Composition having anti-oxidant, anti-aging, and autophagy activities and uses thereof

Abstract

Provided are a composition having anti-oxidant, anti-aging, and autophagy activities to thereby be useful for preventing, alleviating, or treating age-related metabolic diseases including neurodegenerative diseases or type 2 diabetes, and uses thereof.

Claims

1. A cosmetic composition comprising a compound of the following Structural Formula 1 or a cosmetically acceptable salt thereof as an active ingredient: ##STR00008##

2. A pharmaceutical composition comprising a compound of the following Structural Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient: ##STR00009##

3. The pharmaceutical composition according to claim 2, wherein the composition increases anti-oxidant and autophagy activities of cells.

4. A food composition comprising a compound of the following Structural Formula 1 or a cytologically acceptable salt thereof as an active ingredient: ##STR00010##

5. The food composition according to claim 4, wherein the composition increases anti-oxidant and autophagy activities of cells.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates a result obtained by performing high performance liquid chromatography (HPLC) assay on a compound represented by Structural Formula 1.

(2) FIG. 2 illustrates a result obtained by performing liquid chromatography (LC)/mass spectrometry (MS) assay on the compound represented by Structural Formula 1.

(3) FIG. 3 illustrates a result obtained by confirming anti-oxidant activities of the compound represented by Structural Formula 1, vitamin C, and pyrrolidone carboxylic acid (PCA), respectively, using 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging assay.

(4) FIG. 4 illustrates a result obtained by confirming an intracellular anti-oxidant activity of the compound represented by Structural Formula 1 inhibiting formation of intracellular reactive oxygen species (ROS) using 6-carboxy-2,7-dichlorofluorescein diacetate (DCFH-DA) dye assay and fluorescence activated cell sorter (FACS) scanning. A horizontal axis means a fluorescence intensity detected in each cell, and a vertical axis means a cell count. A (A) line indicates a non-treated control group, a (B) line indicates a group treated with H.sub.2O.sub.2, a (C) line indicates a group treated with H.sub.2O.sub.2 and vitamin C, and a (D) line indicates a group treated with H.sub.2O.sub.2 and the compound represented by Structural Formula 1. The (B) line indicates the group treated only with H.sub.2O.sub.2, and a right shift from the (A) line (the non-treated group) to the (C) line means a state in which the intracellular ROS was formed, and a left shift to the (D) line by treatment of the compound means that the intracellular ROS was decreased.

(5) FIG. 5 illustrates a western block result obtained by measuring changes in expression amounts of aging-related proteins at the time of treating cells with the compound represented by Structural Formula 1.

(6) FIG. 6 illustrates a western block result obtained by measuring changes in expression amounts of autophagy-related proteins at the time of treating cells with the compound represented by Structural Formula 1.

(7) FIG. 7 illustrates a result obtained by confirming activation of autophagy by confirming whether or not LC3 puncta is formed in cases in which cells were treated with the compound represented by Structural Formula 1, rapamycin, or the cells were not treated.

(8) FIG. 8 are photographs obtained by analyzing cell images through an electron microscope after treating human dermal fibroblast cells with the compound represented by Structural Formula 1 and culturing the human dermal fibroblast cells for 24 hours.

(9) FIG. 9 illustrates a result obtained by quantitatively analyzing the numbers of intracellular phagophore, autophagosome, and autolysosome in an electron microscope photograph.

(10) FIG. 10 illustrates cell viability (%) at the time of inducing oxidative stress using H.sub.2O.sub.2 after pre-treating cells with the compound represented by Structural Formula 1 or vitamin C for 24 hours.

(11) FIG. 11 illustrates cell viability (%) at the time of inducing oxidative stress using H.sub.2O.sub.2 after pre-treating cells with the compound represented by Structural Formula 1 or vitamin C for 48 hours.

DETAILED DESCRIPTION OF EMBODIMENTS

(12) Hereinafter, the present invention will be described in detail by Examples. However, the following Examples are to illustrate the present invention, and the scope of the present invention is not limited to the following Examples.

Example 1. Synthesis of Compound Represented by Structural Formula 1

(13) A compound represented by Structural Formula 1 according to the present invention was synthesized through a H-Lys-Lys-OH backbone synthesis process (first process), an alkylation reaction process (second process), a hydrolysis reaction process (third process), and a protection group removal reaction process (final process). A detailed description thereof is as follows.

Example 1-1. Synthesis of H-Lys-Lys-OH (Compound 1c)

1. Synthesis of Boc-Lys(Boc)-Lys(Boc)-OMe (Compound 1a)

(14) ##STR00002##

(15) 50 g of Boc-Lys(Boc)-OH (1 equivalent: 144.35 mmol) and 45.09 g of H-Lys(Boc)-OMe (1.2 equivalents) were put into a 2 L round flask and dissolved in 500 ml of dichloromethane (MC). 1.76 g of N,N-dimethylaminopyridine (DMAP, 0.1 equivalents) was added thereto and a temperature of a reactant was lowered to 0 C. using an ice bath. 36 g of N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC, 1.3 equivalents) was dissolved in 300 ml of MC and slowly added thereto. When addition of EDC was terminated, the temperature of the reactant was slowly raised to room temperature, and a reaction was carried out for 12 hours. The reaction was confirmed by thin layer chromatography (TLC, hexane:ethyl acetate=1:1, Rf=0.4). The solvent was removed using a concentrator. After adding 500 ml of ethyl acetate (EA) and 500 ml of an aqueous 1N HCl solution, an EA layer was extracted using a separatory funnel. After 500 ml of an aqueous 5% NaHCO.sub.3 solution was added thereto, an EA layer was extracted using a separatory funnel. After 20 ml of a saturated aqueous NaCl solution was added thereto, an EA layer was extracted using a separatory funnel. After adding Na.sub.2SO.sub.4 thereto to remove water, the resultant was concentrated. 76.4 g of Compound 1a was obtained (yield: 90%).

2. Synthesis of Boc-Lys(Boc)-Lys(Boc)-OH (Compound 1b)

(16) ##STR00003##

(17) 50 g of Compound 1a (84.98 mmol, 1 equivalent) was put into a 2 L round flask, and dissolved by adding 500 ml of methanol thereto. 170 ml (1 equivalent) of an aqueous 1N NaOH solution was added thereto, and a reaction was carried out at room temperature for 12 hours. The reaction was confirmed by thin layer chromatography (TLC, MC:MeOH=10:1, Rf=0.4). Methanol was removed using a concentrator. After adding 500 ml of water thereto to dissolve the resultant, a pH of an aqueous solution was adjusted to 5 by adding sulfuric acid thereto. After adding 500 ml of MC, a MC layer was extracted using a separatory funnel. After adding Na.sub.2SO.sub.4 thereto to remove water, the resultant was concentrated. 46.4 g of Compound 1b was obtained (yield: 95%).

3. Synthesis of H-Lys-Lys-OH (Compound 1c)

(18) ##STR00004##

(19) 40 g of Compound 1B (69.64 mmol, 1 equivalent) was put into a 2 L round flask, and dissolved in 200 ml of MC. 200 ml of trifluoroacetic acid (TFA) was added thereto, and a reaction was carried out at room temperature for 4 hours. After MC was removed using a concentrator, 500 ml of diethyl ether was added thereto to precipitate a product. A solid product was collected by filtering, and the collected solid product was washed with 300 ml of diethyl ether. After dissolving the obtained solid product in 300 ml of water, followed by freeze drying, thereby obtaining 31.5 g of Compound 1c in a TFA salt form.

Example 1-2. Alkylation Reaction

1. Synthesis of (tert-butoxycarbonylmethyl)2-Lys(tert-butoxycarbonylmethyl)2-Lys(tert-butoxycarbonylmethyl)2-O(tert-butoxycarbonylmethyl) (Compound 1d)

(20) ##STR00005##

(21) 30 g of Compound 1c (59.74 mmol, 1 equivalent) was put into a 2 L round flask, and dissolved in 400 ml of water. 125 ml of N,N-diisopropylethylamine (DIPEA, 12 equivalents) was added thereto, and 400 ml of N,N-dimethylformamide (DMF) was added thereto. Finally, 61.3 ml of tert-butylbromoacetate (7 equivalents) was added thereto, and a reaction was carried out at room temperature for 3 days. The reaction was confirmed by thin layer chromatography (TLC, hexane:ethyl acetate=2:1, Rf=0.3). After adding 500 ml of EA, an EA layer was extracted using a separatory funnel. After 500 ml of an aqueous 1N HCl solution was added thereto, an EA layer was extracted using a separatory funnel (extraction was repeated six times). After 500 ml of an aqueous 5% NaHCO.sub.3 solution was added thereto, an EA layer was extracted using a separatory funnel. After 20 ml of a saturated aqueous NaCl solution was added thereto, an EA layer was extracted using a separatory funnel. After adding Na.sub.2SO.sub.4 to remove water, the resultant was concentrated. 38.5 g of Compound 1d was obtained (yield: 60%).

Example 1-3. Hydrolysis Reaction

1. Synthesis of (tert-butoxycarbonylmethyl)2-Lys(tert-butoxycarbonylmethyl)2-Lys(tert-butoxycarbonylmethyl)2-OH (Compound 1e)

(22) ##STR00006##

(23) 30 g of Compound 1d (27.95 mmol, 1 equivalent) was put into a 2 L round flask, and dissolved by adding 600 ml of tetrahydrofuran (THF) thereto. After adding 216 ml of water thereto, 84 ml of an aqueous 1N NaOH solution (3 equivalents) was added thereto, and a reaction was carried out at room temperature for 12 hours. The reaction was confirmed by thin layer chromatography (TLC, MC:MeOH=10:1, Rf=0.4). THF was removed using a concentrator. After adding 500 ml of water thereto to dissolve the resultant, a pH of an aqueous solution was adjusted to 5 by adding sulfuric acid. After adding 500 ml of MC thereto, a MC layer was extracted using a separatory funnel. After adding Na.sub.2SO.sub.4 to remove water, the resultant was concentrated. 25.5 g of Compound 1e was obtained (yield: 95%).

Example 1-4. Protection Group Removal Reaction

1. Synthesis of Compound Represented by Structural Formula 1

(24) ##STR00007##

(25) 20 g of Compound 1e (20.85 mmol, 1 equivalent) was put into a 1 L round flask, and dissolved by adding 200 ml of dioxane. 200 ml of an aqueous 35% HCl solution was added thereto, and a reaction was carried out at room temperature for 12 hours. The reaction was confirmed by HPLC (Shimadzu LC-20AD, GraceSmart RP C18 5u 120A 4.6*250 mm, Rt=3.668 min, 0.01% TFA containing acetonitrile (various concentration gradients: 0% to 20%)/water, 20 minutes) and LC/MS (Waters SQD2, MS(ESI)m/e, [M+H]+=623.03, [M+H]+/2=312.11). Water and dioxane were removed using a concentrator. After adding 300 ml of water thereto to dissolve the resultant, the resultant was concentrated again (this process was repeated four times). After adding 300 ml of water to dissolve the resultant, the resultant was freeze-dried. 12.3 g of the compound represented by Structural formula 1 was obtained (yield: 95%). HPLC assay (FIG. 1) and LC/MS assay (FIG. 2) were performed on the obtained compound represented by Structural Formula 1.

Example 2. Measurement of In-Vitro Anti-Oxidant Activity by Compound Represented by Structural Formula 1

(26) In order to measure in-vitro anti-oxidant activity of the compound represented by Structural Formula according to the present invention, ABTS radical scavenging assay was performed. When 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) reacts with potassium persulfate, ABTS.sup.+, which is an active cation, is formed, but in the case in which an anti-oxidant material is present in a test sample, ABTS.sup.+ is removed by anti-oxidant activity, a greenish blue color, which is a peculiar color of ABTS.sup.+, is decolorized, which may be measured as an absorbance value, thereby making it possible to evaluate the anti-oxidant activity.

(27) As a specific experimental method, after mixing ABTS diammonium salt (7 mM) and potassium persulfate (2.45 mM) with each other in distilled water in order to activate stabilized ABTS diammonium salt, a reaction was carried out at room temperature for 16 hours, thereby forming an ABTS radical. After diluting the resultant in a phosphate buffer solution (PBS) to prepare a reaction solution so that an OD734 value was about 1.5, the reaction solution was mixed with vitamin C (Vit C), pyrrolidone carboxylic acid (PCA), or the compound represented by Structural Formula 1 disclosed in the present invention to induce a reaction. After 30 minutes, an amount of the remaining activated ABTS radical was analyzed by measuring absorbance at OD734.

(28) As a result, vitamin C and the compound represented by Structural Formula 1 exhibited a concentration-dependent scavenging activity on the activated ABTS radical as illustrated in FIG. 3.

Example 3. Measurement of Intracellular Anti-Oxidant Activity by Compound Represented by Structural Formula 1

(29) In order to measure an intercellular anti-oxidant activity of the compound represented by Structural Formula 1 according to the present invention, an amount of intracellular reactive oxygen species (ROS) was measured using 6-carboxy-2,7-dichlorofluorescein diacetate (DCFH-DA) dye and fluorescence activated cell sorter (FACS) scanning. When DCFH-DA is introduced into cells through a cell membrane, DCFH-DA is hydrolyzed to 6-carboxy-2,7-dichlorofluorescein (DCFH) by esterase existing in a cytoplasm, and when there is an intracellular ROS, DCFH is oxidized to fluorescent dichlorofluorescein (DCF), thereby exhibiting green fluorescence with a high fluorescence intensity (excitation at 485 nm, emission at 515 nm). Therefore, since in the case of pre-treating cells with a test material, inducing oxidative stress using hydrogen peroxide (H.sub.2O.sub.2), or the like, and treating the cells with DCFH-DA, a fluorescence intensity of DCF is differently exhibited depending on a degree of an anti-oxidative capacity of the test material, an intracellular anti-oxidant activity of the test material may be measured using the fluorescence intensity as described above.

(30) As a specific experimental method, human epidermal keratinocyte cells (Gibco BRL) were uniformly seeded onto a 600 culture dish (310.sup.5 cells/dish) and cultured in an epilife medium (Gibco BRL) containing human keratinocyte growth supplement (HKGS, Gibco BRL) at 37 C. in a 5% CO.sub.2 incubator for 24 hours. Thereafter, the test materials (H.sub.2O.sub.2, vitamin C, and the compound represented by Structural Formula 1) were each dissolved in water at a concentration of 10 mM to prepare a concentrate, the prepared concentrate was diluted so as to have a concentration of 100 uM using the medium and put into a culture dish to treat the cells. Then, culturing was performed for 48 hours, and after the culturing was terminated, the medium was removed and the resultant was washed, thereby preparing a subsequent experiment. Next, 10 uM hydrogen peroxide was added thereto, the culturing was performed for 10 minutes, the resultant was washed again. After 10 uM DCFH-DA was added thereto again, and additional culturing was performed for 60 minutes, entire cells were obtained by treating the resultant with trypsin. Thereafter, florescence intensities of individual cells were confirmed (excitation at 485 nm, emission at 515 nm) using FACS caliber (Becton Dickinson).

(31) As a result, it may be confirmed that in the case of pre-treatment with vitamin C for 48 hours, an amount of intracellular ROS formed by treatment with treatment of H.sub.2O.sub.2 was not decreased, but in the case of pre-treatment with the compound represented by Structural Formula 1 for 48 hours, intracellular ROS formed by treatment with H.sub.2O.sub.2 almost disappeared as illustrated in FIG. 4.

Example 4. Measurement of Expression Amounts of Anti-Oxidant Protein and Autophagy-Related Protein by Compound Represented by Structural Formula 1

(32) In order to find a cause of increasing an intracellular anti-oxidant activity by treatment with the compound represented by Structural Formula 1, cDNA microarray was performed, thereby confirming that transcription of several kinds of important genes associated with anti-oxidation and autophagy was increased. Proteins for the selected genes were quantified by western blot.

(33) As a specific experimental method, after treating human epidermal keratinocyte cells (Gibco BRL) with the compound represented by Structural Formula 1 at a concentration of 50, 100, or 200 um while culturing the cells in a 600 culture dish, culturing was performed for 48 hours. After the culturing was terminated, a medium was removed, a RIPA lysis buffer containing protease inhibitor cocktail (2 ug/ml aprotinin, 5 ug/ml leupeptin, 1 ug/ml pepstatin A, 1 mM PMSF, 5 mM EDTA and 1 mM EGTA) was added thereto, thereby obtaining an entire cell homogenate. Thereafter, total proteins were quantified through microBCA assay (SMART micro BCA protein assay kit, Intron biotechnology), the cell homogenate (10 to 20 ug) was loaded on a SDS-PAGE gel, and respective proteins were isolated by electrophoresis and moved to a nitrocellulose membrane. Then, non-specific binding was removed by a blocking buffer, and after reacting antibodies (anti-Prx2, anti-Prx3, anti-Grp75, anti-HSP90B1, anti-EPHX2, anti-Stanniocalcin2, anti-Bip, anti-Foxo1, anti-Sirt5 and anti-n actin) for the respective proteins and HRP-bound secondary antibodies (anti-rabbit IgG HRP(sigma), anti-mouse IgG HRP(sigma)) thereof with each other, the reaction resultant was exposed to a photosensitive film by an enhanced chemiluminescence (ECL) method using a WestSave STAR ECL kit (Abfrontier), thereby confirming presence of the respective proteins and quantifying the respective proteins.

(34) As a result, it may be confirmed that at the time of treating the cells with the compound represented by Structural Formula 1, expression of proteins having high anti-oxidant activities such as a peroxiredoxin2 monomer (Prx2 monomer), peroxiredoxin3 (Prx3), HSP70 based Grp75, HSP90B1, epoxide dehydrogenase2 (EPHX2), stanniocalcin2, and the like, was increased as illustrated in FIG. 5. Further, it may be confirmed that expression of proteins important in autophagy and detoxification activities such as BiP, FoxO1, and sirtuin5 was increased as illustrated in FIG. 6.

Example 5. Measurement of Activation of Autophagy by Compound Represented by Structural Formula 1 Through LC3 Puncta Assay

(35) A possibility of activating autophagy in cells by treatment with the compound represented by Structural Formula 1 was confirmed through the result in Example 4, and in order to prove activation of autophagy, formation of light chain3 (LC3) puncta was confirmed.

(36) As a specific experimental method, after 200 ul of poly-L-lysine (sigma) was put into each well of a 15 ul slide glass (Ibidi) and a reaction was carried out at room temperature for 30 minutes, followed by coating, human epidermal keratinocyte cells (Gibco BRL) were cultured and treated with respective test materials (rapamycin, compound represented by Structural Formula 1, and vitamin C) at a concentration of 100 uM. After the culturing for 48 hours was terminated, a medium was removed, and the cultured cells were fixed and blocked using formalin. Thereafter, the cells were labeled with anti-LC3 (Abfrontier) and anti-rabbit IgG-FITC (sigma), nucleus was stained with 4,6-diamidine-2-phenylindole dihydrochloride (DAPI), and then, whether or not a puncta was formed in the cells was confirmed using a confocal microscope.

(37) As a result, it may be confirmed that similarly to rapamycin known as a material inducing autophagy, at the time of treating the cells with the compound represented by Structural Formula 1, a large amount of a LC puncta (a spot considered as a autophagosome) was formed in the cells as illustrated in FIG. 7. On the contrary, formation of the LC3 puncta was not increased by treatment with vitamin C.

(38) The light chain 3 (LC3) is a protein similar to ubiquitin, and LC3-I and phosphatydilethanolamine (PE) are bound to each other to form LC3-II. LC3-II is inserted into a membrane of the autophagosome to form a structure of the autophagosome. When the autophagosome is formed, insertion of LC3-II into the membrane of the autophagosome is an important step which consistently occurs, and an amount of LC3 reflects a relative amount of the autophagosome in cells. When the LC3-II protein is bound to the autophagosome, the LC3-II protein seems to be a puncta like a vesicle at the time of observation using a cell immunostaining method.

(39) Therefore, the large amount of LC puncta was formed by treatment with the compound represented by Structural Formula 1 according to the present invention, which is an important basis for the fact that the compound activates autophagy in cells.

Example 6. Measurement of Activation of Autophagy by Compound Represented by Structural Formula 1 Through Electron Microscopy Assay

(40) The activation of autophagy in cells by treatment with the compound represented by Structural Formula 1 was confirmed by the result in Example 5, and in order to more clearly prove this result, a structure in a cell was analyzed by an electron microscope.

(41) As a specific experimental method, after treating human dermal fibroblast cells with the compound represented by Structural Formula 1 at a concentration of 50 uM while culturing the cells, the culturing was performed for 24 hours. After the culturing was terminated, the cells were fixed, a paraffin block was prepared, and then, cell images were analyzed using an electron microscope.

(42) As a result, it was confirmed that at the time of treating the cells with the compound represented by Structural Formula 1, large amounts of phagophore, autophagosome, and autophagolysosome were formed as illustrated in FIGS. 8 and 9. As a result of measuring the numbers of phagophore, autophagosome, and autophagolysosome in cells in a large number of electron microscope photographs for quantitative analysis, it was observed that the number of autophagosome was significantly increased about 2 times or more by treatment with the compound represented by Structural Formula 1, and the numbers of phagophore and autophagolysosome were also significantly increased.

(43) An autophagy pathway starts from formation of an isolated precursor membrane having a cup shape, referred to as phagophore. The phagophore may form a double membrane endoplasmic reticulum known as autophagosome through initiation, nucleation, elongation, and closure processes. The autophagosome carries condensed proteins, organelles or pathogenic bacteria in damaged cells to lysosome in a captured state, and fused with lysosome to form autophagolysosome. The autophagolysosome decomposes the captured constituents in an acidic environment mediated by acid hydrolase.

(44) Therefore, phagophore, autophagosome, and autophagolysosome were significantly increased by treatment with the compound represented by Structural Formula 1 in the present Example, which is an important basis for the fact that the compound activates autophagy in cells.

Example 7. Measurement of Cell Protection Effect by Compound Represented by Structural Formula 1

(45) Through the results in Examples 2 to 6, it was confirmed that expression of the anti-oxidant proteins and the autophagy-related proteins in cells was increased by the compound represented by Structural Formula 1, and thus, autophagy in the cells was activated. Therefore, in order to confirm whether or not activation of autophagy as described above may protect cells from cytotoxicity caused by ROS, follow-up research into a cell protection effect was conducted.

(46) As a specific experiment method, after treating human epidermal keratinocyte cells (Gibco BRL) with respective test materials (vitamin C and the compound represented by Structural Formula 1) at 0.1 mM or 1 mM, respectively, while culturing the cells in a 96-well culture dish, the cells were cultured for 24 hours or 48 hours, the resultant was washed, and 1 mM H.sub.2O.sub.2 was added thereto, and additional culturing was performed for 4 hours. Thereafter, each of the wells was washed, 10 ul of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dissolved in PBS (5 mg/ml) was added to each of the wells, and a reaction was carried out at 37 C. under a 5% CO.sub.2 condition. Thereafter, a supernatant was carefully removed, MTT formazan precipitated in the cells was dissolved in 100 ul of DMSO, and then absorbance at OD570 was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

(47) As a result, it may be confirmed that in the case of vitamin C known as an anti-oxidant, at a concentration of 0.1 mM, at the time of pre-treatment for 24 hours recovered cell viability closely to 95%, and at the time of pre-treatment for 48 hours, recovery of cell viability was hardly observed, and at a concentration of 1 mM, at the time of pre-treatment for 24 hours or 48 hours, cell viability was rather deteriorated as illustrated in FIGS. 10 and 11. On the contrary, it may be confirmed that in the case of the compound represented by Structural Formula 1, at the time of pre-treatment for 24 hours or 48 hours, cell viability was improved.

(48) The formulation containing the compound according to the present invention may activate autophagy, protect cells from oxidative stress, and protect cells, tissue, and an individual from various phenomena and diseases occurring due to the oxidative stress by increasing expression of the anti-oxidant proteins and autophagy-related proteins at the time of being applied to human-derived cells. Therefore, finally, the compound according to the present invention may be expected to treat or alleviate the aging phenomenon and diseases and suppress the aging phenomenon and diseases from being worsened, such that the compound may be evenly applied to formulations of various cosmetics, quasi-drugs, drugs, and food.