KR102469932B1 - Method For Extracting Superoxide Dismutase From Wormwood - Google Patents

Abstract

The present invention relates to a method for extracting antioxidant enzymes from perennial artemisia, which enables extraction of high levels of antioxidant enzymes while being applied to mass production. The method for extracting antioxidant enzymes from perennial artemisia of the present invention comprises: a step (A) of preparing perennial artemisia; a step (B) of putting perennial artemisia in a grinder and crushing the same; a step (C) of immersing the pulverized perennial artemisia in a purified water container filled with purified water for a first set time; a step (D) of extracting the solution by putting the perennial artemisia of the step (C) in a stirring container and first stirring for a second set time; a step (E) of first filtering the solution extracted in the step (D) with a mesh screen; a step (F) of secondary filtering the primary filtrate extracted in the step (E) with a Buchner filter; a step (G) of adding ammonium sulfate to the secondary filtrate extracted in the step (F); a step (H) of extracting solution by putting the mixed solution extracted in the step (G) into the stirring container and stirring the same for a third set time; a step (I) of centrifuging the solution extracted in the step (H) with a centrifuge to remove the supernatant, and then harvesting the hardened antioxidant enzyme; a step (J) of preparing a solution by putting the antioxidant enzyme harvested in the step (I) into a purified water container filled with purified water and performing a second stirring; and a step (K) of collecting the highly active antioxidant enzyme after putting the solution prepared in the step (J) into a container, sealing the same, sterilizing the same by irradiating gamma rays.

Description

Method for Extracting Antioxidant Enzymes from Perennial Wormwood {Method For Extracting Superoxide Dismutase From Wormwood}

The present invention relates to a method for extracting antioxidant enzymes from plants.

SOD becomes an antioxidant enzyme that removes active oxygen. When SOD meets active oxygen, it receives electrons and becomes hydrogen peroxide (H2O2). This hydrogen peroxide meets catalase and glutathione to turn into water (H2O) and then is released. Through this action, SOD is becoming an important factor in preventing diseases such as lung damage, vascular disease, and arthritis, as well as preventing skin aging.

In particular, according to the international scientific journal 'PloSONE', as a result of animal experiments, SOD deficiency thickens cerebral blood vessels and is an important cause of vasomotor disorders.

Currently, as many people are interested in anti-aging, blood vessels and blood flowing through blood vessels, interest in foods that prevent aging and healthy blood vessels and SOD contained in health foods are also increasing. Accordingly, research on how to extract SOD from plants is being actively conducted in the laboratory.

However, many methods of extracting SOD developed so far can extract a large amount of antioxidant enzymes, but have a problem in that the purity of the extracted antioxidant enzymes is not high. That is, there is a problem that high-quality antioxidant enzymes cannot be extracted.

Republic of Korea Patent Publication No. 10-2022-0010845 (published date: 2022.01.27)

Thus, the problem to be solved by the present invention is to solve the problem that high-quality antioxidant enzymes are not extracted when a large amount of antioxidant enzymes are extracted.

The problem to be solved by the present invention is not limited to the problems mentioned above, and other technical problems not mentioned will be clearly understood by those skilled in the art from the following description.

The method of extracting SOD from iron sand mugwort of the present invention for achieving the above technical problem is step (A) of preparing iron sand mugwort, step (B) of grinding iron sand mugwort in a grinder, grinding in a purified water bucket filled with purified water (C) step of immersing the fermented sandwormwood for a first set time, (D) step of extracting a solution by first stirring for a second set time by putting the iron sandwormwood of step (C) in a stirring barrel, and using a mesh screen ( (E) step of firstly filtering the solution extracted in step D), (F) step of secondarily filtering the first filtrate extracted in step (E) with a Buchner filter, and (G) step of adding ammonium sulfate to the tea filtrate, (H) step of extracting the solution by putting the mixed solution extracted in step (G) into a stirring bucket and stirring for a third set time, (H) step with a centrifugal separator After removing the supernatant by centrifuging the solution extracted from the step (I) to harvest the hardened antioxidant enzyme, put the antioxidant enzyme harvested in step (I) into a purified water container filled with purified water and agitate the second solution A step (J) of preparing, a step (K) of collecting the highly active antioxidant enzyme after putting the solution prepared in step (J) into a container, sealing it, sterilizing it by irradiating gamma rays.

Here, the iron sand mugwort in step (A) is 100 kg, the grinder in step (B) outputs a power of 7 horsepower or more and crushes sand iron sand mugwort, and the purified water container in step (C) accommodates 2000 liters and crushes sand iron Wormwood can be soaked.

And in step (D), the solution can be extracted by rotating the stirring barrel at 100 rpm for 3 hours, and in step (G), 250 kg or more of ammonium sulfate can be added.

And, in step (H), the stirring barrel rotates at 100 rpm for 18 hours to extract the solution, and in step (I), the centrifugal separator can rotate at 10000 rpm for 1 hour.

And step (J) may include adding purified water to the washing water container so that the concentration of the antioxidant enzyme harvested in step (I) is 500 mg / mL, and adding 1,2-hexanediol. And step (K) can be sterilized by applying 15 to 20 kg of gamma rays.

The present invention relates to a method for enabling high-purity antioxidant enzyme extraction while being applied to mass production. In addition, the enzyme of the antioxidant extracted through the method of the present invention is high in purity and can be applied to various fields.

1 is a flow chart of a method for extracting antioxidant enzymes from sandwormwood in accordance with an embodiment of the present invention.
Figure 2 is a diagram showing the solvent selection test results for mass production application.
3 is a view showing the temperature selection test results for mass production application.
4 is a view showing the extraction time selection test results for mass production application.
5 is a diagram showing antioxidant enzymes precipitated after salting out ammonium sulfate at a concentration of 30%, ammonium sulfate at a concentration of 50%, and ammonium sulfate at a concentration of 70%.
Figure 6 is a table showing the content of antioxidant enzymes through the optimized extraction method.

Advantages and features of embodiments of the present invention will become clear through the description of the present invention in conjunction with the accompanying drawings. However, the present invention made clear through the drawings and description is not limited to the embodiments disclosed below. The invention is defined solely by the claims and is limited according to the subject matter stated in the claims.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art to fully understand the present invention.

Referring to Figure 1, a method for extracting antioxidant enzymes from sandwormwood according to an embodiment of the present invention will be described in detail.

1 is a perspective view of a method for extracting antioxidant enzymes from sandwormwood in accordance with an embodiment of the present invention.

The method (1) for extracting antioxidant enzymes from sandworm mugwort relates to a method for enabling high-purity antioxidant enzyme extraction while being applied to mass production. In addition, the antioxidant enzyme extracted through this method has high purity and can be applied to various fields.

The method (1) for extracting antioxidant enzymes from sandalwood mugwort having such characteristics can smoothly exhibit the above-described characteristics by proceeding from step (A) to step (K) in a series of steps.

Hereinafter, the progress steps of the method (1) for extracting antioxidant enzymes from sandworm mugwort will be described in detail. Step (A) is a step (S110) of preparing sandalwood mugwort. This step (A) is the starting step of method (1) for extracting antioxidant enzymes. At this time, sandwormwood becomes 100 kg. After step (A), it proceeds to step (B) (S120) of putting iron sand mugwort in a grinder and grinding it. At this time, the crusher outputs a power of 7 horsepower or more and crushes sandwormwood.

After step (B), step (C) (S130) of immersing the pulverized sandalwood mugwort in a purified water container filled with purified water for a first set time is performed. At this time, the purified water container in step (C) can accommodate 2000 liters in an environment of 20 degrees and immerse the pulverized iron sand mugwort for 1 hour. Thereafter, step (D) (S140) of extracting a solution by first stirring for a second set time at 20 degrees by putting sandwormwood from step (C) in a stirring barrel (S140). At this time, the stirring barrel rotates at 100 rpm (Revolution Per Minute) in an environment of 30 degrees. And the second set time is 3 hours. Thereafter, the step (E) of firstly filtering the solution extracted in step (D) with a mesh screen (S150) and the second step of filtering the first filtrate extracted in step (E) with a Buchner filter (F) It proceeds to (S160). At this time, in step (F), the first filtrate is subjected to secondary filtration using diatomaceous earth without pressurization. Thereafter, it proceeds to step (G) (S170) of adding ammonium sulfate to the secondary filtrate extracted in step (F). At this time, ammonium sulfate becomes 250 kg. This ammonium sulfate becomes an electrolyte and precipitates the secondary solution to become an organic compound.

Thereafter, the mixed solution extracted in step (G) is put into a stirring container and stirred for a third set time to proceed to step (H) (S180) of extracting the solution. At this time, the agitator is rotated at 100 rpm per minute for 12 hours in an environment of 4 degrees, and the extracted solution is stirred. Thereafter, the solution extracted in step (H) is centrifuged with a centrifuge to remove the supernatant, and then proceeds to step (I) (S190) of harvesting the hardened antioxidant enzyme. At this time, the centrifuge is rotated at 10000 rpm for 1 hour to float the supernatant in the extracted solution.

After such step (I), the harvested antioxidant enzyme is put in a purified water container filled with purified water and a second stirring is performed to prepare a solution (J) step (S200). At this time, in step (J), purified water is added to the washing water container so that the concentration of the antioxidant enzyme harvested in step (I) is 500 mg/mL, and 1,2-hexanediol is added. Here, 1,2-hexanediol has almost no toxicity or irritation and has both hydrophobic and hydrophilic properties, so it has a structure of a surfactant. When such 1,2-hexanediol is used together with glycols such as glycerin and butylene glycol, high-efficiency antiseptic efficacy can be exhibited.

After the solution prepared in step (J) is placed in a container and sealed (S220), sterilized by irradiation with gamma rays (S230), the process proceeds to step (K) of collecting antioxidant enzymes (S240). At this time, the antioxidant enzyme to be collected may be a high-purity enzyme. And gamma rays become 15 to 20 kg of gamma rays to kill bacillus bacteria and fungi of antioxidant enzymes so that antioxidant enzymes become sterilized for more than one year at room temperature. The high-purity antioxidant enzyme will be described in detail below.

Hereinafter, with reference to FIGS. 2 to 6 , extraction conditions for antioxidant enzymes with high activity will be described in detail.

Figure 2 is a diagram showing the solvent selection test results for mass production application. And Figure 3 is a view showing the temperature selection test results for mass production application, Figure 4 is a view showing the extraction time selection test results for mass production application.

The method for extracting antioxidant enzymes from sandworm mugwort (1) was carried out through several factor tests for mass production application.

Hereinafter, the test applied to steps (C) to (F) of the method (1) for extracting antioxidant enzymes from sandworm mugwort will be described in detail.

In order to determine the suitability of the purified water set in step (C), a comparative experiment was conducted between purified water and ethanol.

As shown in Table 1 below, in order to extract high-purity antioxidant enzymes, purified water and ethanol were mixed with 25 times, 125 times, and 625 times of pulverized temple mugwort to measure the activity of antioxidant enzymes in the extract.

When purified water and pulverized temple mugwort were mixed, the activity of antioxidant enzymes extracted from the mixture was 625 times as high as purified water, and antioxidant enzymes with an activity of 41.7% were extracted. And when the purified water is 125 times, antioxidant enzymes with 83.4% activity are extracted. And when the purified water is 25 times, antioxidant enzymes with 100% activity are extracted.

And when ethanol and pulverized temple mugwort were mixed, the activity of antioxidant enzymes extracted from the mixture was 22.2% when ethanol was 625 times more active. And when ethanol is 125 times, antioxidant enzymes with 61.7% activity are extracted. And when ethanol is 25 times, antioxidant enzymes with 82.3% activity are extracted.

As such, the method of extracting antioxidant enzymes using clean water can extract antioxidant enzymes having higher activity than the extraction method using ethanol.

The pulverized sandstone mugwort was mixed with 25 times more purified water than the pulverized temple mugwort and stirred at 25℃, 40℃, 55℃, 70℃ for 1 hour at 100 rpm to extract antioxidant enzymes and measure their activity.

At this time, when the agitator was stirred at 100 rpm for 1 hour in a 25 ° C environment, an antioxidant enzyme with an activity of 90.7% was confirmed. In addition, when the stirring barrel was stirred at 100 rpm for 1 hour in a 40 ° C environment, antioxidant enzymes with 85.3% activity were confirmed, and when the stirring barrel was stirred at 100 rpm for 1 hour in a 55 ° C environment, antioxidant enzymes with 90.7% activity were found. enzymes are identified.

As such, the activity of antioxidant enzymes at 25 ° C and 40 ° C showed almost similar tendencies, and at 55 ° C and 70 ° C, the activity decreased.

The reason why the activities of antioxidant enzymes at 25 ° C and 40 ° C showed almost similar tendencies is that antioxidant enzymes have protein properties, which result in transformation of tertiary structure by heat, resulting in less activity decrease at room temperature.

After the first filtration of the extracted solution with a mesh screen, the second filtration using a Buchner filter and filter paper, and then the extracted mixed solution was put into a stirring bucket at 25 ° C and 100 rpm for 1, 2, 3, 6, 12 The activity of the solution extracted by stirring for a period of time was measured.

At this time, after mixing 25 times more purified water than pulverized temple mugwort with crushed sandstone mugwort, put it in a stirring barrel, and then stir the stirring barrel at 100 rpm for 1 hour in a 25 ° C environment. Antioxidant enzyme with 80.7% activity was confirmed. It became. And when stirred for 2 hours, an antioxidant enzyme with an activity of 91.3% was confirmed. And when stirred for 3 hours, an antioxidant enzyme with an activity of 99.8% was confirmed, and when stirred for 6 hours, an antioxidant enzyme with an activity of 97.9% was confirmed. And when stirred for 12 hours, an antioxidant enzyme with an activity of 96.9% was confirmed.

As such, the activity of the antioxidant enzyme was the highest in the 3-hour extraction condition, and thereafter, no significant increase in activity was observed with the increase of time.

Hereinafter, with reference to FIGS. 5 and 6, experiments such as a concentration test and a protein precipitation test for increasing the purity of antioxidant enzymes in the extract will be described in detail.

Figure 5 is a picture showing the antioxidant enzymes precipitated after salting out ammonium sulfate at a concentration of 30%, ammonium sulfate at a concentration of 50% and ammonium sulfate at a concentration of 70%, and Figure 6 is a table showing the content of antioxidant enzymes through an optimized extraction method to be.

The method for extracting antioxidant enzymes from perennial mugwort (1) was conducted through a test to increase the content of antioxidant enzymes in various extracts.

Among them, the protein precipitation test for increasing the SOD content of the extract was conducted by the method described below.

First, ammonium sulfate corresponding to 30%, 50%, and 70% concentration was added to the extracted solution at 25 ° C, 100 rpm, 3 hours by adding 20 times the volume of water, and then stirred at 4 ° C for 1 hour and thoroughly The process of dissolution was carried out. Thereafter, the required amount of ammonium sulfate (100mL) for each concentration: 0% -> 30% = 16.4g, 30% -> 50% = 11.7g, 50% -> 70% = 12.5g were added.

Then, transfer to a 50mL tube, stand at 4°C for 1 hour -> centrifuge at 4,000 rpm, 4°C for 1 hour, transfer the supernatant to a new tube, and precipitate 40 μg of protein each through BCA protein quantification It was dissolved in tertiary distilled water to a concentration and proceeded to the step of using it as a sample. Thereafter, the activity evaluation of antioxidant enzymes for the extracted solution was confirmed by NBT assay through native-PAGE.

At this time, when 30% of ammonium sulfate is added, proteins as shown in (A) of FIG. 5 are precipitated, and when 50% of ammonium sulfate is added, proteins as shown in (B) of FIG. 5 are precipitated. And when 70% of ammonium sulfate was added, proteins as shown in (C) of FIG. 5 were precipitated.

In addition, 20 times the volume of water is added to 200 g of the sample, and ammonium sulfate corresponding to a concentration of 50% is added to the extracted solution at 25 ° C., 100 rpm, for 3 hours. Thereafter, the mixture was completely dissolved while stirring at 4° C. for 1 hour. Then, after standing at 4 °C for 1 hour -> centrifuged at 4,000 rpm, 4 °C for 1 hour, the precipitate is dissolved in tertiary distilled water and used as a sample.

As a result of proceeding with 1/100, 1/200, 1/500, 1/1000, 1/2000, 1/5000 dilution of the salting out precipitation concentrate, the activity of antioxidant enzymes is measured in samples diluted 1000 times.

At this time, an antioxidant enzyme with an activity of 93.9% was found in the 1/100 saltout precipitate concentrate, and an antioxidant enzyme with an activity of 84.8% was identified in the 1/200 saltout precipitate concentrate. In addition, antioxidant enzymes with an activity of 67.5% were found in the 1/500 salting out precipitate concentrate, and antioxidant enzymes with an activity of 63.5% were identified in the 1/1000 salting out precipitate concentrate. In addition, antioxidant enzymes with an activity of 34.1% were found in the 1/2000 salt-out precipitate concentrate, and antioxidant enzymes with an activity of 15.2% were identified in the 1/5000 salt-out precipitate concentrate.

Although the embodiments of the present invention have been described with reference to the accompanying drawings, those skilled in the art to which the present invention pertains can be implemented in other specific forms without changing the technical spirit or essential features of the present invention. you will be able to understand Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

1: Method for extracting antioxidant enzymes from sandwormwood

Claims (7)

Step (A) of preparing perennial mugwort;
(B) step of pulverizing iron sand mugwort into a grinder;
(C) immersing the pulverized sandalwood mugwort in a purified water container filled with purified water for a first set time;
(D) step of extracting a solution by first stirring for a second set time by putting sandwormwood from step (C) in a stirring barrel;
(E) step of first filtering the solution extracted in step (D) with a mesh screen;
(F) step of secondary filtration of the primary filtrate extracted in step (E) with a Buchner filter;
(G) adding ammonium sulfate to the secondary filtrate extracted in step (F);
(H) step of extracting the solution by putting the mixed solution extracted in step (G) into a stirring container and stirring for a third set time;
(I) step of centrifuging the solution extracted in step (H) with a centrifuge, removing the supernatant, and then harvesting the hardened antioxidant enzyme;
(J) preparing a solution by putting the antioxidant enzyme harvested in step (I) into a purified water container filled with purified water and a second stirring;
A method for extracting antioxidant enzymes from sandwormwood, comprising the step (K) of putting the solution prepared in step (J) into a container, sealing it, sterilizing it by irradiating gamma rays, and collecting antioxidant enzymes. According to claim 1,
The sandwormwood in step (A) is 100 kg,
The crusher in step (B) outputs more than 7 horsepower and crushes iron sand mugwort,
(C) A method of extracting antioxidant enzymes from sandwormwood, in which 2000 liters of the purified water container are immersed in the crushed sandstone mugwort. According to claim 2,
Step (D) is,
A method for extracting antioxidant enzymes from perennial mugwort, in which the agitator is rotated at 100 rpm for 3 hours to extract the solution. According to claim 3,
Step (G) is,
A method for extracting antioxidant enzymes from sandwormwood in which 250 kg or more of ammonium sulfate is added. According to claim 4,
Step (H) is,
The agitator was rotated at 100 rpm for 18 hours to extract the solution,
Step (I) is,
A method for extracting antioxidant enzymes from perennial mugwort in which a centrifuge is rotated at 10000 rpm for 1 hour. According to claim 5,
Step (J) is,
Add purified water to the washing water bottle so that the concentration of the antioxidant enzyme harvested in step (I) is 500 mg / mL,
A method for extracting antioxidant enzymes from perennial mugwort by adding 1,2-hexanediol. According to claim 6,
(K) step,
A method of extracting antioxidant enzymes from perennial mugwort, which is sterilized by exposing 15 to 20 kg of gamma rays.

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