CN113461833A - Method for extracting astragalus mongholicus polysaccharide - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract description 56
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 252
- 238000000605 extraction Methods 0.000 claims abstract description 204
- 241001061264 Astragalus Species 0.000 claims abstract description 64
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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Abstract
本发明涉及植物提取技术领域，具体提供了一种蒙古黄芪多糖的提取方法。本发明采用水提醇沉的方提取蒙古黄芪多糖；其中水浸提的液料比为(10～15)mL:1g，提取温度为80～90℃，提取时间为90～120min；醇沉用乙醇的体积分数为90～95％。本发明提供的方法步骤简单，容易操作，对设备的要求低，提取率高；进一步的，本发明通过响应面法确定蒙古黄芪多糖的提取条件，能够充分考虑各个因素及两两因素相互作用对提取率的影响，避免单因素试验处理之后的正交设计的片面性，最终确定的提取条件高效、稳定、可行，提取率高，具有较高的推广应用价值。
The invention relates to the technical field of plant extraction, and specifically provides an extraction method of Astragalus mongolica polysaccharide. The present invention adopts the formula of water extraction and alcohol precipitation to extract Mongolian Astragalus polysaccharide; wherein the liquid-material ratio of water extraction is (10-15) mL:1 g, the extraction temperature is 80-90° C., and the extraction time is 90-120 min; The volume fraction of ethanol is 90-95%. The method provided by the invention has simple steps, easy operation, low requirements on equipment and high extraction rate; further, the invention determines the extraction conditions of Astragalus mongolica polysaccharide by response surface method, which can fully consider various factors and the interaction between two factors. The influence of the extraction rate is avoided, and the one-sidedness of the orthogonal design after the single-factor experiment is avoided. The extraction conditions finally determined are efficient, stable and feasible, and the extraction rate is high, which has a high value of popularization and application.
Description
Technical Field
The invention relates to the technical field of plant extraction, and particularly relates to an extraction method of astragalus mongholicus polysaccharide.
Background
Radix astragali is the dried root of Astragalus membranaceus bge or Astragalus membranaceus bge of Leguminosae, and contains substances beneficial to human health such as polysaccharide, glycoside, and flavone. The main component of the astragalus contains water-soluble astragalus polysaccharide, has wide pharmacological action, and has the effects of regulating the immunologic function of a human body, enhancing the metabolic capability of cells, reducing blood sugar and blood pressure, resisting inflammation, protecting the liver and the like. Recent researches show that astragalus polysaccharide can slow down the progress of pulmonary fibrosis, induce anti-inflammatory reaction in intestinal tracts, relieve metabolic disorder caused by high-fat diet and relieve the development of Parkinson's disease. Therefore, the optimization of the extraction process of the astragalus mongholicus polysaccharide has important significance.
In recent years, new technologies such as an enzymolysis method, a microwave-assisted extraction method, an ultrasonic-assisted extraction method and the like exist in the extraction process of astragalus polysaccharide, but the methods have high requirements on equipment and are complex to operate.
Disclosure of Invention
In view of the above, the invention provides an extraction method of astragalus mongholicus polysaccharide, which is simple to operate, low in equipment requirement and high in polysaccharide extraction rate.
In order to achieve the purpose, the invention provides the following scheme:
a method for extracting Mongolian astragalus polysaccharide comprises the following steps:
leaching Mongolian astragalus powder with water to obtain a leaching solution;
centrifuging the leaching solution to obtain a supernatant;
mixing ethanol and the supernatant, and precipitating with ethanol to obtain crude Mongolian radix astragali polysaccharide;
wherein the liquid-material ratio of the water extraction is (10-15) mL:1g, the extraction temperature is 80-90 ℃, and the extraction time is 90-120 min; the volume fraction of the ethanol is 90-95%.
Preferably, the method for determining the liquid-material ratio of water extraction, the extraction temperature, the extraction time and the volume fraction of ethanol used for alcohol precipitation comprises the following steps:
adopting a single variable method to obtain each single factor value of the water leaching and alcohol precipitation process corresponding to the maximum extraction rate of the astragalus mongholicus polysaccharide; the single factor values comprise the liquid-material ratio of water extraction, the extraction temperature, the extraction time or the volume fraction of ethanol used for alcohol precipitation;
taking each single factor value corresponding to the maximum extraction rate of the Mongolian astragalus polysaccharide as an independent variable, taking the extraction rate of the Mongolian astragalus polysaccharide as a response value, and carrying out response surface method optimization through Design-Expert11 software to obtain a regression equation of the extraction rate Y of the Mongolian astragalus polysaccharide as shown in the formula I:
Y＝3.32+0.8334A+0.0435B-0.0047C-0.1158D-0.1960AB+0.1680AC+0.0263AD-0.0493BC+0.0103BD+0.0002CD-1.20A2-0.3048B2-0.0573C2+0.1336D2formula I;
in formula I: a is liquid-material ratio, B is extraction temperature, C is volume fraction of ethanol, and D is extraction time;
analyzing by Design-Expert11 software to obtain the maximum value of the extraction rate of the astragalus mongholicus polysaccharide, the optimal liquid-material ratio, the extraction time, the extraction temperature and the volume fraction of ethanol under the interaction of four factors, namely the liquid-material ratio, the extraction time, the extraction temperature and the volume fraction of ethanol.
Preferably, in the single variable method, the value range of each single factor is as follows: the liquid-material ratio is (5-50) mL:1 g; the extraction time is 30-180 min, the extraction temperature is 50-100 ℃, and the volume fraction of ethanol is 75-100%.
Preferably, when the response surface optimization is carried out, a four-factor three-level test design is carried out according to the Box-Benhnken design principle.
Preferably, the liquid-material ratio of the water extraction is 11mL:1g, the extraction temperature is 80 ℃, and the extraction time is 100 min; the volume fraction of ethanol is 95%.
Preferably, the particle size of the Mongolian astragalus powder is less than or equal to 0.25 mm.
Preferably, the rotating speed of the centrifugation is 3000-4000 r/min, and the time is 10-15 min.
Preferably, the volume ratio of the ethanol to the supernatant is 2-3: 1; and the alcohol precipitation time is 10-14 h.
Preferably, after the alcohol precipitation is finished, the method further comprises the steps of centrifuging the obtained alcohol precipitation liquid to obtain a precipitate, and washing the precipitate by using absolute ethyl alcohol to obtain the crude astragalus mongholicus polysaccharide.
The invention provides an extraction method of astragalus mongholicus polysaccharide, which comprises the following steps: leaching Mongolian astragalus powder with water to obtain a leaching solution; centrifuging the leaching solution to obtain a supernatant; precipitating the supernatant with ethanol to obtain crude Mongolian radix astragali polysaccharide; wherein the liquid-material ratio of the water extraction is (10-15) mL:1g, the extraction temperature is 80-90 ℃, and the extraction time is 90-120 min; the volume fraction of the ethanol is 90-95%. The method provided by the invention adopts a water extraction and alcohol precipitation method to extract the astragalus mongholicus polysaccharide, and has the advantages of simple steps, easiness in operation, low requirement on equipment and high extraction rate.
Furthermore, the extraction parameters are obtained by a response surface optimization method, the influence of 4 different single factors on the extraction rate of the astragalus mongholicus polysaccharide by the liquid-material ratio, the extraction temperature, the ethanol volume fraction and the extraction time is respectively inspected by a single variable experiment, and then the response surface method is adopted to optimize the extraction conditions of the astragalus mongholicus polysaccharide on the basis of the single factor experiment so as to determine the optimal process conditions for extracting the astragalus mongholicus polysaccharide. The method determines the extraction conditions of the astragalus mongholicus polysaccharides by a response surface method, can fully consider the influence of each factor and the interaction of every two factors on the extraction rate, avoids one-sidedness of orthogonal design after single-factor test treatment, and has the advantages of high efficiency, stability, feasibility, high extraction rate and higher popularization and application values.
Drawings
FIG. 1 is a graph showing the effect of liquid-to-liquid ratio on the extraction rate of Astragalus mongholicus polysaccharides in a single-factor experiment;
FIG. 2 is a graph showing the effect of extraction temperature on the extraction rate of Astragalus polysaccharides from Mongolia in a single factor experiment;
FIG. 3 is a graph showing the effect of ethanol volume fraction on the extraction rate of Astragalus polysaccharides from Mongolia in a single factor experiment;
FIG. 4 is a graph showing the effect of extraction time on the extraction rate of Astragalus polysaccharides from Mongolia in a single factor experiment;
FIG. 5 is a graph of the liquid-to-material ratio and extracted temperature interaction response surface and contour plot;
FIG. 6 is a surface graph and contour plot of the interaction response of liquid-to-material ratio and ethanol volume fraction;
FIG. 7 is a plot of liquid-to-material ratio and extraction time interaction response surface and contour plot;
FIG. 8 is a plot of extraction temperature and ethanol volume fraction interaction response surface and contour plot;
FIG. 9 is a graph of extracted temperature and extracted time interaction response surfaces and contour plots;
FIG. 10 is an ethanol volume fraction and extraction time interaction response surface plot and contour plot.
Detailed Description
The invention provides an extraction method of astragalus mongholicus polysaccharide, which comprises the following steps:
leaching Mongolian astragalus powder with water to obtain a leaching solution;
centrifuging the leaching solution to obtain a supernatant;
mixing ethanol and the supernatant, and precipitating with ethanol to obtain crude Mongolian radix astragali polysaccharide;
wherein the liquid-material ratio of the water extraction is (10-15) mL:1g, the extraction temperature is 80-90 ℃, and the extraction time is 90-120 min; the volume fraction of the ethanol is 90-95%.
The invention carries out water leaching on Mongolian astragalus powder to obtain a leaching solution. In the invention, the grain diameter of the Mongolian astragalus powder is preferably less than or equal to 0.25mm, and preferably less than or equal to 0.2 mm; according to the invention, Mongolian milkvetch root is preferably crushed and sieved, and the obtained undersize is dried to obtain Mongolian milkvetch root powder; the comminution is preferably carried out in a mill; the mesh number of the screen for sieving is preferably 60 meshes, and the drying temperature is preferably 65 ℃.
In the invention, the liquid-material ratio of water extraction is (10-15) mL:1g, preferably 11mL:1g, the extraction temperature is 80-90 ℃, preferably 80 ℃, and the extraction time is 90-120 min, preferably 100 min; the invention has no special requirement on water used for water leaching, and the water well known to those skilled in the art can be used, such as deionized water; in the embodiment of the invention, water and astragalus mongholicus powder are preferably mixed according to a liquid-material ratio, and then leaching is carried out under a water bath condition, and stirring is preferably carried out once every half hour in the leaching process.
After obtaining the leaching liquor, the invention centrifuges the leaching liquor to obtain the supernatant. In the invention, the rotation speed of the centrifugation is preferably 3000-4000 r/min, more preferably 3000-3500 r/min, and the time is preferably 10-15 min, more preferably 10-12 min.
After obtaining the supernatant, mixing ethanol and the supernatant for alcohol precipitation to obtain the crude astragalus mongholicus polysaccharide. In the invention, the volume fraction of the ethanol is 90-95%, preferably 95%; the volume ratio of the ethanol to the supernatant is preferably 2-3: 1, and more preferably 2.5-3: 1; the alcohol precipitation time is preferably 10-14 h, and more preferably 12 h; in the embodiment of the present invention, it is preferable that ethanol and the supernatant are mixed and then left to stand at 4 ℃ for alcohol precipitation.
After the alcohol precipitation is finished, the obtained alcohol precipitation liquid is preferably centrifuged to obtain a precipitate, and the precipitate is washed by using absolute ethyl alcohol to obtain the crude Mongolian milkvetch root polysaccharide. In the invention, the rotation speed of the centrifugation is preferably 3000r/min, and the time is preferably 2 min; after the absolute ethyl alcohol is used for washing the precipitate, the invention preferably carries out natural volatilization on the residual ethyl alcohol in the precipitate, and after the volatilization is finished, crude astragalus mongholicus polysaccharide is obtained.
In the invention, the method for determining the liquid-material ratio of water extraction, the extraction temperature, the extraction time and the volume fraction of ethanol used for alcohol precipitation comprises the following steps:
adopting a single variable method to obtain each single factor value of the water leaching and alcohol precipitation process corresponding to the maximum extraction rate of the astragalus mongholicus polysaccharide; the single factor values comprise the liquid-material ratio of water extraction, the extraction temperature, the extraction time or the volume fraction of ethanol used for alcohol precipitation;
taking each single factor value corresponding to the maximum extraction rate of the Mongolian astragalus polysaccharide as an independent variable, taking the extraction rate of the Mongolian astragalus polysaccharide as a response value, and carrying out response surface method optimization through Design-Expert11 software to obtain a regression equation of the extraction rate Y of the Mongolian astragalus polysaccharide as shown in the formula I:
Y＝3.32+0.8334A+0.0435B-0.0047C-0.1158D-0.1960AB+0.1680AC+0.0263AD-0.0493BC+0.0103BD+0.0002CD-1.20A2-0.3048B2-0.0573C2+0.1336D2formula I;
in formula I: a is liquid-material ratio, B is extraction temperature, C is volume fraction of ethanol, and D is extraction time;
analyzing by Design-Expert11 software to obtain the maximum value of the extraction rate of the astragalus mongholicus polysaccharide, the optimal liquid-material ratio, the extraction time, the extraction temperature and the volume fraction of ethanol under the interaction of four factors, namely the liquid-material ratio, the extraction time, the extraction temperature and the volume fraction of ethanol.
The invention adopts a single variable method to obtain each single factor value of the water leaching and alcohol precipitation process corresponding to the maximum extraction rate of the astragalus mongholicus polysaccharide. In the invention, the value range of each single factor in the single variable method is preferably as follows: the liquid-material ratio is (5-50) mL:1 g; the extraction time is 30-180 min, the extraction temperature is 50-100 ℃, and the volume fraction of ethanol is 75-100%; in a specific embodiment of the invention, the liquid-to-material ratio is 5mL:1g, 10mL:1g, 20mL:1g, 30mL:1g, 40mL:1g and 50mL:1 g; the extraction time is 30min, 60min, 90min, 120min, 150min and 180 min; the extraction temperature is 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃ and 100 ℃; the volume fraction of the ethanol is 75%, 80%, 85%, 90%, 95% and 100%. The present invention has no special requirements for the specific experimental design and operation of the single variable method, and the design and operation are well known to those skilled in the art. In a specific embodiment of the present invention, the specific experimental design of the single-variable method is preferably: extracting Astragalus polysaccharides at 90 deg.C for 120min with ethanol volume fraction of 95% at liquid-to-material ratio of 5mL:1g, 10mL:1g, 20mL:1g, 30mL:1g, 40mL:1g and 50mL:1g respectively; extracting Astragalus polysaccharides at 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C, 90 deg.C and 100 deg.C respectively under conditions of liquid-material ratio of 10mL:1g, extraction time of 120min and volume fraction of ethanol of 95%; extracting radix astragali polysaccharide with ethanol integral fraction of 75%, 80%, 85%, 90%, 95% and 100% respectively at liquid-material ratio of 10mL:1g, extraction time of 120min and extraction temperature of 80 deg.C; extracting radix astragali polysaccharide with 10 mL/1 g of ethanol at 80 deg.C for 30min, 60min, 90min, 120min, 150min and 180min respectively.
After single variable experiments are carried out according to the method, a plurality of groups of crude Mongolian astragalus polysaccharide products are obtained, the extraction rate of Mongolian astragalus polysaccharide is calculated by testing the polysaccharide content in the obtained crude Mongolian astragalus polysaccharide products, and the value of each single variable when the extraction rate of Mongolian astragalus polysaccharide is maximum is determined. In the present invention, the extraction rate of the astragalus mongholicus polysaccharide is preferably measured by the following method:
providing a glucose gradient standard solution, measuring the absorbance of the glucose gradient standard solution at 490nm by adopting a phenol-sulfuric acid method, and drawing a standard curve by taking the glucose concentration as an abscissa and the absorbance value as an ordinate;
dissolving the crude astragalus mongholicus polysaccharide product to be detected by using distilled water to obtain a liquid to be detected, then determining the absorbance of the liquid to be detected at 490nm by using a phenol-sulfuric acid method, calculating the polysaccharide content in the astragalus mongholicus polysaccharide product according to the obtained absorbance value and the standard curve, and further calculating the polysaccharide extraction rate according to the polysaccharide content.
The preparation method of the glucose standard solution and the specific operation method for measuring the absorbance by the phenol-sulfuric acid method have no special requirements, and the method known by the technicians in the field can be adopted.
After determining each single factor value corresponding to the maximum extraction rate of the Mongolian astragalus polysaccharides by using a single variable method, the invention takes each determined single factor value as an independent variable and the extraction rate of the Mongolian astragalus polysaccharides as a response value, and carries out response surface method optimization by using Design-Expert11 software to obtain a regression equation of the extraction rate Y of the Mongolian astragalus polysaccharides as shown in formula I (see the above). In the invention, when the response surface is optimized, a four-factor three-level test design is preferably carried out according to a Box-Benhnken design principle; in the specific embodiment of the invention, when the response surface is optimized, the liquid-material ratio is 5mL:1g, 10mL:1g and 15mL:1g, the extraction temperature is 70 ℃, 80 ℃ and 90 ℃, the ethanol volume fraction is 90%, 95% and 100%, and the extraction time is 90min, 120min and 150 min.
After obtaining the regression equation shown in the formula I, the method analyzes through Design-Expert11 software to obtain the maximum value of the extraction rate of the astragalus mongholicus polysaccharide under the interaction of four factors of liquid-material ratio, extraction time, extraction temperature and ethanol volume fraction, and the optimal liquid-material ratio, extraction time, extraction temperature and ethanol volume fraction.
In the invention, the following results are obtained through response surface method analysis: when the liquid-material ratio of water extraction is 11.37mL:1g, the extraction temperature is 80.64 ℃, the volume fraction of ethanol is 94.22%, and the extraction time is 100.93min, the extraction rate of Mongolian astragalus polysaccharide is the highest, and the predicted value of the extraction rate of astragalus polysaccharide under the process condition is 3.56%. By referring to the best fitting extraction conditions and combining the practical situation of a laboratory, the invention determines the best process conditions for extracting the astragalus mongholicus polysaccharide as follows: the liquid-material ratio is 11mL:1g, the extraction temperature is 80 ℃, the volume fraction of ethanol is 95%, the extraction time is 100min, and the extraction rate of the astragalus mongholicus polysaccharide under the optimized condition is 3.48% and is very close to the predicted value of 3.56% through experimental verification. In addition, experimental results show that the polysaccharide extraction rate is high when the liquid-material ratio of water extraction is (10-15) mL:1g, the extraction temperature is 80-90 ℃, the extraction time is 90-120 min, and the volume fraction of ethanol is 90-95%.
According to the method, the extraction conditions of the astragalus mongholicus polysaccharides are determined through the response surface, the influence of each factor and the interaction of every two factors on the extraction rate can be fully considered, the one-sidedness of a single factor test is avoided, and the finally determined extraction conditions are efficient and stable and the extraction rate is high.
In order to better understand the present invention, the following examples are further provided to illustrate the present invention, but the present invention is not limited to the following examples.
The method for measuring the polysaccharide extraction rate in the embodiment of the invention comprises the following steps:
drawing a glucose standard curve: drying glucose in an oven at 105 ℃ to constant weight, dissolving 0.1g of glucose in water, and fixing the volume by using a 100ml volumetric flask to obtain a glucose standard solution; 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5mL of the standard glucose solution was pipetted and diluted to 100mL with water to obtain a glucose gradient standard solution. 2mL of glucose solution with different concentrations is taken, 1.0mL of phenol solution (6%) and 5.0mL of concentrated sulfuric acid are added, mixed uniformly immediately, placed in a water bath kettle at 40 ℃ for 40min in a water bath, cooled to room temperature, and then a visible spectrophotometer is used for testing the absorbance of the solution at the lambda of 490 nm. A standard curve was drawn with the glucose concentration (. mu.g/mL) as the abscissa and the absorbance value as the ordinate.
The method for measuring the polysaccharide content in the astragalus mongholicus by using a phenol-sulfuric acid method comprises the following steps: dissolving Mongolian polysaccharide astragalus into distilled water to obtain a solution to be detected, sucking 200 mu L of the solution to be detected into a dry test tube, adding 1800 mu L of pure water, 1.0mL of phenol solution (6%) and 5mL of concentrated sulfuric acid, uniformly mixing, carrying out water bath in a water bath kettle at 40 ℃ for 40min, cooling to room temperature, and testing the absorbance of the solution at the lambda-490 nm position by using a visible spectrophotometer; substituting the absorbance value into the glucose standard curve to obtain the content of Mongolian radix astragali polysaccharide; further calculating to obtain the extraction rate of the Mongolian astragalus polysaccharide, wherein the formula for calculating the extraction rate of the Mongolian astragalus polysaccharide is as follows:
polysaccharide extraction rate/%, polysaccharide content (g)/raw material mass (g) × 100% formula I;
in formula I: the polysaccharide content is the content of the Mongolian astragalus polysaccharide in the product obtained by calculation through a standard curve, and the raw material quality is the quality of Mongolian astragalus powder.
Example 1
1. The general extraction process of this embodiment is: crushing Mongolian milkvetch roots by a crusher, sieving by a 60-mesh sieve, drying at 65 ℃, precisely weighing 10g of milkvetch root powder, carrying out water extraction according to the corresponding liquid-material ratio, extraction temperature and extraction time, centrifuging the obtained leaching liquor at the rotating speed of 3000r/min for 10min to obtain supernatant, uniformly mixing ethanol with the supernatant at the volume of 3 times of that of the supernatant, standing at-4 ℃ for alcohol precipitation for 12h, centrifuging at the rotating speed of 3000r/min for 2min, leaving precipitate, washing the precipitate by a proper amount of absolute ethanol, and naturally volatilizing the ethanol to obtain crude Mongolian milkvetch root polysaccharide.
2. Single factor tests were performed with the liquid-to-material ratio, extraction temperature, ethanol concentration, and extraction time as 4 influencing factors of the astragalus polysaccharide extraction rate.
(1) Single-factor experiment for influencing extraction rate of astragalus mongholicus polysaccharide by liquid-material ratio
Extracting Astragalus polysaccharides at 90 deg.C for 120min, with ethanol volume fraction of 95%, setting liquid-material ratio at 5:1, 10:1, 20:1, 30:1, 40:1, and 50:1(mL: g), respectively determining polysaccharide content in the product, and calculating extraction rate.
The measurement results are shown in FIG. 1. As can be seen from FIG. 1, under the same conditions of extraction temperature, extraction time and alcohol precipitation concentration, the extraction rate of Astragalus polysaccharides increases from 5:1 to 10:1, reaches the maximum value at 10:1, and then gradually decreases with the increase of the liquid-to-material ratio. Thus, the preferred liquid-to-feed ratio for this experiment was 10:1(mL: g).
(2) Single factor experiment of temperature influence on extraction rate of astragalus mongholicus polysaccharide
Extracting Astragalus polysaccharides with a liquid-material ratio of 10:1(mL: g) for 120min under 95% ethanol precipitation conditions, performing experiments at 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C, 90 deg.C, and 100 deg.C, measuring polysaccharide content, and calculating extraction rate.
The measurement results are shown in FIG. 2. As can be seen from FIG. 2, under the condition of the same liquid-material ratio, extraction time and alcohol precipitation concentration, when the extraction temperature is 50 ℃ to 80 ℃, the extraction rate of astragalus polysaccharide is positively correlated with the temperature, and reaches the maximum value at 80 ℃, and then the extraction rate is reduced along with the increase of the extraction temperature. Therefore, 80 ℃ is selected as the better extraction temperature in the experiment.
(3) Single-factor experiment for influencing extraction rate of astragalus mongholicus polysaccharide by volume fraction of ethanol
Extracting radix astragali polysaccharide at liquid-to-material ratio of 10:1(mL: g) for 120min at 80 deg.C, setting ethanol volume fraction for alcohol precipitation at 75%, 80%, 85%, 90%, and 95%, respectively determining polysaccharide content and calculating extraction rate.
The measurement results are shown in FIG. 3. As can be seen from FIG. 3, under the same conditions of liquid-to-material ratio, extraction time and extraction temperature, the polysaccharide extraction rate increased from 75% to 95% of ethanol concentration, reached the highest point of extraction rate at 95% of ethanol concentration, and then decreased with the increase of ethanol concentration. Therefore, the concentration of the ethanol selected in the experiment is preferably 95%.
(4) Single-factor experiment for influencing extraction rate of astragalus mongholicus polysaccharide by extraction time
Extracting Astragalus polysaccharides at a liquid-material ratio of 10:1(mL: g) at 80 deg.C under 95% ethanol precipitation, performing water bath extraction for 30min, 60min, 90min, 120min, 150min, and 180min respectively, measuring polysaccharide content, and calculating extraction rate.
The measurement results are shown in FIG. 4; as can be seen from FIG. 4, under the conditions of liquid-to-material ratio, extraction temperature, and 95% ethanol precipitation, the extraction rate of Astragalus mongholicus polysaccharides in Mongolia showed an increasing trend in the gradient of water bath time from 30min to 120min, and reached the maximum value when the water bath time was 120min, and the increase of the extraction time did not increase the extraction rate of polysaccharides, but rather the extraction rate gradually stabilized. Therefore, the preferred extraction time for this experiment was 120 min.
3. Response surface optimal design: after the single-factor test is completed, the single-factor results obtained by the single-factor test are planned to be the optimal levels, meanwhile, response surface optimization Design is carried out, Design-expert11 software is used for carrying out four-factor three-level test Design according to the Box-Benhnken Design principle, the specific level setting is shown in table 1, and the test scheme and the response surface result are shown in table 2.
TABLE 1 factor level settings table
TABLE 2 response surface design and response values
Performing multiple regression fitting on each item of data (table 2) obtained by the experiment by using Design-expert11 software to obtain a quadratic multiple regression simulation equation of the extraction rate (Y) of the Mongolian astragalus polysaccharides: y is 3.32+0.8334A +0.0435B-0.0047C-0.1158D-0.1960AB +0.1680AC +0.0263AD-0.0493BC +0.0103BD +0.0002CD-1.20A2-0.3048B2-0.0573C2+0.1336D2，R2＝0.9295。
The results of the analysis of variance of the regression modeling equation are shown in Table 3.
TABLE 3 analysis of variance of the Astragalus polysaccharides regression simulation equation
| Sources of variance | Sum of squares | Degree of freedom | Mean square | F value | P value |
| Model (model) | 19.32 | 14 | 1.38 | 13.19 | <0.0001 |
| A | 8.34 | 1 | 8.34 | 79.67 | <0.0001 |
| B | 0.0227 | 1 | 0.0227 | 0.217 | 0.6485 |
| C | 0.0003 | 1 | 0.0003 | 0.0025 | 0.9608 |
| D | 0.1608 | 1 | 0.1608 | 1.54 | 0.2355 |
| AB | 0.1537 | 1 | 0.1537 | 1.47 | 0.2456 |
| AC | 0.1129 | 1 | 0.1129 | 1.08 | 0.3165 |
| AD | 0.0028 | 1 | 0.0028 | 0.0263 | 0.8734 |
| BC | 0.0097 | 1 | 0.0097 | 0.0927 | 0.7652 |
| BD | 0.0004 | 1 | 0.0004 | 0.0040 | 0.9504 |
| CD | 2.5E-07 | 1 | 2.5E-07 | 2.39E-06 | 0.9988 |
| A2 | 9.40 | 1 | 9.40 | 89.82 | <0.0001 |
| B2 | 0.6025 | 1 | 0.6025 | 5.76 | 0.0309 |
| C2 | 0.0213 | 1 | 0.0213 | 0.2033 | 0.6590 |
| D2 | 0.1158 | 1 | 0.1158 | 1.11 | 0.3106 |
| Residual error | 1.46 | 14 | 0.1046 | ||
| Missimilitude term | 1.29 | 10 | 0.1294 | 3.03 | 0.1484 |
| Pure error | 0.1708 | 4 | 0.0427 | ||
| Sum of | 20.78 | 28 |
As can be seen from Table 3, the regression simulation equation has high fitting degree and small error, and can be used for optimizing the extraction conditions of the Mongolian astragalus polysaccharides.
Analyzing by Design-expert11 software, analyzing and comparing the influence of the extraction rate of the Mongolian astragalus polysaccharides by taking the A liquid-material ratio, the B extraction temperature, the C ethanol concentration and the D extraction time as significant influence factors and the interaction of any two factors, and drawing a response surface graph and a contour graph as shown in figures 5-10: a represents the liquid-to-material ratio, B represents the extraction temperature, C represents the volume fraction of ethanol, and D represents the extraction time. The larger the gradient of the response curved surface is, the more sensitive the response value is to the change of the influence factors, and as can be seen from the steepness of the response curved surface in fig. 5-10, the liquid-material ratio has the most significant influence on the total polysaccharide extraction rate and is consistent with the analysis result of variance.
The optimal process conditions of the extraction rate of the crude astragalus mongholicus polysaccharide are obtained by combination of Design-expert11 software, single-factor experiment results and established mathematical model analysis fitting: liquid-material ratio 11.37: 1(mL: g), the extraction temperature is 80.64 ℃, the volume fraction of ethanol is 94.22 percent, the extraction time is 100.93min, and the predicted value of the extraction rate of the astragalus polysaccharide under the optimized process condition is 3.56 percent. And (3) determining the final optimal extraction conditions by referring to the optimal fitting extraction conditions and combining the actual conditions of a laboratory: liquid-material ratio of 11: 1(mL: g), the extraction temperature is 80 ℃, the ethanol volume fraction is 95%, the extraction time is 100min, and the extraction rate of the astragalus mongholicus polysaccharide under the optimized condition is 3.48% and is very close to the predicted value of 3.56%.
Example 2
Crushing Mongolian milkvetch root by a crusher, sieving by a 60-mesh sieve, drying the obtained undersize at 65 ℃ to obtain Mongolian milkvetch root powder, precisely weighing 10g of Mongolian milkvetch root powder, and performing water leaching according to the liquid-material ratio of 10mL to 1g, the extraction temperature of 80 ℃ and the extraction time of 90min to obtain a leaching solution; centrifuging the obtained leaching solution at a rotating speed of 3000r/min for 10min, collecting supernatant, mixing the supernatant with 90% ethanol with volume 3 times of the supernatant, standing for precipitating with ethanol for 12h, centrifuging the ethanol precipitation solution at a rotating speed of 3000r/min for 2min, collecting precipitate, washing the precipitate with appropriate amount of anhydrous ethanol, and naturally volatilizing the ethanol to obtain crude Astragalus mongholicus polysaccharide. The yield of astragalus polysaccharide is 3.409%.
Example 3
Crushing Mongolian milkvetch root by a crusher, sieving by a 60-mesh sieve, drying the obtained undersize at 65 ℃ to obtain Mongolian milkvetch root powder, precisely weighing 10g of Mongolian milkvetch root powder, and performing water leaching according to the liquid-material ratio of 15mL to 1g, the extraction temperature of 80 ℃ and the extraction time of 90min to obtain a leaching solution; centrifuging the obtained leaching solution at a rotating speed of 3000r/min for 10min, collecting supernatant, mixing the supernatant with 95% ethanol with volume 3 times of that of the supernatant, standing for precipitating with ethanol for 12h, centrifuging the ethanol precipitation solution at a rotating speed of 3000r/min for 2min, collecting precipitate, washing the precipitate with appropriate amount of anhydrous ethanol, and naturally volatilizing the ethanol to obtain crude Astragalus mongholicus polysaccharide. The yield of astragalus polysaccharide is 3.402%.
Example 4
Crushing Mongolian milkvetch root by a crusher, sieving by a 60-mesh sieve, drying the obtained undersize at 65 ℃ to obtain Mongolian milkvetch root powder, precisely weighing 10g of Mongolian milkvetch root powder, and performing water leaching according to the liquid-material ratio of 10mL to 1g, the extraction temperature of 80 ℃ and the extraction time of 120min to obtain a leaching solution; centrifuging the obtained leaching solution at a rotating speed of 3000r/min for 10min, collecting supernatant, mixing the supernatant with 95% ethanol with volume 3 times of that of the supernatant, standing for precipitating with ethanol for 12h, centrifuging the ethanol precipitation solution at a rotating speed of 3000r/min for 2min, collecting precipitate, washing the precipitate with appropriate amount of anhydrous ethanol, and naturally volatilizing the ethanol to obtain crude Astragalus mongholicus polysaccharide. The yield of astragalus polysaccharide is 3.169%.
The principle and the implementation mode of the invention are explained by applying a specific example, and the description of the embodiment is only used for helping to understand the method and the core idea of the invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, the specific embodiments and the application range may be changed. In view of the above, the present disclosure should not be construed as limiting the invention.
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