KR102357449B1 - Composition for preventing or protecting liver damage comprising dieckol against methylglyoxal - Google Patents

Abstract

The present invention relates to a composition for a screening method, a liver damage prevention, or a liver protection of a substance that prevents liver damage or protects the liver, wherein by allowing the diecol to combine with the methylglyoxal to form a methylglyoxal adduct, the present invention has a scavenging activity of methylglyoxal, and can protect the liver by preventing liver damage caused by the methylglyoxal. The screening method comprises: a processing step; a measuring step; and a comparing step.

Description

Composition for preventing or protecting liver damage against methylglyoxal, including dieckol, comprising dieckol against methylglyoxal

The present invention relates to a method for screening a substance for preventing or protecting the liver, and a composition for preventing or protecting the liver.

Methylglyoxal is a viscous yellow liquid with a pungent odor. Methylglyoxal is known to have a strong antibacterial action, and consumption of Manuka honey has been increased because it is contained in Manuka honey. However, recently, it has been reported that rats chronically exposed to methylglyoxal gain abnormal weight and develop early insulin resistance and type 2 diabetes. Recently, there has been a report that the cause of diabetes is caused by methylglyoxal, not by the previously known hyperglycemia. Type 2 diabetes that occurs in middle age or old age causes a number of complications such as increased risk of heart disease and stroke, problems with blood flow to the lower extremities, and damage to eyes, nerves, and kidneys. It has been reported that methylglyoxal is elevated in type 2 diabetes.

On the other hand, the liver is the organ in which metabolism in the body occurs most actively among human body organs. . Therefore, the liver has a higher risk of being exposed to many toxic substances in addition to nutrients than other organs, so the possibility of damage is very high. On the other hand, the liver is an organ with excellent regenerative ability, and if there is a slight damage, it recovers sufficiently to normal, but if the damage continues, a part of the liver tissue is completely destroyed and the liver function is reduced. becomes As a result of liver damage, it leads to various diseases such as hepatitis, fatty liver, liver cancer, and cirrhosis.

Recently, it has been reported that methylglyoxal causes damage to mitochondrial function and toxicity of liver cells.

Accordingly, it is necessary to develop a substance that prevents liver damage induced by methylglyoxal and protects the liver.

Republic of Korea Patent Publication No. 10-2014-0088969

An object of the present invention is to provide a screening method for a substance that prevents liver damage or protects the liver.

Another object of the present invention is to provide a composition for preventing liver damage from methylglyoxal or for protecting the liver, comprising the material screened by the screening method as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating liver damage caused by methylglyoxal comprising diecol as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or improving liver damage from methylglyoxal comprising diecol as an active ingredient.

Another object of the present invention is to provide a method for reducing damage to hepatocytes induced by methylglyoxal.

In order to achieve the above object, the present invention provides a screening method for a substance that prevents liver damage or protects the liver in one aspect.

The screening method of the present invention comprises the steps of treating a candidate substance and methylglyoxal in an experimental group; measuring the level of methylglyoxal, a candidate substance or a methylglyoxal adduct in the experimental group treated with the candidate substance; and comparing the level of the untreated control 1, the control 2 treated with methylglyoxal, or the control 3 treated with a candidate substance.

As used herein, the term "candidate material" refers to an isolated organ, tissue, cell, or non-human animal that combines with methylglyoxal to form a methylglyoxal adduct to reduce (clear) methylglyoxal. presumed substance.

The candidate substances include extracts of natural products, or fractions thereof, synthetic compounds, natural compounds, low molecular weight compounds, high molecular weight compounds, nucleic acid molecules (eg, DNA, RNA, PNA and aptamers), proteins, sugars and lipids, etc. , but is not limited thereto.

As used herein, the term "test group" refers to an isolated organ, tissue, cell, or animal other than humans treated with a candidate substance and methylglyoxal. The candidate material and methylglyoxal may be treated simultaneously, or the candidate material may be pretreated and then post-treated with methylglyoxal, or the candidate material may be post-treated after pretreatment of methylglyoxal.

As used herein, the term "control" refers to an isolated organ, tissue, cell or animal other than a human that has not been treated with a candidate substance, methylglyoxal, or a candidate substance and methylglyoxal. An isolated organ, tissue, cell or non-human animal belonging to a parallel relationship. Specifically, control 1 not treated with both the candidate substance and methylglyoxal, control 2 treated with methylglyoxal without the candidate substance, or control 3 treated with the candidate substance without treatment with methylglyoxal have.

The isolated organ, tissue, or cell may be isolated from the liver.

"Methylglyoxal" (methylglyoxal, MGO) used in the present invention has a chemical formula of C ₃ H ₄ O ₂ , has a molecular weight of 72.1, and is a yellow liquid with a pungent odor. generate steam.

In the present invention, "measuring the level of methylglyoxal, a candidate substance or a methylglyoxal adduct" means measuring the content of these substances.

The methylglyoxal adduct refers to a compound formed by combining a candidate substance with methylglyoxal.

The "content measurement" refers to an experimental group treated with a candidate substance and methylglyoxal or a control group 1 that is not treated with both the candidate substance and methylglyoxal, a control group 2 treated with methylglyoxal without treating the candidate substance, or methylglyoxal The amount of these compounds is measured by checking the presence of methylglyoxal, a candidate substance, or a methylglyoxal adduct in Control 3 treated with a candidate substance without oxal treatment. As an analysis method for this, High-Performance Liquid Chromatograph (HPLC), Mass Spectrometry (MS), Liquid Chromatography-Mass Spectrometry (LC-MS), etc. can be used, but a known method can be used for compound analysis or content measurement. can

In one embodiment of the present invention, through LC-MS analysis, an MGO adduct having a structure in which 1 to 4 MGOs are bonded to one diecohol molecule was observed, and in fact, 11 MGOs can be bonded to one diecol molecule.

When the level of the methylglyoxal adduct of the experimental group treated with the candidate substance and methylglyoxal is higher than that of Controls 1 to 3, the candidate substance may be selected as a substance for preventing liver damage or protecting the liver.

When the level of methylglyoxal in the experimental group treated with the candidate substance and methylglyoxal is lower than that of Control 2 treated with methylglyoxal, the candidate substance may be selected as a substance for preventing liver damage or protecting the liver.

When the level of the candidate material in the experimental group treated with the candidate material and methylglyoxal is reduced compared to the control group 3 treated with the candidate material without methylglyoxal, the candidate material is used to prevent liver damage or to protect the liver can be selected as

In addition, in the experimental group treated with the candidate substance and methylglyoxal, the level of the methylglyoxal adduct was increased than that of Controls 1 to 3, the level of methylglyoxal was decreased than that of Control 2, and the level of the candidate substance was decreased than that of Control 3 When it is further reduced, the candidate substance may be selected as a substance that prevents liver damage or protects the liver.

The screening method for a substance that prevents liver damage or protects the liver of the present invention reduces the toxicity of cells in the experimental group treated with the candidate substance and methylglyoxal compared to the control group 2 treated with methylglyoxal and at the same time methylglyoxal By confirming that the level of the oxal adduct is increased by the binding of methylglyoxal to the candidate substance, untreated control 1, methylglyoxal-treated control 2, or candidate-treated control 3 and generated methylglyoxal It is designed as a way to compare the level (content) of the adduct.

In the present invention, the candidate material may be diecol.

The diecol can be isolated from Ecklonia cava .

The diecol has the following chemical formula.

[Formula 1]

In one embodiment of the present invention, when diecol was used as a candidate material and treated with methylglyoxal, it was confirmed that the toxicity of the hepatocyte line was reduced, and at the same time, diecol was combined with methylglyoxal to form a methylglyoxal adduct. As it was confirmed that the formation of When the level of glyoxal adduct is increased, the candidate can be selected as a substance that prevents liver damage or protects the liver.

The methylglyoxal adduct has the following chemical formula.

[Formula 2]

In another aspect, the present invention provides a composition for preventing liver damage or protecting the liver from methylglyoxal, comprising the material screened by the screening method as an active ingredient.

In the present invention, the description of the term methylglyoxal and the screening method is the same as described above.

In the present invention, the term "liver damage" may be liver damage induced by methylglyoxal, and liver diseases that can be treated by the composition of the present invention are included without limitation, but include, for example, non-alcoholic fatty liver, alcoholic fatty liver, Non-alcoholic steatohepatitis, fatty liver hepatitis, non-steatohepatitis, end-stage fibrotic liver disease, non-viral chronic hepatitis, cirrhosis, acute liver injury, liver cancer, etc. may be present.

As described above, since the substance induced by the screening method has an effect of preventing liver damage induced by methylglyoxal and protecting the liver, the substance screened by the screening method, for example, diecol, etc. It can be used as a composition for preventing liver damage or protecting the liver.

In the present invention, the composition may be a pharmaceutical composition.

In the present invention, when the composition for preventing liver damage or protecting the liver is used as a pharmaceutical composition, it may further include an appropriate carrier, excipient or diluent commonly used in the preparation of the pharmaceutical composition. At this time, the screened material included in the composition, for example, diecol is not particularly limited thereto, but may be 0.0001% by weight or more, specifically 0.001% by weight or more, and 80% by weight or less, specifically, based on the total weight of the composition. It may be 50 wt% or less, but is not limited thereto.

The pharmaceutical composition is any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-drying agents and suppositories It may have a dosage form, and may be oral or parenteral various dosage forms. In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin. It can be prepared by mixing and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like may also be used. Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is dependent on the subject's type and severity, age, sex, and disease. It may be determined according to the type, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, and can be easily determined by those skilled in the art.

The pharmaceutical composition of the present invention is not particularly limited as long as it is an individual for the purpose of preventing liver damage or protecting the liver, and any one is applicable. For example, any of non-human animals such as monkey, dog, cat, rabbit, guinea pig, rat, mouse, cow, sheep, pig, goat, human, bird and fish may be used, and the pharmaceutical composition may be administered parenterally or subcutaneously. , intraperitoneally, intrapulmonary and intranasally, and for local treatment, if necessary, by any suitable method including intralesional administration. The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and weight of the individual, the degree of disease, the drug form, the route of administration, and the duration, but may be appropriately selected by those skilled in the art. For example, it may be administered by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebrovascular injection, but is not limited thereto.

A suitable total daily amount can be determined by a treating physician within the scope of sound medical judgment, and is generally in an amount of 0.001 to 1000 mg/kg, preferably 0.05 to 200 mg/kg, more preferably 0.1 to 100 mg/kg. The amount of kg can be administered in divided doses from once to several times a day.

In addition, in the present invention, the composition may be a food composition, specifically, may be a health functional food.

In the present invention, when the composition for preventing liver damage or protecting the liver is used as a food composition, the food composition of the present invention may include the form of pills, powders, granules, needles, tablets, capsules or liquids, etc., and the type of food There are no restrictions on Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, Alcoholic beverages and vitamin complexes are available.

Other ingredients may be added to the food composition in addition to the screened material such as diecol, and the type is not particularly limited. For example, it may contain, as an additional ingredient, various herbal extracts, food-logically acceptable food supplements or natural carbohydrates, such as conventional food, but is not limited thereto.

As used herein, the term "food supplement additive" refers to a component that can be supplementally added to food, and is added to prepare food of each formulation, and those skilled in the art can appropriately select and use it. Examples of food supplement additives include various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners , pH adjuster, stabilizer, preservative, glycerin, alcohol, carbonation agent used in carbonated beverages, etc., but the above examples are not limited to the type of food supplement additive of the present invention.

Examples of the natural carbohydrate include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Hygin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used.

The food composition of the present invention may include a health functional food. The term "health functional food" used in the present invention refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and pills using raw materials or ingredients useful for the human body. Here, the term "functionality" refers to obtaining a useful effect for health purposes such as regulating nutrients or physiological action with respect to the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the art, and during the manufacture, it can be prepared by adding raw materials and components commonly added in the art. In addition, unlike general drugs, there are no side effects that may occur when taking the drug for a long time by using food as a raw material, and it can be excellent in portability.

The mixed amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, when preparing food, the active ingredient of the present invention may be added in an amount of 1 to 50% by weight, preferably 5 to 10% by weight of the raw material composition, but is not limited thereto. However, in the case of long-term intake for health and hygiene purposes or for health control, the above amount may be used below the above range.

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating liver damage caused by methylglyoxal comprising diecol as an active ingredient.

In the present invention, diecol, methylglyoxal, liver damage, the description of the pharmaceutical composition is the same as described above.

In one embodiment of the present invention, it was confirmed that diecol prevents liver cell damage, such as inhibiting hepatocyte toxicity induced by methylglyoxal, and has a protective effect on hepatocytes. The composition of the present invention prevents liver damage Or it can be used as a therapeutic pharmaceutical composition.

In the present invention, the diecol binds with methylglyoxal to form a methylglyoxal adduct and has scavenging activity of methylglyoxal, so it can protect the liver by preventing liver damage caused by methylglyoxal. .

In another aspect, the present invention provides a food composition for preventing or improving liver damage from methylglyoxal comprising diecol as an active ingredient.

In the present invention, diecol, methylglyoxal, liver damage, the description of the food composition is the same as described above.

In one embodiment of the present invention, it was confirmed that diecol prevents liver cell damage, such as inhibiting hepatocyte toxicity induced by methylglyoxal, and has a protective effect on hepatocytes. The composition of the present invention prevents liver damage Or it can be used as a food composition for improvement.

In the present invention, the diecol binds with methylglyoxal to form a methylglyoxal adduct and has scavenging activity of methylglyoxal, so it can protect the liver by preventing liver damage caused by methylglyoxal. .

In another aspect, the present invention provides a method for reducing damage to hepatocytes induced by methylglyoxal.

The method for reducing damage to hepatocytes of the present invention comprises the steps of treating hepatocytes with methylglyoxal and diecol in an animal except for humans or in vitro; and forming a methylglyoxal adduct.

In the present invention, the description of diecol, methylglyoxal, and methylglyoxal adducts are the same as described above.

As described above, a methylglyoxal adduct is formed by the combination of diecol and methylglyoxal, and the diecol binds to methylglyoxal to form a methylglyoxal adduct, thereby enhancing the scavenging activity of methylglyoxal. Therefore, it can protect the liver by preventing liver damage caused by methylglyoxal.

The present invention relates to a method for screening a substance for preventing or protecting the liver, and a composition for preventing or protecting the liver, wherein diecol combines with methylglyoxal to form a methylglyoxal adduct, Because it has scavenging activity, it can protect the liver by preventing liver damage caused by methylglyoxal.

1 shows the evaluation of cytotoxicity by treating a HepG2 hepatocyte line with dieckol (DK) for each concentration.
Figure 2 shows the evaluation of cytotoxicity by treating each concentration of methylglyoxal (MGO) in HepG2 hepatocyte line.
Figure 3 shows the evaluation of cytotoxicity by treating the HepG2 hepatocyte line with diekol (DK) and methylglyoxal (MGO).
Figure 4 is through HPLC, diekol (DK) decreases over time, diekol (DK) and methylglyoxal (methylglyoxal, MGO) product of MGO adduct due to the combination It is confirmed that this increases.
Figure 5 is performed by HPLC for 18 hours to confirm the production amount of diekol (dieckol, DK) and MGO adduct.
Figure 6 shows the structure of the MGO adduct generated by the combination of diekol (dieckol, DK) and methylglyoxal (methylglyoxal, MGO).
7 is a confirmation of the MGO adduct produced by the combination of dieckol (DK) and methylglyoxal (MGO) by LC-MS.
8 shows a site at which methylglyoxal binds to diecol.
Figure 9 shows the survival rate after treatment with methylglyoxal (methylglyoxal, MGO) in zebrafish embryos.
Figure 10 shows the survival rate after treatment with diekol (dieckol, DK) in zebrafish embryos.
11 shows the survival rate after treatment with diekol (DK) and methylglyoxal (MGO) in zebrafish embryos.
Figure 12 shows the survival rate after treatment with diekol (DK) and methylglyoxal (MGO) in the zebrafish embryo as the intensity of acridine orange.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those of ordinary skill in the art can easily carry out the present invention. However, the present invention may be embodied in several different forms and is not limited to the embodiments described herein.

Example 1. Preparation of diecol and methylglyoxal

Ecklonia cava was collected from the coast of Jeju. After washing with water 3 times to remove foreign substances from the surface of Ecklonia cava, it was stored at -20 ℃ for use. After freeze-drying and pulverizing Ecklonia cava to prepare dry Ecklonia coli powder, an 80% aqueous ethanol solution was added to the dried Ecklonia cava powder to perform extraction, followed by filtering to remove solids, and reduced pressure at 40 ° C. got it The ethanol extract of Ecklonia cava was suspended in water, and ethyl acetate was added thereto to obtain an ethyl acetate fraction. This ethyl acetate fraction was separated using reversed-phase HPLC using a Shephadex LH-20 column, and as a result, it was analyzed by nuclear magnetic resonance (NMR) and mass spectrometer (MS). As a result, diecol (dieckol, DK) was confirmed and used in the following experiments.

Methylglyoxal (MGO) was purchased from Merck KGaA (Darmstadt, Germany) and used.

Example 2. Cytotoxicity (cytoprotective effect) evaluation of diecol and methylglyoxal on hepatocytes

To evaluate the cytotoxicity, HepG2 hepatocytes were inoculated into a 96 well plate at 1Х10 ⁵ cells/ml and cultured for 24 hours. Then, each well was treated with diecol, methylglyoxal, diecol and methylglyoxal for 24 hours. cultured. Next, 100 μl of MTT solution (2 mg/ml) was treated in each well, and after reacting for 4 hours, the medium was removed. After dissolving by adding 200 μl of DMSO to the formazan crystal of each well, absorbance was measured at 540 nm to evaluate cytotoxicity.

First, HepG2 hepatocytes were treated with diecol and methylglyoxal at different concentrations, and then cytotoxicity (cytoprotective effect) to hepatocytes was confirmed.

1 shows the evaluation of cytotoxicity by treating a HepG2 hepatocyte line with dieckol (DK) by concentration. Diecol was used by dissolving in DMSO. As a result, it was confirmed that the treatment with diecohol 2.5 μM to 50 μM did not induce cytotoxicity of hepatocytes.

Figure 2 shows the evaluation of cytotoxicity by treating the HepG2 hepatocyte line with methylglyoxal (MGO) for each concentration. As a result, it was confirmed that, at a concentration of 0.63 mM to 25 mM of methylglyoxal, the cytotoxicity of the hepatocyte line increased as the concentration increased.

Next, HepG2 hepatocytes were treated with diekol (DK) and methylglyoxal (MGO), and then cytotoxicity was evaluated. Figure 3 shows the evaluation of cytotoxicity by treating the HepG2 hepatocyte line with diekol (DK) and methylglyoxal (MGO). As a result, it was confirmed that the toxicity of the hepatocyte line induced by treatment with 25 mM methylglyoxal was significantly reduced by treatment with 12.5 μM to 50 μM of diecol.

From the above results, it was found that diecol prevents damage to hepatocytes and protects hepatocytes, such as inhibiting the toxicity of hepatocytes caused by methylglyoxal.

Example 3. Confirmation of MGO adduct production using HPLC

The purpose of this study was to determine the mechanism by which diecol inhibits the toxicity of hepatocytes by methylglyoxal. First, by performing HPLC, the change in diecol and methylglyoxal content with time was confirmed.

To perform HPLC, diecol and methylglyoxal were mixed in a ratio of 1:1, and the reaction was carried out at 25° C. for 65 minutes using acetonitrile and water as the transferred solvent. Reaction conditions are as follows.

Column: Sunfire C18 5 um, 4.6 x 150 mm column

Flow rate: 0.25 mL/min

Sample conc.: 1 mg/mL, DK 0.5 mg/mL+MGO 2.5 ppm

UV range: 284 nm

Injection volume: 10 μL

Figure 4 shows that the product of the MGO adduct formed by the combination of dieckol (DK) and diekol (DK) and methylglyoxal (MGO) increases with time through HPLC. it has been confirmed As shown in FIG. 4 , it was confirmed that diecol decreased as time elapsed, and MGO adduct formed by the combination of dieckol (DK) and methylglyoxal (MGO) was generated.

Next, in order to confirm the decrease in diecol and the generation of MGO adduct for a longer period of time, HPLC was performed for more than 18 hours. Figure 5 is performed by HPLC for 18 hours to confirm the production amount of diekol (dieckol, DK) and MGO adduct. As a result, it was confirmed that dieckol (DK) decreased as time passed, and it was confirmed that diekol was combined with MGO to generate MGO adduct. In FIG. 5 , the gray graph (MGOadduct_2) shows 3 molecules of MGO bound to one molecule of diecol, and the blue graph (MGOadduct_1) shows 2 molecules of MGO bound to one molecule of diecohol.

Figure 6 shows the structure of the MGO adduct generated by the combination of diekol (dieckol, DK) and methylglyoxal (methylglyoxal, MGO).

Example 4. Confirmation of MGO adduct production using LC-MS

Next, through LC-MS analysis, the MGO adduct produced by the combination of dieckol (DK) and methylglyoxal (MGO) was confirmed.

7 is an LC-MS confirming the MGO adduct resulting from the combination of dieckol (DK) and methylglyoxal (MGO). In the present invention, MGO adduct was analyzed and confirmed through LC/DAD-ESI/MS under the following MS conditions and LC conditions. The analysis conditions are as follows.

<MS condition>

positive cone voltage(V): 30, 45, 60, negative cone voltage: 25, 35, 45, capilary: 3.5 kV, extractor: 3 V, RF Lens: 0.1 V, source temperature: 120℃, desolvation temperature: 350℃ N2 gas flow (L/hr), desolvation: 900, cone: 50.

<LC condition>

10-100% ACN linear gradient for 40 min, C18 5um, 2.0×150mm (INNO, Young Jin biochrom).

As a result, as confirmed in FIG. 7 , it was observed that as time passed, the molecular weight was higher than that of methylglyoxal (molecular weight about 72.06) and diecol (molecular weight about 742.52). This means that the MGO adduct formed by the combination of dieckol (DK) and methylglyoxal (MGO) was generated.

Specifically, molecular weights of 814, 886, 958, and 1030 with 1 to 4 molecules of MGO (MW 72) attached to diecol (DK) (MW 742) were shown. Of these, in the case of MGO adducts with 2 and 4 MGO attached, 908.9 and 1053.1 with one Na ⁺ were present in the positive ion, and 855.0 and 1029.1 with one H+ missing from the negative ion were present.

8 shows the bonding position of diecol and methylglyoxal. When looking at their structural characteristics, it can be seen that 11 MGOs are bound to diecol. 8 shows the positions where methylglyoxal binds to one molecule of diecol, and up to 11 bonds are possible.

Example 5. Analysis of the protective effect on MGO in zebrafish embryos

Zebrafish embryos were treated with MGO or dieckol (DK) and methylglyoxal (MGO), and cytotoxicity in zebrafish embryos was analyzed using acridine orange (Acridinorange). As a result, as shown in FIG. 9 , it was confirmed that the cytotoxicity was significantly increased in the zebrafish embryo by MGO treatment, and the survival rate was significantly reduced.

In addition, in FIG. 10, it was confirmed whether diecol treatment induces cytotoxicity in zebrafish embryos. As a result, it was confirmed that there was no cytotoxicity up to 100 μM, but at high concentrations such as 200 M and 400 μM, diecol induced cytotoxicity, thereby reducing the survival rate of zebrafish embryos. Therefore, from the next experiment, up to 100 μM of diecol was used.

Next, when dieckol (DK) and methylglyoxal (MGO) were treated together in FIG. 11 , it was evaluated whether the cytotoxicity induced by MGO was reduced. As a result, it was confirmed that the cytotoxicity induced by MGO was significantly reduced in zebrafish embryos when 100 μM of diecol 25 was treated. That is, as confirmed in FIG. 12 , the intensity of acridine orange was strongly detected in the zebrafish embryo by MGO, confirming that cytotoxicity was high, and when dieckol (DK) was treated together, diecol treatment As the concentration increased, the intensity of acridine orange decreased. Therefore, it was confirmed that dieckol (DK) significantly reduced the cytotoxicity caused by MGO to protect the zebrafish (FIG. 12).

In the above, the technical idea of the present invention has been described along with the accompanying drawings, but the preferred embodiment of the present invention is exemplarily described and does not limit the present invention. In addition, it is a clear fact that various modifications and imitations are possible without departing from the scope of the technical spirit of the present invention by anyone having ordinary knowledge in the technical field to which the present invention pertains.

Claims (18)

Treating the experimental group with diecol and methylglyoxal as candidates;
measuring the level of methylglyoxal, a candidate substance or a methylglyoxal adduct in the experimental group treated with the candidate substance; and
Comprising the step of comparing the level of untreated control 1, control 2 treated with methylglyoxal, or control 3 treated with a candidate substance,
When the level of the methylglyoxal adduct of the experimental group treated with the candidate substance and methylglyoxal increases compared to Controls 1 to 3, the step of selecting the candidate substance as a substance that prevents liver damage or protects the liver that is,
A method of screening for substances that prevent liver damage or protect the liver. According to claim 1,
The candidate substance is to form a methylglyoxal adduct by combining with methylglyoxal, a screening method of a substance that prevents liver damage or protects the liver. According to claim 1,
The methylglyoxal adduct is a screening method of a substance that prevents liver damage or protects the liver, which is formed by binding of a candidate substance and methylglyoxal. delete delete According to claim 1,
When the level of methylglyoxal in the experimental group treated with the candidate substance and methylglyoxal is reduced than that of control 2, the method further comprising the step of selecting the candidate substance as a substance for preventing liver damage or protecting the liver, A method of screening for substances that prevent liver damage or protect the liver. According to claim 1,
When the level of the candidate material in the experimental group treated with the candidate material and methylglyoxal is reduced than that of the control group 3, further comprising the step of selecting the candidate material as a material for preventing liver damage or protecting the liver, A method of screening for substances that prevent damage or protect the liver. According to claim 1,
In the experimental group treated with the candidate substance and methylglyoxal, the level of the methylglyoxal adduct was increased than that of Controls 1 to 3, the level of methylglyoxal was decreased than that of Control 2, and the level of the candidate substance was decreased than that of Control 3. If so, the screening method of a substance that prevents liver damage or protects the liver, further comprising the step of selecting the candidate substance as a substance that prevents liver damage or protects the liver. delete delete delete delete delete delete delete delete delete delete

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