CN114887350A - Method for extracting rosemary extract serving as strong antioxidant - Google Patents
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Abstract
The invention relates to a method for extracting and preparing rosemary extract serving as a strong antioxidant, which utilizes a response surface method and determines an optimal extraction process according to Design expert12.0 software Design. The method comprises the following steps: (1) preparing a sample; (2) detecting the oxidation resistance; (3) optimization design of a response surface method: according to the test result, drawing analysis is carried out by using Design expert12.0 software, and a curved surface response and contour map of a regression equation are drawn by interaction of two factors of 6 factors such as the average value of the oxidation resistance APC index, the feed-liquid ratio, the concentration of an ethanol solution of an extraction solvent, the extraction temperature, the ultrasonic power, the extraction time and the extraction times. Determining the APC index average range of the rosemary extract by taking the oxidation resistance APC index average value of the conventional antioxidant as a standard, and obtaining the optimal extraction factor ratio of the strong oxidation resistance extracting solution through a fitting result. The method is simple and easy to operate, has low cost and is suitable for expanded production.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for extracting a rosemary extract serving as a strong antioxidant.
Background
Rosemary (Rosemary) is a shrub of erect or creeping type of Rosmarinus genus of Labiatae family, originated from Mediterranean region, and mainly originated from Morocco, Spain, former south Africa, Bulgaria and Tuniss. Rosemary has a long history of cultivation in China, and has been introduced and cultivated as early as three countries. Rosemary is an important aromatic economic plant which is widely used not only in cooking for changing and enhancing flavor but also in conventional medicine, and is a promising medicinal plant for preventing and treating cold, rheumatism, muscle and joint pain. It is one of the most popular natural bioactive compound sources at present, and has the functions of antioxidation, antitumor, anti-inflammatory, antibiosis and the like.
Natural antioxidants from plants are becoming more and more important, and have wide application prospects in the fields of food, medicine, daily chemicals and the like. Rosemary antioxidant is one of the most widely used natural antioxidants in industrial production, and is widely and directly used in grease, oil products, meat and poultry products, marine products, leisure food, spices and the like, and international public believes that the third generation green food additive is a green food additive, wherein the antioxidant components of the additive are mainly carnosic acid, rosmarinic acid, carnosol, flavonoid and the like, and the antioxidant property of the additive is obviously higher than that of conventional oxidants such as BHA, vitamin E and the like. At present, researches on the oxidation resistance of rosemary generally focus on the oxidation resistance comparison and single-component extraction of active ingredients such as carnosic acid, rosmarinic acid and the like, and documents on aspects such as the oxidation resistance research of composite ingredients and the optimization of extraction conditions by using a response surface method are not reported.
Disclosure of Invention
In order to solve the problems, the invention provides a method for extracting rosemary extract serving as a strong antioxidant. The invention utilizes a response surface method to fit a single-factor experiment result to obtain the optimal extraction factor ratio of the strong oxidation resistance extracting solution, and mainly comprises the following steps:
1) cleaning fresh rosemary leaves, putting the fresh rosemary leaves into a freeze dryer for freeze drying, grinding, crushing and sieving, extracting under different material-liquid ratios, the concentration of an ethanol solution as an extraction solvent, the extraction temperature, the ultrasonic power, the extraction time and the extraction times, and filtering an extracting solution by using a filter membrane after suction filtration to obtain a sample to be detected;
2) respectively detecting the antioxidant indexes of DPPH, PTIO, ABTS and FRAP of each sample, calculating the APC index according to the following formula, and calculating the average value of the APC index:
3) and (3) taking the APC index mean value obtained by calculation as a standard, and optimally designing by a response surface method to obtain the optimal feed-liquid ratio, the concentration of an extraction solvent ethanol solution, the extraction temperature, the ultrasonic power, the extraction time and the extraction times.
Wherein the temperature of the cold trap of the freeze dryer is-50 ℃, and the freezing time is freeze drying to room temperature.
Wherein, with a sample: adding ethanol solution into the mixture according to the volume ratio of 1: 10-25 of ethanol.
Wherein the ultrasonic power is 0-500W.
Wherein the extraction time is 20-100 min.
Wherein the extraction temperature is 30-60 ℃.
Wherein the extraction times are 1-3 times.
Wherein, a polyvinylidene fluoride organic microporous filter membrane with the diameter of 0.22 mu m is adopted for filtration.
The response surface method optimization design specifically comprises the following steps: according to the test result, drawing and analyzing by using Design expert12.0 software, drawing a curved surface response and a contour map of a regression equation by using the oxidation resistance APC index mean value as a standard and the interaction of two factors of 6 factors of a material-liquid ratio, an extraction solvent ethanol solution concentration, an extraction temperature, an ultrasonic power, an extraction time and an extraction frequency, selecting conventional antioxidants such as BHA, BHT, vitamin E and the like and a monomer carnosic acid prepared in the extract, performing oxidation resistance detection such as DPPH, PTIO, ABTS, FRAP and the like according to the dosage requirement of a food additive use standard (GB 2760-: the ratio of the material to the liquid is 1: 12-15, the concentration of an ethanol solution serving as an extraction solvent is 60% -90%, the extraction temperature is 50-60 ℃, the ultrasonic power is 200-300W, the extraction time is 50-60 min, and the extraction frequency is 2-3 times.
In one embodiment of the present invention, a method for extracting rosemary extract as a strong antioxidant comprises the following steps:
(1) sample preparation: cleaning fresh rosemary leaves, placing the cleaned fresh rosemary leaves into a freeze dryer, setting the temperature of a cold trap at minus 50 ℃, stopping freeze drying until the temperature reaches room temperature, then crushing the fresh rosemary leaves, sieving the crushed fresh rosemary leaves by a 60-mesh sieve, adding 0-100% ethanol solution according to the ratio of material to liquid of 1: 10-25, extracting the mixture for 20-100 min under the ultrasonic power of 0-500W, wherein the extraction temperature is 30-60 ℃, centrifuging the mixture at the speed of 5000 r/min after the extraction is finished, pouring the supernatant extracting solution into a container, adding the ethanol solution again for repeated extraction for 1-3 times, finally carrying out suction filtration on all the supernatant extracting solution, and filtering the filtrate by using a polyvinylidene fluoride organic microporous filter membrane with the diameter of 0.22 mu m to finish the sample preparation.
(2) And (3) oxidation resistance detection: and (3) respectively detecting antioxidant indexes such as DPPH, PTIO, ABTS, FRAP and the like of each sample, comparing the antioxidant activities of the samples by using an antioxidant activity comprehensive (APC) index method, calculating an APC index according to the following formula, and calculating an APC index mean value (see Table 1).
(3) Response surface method optimal design: according to the test result, drawing analysis is carried out by using Design expert12.0 software, and a curved surface response and a contour map of a regression equation are drawn by interaction of two factors of 6 factors such as a material-liquid ratio, an extraction solvent ethanol solution concentration, an extraction temperature, ultrasonic power, extraction time, extraction times and the like with an antioxidant activity comprehensive (APC) index mean value as a standard (see figure 1). Selecting conventional antioxidants such as BHA, BHT, vitamin E and the like and monomer carnosic acid prepared in the extract, carrying out antioxidant resistance tests such as DPPH, PTIO, ABTS, FRAP and the like according to the dosage requirement of a food additive use standard (GB 2760-. Determining the APC index mean range of the rosemary extract by taking the above as a standard, and obtaining the optimal extraction factor ratio of the strong oxidation resistance extracting solution by a fitting result: the ratio of the material to the liquid is 1: 12-15, the concentration of an ethanol solution serving as an extraction solvent is 60% -90%, the extraction temperature is 50-60 ℃, the ultrasonic power is 200-300W, the extraction time is 50-60 min, and the extraction frequency is 2-3 times.
Compared with the prior art, the invention has the following advantages:
1. the extract is a rosemary extract (mixed component), but the research on the oxidation resistance of rosemary generally focuses on the oxidation resistance comparison and single-component extraction of active components such as carnosic acid, rosmarinic acid and the like at present, while the report on the composite components of the oxidation resistance is less, and the experimental data of the invention show that the comprehensive (APC) index of the oxidation resistance activity of the extract is obviously higher than that of the single component.
2. Rosemary as a natural antioxidant is widely applied to industrial production, the invention firstly adopts the mean value of the antioxidant activity comprehensive (APC) index of active ingredients as a standard, designs the interaction of two factors of 6 factors of the feed-liquid ratio, the concentration of an ethanol solution of an extraction solvent and the extraction temperature in an extraction mode to draw a curved surface response and a contour map of a regression equation, and adopts a conventional antioxidant as the standard, thereby obtaining the extraction factor ratio of active substances with strong antioxidant property, avoiding the problem that the antioxidant effect is neglected when the extraction is carried out by only taking the content of a single ingredient as the standard.
Drawings
FIG. 1 is a graph showing the response surface and contour of each factor interacting with each other in the extraction method.
FIG. 2 shows the combined antioxidant assay for several conventional antioxidants. The dosage of the antioxidant is as follows: according to the dosage requirements of the food additive use standard (GB 2760-2014), the maximum use amount in the vegetable oil is 0.2g/kg of BHA and 0.2g/kg of BHT, the vitamin E is used in a proper amount according to the production requirement (the use amount refers to the use amount of the rosemary extract in the use amount of the invention), and the carnosic acid (the rosemary extract) is 0.7 g/kg.
Detailed Description
In order to explain the technical contents of the present invention in detail, specific examples are provided below. It is to be understood that the following detailed description is intended to further illustrate the present invention, but the scope of the methods and protection described herein is not limited thereto.
The reagents and methods used in the following examples, unless otherwise specified, were formulated and operated in a conventional manner; the reagents used are commercially available.
Example 1: method for extracting rosemary extract serving as strong antioxidant
(1) Sample preparation: cleaning fresh rosemary leaves, placing the fresh rosemary leaves into a freeze dryer, setting the temperature of a cold trap to be 50 ℃ below zero, freeze-drying the fresh rosemary leaves to room temperature, stopping the freeze-drying, then crushing the fresh rosemary leaves, sieving the crushed rosemary leaves by a 60-mesh sieve, accurately weighing 1g of rosemary powder, placing the rosemary powder into a centrifuge tube, adding 80% ethanol solution according to the material-liquid ratio of 1:10, 1: 15, 1: 20 and 1: 25, extracting the rosemary powder for 60min under the ultrasonic power of 300W, wherein the extraction temperature is 50 ℃, centrifuging the rosemary powder at the speed of 5000 r/min after the extraction is finished, taking supernatant extracting solution, carrying out suction filtration by using a polyvinylidene fluoride organic microporous filter membrane with the diameter of 0.22 mu m, and finishing the preparation of samples.
(2) And (3) oxidation resistance detection: and (3) respectively detecting antioxidant indexes such as DPPH, PTIO, ABTS, FRAP and the like of each sample.
Example 2: method for extracting rosemary extract serving as strong antioxidant
(1) Sample preparation: cleaning fresh rosemary leaves, putting the fresh rosemary leaves into a freeze dryer, setting the temperature of a cold trap to be 50 ℃ below zero, stopping freeze drying until the temperature reaches room temperature, then crushing, sieving by a 60-mesh sieve, accurately weighing 1g of rosemary powder, putting the rosemary powder into a centrifugal tube, and mixing the rosemary powder with the liquid-material ratio of 1: 15, adding 0%, 30%, 50%, 80% and 100% ethanol solution respectively, extracting at 50 deg.C under 300W ultrasonic power for 60min, centrifuging at 5000 rpm, taking supernatant, vacuum filtering, and filtering with 0.22 μm polyvinylidene fluoride organic microporous membrane to obtain the final product.
(2) And (3) oxidation resistance detection: and (3) respectively detecting antioxidant indexes such as DPPH, PTIO, ABTS, FRAP and the like of each sample.
Example 3: method for extracting rosemary extract serving as strong antioxidant
(1) Sample preparation: cleaning fresh rosemary leaves, putting the cleaned rosemary leaves into a freeze dryer, setting the temperature of a cold trap to be 50 ℃ below zero, stopping freeze drying until the temperature is room temperature, then crushing, sieving by using a 60-mesh sieve, accurately weighing 1g of rosemary powder, putting the rosemary powder into a centrifuge tube, and mixing the rosemary powder with a liquid-solid ratio of 1: 15, adding 80% ethanol solution, extracting for 20min, 40min, 60min, 80mim and 100min under the ultrasonic power of 300W respectively, wherein the extraction temperature is 50 ℃, centrifuging at the speed of 5000 r/min after extraction is finished, taking supernatant extract, performing suction filtration, and filtering by using a polyvinylidene fluoride organic microporous filter membrane of 0.22 mu m to finish sample preparation.
(2) And (3) oxidation resistance detection: and (3) respectively detecting antioxidant indexes such as DPPH, PTIO, ABTS, FRAP and the like of each sample.
Example 4: method for extracting rosemary extract serving as strong antioxidant
(1) Sample preparation: cleaning fresh rosemary leaves, putting the fresh rosemary leaves into a freeze dryer, setting the temperature of a cold trap to be 50 ℃ below zero, stopping freeze drying until the temperature reaches room temperature, then crushing, sieving by a 60-mesh sieve, accurately weighing 1g of rosemary powder, putting the rosemary powder into a centrifugal tube, and mixing the rosemary powder with the liquid-material ratio of 1: 15, adding 80% ethanol solution, extracting for 60min under the ultrasonic power of 300W, respectively setting the extraction temperature at 30 ℃, 40 ℃, 50 ℃ and 60 ℃, centrifuging at the speed of 5000 r/min after extraction is finished, taking supernatant extracting solution, carrying out suction filtration, and filtering by adopting a polyvinylidene fluoride organic microporous filter membrane of 0.22 mu m to finish sample preparation.
(2) And (3) oxidation resistance detection: and (3) respectively detecting antioxidant indexes such as DPPH, PTIO, ABTS, FRAP and the like of each sample.
Example 5: method for extracting rosemary extract serving as strong antioxidant
(1) Sample preparation: cleaning fresh rosemary leaves, putting the cleaned rosemary leaves into a freeze dryer, setting the temperature of a cold trap to be 50 ℃ below zero, stopping freeze drying until the temperature is room temperature, then crushing, sieving by using a 60-mesh sieve, accurately weighing 1g of rosemary powder, putting the rosemary powder into a centrifuge tube, and mixing the rosemary powder with a liquid-solid ratio of 1: 15, adding 80% ethanol solution, extracting for 60min under the ultrasonic power of 0W, 100W, 200W, 300W, 400W and 500W respectively, setting the extraction temperature to be 50 ℃, centrifuging at the speed of 5000 r/min after extraction is finished, taking supernatant extracting solution, performing suction filtration, and filtering by adopting a polyvinylidene fluoride organic microporous filter membrane of 0.22 mu m to finish sample preparation.
(2) And (3) oxidation resistance detection: and (3) respectively detecting antioxidant indexes such as DPPH, PTIO, ABTS, FRAP and the like of each sample.
Example 6: method for extracting rosemary extract serving as strong antioxidant
(1) Sample preparation: cleaning fresh rosemary leaves, putting the fresh rosemary leaves into a freeze dryer, setting the temperature of a cold trap to be 50 ℃ below zero, stopping freeze drying until the temperature reaches room temperature, then crushing, sieving by a 60-mesh sieve, accurately weighing 1g of rosemary powder, putting the rosemary powder into a centrifugal tube, and mixing the rosemary powder with the liquid-material ratio of 1: 15, adding 80% ethanol solution, extracting for 60min under 300W of ultrasonic power respectively, setting the extraction temperature to be 50 ℃, centrifuging at the speed of 5000 r/min after extraction is finished, pouring the supernatant extracting solution into a container, adding the ethanol solution into a centrifugal tube again for repeated extraction for 1-3 times, and finally filtering all the supernatant extracting solution by using a polyvinylidene fluoride organic microporous filter membrane of 0.22 mu m after suction filtration to finish sample preparation.
(2) And (3) oxidation resistance detection: and (3) respectively detecting antioxidant indexes such as DPPH, PTIO, ABTS, FRAP and the like of each sample.
Based on the oxidation resistance results of the above 6 groups of examples, the APC index was calculated as follows, and the APC index mean was calculated (see table 1).
And performing drawing analysis by using Design expert12.0 software, and drawing a curved surface response and a contour map of a regression equation by using the mean value of the oxidation resistance APC index as a standard and the interaction of two factors of 6 factors such as a material-liquid ratio, the concentration of an ethanol solution of an extraction solvent, an extraction temperature, ultrasonic power, extraction time, extraction times and the like. Selecting antioxidants such as BHA, BHT, vitamin E and monomer carnosic acid prepared in the extract, carrying out antioxidant resistance detection such as DPPH, PTIO, ABTS, FRAP and the like according to the dosage requirement of a food additive use standard (GB 2760-. Determining the APC index mean range of the rosemary extract by taking the above as a standard, and obtaining the optimal extraction factor ratio of the strong oxidation resistance extracting solution by a fitting result: the ratio of the material to the liquid is 1: 12-15, the concentration of an ethanol solution serving as an extraction solvent is 60% -90%, the extraction temperature is 50-60 ℃, the ultrasonic power is 200-300W, the extraction time is 50-60 min, and the extraction frequency is 2-3 times.
Claims (10)
1. A method for extracting rosemary extract serving as a strong antioxidant is characterized by comprising the following steps: the method comprises the following steps:
1) cleaning fresh rosemary leaves, putting the fresh rosemary leaves into a freeze dryer for freeze drying, grinding, crushing and sieving, extracting under different material-liquid ratios, the concentration of an ethanol solution as an extraction solvent, the extraction temperature, the ultrasonic power, the extraction time and the extraction times, and filtering an extracting solution by using a filter membrane after suction filtration to obtain a sample to be detected;
2) respectively detecting the antioxidant indexes of DPPH, PTIO, ABTS and FRAP of each sample, calculating the APC index according to the following formula, and calculating the average value of the APC index:
3) and (3) taking the APC index mean value obtained by calculation as a standard, and optimally designing by a response surface method to obtain the optimal feed-liquid ratio, the concentration of an extraction solvent ethanol solution, the extraction temperature, the ultrasonic power, the extraction time and the extraction times.
2. The extraction method according to claim 1, characterized in that: the temperature of the cold trap of the freeze dryer is-50 ℃, and the freezing time is to freeze and dry the mixture to room temperature.
3. The extraction method according to claim 1, characterized in that: adding an ethanol solution into the sample according to the volume ratio of the sample to the ethanol of 1: 10-25.
4. The extraction method according to claim 1, characterized in that: the concentration of the ethanol solution is 0-100%.
5. The extraction method according to claim 1, characterized in that: the ultrasonic power is 0-500W.
6. The extraction method according to claim 1, characterized in that: the extraction temperature is 30-60 ℃.
7. The extraction method according to claim 1, characterized in that: the extraction time is 20-100 min.
8. The extraction method according to claim 1, characterized in that: the extraction times are 1-3 times.
9. The extraction method according to claim 1, characterized in that: filtering with polyvinylidene fluoride organic microporous filter membrane of 0.22 μm.
10. The extraction method according to claim 1, characterized in that: the response surface method is specifically designed as follows: according to the test result, drawing and analyzing by using Design expert12.0 software, drawing a curved surface response and a contour map of a regression equation by using the oxidation resistance APC index mean value as a standard and the interaction of two factors of 6 factors of a material-liquid ratio, an extraction solvent ethanol solution concentration, an extraction temperature, an ultrasonic power, an extraction time and an extraction frequency, selecting conventional antioxidants such as BHA, BHT, vitamin E and the like and a monomer carnosic acid prepared in the extract, performing oxidation resistance detection such as DPPH, PTIO, ABTS, FRAP and the like according to the dosage requirement of a food additive use standard (GB 2760-: the ratio of the material to the liquid is 1: 12-15, the concentration of an ethanol solution serving as an extraction solvent is 60% -90%, the extraction temperature is 50-60 ℃, the ultrasonic power is 200-300W, the extraction time is 50-60 min, and the extraction frequency is 2-3 times.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102987519A (en) * | 2013-01-09 | 2013-03-27 | 长沙学院 | Antioxidant compound and application thereof |
| CN103416165A (en) * | 2012-05-21 | 2013-12-04 | 中国科学院植物研究所 | Method for collecting rosemary and method for preparing rosemary antioxidant extractives |
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| EP3922107A2 (en) * | 2020-05-19 | 2021-12-15 | Laboratoires Arkopharma | Eutectic solvent of natural origin, use thereof and extraction method implementing same |
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| CN103416165A (en) * | 2012-05-21 | 2013-12-04 | 中国科学院植物研究所 | Method for collecting rosemary and method for preparing rosemary antioxidant extractives |
| CN102987519A (en) * | 2013-01-09 | 2013-03-27 | 长沙学院 | Antioxidant compound and application thereof |
| EP3922107A2 (en) * | 2020-05-19 | 2021-12-15 | Laboratoires Arkopharma | Eutectic solvent of natural origin, use thereof and extraction method implementing same |
| CN112168873A (en) * | 2020-11-13 | 2021-01-05 | 安顺职业技术学院 | Response surface method optimized extraction process of total flavonoids of Asparagus racemosus |
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