A method for extracting and separating ginsenosides and polysaccharides from American ginseng using a deep eutectic solvent aqueous two-phase system

Abstract

The invention discloses a method for extracting and separating ginsenosides and polysaccharides from American ginseng using a low eutectic solvent double aqueous phase system, adding American ginseng powder to a low eutectic solvent aqueous solution, ultrasonically extracting for 20-50 min at 20-80°C and 50-200W, centrifuging, taking the supernatant, and obtaining a crude extract of ginsenosides and polysaccharides; then adding potassium dihydrogen phosphate to the crude extract to form two phases, wherein the low eutectic solvent phase is ginsenosides, and the potassium dihydrogen phosphate phase is polysaccharides. The low eutectic solvent in the low eutectic solvent aqueous solution is mixed with a hydrogen bond donor and a hydrogen bond acceptor and / or deionized water. The invention provides an efficient and green method for extracting and separating ginsenosides and polysaccharides from American ginseng using a low eutectic solvent, the method having the characteristics of short required time, low energy consumption, and green environmental protection. The invention prepares a Chinese medicine extract by introducing a low eutectic solvent, providing a new research and development idea for the extraction of active ingredients of Chinese medicine and the preparation of Chinese medicine.

Description

Technical Field

[0001] The invention relates to a method for preparing an American ginseng extract, and in particular to a method for extracting and separating ginsenosides and polysaccharides in American ginseng by using a low eutectic solvent double aqueous phase system. Background Art

[0002] American ginseng, also known as Western ginseng, has the effects of nourishing, promoting body fluid, replenishing qi and nourishing yin. Studies have shown that American ginseng contains saponins, polysaccharides, proteins, fats and other physiologically active components. Among them, saponins are important active substances in American ginseng, which have the effects of protecting cardiovascular function, anti-tumor, and enhancing immunity.

[0003] Extraction preparation is one of the key links in the production process of traditional Chinese medicine, and the selection of extraction solvent plays a decisive role in the effect of this link. The extraction solvent is not only related to the extraction efficiency of active ingredients, but also plays an important role in the quality and safety of traditional Chinese medicine. At present, in the production and research of traditional Chinese medicine, the commonly used extraction solvents are water and alcohol solvents (ethanol in different proportions). These two types of solvents have the advantages of easy availability of raw materials and low toxicity and side effects, but there are also problems such as narrow polarity range and weak extraction ability of certain types of active ingredients that need to be solved urgently. Summary of the invention

[0004] The purpose of the present invention is to provide an efficient and green method for extracting and separating ginsenosides and polysaccharides in American ginseng using a low eutectic solvent two-phase aqueous system. The method prepares Chinese medicine extracts by introducing a low eutectic solvent, has the characteristics of short time required, low energy consumption, and green environmental protection, and provides a new research and development idea for the extraction of active ingredients of Chinese medicine and the preparation of Chinese medicine.

[0005] The technical solution adopted by the present invention is:

[0006] The invention provides a method for extracting and separating ginsenosides and polysaccharides in American ginseng by using a low eutectic solvent aqueous two-phase system. The method comprises the following steps: adding American ginseng powder to an aqueous solution of a low eutectic solvent, ultrasonically extracting for 20-50 minutes at 20-80°C and 50-200W, centrifuging, taking a supernatant, and obtaining a crude mixed extract of ginsenosides and polysaccharides; and then adding potassium dihydrogen phosphate to the mixed crude extract to form an aqueous two-phase extraction system, wherein the low eutectic solvent phase is ginsenosides and the potassium dihydrogen phosphate phase is polysaccharides.

[0007] The low eutectic solvent is prepared by mixing a hydrogen bond donor (HBD) with a hydrogen bond acceptor (HBA) and / or deionized water (i.e., the low eutectic solvent is prepared by mixing and heating a hydrogen bond donor and a hydrogen bond acceptor, or the low eutectic solvent is prepared by mixing and heating a hydrogen bond donor, a hydrogen bond acceptor and deionized water), the hydrogen bond donor is selected from one or more of lactic acid, ethylene glycol, 1,2-propylene glycol, acetamide, urea, and N,N'-dimethylurea, and the hydrogen bond acceptor is choline chloride.

[0008] Further, two-phase aqueous extraction: potassium dihydrogen phosphate was added to the crude extract and mixed thoroughly, the mass volume ratio of potassium dihydrogen phosphate to the crude extract was 3:10 (m / V), after the potassium dihydrogen phosphate was completely dissolved, it was allowed to stand for 60 minutes, and centrifuged at 4000×g for 10 minutes to form a two-phase aqueous extraction system.

[0009] Furthermore, the low eutectic solvent is prepared by mixing and heating a hydrogen bond donor and a hydrogen bond acceptor in a molar ratio of 1:1-4.

[0010] Furthermore, the low eutectic solvent is prepared by mixing and heating a hydrogen bond donor, a hydrogen bond acceptor and deionized water in a molar ratio of 1:1-4:5.

[0011] Furthermore, the low eutectic solvent is preferably prepared as follows: after mixing the hydrogen bond donor with the hydrogen bond acceptor and / or deionized water, heating and stirring at 80-100° C. for 0.5-2 hours (preferably 80° C., 1 hour) to form a clear and uniform low eutectic solvent.

[0012] Furthermore, the volume concentration of the low eutectic solvent in the low eutectic solvent aqueous solution is 25-100%, and the weight of the American ginseng powder is 25-200 mg / mL based on the volume of the low eutectic solvent aqueous solution.

[0013] Furthermore, the extraction conditions are preferably: ultrasonic extraction at 60°C and 200W for 30 minutes.

[0014] Furthermore, the low eutectic solvent is preferably prepared by mixing choline chloride and N,N'-dimethylurea in a molar ratio of 1:1 and then heating at 80°C for 1 hour.

[0015] The deep eutectic solvent / salt two-phase aqueous system is to mix a certain amount of deep eutectic solvent and salt solution. When the two reach a certain concentration, the deep eutectic solvent and salt form two phases due to the salting-out effect. When the active ingredients in plants are extracted using the deep eutectic solvent / salt two-phase aqueous system, the target product and a small amount of impurities are distributed in one phase, while most of the impurities are distributed in the other phase, which can achieve the purpose of preliminary separation and purification. This technology is a simple, fast, efficient, low-cost, easy to scale up method that does not require chromatography.

[0016] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0017] (1) The present invention provides an efficient and green method for extracting and separating ginsenosides and polysaccharides from American ginseng using a deep eutectic solvent aqueous two-phase system. The method has the characteristics of short time, low energy consumption, and green environmental protection. The present invention introduces a deep eutectic solvent to prepare Chinese medicine extracts, providing a new research and development idea for the extraction of active ingredients of Chinese medicine and the preparation of Chinese medicine.

[0018] (2) This method combines the advantages of low eutectic solvents and aqueous two-phase extraction and is a greener extraction and separation method. DETAILED DESCRIPTION

[0019] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0020] The following is a further explanation of the extraction and separation method of ginsenosides and polysaccharides, the effective components of American ginseng, described in the present invention, in conjunction with the examples, to help those skilled in the art have a more complete, accurate and in-depth understanding of the inventive concept and technical solution of the present invention.

[0021] The American ginseng used in the embodiments of the present invention was collected and prepared in accordance with the 2020 edition of the Chinese Pharmacopoeia.

[0022] Example 1 DES-1 is the solvent (1 mL)

[0023] Choline chloride (0.70 g) and N,N'-dimethylurea (0.44 g) were mixed at a molar ratio of 1:1, heated and stirred at 80°C for 1 hour to form a clear and uniform low eutectic solvent, which was recorded as DES-1. 750 μL of DES-1 was mixed with 250 μL of water to obtain 1 ml of DES-1 aqueous solution, which was added to a 1.5 mL centrifuge tube, and 100.0 mg of American ginseng (origin Shandong) powder was accurately weighed and added thereto, and ultrasonic extraction was performed at 60°C for 30 minutes with an ultrasonic power of 200 W. After ultrasonic extraction, centrifuge at 16200×g for 10min, take the supernatant, and obtain ginsenoside and polysaccharide crude extract; then add potassium dihydrogen phosphate to the crude extract to form two phases, the mass volume ratio of potassium dihydrogen phosphate and crude extract is 3:10 (m / V), after potassium dihydrogen phosphate is completely dissolved, stand for 60min, centrifuge at 4000×g for 10min, form a two-phase extraction system, in which the low eutectic solvent phase is ginsenoside and the potassium dihydrogen phosphate phase is polysaccharide. The supernatant is diluted 10 times with a 50% methanol aqueous solution by volume and then tested by high performance liquid chromatography (HPLC). According to the standard curve of ginsenoside, the types and contents of compounds contained in the American ginseng extract are obtained, and the extraction rate = compound content / American ginseng raw material weight (mg / g); the content of ginsenoside in the American ginseng extract is measured to be 76.73mg / g, and the extraction rate can reach 92%.

[0024] HPLC detection conditions are as follows:

[0025] Agilent 5HC-C18 (4.6×150 mm, 5 μm); mobile phase was acetonitrile (A)-0.1% phosphoric acid aqueous solution (B), gradient elution: 0-25 min, 19%-20% A; 25-60 min, 20%-40% A; 60-90 min, 40%-55% A; 90-100 min, 55%-60% A. Detection wavelength 203 nm; column temperature 40°C; flow rate 1.0 mL / min.

[0026] Example 2 DES-2 is the solvent (1 mL)

[0027] The DES-1 in Example 1 was replaced by choline chloride (0.70 g) and acetamide (0.30 g) in a molar ratio of 1:1, and heated and stirred at 80° C. for 1 hour to form a clear and uniform low eutectic solvent, which was recorded as DES-2. The other operations were the same as those in Example 1, and the content of ginsenoside in the American ginseng extract was measured to be 72.35 mg / g, and the extraction rate was 87%.

[0028] Example 3 DES-1 is solvent (10 g American ginseng, 100 mL aqueous solution)

[0029] Choline chloride (70 g) and N,N'-dimethylurea (44 g) were mixed at a molar ratio of 1:1 and heated and stirred at 80°C for 1 hour to form a clear and uniform low eutectic solvent, which was recorded as DES-1. 25 mL of the low eutectic solvent DES-1 was mixed with 75 mL of water to obtain 100 ml of DES-1 aqueous solution, which was added to a 1000 mL conical flask, and 10.00 g of American ginseng (origin Shandong) powder was accurately weighed and added thereto, and ultrasonic extraction was performed at 60°C for 1 hour, and the ultrasonic power was 200 W. After ultrasonic extraction, centrifuge at 16200×g for 20min, take the supernatant, take the supernatant, and obtain ginsenoside and polysaccharide crude extract; then add potassium dihydrogen phosphate to the crude extract to form two phases, the mass volume ratio of potassium dihydrogen phosphate and crude extract is 3:10 (m / V), after potassium dihydrogen phosphate is completely dissolved, stand for 60min, centrifuge at 4000×g for 10min, form a two-phase extraction system, in which the low eutectic solvent phase is ginsenoside and the potassium dihydrogen phosphate phase is polysaccharide. The content of ginsenoside in the extract measured by the method of Example 1 is 75.68mg / g, and the extraction rate is 91%.

[0030] Example 4 Effect of the type of deep eutectic solvent on the extraction effect

[0031] The low eutectic solvent in Example 1 was changed to that described in Table 1, which consisted of a hydrogen bond acceptor, a hydrogen bond donor and / or deionized water. The amount of American ginseng powder added was 0.1 g / mL based on the total volume of the low eutectic solvent aqueous solution. Other operations were the same as in Example 1. The total amount of ginsenosides in the obtained American ginseng extract is shown in Table 1. The results showed that DES-1 had the best comprehensive extraction performance.

[0032] Table 1. Investigation of the relationship between the type of deep eutectic solvent and the extraction results in step 1 (extraction conditions are the same as in Example 1)

[0033]

[0034] Example 5 Effect of the Volume Concentration of Deep Eutectic Solvent in the Deep Eutectic Solvent Aqueous Solution on the Extraction Effect

[0035] The volume concentration of DES-1 in Example 1 was changed to that shown in Table 2. Other operations were the same as in Example 1. The ginsenoside content in the obtained American ginseng extract was shown in Table 2. The results showed that 75% DES-1 had the best extraction effect.

[0036] Table 2. Effect of deep eutectic solvent concentration on the extraction effect of American ginseng

[0037]

[0038] Example 6 Effect of solid-liquid ratio on extraction effect

[0039] The amount of American ginseng (mg) in Example 1 was changed to the total volume (ml) of DES-1 and water as shown in Table 3. Other operations were the same as in Example 1. The content of ginsenosides in the obtained American ginseng extract is shown in Table 3. The results showed that 100 mg / mL was the optimal solid-liquid ratio for extraction.

[0040] Table 3. Effect of solid-liquid ratio on extraction effect

[0041]

[0042] Example 7 Effect of extraction temperature on extraction effect

[0043] The temperature in Example 1 was changed to that shown in Table 4, and other operations were the same as in Example 1. The content of ginsenoside in the obtained American ginseng extract is shown in Table 4. The results showed that 60°C was the optimal extraction temperature.

[0044] Table 4. Effect of extraction temperature on extraction effect

[0045]

[0046] Example 8 Effect of extraction power on extraction effect

[0047] The power in Example 1 was changed to that shown in Table 5. Other operations were the same as in Example 1. The content of ginsenoside in the obtained American ginseng extract was shown in Table 5. The results showed that 200 W was the optimal extraction power.

[0048] Table 5 Effect of power on extraction effect

[0049]

[0050] Example 9 Effect of extraction time on extraction effect

[0051] The extraction time in Example 1 was changed to that shown in Table 6, and other operations were the same as in Example 1. The content of ginsenoside in the obtained American ginseng extract was shown in Table 6. The results showed that 30 min was the optimal extraction time.

[0052] Table 6. Effect of extraction time on extraction effect

[0053]

[0054] Comparative Example 1 Water as solvent (1 mL)

[0055] Accurately weigh 100.0 mg of American ginseng (origin Shandong) powder and add it to a 1.5 mL centrifuge tube, then add 1.0 mL of deionized water. Ultrasonic extraction was performed at 60°C for 30 min, and the ultrasonic power was 200 W. After ultrasonic extraction, centrifuge at 16200 × g for 10 min, and the supernatant was taken as the prepared American ginseng water extract. The ginsenoside content in American ginseng was measured to be 52.55 mg / g by the method described in Example 1.

[0056] It can be seen that the extraction rate of ginsenosides in the American ginseng extract extracted by the low eutectic solvent two-phase system is 46.0% higher than that of the water extract.

Claims

1. A method for extracting and separating ginsenosides and polysaccharides from American ginseng using a deep eutectic solvent aqueous two-phase system, characterized in that: The method comprises: adding American ginseng powder to a low eutectic solvent aqueous solution, ultrasonically extracting at 60-80°C and 200W for 30 minutes, centrifuging, taking the supernatant to obtain a mixed crude extract of ginsenosides and polysaccharides, and then adding potassium dihydrogen phosphate to the mixed crude extract to form a two-phase aqueous extraction system, wherein the low eutectic solvent phase is ginsenosides and the potassium dihydrogen phosphate phase is polysaccharides; The deep eutectic solvent in the deep eutectic solvent aqueous solution is a mixture of a hydrogen bond donor and a hydrogen bond acceptor and / or deionized water, the hydrogen bond donor is N,N'-dimethylurea, and the hydrogen bond acceptor is choline chloride; The deep eutectic solvent aqueous solution is prepared by mixing and heating a hydrogen bond donor, a hydrogen bond acceptor and deionized water in a molar ratio of 1:1-2:5; Aqueous two-phase extraction system: Add potassium dihydrogen phosphate to the crude extract and mix thoroughly. The mass volume ratio of potassium dihydrogen phosphate to the crude extract is 3:

10. After the potassium dihydrogen phosphate is completely dissolved, let it stand for 60 minutes and centrifuge at 4000×g for 10 minutes to form an aqueous two-phase extraction system.

2. The method according to claim 1, characterized in that The deep eutectic solvent is prepared as follows: after mixing the hydrogen bond donor with the hydrogen bond acceptor and / or deionized water, the mixture is heated and stirred at 80-100° C. for 0.5-2 hours to form a clear and uniform deep eutectic solvent.

3. The method according to claim 1, characterized in that The low eutectic solvent is prepared by mixing choline chloride and N,N'-dimethylurea in a molar ratio of 1:1 and heating the mixture at 80°C for 1 hour.

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