Optimization method of extraction process of total polyphenol in non-medicinal part of golden cypress

Abstract

The invention discloses a method for optimizing a process for extracting total polyphenols from non-medicinal parts of golden cypress by utilizing a response surface. The extraction method specifically comprises the following steps: 1) pretreatment: crushing leaves of non-medicinal parts of golden cypress, and sieving to obtain golden cypress leaf powder; 2) single factor experiment: performing ultrasonic extraction on the powder sample; 3) optimization: optimizing extraction conditions by adopting a response surface method; (4) centrifuging: centrifuging the extracted extracting solution to obtain supernate; and 5) detection: carrying out antioxidant capacity detection on the extracting solution. The method for extracting the amur corktree leaf total polyphenol obtained through response surface method optimization is stable and feasible, the technological operation is simple and easy to implement, the repeatability is good, the extraction efficiency is high, the yield of the extracted polyphenol is high, the amur corktree leaf total polyphenol has a good anti-oxidation effect, and a new thought is provided for utilization of non-medicinal waste parts of plants.

Description

Technical Field

[0001] The invention belongs to the technical field of total polyphenol extraction from non-medicinal parts of plants, and particularly relates to a method for extracting total polyphenol from phellodendron amurense leaves optimized by response surface methodology. Background Art

[0002] The non-medicinal parts of traditional Chinese medicine refer to the tissue structures other than the parts used as medicine in traditional Chinese herbal medicine. For example, the dried bark of Phellodendron chinense is usually used as medicine, and the leaves are the non-medicinal parts. Every year in China, a large number of non-medicinal parts are treated as waste and cannot be used scientifically and rationally; Cork ( Phellodendron amurense Rupr.) is a deciduous tree of the genus Phellodendron in the Rutaceae family. It is also known as yellow pineapple, phellodendron, Guanhuangbo, etc. The inner layer of the bark is processed and used as medicine, called phellodendron. It tastes bitter and is cold in nature. Phellodendron has traditional effects such as clearing away heat and detoxifying, purging fire and drying dampness. It also has modern pharmacological effects such as treating acute bacterial dysentery, acute enteritis, acute icteric hepatitis, and urinary tract infections. "Populus" was already recorded in "Shennong's Herbal Classic". In addition to the official name "Populus", the herbals in the Tang and Song dynasties also recorded the additional name "Tanhuan" and the alias "Phellodendron". Phellodendron is distributed in the provinces of Northeast China and North China, and is also found in Central Asia and Eastern Europe. At present, due to the over-exploitation of mountainous areas, destruction of natural vegetation, high degree of reclamation, and low forest vegetation coverage, the community structure of wild Phellodendron amurense has been seriously disturbed by human activities, resulting in a shortage of wild Phellodendron amurense resources. At the same time, there are few studies on the non-medicinal part of Phellodendron amurense, namely Phellodendron amurense leaves. Therefore, we studied the non-medicinal part resources of Phellodendron amurense leaves, which is of great significance for the development of abandoned resources of Phellodendron amurense. There are few reports on the extraction process of Phellodendron amurense leaf compounds, and there are no relevant reports on the optimization of the extraction process of total polyphenols from Phellodendron amurense leaves and their antioxidant activity by response surface methodology. At present, the main methods for extracting polyphenols are: organic solvent extraction, ultrasonic extraction, microwave extraction, etc., but the extraction rate is low, and sometimes accompanied by the decomposition of the substance, affecting the quality of the extract. Response surface methodology can find a clear function expression between factors and response values ​​within a given range using less experimental data, thereby obtaining the best factor combination and the optimal response value. Therefore, it has obvious advantages over other optimization methods. In addition, the ultrasonic extraction method has low equipment cost, simple operation process, short extraction time, and high extraction rate of polyphenol compounds. With the rapid development of the traditional Chinese medicine industry in recent years, it is necessary to study the relationship between the development and sustainable use of medicinal plant resources. At present, the excessive mining of wild Chinese medicinal resources has affected the development of the pharmaceutical industry. If Phellodendron chinense leaves can be used as a new medicinal part, the waste of resources will be greatly reduced, and it will have important significance for drug development and sustainable development. The invention aims to research and develop the non-medicinal parts of Phellodendron chinense. Summary of the invention

[0003] The purpose of the present invention is to provide a response surface method for optimizing the extraction of waste resources from non-medicinal parts of Phellodendron amurense. Based on a single factor experiment, a four-factor three-level experiment was designed according to the central composite experimental design principle of Box-Behnken, and multiple regression analysis was performed using the software Design expert to determine the optimal ultrasonic extraction process for total polyphenols from Phellodendron amurense leaves; the content of total polyphenols in the Phellodendron amurense leaf extract before and after optimization was determined by ultraviolet spectrophotometry. The method for extracting total polyphenols from Phellodendron amurense leaves is stable, simple to operate, has good repeatability, and a high yield of total polyphenols, which lays a foundation for the industrial extraction of total polyphenols from Phellodendron amurense leaves. In addition, total polyphenols from Phellodendron amurense leaves have good antioxidant effects, which also provides a new method for the development and utilization of waste resources from Phellodendron amurense leaves.

[0004] To achieve the above object, the present invention adopts the following technical solution: A method for extracting total polyphenols from Phellodendron amurense leaves optimized by response surface methodology is disclosed. According to the Box-Behnken experimental design principle, the process conditions of ultrasonic time, solid-liquid ratio, ethanol concentration and ultrasonic power for extracting total polyphenols from Phellodendron amurense leaves are designed and optimized using Design Expert software. The extraction method is as follows: weigh an appropriate amount of Phellodendron amurense leaves, rinse them with pure water, dry them in a 50°C oven for 10-15 min, crush them, sieve them with a 40-mesh sieve, weigh 3 g of Phellodendron amurense leaf powder, and then add ethanol for ultrasonic extraction. The extraction process conditions are as follows: ultrasonic time (30, 60, 90, 120, 150 min), solid-liquid ratio (1:10, 1:20, 1:30, 1:40, 1:50 w / v), ethanol concentration (40%, 50%, 60%, 70%, 80%) and ultrasonic power (100, 150, 200, 250, 300 W).

[0005] The present invention utilizes response surface methodology and the Box-Behnken experimental design principle and utilizes Design Expert software to design and optimize the process conditions of ultrasonic time, solid-liquid ratio, ethanol concentration and ultrasonic power for extracting total polyphenols from Phellodendron amurense leaves. The optimal extraction process conditions for extracting total polyphenols from Phellodendron amurense leaves are: ultrasonic time (90 min), solid-liquid ratio (1:30 w / v), ethanol concentration (60%) and ultrasonic power (200 W).

[0006] The specific steps of the response surface methodology include: ① Grind the dried Phellodendron amurense leaves, pass through a 40-mesh sieve, and obtain Phellodendron amurense leaf powder for later use; ② Single factor experiment: Four variables were tested at three levels of four factors. One factor was changed in each experiment to determine the initial range of factors affecting the extraction rate. The specific extraction conditions are as follows. The effects of four factors, including ultrasonic time (30, 60, 90, 120, 150 min), solid-liquid ratio (1:10, 1:20, 1:30, 1:40, 1:50 w / v), ethanol concentration (40%, 50%, 60%, 70%, 80%) and ultrasonic power (100, 150, 200, 250, 300 W) on total polyphenols in Phellodendron amurense leaves were studied;

[0007] ③Response surface methodology optimization: Taking the total polyphenol extraction rate as an indicator, the Box-Behnken experimental design was carried out on the four factors affecting the total polyphenol extraction rate of Phellodendron amurense leaves, namely, ultrasonic time, solid-liquid ratio, ethanol concentration and ultrasonic power, through the experimental results of step ①. The Box-Behnken experiment was optimized by the response surface method with four factors and three levels. The equation is: the response value Y of the total polyphenol extraction rate of Phellodendron amurense leaves and the ultrasonic time A, solid-liquid ratio B, ethanol concentration C and ultrasonic power D. The quadratic polynomial regression equation of the four investigated factors is: Y=+18.88+0.1342×A+1.1175×B+0.6042×C-0.0642×D+0.905×AB+1.195×AC-0.5375×AD-0.37×BC-1.0475×BD-0.0425×CD-0.3892×A 2 -2.5667×B 2 -0.1142×C 2 -0.3592×D 2 ; ④The optimal extraction process conditions for total polyphenols from Phellodendron amurense leaves were obtained by solving the regression equation using Design-Expert software.

[0008] The above-mentioned method for extracting total polyphenols from Phellodendron amurense leaves optimized by response surface methodology, according to the Box-Behnken experimental design principle, uses Design Expert software to design and optimize the ultrasonic time, solid-liquid ratio, ethanol concentration and ultrasonic power process conditions for extracting total polyphenols from Phellodendron amurense leaves. The extraction method is as follows: the dried Phellodendron amurense leaves are crushed and passed through a 40-mesh sieve to obtain Phellodendron amurense leaf powder, and then ethanol is added for ultrasonic extraction. The extraction process conditions are: ultrasonic time 30 min, solid-liquid ratio 1:25 g / mL, ethanol concentration 40%, and ultrasonic power 200 W.

[0009] Another object of the present invention is to provide the method for extracting total polyphenols from Phellodendron amurense leaves optimized by the response surface method, and to use the total polyphenols from Phellodendron amurense leaves extracted by the method for extracting total polyphenols from Phellodendron amurense leaves in the preparation of antioxidant drugs and food antioxidants, wherein the antioxidant drugs and food antioxidants have the ability to scavenge free radicals. The present invention has the following advantages: (1) The process of the present invention is simple and easy to operate, and the selected raw material is waste Phellodendron amurense leaves, which develops the utilization value of waste resources Phellodendron amurense leaves; (2) The present invention optimizes the extraction process of total polyphenols from Phellodendron amurense leaves by using the response surface method, takes the total polyphenol extraction rate as an indicator, investigates the four factors of ultrasonic time, solid-liquid ratio, ethanol concentration and ultrasonic power, and optimizes the experiment by using the Box-Behnken response surface method with four factors and three levels, thereby obtaining a method for extracting total polyphenols from Phellodendron amurense leaves, which overcomes the disadvantage of low precision of orthogonal experiments and ensures the scientificity and stability of the extraction process. (3) Compared with the existing extraction method of polyphenols, the amount of organic solvent used in the extraction method of the present invention is greatly reduced, and the extraction agent has the advantages of being environmentally friendly and having low extraction cost. At the same time, the operating conditions are mild, and the extraction of low-content polyphenols is suitable. The present invention provides a reference for the evaluation of the intrinsic quality of Phellodendron chinense leaves. (4) The present invention also determines the antioxidant activity of the extracted total polyphenols of Phellodendron chinense leaves, and the results show that the total polyphenols of Phellodendron chinense leaves have good antioxidant effects. The method for extracting total polyphenols of Phellodendron chinense leaves optimized by response surface methodology provided by the present invention and its application lay a foundation for the industrial extraction and clinical development and utilization of total polyphenols of Phellodendron chinense leaves. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Gallic acid absorbance standard curve; Figure 2 Effect of different ultrasonic time on extraction rate; Figure 3 Effect of different solid-liquid ratios on extraction efficiency; Figure 4 Effect of different ethanol concentrations on extraction yield; Figure 5 Effect of different ultrasonic powers on extraction rate; Figure 6 The interactive response surface diagram and interactive contour diagram of ultrasonic power and solid-liquid ratio on the total polyphenol content of Phellodendron amurense leaves; Figure 7 The interactive response surface diagram and interactive contour diagram of ultrasound power and ultrasound time on the total polyphenol content of Phellodendron amurense leaves; Figure 8 The interactive response surface diagram and interactive contour diagram of ultrasound power and ethanol concentration on the total polyphenol content of Phellodendron amurense leaves; Fig. 9 The interactive response surface diagram and interactive contour diagram of ethanol concentration and ultrasound time on the total polyphenol content of Phellodendron amurense leaves; Fig.10Response surface diagram and interaction contour diagram of the interaction between solid-liquid ratio and ethanol concentration on the total polyphenol content of Phellodendron amurense leaves; Fig.11 Response surface diagram and interaction contour diagram of the interaction between solid-liquid ratio and ultrasonic time on the total polyphenol content of Phellodendron amurense leaves; Fig.12 DPPH free radical scavenging ability; Fig.13 ABTS free radical scavenging ability; Fig.14 OH radical scavenging ability; Fig.15 Total antioxidant capacity of Phellodendron amurense leaves. Specific implementation method; The following is a method for optimizing the ultrasonic extraction process of total polyphenols from Phellodendron amurense leaves based on a response surface, and the steps are as follows: (1) Pretreatment: Weigh an appropriate amount of Phellodendron amurense leaves, rinse them with pure water, dry them in a 50°C oven for 10-15 min, crush them, sieve them with a 40-mesh sieve, and weigh 3 g of Phellodendron amurense leaf powder; (2) Single factor experiment: The powder sample was subjected to ultrasonic extraction under the extraction conditions of 30-150 min ultrasonic time, 1:10-1:50 w / v solid-liquid ratio, 40%-80% ethanol solution, and 100-300 W ultrasonic power, to determine the optimal levels of ultrasonic time, solid-liquid ratio, ethanol concentration, and ultrasonic power; (3) Optimization: Two levels were selected symmetrically on both sides of the optimal level, and a four-factor three-level experimental design was performed according to the Box-Behnken design principle. Ultrasonic time, solid-liquid ratio, ethanol concentration, and ultrasonic power were used as independent variables, and total polyphenol content was used as the response value. The multivariate quadratic equation for total polyphenol content was established as follows: Y = 18.88 + 0.1342 × A + 1.1175 × B + 0.6042 × C - 0.0642 × D + 0.905 × AB + 1.195 × AC - 0.5375 × AD - 0.37 × BC - 1.0475 × BD - 0.0425 × CD - 0.3892 × A 2 -2.5667×B 2 -0.1142×C 2 -0.3592×D 2The response value in the formula is the total polyphenol content of Phellodendron amurense leaves, the variable parameter A is the ultrasonic time, the variable parameter B is the solid-liquid ratio, the variable parameter C is the ethanol concentration, and the variable parameter D is the ultrasonic power. The optimal extraction process was finally determined as follows: the ultrasonic time was 80 min, the solid-liquid ratio was 1:34 g / mL, the ethanol concentration was 60%, the ultrasonic power was 173 W, and the total polyphenol content of Phellodendron amurense leaves was 25.22 mg / mL. According to the variable parameters, the Phellodendron amurense leaves pretreated in step (1) were weighed, and an ethanol solution was added to the powder, with an ethanol concentration of 40%, a solid-liquid ratio of 1:25 w / v, and an ultrasonic power of 200 W. The powder was placed in an ultrasonic cleaner, and the ultrasonic time was 30 min. The total polyphenols were extracted by ultrasonication, and then extracted again with an ethanol solution of the same concentration. The filtrates were combined, and a yellow viscous substance was obtained after the ethanol evaporated. The yellow viscous substance was transferred to a 25 mL volumetric flask with an ethanol solution of the same concentration.

[0012] Determination of total polyphenol content: Standard curve drawing: Use 70% ethanol solution to dilute 1 mg / mL gallic acid solution into 5 standard solutions with concentration gradients of 0.02 mg / mL, 0.04 mg / mL, 0.06 mg / mL, 0.08 mg / mL, and 0.1 mg / mL, take 20 μL of each gradient standard solution and put it into a 2 mL centrifuge tube, then add 10 μL of 1 mol / L Folin phenol reagent to each tube and mix well; after placing in the dark for 5 min, add 40 μL of 20% Na2CO3 solution and 430 μL of distilled water, mix well and place at room temperature in the dark for 1 h to prepare the standard curve test solution. Take 200 μL of each gradient standard curve test solution to the standard curve detection well of the 96-well plate, detect the absorbance at a wavelength of 760 nm, repeat each experiment three times to draw the standard curve, the standard curve diagram is shown in the figure. Figure 1 The standard curve equation is: y = 0.03800 + 101.00000*x ( r 2= 0.99883). The present invention adopts an ultrasonic extraction method, and establishes a mathematical model of the interaction between the four influencing factors of ultrasonic time, liquid-to-solid ratio, ethanol concentration, and ultrasonic power and the total polyphenol content response value through the Box-Behnken design principle on the basis of a single factor experiment. The specific operation process is: in the invention, a single factor experiment is used to change one factor in each experiment among the four factors to determine the initial range of the factors affecting the extraction rate. The specific extraction conditions are as follows: The influence of the four factors on the total polyphenols of Phellodendron chinense leaves was studied. The effects of ultrasonic time (30, 60, 90, 120, 150 min), liquid-to-solid ratio (1:10, 1:20, 1:30, 1:40, 1:50 w / v), ethanol concentration (40%, 50%, 60%, 70%, 80%) and ultrasonic power (100, 150, 200, 250, 300 W) were studied.

[0013] Example 1 Effect of ultrasonic time on total polyphenol content of Phellodendron amurense leaves: Weigh 3 g of Phellodendron amurense leaf powder pretreated in step (1) and use ultrasonic time of 30, 60, 90, 120, and 150 min as experimental points to investigate the effect of ultrasonic time on the total polyphenol content of Phellodendron amurense leaves. The following results are obtained: Figure 1 The graph showing the extraction rate of total polyphenols from Phellodendron amurense leaves changing with ultrasound time;

[0014] Depend on Figure 2 It can be seen that, under the condition that other conditions remain unchanged, with the increase of ultrasonic time, the total polyphenol content first increases and then decreases. The total polyphenol content is the highest when the ultrasonic time is 90 min. When the ultrasonic time is greater than 90 min, the total polyphenol content decreases with the increase of ultrasonic time. This is because the ultrasonic time will affect the contact between the extraction solvent and the active substances in the cells. The longer the ultrasonic time, the more fully the solvent can penetrate into the cells, and then contact and dissolve with the active substances, thereby increasing the polyphenol content; while too long an ultrasonic time will cause the temperature in the cells to be too high, causing oxidation of the extract, thereby reducing the extraction rate of total polyphenols. Therefore, from the perspective of the total polyphenol extraction rate, it is appropriate to select an ultrasonic time of 90 min. Example 2 Effect of the solid-liquid ratio on the total polyphenol content of Phellodendron chinense leaves: Weigh 3 g of Phellodendron amurense leaf powder pretreated in step (1) and use the solid-liquid ratio of 1:10, 1:20, 1:30, 1:40, and 1:50 w / v as experimental points to investigate the effect of different solid-liquid ratios on the total polyphenol content of Phellodendron amurense leaves, and draw a graph as shown in the figure. Figure 2 The graph showing the total polyphenol content of Phellodendron amurense leaves as a function of the solid-liquid ratio;

[0015] Depend on Figure 3It can be seen that, with other conditions unchanged, as the ratio of solid to liquid increases, the extraction rate of total polyphenols increases. When the ratio of solid to liquid is 1:30 w / v, the content of total polyphenols is the highest, and then the increase gradually decreases. This may be because the increase in the ratio of solid to liquid will increase the contact area between the material and the solvent, allowing polyphenols to be dissolved more fully, but the total polyphenol extraction rate will no longer increase when the ratio of solid to liquid is greater than 1:30 w / v. Therefore, from the perspective of polyphenol content, it is appropriate to select a ratio of 1:30 w / v.

[0016] Example 3 Effect of ethanol concentration on total polyphenol content of Phellodendron amurense leaves: Weigh 3 g of Phellodendron amurense leaf powder pretreated in step (1) and use 40%, 50%, 60%, 70%, and 80% ethanol concentrations as experimental points to investigate the effect of ethanol concentration on the total polyphenol content of Phellodendron amurense leaves, and draw a graph as shown in the figure. Figure 3 The graph showing the total polyphenol content of Phellodendron amurense leaves following changes in ethanol concentration;

[0017] Depend on Figure 4 It can be seen that, under the condition that other conditions remain unchanged, with the increase of ethanol concentration, the total polyphenol content first increases and then decreases. The total polyphenol content is the highest when the ethanol concentration is 60%, and the total polyphenol content decreases with the increase of ethanol concentration when the ethanol concentration is greater than 60%. It may be because the ethanol concentration is related to the polarity of the total polyphenols in Phellodendron chinense leaves. When the polarities of the two are close, it is conducive to the dissolution of total polyphenols in the solvent, so that the total polyphenol content increases. However, when the ethanol concentration is too high, the polarity of the solvent is too small, which makes the total polyphenol content decrease. Therefore, the ethanol concentration of 60% is the most suitable.

[0018] Example 4 Effect of Ultrasonic Power on Polyphenol Content in Phellodendron amurense Leaves: Weigh 3 g of the Phellodendron amurense leaf powder pretreated in step (1) and use ultrasonic powers of 100, 150, 200, 250, and 300 W as experimental points to investigate the effect of different ultrasonic powers on the total polyphenol content of Phellodendron amurense leaves, and draw a graph as shown in Figure 2. Figure 4 The graph showing the total polyphenol content of Phellodendron amurense leaves following changes in ultrasound power;

[0019] Depend on Figure 5 It can be seen that, with other conditions unchanged, as the ultrasonic power increases, the total polyphenol content of Phellodendron chinense leaves shows a trend of first increasing and then decreasing. The total polyphenol content is highest when the ultrasonic power is 200 W, and then the total polyphenol content gradually decreases. This may be because the increase in ultrasonic power causes the increase in total polyphenol content, but when the ultrasonic power exceeds a certain threshold, excessive molecular vibration and thermal effects may cause enzyme molecule destruction, thereby affecting the total polyphenol content. Therefore, the ultrasonic power of 200 W is selected as the most appropriate.

[0020] Response surface design and analysis of variance The Box-Behnken test design was conducted based on the single factor test results to investigate the four factors affecting the extraction rate of total polyphenols from Phellodendron amurense leaves: ultrasonic time (A), solid-liquid ratio (B), ethanol concentration (C) and ultrasonic power (D).

[0021] The optimal levels of ultrasonic time (A), material-liquid ratio (B), ethanol concentration (C) and ultrasonic power (D) were determined according to the results of the single factor test, and two levels were selected symmetrically on both sides of the optimal level. The specific factor level settings are shown in Table 1. Table 1 Factor level setting table level Ultrasonic time (min) Material-liquid ratio (w / v) Ethanol concentration (%) Ultrasonic power (W) -1 60 20 50 150 0 90 30 60 200 1 120 40 70 250 The four-factor three-level experimental design was carried out using Design expert software according to the Box-Behnken design principle. The response surface design scheme and response value results are shown in Table 2. The variance analysis of the regression equation is shown in Table 3;

[0022] Table 2 Response surface design scheme and response values Experiment No. Ultrasonication time (min) Material-liquid ratio (w / v) Ethanol concentration (%) Ultrasonic power (W) Extraction rate (%) 1 60 20 60 200 7.73 2 120 30 70 200 9.04 3 120 30 50 200 10.57 4 90 40 50 200 14.58 5 90 40 60 250 11.31 6 60 30 60 150 17.31 7 90 20 50 200 8.23 8 90 30 50 150 10.28 9 90 30 70 150 18.76 10 60 30 60 250 9.47 11 90 20 60 150 9.68 12 120 40 60 200 10.37 13 120 30 60 150 13.28 14 60 30 70 200 10.34 15 90 30 60 200 22.64 16 90 30 70 250 8.86 17 90 30 60 200 24.32 18 90 30 60 200 24.64 19 90 20 70 200 11.54 20 90 30 50 250 13.04 21 120 30 60 150 12.86 22 90 20 60 150 11.18 23 90 40 60 150 23.92 24 90 30 60 200 24.43 25 60 30 50 200 12.42 26 60 40 60 200 17.54 27 90 30 60 200 24.87 28 90 40 70 200 12.86 29 120 20 60 200 9.23 Response Surface Analysis and Optimization The experimental data were analyzed by multiple regression using the statistical software Design expert to obtain the regression equation of the influence of experimental factors on the response value. Y = 18.88 + 0.1342 × A + 1.1175 × B + 0.6042 × C - 0.0642 × D + 0.905 × AB + 1.195 × AC - 0.5375 × AD - 0.37 × BC - 1.0475 × BD - 0.0425 × CD - 0.3892 × A 2 -2.5667×B 2 -0.1142×C 2 -0.3592×D 2 The absolute values ​​of the coefficients in the equation reflect the influence of each factor on the response value. The results of the response surface variance analysis of the experimental data are shown in Table 3.

[0023] Sources of variance sum of squares Degrees of Freedom Mean Square Model 909.65 14 64.98 47.69 <0.0001 A 16.73 1 16.73 12.28 0.0035 B 128.81 1 128.81 94.55 <0.0001 C 33.77 1 33.77 24.79 0.0002 D 55.39 1 55.39 40.66 <0.0001 AB 18.79 1 18.79 13.79 0.0023 AC 0.0756 1 0.0756 0.0555 0.8171 AD 5.4 1 5.4 3.96 0.0665 BC 6.33 1 6.33 4.64 0.0491 BD 44.08 1 44.08 32.36 <0.0001 CD 40.07 1 40.07 29.41 <0.0001 A² 303.92 1 303.92 223.09 <0.0001 B² 179.84 1 179.84 132.01 <0.0001 C² 284.87 1 284.87 209.11 <0.0001 D² 89.25 1 89.25 65.51 <0.0001 Residual 19.07 14 1.36 Loss of orientation 14.72 8 1.84 2.53 0.1367 Pure Error 4.35 6 0.7258 Total error 928.72 28 Note: A is the ultrasonic time; B is the material-liquid ratio; C is the ethanol concentration; D is the ultrasonic power R² =0.9795, R Adj ² =0.9589 CV%=8.15%( P <0.01) indicates an extremely significant level; P <0.05) indicates a significant level It can be seen from Table 3 that P = 0.0001 < 0.001 indicates that the quadratic regression equation model is extremely significant and the lack of fit termP =0.1367 is not significant, indicating that the equation fits the experiment well, and the model can be used to replace the real experimental points to predict and analyze the results of the experiment on the total polyphenol content of Phellodendron amurense leaves under different reaction conditions. The variance analysis results of the regression model equation also show that the effects of ultrasonic time, solid-liquid ratio, ethanol concentration and ultrasonic power on the total polyphenol content are extremely significant, and the effects of the quadratic terms are also significant, indicating that the equation fits well;

[0024] The effect of the interaction among the four factors on the total polyphenol extraction rate was analyzed by response surface analysis. Figure 6-11 . The response surface and contour plots can be used to analyze the effects of the interactions between the four process factors of ultrasonic time (A), solid-liquid ratio (B), ethanol concentration (C) and ultrasonic power (D) on the total polyphenol extraction rate. When the slope of the response surface of the interaction between the two factors under investigation is steeper, it indicates that the response value is very sensitive to changes in the interaction, and the interaction has a significant effect on the response value. On the contrary, if the slope of the response surface is relatively gentle, it indicates that the response value has little effect on changes in the interaction, and the interaction has no significant effect on the response value.

[0025] In order to intuitively analyze the results, the Design Expert software was used to draw a response surface analysis diagram, such as Figure 6-11 .according to Figure 6 It can be seen that the total polyphenol extraction rate of Phellodendron amurense leaves has an obvious quadratic parabola relationship with the ultrasonic power and the solid-liquid ratio, and there is an obvious interaction between the two. The polyphenol yield reaches its maximum value when the ultrasonic power is 180-200 W and the solid-liquid ratio is between 30-40 g / mL. Figure 7 It can be seen that the total polyphenol yield of Phellodendron amurense leaves changes with the increase of ultrasonic power and ultrasonic time. The maximum value of total polyphenol yield of Phellodendron amurense leaves is reached when the ultrasonic power is 180-200 W and the ultrasonic time is 80-100 min. Figure 8 It can be seen that the total polyphenol extraction rate of Phellodendron amurense leaves has a quadratic parabola relationship with ethanol concentration and ultrasonic power, and the interaction between ethanol concentration and solid-liquid ratio is obvious. According to the contour map, the total polyphenol extraction rate is the highest when the ethanol concentration is 60-70% and the ultrasonic power is 180-200 W. Fig. 9 It can be seen that the interaction between ethanol concentration and ultrasonic time is not obvious, the response surface is flat, and the total polyphenol yield is at its maximum when the ethanol concentration is 60-70% and the ultrasonic time is 80-100 min. Fig.10 It can be seen that the interaction between ethanol concentration and solid-liquid ratio is obvious, the response surface is steep, and the maximum value appears when the ethanol concentration is 60-70% and the solid-liquid ratio is between 30-40 g / mL.

[0026] Antioxidant activity of total polyphenols from Phellodendron amurense leaves DPPH free radical scavenging ability test The DPPH free radical scavenging activity of the total polyphenol extract of Phellodendron amurense leaves was determined, and the scavenging rate was calculated using VC of the same concentration as a positive control. Fig.12 ;

[0027] When the concentration is in the range of 0.4-100 mg / mL, the ability to scavenge DPPH increases with the increase of concentration, showing an obvious dose-effect linear relationship. When the concentration reaches 100 mg / mL, the scavenging rate of Phellodendron amurense leaf total polyphenols on DPPH free radicals is close to VC. Therefore, Phellodendron amurense leaf total polyphenols have a certain scavenging effect on DPPH free radicals, indicating that Phellodendron amurense leaf total polyphenols have strong antioxidant activity.

[0028] ABTS free radical scavenging ability test The ABTS free radical scavenging activity of the total polyphenol extract of Phellodendron amurense leaves was determined, and the scavenging rate was calculated using VC of the same concentration as a positive control. Fig.13 ;

[0029] When the concentration is in the range of 0.4-100 mg / mL, the ability to scavenge ABTS free radicals increases with the increase of concentration, showing an obvious dose-effect linear relationship. When the concentration reaches 100 mg / mL, the scavenging rate of Phellodendron amurense leaf total polyphenols on ABTS free radicals is infinitely close to VC. Therefore, Phellodendron amurense leaf total polyphenols have a strong scavenging effect on ABTS free radicals, indicating that Phellodendron amurense leaf total polyphenols have strong antioxidant activity.

[0030] OH free radical scavenging ability test The OH free radical scavenging activity of the total polyphenol extract of Phellodendron amurense leaves was determined, and the scavenging rate was calculated using VC of the same concentration as a positive control. Fig.14 ;

[0031] When the concentration is in the range of 0.4-100 mg / mL, the ability to remove OH radicals increases slowly with the increase of concentration. When the concentration reaches 100 mg / mL, the removal of OH radicals by total polyphenols from Phellodendron amurense leaves reaches 80%. Therefore, total polyphenols from Phellodendron amurense leaves have a certain scavenging effect on OH radicals, indicating that total polyphenols from Phellodendron amurense leaves have antioxidant activity.

[0032] Total antioxidant capacity test The total antioxidant capacity of the total polyphenol extract of Phellodendron amurense leaves was determined, and VC of the same concentration was used as a positive control. Fig.15 When the concentration was in the range of 0.4-100 mg / mL, the total antioxidant capacity increased slowly with the increase of concentration, indicating that total polyphenols in Phellodendron amurense leaves had antioxidant activity.

Claims

1. A method for optimizing the extraction process of total polyphenols from non-medicinal parts of Phellodendron chinense, characterized in that The invention provides a new idea for the development and utilization of the non-medicinal parts of Phellodendron chinense leaves, and the method is stable and feasible, the process operation is simple and easy, the repeatability is good, the extraction efficiency is high, and the polyphenols obtained by extraction have a high yield, and the steps are as follows: Step 1) Pretreatment of Phellodendron amurense leaves: Phellodendron amurense leaf samples were collected from the Phellodendron amurense artificial cultivation base of Dongping Seedling Planting Professional Cooperative in Jingyu County, Jilin Province using a five-point sampling method to ensure uniformity; appropriate amount of Phellodendron amurense leaves were weighed, rinsed, dried in an oven, crushed, sieved, and 3 g of Phellodendron amurense leaf powder was weighed; Step 2) Single factor experiment: Extraction was performed using a KQ5200DE (40 KHz) numerically controlled ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., Kunshan, Jiangsu); the initial conditions were ultrasonic time 30 min, solid-liquid ratio 1:25 g / mL, ethanol concentration 40%, and ultrasonic power 200 W. Then, only one extraction condition was changed in the experiment, and the other extraction conditions were kept constant to obtain the single factor experimental results; Step 3) Optimization: Optimizing the extraction conditions by using response surface methodology to determine the optimal extraction process parameters for total polyphenols from Phellodendron amurense leaves, and then ultrasonically extracting according to the optimal extraction process parameters; Step 4) Centrifugation: centrifuging the extracted solution to obtain a supernatant; Step 5) Detection: The extract is subjected to an antioxidant capacity detection.

2. The method according to claim 1, characterized in that In step (1), the cleaning is: cleaning with pure water.

3. The method according to claim 1, characterized in that In step (1), the drying is performed at 50°C for 10-15 minutes.

4. The method according to claim 1, characterized in that In step (1), the crushing and screening is: crushing by a crusher through a 40-mesh sieve.

5. The method according to claim 1, characterized in that In step (2), the extraction conditions are: 30-150 min ultrasonic time, 1:10-1:50 w / v solid-liquid ratio, 40%-80% ethanol solution, and 100-300 W ultrasonic power.

6. The method according to claim 1, characterized in that In step (3), the optimal extraction process parameters are: ultrasonic time of 80 min, solid-liquid ratio of 1:34 g / mL, ethanol concentration of 60%, and ultrasonic power of 173 W.

7. The method according to claim 1, characterized in that In step (4), the centrifugation condition is: 3000-5000 r / min, centrifugation for 10-20 min.

8. The method according to claim 1, characterized in that In step (5), the antioxidant capacity is detected by using an ultraviolet spectrophotometer to respectively detect the DPPH free radical scavenging capacity, ABTS free radical scavenging capacity, OH free radical scavenging capacity and total antioxidant capacity.

9. Use of total polyphenols from non-medicinal parts of Phellodendron chinense obtained by the extraction method according to claim 1 in the preparation of antioxidant drugs and food antioxidants.

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