CN113773332A - Method for extracting ellagic acid from blue brave by using ultrasonic-assisted deep eutectic solvent - Google Patents
Method for extracting ellagic acid from blue brave by using ultrasonic-assisted deep eutectic solvent Download PDFInfo
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- 229960002852 ellagic acid Drugs 0.000 title claims abstract description 76
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- 239000000243 solution Substances 0.000 claims description 18
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- 235000021014 blueberries Nutrition 0.000 claims description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 6
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
- C07D493/06—Peri-condensed systems
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Abstract
The invention discloses a method for extracting ellagic acid from a blue cloth by using an ultrasonic-assisted deep eutectic solvent, which adopts the ultrasonic-assisted deep eutectic solvent to extract the ellagic acid from the blue cloth, takes the yield of the ellagic acid from the blue cloth as an index, and researches the following five factors: the influence of the positive water content (A) of the blue cloth, the extraction time (B), the material-liquid ratio (C), the extraction temperature (D) and the extraction power (E) on the yield of the positive ellagic acid of the blue cloth is tested by using an experimental scheme obtained by a response surface design method so as to obtain the optimal extraction condition parameters of the yield of the positive ellagic acid of the blue cloth. Experimental results show that the optimal extraction process is adopted, namely the positive water content of the blue cloth is 47%, the deep eutectic solvent is added according to the mass-to-volume ratio of 10:1, and ultrasonic extraction is carried out for 31min at 70 ℃ and the extraction power of 300W. The method is stable and reliable, has high extraction efficiency, and can be used for extracting ellagic acid from herba Blumeae Laciniatae.
Description
Technical Field
The invention relates to a method for extracting effective components of traditional Chinese medicines, in particular to a method for extracting ellagic acid from a blue berry by using an ultrasonic-assisted deep eutectic solvent.
Background
Blue cloth, also known as Zhuifeng Qin, is a dry whole plant of Rosaceae plant Geum aleppicum Jacq or Geum japonicum Thunb var chinensis Bolle, sweet, slightly bitter and cool, and enters liver, spleen and lung meridians. It belongs to one of Qinling seven drugs, is widely distributed in Shanxi, Anhui, Hubei, Gansu, Shandong, Henan, Jiangsu and other areas, has the efficacies of strengthening spleen and replenishing qi, moistening and nourishing skin, nourishing yin and moisturizing lung, relieving cough and reducing sputum and the like, and can be used for treating deficiency of both qi and blood, lung qi deficiency, chronic cough, spleen failure in transportation, dampness accumulation and lower energizer and the like. The main pharmacological actions of the traditional Chinese medicine comprise dispelling wind and eliminating dampness, enriching blood, resisting inflammation, resisting hypertension, eliminating phlegm and relieving asthma, reducing blood sugar and the like. The main component of the blue cloth is ellagic acid, and has the effects of dissipating and eliminating in vivo blood stasis, inhibiting growth of tumor cells, effectively inhibiting oxidation reaction of free radicals, etc. As is known from the conventional studies, a heating agitation extraction method, an ultrasonic extraction method, a microwave extraction method, a supercritical fluid extraction method, and the like can be used as the method for extracting ellagic acid, and the conventional extraction method has a low extraction efficiency of ellagic acid.
At present, no method for extracting the ellagic acid from the root of the blueberries by adopting an ultrasonic-assisted deep eutectic solvent is reported.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to overcome the defects in the prior art, screens out an optimal extraction process by a response surface design method, and provides a method for extracting neutral ellagic acid from a blue-cloth medium by using an ultrasonic-assisted deep eutectic solvent, wherein the method can improve the yield of the ellagic acid.
The invention also aims to provide a method for measuring the content of ellagic acid, and the detection method provided by the invention has the advantages of high sensitivity, good stability and great significance for controlling quality and ensuring curative effect.
The technical scheme is as follows: in order to achieve the above purpose, the invention adopts the technical scheme that:
a method for extracting ellagic acid from a blue-berry by using an ultrasonic-assisted deep eutectic solvent comprises the following steps:
(1) preparation of deep eutectic solvent
Weighing a certain amount of choline chloride and oxalic acid, mixing, placing in a water bath for heating, stirring, taking out when the liquid is colorless and transparent and has no bubbles, and cooling to room temperature to obtain a deep eutectic solvent;
(2) and (2) putting the blue cloth into the deep eutectic solvent in the step (1) for ultrasonic extraction, filtering to obtain filtrate, and concentrating to obtain the blue cloth.
Preferably, the method for extracting ellagic acid from the root of common blueberries by using the ultrasonic-assisted deep eutectic solvent comprises the following steps:
(1) preparation of deep eutectic solvent
Weighing choline chloride and oxalic acid in a molar ratio of 1-3: 1-3, mixing, heating in a water bath at 40-60 ℃ for 1-10 h, stirring, taking out when the liquid is colorless and transparent and has no bubbles, and cooling to room temperature to obtain a deep eutectic solvent;
(2) adding the blue cloth into the deep eutectic solvent in the step (1) according to the mass-to-volume ratio of 5-20: 1, performing ultrasonic extraction at 60-80 ℃ for 20-60 min, filtering after extraction to obtain filtrate, and concentrating to obtain the blue cloth.
Preferably, the method for extracting ellagic acid from the root of common blueberries by using the ultrasonic-assisted deep eutectic solvent comprises the following steps:
(1) preparation of deep eutectic solvent
Weighing choline chloride and oxalic acid with a molar ratio of 1:1, mixing, heating in a water bath at 55 ℃ for 4-6 h, stirring, taking out when the liquid is colorless and transparent and has no bubbles, and cooling to room temperature to obtain a deep eutectic solvent;
(2) adding blue cloth with the water content of 47-60% into the deep eutectic solvent in the step (1) according to the mass-to-volume ratio of 10:1, performing ultrasonic extraction at 70 ℃ and the extraction power of 300W for 31min, filtering after extraction to obtain filtrate, and concentrating to obtain the blue cloth.
The method for measuring the content of the ellagic acid prepared by the method comprises the following steps:
(1) preparation of control solution
Precisely weighing ellagic acid reference substance, adding methanol to obtain ellagic acid reference substance solution with concentration of 0.25mg/mL, filtering with 0.45 μm filter membrane, and storing in refrigerator at 4 deg.C;
(2) preparation of test solution
Ultrasonic-assisted deep eutectic solvent is taken to extract ellagic acid obtained from blue cloth, methanol is added for dilution, and the solution is filtered through a 0.45-micrometer filter membrane to obtain a test solution;
(3) and (3) respectively taking the reference substance solution in the step (1) and the test solution in the step (2), injecting the reference substance solution and the test solution into a high performance liquid chromatograph, and measuring the content of the ellagic acid in the test solution according to an external standard one-point method or a standard curve method.
Wherein the chromatographic conditions of the step (3) are as follows: the mobile phase A is methanol water solution, and the mobile phase B is 0.7% phosphoric acid water solution; the gradient elution sequence was as follows: 0-5 min, 8% of A and 92% of B; 5-10 min, 20% of A and 80% of B; 10-20 min, 40% of A and 60% of B; 20-25 min, 75% of A and 25% of B; 25-30 min, 90% of A and 10% of B; 30-35 min, 8% of A and 92% of B; chromatographic column Waters SunFire C18Specification 4.6mm × 150mm, 5 μm; the flow rate is 1 mL/min-1(ii) a The detection wavelength is 253.4 nm; the column temperature is 30 ℃; the amount of sample was 10. mu.L.
Has the advantages that: compared with the prior art, the method for extracting the ellagic acid from the blue-cloth mid-tree by using the ultrasonic-assisted deep eutectic solvent has the following advantages:
the invention adopts the ultrasonic-assisted deep eutectic solvent to extract the positive ellagic acid of the blue cloth, takes the yield of the positive ellagic acid of the blue cloth as an index, and researches the following five factors: the influence of the positive water content (A) of the blue cloth, the extraction time (B), the material-liquid ratio (C), the extraction temperature (D) and the extraction power (E) on the yield of the positive ellagic acid of the blue cloth is tested by using an experimental scheme obtained by a response surface design method so as to obtain the optimal extraction condition parameters of the yield of the positive ellagic acid of the blue cloth. The experimental result shows that the optimal extraction process is adopted, namely the positive water content of the blue cloth is 47%, the deep eutectic solvent is added according to the mass-to-volume ratio of 10:1, ultrasonic extraction is carried out for 31min at 70 ℃ and the extraction power is 300W, and the yield of the extracted ellagic acid in the positive blue cloth is 3.2142mg g-1The extraction rate is 142% higher than that of pure water, 41% higher than that of 70% acetone aqueous solution and 76% higher than that of 50% methanol aqueous solution.
The method is stable and reliable, has high extraction efficiency, and can be used for extracting ellagic acid from herba Blumeae Laciniatae.
In addition, the invention establishes a method for detecting the content of ellagic acid in the euonymus japonicus extractive, and the methodology shows that the detection method provided by the invention has the advantages of high precision, good stability, good repeatability and high accuracy.
Drawings
FIG. 1 is a response surface diagram of the interaction of water cut and extraction time.
FIG. 2 is a response surface diagram of the interaction of water content and feed-liquid ratio.
FIG. 3 is a response surface plot of the interaction of water cut with extraction temperature.
FIG. 4 is a response surface plot of the interaction of water cut and extracted power.
FIG. 5 is a response surface plot of the interaction of extraction time and feed-to-liquid ratio.
FIG. 6 is a response plot of the interaction of extraction time and extraction temperature.
FIG. 7 is a response plot of the interaction of extraction time with extraction power.
FIG. 8 is a response surface plot of the interaction of feed-to-liquid ratio and extraction temperature.
FIG. 9 is a response plot of the interaction of feed-to-liquid ratio and extracted power.
FIG. 10 is a response plot of the interaction of extraction temperature with extraction power.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be purely exemplary and are not intended to limit the scope of the invention, as various equivalent modifications of the invention will occur to those skilled in the art upon reading the present disclosure and fall within the scope of the appended claims.
Example 1 deep eutectic solvent extraction of Canada ellagic acid from blue Brachycarpa
1. Experimental materials, instruments and reagents
1.1 Experimental materials
Blue cloth (product lot 20190312, available from Tongling He Tian Chinese medicinal decoction pieces Co., Ltd.), and further separately collecting 24 batches of blue cloth crude medicinal decoction pieces with different production places and lots, removing impurities mixed in the decoction pieces, pulverizing for 2min with a Chinese herbal medicine pulverizer, sieving with 50 mesh sieve, and storing in dark place for use.
4.1.2 Experimental instruments
| Laboratory apparatus | Production company |
| MiLLI-QIQ7000 water purifier | Mirabbo China Co., Ltd |
| Electric heating constant temperature water bath | Shanghai science Hengshicheng development Co Ltd |
| KQ-500DE type numerical control ultrasonic cleaner | KUNSHAN ULTRASONIC INSTRUMENTS Co.,Ltd. |
| Chinese herbal medicine crusher | TIANJIN TAISITE INSTRUMENT Co.,Ltd. |
| Hersche 3H16RI refrigerated centrifuge | Hunan Hexi Instrument Equipment Co.,Ltd. |
| Acquisytare ultra-high performance liquid chromatograph | Waters Inc. of USA |
| Microfuge20R Microfuge centrifuge | Beckmann Coulter Ltd, USA |
| Mettler MS105DU electronic balance | Shanghai Mei Teller-Tollido laboratory |
1.2 Experimental reagents
| Experimental Material | Chemical formula (II) | Manufacturer of the product |
| Choline chloride | C5H11NO2 | Sigma Aldrich trade company |
| Oxalic acid | C2H2O4 | Sigma Aldrich trade company |
| Methanol (analytically pure) | CH3OH | Tianjin chemical reagent Co Ltd |
| Ellagic acid standard substance | C14H6O8 | Vickqi Biotech Co., Ltd, Sichuan province |
2. Experimental methods
2.1 preparation of deep eutectic solvents
Respectively weighing a certain amount of choline chloride and oxalic acid (the molar ratio is 1:1) and placing the choline chloride and the oxalic acid in a beaker, heating the choline chloride and the oxalic acid in a water bath at 55 ℃ for 4-6 hours, stirring the choline chloride and the oxalic acid with a glass rod at intervals, taking the choline chloride and the oxalic acid out when the liquid is colorless and transparent and has no bubbles, cooling the choline chloride and the oxalic acid to room temperature to obtain a deep eutectic solvent, sealing the eutectic solvent and storing the choline chloride and the oxalic acid in a refrigerator at 4 ℃ for later use.
2.2 method for extracting ellagic acid from blue cloth
Accurately weighing a certain amount of powder of herba Gei Piloselli (product batch No. 20190312, available from Torilis japonica field Chinese medicinal decoction pieces Co., Ltd.), placing into a 10mL EP tube, adding 2.5mL of deep eutectic solvent, performing ultrasonic extraction on ellagic acid in the herba Gei Piloselli according to five factors of experimental design, and preheating to a specified temperature half an hour in advance when two centrifuges are used. After completion of the extraction, the rotation speed of the first refrigerated centrifuge was set to 8000rpm, the temperature was set to 30 ℃, and centrifugation was performed for 20 min. After centrifugation, 1mL of the supernatant was taken out, transferred to a 2mL EP tube, added with 1mL of ultrapure water, and subjected to secondary centrifugation at a speed of 10000rpm in a micro-freezing centrifuge and at a temperature of 20 ℃ for 10 min. And slowly taking out after the second centrifugation is finished, transferring 1mL of supernate into a liquid phase small bottle by using a liquid transfer gun, and analyzing in an ultra-high performance liquid chromatograph, wherein each sample is detected twice.
Chromatographic conditions are as follows: the mobile phase (A) is methanol water solution, and the mobile phase (B) is 0.7 percent phosphoric acid water solution; the sample is developed and injected for analysis, and the gradient elution sequence is as follows: 0-5 min, 8% of A and 92% of B; 5-10 min, 20% of A and 80% of B; 10-20 min, 40% of A and 60% of B; 20-25 min, 75% of A and 25% of B; 25-30 min, 90% of A and 10% of B; 30-35 min, 8% of A and 92% of B; chromatographic column Waters SunFire C18(4.6 mm. times.150 mm, 5 μm); the flow rate is 1 mL/min-1(ii) a The detection wavelength is 253.4 nm; the column temperature is 30 ℃; the amount of sample was 10. mu.L.
The formula for calculating the yield of the ellagic acid in the blue cloth is as follows:
V1: volume of the sample after constant volume, unit: mL; v2: volume and unit of the sample to be measured after constant volume: mL; x: concentration corresponding to the standard curve when detecting the sample by high performance liquid chromatography, unit: mg.mL-1(ii) a m: positive mass of blue cloth, unit: g; w: total ellagic acid yield, unit: mg.g-1。
2.3 Box-Behnken Experimental design
According to the single-factor experiment result and analysis and the Box-Behnken experiment design principle, DES water content, extraction time, material-liquid ratio, extraction temperature and extraction power in the experiment are selected as 5 factors of the Box-Behnken experiment design, and a design scheme with 5 factors and 3 levels is adopted by combining the design principle of a response surface method, and the design scheme is shown in Table 1.
TABLE 1 response surface analysis factors and level design
2.4 determination of mid-ellagic acid yield in blue-cloth of different batches at different places of production
Taking 24 batches of the blue-cloth upright medicinal material decoction pieces with different production places and different batches of numbers respectively, carrying out experimental operation according to optimal extraction condition parameters obtained by a response surface design method experiment to obtain a blue-cloth upright ellagic acid yield determination result, and detecting each sample for three times.
3. Methodology investigation
3.1 preparation of control solutions
Precisely weighing 0.25mg of ellagic acid reference substance, preparing into solution containing 0.25mg of ellagic acid reference substance per 1mL of methanol solution, and storing in refrigerator at 4 deg.C.
3.2 preparation of test solution
Weighing choline chloride and oxalic acid according to a certain molar ratio (1:1), putting the choline chloride and the oxalic acid into a beaker, heating the beaker in a water bath at 55 ℃ for 4-6 hours, stirring the beaker without stirring, taking the beaker out to wait for cooling the beaker to room temperature when the liquid in the beaker is colorless and transparent and has no bubbles, obtaining a deep eutectic solvent, and putting the deep eutectic solvent into a refrigerator at 4 ℃ for storage for later use.
3.3 Experimental Condition parameters
A(50％)、B(30min)、C(20mg·mL-1) D (50 ℃) and E (300W).
3.4 repeatability experiments
And (4) taking blue cloth powder of the same batch, accurately weighing, and paralleling 6 parts for later use. Preparing a test solution, injecting 10 mu L of the test solution into a high performance liquid chromatograph according to the chromatographic conditions, measuring the peak area of the ellagic acid, and inspecting the repeatability of the ellagic acid in the middle of the blue cloth.
3.5 precision test
The sample solution was prepared according to the method of "3.2", taking 0.125 mg. multidot.mL that had been prepared in advance-1According to the chromatographic conditions, the sample solution is analyzed in a high performance liquid chromatograph, 10 mu L of sample injection is carried out each time, and the sample injection is continuously carried out for 6 times, so as to determine the peak area of the ellagic acid.
3.6 stability test
And (4) taking blue cloth powder of the same batch, accurately weighing, and paralleling 6 parts for later use. Preparing a test solution, and cooling to room temperature. The stability of the sample solution was investigated by measuring the peak area of ellagic acid in the sample solution under the above-mentioned chromatographic conditions by introducing 10. mu.L of each sample in a high performance liquid chromatograph for 0, 2, 4, 6, 8, 10, 12, and 24 hours.
3.7 sample application recovery test
A sample with a known content of 0.025g is precisely weighed and is divided into 6 portions for standby. Precisely adding 0.06275mg ellagic acid reference substance, respectively, injecting sample solution into high performance liquid chromatograph according to the above chromatographic conditions, measuring, injecting 10 μ L sample, and calculating recovery rate by measuring peak area of ellagic acid.
3.8 solvent comparison experiment
And (3) taking blue cloth powder of the same batch, accurately weighing, and paralleling for standby application. Adding pure water, 70% acetone, and 50% methanol to obtain sample solution, injecting 10 μ L, injecting into high performance liquid chromatograph under the above chromatographic conditions to determine peak area of ellagic acid, and comparing extraction efficiency of different solvents.
4. Results and analysis of the experiments
4.1 Box-Behnken experiment design optimization extraction process
4.1.1 response surface experimental design and results, see Table 2.
TABLE 2 response surface analysis protocol and results
4.1.2 model equation establishment and significance test
After analyzing the experimental results in table 2, regression equation (1) of the ellagic acid yield (Y) and factors of A, B, C, D and E is obtained:
Y＝2.83-0.0337A+0.0608B-0.0297C+0.2217D-0.0173E-0.0059AB-0.0321AC-0.0633AD+0.0072AE+0.0831BC+0.0467BD+0.0255BE-0.0434CD-0.0110CE-0.0106DE-0.1191A2-0.1130B2-0.0204C2+0.0692D2-0.1374E2
table 3 shows the results of the model regression analysis of variance. Correlation coefficient R of the above regression equation20.8698, indicating that the equation has a high degree of fit; the correction factor is 76.57%, indicating that the model can interpret 76.57% of the experimental data. The mismatching term can represent the difference of data in the model, and as can be seen from table 3, the regression model mismatching term F is 8.35, and P is less than 0.0001, which indicates that the regression model has reached a very significant level, the model is very suitable for practical experiments, and the experimental error is relatively small, and the relationship between the ellagic acid yield and the DES water content, the extraction time, the feed-liquid ratio, the extraction temperature and the extraction power can be reflected. The mismatching term F2.94 and P0.1175 > 0.05 in the model equation indicates that the regression model is not a poor fit and fits well to the experiment. Comparing the P value with the F value, the result shows that the influence of all factors on the ellagic acid yield is as follows: d > B > A > CIs > E. Wherein, B, D, A2、B2、D2、E2The term (P < 0.05) has a significant effect on the extraction of ellagic acid. Therefore, the extraction condition of the ellagic acid in the middle of the blue bramble can be analyzed and predicted by using the model.
TABLE 3 analysis of variance of regression model equations
Note: *. P < 0.05, with significant difference; p < 0.01 was very significantly different.
4.1.3 interaction of factors
The experimental results are shown in table 4 and fig. 1 to 10.
TABLE 4 response surface design optimization of interaction experiment results of various factors
Note: denotes the same factors; the values are determined for each factor.
Generally, in the response surface design method, a response surface map and a contour map are used to directly reflect the influence of interaction between factors on a response value. The shape of the contour lines may reflect the strength of the interaction, and is classified into a circle (weak interaction) and an ellipse (significant interaction). It can be seen that the elliptical shape of II, IX in FIG. 1 is more pronounced, indicating a stronger interaction between AC and CE.
4.1.4 verification of the results
After equation (1) is solved through software Design-Expert 10, the optimal extraction condition parameters are obtained: a (47%), B (31min), C (10 mg. mL)-1) D (70 ℃) and E (300W). Under the condition, the yield of the ellagic acid predicted by a response surface design method is 3.188mg g-1. In order to test whether the model given by the response surface design method is real and credible, the obtained optimal extraction condition parameters are adopted to carry out three verification experiments, and the average yield of the ellagic acid in the three verification experiments is 3.2142mg g-1The relative standard deviation from the theoretical prediction was 0.58%. In conclusion, it can be seen that the model is reliable, and therefore the response surface design method is suitable for regression analysis and condition parameter improvement of the ellagic acid extraction process in the middle of blue cloth.
4.2 measurement of ellagic acid in different lots of blue cloths in different production places, the results are shown in Table 5.
TABLE 5 results of the experiment
4.3 methodological investigation of the results
The results are shown in tables 6 and 7.
TABLE 6 results of the experiment
| Experimental project | Ellagic acid peak area RSD (%) | Results |
| Repeatability test | 2.60 | The extraction method has good repeatability |
| Precision experiment | 0.4 | The precision of the instrument is good |
| Stability test | 0.85 | The test solution is stable within 24h |
TABLE 7 sample recovery test results
4.3 comparative Experimental results of ultrasonic extraction with different extraction solvents
TABLE 8 results of solvent comparison experiments
Taking a blue cloth as an extraction raw material, taking a deep eutectic solvent as an extraction solvent, extracting ellagic acid in the blue cloth by adopting ultrasonic, investigating the process conditions such as DES (data encryption standard) water content, extraction time, material-liquid ratio, extraction temperature and extraction power, and analyzing the experiment by utilizing a response surface method to establish a model, wherein when the blue cloth (DES) water content is 47%, the extraction time is 31min and the material-liquid ratio is 10 mg/mL-1The highest ellagic acid yield is 3.3186mg g when the extraction temperature is 70 ℃ and the extraction power is 300W-1The extraction rate of the ellagic acid is 142% higher than that of pure water, 41% higher than that of 70% acetone aqueous solution and 76% higher than that of 50% methanol aqueous solution, which shows that the method can obviously improve the yield of the ellagic acid by adopting the deep eutectic solvent and obtain good technical effect.
The result of the measurement of the ellagic acid yield of 24 batches of the blue-cloth crude drugs shows that the ellagic acid yield of the Guizhou and Fujian medicinal materials is higher. The significant difference of the ellagic acid yield of different lot numbers of blue cloth Zheng of Fujian province and Y44 of Guizhou province and other four blue cloth Zheng exists, and the significant difference may be caused by factors such as climate, geographical position, harvest season and the like. Therefore, the ellagic acid yield is used as an index for investigating effective chemical components in the positive blue bracteatum, and scientific basis is provided for the subsequent quality research of the positive blue bracteatum medicinal materials.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (5)
1. A method for extracting ellagic acid from a blue-berry by using an ultrasonic-assisted deep eutectic solvent is characterized by comprising the following steps:
(1) preparation of deep eutectic solvent
Weighing a certain amount of choline chloride and oxalic acid, mixing, placing in a water bath for heating, stirring, taking out when the liquid is colorless and transparent and has no bubbles, and cooling to room temperature to obtain a deep eutectic solvent;
(2) and (2) putting the blue cloth into the deep eutectic solvent in the step (1) for ultrasonic extraction, filtering to obtain filtrate, and concentrating to obtain the blue cloth.
2. The method for extracting ellagic acid from root of blueberries by using the ultrasonic-assisted deep eutectic solvent according to claim 1, comprising the following steps:
(1) preparation of deep eutectic solvent
Weighing choline chloride and oxalic acid in a molar ratio of 1-3: 1-3, mixing, heating in a water bath at 40-60 ℃ for 1-10 h, stirring, taking out when the liquid is colorless and transparent and has no bubbles, and cooling to room temperature to obtain a deep eutectic solvent;
(2) adding the blue cloth into the deep eutectic solvent in the step (1) according to the mass-to-volume ratio of 5-20: 1, performing ultrasonic extraction at 60-80 ℃ for 20-60 min, filtering after extraction to obtain filtrate, and concentrating to obtain the blue cloth.
3. The method for extracting ellagic acid from root of blueberries by using the ultrasonic-assisted deep eutectic solvent according to claim 2, comprising the following steps:
(1) preparation of deep eutectic solvent
Weighing choline chloride and oxalic acid with a molar ratio of 1:1, mixing, heating in a water bath at 55 ℃ for 4-6 h, stirring, taking out when the liquid is colorless and transparent and has no bubbles, and cooling to room temperature to obtain a deep eutectic solvent;
(2) adding blue cloth with the water content of 47-60% into the deep eutectic solvent in the step (1) according to the mass-to-volume ratio of 10:1, performing ultrasonic extraction at 70 ℃ and the extraction power of 300W for 31min, filtering after extraction to obtain filtrate, and concentrating to obtain the blue cloth.
4. A method for measuring the content of ellagic acid produced by the method according to any one of claims 1 to 3, comprising the steps of:
(1) preparation of control solution
Precisely weighing ellagic acid reference substance, adding methanol to obtain ellagic acid reference substance solution with concentration of 0.25mg/mL, filtering with 0.45 μm filter membrane, and storing in refrigerator at 4 deg.C;
(2) preparation of test solution
Diluting ellagic acid prepared by the method of any one of claims 1 to 3 with methanol, and filtering with 0.45 μm filter membrane to obtain a test solution;
(3) and (3) respectively taking the reference substance solution in the step (1) and the test solution in the step (2), injecting the reference substance solution and the test solution into a high performance liquid chromatograph, and measuring the content of the ellagic acid in the test solution according to an external standard one-point method or a standard curve method.
5. The method for measuring the content of ellagic acid produced by the method according to claim 4, wherein the chromatographic conditions of step (3) are: the mobile phase A is methanol water solution, and the mobile phase B is 0.7% phosphoric acid water solution; the gradient elution sequence was as follows: 0-5 min, 8% of A and 92% of B; 5-10 min, 20% of A and 80% of B; 10-20 min, 40% of A and 60% of B; 20-25 min, 75% of A and 25% of B; 25-30 min, 90% of A and 10% of B; 30-35 min, 8% of A and 92% of B; chromatographic column Waters SunFire C18Specification 4.6mm × 150mm, 5 μm; the flow rate is 1 mL/min-1(ii) a Detection wavelength is 253.4 nm; the column temperature is 30 ℃; the amount of sample was 10. mu.L.
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