Response surface methodology-based process method for optimizing extraction of total flavonoids and total phenolic acids from Yao-nationality medicinal material common gendarussa herb

Abstract

The invention discloses a process method for optimizing total flavone and total phenolic acid extraction of gendarussa ventricosa based on a multi-index comprehensive scoring method in combination with a response surface method. A single factor experiment is carried out by taking the material-liquid ratio, the extraction temperature, the extraction time and the ethanol concentration as investigation factors, the total flavone content and the total phenolic acid content are taken as response values, the extraction process is optimized through a response surface design test, and the optimal extraction process of the total flavone of the gendarussa vulgaris is obtained: the material-liquid ratio is 1: 30, the extraction temperature is 60 DEG C, the extraction time is 45 minutes, the ethanol concentration is 70%, and the total flavone content is 2.487 + / -0.1332 mg / g; according to the optimal extraction process of the total phenolic acid, the material-liquid ratio is 1: 20, the extraction temperature is 55 DEG C, the extraction time is 40 min, the ethanol concentration is 70%, and the content of the obtained total phenolic acid is 10.625 + / -0.285 mg / g. The process method provided by the invention is stable, feasible, simple and easy to operate, good in repeatability and relatively high in extraction efficiency.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant extraction, and relates to a process for optimizing extraction of total flavonoids and total phenolic acids from Gendarussa vulgaris. BACKGROUND

[0002] Gendarussa vulgaris Nees. is a dry aboveground part of Gendarussa vulgaris Nees. in Labiatae. In the first volume of Quality Standard for Yao Medicinal Materials of Guangxi Zhuang Autonomous Region, it is recorded as Jieguangfeng (Mengjiejinchong) and has the effects of continuing bone and muscle, removing blood stasis, and relieving swelling and pain. In addition, relevant records can also be found in Guangxi Medicinal Plant List, Medicinal Plant Resources in Gongcheng Yao Nationality Dragon Boat Festival Medicine Market of Guangxi, and Volume 9 of Guangdong Flora. In Yao medicine, it is often used for treating Pochong (trauma), Nengnao (fracture), Bengkui Meng (rheumatoid arthritis), and Muming Zongdu (unknown toxic infection, abscess and toxic infection). In Lingnan Medicinal Herbology, it is also recorded that Gendarussa vulgaris has the effects of promoting blood circulation and removing blood stasis, and is suitable for use in the middle stage of fracture (periosteal formation stage).

[0003] Flavonoids and phenolic acids are widely present in plants and are important components of plant secondary metabolites. Chen Yanxia et al. have determined the antioxidant activity of water extract and alcohol extract of stems and leaves of Gendarussa vulgaris. KRISHNA et al. found that the content of flavonoids and phenols in the methanol component of Gendarussa vulgaris is closely related to the antioxidant activity. MRUTHUNJAYA et al. found that the 70% ethanol extract of Gendarussa vulgaris leaves has good antioxidant activity, and it is speculated that this may be related to flavonoids. The medicinal part of Gendarussa vulgaris is the aboveground part according to the 2025 edition of Chinese Pharmacopoeia. Through the arrangement of researches on chemical components and pharmacological effects of Gendarussa vulgaris in recent years, Shupenghua et al. determined that the main active ingredient of the aboveground part of Gendarussa vulgaris for antioxidant is flavonoids. In recent years, researchers often use ultrasonic-assisted extraction method to extract flavonoids and phenolic acids by combining ultrasonic cavitation effect with solvent extraction, which can significantly improve the extraction efficiency. Therefore, the present application also uses ultrasonic method to extract total flavonoids and total phenolic acids.

[0004] At present, there is no index for quality evaluation of Gendarussa vulgaris medicinal materials. Therefore, the present application adopts the principle of Box-Behnken Design to design a four-factor three-level experiment, and uses response surface analysis method to optimize the extraction process of Gendarussa vulgaris, and determines the content of total flavonoids and total phenolic acids in the aboveground part of Gendarussa vulgaris as the index. SUMMARY

[0005] The present application aims to provide a process for optimizing extraction of total flavonoids and total phenolic acids from Gendarussa vulgaris based on response surface method.

[0006] A method for optimizing the extraction of total flavonoids and total phenolic acids from the Yao medicinal material Xiaobogu based on response surface methodology comprises the following steps:

[0007] (1) Grind the above-ground part of the Yao medicinal material Xiaobogu into powder to obtain Xiaobogu medicinal material powder.

[0008] (2) Weigh 2 g of the Yao medicinal material powder of the small bone bone and place it in a 100 mL conical flask. Add different concentrations of ethanol at different material-liquid ratios and obtain the crude extract of the small bone bone at different extraction temperatures and times.

[0009] (3) Four factors, namely, solid-liquid ratio, extraction temperature, extraction time and ethanol concentration, were selected for single-factor experiments. Response surface design was used to perform multiple regression fitting on each factor, with total flavonoids and total phenolic acid content as response values. The quadratic polynomial regression equation was obtained:

[0010] Y 总黄酮含量 =2.47+0.065*A+0.21*B+0.06*C-0.051*D-0.038*AB-0.12*AC-0.098*AD+0.092*BC-0.25*BD-0.024*CD-0.41*A 2 -0.53*B 2 -0.31*C 2 -0.26*D 2

[0011] Y 总酚酸含量 =10.25+0.33*A+0.22*B+0.31*C-1.23*D-0.063*AB-0.06*AC-0.34*AD+0.16*BC+0.5*BD+0.33*CD-0.88*A 2 -0.71*B 2 -0.73*C 2 -1.74*D 2

[0012] Wherein, Y-total flavonoids and total phenolic acid content, A-solid-liquid ratio, B-extraction temperature, C-extraction time, D-ethanol concentration, and the optimal extraction process conditions of total flavonoids and total phenolic acids from the small bone were calculated by the above regression equation.

[0013] In step (3), the extraction range of the single factor experiment is: solid-liquid ratio (1:15 to 1:40 g / mL), extraction temperature (30 to 70° C.), extraction time (10 to 60 min), and ethanol concentration (30 to 90%).

[0014] Through response surface design experimental method optimization, the optimal extraction process of total flavonoids from Bone Marrow was obtained: solid-liquid ratio 1:30, extraction temperature 60℃, extraction time 45min, ethanol concentration 70%; the optimal extraction process of total phenolic acid was: solid-liquid ratio 1:20, extraction temperature 55℃, extraction time 40min, ethanol concentration 70%.

[0015] Beneficial effects of the present invention:

[0016] The research results show that, within the parameter range investigated, when the material-liquid ratio reaches 1:30, the temperature is 60°C, the time is 45 min, and the ethanol concentration is 70%, the experiment is repeated 3 times under these conditions, and the average total flavonoid content is 2.487±0.132 mg / g, which is close to the model predicted value of 2.515 mg / g, indicating that the process parameters optimized by the model are reliable; when the material-liquid ratio reaches 1:20, the temperature is 55°C, the time is 40 min, and the ethanol concentration is 70%, the experiment is repeated 3 times under these conditions, and the average total phenolic acid content is 10.625±0.285 mg / g, which is within the deviation range of 5% from the model predicted value of 10.539 mg / g, also indicating that the process parameters optimized by the model are reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Standard curve of total flavonoids

[0018] Figure 2 Standard curve of total phenolic acids

[0019] Figure 3 Curve diagram of the effect of material-liquid ratio on the content of total flavonoids and total phenolic acids in small bone

[0020] Figure 4 Curve diagram of the effect of extraction temperature on the content of total flavonoids and total phenolic acids in Ossobuco

[0021] Figure 5 Curve diagram of the effect of extraction time on the content of total flavonoids and total phenolic acids in Ossobuchus chinensis

[0022] Figure 6 Curve diagram of the effect of ethanol concentration on the content of total flavonoids and total phenolic acids in Ossobuco

[0023] Figure 7 Response surface plot of the interaction between solid-liquid ratio and temperature on the total flavonoids content

[0024] Figure 8 Response surface plot of the interaction between solid-liquid ratio and time on the total flavonoids content

[0025] Figure 9 Response surface plot of the interaction between solid-liquid ratio and ethanol concentration on the total flavonoids content

[0026] Figure 10Response surface plot of the interaction effect of temperature and time on total flavonoids content

[0027] Figure 11 Response surface plot of the effects of temperature and ethanol concentration on total flavonoids content

[0028] Figure 12 Response surface plot of the effects of time and ethanol concentration on total flavonoids content

[0029] Figure 13 Response surface plot of the interaction between solid-liquid ratio and temperature on the total phenolic acid content

[0030] Figure 14 Response surface plot of the interaction between solid-liquid ratio and time on total phenolic acid content

[0031] Figure 15 Response surface plot of the interaction between solid-liquid ratio and ethanol concentration on total phenolic acid content

[0032] Figure 16 Response surface plot of the effects of temperature and time on total phenolic acid content

[0033] Figure 17 Response surface plot of the effects of ethanol concentration and temperature on total phenolic acid content

[0034] Figure 18 Response surface plot of the effects of ethanol concentration and time on total phenolic acid content

[0035] Figure 19 Comparison of total flavonoids and total phenolic acid contents in 24 batches of Yao medicinal material Xiaobogu DETAILED DESCRIPTION

[0036] The present invention provides a process for optimizing the extraction of total flavonoids and total phenolic acids from the Yao medicinal material Xiaobogu based on response surface methodology, and the steps are as follows:

[0037] (1) Grind the dried aerial part of the Yao medicinal material Xiaobogu into powder to obtain Xiaobogu medicinal material powder.

[0038] (2) Weigh 2 g of the Yao medicinal material powder of the small bone bone and place it in a 100 mL conical flask. Add different concentrations of ethanol at different material-liquid ratios and obtain the crude extract of the small bone bone at different extraction temperatures and times.

[0039] (3) Four factors, namely, material-liquid ratio, extraction temperature, extraction time and ethanol concentration, were selected for single-factor experiments. Response surface design experiments were adopted, with total flavonoids and total phenolic acid contents as response values, respectively. A four-factor three-level experimental scheme was designed to experimentally design the extraction process of total flavonoids and total phenolic acids in small ribs.

[0040] (4) Based on the data from step (3), multiple regression analysis was performed on each factor to determine the optimal extraction process parameters for total flavonoids and total phenolic acids in small sclerotium.

[0041] Example 1

[0042] 1. Materials and Instruments

[0043] Dried aerial parts of the Yao medicinal material Xiaobogu, sodium nitrite, aluminum nitrate, sodium hydroxide, anhydrous ethanol, rutin reference substance. UV spectrophotometer, refrigerated centrifuge, electric constant temperature water bath, electric blast drying oven, CNC ultrasonic cleaner, analytical balance.

[0044] 2. Experimental method for determination of total flavonoids content

[0045] (1) Preparation of small bone herb powder: grind the small bone herb into powder using a grinder, sieve it, and store it in a sealed bag for later use.

[0046] (2) Preparation of rutin reference solution: Accurately weigh 10.40 mg of rutin reference solution, add 75% ethanol and ultrasonicate for 30 min to dissolve it, and dilute to a 10 mL volumetric flask. Shake well to obtain a rutin reference stock solution with a concentration of 1.04 mg / mL.

[0047] (3) Preparation of test solution: Take 2.00 g of Yao medicinal material Xiaobogu powder, accurately weigh it, place it in a 100 mL conical flask, accurately add 50 mL of 75% ethanol, seal it tightly, weigh it, ultrasonicate it in constant water at 50°C for 40 minutes, make up the weight, filter it, and use it as the test solution.

[0048] (4) Total flavonoid color development and determination conditions: Accurately pipette 1 mL of the test solution into a centrifuge tube, add 0.4 mL of freshly prepared 5% NaNO2, let it stand for 6 min, then add 0.4 mL of freshly prepared 10% Al(NO3)3, let it stand for 6 min, add 4 mL of freshly prepared 4% NaOH, let it stand for 15 min, and measure the absorbance at 510 nm.

[0049] (5) The total flavonoid content was calculated according to the formula: M = (c*v*n) / m

[0050] Where M is the flavonoid content, mg / g; c is the concentration, mg / L; V is the volume, mL; n is the multiple; m is the mass, g.

[0051] (6) Drawing of the standard curve of total flavonoids

[0052] Accurately measure 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL, and 1.2 mL of rutin reference solution with a concentration of 1.04 mg / mL, respectively, and place them in 10 mL volumetric flasks. Measure the absorbance according to step (4). Use mass concentration as the abscissa and absorbance as the ordinate to construct the standard curve equation, and obtain the linear regression equation y = 0.0061x + 0.0117, R 2 =0.9998. The reference solution has a good linear relationship in the range of 20.8124.8 mg / L. Figure 1 .

[0053] 3. Single-factor experiment

[0054] (1) Effect of material-liquid ratio on content: Under the conditions of extraction temperature of 50℃, extraction time of 30min and ethanol concentration of 75%, the effects of different material-liquid ratios (1:15, 1:20, 1:25, 1:30, 1:40) on the total flavonoid content in the extract of small rib bone were investigated, and the optimal material-liquid ratio of total flavonoids was 1:25. Figure 3 .

[0055] (2) Effect of temperature on content: Under the conditions of solid-liquid ratio of 1:20, extraction time of 30 min, and ethanol concentration of 75%, the effects of different extraction temperatures (30, 40, 50, 60, 70°C) on the total flavonoid content in the extract of small rib bone were investigated, and the optimal extraction temperature for total flavonoids was 60°C. Figure 4 .

[0056] (3) Effect of time on content: Under the conditions of solid-liquid ratio of 1:20, extraction temperature of 50℃, extraction time of 30min, and ethanol concentration of 75%, the effects of different extraction times (10, 20, 30, 40, 60min) on the total flavonoid content in the extract of small bone were investigated, and the optimal extraction time was 40min. Figure 5 .

[0057] (4) Effect of ethanol concentration on content: Under the conditions of solid-liquid ratio of 1:20, extraction temperature of 50℃, and extraction time of 30min, the effects of different ethanol concentrations (30%, 45%, 60%, 75%, and 90%) on the total flavonoid content in the extract of small rib bone were investigated, and the optimal ethanol concentration was 75%. Figure 6 .

[0058] 4. Response surface analysis of total flavonoids

[0059] The optimal total flavonoids content was determined under the conditions of single-factor experiment. The four factors of solid-liquid ratio, temperature, time and ethanol concentration were taken as the main influencing factors, and the total flavonoids content was taken as the response value. The response surface test method was used to optimize the process. The Box-Behnken experimental design is shown in Table 1. Table 1 Experimental design of response surface analysis of total flavonoids with four factors and three levels

[0060] 5. Experimental Results

[0061] (1) Single factor investigation results: The optimal extraction method for total flavonoids from Rhizoma Cistanche deserticola was a solid-liquid ratio of 1:25, a temperature of 60°C, an extraction time of 40 min, and an ethanol concentration of 75%.

[0062] (2) Response surface test results of total flavonoids: Table 2 Experimental design and results of response surface optimization for total flavonoid content Table 2 Response surface optimization experimental design and results of total flavonoids content

[0063] (3) Establishment and analysis of total flavonoid content model:

[0064] Assuming that the material-liquid ratio, temperature, time and ethanol concentration are A, B, C and D respectively, and taking the total flavonoids content as the response value, a multivariate regression fitting is performed to obtain the quadratic polynomial regression equation:

[0065] Y 总黄酮含量 =2.47+0.065*A+0.21*B+0.06*C-0.051*D-0.038*AB-0.12*AC-0.098* AD+0.092*BC-0.25*BD-0.024*CD-0.41*A2-0.53*B 2 -0.31*C 2 -0.26*D 2 Table 3 Regression analysis results of total flavonoid content model and regression coefficient Table 3 Regression analysis results of total flavonoid content model and regression coefficient Note: P<0.01 is extremely significant, indicated by **; P<0.05 is significant, indicated by *; P>0.05 is not significant, indicated by ns

[0066] The difference in the model was extremely significant (P < 0.0001), and the coefficient of determination R 2 The error of the model is small, and the coefficient of determination RAdj is 0.9773, which indicates that the model has a good degree of fit and can fit the test results more intuitively. 2It is 0.9546, indicating that the model has good relevance and explanatory power, and can be used for theoretical analysis and prediction.

[0067] Analysis of the effects of various factors on the total flavonoid content shows that the primary term temperature has a very significant effect on the total flavonoid content (P < 0.01), while the material-liquid ratio, ethanol concentration, and time have significant effects on the total flavonoid content (P < 0.05). Among the four influencing factors, temperature has the greatest impact on the total flavonoid content, followed by the material-liquid ratio, and then time, while ethanol concentration has the least impact. 2 、C 2 、D 2 The effect on the total flavonoid content was extremely significant (P < 0.01), indicating that the four factors had a nonlinear effect on the total flavonoid content. The interaction terms BD and AC had an extremely significant effect on the total flavonoid content (P < 0.01), and AD and BC had a significant effect on the total flavonoid content (P < 0.05).

[0068] (4) Optimal process and verification test results

[0069] Taking the maximum total flavonoids content of small ribs as the optimization target, the experiment was optimized by Design-Expert 10.0.3 software, and the predicted total flavonoids content was 2.515 mg / g. The predicted values ​​of the four factors were solid-liquid ratio of 1:28.481, temperature of 62.69℃, time of 46.939 min, and ethanol concentration of 71.190%. In order to determine the accuracy of the model, the optimized parameters were used for verification experiments. For ease of operation, the condition parameters were set as solid-liquid ratio of 1:30, temperature of 60℃, time of 45 min, and ethanol concentration of 70%. The experiment was repeated 3 times under these conditions, and the average total flavonoids content was measured to be 2.487±0.132 mg / g, which was close to the model predicted value of 2.515 mg / g, indicating that the process parameters optimized by the model are reliable.

[0070] Example 2

[0071] 1. Materials and Instruments

[0072] Dried aerial parts of the Yao medicinal material Xiaobogu, anhydrous sodium carbonate, folin, anhydrous ethanol, and a gallic acid reference substance. Also included: a UV spectrophotometer, a refrigerated centrifuge, an electric constant-temperature water bath, an electric blast drying oven, a CNC ultrasonic cleaner, and an analytical balance.

[0073] 2. Experimental method for determination of total phenolic acid content

[0074] (1) Preparation of small bone medicinal material powder:

[0075] The method is the same as Example 1

[0076] (2) Preparation of Gallic Acid Reference Substance

[0077] Accurately weigh 10.20 mg of gallic acid, add distilled water, ultrasonically dissolve and dilute to a 10 mL volumetric flask, and shake well to obtain a gallic acid reference stock solution with a concentration of 1.02 mg / mL.

[0078] (3) Preparation of test solution

[0079] The method is the same as Example 1.

[0080] (4) Total phenolic acid color development and determination conditions

[0081] Accurately pipette 0.7 mL of the test solution into a centrifuge tube, add 0.5 mL of forlin solution and 1 mL of freshly prepared 20% NaCO3 solution, dilute to 10 mL with distilled water, react in a 70°C water bath for 30 min, let stand for 2 h, and measure the absorbance at 765 nm.

[0082] The total phenolic acid content was calculated according to the formula:

[0083] M=(c*v*n) / m

[0084] Where M is the phenolic acid content, mg / g; c is the concentration, mg / L; V is the volume, mL; n is the multiple; m is the mass, g.

[0085] (5) Drawing of standard curve

[0086] Accurately measure 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL, and 1.2 mL of 1.02 mg / mL gallic acid reference solution, respectively, and place them in 10 mL volumetric flasks. Measure the absorbance according to step (4). Use concentration as the abscissa and absorbance as the ordinate to construct the standard curve equation, and obtain the linear regression equation y = 0.0068x + 0.0172, R 2 =0.9999. The reference solution has a good linear relationship in the range of 20.4~122.4mg / L. Figure 2 .

[0087] 3. Single-factor experiment

[0088] (1) Effect of material-liquid ratio on content

[0089] The method is the same as that in Example 1, and the optimal solid-liquid ratio of total phenolic acid is 1:20. Figure 3 .

[0090] (2) Effect of temperature on content

[0091] The method was the same as in Example 1, and the optimal extraction temperature for total phenolic acids was 50°C. Figure 4 .

[0092] (3) Effect of time on content

[0093] The method is the same as that in Example 1, and the optimal extraction time is 40 min. Figure 5 .

[0094] (4) Effect of ethanol concentration on content

[0095] The method is the same as that in Example 1, and the optimal ethanol concentration is 75%. Figure 6 .

[0096] 4. Response surface experiments on total phenolic acids:

[0097] With the material-liquid ratio, temperature, time and ethanol concentration as the main influencing factors and the total phenolic acid content of small bone as the response value, the response surface experiment method was used to optimize the process. The Box-Behnken experimental design is shown in Table 4. Table 4 Experimental design of response surface analysis of total phenolic acid with four factors and three levels

[0098] 5. Experimental Results

[0099] (1) Single factor investigation results: The optimal extraction method for total phenolic acids of small bone is a solid-liquid ratio of 1:20, a temperature of 50℃, an extraction time of 40 minutes, and an ethanol concentration of 75%.

[0100] (2) Response surface test results of total phenolic acids: Table 5 Experimental design and results of response surface optimization for total phenolic acid content

[0101] (3) Model establishment and analysis of total phenolic acid content:

[0102] Assuming that the material-liquid ratio, temperature, time and ethanol concentration are A, B, C and D respectively, and taking the total phenolic acid content as the response value, a multivariate regression fitting is performed to obtain the quadratic polynomial regression equation:

[0103] Y 总酚酸含量 =10.25+0.33*A+0.22*B+0.31*C-1.23*D-0.063*AB-0.06*AC-0.34*AD+0.16*BC+0.5*BD+0.33*CD-0.88*A 2 -0.71*B 2 -0.73*C 2 -1.74*D 2 Table 6 Regression analysis results of total phenolic acid content model and regression coefficient Note: P < 0.01 is extremely significant, indicated by **, P < 0.05 is significant, indicated by *, and P > 0.05 is not significant, indicated by ns.

[0104] As shown in Table 6, the model showed a significant difference (P < 0.001), and the coefficient of determination R 2 The error of the model is small, and the coefficient of determination RAdj is 0.9812, which indicates that the model fits well and can fit the test results more intuitively. 2 It is 0.9624, indicating that the model has good relevance and explanatory power, and can be used for theoretical analysis and prediction.

[0105] The size of the F value is an important indicator for evaluating the degree of influence of each variable on the response value. The larger the F value, the higher the contribution of the relevant model component to the response. When the significance test probability P<0.05, it reveals that the variable has a significant effect on the response value and has mathematical and statistical significance. Analysis of the influence of each factor on the total phenolic acid content shows that the first-order terms temperature, material-liquid ratio, time, and ethanol concentration have extremely significant effects on the total phenolic acid content (P<0.01). Among the four influencing factors, the degree of influence on the total phenolic acid content is D>A>C>B, that is, ethanol concentration>material-liquid ratio>time>temperature, and the second-order terms A>B>C>B ... 2 、B 2 、C 2 、D 2 The effect on total phenolic acid content was extremely significant (P < 0.01), indicating that these four factors had a nonlinear effect on total phenolic acid content. The interaction term BD had an extremely significant effect on total phenolic acid content (P < 0.01), and AD and CD had a significant effect on total phenolic acid content (P < 0.05).

[0106] (4) Optimal process and verification test results

[0107] With the maximum total phenolic acid content as the optimization target, the experiment was optimized using Design-Expert 10.0.3 software, resulting in a predicted total phenolic acid content of 10.539 mg / g. The predicted values ​​for the four factors were: a solid-liquid ratio of 1:21.268, a temperature of 55.470°C, a time of 42.505 min, and an ethanol concentration of 69.549%. To confirm the accuracy of the model, a validation experiment was conducted using the optimized parameters. For ease of operation, the parameters were set to a solid-liquid ratio of 1:20, a temperature of 55°C, a time of 40 min, and an ethanol concentration of 70%. The experiment was repeated three times under these conditions, and the average total phenolic acid content was 10.625±0.285 mg / g, which was within 5% of the model-predicted value of 10.539 mg / g, demonstrating the reliability of the process parameters optimized by the model.

[0108] (5) Determination results of small bone content from different origins Table 7 Determination results of Yao medicinal material small bone samples from different origins Table 7 Determination results of Yao medicinal material small bone samples from different origins Table 7 Determination results of Yao medicinal material small bone samples from different origins

[0109] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any manner. Persons skilled in the art may make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Therefore, to the extent such modifications and variations fall within the scope of the claims and their equivalents, the present invention is intended to encompass such modifications and variations.

Claims

1. A method for optimizing the extraction of total flavonoids and total phenolic acids from the Yao medicinal material Xiaobogu based on response surface methodology, characterized in that: The following steps are involved: (1) Grind the above-ground part of the Yao medicinal material Xiaobogu into powder to obtain Xiaobogu medicinal material powder. (2) Weigh 2 g of the Yao medicinal material powder of the small bone bone and place it in a 100 mL conical flask. Add different concentrations of ethanol at different material-liquid ratios and obtain the crude extract of the small bone bone at different extraction temperatures and times. (3) Four factors, namely, solid-liquid ratio, extraction temperature, extraction time and ethanol concentration, were selected for single-factor experiments. Response surface design was used to perform multiple regression fitting on each factor, with total flavonoids and total phenolic acid content as response values. The quadratic polynomial regression equation was obtained: Y 总黄酮含量 =2.47+0.065*A+0.21*B+0.06*C-0.051*D-0.038*AB-0.12*AC-0.098*AD+0.092*BC-0.25*BD-0.024*CD-0.41*A2-0.53*B 2 -0.31*C 2 -0.26*D 2 Y 总酚酸含量 =10.25+0.33*A+0.22*B+0.31*C-1.23*D-0.063*AB-0.06*AC-0.34*AD+0.16*BC+0.5*BD+0.33*CD-0.88*A 2 -0.71*B 2 -0.73*C 2 -1.74*D 2 Wherein, Y-total flavonoids and total phenolic acid content, A-solid-liquid ratio, B-extraction temperature, C-extraction time, D-ethanol concentration, and the optimal extraction process conditions of total flavonoids and total phenolic acids from the small bone were calculated by the above regression equation.

2. The response surface optimization method for extracting total flavonoids and total phenolic acids from Ossobuco serrata according to claim 1, characterized in that: In step (3), the extraction range of the single factor experiment is: solid-liquid ratio (1:15 to 1:40 g / mL), extraction temperature (30 to 70° C.), extraction time (10 to 60 min), and ethanol concentration (30 to 90%). The response surface optimization method for extracting total flavonoids and total phenolic acids from Ossobuco serrata according to claim 1 or 2, characterized in that: Through response surface design experimental method optimization, the optimal extraction process of total flavonoids from Bone Marrow was obtained: solid-liquid ratio 1:30, extraction temperature 60℃, extraction time 45min, ethanol concentration 70%; the optimal extraction process of total phenolic acid was: solid-liquid ratio 1:20, extraction temperature 55℃, extraction time 40min, ethanol concentration 70%.

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