A method for extracting total saponins from radix ophiopogonis by using a eutectic solvent

Abstract

The present application relates to the technical field of steroid saponin extraction, and discloses a method for extracting total ophiopogon saponins by using a eutectic solvent, which comprises the steps of sample pretreatment, eutectic solvent preparation, ultrasonic extraction and the like; the present application has high extraction rate, the process parameters are optimized by the response surface method, the extraction rate of total ophiopogon saponins can reach 6.22%, which is significantly higher than that of the traditional ethanol extraction method; the present application is green and environmentally friendly, the natural eutectic solvent is used to replace the organic solvent, the environmental pollution is reduced, and the green production concept is met; a group of eutectic solvents with high extraction rate is obtained by screening different components, which is choline chloride-lactic acid, and the effects of ultrasonic time, water content, solid-liquid ratio and ultrasonic power on the extraction efficiency are investigated, and the optimal extraction conditions are screened by the response surface method.

Description

Technical Field

[0001] This invention relates to the field of steroidal saponin extraction technology, specifically a method for extracting total saponins from Ophiopogon japonicus using a eutectic solvent. Background Technology

[0002] Ophiopogon japonicus is a perennial herb belonging to the genus Ophiopogon japonicus in the family Liliaceae. It has a sweet and slightly bitter taste, is slightly cold in nature, and enters the heart, lung, and stomach meridians. It nourishes yin and generates fluids, moistens the lungs and clears the heart, and can be used for symptoms such as heat-related illnesses causing fluid depletion, irritability, and thirst. Ophiopogon japonicus is rich in various chemical components, mainly including steroidal saponins, isoflavones, sterols, and polysaccharides. Saponins are one of its main active ingredients, exhibiting anti-tumor proliferation and apoptosis effects, and also showing potential value in improving insulin resistance and regulating lipid metabolism. The extraction of steroidal saponins mostly adopts traditional methods, such as solvent reflux extraction and soaking. These methods consume a large amount of solvent and are difficult to recover. They also have low extraction efficiency, are time-consuming, and energy-intensive. Furthermore, high temperatures can easily destroy the total saponin components and increase the dissolution of impurities. The use of large amounts of organic solvents poses a risk of flammability and explosion, and the discharge of waste liquid can easily cause environmental pollution. Summary of the Invention

[0003] The purpose of this invention is to provide a method for extracting total saponins from Ophiopogon japonicus using a eutectic solvent, which has the advantages of high extraction rate and being environmentally friendly.

[0004] The above-mentioned technical objective of the present invention is achieved through the following technical solution: a method for extracting total saponins from Ophiopogon japonicus using a eutectic solvent, comprising the following steps: S1: Sample pretreatment: After drying the Ophiopogon japonicus raw material, crush it into small pieces and grind it into powder. Store it in a desiccator for later use. S2: Preparation of eutectic solvent: Eutectic solvent is prepared by heating and stirring. Hydrogen bond acceptor and hydrogen bond donor are mixed in a certain molar ratio and stirred and melted in a 60°C water bath. After forming a stable, homogeneous and transparent liquid, 30% water by mass of the solution is added and stored at room temperature for later use. S3: Ultrasonic extraction. Accurately weigh 0.25g of ground Ophiopogon japonicus and add a eutectic solvent with a water content of 30% and a material-to-liquid ratio of 1:80μg / mL. Extract with ultrasonic assistance at 60℃ for 30min using a power of 120W.

[0005] A further configuration of the present invention is as follows: the hydrogen bond acceptor is divided into quaternary ammonium salts, zwitterions, and amino acids, wherein the quaternary ammonium salt is choline chloride, the zwitterion is betaine, and the amino acid is proline.

[0006] A further configuration of the present invention is as follows: the hydrogen bond donor is divided into alcohols, glycosyl groups, amino acids, carboxylic acids, and amides, wherein the amide is urea, the glycosyl group is glucose, the carboxylic acid is lactic acid, and the carboxylic acid is ethylene glycol, 1,3-butanediol, or 1,2-propanediol.

[0007] A further feature of the present invention is that during the preparation of the eutectic solvent in step S2, the stirring rate is 300-500 r / min and the melting time is 30-60 min, so as to ensure the formation of a uniform and transparent liquid.

[0008] A further feature of the present invention is that the ultrasonic extraction in step S3 uses a single-frequency ultrasonic cleaner with a working frequency of 40kHz.

[0009] In summary, the present invention has the following beneficial effects: 1. High extraction rate: By optimizing process parameters using response surface methodology, the total saponin extraction rate of Ophiopogon japonicus can reach 6.22%, which is significantly higher than that of traditional ethanol extraction. 2. Green and environmentally friendly, using natural eutectic solvents to replace organic solvents, reducing environmental pollution and conforming to the concept of green production; 3. By screening different eutectic solvents, the group with the highest extraction rate was identified as choline chloride-lactic acid. The effects of ultrasonic time, water content, solid-liquid ratio, and ultrasonic power on the extraction efficiency were investigated, and the optimal extraction conditions were screened using response surface methodology. The results showed that the optimal extraction conditions were: ultrasonic power of 120W, eutectic solvent of choline chloride-lactic acid with 50% water content, liquid-liquid ratio of 1:170μg / mL, ultrasonic time of 30 min, and total saponin extraction rate of 14.7%. Attached Figure Description

[0010] Figure 1 This is the standard curve of the saponins of this invention; Figure 2 The effect of the type of eutectic solvent on the total saponin extraction rate in this invention; Figure 3 This invention relates to the effect of moisture content on the extraction rate of total saponins from Ophiopogon japonicus. Figure 4 This invention relates to the effect of the material-to-liquid ratio on the extraction rate of total saponins from Ophiopogon japonicus. Figure 5 This invention relates to the effect of extraction time on the extraction rate of total saponins from Ophiopogon japonicus. Figure 6 This invention relates to the effect of ultrasonic power on the extraction rate of total saponins from Ophiopogon japonicus. Figure 7 This is a response surface methodology diagram showing the effect of extraction temperature and moisture content on the extraction rate of total saponins from Ophiopogon japonicus in this invention. Figure 8This is a response surface methodology diagram showing the effect of extraction time and solid-liquid ratio on the extraction rate of total saponins from Ophiopogon japonicus in this invention. Figure 9 This is a response surface methodology diagram showing the effect of extraction time and ultrasonic power on the extraction rate of total saponins from Ophiopogon japonicus in this invention. Figure 10 This is a response surface methodology diagram showing the effect of water content and material-to-liquid ratio on the extraction rate of total saponins from Ophiopogon japonicus in this invention. Figure 11 This is a response surface methodology diagram showing the effect of water content and ultrasonic power on the extraction rate of total saponins from Ophiopogon japonicus in this invention. Figure 12 This is a response surface methodology diagram showing the effect of the material-liquid ratio and ultrasonic power on the extraction rate of total saponins from Ophiopogon japonicus in this invention. Detailed Implementation

[0011] The present invention will be further described below with reference to the accompanying drawings in the embodiments of the present invention.

[0012] Example 1: Determination of Linearity A suitable amount of protodiosgenin standard was dissolved in anhydrous ethanol to prepare a 1 μg / mL saponin standard solution. Five samples were prepared from this solution, with concentrations of 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL, and diluted to 1.0 mL with ethanol. 0.2 mL of freshly prepared 5% vanillin-glacial acetic acid solution and 0.8 mL of perchloric acid were added to each sample, and the mixture was shaken until thoroughly mixed. The mixed solution was heated in a 60°C water bath for 5 to 10 minutes, then cooled in an ice bath. 5 mL of glacial acetic acid was then added to each sample, and the mixture was stirred again until homogeneous. The maximum absorption wavelength was measured using a UV-Vis spectrophotometer at 450 nm. The absorbance of different samples was then measured at 450 nm, and linear regression analysis was performed to obtain the results. Figure 1 The x-axis represents the solution concentration (μg / mL), and the y-axis represents the absorbance value. The regression equation is as follows: y = 1.9264x + 0.0022, with a correlation coefficient of R² = 0.9992.

[0013] Example 2: Taking DESs-6 as an example The dried Ophiopogon japonicus raw material was crushed into small pieces and then ground into powder. The powder was then sealed in a desiccator and stored at room temperature for later use. 0.25 g of the ground Ophiopogon japonicus was accurately weighed and added to a eutectic solvent with a water content of 30% and a material-to-liquid ratio of 1:80 μg / mL. Extraction was performed at 60 ℃ using ultrasound-assisted extraction at 120 W for 30 min. After extraction, the extract was centrifuged at 4000 r / min for 20 minutes to separate the material and liquid. 1 mL of the supernatant was added to 0.2 mL of freshly prepared 5% vanillin-glacial acetic acid solution, followed by 0.8 mL of perchloric acid, and mixed thoroughly. The mixture was heated in a water bath at 60 ℃ for 5-10 min, followed by cooling in an ice bath for 3 min. 5 mL of glacial acetic acid was added and mixed thoroughly. The absorbance was measured, and the DESs-6 extraction rate was calculated using a linear regression equation.

[0014] Example 3: Screening of natural eutectic solvents Replace DESs-6 in Example 2 with other eutectic solvents in Table 1, and proceed as in Example 2, based on... Figure 2 DESs-6 is known to have the best extraction effect on total saponins from Ophiopogon japonicus, and is therefore the best solvent extractant.

[0015] Table 1 Different types of DES solvents

[0016] Example 4: Effect of moisture content on the extraction rate of total saponins from Ophiopogon japonicus The water content in Example 2 was changed to Figure 3 As shown, the material-to-liquid ratio was set to 1:80 μg / mL, the ultrasonic time to 30 min, and the extraction power to 120 W. Other operations were the same as in Example 2. Figure 3 As shown, a moisture content of 10% is optimal, at which point the extraction rate of total saponins from Ophiopogon japonicus is 9.10%.

[0017] Example 5: Effect of material-to-liquid ratio on the extraction rate of total saponins from Ophiopogon japonicus The material-liquid ratio in Example 2 was changed to... Figure 4 As shown, the settings were: water content 10%, ultrasonic time 30 min, extraction power 120 W, and other operations as in Example 1. Figure 4 As shown, the extraction rate of total saponins from Ophiopogon japonicus was the highest, at 11.33%, when the material-to-liquid ratio was 1:140 μg / mL.

[0018] Example 6: Effect of ultrasound time on the extraction rate of total saponins from Ophiopogon japonicus The ultrasound time in Example 2 was changed to Figure 5 As shown, the settings were: water content 10%, material-to-liquid ratio 1:140 μg / mL, extraction power 120 W, and other operations as in Example 1. Figure 5As shown, the extraction rate of total saponins from Ophiopogon japonicus was highest when the ultrasonic time was 30 min, with an extraction rate of 11.33%.

[0019] Example 7: Effect of ultrasonic power on the extraction rate of total saponins from Ophiopogon japonicus The ultrasonic power in Example 2 was changed to Figure 6 As shown, the settings were: water content 10%, material-to-liquid ratio 1:140 μg / mL, ultrasonic time 30 min, and other operations as in Example 1. Figure 6 As shown, the extraction rate of total saponins from Ophiopogon japonicus reached its maximum of 11.41% when the extraction power was 150W.

[0020] Example 8: Response Surface Methodology Results Using Design Expert 13 software, experimental design and regression fitting were performed on the selected factors and levels. The regression equation for the total saponin yield Y (mg / g) of Ophiopogon japonicus with four factors—ultrasonic time (A, min), water content (B, %), material-to-liquid ratio (C, μg / mL), and ultrasonic power (D, W)—was obtained as follows: Y = 11.65 + 0.6174A - 0.4988B + 0.0839C - 0.3880D - 0.6502AB + 0.5456AC + 0.4043AD + 0.1715BC + 1.04BD + 0.0394CD - 1.92A² - 2.45B² - 2.48C² - 1.98D².

[0021] Table 2 Response Surface Analysis Scheme and Results

[0022] Example 9: Response Surface Analysis of Variance According to the analysis results in Table 3, the model has a P < 0.0001, indicating a highly significant difference. The lack-of-fit term has a P > 0.05, indicating no significant difference. Meanwhile, R² = 0.9250, R²adj = 0.8499, and R²pre = 0.6520. The difference between the latter two is less than 0.2, and the precision is > 4, indicating that the model has no significant error and is relatively reliable, and can be used for subsequent analysis.

[0023] Table 3. Analysis of Variance for Regression Models

[0024] Example 10: Interaction Analysis In response surface methodology (RSM), the strength of the interaction between any two factors is related to the slope of the corresponding response surface curve. The stronger the interaction between the two factors, the steeper the surface slope; conversely, the weaker the interaction, the shallower the surface slope. Figures 7 to 12Table 3 shows that the effects of the four factors on the total saponin extraction rate of Ophiopogon japonicus are in descending order as follows: extraction time (A) > water content (B) > ultrasonic power (D) > material-liquid ratio (C). The response surfaces of the interaction terms between AB, AC, and BD are relatively steep, indicating that the interaction between the two factors is relatively significant. However, the response surfaces of the interaction terms between CD, AD, and BC are relatively flat, indicating that the interaction is not significant. The above analysis is consistent with the variance results.

[0025] Example 11: Experimental Verification Based on Desigin-Expert13, the above analysis and Table 3, and the actual parameters were adjusted, it was finally concluded that the highest extraction rate of total saponins from Ophiopogon japonicus was achieved under the conditions of ultrasonic power of 150W, ultrasonic time of 30min, water content of 12.5%, and material-liquid ratio of 1:150μg / mL.

[0026] The above description is only a preferred embodiment of the present invention. Therefore, all equivalent changes or modifications made to the structure, features and principles described in the claims of this patent application are included in the scope of this patent application.

Claims

1. A method for extracting total saponins from Ophiopogon japonicus using a eutectic solvent, characterized in that: Includes the following steps: S1: Sample pretreatment: After drying the Ophiopogon japonicus raw material, crush it into small pieces and grind it into powder. Store it in a desiccator for later use. S2: Preparation of eutectic solvent: Eutectic solvent is prepared by heating and stirring. Hydrogen bond acceptor and hydrogen bond donor are mixed in a certain molar ratio and stirred and melted in a 60°C water bath. After forming a stable, homogeneous and transparent liquid, 30% water by mass of the solution is added and stored at room temperature for later use. S3: Ultrasonic extraction. Accurately weigh 0.25g of ground Ophiopogon japonicus and add a eutectic solvent with a water content of 30% and a material-to-liquid ratio of 1:80μg / mL. Extract with ultrasonic assistance at 60℃ for 30min using a power of 120W.

2. The method for extracting total saponins from Ophiopogon japonicus using a eutectic solvent according to claim 1, characterized in that: The hydrogen bond acceptors are classified into quaternary ammonium salts, zwitterions, and amino acids. The quaternary ammonium salt is choline chloride, the zwitterion is betaine, and the amino acid is proline.

3. The method for extracting total saponins from Ophiopogon japonicus using a eutectic solvent according to claim 1, characterized in that: The hydrogen bond donors are classified into alcohols, glycosyl groups, amino acids, carboxylic acids, and amides. The amides are urea, the glycosyl groups are glucose, the carboxylic acids are lactic acid, and the carboxylic acids are ethylene glycol, 1,3-butanediol, and 1,2-propanediol.

4. The method for extracting total saponins from Ophiopogon japonicus using a eutectic solvent according to claim 1, characterized in that: In step S2, during the preparation of the eutectic solvent, the stirring rate is 300-500 r / min and the melting time is 30-60 min to ensure the formation of a uniform and transparent liquid.

5. The method for extracting total saponins from Ophiopogon japonicus using a eutectic solvent according to claim 1, characterized in that: In step S3, ultrasonic extraction is performed using a single-frequency ultrasonic cleaner with a working frequency of 40kHz.

Images