KR101696684B1 - Methods for screening inhibitor of reactive carbonyl species - Google Patents

Abstract

(A) reacting ubiquitin, an activated carbonyl group, and an activated carbonyl group inhibitor candidate; (b) performing mass chromatography on the reactant to measure the mass / charge (m / z) of the modified ubiquitin bound to the activated carbonyl group and ubiquitin; And (c) the mass / charge (m / z) of the modified ubiquitin in step (b) and the mass / charge (m / z) of the modified ubiquitin of the ubiquitin and activated carbonyl group reactants, which are controls under the absence of the active carbonyl group inhibitor candidate, z) to determine that the candidate substance is an active carbonyl group inhibitor if the mass / charge (m / z) of the modified ubiquitin of the reactant in the presence of the inhibitor candidate is decreased .
According to the present invention, an active carbonyl group inhibitor can be screened to prevent, ameliorate or treat diseases related to oxidative stress, or to use in foods, cosmetics and medicines.

Description

Methods for Screening Activated Carbonyl Inhibitors < RTI ID = 0.0 >

The present invention relates to a method for screening for an active carbonyl group inhibitor.

Active species play an important role in immune responses that control cell shrinkage and cell defense against a variety of environmental changes. Reactive species (RS) are constantly produced and removed from various organisms, ranging from bacteria to humans. Under normal physiological conditions, the concentration of the active species in the steady state is maintained within a certain range, Parameter. Active oxygen species (ROS) and reactive carbonyl species (RCS) are involved in active species, and active carbonyl groups are known to be formed downstream of reactive oxygen species.

These active oxygen species and activated carbonyl groups are increased by carbonyl stress or oxidative stress and are known to exhibit various damaging effects. In particular, it destroys the structure and function of proteins, nucleic acids, lipids, and carbohydrates, causing undesirable effects and loss of function at cell and organism levels, and even affecting survival.

Recent studies have shown that reactive oxygen species are increased by carbonyl stress and that active carbonyl groups formed downstream of reactive oxygen species are increased by oxidative stress. Among these, active carbonyl groups which are increased by oxidative stress are known to be involved in various chronic diseases and degenerative diseases. Among them, metabolic diseases, diabetes, obesity, kidney disease, heart disease, arteriosclerosis and neurodegenerative diseases are involved .

The pathogenesis of this pathogenesis of oxidative stress is oxidative stress, which causes oxidation of sugars or unsaturated fatty acids to produce reactive carbonyl species (RCS). These activated carbonyl groups are electrophilic, The protein or DNA easily binds to the protein, DNA damage caused by the degradation of the physiological function and organ damage caused by a variety of diseases are involved. In addition, it is known that the function of the protein or DNA is impaired by the function of the cell, resulting in apoptosis and necrosis, thereby causing cell death or disease progression (Fig. 1).

Antioxidation refers to the prevention of various oxidative effects in the body. The lipid present as a biological membrane or lipoprotein is attacked by free radicals generated in the body to form various kinds of peroxides. Peroxides and Decomposition products and the like are highly reactive, thereby converting the structure and function of the surrounding biomolecules, resulting in aging and various chronic diseases. There are many antioxidant defense mechanisms in the body that can neutralize these free radicals and protect the body. O2 generated in human disease, and aging the body metabolic processes ^{- (superoxide), NO (nitric} oxide), NO2 (nitrogen dioxide), OH (hydroxyl), ROO (proxyl), RO (alkoxyl), HO2 (hydroperoxyl) radical and superoxide dismutase (SOD), which is an antioxidant enzyme as a means for protecting the living body from free radicals such as radicals, is a superoxide radical (superoxide radical) Is converted to H ₂ O ₂ and the produced H ₂ O ₂ and the like are reacted with antioxidant enzymes such as catalase (CAT) and glutathione peroxidase (GSH-px), vitamin E and glutathione The presence of antioxidants protects the body from oxidative stress by eliminating free radicals (Ishinaga M, et al., Daily intake of fatty acids, sterols, and phospholipids by Japanese women and serum cholesterol, J. Nutr. Sci. 40, pp. 557-567, 1994).

However, when such an antioxidant defense system declines to such an extent that free radical formation can not be controlled, the oxidative stress and damage of the tissue are promoted, and the excess free radicals and lipid peroxides produced in the body are oxidized by oxidative stress such as protein oxidation and DNA damage .

Therefore, many synthetic or natural antioxidants have been developed to reduce such oxidative stress, but synthetic antioxidants have been found to be poor in safety. In recent years, the use of antioxidants such as carotenoids, flavonoids, and polyphenols in various herbal medicines and edible plant extracts, as well as vitamins A, C and E, We are working hard.

Accordingly, the present inventors have found that a substance capable of inhibiting oxidative stress and inhibiting the formation of an activated carbonyl group inhibits the production of an activated carbonyl group, and as a result, And thus the present invention has been completed.

It is an object of the present invention to provide a method for screening an active carbonyl group inhibitor.

In one aspect to accomplish the above object, the present invention provides a process for the production of a pharmaceutical composition comprising: (a) reacting ubiquitin, an activated carbonyl group, and an activated carbonyl group inhibitor candidate; (b) performing mass chromatography on the reactant to measure the mass / charge (m / z) of the modified ubiquitin bound to the activated carbonyl group and ubiquitin; And (c) the mass / charge (m / z) of the modified ubiquitin in step (b) and the mass / charge (m / z) of the modified ubiquitin of the ubiquitin and activated carbonyl group reactants, which are controls under the absence of the active carbonyl group inhibitor candidate, z) to determine that the candidate substance is an active carbonyl group inhibitor if the mass / charge (m / z) of the modified ubiquitin of the reactant in the presence of the inhibitor candidate is decreased . ≪ / RTI >

(A) of the present invention relates to the step of reacting ubiquitin, an activated carbonyl group, and an activated carbonyl group inhibitor candidate.

In the present invention, the ubiquitin is universally present in cells, and the ubiquitin protein is composed of 76 amino acids which are very well preserved. It is present in all eukaryotes, and is involved in regulation of cell cycle, regulation of transcription, It is known to be involved in many intracellular reactions such as pathways, presentation of antigens, and abnormal protein degradation. The mass / charge of the ubiquitin in step (a) is 773 m / z to 782 m / z.

In the present invention, the activated carbonyl group is increased by oxidative stress. The activated carbonyl group oxidizes and destroys cells in the body, thereby exposing the human body to various diseases.

The active carbonyl group inhibitor candidate substance means a substance capable of inhibiting the activity of an activated carbonyl group.

The active carbonyl group may be 4-hydroxy-2-nonenal (HNE), acrolein (ACR), malondialdehyde (MDA), glyoxal (GO), methylglyoxal (MGO) or 4-oxo-nonenal But not limited to, α, β-unsaturated aldehydes, di-aldehydes, and keto-aldehydes, which are capable of binding to proteins and cause toxicity, and advanced glycoxidation end products and lipoxidation end products Lt; RTI ID = 0.0 > ALEs). ≪ / RTI > The α, β-unsaturated aldehydes bind histidine, cysteine and lysine of proteins to form advanced glycoxidation end products (AGEs) and lipoxidation end products (ALEs), which are toxic protein conjugates. Representative α, β-unsaturated aldehydes include 4-hydroxy-2-nonenal (HNE) and acrolein (ACR).

The step (a) of the present invention is a step of reacting a ubiquitin, an activated carbonyl group, and an activated carbonyl group inhibitor candidate substance, wherein the mass / charge amount of ubiquitin is reacted with the mass carbonyl group Lt; RTI ID = 0.0 > m / z < / RTI >

The step (b) of the present invention comprises performing mass chromatography on the reactant to measure the mass / charge (m / z) of the modified ubiquitin bound to the activated carbonyl group and the ubiquitin.

In the step (b) of the present invention, mass spectrometry of the reactant of step (a) is performed to determine the mass / charge (m / z) of the modified ubiquitin by measuring the binding of the activated carbonyl group and ubiquitin.

In one embodiment of the present invention, HNE, an active carbonyl group, and ubiquitin, a widely distributed protein in the human body, were selected and reacted with HNE. As a result, it was confirmed by HPLC-Ms / Ms analysis that HNE binds to ubiquitin and denatured in 15 peptides of ubiquitin to form HNE michael adduct (FIG. 2). The precursor ion of ubiquitin before binding to HNE was 779 m / z and the precursor ion of the ubiquitin modified form of HNE michael adduct was 793 m / z (Example 4). At this time, the protein molecular weight increase by HNE was found to be 156 Da.

In addition, it was confirmed that the amount of ubiquitin produced as a result of the concentration of HNE increases in proportion to the concentration of HNE (FIG. 3). It was also confirmed that the content of the denatured ubiquitin increases with the reaction time (FIG. 4).

After reacting various activated carbonyl groups with ubiquitin, the resulting modified ubiquitin was analyzed by HPLC-Ms / Ms. In the case of 4-hydroxy-2-nonenal (HNE), ubiquitin was identified at 793 m / z. Glyoxal (GO) and ubiquitin were identified at 784 m / z and 790 m / z. Methylglyoxal 784, 786, 791, and 792 m / z, respectively. The reaction with malondialdehyde (MDA) confirmed that the modified ubiquitin was identified at 784 and 789 m / z (FIG. 5) .

In the present invention, the mass / charge amount is measured by performing mass chromatography and can be analyzed by a mass spectrometer of ESI / MS / MS, LC-MS / MS or MALDI-TOF, MS mass spectrometer, but are not limited thereto.

The step (c) of the present invention is characterized in that the mass / charge (m / z) of the modified ubiquitin of step (b) and the mass / volume of the modified ubiquitin of the ubiquitin and the activated carbonyl group reactant, which are control groups in the absence of the active carbonyl group- Comparing the charge amount (m / z) and determining that the candidate substance is an active carbonyl group inhibitor when the mass / charge (m / z) of the modified ubiquitin of the reactant in the presence of the inhibitor candidate substance decreases.

The step (c) of the present invention may further comprise the step of mixing the mass / charge (m / z) of the modified ubiquitin measured in step (b) with the mass / mass of the modified ubiquitin of the ubiquitin and the activated carbonyl group reactant under the absence of the active carbonyl group- When the mass / charge (m / z) of the reacted ubiquitin in the presence of the inhibitor candidate material is decreased by comparing the charge amount (m / z), the candidate substance is judged to be an activated carbonyl group inhibitor.

Under the absence of the active carbonyl group inhibitor candidate of the present invention, the mass / charge (m / z) of the ubiquitin-modified ubiquitin in the reactants of ubiquitin and activated carbonyl groups was measured by reacting the ubiquitin protein with 4-Hydroxynonenal (HNE) , The content of modified ubiquitin is increased by binding of ubiquitin to an activated carbonyl group, and the mass / charge amount of modified ubiquitin is 783 m / z to 795 m / z.

However, the presence of an activated carbonyl group inhibitor inhibits the production of ubiquitin and activated carbonyl group reactants, thereby reducing the content of modified ubiquitin and the mass / charge (m / z) of most ubiquitin from 773 m / z to 782 m / z range.

In one embodiment of the present invention, it was confirmed that the water extract of snowballs (bovine goby goby) extract, preferably the goby goby, was a candidate substance capable of inhibiting the activated carbonyl group. Specifically, in the case of the reaction with ubiquitin and HNE, the degenerated HNE michael adduct, which is a combination of ubiquitin and HNE, decreased in proportion to the concentration of the eye shadow extract, effectively inhibiting the activated carbonyl group . Also, it was confirmed that the mass / charge (m / z) of the modified ubiquitin bound to the activated carbonyl group and ubiquitin decreased from 793 m / z to 779 m / z (Fig. 8).

In the present invention, the term " macrography giant embryo "is rice having a black waxy giant embryo. The giant embryo of the present invention is characterized in that it is a giant embryo which is black and glutinous rice. For example, it is a black giant giant embryo developed by artificial interbreeding of an old black rice and a giant bacilli (YR23517Acp79) And Mokyang 263). Herein, it means that the amylose is composed of 2 to 10% of amylose and 90 to 98% of amylopectin. The giant embryo is a variant of mutant breeding which is 3 to 5 times larger than that of the conventional rice varieties, Rice cultivated more than 80% of rice is 3 ~ 5 times bigger than general brown rice, so rice is rich in nutrients.

In the present invention, the giant-bony giant embryo may be, but is not limited to, a black-and-white graphite (Milyang 263). The above-mentioned giant-ray giant embryonic rice mammalian 263 and the breeding method thereof are disclosed in Korean Patent No. 10-1201436 and deposited with KACC98009P on Jun. 30, 2010 at the National Agricultural Research Center of Agricultural Gene Center.

In the present invention, the "extract of macrophage macrobiotech" can be collectively referred to as a concentrate obtained by squeezing each of the macroglobulin giant embryos with a suitable leaching solution and evaporating the leaching solution. Preferably, the extract of the macrophage macrophage can be obtained by extracting the brown rice of the macrophage macrophage.

The brown rice refers to rice in which harvested rice is dried and thawed, and rice husked off by a machine made of a rubber roller. In the present invention, the brown rice refers to rice which has been dried and thawed and then the rice husk has been removed, but the present invention is not limited thereto.

The extract may be obtained by an extraction process using water, an organic solvent, or a mixed solvent thereof, and may be a dry powder of an extract, a diluted or concentrated liquid thereof, or an extract, It can include all forms. In addition, in the extraction method, methods such as hot water extraction, hot water extraction, cold extraction, hot extraction, pressure extraction, reflux cooling extraction, ultrasonic extraction, or supercritical extraction may be used.

In order to obtain the above extract, it is preferably extracted with water, an organic solvent or a mixed solvent thereof. In the case of extraction using an organic solvent, a water-soluble solvent containing C1 to C4 alcohols such as methanol, ethanol, isopropanol, and butanol; Or a fat-soluble solvent including benzene, toluene, acetone, carbon tetrachloride, hexane, dichloromethane, chloroform, ether, methylene, ethyl acetate and the like. In addition, the active ingredient such as protein, free sugar, amino acid, and GABA can be extracted by room temperature or warming under conditions that are not destroyed or minimized. The amount of the extraction solvent may be 1 to 50 times as much as the weight of the micro-goby goby or the pulverized product thereof, but is not limited thereto.

In the present invention, the water-soluble extract is an extract obtained by extracting with a water-soluble solvent such as C1 to C4 alcohols such as water, methanol, ethanol, isopropanol, butanol and the like and a mixed solvent thereof, and the oil-soluble extract is selected from benzene, toluene, acetone, And extracted with a lipid soluble solvent such as hexane, dichloromethane, chloroform, ether, methylene, ethyl acetate and the like.

The extract may further comprise a step of filtering the extract to remove suspended solid particles. Nylon, etc., or ultrafiltration, freezing filtration, centrifugation, etc. may be used.

Concentration of the extract can be carried out by reduced-pressure concentration, reverse osmosis concentration, or the like. The concentration and drying step includes freeze drying, vacuum drying, hot air drying, spray drying, vacuum drying, foam drying, high frequency drying, or infrared drying. In some cases, it may further comprise a step of pulverizing the finally dried extract. In addition, the extract may be subjected to an additional fractionation process.

In Example 1 of the present invention, the free amino acid content was higher than that of the Japanese rice (Example 1), and the cytotoxicity was not observed in both the water-soluble extract and the oil-soluble extract of the black rice blotch (Example 3). In addition, when ubiquitin and HNE were reacted with ubiquitin and HNE, the production of substances combined with ubiquitin and HNE decreased as the concentration of the eye shadow extract increased, thus effectively inhibiting the activated carbonyl group (Example 5). Thus, the antioxidative effect of the present invention by inhibiting the production of an activated carbonyl group, which is known to be increased by oxidative stress, can be achieved by the removal of the active carbonyl group.

The term "a disease caused by oxidative stress" as used herein refers to a disease caused by an oxidative stress caused by the reaction of oxygen with a nutrient, The diseases caused by oxidative stress induced by activated carbonyls or free radicals (free radicals) include lipid metabolic diseases such as hyperlipidemia, arteriosclerosis, heart failure, hypertensive heart disease, arrhythmia, myocardial infarction, Cardiovascular diseases such as angina pectoris, aging, immune diseases and cancer, but the present invention is not limited thereto.

In the present invention, the active carbonyl group inhibitor may be a food composition for antioxidation, and the term "antioxidant" means an oxidation inhibiting action. The human body may contain a prooxidant and an antioxidant However, due to various factors, such an equilibrium state becomes imbalanced, and when it is inclined toward the promotion of oxidation, oxidative stress is induced, leading to potential cell damage and pathological disease. The reactive oxygen species (ROS), which is known to be a direct cause of oxidative stress, is unstable and highly reactive. It reacts easily with various bio-materials, attacking the body's polymers, causing irreversible damage to cells and tissues, Cytotoxicity and carcinogenesis. The active oxygen species is increased by carbonyl stress and the active carbonyl group formed downstream of the active oxygen species is increased by oxidative stress. Such active carbonyl groups oxidize and destroy cells in the body, thereby exposing them to various diseases. Therefore, the antioxidant effect can be attained by incorporating the giant-bloom giant embryo of the present invention into the food, thereby contributing to health promotion.

In addition, the active carbonyl group inhibitor may be an antioxidant cosmetic composition. Since the extract of the present invention has an antioxidative effect, it may be added to a cosmetic composition for the purpose of antioxidation.

According to the present invention, an active carbonyl group inhibitor can be screened to prevent, ameliorate or treat diseases related to oxidative stress, or to use in foods, cosmetics and medicines.

Figure 1 shows the production of activated carbonyl groups by oxidative stress and the mechanism of cell damage.
FIG. 2 shows that an activated carbonyl group (HNE) is combined with ubiquitin to form HNE michael adduct.
Figure 3 shows the amount of modified ubiquitin produced according to the concentration of HNE.
FIG. 4 shows the amount of modified ubiquitin produced according to the reaction time.
Figure 5 shows the HPLC-Ms / Ms analysis of the modified ubiquitin produced when various activated carbonyl groups were reacted with ubiquitin.
FIG. 6 is a schematic view of an experimental method of removing the active carbonyl group of the eye shadow extract.
FIG. 7 shows the result of inhibition of the activated carbonyl group when 100% of the extract from the common product was mixed with ubiquitin and HNE.
FIG. 8 shows the result of inhibition of the activated carbonyl group by mixing the ubiquitin and HNE in a water-blotted water-extract of the eyes.

Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are illustrative of the present invention, and the scope of the present invention is not limited by the following examples.

Example  One: Snowball ( Giant biomass , Milyang 263 ) And Japan

Example  1-1: Snowball  Analysis of major characteristics of Japan and Japan

The characteristics of the eyeballs of the present invention were analyzed and the results are shown in Table 1 below.

A blind eye  Brown rice characteristics division Brown rice heaven
(g) Weight
(g / 1000 lips) Filling weight
(g / 1000 lips) Percentage of Brown Rice (%) Main characteristics Snowball 19.4 1.464 17.96 7.5 Black Chagger Japan 22.8 0.616 22.18 2.7 General Mamba

As a result, the grains of the black chrysanthemum rhizome were larger than those of the ordinary rice, and the weight was also significant (Table 1).

Example  1-2: Snowball  And nutrient analysis of Japanese and American

The contents of anthocyanin, amylopectin, protein and inorganic components were analyzed in order to compare the nutritional components of the brown rice of the present invention.

Kinds Anthocyanin
(mg / 100g) Amylopectin
(%) protein
(%) Inorganic component (mg / kg) Ca Mg Fe Snowball 129.4 94.5 8.98 338.1 1366.0 45.7 Japan - 83.5 7.45 164.0 875.0 30.3

As a result, it was found that the brown rice powder contains a large amount of anthocyanin which is not contained in the Japanese rice. In addition, the amylopectin and protein content were also higher than that of Japan. Inorganic components such as calcium, magnesium, and iron also contained a considerable amount as compared with Japan (Table 2).

Example  1-3: Snowball  And free sugar content analysis of Japan

In order to compare the content of brown rice in the eyes of the present invention, a sugar analysis of brown rice was carried out, and the results are shown in Table 3 below.

Glucose
(mg / 100g) Sucrose
(mg / 100g) Raffinose
(mg / 100g) Fructose
(mg / 100g) Total sugar content
(%) Snowball 2147.7 799.8 0.0 31.7 3.00.2 Japan 2167.1 602.2 72.4 51.9 2.90.3

As a result, the content of glucose was not significantly different from that of black eyes, but the black eyes contained a large amount of sucrose and did not contain raffinose. The overall total free sugar content of the eyeshadows was about 3.0%, which is about 0.1% more than that of Japan.

Example  1-4: Snowball  And amino acid composition analysis

Table 4 shows the amino acid composition of the brown rice of the black-and-white photoperiod, which was analyzed by comparing with the general amino acids.

Snowball  Amino acid composition of brown rice glass mg / g) Name
(mg / g) Snowball
(Macrophyte giant embryo) Japan
(General) His 0.0605 0.0218 Ser 0.0951 0.0379 Arg 0.2220 0.0371 Gly 0.0628 0.0154 Asp 0.3023 0.0777 Glu 0.2503 0.1604 Thr 0.0476 0.0062 Ala 0.1188 0.0408 Pro 0.0798 0.0208 Cys 0.0000 0.0013 Lys 0.1145 0.0030 Tyr 0.0596 0.0073 Met 0.0273 0.0011 Val 0.0567 0.0039 Ile 0.0307 0.0056 Leu 0.0644 0.0024 Phe 0.0368 0.0042 Trp 0.0366 0.0255 GABA 0.247 0.033 Total amino acid 1.912 1.086

As a result, the total amino acid content was found to be abundantly contained in the brown rice of the eyeballs, and the content of all amino acids except Cys was found to be contained in the brown rice of the eyes.

Example  2: Snowball  Yield analysis of brown rice extract

In order to obtain water-soluble and liposoluble extracts of brown rice, the extracts of black rice and brown rice isolated in Example 1 were respectively extracted with 80% methanol (water-soluble extract) and hexane (oil-soluble extract) The results are shown in Table 5 below.

Snowball  The yield of extracts according to the solvent of brown rice kind Water-soluble extract
(100% water, g / 100 g) Water-soluble extract
(80% methanol, g / 100 g) Liposoluble extract
(Hexane, g / 100 g) Snowball 1.30 2.95 2.47 Japan 1.30 0.86 0.49

As a result, the extracts of 100% water showed 1.3 g, and the yield of 80% methanol aqueous extract was higher than that of Japan, and the yield of the extract was higher than that of Japan. The yield of the extract was significantly higher than that of Japan.

Example  3: Snowball  Evaluation of cytotoxicity of extracts

To evaluate the toxicity of the water-soluble extracts and liposoluble extracts of the present invention, the cytotoxicity of 200 ppm extracts was evaluated using adipocytes (3T3-L1 cells).

Snowball  Evaluation of cytotoxicity of brown rice extract Varieties and samples Water-soluble extract
[80% methanol, cell viability (% of control)] Liposoluble extract
[Hexane, cell viability (% of control)] contrast
(No treatment) 100.0 8.7 100.0 8.7 Snowball 106.7 7.4 107.5 8.7 Japan 103.4 6.1 100.4 3.1

As a result, as shown in Table 6, the cytotoxicity was not observed in both the water-soluble extract and the liposoluble extract of the eyeshadows, and the extract of 200 ppm was found to be effective concentration.

Example  4: active Carbonyl group  Inhibitory substance assay method

Α, β-unsaturated aldehydes, one of the active carbonyl groups, bind to histidine, cysteine and lysine of proteins and form lipidation end products (ALEs) with advanced glycoxidation end products (AGEs). Representative α, β-unsaturated aldehydes include 4-hydroxy-2-nonenal (HNE) and acrolein (ACR). To test the production of toxic proteins by HNE, ubiquitin, a widely distributed protein in the human body, was selected and reacted with HNE.

As a result, it was confirmed by HPLC-Ms / Ms analysis that HNE binds to ubiquitin and denatured in 15 peptides of ubiquitin to form HNE michael adduct (FIG. 2). The precursor ion of ubiquitin before binding to HNE was 779 m / z, and the precursor ion of the ubiquitin modified form of HNE michael adduct was 793 m / z. At this time, the protein molecular weight increase by HNE was found to be 156 Da.

In addition, it was confirmed that the amount of ubiquitin produced as a result of the concentration of HNE increases in proportion to the concentration of HNE (FIG. 3). It was also confirmed that the content of the denatured ubiquitin increases with the reaction time (FIG. 4).

After reacting various activated carbonyl groups with ubiquitin, the resulting modified ubiquitin was analyzed by HPLC-Ms / Ms. In case of reacting ubiquitin with 4-hydroxy-2-nonenal (HNE), it was identified at 793 m / z. When Glyoxal (GO) was reacted with ubiquitin, it was identified at 784 m / z and 790 m / z. Methylglyoxal 784, 786, 791, and 792 m / z, respectively. The reaction with malondialdehyde (MDA) confirmed that the modified ubiquitin was identified at 784 and 789 m / z (FIG. 5) .

Example  5: The giant embryo  Active activity Carbonyl group  Inhibitory substance assay method

In order to investigate whether the test substance, snowballs, can remove the toxic active carbonyl group in the body, the reaction product of the ubiquitin protein used in Example 4 with the toxic active carbonyl group 4-Hydroxynonenal (HNE) And the content thereof was measured.

Afterwards, it was confirmed whether the ubiquitin protein and the toxic active carbonyl group 4-Hydroxynonenal (HNE) were able to inhibit the production of the reaction product.

For this, as shown in the schematic diagram of the experimental method of removing the activated carbonyl group shown in FIG. 6, ubiquitin and HNE were mixed with 100% water extract of snow flake and reacted with high-resolution LC-MS / MS to determine the content of ubiquitin The content of ubiquitin-HNE binding substance (HNE michael adduct), which is a modified ubiquitin, was assayed.

As a result, it was found that when the ubiquitin and HNE were mixed with 100% extract of general food, they could not inhibit the binding of ubiquitin with HNE, which is an active carbonyl group, regardless of the concentration of general extract. It was confirmed that the precursor ion was identified at the same content at 793 m / z as in Example 4 (Fig. 7). The results showed that the extracts of the common mushroom were not effective in inhibiting the activity of the activated carbonyl group.

On the other hand, in the case of the reaction with ubiquitin and HNE, it was found that the degenerated HNE michael adduct formed with ubiquitin and HNE decreased in proportion to the treatment concentration of eyeshadow extract, effectively inhibiting the activated carbonyl group. In addition, the inhibition rate of 15.4% at 10 mg / ml of the control group was 36.2% at 25 mg / ml and 58.4% at 50 mg / ml, and the inhibition rate of the activated carbonyl group (Fig. 8).

HNE, which is an activated carbonyl group, is known to bind to a protein or a peptide. Through this Example 5, it can be seen that a specific protein or a specific peptide contained in a black spot extract is combined with an activated carbonyl group to inhibit the activity of an activated carbonyl group Can be predicted.

Claims (9)

(a) reacting ubiquitin, an activated carbonyl group, and an activated carbonyl group inhibitor candidate;
(b) performing mass chromatography on the reactant to measure the mass / charge (m / z) of the modified ubiquitin bound to the activated carbonyl group and ubiquitin; And
(c) the mass / charge (m / z) of the modified ubiquitin in the step (b) and the mass / charge (m / z) of the modified ubiquitin in the control group of ubiquitin and the activated carbonyl group reactant under the absence of the active carbonyl group- ), Determining that the candidate substance is an active carbonyl group inhibitor when the mass / charge (m / z) of the modified ubiquitin in the reaction product in the presence of the inhibitor candidate substance decreases,
Wherein the mass / charge amount of the modified ubiquitin of the ubiquitin and the activated carbonyl group reactant under the absence of the inhibitor candidate substance is 783 m / z to 795 m / z, and the reduced mass / weight ratio of the modified ubiquitin of the reactant in the presence of the inhibitor candidate substance, Wherein the charge amount is from 773 m / z to 782 m / z.
2. The method of claim 1, wherein the active carbonyl group inhibitor is a therapeutic agent for a disease caused by oxidative stress.
2. The method of claim 1, wherein the active carbonyl group inhibitor is a food composition for antioxidant.
2. The method of claim 1, wherein the active carbonyl group inhibitor is an antioxidant cosmetic composition.
2. The method of claim 1, wherein the inhibitor is a water extract of a macrophage giant embryo.
The method of claim 1, wherein the mass / charge amount is analyzed by a mass spectrometer of ESI / MS / MS, LC-MS / MS or MALDI-TOF.
delete delete The method according to claim 1,
Wherein the activated carbonyl group is at least one selected from the group consisting of 4-hydroxy-2-nonenal (HNE), glyoxal (GO), methylglyoxal (MGO) and malondialdehyde (MDA).

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