KR102404228B1 - Composition including reduced lipoic acid-peptide derivative that increases the antioxidant effect and method of preparing the same - Google Patents

Abstract

The present invention relates to a cosmetic and/or pharmaceutical composition including a lipoic acid-peptide derivative and/or a salt thereof, which has excellent antioxidant effect and retention force.

Description

Composition including reduced lipoic acid-peptide derivative that increases the antioxidant effect and method of preparing the same

The present invention relates to a composition for antioxidants, and more particularly, to a cosmetic and/or pharmaceutical composition comprising a reduced lipoic acid-peptide derivative and/or a salt thereof.

Recently, various respiratory diseases, skin aging, and inflammatory reactions are occurring due to fine dust generated in China and air pollutants emitted from factories and automobiles. This phenomenon is caused by reactive oxygen species (ROS), reactive nitrogen species (RNS), and polyaromatic hydrocarbons (PAH) in fine dust.

Antioxidant is a substance that can prevent skin cell aging, melanin production, and autoimmune diseases caused by reduced immunity by suppressing free radicals or reactive oxygen species generated by the external environment. is known

Our body has an antioxidant system that can remove continuously generated free radicals and restore damaged cells. That is, it contains various enzymes that can remove active oxygen (superoxide dismutase, catalase, heme oxygenase-1, etc.) and antioxidants (vitamins A, C, E, glutathione, melatonin, lipoic acid, polyphenols, etc.) Among them, glutathione (γ-Glu-Cys-Gly) is a powerful antioxidant produced by the body itself, and is produced by glutamate cysteine ligase (GCL) and glutathione synthesis (GS) synthetase. In addition, glutathione material has functions such as immunity enhancement, detoxification, body fat reduction, and whitening effect, so it is used in various fields, but because it can be easily oxidized, it has the disadvantage that it does not last long. As a result, the need to correct the problem is emerging.

The present inventors conducted research focusing on the development of new antioxidant materials. By binding an antioxidant peptide to the reduced lipoic acid material, a novel compound was synthesized, and a candidate compound was selected through evaluation of the antioxidant efficacy of three methods. At this time, considering hydrogen atom transfer (HAT) and single electron transfer (SET), which are antioxidant reaction mechanisms that scavenge free radicals, DPPH (2,2-diphenyl-1-picrylhydrazyl), FRAP (ferric reducing antioxidant power) and ORAC (oxygen radical absorbance capacity) methods were used.

Under the above background, the present inventors confirmed the antioxidant efficacy and durability by binding an antioxidant peptide to an antioxidant substance (reduced lipoic acid). By confirming, the present invention was completed.

The present invention includes a peptide derivative or a salt thereof in which 3 to 10 types of amino acids are bound to lipoic acid,

The 3 to 10 amino acids are γ-glutamic acid (γ-Glutamic acid; γ-Glu; γ-E), glutamic acid (Glu; E), aspartic acid (Asp; D), glutamine (Glutamine; Gln; Q), Asparagine (Asparagine; Asn; N), Histidine (Histidine; His; H), Lysine (Lysine; Lys; K), Arginine (Arginine; Arg; R), Serine (Serine; Ser; S), Cysteine (Cys; C), Beta-Alanine (β-Alanine; β-Ala; β-A), Alanine (Alanine; Ala; A), Glycine (Gly; Gly; G), Leucine ; Leu; L), isoleucine (Isoleucine; Ile; I), valine (Valine; Val; V), threonine (Threonine; Thr; T), methionine (Methionine; Met; M), proline (Proline; Pro; P) , phenylalanine (Phenylalanine; Phe; F), tyrosine (Tyrosine; Tyr; Y) and tryptophan (Tryptophan; Trp; W) selected from the group consisting of, provides a cosmetic and / or pharmaceutical composition for antioxidants.

The present invention includes a peptide derivative comprising a peptide comprising 3 to 10 amino acids or a salt thereof,

The 3 to 10 amino acids are γ-glutamic acid (γ-Glutamic acid; γ-Glu; γ-E), glutamic acid (Glu; E), aspartic acid (Asp; D), glutamine (Glutamine; Gln; Q), Asparagine (Asparagine; Asn; N), Histidine (Histidine; His; H), Lysine (Lysine; Lys; K), Arginine (Arginine; Arg; R), Serine (Serine; Ser; S), Cysteine (Cys; C), Beta-Alanine (β-Alanine; β-Ala; β-A), Alanine (Alanine; Ala; A), Glycine (Gly; Gly; G), Leucine ; Leu; L), isoleucine (Isoleucine; Ile; I), valine (Valine; Val; V), threonine (Threonine; Thr; T), methionine (Methionine; Met; M), proline (Proline; Pro; P) , phenylalanine (Phenylalanine; Phe; F), tyrosine (Tyrosine; Tyr; Y) and tryptophan (Tryptophan; Trp; W) selected from the group consisting of, provides a cosmetic and / or pharmaceutical composition for antioxidants.

The peptide derivative has a structure of Formula 1, Formula 2, Formula 3, or Formula 4,

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

wherein Y ₁ , Y ₂ and Y ₃ are each independently γ-glutamic acid, glutamic acid, aspartic acid, glutamine, asparagine, histidine, lysine, arginine, serine, cysteine, beta alanine, alanine, glycine, leucine, isoleucine, valine , may be selected from the group consisting of threonine, methionine, proline, phenylalanine, tyrosine and tryptophan,

wherein X is selected from the group consisting of Fmoc-mini-PEG-OH (Fmoc-8-amino-3,6-dioxaoctanoic acid; mini-PEG), 6-aminohexanoic acid (AHA), and Gly-Gly-Gly. may, but is not limited thereto.

In the present invention, the antioxidant use of the peptide derivative and/or its salt was confirmed.

Accordingly, an example includes a peptide derivative or a salt thereof comprising a peptide comprising 3 to 10 amino acids,

The 3 to 10 amino acids are γ-glutamic acid (γ-Glutamic acid; γ-Glu; γ-E), glutamic acid (Glu; E), aspartic acid (Asp; D), glutamine (Glutamine; Gln; Q), Asparagine (Asparagine; Asn; N), Histidine (Histidine; His; H), Lysine (Lysine; Lys; K), Arginine (Arginine; Arg; R), Serine (Serine; Ser; S), Cysteine (Cys; C), Beta-Alanine (β-Alanine; β-Ala; β-A), Alanine (Alanine; Ala; A), Glycine (Gly; Gly; G), Leucine ; Leu; L), isoleucine (Isoleucine; Ile; I), valine (Valine; Val; V), threonine (Threonine; Thr; T), methionine (Methionine; Met; M), proline (Proline; Pro; P) , phenylalanine (Phenylalanine; Phe; F), tyrosine (Tyrosine; Tyr; Y) and tryptophan (Tryptophan; Trp; W) selected from the group consisting of, provides a cosmetic and / or pharmaceutical composition for antioxidants.

One example comprises the step of binding 3 to 10 types of amino acids to lipoic acid,

The 3 to 10 amino acids are γ-glutamic acid (γ-Glutamic acid; γ-Glu; γ-E), glutamic acid (Glu; E), aspartic acid (Asp; D), glutamine (Glutamine; Gln; Q), Asparagine (Asparagine; Asn; N), Histidine (Histidine; His; H), Lysine (Lysine; Lys; K), Arginine (Arginine; Arg; R), Serine (Serine; Ser; S), Cysteine (Cys; C), Beta-Alanine (β-Alanine; β-Ala; β-A), Alanine (Alanine; Ala; A), Glycine (Gly; Gly; G), Leucine ; Leu; L), isoleucine (Isoleucine; Ile; I), valine (Valine; Val; V), threonine (Threonine; Thr; T), methionine (Methionine; Met; M), proline (Proline; Pro; P) , phenylalanine (Phenylalanine; Phe; F), tyrosine (Tyrosine; Tyr; Y) and tryptophan (Tryptophan; Trp; W) selected from the group consisting of, provides a method for preparing a cosmetic and / or pharmaceutical composition for antioxidants.

The lipoic acid may be in a reduced form, but is not limited thereto.

The step of coupling 3 to 10 types of amino acids to the lipoic acid may be performed using a linker, but is not limited thereto.

In another example, the peptide derivative has a structure of Formula 1 or Formula 3,

[Formula 1]

[Formula 3]

wherein Y ₁ , Y ₂ and Y ₃ are each independently γ-glutamic acid, glutamic acid, aspartic acid, glutamine, asparagine, histidine, lysine, arginine, serine, cysteine, beta alanine, alanine, glycine, leucine, isoleucine, valine , may be selected from the group consisting of threonine, methionine, proline, phenylalanine, tyrosine and tryptophan, but is not limited thereto.

In another example, the peptide derivative has a structure of Formula 2 or Formula 4,

[Formula 2]

[Formula 4]

In the above formula, Y ₁ , Y ₂ and Y ₃ are each independently γ-glutamic acid, glutamic acid, aspartic acid, glutamine, asparagine, histidine, lysine, arginine, serine, cysteine, beta-alanine, alanine, glycine, leucine, isoleucine, selected from the group consisting of valine, threonine, methionine, proline, phenylalanine, tyrosine and tryptophan,

wherein X is selected from the group consisting of Fmoc-mini-PEG-OH (Fmoc-8-amino-3,6-dioxaoctanoic acid; mini-PEG), 6-aminohexanoic acid (AHA), and Gly-Gly-Gly. may, but is not limited thereto.

As a specific example, the Y ₁ , Y ₂ and Y ₃ may each independently be selected from the group consisting of γ-glutamic acid, serine, glycine, glutamic acid, histidine, tyrosine, leucine and beta-alanine. not.

X may be a linker, but is not limited thereto.

As a specific example, Y ₁ may be γ-glutamic acid, glutamic acid, or beta-alanine, Y ₂ may be serine, glutamic acid or histidine, and Y ₃ may be glycine, histidine or leucine, and is limited thereto. not.

Hereinafter, the present invention will be described in more detail.

As used herein, “lipoic acid” is an organosulfur compound derived from caprylic acid, which is an essential substance for aerobic metabolism, and may have the following Chemical Formula 5, but is not limited thereto.

[Formula 5]

In the present specification, “salt” may mean a physiologically acceptable salt among salts, which are substances in which cations and anions are combined by electrostatic attraction, for example, pharmaceutically acceptable salts, cosmetically acceptable salts. , and/or food-acceptable salts. For example, the salt may be at least one selected from the group consisting of a metal salt, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid, a salt with a basic or acidic amino acid, and the like. In one example, the metal salt may be at least one selected from the group consisting of alkali metal salts (sodium salts, potassium salts, etc.), alkaline earth metal salts (calcium salts, magnesium salts, barium salts, etc.), aluminum salts, and the like; Salts with organic bases include triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N-dibenzylethylenediamine, etc. It may be at least one selected from the group consisting of salts and the like; The salt with an inorganic acid may be at least one selected from the group consisting of salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like; The salt with organic acid is at least one selected from the group consisting of salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. can; The salt with a basic amino acid may be at least one selected from the group consisting of salts with arginine, lysine, ornithine, etc.; The salt with the acidic amino acid may be at least one selected from the group consisting of salts with aspartic acid, glutamic acid, and the like.

In the present specification, the “antioxidant” effect inhibits the oxidation of cells by highly reactive free radicals or reactive oxygen species (ROS) depending on intracellular metabolism or oxidative stress caused by the influence of ultraviolet rays. Including the removal of free radicals or reactive oxygen species, thereby reducing the damage to cells, and preventing or inhibiting oxidation, the opposite concept of oxidation is “reduction”, and “reducing power” is antioxidant It can be a measure of measure.

As used herein, "oxidation" means a case of bonding with oxygen, separation of hydrogen, and an increase in the oxidation number (reducing the number of electrons), and "reduction" means separation from oxygen, bonding with hydrogen, and reduction of the oxidation number ( The number of electrons increases), and when one element is oxidized, the other element is reduced, so it can be seen that it always occurs together.

In the present specification, the “linker” serves to indirectly connect the reduced lipoic acid and the antioxidant peptide, and may affect the physical properties of the overall compound.

The antioxidant cosmetic and/or pharmaceutical composition of the present invention contains the peptides 0.01 μM to 10,000 μM, 0.01 μM to 5,000 μM, 0.01 μM to 3,000 μM, 0.01 μM to 2,000 μM, 0.01 μM to 1,500 μM, 0.01 μM to 1,200 μM, 0.01 μM to 1,000 μM, 0.1 μM to 10,000 μM, 0.1 μM to 5,000 μM, 0.1 μM to 3,000 μM, 0.1 μM to 2,000 μM, 0.1 μM to 1,500 μM, 0.1 μM to 1,200 μM, 0.1 μM to 1,000 μM, 1 μM to 10,000 μM, 1 μM to 5,000 μM, 1 μM to 3,000 μM, 1 μM to 2,000 μM, 1 μM to 1,500 μM, 1 μM to 1,200 μM, 1 μM to 1,000 μM, 10 μM to 10,000 μM, 10 μM to 5,000 μM, 10 μM to 3,000 μM, 10 μM to 2,000 μM, 10 μM to 1,500 μM, 10 μM to 1,200 μM, 10 μM to 1,000 μM, 100 μM to 10,000 μM, 100 μM to 5,000 μM, 100 μM to 3,000 μM, 100 μM to 2,000 μM, 100 μM to 1,500 μM, 100 μM to 1,200 μM, 100 μM to 1,000 μM, 500 μM to 10,000 μM, 500 μM to 5,000 μM, 500 μM to 3,000 μM, 500 μM to 2,000 μM, 500 μM to 1,500 μM, 500 μM to 1,200 μM, 500 μM to 1,000 μM, 800 μM to 10,000 μM, 800 μM to 5,000 μM, 800 μM to 3,000 μM, 800 μM to 2,000 μM, 800 μM to 1,500 μM, 800 μM to 1,200 μM, 800 μM to 1,000 μM, 1,000 μM to 10,000 μM, 1,000 μM to 5,000 μM, 1,000 μM to 3,000 μM, 1,000 μM to 2,000 μM, 1,000 μM to 1,500 μM, or 1,000 μM to 1,200 μM, 1,000 μM to 1,000 μM and may include, for example, a concentration of 1, 10, 100, or 1,000 μM, but is not limited thereto.

The cosmetic and/or pharmaceutical composition for antioxidants of the present invention may have antioxidant persistence, DPPH radical scavenging activity, and/or active oxygen absorption activity, but is not limited thereto.

The peptide derivative or salt thereof included in the antioxidant cosmetic and/or pharmaceutical composition may have antioxidant persistence, DPPH radical scavenging activity, and/or active oxygen absorption activity, but is not limited thereto.

The cosmetic and/or pharmaceutical composition for antioxidant of the present invention has excellent antioxidant activity, and can be used as an antioxidant.

Specifically, the antioxidant activity of the composition was measured through the DPPH (1,1-Diphenyl-2-picryhydrazyl) method, and can be measured by calculating the free radical activity inhibition rate through Equation 1 below, but is not limited thereto .

[Equation 1]

Free radical activity inhibition rate (%) = 100 - [(reaction absorbance of sample / reaction absorbance of blank) X 100]

In Equation 1, the sample means the measurement target for which the free radical activity inhibition rate is to be measured, and the blank sample means that the sample is not treated. The free radical activity inhibition rate of the composition is 1% or more, 2% or more, 3% or more, 4% or more, 10% or more, 13% or more, 20% or more, 30% when the peptide is included at a concentration of 1,000 μM. or more, 40% or more, 44% or more, 50% or more, 55% or more, 60% or more, 70% or more, 72% or more, 99.9% or less, 99% or less, for example, It may be measured as 4.7%, 44.9%, 55.4%, or 72.8%, but is not limited thereto.

Specifically, the antioxidant activity of the composition was measured by ORAC (Oxygen radical absorbance capacity; active oxygen absorption capacity) method, and by calculating the area under the curve (AUC) of the fluorescence reduction curve through Equation 2 below. Measurable, but not limited thereto.

[Equation 2]

AUC = 1+ f1/f0 + ... + fi/f0 + ... + f60/f0

In Equation 2, f0 denotes a fluorescence value at 0 min, and fi denotes a fluorescence value at i min.

When the composition contains the peptide at a concentration of 100 μM, the AUC is -5 or more, -3 or more, -1 or more, 1 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 41 or more, Or 43 or more, 10,000 or less, 1,000 or less, 100 or less, for example, it may be measured as -0.5, 5.8, 41.4 or 43.0, but is not limited thereto.

Specifically, the antioxidant activity of the composition was measured through the FRAP (Ferric reducing ability of plasma) method, it can be measured by calculating the FRAP value (μM) through Equation 4 below, but is not limited thereto.

[Equation 4]

FRAP value (μM) = (ΔA593nm - intercept) / slope

In Equation 4, ΔA593nm is the absorbance measured at a wavelength of 593nm, and intercept and slope mean the slope and Y-intercept values of the linear regression equation of the standard curve obtained by using Fe ²⁺ for each concentration, respectively.

The cosmetic and/or pharmaceutical composition for antioxidant of the present invention has no cytotoxicity, it can be confirmed using the WST-1 (Water soluble tetrazolium salt) method, and has a level similar to that of glutathione (GSH), which is known to have an antioxidant effect. It can be confirmed that it has cell viability, so it can be safely used as a cosmetic or drug.

A cosmetic composition comprising a peptide derivative or a salt thereof comprising the peptide is a skin, lotion, solution, ointment, cream, foam, nourishing lotion, soft lotion, pack, soft water, emulsion, makeup base, essence, soap, detergent, Liquid detergent, bath products, sunscreen cream, sun oil, suspension, emulsion, paste, gel, lotion, powder, soap, surfactant-containing cleansing, oil, foundation, powder foundation, emulsion foundation, wax foundation, patch, And it may be prepared in a formulation selected from the group consisting of a spray, but is not limited thereto.

The cosmetic composition may further include one or more cosmetically acceptable carriers to be formulated in general skin cosmetics, and as common ingredients, for example, oil, water, surfactant, humectant, lower alcohol, thickener, chelate Agents, colors, preservatives, fragrances, etc. may be appropriately mixed, but the present invention is not limited thereto.

The cosmetically acceptable carrier included in the cosmetic composition varies depending on the formulation of the cosmetic composition.

When the formulation of the cosmetic composition is an ointment, paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide as a carrier component and the like may be used, but is not limited thereto. These may be used alone or in combination of two or more.

When the formulation of the cosmetic composition is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydro propellants such as, but not limited to, carbon, propane/butane or dimethyl ether. These may be used alone or in combination of two or more.

When the formulation of the cosmetic composition is a solution or emulsion, a solvent, solubilizer, or emulsifier may be used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate. , propylene glycol, 1,3-butyl glycol oil, etc. may be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic ester, polyethylene glycol or fatty acid of sorbitan Esters may be used, but are not limited thereto. These may be used alone or in combination of two or more.

When the formulation of the cosmetic composition is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester; Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth may be used, but is not limited thereto. These may be used alone or in combination of two or more.

When the formulation of the cosmetic composition is surfactant-containing cleansing, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid as carrier components Amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linoline derivative or ethoxylated glycerol fatty acid ester may be used, but is not limited thereto. These may be used alone or in combination of two or more.

In the cosmetic composition of the cosmetic composition, in addition to the above essential components, other components normally blended in cosmetics may be blended as needed. Specifically, as blending ingredients that may be added, fats and oils, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, UV absorbers, preservatives, disinfectants, antioxidants, plant extracts, pH adjusters, alcohols, pigments, fragrances, blood circulation promoters, cooling agents, limiting agents, purified water, and the like, but are not limited thereto.

A pharmaceutical composition comprising a peptide derivative or a salt thereof containing the peptide is used for primates such as humans and monkeys, rodents such as mice, rats and rabbits, and dogs, cats, cattle, pigs, sheep, horses, goats, etc. It can be administered to an administration target selected from vertebrates, such as mammals, chickens, ducks, birds including geese, snakes, lizards, turtles, and vertebrates including crocodiles, amphibians, and fish.

The pharmaceutical composition may have an effect of treating one or more diseases selected from the group consisting of type 2 diabetes, myocardial infarction, stroke and non-melanoma skin cancer, but is not limited thereto.

The method of administration of the pharmaceutical composition may be any method commonly used, for example, oral administration, or intravenous, intramuscular, subcutaneous, or intraperitoneal injection, or parenteral administration such as transdermal administration. However, the present invention is not limited thereto.

The pharmaceutical composition can be formulated and used in oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, or parenteral formulations in the form of sterile injection solutions, respectively, according to conventional methods. have.

In addition to the active ingredients described above, the pharmaceutical composition is generally selected from the group consisting of pharmaceutically and/or physiologically acceptable carriers, excipients, and diluents selected in consideration of administration methods and standard pharmaceutical practice. It may be administered in admixture with one or more selected adjuvants. For example, the pharmaceutically and/or physiologically acceptable carrier may be lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate, mineral oil, etc. may be one containing at least one selected from the group consisting of However, it is not limited thereto, and may be any carrier commonly used in the pharmaceutical field.

The pharmaceutically and/or physiologically acceptable diluents and/or excipients are all fillers, bulking agents, binders, wetting agents, disintegrants, lubricants, surfactants, etc. commonly used in the proper formulation of pharmaceutical compositions from the group consisting of It may be one or more selected.

For example, in the case of a solid preparation for oral administration such as tablets, pills, powders, granules, or capsules, the excipient is at least one or more substances for formulating the solid preparation, such as starch, calcium carbonate , sucrose (Sucrose), lactose (Lactose), may be at least one selected from the group consisting of gelatin. In addition, lubricants such as magnesium stearate talc and the like may also be used. In addition, in the case of formulation of liquid formulations for oral administration, such as suspensions, internal solutions, emulsions, and syrups, at least one selected from the group consisting of water, liquid paraffin, etc., which are commonly used simple diluents, may be used, and in addition , various commonly used excipients, for example, wetting agents, sweetening agents, flavoring agents, and/or preservatives may be included, but are not limited thereto.

For example, the pharmaceutical composition may be administered orally or orally in the form of a tablet containing starch or lactose, or in the form of a capsule containing an appropriate excipient, or in the form of an elixir or suspension containing a flavoring or coloring chemical. It may be administered intranasally or sublingually. Such liquid formulations may contain suspending agents (eg, methylcellulose, semisynthetic glycerides such as Witepsol or mixtures of Apricot kernel oil and PEG-6 esters or PEG-8 and caprylic/capric It may be formulated with a pharmaceutically acceptable excipient such as a mixture of glycerides, such as a mixture of glycerides, but is not limited thereto.

In addition, the pharmaceutical composition may be formulated in the form of injection, and pharmaceutical adjuvants such as solvents, preservatives, stabilizers, wetting agents, emulsification accelerators, suspending agents, isotonic agents and/or buffers, and other therapeutically useful substances may be added. may include When the pharmaceutical composition of the present invention is formulated and administered in the form of an injection due to the characteristic of exhibiting the efficacy of preventing hair loss or promoting hair growth, it may be a subcutaneous injection, but is not limited thereto.

The present invention relates to a cosmetic and/or pharmaceutical composition comprising a peptide derivative and/or a salt thereof, and is characterized in that it has an antioxidant effect.

1 is a schematic diagram showing the production process of one of the reduced lipoic acid-peptide derivatives.
2 is a graph showing the inhibition rate of free radical activity of the reduced lipoic acid-peptide derivative by the DPPH method. NT denotes a negative control (measured without addition of the derivative), GSH denotes a glutathione (GSH)-treated group as a positive control, the concentration of the compound used was 1000 μM, and 1 to 10 denote compounds 1 to 10, respectively.
3 is a result of measuring the reduced lipoic acid-peptide derivative by the ORAC method, and is a graph measuring the net area (netAUC) of the negative control group (measured without the addition of the derivative) from the AUC value. NT denotes a negative control group and GSH denotes a glutathione (GSH)-treated group, which is a positive control group, the concentration of the compound used was 100 μM, and 1 to 10 denote compounds 1 to 10, respectively.
4 is a graph showing the results of measuring the cell viability of the reduced lipoic acid-peptide derivatives (four candidate compounds). NT denotes no addition of the derivative, GSH denotes a glutathione (GSH) treated group as a positive control, and 2 to 5 denote compounds 2 to 5, respectively.
5 is a graph showing the inhibition rate of free radical activity of reduced lipoic acid-peptide derivatives (four candidate compounds) by the DPPH method. NT denotes no addition of the derivative, GSH denotes a glutathione (GSH) treated group as a positive control, and 2 to 5 denote compounds 2 to 5, respectively.
6 is a graph of measuring the net area (netAUC) of the reduced lipoic acid-peptide derivative (four candidate compounds) by the ORAC method by deducting the AUC value of the negative control from the AUC value. NT denotes no addition of the derivative, GSH denotes a positive control group treated with glutathione (GSH), and 2 to 5 denote compounds 2 to 5, respectively.
7 is a graph showing the FFAP value as a result of measuring the reduced lipoic acid-peptide derivative (four candidate compounds) by the FRAP method. NT denotes no addition of the derivative, GSH denotes a positive control group treated with glutathione (GSH), and 2 to 5 denote compounds 2 to 5, respectively.
8 is a graph showing the inhibition rate of free radical activity of reduced lipoic acid (LA), reduced lipoic acid (reLA), YEG peptide and lipoic acid-peptide compound (3) by the DPPH method.
Figure 9 is the result of measuring reduced lipoic acid (LA), reduced lipoic acid (reLA), YEG peptide and lipoic acid-peptide compound (3) by the ORAC method, the net area of the negative control from the AUC value limiting the AUC value (netAUC) is a measured graph.
10 is a fluorescence analysis (excitation 485 nm, mission 528 nm) of reduced lipoic acid (LA), reduced lipoic acid (reLA), YEG peptide and lipoic acid-peptide compound (3) every 1 minute, 60 This is a graph showing the results of the minutes.

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples only illustrate the present invention, and the present invention is not limited by the following examples.

Example 1. Synthesis of reduced lipoic acid-peptide derivatives

Solid-phase peptide synthesis (SPPS) (COIN, Irene; BEYERMANN, Michael; BIENERT, Michael. Solid-phase peptide synthesis: from standard procedures to The peptide was synthesized using the protocol described in the synthesis of difficult sequences.Nature protocols , 2007, 2(12), 3247-3256.).

Specifically, each amino acid coupling reaction was carried out for each amino acid/N-Hydroxybenzotriazole (HOBt)/Hexafluorophosphate Benzotriazole Tetramethyl Uronium (HBTU)/N,N-Diisopropylethylamine (DIPEA) (substitution basis, 3 equivalents / 3 equivalents / 3 equivalents / The reaction was carried out under the conditions of 6 equivalents) for 6 hours, and the reaction of binding the peptide to lipoic acid was carried out under the conditions of DIC/HOBt/DIPEA (2 equivalents / 2 equivalents / 2 equivalents based on the substitution rate) for 16 hours. Trifluoroacetic acid (TFA)/H ₂ O=95:5 (v/v), the reduction reaction of the lipoic acid-peptide compound is in 0.25N NaHCO ₃ /MeOH=3:1 (v/v) solution phase , the temperature was lowered to 0 °C, sodium borohydride (NaBH ₄ , 4 equivalents) was slowly added, and the reaction was carried out at room temperature for 2 hours. The peptides used in the preparation of the compound are γ-glutamic acid (γ-Glutamic acid; γ-Glu; γ-E), serine (Serine; Ser; S), glycine (Glycine; Gly; G), glutamic acid (Glutamic acid; Glu) ; E), histidine (Histidine; His; H), tyrosine (Tyr; Y), leucine (Leu; L), or beta-alanine (β-Alanine; β-Ala; β-A) did In addition, a linker can be used in the separation step of the lipoic acid-peptide compound, and the Fmoc-mini-PEG-OH (Fmoc-8-amino-3,6-dioxaoctanoic acid; mini-PEG) compound used as a linker is from Bioorg . . Med. Chem. Lett. It was synthesized with reference to 1995, 5(6), 573.

The peptide was purified by preparative high-performance liquid chromatography (Prep. HPLC, Shimazu, Corp., model name: 10A), and the molecular weight and purity of the peptide were confirmed by liquid chromatography-mass spectrometry (LC-Mass, Waters, Inc.). , model name: SQD2) and high performance liquid chromatography (HPLC, Shimazu, Corp., model name: 20A) were used to confirm. The compound prepared by the above reaction is represented by the following Chemical Formulas 1 and 2, and the composition of Compound 1 is shown in Table 1 below and the composition of Compound 2 according to whether the peptide and linker used are used in Table 2 below.

[Formula 1]

[Formula 2]

compound name Y ₁ Y ₂ Y ₃ Theoretical molecular weight empirical molecular weight compound 1 (γ-E) S G 481.6 482.3 compound 2 E S H 561.7 562.4 compound 3 Y E G 557.7 558.4 compound 4 Y H L 621.8 622.5 compound 5 (β-A) H - 416.5 417.2

compound name X Y ₁ Y ₂ Y ₃ Theoretical molecular weight empirical molecular weight compound 6 mini-PEG (γ-E) S G 626.7 627.3 compound 7 mini-PEG E S H 706.8 707.5 compound 8 mini-PEG Y E G 702.8 703.5 compound 9 mini-PEG Y H L 766.9 767.5 compound 10 mini-PEG (β-A) H - 561.7 562.3

During the amino acid coupling reaction, the reaction was carried out in the order of the amino acids Y ₃ , Y ₂ and Y ₁ , and the preparation process of Compound 1 by the preparation method is shown in FIG. 1 .

Example 2. Reduced lipoic acid-derivation of antioxidative candidate compounds of peptide derivatives

In order to confirm the antioxidant efficacy of the reduced lipoic acid-peptide derivatives (Compounds 1 to 10) prepared in Example 1, DPPH (1,1-Diphenyl-2-picryhydrazyl) and ORAC (Oxygen radical absorbance capacity; active oxygen absorption) ability) method, the antioxidant ability was confirmed.

Specifically, in the DPPH test method, the reduced lipoic acid-peptide derivative compounds 1 to 10 prepared in Example 1 were each added to a solvent of acetonitrile: water = 1:1 (v/v) by concentration (1, 10). , 100, 1000uM), 40 μl of each concentration sample and 160 μl of 0.1 mM DPPH were reacted at room temperature for 30 minutes, and absorbance was measured at 517 nm wavelength using ELISA equipment. As a positive control, glutathione (GSH) at the same concentration as the derivative was prepared by reacting with DPPH instead of the derivative, and as a negative control, DPPH without the addition of the derivative was prepared and the experiment was carried out. The antioxidant ability was measured by Equation 1 below, and the experimental results performed at a sample concentration of 1000 μM are shown in Table 3 and FIG. 2 below.

[Equation 1]

Free radical activity inhibition rate (%) = 100 - [(reaction absorbance of sample / reaction absorbance of blank) X 100]

(In Equation 1, the blank sample means DPPH that is not treated with the derivative)

material to be treated DPPH scavenging ability (%) positive control 13.2 negative control - compound 1 10.3 compound 2 55.4 compound 3 72.8 compound 4 4.7 compound 5 44.9 compound 6 13.3 compound 7 9.1 compound 8 5.1 compound 9 7.2 compound 10 8.1

In addition, the ORAC experimental method is a solution of sodium fluorescein diluted to 800 nM concentration in 75 mM phosphate buffer solution (pH 7.4) with 125 μl and compounds 1 to 10 prepared in Example 1 by concentration (1, 10, 100, 1000uM), mix each, add 50 μL of AAPH [75 mM, 2,2′-Azobis(2-methylpropionamidine) dihydrochloride)] to make a mixed solution, and then react at 37℃ for 60 minutes, Fluorescence analysis (excitation 485nm, emission 528nm) was performed every 1 minute to measure. As a positive control, glutathione (GSH) at the same concentration as the derivative was prepared by mixing with AAPH instead of the derivative, and as a negative control, AAPH to which the derivative was not added was prepared and the experiment was carried out. Using the measured fluorescence value, the area under the curve (AUC) of the fluorescence reduction curve was calculated by Equation 2 below.

[Equation 2]

AUC = 1+ f1/f0 + ... + fi/f0 + ... + f60/f0

(In Equation 2, f0 denotes a fluorescence value at 0 min, and fi denotes a fluorescence value at i min.)

The antioxidant activity was calculated as the net area (netAUC) by subtracting the AUC value of the negative control from the measured AUC value (netAUC = AUC value of the sample - AUC value of the negative control group), and the experimental results performed at 100 μM concentration of the compound It is shown in Table 4 and FIG. 3 below.

test substance netAUC positive control 17.2 negative control - compound 1 -2.1 compound 2 5.8 compound 3 43.0 compound 4 41.4 compound 5 -0.5 compound 6 -3.4 compound 7 0.8 compound 8 42.1 compound 9 41.0 compound 10 -0.9

As shown in Table 3, Table 4, Figures 2 and 3, when the experimental results of the measured DPPH and ORAC are summarized, reduced lipoic acid-compounds 2, 3, 4 and 5 among 10 derivatives of the peptide Four types of compounds were measured to have excellent antioxidant efficacy, and were selected as candidate compounds.

Example 3. Confirmation of toxicity in human skin fibroblasts of 4 candidate compounds

In order to confirm the cytotoxicity in human dermal fibroblasts (HDF) of the four peptides selected in Example 2, WST-1 (Water soluble tetrazolium salt; Protocol Guide: WST-1 Assay for Cell Proliferation and Viability https://www.sigmaaldrich.com/KR/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/cell-counting-and-health-analysis/cell-proliferation-reagent-wst -1) Cell viability was confirmed using EZ-cytox (http://www.dogenbio.com/shop/item.php?it_id=1490923054) as a reagent to which the method was applied.

Specifically, HDF cells (1X10 ⁴ cell/well) were treated with a sample diluted with the four peptides selected in Example 2 by concentration (1, 10, 100, 1000 μM), and then cultured for 24 hours. , WST was treated. As a positive control, glutathione (GSH) at the same concentration as the peptide was prepared by treating HDF cells instead of the peptide, and as a negative control, the peptide was prepared without treatment and the experiment was carried out. After 3 hours had elapsed, absorbance was measured at 450 nm, and cell viability was calculated and confirmed by Equation 3 below, and the results are shown in Table 5 and FIG. 4 .

[Equation 3]

Cell viability (%) = (absorbance of experimental group / absorbance of control group) x 100

(In Equation 3, the control means a sample not treated with the peptide)

processing material Cell viability (%) 10% FBS 0 μM 1 μM 10 μM 100 μM 1000 μM positive control 136.4 100.00 112.5 109.7 108.2 104.9 compound 2 136.4 100.00 112.5 109.3 108.1 105.2 compound 3 136.4 100.00 113.1 112.8 110.4 95.4 compound 4 136.4 100.00 117.9 111.2 107.8 100.9 compound 5 136.4 100.00 129.3 125.1 127.3 115.7

As shown in Table 5 and Figure 4, GSH and cell viability, known as antioxidants, were measured almost similarly, and it was confirmed that there was no toxicity in the four samples.

Example 4. Antioxidant efficacy experiment according to the concentration of 4 candidate compounds

In order to confirm the antioxidant capacity according to the treatment concentration (1, 10, 100, 1000 μM) of the four peptide compounds selected in Example 2, DPPH (1,1-Diphenyl-2-picryhydrazyl), ORAC (Oxygen radical absorbance capacity) ), FRAP (Ferric reducing ability of plasma) was confirmed by three methods.

Specifically. The DPPH and ORAC experiments were conducted in substantially the same manner as the DPPH and ORAC experiments of Example 2, and the results are shown in Tables 6 and 5, and Tables 7 and 6, respectively.

processing material DPPH scavenging ability (%) 1 μM 10 μM 100 μM 1000 μM negative control 0 0 0 0 positive control 2.74 0.39 6.06 27.17 compound 2 -0.39 -0.10 1.86 3.81 compound 3 0.00 -0.29 2.83 26.39 compound 4 2.35 0.20 1.76 6.55 compound 5 3.62 -0.88 1.08 5.47

processing material netAUC 1 μM 10 μM 100 μM 1000 μM negative control 0 0 0 0 positive control 5.38 4.82 17.35 48.45 compound 2 1.55 3.49 6.01 37.07 compound 3 2.51 13.72 46.46 50.89 compound 4 2.40 11.37 44.06 50.28 compound 5 -0.44 1.49 1.15 31.27

In addition, the FRAP experiment was performed by dissolving TPTZ (2,4,6-Tris(2-pyridyl)-s-triazine) at a concentration of 10 mM in 30 mM acetate buffer solution (pH 3.6) and 40 mM HCl solution and 20 mM ferric The chloride solution was mixed in a ratio of 10:1:1 (v/v/v) to make a FRAP reagent. 220 μl of the FRAP reagent and 10 μl of a sample diluted by concentration (1, 10, 100, 1000 μM) of the four peptides selected in Example 2 were reacted for 4 minutes, and then absorbance was measured at 593 nm wavelength, reducing power (reducing power) power) was confirmed. As a positive control, glutathione (GSH) at the same concentration as the peptide was prepared by reacting with a FRAP reagent instead of the peptide, and as a negative control, the peptide was prepared without treatment and the experiment was carried out. The FRAP value (μM) was calculated by Equation 4 below based on the Fe ²⁺ concentration, and the results are shown in Table 8 and FIG. 7 below. [Equation 4]

FRAP value (μM) = (ΔA593nm - intercept) / slope

(In Equation 4, ΔA593nm is absorbance measured at a wavelength of 593nm, and intercept and slope mean the slope and Y-intercept value of the linear regression equation of the standard curve obtained by using Fe ²⁺ for each concentration, respectively)

material to be treated reducing power (μM) 1 μM 10 μM 100 μM 1000 μM negative control 2.06 2.06 2.06 2.06 positive control 3.72 3.17 4.83 33.72 compound 2 3.17 2.61 5.94 27.06 compound 3 3.72 3.17 13.17 58.72 compound 4 3.72 4.28 5.94 34.28 compound 5 4.28 3.17 3.72 15.39

As shown in Tables 6 to 8 and Figs. 5 to 7, as a result of evaluating the antioxidant efficacy by concentration for the four compounds, the DPPH scavenging ability, netAUC value and reducing power of compound 3 were measured higher than other compounds, and the antioxidant effect was was found to be the best.

Example 5. Comparison of antioxidant efficacy of lipoic acid, reduced lipoic acid and lipoic acid-peptide compound

Compound 3, which confirmed the most excellent antioxidant effect in Example 4, is a compound to which reduced lipoic acid-YEG (Tyrosine-Glutamic acid-Glycine) peptide is bound. In order to verify the efficacy of the reduced lipoic acid and YEG peptide bound compound, lipoic acid (LA) (TCI, L0058), reduced lipoic acid (reLA) (Aldrich, T8260), YEG peptide (synthesized by itself) , the antioxidant experiment for compound 3 was carried out by concentration.

Specifically, the antioxidant effect of the material was measured in substantially the same manner as the DPPH and ORAC methods of Example 4, and each experiment was conducted without treating the materials as a control (negative control group), and the results are shown below. Tables 9 and 10 and FIGS. 8 and 9 are shown.

In addition, each sample (0.1 mM) was subjected to fluorescence analysis (excitation 485 nm, emission 528 nm) for 60 minutes to measure the intensity of fluorescence. shown in

material to be treated DPPH scavenging ability (%) 1 μM 10 μM 100 μM 1000 μM negative control 0 0 0 0 LA 0.79 0.91 2.38 3.52 reLA 2.50 14.87 72.99 80.25 YEG -0.79 -0.34 0.91 4.31 compound 3 2.27 9.42 48.58 81.27

material to be treated netAUC 1 μM 10 μM 100 μM 1000 μM negative control 0 0 0 0 LA 5.17 -5.74 1.40 22.50 reLA 4.97 -2.25 27.25 40.97 YEG 4.59 8.53 23.50 37.06 compound 3 4.11 11.97 39.91 44.19

As shown in Tables 9, 10, 8, 9 and 10, it was confirmed that compound 3 was superior to the reduced antioxidant effect and lasting effect than the reduced lipoic acid compound.

Claims (5)

Including a peptide derivative or a salt thereof comprising a peptide comprising three amino acids,
The peptide derivative has the structure of Formula 1 below,
[Formula 1]

In Formula 1 above
1) Y1 is glutamic acid (Glutamic acid; Glu; E), Y2 is serine (Serine; Ser; S), Y3 is histidine (Histidine; His; H);
2) Y1 is tyrosine (Tyrosine; Tyr; Y), Y2 is glutamic acid (Glu; E), and Y3 is glycine (Glycine; Gly; G); or
3) Y1 is tyrosine (Tyrosine; Tyr; Y), Y2 is histidine (Histidine; His; H), and Y3 is leucine (Leucine; Leu; L). A peptide derivative comprising a peptide comprising two amino acids or a salt thereof,
The peptide derivative has the structure of Formula 3 below,
[Formula 3]

In the above formula (3)
wherein Y1 is beta-alanine (β-Alanine; β-Ala; β-A), and Y2 is histidine (Histidine; His; H). The cosmetic composition for antioxidant according to claim 1 or 2, comprising the peptide at a concentration of 0.01 μM to 10,000 μM. The cosmetic composition for antioxidant according to claim 1 or 2, having DPPH radical scavenging activity. The cosmetic composition for antioxidant according to claim 1 or 2, having active oxygen absorption activity.

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