Method for optimizing extraction conditions of fructus aurantii immaturus polysaccharide by response surface method, fructus aurantii immaturus polysaccharide and application of fructus aurantii immaturus polysaccharide

Abstract

The invention discloses a method for optimizing extraction conditions of fructus aurantii immaturus polysaccharide by using a response surface method, the fructus aurantii immaturus polysaccharide and application of the fructus aurantii immaturus polysaccharide. The method comprises the following steps: S1, pretreatment of a fructus aurantii immaturus raw material: selecting high-quality fructus aurantii immaturus, cleaning, drying, crushing and sieving to obtain fructus aurantii immaturus powder; s2, single-factor test: determining three factors, namely a solid-liquid ratio, extraction time and extraction temperature, and determining the influence of a certain factor on the extraction rate of the immature bitter orange polysaccharide from the immature bitter orange powder under the condition that other conditions are not changed; s3, response surface test design: adopting a Box-Behnken response surface design method, taking the extraction rate of the immature bitter orange crude polysaccharide as a response value on the basis of a single factor experiment, performing polynomial fitting regression on the corresponding variables to obtain a regression equation, and calculating the regression equation to obtain the extraction condition of the immature bitter orange crude polysaccharide after response surface optimization, the regression equation is Y = 17.54 + 0.59 X < 1 > + 0.5875 X < 2 > + 3150 X < 3 >-1.60 X < 1 > X < 2 > + 1.04 X < 1 > X < 3 >-1.65 X < 2 > X < 3 >-2.99 X < 12 >-3.98 X < 22 >-3.47 X < 32 >, Y is the extraction rate of the immature bitter orange 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. After the response surface is optimized, the optimal extraction condition of the immature bitter orange can be quickly and accurately determined, the extraction rate of the immature bitter orange polysaccharide is improved, the extraction process is simplified, and the production cost is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine extraction, and in particular to a method for optimizing the extraction conditions of polysaccharide from Citrus aurantium using response surface methodology, the polysaccharide from Citrus aurantium and its application. Background Art

[0002] Citrus aurantium (Fructus Aurantii Immaturus) is the dried young fruit of the Rutaceae plant, Citrus aurantium and its cultivated varieties, or Citrus citrus sweet. Primarily produced in Sichuan and Jiangxi, it is a widely used traditional Chinese medicinal herb with a long history. It has a firm texture, a delicate fragrance, and a bitter, slightly sour flavor. Its bitter taste has the properties of dispersing qi and eliminating stagnation, dissolving phlegm and dampness, and relieving chest tightness. It is effective in treating symptoms such as chest pain, heartache, and chest and flank distension caused by phlegm obstruction and qi stagnation. Modern research has shown that the main components of Citrus aurantium, including polysaccharides, flavonoids, glycosides, triterpenes, and inorganic ions, contain a variety of physiologically active substances, exhibiting antiviral, anticoagulant, lipid-lowering, anti-tumor, immunomodulatory, and anti-aging activities.

[0003] The main methods for extracting polysaccharides from Citrus aurantium immaturus include water extraction, ultrasonic-assisted extraction, microwave-assisted extraction, enzyme extraction, and supercritical fluid extraction. However, compared with water extraction, these other methods have problems such as complex processes and high costs. Currently, the process conditions for water extraction of polysaccharides from Citrus aurantium immaturus remain limited to the process parameters (material-liquid ratio, extraction temperature, extraction time, etc.) found by the single-factor control variable method. There is a lack of research using response surface methodology to establish a continuous variable surface model to optimize the water extraction conditions of polysaccharides from Citrus aurantium immaturus. Summary of the Invention

[0004] Under these background conditions, the present invention proposes a method for optimizing the extraction conditions of Citrus aurantium polysaccharides using response surface methodology, Citrus aurantium polysaccharides, and a method for preparing the same. Suitable Citrus aurantium raw materials are selected and pretreated. Then, through single-factor experiments, the effects of extraction temperature, extraction time, and solid-liquid ratio on the extraction rate of Citrus aurantium polysaccharides are investigated. With the optimal point of the single-factor experiment as the center, a level value above and below the optimal point is taken as the approximate range of each factor. Next, based on the results of the single-factor experiment, a response surface experiment scheme is designed. Taking the polysaccharide extraction rate as the response value, the three factors of extraction temperature, extraction time, and solid-liquid ratio are optimized. Statistical analysis software is used to analyze the response surface experiment data, establish a mathematical model, and obtain the optimal combination of each factor.

[0005] In order to overcome at least one of the above-mentioned shortcomings of the prior art, the present invention provides a method for optimizing the extraction conditions of polysaccharides from Citrus aurantium immaturum using response surface methodology, comprising the following steps: S1 Pretreatment of the raw material of Citrus aurantium: Select high-quality Citrus aurantium, wash and dry it, and then crush and sieve it to obtain Citrus aurantium 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 polysaccharides from Citrus aurantium powder 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, the three factors of solid-liquid ratio, extraction time and extraction temperature were selected as the corresponding variables. The optimal point of the single factor experiment was taken as the center. A level value above and below the optimal point was taken as the level of the response surface. The extraction rate of crude polysaccharide of Citrus aurantium was used 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 crude polysaccharide of Citrus aurantium after the response surface optimization. The regression equation is: Y=17.54+0.59X1+0.5875X2+3150X3-1.60X1X2+1.04X1X3-1.65X2X3-2.99X1 2 -3.98X2 2 -3.47X3 2 Among them, Y is the extraction rate of crude polysaccharide of Citrus aurantium, variable parameter X1 is the solid-liquid ratio, X2 is the extraction time, and X3 is the extraction temperature.

[0006] Furthermore, the selection ranges of the three factors are material-liquid ratio 1:20-1:60; extraction time 1-5 hours; and extraction temperature 60°C-100°C.

[0007] Furthermore, the step S2 specifically includes: Step 1: Water extraction: extracting the Citrus aurantium powder with water, centrifuging, filtering, and concentrating by rotary evaporation to obtain a concentrated solution of crude polysaccharides from Citrus aurantium; Step 2: alcohol precipitation: the concentrated solution of the crude polysaccharide of Citrus aurantium is precipitated with alcohol overnight, the solid is separated, the solid precipitated with alcohol is dissolved, and then freeze-dried to obtain the crude polysaccharide of Citrus aurantium; Step 3: removing protein: removing impurity proteins in the crude polysaccharide of Citrus aurantium; Step 4: impurity removal: remove small molecular impurities in the crude polysaccharide of Citrus aurantium to obtain purified polysaccharide of Citrus aurantium.

[0008] Furthermore, in the step one, the centrifugation conditions are: 7000~8000rpm, 8~10min; the rotary evaporation conditions are: 50~55℃, 60~90rpm, and the rotary evaporation is completed when the remaining liquid is 1 / 3 of the original volume, thereby obtaining the concentrated polysaccharide solution of Citrus aurantium immaturus; in the step two, the alcohol precipitation conditions are: adding 4 times the volume of anhydrous ethanol to the concentrated polysaccharide solution of Citrus aurantium immaturus, and then placing it in a refrigerator at 4℃ for alcohol precipitation overnight; in the step three, the method for removing protein from the crude polysaccharide solution of Citrus aurantium immaturus is the Sevage method for removing protein.

[0009] Furthermore, the specific method of the Sevage method for removing protein from the crude polysaccharide of Citrus aurantium is as follows: using distilled water to dissolve the dried crude polysaccharide of Citrus aurantium, adding 1 / 4 volume of Sevage reagent (chloroform: n-butanol = 4:1 / V:V), and fully oscillating on a constant temperature oscillator at room temperature for 30 minutes (using vortex), and then centrifuging, the centrifugation conditions are: 8000 rpm, 10 minutes; 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.

[0010] Furthermore, in the step 4, the method used to remove small molecular impurities in the crude polysaccharide of Citrus aurantium is dialysis.

[0011] Furthermore, the specific method of removing small molecular impurities in the crude polysaccharide of Citrus aurantium by dialysis is as follows: placing the crude polysaccharide solution of Citrus aurantium after deproteinization by the Sevage method in a dialysis bag, adding deionized water to the environment in which the dialysis bag is located, changing the water every half a day, collecting the solution in the bag after 24 hours, and obtaining Citrus aurantium polysaccharide after freeze-drying.

[0012] 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 were obtained as follows: solid-liquid ratio 1:49.7, extraction time 3.4 hours, and extraction temperature 90.5°C.

[0013] The second aspect of the present invention provides a polysaccharide of Citrus aurantium extracted by the above method.

[0014] The third aspect of the present invention provides an application of the above-mentioned polysaccharide from Citrus aurantium in terms of antioxidant activity.

[0015] The beneficial effect of the present invention is that, on the basis of a single factor experiment, the response surface methodology is used to optimize the extraction process conditions of Citrus aurantium polysaccharide. By determining that the three conditions of solid-liquid ratio, extraction temperature and extraction time have a more significant effect on the extraction rate of Citrus aurantium polysaccharide, a three-factor three-level orthogonal experiment is designed on the basis of the single factor experiment, and the response surface methodology is used to optimize the extraction conditions of Citrus aurantium polysaccharide. The optimal extraction conditions can be determined quickly and accurately, thereby improving the extraction rate of Citrus aurantium polysaccharide, simplifying the extraction process, and reducing the production cost, which is beneficial to the industrial production and further application of Citrus aurantium polysaccharide. Finally, the optimal process conditions for extracting Citrus aurantium polysaccharide are determined: solid-liquid ratio 1:49.7, extraction time 3.4 hours, extraction temperature 90.5°C, providing a reference basis for the development and utilization of Citrus aurantium polysaccharide. Experimental verification shows that the Citrus aurantium polysaccharide provided by this method has significant antioxidant effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention will be further described below with reference to the accompanying drawings and examples.

[0017] Figure 1 This is a graph showing the effect of the solid-liquid ratio on the extraction rate of polysaccharides from Citrus aurantium in an embodiment of the present invention; Figure 2 This is a graph showing the effect of extraction time on the extraction rate of polysaccharides from Citrus aurantium in an embodiment of the present invention; Figure 3 Graph showing the effect of extraction temperature on the extraction rate of polysaccharides from Citrus aurantium in an embodiment of the present invention; Figure 4 This is a graph showing the effects of the solid-liquid ratio and extraction time on the extraction rate of polysaccharides from Citrus aurantium in an embodiment of the present invention; Figure 5 Graph showing the effects of solid-liquid ratio and extraction temperature on the extraction rate of polysaccharides from Citrus aurantium immaturum in an embodiment of the present invention; Figure 6 Graph showing the effects of extraction time and temperature on the extraction rate of polysaccharides from Citrus aurantium in an embodiment of the present invention; Figure 7 This is a data graph of the ability to scavenge hydroxyl radicals (·OH) in an embodiment of the present invention; Figure 8 This is a data diagram of the ability to remove superoxide anions in an embodiment of the present invention. DETAILED DESCRIPTION

[0018] Before discussing the exemplary embodiments in more detail, it should be mentioned that some exemplary embodiments are described as processes or methods depicted as flow charts. Although the flow charts describe the various operations as sequential processes, many of the operations therein can be implemented in parallel, concurrently, or simultaneously. In addition, the order of the various 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.

[0019] It should be understood that although the terms "first," "second," and the like may be used herein to describe various elements, these elements should not be limited by these terms. These terms are used solely to distinguish one element from another. For example, a first element may be referred to as a second element, and similarly, a second element may be referred to as a first element, without departing from the scope of the exemplary embodiments. The term "and / or" as used herein includes any and all combinations of one or more of the listed associated items.

[0020] The present invention provides a method for optimizing the extraction conditions of polysaccharides from Citrus aurantium using response surface methodology, comprising: S1 Pretreatment of the raw material of Citrus aurantium: Select high-quality Citrus aurantium, wash and dry it, and then crush and sieve it to obtain Citrus aurantium 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 polysaccharides from Citrus aurantium powder 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, the three factors of solid-liquid ratio, extraction time and extraction temperature were selected as the corresponding variables. The optimal point of the single factor experiment was taken as the center. A level value above and below the optimal point was taken as the level of the response surface. The extraction rate of crude polysaccharide of Citrus aurantium was used 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 crude polysaccharide of Citrus aurantium after the response surface optimization. The regression equation is: Y=17.54+0.59X1+0.5875X2+3150X3-1.60X1X2+1.04X1X3-1.65X2X3-2.99X1 2 -3.98X2 2 -3.47X3 2 Among them, Y is the extraction rate of crude polysaccharide of Citrus aurantium, variable parameter X1 is the solid-liquid ratio, X2 is the extraction time, and X3 is the extraction temperature.

[0021] Step S2 also includes: step 1, water extraction: extracting the Citrus aurantium powder with water, centrifuging, filtering, and concentrating by rotary evaporation after the water extraction to obtain a concentrated solution of crude polysaccharides from Citrus aurantium; Step 2, alcohol precipitation: After the concentrated solution of the crude polysaccharide of Citrus aurantium was precipitated with alcohol overnight, the solid after the alcohol precipitation was dissolved and then freeze-dried to obtain the crude polysaccharide of Citrus aurantium; Step 3, protein removal: removing impurity proteins in the crude polysaccharide of Citrus aurantium; Step 4, impurity removal: removing small molecular impurities in the crude polysaccharide of Citrus aurantium to obtain purified polysaccharide of Citrus aurantium.

[0022] Example 1 Pretreatment of the raw material of Citrus aurantium: Select high-quality Citrus aurantium, wash and dry it, then crush and sieve it to obtain Citrus aurantium powder; Single factor experiment: Extract the Citrus aurantium powder at a solid-liquid ratio of 1:20~1:60g / ml at 60℃~100℃ for 1~5h, and determine the effect of a certain factor on the extraction rate of Citrus aurantium polysaccharide while keeping other conditions unchanged.

[0023] Effect of solid-liquid ratio on the extraction rate of polysaccharides from Citrus aurantium: The pretreated Citrus aurantium powder was weighed and mixed at a solid-liquid ratio of 1:20, 1:30, 1:40, 1:50, and 1:60 (the extraction liquid was distilled water). The mixture was extracted at 90°C for 4 hours, centrifuged at 8000 r / min for 10 minutes, and the supernatant was collected. Anhydrous ethanol was added until the concentration of the precipitation reached 80%, and the mixture was precipitated at 4°C overnight. The solid was collected after centrifugation, redissolved with deionized water, and freeze-dried to obtain crude polysaccharides from Citrus aurantium. The extraction rate of crude polysaccharides from Citrus aurantium was calculated.

[0024] The formula for calculating the extraction rate of Citrus aurantium polysaccharides is: Y (%) = Citrus aurantium polysaccharide mass concentration * fixed volume * dilution factor / Citrus aurantium raw material powder mass × 100%. The ratio of raw material to extractant (water) is a parameter that significantly affects the extraction rate.

[0025] Crude polysaccharides from Citrus aurantium immaturus were dissolved in water, and the protein content of the solution was removed using the Sevage method: dry crude polysaccharides from Citrus aurantium immaturus were dissolved in distilled water, and 1 / 4 volume of Sevage reagent (chloroform:n-butanol = 4:1 / v:v) was added. The mixture was thoroughly shaken on a thermostatic shaker at room temperature for 30 minutes (using vortex flow), followed by centrifugation at 8000 rpm for 10 minutes. The organic and aqueous phases were separated, and 1 / 4 volume of Sevage reagent was added to the aqueous phase. This process was repeated until no white precipitate remained at the interface of the organic and aqueous layers after centrifugation, indicating complete protein removal.

[0026] The dialysis method is used to remove small molecular impurities in the crude polysaccharide of Citrus aurantium, and the polysaccharide of Citrus aurantium is obtained after freeze-drying: the crude polysaccharide solution of Citrus aurantium after deproteinization by the 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 polysaccharide of Citrus aurantium is obtained after freeze-drying.

[0027] Purity detection of polysaccharide from Citrus aurantium: The purified polysaccharide from Citrus aurantium was detected using the phenol-sulfuric acid method.

[0028] Effect of solid-liquid ratio on the extraction rate of polysaccharides from Citrus aurantium: Weigh 1 g of pretreated Citrus aurantium powder and mix it at solid-liquid ratios of 1:20, 1:30, 1:40, 1:50, and 1:60. Extract it at 90°C for 4 hours, centrifuge it (8000 r / min, 10 minutes), collect the supernatant, add anhydrous ethanol to the alcohol precipitation concentration reaching 80%, and precipitate it at 4°C overnight. Collect the solid after centrifugation, redissolve it with deionized water, and freeze-dry it to obtain crude polysaccharides from Citrus aurantium. The extraction rate of crude polysaccharides from Citrus aurantium was calculated. Figure 1The following figure shows the effect of the solid-liquid ratio on the extraction yield of Citrus aurantium polysaccharides. Analysis shows that when the raw material to liquid ratio is 1:50, the extraction yield of Citrus aurantium polysaccharides reaches a maximum of 15.11%. This may be because as the extraction solid-liquid ratio increases, the mass transfer dynamics increase, increasing the contact area between the Citrus aurantium raw material powder and the aqueous solvent, and increasing the amount of polysaccharides that diffuse into the solvent, leading to the dissolution of water-soluble polysaccharides. The reason why the polysaccharide extraction yield decreases when the ratio is higher than 1:50 may be that the Citrus aurantium polysaccharides are essentially dissolved in water at 1:50. At this point, increasing the amount of water will increase the solution volume during the subsequent alcohol precipitation, increasing the loss of Citrus aurantium polysaccharides.

[0029] Effect of extraction time on the extraction rate of polysaccharides from Citrus aurantium: 1 g of pretreated Citrus aurantium powder was weighed and mixed at a solid-liquid ratio of 1:50. The mixture was extracted at 90°C for 1 h, 2 h, 3 h, 4 h, and 5 h. The mixture was centrifuged (8000 r / min, 10 min), the supernatant was collected, and anhydrous ethanol was added until the alcohol precipitation concentration reached 80%. The mixture was precipitated at 4°C overnight. The solid was collected after centrifugation, redissolved in deionized water, and freeze-dried to obtain crude polysaccharides from Citrus aurantium. The extraction rate of crude polysaccharides from Citrus aurantium was calculated. Figure 2 The following figure shows the effect of extraction time on the extraction yield of Citrus aurantium polysaccharides. Analysis shows that the extraction time for Citrus aurantium polysaccharides increases from 1 to 4 hours; the extraction yield reaches a maximum of 13.46% at 4 hours; and at 5 hours, the extraction yield decreases over time. This decrease in extraction yield with increasing extraction time may be due to the dissolution of other components of plant cells, such as proteins, pigments, and tannins, in addition to polysaccharides. These impurities interact with polysaccharides, potentially forming complexes or colloids. This increases the viscosity and complexity of the extract, hindering the dissolution and separation of polysaccharides and reducing the extraction yield. Furthermore, prolonged exposure to high temperatures and water can hydrolyze the glycosidic bonds of polysaccharides, disrupting the previously intact polysaccharide structure and making complete extraction difficult, thus reducing the extraction yield.

[0030] Effect of extraction temperature on the extraction rate of polysaccharides from Citrus aurantium: Weigh 1 g of pretreated Citrus aurantium powder, mix it at a solid-liquid ratio of 1:50, extract it at 60℃, 70℃, 80℃, 90℃, and 100℃ for 4 hours, centrifuge it (8000r / min, 10min), collect the supernatant, add anhydrous ethanol to the alcohol precipitation concentration reaching 80%, precipitate it at 4℃ overnight, collect the solid after centrifugation, redissolve it with deionized water, and freeze-dry it to obtain crude polysaccharides from Citrus aurantium, and calculate the extraction rate of crude polysaccharides from Citrus aurantium. Figure 3The following figure shows the effect of extraction temperature on the extraction rate of polysaccharides from Citrus aurantium. Analysis shows that the extraction rate of polysaccharides from Citrus aurantium gradually increases within the range of 60-90°C, reaching a maximum of 13.21% at 90°C. When the temperature exceeds 90°C, the extraction rate of polysaccharides from Citrus aurantium slightly decreases. This result may be due to the acceleration of molecular motion and increased solubility as the temperature increases, which in turn increases the extraction rate. When the temperature exceeds 90°C, the high temperature may be detrimental to the dissolution of polysaccharide molecules, destroying the polysaccharide structure and resulting in a decrease in the polysaccharide extraction rate.

[0031] Using the optimal conditions determined by the above single factors, a material-liquid ratio of 1:50, an extraction time of 4 hours, an extraction temperature of 90°C, and other conditions remaining unchanged, an extraction experiment was conducted and the extraction yield obtained was 15.96%.

[0032] S3 response surface experimental design: Based on the results of the single-factor experiment, the solid-liquid ratio, extraction time and extraction temperature were selected as independent variables, and the extraction rate of polysaccharides from Citrus aurantium was used as the response value. The response surface experimental data were subjected to regression analysis using DesignExpert software, and a quadratic polynomial model was established to obtain the multivariate quadratic regression equation: Y=17.54+0.59X1+0.5875X2+3150X3-1.60X1X2+1.04X1X3-1.65X2X3-2.99X1 2 -3.98X2 2 -3.47X3 2 Where Y is the extraction rate of polysaccharides from Citrus aurantium immaturus, 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 analysis of variance and other methods. The effects of the interactions between the various factors on the extraction rate were analyzed to determine the optimal level combination of each factor. The optimal extraction conditions were obtained as follows: solid-liquid ratio 1:49.7, extraction time 3.4 hours, and extraction temperature 90.5°C. Based on single-factor experiments, the optimal values ​​for each extraction condition were determined. A three-factor, three-level response surface analysis was conducted, with the solid-liquid ratio, extraction time, and temperature as independent variables and the extraction rate of crude polysaccharides from Citrus aurantium as the response value. The Box-Behnken method was used to optimize the extraction conditions for polysaccharides from Citrus aurantium. The experimental factor level design is shown in Table 1.

[0033] Table 1 Factors and levels of response surface analysis of polysaccharides from Citrus aurantium

[0034] According to the response surface software design, there were 17 groups of optimization experiments for the extraction conditions of polysaccharides from Citrus aurantium immaturus, including 5 groups of center point replicate experiments. The experimental design and response value results are shown in Table 2.

[0035] Table 2 Response surface experimental design and results

[0036] The data in Table 2 were subjected to variance analysis and multiple regression fitting using Design-Expert13 software, with the extraction rate of crude polysaccharides from Citrus aurantium as the response value Y. The results are shown in Table 3.

[0037] Table 3 ANOVA results of response surface quadratic model

[0038] Note: * indicates a significant effect (p < 0.05), ** indicates an extremely significant effect (p < 0.01), and *** indicates a highly significant effect (p < 0.001).

[0039] Effects of solid-liquid ratio and extraction time on the extraction rate of polysaccharides from Citrus aurantium Figure 4 The effects of solid-liquid ratio and extraction temperature on the extraction rate of polysaccharides from Citrus aurantium are shown in Figure 5 The effects of extraction time and temperature on the extraction rate of polysaccharides from Citrus aurantium are shown in Figure 6 shown.

[0040] From the quadratic multinomial model and variance analysis results in Table 3, it can be seen that the linear terms A, B, and C of the regression model are significant, the quadratic terms A², B², and C² are highly significant, the interaction terms AB and AC are extremely significant, and BC is highly significant, indicating that there is no simple linear relationship between different extraction conditions and the extraction rate of polysaccharides from Citrus aurantium. After regression analysis of the experimental data, the quadratic multivariate regression equation was obtained: Y=17.54+0.59X1+0.5875X2+3150X3-1.60X1X2+1.04X1X3-1.65X2X3-2.99X1 2 -3.98X2 2 -3.47X3 2 The regression equation model is extremely significant (p < 0.001); the lack of fit term p = 0.9362 > 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 is R² = 0.9884, and the adjusted coefficient of determination is AdjustedR 2 =0.9734, indicating that the model can explain 97.34% 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 polysaccharides from Citrus aurantium.

[0041] Verification test Based on the results of the response surface experiment and a quadratic polynomial regression equation, and using Design-Expert 13 software to analyze the data, the optimal extraction conditions for the highest polysaccharide extraction rate from Citrus aurantium immaturus were: a solid-liquid ratio of 1:49.7, an extraction time of 3.4 hours, and an extraction temperature of 90.5°C. Under these conditions, the extraction rate of crude polysaccharides from Citrus aurantium immaturus was 15.8%. Validation experiments were conducted on the optimized extraction conditions using the response surface method, repeated three times. The results are shown in Table 4.

[0042] Table 4

[0043] The average value of the three groups of experiments under the above-mentioned optimized extraction conditions (i.e., when the solid-liquid ratio was 1:49.7, the extraction time was 3.4 hours, and the extraction temperature was 90.5°C) was 16.02%, and the relative error from the 15.8% predicted by the regression equation was 0.22%, 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 polysaccharides from Citrus aurantium was relatively reliable.

[0044] Take 2 grams of crude polysaccharide from the above verification experiment and remove protein and impurities. The specific operation is as follows: 1. Experimental Principle The Sevage method is a common method for removing protein impurities from biological samples and is widely used in the purification processes of polysaccharides, nucleic acids, natural product extracts, etc. It uses organic solvents to denature and precipitate proteins to effectively separate target components from protein impurities. For example, in the polysaccharide extraction process, this method can be used to preliminarily remove free proteins or bound proteins in crude polysaccharide solutions, evaluate the protein removal effect, and provide a basis for the subsequent acquisition of high-purity target products.

[0045] The principle of the Sevage method for removing proteins is to use the emulsification effect produced by the vigorous shaking of a mixed solvent of chloroform and amyl alcohol (or isoamyl alcohol) to denature and precipitate the protein. Taking the classic chloroform-amyl alcohol method as an example, a certain volume ratio of chloroform and amyl alcohol mixture (usually 4:1) is added to the aqueous solution of the protein sample. After sufficient shaking and mixing, the mixed solvent acts on the protein molecules, destroying the hydration film on its surface and interfering with its stable structure (such as hydrogen bonds, hydrophobic interactions), causing protein denaturation; at the same time, the emulsion formed during the shaking process greatly increases the contact area between the solvent and the protein, accelerating the denaturation process; the denatured protein loses its solubility and condenses into a gel-like precipitate at the interface of the two solvents or in the aqueous phase. Subsequently, the emulsified state is destroyed by centrifugation, and the solution will be separated into: a lower organic solvent phase (chloroform phase), a middle denatured protein gel layer, and an upper aqueous phase containing the target component (such as polysaccharides); by separating and collecting the upper aqueous phase, effective removal of protein impurities can be achieved. 2. Experimental Methods Dissolve 20 mL of the crude polysaccharide solution in deionized water, then add 5 mL (approximately 1 / 4 the volume of the crude polysaccharide solution) of sevage reagent. Mix thoroughly, then centrifuge at 8000 rpm for 15 minutes. Discard the lower organic phase and the gelatinous denatured protein layer in the middle. Collect the supernatant and deproteinize it multiple times until no significant precipitate forms. Collect the supernatant and determine its protein concentration.

[0046] 3. Experimental Results Pipette 0.5 ml of a 5 mg / mL polysaccharide solution. Determine the protein content using the Coomassie Brilliant Blue method. Measure the absorbance at 590 nm using bovine serum albumin as a standard to calculate the protein content in the sample.

[0047]

[0048] This shows that after deproteinization by the sevage method, the protein concentration in the polysaccharide solution is lower. Zhishi dialysis process 1. Experimental Principle Dialysis is a classic technology for separating and purifying biomacromolecules (such as polysaccharides, proteins, and nucleic acids). It is widely used in the removal of salt, small molecule impurities, and small molecule pigments from crude polysaccharide solutions. It effectively separates target macromolecules from impurity molecules by utilizing the selective permeability of semipermeable membranes. For example, in the polysaccharide purification process, this method can be used to remove dialyzable impurities introduced during the extraction process, reduce the sample ionic strength, and provide high-purity polysaccharide samples for subsequent chromatographic analysis or activity studies.

[0049] The principle of dialysis impurity removal is based on the osmotic pressure difference generated by the concentration difference between the two sides of a semipermeable membrane, which drives the diffusion and migration of small molecules while retaining target macromolecules. For example, in the treatment of a crude polysaccharide solution, a solution containing the target polysaccharide (high molecular weight) and small molecule impurities (such as salt ions, monosaccharides, oligosaccharides, small molecule pigments, and organic solvents) is placed in a sealed dialysis bag with a specific pore size. The bag is then immersed in a continuously changing volume of low-ionic strength dialysate (such as distilled water, deionized water, or a specific buffer). Driven by the concentration gradient, small molecule impurities (with molecular weights below the membrane cutoff) within the bag continuously diffuse through the pores of the semipermeable membrane into the dialysate. However, the target polysaccharide molecules (with molecular weights above the membrane cutoff) are confined within the dialysis bag due to their large size and cannot pass through. By continuously and repeatedly changing the external dialysate to maintain a high concentration difference between the small molecule impurities inside and outside the bag, the small molecule impurities within the bag are continuously diffused into the dialysate outside the bag and removed, until the concentrations of the small molecule impurities inside and outside the bag reach a dynamic equilibrium (generally considered to have been fully removed). Ultimately, the solution in the bag becomes a relatively pure polysaccharide solution with small molecule impurities removed, laying the foundation for subsequent concentration, freeze-drying, and precise quantitative analysis or activity evaluation.

[0050] 2. Experimental Methods The crude polysaccharide solution after protein removal using the Sevage method is placed in a dialysis bag (MW: 3500Da). Deionized water is added to the environment of the dialysis bag and the water is changed every half a day to ensure sufficient diffusion of impurities. After 24 hours, the solution in the bag is collected and freeze-dried to obtain the Citrus aurantium polysaccharide.

[0051] 3. Experimental Results Dissolve 0.01 g of Citrus aurantium polysaccharide powder in deionized water and dilute to 10 ml. Measure the absorbance at 490 nm using the phenol-sulfuric acid method to calculate the polysaccharide content in the sample.

[0052]

[0053] Purity detection of Citrus aurantium polysaccharide: The phenol-sulfuric acid method was used to quantify the purity of the purified Citrus aurantium polysaccharide extracted from the crude polysaccharide optimized by response surface methodology, and the purity could reach 81.14%.

[0054] The following is the in vitro antioxidant activity test of the polysaccharide (after purification) extracted from the above method The following is the in vitro antioxidant activity test of the polysaccharide (after purification) extracted from the above method In vitro antioxidant assay of Citrus aurantium extract Ability to scavenge hydroxyl radicals (·OH) 1) Experimental Principle The hydroxyl radical scavenging assay can be used to evaluate the ability of antioxidants to scavenge hydroxyl radicals. Hydroxyl radicals are a highly oxidizing reactive oxygen species in organisms that can cause lipid peroxidation and DNA damage. This assay uses deoxyribose oxidative damage as a model. Based on the Fenton reaction, hydroxyl radicals are generated in the presence of ferrous ions and hydrogen peroxide. When deoxyribose is added to the system, hydroxyl radicals attack the deoxyribose, oxidizing it and producing decomposition products including malondialdehyde. MDA reacts with thiobarbituric acid to form a red complex with a maximum absorption wavelength around 532 nm. Antioxidants can scavenge hydroxyl radicals, reducing deoxyribose oxidative damage and malondialdehyde production. By measuring the absorbance change at 532 nm, the hydroxyl radical scavenging rate is calculated, thereby assessing the scavenging ability of antioxidants. This assay is simple to perform and provides intuitive results, making it suitable for screening and evaluating antioxidants.

[0055] 2) Experimental methods To determine the hydroxyl radical (OH) scavenging ability, 1.0 mL of polysaccharide aqueous solution at concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL was added, in order, to 1.0 mL each of a 9 mmol / L FeSO4 solution, a 9 mmol / L salicylic acid ethanol solution, and a 9 mmol / L H2O2 solution. After mixing, the mixture was reacted at 37°C for 30 minutes, and the absorbance was measured at 510 nm. Deionized water was used instead of the polysaccharide solution for the blank control, and deionized water was used instead of the H2O2 solution for the background control. VC solutions with concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL were treated in the same manner and served as positive controls. The OH scavenging efficiency of the polysaccharide solution was calculated according to the formula.

[0056] Where:

[0057] A0——blank control absorbance; A a ——Measurement tube absorbance; A b ——Measure the background absorbance of the tube.

[0058] 3) Experimental results See Figure 7 .

[0059] 4) Experimental Conclusion ·OH is the most chemically active free radical among reactive oxygen species. It reacts extremely rapidly with nearly any biomolecule in living cells, making it the most harmful free radical to the body. Excessive ·OH can damage cells, kill red blood cells, degrade DNA, and lead to various diseases. As shown in the figure, with increasing concentration of Citrus aurantium polysaccharide, its ·OH scavenging capacity increases in a dose-dependent manner, demonstrating that Citrus aurantium polysaccharide has a certain ·OH scavenging capacity.

[0060] 2. Ability to scavenge superoxide anions 1) Experimental Principle The superoxide anion free radical scavenging experiment is used to detect the ability of a sample to scavenge superoxide anion free radicals. Superoxide anion free radicals are important reactive oxygen species in organisms and can cause oxidative damage. This experiment can be used for antioxidant research. The experimental principle is based on the xanthine-xanthine oxidase system to produce superoxide anion free radicals. The system contains nitro blue tetrazolium. Superoxide anion free radicals can reduce nitro blue tetrazolium to blue formazan. The color depth is related to the number of free radicals. When the sample contains antioxidant ingredients, it can scavenge superoxide anion free radicals. The superoxide anion free radical scavenging rate is calculated by measuring the change in absorbance at 560 nm. The higher the scavenging rate, the stronger the antioxidant capacity of the sample. This experiment simulates the in vivo environment and effectively evaluates the ability of the sample to scavenge superoxide anion free radicals. It is suitable for antioxidant substance screening and antioxidant mechanism research. 2) Experimental methods To assess superoxide anion scavenging ability, 200 μl of each of various concentrations of Citrus aurantium polysaccharide and vitamin C solutions (0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL) were placed in stoppered test tubes. 3 mL of TrisHCl solution (0.1 mol / L) was added and the mixture was heated in a 37°C water bath for 10 minutes. 12 μl of pyrogallol solution (30 mmol / L) was added and allowed to react for 4 minutes. The reaction was immediately terminated by the addition of 0.5 mL of concentrated hydrochloric acid. The absorbance was measured at 320 nm. Each experiment was performed in triplicate, using vitamin C as a positive control. The scavenging rate of each free radical was calculated using the formula.

[0061] Where:

[0062] A0——blank control absorbance; A a ——Measurement tube absorbance; A b ——Measure the background absorbance of the tube.

[0063] 3) Experimental results See Figure 8 .

[0064] 4) Experimental Conclusion Superoxide anions are the most common free radicals in the human body. They have dual properties. When present in small quantities, they can kill harmful bacteria and promote health. However, when they accumulate in large quantities, they can damage healthy cells, leading to aging and various diseases. Promptly removing accumulated superoxide anions from the body is crucial. As shown in the figure, as the concentration of Citrus aurantium polysaccharides increases, their superoxide anion scavenging capacity increases in a dose-dependent manner.

[0065] The above is only an embodiment of the present invention. Common knowledge such as the known specific structures and characteristics in the scheme is not described in detail here. Ordinary technicians in the field are aware of all common technical knowledge in the technical field of the invention before the application date or priority date, can obtain all existing technologies in the field, and have the ability to apply conventional experimental means before that date. Ordinary technicians in the field can improve and implement this scheme in combination with their own abilities under the inspiration given by this application. Some typical known structures or known methods should not become obstacles for ordinary technicians in the 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 variations and improvements can be made, which should also be regarded as the scope of protection of the present invention. These 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 polysaccharides from Citrus aurantium using response surface methodology, characterized in that: The steps include: S1 Pretreatment of the raw material of Citrus aurantium: Select high-quality Citrus aurantium, wash and dry it, and then crush and sieve it to obtain Citrus aurantium 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 polysaccharides from Citrus aurantium powder 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, the three factors of solid-liquid ratio, extraction time and extraction temperature were selected as the corresponding variables. The optimal point of the single factor experiment was taken as the center. A level value above and below the optimal point was taken as the level of the response surface. The extraction rate of crude polysaccharide of Citrus aurantium was used 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 crude polysaccharide of Citrus aurantium after the response surface optimization. The regression equation is: Y=17.54+0.59X1+0.5875X2+3150X3-1.60X1X2+1.04X1X3-1.65X2X3-2.99X1 2 -3.98X2 2 -3.47X3 2 Among them, Y is the extraction rate of crude polysaccharide of Citrus aurantium, 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 polysaccharides from Citrus aurantium using response surface methodology according to claim 1, wherein: The selection ranges of the three factors are: solid-liquid ratio 1:20-1:60; extraction time 1-5 hours; extraction temperature 60°C-100°C.

3. The method for optimizing the extraction conditions of polysaccharides from Citrus aurantium using response surface methodology according to claim 1, wherein: The step S2 specifically includes: Step 1: water extraction: soaking the Citrus aurantium powder, water extraction after soaking, centrifugation, suction filtration, and rotary evaporation to obtain a concentrated solution of crude polysaccharides from Citrus aurantium; Step 2: alcohol precipitation: the concentrated solution of the crude polysaccharide of Citrus aurantium is precipitated with alcohol overnight, the solid is separated, the solid after alcohol precipitation is redissolved, and then freeze-dried to obtain the crude polysaccharide of Citrus aurantium; Step 3: removing protein: removing impurity proteins in the crude polysaccharide of Citrus aurantium; Step 4: impurity removal: removing small molecular impurities in the crude polysaccharide of Citrus aurantium to obtain purified polysaccharide of Citrus aurantium.

4. The method for optimizing the extraction conditions of polysaccharides from Citrus aurantium using response surface methodology according to claim 3, wherein: In the step 1, the centrifugation conditions are: 7000~8000rpm, 8~10min; the rotary evaporation conditions are: 50~55℃, 60~90rpm, and the rotary evaporation is completed when the remaining liquid is 1 / 3 of the original volume, thereby obtaining the concentrated polysaccharide solution of Citrus aurantium immaturus; in the step 2, the alcohol precipitation conditions are: adding 4 times the volume of anhydrous ethanol to the concentrated polysaccharide solution of Citrus aurantium immaturus, and then placing it in a refrigerator at 4℃ for alcohol precipitation overnight; in the step 3, the method for removing protein from the crude polysaccharide solution of Citrus aurantium immaturus is the Sevage method for removing protein.

5. The method for optimizing the extraction conditions of polysaccharides from Citrus aurantium using response surface methodology according to claim 4, characterized in that: The specific method of the Sevage method for removing protein from the crude polysaccharide of Citrus aurantium is as follows: using distilled water to dissolve the dried crude polysaccharide of Citrus aurantium, 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: 8000 rpm, 10 minutes; 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, indicating that the protein has been completely removed.

6. The method for optimizing the extraction conditions of polysaccharides from Citrus aurantium using response surface methodology according to claim 3, wherein: In the step 4, the method used to remove small molecular impurities in the crude polysaccharide of Citrus aurantium is dialysis.

7. The method for optimizing the extraction conditions of polysaccharides from Citrus aurantium using response surface methodology according to claim 6, wherein: The specific method of removing small molecular impurities in the crude polysaccharide of Citrus aurantium by dialysis is as follows: placing the crude polysaccharide solution of Citrus aurantium after deproteinization 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 polysaccharide of Citrus aurantium after freeze-drying.

8. The method for optimizing the extraction conditions of polysaccharides from Citrus aurantium using response surface methodology according to claim 3, wherein: 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 impact 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 were obtained as follows: solid-liquid ratio 1:49.7, extraction time 3.4 hours, and extraction temperature 90.5°C.

9. A polysaccharide extracted from Citrus aurantium immaturum using the method for optimizing the extraction conditions of Citrus aurantium immaturum polysaccharide using response surface methodology according to any one of claims 1 to 8.

10. A use of the polysaccharide of Citrus aurantium according to claim 9, characterized in that: Used for the preparation of antioxidant drugs.

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