CN111253497A - Extraction method and application of Grateloupia filicina polysaccharide - Google Patents
Extraction method and application of Grateloupia filicina polysaccharide Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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Abstract
The invention discloses an extraction method and application of Grateloupia filicina polysaccharide, belonging to the technical field of polysaccharide extraction, and the method comprises the following steps: a. placing Grateloupia filicina powder in a Soxhlet extractor, refluxing with petroleum ether and ethanol respectively, and drying; b. adding the dried Grateloupia filicina powder into deionized water, extracting under the auxiliary action of ultrasonic waves, and centrifuging to obtain Grateloupia filicina polysaccharide extract; extraction conditions are as follows: extracting at 60-100 deg.C for 1-5h with a feed-liquid ratio of 1: 70-110; c. concentrating the Grateloupia filicina polysaccharide extract, precipitating with ethanol, centrifuging to separate precipitate, and air drying to obtain Grateloupia filicina polysaccharide. The invention takes uniformly crushed and stable grateloupia filicina powder as a raw material, uses deionized water as an extracting solution, adopts a hot water extraction method to extract grateloupia filicina polysaccharide, and shortens the extraction time and improves the extraction rate by matching the extraction temperature, the material-liquid ratio and the extraction time with the ultrasonic action.
Description
Technical Field
The invention relates to the technical field of polysaccharide extraction, in particular to an extraction method and application of Grateloupia filicina polysaccharide.
Background
Grateloupia filicina C.Ag, belongs to algae of Grateloupia, Euchrysophyceae, Cryptonemales, Hymenochaetaceae, Grateloupia, and its algae are red or purple, and have soft or slightly hard texture and clumpy appearance, and mostly grow on rock reef at the edge of sea tide line or in harmony marshland. The seaweed has specific requirements on the growth environment, and grows in the marine environment with high pressure, high salt content, low light content, low oxygen content and the like. The Chinese medicinal composition is used as a marine Chinese medicament since ancient times, has the effects of clearing away heat and toxic materials and expelling parasites, is rich in resources, is widely distributed on the coast of China, and is eaten by local people. The polysaccharide is used as a main chemical component of the Grateloupia filicina and mainly takes galactan sulfate, and the research at home and abroad fully shows that the Grateloupia filicina polysaccharide has various biological activities of anticoagulation, antivirus, antitumor and the like, thereby showing that the Grateloupia filicina polysaccharide has greater development and utilization values. In the development and utilization of algal polysaccharides, the extraction process has great influence on the extraction rate, purity, chemical structure and biological activity of the polysaccharides and industrial application. In the production and extraction process of algal polysaccharide, people continuously innovate and develop new technology. At present, the methods for extracting crude polysaccharide from seaweed mainly adopt solvent extraction methods such as hot water extraction, weak acid extraction, weak base extraction and the like at home and abroad. With the progress of separation and extraction technology, many new extraction technologies, such as ultrasonic, microwave, enzymolysis, etc., are developed, but not limited to the traditional extraction method.
At present, the hot water extraction method is still a common polysaccharide extraction method, but the method has high extraction temperature, low yield and time-consuming operation. Therefore, it is necessary to provide a method for extracting Grateloupia filicina polysaccharide to shorten the extraction time and improve the Grateloupia filicina polysaccharide yield.
Meanwhile, oxidative stress often causes an increase in the free radical content in the body, thereby having a detrimental effect on proteins, lipids and deoxyribonucleic acid. In addition, it also has effects on various pathophysiological processes such as neurodegenerative diseases, cancer, cardiovascular diseases, inflammatory diseases and aging. The antioxidant can reduce oxidative stress, and is beneficial to health. Therefore, in recent years, antioxidant products have become a new favorite for health care and cosmetic enterprises.
Disclosure of Invention
The invention aims to provide an extraction method and application of Grateloupia filicina polysaccharide to solve the problems in the prior art.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a method for extracting Grateloupia filicina polysaccharide, which comprises the following steps:
a. placing Grateloupia filicina powder in a Soxhlet extractor, refluxing with petroleum ether and ethanol respectively, and drying;
b. adding the dried Grateloupia filicina powder into deionized water, extracting under the auxiliary action of ultrasonic waves, and centrifuging to obtain Grateloupia filicina polysaccharide extract;
extraction conditions are as follows: extracting at 60-100 deg.C for 1-5h with a feed-liquid ratio of 1: 70-110;
c. concentrating the Grateloupia filicina polysaccharide extract, precipitating with ethanol, centrifuging to separate precipitate, and air drying to obtain Grateloupia filicina polysaccharide.
Furthermore, the Grateloupia filicina powder is obtained by drying Grateloupia filicina, pulverizing, and sieving with 40 mesh sieve.
Further, the boiling range of the petroleum ether is 60-90 ℃, and the reflux time of the petroleum ether is 2 hours.
Further, the ethanol is ethanol with the volume fraction of 95%, and the ethanol refluxing time is 2 h.
Further, the drying is drying at 60 ℃ for 12 h.
Furthermore, the ultrasonic wave is divergent ultrasonic, the frequency is 40kHz, and the power is 0-800W.
Further, in step b, the centrifugation condition is 60000rpm for 15 min.
Further, the concentration in the step c is 1/2 of the volume of the original extracting solution after vacuum-pumping rotary evaporation and concentration; and the alcohol precipitation is to add an ethanol solution with the volume 2-3 times of the volume of the concentrated extracting solution, and to stand the mixture for 12-24 hours at the temperature of 4-10 ℃, wherein the concentration of the ethanol solution is 90-100% of the volume fraction.
The invention also provides the Grateloupia filicina polysaccharide extracted by the extraction method. The invention also provides application of the Grateloupia filicina polysaccharide in antioxidant activity and preparation of a natural antioxidant.
The invention discloses the following technical effects:
according to the invention, the grateloupia filicina polysaccharide is extracted by using uniformly crushed and stable grateloupia filicina powder as a raw material and deionized water as an extracting solution and using a hot water extraction method, and the cavitation action of ultrasonic waves is matched with the action of ultrasonic waves, so that the broken cell walls and the whole organism are broken due to the extremely high pressure generated by the cavitation action of the ultrasonic waves, and the whole breaking process is completed instantly, thereby being beneficial to the dissolution of the polysaccharide; and the extraction time is shortened and the extraction rate is improved by matching the extraction temperature, the feed-liquid ratio and the extraction time. Meanwhile, the invention verifies the antioxidant activity of the Grateloupia filicina polysaccharide and provides a good foundation for research and application of the Grateloupia filicina polysaccharide in the aspect of antioxidant property.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a glucose standard curve of example 1;
FIG. 2 is a graph of extraction versus temperature for example 1;
FIG. 3 is a graph of the extraction ratio versus the feed-to-liquid ratio of example 1;
FIG. 4 is a graph of extraction rate versus extraction time for example 1;
FIG. 5 is a graph of DPPH radical clearance rate for example 2;
FIG. 6 is a graph of hydroxyl radical clearance for example 2;
FIG. 7 is a plot of superoxide anion radical clearance rate for example 2;
FIG. 8 is a graph showing the reducing power in example 2.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
In the examples of the present invention, Grateloupia filicina was purchased from Guangdong Shanshen shop. The reagents used are analytical grade, and the source of the reagents used and the source of the apparatus used can be selected by the person skilled in the art on the basis of experience or limited experimentation.
The embodiment of the invention provides an extraction method of Grateloupia filicina polysaccharide, which comprises the following steps:
a. placing Grateloupia filicina powder in a Soxhlet extractor, refluxing with petroleum ether and ethanol respectively, and drying;
b. adding the dried Grateloupia filicina powder into deionized water, extracting under the auxiliary action of ultrasonic wave, and centrifuging to obtain Grateloupia filicina polysaccharide extract with extraction rate of 50%;
extraction conditions are as follows: extracting at 60-100 deg.C for 1-5h with a feed-liquid ratio of 1: 70-110;
c. concentrating the Grateloupia filicina polysaccharide extract, precipitating with ethanol, centrifuging to separate precipitate, and air drying to obtain Grateloupia filicina polysaccharide.
The Grateloupia filicina powder is prepared by drying Grateloupia filicina, pulverizing, and sieving with 40 mesh sieve.
The boiling range of the petroleum ether is 60-90 ℃, and the reflux time of the petroleum ether is 2 h.
The ethanol is 95% ethanol, and the ethanol reflux time is 2 h.
The drying is carried out for 12h at 60 ℃.
The ultrasonic wave is divergent ultrasonic, the frequency is 40kHz, and the power is 0-800W.
In step b, the centrifugation condition is 60000rpm centrifugation for 15 min.
The concentration in the step c is 1/2 of the volume of the original extracting solution after vacuum-pumping rotary evaporation and concentration; and the alcohol precipitation is to add an ethanol solution with the volume 2-3 times of the volume of the concentrated extracting solution, and to stand the mixture for 12-24 hours at the temperature of 4-10 ℃, wherein the concentration of the ethanol solution is 90-100% of the volume fraction.
The embodiment of the invention also provides the Grateloupia filicina polysaccharide extracted by the extraction method.
The embodiment of the invention also provides application of the Grateloupia filicina polysaccharide or the Grateloupia filicina polysaccharide extracted by the extraction method in antioxidant activity and preparation of natural antioxidants. In the natural antioxidant, the Grateloupia filicina polysaccharide can be directly prepared into the natural antioxidant, or can be mixed with auxiliary materials acceptable in application to prepare the natural antioxidant, and then the natural antioxidant is packaged according to a conventional method. The dosage of the Grateloupia filicina polysaccharide can be properly changed according to the use scene, the use mode or different preparations of the natural antioxidant, but the premise is to ensure that the natural antioxidant achieves effective antioxidant concentration in the use scene.
The extraction rate of the Grateloupia filicina polysaccharide can be influenced by various factors, and in order to explore the mutual relation, the material-liquid ratio, the extraction time and the extraction temperature are set as three factors. One factor was changed and the others were unchanged. The process and embodiments of the present invention are further explained below.
Example 1 Synthesis of Grateloupia filicina polysaccharide and determination of extraction yield
1. Preparation of glucose Standard Curve
1.1 preparation of glucose standards of different concentrations
Accurately weighing 0.025g of glucose standard. Adding a proper amount of deionized water to dissolve the glucose solution, transferring the glucose solution to a 50mL volumetric flask, and fixing the volume to a scale mark to obtain a 0.500mg/mL glucose reference substance solution. Precisely sucking 0.500mg/mL glucose standard solution 2.00, 3.00, 4.00, 5.00, 6.00mL respectively in a 25mL volumetric flask to constant volume to obtain 0.04, 0.06, 0.08, 0.10, 0.12mg/mL glucose solution.
1.2 phenol-sulfuric acid color development
1.0mL of the above different concentration solution was pipetted into a test tube, and 1.0mL of a 5% phenol solution was slowly added to the tube in an ice-water bath and shaken well. 5mL of concentrated sulfuric acid solution is quickly added into the test tube, shaken for 5min and placed in a boiling water bath for 10 min. After heating, the material was taken out and cooled for 20 min. The same procedure was used as a blank in place of the glucose solution with deionized water while the procedure was being carried out.
1.3 content determination and drawing of Standard Curve
The absorbance values were measured at 485nm and a standard curve was generated, as shown in FIG. 1: the horizontal axis is the concentration of glucose, the vertical axis is the absorbance value, and the regression equation Y is calculated to be 9.9812X + 0.0095.
2. Determination of Grateloupia filicina polysaccharide extraction rate
The same procedure as that of the phenol-sulfuric acid color development method in 1.3 was repeated except that 1.0mL of Grateloupia filicina polysaccharide extract was used in place of the glucose standard solution in 1.2. Substituting the obtained absorbance value into regression equation to obtain the mass concentration (mg/mL) of polysaccharide compound in the extractive solution, and calculating polysaccharide extraction rate according to formula (1-1).
In the formula: c is the concentration (mg/mL) of the Grateloupia filicina polysaccharide extract; v is the constant volume (mL); w is the mass (g) of the Grateloupia filicina powder.
3. Determination of optimum extraction process of Grateloupia filicina polysaccharide
3.1 Single factor test
The extraction test of the Grateloupia filicina polysaccharide comprises the following steps:
a. drying Grateloupia filicina, pulverizing, sieving with 40 mesh sieve to obtain Grateloupia filicina powder, placing Grateloupia filicina powder in Soxhlet extractor, refluxing with petroleum ether with boiling range of 60-90 deg.C for 2 hr, refluxing with 95% ethanol for 2 hr, and drying at 60 deg.C for 12 hr;
b. adding the dried Grateloupia filicina powder into deionized water, extracting under the auxiliary action of divergent ultrasonic wave at frequency of 40kHz and power of 400W, and centrifuging at 60000rpm for 15min to obtain Grateloupia filicina polysaccharide extractive solution; extraction conditions are as follows: extracting at 60-100 deg.C for 1-5h with a feed-liquid ratio of 1: 70-110;
c. vacuum-pumping, rotary evaporating and concentrating Grateloupia filicina polysaccharide extractive solution to 1/2 volume of original extractive solution, adding 95% ethanol solution with volume fraction 3 times of the volume of the concentrated extractive solution, standing at 8 deg.C for 18h, centrifuging at 6000r for 20min, separating precipitate, and air drying to obtain Grateloupia filicina polysaccharide.
The extraction rate of the Grateloupia filicina polysaccharide can be influenced by various factors, and in order to explore the mutual relation, the material-liquid ratio, the extraction time and the extraction temperature are set as three factors. One factor was changed and the others were unchanged.
3.1.1 temperature
In order to investigate the relationship between the change of temperature (DEG C) and the extraction rate of Grateloupia filicina polysaccharide, the feed-liquid ratio was set to be 1:70(g/mL), and heating was carried out for 1 h. The temperature was changed to 60 deg.C, 70 deg.C, 80 deg.C, 90 deg.C, 100 deg.C.
The operation is as follows:
(1) the reaction mixture was placed in a thermostatically heated magnetic stirrer and set at the different temperatures described above.
(2) After the reaction is finished, the reactants are separated under a high-speed centrifuge. The supernatant is polysaccharide crude extract.
(3) The extract was measured for absorbance by the method of 3.3. And calculating the extraction rate.
As shown in FIG. 2, it is understood from FIG. 2 that the extraction rate is slightly decreased when the temperature is increased from 60 ℃ to 70 ℃ but the difference is not so large. When the temperature is from 70 ℃ to 100 ℃, the extraction rate of the Grateloupia filicina polysaccharide is increased and then decreased along with the increase of the extraction temperature. At 90 ℃, the extraction rate of polysaccharide reaches 46.76 percent, which is improved by 69.11 percent compared with 27.65 percent at 70 ℃.
The results show that increasing the temperature increases the extraction yield of the polysaccharide. However, too high a temperature may result in a change in the structure of the polysaccharide, leading to a reduction or even inactivation of the biological activity of the polysaccharide. Therefore, the extraction temperature range is chosen to be around 90 ℃ with other factors fixed.
3.1.2 feed-to-liquid ratio
In order to explore the relationship between the ratio of feed to liquid and the extraction rate of Grateloupia filicina polysaccharide, extraction was performed at 90 deg.C for 1 h. The feed-to-liquid ratio (g/mL) was varied to 1:70, 1:80, 1:90, 1:100, 1: 110. The operation is as 3.1.1. As shown in FIG. 3, it is understood from FIG. 3 that the increase in the extraction rate is significant when the feed-to-liquid ratio is from 1:70 to 1:90, and is gradual from 1:90 to 1: 110. The extraction rate reaches the highest value of 42.01% at the ratio of 1:100, which is 24.80% higher than 33.66% at the ratio of 1: 70.
The result shows that the water quantity is increased, more space can be provided for dissolving the polysaccharide, and the extraction rate of the polysaccharide is further improved. The polysaccharide content is gradually saturated and the extraction rate is slowly increased along with the increase of the feed-liquid ratio. In addition, the material-liquid ratio is increased, the subsequent workload and cost are also increased, and the production efficiency and the working cost are considered. Therefore, under the condition of fixing other factors, the range of the ratio of the material to the liquid is selected to be about 1: 100.
3.1.3 extraction time
To investigate the relationship between time (h) and Grateloupia filicina polysaccharide extraction rate, the feed-to-liquid ratio was set at 1:70(g/mL), and heating was carried out at 90 ℃. The heating time was varied to 1h, 2h, 3h, 4h, 5 h. The operation is as 3.1.1. As a result, as shown in fig. 4, it is understood from fig. 4 that the extraction rate gradually increases as the heating time increases. When the time is increased from 1h to 3h, the extraction rate is increased from 34.45% to 39.06%. When the time is 3h to 5h, the extraction rate tends to be flat, and the extraction rate reaches 40.25 percent at 5h, which is 16.69 percent higher than that of 1 h.
The results show that increasing the heating time increases the polysaccharide extraction rate, but the trend is slow. Increasing the time allows the polysaccharide to penetrate into the extraction solution as much as possible. The trend becomes slower as time goes and the concentration rises towards saturation. Considering also the process cost and the time cost, the extraction time range is selected to be around 4h with the highest possible extraction rate and with other single factors fixed.
3.2 orthogonal test
Selecting proper condition range according to the test result of single factor, and selecting L9 (3)4) The orthogonal table is used for carrying out orthogonal tests of 3 factors and 3 levels of temperature, feed-liquid ratio and extraction time to determine the optimal extraction conditions. The design and results are shown in table 1.
TABLE 1
Analysis in combination with table 1 shows that the influence of different single factors on the extraction rate of Grateloupia filicina polysaccharide is from large to small: temperature > extraction time > material-to-liquid ratio. To obtain significance, the analysis was re-performed using SPSS software, and the ANOVA table is shown in Table 2.
TABLE 2
a.R Fang (0.996 (adjustment R Fang (0.982))
As can be seen from Table 2, the p-value of the temperature factor is equal to 0.01, indicating a very significant difference; the P value of the feed-liquid ratio and the time factor is less than 0.05, which shows that the difference is obvious. The optimal process parameters are as follows: extracting at 90 deg.C for 5 hr at a ratio of 1:100 (g/mL). Under the condition, the extraction rate of the Grateloupia filicina polysaccharide compound is 50.34%, and the content of crude polysaccharide is 83.46% (0.417g/mL) measured by a phenol-sulfuric acid method.
3.3 validation test
In order to check whether the calculated optimum condition is the same as the true condition, an approximate verification experiment is performed. The Grateloupia filicina polysaccharide was subjected to 3 parallel tests under the optimal extraction process conditions. And (3) measuring the extraction rate of the extracting solution according to the method in the step 2, and obtaining the average extraction rate of the Grateloupia filicina polysaccharide of 50.07%. Compared with a theoretical predicted value, the relative deviation is 0.27%, the repeatability is better, and the result is reliable.
Example 2 Grateloupia filicina polysaccharide anti-oxidation test
1 DPPH radical scavenging experiment
1.1 preparation of the solution
(1) Accurately weighing 0.0025g of 1, 1-diphenyl-2-trinitrophenylhydrazine (DPPH), dissolving with a small amount of 70% ethanol, transferring into a 50ml brown volumetric flask, fixing the volume with 70% ethanol to obtain 0.05mg/mLDPPH test solution, storing in a refrigerator, and using when the test solution is prepared;
(2) taking the crude polysaccharide solution of the Grateloupia filicina, and preparing the crude polysaccharide solution into solutions with the concentrations of 0.5, 1.0, 1.5, 2.0 and 2.5mg/mL respectively.
1.2 determination of the clearance
5.00mL of each sample solution was precisely aspirated, 5.00mL of a DPPH solution (0.05mg/mL) was added thereto, the mixture was sufficiently shaken, and the mixture was allowed to stand in the dark for 30 min. Determination of the absorbance A of the test solution at 517nmi. After 5.00mL of the sample solution and 5.00mL of the solvent were sufficiently mixed, the absorbance A at 517nm was measuredj. After 5.00mL of DPPH solution and 5.00mL of solvent were thoroughly mixed, the absorbance at 517nm was measuredAc。
And (5) sorting and substituting the obtained A value into a formula (2-2) to calculate the DPPH free radical clearance.
In the formula AiThe absorbance of the mixed solution of the sample solution and the DPPH solution; a. thejThe absorbance of the mixed solution of the sample solution and the solvent of 70 percent ethanol; a. thecThe absorbance of the mixture of DPPH solution and 70% ethanol solvent is shown.
The results are shown in FIG. 5, and it can be seen from FIG. 5 that: (1) the clearance rate increases with increasing concentration. (2) The clearance of DPPH by polysaccharide is gradually flat after the concentration is increased. At a polysaccharide concentration of 2mg/mL, the clearance rate reached a maximum of 12.88%.
2 hydroxy radical scavenging experiments
2.1 preparation of the solution
(1)6mmol/L FeSO4Solution: accurately weighing FeSO4·7H20.0840g of O powder, which is dissolved in deionized water serving as a solvent by adding a small amount of deionized water, is transferred into a 50ml brown volumetric flask, is subjected to constant volume to a scale by using the deionized water, and is stored in a dark place to be prepared for use;
(2)6mmol/L salicylic acid-ethanol solution: accurately weighing 0.0420g of salicylic acid powder, taking absolute ethyl alcohol as a solvent, adding a small amount of absolute ethyl alcohol for dissolving, transferring into a 50mL volumetric flask, and fixing the volume to a scale by using the absolute ethyl alcohol to obtain the salicylic acid powder;
(3)6mmol/LH2O2solution: precisely absorb 30% H2O2Putting 4.60mL of the solution in a 25mL volumetric flask, and fixing the volume to a scale with deionized water to obtain the product;
(4) grateloupia filicina crude polysaccharide solution, prepared into concentration of 0.00, 0.25, 0.5, 0.75 and 1.00mg/mL solution.
2.2 determination of clearance
Accurately absorb 6mmol/L Fe2+2.00mL of the solution and 2.00mL of a 6mmol/L salicylic acid-ethanol solution were placed in a test tube. Respectively adding 2mL of sample solution into the test tubes, fully shaking, and standing for 10 min. 6mmol/L H was added2O2The reaction was started with 2.00mL of the solution and immediately placed in a 37 ℃ water bath for 30 min. And (6) cooling. Measuring absorbance value Ai at 510 nm; replacement of H with deionized water2O2The solution is not changed, and the absorbance value is measured to be Ai0. Replacing sample liquid with deionized water, operating, and measuring absorbance value A0。
And (4) sorting the obtained numerical values and substituting the numerical values into a calculation formula of the clearance rate, such as a formula (2-3).
In the formula AiIs the absorbance value of the sample solution; a. thei0Blank control absorbance value of the sample solution; a. the0Is a blank solution absorbance value.
The results are shown in FIG. 6, and FIG. 6 shows that: the ability of polysaccharides to scavenge hydroxyl radicals increases with increasing concentration.
3 superoxide anion radical scavenging experiment
3.1 preparation of the solution
(1) pH 8.2Tris-HCl buffer solution: 1.2114g of 2-amino-2- (hydroxymethyl) -1, 3-propanediol (Tris) is precisely weighed, and the volume is adjusted to 100mL by deionized water, so as to obtain a Tris solution (0.1 mol/L). Putting 50mL of the Tris solution into a 100mL volumetric flask, adding 22.9mL of 0.1mol/L hydrochloric acid solution, fully and uniformly mixing, and then fixing the volume to a scale with deionized water to obtain the Tris-base aqueous solution;
(2)3mmol/L pyrogallol solution: accurately weighing 0.0191g of pyrogallol, adding a small amount of 10mmol/L HCL solution for dissolving, transferring to a 50mL brown volumetric flask, and fixing the volume to the scale by using 10mmol/L HCL to obtain the pyrogallol;
(3) grateloupia filicina crude polysaccharide solution, prepared into concentration of 0.050, 0.075, 0.100, 0.125 and 0.150mg/mL solution.
3.2 determination of clearance
Taking a test tube, adding 4.50mL of Tris-HCL buffer solution (pH 8.2) and 4.20mL of sample solutions with different concentrations, and uniformly mixing. The mixture was transferred to a 25 ℃ water bath for 20 min. After water bath, 0.30mL of pyrogallol solution with concentration of 3mmol/L preheated to 25 ℃ is added, and the mixture is quickly and fully shaken. Quickly poured into a cuvette, and the absorbance of the cuvette was measured at 325nm against a 0.1mol/L HCl solution. The measurement is carried out every 30s for 5min continuously. The sample was replaced with deionized water and the rest of the procedure was unchanged as a control.
3.3 calculate the slope of wavelength and time by plotting the wavelength as ordinate and the time as abscissa, and the experimental group is KbThe control group is K0。
The obtained value is substituted into the calculation formula of the superoxide radical clearance rate, and the calculation formula is shown as (2-4):
the results are shown in FIG. 7, and the analysis in FIG. 7 reveals that: the inhibition rate of the polysaccharide on superoxide anions increases with increasing concentration.
4 reduction force test
4.1 preparation of the solution
(1) pH6.6 phosphoric acid buffer solution: 3.121g of sodium dihydrogen phosphate is precisely weighed, dissolved by deionized water and transferred to a 100mL volumetric flask, and the deionized water is used for constant volume; 7.164g of disodium hydrogen phosphate is precisely weighed, dissolved by deionized water and transferred to a 100mL volumetric flask, and the volume is determined by the deionized water; putting 62.5mL of sodium dihydrogen phosphate and 37.5mL of disodium hydrogen phosphate into a 100mL volumetric flask, and adjusting the pH value to obtain the product;
(2) 1% potassium ferricyanide solution: accurately weighing 1.00g of potassium ferricyanide, dissolving the potassium ferricyanide with deionized water, transferring the potassium ferricyanide into a 100mL volumetric flask, and fixing the volume with the deionized water to obtain the potassium ferricyanide solution;
(3) preparation of 10% trichloroacetic acid solution: accurately weighing 10.00g of trichloroacetic acid, dissolving with deionized water, transferring into a 100mL volumetric flask, and diluting with deionized water to a constant volume to obtain the trichloroacetic acid solution;
(4) 0.1% ferric chloride solution: accurately weighing 0.10g of ferric trichloride, dissolving with deionized water, transferring into a 100mL volumetric flask, and diluting with deionized water to a constant volume to obtain the ferric trichloride solution;
(5) grateloupia filicina crude polysaccharide solution, prepared into concentration of 0.050, 0.075, 0.100, 0.125 and 0.150mg/mL solution.
4.2 reduction force measurement
2.5mL of the sample solution, 2.5mL of pH6.6 phosphate buffer solution and 2.5mL of 1% potassium ferricyanide solution were placed in a test tube and mixed well. Water bath at 50 deg.c for 20 min. And (5) rapidly cooling. Then 2.5mL of 10% trichloroacetic acid is added and shaken up, and centrifuged at 3000r/min for 10 min. And adding 4mL of deionized water and 1mL of 0.1% ferric trichloride into 5mL of supernatant, uniformly mixing, and reacting for 10 min. The absorbance was measured at a wavelength of 700 nm.
The reducing power is related to the absorbance value. The larger the absorbance, the stronger the reducing power, and thus the comparison.
As a result, as shown in FIG. 8, the antioxidant (reducing agent) scavenges radicals by giving electrons by reducing action of itself, and the stronger the reducing ability is, the stronger the oxidation resistance is. As can be seen from fig. 8: the reducing power of the polysaccharide in the concentration range is increased and then decreased along with the increase of the concentration.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (10)
1. The extraction method of Grateloupia filicina polysaccharide is characterized by comprising the following steps:
a. placing Grateloupia filicina powder in a Soxhlet extractor, refluxing with petroleum ether and ethanol respectively, and drying;
b. adding the dried Grateloupia filicina powder into deionized water, extracting under the auxiliary action of ultrasonic waves, and centrifuging to obtain Grateloupia filicina polysaccharide extract;
extraction conditions are as follows: extracting at 60-100 deg.C for 1-5h with a feed-liquid ratio of 1: 70-110;
c. concentrating the Grateloupia filicina polysaccharide extract, precipitating with ethanol, centrifuging to separate precipitate, and air drying to obtain Grateloupia filicina polysaccharide.
2. The extraction method of Grateloupia filicina polysaccharide as claimed in claim 1, wherein Grateloupia filicina powder is obtained by drying Grateloupia filicina, pulverizing, and sieving with 40 mesh sieve.
3. The extraction method of Grateloupia filicina polysaccharide according to claim 1, wherein the boiling range of petroleum ether is 60-90 ℃, and the reflux time of petroleum ether is 2 h.
4. The extraction method of Grateloupia filicina polysaccharide as claimed in claim 1, wherein the ethanol is 95% ethanol by volume fraction, and the time of ethanol reflux is 2 h.
5. The extraction method of Grateloupia filicina polysaccharide as claimed in claim 1, wherein the drying is performed at 60 deg.C for 12 h.
6. The extraction method of Grateloupia filicina polysaccharide as claimed in claim 1, wherein the ultrasonic wave is divergent ultrasonic wave with frequency of 40kHz and power of 0-800W.
7. The extraction method of Grateloupia filicina polysaccharide as claimed in claim 1, wherein in step b, the centrifugation condition is 60000rpm for 15 min.
8. The extraction method of Grateloupia filicina polysaccharide as claimed in claim 1, wherein the concentration in step c is 1/2 by vacuum rotary evaporation to the volume of the original extract; and the alcohol precipitation is to add an ethanol solution with the volume 2-3 times of the volume of the concentrated extracting solution, and to stand the mixture for 12-24 hours at the temperature of 4-10 ℃, wherein the concentration of the ethanol solution is 90-100% of the volume fraction.
9. A Grateloupia filicina polysaccharide extracted by the extraction method of Grateloupia filicina polysaccharide of any one of claims 1-8.
10. The use of the Grateloupia filicina polysaccharide of claim 9 in antioxidant activity and preparation of natural antioxidants.
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| CN109608561A (en) * | 2019-02-01 | 2019-04-12 | 中国水产科学研究院南海水产研究所 | A kind of extracting method of centipede polysaccharides |
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| CN109608561A (en) * | 2019-02-01 | 2019-04-12 | 中国水产科学研究院南海水产研究所 | A kind of extracting method of centipede polysaccharides |
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