A method for extracting American ginsenoside

Abstract

The invention relates to the technical field of saponin extraction, and discloses a method for extracting American ginseng saponin. 5-bromo-insiditin, 3-amino-5-boronic acid, chloromethylated macroporous resin and the like are used as raw materials to obtain a functionalized macroporous resin; American ginseng fibrous roots are subjected to crushing and screening, chloroform soaking, ultrahigh pressure extraction, functionalized macroporous resin adsorption and column chromatography purification to obtain American ginseng saponin; the ultrahigh pressure extraction can increase the osmotic pressure difference, improve the penetration and diffusion rates, shorten the dissolution equilibrium time, increase the solubility of the effective components, and thus improve the extraction yield; the functionalized macroporous resin contains an indolequinone structure, and the amide group and the carbonyl structure in the structure can generate hydrogen bonding with the phenolic hydroxyl group in the saponin, so that the American ginseng saponin can be quickly and specifically adsorbed, and a large amount of impurities can be removed at the same time, so that the content of the target substance is increased to 98.0%. The method is simple and feasible, has low production cost, and is suitable for industrial production.

Description

Technical Field

[0001] The invention relates to the technical field of saponin extraction, in particular to a method for extracting American ginseng saponin. Background Art

[0002] American ginseng was once called "green gold". It has the effects of nourishing yin and qi, promoting body fluid and quenching thirst, eliminating irritability, clearing away internal heat, strengthening the body's vital energy, and resisting fatigue. Modern research shows that American ginseng has the properties of anti-aging, enhancing immunity, protecting against brain damage and myocardial ischemia, and anti-tumor. The main component of American ginseng is ginsenoside, and the five types of saponins that have been isolated are: ginsenoside R0, R b1 , Rg1, R e and pseudoginsenoside F 11 The active ingredients contained in American ginseng are basically the same as the monomer saponins of ginseng, and even the sapogenins contained are exactly the same, all of which are oleanolic acid, ginsenodiol and ginsenotriol, all of which have high development and utilization value. Therefore, the extraction of American ginseng saponins has very important economic and social value.

[0003] There are many chemical components in American ginseng, nitrogen-containing compounds account for 12-16%, including proteins, amino acids, polypeptides, nucleic acids, and alkaloids; fat-soluble components account for 2%, mainly lipids, fatty acids, volatile oils, etc.; vitamins account for 0.05%; carbohydrates account for 60-70%, including monosaccharides, disaccharides, trisaccharides, polysaccharides, fructose and crude fiber; ash accounts for 4%, mainly inorganic matter; water accounts for 9-11%, and its main active ingredient is ginsenoside, which is also the substance with the most significant physiological activity, and its content is generally 3-6%; the main structure of ginsenoside is ginsenoside diol type and ginsenoside triol type.

[0004] Traditional ginsenoside extraction methods include reflux method and decoction method. These two methods take a long time to extract, the extraction process is cumbersome, the amount of organic solvent used is large, and it is easy to cause structural changes when heated, and the extraction rate is not high; microwave extraction method uses less solvent and has a high extraction rate, but there is uneven radiation, which can easily cause local overtemperature and lead to denaturation and loss of effective ingredients; compared with traditional extraction methods, the biggest advantage of supercritical CO2 extraction is that it can be extracted and separated under near-normal temperature conditions, and almost all effective ingredients in the product are retained, but it is only suitable for extracting lipophilic and small molecular weight substances. For substances with high polarity and large molecular weight, a carrier needs to be added or the extraction needs to be carried out under very high pressure; the application of enzyme method can improve the extraction rate of effective ingredients in American ginseng, but the main disadvantage of enzyme degradation is that the action time is long, generally taking several days. In addition, it is necessary to strictly control miscellaneous bacteria, and the process conditions are very strict.

[0005] Macroporous adsorption resin is a kind of organic polymer adsorbent developed in modern times. It works by physical adsorption through its huge specific surface area by relying on the van der Waals attraction between it and the adsorbed molecules. Currently, there are relatively few varieties of adsorption resins, and in fact, there are only two types, polar and non-polar, which cannot meet the needs of extraction and separation of effective components of American ginseng. The present invention aims to synthesize a new functional macroporous resin that can effectively and selectively adsorb ginsenosides, and adopt ultra-high pressure extraction technology to separate the saponin components in American ginseng through column chromatography. The extraction yield is high, the method is simple and controllable, the production cycle is short, and it is suitable for industrial extraction of American ginseng saponins. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for extracting American ginsenosides, which improves the dissolution rate of saponins and can quickly and efficiently extract ginsenosides.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] A method for extracting American ginsenosides is carried out according to the following steps:

[0009] Step (1), using dried American ginseng root as raw material, crushing through a 60-100 mesh sieve, adding 10-20 times the weight of chloroform to the raw material powder, boiling at 60-80° C. for 2-5 hours, filtering, adding chloroform to the filter residue, boiling again for 2-3 times, filtering to remove the solvent, and obtaining defatted American ginseng.

[0010] Step (2), mixing defatted American ginseng with an ethanol aqueous solution at a solid-liquid ratio of 1 g: (50-100) mL, adding the mixture to an ultra-high pressure isostatic press for ultra-high pressure extraction, filtering to remove the residue, and concentrating the filtrate to obtain a crude American ginseng saponin extract.

[0011] Step (3), adding the crude American ginseng saponin extract to a methanol-water solution for dissolution, applying it to a functionalized macroporous resin chromatography column, adsorbing it for 12-24 hours, washing it with deionized water and ethanol in sequence, collecting the eluate, recovering the ethanol phase, and obtaining a crude American ginseng saponin product.

[0012] Step (4), dissolving the crude American ginsenoside with methanol aqueous solution, mixing with glue, drying and adding to a silica gel column for column chromatography, eluting with ethanol aqueous solution of different mass fractions, collecting the eluate, concentrating under reduced pressure, adding tetrahydrofuran aqueous solution to dissolve, reflux at 60-80° C. for 4-8 hours, cooling to room temperature, freezing and crystallizing, filtering under reduced pressure, and drying to obtain American ginsenoside.

[0013] Furthermore, the mass fraction of the ethanol aqueous solution in step (2) is 60-80%.

[0014] Furthermore, in step (2), the ultrahigh pressure extraction pressure is 150-350 MPa, and the extraction time is 1-3 min.

[0015] Furthermore, in step (3), the ratio of the crude American ginseng saponin extract to the functionalized macroporous resin is 1 g:(30-50) g.

[0016] Furthermore, in step (4), the mass fraction of the ethanol aqueous solution is 10-70%, and the mass fraction of the tetrahydrofuran aqueous solution is 75-90%.

[0017] Furthermore, the preparation method of the functionalized macroporous resin in step (4) is carried out according to the following steps:

[0018] Step S1: In a nitrogen atmosphere, 5-bromoisatin, ethylene glycol dimethyl ether and deionized water are added to a reaction flask, and after stirring, 3-amino-5-boronic acid, tetrakis(triphenylphosphine)palladium and sodium carbonate are added, and the mixture is placed in a microwave device for microwave reaction. After the reaction is completed, the reaction is purified by column chromatography to obtain an aminocarboxylic acid indolequinone intermediate. The preparation reaction formula is as follows:

[0019]

[0020] Step S2, under a nitrogen atmosphere, add chloromethylated macroporous resin and N,N-dimethylformamide to a reaction flask, stir evenly, add aminocarboxylic acid indolequinone intermediate and sodium hydroxide, react at 65-90° C. for 24-48 hours, wash with ethanol and deionized water, and dry to obtain a functionalized macroporous resin.

[0021] Furthermore, in step S1, the ratio of 5-bromoisatin, ethylene glycol dimethyl ether, deionized water, 3-amino-5-boronic acid, tetrakis(triphenylphosphine)palladium, and sodium carbonate is 1 g:(10-25) mL:(2-5) mL:(0.8-0.95) g:(0.12-0.25) g:(0.7-1.2) g.

[0022] Furthermore, in step S1, the microwave reaction temperature is 120-140° C., and the microwave reaction time is 10-20 min.

[0023] Furthermore, in step S2, the ratio of chloromethylated macroporous resin, N,N-dimethylformamide, aminocarboxylic acid indolequinone intermediate, and sodium hydroxide is 1 g: (10-20) mL: (0.4-0.6) g: (0.5-0.8) g.

[0024] By adopting the above technical solution, the beneficial effects of the present invention are:

[0025] The invention provides a method for obtaining an aminocarboxylic acid indolequinone intermediate by coupling reaction of 5-bromoindigo carboxylic acid and 3-amino-5-boronic acid under the action of a palladium catalyst, and then obtaining a functionalized macroporous resin by substitution reaction with a chloromethylated macroporous resin; crushing and sieving the fibrous roots of American ginseng, and then boiling them with chloroform to obtain defatted American ginseng; using ultra-high pressure extraction to obtain a crude American ginseng saponin extract, and then applying a functionalized macroporous resin chromatography column to obtain a crude American ginseng saponin extract by static adsorption; and finally purifying the extract by column chromatography to obtain the American ginseng saponin.

[0026] Under the action of ultra-high pressure, the rate at which the solvent penetrates into the American ginseng tissue is relatively fast. In the process of saponins diffusing outward across the cell membrane, the mass transfer power provided by the ultra-high pressure is very large, so the diffusion rate is relatively fast, resulting in a very short ultra-high pressure extraction time, which is beneficial to improving the extraction efficiency; it can increase the osmotic pressure difference, increase the penetration and diffusion rates, shorten the dissolution equilibrium time, and at the same time destroy the structure of various organelles such as the cell membrane, increase the contact area between the solvent and the solute, reduce the mass transfer resistance, shorten the extraction time, and at the same time, the solubility of the effective ingredients under ultra-high pressure increases, thereby improving the extraction yield, so that the total saponins of American ginseng can be extracted quickly and to the greatest extent.

[0027] The functionalized macroporous resin contains an indolequinone structure, the amide group in the structure can generate hydrogen bonds with the phenolic hydroxyl group of American ginseng to form a stable ring structure, the additional carbonyl structure can form hydrogen bonds with other phenolic hydroxyl groups, the benzene ring can form π-π conjugation with the benzene ring of phenol, the carboxyl group and the phenolic hydroxyl group can also generate covalent bonds, and the charge effect of the amide group and the dissociated phenolic hydroxyl group can enhance the adsorption capacity of the resin; the functionalized macroporous resin can rapidly and specifically adsorb American ginseng saponins with a large adsorption amount, and at the same time remove a large amount of impurities, thereby increasing the content of the target substance, and after a column chromatography purification, the content of the target substance is increased to 98.0%, the method is simple and feasible, the recovered liquid can be reused, the production cost is low, and it is suitable for industrial production. DETAILED DESCRIPTION

[0028] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0029] Preparation of chloromethylated macroporous resin: Add 24 g of dried D101 resin into a three-necked flask equipped with a stirrer and a thermometer, stir evenly, add 80 mL of 1,4-di(chloromethoxy)-butane and 280 mL of carbon tetrachloride, let stand for swelling for 12 h, add 48 g of anhydrous zinc chloride, stir at 50 ° C for 14 h, wash the product with 1 mol / L dilute hydrochloric acid, ethanol and deionized water in turn, add silver nitrate solution to produce a precipitate, and then dry to obtain a chloromethylated macroporous resin.

[0030] Example 1

[0031] (1) Under a nitrogen atmosphere, 25 g of 5-bromoisatin, 400 mL of ethylene glycol dimethyl ether and 80 mL of deionized water were added to a reaction flask, and after stirring, 22 g of 3-amino-5-boronic acid, 4.5 g of tetrakis(triphenylphosphine)palladium and 26.2 g of sodium carbonate were added, and the mixture was placed in a microwave device for microwave reaction. The reaction temperature was 130° C., and the microwave reaction time was 15 min. After column chromatography purification, the mobile system was petroleum ether and acetone in a volume ratio of 20:1, to obtain an aminocarboxylic acid indolequinone intermediate.

[0032] (2) Under nitrogen atmosphere, 20 g of chloromethylated macroporous resin and 320 mL of N,N-dimethylformamide were added to a reaction flask. After stirring, 9 g of aminocarboxylic acid indolequinone intermediate and 13 g of sodium hydroxide were added. The mixture was reacted at 80 °C for 36 h. The mixture was washed with ethanol and deionized water and dried to obtain a functionalized macroporous resin.

[0033] (3) Using dried American ginseng root as raw material, the root was crushed through an 80-mesh sieve, 15 times the weight of chloroform was added to the raw material powder, and the mixture was boiled at 70°C for 3 h, filtered, and chloroform was added to the residue, and the mixture was boiled for another 3 times, filtered to remove the solvent, and defatted American ginseng was obtained.

[0034] (4) 18 g of defatted American ginseng was mixed evenly with 1170 mL of 70% ethanol aqueous solution, and added into an ultra-high pressure isostatic press for ultra-high pressure extraction at an extraction pressure of 150 MPa for 2 min. The residue was removed by filtration, and the filtrate was concentrated to obtain a crude American ginseng saponin extract.

[0035] (5) After 10 g of American ginseng crude saponin extract was dissolved in methanol-water solution, it was applied to a 300 g functionalized macroporous resin chromatography column and adsorbed for 16 h. The column was washed with deionized water and ethanol in turn, the eluate was collected, and the ethanol phase was recovered to obtain the crude American ginseng saponin.

[0036] (6) The crude American ginsenosides were dissolved in a methanol aqueous solution, mixed with a gel, dried, and added to a silica gel column for column chromatography, eluted with a 40% by mass ethanol aqueous solution, the eluate was collected, concentrated under reduced pressure, and dissolved in an 80% by mass tetrahydrofuran aqueous solution, refluxed at 70° C. for 5 h, cooled to room temperature, frozen for crystallization, filtered under reduced pressure, and dried to obtain American ginsenosides.

[0037] Example 2

[0038] (1) Under a nitrogen atmosphere, 45 g of 5-bromoisatin, 450 mL of ethylene glycol dimethyl ether and 90 mL of deionized water were added to a reaction flask, and after stirring, 36 g of 3-amino-5-boronic acid, 5.4 g of tetrakis(triphenylphosphine)palladium and 31.5 g of sodium carbonate were added, and the mixture was placed in a microwave device for microwave reaction. The reaction temperature was 140° C., and the microwave reaction time was 10 min. After column chromatography purification, the mobile system was petroleum ether and acetone in a volume ratio of 20:1, to obtain an aminocarboxylic acid indolequinone intermediate.

[0039] (2) Under nitrogen atmosphere, 40 g of chloromethylated macroporous resin and 400 mL of N,N-dimethylformamide were added to a reaction flask. After stirring, 16 g of aminocarboxylic acid indolequinone intermediate and 20 g of sodium hydroxide were added. The mixture was reacted at 90 °C for 24 h, washed with ethanol and deionized water, and dried to obtain a functionalized macroporous resin.

[0040] (3) Using dried American ginseng root as raw material, crush it through a 100-mesh sieve, add 10 times the weight of chloroform to the raw material powder, cook it at 80°C for 2 hours, filter, add chloroform to the residue, cook it again for 2 times, filter to remove the solvent, and obtain defatted American ginseng.

[0041] (4) 35 g of defatted American ginseng was mixed evenly with 1750 mL of 60% ethanol aqueous solution, and added into an ultra-high pressure isostatic press for ultra-high pressure extraction at an extraction pressure of 200 MPa for 1 min. The residue was removed by filtration, and the filtrate was concentrated to obtain a crude American ginseng saponin extract.

[0042] (5) After 10 g of American ginseng crude saponin extract was dissolved in methanol-water solution, it was applied to a 350 g functionalized macroporous resin chromatography column and adsorbed for 12 h. The column was washed with deionized water and ethanol in turn, the eluate was collected, and the ethanol phase was recovered to obtain the crude American ginseng saponin.

[0043] (6) The crude American ginsenosides were dissolved in a methanol aqueous solution, mixed with a gel, dried, and added to a silica gel column for column chromatography, eluted with a 10% by mass ethanol aqueous solution, the eluate was collected, concentrated under reduced pressure, and dissolved in a 75% by mass tetrahydrofuran aqueous solution, refluxed at 80° C. for 4 h, cooled to room temperature, frozen for crystallization, filtered under reduced pressure, and dried to obtain American ginsenosides.

[0044] Example 3

[0045] (1) Under a nitrogen atmosphere, 30 g of 5-bromoisatin, 750 mL of ethylene glycol dimethyl ether and 150 mL of deionized water were added to a reaction flask, and after stirring, 28.5 g of 3-amino-5-boronic acid, 7.5 g of tetrakis(triphenylphosphine)palladium and 36 g of sodium carbonate were added, and the mixture was placed in a microwave device for microwave reaction. The reaction temperature was 120° C., and the microwave reaction time was 20 min. After column chromatography purification, the mobile system was petroleum ether and acetone in a volume ratio of 20:1, to obtain an aminocarboxylic acid indolequinone intermediate.

[0046] (2) Under nitrogen atmosphere, 28 g of chloromethylated macroporous resin and 560 mL of N,N-dimethylformamide were added to a reaction flask. After stirring, 16.8 g of aminocarboxylic acid indolequinone intermediate and 22.4 g of sodium hydroxide were added. The mixture was reacted at 65 °C for 48 h. The mixture was washed with ethanol and deionized water and dried to obtain a functionalized macroporous resin.

[0047] (3) Using dried American ginseng root as raw material, the root was crushed through a 60-mesh sieve, 20 times the weight of chloroform was added to the raw material powder, and the mixture was boiled at 60°C for 5 h, filtered, and chloroform was added to the residue, and the mixture was boiled for three more times. The solvent was removed by filtration to obtain defatted American ginseng.

[0048] (4) 25 g of defatted American ginseng was mixed evenly with 2500 mL of 60% ethanol aqueous solution, and added into an ultra-high pressure isostatic press for ultra-high pressure extraction at an extraction pressure of 250 MPa for 3 min. The residue was removed by filtration, and the filtrate was concentrated to obtain a crude American ginseng saponin extract.

[0049] (5) After 10 g of American ginseng crude saponin extract was dissolved in methanol-water solution, it was applied to a 400 g functionalized macroporous resin chromatography column and adsorbed for 12 h. The column was washed with deionized water and ethanol in turn, the eluate was collected, and the ethanol phase was recovered to obtain the crude American ginseng saponin.

[0050] (6) The crude American ginsenosides were dissolved in a methanol aqueous solution, mixed with a gel, dried, and added to a silica gel column for column chromatography, eluted with a 70% by mass ethanol aqueous solution, the eluate was collected, concentrated under reduced pressure, and dissolved in a 90% by mass tetrahydrofuran aqueous solution, refluxed at 60° C. for 8 h, cooled to room temperature, frozen for crystallization, filtered under reduced pressure, and dried to obtain American ginsenosides.

[0051] Example 4

[0052] (1) Under a nitrogen atmosphere, 20 g of 5-bromoisatin, 320 mL of ethylene glycol dimethyl ether and 65 mL of deionized water were added to a reaction flask, and after stirring, 17 g of 3-amino-5-boronic acid, 4.4 g of tetrakis(triphenylphosphine)palladium and 19 g of sodium carbonate were added, and the mixture was placed in a microwave device for microwave reaction. The reaction temperature was 135° C., and the microwave reaction time was 12 min. After column chromatography purification, the mobile system was petroleum ether and acetone in a volume ratio of 20:1 to obtain an aminocarboxylic acid indolequinone intermediate.

[0053] (2) Under nitrogen atmosphere, 32 g of chloromethylated macroporous resin and 480 mL of N,N-dimethylformamide were added to a reaction flask. After stirring, 14.5 g of aminocarboxylic acid indolequinone intermediate and 20.8 g of sodium hydroxide were added. The mixture was reacted at 80 °C for 36 h. The mixture was washed with ethanol and deionized water and dried to obtain a functionalized macroporous resin.

[0054] (3) Using dried American ginseng root as raw material, the root was crushed through a 90-mesh sieve, 15 times the weight of chloroform was added to the raw material powder, and the mixture was boiled at 75°C for 4 h, filtered, and chloroform was added to the residue, and the mixture was boiled for another 3 times, filtered to remove the solvent, and defatted American ginseng was obtained.

[0055] (4) 18 g of defatted American ginseng was mixed evenly with 1150 mL of 75% ethanol aqueous solution, and added into an ultra-high pressure isostatic press for ultra-high pressure extraction at an extraction pressure of 300 MPa for 2 min. The residue was removed by filtration, and the filtrate was concentrated to obtain a crude American ginseng saponin extract.

[0056] (5) After 10 g of American ginseng crude saponin extract was dissolved in methanol-water solution, it was applied to a 450 g functionalized macroporous resin chromatography column and adsorbed for 15 h. The column was washed with deionized water and ethanol in turn, the eluate was collected, and the ethanol phase was recovered to obtain the crude American ginseng saponin.

[0057] (6) The crude American ginsenosides were dissolved in a methanol aqueous solution, mixed with a gel, dried, and added to a silica gel column for column chromatography, eluted with a 60% by mass ethanol aqueous solution, the eluate was collected, concentrated under reduced pressure, and dissolved in an 85% by mass tetrahydrofuran aqueous solution, refluxed at 75° C. for 6 h, cooled to room temperature, frozen for crystallization, filtered under reduced pressure, and dried to obtain American ginsenosides.

[0058] Example 5

[0059] (1) Under a nitrogen atmosphere, 60 g of 5-bromoisatin, 1100 mL of ethylene glycol dimethyl ether and 215 mL of deionized water were added to a reaction flask, and after stirring, 51.5 g of 3-amino-5-boronic acid, 12.6 g of tetrakis(triphenylphosphine)palladium and 51 g of sodium carbonate were added, and the mixture was placed in a microwave device for microwave reaction. The reaction temperature was 125° C., and the microwave reaction time was 18 min. After column chromatography purification, the mobile system was petroleum ether and acetone in a volume ratio of 20:1 to obtain an aminocarboxylic acid indolequinone intermediate.

[0060] (2) Under nitrogen atmosphere, 50 g of chloromethylated macroporous resin and 800 mL of N,N-dimethylformamide were added to a reaction flask. After stirring, 22.5 g of aminocarboxylic acid indolequinone intermediate and 34 g of sodium hydroxide were added. The mixture was reacted at 80 °C for 32 h, washed with ethanol and deionized water, and dried to obtain a functionalized macroporous resin.

[0061] (3) Using dried American ginseng root as raw material, the root was crushed through a 70-mesh sieve, 16 times the weight of chloroform was added to the raw material powder, and the mixture was boiled at 75°C for 5 h, filtered, and chloroform was added to the residue, and the mixture was boiled for three more times. The solvent was removed by filtration to obtain defatted American ginseng.

[0062] (4) 40 g of defatted American ginseng was mixed evenly with 3400 mL of 70% ethanol aqueous solution, and added into an ultra-high pressure isostatic press for ultra-high pressure extraction at an extraction pressure of 350 MPa for 3 min. The residue was removed by filtration, and the filtrate was concentrated to obtain a crude American ginseng saponin extract.

[0063] (5) After 10 g of American ginseng crude saponin extract was dissolved in methanol-water solution, it was applied to a 500 g functionalized macroporous resin chromatography column and adsorbed for 24 h. The column was washed with deionized water and ethanol in turn, the eluate was collected, and the ethanol phase was recovered to obtain the crude American ginseng saponin.

[0064] (6) The crude American ginsenosides were dissolved in a methanol aqueous solution, mixed with a gel, dried, and added to a silica gel column for column chromatography, eluted with a 60% by mass ethanol aqueous solution, the eluate was collected, concentrated under reduced pressure, and dissolved in an 85% by mass tetrahydrofuran aqueous solution, refluxed at 65° C. for 8 h, cooled to room temperature, frozen for crystallization, filtered under reduced pressure, and dried to obtain American ginsenosides.

[0065] Comparative Example 1

[0066] (1) 10 g of defatted American ginseng (prepared in Example 1) was dissolved in a methanol-water solution, and then applied to a 300 g functionalized macroporous resin chromatography column for adsorption for 16 h. The column was washed with deionized water and ethanol in turn, the eluate was collected, and the ethanol phase was recovered to obtain a crude product of American ginsenosides.

[0067] (2) The crude American ginsenosides are dissolved in a methanol aqueous solution, mixed with a gel, dried, and added to a silica gel column for column chromatography, eluted with a 40% by mass ethanol aqueous solution, the eluate is collected, concentrated under reduced pressure, and dissolved in an 80% by mass tetrahydrofuran aqueous solution, refluxed at 70° C. for 5 h, cooled to room temperature, frozen for crystallization, filtered under reduced pressure, and dried to obtain American ginsenosides.

[0068] Comparative Example 2

[0069] (1) 10 g of crude American ginseng saponin extract (prepared in Example 1) was dissolved in methanol-water solution, and then applied to a 300 g D101 resin chromatography column for adsorption for 16 h. The column was washed with deionized water and ethanol in turn, the eluate was collected, and the ethanol phase was recovered to obtain crude American ginseng saponin.

[0070] (2) The crude American ginsenosides are dissolved in a methanol aqueous solution, mixed with a gel, dried, and added to a silica gel column for column chromatography, eluted with a 40% by mass ethanol aqueous solution, the eluate is collected, concentrated under reduced pressure, and dissolved in an 80% by mass tetrahydrofuran aqueous solution, refluxed at 70° C. for 5 h, cooled to room temperature, frozen for crystallization, filtered under reduced pressure, and dried to obtain American ginsenosides.

[0071] Comparative Example 3

[0072] The defatted American ginseng (prepared in Example 1) was dissolved in a methanol aqueous solution, mixed with a material glue, dried, and added to a silica gel column for column chromatography, eluted with a 40% by mass ethanol aqueous solution, the eluate was collected, concentrated under reduced pressure, and dissolved in an 80% by mass tetrahydrofuran aqueous solution, refluxed at 70°C for 5h, cooled to room temperature, frozen and crystallized, filtered under reduced pressure, and dried to obtain American ginseng saponins.

[0073] Static saturated adsorption capacity test: The macroporous resin prepared in each embodiment and comparative example was placed in a 250 mL conical flask, and 200 mL of American ginseng crude saponin extract and defatted American ginseng were added according to the steps of the embodiment and comparative example, respectively. The mixture was shaken at 100 r / min for 24 h to allow sufficient adsorption, and the adsorption capacity of American ginseng saponins by the macroporous resin was tested.

[0074] Adsorption amount (mg / g) = [(concentration before adsorption - concentration after adsorption) × solution volume] / resin mass.

[0075] Static desorption rate test: After 24 hours of static adsorption, rinse the resin with clean liquid, accurately add 200 mL of 80% ethanol by mass, and continuously oscillate at room temperature for 12 hours to allow the saponin to be fully desorbed. Filter, take the filtrate, determine the saponin content, and calculate the desorption rate.

[0076] Desorption rate (%) = [(concentration of solution after desorption × volume of solution after desorption) / (concentration of solution before adsorption × volume of adsorbed solution - concentration of solution after adsorption × volume of adsorbed solution)] × 100%.

[0077] Table 1 Adsorption capacity and desorption rate test

[0078] Adsorption capacity (mg / g) Desorption rate (%) Example 1 53.02 85.21 Example 2 68.16 90.57 Example 3 80.24 94.62 Example 4 95.10 96.54 Example 5 100.62 96.09 Comparative Example 1 50.65 85.43 Comparative Example 2 35.32 73.62 Comparative Example 3 - -

[0079] It can be seen from the test data in the above table that with the increase of the content of the functionalized macroporous resin, the adsorption amount of the crude American ginseng saponin gradually increases. The adsorption amount in Example 5 reaches 100.62 mg / g, and the desorption rate is 96.09%, which has good desorption performance. This is because, on the one hand, the crude American ginseng saponin extract obtained by ultra-high pressure extraction increases the content of American ginseng saponin; on the other hand, the functionalized macroporous resin contains an indolequinone structure, and the amide group in its structure can produce hydrogen bonds with the phenolic hydroxyl group of American ginseng to form a stable ring structure. The additional carbonyl structure can form hydrogen bonds with other phenolic hydroxyl groups, and the benzene ring can form a π-π conjugation with the benzene ring of phenol. The carboxyl group and the phenolic hydroxyl group can also produce a covalent bond. The charge effect of the amide group and the dissociated phenolic hydroxyl group will enhance the adsorption capacity of the resin.

[0080] In Comparative Example 1, ultrahigh pressure extraction was not performed, resulting in low purity of crude American ginsenosides and limited adsorption capacity of the macroporous resin; in Comparative Example 2, ordinary D101 resin was added, which does not contain an indolequinone structure and has no adsorption specificity; in Comparative Example 3, ultrahigh pressure extraction was not performed and no macroporous resin was added, so it has no adsorption performance.

[0081] Table 2 American ginsenoside content and yield test

[0082] Ginsenoside content (%) American ginsenoside yield (%) Example 1 85.6 74.5 Example 2 90.1 81.2 Example 3 94.5 85.6 Example 4 96.9 88.7 Example 5 98.0 90.3 Comparative Example 1 80.3 72.5 Comparative Example 2 74.8 65.1 Comparative Example 3 67.6 50.3

[0083] It can be seen from the test results in the above table that the combination of ultra-high pressure extraction and functionalized macroporous resin adsorption can significantly improve the content and yield of American ginseng saponins extracted. The ultra-high pressure method for extracting American ginseng saponins can increase the osmotic pressure difference, increase the penetration and diffusion rate, shorten the dissolution equilibrium time, and at the same time destroy the structure of various organelles such as cell membranes, increase the contact area between solvents and solutes, reduce mass transfer resistance, and shorten the extraction time, so that the total saponins of American ginseng can be extracted quickly and to the greatest extent; the functionalized macroporous resin can quickly adsorb American ginseng saponins in a targeted manner, and the adsorption amount is large. At the same time, a large amount of impurities are removed, and the content of the target substance is increased. After another column chromatography purification, the content of the target substance is increased to 98.0%. The method is simple and feasible, with low production cost, and is suitable for industrial production.

[0084] The above embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention. The protection scope of the present invention is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present invention within the essence and protection scope of the present invention, and such modifications or equivalent substitutions shall also be deemed to fall within the protection scope of the present invention.

Claims

1. A method for extracting American ginsenosides, characterized in that: The extraction method is carried out according to the following steps: Step (1), using dried American ginseng root as raw material, crushing through a 60-100 mesh sieve, adding 10-20 times the weight of chloroform to the raw material powder, boiling at 60-80° C. for 2-5 hours, filtering, adding chloroform to the residue, boiling again for 2-3 times, filtering to remove the solvent, and obtaining defatted American ginseng; Step (2), mixing defatted American ginseng with an ethanol aqueous solution at a solid-liquid ratio of 1 g: (50-100) mL, adding the mixture to an ultra-high pressure isostatic press for ultra-high pressure extraction, filtering to remove the residue, and concentrating the filtrate to obtain a crude American ginseng saponin extract; Step (3), adding the crude American ginseng saponin extract to a methanol aqueous solution for dissolution, applying it to a functionalized macroporous resin chromatography column, adsorbing it for 12-24 hours, washing it with deionized water and ethanol in sequence, collecting the eluate, recovering the ethanol phase, and obtaining a crude American ginseng saponin product; Step (4), dissolving the crude American ginsenoside with methanol aqueous solution, mixing with glue, drying and adding to a silica gel column for column chromatography, eluting with ethanol aqueous solution of different mass fractions, collecting the eluate, concentrating under reduced pressure, adding tetrahydrofuran aqueous solution to dissolve, reflux at 60-80° C. for 4-8 hours, cooling to room temperature, freezing and crystallizing, filtering under reduced pressure, and drying to obtain American ginsenoside; The method for preparing the functionalized macroporous resin in step (3) is carried out according to the following steps: Step S1, under a nitrogen atmosphere, add 5-bromoisatin, ethylene glycol dimethyl ether and deionized water into a reaction flask, stir evenly, add 3-amino-5-boronic acid, tetrakis(triphenylphosphine)palladium and sodium carbonate, place in a microwave device for microwave reaction, and purify by column chromatography after the reaction to obtain an aminocarboxylic acid indolequinone intermediate; Step S2, under a nitrogen atmosphere, add chloromethylated macroporous resin and N,N-dimethylformamide to a reaction flask, stir evenly, add aminocarboxylic acid indolequinone intermediate and sodium hydroxide, react at 65-90° C. for 24-48 hours, wash with ethanol and deionized water, and dry to obtain a functionalized macroporous resin.

2. The method for extracting American ginsenosides according to claim 1, characterized in that: The mass fraction of the ethanol aqueous solution in step (2) is 60-80%.

3. The method for extracting American ginsenosides according to claim 1, characterized in that: In the step (2), the ultrahigh pressure extraction pressure is 150-350 MPa, and the extraction time is 1-3 min.

4. The method for extracting American ginsenosides according to claim 1, characterized in that: In the step (3), the ratio of the crude American ginseng saponin extract to the functionalized macroporous resin is 1 g: (30-50) g.

5. The method for extracting American ginsenosides according to claim 1, characterized in that: In the step (4), the mass fraction of the ethanol aqueous solution is 10-70%, and the mass fraction of the tetrahydrofuran aqueous solution is 75-90%.

6. The method for extracting American ginsenosides according to claim 1, characterized in that: In the step S1, the ratio of 5-bromoisatin, ethylene glycol dimethyl ether, deionized water, 3-amino-5-boronic acid, tetrakis(triphenylphosphine)palladium, and sodium carbonate is 1 g: (10-25) mL: (2-5) mL: (0.8-0.95) g: (0.12-0.25) g: (0.7-1.2) g.

7. The method for extracting American ginsenosides according to claim 1, characterized in that: In step S1, the microwave reaction temperature is 120-140° C., and the microwave reaction time is 10-20 min.

8. The method for extracting American ginsenosides according to claim 1, characterized in that: In the step S2, the ratio of the chloromethylated macroporous resin, N,N-dimethylformamide, aminocarboxylic acid indolequinone intermediate, and sodium hydroxide is 1 g: (10-20) mL: (0.4-0.6) g: (0.5-0.8) g.

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