Method for optimizing lysimachia christinae hance polysaccharide extraction conditions by using response surface method

Abstract

The invention discloses a method for optimizing lysimachia christinae hance polysaccharide extraction conditions by using a response surface method.The method comprises the following steps that S1, lysimachia christinae hance raw materials are pretreated, specifically, high-quality lysimachia christinae hance is selected, washed, dried, smashed and sieved, and lysimachia christinae S2, single-factor test: determining three factors, namely a material-liquid ratio, extraction time and extraction temperature, and determining the influence of a certain factor on the extraction rate of the lysimachia christinae hance polysaccharide from the lysimachia christinae hance powder under the condition that other conditions are not changed; s3, response surface test design: adopting a Box-Behnken response surface design method, on the basis of a single-factor experiment, taking the extraction rate of the christina loosestrife herb crude polysaccharide as a response value, performing polynomial fitting regression on the corresponding variables to obtain a regression equation, and calculating the regression equation to obtain the extraction conditions of the christina loosestrife herb crude polysaccharide after response surface optimization, the regression equation is Y = 7.71 + 0.96 A + 0.5575 B + 0.5575 C-0.195 AB + 0.655 AC + 0.17 BC-1.13 A2-1.48 B2-3.76 C, Y is the extraction rate of the christina loosestrife herb crude polysaccharide, the variable parameter X1 is the material-liquid ratio, the variable parameter X2 is the extraction time, and the variable parameter X3 is the extraction temperature. The optimal extraction condition can be quickly and accurately determined, the extraction rate of the lysimachia christinae hance polysaccharide is improved, the extraction process is simplified, and the production cost is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine extraction, and in particular to a method for optimizing extraction conditions of Lysimachia sibiricum polysaccharide by utilizing response surface methodology. Background Art

[0002] The herb of Lysimachia christinae Hance is the dried whole herb of Lysimachia christinae Hance, a plant of the genus Lysimachia in the family Primulaceae. Herb of Lysimachia is also known as the herb of the herb of Lysimachia christinae, the small lantern, the small money, the small copper coin grass, the horse hoof gold, the golden horse hoof grass, and a string of money. Herb of Lysimachia is used as medicine with the whole herb. It is a traditional Chinese herbal medicine in my country. It tastes sweet and salty, is slightly cold in nature, and has the effects of clearing dampness and heat, relieving stranguria, and reducing swelling. It is commonly used to treat hot stranguria, sand stranguria, painful urination, jaundice and red urine, hepatobiliary stones, urinary tract stones, etc. Existing pharmacological studies have shown that the components of Herb of Lysimachia include polysaccharides and flavonoids. Herb of Lysimachia polysaccharide is one of the main active ingredients of Herb of Lysimachia, and it has important physiological activities in anti-oxidation, promoting urination, regulating immunity, and anti-tumor.

[0003] The main methods for extracting Lysimachia polysaccharides include water extraction, ultrasonic-assisted extraction, microwave-assisted extraction, enzyme extraction, and supercritical fluid extraction, but compared with water extraction, other methods have problems such as complex process and high cost. At present, the process conditions of water extraction remain at the process parameters (solid-liquid ratio, extraction temperature, extraction time, etc.) found by the single factor control variable method, and there is a lack of using the response surface method to establish a continuous variable surface model to optimize the water extraction conditions of Lysimachia polysaccharides. Summary of the invention

[0004] Under this background condition, the present invention proposes a preparation method of a method for optimizing the extraction conditions of Lysimachia sibiricum polysaccharide by response surface methodology, selects suitable Lysimachia sibiricum raw material and performs pre-treatment, then, by single factor test, investigates the influence of extraction temperature, extraction time, solid-liquid ratio factors on the extraction rate of Lysimachia sibiricum polysaccharide, takes the single factor test optimal point as the center, takes a level value around the optimal point as the approximate range of each factor. Then, according to the single factor test result, design response surface test scheme, take polysaccharide extraction rate as response value, optimize extraction temperature, extraction time, solid-liquid ratio three factors. Statistical analysis software is used to analyze response surface test data, establish mathematical model, and obtain the optimal combination of each factor.

[0005] In order to overcome at least one of the above disadvantages of the prior art, the present invention provides a method for optimizing the extraction conditions of Lysimachia sibiricum polysaccharides using response surface methodology, comprising the following steps:

[0006] S1 Pretreatment of the Lysimachia sibiricum raw material: Select high-quality Lysimachia sibiricum, wash and dry it, and then crush and sieve it to obtain Lysimachia sibiricum powder;

[0007] S2 single factor test: determine the three factors of solid-liquid ratio, extraction time and extraction temperature, and measure the effect of one factor on the extraction rate of Lysimachia polysaccharide while keeping other conditions unchanged.

[0008] S3 response surface experimental design: The Box-Behnken response surface design method was used. On the basis of the single factor experiment, three factors, namely, solid-liquid ratio, extraction time and extraction temperature, were selected as corresponding variables. The optimal point of the single factor experiment was taken as the center. A level value was taken above and below the optimal point as the level of the response surface. The extraction rate of Lysimachia chinensis crude polysaccharide was taken as the response value. Polynomial fitting regression was performed on the corresponding variables to obtain the regression equation. The regression equation was calculated to obtain the extraction conditions of Lysimachia chinensis crude polysaccharide after the response surface optimization.

[0009] The regression equation is:

[0010] Y=Y=7.71+0.96A+0.5575B+0.5575C-0.195AB+0.655AC+0.17BC-1.13A 2 -1.48B 2 -3.76C,

[0011] Wherein, Y is the extraction rate of Lysimachia sibiricum polysaccharide, variable parameter X1 is the solid-liquid ratio, X2 is the extraction time, and X3 is the extraction temperature.

[0012] Furthermore, the selection ranges of the three factors are solid-liquid ratio 1:20 to 1:100; extraction time 1 to 6 hours; extraction temperature 40°C to 90°C.

[0013] Furthermore, the step S2 specifically includes:

[0014] Step 1, water extraction: extracting the Lysimachia sibiricum powder with water, centrifuging, filtering, and concentrating by rotary evaporation to obtain a Lysimachia sibiricum crude polysaccharide concentrate;

[0015] Step 2, alcohol precipitation: subjecting the concentrated solution of Lysimachia sibiricum polysaccharide to alcohol precipitation overnight, dissolving the solid after the alcohol precipitation, and then freeze-drying to obtain Lysimachia sibiricum polysaccharide;

[0016] Step 3, removing protein: removing impurity protein in the crude polysaccharide of Lysimachia sibiricum;

[0017] Step 4, impurity removal: removing small molecular impurities in the crude Lysimachia herba polysaccharide to obtain purified Lysimachia herba polysaccharide.

[0018] Furthermore, in the step 1, the centrifugation conditions are: 7000-8000rpm, 5-8min; the rotary evaporation conditions are: 40-45°C, 100-120rpm, and the rotary evaporation is completed when the remaining liquid is 1 / 3 of the original volume to obtain the Lysimachia sibiricum crude polysaccharide concentrate.

[0019] Furthermore, in the step 2, the alcohol precipitation condition is to add 4 times the volume of anhydrous ethanol to the Lysimachia sibiricum crude polysaccharide concentrate, and then put it in a 4°C refrigerator for alcohol precipitation overnight.

[0020] Furthermore, the method for removing protein from the crude polysaccharide of Lysimachia sibiricum in step three is the Sevage method for removing protein.

[0021] Further, the Sevage method removes the specific method of protein in the crude polysaccharide of Lysimachia sibiricum by distilled water: use distilled water to dissolve the dried crude polysaccharide of Lysimachia sibiricum, add 1 / 4 volume of Sevage reagent (chloroform: n-butanol=4:1 / V:V), fully oscillate 30min (using vortex) at room temperature on a constant temperature oscillator, and then centrifuge, and the centrifugation condition is: 9000rpm, 15min; After separating the organic phase and the aqueous phase, the aqueous phase is added with a Sevage reagent equivalent to 1 / 4 of its volume, and the above process is repeated until there is no white precipitation at the junction of the organic layer and the aqueous layer after centrifugation, then it is proved that the protein is removed cleanly. Further, in the step 4, the method used for removing the small molecule impurities in the crude polysaccharide of Lysimachia sibiricum is dialysis.

[0022] Furthermore, the specific method of removing small molecular impurities in the crude polysaccharide of Lysimachia chinensis by dialysis is as follows: placing the crude polysaccharide solution of Lysimachia chinensis after protein removal by the Sevage method in a dialysis bag, adding deionized water to the environment of the dialysis bag, changing the water every half a day, collecting the solution in the bag after 24 hours, and obtaining the Lysimachia chinensis polysaccharide after freeze-drying.

[0023] Furthermore, the S3 response surface experimental design also includes: evaluating the significance and goodness of fit of the model through the variance analysis method, analyzing the influence of the interaction between the three factors on the extraction rate, and determining the optimal level combination of each factor. After analysis, the optimal extraction conditions are: solid-liquid ratio 1:64, extraction time 7.2 hours, and extraction temperature 71.2°C.

[0024] The invention has the beneficial effects of optimizing the extraction process conditions of Lysimachia polysaccharide by using the response surface method on the basis of the single factor experiment. The influence of the three conditions of solid-liquid ratio, extraction temperature and extraction time on the extraction rate of Lysimachia polysaccharide is more significant. On the basis of the single factor experiment, a three-factor three-level orthogonal experiment is designed, and the extraction conditions of Lysimachia polysaccharide are optimized by using the response surface method, so that the optimal extraction conditions can be determined quickly and accurately, the extraction rate of Lysimachia polysaccharide is improved, the extraction process is simplified, the production cost is reduced, and the industrial production and further application of Lysimachia polysaccharide are facilitated. Finally, the optimal process conditions for extracting Lysimachia polysaccharide are determined: solid-liquid ratio 1:64, extraction time 7.2 hours, extraction temperature 71.2°C, which provides a reference basis for the development and utilization of Lysimachia polysaccharide. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0026] Figure 1 This is a graph showing the effect of the solid-liquid ratio on the extraction rate of Lysimachia sibiricum polysaccharide in an embodiment of the present invention;

[0027] Figure 2 This is a graph showing the effect of extraction time on the extraction rate of Lysimachia sibiricum polysaccharide in an embodiment of the present invention;

[0028] Figure 3 This is a graph showing the effect of extraction temperature on the extraction rate of Lysimachia sibiricum polysaccharide in an embodiment of the present invention;

[0029] Figure 4 This is a graph showing the effect of the solid-liquid ratio and the extraction time on the extraction rate of Lysimachia sibiricum polysaccharide in the embodiment of the present invention;

[0030] Figure 5 This is a graph showing the influence of the solid-liquid ratio and the extraction temperature on the extraction rate of Lysimachia sibiricum polysaccharide in the embodiment of the present invention;

[0031] Figure 6 This is a graph showing the effects of extraction time and extraction temperature on the extraction rate of Lysimachia sibiricum polysaccharide in an embodiment of the present invention. DETAILED DESCRIPTION

[0032] It should be mentioned before discussing the exemplary embodiments in more detail that some exemplary embodiments are described as processes or methods depicted as flow charts. Although the flow charts describe the operations as sequential processes, many of the operations therein can be implemented in parallel, concurrently or simultaneously. In addition, the order of the operations can be rearranged. The process can be terminated when its operation is completed, but can also have additional steps not included in the accompanying drawings. The process can correspond to a method, function, procedure, subroutine, subprogram, etc.

[0033] It should be understood that, although the terms "first", "second", etc. may be used herein to describe various units, these units should not be limited by these terms. These terms are used only to distinguish one unit from another unit. For example, without departing from the scope of the exemplary embodiments, the first unit may be referred to as the second unit, and similarly the second unit may be referred to as the first unit. The term "and / or" used herein includes any and all combinations of one or more of the listed associated items.

[0034] The present invention provides a method for optimizing the extraction conditions of Lysimachia sibiricum polysaccharide by using response surface methodology, comprising:

[0035] S1 Pretreatment of the Lysimachia sibiricum raw material: Select high-quality Lysimachia sibiricum, wash and dry it, and then crush and sieve it to obtain Lysimachia sibiricum powder;

[0036] S2 single factor test: determine the three factors of solid-liquid ratio, extraction time and extraction temperature, and measure the effect of one factor on the extraction rate of Lysimachia polysaccharide while keeping other conditions unchanged.

[0037] S3 response surface experimental design: The Box-Behnken response surface design method was used. On the basis of the single factor experiment, three factors, namely, solid-liquid ratio, extraction time and extraction temperature, were selected as corresponding variables. The optimal point of the single factor experiment was taken as the center. A level value was taken above and below the optimal point as the level of the response surface. The extraction rate of Lysimachia chinensis crude polysaccharide was taken as the response value. Polynomial fitting regression was performed on the corresponding variables to obtain the regression equation. The regression equation was calculated to obtain the extraction conditions of Lysimachia chinensis crude polysaccharide after the response surface optimization.

[0038] The regression equation is:

[0039] Y=Y=7.71+0.96A+0.5575B+0.5575C-0.195AB+0.655AC+0.17BC-1.13A 2 -1.48B 2 -3.76C,

[0040] Wherein, Y is the extraction rate of Lysimachia sibiricum polysaccharide, variable parameter X1 is the solid-liquid ratio, X2 is the extraction time, and X3 is the extraction temperature.

[0041] Step S2 also includes: step 1, water extraction: extracting the Lysimachia sibiricum powder with water, centrifuging, filtering, and concentrating by rotary evaporation after water extraction to obtain a Lysimachia sibiricum crude polysaccharide concentrate;

[0042] Step 2, alcohol precipitation: subjecting the concentrated solution of Lysimachia sibiricum polysaccharide to alcohol precipitation overnight, dissolving the solid after the alcohol precipitation, and then freeze-drying to obtain Lysimachia sibiricum polysaccharide;

[0043] Step 3, removing protein: removing impurity protein in the crude polysaccharide of Lysimachia sibiricum;

[0044] Step 4, impurity removal: removing small molecular impurities in the crude Lysimachia herba polysaccharide to obtain purified Lysimachia herba polysaccharide.

[0045] Example 1

[0046] S1 Pretreatment of the Lysimachia sibiricum raw material: Select high-quality Lysimachia sibiricum, wash and dry it, and then crush and sieve it to obtain Lysimachia sibiricum powder;

[0047] S2 single factor test: extract the Lysimachia sibiricum powder at a solid-liquid ratio of 1:15-1:35 g / ml at 30℃-70℃ for 0.5-2.5h, and determine the effect of a certain factor on the extraction rate of Lysimachia sibiricum polysaccharide while keeping other conditions unchanged.

[0048] Effect of solid-liquid ratio on the extraction rate of Lysimachia chinensis polysaccharides: Weigh the pretreated Lysimachia chinensis powder, mix it in a solid-liquid (extracting liquid is water) ratio of 1:20, 1:40, 1:60, 1:80, and 1:100, extract it at 70°C for 2h, centrifuge it (8000r / min, 5min), collect the supernatant, add anhydrous ethanol to the alcohol precipitation concentration reaching 80%, precipitate it at 4°C overnight, filter it, collect the filter cake, and freeze-dry it to obtain crude Lysimachia chinensis polysaccharides, and calculate the extraction rate of crude Lysimachia chinensis polysaccharides.

[0049] The calculation formula of the extraction rate of Lysimachia polysaccharide is: Y (%) = Lysimachia polysaccharide mass / Lysimachia raw material powder mass × 100%. The ratio of raw material to extract (water) is a parameter that significantly affects the extraction rate.

[0050] The crude polysaccharide of Lysimachia was dissolved in water, and the impurity protein in the crude polysaccharide solution of Lysimachia was removed by Sevage method: the dried crude polysaccharide of Lysimachia was dissolved in distilled water, 1 / 4 volume of Sevage reagent (chloroform: n-butanol = 4: 1 / V: V) was added, and the mixture was fully oscillated at room temperature for 30 min on a constant temperature oscillator (using vortex), and then centrifuged, and the centrifugation conditions were: 9000 rpm, 15 min. The organic phase and the aqueous phase were separated, and then the aqueous phase was added with Sevage reagent equivalent to 1 / 4 of its volume, and the above process was repeated until there was no white precipitate at the junction of the organic layer and the aqueous layer after centrifugation, indicating that the protein was completely removed.

[0051] The small molecule impurities in the crude polysaccharide of Lysimachia chinensis are removed by dialysis, and the Lysimachia chinensis polysaccharide is obtained after freeze-drying: the crude polysaccharide solution of Lysimachia chinensis after protein removal by Sevage method is placed in a dialysis bag (MW: 3500Da), deionized water is added to the environment of the dialysis bag, the water is changed every half a day, the solution in the bag is collected after 24 hours, and the Lysimachia chinensis polysaccharide is obtained after freeze-drying.

[0052] Purity detection of Lysimachia chinensis polysaccharide: The purified Lysimachia chinensis polysaccharide was detected using the phenol-sulfuric acid method.

[0053] Figure 1 The figure below is the effect of the material-liquid ratio on the extraction rate of Lysimachia polysaccharides. Analysis shows that when the ratio of raw material to liquid is 1:60, the extraction rate of Lysimachia polysaccharides reaches a maximum value of 5.99%. The possible reason is that with the increase of the extraction material-liquid ratio, the mass transfer power will increase, making the contact area between the Lysimachia raw material powder and the water solvent larger, and the polysaccharides diffused into the solvent will also increase, resulting in the dissolution of water-soluble polysaccharides. The reason why the polysaccharide extraction rate decreases when the ratio is higher than 1:60 may be that the Lysimachia polysaccharides are basically dissolved in water at 1:60. At this time, the increase in the amount of water will increase the amount of solution in the subsequent alcohol precipitation, increasing the loss of Lysimachia polysaccharides.

[0054] Effect of extraction time on the extraction rate of Lysimachia chinensis polysaccharide: Weigh 1 g of pretreated Lysimachia chinensis powder, mix it at a solid-liquid ratio of 1:60, extract it at 70°C for 1h, 2h, 3h, 4h, 5h, and 6h, centrifuge it (8000r / min, 6min), collect the supernatant, add anhydrous ethanol to the alcohol precipitation concentration reaching 80%, precipitate it at 4°C overnight, filter it, collect the filter cake, and freeze-dry it to obtain Lysimachia chinensis polysaccharide, and calculate the extraction rate of Lysimachia chinensis polysaccharide. Figure 2 The figure below shows the effect of extraction time on the extraction rate of Lysimachia polysaccharide. Analysis shows that the extraction time of Lysimachia polysaccharide shows an upward trend when it is 1 to 3 hours; the extraction rate reaches the maximum level of 5.17% at 3 hours; and the extraction rate decreases with time when it is 3 to 6 hours. The possible reason for the decrease in extraction rate with the extension of extraction time is that with the extension of extraction time, in addition to polysaccharides, other components in plant cells such as proteins, pigments, tannins, etc. will also be dissolved in large quantities. These impurities will interact with polysaccharides and may form complexes or colloids, increase the viscosity and complexity of the extract, hinder the dissolution and separation of polysaccharides, and reduce the extraction rate of polysaccharides. In addition, long-term high temperature and water will hydrolyze the glycosidic bonds of polysaccharides, destroying the original complete polysaccharide structure and making it difficult to be completely extracted, thereby reducing the extraction rate.

[0055] Effect of temperature on the extraction rate of Lysimachia chinensis polysaccharide: Weigh 1 g of pretreated Lysimachia chinensis powder, mix it at a solid-liquid ratio of 1:60, extract it at 40°C, 50°C, 60°C, 70°C, 80°C, and 90°C for 3 h, centrifuge it (8000 r / min, 8 min), collect the supernatant, add anhydrous ethanol to the alcohol precipitation concentration reaching 80%, precipitate it at 4°C overnight, filter it, collect the filter cake, and freeze-dry it to obtain Lysimachia chinensis polysaccharide, and calculate the extraction rate of Lysimachia chinensis polysaccharide. Figure 3 The figure shows the effect of material extraction temperature on the extraction rate of Lysimachia polysaccharide. It can be seen from the analysis that the extraction rate of Lysimachia polysaccharide gradually increases in the range of 40-70℃, reaching a maximum value of 6.54% at 70℃. When the temperature exceeds 70℃, the extraction rate of Lysimachia polysaccharide decreases slightly. The reason for this result may be that as the temperature increases, the molecular movement accelerates, the solubility increases, and the corresponding extraction rate also increases. When the temperature exceeds 50℃, it may be unfavorable for the dissolution of polysaccharide molecules, resulting in a decrease in the polysaccharide extraction rate.

[0056] Using the optimal conditions determined by the above single factors, a solid-liquid ratio of 1:60, an extraction time of 3 hours, an extraction temperature of 70°C, and other conditions remaining unchanged, an extraction experiment was conducted and the extraction rate obtained was 6.61%.

[0057] S3 response surface test design: According to the results of the single factor test, the solid-liquid ratio, extraction time and extraction temperature were selected as independent variables, and the extraction rate of Lysimachia sibiricum polysaccharide was used as the response value. The response surface test data were regressed using the Design Expert software, and a quadratic polynomial model was established to obtain the multivariate quadratic regression equation:

[0058] Y = 21.58-1.15X1 + 3.95X2 + 3.5X3 + 0.2X1X2 - 0.7X1X3 - 1.70X2X3 - 4.69X1X1 - 3.89X2X2 - 5.99X3X3; where Y is the extraction rate of Lysimachia polysaccharide, variable parameter X1 is the solid-liquid ratio, X2 is the extraction time, and X3 is the extraction temperature. The significance and goodness of fit of the model were evaluated by variance analysis and other methods. The effects of the interactions between the factors on the extraction rate were analyzed to determine the optimal level combination of the factors. The optimal extraction conditions obtained by analysis were: solid-liquid ratio 1:64, extraction time 3.2 hours, and extraction temperature 71.2 °C.

[0059]

[0060] The optimal values ​​of each extraction condition were determined on the basis of single factor experiments. The three-factor three-level response surface analysis was performed with the solid-liquid ratio, extraction time and extraction temperature as independent variables and the extraction rate of Lysimachia polysaccharide as the response value. The Box-Behnken method was used to optimize the extraction conditions of Lysimachia polysaccharide. The experimental factor level design is shown in Table 1.

[0061] Table 1 Factors and levels of response surface analysis of Lysimachia sibiricum polysaccharides

[0062] According to the response surface software design, there were 17 groups of optimization experiments on the extraction conditions of Lysimachia sibiricum polysaccharides, including 5 groups of center point replicate experiments. The experimental design and its response value results are shown in Table 2.

[0063] Table 2 Response surface experimental design and results

[0064]

[0065] Design-Expert 13 software was used to perform variance analysis and multiple regression fitting on the data in Table 2, with the extraction rate of crude polysaccharides from Lysimachia scabra as the response value Y. The results are shown in Table 3.

[0066] Table 3 ANOVA results of response surface quadratic model

[0067]

[0068] Note: * indicates significant effect (p < 0.05), ** indicates extremely significant effect (p < 0.01), and *** indicates highly significant effect (p < 0.001).

[0069] Effects of solid-liquid ratio and extraction time on the extraction rate of Lysimachia sibiricum polysaccharide Figure 4 The effects of solid-liquid ratio and extraction temperature on the extraction rate of Lysimachia sibiricum polysaccharide are shown in Figure 5 The effects of extraction time and extraction temperature on the extraction rate of Lysimachia sibiricum polysaccharide are shown in Figure 6 shown.

[0070] From the quadratic multinomial model and variance analysis results in Table 3, we can see that the linear term A of the regression model is highly significant, B and C are extremely significant, and the quadratic term A is 2 Very significant, B 2 , C 2 The results are highly significant, the interaction terms AC are significant, and AB and BC are not significant, indicating that there is no simple linear relationship between different extraction conditions and the extraction rate of Lysimachia polysaccharide. After regression analysis of the experimental data, the quadratic multiple regression equation is obtained:

[0071] Y=7.71+0.96A+0.5575B+0.5575C-0.195AB+0.655AC+0.17BC-1.13A 2 -1.48B 2 -3.76C 2

[0072] The regression equation model is highly significant (p < 0.001); the lack of fit term p = 0.9059 > 0.05, indicating that the lack of fit term is not significant, that is, the model fits this experiment well. The correlation coefficient of the model R 2 =0.9862, adjusted coefficient of determination AdjustedR 2 =0.9685, indicating that the model can explain 96.85% of the response value changes, that is, the model has a good fit with the actual experiment, the experimental error is small, and it is feasible to use this model to analyze the extraction conditions of Lysimachia sibiricum polysaccharides.

[0073] Verification test

[0074] According to the results obtained from the response surface test and the quadratic polynomial regression equation, and using Design-Expert 13 software to analyze the data, the optimal extraction conditions of each factor for the highest extraction rate of Lysimachia polysaccharide in this experiment were obtained as follows: solid-liquid ratio of 1:64, extraction time of 3.2h, extraction temperature of 71.2℃, under which the extraction rate of Lysimachia crude polysaccharide was 8.01%. The extraction conditions after response surface optimization were verified and repeated 3 times. The test results are shown in Table 4.

[0075] Table 4

[0076]

[0077] The average value of the three groups of experiments under the above-mentioned optimized extraction conditions (i.e., when the solid-liquid ratio was 1:64, the extraction time was 3.2 h, and the extraction temperature was 71.2°C) was 7.64%, and the relative error with the 8.01% predicted by the regression equation was 4.62%, which was within the allowable error range (20%), indicating that the regression model of the response surface methodology in the optimization experiment of the extraction conditions of Lysimachia scabra polysaccharides was more reliable.

[0078] Purity detection of Lysimachia chinensis polysaccharide: The crude polysaccharide extracted by optimizing the extraction process of Lysimachia chinensis polysaccharide using response surface methodology was purified using the phenol-sulfuric acid method, and the resulting Lysimachia chinensis polysaccharide was quantified, with a purity of up to 83.71%.

[0079] The above is only an embodiment of the present invention. The common sense such as the known specific structure and characteristics in the scheme is not described in detail here. The ordinary technicians in the relevant field know all the common technical knowledge in the technical field of the invention before the application date or priority date, can know all the existing technologies in the field, and have the ability to apply the conventional experimental means before that date. The ordinary technicians in the relevant field can improve and implement this scheme in combination with their own abilities under the enlightenment given by this application. Some typical known structures or known methods should not become obstacles for ordinary technicians in the relevant field to implement this application. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, several deformations and improvements can be made, which should also be regarded as the scope of protection of the present invention, which will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A method for optimizing the extraction conditions of Lysimachia sibiricum polysaccharides using response surface methodology, characterized in that: The steps include: S1 Pretreatment of the Lysimachia sibiricum raw material: Select high-quality Lysimachia sibiricum, wash and dry it, and then crush and sieve it to obtain Lysimachia sibiricum powder; S2 single factor test: determine the three factors of solid-liquid ratio, extraction time and extraction temperature, and measure the effect of one factor on the extraction rate of Lysimachia polysaccharide while keeping other conditions unchanged. S3 response surface experimental design: The Box-Behnken response surface design method was used. On the basis of the single factor experiment, three factors, namely, solid-liquid ratio, extraction time and extraction temperature, were selected as corresponding variables. The optimal point of the single factor experiment was taken as the center. A level value was taken above and below the optimal point as the level of the response surface. The extraction rate of Lysimachia chinensis crude polysaccharide was taken as the response value. Polynomial fitting regression was performed on the corresponding variables to obtain the regression equation. The regression equation was calculated to obtain the extraction conditions of Lysimachia chinensis crude polysaccharide after the response surface optimization. The regression equation is: Y=7.71+0.96A+0.5575B+0.5575C-0.195AB+0.655AC+0.17BC-1.13A2-1.48B2-3.76C, Among them, Y is the extraction rate of crude polysaccharide of Lysimachia scabra, variable parameter X1 is the solid-liquid ratio, X2 is the extraction time, and X3 is the extraction temperature.

2. The method for optimizing the extraction conditions of Lysimachia sibiricum polysaccharides using response surface methodology according to claim 1, characterized in that: The selection ranges of the three factors are solid-liquid ratio 1:20-1:100; extraction time 1-6h; extraction temperature 40°C-90°C.

3. The method for optimizing the extraction conditions of Lysimachia sibiricum polysaccharides using response surface methodology according to claim 1, characterized in that: The step S2 specifically includes: Step 1, water extraction: extracting the Lysimachia sibiricum powder with water, centrifuging, filtering, and concentrating by rotary evaporation to obtain a Lysimachia sibiricum crude polysaccharide concentrate; Step 2, alcohol precipitation: subjecting the concentrated solution of Lysimachia sibiricum polysaccharide to alcohol precipitation overnight, dissolving the solid after the alcohol precipitation, and then freeze-drying to obtain Lysimachia sibiricum polysaccharide; Step 3, removing protein: removing impurity protein in the crude polysaccharide of Lysimachia sibiricum; Step 4, impurity removal: removing small molecular impurities in the crude Lysimachia herba polysaccharide to obtain purified Lysimachia herba polysaccharide.

4. The method for optimizing the extraction conditions of Lysimachia sibiricum polysaccharides using response surface methodology according to claim 3, characterized in that: In the step 1, the centrifugation conditions are: 7000-8000rpm, 5-8min; the rotary evaporation conditions are: 40-45°C, 100-120rpm, and the rotary evaporation is completed when the remaining liquid is 1 / 3 of the original volume to obtain the Lysimachia sibiricum crude polysaccharide concentrate.

5. The method for optimizing the extraction conditions of Lysimachia sibiricum polysaccharides using response surface methodology according to claim 3, characterized in that: In the step 2, the alcohol precipitation condition is to add 4 times the volume of anhydrous ethanol to the Lysimachia sibiricum crude polysaccharide concentrate, and then put it in a 4° C. refrigerator for alcohol precipitation overnight.

6. The method for optimizing the extraction conditions of Lysimachia sibiricum polysaccharides using response surface methodology according to claim 3, characterized in that: The method for removing protein from the crude polysaccharide of Lysimachia scabra in the step 3 is the Sevage method for removing protein.

7. The method for optimizing the extraction conditions of Lysimachia sibiricum polysaccharides using response surface methodology according to claim 6, characterized in that: The specific method of the Sevage method for removing protein from the crude polysaccharide of Lysimachia chinensis is as follows: using distilled water to dissolve the dried crude polysaccharide of Lysimachia chinensis, adding 1 / 4 volume of Sevage reagent (chloroform: n-butanol = 4:1 / V:V), fully oscillating on a constant temperature oscillator at room temperature for 30 minutes (using vortex), and then centrifuging, the centrifugation conditions are: 9000rpm, 15min; after separating the organic phase and the aqueous phase, adding Sevage reagent equivalent to 1 / 4 of its volume to the aqueous phase, repeating the above process until there is no white precipitate at the junction of the organic layer and the aqueous layer after centrifugation, which proves that the protein is completely removed.

8. The method for optimizing the extraction conditions of Lysimachia sibiricum polysaccharides using response surface methodology according to claim 3, characterized in that: In the step 4, the method used to remove small molecule impurities in the crude polysaccharide of Lysimachia scabra is dialysis.

9. The method for optimizing the extraction conditions of Lysimachia sibiricum polysaccharides using response surface methodology according to claim 8, characterized in that: The specific method of removing small molecular impurities in the Lysimachia crude polysaccharide by dialysis is as follows: placing the Lysimachia crude polysaccharide solution after protein removal by Sevage method in a dialysis bag, adding deionized water to the environment of the dialysis bag, changing the water every half a day, collecting the solution in the bag after 24 hours, and obtaining the Lysimachia polysaccharide after freeze-drying.

10. The method for optimizing the extraction conditions of Lysimachia sibiricum polysaccharides using response surface methodology according to claim 3, characterized in that: The S3 response surface experimental design also includes: evaluating the significance and goodness of fit of the model through the variance analysis method, analyzing the influence of the interaction between the three factors on the extraction rate, and determining the optimal level combination of each factor. After analysis, the optimal extraction conditions are: solid-liquid ratio 1:64, extraction time 7.2 hours, and extraction temperature 71.2°C.

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