Method for optimizing total yield of polyphyllin VII, polyphyllin II and polyphyllin I based on response surface method

Abstract

The invention discloses a method for optimizing total yield of polyphyllin VII, polyphyllin II and polyphyllin I. A content determination method of the polyphyllin VII, the polyphyllin II and the polyphyllin I is established, methodological investigation is carried out, the total yield of the three saponins is used as an evaluation index, and the solid-liquid ratio, the extraction time, the temperature and the ethanol concentration are used as influence factors, so that the total yield of the polyphyllin VII, the polyphyllin II and the polyphyllin I is optimized. A response surface method is applied to design an experiment, variance analysis and optimal process prediction and verification are carried out, and extraction process parameters for optimizing the total yield of the polyphyllin VII, the polyphyllin II and the polyphyllin I are determined. The extraction conditions of the total yield of the polyphyllin VII, the polyphyllin II and the polyphyllin I are optimized and researched on the basis of the response surface method, and the method is high in pertinence, simple and convenient to operate, high in accuracy, scientific and reasonable in process and suitable for measuring the content of effective components of medicinal materials.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine extraction technology, specifically involving a method for optimizing the total yield of Paris polyphylla saponin VII, Paris polyphylla saponin II, and Paris polyphylla saponin I based on response surface methodology. Background Technology

[0002] Paris rhizome, the dried rhizome of *Paris polyphylla* or *Paris yunnanensis*, plants belonging to the genus *Paris* in the family Liliaceae, possesses antitumor, anti-inflammatory, antibacterial, swelling-reducing, analgesic, and immunomodulatory effects. Clinically, it is often used to treat boils, carbuncles, and infantile convulsions. More than 80 drugs have been developed based on Paris rhizome or its active ingredients. In antitumor applications, it is mainly used as part of traditional Chinese medicine compound prescriptions for adjuvant therapy. Currently, more than 300 compounds have been isolated from plants of the *Paris* genus, including steroidal compounds, flavonoids, and phytosterols. Steroidal saponins have been confirmed as the main chemical components and active substances of *Paris* plants, and pharmacological activity studies have shown that they have antitumor, anti-inflammatory, and hemostatic activities. Paris saponin VII, Paris saponin II, and Paris saponin I have shown strong antitumor activity in various cancers, such as breast cancer, lung cancer, and liver cancer. To meet the national pharmacopoeia standard, the total content of the three saponins (Parisin VII, Parisin II, and Parisin I) in the Chinese herb Paris must be greater than 0.6%. Furthermore, the content of the active ingredients in Paris can vary depending on the place of origin, harvesting year, planting method, and storage. Therefore, it is necessary to establish an extraction method to extract as many saponins as possible.

[0003] Many methods are commonly used in laboratories to optimize extraction processes, including single-factor experiments, orthogonal experiments, and Box-Behnken response surface methodology. Compared with other methods, response surface methodology can achieve continuous-level optimization, has better predictive ability, and the visualization of data makes the experimental results more intuitive, clearly revealing the interactions between various factors. It is widely used in the extraction research of traditional Chinese medicine. Summary of the Invention

[0004] To address the above problems, the present invention aims to provide a method for optimizing the total yield of Paris polyphylla saponin VII, Paris polyphylla saponin II, and Paris polyphylla saponin I based on response surface methodology.

[0005] The specific technical solution is as follows:

[0006] A method for optimizing the total yield of Paris polyphylla saponin VII, Paris polyphylla saponin II, and Paris polyphylla saponin I based on response surface methodology includes the following steps:

[0007] 1) Prepare reference solutions and plot standard curves: Weigh Paris polyphylla saponin VII, Paris polyphylla saponin II, and Paris polyphylla saponin I, dissolve them, prepare reference stock solutions, transfer different reference stock solutions to mix, dilute to obtain mixed reference solutions of different mass concentrations, analyze them by ultra-high performance liquid chromatography, and plot standard curves.

[0008] 2) Screening conditions: Four factors affecting the total yield Y during the extraction experiment were determined. A single-factor variable experiment was adopted. The powder of Paris polyphylla was weighed, ethanol was added, weighed, heated to reflux, cooled, filtered, weighed, and the weight loss was made up with ethanol. The mixture was shaken, filtered, and the filtrate was collected. Ultra-high performance liquid chromatography was used to analyze the filtrate and determine the three levels of data for each influencing factor.

[0009] 3) Response surface methodology: Based on the data from step 2), using the total yield Y of Paris polyphylla saponin VII, Paris polyphylla saponin II, and Paris polyphylla saponin I as the evaluation index, a response surface methodology experiment with three levels of experimental points was designed using the Box-Behnken model to analyze all influencing factors. The Design-Expert13 software was used to perform a multivariate fitting analysis on the total yield Y and the influencing factors, obtaining the binary regression equation model and variance analysis results of the total yield Y and the influencing factors. Based on the results, the optimal extraction process was predicted and a verification experiment was conducted.

[0010] Furthermore, the chromatographic conditions for ultra-high performance liquid chromatography are: InertSustain An AQ-C18 column, 2.1 × 100 mm in size and 1.9 μm in diameter, was used. The flow rate was 0.2 ml / min, the column temperature was 25 ℃, the detection wavelength was 203 nm, and the injection volume was 2 μL. The gradient elution program was as follows: the initial mobile phase ratio was water-acetonitrile 90:10; at 5 min, the mobile phase ratio was reduced to water-acetonitrile 84:16; at 10 min, it was reduced to water-acetonitrile 75:25; at 17 min, it was reduced to water-acetonitrile 60:40; at 23 min, it was reduced to water-acetonitrile 54:46 and held for 5 min; at 35 min, it was reduced to water-acetonitrile 40:60; at 40 min, it was reduced to water-acetonitrile 10:90 and held for 10 min; at 53 min, the mobile phase ratio was increased to water-acetonitrile 90:10 and held for 2 min.

[0011] Furthermore, the influencing factors in step 2) are ethanol concentration, material-to-liquid ratio, extraction time, and reflux temperature.

[0012] Furthermore, the three levels of ethanol concentration were 45%, 60%, and 75%; the three levels of material-liquid ratio were 1:20 g / mL, 1:30 g / mL, and 1:40 g / mL; the three levels of extraction time were 60 min, 90 min, and 120 min; and the three levels of temperature were 65℃, 75℃, and 85℃. The three levels of each influencing factor were set from smallest to largest as -1, 0, and 1.

[0013] Further, the binary regression equation model obtained in step 3) is Y(%) = 1.59 + 0.0182A + 0.2310B + 0.0040C + 0.0255D + 0.0055AB - 0.0070AC - 0.0067AD - 0.0117BC - 0.0181BD - 0.0019CD - 0.0539A 2 -0.4274B 2 -0.0240C 2 -0.0300D 2 In the formula, Y represents the total yield, A represents the extraction time (min), B represents the solid-liquid ratio (g / mL), C represents the temperature (°C), and D represents the ethanol concentration (%).

[0014] The beneficial effects of this invention are as follows:

[0015] 1) This invention establishes a method for determining the content of index components Paris saponin VII, Paris saponin II, and Paris saponin I in the traditional Chinese medicine Paris polyphylla and conducts methodological investigation; taking extraction time, ethanol concentration, material-liquid ratio, and temperature as influencing factors, the experiment is designed using response surface methodology, a multiple quadratic regression model of yield and investigated factors is established, variance analysis is performed, optimal process prediction and verification are conducted, and finally the optimized extraction process parameters for the total yield of Paris saponin VII, Paris saponin II, and Paris saponin I in the traditional Chinese medicine Paris polyphylla are determined.

[0016] 2) This invention specifically relates to the optimization of extraction conditions for the total yield of Paris polyphylla saponins VII, II, and I based on response surface methodology. This method is highly targeted, simple to operate, accurate, and scientifically sound, making it particularly suitable for the determination of the content of effective components in medicinal materials. The content determination index components selected in this invention have pharmacological and efficacy effects consistent with anti-tumor therapeutic effects and are commonly used indicators in process research. Using the total yield of Paris polyphylla saponins VII, II, and I as the response value, the optimized process is scientifically sound and highly accurate.

[0017] 3) Response surface methodology is suitable for the design and optimization of experiments involving multiple factors and indicators. It can accurately predict function models and reflect the mathematical relationship between each factor and the response value. Compared with orthogonal design, it can more intuitively analyze the impact of each interaction factor on the experimental results. This invention applies response surface methodology to design experiments and determines the optimal extraction process parameters by analyzing the regression equation model. Compared with existing methods, it is more scientific, rigorous, and simple to implement.

[0018] 4) This invention provides a theoretical basis and technical support for the later development of Paris polyphylla and the research on the medicinal value of related medicinal materials. This provides a reference for the optimization of Chinese medicine extraction processes and has a good application prospect. Attached Figure Description

[0019] Figure 1 The results of the linear relationship investigation of Paris polyphylla saponin VII;

[0020] Figure 2 The results of the linear relationship study of Paris polyphylla saponin II;

[0021] Figure 3 The results of the linear relationship study of Paris polyphylla saponin I;

[0022] Figure 4 The response surface of the interaction between time and feed-liquid ratio;

[0023] Figure 5 The response surface methodology for the interaction between the feed-to-liquid ratio and the ethanol concentration;

[0024] Figure 6 The response surface is the interaction between time and ethanol concentration. Detailed Implementation

[0025] The present invention will be further described below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited thereto.

[0026] Chromatographic conditions for Paris saponin VII, Paris saponin II, and Paris saponin I: The chromatographic column was InertSustain. The AQ-C18 column has dimensions of 2.1 × 100 mm and a diameter of 1.9 μm. The flow rate is 0.2 ml / min, the column temperature is 25℃, the detection wavelength is 203 nm, and the injection volume is 2 μL. The gradient elution program is as follows: the initial mobile phase ratio is water-acetonitrile 90:10; at 5 min, the ratio decreases to water-acetonitrile 84:16; at 10 min, it decreases to water-acetonitrile 75:25; at 17 min, it decreases to water-acetonitrile 60:40; at 23 min, it decreases to water-acetonitrile 54:46 and holds for 5 min; at 35 min, it decreases to water-acetonitrile 40:60; at 40 min, it decreases to water-acetonitrile 10:90 and holds for 10 min; at 53 min, the ratio increases to water-acetonitrile 90:10 and holds for 2 min.

[0027] Example 1

[0028] 1. Establishment of content determination method

[0029] 1) Preparation of reference solution

[0030] Accurately weigh Paris saponin VII, Paris saponin II, and Paris saponin I, dissolve them in methanol to prepare reference stock solutions, accurately transfer the reference stock solutions, dilute with methanol to obtain a series of reference solutions of different mass concentrations for later use, as shown in Table 1.

[0031] Table 1 shows a series of mixed reference solutions with varying mass concentrations.

[0032] series Paris saponin VII μg / mL Paris saponin II μg / mL Paris saponins I μg / mL 1 400 7 10 2 500 8.33 12.5 3 800 10 20 4 1000 12.5 50 5 2000 20 100

[0033] 2) Preparation of test solution

[0034] Accurately weigh 2g of Paris polyphylla powder and place it in a round-bottom flask. Accurately add 100ml of ethanol, weigh the mixture, heat under reflux for 30 minutes, cool, weigh the mixture again, replenish the lost weight with ethanol, shake well, filter, and collect the filtrate to obtain the test solution.

[0035] 2. Methodological Validation

[0036] 1) Linearity and Range

[0037] Inject the mixed reference solution for analysis, and plot the standard curve with peak area (Y) on the ordinate and concentration (X μg / mL) on the abscissa. Figures 1 to 3 The results showed that the regression equations for Paris polyphylla saponin VII, Paris polyphylla saponin II, and Paris polyphylla saponin I were Y = 2863.4X - 195957(R). 2=0.9997), Y=2820.2x-693.79(R 2 =0.9990), Y=3456.1x-3652.9(R 2 =0.9998), with linear ranges of 400-2000 μg / mL, 7.14-20.00 μg / mL, and 10.00-100.00 μg / mL, respectively.

[0038] 2) Precision

[0039] Using the same mixed reference standard, six injections were performed consecutively under the chromatographic conditions described above. The peak areas were measured, and the RSDs were calculated. The results showed that the RSDs of the peak areas of Paris polyphylla VII, Paris polyphylla II, and Paris polyphylla I were 0.43%, 0.91%, and 0.18% (n=6), respectively, indicating good instrument precision.

[0040] 3) Stability

[0041] The prepared test solution was injected at 0h, 3h, 6h, 9h, and 12h according to the chromatographic conditions, and the peak area was measured and RSD was calculated. The results showed that the RSDs of the peak areas of Paris polyphylla VII, Paris polyphylla II, and Paris polyphylla I were 0.01%, 1.90%, and 1.62% (n=6), respectively, indicating that the test solution had good stability.

[0042] 4) Reproducibility

[0043] The test solution was prepared in parallel to a sample of six. Each sample was injected and analyzed under the chromatographic conditions described above. The peak area was measured, and the RSD was calculated. The results showed that the RSDs of the peak areas of Paris polyphylla VII, Paris polyphylla II, and Paris polyphylla I were 0.10%, 1.90%, and 1.71% (n=6), respectively, indicating good reproducibility of the method.

[0044] 5) Recovery rate

[0045] Six portions of Paris polyphylla powder were accurately weighed and used to prepare six test solutions. Three reference solutions of the same concentration were added to each solution. The chromatograms were integrated under the chromatographic conditions described above, and the results were substituted into the accompanying standard curve to calculate the recovery rate and RSD of each component. The results showed that the recoveries of Paris polyphylla saponin VII, II, and I were between 80% and 120%, with RSD ≤ 25%. This method is accurate and reliable. The results are shown in Table 2.

[0046] Table 2. Results of the recovery tests for Paris polyphylla saponin VII, Paris polyphylla saponin II, and Paris polyphylla saponin I (n=6)

[0047]

[0048]

[0049] The following examples screen extraction time, ethanol concentration, solid-liquid ratio, and temperature.

[0050] Example 2

[0051] Weigh out the powdered Paris polyphylla and place it in a round-bottom flask. Add 50 times the amount of 75% ethanol, weigh the mixture, heat to 85℃ under reflux, cool, filter, weigh again, replenish the lost weight with ethanol, shake well, filter, and collect the filtrate. The effects of different extraction times on the extraction of Paris polyphylla saponins VII, II, and I from the traditional Chinese medicine Paris polyphylla were investigated under the above chromatographic conditions. The reflux times were 30 min, 60 min, 90 min, 120 min, and 150 min, respectively.

[0052] Based on the total yields of Paris saponin VII, Paris saponin II, and Paris saponin I, and after comprehensive consideration, the single-factor experiment selected an extraction time of 90 min, and the Box-Behnken response surface methodology was conducted at three levels: 60 min, 90 min, and 120 min.

[0053] Example 3

[0054] Weigh out the powdered Paris polyphylla and place it in a round-bottom flask. Add 75% ethanol, weigh the mixture, heat to reflux at 85℃ for 90 min, cool, filter, weigh again, replenish the lost weight with ethanol, shake well, filter, and collect the filtrate. The effects of different ethanol addition amounts on the extraction of Paris polyphylla saponins VII, II, and I from the traditional Chinese medicine Paris polyphylla were investigated under the above chromatographic conditions. The ethanol addition amounts were 10, 20, 30, 40, and 50 times, respectively.

[0055] Based on the total yields of Paris saponin VII, Paris saponin II, and Paris saponin I, and after comprehensive consideration, the single-factor experiment selected a material-to-liquid ratio of 1:30 g / mL, and three levels of material-to-liquid ratios of 1:20 g / mL, 1:30 g / mL, and 1:40 g / mL for Box-Behnken response surface methodology.

[0056] Example 4

[0057] Weigh out the powdered Paris polyphylla and place it in a round-bottom flask. Add 30 times the amount of 75% ethanol, weigh the mixture, heat under reflux for 90 min, cool, filter, weigh again, replenish the lost weight with ethanol, shake well, filter, and collect the filtrate. The effects of different temperatures on the extraction of Paris polyphylla saponins VII, II, and I from the traditional Chinese medicine Paris polyphylla were investigated under the above chromatographic conditions: 55℃, 65℃, 75℃, 85℃, and 95℃.

[0058] Based on the total yields of Paris saponin VII, Paris saponin II, and Paris saponin I, and after comprehensive consideration, the Box-Behnken response surface methodology was conducted at three temperature levels: 75℃, 65℃, 75℃, and 85℃.

[0059] Example 5

[0060] Weigh out the powdered Paris polyphylla and place it in a round-bottom flask. Add 30 times the amount of ethanol, weigh the mixture, heat under reflux at 75℃ for 90 min, cool, filter, weigh again, and replenish the lost weight with ethanol. Shake well, filter, and collect the filtrate. The effects of different ethanol concentrations on the extraction of Paris polyphylla saponins VII, II, and I from the herb Paris polyphylla were investigated under the above chromatographic conditions. The ethanol concentrations were 45%, 60%, 75%, 90%, and 100%, respectively.

[0061] Based on the total yields of Paris saponin VII, Paris saponin II, and Paris saponin I, and after comprehensive consideration, the Box-Behnken response surface methodology was conducted using three ethanol concentration levels: 60%, 45%, 60%, and 75%.

[0062] Example 6

[0063] Box-Behnken response surface methodology

[0064] (1) Experimental Design and Results of Box-Behnken Response Surface

[0065] Based on the results of the single-factor experiments, the total yield Y of Paris polyphylla saponin VII, Paris polyphylla saponin II, and Paris polyphylla saponin I was used as the evaluation index, and extraction time (A), material-to-liquid ratio (B), temperature (C), and ethanol concentration (D) were used as influencing factors. The Box-Behnken response surface methodology with 4 factors and 3 levels was used for the experiment. The factors and levels of the Box-Behnken response surface experimental design are shown in Table 3, and the scheme and results are shown in Table 4.

[0066] Table 3. Factors and Levels in the Box-Behnken Response Surface Experiment Design

[0067] Table 4. Experimental Design and Results of Box-Behnken Response Surface Methodology

[0068]

[0069]

[0070] (2) Model Fitting and Analysis of Variance

[0071] The Design-Expert 13 software was used to perform multiple regression fitting and analysis of variance on the data in Table 4. The results are shown in Table 5. The quadratic multinomial regression equation between factors A, B, C, and D and the yield Y is Y(%) = 1.59 + 0.0182A + 0.2310B + 0.0040C + 0.0255D + 0.0055AB - 0.0070AC - 0.0067AD - 0.0117BC - 0.0181BD - 0.0019CD - 0.0539A 2 -0.4274B 2 -0.0240C 2 -0.0300D 2 The coefficients show that the effects of the four factors on the total yield of Paris polyphylla saponins VII, II, and I in the traditional Chinese medicine Paris polyphylla are: material-to-liquid ratio > ethanol concentration > extraction time > temperature. Therefore, material-to-liquid ratio, ethanol concentration, and extraction time were selected for response surface methodology (RSM). Choosing the three most influential factors for RSM ensures that their weights and directions of influence on the response value (yield) are larger, making them key variables. Temperature has a smaller impact; fixing the temperature at its optimal level eliminates random interference, allowing the RSM model to more clearly reveal the true interactions between key factors. Table 5 shows that the model P < 0.05, and the lack-of-fit term P = 0.0508, indicating that the model is significant, while the lack-of-fit term is not significant. This suggests that the unknown factors have little impact on the experiment, and the model can be used to predict experimental results. The model correlation coefficient R0 is also shown. 2 =0.9979, adjusted R 2 =0.9958, indicating that the model's simulation is good and the method is reliable.

[0072] Table 5. Results of ANOVA for the regression model

[0073]

[0074]

[0075] (1) Response surface analysis and prediction

[0076] Box-Behnken response surface analysis was performed on the model equations using Design-Expert13 software, and the response surface plots were obtained. The results are shown below. Figures 4-6Based on the model fitting results, the optimal extraction process for the total yield of Paris polyphylla saponins VII, II, and I was predicted to be: extraction time: 83.649 min, solid-liquid ratio: 1:32.428 g / mL, temperature: 82.478℃, and ethanol concentration: 61.055%, with a predicted value of 1.602%. Further adjustments based on actual operation yielded the optimal extraction process to: extraction time: 84 min, solid-liquid ratio: 1:32 g / mL, temperature: 82℃, and ethanol concentration: 61%.

[0077] (2) Validation of extraction process for Paris saponin VII, Paris saponin II and Paris saponin I

[0078] The optimal extraction process was repeated three times for validation. The results showed that the average yield of the three validation experiments was 1.599%, and the RSD was 0.05%, indicating that the established extraction process for Paris polyphylla saponins VII, II, and I was stable and reproducible. The deviation between the actual values ​​and the predicted values ​​(1.602%) obtained by the Box-Behnken response surface methodology was 0.03%, indicating that the model had good predictive ability. The results are shown in Table 6.

[0079] Table 6. Validation results of the extraction process for Paris polyphylla saponin VII, Paris polyphylla saponin II, and Paris polyphylla saponin I.

[0080]

[0081]

Claims

1. A method for optimizing the total yield of Paris polyphylla saponin VII, Paris polyphylla saponin II, and Paris polyphylla saponin I based on response surface methodology, characterized in that, Includes the following steps: 1) Prepare reference solutions and plot standard curves: Weigh Paris polyphylla saponin VII, Paris polyphylla saponin II, and Paris polyphylla saponin I, dissolve them, prepare reference stock solutions, transfer different reference stock solutions to mix, dilute to obtain mixed reference solutions of different mass concentrations, analyze them by ultra-high performance liquid chromatography, and plot standard curves. 2) Screening conditions: Four factors affecting the total yield Y during the extraction experiment were determined. A single-factor variable experiment was adopted. The powder of Paris polyphylla was weighed, ethanol was added, weighed, heated to reflux, cooled, filtered, weighed, and the weight loss was made up with ethanol. The mixture was shaken, filtered, and the filtrate was collected. Ultra-high performance liquid chromatography was used to analyze the filtrate and determine the three levels of data for each influencing factor. 3) Response surface methodology: Based on the data from step 2), using the total yield Y of Paris polyphylla saponin VII, Paris polyphylla saponin II, and Paris polyphylla saponin I as the evaluation index, a response surface methodology experiment with three levels of experimental points was designed using the Box-Behnken model to analyze all influencing factors. The Design-Expert13 software was used to perform a multivariate fitting analysis on the total yield Y and the influencing factors, obtaining the binary regression equation model and variance analysis results of the total yield Y and the influencing factors. Based on the results, the optimal extraction process was predicted and a verification experiment was conducted.

2. The method for optimizing the total yield of Paris polyphylla saponin VII, Paris polyphylla saponin II, and Paris polyphylla saponin I based on response surface methodology as described in claim 1, characterized in that, The chromatographic conditions for ultra-high performance liquid chromatography are as follows: An InertSustain AQ-C18 column, 2.1 × 100 mm in size and 1.9 μm in diameter, was used. The flow rate was 0.2 ml / min, the column temperature was 25 ℃, the detection wavelength was 203 nm, and the injection volume was 2 μL. The gradient elution program was as follows: the initial mobile phase ratio was water-acetonitrile 90:10; at 5 min, the mobile phase ratio was reduced to water-acetonitrile 84:16; at 10 min, it was reduced to water-acetonitrile 75:25; at 17 min, it was reduced to water-acetonitrile 60:40; at 23 min, it was reduced to water-acetonitrile 54:46 and held for 5 min; at 35 min, it was reduced to water-acetonitrile 40:60; at 40 min, it was reduced to water-acetonitrile 10:90 and held for 10 min; at 53 min, the mobile phase ratio was increased to water-acetonitrile 90:10 and held for 2 min.

3. The method for optimizing the total yield of Paris polyphylla saponin VII, Paris polyphylla saponin II, and Paris polyphylla saponin I based on response surface methodology as described in claim 1, characterized in that, The influencing factors in step 2) are ethanol concentration, solid-liquid ratio, extraction time, and reflux temperature.

4. The method for optimizing the total yield of Paris polyphylla saponin VII, Paris polyphylla saponin II, and Paris polyphylla saponin I based on response surface methodology as described in claim 3, characterized in that, The three levels of ethanol concentration were 45%, 60%, and 75%; the three levels of solid-liquid ratio were 1:20 g / mL, 1:30 g / mL, and 1:40 g / mL; the three levels of extraction time were 60 min, 90 min, and 120 min; and the three levels of temperature were 65℃, 75℃, and 85℃. The three levels of each influencing factor were set from smallest to largest as -1, 0, and 1.

5. The method for optimizing the total yield of Paris polyphylla saponin VII, Paris polyphylla saponin II, and Paris polyphylla saponin I based on response surface methodology as described in claim 4, characterized in that... The binary regression equation model obtained in step 3) is Y(%) = 1.59 + 0.0182A + 0.2310B + 0.0040C + 0.0255D + 0.0055AB - 0.0070AC - 0.0067AD - 0.0117BC - 0.0181BD - 0.0019CD - 0.0539A 2 -0.4274B 2 -0.0240C 2 -0.0300D 2 In the formula, Y represents the total yield, A represents the extraction time (min), B represents the solid-liquid ratio (g / mL), C represents the temperature (°C), and D represents the ethanol concentration (%).

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