阅读说明：本技术 利用响应面法优化水提小蓝粉总皂苷的提取方法 (Method for optimizing extraction of total saponins of water-extracted small blueberries by using response surface method ) 是由 刘全宏 王布雷 殷刚 于 2019-11-28 设计创作，主要内容包括：本发明属于植物提取技术领域,尤其涉及一种利用响应面法优化小蓝粉总皂苷的提取方法,具体包括(1)制备小蓝粉样品溶液；(2)使用香草醛-冰醋酸法测定样品溶液皂苷含量；(3)试验设计与统计分析；(4)数据处理。本发明方法具有得率高,提取工艺准确、可靠等优点,同时简单易行,提取成本较低,响应面法的采用,保证了最优提取条件的科学性,可以带来良好的经济效益和社会效益。(The invention belongs to the technical field of plant extraction, and particularly relates to an extraction method for optimizing total saponins of small blue powder by using a response surface method, which specifically comprises the following steps of (1) preparing a small blue powder sample solution; (2) measuring the saponin content of the sample solution by using a vanillin-glacial acetic acid method; (3) experimental design and statistical analysis; (4) and (6) data processing. The method has the advantages of high yield, accurate and reliable extraction process, simplicity, practicability, low extraction cost and the like, ensures the scientificity of the optimal extraction condition by adopting a response surface method, and can bring good economic benefit and social benefit.)

1. A method for optimizing the extraction of the total saponins of the small blue powder by using a response surface method is characterized by comprising the following steps of:

s1: preparing a small blue powder sample solution;

s2: measuring the saponin content of the sample solution by using a vanillin-glacial acetic acid method;

s201: drawing a standard curve by taking the gypenoside standard substance as a reference, and calculating a regression equation;

s202: substituting the light absorption value of the small blue powder sample solution into the regression equation to calculate the saponin concentration, and then substituting the saponin concentration into the following formula to obtain the saponin yield:

the saponin yield (%) is that the saponin concentration in the sample liquid is multiplied by the volume of the sample liquid and multiplied by the dilution ratio/the initial gynostemma pentaphylla mass/1000 times 100;

s3: experimental design and statistical analysis;

s301: sequentially investigating the influence of single factors such as liquid-material ratio, extraction time and extraction temperature on the saponin yield by taking the saponin yield in the step S202 as an index;

s302: on the basis of a single-factor test result, a three-factor three-horizontal response surface test is designed by taking the yield of the gypenoside as a response value and taking three factors, namely a material-liquid ratio, extraction time and extraction temperature as independent variables, and the optimal extraction process conditions of the small blue powder saponin are analyzed;

s4: data processing: the extraction process is optimized by taking the saponin yield as the yield, a regression equation is established through multivariate regression and binomial fitting of three factors of liquid-material ratio, extraction time and extraction temperature to obtain a two-dimensional and three-dimensional response surface map, and the optimal extraction process range of the small blue powder saponin is determined through analysis of test results.

2. The method for extracting total saponins of blue grass powder optimized by the response surface method as claimed in claim 1, wherein the influence of each factor on the saponin yield in step S301 is extraction temperature > feed-liquid ratio > extraction time.

3. The method for extracting total saponins of blue powder optimized by using the response surface method as claimed in claim 2, wherein the optimum extraction process range analyzed in the step S302 is as follows: the liquid-material ratio is 30: 1-50: 1ml/g, the extraction time is 60-120 min, and the extraction temperature is 50-70 ℃.

4. The method for extracting total saponins of blue powder optimized by using the response surface method as claimed in claim 3, wherein the optimal extraction process determined in step S4 is as follows: the liquid-material ratio is 45ml/g, the extraction time is 105min, the extraction temperature is 70 ℃, and the saponin yield is 5.02%.

Technical Field

The invention relates to the technical field of plant extraction, in particular to an extraction method for optimizing total saponins of small blue powder by using a response surface method.

Background

Gynostemma pentaphylla is perennial herbaceous vine of Gynostemma of Cucurbitaceae, and is also called as radix Et caulis Acanthopanacis Senticosi or radix Et rhizoma Rhei Palmati. It was recorded in the Ben Cao Ji ban of Ming Dynasty, Zhong Ben Cao, TCM dictionary and Chinese herbal medicine compilation. Gynostemma pentaphylla has the effects of diminishing inflammation, removing toxicity, relieving cough and eliminating phlegm, and is mainly used for treating diseases such as chronic bronchitis, infectious hepatitis, nephritis, gastroenteritis and the like. Modern pharmacological studies have demonstrated that: the saponin (Gyp) contained in gynostemma pentaphylla is the main active component, Gyp has a tetracyclic triterpene dammar type structure similar to ginsenoside, and gynostemma pentaphylla is a plant which only contains the saponin resource except araliaceae, so that the reputation of 'southern ginseng' is improved.

Gyp it is popular among researchers because of its series of biological activities such as anti-tumor, anti-oxidation, anti-aging, anti-inflammatory and immunoregulation, according to the principle of similarity and compatibility, the scholars mainly use ethanol reflux extraction Gyp, for example, Chinese patent CN201810458420.2 discloses a method for extracting gypenoside, which comprises the steps of crushing medicinal materials, ethanol extraction and macroporous resin purification, wherein ethanol extraction is a key step, however, the introduction of ethanol increases the production cost of saponin on the one hand, and also brings organic reagent pollution on the other hand, and the development of the current gynostemma pentaphylla resource mainly takes tea drinks as the main material, the tea soup is the water extract of gynostemma pentaphylla, and the theoretical research and practical application are greatly disjointed.

Disclosure of Invention

In order to solve the problems, the invention provides a method for optimizing the extraction of the total saponins of the gynostemma pentaphylla powder by using a response surface method, which takes the yield of the total saponins of the gynostemma pentaphylla as an index for the first time and improves the total content of the saponins in the gynostemma pentaphylla tea, and comprises the following steps:

s1: preparing a small blue powder sample solution;

s2: measuring the saponin content of the sample solution by using a vanillin-glacial acetic acid method;

s201: drawing a standard curve by taking the gypenoside standard substance as a reference, and calculating a regression equation;

s202: substituting the light absorption value of the small blue powder sample solution into the regression equation to calculate the saponin concentration, and then substituting the saponin concentration into the following formula to obtain the saponin yield:

the saponin yield (%) is that the saponin concentration in the sample liquid is multiplied by the volume of the sample liquid and multiplied by the dilution ratio/the initial gynostemma pentaphylla mass/1000 times 100;

s3: experimental design and statistical analysis;

s301: sequentially investigating the influence of single factors such as liquid-material ratio, extraction time and extraction temperature on the saponin yield by taking the saponin yield in the step S202 as an index;

s302: on the basis of a single-factor test result, a three-factor three-horizontal response surface test is designed by taking the yield of the gypenoside as a response value and taking three factors, namely a material-liquid ratio, extraction time and extraction temperature as independent variables, and the optimal extraction process conditions of the small blue powder saponin are analyzed;

s4: data processing: the extraction process is optimized by taking the saponin yield as the yield, a regression equation is established through multivariate regression and binomial fitting of three factors of liquid-material ratio, extraction time and extraction temperature to obtain a two-dimensional and three-dimensional response surface map, and the optimal extraction process range of the small blue powder saponin is determined through analysis of test results.

As a further explanation of the above scheme, the influence of each factor on the saponin yield in step S301 is extraction temperature > material-to-liquid ratio > extraction time.

As a further illustration of the above scheme, the optimal extraction process range analyzed in step S302 is: the liquid-material ratio is 30: 1-50: 1ml/g, the extraction time is 60-120 min, and the extraction temperature is 50-70 ℃.

As a further explanation of the above scheme, in step S4, the determined optimal extraction process is: the liquid-material ratio is 45ml/g, the extraction time is 105min, and the extraction temperature is 70 ℃.

The invention has the beneficial effects that:

1. in order to avoid pollution caused by ethanol and meet the requirement of practical development, the method selects water as an extraction solvent for the first time without using any organic reagent, optimizes the extraction process of the small blue powder saponin by using a response surface method on the basis of a single-factor test, and provides theoretical basis and process basis for further development of the small blue powder tea. The optimal process is obtained through Design-Expert 8.0 software and BBD test Design: the liquid-material ratio (45:1ml/g), the extraction time (105min), the extraction temperature (70 ℃) and the saponin yield are 5.02 percent.

2. Compared with the orthogonal method, the BBD response surface test design adopted by the invention can obtain the optimal result by using 3 change factors, 3 levels and a small amount of experimental groups (only 17 groups), thereby improving the yield.

3. The invention has the advantages of high yield, accurate and reliable extraction process, simple and easy operation, low extraction cost, adoption of a response surface method, guarantee of scientificity of optimal extraction conditions and bringing of good economic and social benefits.

Drawings

FIG. 1: standard curve of small blue powder saponin.

FIG. 2: single factor experiment shows the influence of liquid-material ratio, extraction time and temperature on the yield of small blue powder saponin.

FIG. 3: response surface and contour plot of factor interaction on saponin yield.

Detailed Description

In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments of the specification, the small blue powder of the present invention is a gynostemma pentaphyllum ultra-fine powder prepared by using an ultra-fine powder process and using gynostemma pentaphyllum as a raw material, and the method for extracting total saponins from the small blue powder optimized by using a response surface method provided in this embodiment is performed in the following manner:

in a first step, a sample solution is prepared.

Accurately weighing 1g of herba Cymbopogonis Citrari powder, adding water, soaking, centrifuging at 5000rpm for 10min, collecting supernatant, determining according to vanillin-glacial acetic acid method, and calculating total saponin content in the extractive solution.

And secondly, measuring and calculating the content of the total saponin in the extracting solution by a vanillin-glacial acetic acid method.

Firstly, making a standard curve: accurately weighing 10mg of gypenoside standard substance, preparing into 1mg/mL solution, diluting to 0.2,0.4,0.5,0.6,0.8mg/mL respectively, and using distilled water as reference; taking 100 mu L of standard solutions with different concentrations, adding 100 mu L of vanillin (5%) and 500 mu L of perchloric acid, reacting in a water bath at 65 ℃ for 15min, cooling the reaction solution to room temperature, adding 1mL of glacial acetic acid, and mixing uniformly; transferring 200 mu L of the solution to detect the light absorption value at 550 nm; drawing a standard curve by taking the saponin concentration mg/mL as an abscissa and the absorbance as an ordinate, wherein the obtained regression equation is that y is 0.0006x +0.0179, and R2 is 0.09954 (refer to fig. 1); and then, measuring the content of the saponin in the sample: sampling 100 mu L of sample liquid, wherein the rest operations are the same as the steps for preparing a standard curve, and obtaining the concentration of the saponin in the sample by using a regression equation of the standard curve; finally, substituting the concentration of the saponin in the sample into a formula: the saponin yield (%) is obtained by multiplying the saponin concentration in the sample solution by the volume of the sample solution by the dilution factor/the initial gynostemma pentaphylla mass/1000 × 100.

The third step: the optimum extraction process of saponin is analyzed through experimental design and statistics.

First, a single factor test was performed: sequentially examining the influence of 3 factors of liquid-material ratio (10:1, 20:1, 30:1, 40:1, 50:1, 60:1ml/g, fixed time 60min, temperature 70 ℃), extraction time (30, 60, 90, 120, 150, 180min, fixed liquid-material ratio 40:1, temperature 70 ℃), extraction temperature (30, 40, 50, 60, 70, 80 ℃, fixed liquid-material ratio 40:1, time 100min) on the saponin yield by taking the saponin yield as an index (refer to figure 2);

then, a response surface optimization test method is carried out: on the basis of the single-factor test result, designing test conditions by adopting Design-Expert 8.0(RSM) software, selecting a Box-Behnken (BBD) model, designing a three-factor three-level response surface test (see table 1) by taking the yield of the saponin as a response value (R) and 3 factors of a material-liquid ratio (A), extraction time (B) and extraction temperature (C) as independent variables, and predicting the optimal extraction process (see table 2) of the saponin; and (3) carrying out multiple linear regression fitting on the result to obtain a quadratic polynomial regression equation model of the yield of the gypenoside to the material-liquid ratio (A), the extraction time (B) and the extraction temperature (C):

Y＝4.92+0.096×A+6.250×10^-3×B+0.23×C-0.23×AB+0.060×AC+0.18×BC-0.36×A²-0.27×B²-0.17×C²

TABLE 1 response surface analysis factors and horizon table

Table 2 response surface test protocol design results

Where a is the average of three trials.

And finally, carrying out variance analysis and significance test on the regression model: the results of the response surface model analysis of variance for the quadratic regression equation are shown in table 3, from which it can be seen that the model is very significant (P <0.01), and the misfit is not significant (P > 0.05); meanwhile, R2 is 0.9814, and the corrected R2 is 0.9603, which shows that the fitting degree of the model is better; CV is 1.52, and the value is small, so that the test operation has reliability; therefore, the model can be used for guiding and optimizing the water extraction process of the small blue powder.

TABLE 3 ANOVA TABLE

a pole significant P <0.01

b significant P <0.05

c is not significant P >0.05

Analysis of variance of the regression equations indicates: the most significant is the extraction temperature and the liquid-material ratio, and the saponin yield influence in the extraction time is not significant; in the interactive effect among factors, only the interactive effect of the material-liquid ratio and the extraction time is not obvious; the quadratic effect of the three factors is obvious; according to the F value, the influence of various factors on the saponin can be obtained, namely the extraction temperature is more than the material-liquid ratio and the extraction time is more than the F value.

And fourthly, processing data.

Drawing a chart by using Origin 9.0 and analyzing data by using SPSS 17.0, repeating all the tests for three times, and finally obtaining the optimal extraction process of the small blue powder saponin by optimizing through a response surface method, wherein the optimal extraction process comprises the following steps: liquid-material ratio (45:1ml/g), extraction time (105min), extraction temperature (70 ℃), and final saponin yield of 5.02%.

The above-mentioned embodiments are only specific embodiments of the present invention, and are used to illustrate the technical solutions of the present invention, but not to limit the present invention, and the protection scope of the present invention is not limited thereto, and those skilled in the art should understand that: any person skilled in the art can modify or easily conceive the technical solutions described in the foregoing embodiments or equivalent substitutes for some technical features within the technical scope of the present disclosure; the modifications, changes or substitutions do not cause the essence of the corresponding technical solutions to depart from the spirit and scope of the technical solutions of the embodiments of the present invention, and the scope of the present invention should be determined by the scope of the claims.