CN114149982B - Extraction method of rosa roxburghii tratt superoxide dismutase - Google Patents
Extraction method of rosa roxburghii tratt superoxide dismutase Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y115/00—Oxidoreductases acting on superoxide as acceptor (1.15)
- C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
- C12Y115/01001—Superoxide dismutase (1.15.1.1)
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Abstract
The invention provides a method for extracting superoxide dismutase from roxburgh rose, which comprises the following steps: removing cores of fructus Rosae Normalis, pulverizing, mixing with the extractive salt solution, extracting, centrifuging, filtering, and decolorizing with active carbon to obtain crude extractive solution; precipitating the crude extract at 50-55 ℃ and pH value of 5.0, adding 10% absolute ethyl alcohol by volume to obtain a purified solution; vacuum concentrating the purified solution at 45-50 ℃ under reduced pressure until the concentration of extracted salt is 20%, cooling, standing, centrifuging, washing and drying to obtain the superoxide dismutase. The extraction method of the rosa roxburghii superoxide dismutase provided by the invention does not need to carry out special treatment on rosa roxburghii raw materials, and the reagents used in the extraction process are safe, so that the obtained superoxide dismutase has high activity.
Description
Technical Field
The invention belongs to the technical field of enzyme extraction, and relates to a method for extracting superoxide dismutase from roxburgh rose.
Background
Rosa roxburghii (Rosa roxbunghii) is a fruit of a rose perennial fallen leaf bush nest flower, which is grown in sunny hillsides, valleys, roadsides and bushes with the altitude of 500-2500 m, is a natural wild fruit in areas such as Guizhou, jaw mountain areas, xiangxi, cold mountain, crown mountain areas and the like, and is artificially planted in a large area in the unsealed market of Guizhou province and Henan province. The roxburgh rose has good edible value and medicinal value, and the roxburgh rose contains various components such as rich B vitamins, VC, flavone, organic acid and the like, can protect the heart, and has the effects of relieving fatigue, enhancing myocardial activity, reducing blood pressure, enhancing immunity, delaying aging, resisting cancer and the like. The harvesting period of the rosa roxburghii is short, and meanwhile, the fruit with the highest superoxide dismutase content is found at present, so the method for extracting the superoxide dismutase from the rosa roxburghii has wide prospect in industry.
Superoxide dismutase (Superoxide Dismutase, SOD), which is a metalloenzyme with superoxide anion radical as a substrate, is widely found in animals, plants and microorganisms. SOD can remove free radicals, plays an important role in protecting against oxygen toxicity, resisting radiation injury, preventing aging and the like, is widely used clinically as a biochemical new drug, and is widely applied to daily chemical products such as health-care food, cosmetics, toothpaste and the like as a bioactive component. The SOD products are all extracted from human blood or animal blood at present, but the international spreading of infectious diseases such as mad cow disease, foot-and-mouth disease and the like, and the use of SOD extracted from animal blood brings a plurality of dangerous factors to people. SOD is extracted from plants, especially pollution-free vegetables, melons and fruits, wild plants and grains which are eaten daily by people, such as cactus, garlic, shizandra berry, pollen, white spirit and other fruits and vegetables, and the grains such as mung beans, corn and the like also contain rich SOD, so that the plant SOD has rich resources, high use safety, avoids possible cross infection and low cost.
The development of the extraction and separation method of the superoxide dismutase of the roxburgh rose not only can improve the added value of the roxburgh rose, but also is beneficial to promoting the deep processing of the roxburgh rose and prolonging the industrial chain of the roxburgh rose. At present, the extraction method of the superoxide dismutase of the roxburgh rose mainly adopts ultrasonic-assisted extraction, a thermal denaturation method, an isoelectric point method, an organic solvent precipitation method and the like, and the common extraction method has the problems of low extraction rate or limitation of organic solvent pollution, high cost and the like.
Disclosure of Invention
The invention aims to provide a method for extracting superoxide dismutase from roxburgh rose, which has high enzyme activity and low process cost.
Based on the above objects, the present application addresses this need in the art by providing a method for extracting superoxide dismutase from Rosa roxburghii.
In one aspect, the invention relates to a method for extracting superoxide dismutase from roxburgh rose, which comprises the following steps: removing cores of fructus Rosae Normalis, pulverizing, mixing with the extractive salt solution, extracting, centrifuging, filtering, and decolorizing with active carbon to obtain crude extractive solution; precipitating the crude extract at 50-55 ℃ and pH value of 5.0, adding 10% absolute ethyl alcohol by volume to obtain a purified solution; vacuum concentrating the purified solution at 45-50 ℃ under reduced pressure until the concentration of extracted salt is 20%, cooling, standing, centrifuging, washing and drying to obtain superoxide dismutase; the mixing ratio of the roxburgh rose to the extracted saline water is 1:3-5 in terms of g/mL.
In the extraction method of the rosa roxburghii superoxide dismutase provided by the invention, the extraction salt is NaCl, and the extraction salt solution is 3% NaCl solution.
Further, in the extraction method of the rosa roxburghii tratt superoxide dismutase provided by the invention, the added volume of absolute ethyl alcohol is 10% of the volume of the purified liquid.
Specifically, the extraction method of the rosa roxburghii superoxide dismutase provided by the invention comprises the following steps:
Removing cores of Rosa roxburghii, crushing, stirring with 3% NaCl solution at room temperature, wherein the mixing ratio of the Rosa roxburghii and the 3% NaCl solution is 1:3-5 in terms of g: mL, filtering with a 100-mesh screen, centrifuging the filtrate, filtering the centrifugate with filter paper, decolorizing with active carbon, and preparing into crude extract; after the crude extract is subjected to water bath for 30min at 50-55 ℃, rapidly cooling and filtering to remove precipitated impurities, and obtaining a first supernatant; regulating the pH of the first supernatant to 5, stirring, standing at room temperature for 15min, pouring into a centrifuge tube, centrifuging, and removing precipitated impurities to obtain a second supernatant; adding 10% absolute ethyl alcohol into the second supernatant, standing at room temperature for 10min, and centrifuging to obtain supernatant, namely the purified solution; concentrating the purified solution at 45-50 ℃ under reduced pressure and vacuum until the NaCl concentration is 20%, cooling to room temperature and standing for 12 hours to obtain superoxide dismutase precipitation solution, centrifuging to obtain solid, washing with pure water, centrifuging to obtain solid to obtain superoxide dismutase wet product, and freeze-drying to obtain the superoxide dismutase; preferably, the temperature of freezing the roxburgh rose before stoning and crushing is 4 ℃.
Further, in the extraction method of the rosa roxburghii superoxide dismutase provided by the invention, the centrifugation condition is 5000r/min, and the time is 20min.
Further, in the extraction method of the rosa roxburghii superoxide dismutase provided by the invention, the reagents used for adjusting the pH are 3.5% hydrochloric acid and 3.5% sodium hydroxide.
Compared with the prior art, the invention has the following beneficial effects or advantages:
(1) The invention provides a method for extracting superoxide dismutase from roxburgh rose, which realizes the efficient extraction of the superoxide dismutase in the roxburgh rose, and the obtained superoxide dismutase has high activity;
(2) The invention provides a method for extracting superoxide dismutase from roxburgh rose, which is used for removing cores from roxburgh rose, so that the influence of impurities in the cores on the extraction of the superoxide dismutase is avoided.
(3) The invention provides a method for extracting the superoxide dismutase of the roxburgh rose, which avoids the pollution of an organic solvent to the environment, and all reagents are common inorganic reagents and have the advantage of cost.
Detailed Description
The following describes the technical aspects of the present invention with reference to examples, but the present invention is not limited to the following examples.
Example 1
The embodiment provides a test of an extraction method of the rosa roxburghii tratt superoxide dismutase.
Removing cores of 50g of roxburgh rose, crushing, stirring for 5h with 150mL of 3% NaCl solution at room temperature, filtering with a 100-mesh screen, centrifuging the filtrate, filtering the centrifugate with filter paper and decolorizing with active carbon to obtain a crude extract; after the crude extract is subjected to water bath for 30min at 50-55 ℃, rapidly cooling and filtering to remove precipitated impurities, and taking a first supernatant; adjusting the pH of the first supernatant to 5 by using a 3.5% NaOH solution, stirring, standing at room temperature for 15min, pouring into a centrifuge tube for centrifugation, and removing precipitated impurities to obtain a second supernatant; adding 10% absolute ethyl alcohol into the second supernatant, standing at room temperature for 10min, and centrifuging to obtain supernatant, namely the purified solution; concentrating the purified solution at 45-50 ℃ under reduced pressure and vacuum until the NaCl concentration is 20%, cooling to room temperature and standing for 12 hours to obtain superoxide dismutase precipitation liquid, centrifuging to obtain solid, washing with pure water, centrifuging to obtain solid to obtain superoxide dismutase wet product, and freeze-drying to obtain the superoxide dismutase.
Example 2
The embodiment provides a test of an extraction method of the rosa roxburghii tratt superoxide dismutase.
Removing 50g of roxburgh rose, crushing, stirring for 5h with 200mL of 3% NaCl solution at room temperature, filtering with a 100-mesh screen, centrifuging the filtrate, filtering the centrifugate with filter paper and decolorizing with active carbon to obtain a crude extract; after the crude extract is subjected to water bath for 30min at 50-55 ℃, rapidly cooling and filtering to remove precipitated impurities, and taking a first supernatant; adjusting the pH of the first supernatant to 5 by using a 3.5% NaOH solution, stirring, standing at room temperature for 15min, pouring into a centrifuge tube for centrifugation, and removing precipitated impurities to obtain a second supernatant; adding 10% absolute ethyl alcohol into the second supernatant, standing at room temperature for 10min, and centrifuging to obtain supernatant, namely the purified solution; concentrating the purified solution at 45-50 ℃ under reduced pressure and vacuum until the NaCl concentration is 20%, cooling to room temperature and standing for 12 hours to obtain superoxide dismutase precipitation liquid, centrifuging to obtain solid, washing with pure water, centrifuging to obtain solid to obtain superoxide dismutase wet product, and freeze-drying to obtain the superoxide dismutase.
Example 3
The embodiment provides a test of an extraction method of the rosa roxburghii tratt superoxide dismutase.
Removing cores of 50g of roxburgh rose, crushing, stirring with 250mL of 3% NaCl solution at room temperature for 5h, filtering with a 100-mesh screen, centrifuging the filtrate, filtering the centrifugate with filter paper and decolorizing with active carbon to obtain a crude extract; after the crude extract is subjected to water bath for 30min at 50-55 ℃, rapidly cooling and filtering to remove precipitated impurities, and taking a first supernatant; adjusting the pH of the first supernatant to 5 by using a 3.5% NaOH solution, stirring, standing at room temperature for 15min, pouring into a centrifuge tube for centrifugation, and removing precipitated impurities to obtain a second supernatant; adding 10% absolute ethyl alcohol into the second supernatant, standing at room temperature for 10min, and centrifuging to obtain supernatant, namely the purified solution; concentrating the purified solution at 45-50 ℃ under reduced pressure and vacuum until the NaCl concentration is 20%, cooling to room temperature and standing for 12 hours to obtain superoxide dismutase precipitation liquid, centrifuging to obtain solid, washing with pure water, centrifuging to obtain solid to obtain superoxide dismutase wet product, and freeze-drying to obtain the superoxide dismutase.
Comparative example 1
The present example provides a comparative test of the extraction method of the superoxide dismutase of the rosa roxburghii without being treated by the active carbon.
Removing cores of 50g of fructus Rosae Normalis, pulverizing, stirring with 250mL of 3% NaCl solution at room temperature for 5h, filtering with 100 mesh sieve, centrifuging the filtrate, and filtering the centrifugate with filter paper to obtain crude extract; after the crude extract is subjected to water bath for 30min at 50-55 ℃, rapidly cooling and filtering to remove precipitated impurities, and taking a first supernatant; adjusting the pH of the first supernatant to 5 by using a 3.5% NaOH solution, stirring, standing at room temperature for 15min, pouring into a centrifuge tube for centrifugation, and removing precipitated impurities to obtain a second supernatant; adding 10% absolute ethyl alcohol into the second supernatant, standing at room temperature for 10min, and centrifuging to obtain supernatant, namely the purified solution; concentrating the purified solution at 45-50 ℃ under reduced pressure and vacuum until the NaCl concentration is 20%, cooling to room temperature and standing for 12 hours to obtain superoxide dismutase precipitation liquid, centrifuging to obtain solid, washing with pure water, centrifuging to obtain solid to obtain superoxide dismutase wet product, and freeze-drying to obtain the superoxide dismutase.
Comparative example 2
The example provides a comparative test of a method for extracting superoxide dismutase from Rosa roxburghii using propanol as the purifying agent.
Removing cores of 50g of roxburgh rose, crushing, stirring with 250mL of 3% NaCl solution at room temperature for 5h, filtering with a 100-mesh screen, centrifuging the filtrate, filtering the centrifugate with filter paper and decolorizing with active carbon to obtain a crude extract; after the crude extract is subjected to water bath for 30min at 50-55 ℃, rapidly cooling and filtering to remove precipitated impurities, and taking a first supernatant; adjusting the pH of the first supernatant to 5 by using a 3.5% NaOH solution, stirring, standing at room temperature for 15min, pouring into a centrifuge tube for centrifugation, and removing precipitated impurities to obtain a second supernatant; adding 10% of anhydrous propanol into the second supernatant, standing at room temperature for 10min, and centrifuging to obtain supernatant, namely the purified solution; concentrating the purified solution at 45-50 ℃ under reduced pressure and vacuum until the NaCl concentration is 20%, cooling to room temperature and standing for 12 hours to obtain superoxide dismutase precipitation liquid, centrifuging to obtain solid, washing with pure water, centrifuging to obtain solid to obtain superoxide dismutase wet product, and freeze-drying to obtain the superoxide dismutase.
Comparative example 3
This example provides a comparative test of a method for extracting superoxide dismutase from Rosa roxburghii, wherein the purified solution is concentrated under reduced pressure at 45-50 ℃ to a NaCl concentration of 30%, and analysis of the extraction results of superoxide dismutase in examples 1-3 and comparative examples 1-3.
The embodiment provides a test of an extraction method of the rosa roxburghii tratt superoxide dismutase.
Removing cores of 50g of roxburgh rose, crushing, stirring with 250mL of 3% NaCl solution at room temperature for 5h, filtering with a 100-mesh screen, centrifuging the filtrate, filtering the centrifugate with filter paper and decolorizing with active carbon to obtain a crude extract; after the crude extract is subjected to water bath for 30min at 50-55 ℃, rapidly cooling and filtering to remove precipitated impurities, and taking a first supernatant; adjusting the pH of the first supernatant to 5 by using a 3.5% NaOH solution, stirring, standing at room temperature for 15min, pouring into a centrifuge tube for centrifugation, and removing precipitated impurities to obtain a second supernatant; adding 10% absolute ethyl alcohol into the second supernatant, standing at room temperature for 10min, and centrifuging to obtain supernatant, namely the purified solution; concentrating the purified solution at 45-50 ℃ under reduced pressure and vacuum until the NaCl concentration is 30%, cooling to room temperature and standing for 12 hours to obtain superoxide dismutase precipitation liquid, centrifuging to obtain solid, washing with pure water, centrifuging to obtain solid to obtain superoxide dismutase wet product, and freeze-drying to obtain the superoxide dismutase.
Comparative example 4
The present example provides a comparative test of a method for extracting superoxide dismutase from Rosa roxburghii without denucleation.
Crushing 50g of roxburgh rose, stirring with 250mL of 3% NaCl solution at room temperature, filtering with a 100-mesh screen, centrifuging the filtrate at room temperature, filtering the centrifugate with filter paper and decolorizing with active carbon to obtain a crude extract; after the crude extract is subjected to water bath for 30min at 50-55 ℃, rapidly cooling and filtering to remove precipitated impurities, and taking a first supernatant; adjusting the pH of the first supernatant to 5 by using a 3.5% NaOH solution, stirring, standing at room temperature for 15min, pouring into a centrifuge tube, centrifuging at room temperature, and removing precipitated impurities to obtain a second supernatant; adding 10% absolute ethyl alcohol into the second supernatant, standing at room temperature for 10min, and centrifuging to obtain supernatant, namely the purified solution; vacuum concentrating the purified solution at 45-50 ℃ until the NaCl concentration is 20%, cooling to room temperature, standing for 12h to obtain superoxide dismutase precipitation solution, centrifuging to obtain solid, washing with pure water, centrifuging to obtain solid to obtain superoxide dismutase wet product, and freeze drying to obtain the superoxide dismutase.
Comparative example 5
This example provides a comparative experiment of a method for extracting superoxide dismutase from Rosa roxburghii in a direct salting-out comparative experiment and analysis of the extraction results of superoxide dismutase in examples 1-3 and comparative examples 1-5.
Removing 50g of roxburgh rose core, crushing, stirring with 250mL of 3% NaCl solution at room temperature, filtering with a 100-mesh screen, centrifuging the filtrate at room temperature, filtering the centrifugate with filter paper and decolorizing with active carbon to obtain crude extract; adding NaCl into the crude extract until the overall salinity of the solution reaches 20%, obtaining superoxide dismutase precipitation liquid, centrifuging to obtain solid, washing with pure water, centrifuging to obtain solid to obtain superoxide dismutase wet product, and freeze-drying to obtain the superoxide dismutase.
The specific activities of the superoxide dismutase obtained in examples 1 to 3 and comparative examples 1 to 5 were measured by the pyrogallol autoxidation method (the unit of enzyme activity is defined as 1 unit of enzyme activity, and the amount of enzyme inhibiting the pyrogallol autoxidation rate to 50% in 1mL of the reaction solution at a constant temperature of 25 ℃ C., for 1 min) as shown in Table 1.
TABLE 1 specific enzyme Activity of Rosa roxburghii superoxide dismutase
| Specific activity of enzyme (U/g) | |
| Example 1 | 21636 |
| Example 2 | 23948 |
| Example 3 | 21970 |
| Comparative example 1 | 8161 |
| Comparative example 2 | 18020 |
| Comparative example 3 | 2243 |
| Comparative example 4 | 9400 |
| Comparative example 5 | 2174 |
As shown in Table 1, the specific activity of the superoxide dismutase obtained by the extraction method of the rosa roxburghii tratt superoxide dismutase provided by the invention can reach 23948U/g at the highest. The methods of activated carbon treatment, purifying agent selection, vacuum concentration, stoning treatment and the like in the extraction method of the rosa roxburghii superoxide dismutase provided by the invention can effectively improve the enzyme activity of the rosa roxburghii superoxide dismutase extraction.
The present invention may be better implemented as described above, and the above examples are merely illustrative of preferred embodiments of the present invention and not intended to limit the scope of the present invention, and various changes and modifications made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the present invention without departing from the spirit of the design of the present invention.
Claims (2)
1. The extraction method of the rosa roxburghii tratt superoxide dismutase is characterized by comprising the following steps:
Removing cores of fructus Rosae Normalis, pulverizing, mixing with the extractive salt solution, extracting, centrifuging, filtering, and decolorizing with active carbon to obtain crude extractive solution; precipitating the crude extract at 50-55 ℃ and pH value of 5.0, adding 10% absolute ethyl alcohol by volume to obtain a purified solution;
the pH adjusting agent is 3.5% hydrochloric acid and 3.5% sodium hydroxide;
Removing cores of Rosa roxburghii, crushing, stirring with 3% NaCl solution at room temperature, wherein the mixing ratio of the Rosa roxburghii and the 3% NaCl solution is 1:3-5 in terms of g: mL, filtering with a 100-mesh screen, centrifuging the filtrate, filtering the centrifugate with filter paper, decolorizing with active carbon, and preparing into crude extract;
after the crude extract is subjected to water bath for 30min at 50-55 ℃, rapidly cooling and filtering to remove precipitated impurities, and obtaining a first supernatant; regulating the pH of the first supernatant to 5, stirring, standing at room temperature for 15min, pouring into a centrifuge tube, centrifuging, and removing precipitated impurities to obtain a second supernatant; adding 10% absolute ethyl alcohol into the second supernatant, standing at room temperature for 10min, and centrifuging to obtain supernatant, namely the purified solution;
concentrating the purified solution at 45-50 ℃ under reduced pressure and vacuum until the NaCl concentration is 20%, cooling to room temperature and standing for 12 hours to obtain superoxide dismutase precipitation liquid, centrifuging to obtain solid, washing with pure water, centrifuging to obtain solid to obtain superoxide dismutase wet product, and freeze-drying to obtain the superoxide dismutase.
2. The method of claim 1, wherein the centrifugation conditions are 5000r/min for 20min.
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