sevage reagent are determined according to the mass ratio and volume ratio described in Example 1, respectively. Other process conditions are the same as in Example 1.

[0039] After alcohol precipitation and vacuum freeze drying, 717 mg of Astragalus polysaccharide powder was obtained.

[0040] Example 3: This embodiment provides a method for separating Astragalus polysaccharides from Astragalus slices, and adds an ultrafiltration concentration step after enzymatic protein desorption and before Sevage extraction for protein removal, as detailed below: (1) Degreasing treatment: Astragalus root slices were dried, pulverized, and passed through a 60-mesh sieve. 1400 g of the resulting Astragalus powder was weighed out and approximately 8 L of anhydrous ethanol was added to submerge the powder. The powder was then sonicated for 20 min, filtered, and the solid was collected. Approximately 8 L of anhydrous ethanol was added again to submerge the solid, and the powder was sonicated for 10 min. The solid was then filtered and collected. The resulting solid was air-dried at room temperature for 24 h to obtain defatted Astragalus powder for later use.

[0041] (2) Water extraction and solid-liquid separation: Approximately 1280g of defatted Astragalus membranaceus powder was collected and added to 12.8L of ultrapure water at a material-to-liquid ratio of 1g:10mL. A first constant-temperature water extraction was performed at 85℃ for 90 min, followed by filtration through a 120-mesh filter to collect the first extract. Another 12.8L of ultrapure water was added to the filter residue at a material-to-liquid ratio of 1g:10mL, and a second constant-temperature water extraction was performed at 85℃ for 60 min. The second extract was then collected after filtration through a 120-mesh filter. The two extracts were combined and filtered through a 200-mesh filter. The mixture was then centrifuged at 4000rpm for 20 min in a large-capacity industrial centrifuge to obtain 22L of coarse filtrate.

[0042] (3) Gradient microfiltration: The coarse filtrate obtained in step (2) was continuously passed through microfiltration membranes with pore sizes of 10 μm, 5 μm and 0.45 μm, and the clarified liquid was collected.

[0043] (4) First ultrafiltration concentration: Using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, the clarified liquid obtained in step (3) was concentrated by ultrafiltration under the conditions of a transmembrane pressure of 0.25 MPa and room temperature. About 22 L of the clarified liquid was concentrated to about 4.2 L, and the membrane retentate was collected to obtain the first concentrate.

[0044] (5) Enzymatic decomposition of proteins: Add 1.5% of papain by mass to the first concentrate obtained in step (4), and perform enzymatic hydrolysis at 55°C for 3 h.

[0045] The enzyme was then inactivated by heating to 85°C and holding for 15 minutes, followed by centrifugation at 10,000 rpm for 10 minutes, and the first supernatant was collected.

[0046] (6) Second ultrafiltration concentration: The first supernatant obtained in step (5) was concentrated by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to a total volume of 750 mL, resulting in a polysaccharide concentrate.

[0047] (7) Sevage extraction for protein removal: With a volume ratio of polysaccharide concentrate to Sevage reagent of 4:1, 187.5 mL of Sevage reagent was added to the 750 mL polysaccharide concentrate obtained in step (6), and the mixture was extracted by shaking at 200 r / min for 50 min. After extraction, the mixture was centrifuged to separate the layers, and the upper aqueous phase was collected to obtain the second supernatant.

[0048] (8) Ultrafiltration washing, desalination, and concentration: The second supernatant obtained in step (7) was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and ultrapure water was added simultaneously in equal volumes. When the cumulative volume of ultrafiltration permeate reached 5 times the initial volume of the second supernatant, the addition of ultrapure water was stopped, and ultrafiltration concentration was continued until the membrane filtration volume was approximately 600 mL, thus obtaining the second concentrate.

[0049] (9) Alcohol precipitation and freeze drying: Add four times the volume of anhydrous ethanol to the second concentrate obtained in step (8), and allow it to stand at 4 °C for 12 h for alcohol precipitation. Centrifuge at 10000 r / min for 15 min, collect the precipitate, and add an appropriate amount of ultrapure water to completely dissolve the precipitate. Pre-freeze the resulting reconstituted solution at -80 °C for 10 h, and then freeze-dry under vacuum for 48 h to obtain 11.12 g of Astragalus polysaccharide product.

[0050] Comparative Example 1: This comparative example provides a method for separating astragalus polysaccharides from astragalus, which is basically the same as that in Example 1, except that only enzymatic deproteinization is performed, and Sevage extraction is not performed to remove protein. The process conditions for the remaining steps are the same as those in Example 1, and the feed amount is 50 g of crushed astragalus slices.

[0051] After alcohol precipitation and vacuum freeze-drying, 852 mg of Astragalus polysaccharide powder was obtained. Analysis showed that the polysaccharide content was 82.5%, the protein content was 6.8%, and the product was a pure white, loose powder.

[0052] Comparative Example 2: This comparative example provides a method for separating astragalus polysaccharides from astragalus, which is basically the same as that in Example 1, except that: enzymatic deproteinization is not performed, only Sevage extraction is performed to remove protein, and the process conditions of the remaining steps are the same as those in Example 1, with a feed amount of 50 g of crushed astragalus slices.

[0053] After alcohol precipitation and vacuum freeze-drying, 112 mg of Astragalus polysaccharide powder was obtained. Analysis showed that the polysaccharide content was 78.2%, the protein content was 5.7%, and the product was a pure white, loose powder.

[0054] Comparative Example 3: This comparative example uses a method that separates Astragalus polysaccharides without gradient microfiltration, enzymatic deproteinization, Sevage extraction for deproteinization, and ultrafiltration washing and desalting. The specific steps are as follows: (1) Degreasing treatment: The defatting process was carried out according to the method described in step (1) of Example 1, except that the amount of crushed Astragalus membranaceus slices was 200 g and the amount of ethanol was adjusted accordingly based on the amount of material added.

[0055] (2) Water extraction and solid-liquid separation: Astragalus root slices were added to ultrapure water at a ratio of 1 g: 20 mL, and the mixture was extracted at a constant temperature of 80 °C for 2.5 h. The extract was then filtered through a 200-mesh sieve, collected, and centrifuged at 5000 r / min for 10 min to collect the supernatant.

[0056] (3) Reduced pressure concentration: The supernatant obtained in step (2) was concentrated under reduced pressure at 50 °C to about 1 / 6 of its original volume to obtain the concentrate.

[0057] (4) Alcohol precipitation: Add 4 times the volume of anhydrous ethanol to the concentrate obtained in step (3), and let it stand at 4 °C for 14 h for alcohol precipitation. After the alcohol precipitation is completed, centrifuge at 5000 r / min for 10 min, collect the precipitate, and add an appropriate amount of ultrapure water to completely dissolve the precipitate to obtain a polysaccharide complex solution.

[0058] (5) Dialysis desalination: The polysaccharide complex solution obtained in step (4) was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in tap water for 8 h, with the tap water being changed every 2 h; then it was transferred to ultrapure water and dialyzed for 16 h, with the ultrapure water being changed every 4 h.

[0059] (6) Freeze-drying: The dialyzed polysaccharide solution was pre-frozen at -80 °C for 10 h, and then freeze-dried under vacuum for 48 h to obtain 3.78 g of Astragalus polysaccharide powder.

[0060] Comparative Example 4: This comparative example used multiple Sevage extractions for deproteinization, but did not undergo gradient microfiltration, enzymatic deproteinization, or ultrafiltration washing and desalting. The specific steps are as follows:

[0061] (1) Degreasing treatment: The defatting process was carried out according to the method described in step (1) of Example 1, except that the amount of crushed Astragalus membranaceus slices was 200 g and the amount of ethanol was adjusted accordingly based on the amount of material added.

[0062] (2) Water extraction and solid-liquid separation: Water extraction and solid-liquid separation were performed according to step (2) of Example 1, except that the amount of Astragalus membranaceus slices crushed was 200 g, and the amount of ultrapure water was adjusted accordingly based on the amount of material added. The two extracts were combined, filtered through a 200-mesh filter, and centrifuged at 5000 r / min for 10 min, and the supernatant was collected. This comparative example did not undergo gradient microfiltration.

[0063] (3) Reduced pressure concentration: The supernatant obtained in step (2) was concentrated under reduced pressure at 50 °C to about 1 / 6 of its original volume to obtain the concentrate.

[0064] (4) Sevage extraction to remove protein: Add Sevage reagent to the concentrate obtained in step (3) at a volume ratio of 4:1 between the concentrate and Sevage reagent, shake at 200 r / min for 20 min, then centrifuge at 5000 rpm for 10 min, and collect the upper aqueous phase after the liquid has separated into layers.

[0065] The volume ratio of the obtained upper aqueous phase to the newly added Sevage reagent was 4:1. The above shaking extraction and centrifugation separation operations were repeated. The total number of Sevage extractions was 4. The final obtained upper aqueous phase was collected.

[0066] (5) Alcohol precipitation: Add 4 times the volume of anhydrous ethanol to the upper aqueous phase obtained in step (4), let it stand at 4 °C for 14 h to precipitate the ethanol, centrifuge and collect the precipitate, and add ultrapure water to dissolve the precipitate.

[0067] (6) Dialysis desalination: The polysaccharide complex solution obtained in step (5) was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in tap water for 8 hours, with the tap water being changed every 2 hours; then it was transferred to ultrapure water and dialyzed for another 16 hours, with the ultrapure water being changed every 4 hours.

[0068] (7) Freeze-drying: The dialyzed polysaccharide solution was pre-frozen at -80 °C for 10 h, and then freeze-dried under vacuum for 48 h to obtain 3.26 g of Astragalus polysaccharide powder.

[0069] Astragalus polysaccharide product detection and characterization: To evaluate the performance of the Astragalus polysaccharide products prepared by the method of the present invention, the polysaccharide content, protein residue, and appearance of the Astragalus polysaccharide products obtained in Examples 1-3 and Comparative Examples 1-4 were evaluated; and the molecular weight and microstructure of the Astragalus polysaccharide product obtained in Example 1 were characterized as a representative.

[0070] (1) Detection of polysaccharide content and protein residue: The total polysaccharide content in the Astragalus polysaccharide products was determined using the phenol-sulfuric acid method; the residual protein content was determined using the BCA protein quantification method. The Astragalus polysaccharide products obtained in Examples 1-3 and Comparative Examples 1-4 were tested, and a comprehensive evaluation was conducted based on the product appearance. The results are shown in Table 1.

[0071] Table 1: Test Results of Astragalus Polysaccharide Products

[0072] As shown in Table 1, Comparative Example 3, using the traditional water extraction and alcohol precipitation process without gradient microfiltration, enzymatic protein removal, Sevage extraction for protein removal, and ultrafiltration washing and desalting, yielded a product with a polysaccharide content of only 47.49% and a protein content as high as 24.70%. Furthermore, the product was a pale yellow, fluffy sponge. This was mainly because a large amount of protein, pigments, and other impurities in the Astragalus water extract were not removed, and they co-precipitated with the Astragalus polysaccharides during alcohol precipitation, resulting in a significant decrease in product purity and discoloration. Comparative Example 4, using only multiple Sevage extractions for protein removal without gradient microfiltration, enzymatic protein removal, and ultrafiltration washing and desalting, still yielded a product with a protein content of 9.10% and a polysaccharide content of [missing information]. The protein content was 67.70%, indicating that repeated Sevage extraction alone could not completely remove the protein, and impurities such as suspended particles, colloids, and small molecule pigments were not effectively removed. Comparative Examples 1 and 2 used only enzymatic deproteinization or Sevage extraction to remove the protein, respectively, and the protein content in the products was 6.8% and 5.7%, respectively. This shows that both single deproteinization methods have the defect of insufficient protein removal. Although papain enzymatic hydrolysis can degrade most proteins into small molecule peptides under mild conditions, it has limited removal of residual proteins that are tightly bound to polysaccharides. Although Sevage reagent can denature proteins and remove them by precipitation at the interface, it is difficult to remove them completely in a single extraction.

[0073] In contrast, Examples 1-3, based on gradient microfiltration and ultrafiltration concentration, employed a two-step combined deproteinization process using papain enzymatic hydrolysis and Sevage extraction, supplemented by ultrafiltration washing and desalting to remove small molecules and inorganic salts. The protein content of the resulting Astragalus polysaccharide products was all below the detection limit of the BCA protein quantification method, and the total polysaccharide content reached 98.8%-99.3%. The products were pure white loose powder or fluffy sponge.

[0074] (2) Detection of molecular weight and molecular weight distribution: Using the Astragalus polysaccharide product obtained in Example 1 as the sample, high-performance gel permeation chromatography-multi-angle laser light scattering (HPGFC-MALLS) was employed for detection. An Agilent 1260 high-performance liquid chromatography system equipped with a multi-angle laser light scattering detector was used. The chromatographic column was a Shodex OHpak SB-805 HQ gel column, with 0.1 mol / L sodium nitrate solution as the mobile phase, a flow rate of 0.5 mL / min, and a column temperature of 40 ℃. 50 mg of lyophilized Astragalus polysaccharide sample was weighed, fully dissolved in the mobile phase, and brought to a final volume of 10 mL. After complete dissolution, the sample was injected for detection. Data processing was performed based on the light scattering signal and sample concentration to calculate the weight-average molecular weight (Mw), peak molecular weight (Mp), and dispersion factor (Mw / Mn).

[0075] The test results showed that the main molecular weight component of the Astragalus polysaccharide obtained in Example 1 was 99.1% by mass, and the weight-average molecular weight (Mw) was 5.073 × 10⁻⁶. 4 g / mol, peak molecular weight Mp is 2.812 × 10⁻⁶ g / mol. 4 g / mol, dispersion coefficient Mw / Mn is 1.519, and the detection results are as follows: Figure 2 As shown.

[0076] (3) Microscopic morphological characterization: An appropriate amount of freeze-dried Astragalus polysaccharide (APS) sample powder was uniformly dispersed on the surface of a sample stage coated with conductive adhesive. After gold sputtering, the sample was observed using a scanning electron microscope. Images were acquired at magnifications of 500×, 2000×, 5000×, and 10000× to observe the surface morphology, particle state, and microstructure characteristics of the Astragalus polysaccharide sample obtained in Example 1. The results are as follows: Figure 3 As shown.

[0077] The above results demonstrate that the present invention removes suspended particles and impurities such as colloids, proteins, small molecules, inorganic salts, and pigments stepwise through a cascade treatment of microfiltration, ultrafiltration concentration, combined deproteinization, ultrafiltration washing and desalting, and alcohol precipitation. Each step works synergistically: gradient microfiltration reduces the subsequent membrane fouling load and protects the ultrafiltration membrane; ultrafiltration achieves polysaccharide retention and enrichment; enzymatic hydrolysis and Sevage extraction complement each other to ensure complete deproteinization; and washing and desalting reduce the co-precipitation of impurities during alcohol precipitation. Thus, while improving the purity of Astragalus polysaccharides and reducing protein residue, the process maintains a good product appearance. The process effect is significantly better than existing methods that only use single deproteinization or do not perform cascaded impurity removal.

[0078] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A cascade separation and purification method for isolating astragalus polysaccharides from astragalus, characterized in that, Includes the following steps: (1) The coarse filtrate obtained by water extraction and solid-liquid separation of Astragalus membranaceus slices is sequentially filtered through multiple microfiltration membranes with progressively smaller pore sizes, and the clear liquid is collected. (2) The clarified liquid is concentrated by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 5~10 kDa, and the retentate is collected to obtain the first concentrated liquid; (3) Papain was added to the first concentrate to enzymatically decompose the protein. After inactivation and centrifugation, the first supernatant was collected. Add Schwager's reagent to the first supernatant and extract by shaking. After centrifugation, collect the second supernatant. (4) Use an ultrafiltration membrane with a molecular weight cutoff of 5~10 kDa to ultrafilter the second supernatant, and add an equal volume of ultrapure water at the same time; when the cumulative volume of ultrafiltration permeate reaches 5~10 times the initial volume of the second supernatant, stop adding ultrapure water and continue ultrafiltration concentration to obtain the second concentrate. (5) Add ethanol to the second concentrate for alcohol precipitation, separate and collect the precipitate, redissolve the precipitate and dry it to obtain Astragalus polysaccharide.

2. The cascade separation and purification method according to claim 1, characterized in that, In step (1), the coarse filtrate is prepared according to the following method: The Astragalus membranaceus slices were crushed and added to ethanol or an ethanol-water solution with a volume fraction of 70% to 95%. The mixture was then subjected to a defatting treatment at an ultrasonic power of 200 to 500 W for 1 to 3 times, each time for 10 to 30 minutes. Collect the defatted solids, and add ultrapure water in 1 to 3 portions based on the initial mass of Astragalus membranaceus slices. The ratio of the mass of Astragalus membranaceus slices to the cumulative volume of ultrapure water added is 1 g: (10 to 20) mL. Extract in a water bath at 70 to 90 ℃ for 60 to 90 min each time. The combined extracts were then subjected to compression filtration and centrifugation to obtain the coarse filtrate.

3. The cascade separation and purification method according to claim 1, characterized in that, In step (1), the coarse filtrate is sequentially filtered through microfiltration membranes with pore sizes of 10~20 μm, 3~5 μm and 0.2~0.65 μm.

4. The cascade separation and purification method according to claim 1, characterized in that, In step (2), the transmembrane pressure of the ultrafiltration concentration is 0.1~0.3 MPa, and the clarified liquid is concentrated to 1 / 10~1 / 4 of the initial volume to obtain the first concentrated liquid.

5. The cascade separation and purification method according to claim 1, characterized in that, In step (3), the added papain is 1% to 3% of the mass of the first concentrate; the enzymatic hydrolysis of the protein is carried out at 55 to 60 °C for 3 to 4 h, and after the enzymatic hydrolysis is completed, the temperature is raised to 70 to 90 °C and held for 10 to 15 min to inactivate the papain, and then centrifuged at 8000 to 10000 r / min for 10 to 20 min.

6. The cascade separation and purification method according to claim 1, characterized in that, In step (3), the Chevalier reagent is prepared by mixing chloroform and n-butanol in a volume ratio of 4:1; the volume ratio of the first supernatant to the Chevalier reagent is (4~5):1, the shaking extraction speed is 200 r / min, and the time is 30~50 min.

7. The cascade separation and purification method according to claim 1, characterized in that, In step (3), after collecting the first supernatant and before adding the Schwager reagent, the first supernatant is further concentrated by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 5-10 kDa.

8. The cascade separation and purification method according to claim 1, characterized in that, In step (4), the cumulative replenishment volume of ultrapure water during ultrafiltration is 5 to 10 times the volume of the second supernatant.

9. The cascade separation and purification method according to claim 1, characterized in that, In step (5), the volume of ethanol added is 2 to 4 times the volume of the second concentrate; after adding ethanol, it is allowed to stand at 4 °C for 10 to 18 h, and then centrifuged at 8000 to 10000 r / min for 10 to 15 min to collect the precipitate.

10. The cascade separation and purification method according to claim 1, characterized in that, In step (5), the drying is vacuum freeze drying, which includes: pre-freezing the reconstituted precipitate solution at -80 ℃ for 4~10 h, and then vacuum freeze drying for 24~48 h under the conditions that the condenser temperature is not higher than -50 ℃ and the vacuum degree is not higher than 20 Pa.

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