Ultrasonic extraction method of polygonatum sibiricum saponin

Abstract

The invention provides an ultrasonic extraction method of polygonatum sibiricum saponin, which comprises the following steps: (1) taking polygonatum sibiricum powder, adding an ethanol extracting solution, uniformly dipping, and carrying out ultrasonic extraction; wherein the concentration of the ethanol is 80.560%, the liquid-material ratio of the rhizoma polygonati powder to the ethanol extracting solution is 20: 1mL / g, the ultrasonic power is 337.316 W, and the ultrasonic time is 35 minutes; and (2) centrifuging and collecting supernate. The invention also provides another ultrasonic extraction method of polygonatum sibiricum saponin. The ultrasonic extraction parameters are as follows: the ethanol concentration is 80%, the liquid-material ratio of polygonatum sibiricum powder to an ethanol extracting solution is 20: 1mL / g, the ultrasonic power is 340W, and the ultrasonic time is 35min. According to the method, the concentration, the material-to-liquid ratio, the ultrasonic power and the ultrasonic time of the extracting solution are optimized by combining a single-factor experiment with response surface analysis and the like to obtain optimal ultrasonic extraction parameters, so that the polygonatum sibiricum saponin extraction method which is simple to operate, short in extraction time and high in extraction efficiency is obtained.

Description

Technical Field

[0001] The invention relates to the technical field of plant secondary metabolite extraction, in particular to an ultrasonic extraction method for polygonatum saponin. Background Art

[0002] Polygonatum sibiricum, belonging to the genus Polygonatum of the Liliaceae family, contains a variety of bioactive ingredients, and its rhizome is the main part used as medicine. According to the 2020 edition of the Chinese Pharmacopoeia (Volume 1), Polygonatum sibiricum is a traditional Chinese medicine and food substance that has been used in China for more than two thousand years. Polygonatum sibiricum has the effects of replenishing qi and benefiting the kidneys, and has obvious functional characteristics in enhancing immunity, regulating blood sugar and blood lipids, delaying aging, anti-oxidation and anti-tumor.

[0003] Polygonatum saponin is one of the effective ingredients of Polygonatum sibiricum, which has many physiological functions such as anti-fatigue, anti-oxidation, lowering blood sugar, lowering blood lipids, enhancing immunity, anti-inflammatory, anti-viral, etc., and has shown broad application prospects in the fields of medicine, food, cosmetics, etc. As a natural antioxidant, Polygonatum saponin can remove excessive free radicals in the body and protect cells from oxidative stress damage, and has high potential value in the prevention and treatment of oxidative stress-related diseases.

[0004] At present, the common extraction methods of polygonatum saponins are mostly based on water extraction and alkali extraction, and ultrasonic extraction, enzyme extraction, microwave extraction, etc. are auxiliary extraction methods. Among them, ultrasonic extraction can destroy the cell wall structure, promote the release and dissolution of active ingredients, thereby improving the extraction efficiency. However, the differences in ultrasonic time, temperature, ultrasonic power and extraction reagents will lead to different extraction efficiencies of the target product. Such as too long extraction time, too large extraction power and temperature, destroy the target product structure and reduce productivity. And short ultrasonic time, low ultrasonic power and temperature, lead to incomplete extraction of the target product, causing waste. Therefore, ultrasonic extraction can significantly improve the extraction efficiency of saponins, but the key factors in the extraction process need to be optimized. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a new method for ultrasonic extraction of polygonatum saponin, optimize ultrasonic parameters by single factor experiment combined with response surface analysis, and finally obtain a polygonatum saponin extraction method with simple operation, short extraction time and high extraction efficiency.

[0006] The present invention adopts the following technical solutions to solve the above technical problems:

[0007] A method for ultrasonically extracting polygonatum saponin comprises the following steps:

[0008] (1) Take polygonatum powder, add ethanol extract, soak evenly, and extract by ultrasonic; wherein the ethanol concentration is 80.560%, the liquid-to-material ratio of polygonatum powder to ethanol extract is 20:1 mL / g, the ultrasonic power is 337.316 W, and the ultrasonic time is 35 min;

[0009] (2) After the ultrasound is completed, centrifuge and collect the supernatant, which is the polygonatum saponin extract.

[0010] As one of the preferred embodiments of the present invention, in step (1), the ethanol concentration, liquid-to-solid ratio, ultrasonic power and ultrasonic time are all determined by single factor experiment combined with response surface analysis.

[0011] As one of the preferred embodiments of the present invention, first, the initial ultrasonic parameters of polygonatum saponin are optimized using a single factor experiment, and the ultrasonic parameters after single factor optimization are: ethanol concentration 75% to 85%, liquid-to-solid ratio (10 to 20): 1 mL / g, ultrasonic power 300 to 380 W, and ultrasonic time 35 min; then, taking the extraction conditions optimized by the single factor as the central value and the extraction rate of polygonatum saponin as the response value, the ultrasonic parameters of polygonatum saponin are further optimized using the response surface analysis method, and the final optimized ultrasonic parameters are: ethanol concentration of 80.560%, liquid-to-solid ratio of polygonatum powder to ethanol extract of 20:1 mL / g, ultrasonic power 337.316 W, and ultrasonic time 35 min.

[0012] As one of the preferred embodiments of the present invention, in the process of optimizing the ultrasonic parameters by the response surface analysis method, the quadratic multivariate regression equation model established is:

[0013] Y=3.632+0.05625A+0.045B+0.01125C+0.005AB+0.0025AC+0.04BC+0.11225A 2 +0.18975B 2 +0.13725C 2 ; Wherein, Y is the extraction rate of polygonatum saponin, A is the liquid-to-solid ratio, B is the ethanol concentration, and C is the ultrasonic power.

[0014] As one of the preferred embodiments of the present invention, in step (2), the centrifugation condition is 4000 r / min for 10 min.

[0015] As one of the preferred embodiments of the present invention, in step (2), the obtained polygonatum saponin extract has a scavenging effect on DPPH free radicals and ABTS free radicals, and has a protective effect on bovine serum albumin in the metal-catalyzed oxidative damage system.

[0016] A method for ultrasonically extracting polygonatum saponin comprises the following steps:

[0017] (1) Take polygonatum powder, add ethanol extract, soak evenly, and perform ultrasonic extraction; wherein the ethanol concentration is 80%, the liquid-to-material ratio of polygonatum powder to ethanol extract is 20:1 mL / g, the ultrasonic power is 340 W, and the ultrasonic time is 35 min;

[0018] (2) After the ultrasound is completed, centrifuge and collect the supernatant, which is the polygonatum saponin extract.

[0019] As one of the preferred embodiments of the present invention, in step (1), the ethanol concentration, liquid-to-solid ratio, ultrasonic power and ultrasonic time are determined by single factor experiment combined with response surface analysis and actual operation.

[0020] As one of the preferred embodiments of the present invention, firstly, the initial ultrasonic parameters of polygonatum saponin are optimized by single factor experiment, and the ultrasonic parameters after single factor optimization are: ethanol concentration 75% to 85%, liquid-to-solid ratio (10 to 20): 1 mL / g, ultrasonic power 300 to 380 W, and ultrasonic time 35 min; then, taking the extraction conditions optimized by single factor as the central value and the extraction rate of polygonatum saponin as the response value, the ultrasonic parameters of polygonatum saponin are further optimized by response surface analysis, and the ultrasonic parameters obtained by response surface optimization are: ethanol concentration of 80.560%, liquid-to-solid ratio of polygonatum powder to ethanol extract of 20:1 mL / g, ultrasonic power of 337.316 W, and ultrasonic time of 35 min; finally, combined with the convenience and feasibility of actual operation, the response surface optimization parameters are rounded, that is, liquid-to-solid ratio of 20:1 mL / g, ethanol concentration of 80%, ultrasonic power of 340 W, and ultrasonic time of 35 min are selected as the final optimization parameters.

[0021] As one of the preferred embodiments of the present invention, in the process of optimizing the ultrasonic parameters by the response surface analysis method, the quadratic multivariate regression equation model established is:

[0022] Y=3.632+0.05625A+0.045B+0.01125C+0.005AB+0.0025AC+0.04BC+0.11225A 2 +0.18975B 2 +0.13725C 2 ; Wherein, Y is the extraction rate of polygonatum saponin, A is the liquid-to-solid ratio, B is the ethanol concentration, and C is the ultrasonic power.

[0023] As one of the preferred embodiments of the present invention, in step (2), the centrifugation condition is 4000 r / min for 10 min.

[0024] As one of the preferred embodiments of the present invention, in the step (2), the extraction rate of polygonatum saponin is 3.59%.

[0025] The advantages of the present invention compared to the prior art are:

[0026] (1) The present invention optimizes the factors affecting the extraction of polygonatum saponin, such as the concentration of the extract, the solid-liquid ratio, the ultrasonic power, and the ultrasonic time, by combining single-factor experiments with the response surface analysis method, and obtains the optimal ultrasonic extraction parameters; based on the above-mentioned optimal ultrasonic extraction parameters, the present invention obtains a polygonatum saponin extraction method with simple operation, short extraction time, and high extraction efficiency, which provides technical method guidance for the development and application of polygonatum, and also provides a reference for achieving efficient extraction and energy saving;

[0027] (2) The polygonatum saponin extract obtained by the method of the present invention has a good free radical scavenging ability, has a significant scavenging effect on DPPH free radicals and ABTS free radicals, and has a protective effect on bovine serum albumin (BSA) in the metal catalyzed oxidative damage system (MCO). BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the standard curve of polygonatum saponin in Example 3;

[0029] Figure 2 This is the result diagram of the effect of liquid-to-solid ratio on the extraction rate of polygonatum saponin in Experimental Example 1;

[0030] Figure 3 This is the result diagram of the effect of ethanol concentration on the extraction rate of polygonatum saponin in Experimental Example 1;

[0031] Figure 4 This is a result diagram showing the effect of ultrasonic power on the extraction rate of polygonatum saponin in Experimental Example 1;

[0032] Figure 5 This is the result diagram of the effect of ultrasonic time on the extraction rate of polygonatum saponin in Experimental Example 1;

[0033] Fig. 6A is a graph showing the interaction between liquid-to-solid ratio and ultrasonic power in Experimental Example 1;

[0034] Figure 6B is a graph showing the interaction between ethanol concentration and ultrasonic power in Experimental Example 1;

[0035] Figure 6C is a graph showing the interaction between the liquid-to-solid ratio and the ethanol concentration in Experimental Example 1;

[0036] Figure 7 This is a graph showing the results of the determination of the iron reducing power of saponins in Experimental Example 2;

[0037] Figure 8 This is a graph showing the effect of polygonatum saponin extract on DPPH free radical levels in Experimental Example 3;

[0038] Fig. 9This is a graph showing the effect of polygonatum saponin extract on ABTS free radical levels in Experimental Example 3;

[0039] Fig.10 is a diagram of the experimental results of establishing the protein metal catalyzed oxidative damage system in Experimental Example 4;

[0040] Fig.11 This is a graph showing the protective effect of Polygonatum sibiricum saponin extract on bovine serum albumin (BSA) in the metal-catalyzed oxidative damage system (MCO) in Experimental Example 4. DETAILED DESCRIPTION

[0041] The following is a detailed description of the embodiments of the present invention. The embodiments are implemented based on the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments. At the same time, the experimental methods used in the following embodiments are conventional methods unless otherwise specified; the materials, reagents, etc. used can be obtained from commercial channels unless otherwise specified.

[0042] Example 1

[0043] The ultrasonic extraction method of polygonatum saponin of the present embodiment comprises the following steps:

[0044] (1) Take the dried powder of the rhizome of Polygonatum sibiricum, add ethanol extract, soak evenly, and perform ultrasonic extraction; wherein the ethanol concentration is 80.560%, the liquid-to-solid ratio of Polygonatum sibiricum powder to ethanol extract is 20:1 mL / g, the ultrasonic power is 337.316 W, and the ultrasonic time is 35 min.

[0045] (2) After the ultrasonic treatment, centrifuge at 4000 r / min for 10 min and collect the supernatant, which is the polygonatum saponin extract.

[0046] Example 2

[0047] The ultrasonic extraction method of polygonatum saponin of the present embodiment comprises the following steps:

[0048] (1) Take the dried powder of the rhizome of Polygonatum sibiricum, add ethanol extract, soak evenly, and perform ultrasonic extraction; wherein the ethanol concentration is 80%, the liquid-to-material ratio of Polygonatum sibiricum powder to ethanol extract is 20:1 mL / g, the ultrasonic power is 340 W, and the ultrasonic time is 35 min;

[0049] (2) After the ultrasonic treatment, centrifuge at 4000 r / min for 10 min and collect the supernatant, which is the polygonatum saponin extract.

[0050] Example 3

[0051] A method for determining the content of polygonatum saponin in the above embodiment of the present embodiment:

[0052] (1) Draw a standard curve

[0053] Accurately weigh 2.5 mg of diosgenin reference substance, place it in a 10 mL volumetric flask, and dilute with ethanol to obtain a reference substance solution containing 0.25 mg of diosgenin per 1 mL, and refrigerate at 4°C for later use. Take an appropriate amount of vanillin and an appropriate amount of glacial acetic acid and place them in a beaker, stir evenly with a glass rod, and prepare a 5% vanillin glacial acetic acid solution, which is sealed and stored for later use. Take 4 mL of freshly prepared 5% vanillin glacial acetic acid solution and 16 mL of perchloric acid and place them in a beaker, mix evenly, and place them in a reagent bottle, seal and store for later use. Accurately pipette 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, and 0.6 mL of the above reference substance solution into a 10 mL stoppered test tube and evaporate the solvent in a 60°C water bath overnight. Add 1 mL of freshly prepared vanillin glacial acetic acid-perchloric acid solution to each test tube, shake well, and develop color in a 60°C water bath for 25 min. After cooling in an ice-water bath for 5 min, add 5 mL of glacial acetic acid, shake well, let stand for 5 min, and measure the absorbance at 560 nm.

[0054] With the different masses (x) of diosgenin standard as the abscissa and the corresponding absorbance values ​​(y) as the ordinate, a graph is plotted as follows: Figure 1 As shown, the standard curve equation is: y = 4.6629x + 0.0153, R 2 =0.9958.

[0055] (2) Determination of Polygonatum sibiricum saponin content

[0056] Take 1mL of polygonatum saponin extract, put it into a 10mL volumetric flask, and make up to volume. Take 0.15mL of the constant volume solution, put it into a 10mL test tube, and evaporate the solvent in a 60℃ water bath overnight. Draw the same operation steps as the standard curve, measure the absorbance at 560nm, and calculate the total saponin content.

[0057] Experimental Example 1

[0058] This experimental example is used to illustrate the optimization process and results of the best ultrasonic extraction parameters of the present invention.

[0059] 1. Single factor experiment to optimize ultrasonic extraction parameters of polygonatum saponins

[0060] The liquid-to-solid ratio, ethanol concentration, ultrasonic power and ultrasonic time were selected for single factor experiments, and the content of polygonatum saponin under each single factor experiment was determined with reference to Example 3, and the relationship between each factor and the extraction rate of polygonatum saponin was analyzed.

[0061] (1) Effect of liquid-to-solid ratio on the extraction rate of polygonatum saponin

[0062] Under the fixed conditions of "ethanol concentration 80%, ultrasonic power 300 W, ultrasonic time 40 min", different levels of liquid-to-solid ratios (5:1 mL / g, 10:1 mL / g, 15:1 mL / g, 20:1 mL / g, 25:1 mL / g) were set to carry out the experiment.

[0063] The results are as follows Figure 2 As shown. The results show that when the liquid-to-solid ratio is 5:1-15:1 mL / g, the extraction rate of polygonatum saponin gradually increases with the increase of the liquid-to-solid ratio; when the liquid-to-solid ratio reaches 15:1 mL / g, the extraction rate of polygonatum saponin reaches a maximum of 3.53%; after the liquid-to-solid ratio reaches 15:1 mL / g, as the liquid-to-solid ratio gradually increases, the extraction rate of polygonatum saponin begins to decrease. Therefore, in order to achieve the optimization of cost-effectiveness and extraction efficiency, the appropriate liquid-to-solid ratio for polygonatum saponin extraction is 15:1 mL / g.

[0064] (2) Effect of ethanol concentration on the extraction rate of polygonatum saponin

[0065] Under the fixed conditions of "liquid-to-solid ratio 15:1 mL / g, ultrasonic power 300 W, ultrasonic time 40 min", different levels of ethanol concentration (70%, 75%, 80%, 85%, 90%) were set for experiment.

[0066] The results are as follows Figure 3 As shown. The results show that: when the ethanol concentration increases from 70% to 80%, the extraction rate of polygonatum saponin increases significantly with the increase of ethanol concentration; when the ethanol concentration is 80%, the extraction rate of polygonatum saponin reaches a maximum value of 3.54%; after the ethanol concentration reaches 80%, the extraction rate of polygonatum saponin begins to gradually decrease with the increase of ethanol concentration. Therefore, when the ethanol concentration is 80%, the extraction rate of polygonatum saponin reaches its maximum value.

[0067] (3) Effect of ultrasonic power on the extraction rate of polygonatum saponin

[0068] Under the fixed conditions of "liquid-to-solid ratio 15:1 mL / g, ethanol concentration 80%, ultrasonic time 40 min", different levels of ultrasonic power (220 W, 260 W, 300 W, 340 W, 380 W) were set for experiments.

[0069] The results are as follows Figure 4 The results show that when the ultrasonic power is 220-340W, the extraction rate of polygonatum saponin is significantly improved; when the ultrasonic power is 340W, the extraction rate of polygonatum saponin reaches a maximum value of 3.69%; when the ultrasonic power is 340-380W, the extraction rate of polygonatum saponin continues to decrease. Therefore, when the ultrasonic power is 340W, the extraction rate of polygonatum saponin reaches the maximum value.

[0070] (4) Effect of ultrasonic time on the extraction rate of polygonatum saponin

[0071] Under the fixed conditions of "liquid-to-solid ratio of 15:1 mL / g, ethanol concentration of 80%, and ultrasonic power of 300 W", different levels of ultrasonic time (20 min, 25 min, 30 min, 35 min, 40 min) were set for the experiment.

[0072] The results are as follows Figure 5 The results show that when the ultrasonic time is 30-35 minutes, the extraction rate of polygonatum saponin gradually increases with the increase of time, from 3.22% to 3.52%; however, when the time reaches 40 minutes, the extraction rate of polygonatum saponin shows a relatively gentle trend of first increasing and then decreasing. Considering the extraction efficiency, quality and energy consumption, the ultrasonic time in the subsequent response surface optimization is 35 minutes.

[0073] 2. Optimization of Ultrasonic Extraction Parameters by Response Surface

[0074] (1) Response surface experimental design

[0075] According to the above single factor experimental results, the extraction rate of polygonatum saponin was taken as the response value, and three factors with greater influence on it were selected: liquid-to-solid ratio, ethanol concentration, and ultrasonic power. A response surface analysis experiment was designed (Table 1), and the corresponding polygonatum saponin extraction optimization experiment and variance analysis were carried out. The optimal extraction parameters obtained by equation fitting were verified by experiments (Table 2).

[0076] Table 1 Response surface optimization coding table for extracting polygonatum saponins by ultrasonic method

[0077]

[0078] Table 2 Box-Behnken experimental design and results of ultrasonic extraction of polygonatum saponins

[0079]

[0080]

[0081] (2) Regression model establishment and variance analysis

[0082] Using Design-Expert 12 software to perform regression fitting on the data in the model, the regression equation between the three factors (A, B, C) and the extraction rate of polygonatum saponin (Y) was obtained:

[0083] Y=3.632+0.05625A+0.045B+0.01125C+0.005AB+0.0025AC+0.04BC+0.11225A 2 +0.18975B 2 +0.13725C2 ;

[0084] Wherein, Y is the extraction rate of polygonatum saponin, A is the liquid-to-solid ratio, B is the ethanol concentration, and C is the ultrasonic power.

[0085] From the results of variance analysis (Table 3), it can be seen that in the significance verification of the model, F = 40.44, p < 0.0001 is statistically significant; while in the lack of fit item, F = 0.5806, p > 0.05 indicates that the lack of fit item is not statistically significant. The adjusted determination coefficient R of the model 2 Adj =0.9569, indicating that the experimental model has a good fit for this experiment and can better reflect the effects of liquid-to-solid ratio, ethanol concentration, and ultrasonic power on the extraction rate of polygonatum saponin. From the F value, we can see that A>B>C, indicating that the order of influence of each factor on the experimental results is: liquid-to-solid ratio>ethanol concentration>ultrasonic power. The 3D response surface diagram of polygonatum saponin is shown in Fig. 6A , 6B , as shown in 6C.

[0086] Table 3 Regression analysis results of polygonatum saponin extraction rate

[0087]

[0088]

[0089] (3) Model verification

[0090] According to the analysis of Design Expert software, the optimized extraction parameters of polygonatum saponin are "liquid-to-solid ratio 20:1mL / g, ethanol concentration 80.560%, ultrasonic power 337.316W", and the predicted value is 3.579%.

[0091] However, combined with the actual operation (limited by the accuracy of instrument settings), "liquid-to-solid ratio 20:1 mL / g, ethanol concentration 80%, ultrasonic power 340 W, and ultrasonic time 35 min" were selected as the optimized extraction process.

[0092] Based on the above analysis results, the optimized parameter conditions for ultrasonic extraction of polygonatum saponin were obtained. And through three parallel experiments, the average extraction rate of polygonatum saponin was determined to be 3.59%, which is close to the simulation prediction value, indicating that the optimized process selected by the model is true and reliable and has practical significance.

[0093] Experimental Example 2

[0094] This experimental example is used to verify the iron reducing power of Polygonatum sibiricum saponins.

[0095] Referring to the method of Example 2 (liquid-to-solid ratio of 20:1 mL / g, ethanol concentration of 80%, ultrasonic power of 340 W, ultrasonic time of 35 min), the polygonatum saponins of the selected variety 'Fulin Polygonatum No. 1' (provided by Anhui Engineering Technology Center for the Development and Utilization of Local Characteristic Plant Resources of Anhui Agricultural University) and the control variety 'Qingyang County's main local planted multi-flowered polygonatum' (provided by the standardized planting base of polygonatum of Qingyang County Huayao Agricultural Development Co., Ltd.) were extracted respectively, and the iron reducing power of the saponins was determined respectively.

[0096] The results are as follows Figure 7 As shown. Figure 7 It can be seen that: within the range of saponin concentration of 1.0-5.0 mg / mL, the iron reducing power of saponin of the selected variety 'Fulin Huangjing No. 1' gradually increased from 0.053 to 0.420, and the reducing power of saponin of the control variety Huangjing increased from 0.052 to 0.276. It can be seen that the saponins of the selected variety 'Fulin Huangjing No. 1' and the control variety 'Qingyang County's local main plant Polygonatum cyrtonema' both have a reducing effect on trivalent iron, showing concentration dependence, and are significantly weaker than Vc; under the same concentration conditions, the reducing power of saponin of 'Fulin Huangjing No. 1' is slightly better than that of 'Qingyang County's local main plant Polygonatum cyrtonema'. Because the absorbance values ​​of the iron reducing power of Vc measured under experimental conditions are all greater than 1, the absorbance values ​​of the iron reducing power of Vc are uniformly recorded as 1.

[0097] Experimental Example 3

[0098] This experimental example is used to verify the antioxidant activity of polygonatum saponins at the free radical level.

[0099] 1. DPPH free radical scavenging test of saponin

[0100] Referring to the method of Example 2 (liquid-to-solid ratio 20:1 mL / g, ethanol concentration 80%, ultrasonic power 340 W, ultrasonic time 35 min), polygonatum saponins of the selected variety 'Fulin Polygonatum No. 1' (provided by Anhui Engineering Technology Center for the Development and Utilization of Local Characteristic Plant Resources of Anhui Agricultural University) and the control variety 'Qingyang County's local main planted multi-flowered polygonatum' (provided by the standardized polygonatum planting base of Qingyang County Huayao Agricultural Development Co., Ltd.) were extracted respectively, and the saponins were tested for scavenging DPPH free radicals.

[0101] The results are as follows Figure 8 As shown. Figure 8 It can be seen that within the concentration range of 0.25-1.25 mg / mL, the saponins of the selected variety 'Fulin Huangjing No. 1' and the control variety 'Qingyang County Local Main Plant Polygonatum Multiflorum' have a certain ability to scavenge DPPH free radicals. The scavenging rate of saponins of the selected variety on DPPH free radicals gradually increased from 15.47% to 25.68% with the increase of saponin concentration, while the scavenging rate of saponins of the control variety on DPPH free radicals increased from 9.09% to 23.44%.

[0102] The experimental results showed that the scavenging rate of saponins from the two varieties of Polygonatum cyrtonema increased gradually with the increase of saponin concentration, and both were weaker than the positive control Vc. Within the experimental concentration range, the scavenging rate of saponins from 'Fulin Polygonatum 1' on DPPH free radicals was better than that of the control variety 'Polygonatum cyrtonema', the main local plant in Qingyang County.

[0103] 2. Determination of the ability of saponins to scavenge ABTS free radicals

[0104] Referring to the method of Example 2 (liquid-to-solid ratio 20:1 mL / g, ethanol concentration 80%, ultrasonic power 340 W, ultrasonic time 35 min), polygonatum saponins of the selected variety 'Fulin Polygonatum No. 1' and the control variety 'Qingyang County's local main cultivated multi-flowered polygonatum' were extracted respectively, and the saponins were tested for the ABTS free radical scavenging ability.

[0105] The results are as follows Fig. 9 As shown. Fig. 9 It can be seen that within the concentration range of 0.25-1.25 mg / mL, the saponins of the selected variety 'Fulin Huangjing No. 1' and the control variety 'Qingyang County's local main plant Polygonatum cyrtonema' have a certain ability to scavenge ABTS free radicals, and their scavenging ability gradually increases with the increase of saponin concentration, but the scavenging ability is weaker than Vc. Under the experimental conditions, the scavenging rate of ABTS free radicals of 'Fulin Huangjing No. 1' increased from 8.69% to 22.00%, and the scavenging rate of ABTS free radicals of the control variety 'Qingyang County's local main plant Polygonatum cyrtonema' increased from 6.10% to 19.41%. The scavenging ability of saponins of the selected variety 'Fulin Huangjing No. 1' on ABTS free radicals is better than that of the control variety.

[0106] Experimental Example 4

[0107] This experimental example was used to verify the antioxidant activity of Polygonatum sibiricum saponins at the protein level.

[0108] 1. Establishment of protein metal-catalyzed oxidative damage system

[0109] In a volume of 6 to 16 μL of Cu 2+ / H2O2-induced damage, bovine serum albumin was damaged to a certain extent, and with the 2+ / H2O2 induced an increase in the injury volume and a gradual decrease in the content of BSA bands ( Fig.10 ). When the volume of the induced damage system was increased to 14 μL, the band content of bovine serum albumin decreased to 49.82% ± 0.48%, and the damage rate was close to 50%. Therefore, 14 μL of Cu 2+ / H2O2-induced injury volume was used as the model group.

[0110] 2. Protective effect of saponins on metal-catalyzed oxidative damage of bovine serum albumin

[0111] Referring to the method of Example 2 (liquid-to-solid ratio of 20:1 mL / g, ethanol concentration of 80%, ultrasonic power of 340 W, ultrasonic time of 35 min), polygonatum saponins of the selected variety 'Fulin Polygonatum No. 1' (provided by Anhui Engineering Technology Center for Development and Utilization of Local Characteristic Plant Resources of Anhui Agricultural University) and the control variety 'Polygonatum cyrtonema mainly cultivated locally in Qingyang County' (provided by the standardized planting base of Polygonatum cyrtonema of Qingyang County Huayao Agricultural Development Co., Ltd.) were extracted respectively, and their protective effects on metal-catalyzed oxidative damage of bovine serum albumin were tested within the experimental concentration range.

[0112] The results are as follows Fig.11 As shown. Fig.11 It can be seen that: within the experimental concentration range, saponins from the selected variety 'Fulin Polygonatum 1' and the control variety 'Polygonatum sibiricum, the main local plant in Qingyang County' both showed a certain effect in inhibiting the metal-catalyzed oxidative damage of bovine serum albumin; compared with the model group, under the action of these two Polygonatum saponins, the concentration of the protein band gradually increased with the increase of saponin concentration, that is, the protective effect of these two saponins on the metal-catalyzed oxidative damage of bovine serum albumin increased with the increase of concentration.

[0113] Further comparative analysis showed that at the same saponin concentration, there were differences in the effects of saponins from the selected variety 'Fulin Huangjing No. 1' and the control variety in inhibiting metal-catalyzed oxidative damage to bovine serum albumin. Under the action of saponins from 'Fulin Huangjing No. 1', the concentrations of the protein bands were 54.90%, 59.23%, 61.78%, 72.58%, and 76.99% in the concentration range of 1-5 mg / mL; while under the action of saponins from the control variety Polygonatum cyrtonema, the concentrations of the protein bands were 53.00%, 58.12%, 60.63%, 65.75%, and 71.75%, respectively. Compared with the saponins from the control variety Polygonatum cyrtonema, the main local plant in Qingyang County, the saponins from the selected variety 'Fulin Huangjing No. 1' have a more superior protective effect on metal-catalyzed oxidative damage to bovine serum albumin.

[0114] In summary, the ultrasonic extraction method of the present invention is applicable to both the existing 'Fulin Huangjing No. 1' variety and the main cultivated varieties. The extracted saponins have good free radical scavenging ability, have a significant scavenging effect on DPPH free radicals and ABTS free radicals, and have a protective effect on bovine serum albumin (BSA) in the metal catalyzed oxidative damage system (MCO), and the application result on the 'Fulin Huangjing No. 1' variety is the best.

[0115] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for ultrasonic extraction of polygonatum saponins, characterized in that: The steps include: (1) Take polygonatum powder, add ethanol extract, soak evenly, and extract by ultrasonic; wherein the ethanol concentration is 80.560%, the liquid-to-material ratio of polygonatum powder to ethanol extract is 20:1 mL / g, the ultrasonic power is 337.316 W, and the ultrasonic time is 35 min; (2) After the ultrasound is completed, centrifuge and collect the supernatant, which is the polygonatum saponin extract.

2. The ultrasonic extraction method of polygonatum saponin according to claim 1, characterized in that: In the step (1), the ethanol concentration, liquid-to-solid ratio, ultrasonic power and ultrasonic time are all determined by single factor experiment combined with response surface analysis.

3. The ultrasonic extraction method of polygonatum saponin according to claim 2, characterized in that: Firstly, the initial ultrasonic parameters of polygonatum saponins were optimized using a single factor experiment. The ultrasonic parameters after single factor optimization were: ethanol concentration 75%-85%, liquid-to-solid ratio (10-20): 1mL / g, ultrasonic power 300-380W, and ultrasonic time 35min; then, taking the extraction conditions optimized by single factor as the central value and the extraction rate of polygonatum saponins as the response value, the response surface analysis method was used to further optimize the ultrasonic parameters of polygonatum saponins. The final optimized ultrasonic parameters were: ethanol concentration of 80.560%, liquid-to-solid ratio of polygonatum powder and ethanol extract of 20:1mL / g, ultrasonic power of 337.316W, and ultrasonic time of 35min.

4. The ultrasonic extraction method of polygonatum saponin according to claim 3, characterized in that: In the process of optimizing ultrasonic parameters by response surface analysis, the quadratic multiple regression equation model established is: Y=3.632+0.05625A+0.045B+0.01125C+0.005AB+0.0025AC+0.04BC+0.11225A 2 +0.18975B 2 +0.13725C 2 ; In the formula, Y is the extraction rate of polygonatum saponin, A is the liquid-to-solid ratio, B is the ethanol concentration, and C is the ultrasonic power.

5. The ultrasonic extraction method of polygonatum saponin according to claim 1, characterized in that: In the step (2), the centrifugation condition is 4000 r / min for 10 min.

6. The ultrasonic extraction method of polygonatum saponin according to claim 1, characterized in that: In the step (2), the obtained polygonatum saponin has a scavenging effect on DPPH free radicals and ABTS free radicals, and has a protective effect on bovine serum albumin in the metal-catalyzed oxidative damage system.

7. A method for ultrasonic extraction of polygonatum saponins, characterized in that: The steps include: (1) Take polygonatum powder, add ethanol extract, soak evenly, and perform ultrasonic extraction; wherein the ethanol concentration is 80%, the liquid-to-material ratio of polygonatum powder to ethanol extract is 20:1 mL / g, the ultrasonic power is 340 W, and the ultrasonic time is 35 min; (2) After the ultrasound is completed, centrifuge and collect the supernatant, which is the polygonatum saponin extract.

8. The ultrasonic extraction method of polygonatum saponin according to claim 7, characterized in that: In the step (1), the ethanol concentration, liquid-to-solid ratio, ultrasonic power and ultrasonic time are determined by single factor experiment combined with response surface analysis and actual operation.

9. The ultrasonic extraction method of polygonatum saponin according to claim 8, characterized in that: Firstly, the initial ultrasonic parameters of polygonatum saponins were optimized using a single factor experiment. The ultrasonic parameters after single factor optimization were: ethanol concentration 75%-85%, liquid-to-solid ratio (10-20): 1mL / g, ultrasonic power 300-380W, and ultrasonic time 35min; then, taking the extraction conditions optimized by single factor as the central value and the extraction rate of polygonatum saponins as the response value, the response surface analysis method was used to further optimize the ultrasonic parameters of polygonatum saponins. The ultrasonic parameters obtained by response surface optimization were: ethanol concentration of 80.560%, liquid-to-solid ratio of polygonatum powder and ethanol extract of 20:1mL / g, ultrasonic power of 337.316W, and ultrasonic time of 35min; finally, the response surface optimization parameters were rounded, that is, liquid-to-solid ratio of 20:1mL / g, ethanol concentration of 80%, ultrasonic power of 340W, and ultrasonic time of 35min were selected as the final optimization parameters.

10. The ultrasonic extraction method of polygonatum saponin according to claim 7, characterized in that: In the step (2), the extraction rate of polygonatum saponin is 3.59%.

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