Method for extracting pulsatilla chinensis polysaccharide by using ternary eutectic solvent (DESs) system

Abstract

The invention discloses a method for extracting pulsatilla chinensis polysaccharide based on a ternary eutectic solvent (DESs) system. Comprising the following steps: firstly, determining the composition of a ternary eutectic solvent, and optimizing an extraction process through a single-factor experiment; five different ternary eutectic solvents are selected under the same condition. The extraction yield of the pulsatilla polysaccharide is compared and analyzed according to the water content, the extraction time, the temperature and the material-liquid ratio. Experimental results show that when the composition of the ternary eutectic solvent is glycerol-choline chloride-acetic acid, the water content is 30wt%, the extraction time is 1.5 h, the extraction temperature is 90 DEG C, and the material-liquid ratio is 1: 50 (w / v), the extraction effect is optimal. Compared with an existing ternary eutectic solvent extraction method, the extraction efficiency of the method is remarkably superior to that of traditional water solvent extraction. The extraction activity is higher than that of a traditional method. Moreover, the equipment used in the invention is simple, the production cost is lower, continuous operation can be realized, and the method has higher practicability and operability.

Description

Technical Field

[0001] The present invention belongs to the technical field of extraction and separation of Pulsatilla polysaccharides, and relates to a method for extracting Pulsatilla polysaccharides using ternary deep eutectic solvents (DESs). Background Art

[0002] 1. Pulsatilla chinensis is one of the traditional Chinese medicinal herbs, named after its rhizome resembling an old man with white hair. Li Shizhen recorded it in "Compendium of Materia Medica" and found that Pulsatilla chinensis has the medicinal value of "clearing heat and detoxifying, cooling blood and stopping dysentery", and included it in the classic prescription "Pulsatilla Decoction" for treating heat-toxicity bloody dysentery. To this day, this prescription is still widely used in the treatment of diseases such as acute enteritis and bacillary dysentery. Modern medical research shows that Pulsatilla chinensis extracts have many pharmacological effects, including anti-tumor, anti-aging, antibacterial, antiviral, hypoglycemic and other multiple effects. As one of the important active ingredients in Pulsatilla chinensis, the medicinal potential of polysaccharides has not been fully explored. Therefore, choosing a suitable extraction method to maximize the biological activity of Pulsatilla polysaccharides has become an important research topic in current research.

[0003] 2. In the research process of Chinese medicine polysaccharides, choosing a suitable extraction method is crucial for maintaining the biological activity of polysaccharides, because different extraction techniques will have a significant impact on the yield, properties and biological activity of polysaccharides. Facing the complexity of raw materials, traditional extraction methods usually face different economic and environmental costs, each with its own advantages and disadvantages. For example, water extraction method, alkaline extraction method and enzyme extraction method often extract other components such as proteins, glycosides and pigments at the same time, which not only increases the difficulty of separation and purification, but also may affect the final quality of polysaccharides. Although ultrasonic and microwave-assisted extraction methods have advantages in improving extraction efficiency, they require the use of relatively expensive equipment, thus increasing the cost. At the same time, some toxic organic solvents are used in some traditional extraction methods, which will increase the potential harm to the environment and human body. Therefore, it is necessary to develop a green and pollution-free extraction technology that can not only improve the extraction efficiency but also maximize the retention of biological activity. Compared with traditional methods, ternary deep eutectic solvents (DESs), as a new type, green and sustainable extraction system, have shown great application potential in the enrichment of bioactive substances such as plant polysaccharides, polyphenols and saponins. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for extracting Pulsatilla polysaccharides using ternary deep eutectic solvents (DESs).

[0005] The technical solution of the present invention is summarized as follows:

[0006] 1 Determination of ternary deep eutectic solvents

[0007] Prepare a ternary eutectic solvent with a water content of 30%. According to the molar ratio (n1:n2:n3) of 1:2:1, prepare five groups of ternary eutectic solvents with different components. Respectively, the first group: 1,2-propanediol, choline chloride, acetic acid; the second group: 1,4-butanediol, choline chloride, acetic acid; the third group: isopropanol, choline chloride, acetic acid; the fourth group: ethylene glycol, choline chloride, acetic acid; the fifth group: glycerol, choline chloride, acetic acid. Heat to 70 °C and continuously stir with a glass rod until the solution is completely mixed evenly. Take 300 mL of the prepared ternary eutectic solvent to extract pulsatilla polysaccharide. Measure the extraction rates of pulsatilla polysaccharide in the five groups of experiments respectively, and finally determine the ternary eutectic solvent.

[0008] 2 Results of single-factor experiments

[0009] To optimize the extraction process of Pulsatilla chinensis and improve the extraction rate of polysaccharide, in this study, glycerol-choline chloride-acetic acid was finally used as the extraction system. Under the same conditions of ternary eutectic solvent, single-factor experiments were carried out. The experiments mainly investigated the effects of factors such as the water content of the ternary eutectic solvent, extraction time, extraction temperature, and solid-liquid ratio on the polysaccharide yield.

[0010] (1) Water content of ternary eutectic solvent

[0011] Experimental conditions: Extraction time 2 h, temperature 90 °C, solid-liquid ratio 1:30 (w / v). Set the water content of the ternary eutectic solvent to 20%, 25%, 30%, 35%, and 40% respectively for five parallel experiments to measure the yield of pulsatilla polysaccharide.

[0012] (2) Extraction time

[0013] Experimental conditions: Water content of ternary eutectic solvent 30%, temperature 90 °C, solid-liquid ratio 1:30 (w / v). Set the extraction time to 1 h, 1.5 h, 2 h, 2.5 h, and 3 h respectively for five parallel experiments to measure the yield of pulsatilla polysaccharide.

[0014] (3) Extraction temperature

[0015] Experimental conditions: Water content of ternary eutectic solvent 30%, extraction time 1.5 h, solid-liquid ratio 1:30 (w / v). Set the extraction temperature to 60 °C, 70 °C, 80 °C, 90 °C, and 100 °C respectively for five parallel experiments to measure the yield of pulsatilla polysaccharide.

[0016] (4) Solid-liquid ratio

[0017] Experimental conditions: Water content of ternary eutectic solvent 30%, extraction time 1.5 h, temperature 90 °C. Set the solid-liquid ratio to 1:10, 1:20, 1:30, 1:40, and 1:50 (w / v) respectively for five parallel experiments to measure the yield of pulsatilla polysaccharide.

[0018] Five different ternary deep eutectic solvents (DESs) set by experiments were used to compare the yields of Pulsatilla polysaccharide extraction under the same water content, time, temperature, and solid-liquid ratio of the ternary DESs. It was found that the highest yield of polysaccharide extraction was obtained when the composition of the ternary DES was glycerol-choline chloride-acetic acid. According to the single-factor experiments, the optimal extraction conditions were determined as follows: the water content of the ternary DES was 30%, the extraction time was 1.5 h, the extraction temperature was 90 °C, and the solid-liquid ratio was 1:50 (w / v).

[0019] Specifically, the method for extracting Pulsatilla polysaccharide using a ternary deep eutectic solvent (DESs) system according to the present invention includes the following steps:

[0020] (1) Pretreatment: Add petroleum ether to dry Pulsatilla, and extract at a certain temperature under reflux condensation to obtain a sample. After the sample is cooled, centrifuge to remove the organic solvent to obtain defatted Pulsatilla powder. Put the defatted Pulsatilla powder into an oven to dry and obtain defatted Pulsatilla.

[0021] (2) Preparation of the ternary deep eutectic solvent: Take glycerol: 117.8 mL, choline chloride: 450.4 g, acetic acid: 92.1 mL, and ddH2O: 89.95 mL, put them into a beaker, stir and heat until all are melted into a liquid to obtain a ternary deep eutectic solvent with a water content of 30 wt%.

[0022] (3) In a 1000 mL round-bottom flask, add 500 mL of the ternary deep eutectic solvent with a water content of 30 wt% and 10 g of the defatted Pulsatilla obtained in step (1).

[0023] (4) Perform reflux extraction with temperature controlled at 90 °C and extraction time of 1.5 h.

[0024] (5) After the extraction reaction is completed, centrifuge, take the supernatant, and slowly pour 4 times the volume of anhydrous ethanol of the supernatant into the supernatant while stirring.

[0025] (6) Place it in a 4 °C refrigerator for alcohol precipitation for 24 h.

[0026] (7) After 24 h of alcohol precipitation, centrifuge, discard the supernatant, and freeze-dry the obtained precipitate to obtain crude Pulsatilla polysaccharide.

[0027] Further, for the pretreatment, 250 g of dry Pulsatilla is added with 750 mL of petroleum ether, and extracted at 66 °C for 4 h under reflux condensation to obtain a sample. After the sample is cooled, centrifuge to remove the organic solvent to obtain defatted Pulsatilla powder. Put the defatted Pulsatilla powder into an oven to dry and obtain defatted Pulsatilla.

[0028] Advantages of the present invention: The present invention establishes a method for extracting pulsatilla polysaccharide with ternary deep eutectic solvents (DESs). The results show that in the ternary deep eutectic solvents (DESs), the solvent components are: glycerol-choline chloride-acetic acid system, with a molar ratio (n1:n2:n3) of 1:2:1, the water content of the ternary deep eutectic solvent is 30 wt%, the extraction time is 1.5 h, the extraction temperature is 90 °C, and the solid-liquid ratio is 1:50 (w / v). Under these conditions, the extraction yield of pulsatilla polysaccharide is 51.7%. Description of the Drawings

[0029] Figure 1 It is a system diagram of the ternary deep eutectic solvent (glycerol-choline chloride-acetic acid) described in the present invention;

[0030] Figure 2 It is a diagram of extracting pulsatilla polysaccharide with the ternary deep eutectic solvent (glycerol-choline chloride-acetic acid) described in the present invention;

[0031] Figure 3 It is a diagram of the qualitative result of the crude pulsatilla polysaccharide extracted by the phenol-sulfuric acid method. Detailed Embodiments

[0032] The following further detailed description of the present invention is made in conjunction with specific embodiments and drawings, so that those skilled in the art can implement it according to the text of the specification. However, the present invention is not limited to the following embodiments, and the conditions adopted in the embodiments can be adjusted according to specific situations.

[0033] Embodiment

[0034] 1 Determination of ternary deep eutectic solvent

[0035] Prepare a ternary deep eutectic solvent with a water content of 30 wt% and a molar ratio (n1:n2:n3) of 1:2:1, and take 300 mL. Set five groups of experiments on ternary deep eutectic solvents with different components, namely the first group: 1,2-propanediol: choline chloride: acetic acid; the second group: 1,4-butanediol: choline chloride: acetic acid; the third group: isopropanol: choline chloride: acetic acid; the fourth group: ethylene glycol: choline chloride: acetic acid; the fifth group: glycerol: choline chloride: acetic acid. Continuously stir with a glass rod at 70 °C until a homogeneous liquid is obtained (see Table 1). Finally, it is confirmed that the ternary deep eutectic solvent composed of glycerol-choline chloride-acetic acid is used.

[0036] Table 1 Determination of ternary deep eutectic solvent

[0037]

[0038] 2 Results of single-factor experiments

[0039] To optimize the extraction process of Pulsatilla polysaccharide and obtain a higher polysaccharide extraction yield, a ternary deep eutectic solvent was composed of glycerol-choline chloride-acetic acid with a molar ratio (n1:n2:n3) of 1:2:1. Under the same ternary deep eutectic solvent composed of glycerol-choline chloride-acetic acid with a molar ratio of 1:2:1, single-factor experiments were carried out to investigate the effects of the water content of the ternary deep eutectic solvent, extraction time, extraction temperature, and solid-liquid ratio on the polysaccharide yield.

[0040] (1) Water content of the ternary deep eutectic solvent

[0041] The extraction time was controlled at 2 h, the temperature at 90 °C, and the solid-liquid ratio at w / v (weight-volume ratio) = 1:30. Five parallel experiments were set up with the water content of the ternary deep eutectic solvent composed of glycerol-choline chloride-acetic acid with a molar ratio of 1:2:1 being 20 wt%, 25 wt%, 30 wt%, 35 wt%, and 40 wt% respectively to measure the yield of Pulsatilla polysaccharide (see Table 2). The experimental results showed that the extraction effect was the best when the water content of the ternary deep eutectic solvent was 30 wt%.

[0042] Table 2 Investigation of the influence of the water content of the ternary deep eutectic solvent

[0043]

[0044] (2) Extraction time

[0045] The water content of the ternary deep eutectic solvent composed of glycerol-choline chloride-acetic acid with a molar ratio of 1:2:1 was controlled at 30 wt%, the temperature at 90 °C, and the solid-liquid ratio at 1:30 (w / v). Five parallel experiments were set up with the extraction time being 1 h, 1.5 h, 2 h, 2.5 h, and 3 h respectively to measure the yield of Pulsatilla polysaccharide (see Table 3). The experimental results showed that the extraction effect was the best when the extraction time was 1.5 h.

[0046] Table 3 Investigation of the influence of extraction time

[0047]

[0048]

[0049] (3) Extraction temperature

[0050] The water content of the ternary deep eutectic solvent composed of glycerol-choline chloride-acetic acid with a molar ratio of 1:2:1 was controlled at 30 wt%, the extraction time at 1.5 h, and the solid-liquid ratio at 1:30 (w / v). Five parallel experiments were set up with the extraction temperature being 60 °C, 70 °C, 80 °C, 90 °C, and 100 °C respectively to measure the yield of Pulsatilla polysaccharide (see Table 4). The experimental results showed that the extraction effect was better when the extraction temperature was 90 °C.

[0051] Table 4 Investigation of the influence of extraction temperature

[0052]

[0053] (4) Solid-liquid ratio

[0054] In the experiment, the water content of the ternary eutectic solvent composed of glycerol-choline chloride-acetic acid was controlled at 30 wt%, the extraction time was 1.5 h, and the temperature was 90 °C. Five parallel experiments were set with solid-liquid ratios of 1:10, 1:20, 1:30, 1:40, and 1:50 (w / v) respectively to measure the yield of pulchinenoside (see Table 5). The experimental results show that the extraction effect is better when the solid-liquid ratio is 1:50 (w / v).

[0055] Table 5 Investigation of solid-liquid ratio

[0056]

[0057] Through the five different ternary eutectic solvents set in the experiments described in Table 5, under the same conditions, the effects of factors such as the water content, time, temperature, and solid-liquid ratio of the ternary eutectic solvent on the yield of pulchinenoside extraction were studied. The experimental results show that when the ternary eutectic solvent composition system is glycerol-choline chloride-acetic acid with a molar ratio of 1:2:1, the water content of the ternary eutectic solvent is 30 wt%, the extraction time is 1.5 h, the extraction temperature is 90 °C, and the solid-liquid ratio is 1:50 (w / v), these are the optimal extraction conditions.

[0058] Optimal example

[0059] (1) Pretreatment: Weigh 250 g of dry Pulsatilla chinensis, add 750 mL of petroleum ether, and extract at 66 °C for 4 h in a condensing reflux. After the sample is cooled, centrifuge to remove the organic solvent to obtain defatted Pulsatilla chinensis powder. Then put the defatted Pulsatilla chinensis powder into an oven to dry and obtain defatted Pulsatilla chinensis.

[0060] (2) Preparation of the ternary eutectic solvent, prepared according to the optimal extraction conditions. Select the glycerol-choline chloride-acetic acid system and prepare it according to the molar ratio (n1:n2:n3) of 1:2:1 with a water content of 30 wt% in the ternary eutectic solvent: (glycerol: 117.8 mL, choline chloride: 450.4 g, acetic acid: 92.1 mL, ddH2O: 89.95 mL) are put into a beaker and heated with stirring until all are melted into a liquid.

[0061] (3) Solid-liquid ratio of 1∶50 (w / v): In a 1000 mL round-bottom flask, add 500 mL of the ternary eutectic solvent composed of glycerol-choline chloride-acetic acid with a molar ratio of 1:2:1 and 10 g of the defatted Pulsatilla chinensis obtained in step (1).

[0062] (4) Carry out condensation reflux extraction, control the temperature at 90 °C, and the extraction time is 1.5 h.

[0063] (5) After the extraction reaction is completed, carry out centrifugation (3500 rpm, 30 min), take the supernatant, and slowly pour 4 times the volume of anhydrous ethanol of the supernatant into the supernatant while stirring.

[0064] (6) Place it in a 4 °C refrigerator for alcohol precipitation for 24 h.

[0065] (7) After 24 h of alcohol precipitation, carry out centrifugation (3500 rpm, 30 min), discard the supernatant, and freeze-dry the obtained precipitate to obtain crude polysaccharide of Pulsatilla chinensis. The extraction yield of Pulsatilla chinensis polysaccharide is 51.7%.

[0066] (8) Use the phenol-sulfuric acid method to qualitatively analyze the crude polysaccharide of Pulsatilla chinensis extracted in step (7). Weigh an appropriate amount of the freeze-dried crude polysaccharide of Pulsatilla chinensis accurately, dissolve it in ddH2O, and prepare polysaccharide solutions of 100 μg / mL, 50 μg / mL, and 10 μg / mL respectively. Prepare test tubes numbered 1-4. Among them, the first test tube is added with 2 mL of ddH2O as a blank control solution, and the 2nd-4th test tubes are respectively added with 2 mL of different concentration polysaccharide solutions of 100 μg / mL, 50 μg / mL, and 10 μg / mL. After standing for 10 minutes, add 1 mL of 6 wt% phenol solution to each test tube, shake well quickly, and then slowly add 5 mL of concentrated sulfuric acid along the wall of the test tube. Finally, place the test tube in a constant temperature boiling water bath and heat for 15 min. During this process, observe the color reaction, and the experimental results Figure 3 , from Figure 3 [[ID=ID=16]]As can be seen from the figure: the color developed in the first test tube is colorless, the color developed in the second test tube is dark brown, the third test tube is light brown, and the color of the fourth test tube is light brown; it shows that the product extracted in step (7) of the present invention is crude polysaccharide of Pulsatilla chinensis.

Claims

1. A method for extracting pulsatilla polysaccharide using a ternary deep eutectic solvent (DESs) system, comprising the following steps: (1) Pretreatment: Add petroleum ether to dry pulsatilla. Extract the sample at a certain temperature under reflux condensation. After the sample cools, centrifuge to remove the organic solvent to obtain defatted pulsatilla powder. Put the defatted pulsatilla powder into an oven to dry and obtain defatted pulsatilla. (2) Preparation of ternary deep eutectic solvent: Take glycerol: 117.8 mL, choline chloride: 450.4 g, acetic acid: 92.1 mL, ddH2O: 89.95 mL. Put them into a beaker and heat with stirring until all melt into a liquid to obtain a ternary deep eutectic solvent with a water content of 30 wt%. (3) In a 1000 mL round-bottom flask, add 500 mL of the ternary deep eutectic solvent with a water content of 30 wt% and 10 g of the defatted pulsatilla obtained in step (1). (4) Carry out reflux extraction with the temperature controlled at 90 °C and the extraction time of 1.5 h. (5) After the extraction reaction is completed, centrifuge and take the supernatant. Slowly pour 4 times the volume of anhydrous ethanol of the supernatant into the supernatant while stirring. (6) Place it in a 4 °C refrigerator for alcohol precipitation for 24 h. (7) After 24 h of alcohol precipitation, centrifuge, discard the supernatant, and freeze-dry the obtained precipitate to obtain crude pulsatilla polysaccharide.

2. The method for extracting pulsatilla polysaccharide by a ternary deep eutectic solvent (DESs) system according to claim 1, characterized in that The pretreatment is to add 750 mL of petroleum ether to 250 g of dry pulsatilla, extract the sample at 66 °C under reflux condensation for 4 h. After the sample cools, centrifuge to remove the organic solvent to obtain defatted pulsatilla powder. Put the defatted pulsatilla powder into an oven to dry and obtain defatted pulsatilla.

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