Method for optimizing bletilla striata neutral polysaccharide extraction and separation process by adopting response surface method

Abstract

The invention relates to a method for optimizing a bletilla striata neutral polysaccharide extraction and separation process by adopting a response surface method.The method comprises the following steps that 1, a bletilla striata medicinal material is taken and subjected to water extraction, filtrate is concentrated, absolute ethyl alcohol is added, a mixture is obtained, after the mixture is filtered, sediment is dried, then water is added for dissolution again, and a head product is obtained; 2, dialyzing the initial product with distilled water, and concentrating and drying to obtain rhizoma bletillae raw sugar; 3, multiple regression fitting analysis is conducted through a single-factor method in combination with Design-Expert software, and therefore a fitting full-variable quadratic regression equation is obtained, and optimal technological parameters are optimized through a Box-Behnken response surface method; step 4, dissolving the rhizoma bletillae raw sugar in distilled water, separating and purifying by using an anion exchange column, and concentrating and freeze-drying to obtain first neutral polysaccharide; and 5, separating and purifying the first neutral polysaccharide by using a gel column, concentrating and freeze-drying to obtain the first neutral polysaccharide.

Description

Technical Field

[0001] The invention relates to the technical field of biopharmaceuticals, in particular to the field of IPC C08B 37, and more specifically to a method for optimizing the extraction and separation process of neutral polysaccharides from Bletilla striata by using a response surface methodology. Background Art

[0002] The chemical structure of macromolecular polysaccharides is extremely complex. At present, the research on the relationship between polysaccharide structure and function is also deepening, but no comprehensive and authoritative conclusion has been obtained. Therefore, this field is still in the research and exploration stage and needs further development. Bletilla striata (Thunb.) Reichb.f. is the dried tuber of Bletilla striata, a traditional Chinese medicine of the orchid family. The main active ingredient with a large content is Bletilla striata polysaccharide, followed by polyphenols and amino acids. At present, the extraction methods of Bletilla striata polysaccharide mainly include solvent extraction, ultrasonic extraction, microwave extraction, enzyme-assisted extraction, etc., and different methods have their own advantages and disadvantages. Polysaccharides are divided into primary structure and higher structure, and different structures have different characterizations. Among them, the primary structure belongs to the primary structure of polysaccharides, and the remaining secondary, tertiary and quaternary structures are the higher structures of polysaccharides. Among them, the structure of polysaccharides with primary structure is very complex, so it is difficult to identify the structure of polysaccharides with primary structure, which is not conducive to subsequent experimental research.

[0003] CN 108329398 B A polysaccharide from Bletilla striata and an extraction method thereof. The method comprises the following steps: 1) before extraction, ultrasonically pulverizing Bletilla striata in a frozen state to obtain solid particles of Bletilla striata; and may further comprise: 2) subjecting the obtained solid particles of Bletilla striata to continuous dynamic countercurrent extraction to obtain an extract; and 3) purifying and crystallizing the extract. The method of the invention is simple, and increases the content of Bletilla striata polysaccharide in the extract; improves production efficiency, reduces solvent usage, reduces extraction temperature, reduces energy consumption, and improves industrial investment efficiency. However, the invention still uses organic solvents such as trichloroacetic acid in the extraction process, which does not conform to the concept of green environmental protection, and limits the development and application in medicine and daily use. Summary of the invention

[0004] This study aims to provide a research basis for the extraction process of Bletilla striata polysaccharide products, and to ensure that Bletilla striata polysaccharide components with high quality and large quantity are extracted more efficiently under the premise of green environmental protection. The present invention provides a method for optimizing the extraction and separation process of Bletilla striata neutral polysaccharide by using response surface methodology, comprising the following steps:

[0005] Step 1: Take the medicinal material of Bletilla striata, extract it with water, concentrate the filtrate and add anhydrous ethanol to obtain a mixture, filter the mixture, dry the sediment, and then add water to dissolve it again to obtain the initial product;

[0006] Step 2: dialyze the initial product with distilled water, concentrate and dry it to obtain white radix schizonepetae crude sugar;

[0007] Step 3: The influence of different water extraction processes on the extraction rate of white ginseng raw sugar was investigated by single factor method, and then multiple regression fitting analysis was performed using Design-Expert software to obtain the quadratic regression equation for all variables, and the optimal process parameters were selected by Box-Behnken response surface method;

[0008] Step 4: using the process parameters obtained in step 3 to sequentially perform steps 1 and 2 to extract and obtain white radix schizonepetae crude sugar, dissolving the white radix schizonepetae crude sugar in distilled water and separating and purifying the white radix schizonepetae crude sugar using an anion exchange column, and concentrating and freeze-drying the raw sugar to obtain a first neutral polysaccharide;

[0009] Step 5: Separate and purify the first neutral polysaccharide using a gel column, concentrate and freeze-dry to obtain the polysaccharide.

[0010] The water extraction temperature is 50-90°C.

[0011] The water extraction time is 50-150 min.

[0012] The solid-liquid ratio of the water extraction is 1:(5-30).

[0013] Preferably, the water extraction temperature is 60-80°C, the water extraction time is 60-120 min, and the material-liquid ratio of the water extraction is 1:(10-20).

[0014] The water extraction is performed 2-5 times.

[0015] Preferably, the water extraction is performed 2-4 times.

[0016] The volume after concentration in step 1 is 1 / 4-1 / 6 of the volume before concentration.

[0017] The content of anhydrous ethanol in the mixture is 70-90 wt %.

[0018] Preferably, the content of anhydrous ethanol in the mixture is 75-90 wt %.

[0019] The Box-Behnken response surface method selects a three-factor three-level response surface analysis scheme.

[0020] The three factors are: the water extraction temperature, the water extraction time and the material-liquid ratio of the water extraction.

[0021] For the extraction of Bletilla striata polysaccharide, the most widely used extraction methods are ultra-high pressure method, ultrasonic extraction method, etc. Although the extraction rate is higher, it is difficult to achieve batch production of Bletilla striata polysaccharide due to the severe conditions and high experimental requirements, and it is not ruled out that the polysaccharide structure will be damaged or changed during the extraction process. The crude Bletilla striata polysaccharide extracted from Bletilla striata medicinal material powder by water extraction and alcohol precipitation method is safe and reliable and meets the requirements of economy and green environmental protection, but there are many influencing parameters in the extraction process and it is impossible to extract efficiently. The research of this application found that compared with the optimization methods such as uniform design, orthogonal design, and star point design commonly used in current related research, the Box-Behnken response surface method selected in this experiment adopts multivariate linear and quadratic model fitting, thereby effectively improving the accuracy of the experiment and predicting the optimal extraction conditions. Further combining anion exchange column separation and purification and gel column separation and purification can maximize the structural stability of polysaccharides and reduce the introduction of impurities, making the extraction process simple to operate and low-cost and efficient.

[0022] In the process research of this experiment, the optimal extraction conditions of Bletilla striata polysaccharide were: time 94 min, extraction temperature 68°C, solid-liquid ratio 1:14. In actual application, appropriate adjustments can be made according to the degree of influence of these three conditions of time, temperature and solid-liquid ratio on the extraction rate of Bletilla striata polysaccharide to ensure a higher polysaccharide extraction rate. For example, based on the actual situation, one of the single factor conditions may be preferentially satisfied so that other factors can be appropriately reduced, but at the same time, the extraction efficiency of Bletilla striata polysaccharide is ensured to be at a better level, thereby obtaining maximum benefits.

[0023] Beneficial effects:

[0024] 1. The crude polysaccharide of Bletilla striata extracted from the powder of Bletilla striata medicinal material by water extraction and alcohol precipitation is safe and reliable and meets the requirements of economy and green environmental protection.

[0025] 2. When using the water alcohol precipitation method to extract Bletilla striata polysaccharides, the factors affecting its extraction efficiency are ranked in order of the size of the impact on the efficiency: extraction time > extraction temperature > solid-liquid ratio.

[0026] 3. Compared with the optimization methods commonly used in current related research, such as uniform design, orthogonal design, and star-point design, the Box-Behnken response surface method selected in this experiment uses multivariate linear and quadratic model fitting, which effectively improves the accuracy of the experiment and predicts the optimal extraction conditions.

[0027] 4. The optimal process parameters selected by Box-Behnken response surface methodology are: extraction temperature 68°C, extraction time 94 min, solid-liquid ratio 1:14, and the relative deviation compared with the actual experiment is less than 0.4%.

[0028] 5. The present application combines anion exchange column separation and purification with gel column separation and purification to ensure the structural stability of the polysaccharide to the greatest extent and reduce the introduction of impurities, making the extraction process simple to operate with low cost and high efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the response surface diagram of the interaction effect of A (water extraction temperature) and B (water extraction time) in Example 1.

[0030] Figure 2 This is the response surface diagram of the interaction between A (water extraction temperature) and C (solid-to-liquid ratio of water extraction) in Example 1.

[0031] Figure 3 This is the response surface diagram of the interaction effect of B (water extraction time) and C (solid-to-liquid ratio of water extraction) in Example 1. DETAILED DESCRIPTION

[0032] Example 1

[0033] A method for optimizing the extraction and separation process of neutral polysaccharides from Bletilla striata by using response surface methodology comprises the following steps:

[0034] Step 1: Take the medicinal material of Bletilla striata, extract it with water, concentrate the filtrate and add anhydrous ethanol, add anhydrous ethanol until the final mass fraction reaches 85%, place it in a cabinet at 4°C overnight, filter it, and freeze-dry the sediment to obtain the initial product;

[0035] Step 2: The primary product is dialyzed with distilled water, placed in a dialysis bag with a number average molecular weight of 7000Da, and dialyzed with distilled water, then the solution in the bag is collected, centrifuged at 4000r / min for 20 minutes, and the supernatant is concentrated and dried to obtain white schizonepeta crude sugar, named: BSP;

[0036] Step 3: The influence of different water extraction processes on the extraction rate of white ginseng raw sugar was investigated by single factor method (performance test: methodological investigation), and then multiple regression fitting analysis was performed using Design-Expert8.0.5 software to obtain the quadratic regression equation for all variables, and the optimal process parameters were selected by Box-Behnken response surface method;

[0037] Step 4: using the process parameters obtained in step 3 to sequentially perform steps 1 and 2 to extract and obtain white radix schizonepetae crude sugar, dissolving the white radix schizonepetae crude sugar in distilled water and separating and purifying the white radix schizonepetae crude sugar using an anion exchange column, and concentrating and freeze-drying the raw sugar to obtain a first neutral polysaccharide;

[0038] Step 5: Separate and purify the first neutral polysaccharide using a gel column, concentrate and freeze-dry to obtain;

[0039] According to step 1 and step 2, 17 groups of experiments were set up, and the specific process parameters were shown in Table 1. The extraction rates (W) of the 17 groups of experiments were tested according to the test method in the performance test section, as shown in Table 2.

[0040] Combined with the data in Tables 1 and 2, the water extraction temperature, water extraction time and water extraction solid-liquid ratio were selected as three factors through single factor comparison, represented by A, B, and C respectively. Three levels were designed for each factor. The extraction rate of Bletilla striata polysaccharide content was used as the evaluation index, and a response surface analysis scheme with three factors and three levels was designed.

[0041] The experimental results were analyzed by multiple regression fitting using Design-Expert 8.0.5 software, and the quadratic regression equation of all variables was obtained: W = 55.8-4.07A + 4.34B + 3.86C + 1.99AB + 0.5AC-0.47BC-8.47A2-13.07B2-6.62C2. The judgment index is the size of the F value. The order of influence of the three single factor indicators selected in the experiment on the extraction rate of Bletilla striata polysaccharide is: B>A>C, see Table 1 and Table 2.

[0042] The relationship between the three selected factors was analyzed, and the maximum value of the relationship curve between each factor and each factor was obtained. The optimal process parameters selected are: A 2 , B 2 , C 2 The extraction rate of polysaccharide from Bletilla striata was significantly affected, while AB, BC and AC had no significant effect on the extraction rate of polysaccharide from Bletilla striata. On this basis, the maximum extraction rate was optimized by using the effect surface method, and the water extraction temperature was determined to be 67.86℃, the time was 94.35min, and the solid-liquid ratio was 1:14. At this time, the extraction rate of polysaccharide content in Bletilla striata reached 57.11%. Figures 1 to 3 .

[0043] The step 4 is specifically as follows: 1.5 kg of dried medicinal materials of Bletilla striata are taken, and Bletilla striata polysaccharides are extracted according to the optimized optimal process, that is, the optimal conditions: distilled water is added at a mass volume ratio of 1:14 (g:mL), and constant temperature extraction is performed in a water bath at 68°C for 3 times, each time for 94 minutes, and the filtrate is filtered through four layers of gauze and then combined. The filtrate is concentrated by rotary evaporation to about 1 / 5 of the original. Anhydrous alcohol is added until the final mass fraction reaches 85%, and it is placed in a refrigerator at 4°C overnight. After filtration, the sediment is freeze-dried, and then an appropriate amount of distilled water is added to dissolve it again, and it is fully stirred at room temperature until it is completely dissolved. The sample solution is placed in a dialysis bag with a molecular weight of 7000Da, dialyzed with distilled water, and then the solution in the bag is collected, centrifuged at 4000r / min for 20 minutes, and the supernatant is concentrated and dried to obtain the BSP of Bletilla striata and crude sugar components; after weighing, it is stored at -20°C for standby use.

[0044] The step 5 is specifically as follows: 2 grams of Bletilla striata polysaccharide BSP is added to an appropriate amount of distilled water and mixed to dissolve it in water, and then centrifuged at 4000r / min for 15 minutes to remove the insoluble matter, and then the precipitate is collected and about 5mL of supernatant is taken out; the sample is loaded onto a filled anion exchange column (DEAE-650M, 55mm x 30cm), rinsed with water, measured with phenol, and graded and recovered to obtain the first neutral polysaccharide, named: BSP-1, and BSP-1 is concentrated and freeze-dried.

[0045] Add an appropriate amount of pure water to 500 mg of BSP-1 and completely dissolve it at room temperature, then put it into a dialysis bag with a molecular weight of 7000Da; after dialysis with distilled water, collect the solvent in the bag, 4000r / min, 20min, concentrate the supernatant, and then concentrate the supernatant to a certain volume, add it to a uniformly filled gel chromatography (Sepharose 6B, 45mmx90 cm), and then elute with H2O, 14mL in each small tube, and then use the sulfuric acid phenol method to determine the sugar-containing components, and collect until there is no sugar. According to the elution curve drawn by the elution amount, it is combined, concentrated and dialyzed; after freeze-drying, a neutral polysaccharide of Bletilla striata is obtained, named: BSP-2.

[0046] Table 1

[0047]

[0048]

[0049] Performance Testing Methods and Data

[0050] 1. Methodological Investigation

[0051] Establishment of regression equation and determination of extraction rate (W): Accurately weigh 10.00 mg of Bletilla striata polysaccharide in a 100 mL volumetric flask, thoroughly shake and dissolve with distilled water, and then dilute to the mark to obtain a 0.10 mg / mL Bletilla striata sample solution. Take 1 mL of the sample solution in a test tube, add 1.2 mL of phenol-sulfuric acid test solution (0.2 mL 5% phenol solution, 1 mL concentrated sulfuric acid), shake well, let stand for 20 minutes, and use an ultraviolet spectrophotometer to measure the absorbance of the solution at 490 nm and 280 nm, and record.

[0052] Weigh 25.00 mg of glucose standard into a 100 mL volumetric flask, add an appropriate amount of distilled water to make the solution volume to the mark, and obtain a 0.25 mg / mL standard solution. Take 0.1, 0.3, 0.5, 0.7, and 0.9 mL of glucose standard solution into test tubes, add 0.9, 0.7, 0.5, 0.3, and 0.1 mL of distilled water respectively, and then add 1.2 mL of phenol-sulfuric acid test solution (0.2 mL of 5% phenol solution, 1 mL of concentrated sulfuric acid), shake well, and let stand for 20 minutes, and use distilled water as a control, and use a UV spectrophotometer to measure the absorbance of the solution at 490 nm. With absorbance as the ordinate (Y) and the concentration of the glucose standard solution as the abscissa (X, μg / mL), a standard curve was drawn, and the regression equation was obtained: Y=0.0455X+0.0275, r=0.9987. The linear relationship of the regression equation was good within the concentration range of 0.025-0.0225 mg / mL. Extraction rate of polysaccharide content in Bletilla striata (W):

[0053] W (%) = (sample concentration × volume of sample after constant volume / mass of sample) × 100;

[0054] 1. Precision experiment

[0055] Accurately take 1 mL of glucose standard solution of known concentration into a volumetric flask (50 mL), add distilled water to 50 mL, and measure the absorbance 6 times in parallel after treatment according to the above method. The absorbance values ​​are 0.687, 0.69, 0.691, 0.689, 0.692, and 0.688, respectively. The calculated absorbance RSD value is 0.31%. The results show that the instrument has good precision.

[0056] 2. Repeatability experiment

[0057] The corresponding Bletilla striata test sample solutions were prepared according to the above method, and then the absorbance of the 6 groups of test samples was measured in parallel. The absorbance values ​​were 0.341, 0.348, 0.343, 0.344, 0.340, and 0.345, respectively. The calculated RSD value was 0.19%, which showed that the method adopted in the experiment had good repeatability.

[0058] 3. Stability test

[0059] The test used the same test solution, and the same UV spectrophotometer was used to measure the absorbance of the test solution after 0, 5, 10, 15, 20, 25, and 30 minutes, respectively. The absorbance values ​​were 0.345, 0.344, 0.338, 0.337, 0.333, and 0.331, respectively. Through calculation, the RSD value of the absorbance was 1.26%. The test results showed that the solution was relatively stable within 30 minutes after color development.

[0060] 4. Sample recovery experiment

[0061] The sample recovery method was used to test, 0.25g of Bletilla striata powder with known total polysaccharide content was taken, 6 samples were accurately weighed, and an appropriate amount of glucose reference substance was accurately added to each sample. The reference solution and the test solution were prepared accordingly, and the recovery rate was calculated. The total polysaccharide content was calculated to be 104.4, 102.5, 103.6, 101.9, 100.3, and 100.9 mg / mL, respectively. The test results showed that the average recovery rate and RSD were 102.23 (n=6) and 2.97%, respectively.

[0062] 2. Verification Experiment and Results

[0063] The optimal extraction conditions of Bletilla striata polysaccharide content were obtained by optimization using response surface methodology. After the optimal condition values ​​were retained as integers considering practical operational factors, three parallel experiments were conducted on steps 4 and 5 of the embodiment. As can be seen from Table 3, the error between the experimental data and the theoretical calculation data is small, and the correlation between the two is good, indicating that the experiment is effective and feasible. It is verified that the optimal process optimized by the response surface method has high accuracy.

[0064] Performance test data

[0065] Table 2

[0066]

[0067]

[0068] Table 3

[0069]

[0070] Table 4

[0071]

Claims

1. A method for optimizing the extraction and separation process of neutral polysaccharides from Bletilla striata using response surface methodology, characterized in that: The following steps are involved: Step 1: Take the medicinal material of Bletilla striata, extract it with water, concentrate the filtrate and add anhydrous ethanol to obtain a mixture, filter the mixture, dry the sediment, and then add water to dissolve it again to obtain the initial product; Step 2: dialyze the initial product with distilled water, concentrate and dry it to obtain white radix schizonepetae raw sugar; Step 3: The influence of different water extraction processes on the extraction rate of white ginseng raw sugar was investigated by single factor method, and then multiple regression fitting analysis was performed using Design-Expert software to obtain the quadratic regression equation of all variables, and the optimal process parameters were selected by Box-Behnken response surface method; Step 4: using the process parameters obtained in step 3, sequentially performing steps 1 and 2 to extract and obtain white radix schizonepetae crude sugar, dissolving the white radix schizonepetae crude sugar in distilled water, separating and purifying the white radix schizonepetae crude sugar using an anion exchange column, and concentrating and freeze-drying the raw sugar to obtain a first neutral polysaccharide; Step 5: Separate and purify the first neutral polysaccharide using a gel column, concentrate and freeze-dry to obtain the polysaccharide.

2. The method for optimizing the extraction and separation process of neutral polysaccharides from Bletilla striata by response surface methodology according to claim 1, characterized in that: The water extraction temperature is 50-90°C.

3. The method for optimizing the extraction and separation process of neutral polysaccharides from Bletilla striata by response surface methodology according to claim 2, characterized in that: The water extraction time is 50-150 min.

4. The method for optimizing the extraction and separation process of neutral polysaccharides from Bletilla striata by response surface methodology according to claim 3, characterized in that: The solid-liquid ratio of the water extraction is 1:(5-30).

5. The method for optimizing the extraction and separation process of neutral polysaccharides from Bletilla striata by response surface methodology according to claim 4, characterized in that: The water extraction temperature is 60-80°C, the water extraction time is 60-120min, and the material-liquid ratio of the water extraction is 1:(10-20).

6. The method for optimizing the extraction and separation process of neutral polysaccharides from Bletilla striata by using response surface methodology according to claim 5, characterized in that: The water extraction is performed 2-5 times.

7. The method for optimizing the extraction and separation process of neutral polysaccharides from Bletilla striata by using response surface methodology according to claim 1 or 6, characterized in that: The volume after concentration in step 1 is 1 / 4-1 / 6 of the volume before concentration.

8. The method for optimizing the extraction and separation process of neutral polysaccharides from Bletilla striata by using response surface methodology according to claim 7, characterized in that: The content of anhydrous ethanol in the mixture is 70-90 wt %.

9. The method for optimizing the extraction and separation process of neutral polysaccharides from Bletilla striata by response surface methodology according to claim 1, characterized in that: The Box-Behnken response surface method selects a three-factor three-level response surface analysis scheme.

10. The method for optimizing the extraction and separation process of neutral polysaccharides from Bletilla striata by using response surface methodology according to claim 9, It is characterized in that The three factors are: the water extraction temperature, the water extraction time and the material-liquid ratio of the water extraction.

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