Extraction method of American ginseng glycoprotein, obtained product and application

Abstract

The invention discloses an extraction method of American ginseng glycoprotein, an obtained product and application, American ginseng is used as a main raw material, a natural eutectic solvent aqueous solution is used as a solvent, warm immersion stirring, ultrasonic and microwave extraction are performed in sequence to obtain an extracting solution, then the extracting solution is subjected to subsequent operations such as alcohol precipitation and free protein removal, and the American ginseng glycoprotein is obtained. The American ginseng glycoprotein is obtained. The American ginseng glycoprotein extraction method is simple in process, high in safety, high in yield and purity and suitable for industrial production, and the obtained American ginseng glycoprotein has liver protection / liver protection and antioxidant activity, can be prepared into drugs, health care products and food, provides clinical guidance and medication basis for patients with liver diseases, further improves the life quality of the patients and has a good clinical application prospect. And the method has good application value.

Description

Technical Field

[0001] The present invention relates to a method for extracting American ginseng glycoprotein, a product obtained therefrom and applications thereof, and belongs to the technical field of natural product extraction and application. Background Art

[0002] American ginseng is a plant of the Araliaceae family ( Panax quinquefolius The dried root of American ginseng (Panax quinquefolius L.) contains saponins, polysaccharides, flavonoids, fatty acids, and polyacetylenes as its main chemical components. It exhibits pharmacological activities such as anti-tumor, anti-fatigue, immunomodulatory, metabolic regulation, and anti-inflammatory properties. Currently, the development and application of American ginseng primarily focuses on small-molecule compounds such as saponins, while research on macromolecular compounds with significant biological activity is insufficient, particularly glycoproteins, for which no relevant reports have been published.

[0003] Glycoproteins are proteins containing oligosaccharide chains linked by covalent bonds. Their structural stability and potential health and medicinal properties have made them a growing research hotspot. As conjugated proteins, glycoproteins possess some of the properties of both proteins and polysaccharides, and therefore their extraction methods are often tailored to the properties of these two groups. Natural deep eutectic solvents are often used to extract proteins and polysaccharides from natural products, and are expected to replace traditional organic solvents and address challenges encountered in the extraction of natural compounds. Glycoproteins have been shown to possess numerous biological activities, and several have been shown to possess significant antioxidant and hepatoprotective properties. These properties make them potentially valuable for the treatment of liver diseases.

[0004] At present, there are no reports on the extraction of glycoproteins from American ginseng. The extraction of glycoproteins from American ginseng is of great significance for the further development and utilization of American ginseng. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for extracting American ginseng extract, more specifically, a method for extracting American ginseng glycoprotein, which adopts a natural low eutectic solvent aqueous solution for extraction and is combined with different auxiliary methods. The method is simple to operate, highly safe, and pollution-free. The obtained extract contains American ginseng glycoprotein components, enriches the active ingredients in American ginseng, and has a high extraction rate of American ginseng glycoprotein.

[0006] The specific technical solutions adopted in the present invention are as follows: A method for extracting an American ginseng extract, wherein the extract is mainly American ginseng glycoprotein, the method comprising the following steps: (1) Grinding American ginseng, mixing the American ginseng powder with an aqueous solution of a natural deep eutectic solvent, extracting the mixture under stirring, and collecting the extract and filter residue; (2) mixing the filter residue from step (1) with an aqueous solution of a natural deep eutectic solvent, extracting the mixture under ultrasound, and collecting the extract and the filter residue; (3) mixing the filter residue from step (2) with an aqueous solution of a natural deep eutectic solvent, extracting the mixture under microwave conditions, and collecting the extract and the filter residue; (4) combining the extracts obtained in steps (1), (2), and (3), and precipitating with alcohol to obtain a crude product of American ginseng glycoprotein; (5) The crude glycoprotein of American ginseng is redissolved in water and free protein is removed to obtain an American ginseng extract, namely, crude glycoprotein of American ginseng. The extract is further purified to obtain a glycoprotein of high purity.

[0007] Furthermore, the present invention uses a natural deep eutectic solvent aqueous solution to extract American ginseng glycoprotein from American ginseng, wherein the natural deep eutectic solvent aqueous solution is composed of a natural deep eutectic solvent and water. The natural deep eutectic solvent aqueous solution is prepared by mixing a hydrogen bond acceptor and a hydrogen bond donor in a molar ratio of 1:1 to 3. The hydrogen bond acceptor is choline chloride, and the hydrogen bond donor is malic acid or urea.

[0008] Furthermore, the preparation method of the natural deep eutectic solvent aqueous solution comprises: uniformly mixing a hydrogen bond acceptor and a hydrogen bond donor according to a ratio, and continuously stirring at 45-55°C until the solution becomes clear and transparent to obtain a natural deep eutectic solvent; and uniformly mixing the natural deep eutectic solvent with water to obtain a natural deep eutectic solvent aqueous solution. Stirring can be achieved using a magnetic stirrer, ultrasonic equipment, microwave synthesizer, etc., with a magnetic stirrer being preferred due to its ease of operation, energy saving, and high efficiency.

[0009] Furthermore, in the present invention, in steps (1), (2), and (3), different methods are used to extract the American ginseng glycoprotein using a natural deep eutectic solvent aqueous solution. The natural deep eutectic solvent aqueous solution used in each step is different. The combined effect of the natural deep eutectic solvent aqueous solution and auxiliary methods such as stirring, ultrasound, and microwaves can improve the extraction rate of the American ginseng glycoprotein.

[0010] Furthermore, in step (1), the hydrogen bond acceptor of the natural deep eutectic solvent is choline chloride, and the hydrogen bond donor is urea. In the natural deep eutectic solvent aqueous solution, the volume concentration of the natural deep eutectic solvent is 60-65%.

[0011] Furthermore, in step (1), the mass ratio of American ginseng powder to the natural deep eutectic solvent aqueous solution is 1:20-30.

[0012] Furthermore, in step (1), the extraction is performed at least once, preferably once. The extraction temperature is 50-90°C, and the extraction time is 40-50 min. Stirring can be performed by conventional stirring methods, such as magnetic stirring, blade stirring, etc. The stirring speed is generally above 650 rpm, such as 650 rpm, 700 rpm, 750 rpm, 800 rpm, 850 rpm, and 900 rpm.

[0013] Furthermore, in step (2), the hydrogen bond acceptor of the natural deep eutectic solvent is choline chloride, and the hydrogen bond donor is urea. In the natural deep eutectic solvent aqueous solution, the volume concentration of the natural deep eutectic solvent is 75-80%.

[0014] Furthermore, in step (2), the mass ratio of American ginseng powder to the natural deep eutectic solvent aqueous solution is 1:20-30.

[0015] Furthermore, in step (2), the extraction is performed at least once, preferably once. The extraction temperature is 50-90°C, and the extraction time is 40-50 minutes. Ultrasonication can be performed using commonly used laboratory ultrasonic equipment.

[0016] Furthermore, in step (3), the hydrogen bond acceptor of the natural deep eutectic solvent is choline chloride, and the hydrogen bond donor is malic acid. In the natural deep eutectic solvent aqueous solution, the volume concentration of the natural deep eutectic solvent is 85-90%.

[0017] Furthermore, in step (3), the mass ratio of American ginseng powder to the natural deep eutectic solvent aqueous solution is 1:10-20.

[0018] Furthermore, in step (3), the extraction is performed at least once, preferably twice.

[0019] Furthermore, in step (3), the extraction is performed twice under microwave conditions. The first microwave extraction is performed at a microwave power of 50-75 W, a microwave temperature of 45-55 °C, and an extraction time of 10-30 min; the second microwave extraction is performed at a microwave power of 75-100 W, a microwave temperature of 95-105 °C, and an extraction time of 10-30 min. Under the technology of microwave assisted natural deep eutectic solvent, the American ginseng glycoprotein is extracted twice, and by gradually increasing the microwave power and temperature, the American ginseng glycoprotein is extracted to the maximum extent.

[0020] Furthermore, in step (4), the alcohol used for alcohol precipitation is ethanol. The extracts obtained in steps (1), (2), and (3) can be directly combined and then subjected to alcohol precipitation, or they can be concentrated and then subjected to alcohol precipitation. Concentrating and then subjecting to alcohol precipitation can effectively reduce alcohol waste and improve the effect of alcohol precipitation. The extracts obtained in steps (1), (2), and (3) can be concentrated first and then combined, or combined first and then concentrated, so that the specific gravity of the final combined extract is 1.02~1.06. Concentration can be carried out using a rotary evaporator.

[0021] Furthermore, in step (4), the number of alcohol precipitations is 1 to 2 times, preferably 2 times. The use of a secondary alcohol precipitation method to separate the American ginseng glycoprotein can not only achieve a better purification effect of the American ginseng glycoprotein, but also effectively remove the residual natural deep eutectic solvent in the sample. The concentration of alcohol in the two alcohol precipitations can be the same or different, and the alcohol precipitation makes the alcohol concentration between 70 and 95 wt%.

[0022] Preferably, during the first alcohol precipitation, alcohol is added to the extract to a concentration of 70-75 wt %, and during the second alcohol precipitation, alcohol is added to the precipitate obtained from the first alcohol precipitation to a concentration of 70-95 wt %. More preferably, during the first alcohol precipitation, alcohol is added to the extract to a concentration of 70-75 wt %, and during the second alcohol precipitation, alcohol is added to the precipitate obtained from the first alcohol precipitation to a concentration of 90-95 wt %.

[0023] Furthermore, in step (5), the free protein is removed by the Sevag method. The Sevag method can be a method commonly used in the prior art. For example, the conditions for removing free protein by the Sevag method can be: adding Sevag reagent (chloroform: n-butanol = 4:1, v / v) at 20-30% (preferably 20%) of the volume of the crude ginseng glycoprotein solution, adding the crude ginseng glycoprotein solution and the Sevag reagent in a corresponding proportion to a separating funnel, after sufficient contact by strong shaking, standing and separating the layers, retaining the upper aqueous solution and discarding the lower layer, repeating the above operation for the aqueous solution, and when no denatured protein appears, collecting the supernatant for freeze-drying; or it can be: adding Sevag reagent (chloroform: n-butanol = 4:1, v / v) at 20-30% (preferably 20%) of the volume of the crude ginseng glycoprotein solution, and stirring at 4°C for 5-10 min (1000 rpm). The emulsion is then centrifuged at 7500 rpm for 5 minutes, retaining the upper aqueous layer and discarding the lower layer. Repeat this process for the aqueous solution three to four times. When no denatured protein is present, collect the supernatant. The deproteinized supernatant is evaporated to dryness using a rotary evaporator, reconstituted, and lyophilized. The second method is preferred, as adjusting the stirring speed improves protein removal efficiency. Centrifuging the emulsion effectively reduces the time required for protein removal and effectively removes the protein layer.

[0024] The present invention also provides a product obtained according to the above method, which is a glycoprotein from American ginseng. It has been verified that the glycoprotein from American ginseng has good hepatoprotective and antioxidant effects. Therefore, the present invention also provides the use of the glycoprotein from American ginseng in the preparation of hepatoprotective and antioxidant products. The hepatoprotective and antioxidant products can be medicines, health products, or foods. The antioxidant products can be health products, foods, or cosmetics.

[0025] The beneficial effects of the present invention are: 1. The present invention discovered the glycoprotein component in American ginseng for the first time through extraction and separation, enriching the active ingredients in American ginseng.

[0026] 2. The present invention uses a natural low eutectic solvent to extract American ginseng glycoprotein, which has the characteristics of environmental protection, high efficiency, low cost, low toxicity and versatility.

[0027] 3. The present invention extracts American ginseng glycoprotein by combining different auxiliary extraction methods, first performing warm immersion and stirring extraction, and then performing ultrasonic and microwave extraction, which can maximize the biological activity of the extract and maximize the extraction rate of American ginseng glycoprotein. It is highly practical and easy to scale up production.

[0028] 4. The present invention removes free protein through the Sevag method, effectively saving time and cost.

[0029] 5. The method of the present invention is easy to operate, highly safe, pollution-free, easy to clean, precise in control, low in noise, wide in application range, capable of automatic control, and energy-saving and high-efficiency.

[0030] 6. The present invention is the first to separate American ginseng glycoprotein from American ginseng. The American ginseng glycoprotein has good antioxidant activity and hepatoprotective activity, has a therapeutic effect on liver disease, provides a new direction for the prevention and treatment of liver disease, and is of great significance for the further development and utilization of American ginseng. DETAILED DESCRIPTION

[0031] The following is a clear and complete description of the technical solutions of the present invention in conjunction with specific embodiments. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] Example 1 S1. Choline chloride is used as a hydrogen bond acceptor, and malic acid and urea are used as hydrogen bond donors, respectively. They are mixed in a molar ratio of hydrogen bond acceptor:hydrogen bond donor = 1:2, placed in a magnetic stirrer at 50°C, and stirred continuously until clear and transparent to obtain a natural deep eutectic solvent. The natural deep eutectic solvent synthesized from choline chloride and urea is then diluted with water to a volume fraction of 65% and 75%, respectively. The natural deep eutectic solvent synthesized from choline chloride and malic acid is also diluted with water to a volume fraction of 85%, to obtain an aqueous solution of the natural deep eutectic solvent.

[0033] S2. Take dried American ginseng, grind it, and pass it through a 30-60 mesh sieve. Add 25 times the mass of a 65% by volume natural deep eutectic solvent aqueous solution synthesized from choline chloride and urea to the American ginseng powder. Extract the mixture in a magnetic stirrer at 90°C (650 rpm) for 45 minutes. Filter and concentrate the filtrate to a specific gravity of 1.04 to obtain American ginseng aqueous extract concentrate 1 and American ginseng residue. S3. The residue from S2 was further added with 25 times the mass of a 75% volume fraction of a natural deep eutectic solvent aqueous solution synthesized from choline chloride and urea, and extracted under ultrasound at 90 °C for 45 min. The filtrate was filtered and concentrated to a specific gravity of 1.04 to obtain American ginseng aqueous extract concentrate 2 and American ginseng residue; S4. The residue from S3 was further added with 15 times the mass of an 85% by volume natural deep eutectic solvent aqueous solution synthesized from choline chloride and malic acid. The mixture was placed in a microwave synthesizer and extracted at a microwave power of 75 W and a microwave temperature of 50°C for 15 min. The mixture was filtered. The residue was then extracted at a microwave power of 100 W and a microwave temperature of 100°C for 15 min. The filtrates were combined and concentrated to a specific gravity of 1.04 to obtain American ginseng aqueous extract concentrate 3 and American ginseng residue. S5. The American ginseng water extract concentrates 1, 2, and 3 from S2, S3, and S4 were combined to obtain American ginseng water extract concentrate 4. High-concentration ethanol was added to the concentrate to achieve an alcohol concentration of 70-75 wt %. The concentrate was allowed to stand at 4°C for 24 hours and filtered to obtain a filtrate and a precipitate. The filtrate was decompressed and the ethanol solution was recovered to obtain a crude American ginseng total saponin extract. The precipitate was subjected to a second alcohol precipitation by adding high-concentration ethanol to achieve an alcohol concentration of 90-95 wt %. The concentrate was then allowed to stand at 4°C for 24 hours and filtered. The resulting precipitate was the crude American ginseng glycoprotein extract. S6. The crude ginseng glycoprotein extract from S5 was reconstituted with water and free protein was removed using the Sevag method. The following conditions were used: Sevag reagent (chloroform: n-butanol = 4:1, v / v) was added to the crude extract aqueous solution at 20% of its volume, and the solution was stirred at 4°C (1000 rpm) for 5 minutes. The resulting mixture was then centrifuged at 7500 rpm for 5 minutes, retaining the upper aqueous layer and discarding the lower layer. Repeat the above free protein removal procedure three to four times for the aqueous solution. When no denatured protein was present, the supernatant was collected and evaporated to dryness using a rotary evaporator. The supernatant was reconstituted with distilled water and lyophilized to obtain ginseng glycoprotein powder.

[0034] Example 2 American ginseng glycoprotein was prepared according to the method of Example 1, except that: in step S2, the extraction temperature was 50°C; in step S3, the extraction temperature was 50°C; in step S4, the microwave power of the first microwave extraction was 50 W, the microwave temperature was 45°C, and the extraction time was 30 min; and the microwave power of the second microwave extraction was 75 W, the microwave temperature was 95°C, and the extraction time was 30 min.

[0035] Example 3 American ginseng glycoprotein was prepared according to the method of Example 1, except that: in step S2, the volume fraction of the natural deep eutectic solvent aqueous solution was 60%; in step S3, the volume fraction of the natural deep eutectic solvent aqueous solution was 80%; and in step S4, the volume fraction of the natural deep eutectic solvent aqueous solution was 90%.

[0036] Example 4 American ginseng glycoprotein was prepared according to the method of Example 1, except that in step S4, extraction was performed twice in a microwave synthesizer, and the extraction conditions for each extraction were: microwave power of 100 W, microwave temperature of 100° C., and extraction time of 30 min.

[0037] Example 5 American ginseng glycoprotein was prepared according to the method of Example 1, except that: in step S5, the American ginseng water extract concentrate 4 was subjected to alcohol precipitation twice, and the condition was that the alcohol concentration reached 70-75 wt%.

[0038] Comparative Example 1 American ginseng glycoprotein was prepared according to the method of Example 1, except that the extraction was performed in the order of microwave extraction, stirring extraction, and ultrasonic extraction, that is, step S4 was performed first, then step S2, and then step S3.

[0039] Comparative Example 2 American ginseng glycoprotein was prepared according to the method of Example 1, except that the extraction was performed in the order of ultrasonic extraction, stirring extraction, and microwave extraction, that is, step S3 was performed first, then step S2, and then step S4.

[0040] Comparative Example 3 S1. Choline chloride is used as a hydrogen bond acceptor and malic acid is used as a hydrogen bond donor. They are mixed in a molar ratio of 1:1 and placed in a microwave oven at 180 W. When the solution becomes clear and transparent, a natural deep eutectic solvent is obtained. The natural deep eutectic solvent is then diluted with water to a volume fraction of 20% to obtain an aqueous natural deep eutectic solvent solution.

[0041] S2. Take dried American ginseng, grind it, and pass it through a 30-60 mesh sieve. Add 25 times the mass of a 20% by volume natural deep eutectic solvent aqueous solution synthesized from choline chloride and malic acid to the American ginseng powder. Place the powder in a microwave synthesizer and extract it at a microwave power of 90 W and a microwave temperature of 90°C for 15 minutes. Filter and concentrate the filtrate to a specific gravity of 1.04 to obtain American ginseng aqueous extract concentrate 1 and American ginseng residue. S3. The residue from S2 was further added with 25 times the mass of a 20% volume fraction of a natural deep eutectic solvent aqueous solution synthesized from choline chloride and malic acid. The mixture was placed in a microwave synthesizer and extracted at a microwave power of 90 W and a microwave temperature of 90°C for 15 min. The mixture was filtered and the filtrate was concentrated to a specific gravity of 1.04 to obtain American ginseng aqueous extract concentrate 2 and American ginseng residue. S4. The residue from S3 was further added with 15 times the mass of a 20% volume fraction of a natural deep eutectic solvent aqueous solution synthesized from choline chloride and malic acid. The mixture was placed in a microwave synthesizer and extracted at a microwave power of 90 W and a microwave temperature of 50°C for 15 min. The mixture was filtered. The residue was then extracted at a microwave power of 90 W and a microwave temperature of 100°C for 15 min. The filtrates were combined and concentrated to a specific gravity of 1.04 to obtain American ginseng aqueous extract concentrate 3 and American ginseng residue. S5. Same as Example 1; S6. Same as Example 1.

[0042] Comparative Example 4 American ginseng glycoprotein was prepared according to the method of Example 1, except that: in step S2, the natural deep eutectic solvent aqueous solution was a natural deep eutectic solvent aqueous solution synthesized by choline chloride and malic acid with a volume fraction of 85%; in step S3, the natural deep eutectic solvent aqueous solution was a natural deep eutectic solvent aqueous solution synthesized by choline chloride and malic acid with a volume fraction of 85%; in step S4, the natural deep eutectic solvent aqueous solution was a natural deep eutectic solvent aqueous solution synthesized by choline chloride and urea with a volume fraction of 85%.

[0043] Comparative Example 5 Choline chloride is used as a hydrogen bond acceptor and ethylene glycol is used as a hydrogen bond donor. They are mixed in a molar ratio of hydrogen bond acceptor: hydrogen bond donor = 1:2, placed in a magnetic stirrer at 50°C, and continuously stirred until clear and transparent to obtain a natural deep eutectic solvent. The natural deep eutectic solvent is then diluted with water to a volume fraction of 85% to obtain a natural deep eutectic solvent aqueous solution.

[0044] American ginseng glycoprotein was prepared according to the method of Example 1, except that in steps S2, S3 and S4, the natural deep eutectic solvent aqueous solution used was replaced by the above-mentioned 85% volume fraction of choline chloride and ethylene glycol natural deep eutectic solvent aqueous solution.

[0045] The American ginseng glycoprotein obtained in the above examples and comparative examples was evaluated in the following manner: 1. Determination of Glycoprotein Quality Fraction of American Ginseng 1.1 Determination of polysaccharide mass fraction in American ginseng glycoprotein 1.1.1 Drawing of the standard curve Accurately pipette 0 μL, 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, and 100 μL of glucose standard solution with a mass concentration of 0.1 mg / mL into a centrifuge tube, add ultrapure water to make up to 100 μL, mix well, and obtain standard solutions with mass concentrations of 0.00 mg / mL, 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, and 0.10 mg / mL, respectively. Add 100 μL of 6% phenol solution, slowly add 500 μL of concentrated sulfuric acid, mix well, and place in a 100°C water bath for 5 min. After the temperature in the water bath drops to room temperature, take out the sample and measure the absorbance at 490 nm. Draw a standard curve of concentration and absorbance to obtain the regression equation (1).

[0046] y=4.2116x+0.143, R 2 =0.9977 (1) 1.1.2 Sample preparation 1000 mg of American ginseng powder was taken respectively, and American ginseng glycoprotein powder was prepared using the methods of the above examples and comparative examples.

[0047] The American ginseng glycoprotein powder prepared in each example and comparative example was respectively diluted to a 10 ml volumetric flask to obtain a sample solution.

[0048] 1.1.3 Determination of sample solution Accurately pipette 100 μL of sample solution into a centrifuge tube, add 100 μL of 6% phenol solution, slowly add 500 μL of concentrated sulfuric acid, mix well, and place in a 100°C water bath for 5 min. After the temperature in the water bath drops to room temperature, take out the sample and measure the absorbance at 490 nm. Substitute it into the regression equation (1) to calculate the mass concentration of polysaccharides in the sample solution, and calculate the mass fraction of polysaccharides in American ginseng (W1) according to equation (2).

[0049] W1 = polysaccharide concentration in sample solution * total volume of sample solution / mass of American ginseng powder * 100% (2) 1.2 Determination of protein mass fraction in American ginseng glycoprotein 1.2.1 Drawing of the standard curve Accurately pipette 0 μL, 1 μL, 2 μL, 4 μL, 8 μL, 12 μL, 16 μL, and 20 μL of 0.5 mg / mL BSA standard solution into a 96-well plate, add distilled water to make up to 20 μL, add 200 μL of BCA working solution to each well, place at 37°C for 30 minutes, cool to room temperature, measure the absorbance at 562 nm, draw a standard curve of absorbance and concentration, and obtain the regression equation (3).

[0050] y=1.0118x+0.1438, R 2 =0.999 (3) 1.2.2 Sample preparation 1000 mg of American ginseng powder was taken respectively, and American ginseng glycoprotein powder was prepared using the methods of the above examples and comparative examples.

[0051] The American ginseng glycoprotein powder prepared in each example and comparative example was respectively diluted to a 10 ml volumetric flask to obtain a sample solution.

[0052] 1.2.3 Determination of sample solution Accurately pipette 20 μL of sample solution into a 96-well plate, add 200 μL of BCA working solution to each well, place at 37 °C for 30 minutes, cool to room temperature, measure the absorbance at 562 nm, and substitute into the regression equation (3) to calculate the protein mass concentration in the sample solution, and calculate the protein mass fraction (W2) in American ginseng according to equation (4).

[0053] 1.3 Calculation of glycoprotein quality score of American ginseng The glycoprotein mass fraction (W) in American ginseng was calculated according to formula (5).

[0054] W=W1+W2 (5) 1.4 Results The results of the American ginseng glycoprotein quality analysis are shown in Table 1. As can be seen from the table below, the American ginseng glycoprotein quality scores of Examples 1-5 are all superior to those of Comparative Examples 1-5, with Example 1 achieving the highest extraction efficiency. This indicates that after treatment with the present method, the American ginseng glycoprotein was extracted to the greatest extent possible.

[0055] 2. In vitro antioxidant assay 2.1 DPPH free radical scavenging ability Prepare a 0.1 mmol / L DPPH alcohol solution in anhydrous ethanol for immediate use and store in a refrigerator away from light.

[0056] Take the American ginseng glycoprotein extract and prepare sample solutions at (10.00, 5.00, 2.50, 1.25, 0.63) mg / ml.

[0057] Take 200 μL of sample solution of different concentrations and add 200 μL of DPPH alcohol solution to form the sample group. Take a blank tube and add 200 μL of sample solvent and then 200 μL of anhydrous ethanol to form the blank control group. Take a blank tube and add 200 μL of sample solvent and then 200 μL of DPPH alcohol solution to form the blank group. Take a blank tube and add 200 μL of sample solution and then 200 μL of anhydrous ethanol to form the sample control group. After each group is thoroughly mixed, stand at room temperature in the dark for 30 minutes, and immediately measure the absorbance at 517 nm. Perform three parallel measurements and calculate the DPPH scavenging rate as follows. Graphpad Prism 9.0 is used to process the data and calculate the scavenging rate IC50.

[0058] Clearance rate (%) = [ (A1-A2) - (A3-A4)] / (A1-A2) * 100% Where: A1: absorbance value of blank group; A2: absorbance value of blank control group; A3: absorbance value of the sample group; A4: Absorbance value of the sample control group.

[0059] 2.2 ABTS free radical scavenging ability Preparation of ABTS working solution: 7.0 mmol / L ABTS solution and 2.45 mmol / L K2S2O8 solution; mix equal volumes of the prepared ABTS solution and K2S2O8 solution, and store in the dark at room temperature for 12-16 hours to obtain the ABTS working solution. Before use, dilute with anhydrous ethanol until the absorbance at 734 nm is 0.70 ± 0.02. Store in the dark until testing.

[0060] Take the American ginseng glycoprotein extract and prepare sample solutions at (10.00, 5.00, 2.50, 1.25, 0.63) mg / ml.

[0061] 50 μL of sample solution at different concentrations was added to 80 μL of ABTS working solution to form the sample group. A blank tube was prepared by adding 50 μL of sample solvent and then 80 μL of anhydrous ethanol to form the blank control group. A blank tube was prepared by adding 50 μL of sample solvent and then 80 μL of ABTS working solution to form the blank control group. A blank tube was prepared by adding 50 μL of sample solution and then 80 μL of anhydrous ethanol to form the sample control group. After thorough mixing, each group was incubated at room temperature in the dark for 6 minutes. The absorbance was immediately measured at 734 nm. Three replicates were performed. The ABTS clearance rate was calculated according to the following formula. Data were processed using Graphpad Prism 9.0 to calculate the clearance IC50 (sample concentration at which the clearance rate is 50%).

[0062] Clearance rate I (%) = [(A1-A2)-(A3-A4)] / (A1-A2)*100% Where: A1: absorbance value of blank group; A2: absorbance value of blank control group; A3: absorbance value of the sample group; A4: Absorbance value of the sample control group.

[0063] 2.3 Results The results of the DPPH and ABTS free radical scavenging activities of American ginseng glycoprotein are shown in Table 2 below. As can be seen from the table below, the antioxidant activity of the American ginseng glycoprotein of Example 1 is superior to that of Comparative Examples 1 to 3. This indicates that after treatment with the present method, the resulting American ginseng glycoprotein maintains the biological activity of the extract to the greatest extent possible.

[0064] 3. Hepatoprotective activity detection 3.1 Sample preparation Take the American ginseng glycoprotein extract and prepare sample solutions of (10.00, 20.00, 40.00, 80.00, 160.00, 320.00, 640.00, 1280.00) μg / ml.

[0065] 3.2 Cell culture and passaging HSC-T6 cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% double-antibody at 37°C in a 5% CO2 humidified incubator. When the cell density was about 80-90%, 1 × 10 6 / ml passage.

[0066] HepG2 cells were cultured in MEM medium supplemented with 10% fetal bovine serum and 1% double-antibody at 37°C in a 5% CO2 humidified incubator. When the cell density was about 80-90%, 1 × 10 6 / ml passage.

[0067] LX2 cells were cultured in 1640 medium supplemented with 10% fetal bovine serum and 1% double-antibody at 37°C in a 5% CO2 humidified incubator. When the cell density was about 80-90%, 1 × 10 6 / ml passage.

[0068] 3.3 MTT colorimetric assay for cell proliferation The concentration was 1 × 10 6 Cells were seeded at 500 μg / ml in a 96-well culture plate. After cells adhered, drugs at varying concentrations were added, with replicate wells, control wells, and blank wells. The plates were incubated at 37°C, 5% CO₂ for the desired time. 20 μL of MTT was added to each well and incubated for another 4 hours. DMSO (100 μL) was added and mixed by vortexing. The absorbance (OD) of each well was measured at 570 nm using a microplate reader. The cell inhibition rate was calculated, and the data were processed using GraphPad Prism 9.0 to calculate the IC₅(50) of cell viability inhibition.

[0069] Cell inhibition rate (%) = (OD control - OD administration) / (OD control - OD blank) * 100%

Claims

1. A method for extracting American ginseng glycoprotein, characterized in that The following steps are involved: (1) crushing American ginseng, mixing the American ginseng powder with an aqueous solution of a natural deep eutectic solvent, extracting under stirring, and collecting the extract and the residue; (2) mixing the filter residue from step (1) with an aqueous solution of a natural deep eutectic solvent, extracting the mixture under ultrasound, and collecting the extract and the filter residue; (3) mixing the filter residue from step (2) with an aqueous solution of a natural low eutectic solvent, extracting the mixture under microwave conditions, and collecting the extract and the filter residue; (4) combining the extracts obtained in steps (1), (2) and (3), and precipitating with alcohol to obtain a crude product of American ginseng glycoprotein; (5) Redissolving the crude American ginseng glycoprotein with water, removing free protein, and obtaining American ginseng glycoprotein.

2. The extraction method according to claim 1, characterized in that: The natural deep eutectic solvent is prepared by mixing a hydrogen bond acceptor and a hydrogen bond donor in a molar ratio of 1:1 to 3. The hydrogen bond acceptors of the natural deep eutectic solvents in steps (1) to (3) are all choline chloride, the hydrogen bond donors of the natural deep eutectic solvents in steps (1) and (2) are all urea, and the hydrogen bond donor of the natural deep eutectic solvent in step (3) is malic acid.

3. The extraction method according to claim 1 or 2, characterized in that: In the natural deep eutectic solvent aqueous solution of step (1), the volume concentration of the natural deep eutectic solvent is 60-65%; in the natural deep eutectic solvent aqueous solution of step (2), the volume concentration of the natural deep eutectic solvent is 75-80%; in the natural deep eutectic solvent aqueous solution of step (3), the volume concentration of the natural deep eutectic solvent is 85-90%.

4. The extraction method according to claim 1, wherein: In step (1), the mass ratio of American ginseng powder to the natural deep eutectic solvent aqueous solution is 1:20-30; in step (2), the mass ratio of the filter residue to the natural deep eutectic solvent aqueous solution is 1:20-30; in step (3), the mass ratio of American ginseng powder to the natural deep eutectic solvent aqueous solution is 1:10-20.

5. The extraction method according to claim 1, characterized in that: In steps (1) to (3), the extraction is performed at least once; preferably, steps (1) and (2) are extracted once, and step (3) is extracted twice.

6. The extraction method according to claim 1, characterized in that: In step (1), the extraction temperature is 50-90°C and the extraction time is 40-50 min; in step (2), the extraction temperature is 50-90°C and the extraction time is 40-50 min.

7. The extraction method according to claim 1, characterized in that: In step (3), the extraction is carried out twice under microwave, the microwave power of the first microwave extraction is 50-75 W, the microwave temperature is 45-55 ° C, and the extraction time is 10-30 min; the microwave power of the second microwave extraction is 75-100 W, the microwave temperature is 95-105 ° C, and the extraction time is 10-30 min.

8. The extraction method according to claim 1, characterized in that: Include at least one of the following conditions: In step (4), the extracts obtained in steps (1), (2) and (3) are first concentrated and then combined, or first combined and then concentrated, so that the specific gravity of the final combined extract is 1.02 to 1.06; In step (4), the extract is subjected to alcohol precipitation for 1 to 2 times. In the first alcohol precipitation, alcohol is added to the extract until the alcohol concentration is 70 to 75 wt %. In the second alcohol precipitation, alcohol is added to the precipitate obtained by the first alcohol precipitation until the alcohol concentration is 70 to 95 wt %, preferably 90 to 95 wt %. In step (5), the Sevag method is used to remove free proteins.

9. American ginseng glycoprotein obtained by the extraction method according to any one of claims 1 to 8.

10. Use of the American ginseng glycoprotein according to claim 9 in the preparation of liver protection products and / or antioxidant products.

Images