A method for purifying panax notoginseng saponins

Abstract

The present disclosure relates to the technical field of total saponin extraction / purification, in particular to a purification method of total saponins of Panax notoginseng; the purification method comprises the following steps: crushing, sieving and enzymolysis of Panax notoginseng to obtain an enzyme hydrolysate of Panax notoginseng; then reflux extraction to obtain an extract of Panax notoginseng; then decolorization treatment to obtain a decolorized solution; preparation of modified adsorption resin; wherein the modified adsorption resin is a styrene-based resin modified by a rosin derivative; finally, the decolorized solution is purified by the modified adsorption resin to obtain total saponins of Panax notoginseng.

Description

Technical Field

[0001] This disclosure relates to the field of total saponin extraction / purification technology, specifically a method for purifying total saponins from Panax notoginseng. Background Technology

[0002] Panax notoginseng is a traditional and precious Chinese medicinal herb with effects such as promoting blood circulation, removing blood stasis, reducing swelling, and relieving pain. Its main active ingredient is total saponins (PNS), including notoginsenoside R1, ginsenoside Rg1, Re, Rb1, and Rd. High-purity PNS has broad application prospects in cardiovascular drugs, health foods, and cosmetics; therefore, efficient separation and purification technology of PNS has always been a research hotspot.

[0003] Adsorption resin methods have become the mainstream technology for PNS purification due to their advantages such as simple operation, low cost, and ease of industrialization. Currently, commercial macroporous adsorption resins such as D101, AB-8, and NKA9 are widely used industrially; however, these resins suffer from limited adsorption capacity and poor selectivity. In recent years, rosin-based adsorption resins have emerged as a novel type of biomass adsorption material. This is because the hydrogenated phenanthrene ring structure in rosin molecules is similar to the triterpenoid saponin skeleton structure of PNS, demonstrating good separation and purification potential.

[0004] Huang Jinfu et al. (Modern Food Science and Technology, Vol. 39, No. 10, 2023; Purification of Total Saponins of Panax notoginseng by Rosin-Based Adsorption Resin and Adsorption Mechanism) prepared a rosin-based adsorption resin (RBAR) by suspension polymerization using hydrogenated rosin (β-acryloyloxyethyl) ester (HRE) and methacrylic acid (MAA) as monomers and divinylbenzene (DVB) as a crosslinking agent. RBAR exhibited good adsorption and purification effects on total saponins of Panax notoginseng. Zeng Zhenfang et al. (Forest Products Chemistry and Industry, Vol. 46, No. 1, February 2026; Study on Purification of Total Saponins of Panax notoginseng by Acylthiourea-Containing Rosin-Based Adsorption Resin) developed an acylthiourea-containing rosin-based adsorption resin (RDMP). Using N-(O-methylbenzoyl)thiourea diacrylate maleic rosin (NMDMR) as a crosslinking agent, thiourea groups were introduced to enhance hydrogen bonding, resulting in good adsorption and purification effects on total saponins of Panax notoginseng.

[0005] However, the above-mentioned rosin-based resins are all directly synthesized by suspension polymerization, using rosin derivatives as monomers or crosslinking agents to participate in the polymerization reaction, which has the following technical defects: (1) The synthesis process is complex and the batch stability is poor. The polymerization of rosin derivatives as monomers requires pre-preparation through multiple steps such as esterification and acylation, which results in a long synthesis route and high cost; and the formation of oil droplets during suspension polymerization is easily affected by the hydrophobicity and viscosity of rosin derivatives, making it difficult to accurately control the particle size distribution. (2) Insufficient mechanical strength and large pressure drop in column chromatography. Rosin-based resins are random crosslinked polymers with large skeleton flexibility, which are prone to deformation in dynamic column chromatography, leading to column bed compaction and reduced flow rate.

[0006] Therefore, developing a novel adsorption resin that retains the advantages of the rosin phenanthrene ring structure while avoiding the aforementioned defects is of great significance for improving PNS purification efficiency and reducing production costs. Summary of the Invention

[0007] This disclosure provides a method for purifying total saponins from Panax notoginseng to address the shortcomings of related technologies.

[0008] According to a first aspect of the present disclosure, a method for purifying total saponins of Panax notoginseng is provided, the purification method comprising the following steps: Step 1: After crushing and sieving Panax notoginseng, obtain Panax notoginseng powder; after enzymatic hydrolysis of Panax notoginseng powder, obtain Panax notoginseng enzymatic hydrolysate; Step 2: The Panax notoginseng enzymatic hydrolysis product obtained in Step 1 is extracted by reflux to obtain Panax notoginseng extract; Step 3: Decolorize the Panax notoginseng extract obtained in Step 2 to obtain a decolorized solution; Step 4: Prepare modified adsorption resin; wherein the modified adsorption resin is a styrene-based resin modified with rosin derivatives; Step 5: Purify the decolorizing solution using the modified adsorption resin prepared in Step 4 to obtain the total saponins of Panax notoginseng.

[0009] In one aspect of this disclosure, step 1 includes: Step 1-1: Grind Panax notoginseng into powder and pass it through a 40-60 mesh sieve to obtain Panax notoginseng powder; then add the Panax notoginseng powder to deionized water, with a material-to-liquid ratio of 1:(10-15), and add glacial acetic acid to adjust the pH of the system to 6.5-6.8. Steps 1-2: Add 1%-1.5% of neutral cellulase by weight of Panax notoginseng powder and enzymatically hydrolyze in a water bath at 45℃-50℃ for 2-3 hours; after enzymatic hydrolysis, raise the temperature to 90℃-95℃ and maintain it for 3-8 minutes to inactivate the enzyme, and then cool to room temperature. Steps 1-3: Add glacial acetic acid to adjust the pH of the system to 6.0-6.2; then add 0.5%-1% of α-amylase by weight of Panax notoginseng powder, and enzymatically hydrolyze in a water bath at 50℃-60℃ for 1-2 hours; after enzymatic hydrolysis, raise the temperature to 90℃-95℃ and maintain it for 3-8 minutes to inactivate the enzyme, and then cool to room temperature to obtain the Panax notoginseng enzymatic hydrolysate.

[0010] In one aspect of this disclosure, step 2 includes: Step 2-1: Add 8-12 times the mass of Panax notoginseng powder in 90% ethanol solution to the Panax notoginseng enzymatic hydrolysate, then heat to 80℃-85℃ and reflux for extraction 3 times. Step 2-2: After the reflux extraction is completed, the extracts are combined, then filtered and concentrated to 1 / 2 of the original volume; the Panax notoginseng extract is obtained.

[0011] In one aspect of this disclosure, step 3 includes: Step 3-1: Add 0.5%-2% (by weight of Panax notoginseng extract) of powdered activated carbon or granular activated carbon to the Panax notoginseng extract, heat to 40℃-60℃, and keep stirring for 1-3 hours. Step 3-2: After removing activated carbon by filtration, the resulting filtrate is filtered through a 0.45μm microporous membrane to obtain the decolorized solution.

[0012] In one aspect of this disclosure, the modified adsorbent resin is a chloromethylated polystyrene resin modified with maleic acrid acid.

[0013] In one aspect of this disclosure, the chloromethylated polystyrene resin modified with maleic acrid acid is prepared by the following steps: Step A-1: ​​Prepare microspheres of chloromethylated polystyrene resin; the chloromethylated polystyrene resin comprises the structure of Formula I-1: , Step A-2: The microspheres of chloromethylated polystyrene resin were added to dichloromethane and allowed to swell overnight. The temperature was then raised to 55°C-65°C, a catalyst was added, and the reaction was allowed to proceed for 3-6 hours. The resin was then removed, washed, and dried to obtain the treated resin. , Step A-3: The treated resin obtained in Step A-2 is added to N,N-dimethylformamide, followed by maleic acrid acid. The mixture is allowed to swell overnight, then heated to 75℃-85℃, and a catalyst is added. The reaction is allowed to proceed for 10-16 hours. The resin is then removed, washed, and dried to obtain maleic acrid acid-modified chloromethylated polystyrene resin. .

[0014] In one aspect of this disclosure, the microsphere-shaped chloromethylated polystyrene resin is prepared by the following steps: Step B-1: Disperse hydroxypropyl methylcellulose in an ethanol solution, then start stirring and add polyvinylpyrrolidone; after the polyvinylpyrrolidone is completely dissolved, add n-hexanol; Step B-2: Add a mixture containing azobisisobutyronitrile, styrene, divinylbenzene and chloromethylstyrene to the solution obtained in step B-1; then, under nitrogen protection, raise the temperature of the system to 60℃-70℃ and react for 0.5-1.5h; remove the nitrogen protection and continue the reaction for 8-12h; after the reaction is completed, filter, wash and dry to obtain the microsphere chloromethylated polystyrene resin.

[0015] In one aspect of this disclosure, in step A-2, the catalyst is selected from anhydrous ferric chloride.

[0016] In one aspect of this disclosure, in step A-3, the catalyst is selected from sodium bicarbonate.

[0017] In one aspect of the present disclosure, in step A-3, the mass ratio of the treated resin obtained in step A-2 to maleic acrid acid is selected from 1:(0.25-0.5).

[0018] In one aspect of the present disclosure, in step B-2, the mass ratio of azobisisobutyronitrile, styrene, divinylbenzene and chloromethylstyrene in the mixture is selected from (0.2-0.4):(6-9):(0.15-0.3):(2-4).

[0019] In one aspect of this disclosure, step 5 includes: Step 5-1: The decolorizing solution is pre-washed with the modified adsorption resin prepared in step 4 using 2-3 BV of deionized water at a flow rate of 1.0-2.0 BV / h. Step 5-2: Use 1-2 BV of 20% ethanol solution for pre-washing at a flow rate of 0.5-1.5 BV / h; Step 5-3: Wash with 4-6 BV of 60% ethanol solution at a flow rate of 0.5-1.5 BV / h; collect the eluent, concentrate and freeze-dry to obtain the total saponins of Panax notoginseng.

[0020] Compared with the prior art, the beneficial effects of this disclosure are: Chloromethylated polystyrene resin possesses advantages such as high mechanical strength, good chemical stability, and uniform particle size. The chloromethyl groups on its surface can be introduced with various functional groups through nucleophilic substitution reactions. Maleic pithiacin, an important derivative of rosin, retains the structure of the hydrogenated phenanthrene ring while introducing carboxyl groups. This disclosure creatively grafts maleic pithiacin onto the surface of chloromethylated polystyrene resin, thus leveraging the mechanical stability and processability of the polystyrene skeleton while preserving the specific affinity of the rosin phenanthrene ring for PNS. Attached Figure Description

[0021] Figure 1This is a SEM image of the modified adsorption resin prepared in Example 1. Detailed Implementation

[0022] The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this disclosure. Rather, they are merely examples of apparatuses and methods consistent with some aspects of this disclosure as detailed in the appended claims.

[0023] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions of this application will be clearly and completely described below in conjunction with embodiments. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. The embodiments described herein are illustrative in nature and are used to provide a basic understanding of this application. The embodiments of this application should not be construed as limiting this application.

[0024] For the sake of brevity, this article only discloses a few specific numerical ranges. However, any lower limit can be combined with any upper limit to form an unspecified range; and any lower limit can be combined with other lower limits to form an unspecified range, just as any upper limit can be combined with any other upper limit to form an unspecified range. Furthermore, each individually disclosed point or single value can itself serve as a lower or upper limit and be combined with any other point or single value or with other lower or upper limits to form an unspecified range.

[0025] In this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0026] In this description, unless otherwise stated, "above" and "below" include the stated number.

[0027] Unless otherwise stated, the terms used in this disclosure have their common meanings as commonly understood by those skilled in the art. Unless otherwise stated, the values ​​of the parameters mentioned in this disclosure can be measured using various measurement methods commonly used in the art (e.g., they can be tested according to the methods given in the embodiments of this disclosure).

[0028] The term "about" is used to describe and indicate small variations. When used in conjunction with an event or situation, the term may refer to examples in which the event or situation occurred precisely or in examples in which the event or situation occurred very approximately. For example, when used in conjunction with numerical values, the term may refer to a range of variation less than or equal to ±10% of the numerical value, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.1%, or less than or equal to ±0.05%. Additionally, quantities, ratios, and other numerical values ​​are sometimes presented in range format herein. It should be understood that such range format is for convenience and brevity and should be interpreted flexibly to include not only numerical values ​​explicitly specified as range limits but also all individual numerical values ​​or subranges covered within the range, as if each numerical value and subrange were explicitly specified.

[0029] The list of items connected by the terms "at least one of," "at least one of," "at least one of," or other similar terms can mean any combination of the listed items. For example, if items A and B are listed, then the phrase "at least one of A and B" means only A; only B; or A and B. In another instance, if items A, B, and C are listed, then the phrase "at least one of A, B, and C" means only A; or only B; only C; A and B (excluding C); A and C (excluding B); B and C (excluding A); or all of A, B, and C. Item A may contain a single component or multiple components. Item B may contain a single component or multiple components. Item C may contain a single component or multiple components.

[0030] The present disclosure will be further described below by way of specific embodiments. Unless otherwise specified, all chemical reagents used in the embodiments of the present disclosure are obtained through conventional commercial means. Unless otherwise specified, all contents mentioned below are mass contents. Unless otherwise specified, it is understood that the process is carried out at room temperature.

[0031] Examples and comparative examples:

[0032] Example 1: Example 1 includes the following steps: 1. Preparation of microspheres of chloromethylated polystyrene resin: 0.45 g of hydroxypropyl methylcellulose (HPMC) was dispersed in an ethanol solution (obtained by mixing 39 mL of anhydrous ethanol and 60 mL of deionized water), then filtered. The filtrate was added to a four-necked flask equipped with a mechanical stirrer, a nitrogen inlet, a thermometer sleeve, and a spherical reflux condenser. Stirring was then started, and 0.45 g of polyvinylpyrrolidone (PVP) was added. After the PVP was completely dissolved, 1.5 mL of n-hexanol was added. The mixture was stirred for a period of time to ensure uniform dispersion. Then, 1.125 g of azobisisobutyronitrile, 21 g of styrene, 0.6 g of divinylbenzene, and 9 g of chloromethylstyrene were mixed to obtain a transparent mixture, which was added to the aforementioned solution. Under nitrogen protection, the temperature of the system was raised to 65 °C and reacted for 1 h. Then, the nitrogen protection was removed, and the reaction was continued for 10 h. After the reaction was completed, the product solution was ultrasonically dispersed, then filtered, washed, and vacuum dried to obtain a white powder, which is the microsphere-shaped chloromethylated polystyrene resin of this embodiment.

[0033] The above steps can be repeated multiple times to prepare enough product for subsequent reactions.

[0034] 2. Preparation of chloromethylated polystyrene resin modified with maleic acrid acid: Weigh 20g of the previously prepared microspheres of chloromethylated polystyrene resin and add it to 100mL of 1,2-dichloroethane. Let it swell overnight, then heat to 60℃ and add 3.2g of anhydrous ferric chloride. React for 4 hours. Then remove the resin; wash it three times each with ethanol and pure water using ultrasonication, and then dry it in a 60℃ oven to constant weight to obtain the treated resin. , Weigh 18g of the pre-prepared treated resin and add it to 120mL of N,N-dimethylformamide. Then add 6g of maleic acrid acid, allow it to swell overnight, then heat to 80℃, add 2g of sodium bicarbonate, and react for 12h. Remove the resin, wash it three times with ethanol and pure water respectively using ultrasonication, and then dry it in a 60℃ oven to constant weight to obtain the modified adsorption resin of this embodiment. Its SEM image is shown below. Figure 1 As shown.

[0035] 3. Extraction and purification of total saponins from Panax notoginseng: Commercially available Panax notoginseng was pulverized and passed through a 60-mesh sieve to obtain Panax notoginseng powder. The powder was then added to deionized water at a ratio of 1:12, and glacial acetic acid was added dropwise to adjust the pH to approximately 6.8. Next, 1% (by weight of the Panax notoginseng powder) of neutral cellulase was added, and the mixture was enzymatically hydrolyzed in a 45°C water bath for 2.5 hours. After hydrolysis, the temperature was raised to 95°C and held for 4 minutes to inactivate the enzyme, then cooled to room temperature. Glacial acetic acid was added dropwise to adjust the pH to approximately 6.0. Then, 0.75% (by weight of the Panax notoginseng powder) of α-amylase was added, and the mixture was enzymatically hydrolyzed in a 55°C water bath for 1.5 hours. After hydrolysis, the temperature was raised to 95°C and held for 4 minutes to inactivate the enzyme, then cooled to room temperature, yielding the Panax notoginseng enzymatic hydrolysate.

[0036] Add 10 times the mass of Panax notoginseng powder to the Panax notoginseng enzymatic hydrolysate and then heat to 85°C and reflux for extraction 3 times. After the reflux extraction is completed, combine the extracts, filter and concentrate to 1 / 2 of the original volume to obtain Panax notoginseng extract.

[0037] Add 0.5% (by weight of Panax notoginseng extract) of powdered activated carbon to the Panax notoginseng extract, heat to 50°C, and stir for 2 hours. Then remove the activated carbon by filtration, and filter the resulting filtrate through a 0.45 μm microporous membrane to obtain a decolorized solution.

[0038] The decolorizing solution was passed through the modified adsorption resin prepared above; a wet packing column was used; the column volume was 50 mL, and the diameter-to-height ratio was 1:4; the column was first pre-washed with 2 BV of deionized water at a flow rate of 2.0 BV / h to remove water-soluble impurities (polysaccharides, proteins, and inorganic salts); then pre-washed with 1 BV of 20% ethanol solution at a flow rate of 0.5 BV / h to remove flavonoids and anthraquinones, which are highly polar pigment impurities; finally, the column was washed with 6 BV of 60% ethanol solution at a flow rate of 1.5 BV / h; the eluent was collected, concentrated, and freeze-dried to obtain the total saponins of Panax notoginseng in this embodiment.

[0039] The total saponins obtained in this example were tested according to the detection method of total saponins in the 2020 edition of the Chinese Pharmacopoeia; the content of total saponins was found to be 99.6%.

[0040] The analysis conditions are as follows: Chromatographic column: HITACHI LaChrom C18 (5 μm); 4.6 mm ID × 150 mm; The mobile phase was A: acetonitrile; B: water (gradient elution); 20% A (0-20 min) → 46% A (45 min) → 55% A (55-60 min) → 20% A (60.1-75 min); The flow rate was 1.5 mL / min; the column temperature was 25℃; the detection wavelength was UV 203 nm; and the injection volume was 10 μL.

[0041] Comparative Example 1: Comparative Example 1 includes the following steps: 1. Preparation of microspheres of chloromethylated polystyrene resin: 0.45 g of hydroxypropyl methylcellulose (HPMC) was dispersed in an ethanol solution (obtained by mixing 39 mL of anhydrous ethanol and 60 mL of deionized water), then filtered. The filtrate was added to a four-necked flask equipped with a mechanical stirrer, a nitrogen inlet, a thermometer sleeve, and a spherical reflux condenser. Stirring was then started, and 0.45 g of polyvinylpyrrolidone (PVP) was added. After the polyvinylpyrrolidone was completely dissolved, 1.5 mL of n-hexanol was added. The mixture was stirred for a period of time to ensure uniform dispersion. Then, 1.125 g of azobisisobutyronitrile, 21 g of styrene, 0.6 g of divinylbenzene and 9 g of chloromethylstyrene were mixed to obtain a transparent mixture, which was added to the aforementioned solution. Under nitrogen protection, the temperature of the system was raised to 65 °C and reacted for 1 h. Then, the nitrogen protection was removed and the reaction continued for 10 h. After the reaction was completed, the product solution was ultrasonically dispersed, filtered, washed, and vacuum dried to obtain a white powder, which is the microsphere-shaped chloromethylated polystyrene resin of this comparative example. This comparative example directly uses the microsphere-shaped chloromethylated polystyrene resin as the adsorption resin.

[0042] The above steps can be repeated multiple times to prepare enough product for subsequent reactions.

[0043] 2. Extraction and purification of total saponins from Panax notoginseng: Commercially available Panax notoginseng was pulverized and passed through a 60-mesh sieve to obtain Panax notoginseng powder. The powder was then added to deionized water at a ratio of 1:12, and glacial acetic acid was added dropwise to adjust the pH to approximately 6.8. Next, 1% (by weight of the Panax notoginseng powder) of neutral cellulase was added, and the mixture was enzymatically hydrolyzed in a 45°C water bath for 2.5 hours. After hydrolysis, the temperature was raised to 95°C and held for 4 minutes to inactivate the enzyme, then cooled to room temperature. Glacial acetic acid was added dropwise to adjust the pH to approximately 6.0. Then, 0.75% (by weight of the Panax notoginseng powder) of α-amylase was added, and the mixture was enzymatically hydrolyzed in a 55°C water bath for 1.5 hours. After hydrolysis, the temperature was raised to 95°C and held for 4 minutes to inactivate the enzyme, then cooled to room temperature, yielding the Panax notoginseng enzymatic hydrolysate.

[0044] Add 10 times the mass of Panax notoginseng powder to the Panax notoginseng enzymatic hydrolysate and then heat to 85°C and reflux for extraction 3 times. After the reflux extraction is completed, combine the extracts, filter and concentrate to 1 / 2 of the original volume to obtain Panax notoginseng extract.

[0045] Add 0.5% (by weight of Panax notoginseng extract) of powdered activated carbon to the Panax notoginseng extract, heat to 50°C, and stir for 2 hours. Then remove the activated carbon by filtration, and filter the resulting filtrate through a 0.45 μm microporous membrane to obtain a decolorized solution.

[0046] The decolorizing solution was passed through the modified adsorption resin prepared above; a wet packing column was used; the column volume was 50 mL, and the diameter-to-height ratio was 1:4; the column was first pre-washed with 2 BV of deionized water at a flow rate of 2.0 BV / h to remove water-soluble impurities (polysaccharides, proteins, and inorganic salts); then pre-washed with 1 BV of 20% ethanol solution at a flow rate of 0.5 BV / h to remove flavonoids and anthraquinones, which are highly polar pigment impurities; finally, the column was washed with 6 BV of 60% ethanol solution at a flow rate of 1.5 BV / h; the eluent was collected, concentrated, and freeze-dried to obtain the total saponins of Panax notoginseng in this comparative example.

[0047] The total saponins of Panax notoginseng obtained in this comparative example were tested according to the detection method of total saponins of Panax notoginseng in the 2020 edition of the Chinese Pharmacopoeia; the content of total saponins of Panax notoginseng was found to be 86.2%.

[0048] Comparative Example 2: Comparative Example 2 includes the following steps: 1. Preparation of microspheres of chloromethylated polystyrene resin: 0.45 g of hydroxypropyl methylcellulose (HPMC) was dispersed in an ethanol solution (obtained by mixing 39 mL of anhydrous ethanol and 60 mL of deionized water), then filtered. The filtrate was added to a four-necked flask equipped with a mechanical stirrer, a nitrogen inlet, a thermometer sleeve, and a spherical reflux condenser. Stirring was then started, and 0.45 g of polyvinylpyrrolidone (PVP) was added. After the PVP was completely dissolved, 1.5 mL of n-hexanol was added. The mixture was stirred for a period of time to ensure uniform dispersion. Then, 1.125 g of azobisisobutyronitrile, 21 g of styrene, 0.6 g of divinylbenzene, and 9 g of chloromethylstyrene were mixed to obtain a transparent mixture, which was added to the aforementioned solution. Under nitrogen protection, the temperature of the system was raised to 65 °C and reacted for 1 h. Then, the nitrogen protection was removed, and the reaction was continued for 10 h. After the reaction was completed, the product solution was ultrasonically dispersed, then filtered, washed, and vacuum dried to obtain a white powder, which is the microsphere-like chloromethylated polystyrene resin of this comparative example.

[0049] The above steps can be repeated multiple times to prepare enough product for subsequent reactions.

[0050] 2. Preparation of acylated modified chloromethylated polystyrene resin: Weigh 20g of the microspheres of chloromethylated polystyrene resin prepared above, add it to 100mL of 1,2-dichloroethane, allow it to swell overnight, then heat it to 60℃, add 3.2g of anhydrous ferric chloride, and react for 4h; then take out the resin; wash it three times with ethanol and pure water respectively by ultrasonication, and then dry it in an oven at 60℃ to constant weight to obtain the treated adsorption resin.

[0051] Weigh 18g of the previously prepared treated resin and add it to 120mL of N,N-dimethylformamide. Then add 6g of maleic anhydride and allow it to swell overnight. Then heat the solution to 80℃, add 2g of sodium bicarbonate, and react for 12h. Then remove the resin and wash it three times with ethanol and pure water, respectively. Then dry it in an oven at 60℃ to constant weight to obtain the modified adsorption resin of this comparative example.

[0052] 3. Extraction and purification of total saponins from Panax notoginseng: The preparation steps of the decolorizing solution in this comparative example are the same as those in Example 1.

[0053] The decolorizing solution was passed through the modified adsorption resin prepared above; a wet packing column was used; the column volume was 50 mL, and the diameter-to-height ratio was 1:4; the column was first pre-washed with 2 BV of deionized water at a flow rate of 2.0 BV / h to remove water-soluble impurities (polysaccharides, proteins, and inorganic salts); then pre-washed with 1 BV of 20% ethanol solution at a flow rate of 0.5 BV / h to remove flavonoids and anthraquinones, which are highly polar pigment impurities; finally, the column was washed with 6 BV of 60% ethanol solution at a flow rate of 1.5 BV / h; the eluent was collected, concentrated, and freeze-dried to obtain the total saponins of Panax notoginseng in this comparative example.

[0054] The total saponins of Panax notoginseng obtained in this comparative example were tested according to the detection method of total saponins of Panax notoginseng in the 2020 edition of the Chinese Pharmacopoeia; the content of total saponins of Panax notoginseng was found to be 83.8%.

[0055] Comparative Example 3: Comparative Example 3 includes the following steps: 1. Preparation of microspheres of chloromethylated polystyrene resin: 0.45 g of hydroxypropyl methylcellulose (HPMC) was dispersed in an ethanol solution (obtained by mixing 39 mL of anhydrous ethanol and 60 mL of deionized water), then filtered. The filtrate was added to a four-necked flask equipped with a mechanical stirrer, a nitrogen inlet, a thermometer sleeve, and a spherical reflux condenser. Stirring was then started, and 0.45 g of polyvinylpyrrolidone (PVP) was added. After the PVP was completely dissolved, 1.5 mL of n-hexanol was added. The mixture was stirred for a period of time to ensure uniform dispersion. Then, 1.125 g of azobisisobutyronitrile, 21 g of styrene, 0.6 g of divinylbenzene, and 9 g of chloromethylstyrene were mixed to obtain a transparent mixture, which was added to the aforementioned solution. Under nitrogen protection, the temperature of the system was raised to 65 °C and reacted for 1 h. Then, the nitrogen protection was removed, and the reaction was continued for 10 h. After the reaction was completed, the product solution was ultrasonically dispersed, then filtered, washed, and vacuum dried to obtain a white powder, which is the microsphere-like chloromethylated polystyrene resin of this comparative example.

[0056] The above steps can be repeated multiple times to prepare enough product for subsequent reactions.

[0057] 2. Preparation of acylated modified chloromethylated polystyrene resin: Weigh 20g of the microspheres of chloromethylated polystyrene resin prepared above, add it to 100mL of 1,2-dichloroethane, allow it to swell overnight, then heat it to 60℃, add 3.2g of anhydrous ferric chloride, and react for 4h; then take out the resin; wash it three times with ethanol and pure water respectively by ultrasonication, and then dry it in an oven at 60℃ to constant weight to obtain the treated adsorption resin.

[0058] Weigh 18g of the pre-prepared treated resin and add it to 120mL of N,N-dimethylformamide. Then add 6g of L-malic acid and allow it to swell overnight. Then heat the solution to 80℃, add 2g of sodium bicarbonate, and react for 12h. Then remove the resin and wash it three times with ethanol and pure water, respectively. Then dry it in an oven at 60℃ to constant weight to obtain the modified adsorption resin of this comparative example.

[0059] 3. Extraction and purification of total saponins from Panax notoginseng: The preparation steps of the decolorizing solution in this comparative example are the same as those in Example 1.

[0060] The decolorizing solution was passed through the modified adsorption resin prepared above; a wet packing column was used; the column volume was 50 mL, and the diameter-to-height ratio was 1:4; the column was first pre-washed with 2 BV of deionized water at a flow rate of 2.0 BV / h to remove water-soluble impurities (polysaccharides, proteins, and inorganic salts); then pre-washed with 1 BV of 20% ethanol solution at a flow rate of 0.5 BV / h to remove flavonoids and anthraquinones, which are highly polar pigment impurities; finally, the column was washed with 6 BV of 60% ethanol solution at a flow rate of 1.5 BV / h; the eluent was collected, concentrated, and freeze-dried to obtain the total saponins of Panax notoginseng in this comparative example.

[0061] The total saponins of Panax notoginseng obtained in this comparative example were tested according to the detection method of total saponins of Panax notoginseng in the 2020 edition of the Chinese Pharmacopoeia; the content of total saponins of Panax notoginseng was found to be 90.6%.

[0062] Example 2: Example 2 includes the following steps: 1. Preparation of maleic acrid acid-modified polysiloxane resin: Add 50g of octamethylcyclotetrasiloxane, 10g of tetramethyltetravinylcyclotetrasiloxane, 17g of aminopropylmethyldiethoxysilane, 5g of hexamethyldisiloxane, and 0.1g of potassium hydroxide to a reaction vessel. Stir and react at 100℃ for 8 hours, then gradually raise the temperature to 180℃ and remove low-boiling substances under vacuum. Cool to 120℃ and add 36g of maleic acrid acid. Stir and react for 4 hours, then raise the temperature to 180℃ and remove low-boiling substances under vacuum. Collect the maleic acrid acid-modified polysiloxane resin while hot under nitrogen protection.

[0063] 2. Extraction and purification of total saponins from Panax notoginseng: The preparation steps of the decolorizing solution in this embodiment are the same as those in Example 1.

[0064] The decolorizing solution was passed through the modified adsorption resin prepared above; a wet packing column was used; the column volume was 50 mL, and the diameter-to-height ratio was 1:4; the column was first pre-washed with 2 BV of deionized water at a flow rate of 2.0 BV / h to remove water-soluble impurities (polysaccharides, proteins, and inorganic salts); then pre-washed with 1 BV of 20% ethanol solution at a flow rate of 0.5 BV / h to remove flavonoids and anthraquinones, which are highly polar pigment impurities; finally, the column was washed with 6 BV of 60% ethanol solution at a flow rate of 1.5 BV / h; the eluent was collected, concentrated, and freeze-dried to obtain the total saponins of Panax notoginseng in this embodiment.

[0065] The total saponins of Panax notoginseng obtained in this comparative example were tested according to the detection method of total saponins of Panax notoginseng in the 2020 edition of the Chinese Pharmacopoeia; the content of total saponins of Panax notoginseng was found to be 86.0%.

[0066] As can be seen, the extraction and purification method provided in this disclosure chemically grafts maleic acrid acid onto the surface of chloromethylated polystyrene resin, which not only leverages the mechanical stability and processability of the polystyrene skeleton but also retains the specific affinity of the rosin phenanthrene ring for PNS, thus exhibiting excellent purification capabilities for PNS. Comparative Examples 1 to 3 did not use maleic acrid acid to graft onto the chloromethylated polystyrene resin, while Example 2 used maleic acrid acid to graft onto the polysiloxane resin; its purification capabilities for PNS were significantly weaker than the resin prepared in Example 1.

[0067] Other embodiments of this disclosure will readily occur to those skilled in the art upon consideration of the specification and practice of the disclosure herein. This disclosure is intended to cover any variations, uses, or adaptations of this disclosure that follow the general principles of this disclosure and include common knowledge or customary techniques in the art not disclosed herein.

Claims

1. A method for purifying total saponins from Panax notoginseng, characterized in that, The purification method includes the following steps: Step 1: After crushing and sieving Panax notoginseng, obtain Panax notoginseng powder; after enzymatic hydrolysis of Panax notoginseng powder, obtain Panax notoginseng enzymatic hydrolysate; Step 2: The Panax notoginseng enzymatic hydrolysis product obtained in Step 1 is extracted by reflux to obtain Panax notoginseng extract; Step 3: Decolorize the Panax notoginseng extract obtained in Step 2 to obtain a decolorized solution; Step 4: Prepare modified adsorption resin; wherein the modified adsorption resin is a styrene-based resin modified with rosin derivatives; Step 5: Purify the decolorizing solution using the modified adsorption resin prepared in Step 4 to obtain the total saponins of Panax notoginseng.

2. The purification method for total saponins of Panax notoginseng according to claim 1, characterized in that, Step 1 includes: Step 1-1: Grind Panax notoginseng into powder and pass it through a 40-60 mesh sieve to obtain Panax notoginseng powder; then add the Panax notoginseng powder to deionized water, with a material-to-liquid ratio of 1:(10-15), and add glacial acetic acid to adjust the pH of the system to 6.5-6.

8. Steps 1-2: Add 1%-1.5% of neutral cellulase by weight of Panax notoginseng powder and enzymatically hydrolyze in a water bath at 45℃-50℃ for 2-3 hours; after enzymatic hydrolysis, raise the temperature to 90℃-95℃ and maintain it for 3-8 minutes to inactivate the enzyme, and then cool to room temperature. Steps 1-3: Add glacial acetic acid to adjust the pH of the system to 6.0-6.2; then add 0.5%-1% of α-amylase by weight of Panax notoginseng powder, and enzymatically hydrolyze in a water bath at 50℃-60℃ for 1-2 hours; after enzymatic hydrolysis, raise the temperature to 90℃-95℃ and maintain it for 3-8 minutes to inactivate the enzyme, and then cool to room temperature to obtain the Panax notoginseng enzymatic hydrolysate.

3. The purification method for total saponins of Panax notoginseng according to claim 1, characterized in that, Step 2 includes: Step 2-1: Add 8-12 times the mass of Panax notoginseng powder in 90% ethanol solution to the Panax notoginseng enzymatic hydrolysate, then heat to 80℃-85℃ and reflux for extraction 3 times. Step 2-2: After the reflux extraction is completed, the extracts are combined, then filtered and concentrated to 1 / 2 of the original volume; the Panax notoginseng extract is obtained.

4. The purification method for total saponins of Panax notoginseng according to claim 1, characterized in that, Step 3 includes: Step 3-1: Add 0.5%-2% (by weight of Panax notoginseng extract) of powdered activated carbon or granular activated carbon to the Panax notoginseng extract, heat to 40℃-60℃, and keep stirring for 1-3 hours. Step 3-2: After removing the activated carbon by filtration, the resulting filtrate is filtered through a 0.45μm microporous membrane to obtain the decolorized solution.

5. The purification method for total saponins of Panax notoginseng according to claim 1, characterized in that, The modified adsorbent resin is a chloromethylated polystyrene resin modified with maleic acrid acid.

6. The purification method for total saponins of Panax notoginseng according to claim 5, characterized in that, The chloromethylated polystyrene resin modified with maleic acrid acid was prepared by the following steps: Step A-1: ​​Prepare microspheres of chloromethylated polystyrene resin; the chloromethylated polystyrene resin comprises the structure of Formula I-1: Step A-2: The microspheres of chloromethylated polystyrene resin were added to dichloromethane and allowed to swell overnight. The temperature was then raised to 55°C-65°C, a catalyst was added, and the reaction was allowed to proceed for 3-6 hours. The resin was then removed, washed, and dried to obtain the treated resin. Step A-3: The treated resin obtained in Step A-2 is added to N,N-dimethylformamide, followed by maleic acrid acid. The mixture is allowed to swell overnight, then heated to 75℃-85℃, and a catalyst is added. The reaction is allowed to proceed for 10-16 hours. The resin is then removed, washed, and dried to obtain maleic acrid acid-modified chloromethylated polystyrene resin. 。 7. The purification method for total saponins of Panax notoginseng according to claim 6, characterized in that, The microsphere-shaped chloromethylated polystyrene resin was prepared by the following steps: Step B-1: Disperse hydroxypropyl methylcellulose in an ethanol solution, then start stirring and add polyvinylpyrrolidone; after the polyvinylpyrrolidone is completely dissolved, add n-hexanol; Step B-2: Add a mixture containing azobisisobutyronitrile, styrene, divinylbenzene and chloromethylstyrene to the solution obtained in step B-1; then, under nitrogen protection, raise the temperature of the system to 60℃-70℃ and react for 0.5-1.5h; remove the nitrogen protection and continue the reaction for 8-12h; after the reaction is completed, filter, wash and dry to obtain the microsphere chloromethylated polystyrene resin.

8. The purification method for total saponins of Panax notoginseng according to claim 6 or 7, characterized in that, The purification method satisfies any one of the following conditions: (1) In step A-2, the catalyst is selected from anhydrous ferric chloride; (2) In step A-3, the catalyst is selected from sodium bicarbonate; (3) In step A-3, the mass ratio of the treated resin obtained in step A-2 to maleic acrid acid is selected from 1:(0.25-0.5); (4) In step B-2, the mass ratio of azobisisobutyronitrile, styrene, divinylbenzene and chloromethylstyrene in the mixture is selected from (0.2-0.4):(6-9):(0.15-0.3):(2-4).

9. The purification method for total saponins of Panax notoginseng according to claim 1, characterized in that, Step 5 includes: Step 5-1: The decolorizing solution is pre-washed with the modified adsorption resin prepared in step 4 using 2-3 BV of deionized water at a flow rate of 1.0-2.0 BV / h. Step 5-2: Use 1-2 BV of 20% ethanol solution for pre-washing at a flow rate of 0.5-1.5 BV / h; Step 5-3: Wash with 4-6 BV of 60% ethanol solution at a flow rate of 0.5-1.5 BV / h; collect the eluent, concentrate and freeze-dry to obtain the total saponins of Panax notoginseng.

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