KR102530047B1 - A Composition for antioxidant screening using protein based photosensitizer and substrate and method thereof - Google Patents

Abstract

This application relates to antioxidant screening compositions, methods and uses thereof. Specifically, the present application relates to a method for screening antioxidants using a protein-based photosensitizer and a substrate whose color is changed by oxidation, a composition, and a use thereof.

Description

A composition for antioxidant screening using protein based photosensitizer and substrate and method thereof}

The present application relates to an antioxidant screening composition comprising a photosensitive protein that generates active oxygen and a substrate capable of inducing a change in color by being oxidized by the active oxygen.

The present application relates to a method for screening antioxidants using an antioxidant screening composition.

This application relates to various uses of the composition.

Reactive oxygen species (or reactive oxygen species, ROS) are chemically reactive molecules of oxygen with strong oxidizing power.

Active oxygen has strong oxidizing power, causing damage and oxidation of normal cells, which causes aging and various diseases.

ROS scavengers that inactivate reactive oxygen species (ROS scavengers) belong to antioxidants used to reduce oxidizing molecules.

Active oxygen scavengers should have an effect of inhibiting the production of active oxygen, but it is known that the method of measuring this can detect active oxygen by a method such as a method using fluorescence and chemiluminescence probes (Analytical Methods, 2018 , DOI: 10.1039/C8AY01339J).

, DAB)의 중합을 촉매화하여 침전물을 생성하는 것에 기반하여 활성산소의 생성을 관찰하고자 하는 인자를 광감각제에 라벨링(labeling)하여 이를 현미경으로 관찰한 연구가 보고되었다(PLoS Biology. 2011, Apr;9(4):e1001041).Recently, the generation of active oxygen can be observed with an electron microscope, and the active oxygen generated by the photosensitizer excited by light is diaminobenzidine (3,3

, DAB) based on the production of precipitates by catalyzing the polymerization, a study was reported in which the photosensitizer was labeled with the factor to be observed for the generation of active oxygen and observed under a microscope (PLoS Biology. 2011, Apr;9(4):e1001041).

′'-Tetramethylbenzidine, TMB)이 일중항산소의 비색반응을 유도하는 물질이라는 보고가 있었다(Chemical Communications, 2015 Oct 4;51(77):14465-8).In addition, tetramethylbenzidine (3,3

It has been reported that ''-Tetramethylbenzidine, TMB) is a substance that induces a colorimetric reaction of singlet oxygen (Chemical Communications, 2015 Oct 4;51(77):14465-8).

Although various methods have been developed, cumbersome methods, low specificity, low sensitivity, expensive equipment, autofluorescence, fast photobleaching, short half-life of fluorescence, etc. There are various problems of In particular, when measuring the degree of generation of active oxygen in antioxidants using a compound, a problem arises in that the compound may interfere with the measurement of active oxygen in antioxidants.

Therefore, in order to overcome the problems of various methods of measuring active oxygen so far, the development of a simple and highly accurate method is required.

1. US Patent US12/741455 2. US Patent US12/766781

1. A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms, PLoS Biol, 2011 Apr;9(4):e1001041, doi: 10.1371/journal.pbio.1001041. 2. Photocatalytic oxidation of TMB with the double stranded DNA-SYBR Green I complex for label-free and universal colorimetric bioassay. Chem Commun (Camb), 2015 Oct 4;51(77):14465-8, doi: 10.1039/c5cc06105a.

An object of the present application is to provide an antioxidant screening composition comprising a protein-based photosensitizer and a substrate capable of inducing a colorimetric reaction.

Another object of the present application is to provide a method for screening antioxidants using the antioxidant screening composition.

Another object of the present application is to provide various uses of the antioxidant screening method.

In order to solve the above problems, the present application uses a protein-based photosensitizer and a color-changing substrate that is oxidized and changes color.

To this end, the present application may provide an antioxidant screening method.

In particular, for this purpose, we provide:

a) a protein-based photosensitizer;

b) chromogenic substrate;

c) antioxidant candidates; and

d) light;

determining whether the antioxidant candidate material is an antioxidant material by confirming a color change of the color substrate; It can provide an antioxidant screening method comprising a. In particular, at this time, a method characterized in that the color change of the color substrate occurs when the antioxidant candidate material removes active oxygen generated by the protein-based photosensitizer.

In another aspect, the present application provides a protein-based photosensitizer; It can provide an antioxidant screening kit comprising a; and a chromogenic substrate. In particular, the kit may be provided with a kit characterized in that it further requires light provided from the outside. In addition, the kit includes different protein-based photosensitizers according to the type of active oxygen generated by the protein-based photosensitizer activated by light; Or / and a substrate; a kit characterized in that it can include two or more can be provided.

According to this application, the following effects are generated.

First, according to the present application, it is possible to provide an antioxidant screening composition including a protein-based photosensitizer and a color substrate capable of inducing a colorimetric reaction.

Second, according to the present application, it is possible to provide an antioxidant screening method using the antioxidant screening composition. In particular, in the method of the present application, the color development is changed because the color substrate is oxidized due to active oxygen generated when the protein-based photosensitizer of the composition is activated by light. Based on this principle, it is possible to provide a method of screening for antioxidants by measuring the active oxygen scavenging ability of antioxidant candidates.

Third, according to the present application, it is possible to provide various uses using the antioxidant screening method.

1 is a diagram showing a schematic diagram of colorimetric detection of active oxygen using active oxygen generating protein and tetramethylbenzidine of the present application.
Figure 2 is a view showing the absorbance measurement result (A) and a fluorescence photograph (B) observed with a transilluminator according to the presence or absence of light irradiation of miniSOG protein and tetramethylbenzidine.
Figure 3 is a result of confirming the effect of inhibiting the production of active oxygen by ROS scavengers DBS (Di-n-butylsulfide), NaN ₃ and mannitol according to the incubation time after irradiating miniSOG protein and tetramethylbenzidine with LED light for 10 minutes. (A) and a graph (B) obtained by measuring the absorbance at 660 nm.
Figure 4 shows the result (A) of confirming the effect of inhibiting the production of active oxygen by DBS, NaN ₃ and mannitol according to the concentration using miniSOG protein and tetramethylbenzidine, and a graph (B) obtained by measuring it at 660 nm absorbance.
5 confirms the inhibitory effect of ROS scavengers DBS (Di-n-butylsulfide), NaN ₃ and mannitol on the generation of active oxygen according to the incubation time after 10-minute LED light irradiation of miniSOG protein and dopamine. This is a diagram showing the result.
6 is a diagram showing the results of measuring the production rate of singlet oxygen for each of DBS, NaN ₃ and mannitol through anthracene-9,10-dipropionic acid (ADPA).
Figure 7 shows the results of confirming the effect of inhibiting the generation of active oxygen of natural products (#1 to #5) extracted from Physallis spp. using miniSOG protein and tetramethylbenzidine (A) and a graph (B) measured at 660 nm absorbance is the drawing shown.
8 is a diagram showing the results of measuring the generation rate of singlet oxygen for each of the natural products (#1 to #5) extracted from Physallis spp. through ADPA (Anthracene-9,10-dipropionic acid). .
9 is a result of confirming the effect of inhibiting the generation of active oxygen according to the concentration of # 3 among natural products extracted from Physallis spp. using miniSOG protein and tetramethylbenzidine (A) and a graph (B) showing it measured at 660 nm absorbance it is a drawing (C) is a diagram showing the chemical structure of #3.
10 is a diagram showing information on each of the natural products (#1 to #5) extracted from Physallis spp. and the results of measuring IC ₅₀ (half maximal inhibitory concentration).

The content of the application will be described in more detail with reference to the accompanying drawings and the following description. The accompanying drawings include embodiments of the application, but do not include all embodiments. In addition, the subject matter of the present application may be embodied in various forms and is not limited to the specific implementations described below. That is, the present application can make various changes and have various embodiments. Although the configuration and characteristics of the present application have been described based on the embodiments according to the present application, the present application is not limited thereto, and it is possible to make various changes or modifications within the spirit and scope of the present application in the technical field to which this application belongs. It will be apparent to those skilled in the art, and thus such changes or modifications are intended to fall within the scope of the appended claims.

0. Definition of Terms

In this application, the term "screening" refers to both obtaining information about an unknown substance or confirming information about a known substance. In this case, the material may be a compound, protein or a complex thereof, but is not limited thereto. The terms search or screening can be used synonymously with screening.

1. Antioxidant screening

Methods and compositions for antioxidant screening may be provided by the present application.

In general, an antioxidant refers to a substance that blocks or inhibits the action of a substance having an oxidizing action. In this application, the term "antioxidant" refers to a substance having a function of removing active oxygen. The term antioxidant can be used interchangeably with free radical scavenger or scavenger.

● Advantages of antioxidant screening

Screening antioxidants capable of removing reactive oxygen species with strong oxidizing power that attack biological tissues and damage cells with compounds of oxygen generated in living organisms has various advantages. Since active oxygen is known to cause various diseases such as aging and cancer, accurate and rapid screening of antioxidants will provide much help in exploring substances that can be used for various diseases. Furthermore, if the degree of generation of active oxygen at a specific site can be quickly measured, antioxidants can be applied by targeting only that site. In addition, if it is known which active oxygen among various active oxygens the antioxidant is targeting, a specific antioxidant may be applied according to the type of active oxygen generated. In this way, antioxidant screening can not only simply search for substances, but can also be applied in various ways depending on the screening method.

● Limitations of antioxidant screening

In general, when screening antioxidants, i) a method using a fluorescent substance; and ii) a method using a fluorescent compound; are used, but have the following limitations.

i) When using a fluorescent substrate, specific equipment must be used, and due to the nature of fluorescence, there is a problem in that specificity is lowered due to a high background.

When using a fluorescent substrate, you must use specific equipment capable of measuring the wavelength range of the fluorescence used. In addition, the half-life is short due to the nature of the fluorescent material, and it has autofluorescence, causing fast photobleaching. Therefore, it is difficult to measure the background value to measure the degree of active oxygen removal of the antioxidant to be actually measured, or there is a limit in that the specificity is lowered.

ii) When a compound is used, there is a problem in that the specificity of detection is lowered because the compound to be screened reacts with the compound.

Using chemical precursors, active oxygen generated as they react is used. So it is cumbersome and complicated experimentally. In addition, it is not clear whether the generated active oxygen is removed due to the influence of the compound or whether the candidate material to be screened removes the generated active oxygen. Therefore, there is a limit in that the specificity of screening for active oxygen substances is unclear, that is, the accuracy of whether antioxidant substances actually function to remove active oxygen is low.

As described above, the present application relates to screening of antioxidants with high specificity by a very simple method. In particular, we tried to overcome the limitations of existing methods, such as equipment dependence, low specificity, and complicated and cumbersome experimental procedures, by using protein-based photosensitizers and color-developing substrates.

● Technical features of this application

i) Since a protein-based photosensitizer is used, the problem of low detection specificity occurring when using a compound can be overcome.

In the case of using a protein-based photosensitizer, the protein-based photosensitizer is characterized by generating active oxygen when light is irradiated from the outside. Therefore, there is an advantage in that specificity does not deteriorate because there is no collision with the antioxidant to be measured.

ii) Since a chromogenic substrate is used, it is necessary to use specific equipment that occurs when using a fluorescent substrate; And it can overcome the problem of low specificity.

In the case of using a color-changing substrate that changes color upon oxidation, it is possible to visually confirm that the color change of the color-developing substrate occurs during oxidation, so there is an advantage in that expensive fluorescence equipment is not required. In addition, since it does not have autofluorescence, photobleaching does not occur. That is, there is an advantage that can overcome the disadvantage of low specificity due to high background during measurement.

Hereinafter, a method for screening antioxidants using protein-based photosensitizers and erectile substances will be described in detail.

Principles of using protein-based photosensitizers and substrates

1 shows an arbitrary embodiment of the antioxidant screening method of the present application.

As shown in FIG. 1, MiniSOG, a protein-based photosensitizer, has a yellow color due to a cofactor called FMN, and at this time, when external light is provided, active oxygen is induced. If MiniSOG cannot remove the reactive oxygen species produced, the substrate is oxidized and turns blue. However, when the active oxygen produced by MiniSOG is removed, the substrate is not oxidized and the color does not change.

Based on this principle, the present application can screen antioxidants by visually checking substrate color change according to whether or not antioxidants remove active oxygen generated by a protein-based photosensitizer.

One aspect of the present application relates to a composition for screening antioxidants including a protein-based photosensitizer and a substrate.

A) Photosensitizer (PS)

The photosensitizer is a substance that is activated by light and generates active oxygen.

The term photosensitizer may be used interchangeably with photosensitizer or photosensitizer. In this case, the term protein-based photosensitizer may be used interchangeably with active oxygen generating protein or photosensitizing protein.

The antioxidant screening method and composition of the present application include a protein-based photosensitizer.

In general, photosensitizers may include protein-based photosensitizers and compound-based photosensitizers.

In the case of using the compound-based photosensitizer, there is a limit in that the specificity of the antioxidant is lowered. Specifically, it is not clear whether the active oxygen produced by the compound-based photosensitizer reacts with light is removed by the antioxidant or the compound-based photosensitizer causes a chemical reaction to remove it. That is, the compound-based photosensitizer collides with the antioxidant, making it difficult to perform accurate screening.

In comparison, when using a protein-based photosensitizer, since it does not collide with antioxidants, the antioxidant screening method of the present application has the advantage of having high specificity.

The protein-based photosensitizer may be any one or more selected from KillerRed, MiniSOG, SOPP, FPFB, SuperNova, mKate2, and KillerOrange, but is not limited thereto. In addition, the protein-based photosensitizer includes mutants such as KillerRed, miniSOG, SOPP, FPFB, SuperNova, mKate2, and Killerorange.

The KillerRed is a variant of Aequorea Victoria-derived green fluorescent protein having a size of about 27 kDa and is known to generate superoxide when exposed to green light.

The MiniSOG is derived from the LOV domain of Arabidopsis phototropin 2, which has a size of about 14 kD, and is known to generate singlet oxygen when exposed to blue light.

The SOPP or FPFB is known to generate singlet oxygen when exposed to light.

The Killer Orange is known to generate superoxide when exposed to light.

The SuperNova or mKate2 is known to generate singlet oxygen or/and superoxide when exposed to light.

The protein-based photosensitizer of the present application may include a partial sequence or the entire sequence of any one sequence selected from KillerRed, miniSOG, SOPP, FPFB, SuperNova, mKate2, and Killerorange.

Any one or more sequences selected from KillerRed, miniSOG, SOPP, FPFB, SuperNova, mKate2, and Killerorange may be known sequences, for example, sequences disclosed in known databases.

The active oxygen is also called reactive oxygen species (ROS), and refers to all chemically reactive molecules including oxygen atoms.

For example, the active oxygen is a superoxide (O ₂ ^- , superoxide), hydroxyl radical (HO ), singlet oxygen ( ¹ O ₂ ), hydrogen peroxide (H ₂ O ₂ ), hypochlorous acid (HOCl), nitric oxide (Nitric oxide, NO), peroxyl radical (ROO·), peroxynitrite (ONOO-), etc., but is not limited thereto.

The protein-based photosensitizer is activated by light, and the active oxygen, for example, any one selected from superoxide, hydroxyl radical, singlet oxygen, hydrogen peroxide, hypochlorous acid, nitrogen monoxide, peroxyl radical, and peroxynitrate You can create more than one.

For example, the antioxidant screening composition of the present application may include any one selected from KillerRed, miniSOG, SOPP, FPFB, SuperNova, mKate2, and Killerorange.

The protein-based photosensitizer may be used by using a commercially available protein or obtained by a known method.

B) Substrate

The antioxidant screening composition of the present application includes the protein-based photosensitizer and the substrate.

This application uses "substrate" as an index for determining the presence or absence of active oxygen.

The substrate is a material that changes color when oxidized.

In particular, the substrate of the present application refers to a material that is oxidized due to active oxygen generated when the protein-based photosensitizer is activated by light.

The substrate recognizes the active oxygen generated when the protein-based photosensitizer is activated by light, and is oxidized thereby to change color. When this is applied to an antioxidant screening composition, the antioxidant candidate material removes the active oxygen, so that the substrate is not oxidized and the color is not changed. If the antioxidant candidate material fails to remove the active oxygen, the substrate is oxidized and the color is changed.

In this way, the substrate is used as an indicator to determine whether or not the antioxidant candidate material has active oxygen scavenging ability in the antioxidant screening composition.

In general, the substrate may include a fluorescent substrate and a chromogenic substrate, but the antioxidant screening method and composition of the present application are characterized in that the chromogenic substrate is included.

In the case of using a fluorescent substrate, expensive special equipment capable of measuring fluorescence is absolutely necessary. Moreover, fluorescence has a limited time to express fluorescence due to its characteristics and has a short half-life. In addition, since it has autofluorescence and causes photobleaching, difficulty in measurement may occur due to a high background value. That is, fluorescent substrates not only require expensive equipment, but also have low specificity, making it difficult to screen antioxidants.

On the other hand, when a color-developing substrate is used, the color change can be immediately checked with the naked eye without expensive equipment and the accuracy is high, so the antioxidant screening method of the present application has the advantage of being very simple and efficient.

), DAD(o-Dianisidine dihydrochloride), OPD(o-Phenylenediamine dihydrochloride), TMB(3,3

′'-Tetramethylbenzidine), BCIP/NBT(5-bromo-4-chloro-3-indolyl-phosphate/ nitro blue tetrazolium), dopamine, serotonin 등 또는 이의 유도체(derivative), 변이체 일 수 있으나, 이에 제한하지 않는다.The color-developing substrate is ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), 5-ASA (5-aminosalicylic acid), 4-CN (4-chloro-1-naphthol), DAB ( 3,3

), DAD (o-Dianisidine dihydrochloride), OPD (o-Phenylenediamine dihydrochloride), TMB (3,3

''-Tetramethylbenzidine), BCIP/NBT (5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium), dopamine, serotonin, etc., or derivatives or variants thereof, but are not limited thereto.

For example, upon oxidation, ABTS is green, 5-ASA and DAB are brown, 4-CN is blue/black, OPD is yellow, TMB is blue, DAD is yellow/orange, BCIP/NBT is blue/purple, Dopamine can turn a brown/black color.

The chromogenic substrate and the protein-based photosensitizer have a volume ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1 :10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 2:1, 2:3 , 2:5, 2:7, 2:9, 2:11, 3:1, 3:2, 3:4, 4:1, 4:3, 4:5, 5:1, 5:2, 5 :3, 5:4, etc., but is not limited thereto.

The active oxygen that affects the color change of the chromogenic substrate is generated when the protein-based photosensitizer is activated by light, and at this time, the type of active oxygen is 1, 2, 3, 4, 5, etc. may, but is not limited thereto.

For example, the color of the TMB may change through an oxidation reaction with singlet oxygen.

For another example, the color of the DAB may be changed by an oxidation reaction with at least one selected from singlet oxygen and hydrogen peroxide.

As another example, the color of the ABTS may change due to an oxidation reaction with hydrogen peroxide.

As another example, the dopamine and serotonin may undergo an oxidation reaction with superoxide to change color.

The antioxidant screening composition may optionally further include a cofactor to promote the activity of the protein-based photosensitizer.

The cofactor is not limited as long as it is a material that helps the action of the protein-based photosensitizer. For example, the action may be generation of active oxygen or promotion of activity.

The cofactor may be a metal ion cofactor, a compound cofactor, and the like, but is not limited thereto.

For example, the metal ion cofactor may be Ca ²⁺ , Cu ²⁺ , Fe ²⁺ , Fe ³⁺ , Mg ²⁺ , Mn ²⁺ , Mo, Ni ²⁺ , Zn ^{2+ ,} etc., but is not limited thereto. .

For example, the compound cofactor is thiamine pyrophosphate (TPP or ThDP), flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide ( nicotinamide adenine dinucleotide (NAD+ or NADH), nicotinamide adenine dinucleotide phosphate (NADP+ or NADPH), coenzyme A (CoA), pyridoxal phosphate (PLP or P5P), biotin or B7), tetrahydro folic acid (THFA or H4FA), dihydrofolic acid (DHFA or H2FA), 5,10-methylenetetrahydrofolate (MTHF), adenosylcobalamin, AdoCbl), methylcobalamin (MeCbl or B12), ascorbic acid, phylloquinone (K1), menaquinone (K2), Coenzyme F420 (8-hydroxy-5-deazaflavin) etc., but is not limited thereto.

For example, when the antioxidant screening composition is used in vitro, a cofactor may be added to the composition so that the protein-based photosensitizer can generate active oxygen. In this case, the cofactor may be mixed with the protein-based photosensitizer in the antioxidant screening composition in a specific ratio.

The specific ratio may refer to a mixing ratio of the cofactor to enable generation of active oxygen when the protein-based photosensitizer is activated by light.

The specific ratio may be a molar ratio, volume ratio, concentration ratio, etc., but is not limited thereto.

For example, the molar ratio of the protein-based photosensitizer and the cofactor is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1 :9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 2:1 , 2:3, 2:5, 2:7, 2:9, 2:11, 3:1, 3:2, 3:4, etc., but is not limited thereto.

In one embodiment, MiniSOG and FMN may be mixed in the antioxidant screening composition at a molar ratio of 1:10 to 1:15.

In one example, the composition for screening antioxidants of the present application may include a protein-based photosensitizer and a color-developing substrate.

In another example, the composition for screening antioxidants of the present application may include a protein-based photosensitizer, a cofactor, and a color-developing substrate.

In certain embodiments, the composition for screening antioxidants may include MiniSOG protein, FMN, and TMB.

In another specific embodiment, the composition for screening antioxidants may include MiniSOG protein, FMN, and dopamine.

In certain embodiments, the composition for screening antioxidants may include KillerRed protein and BCIP/NBT.

In any other embodiment, the composition for screening antioxidants may include SuperNova protein and TMB.

In any other embodiment, the composition for screening antioxidants may include mKate2 protein and TMB.

In certain embodiments, the composition for screening antioxidants may include SOPP protein and TMB.

In any other embodiment, the composition for screening antioxidants may include FPFB protein, FMN, and TMB.

The composition may be in the form of a kit, but is not limited thereto.

The kit may include one, two, three, four, five, etc. of the different compositions, but is not limited thereto.

Another aspect of the present application relates to a method for screening antioxidants using the antioxidant screening composition.

The method of screening antioxidants can be screened by evaluating the active oxygen scavenging ability of antioxidants using a protein-based photosensitizer that is activated by light to generate active oxygen and a chromogenic substrate that changes color upon oxidation.

Specifically, when the protein-based photosensitizer is activated by light, active oxygen is generated, and the color is changed as the color-developing substrate is oxidized due to the generated active oxygen. Since the chromogenic substrate changes color, it functions as a marker. Because of this, when a substance having a function of removing active oxygen among antioxidant candidates removes the active oxygen generated by the protein-based photosensitizer, the color of the chromogenic substrate does not change, so it can be used to evaluate the active oxygen scavenging ability of a substance. there is.

As described above, the method of screening antioxidants of the present application can be conveniently performed in vitro using the composition.

The method of screening the antioxidant may include providing the protein-based photosensitizer, color-developing substrate, light, and antioxidant candidate.

A protein used as the photosensitizer, such as MiniSOG, can be obtained and used as a commercially available protein or by a known method, and can be provided together with or separately from the chromogenic substrate in vitro.

At this time, in order to promote or help active oxygen production of the protein-based photosensitizer, a cofactor may be further provided.

Therefore, a composition for evaluating the antioxidant activity of a candidate antioxidant material is a mixture of a protein-based photosensitizer and a color-developing substrate; or a mixture of a protein-based photosensitizer, a chromogenic substrate, and a cofactor. At this time, the mixing of the protein-based photosensitizer, color substrate, antioxidant candidate, and optionally cofactor may be sequentially, randomly, or simultaneously, but is not limited thereto. When light is provided to the mixture, a protein-based photosensitizer generates active oxygen, and a color change occurs due to oxidation of a chromogenic substrate.

In the method of the present application,

Light may be provided after the antioxidant candidate is provided simultaneously or separately from the preparation of the mixture.

In addition, after providing light to the mixture, an antioxidant candidate material may be provided.

Then, it is determined whether the antioxidant candidate material is an antioxidant material by checking the color change of the chromogenic substrate.

In the antioxidant screening method,

For example, after irradiating light to a protein-based photosensitizer, a color-developing substrate; and antioxidant candidates; may be mixed.

Any other examples of protein-based photosensitizers; And after mixing the chromogenic substrate, after irradiation with light, antioxidant candidates; may be mixed.

For example, after mixing a protein-based photosensitizer, a color-developing substrate, and an antioxidant candidate, light may be provided.

any eg protein-based photosensitizer; And after mixing the antioxidant candidate material, after irradiation with light, a color-developing substrate; may be mixed.

The chromogenic substrate, cofactor, or/and antioxidant candidate may be prepared by diluting, concentrating, or purifying in an appropriate reagent, but is not limited thereto.

The dilution or concentration or purification is not limited thereto as long as it is a method known in the art.

The titration reagent may be selected according to dilution or concentration or purification, but is not limited thereto.

For example, the titration reagent may be a buffer solution, but is not limited thereto.

In this case, the light may be provided from an external light source.

For example, the light source may be a light source device such as an LED or a laser, but is not limited thereto.

The light may be irradiated repeatedly, such as 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, etc., but the number of times the photosensitizer can be activated ramen is not limited

1 minute, 3 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes , 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, 100 minutes, etc. may be investigated, but is not limited thereto.

The antioxidant candidate material is a screening target, and is a material for determining whether or not the material has antioxidant activity, for example, a function as a reactive oxygen scavenger (ROS scavenger).

The antioxidant candidate material may be a single material or a complex material, for example, a compound, a protein, or a complex thereof, but is not limited thereto.

In particular, antioxidant candidates screened in the present application may be ROS scavengers.

The antioxidant candidate material may be prepared by mixing with the protein-based photosensitizer or/and color-developing substrate in a specific ratio. In this case, it may be a molar ratio, a volume ratio, a concentration ratio, etc., but is not limited thereto.

The antioxidant candidate material may be prepared by being mixed with a protein-based photosensitizer or/and a chromogenic substrate or/and light, or may be prepared independently. At this time, the order of mixing is not limited.

The antioxidant candidate material and the protein-based photosensitizer have a volume ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 , 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 2:1, 2 :3, 2:5, 2:7, 2:9, 2:11, 3:1, 3:2, 3:4, etc., but is not limited thereto.

The antioxidant candidate material and the chromogenic substrate have a volume ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 , 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 2:1, 2:3, 2 :5, 2:7, 2:9, 2:11, 3:1, 3:2, 3:4, etc., but is not limited thereto.

The antioxidant candidate material, the protein-based photosensitizer, and the color substrate have a volume ratio of 1:1:1, 1:2:1, 1:2:2, 1:2:3, 1:2:4, 1: 2:5, 1:3:1, 1:3:2, 1:3:3, 1:3:4, 1:3:5, 1:4:1, 1:4:2, 1:4: 3, 1:4:4, 1:4:5, 1:5:1, 1:5:2, 1:5:3, 1:5:4, 1:5:5, etc., but are limited thereto I never do that.

In addition, the method of screening antioxidants includes the step of determining whether an antioxidant candidate is an antioxidant based on a color change of the color substrate.

Determining whether the antioxidant is present; is to determine whether the protein-based photosensitizer can remove active oxygen generated by being activated by light. At this time, it is determined whether or not the antioxidant candidate material is an antioxidant through a color change of the color substrate.

For example, when the antioxidant candidate material removes active oxygen generated when the protein-based photosensitizer is activated by light, the color-developing substrate may not be oxidized and the color may not change. At this time, the antioxidant candidate material may be determined as an antioxidant material.

For another example, when the antioxidant candidate material fails to remove active oxygen generated when the protein-based photosensitizer is activated by light, the color-developing substrate may be oxidized and the color may change. At this time, it can be determined that the antioxidant candidate material is not an antioxidant material.

Examples 2 to 4 and FIGS. 7 to 10 of the present application describe the results of observing that the color changes before and after light irradiation using a composition containing miniSOG as a protein-based photosensitizer, FMN as a cofactor, and TMB as a color developing substrate. are doing

In order to confirm the validity of the screening method of the present application, DBS (Di-n-butylsulfide), NaN ₃ and mannitol, known as ROS scavengers, and natural products extracted from Physallis spp. were used.

When the composition containing miniSOG, FMN, and TMB was irradiated with light, it was confirmed that miniSOG and FMN were activated by light to generate active oxygen, and TMB was oxidized due to the generated active oxygen, and the color changed to green. As a result of reacting various ROS scavengers based on this principle, DBS and NaN ₃ scavenge singlet oxygen or superoxide, which is an active oxygen generated by light from miniSOG and FMN, and TMB becomes an active oxygen that causes an oxidation reaction. It was found that the color of TMB did not change because there was no However, mannitol, which has been reported to inhibit hydroxyl radicals, did not scavenge singlet oxygen or superoxide, so TMB oxidized with active oxygen and the color changed to green. In other words, it suggests that the presence or absence of ROS scavengers can be confirmed by the presence or absence of color change during oxidation of TMB due to singlet oxygen or superoxide generated by miniSOG and FMN.

In addition, as a result of confirming the active oxygen generation inhibitory effect by reacting the composition with natural products (#1 to #5) extracted from Physallis spp. and irradiating with light, it was confirmed that only natural product #3 did not undergo an oxidation reaction of TMB. In other words, based on this principle, it suggests that it is possible to visually confirm which of the five natural products is an antioxidant.

Figure 5 of Example 3 of the present application is DBS (Di-n-butylsulfide) known as ROS scavengers when irradiated with light using a composition containing miniSOG as a protein-based photosensitizer, FMN as a cofactor, and dopamine as a substrate , the results of confirming the effect of inhibiting active oxygen production of NaN ₃ and mannitol are described. As a result of confirming the inhibitory effect of DBS, NaN ₃ and mannitol on the production of reactive oxygen species using dopamine as a substrate, it was confirmed that among the three ROS scavengers, at a concentration of 20 mM, the oxidation reaction of dopamine was greatly suppressed in the order of DBS and NaN ₃ . On the other hand, it was confirmed that Mannitol did not inhibit the dopamine oxidation reaction. That is, it suggests that it can be confirmed with the naked eye that DBS is a substance that induces the ROS scavenger effect of singlet oxygen the most through the color change according to the oxidation reaction of dopamine.

Therefore, through the examples of the present application, it can be seen that antioxidant screening can be simplified with the naked eye based on the color change of the chromogenic substrate oxidized due to the active oxygen generated when the protein-based photosensitizer is activated by light. there was.

In one embodiment, the present application may provide a method for screening antioxidants as follows.

A method for screening antioxidant candidates for in vitro use, the method comprising:

provide;

a) protein-based photosensitizer

b) chromogenic substrate

c) antioxidant candidates; and

d) light;

determining whether the antioxidant candidate material is an antioxidant material by confirming a color change of the color substrate;

including,

At this time, it is possible to provide a method characterized in that the color change of the chromogenic substrate occurs when the antioxidant candidate material removes the active oxygen generated by the protein-based photosensitizer.

In the above method, a) a protein-based photosensitizer;, b) a color-developing substrate;, c) an antioxidant candidate;, d) light; are provided in a mixture of any two or more selected as described above, or individually It may be provided, but is not limited thereto.

In any embodiment, the antioxidant screening method,

Create a mixture of MiniSOG, TMB, and antioxidant candidates;

irradiating the mixture with light;

determining whether the antioxidant candidate is an antioxidant by checking the color change of the TMB;

can include At this time, the mixture may optionally further include a cofactor.

In any other embodiment, the antioxidant screening method,

MiniSOG, making a mixture with TMB mixed;

irradiating the mixture with light;

mixing antioxidant candidates;

determining whether the antioxidant candidate is an antioxidant by checking the color change of the TMB;

can include At this time, the mixture may optionally further include a cofactor.

As described above, the antioxidant screening method of the present application can provide very simple and accurate detection results in vitro.

Meanwhile, one aspect of the antioxidant screening method of the present application may be performed in a cell.

In this case, the protein-based photosensitizer may be introduced into cells in the form of a protein or a vector containing a nucleic acid encoding the same.

The vector may be a viral vector. For example, the viral vector may be at least one selected from the group consisting of retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus, and herpes simplex virus, but is not limited thereto. .

The introduction is one selected from among electroporation, gene gun, lipid-mediated transfection, nanoparticle, calcium phosphate, dendrimer, sonoporation, magnetic injection, microinjection, PTD (Protein translocation domain) fusion protein method It may be performed in the above method, but is not limited thereto.

Other than the protein-based photosensitizer, chromogenic substrates, cofactors, antioxidant candidates, and the like can be introduced into cells for screening using known methods.

Other configurations necessary for performing the method of the present application are as described above.

Since the present application uses a protein-based photosensitizer, it has the advantage that it can be used for various purposes as follows.

For example, since the photosensitizer of the present application is protein-based, it has the advantage of being fixed or/and expressed on various surfaces or matrices within cells.

Using this point, it is possible to induce movement to a specific organelle by fusing a domain or the like that targets an organelle within a cell.

In particular, the domain targeting the intracellular organelle induces the protein-based photosensitizer and the chromogenic substrate near the intracellular organelle, and the color change of the chromogenic substrate occurs due to the active oxygen generated by the intracellular organelle.

Therefore, the ability of the intracellular organelle to generate active oxygen can be measured according to the degree of color change of the chromogenic substrate.

Through this, the antioxidant screening method of the present application can be used to determine the degree of active oxygen production in specific organelles.

The intracellular organelle may be, but is not limited to, mitochondria, ribosomes, endoplasmic reticulum (ER), Golgi complex, and lysosomes.

As another example, the type of active oxygen produced may vary depending on the type of the protein-based photosensitizer of the present application.

Using this point, it is possible to identify which active oxygens can be removed by antioxidants.

For any example,

a) a protein-based photosensitizer that generates singlet oxygen and a color-changing substrate that reacts with singlet oxygen;

b) a protein-based photosensitizer that generates superoxide and a color-changing substrate that reacts with superoxide;

c) a protein-based photosensitizer that generates hydroxyl radicals and a color-changing substrate that reacts with hydroxyl radicals;

d) a protein-based photosensitizer that generates hypochlorous acid and a color-changing substrate that reacts with hypochlorous acid;

e) a protein-based photosensitizer that generates hydrogen peroxide and a color-changing substrate that reacts with hydrogen peroxide;

Active oxygen targeted by antioxidants can be searched using a kit containing. At this time, a) to e) may exist independently in the kit.

When an antioxidant is reacted with a) to e) in the kit, and external light is provided, it can be confirmed whether or not the color of the chromogenic substrate changes.

It is possible to determine which type of active oxygen can be removed by the antioxidant by checking whether the color of the chromogenic substrate changes.

Through this, the antioxidant screening method of the present application can be used for the purpose of exploring which active oxygens can be removed by antioxidants.

[Form for Implementation of Application]

Hereinafter, the present application will be described in more detail through examples.

These examples are only for explaining the present application in more detail, and it will be apparent to those skilled in the art that the scope of the present application is not limited by these examples.

● experimental material

In the embodiments of the present application,

MiniSOG (named MS) as a protein-based photosensitizer;

FMN as a cofactor - a substance known to help generate singlet oxygen when the MiniSOG is activated by light in vitro -;

TMB or dopamine as a chromogenic substrate;

DBS, NaN3 and D-mannitol as ROS scavengers;

MISR-PM3, MISR-PM4, MISR-PM5, MISR-PM6 and MISR-PM7 compounds (named #1 to #5) extracted from Physalis minima L. as candidates for antioxidant activity;

was used.

All materials and reagents used in the experiments were used without further purification and were purchased from commercial sources as follows: anthracene-9,10-dipropionic acid (ADPA, Abcam), flavin mononucleotide (FMN, Sigma-Aldrich), 4, 5-Dihydroxy-1,3-benzenedisulfonic acid disodium salt monohydrate (DBS, Sigma-Aldrich), sodium azide (NaN3, Sigma-Aldrich), D-mannitol (Sigma-Aldrich), transparent 96-well microplate (SPL), tetramethylbenzidine (TMB, Sigma-Aldrich), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Thermo Scientific), and potassium hydroxide (99.9%, KOH, Sigma-Aldrich), Dopamine hydrochloride (Sigma-Aldrich)

● Schematic diagram of this application

A schematic diagram of an embodiment of the present application described below is shown in FIG. 1 .

1 relates to a schematic diagram designing a colorimetric detection method for singlet oxygen and scavenging effects using protein photosensitizers MiniSOG and TMB.

As a cofactor, Flavin mononucleotide (FMN) binds to MiniSOG. Due to this, the solution of miniSOG/FMN shows a yellow color due to the color development of FMN.

(A) of FIG. 1 shows a case where the MiniSOG/FMN solution is irradiated with LEDs. During LED irradiation, MiniSOG/FMN generates singlet oxygen, and TMB is oxidized by singlet oxygen to produce a blue product. Therefore, the resulting green color is due to oxidized TMB (blue) and miniSOG/FMN (yellow).

(B) of FIG. 1 shows a case in which the MiniSOG/FMN solution is irradiated with LEDs in the presence of an active oxygen scavenger. When the active oxygen scavenger removes singlet oxygen generated by MiniSOG/FMN during LED irradiation, the oxidation reaction of TMB is suppressed. Therefore, due to the oxidation-inhibited TMB and miniSOG/FMN (yellow), the resultant color is yellow.

Active oxygen and active oxygen scavenging effects can be easily observed with the naked eye using the method of the schematic diagram shown in FIG. 1, and can also be quantified by measuring their absorbance (wavelength: 660 nm).

● Gene construction and protein expression

The MiniSOG gene was prepared using two primers (forward (SEQ ID No. 1): 5'GAT CCC ATG GAG AAG AGC TTC GTG ATC AC-3'; reverse (SEQ ID No. 2): 5'AAG CTT CTA GCC GTC CAG CTG CAC G-3') was used to amplify with mCherry-miniSOG-N1 (Addgene, No. 54803 plasmid). And it was cloned into the pRSETB plasmid containing the N-terminal His6-tag, and pRSETB-MS was made and used with BamH° and Hind² restriction enzymes.

For protein expression and purification, a plasmid having Escherichia coli BL21 was inoculated into 500 mL Luria broth containing 100 mg/mL ^-1 ampicillin. It was incubated at 37 °C until the optical density at 600 nm (OD600) reached 0.6-0.8. Expression was performed using 1 mM isopropyl β-D-thiogalactoside (IPTG) at 20 °C for 20 hours. Cells were harvested by centrifugation at 7,800 rpm for 20 minutes at 4°C, and resuspended in 20 mL of buffer A (50 mM sodium phosphate containing 300 mM NaCl and 10 mM imidazole; pH 8.0). The resuspended pellet was sonicated in the presence of 40 mg of lysozyme and then centrifuged sequentially at 7,800 rpm for 20 minutes at 4°C and 14,000 rpm for 15 minutes at 4°C. The His6-tagged expressed protein was separated from the supernatant using a Ni ²⁺ -NTA agarose-filled column (Pierce) equilibrated with buffer A. The column was washed three times with buffer B (50 mM sodium phosphate containing 300 mM NaCl and 20 mM imidazole; pH 8.0). Bound proteins were eluted from the column with 5 mL of buffer C (50 mM sodium phosphate containing 300 mM NaCl and 250 mM imidazole; pH 8.0). The buffer in the eluate protein was replaced with 1Х phosphate-buffered saline (PBS; pH 7.4) using a PD-10 desalting column (GE Healthcare).

Produced proteins were identified using SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis). The concentration of the purified fusion protein was determined from the absorbance value at 280 nm based on the extinction coefficient values.

● ^One O ₂ Colorimetric measurement of

Unless otherwise noted, before using MiniSOG, it was pre-incubated with FMN in phosphate-buffered saline (1xPBS, pH 7.4) at a 1:15 molar ratio for 30 minutes at room temperature (hereinafter referred to as MiniSOG/FMN). .

For colorimetric detection of ¹ O ₂ , 100 μl of the MiniSOG/FMN solution was transferred to a transparent 96-well microplate, and then a TMB solution (from 1 mg/mL ^-1 ) dissolved in 50 mM phosphate-citrate buffer (pH 5.0). 1/10 dilution) was added to each well to make a final volume of 200 μl. The final concentrations of miniSOG, FMN and TMB per 200 μl were 5 μM, 75 μM and 50 μg/mL ⁻¹ , respectively.

Example 1: Measurement of colorimetric response between MiniSOG/FMN and TMB

In order to determine whether colorimetric detection of active oxygen is possible, the ability of photoactivated MiniSOG/FMN to react with TMB was measured. To this end, we first tested with or without light irradiation (10 min at a light density of 10 mM cm-1) with or without TMB. The result is shown in Figure 2.

2(A) shows MiniSOG/FMN alone (named as miniSOG), MiniSOG/FMN and TMB without LED irradiation (miniSOG+TMB(named -light)), and MiniSOG/FMN and TMB with LED irradiation. When (named as miniSOG+TMB(+light)), each absorbance spectrum was shown. There was no significant difference between the absorbance spectra of miniSOG and miniSOG+TMB(-light), but the absorbance spectra of miniSOG+TMB(+light) showed absorbance peaks at 370nm and 660nm. In addition, it was confirmed that the color of TMB did not change depending on the presence or absence of light irradiation (picture inserted in FIG. 2 (A)). 2 (A) Experimental results, we chose 660 nm to quantify the oxidized product of TMB because the absorbance value at 370 nm can be affected by other compounds potentially acting as ROS scavengers. .

Figure 2 (B) confirms the color change according to the presence or absence of light irradiation on the 96-well microplate, and the bottom surface of the transparent 96-well microplate was directly irradiated using a custom-made trans illuminator equipped with an LED lamp. When the LED was not irradiated, it was confirmed that the MiniSOG/FMN solution exhibited yellow color, and when the LED was irradiated, it was confirmed that the color change was mediated by TMB (ie, oxidation reaction) and the solution was green.

In other words, due to the singlet oxygen generated by the activation of the yellow MiniSOG/FMN by light, TMB, which has no color, is oxidized and turns blue, and finally appears green.

Through the results of FIG. 2, the photoactivated MiniSOG/FMN designed by the inventors of the present application reacts with TMB to change color (schematic diagram in FIG. It was found that there were advantages that could be observed simply.

● Measurement of active oxygen scavenging effect of ROS scavenger - colorimetric reaction

For the scavenging assay of ¹ O ₂ , ROS scavengers were initially dissolved in DMSO or distilled water, and then each chemical was diluted in PBS to an appropriate concentration (DBS, NaN 3 , and Mannitol each at a final concentration of 10 mM). Afterwards, the chemical solution (20 μl) was mixed with the MiniSOG/FMN solution (80 μl) and transferred to a 96-well microplate. And 100 μl of TMB solution (1 mg/mL ^-1 to 1/10 dilution) dissolved in 50 mM phosphate-citrate buffer (pH 5.0) or dopamine solution (final concentration 10 mM) dissolved in 10 mM Tris-HCl (pH 8.5) was added to each well to make a final volume of 200 μl.

The 96-well microplate was irradiated with LED light (10mW cm ⁻² ) for 10 minutes using an LED lamp, and then the microplate was further incubated at room temperature for each hour without irradiation of light. Absorbance spectra of the solutions were measured at 660 nm using a microplate reader (Varioskan, Thermo Fisher Scientific). To remove background values, each control sample measured without illumination (ABS ₀ ) and the sample with LED illumination (ABS _LED ) were normalized; That is, ABS _LED -ABS ₀ . Fluorescence (FL) images of MiniSOG in the wells were obtained with a 520 nm emission filter under blue LED excitation conditions.

Example 2: Measurement of active oxygen scavenging effect of ROS scavenger using MiniSOG/FMN and TMB

To confirm the scavenging effect of the active oxygen scavenger, the solution containing MiniSOG/FMN and TMB was tested according to the presence or absence of three known ROS scavengers (DBS, NaN3, and D-mannitol). The results are shown in Figures 3 and 4.

DBS, NaN3 and D-mannitol have been reported to inhibit superoxide anion, singlet oxygen and hydroxyl radical, respectively.

Figure 3 shows the results of treating three ROS scavengers at high concentrations (10 mM) in a solution containing MiniSOG/FMN and TMB.

3(A) shows MiniSOG/FMN (named as miniSOG) but no TMB and ROS scavenger (Well No. 1), MiniSOG/FMN (named as miniSOG) and TMB present but ROS scavenger LED light irradiation (10 min at a light density of 10 mM cm-1) and irradiated with LED light (10 mW cm ^-2 ) for 10 minutes, and then microplates were irradiated with no light at different times (10 minutes, 20 minutes, 30 minutes). , 1 hour) showed the result of additional incubation at room temperature.

Since MiniSOG/FMN does not generate active oxygen due to photoactivation when no LED light is irradiated (named No LED light), Well No. No color change was observed in all of 1 to 5 and the yellow color was maintained, and when incubated by time after irradiation with LED light (named 10min, 20min, 30min, 1h), Well No. 2 was observed to turn green from 10 minutes of incubation, which was maintained up to 1 hour. In addition, Well No. 3 containing MiniSOG/FMN, TMB and DBS and Well No. 4 containing MiniSOG/FMN, TMB and NaN3 remained yellow without color change, and MiniSOG/FMN, TMB and D-Mannitol remained yellow. Only Well No. 5 containing the color changed to green.

That is, due to the singlet oxygen produced by the activation of the yellow MiniSOG/FMN by light, TMB, which does not have a color, is oxidized and turned blue, finally showing green. At this time, since the ROS scavenger scavenges the active oxygen generated by the photoactivated MiniSOG/FMN, TMB is not oxidized, so it is finally shown in yellow.

DBS, which is structurally similar to vitamin E, is known as a superoxide scavenger, but it was found to have a similar effect to NaN3, a singlet oxygen scavenger. These results are consistent with the fact that MiniSOG produces both singlet oxygen and superoxide, as previously reported (Free Radical Biology and Medicine 116, 134-140, 2018).

3(B) is a graph obtained by measuring the absorbance of FIG. 3(A) using a microplate reader at 660 nm. Well No.3 containing MiniSOG/FMN, TMB and DBS with ROS scavenger present and MiniSOG/FMN, TMB with ROS scavenger present than Green absorbance with MiniSOG/FMN and TMB without ROS scavenger as in Well No.2. And it can be seen that the yellow absorbance value of Well No. 4 containing NaN3 is low.

3, when MiniSOG/FMN is irradiated with light, singlet oxygen is generated within a relatively short time (10 minutes), and the color changes due to oxidation of TMB depending on the presence or absence of ROS scavenger, thereby reducing active oxygen of ROS scavenger. The elimination effect could be confirmed.

Figure 4 shows the results of treatment with the dog ROS scavenger at different concentrations (10 μM to 10 ⁴ μM) in a solution containing MiniSOG/FMN and TMB. Figure 4 is similar to the experiment of Figure 3, but measured after irradiation of LED light (10mW cm ^-2 ) for 10 minutes for each concentration of ROS scavenger. Specifically, LED light (10 mW cm −2 ) was irradiated for 10 minutes, and then the microplate was further incubated at room temperature for 1 hour without light irradiation.

In (A) of FIG. 4, among the three ROS scavengers, D-mannitol turned green regardless of concentration, and DBS and NaN3 turned green at concentrations of 10 μM and 10 ² μM, but 10 ³ μM and 10 μM respectively. At a concentration of ⁴ μM, it was confirmed that the color did not change and the yellow color was maintained.

That is, due to the singlet oxygen produced by the activation of the yellow MiniSOG/FMN by light, TMB, which does not have a color, is oxidized and turned blue, finally showing green. At this time, since the ROS scavenger scavenges the active oxygen generated by the photoactivated MiniSOG/FMN, TMB is not oxidized, so it is finally shown in yellow.

4(B) is a graph obtained by measuring the absorbance of FIG. 4(A) at 660 nm in a microplate reader. D-Mannitol had a similar absorbance regardless of the concentration, but DBS and NaN3 had lower absorbance values at concentrations of 10 ³ μM and 10 ⁴ μM than those at 10 μM and 10 ² μM.

4, singlet oxygen generated when MiniSOG/FMN is irradiated with light can be prevented from oxidation of TMB even with a low concentration of ROS scavenger, and the active oxygen scavenger effect of ROS scavenger can be changed by color change. I was able to confirm.

Example 3: Measurement of active oxygen scavenging effect of ROS scavenger using MiniSOG/FMN and dopamine

As the ROS scavenger's active oxygen scavenging effect was confirmed using MiniSOG/FMN and TMB, the active oxygen scavenger's active oxygen scavenging effect was measured using Dopamine, which changes color when oxidized similarly to TMB. The result of this is shown in FIG. 5 .

Figure 5 shows the results measured by treating three ROS scavengers by concentration (20 mM and 20 μM) in a solution containing MiniSOG/FMN and Dopamine, respectively.

Figure 5 (A) shows the presence of MiniSOG/FMN (named as miniSOG) but no dopamine and ROS scavenger (Well No. 1), MiniSOG/FMN (named as miniSOG) and dopamine but presence of ROS scavenger LED light irradiation (10 min at a light density of 10 mM cm-1) and irradiated with LED light (10 mW cm ^-2 ) for 10 minutes, and then microplates were irradiated with no light at different times (10 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 4 hours) showed the results of additional incubation at room temperature.

Since MiniSOG/FMN does not generate active oxygen due to photoactivation when LED light is not irradiated (named at 0 min), Well No. In all of 1 to 5, no color change was observed and the yellow color was maintained, and when incubated by time after irradiation with LED light (named 10min, 20min, 30min, 1h, 2h, 4h), MiniSOG/FMN and TMB were included. It was observed that Well No. 2 to be changed to black at 4 hours while changing to light brown from 10 minutes of incubation. Well No. 3 containing MiniSOG/FMN, dopamine, and DBS maintained a yellow color until 2 hours of incubation and then slightly changed to light brown at 4 hours. Well No. 4 containing MiniSOG/FMN, dopamine, and NaN3 turned slightly brown at 2 hours of incubation and brown at 4 hours. Well No.5 containing MiniSOG/FMN, TMB and D-Mannitol turned brown from 30 minutes of incubation and turned black at 4 hours.

The results showed a pattern similar to that when TMB, which is oxidized and color-changed by reaction with active oxygen generated by photoactivated MiniSOG/FMN, was used as a color development substrate. The active oxygen scavenging effect of the three ROS scavengers was found in DBS and NaN3. .

5 (B) shows a group in which MiniSOG/FMN (named as miniSOG) is present but dopamine and ROS scavenger are absent (Well No. 1), MiniSOG/FMN (named as miniSOG) and dopamine are present but ROS scavenger is present LED light irradiation (10 min at a light density of 10 mM cm-1) and irradiated with LED light (10 mW cm ^-2 ) for 10 minutes, and then microplates were irradiated with no light at different times (10 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 4 hours) showed the results of additional incubation at room temperature.

Since MiniSOG/FMN does not generate active oxygen due to photoactivation when LED light is not irradiated (named at 0 min), Well No. In all of 1 to 5, no color change was observed and the yellow color was maintained, and when incubated by time after irradiation with LED light (named 10min, 20min, 30min, 1h, 2h, 4h), MiniSOG/FMN and TMB were included. It was observed that Well No. 2 to be changed to black at 4 hours while changing to light brown from 10 minutes of incubation. Well Nos. 3 to 5 containing MiniSOG/FMN, dopamine, DBS, NaN3, and D-Mannitol, respectively, turned light brown after 30 minutes of incubation and turned black after 4 hours.

In other words, due to the singlet oxygen produced by the activation of yellow MiniSOG/FMN by light, colorless dopamine was oxidized and changed to brown-black, which was finally shown as brown-black. At this time, the ROS scavenger scavenges the active oxygen generated by the photoactivated MiniSOG/FMN, and dopamine is not oxidized, so it is finally shown in yellow.

5, it was found that dopamine also reacts with the photoactivated MiniSOG/FMN and changes color according to the oxidation reaction, which can be used to confirm the active oxygen scavenger effect of the ROS scavenger.

3 to 5 suggest that it is a simple method for screening ROS scavengers using photoactivated MiniSOG/FMN and TMB or dopamine, which are chromogenic substrates that change color upon oxidation.

● Measurement of active oxygen scavenging effect of ROS scavenger - Fluorescence

For in vitro ROS analysis, singlet oxygen was measured using ADPA (Anthracene-9,10-dipropionic acid). First, 10 μM of purified miniSOG protein and 50 μM ADPA were mixed with ADPA reaction buffer (50 mM HEPES-KOH, pH 7.4) to make a mixture with a final volume of 100 μL. The mixture was irradiated with LED light (10 mW cm ^-2 ) for 10 minutes. ROS levels were analyzed by measuring the decrease in fluorescence intensity of ADPA before and after LED light. Fluorescence change of ADPA was measured using a microplate reader set to an excitation wavelength of 380 nm and an emission wavelength of 430 nm. At this time, the excitation wavelength had a minimal effect on the excitation of the miniSOG or the scavenger.

To evaluate the ROS scavenging effect, three ROS scavengers (DBS, NaN3 and D-mannitol) and five plant-extracted compounds from Physalis minima L. (MISR-PM3, MISR-PM4, MISR-PM5, MISR- PM6 and MISR-PM7; designated #1 to #5, respectively) were used. Each compound at an appropriate concentration and miniSOG protein (final concentration, 10 μM) were first mixed in ADPA reaction buffer to form a mixture, and then ADPA (final concentration, 50 μM) was added to the mixture.

Example 4: Measurement of active oxygen scavenging effect of ROS scavenger using MiniSOG/FMN and ADPA

To confirm the scavenging effect of the active oxygen scavenger, the degree of active oxygen scavenging of three known ROS scavengers (DBS, NaN3, and D-mannitol) in a solution containing MiniSOG/FMN and ADPA was measured with a fluorometer. did. The result of this is shown in FIG. 6 .

Figure 6 shows a group containing MiniSOG/FMN and ADPA (named Control), a group containing MiniSOG/FMN, ADPA, and three ROS scavengers (named DBS, NaN3, and Mannitol, respectively) for 10 minutes. LED light (10mW cm ⁻² ) was irradiated, and then the microplate was further incubated at room temperature by time (10 minutes, 20 minutes, 30 minutes) without light irradiation.

Since the control group did not contain the ROS scavenger, the fluorescence intensity of ADPA was high, and among the three ROS scavengers, NaN3 had the lowest ADPA fluorescence intensity, that is, it was observed that the active oxygen scavenger effect was the most excellent. D-Mannitol was observed to have the highest fluorescence intensity of ADPA, that is, no active oxygen scavenging effect.

This result did not match the result of FIG. 3 measured using MiniSOG/FMN and TMB. This discrepancy is due to autofluorescence of DBS at the excitation wavelength of ADPA. However, as in the results of FIG. 3, it can be seen that when MiniSOG / FMN is photoactivated, it is consistent to support the production of singlet oxygen and superoxide.

Through this, it was confirmed that the antioxidant screening composition including the light-sensing protein and the chromogenic substrate of the present application does not have the self-fluorescence problem when measured by ADPA because it uses a chromogenic substrate rather than a fluorescent substrate. .

● Extraction and purification of natural compounds from a Vietnamese plant

The aerial parts of Physalis minima L. were obtained from Nam Dong, Thua Thien Hue Province, Vietnam in September 2018, and Ms. Nguyen Quynh Nga (National Institute of Medicinal Materials, Vietnam). The voucher specimen (MISR2018-18) was deposited in the Herbarium of the Mientrung Institute for Scientific Research, Vietnam VAST.

The dried and powdered aerial parts of P. minima (2.6 kg) were sonicated for 1 hour and extracted with MeOH three times (5 L each). The MeOH extract was concentrated under reduced pressure to obtain a residue (PM, 150.0 g). This residue was suspended in water (1.5 L) and partitioned successively between n-hexane (3 x 2 L), dichloromethane (3 x 2 L) and ethyl acetate (3 x 2 L) to give: n-Hexane (PMH, 31.0 g), dichloromethane (PMD, 15.0 g), ethyl acetate (PME, 8.0 g) and water layer (PMW): Mass listed is after solvent removal.

The PMD fraction was applied to silica gel CC and eluted with dichloromethane/methanol (20/1, v/v) to obtain three sub-fractions (PMD1-PMD3). Partial fraction PMD2 was further purified using solvent RP-18 CC and methanol/water (2/1, v/v) to give compound MISR-PM3 (40 mg). Partial fraction PMD3 was separated by silica gel CC using solvent dichloromethane/methanol (20/1, v/v), and additionally RP-18 CC and acetone/water (1/2, v/v) were separated to form compound MISR -PM4 (20 mg) and MISR-PM7 (50 mg) were obtained.

Water/methanol containing aqueous solutions with increasing concentrations of methanol (25, 50, 75, and 100%; 1.5 L each) was applied to a Diaion HP-20P column to obtain four fractions (PMW1-PMW4).

The PMW2 fraction was immersed in silica gel CC and eluted with dichloromethane/methanol/water (5/1/0.1, v/v/v) to give two sub-fractions (PMW2a-PMW2b). The PMW2a sub-fraction was further purified using the solvents acetone/water (1/2, v/v) and RP-18 CC to obtain the compound MISR-PM5 (9.0 mg). Finally, the PMW2b sub-fraction was further purified using solvents acetone/water (1/2, v/v) and RP-18 CC to obtain compound MISR-PM6 (12.0 mg). The chemical structures of the five isolated compounds were explained based on NMR data as well as comparison with data reported in the literature.

● IC ₅₀ Determination of IC ₅₀ (inhibitory concentration))

For the inhibition assay, the stock solution (32.8 mM) of MISR-PM5 was serially diluted to various concentrations in the reaction buffer, and an equal volume of the compound for each concentration was dissolved in the reaction buffer. A solution containing miniSOG and TMB was mixed.

To calculate the IC50, normalized absorbance values (ABS _LED -ABS ₀ at 660 nm) were plotted as a function of chemical concentration, followed by 4 -parameter logistic equation (dose-response model for ligand binding) was applied.

Example 5: Measurement of active oxygen scavenging effect of 5 unknown compounds extracted from Physalis minima L. - colorimetric reaction

It was found that ROS scavenger screening was possible through Examples 1 to 4, and the active oxygen scavenging effect of the unknown compound was measured using this.

Five natural compounds (MISR-PM3, MISR-PM4, MISR-PM5, MISR-PM6, MISR-PM7) extracted from Physalis minima L. in a solution containing MiniSOG/FMN and TMB at different concentrations (100 μM, 1 mM) The active oxygen scavenging effect according to the presence or absence of light irradiation was measured. The result of this is shown in FIG. 7 .

7(A) shows a group containing MiniSOG/FMN and TMB (named untreated control), a group containing MiniSOG/FMN, TMB and five compounds (#1, #2, #3, #4, respectively). , #5) was not irradiated with LED, so active oxygen was not generated, so the color did not change at both 100μM and 1mM compound concentrations and maintained a yellow color. When the LED was irradiated, only #3 among the five compounds remained yellow without changing color, and #1, #2, #4, and #5 turned green. This was observed at both 100 μM and 1 mM compound concentrations.

That is, due to the singlet oxygen produced by the activation of the yellow MiniSOG/FMN by light, TMB, which does not have a color, is oxidized and turned blue, finally showing green. At this time, among the compounds #1, #2, #3, #4, and #5, if the compound has an active oxygen scavenging effect, TMB is not oxidized by scavenging the active oxygen generated by the photoactivated MiniSOG/FMN. Because it is not, it is finally shown in yellow.

This means that among the 5 compounds, #1, #2, #4, and #5 did not scavenge the active oxygen generated by the photoactivated MiniSOG/FMN, resulting in an oxidation reaction of TMB, resulting in a color change to green. The yellow color was maintained because the photoactivated MiniSOG/FMN scavenged the active oxygen generated and the TMB did not undergo an oxidation reaction. FIG. 7(B) is a graph obtained by measuring the absorbance of FIG. 7(A) at 660 nm with a microplate reader.

It can be seen from FIG. 7 that #3 among the five unknown compounds extracted from Physalis minima L. has an excellent active oxygen scavenging effect.

Example 6: Measurement of the active oxygen scavenging effect of 5 unknown compounds extracted from Physalis minima L. using MiniSOG/FMN and ADPA - Fluorescence

Through Example 5, it was confirmed that the color of the active oxygen scavenging effect of the five compounds was visually changed, and this was confirmed and compared through a fluorescence meter.

The degree of active oxygen scavenging of five unknown compounds (named #1, #2, #3, #4, and #5, respectively) in a solution containing MiniSOG/FMN and ADPA was measured using a fluorometer. The results are shown in FIG. 8 .

Figure 8 shows a group containing MiniSOG/FMN and ADPA (named untreated control), a group containing MiniSOG/FMN, ADPA and five unknown compounds (#1, #2, #3, #4, #5, respectively). Named) was irradiated with LED light (10 mW cm ^-2 ) for 10 minutes, and the microplate was further incubated at room temperature for each hour (10 minutes, 20 minutes, 30 minutes) without irradiation thereafter. showed

Since the unknown control group contained only MiniSOG/FMN and ADPA, the fluorescence intensity of ADPA was high, and the ADPA fluorescence intensity of #3 among the five unknown compounds was the lowest, that is, it was confirmed that the active oxygen scavenging effect was excellent.

This result was consistent with the result of FIG. 7 measured using MiniSOG/FMN and TMB.

Example 7: Determination of reactive oxygen species scavenging IC50 of unknown compound #3 extracted from Physalis minima L. using MiniSOG/FMN and TMB

It was confirmed that among the five compounds extracted from Physalis minima L. through Examples 5 and 6, compound #3 had the most excellent active oxygen scavenging effect.

Therefore, the active oxygen scavenging IC50 concentration of compound #3 was measured. Compound #3 was serially diluted in a solution containing MiniSOG/FMN and TMB, and each concentration was labeled as 0.5 μM, 1 μM, 1.5 μM, 2.5 μM, 4 μM, 6 μM, 10 μM, 15 μM, 25 μM, 40 μM, 65 μM, and 100 μM, respectively. The active oxygen scavenging effect according to the presence or absence of light irradiation was measured. The results are shown in FIG. 9 .

Figure 9 shows the results of measuring the active oxygen scavenging effect according to the presence or absence of LED light irradiation to MiniSOG / FMN, TMB and # 3 compound by serially diluted concentration.

In (A) of FIG. 9 , yellow color was maintained at all concentrations of compound #3 when LED light was not irradiated. When irradiated with light, it was confirmed that the concentration of #3 compound turned green up to 0.5 μM, 1 μM, 1.5 μM, 2.5 μM, 4 μM, 6 μM, and 10 μM, but turned yellow at subsequent concentrations.

FIG. 9(B) is a graph obtained by measuring the absorbance of FIG. 9(A) using a microplate reader at 660 nm to calculate IC50. The IC50 of chemical #3 was found to be 9.8 μM.

Figure 9(C) shows five unknown compounds extracted from Physalis minima L. analyzed, and the chemical structural formula of #3, which has an active oxygen scavenging effect, is shown.

Figure 10 shows the results of analyzing 5 unknown compounds extracted from Physalis minima L., and is the result of investigating the molecular weight and compound name of each compound.

Among the five compounds screened by the method of the present application, #3, which was found to have an active oxygen scavenging effect, was a substance called Rutin.

Rutin, also called rutoside, is a kind of flavonoid-based flavonol glycoside (glycid). It is a compound belonging to the group of polyphenolic flavonoids found mainly in natural plants such as oranges, lemons, grapes and strawberries. In fact, rutin has shown an ROS scavenging effect, and is a substance that has therapeutic potential for ROS-related diseases (Molecules 19, 19036-19049 (2014);, Oxidative Medicine and Cellular Longevity (2018);, Expert Opinion on Investigational Drugs 22 , 1063-1079 (2013); Food & Function 6, 3296-3306 (2015)).

These results are very consistent with the previous report that rutin acts as a singlet oxygen scavenger and is four times stronger than quercetin or aringin in scavenging singlet oxygen (Free Radical Biology and Medicine 9, 19-21 (1990) ;, Journal of Physical Chemistry B 109, 4234-4240 (2005);, PLoS One 7 (2012)).

Summarizing the results of FIGS. 7 to 10, the results of evaluating the active oxygen scavenging effect of each of the five unknown compounds by the method of the present application were obtained consistent with the reverse analysis of the compound later. That is, it suggests that the method of the present application is very suitable for simple and accurate evaluation and screening of the active oxygen scavenging ability of antioxidant candidates.

In the above, the configuration and characteristics of the present application have been described based on the embodiments according to the present application, but the present application is not limited thereto, and various changes or modifications can be made within the spirit and scope of the present application. It is apparent to those skilled in the art, and therefore such changes or modifications are intended to fall within the scope of the appended claims.

<110> INDUSTRY-UNIVERSITY COOPERATION FOUNDATION HANYANG UNIVERSITY <120> A composition for antioxidant screening using protein based photosensitizer and substrate and method thereof <130> CP19-225 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 32 <212> DNA <213> artificial sequence <220> <223> MiniSOG forward primer <400> 1 cgggatccca tggagaagag cttcgtgatc ac 32 <210> 2 <211> 28 <212> DNA <213> artificial sequence <220> <223> MiniSOG reverse primer <400> 2 cccaagcttc tagccgtcca gctgcacg 28

Claims (13)

A method for screening antioxidants performed in vitro, the method comprising:
(1) Provided:
a) a protein-based photosensitizer;
b) chromogenic substrate;
c) antioxidant candidates; and
d) light;
(2) determining whether the antioxidant candidate material is an antioxidant material by checking the color change of the color substrate;
including,
At this time, the chromogenic substrate is 5-ASA (5-aminosalicylic acid), 4-CN (4-chloro-1-naphthol), DAB (3,3′-diaminobenzidine), DAD (o-Dianisidine dihydrochloride), OPD ( At least one selected from o-Phenylenediamine dihydrochloride), TMB (3,3′′,5,5′′-Tetramethylbenzidine), dopamine, serotonin and derivatives and variants thereof,
At this time, the color change is visually confirmed without using a measuring device,
At this time, the color change of the chromogenic substrate is characterized in that the antioxidant candidate material is caused by removing the active oxygen generated by the protein-based photosensitizer.
According to claim 1,
The above (1) provides;
a) a protein-based photosensitizer; b) a color-developing substrate; c) an antioxidant candidate; d) light; A method characterized in that any one or more selected from are provided independently or / and provided in combination.
According to claim 1,
The above (1) provides;
making a mixture of a) a protein-based photosensitizer; b) a color-developing substrate; and c) an antioxidant candidate; d) light is provided to the mixture.
According to claim 1,
The above (1) provides;
making a mixture of a) a protein-based photosensitizer; and c) an antioxidant candidate; d) providing light to the mixture; b) mixing a chromogenic substrate;
According to claim 1,
The protein-based photosensitizer is a method, characterized in that any one or more selected from KillerRed, MiniSOG, SOPP, FPFB, SuperNova, mKate2, KillerOrange.
delete According to claim 1,
The antioxidant is a ROS scavenger that removes active oxygen.
A method characterized by being.
According to claim 1,
The active oxygen is a superoxide (O ₂ ^- , superoxide), hydroxyl radical (HO ), singlet oxygen ( ¹ O ₂ ), hydrogen peroxide (H ₂ O ₂ ), hypochlorous acid (HOCl), nitric oxide (Nitric oxide, NO), peroxyl radical (ROO·), and peroxynitrite (Peroxynitrite, ONOO-).
According to claim 1,
Thiamine pyrophosphate (TPP or ThDP), flavin mononucleotide (FMN), flavin adenine dinucleotide (flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD+ or NADH), nicotinamide adenine dinucleotide phosphate (NADP+ or NADPH), coenzyme A (CoA), pyridoxal phosphate ( Pyridoxal phosphate (PLP or P5P), biotin (Biotin or B7), tetrahydro folic acid (THFA or H4FA), dihydrofolic acid (DHFA or H2FA), 5,10-methylenetetrahydrofolate ( methylentetrahydrofolate (MTHF), adenosylcobalamin (AdoCbl), methylcobalamin (MeCbl or B12), ascorbic acid, phylloquinone (K1), menaquinone (K2), coenzyme F420 ( Coenzyme F420, 8-hydroxy-5-deazaflavin) characterized in that it can optionally further include any one or more selected from.
According to claim 9,
The protein-based photosensitizer and the compound cofactor are 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1: 10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 2:1, 2:3, 2: 5, 2: 7, 2: 9, 2: 11, 3: 1, 3: 2, characterized in that it has a molar ratio (molar ratio) of any one selected from 4.
As a kit for screening antioxidants performed in vitro,
The kit,
A protein-based photosensitizer selected from KillerRed, MiniSOG, SOPP, FPFB, SuperNova, mKate2, and KillerOrange; and
5-ASA(5-aminosalicylic acid), 4-CN(4-chloro-1-naphthol), DAB(3,3′-diaminobenzidine), DAD(o-Dianisidine dihydrochloride), OPD(o-Phenylenediamine dihydrochloride), TMB (3,3′′,5,5′′-Tetramethylbenzidine), dopamine, serotonin and its derivatives (derivative), any one or more selected from variants of the chromogenic substrate;
including,
The kit requires light provided from an external light source,
At this time, the protein-based photosensitizer is activated by light to generate active oxygen,
Depending on whether the active oxygen is removed by the antioxidant candidate for antioxidant screening, color change occurs due to oxidation of the chromogenic substrate,
The kit is characterized in that the active oxygen scavenging ability of the antioxidant candidate material can be confirmed by the color change of the chromogenic substrate.
According to claim 11,
The kit contains thiamine pyrophosphate (TPP or ThDP), flavin mononucleotide (FMN), and flavin adenine dinucleotide ( flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD+ or NADH), nicotinamide adenine dinucleotide phosphate (NADP+ or NADPH), coenzyme A (CoA), pyridoxal Phosphoric acid (Pyridoxal phosphate; PLP or P5P), biotin (Biotin or B7), tetrahydro folic acid (THFA or H4FA), dihydrofolic acid (DHFA or H2FA), 5,10-methylenetetrahydrofol methylentetrahydrofolate (MTHF), adenosylcobalamin (AdoCbl), methylcobalamin (MeCbl or B12), ascorbic acid, phylloquinone (K1), menaquinone (K2), coenzyme F420 (Coenzyme F420, 8-hydroxy-5-deazaflavin) kit characterized in that it can optionally further include any one or more selected from. delete

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