Composition for suppressing accumulation of glycated proteins

Abstract

To provide a new composition for suppressing the accumulation of glycated proteins.SOLUTION: Provided is a composition for suppressing the accumulation of glycated proteins, the composition containing squalene as an active ingredient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition for inhibiting the accumulation of glycated proteins. [Background technology]

[0002] As a typical example of a reaction between biological substances and sugars in the body, it is known that reducing sugars necessary for the body's vital activities and proteins undergo the Maillard reaction to form advanced glycation end-products (hereinafter referred to as AGEs) via intermediates.

[0003] The produced AGEs are either broken down by metabolism or excreted from the body, but as long as the body is active, they accumulate in various tissues within the body. Accumulated AGEs affect biological functions, trigger biological reactions, and are involved in the formation of various pathologies. Typical examples are aging, age-related diseases, and diabetic complications. In the skin, they trigger inflammatory reactions and cause browning and dullness of the skin. For example, Patent Documents 1 and 2 describe ingredients that inhibit the production and accumulation of AGEs. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 7272908 [Patent Document 2] Patent No. 7318082 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a novel composition for inhibiting the accumulation of glycated proteins. [Means for solving the problem]

[0006] The present invention includes the following aspects. [1] A composition for inhibiting the accumulation of glycated proteins, comprising squalene as an active ingredient. [2] The composition according to [1], which is a pharmaceutical composition, a quasi-drug composition, a food composition or a cosmetic composition. [Effects of the Invention]

[0007] According to the present invention, a novel composition for inhibiting the accumulation of glycated proteins can be provided. [Brief explanation of the drawings]

[0008] [Figure 1A] This figure shows the results of detecting methylglyoxal (MG)-H1 using an anti-MG-H1 antibody in Annexin V reacted with MG in Experimental Example 1. MG was used as a highly reactive α-dicarbonyl compound that causes glycation derived from intermediates of glycometabolism, and Annexin V was used as a representative protein. [Figure 1B] FIG. 1 shows the results of detecting MG-H1 using anti-MG-H1 antibody for Annexin V reacted with MG in Experimental Example 1. [Figure 1C] FIG. 1 shows the results of detecting MG-H1 and CEL using anti-MG-H1 antibody and anti-CEL antibody for Annexin V reacted with MG in Experimental Example 1.

[0009] [Figure 2A] 1 shows an image of the fine structure of annexin V reacted with MG taken using a high-speed atomic force microscope in Experimental Example 2. [Figure 2B] 1 shows an image of the fine structure of annexin V reacting with MG taken using a high-speed atomic force microscope in Experimental Example 2. [Figure 2C] 2B is a graph showing the quantification of the number of trimer-deleted annexin V in (B1) to (B7) of FIG. 2B.

[0010] [Figure 3A]FIG. 10 shows the results of an analysis of the inhibitory effect of MG on glycation reaction using anti-MG-H1 antibody in Experimental Example 3, in which annexin V was reacted with squalene and then reacted with annexin V with MG. [Figure 3B] 3B is a graph showing the average signal intensity obtained by performing the analysis of FIG. 3A three times in Experimental Example 3.

[0011] [Figure 4A] FIG. 10 shows the results of analyzing the inhibitory effect of MG on glycation reaction using anti-MG-H1 antibody when annexin V was reacted with squalene and MG simultaneously in Experimental Example 4. [Figure 4B] 4B is a graph showing the average signal intensity obtained by performing the analysis of FIG. 4A three times in Experimental Example 4.

[0012] [Figure 5A] This figure shows the results of an analysis of the effect of squalene on MG-H1 using anti-MG-H1 antibody in Experimental Example 5, in which annexin V was reacted with MG and then annexin V was reacted with squalene. [Figure 5B] 5B is a graph showing the average signal intensities obtained by performing the analysis of FIG. 5A three times in Experimental Example 5. [Figure 5C] FIG. 10 shows the results of quantifying MG-H1 by mass spectrometry in Experimental Example 5, in which annexin V was reacted with MG and then annexin V was reacted with squalene.

[0013] [Figure 6A] This figure shows the results of analyzing the effect of squalene on carboxyethyllysine (CEL) using an anti-CEL antibody in Experimental Example 6, where annexin V was reacted with MG and then reacted with squalene. [Figure 6B] 6B is a graph showing the signal intensity of the analysis of FIG. 6A in Experimental Example 6. [Figure 6C]FIG. 10 shows the results of analyzing the effect of squalene on CEL by mass spectrometry in Experimental Example 6, in which annexin V was reacted with MG and then reacted with squalene.

[0014] [Figure 7A] This figure shows the results of analyzing the effect of squalene on MG-H1 using anti-MG-H1 antibody in Experimental Example 7, where annexin V was reacted with MG, and then MG was completely removed before adding squalene. [Figure 7B] 7B is a graph showing the average signal intensities obtained by performing the analysis of FIG. 7A four times in Experimental Example 7. DETAILED DESCRIPTION OF THE INVENTION

[0015] (composition) The composition according to this embodiment contains squalene as an active ingredient. The composition according to this embodiment is used to inhibit the accumulation of glycated proteins.

[0016] As mentioned above, glycated proteins are produced in vivo through the glycation reaction, in which proteins react with carbonyl compounds, such as reducing sugars. As shown below, in the glycation reaction, the amino group of the amino acid in the protein reacts with the aldehyde group of the reducing sugar to form a Schiff base, followed by the formation of an Amadori rearrangement compound, which then undergoes oxidation, dehydration, condensation, cross-linking, and other reactions to produce advanced glycation end products (AGEs). The following diagram explains the reaction process between glucose and an amino group.

[0017] [ka]

[0018] Examples of reducing sugars include monosaccharides such as glucose, fructose, and galactose, and methylglyoxal, a metabolic intermediate derived from glycolysis. The type of protein to be glycated is not particularly limited.

[0019] Examples of structures possessed by glycated proteins include methylglyoxal-hydroimidazolone (MG-H1), formed by the amino group of an arginine residue and methylglyoxal, and Nε-(carboxyethyl)lysine (CEL), formed by the amino group of a lysine residue and methylglyoxal, as shown in the chemical formulas below. In the structural formula of MG-H1 below, * denotes a bond to the α-carbon of the arginine residue. In the structural formula of CEL below, ** denotes a bond to the α-carbon of the lysine residue.

[0020] [ka]

[0021] As used herein, "suppressing the accumulation of glycated proteins" means that, for example, the amount of glycated protein accumulation in an animal, such as a human, administered with the composition of this embodiment is suppressed compared to the amount of glycated protein accumulation in that animal not administered with the composition of this embodiment.

[0022] The composition of this embodiment can be determined to have activity in suppressing the accumulation of glycated proteins if, for example, the amount of glycated proteins accumulated in the cells of an animal to which the composition has been administered is reduced compared to the amount of glycated proteins accumulated in the cells of an animal to which the composition has not been administered.

[0023] The amount of glycated protein accumulation can be measured, for example, by Western blotting using anti-MG-H1 antibody, anti-CEL antibody, etc. Alternatively, it can be measured by quantifying the amount of MG-H1 and CEL formed in proteins using mass spectrometry.

[0024] <Squalene> Squalene is 2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene. Its CAS registry number is 111-02-4.

[0025] The squalene contained in the composition according to this embodiment may be either a natural product or a synthetic product. Natural squalene may be squalene purified from animals or plants.

[0026] <Other ingredients> The composition according to this embodiment may contain other ingredients in addition to the above-mentioned active ingredients. The other components are not particularly limited as long as the composition exhibits its effects, and examples thereof include excipients, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, solubilizers, antiseptics, flavorings, stabilizers, pH adjusters, antioxidants, preservatives, moisturizers, skin protectants, freshening agents, fragrances, colorants, chelating agents, antiperspirants, plant extract components, animal extract components, oily bases, emulsifiers, emulsion stabilizers, powder components, polymer components, adhesion improvers, film-forming agents, shape retention agents, and lubricants.

[0027] <Content> The content of the active ingredient contained in the composition according to this embodiment is not particularly limited as long as the composition exerts its effect, and is adjusted appropriately depending on the dosage form, etc., and may be, for example, 0.0001% by mass or more and 100% by mass or less, or 0.0001% by mass or more and 50% by mass or less, relative to the total mass (100% by mass) of the composition.

[0028] <Dosage form> The dosage form of the composition according to this embodiment is not particularly limited as long as the effects of the present invention are achieved, and may be any dosage form, such as liquid, powder, granules, or tablets.

[0029] <Target of administration> The subject to which the composition according to this embodiment is administered is not particularly limited as long as the effects of the present invention are achieved, and examples thereof include humans and non-human animals. Examples of non-human animals include pets and livestock. Examples of non-human animals include birds such as chickens and ostriches; rodents such as rats, hamsters, and guinea pigs; lagomorphs such as rabbits; ungulates such as pigs, cows, goats, horses, and sheep; Carnivora such as dogs and cats; and non-human primates such as monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, and chimpanzees.

[0030] <Administration method> The method of administration of the composition of this embodiment is not particularly limited as long as the effect of the present invention is exhibited in the animal to which it is administered. It may be administered orally or parenterally, but oral administration is preferred.

[0031] The administration route may be, for example, intraarterial injection, intravenous injection, subcutaneous injection, intranasal injection, transbronchial injection, intramuscular injection, transdermal injection, or oral administration, which are well known to those skilled in the art.

[0032] Injectable preparations can also be prepared as non-aqueous diluents (e.g., propylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, etc.), suspensions, or emulsions. Such injectable preparations can be sterilized by filtration sterilization or by adding a disinfectant. Injectable preparations can be manufactured in a form ready for use. That is, they can be made into a sterile solid composition by freeze-drying or the like, and then dissolved in distilled water for injection or other solvents before use.

[0033] <Dosage> The dosage of the composition according to this embodiment is adjusted appropriately taking into consideration the age, sex, weight, symptoms, treatment method, administration method, treatment time, etc. of the animal to be administered.

[0034] <Application> The use of the composition according to this embodiment is not particularly limited as long as the effects of the present invention are achieved, and the composition can be used by being incorporated into, for example, pharmaceutical compositions, quasi-drug compositions, food compositions, cosmetic compositions, etc.

[0035] <Pharmaceutical composition, quasi-drug composition> The composition of the above embodiment may be a pharmaceutical composition or a quasi-drug composition. The pharmaceutical composition may comprise an effective amount of an active ingredient and a pharmaceutically acceptable carrier.

[0036] The term "pharmaceutically acceptable carrier" refers to a carrier that does not inhibit the physiological activity of an active ingredient and is not substantially toxic to a recipient. "Not substantially toxic" refers to a component that is not toxic to a recipient at a dose normally used. In the pharmaceutical composition of this embodiment, the pharmaceutically acceptable carrier may be a component that does not inhibit the activity of the composition of the embodiment to inhibit the accumulation of glycated proteins and is not substantially toxic to a recipient.

[0037] Examples of pharmaceutically acceptable carriers include excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, and calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropyl cellulose, gelatin, gum arabic, polyethylene glycol, sucrose, and starch; disintegrants such as starch, carboxymethylcellulose, hydroxypropyl starch, sodium glycol starch, sodium bicarbonate, calcium phosphate, and calcium citrate; lubricants such as magnesium stearate, aerosil, talc, and sodium lauryl sulfate; flavorings such as citric acid, menthol, glycyrrhizin ammonium salt, glycine, and orange powder; preservatives such as sodium benzoate, sodium bisulfite, methylparaben, and propylparaben; stabilizers such as citric acid, sodium citrate, and acetic acid; suspending agents such as methylcellulose, polyvinylpyrrolidone, and aluminum stearate; dispersing agents such as surfactants; diluents such as water, buffer solutions, and saline; and base waxes such as cocoa butter, polyethylene glycol, and white kerosene.

[0038] When the pharmaceutical composition according to this embodiment is to be administered orally, the pharmaceutical composition formulation can be produced by a conventional method using additives commonly used for oral preparations, such as excipients, fillers, binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, solubilizers, antiseptics, flavoring agents, soothing agents, and stabilizers. Examples of usable additives include lactose, fructose, glucose, starch, gelatin, methylcellulose, gum arabic, polyethylene glycol, citric acid, sodium sulfite, sodium phosphate, β-cyclodextrin, and hydroxypropyl-β-cyclodextrin. When the pharmaceutical composition according to this embodiment is to be administered orally, the pharmaceutical composition may be prepared in a dosage form suitable for oral administration, such as capsules, tablets, granules, powders, pills, fine granules, or lozenges.

[0039] When the pharmaceutical composition according to this embodiment is used for parenteral administration, the pharmaceutical composition may contain an antioxidant, a buffer solution, a bacteriostatic agent, an isotonic agent, a suspending agent, a solubilizing agent, a thickener, a stabilizer, a preservative, etc. When the pharmaceutical composition according to this embodiment is used for parenteral administration, the pharmaceutical composition may be sealed in a container such as an ampoule, a vial, or a syringe cartridge in a unit dose or in multiple doses.

[0040] The effective amount of squalene contained in the pharmaceutical composition according to this embodiment is not particularly limited as long as the pharmaceutical composition exerts its effect, and can be appropriately determined by a person skilled in the art. The "effective amount" refers to the amount of squalene that is effective in suppressing the accumulation of glycated proteins.

[0041] The aspects of the quasi-drug are the same as those of the pharmaceutical composition.

[0042] <Food composition> The composition of the above embodiment may be a food composition. Examples of food compositions include health foods, functional foods, health foods, health supplements, supplements, nutritional supplements, foods for specified health uses, nutritionally functional foods, medical foods, foods for the sick, foods for infants, foods for nursing care, and foods for the elderly.

[0043] The food composition may be added to food or drink. The foods and beverages to which the food composition can be added are not particularly limited, but examples include beverages such as tea drinks, soft drinks, milk drinks, vegetable drinks, alcoholic beverages, and energy drinks; food ingredients such as meat, fish, vegetables, grains, dairy products, egg products, and processed products thereof; and seasonings such as sauces, spices, oils, and herbs.

[0044] The food composition may be in the form of, for example, granules, particles, paste, gel, solid, or liquid.

[0045] The food composition may contain other ingredients in addition to squalene, as long as the effects of the present invention are achieved. Examples of other ingredients include binders, disintegrants, thickeners, dispersants, resorption promoters, flavoring agents, buffers, surfactants, solubilizers, preservatives, emulsifiers, tonicity agents, stabilizers, and pH adjusters.

[0046] The food composition may be for humans or non-human animals, including the animals mentioned above.

[0047] <Cosmetic composition> The composition of the above embodiment may be a cosmetic composition. The cosmetic composition can be used in the form of, for example, a cream, emulsion, pack, gel, aerosol, sheet, etc. More specific examples include skin cosmetics such as lotion, serum, whitening agent, moisturizer, face mask, emulsion, foundation, eye shadow, mascara, eyebrow pencil, eyeliner, blush powder, lipstick, lip balm, pack, soap, etc.; and hair cosmetics such as hair rinse, hair conditioner, hair treatment, hair lotion, hair tonic, hair pack, hair cream, conditioning mousse, hair mousse, hair spray, shampoo, leave-on treatment, hair dye, hair styling product, etc.

[0048] The cosmetic composition may contain other ingredients (other ingredients) in addition to squalene, as long as the effects of the present invention are achieved. Examples of other ingredients include emulsifiers, hydrating agents, solvents, emollients, stabilizers, thickeners, preservatives, lubricants, chelating agents, fillers, excipients, powders, fragrances, flavorings, absorbents, dyes, opacifiers, antioxidants, vitamins, and amino acids.

[0049] The composition according to the present embodiment described above contains squalene as an active ingredient, and is therefore capable of suppressing the accumulation of glycated proteins. The composition according to the present embodiment is presumed to have activity for suppressing protein glycation reactions, removing glycated protein structures, and the like. That is, by administering the composition according to this embodiment to an animal or cells in advance, it is possible to prevent protein glycation reactions. Furthermore, by administering the composition according to this embodiment to an animal or cells, it is possible to suppress protein glycation reactions. Furthermore, by administering the composition according to this embodiment to an animal or cells, it is possible to remove glycated protein structures (e.g., MG-H1, CEL, etc.).

[0050] [Other embodiments]

[0051] In one aspect, the present invention provides a method for inhibiting the accumulation of glycated proteins, comprising administering a composition according to the embodiment to a subject animal. The target animal may be a human or a non-human mammal. The dosage of the composition may be any of the dosages described above for the compositions of the embodiments.

[0052] In one aspect, the present invention provides a method for treating a disease, comprising administering to an animal in need thereof an effective amount of a composition according to the embodiments. The target disease is not particularly limited as long as it is a disease caused by glycated proteins. The animals to be treated include humans and non-human mammals. The effective amount of the composition may be an effective amount as described above for the pharmaceutical composition of the embodiment. The method of administering the composition may be the same as that described above for the pharmaceutical composition according to the embodiment.

[0053] In one aspect, the present invention provides the use of squalene for the manufacture of a pharmaceutical composition for treating or preventing a disease caused by a glycated protein. [Example]

[0054] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0055] Materials and Methods <Material> Purified human annexin V protein (hereinafter simply referred to as annexin V) was provided by Dr. Hiroki Konno (Nano Life Science Institute). The amino acid sequence of annexin V is registered as Genbank: AAB40047.1. The annexin V protein was expressed in Escherichia coli, and the crude extract was purified using Profinity eXact Purification Resin (Bio-Rad). As methylglyoxal, a commercially available product (M0252, Sigma Aldrich) was used. The squalene used was 99% pure squalene provided by Nissei Marine Industries Co., Ltd. The purity of squalene was calculated based on the results of analysis by gas chromatography.

[0056] <Method> Glycation reaction For the protein glycation reaction, annexin V recombinant protein was reacted with methylglyoxal in 0.2 M phosphate buffer (pH 7.4) at room temperature.

[0057] Western blotting After the protein glycation reaction, a sample buffer containing 2-mercaptoethanol was added to the sample and heated to 100°C. The heated sample was then developed on a 12% SDA-PAGE (ATTO Corporation) and transferred semi-dry to a PVDF membrane (Millipore). The transferred PVDF membrane was blocked with PBS containing 1% (w / v) BSA and 0.05% Tween 20 (PBS-T) at room temperature for 1 hour. The PVDF membrane was then incubated overnight at 4°C with a primary antibody. The primary antibody used was either an anti-MG-H1 antibody (HM5017, BioConnect BV) diluted 1:500 or an anti-CEL antibody (AEG-M02, Cosmo Bio Co., Ltd.) diluted 1:500. The membrane was then washed five times with PBS-T. The transferred PVDF membrane was then reacted with a secondary antibody at room temperature for 1 hour. The secondary antibody used was anti-mouse IgG IRDye680 antibody (15,000-fold dilution). The PVDF membrane was then washed with PBS-T. Fluorescence intensity was then measured using an Odyssey Infrared Imaging system (LI COR Inc.).

[0058] CBB staining After the transfer, the PVDF membrane was stained with a CBB staining solution (0.025% CBB R-250, 40% methanol) and then destained with 50% methanol.

[0059] High-speed atomic force microscope (HS-AFM) We used an HS-AFM fabricated at WPI-NanoLSI, Kanazawa University. Annexin V protein was placed on mica coated with artificial lipids and observed while adding 10 mM MG. The HS-AFM buffer used was 10 mM HEPES-NaPH (pH 7.0) / 150 mM NaCl / 2 mM CaCl2. The artificial lipid used was 1,2 dioleoyl sn glycero 3 phosphocholine: L α dioleoylphosphatidylserine: 1,2 dioleoyl sn glycero 3 phosphoethanolamine N-(cap biotinyl) = 70:20:10 (weight ratio).

[0060] TOF-MS / MS For mass spectrometry, the sample to be measured was concentrated without ultrafiltration or other methods, dissolved in a buffer containing urea, and then subjected to reductive alkylation and trypsin digestion. The resulting digest was then desalted and purified before being subjected to mass spectrometry. Analysis of amino acid modifications due to glycation reactions was performed using PEAKS (Bioinformatics Solutions Inc., Waterloo, Ontario, Canada).

[0061] (Experimental Example 1) The glycation reaction of annexin V by methylglyoxal (hereinafter sometimes referred to as MG) was analyzed.

[0062] Annexin V was reacted with MG (final concentrations: 0, 10, 100, and 1000 mM) for 24 hours. Annexin V after the reaction was then detected with anti-MG-H1 antibody. The results are shown in Figure 1A.

[0063] As shown in Figure 1A, MG-H1 was not detected when annexin V was not reacted with MG, but was detected when annexin V was reacted with MG. When annexin V was reacted with 1000 mM MG, the mobility of the reaction product changed compared to other reaction conditions. When annexin V was reacted with 100 mM or 1000 mM MG, multiple bands with lower mobility than annexin V were detected. These results indicate that annexin V was glycosylated by MG to form MG-H1.

[0064] Next, the final concentration of MG reacted with annexin V was varied from 0 to 10,000 μM. The reaction time between annexin V and MG was 60 or 120 minutes. The results are shown in Figure 1B. As shown in Figure 1B, the amount of MG-H1 formation increased depending on the MG concentration and reaction time.

[0065] Next, the reaction time between Annexin V and MG was varied to 10, 60, and 120 minutes. The results are shown in Figure 1C. As shown in Figure 1C, MG-H1 was detected even after a 10-minute reaction. Furthermore, detection with an anti-CEL antibody indicated that CEL was formed in the reaction mixture.

[0066] From the above results, it was confirmed that by reacting annexin V with MG, annexin V was glycosylated by MG, resulting in the formation of MG-H1 and CEL in annexin V.

[0067] (Experimental Example 2) The ultrastructural changes of annexin V due to glycation were analyzed using high-speed atomic force microscopy.

[0068] Annexin V that had not reacted with MG was placed on mica coated with artificial lipids and observed using a high-speed atomic force microscope. The results are shown in Figure 2A. The scale bar in Figure 2A (A1)-(A2) indicates 20 nm. As previously reported, the honeycomb structure of annexin V was confirmed, as shown in Figure 2A (A1).

[0069] Next, annexin V reacted with 10 mM MG was observed using a high-speed atomic force microscope. The results are shown in Figure 2A (A2). As shown in Figure 2A (A2), the trimer at the center of the honeycomb structure was confirmed to be missing. This confirmed that the glycation reaction by MG changes the ultrastructure of annexin V.

[0070] Next, 10 mM MG was added while observing annexin V under high-speed atomic force microscopy, and changes in the ultrastructure of annexin V were analyzed. The results are shown in Figure 2B (B1) to (B10). Figure 2C is a graph quantifying the number of trimer-deleted annexin V in each of the images shown in Figure 2B (B1) to (B7). The size of each image was 58.4 nm × 58.4 nm.

[0071] As shown in Figure 2B (B1) to (B7) and Figure 2C, the number of trimer-deleted annexin V increased over time. As shown in Figure 2B (B8) to (B10), the honeycomb structure was lost.

[0072] (Experimental Example 3) Annexin V was reacted with squalene, and then annexin V was reacted with MG, and the inhibitory effect of MG on glycation reaction was analyzed.

[0073] First, various concentrations of squalene (0, 0.3, 1, 10, 100 mM) were reacted with 0.2 μM annexin V for 30 minutes at room temperature with stirring. Next, 10 mM MG was added and the mixture was reacted for 2 hours. MG-H1 was then detected by Western blotting using an anti-MG-H1 antibody. The results are shown in Figure 3A. Similar experiments were performed three times and quantitative analysis was performed. The results are shown in Figure 3B. In Figure 3B, the average MG-H1 detection intensity without squalene was set to 1. In Figure 3B, the p-value was calculated using Student's t-test.

[0074] As shown in Figures 3A and 3B, it was confirmed that the formation of MG-H1 in annexin V was inhibited by reacting annexin V with squalene and then reacting annexin V with MG.

[0075] (Experimental Example 4) When annexin V was reacted with squalene and MG simultaneously, the inhibitory effect of MG on glycation reaction was analyzed.

[0076] Various concentrations of squalene (0, 0.3, 1, 10, 100 mM), 0.2 μM annexin V, and 10 mM MG were mixed and incubated at room temperature for 2 hours. MG-H1 was then detected by Western blotting using an anti-MG-H1 antibody. The results are shown in Figure 4A. Similar experiments were performed three times, and the quantitative analysis results are shown in Figure 4B. In Figure 4B, the average MG-H1 detection intensity without squalene was set to 1. The p-value in Figure 4B was calculated using Student's t-test.

[0077] As shown in Figures 4A and 4B, by reacting annexin V with squalene and MG simultaneously, it was confirmed that squalene inhibited the formation of MG-H1 in annexin V in a concentration-dependent manner.

[0078] (Experimental Example 5) The effect of squalene on MG-H1 was analyzed when MG was reacted with annexin V and then annexin V was reacted with squalene.

[0079] First, 0.2 μM annexin V and 10 mM MG were reacted at room temperature for 2 hours, and MG-H1 was formed from annexin V by glycation. Subsequently, various concentrations of squalene (0, 0.3, 1, 10, and 100 mM) were added and reacted at room temperature for 2 hours. MG-H1 was then detected by Western blotting using an anti-MG-H1 antibody. The results are shown in Figure 5A. Similar experiments were performed three times, and the quantitative analysis results are shown in Figure 5B. In Figure 5B, the average MG-H1 detection intensity without squalene was set to 1. In Figure 5B, p values ​​were calculated using Student's t-test.

[0080] As shown in Figures 5A and 5B, by reacting MG with annexin V and then reacting annexin V with squalene, it was suggested that squalene may act on the already formed MG-H1.

[0081] Next, to quantitatively and qualitatively confirm the results, we analyzed the glycation of arginine residues in annexin V by MG using a mass spectrometer. The results are shown in Figure 5C. In Figure 5C, each number indicates the amino acid residue number of annexin V (Genbank: AAB40047.1).

[0082] As shown in Figure 5C, it was confirmed that MG-H1 formation occurs through the glycation reaction of MG at 16 of the 19 arginine residues in annexin.

[0083] As shown in Figure 5C, even without the addition of MG, annexin V was found to produce a small amount of MG-H1 (R6, R18, R25, R63, R117, R123, R271, and R285). This indicates that recombinant annexin V produces a small amount of MG-H1 during production and purification.

[0084] Furthermore, it was revealed that the addition of 0.3 mM squalene significantly inhibited the formation of MG-H1 by MG at arginine 63 (R63) of annexin V. It was also confirmed that the addition of 0.3 mM squalene reduced the MG-H1 modification formed during production and purification of recombinant annexin V R63.

[0085] These results suggest that squalene may also act on already formed MG-H1.

[0086] (Experimental Example 6) As in Experimental Example 5, annexin V was reacted with MG, and then annexin V was reacted with squalene to analyze the effect of squalene on CEL. CEL was detected by Western blotting using an anti-CEL antibody. The results are shown in Figures 6A and 6B.

[0087] As shown in Figures 6A and 6B, it was suggested that squalene, like MG-H1, may act on already formed CEL.

[0088] The glycation of lysine residues in annexin V by MG was analyzed using a mass spectrometer. The results are shown in Figure 6C. In Figure 6C, each number indicates the amino acid residue number of annexin V (Genbank: AAB40047.1). Analysis using a mass spectrometer confirmed that 11 of the 22 lysine residues in annexin V were modified with CEL by MG.

[0089] As shown in Figure 6C, annexin V was found to be slightly modified with CEL (K26, K29, K76, K101, K310) even without the addition of MG. This indicates that a small amount of CEL is formed in recombinant annexin V during production and purification.

[0090] Furthermore, the addition of 0.3 mM squalene was found to suppress MG-H1 formation by MG (K29, K76, K79, K108, K126, K193, K286, K310).

[0091] These results suggest that squalene may also act on already formed CEL.

[0092] (Experimental Example 7) We analyzed whether squalene has the activity to remove MG-H1 formed by annexin V.

[0093] MG-H1-modified annexin V was prepared by reacting 1 μM annexin V with 10 mM MG at room temperature for 2 hours. To completely stop the MG-mediated glycation reaction, multiple solution changes were performed using an ultrafiltration spin column to completely remove MG. Various concentrations of squalene (0, 0.3, 10 mM) were then added and the mixture was incubated at room temperature for 24 hours. MG-H1 was then detected by Western blotting using an anti-MG-H1 antibody. The results are shown in Figure 7A. Similar experiments were performed four times, and the quantitative analysis results are shown in Figure 7B. In Figure 7B, the mean MG-H1 detection intensity (indicated as "MG") immediately after complete removal of MG was set to 1. p values ​​in Figure 7B were calculated using Student's t-test.

[0094] As shown in Figure 7B (squalene 0 mM), after the complete removal of MG, annexin V was stirred at room temperature for 24 hours without adding squalene, and no new MG-H1 was formed. As shown in Figure 7B (squalene 0.3, 10 mM), after complete removal of MG, squalene was added to annexin V and stirred at room temperature for 24 hours. This confirmed that the previously formed MG-H1 was removed. [Industrial Applicability]

[0095] According to the present invention, a novel composition for inhibiting the accumulation of glycated proteins can be provided. By administering the composition of the present invention to an animal, it is possible to remove structures formed in proteins due to glycation reactions.

Claims

1. A composition for inhibiting the accumulation of glycated proteins, comprising squalene as an active ingredient.

2. The composition according to claim 1, which is a pharmaceutical composition, a quasi-drug composition, a food composition, or a cosmetic composition.

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