CN114149982A - Method for extracting fructus Rosae Normalis superoxide dismutase - Google Patents
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- 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 rosa roxburghii superoxide dismutase, which comprises the following steps: removing core of fructus Rosae Normalis, pulverizing, mixing with 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, and adding 10% of absolute ethyl alcohol by volume for precipitation to obtain a purified solution; vacuum concentrating the purified liquid at 45-50 deg.c to extract salt with concentration of 20%, cooling, standing, centrifuging, washing and drying to obtain SOD. The extraction method of the roxburgh rose superoxide dismutase provided by the invention does not need to carry out special treatment on the roxburgh rose raw material, the reagent used in the extraction process is safe, and 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 roxburgh rose superoxide dismutase.
Background
Rosa roxburghii (Rosa roxbunghii) is a fruit of perennial deciduous shrub silk flower of Rosaceae, grows in sunny hillside, valley, roadside and bush with elevation of 500-2500 m, is a natural wild fruit in Guizhou, Hubei mountain, Hunan west, Liangshan, crown mountain and other places, and has large-area artificial planting in open cities of Guizhou province and Henan province. The roxburgh rose has good edible value and medicinal value, contains various rich components such as B vitamins, VC, flavone, organic acid and the like, can protect the heart, and has the effects of relieving fatigue, enhancing myocardial vitality, reducing blood pressure, enhancing immunity, delaying senescence, resisting cancer and the like. The roxburgh rose has short harvest period and is the fruit with the highest content of superoxide dismutase discovered at present, so that the method for extracting the superoxide dismutase from the roxburgh rose has wide prospect in industry.
Superoxide Dismutase (SOD) widely exists in animals, plants and microorganisms, and is a kind of metalloenzyme taking Superoxide anion free radical as substrate. SOD can eliminate free radicals, so it plays an important role in the aspects of oxygen toxicity prevention, radiation damage resistance, aging prevention and the like, has been widely used clinically as a new biochemical drug, and is widely applied to daily chemical products such as health food, cosmetics, toothpaste and the like as a bioactive component. The SOD products used at present are all extracted from human blood or animal blood, but the spreading of infectious diseases such as mad cow disease, foot and mouth disease and the like in the world, and the use of SOD extracted from animal blood brings many dangerous factors to people. SOD is extracted from plants, especially pollution-free vegetables, melons and fruits, wild plants and grains which are eaten by people in daily life, such as cactus, garlic, schisandra chinensis, pollen, Xiaobailai rice and other fruits and vegetables, and the SOD also contains abundant SOD in grains such as mung beans, corns and the like, has rich resources and very high use safety, avoids possible cross infection and has low cost.
The method for extracting and separating the superoxide dismutase of the roxburgh rose is developed, so that the additional value of the roxburgh rose can be improved, the deep processing of the roxburgh rose can be promoted, and the industrial chain of the roxburgh rose can be prolonged. The prior extraction method of the rosa roxburghii superoxide dismutase mainly adopts an ultrasonic-assisted extraction method, a thermal denaturation method, an isoelectric point method, an organic solvent precipitation method and the like, and common extraction methods have 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.
In view of the above, 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 rosa roxburghii superoxide dismutase, which comprises the following steps: removing core of fructus Rosae Normalis, pulverizing, mixing with 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, and adding 10% of absolute ethyl alcohol by volume for precipitation to obtain a purified solution; vacuum concentrating the purified liquid at 45-50 deg.c to extract salt with concentration of 20%, cooling, standing, centrifuging, washing and drying to obtain SOD; the mixing ratio of the roxburgh rose to the extraction saline water is 1: 3-5 in g: mL.
Further, in the extraction method of the rosa roxburghii superoxide dismutase, the extraction salt is NaCl, and the extraction salt solution is 3% NaCl solution.
Furthermore, in the extraction method of the rosa roxburghii superoxide dismutase, the volume of the added 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 kernels of rosa roxburghii tratt, crushing, stirring at room temperature by using a 3% NaCl solution, mixing the rosa roxburghii tratt and the 3% NaCl solution according to a ratio of g to mL of 1: 3-5, filtering by using a 100-mesh screen, centrifuging the filtrate, filtering the centrifugate by using filter paper and decolorizing by using activated carbon to prepare a crude extract; carrying out water bath on the crude extract at 50-55 ℃ for 30min, and then rapidly cooling and filtering to remove precipitated impurities to obtain a first supernatant; adjusting the pH value 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% volume of absolute ethyl alcohol into the second supernatant, standing at room temperature for 10min, and centrifuging to obtain supernatant, namely purified solution; vacuum concentrating the purified solution at 45-50 ℃ under reduced pressure until the NaCl concentration is 20%, cooling to room temperature, standing for 12h to obtain superoxide dismutase eluate, centrifuging to obtain a solid, washing with pure water, centrifuging to obtain a solid to obtain a wet superoxide dismutase product, and freeze-drying to obtain the superoxide dismutase; preferably, the temperature for freezing before pitting and crushing the rosa roxburghii tratt is 4 ℃.
Furthermore, in the extraction method of the Rosa roxburghii superoxide dismutase, the centrifugation condition is 5000r/min, and the time is 20 min.
Furthermore, in the extraction method of the rosa roxburghii superoxide dismutase, 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 high-efficiency extraction of the superoxide dismutase from 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 kernels of the roxburgh rose and avoiding the influence of nuclear impurities on the extraction of the superoxide dismutase.
(3) The invention provides a method for extracting superoxide dismutase from roxburgh rose, which avoids the pollution of organic solvent to the environment, and the used reagents are common inorganic reagents and have the advantage of cost.
Detailed Description
The following examples are given to illustrate the technical aspects of the present invention, but the present invention is not limited to the following examples.
Example 1
This example provides a test of the extraction method of superoxide dismutase from Rosa roxburghii Tratt.
Removing core of 50g fructus Rosae Normalis, pulverizing, stirring with 150mL 3% NaCl solution at room temperature for 5 hr, filtering with 100 mesh screen, centrifuging the filtrate, filtering the centrifugate with filter paper and decolorizing with active carbon to obtain crude extractive solution; carrying out water bath on the crude extract at 50-55 ℃ for 30min, then quickly cooling and filtering to remove precipitated impurities, and taking a first supernatant; adjusting the pH value 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, and removing precipitated impurities to obtain a second supernatant; adding 10% volume of absolute ethyl alcohol into the second supernatant, standing at room temperature for 10min, and centrifuging to obtain supernatant, namely purified solution; and (3) concentrating the purified liquid at 45-50 ℃ under reduced pressure and in vacuum until the NaCl concentration is 20%, cooling to room temperature, standing for 12h to obtain a superoxide dismutase eluate, centrifuging to obtain a solid, washing with pure water, centrifuging to obtain a solid to obtain a wet superoxide dismutase product, and freeze-drying to obtain the superoxide dismutase.
Example 2
This example provides a test of the extraction method of superoxide dismutase from Rosa roxburghii Tratt.
Removing core of 50g fructus Rosae Normalis, pulverizing, stirring with 200mL of 3% NaCl solution at room temperature for 5 hr, filtering with 100 mesh screen, centrifuging the filtrate, filtering the centrifugate with filter paper and decolorizing with active carbon to obtain crude extractive solution; carrying out water bath on the crude extract at 50-55 ℃ for 30min, then quickly cooling and filtering to remove precipitated impurities, and taking a first supernatant; adjusting the pH value 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, and removing precipitated impurities to obtain a second supernatant; adding 10% volume of absolute ethyl alcohol into the second supernatant, standing at room temperature for 10min, and centrifuging to obtain supernatant, namely purified solution; and (3) concentrating the purified liquid at 45-50 ℃ under reduced pressure and in vacuum until the NaCl concentration is 20%, cooling to room temperature, standing for 12h to obtain a superoxide dismutase eluate, centrifuging to obtain a solid, washing with pure water, centrifuging to obtain a solid to obtain a wet superoxide dismutase product, and freeze-drying to obtain the superoxide dismutase.
Example 3
This example provides a test of the extraction method of superoxide dismutase from Rosa roxburghii Tratt.
Removing core of 50g fructus Rosae Normalis, pulverizing, stirring with 250mL of 3% NaCl solution at room temperature for 5 hr, filtering with 100 mesh screen, centrifuging the filtrate, filtering the centrifugate with filter paper and decolorizing with active carbon to obtain crude extractive solution; carrying out water bath on the crude extract at 50-55 ℃ for 30min, then quickly cooling and filtering to remove precipitated impurities, and taking a first supernatant; adjusting the pH value 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, and removing precipitated impurities to obtain a second supernatant; adding 10% volume of absolute ethyl alcohol into the second supernatant, standing at room temperature for 10min, and centrifuging to obtain supernatant, namely purified solution; and (3) concentrating the purified liquid at 45-50 ℃ under reduced pressure and in vacuum until the NaCl concentration is 20%, cooling to room temperature, standing for 12h to obtain a superoxide dismutase eluate, centrifuging to obtain a solid, washing with pure water, centrifuging to obtain a solid to obtain a wet superoxide dismutase product, and freeze-drying to obtain the superoxide dismutase.
Comparative example 1
This example provides a comparative experiment of the extraction process of superoxide dismutase from Rosa roxburghii Tratt without activated carbon treatment.
Removing core of 50g fructus Rosae Normalis, pulverizing, stirring with 250mL of 3% NaCl solution at room temperature for 5 hr, filtering with 100 mesh screen, centrifuging the filtrate, and filtering the centrifugate with filter paper to obtain crude extractive solution; carrying out water bath on the crude extract at 50-55 ℃ for 30min, then quickly cooling and filtering to remove precipitated impurities, and taking a first supernatant; adjusting the pH value 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, and removing precipitated impurities to obtain a second supernatant; adding 10% volume of absolute ethyl alcohol into the second supernatant, standing at room temperature for 10min, and centrifuging to obtain supernatant, namely purified solution; and (3) concentrating the purified liquid at 45-50 ℃ under reduced pressure and in vacuum until the NaCl concentration is 20%, cooling to room temperature, standing for 12h to obtain a superoxide dismutase eluate, centrifuging to obtain a solid, washing with pure water, centrifuging to obtain a solid to obtain a wet superoxide dismutase product, and freeze-drying to obtain the superoxide dismutase.
Comparative example 2
This example provides a comparative experiment of the extraction of superoxide dismutase from Rosa roxburghii with propanol as the purification agent.
Removing core of 50g fructus Rosae Normalis, pulverizing, stirring with 250mL of 3% NaCl solution at room temperature for 5 hr, filtering with 100 mesh screen, centrifuging the filtrate, filtering the centrifugate with filter paper and decolorizing with active carbon to obtain crude extractive solution; carrying out water bath on the crude extract at 50-55 ℃ for 30min, then quickly cooling and filtering to remove precipitated impurities, and taking a first supernatant; adjusting the pH value 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, and removing precipitated impurities to obtain a second supernatant; adding 10% volume of anhydrous propanol into the second supernatant, standing at room temperature for 10min, centrifuging, and collecting supernatant to obtain purified solution; and (3) concentrating the purified liquid at 45-50 ℃ under reduced pressure and in vacuum until the NaCl concentration is 20%, cooling to room temperature, standing for 12h to obtain a superoxide dismutase eluate, centrifuging to obtain a solid, washing with pure water, centrifuging to obtain a solid to obtain a wet superoxide dismutase product, and freeze-drying to obtain the superoxide dismutase.
Comparative example 3
This example provides a comparative experiment of a method for extracting superoxide dismutase from Rosa roxburghii Tratt, in which the purified solution is vacuum concentrated at 45-50 deg.C under reduced pressure until the NaCl concentration is 30%, and an analysis of the extraction results of superoxide dismutase from examples 1-3 and comparative examples 1-3.
This example provides a test of the extraction method of superoxide dismutase from Rosa roxburghii Tratt.
Removing core of 50g fructus Rosae Normalis, pulverizing, stirring with 250mL of 3% NaCl solution at room temperature for 5 hr, filtering with 100 mesh screen, centrifuging the filtrate, filtering the centrifugate with filter paper and decolorizing with active carbon to obtain crude extractive solution; carrying out water bath on the crude extract at 50-55 ℃ for 30min, then quickly cooling and filtering to remove precipitated impurities, and taking a first supernatant; adjusting the pH value 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, and removing precipitated impurities to obtain a second supernatant; adding 10% volume of absolute ethyl alcohol into the second supernatant, standing at room temperature for 10min, and centrifuging to obtain supernatant, namely purified solution; and (3) concentrating the purified liquid at 45-50 ℃ under reduced pressure and in vacuum until the NaCl concentration is 30%, cooling to room temperature, standing for 12h to obtain a superoxide dismutase eluate, centrifuging to obtain a solid, washing with pure water, centrifuging to obtain the solid to obtain a wet superoxide dismutase product, and freeze-drying to obtain the superoxide dismutase.
Comparative example 4
This example provides a comparative experiment of the extraction of superoxide dismutase from Rosa roxburghii without enucleation.
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 activated carbon to obtain a crude extract; carrying out water bath on the crude extract at 50-55 ℃ for 30min, then quickly cooling and filtering to remove precipitated impurities, and taking a first supernatant; adjusting the pH value 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% volume of absolute ethyl alcohol into the second supernatant, standing at room temperature for 10min, and centrifuging to obtain supernatant, namely purified solution; vacuum concentrating the purified liquid at 45-50 deg.C under reduced pressure until NaCl concentration is 20%, cooling to room temperature, standing for 12 hr to obtain superoxide dismutase eluate, centrifuging to obtain solid, washing with pure water, centrifuging to obtain solid to obtain wet superoxide dismutase, 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 Tratt and an analysis of the extraction results of superoxide dismutase from examples 1-3 and comparative examples 1-5.
Removing core of 50g fructus Rosae Normalis, pulverizing, stirring with 250mL of 3% NaCl solution at room temperature, filtering with 100 mesh screen, centrifuging the filtrate at room temperature, filtering the centrifugate with filter paper and decolorizing with active carbon to obtain crude extractive solution; adding NaCl into the crude extract until the overall salinity of the solution reaches 20% to obtain superoxide dismutase eluate, centrifuging to obtain solid, washing with pure water, centrifuging to obtain solid to obtain wet superoxide dismutase, and freeze drying to obtain the superoxide dismutase.
The enzyme specific activities of the superoxide dismutase obtained in examples 1-3 and comparative examples 1-5 were determined by using pyrogallol autoxidation method (enzyme activity unit is defined as 1 enzyme activity unit, which is the amount of enzyme that inhibits pyrogallol autoxidation rate by 50% in 1mL of reaction solution at 25 ℃ for 1 min), as shown in Table 1.
TABLE 1 enzymatic specific 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 enzymatic specific activity of the superoxide dismutase obtained by the extraction method of the roxburgh rose superoxide dismutase provided by the invention can reach 23948U/g at most. Compared with the embodiments 1-3 and the comparative examples 1-5, the method for extracting the rosa roxburghii superoxide dismutase, which is provided by the invention, can effectively improve the enzyme activity of the rosa roxburghii superoxide dismutase extraction by the methods of active carbon treatment, purifying agent selection, vacuum concentration, denucleation treatment and the like.
As described above, the present invention can be preferably implemented, and the above-mentioned embodiments only describe the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various changes and modifications of the technical solution of the present invention made by those skilled in the art without departing from the design spirit of the present invention shall fall within the protection scope defined by the present invention.
Claims (7)
1. A method for extracting Rosa roxburghii superoxide dismutase is characterized by comprising the following steps:
removing core of fructus Rosae Normalis, pulverizing, mixing with 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, and adding 10% of absolute ethyl alcohol by volume for precipitation to obtain a purified solution; vacuum concentrating the purified liquid at 45-50 deg.c to extract salt with concentration of 20%, cooling, standing, centrifuging, washing and drying to obtain SOD;
the mixing ratio of the roxburgh rose to the extraction saline water is 1: 3-5 in g: mL.
2. The method of claim 1, wherein the extraction salt is NaCl and the extraction salt solution is a 3% NaCl solution.
3. The method according to claim 1, wherein the absolute ethanol is added in an amount of 10% by volume based on the volume of the purified solution.
4. A method according to any one of claims 1 to 3, comprising:
removing kernels of rosa roxburghii tratt, crushing, stirring at room temperature by using a 3% NaCl solution, mixing the rosa roxburghii tratt and the 3% NaCl solution according to a ratio of g to mL of 1: 3-5, filtering by using a 100-mesh screen, centrifuging the filtrate, filtering the centrifugate by using filter paper and decolorizing by using activated carbon to prepare a crude extract;
carrying out water bath on the crude extract at 50-55 ℃ for 30min, and then rapidly cooling and filtering to remove precipitated impurities to obtain a first supernatant; adjusting the pH value 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% volume of absolute ethyl alcohol into the second supernatant, standing at room temperature for 10min, and centrifuging to obtain supernatant, namely purified solution;
and (3) concentrating the purified liquid at 45-50 ℃ under reduced pressure and in vacuum until the NaCl concentration is 20%, cooling to room temperature, standing for 12h to obtain a superoxide dismutase eluate, centrifuging to obtain a solid, washing with pure water, centrifuging to obtain a solid to obtain a wet superoxide dismutase product, and freeze-drying to obtain the superoxide dismutase.
5. The method of claim 4, wherein the centrifugation conditions are 5000r/min for 20 min.
6. The method according to claim 4, wherein the pH adjusting agent is 3.5% hydrochloric acid and 3.5% sodium hydroxide.
7. A superoxide dismutase produced by the method of claim 1.
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