CN112367968A

CN112367968A - Cosmetic composition for removing or adsorbing fine dust comprising peptide complex as active ingredient

Info

Publication number: CN112367968A
Application number: CN201980043233.0A
Authority: CN
Inventors: 姜成珍; 金多恩; 郑有娜
Original assignee: An Yijian Co ltd
Current assignee: An Yijian Co ltd
Priority date: 2018-07-06
Filing date: 2019-07-05
Publication date: 2021-02-12
Anticipated expiration: 2039-07-05
Also published as: KR20200005499A; JP2021531247A; CN112367968B; WO2020009548A1; JP7273073B2; KR102282558B1

Abstract

The present invention relates to a cosmetic composition for removing or adsorbing fine dust comprising a fatty acid-amino acid complex or a fatty acid oligopeptide complex as an active ingredient, which has no skin toxicity and is excellent in the effect of removing fine dust, and thus, can be usefully used as a composition for preventing or treating various diseases caused by fine dust.

Description

Cosmetic composition for removing or adsorbing fine dust comprising peptide complex as active ingredient

Technical Field

The present invention relates to a cosmetic composition for removing or adsorbing fine dusts, comprising a peptide complex as an active ingredient.

Background

Particulate Matter (PM) is a particulate matter containing Nitrate (NO)₃ ^-) Ammonium (NH)₄ ⁺) Sulfate (SO)₄ ^2-) The ionic component, the air contaminant such as a carbon compound or a metal compound, and the like mean a particulate substance having a diameter of 10 μm or less which drifts in the air for a long time. If the particle diameter of the fine dust is less than 10 μm, it is marked as PM10, and if the particle diameter is less than 2.5 μm, it is marked as PM2.5, and it is called ultra fine dust or ultra fine dust. Most of the ultrafine dusts are generated by human factors such as dusts floating in automobile exhaust, industrial processes, roads, and the like.

When exposed to dust for a long time, the immunity is rapidly reduced, so that respiratory diseases such as cold, asthma, tracheitis and the like may be suffered, and various diseases such as cardiovascular diseases, skin diseases, eye diseases and the like may be suffered. It is reported that mote deteriorates respiratory diseases such as asthma and causes a decrease in lung function, and particularly, particles of ultra-mote are extremely fine and thus are not filtered through nasal mucosa but directly permeate into alveoli, thereby increasing the prevalence and early mortality of lung diseases. Further, the fine dust can invade not only into the body through the nose and mouth but also into the body through the skin. The fine dusts are about 20 times larger than the size of pores, and thus, easily invade into the pores and are hardly removed, and the skin immunity may be lowered due to the increase of inflammatory cytokines and active oxygen in vivo caused by the absorbed fine dusts, thereby further accelerating the skin aging phenomenon such as acne, wrinkle formation, skin dryness and pigmentation. Specifically, the fine dust may induce an inflammatory reaction to damage the skin barrier, thus exacerbating atopic dermatitis, generating reactive oxygen species (reactive oxygen species) in mitochondria to reduce collagen synthesis, and increasing collagen decomposition to induce skin aging. Furthermore, Polynuclear Aromatic Hydrocarbons (PAH) attached to fine dust proliferate melanocytes to increase facial pigmentation spots, and particularly, asian people have darker skin color than white people, so that the pigmentation spots caused by fine dust are more likely to increase, and dermatitis, burning, and itching of people with sensitive skin can be increased. In the case of prolonged exposure to fine dust, the skin loses its function of protecting the body from stress factors due to irrecoverable skin damage, and may also cause serious diseases such as skin cancer.

Recently, as the risk and harm of fine dust become serious, the interest in the development of products for preventing fine dust has been increased, the sales of products for cosmetics, health functional foods, air cleaners, masks, and the like related to fine dust have been increased sharply, and in particular, the development of cosmetic compositions that can remove fine dust or significantly prevent the adsorption of fine dust has been continuously performed. A representative material among highly functional biomaterials that meet this trend is a peptide material. According to the arrangement and structure of the amino acid sequence in the peptide, the peptide has physiological activity in vivo or on the skin, and thus can be applied to the development of biological materials in various ways, and also can be applied to the development of quasi-therapeutic products for wrinkle improvement, whitening, allergic skin and improvement of various skin diseases, and thus has an excellent industrial effective value as a cosmetic and pharmaceutical material.

On the other hand, korean patent No. 1723941 discloses "cosmetic composition for adsorbing and removing fine dust containing coral tree and kokura resin extract as an active ingredient", and korean patent No. 1850313 discloses "cosmetic composition for removing heavy metal or fine dust containing chrysanthemum extract as an active ingredient", but does not disclose the cosmetic composition for removing or adsorbing fine dust containing the peptide complex of the present invention as an active ingredient.

Disclosure of Invention

Technical problem

The present inventors have derived from the above-mentioned requirements, and have developed a fatty acid-amino acid complex and a fatty acid oligopeptide complex by binding a saturated fatty acid or an unsaturated fatty acid to the N-terminal, C-terminal or side chain of an amino acid or an oligopeptide, and have confirmed that the above-mentioned complex can reduce cytotoxicity due to dust particles and can aggregate with the dust particles to remove the dust particles, thereby completing the present invention.

Means for solving the problems

In order to solve the above problems, the present invention provides a cosmetic composition for removing or adsorbing fine dust comprising a complex in which a fatty acid is bound to the N-terminus, C-terminus or side chain of an amino acid or oligopeptide as an effective ingredient.

ADVANTAGEOUS EFFECTS OF INVENTION

The peptide complex of the present invention is excellent in the effect of protecting cells from dust particles and the effect of removing dust particles by aggregation or binding with dust particles, and thus, the composition comprising the peptide complex of the present invention can be used as a cosmetic or pharmaceutical composition for the prevention and treatment of various diseases caused by exposure to dust particles.

Drawings

Fig. 1 is a schematic diagram showing a method of binding a fatty acid at the N-terminal (a), C-terminal (B), or side chain (C) of an oligopeptide (pentapeptide).

FIGS. 2 to 4 are the results of analyzing the peptide complex (Table 5-division 9) purified by High Performance Liquid Chromatography (HPLC).

FIGS. 5 to 15 are results of confirming the cell survival rate after keratinocyte cells were treated with ultra fine dust (diesel exhaust particles: fine dust having a diameter of 2.5 μm or less, PM2.5) and the peptide complex of the present invention at the same time. N.C. (negative control group): treatment with 0.2% dimethyl sulfoxide (DMSO)/dalberg's Modified Eagle Medium (DMEM, Dulbecco's Modified Eagle Medium), P.C. (positive control): 10% dimethylsulfoxide/Darber modified Ile-Gel medium.

FIG. 16 is a graph showing cytotoxicity of concentration-treated solutions loaded with ultra-fine dust on human lung cancer epithelial cell line (A549).

Fig. 17 is a result of confirming cell viability after treating a549 cells with ultra-fine dust and a complex in which oligopeptide binds saturated fatty acid. The information for the X-axis sample of the graph is the same as in table 10 below.

FIG. 18 is a photograph of SDS-PAGE gel by silver staining (silver staining) to analyze the agglutination effect of the peptide complex and the ultra fine dust.

Fig. 19 is a graph showing the particle removal efficiency of general masks (HIP's) and a fine dust mask (KF84) sold in the market using the dust trapping (particle removal) efficiency evaluation system.

Fig. 20 and 21 are graphs showing the efficiency of removing ultrafine particles from a general mask not sprayed with any substance, a general mask sprayed with purified water, and a general mask sprayed with a fatty acid oligopeptide complex of 250ppm (diluted with distilled water) by the dust collection (particle removal) efficiency evaluation system.

Detailed Description

In order to achieve the object of the present invention, the present invention provides a cosmetic composition for removing or adsorbing fine dust comprising a complex in which a fatty acid is bound to the N-terminus, C-terminus or side chain of an amino acid or oligopeptide as an effective ingredient.

In the present invention, the term "dust" is a concept including ultra-fine dust, the term "dust" refers to dust having a diameter of 10 μm or less, and the term "dust" refers to dust having a diameter of 2.5 μm or less, and may include, but is not limited to, yellow sand, automobile exhaust gas, factory soot, combustion gas in daily life, and heavy metals.

In the present invention, the term "fine dust adsorption" may be used to remove fine substances remaining in pores or the like in the skin by adsorption. The adsorption force to fine dust is improved by the treatment of the cosmetic composition based on fatty acid, amino acid complex or fatty acid and oligopeptide complex, thereby causing less irritation to the skin and solving skin inflammation or skin allergy, etc., which may occur when fine dust remains.

In the present invention, the terms "N-terminal and C-terminal" refer to amino groups (-NH) in the amino acid sequence of a peptide₂) One side end ofAnd the other terminal end having a carboxyl group (-COOH), the term "side chain" refers to a carbon chain branched from a straight chain carbon atom, wherein the carbon atoms are regularly linked, or an aliphatic carbon chain linked on the ring of a cyclic compound.

In the cosmetic composition for removing or adsorbing fine dust of the present invention, the amino acid may be one or more selected from the group consisting of Alanine (a), Cysteine (C), Aspartic acid (D), Glutamic acid (Glutamic acid, E), Phenylalanine (phenyl Alanine, F), Glycine (Glycine, G), Histidine (histadine, H), Isoleucine (isoleucin, I), Lysine (Lysine, K), Leucine (Leucine, L), Methionine (Methionine, M), Asparagine (Asparagine, N), Lysine (pyrolysine, O), Proline (Proline, P), Glutamine (Glutamine, Q), Arginine (Arginine, R), Serine (Serine, S), Threonine (Threonine, T), Selenocysteine (Serine, U), Valine (Valine, V), Tyrosine (Tyrosine, Y), may be glycine (G), glutamic acid (E) or lysine (K), but is not limited thereto. The amino acids may include, but are not limited to, D-form amino acids and L-form amino acids.

In the cosmetic composition for removing or adsorbing fine dust according to the present invention, the oligopeptide is a peptide comprising 2 or more amino acids, and preferably, may be one selected from the group consisting of AAPV (seq id No. 1), AAP (seq id No. 2), AA (seq id No. 3), GHK (seq id No. 4), GH (seq id No. 5), KTTKS (seq id No. 6), KTTK (seq id No. 7), KTT (seq id No. 8), KT (seq id No. 9), EEMQRR (seq id No. 10), EEMQR (seq id No. 11), EEMQ (seq id No. 12), EEM (seq id No. 13), EE (seq id No. 14), KKKKKKKKK (seq id No. 15), kkkkkkkkkkkkk (seq id No. 16), KKK (seq id No. 17), KK (seq id No. 18), GPO (seq id No. 19), GP (seq id No. 20), GQPR (seq id No. 21), GQP (seq id No. 22), GQ (seq id No. 23), TTKS (seq id No. 24), TKS (seq id No. 25), TK (seq id No. 26), but is not limited thereto. The amino acid of the oligopeptide may be a D-form amino acid or an L-form amino acid, and the amino acid PO of the sequence 19 may be O-hydroxyproline (O-hydroxyproline), but is not limited thereto.

In the cosmetic composition for removing or adsorbing fine dust according to the present invention, the fatty acid may be a saturated fatty acid or an unsaturated fatty acid, but is not limited thereto.

The above-mentioned saturated fatty acid of the present invention may be one selected from the group consisting of Caproic acid (Caproic acid, C6:0), Caprylic acid (Caprylic acid, C8:0), Pelargonic acid (Pelargonic acid, C9:0), Capric acid (Capric acid, C10:0), undecanoic acid (Undecylic acid, C11:0), Lauric acid (Lauric acid, C12:0), Myristic acid (Myrisic acid, C14:0), pentadecanoic acid (Pentadecylic acid, C15:0), Palmitic acid (Palmitic acid, C16:0), Margaric acid (Margaric acid, C17:0), Stearic acid (Stearic acid, C18:0), nonadecanoic acid (Nondelic acid, C19:0), Arachidic acid (Arachidic acid, C20:0), Stearic acid (Stearic acid, C18: 960), eicosanoic acid (C3937: 24), c26:0), heptacosanoic acid (C27: 0), Montanic acid (Montanic acid, C28:0), nonacosanoic acid (nonacosalicylic acid, C29:0), Melissic acid (Melissic acid, C30:0), hentriacontylic acid (C31: 0), lacceric acid (Lacceroic acid, C32:0), tridecanoic acid (Psyllic acid, C33:0), tetratriacontanoic acid (Geddic acid, C34:0), pentacontanoic acid (Ceroplastic acid, C35:0), and hexadecanoic acid (hexatriacontanoic acid, C36:0), preferably, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, heptacosanoic acid, or behenic acid, but not limited thereto.

And, the above unsaturated fatty acid may be one selected from the group consisting of Palmitoleic acid (Palmitoleic acid, C16:1), Oleic acid (Oleic acid, C18:1), Elaidic acid (Elaidic acid, C18:1), Petroselinic acid (Petroselinic acid, C18:1), Vaccenic acid (C18: 1), whale acid (Gondoic acid, C20:1), sinapic acid (Erucic acid, C22:1), Nervonic acid (Nernic acid, C24:1), Linoleic acid (Linoleeic acid, C18:2), Linolenic acid (cis-Linolenic acid, C18:3), Punicic acid (Punicic acid, C18:3), Eleostearic acid (Eleosteic acid, C18:3), stearidonic acid (C63894: 894), Arachidonic acid (Dosinonic acid, C964: 36), Arachidonic acid (Adrenanoic acid, Dosinoic acid, C3637: 22), c22:6) and Ricinoleic acid (Ricinoleic acid, C18:1), preferably oleic acid, linoleic acid, palmitoleic acid, elaidic acid, petroselinic acid, vaccenic acid, macrocephalic acid, sinapic acid, nervonic acid, linolenic acid, punicic acid, eleostearic acid, herring acid or Ricinoleic acid, but not limited thereto.

The cosmetic composition for removing or adsorbing fine dust according to the present invention is a fatty acid-amino acid complex or a fatty acid-oligopeptide complex in which a fatty acid is bound to the N-terminal, C-terminal or side chain of an amino acid or an oligopeptide, wherein the fatty acid-amino acid complex may be formed by binding oleic acid, which is an unsaturated fatty acid, to glycine (G), glutamic acid (E) or lysine (K), but is not limited thereto, and the fatty acid-oligopeptide complex may be formed by binding one of the N-terminal, C-terminal or side chain of an oligopeptide formed from the amino acid sequences of seq id No. 1, seq id No. 4, seq id No. 6, seq id No. 7, seq id No. 8, seq id No. 10, seq id No. 11, seq id No. 15, seq id No. 16, seq id No. 17, seq id No. 19, seq id No. 21, seq id No. 27, seq id No. 28, seq id No., One or more saturated fatty acids selected from the group consisting of stearic acid, behenic acid and behenic acid, or one or more unsaturated fatty acids selected from the group consisting of oleic acid, linoleic acid, palmitoleic acid, elaidic acid, petroselinic acid, vaccenic acid, macrocephalic acid, erucic acid, nervonic acid, linolenic acid, punicic acid, elaeostearic acid, menhaden acid and ricinoleic acid, but not limited thereto.

Preferably, in the cosmetic composition for removing or adsorbing fine dust according to the present invention, the complex in which the fatty acid is bound to the side chain of the oligopeptide may be, but is not limited to, a ninth Lysine (Lysine, K) in the oligopeptide amino acid sequence of SEQ ID No. 30 (LLWIALRKK, L-amino acid; llwilarkk, D-amino acid), a fourth Lysine in the oligopeptide amino acid sequences of SEQ ID No. 6(KTTKS) and SEQ ID No. 7 (KTTKK), and a fifth Lysine in the oligopeptide amino acid sequence of SEQ ID No. 16 (KKKKKKKKK) bound to the fatty acid.

Further, the peptide complex of the present invention may be a peptide complex in which an amino group (-NH) is bonded to the N-terminus or C-terminus₂) Or a carboxyl group (-COOH), specifically NH₂Amino acids-fatty acids-COOH, NH₂Oligopeptide-fatty acid-COOH, COOH-amino acid-fatty acid-NH₂COOH-oligopeptide-fatty acid-NH₂、NH₂-fatty acid-amino acid-COOH, NH₂-fatty acid oligomer peptide-COOH, COOH-fatty acid-amino acid-NH₂And COOH-fatty acid oligomer peptide-NH₂The form is not limited to this.

The compound of the present invention in which a saturated fatty acid or an unsaturated fatty acid is bonded to the N-terminal, C-terminal or side chain of an amino acid or oligopeptide has no toxicity to normal skin, can reduce cytotoxicity caused by fine dust, and has an effect of removing ultrafine dust particles (dust trapping).

Specifically, the capric acid-oligopeptide-binding complex of the present invention, palmitic acid-binding complex of, A complex of binding of heptadecanoic acid to an oligopeptide of seq id No. 15, seq id No. 16, seq id No. 27 or seq id No. 28, a complex of binding of heptadecanoic acid to an oligopeptide of seq id No. 15, seq id No. 16, seq id No. 27, seq id No. 28 or seq id No. 30, a complex of binding of stearic acid to an oligopeptide of seq id No. 15, seq id No. 16, seq id No. 27, seq id No. 28 or seq id No. 30, a complex of binding of behenic acid to an oligopeptide of seq id No. 15, seq id No. 16, seq id No. 27 or seq id No. 30, a complex of binding of behenic acid to an oligopeptide of seq id No. 15, seq id No. 27 or seq id No. 30, a complex of binding of elaidic acid to an oligopeptide of seq id No. 6, seq id No. 15, seq id No. 16, seq id No. 19 or seq id, A complex of vaccenic acid bound to an oligopeptide of sequence 15, sequence 27 or sequence 28, a complex of macrocephalic acid bound to an oligopeptide of sequence 6, sequence 15, sequence 27, sequence 28 or sequence 30, a complex of sinapic acid bound to an oligopeptide of sequence 15, sequence 27, sequence 28 or sequence 30, a complex of nervonic acid bound to an oligopeptide of sequence 4, sequence 6, sequence 15, sequence 16, sequence 27, sequence 28 or sequence 30, a complex of linolenic acid bound to an oligopeptide of sequence 15, sequence 16, sequence 27, sequence 28 or sequence 30, a complex of punicic acid bound to an oligopeptide of sequence 6, 15, 16 or 27, a complex of eleostearic acid bound to an oligopeptide of sequence 6, sequence 15, sequence 27 or sequence 30, a complex of menhaden acid bound to an oligopeptide of sequence 1, sequence 15, sequence 16 or sequence 29, or a complex of ricinoleic acid bound to an oligopeptide of sequence 15, sequence 28, The oligopeptide of sequence 27 or sequence 30 bound complex reduces cytotoxicity increased by ultra-fine dust and increases ultra-fine dust particle removal (dust capture) rate.

Preferably, the cosmetic composition for removing or adsorbing fine dusts of the present invention is in a form selected from one or more of a solution, a suspension, an emulsion, a paste, a gel, a cream, a skin lotion, a powder, a soap, a surfactant-containing cleanser, an oil, a foundation, a wax foundation and a spray, and more preferably, may be in a form selected from the group consisting of a skin ointment, a cream, a softening emulsion, a nourishing emulsion, a mask, a essence, a hair tonic, a shampoo, a hair conditioner, an ointment, a gel, a skin lotion, a skin softener, a skin toner, an astringent, a skin lotion, a moisturizing emulsion, an emulsion, a massage cream, a nourishing cream, an eye cream, a moisturizing cream, a hand cream, a foundation, a nourishing essence, a sunscreen cream, a soap, a cleansing foam, a cleansing emulsion, a cleansing cream, a moisturizing lotion and a cleansing cream, but is not limited thereto. The composition of these respective dosage forms may contain appropriate various bases and additives for formulation of the dosage forms thereof, and the kinds and amounts of these ingredients may be selected by those of ordinary skill in the art to which the present invention pertains.

In the case where the dosage form of the present invention is a paste, cream or gel, as a carrier ingredient, animal fiber, plant fiber, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zincic acid, and the like can be used.

In the case where the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, and polyamide powder can be used as a carrier component, and particularly, in the case of a spray, a propellant such as chlorofluorocarbon or propane/butane dimethyl ether can be additionally used.

In the case where the formulation of the present invention is a solution or emulsion, a solvent, an emulsifier, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol oil, a glycerol aliphatic ester, polyethylene glycol, a fatty acid ester of sorbitan is used as a carrier component.

In the case where the dosage form of the present invention is a suspension, as a carrier ingredient, a liquid diluent such as water, ethanol and propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth, or the like can be used.

When the formulation of the present invention is a surfactant-containing detergent, fatty alcohol sulfate, fatty alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamino betaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, a linoline derivative, and ethoxylated glycerol fatty acid ester can be used as the carrier component.

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

Example 1: preparation of peptide complexes

Amino acid starting materials for complex synthesis were purchased from GLS (GL Biochem, Shanghai), fatty acids from TCI (TCI chemicals, India) and Sigma (Sigma Aldrich, US) (table 1), Dimethylformamide (DMF), diisopropylethylamine (N, N-diisopropyrephyllylamine, DIEA), Dichloromethane (DCM), and piperidine (piperidine).

TABLE 1

Fatty acid purchase information

1-1. resin swelling (shelling)

The synthesis of peptides having carboxyl groups (-COOH) at the end of the complex using a solid-phase (solid-phase) synthesis reactor equipped with a filtration membrane using 2-chlorotrifluoroethylene resin 2-Chlorotritylchloride resin; bead Tech, Korea), conjugated with a peptide bond (-CONH) at the end of the complex₂) For the terminal peptide, a starting resin for polypeptide synthesis (Rink amide resin; GLS, china). The resin was allowed to swell for 20 minutes using dichloromethane and dimethylformamide to prepare the synthesis.

1-2 amino acid Loading (loading)

The synthesis using chlorotrifluoroethylene resin involves a first loading of amino acids into the resin. Under reduced pressure, the solvent was removed by filtration through a membrane. After 3 to 5 equivalents of amino acid are completely dissolved in dimethylformamide, the resulting solution is added to a chlorotrifluoroethylene resin, and diisopropylethylamine in consideration of density is added in an amount corresponding to 3 to 5 equivalents of the chlorotrifluoroethylene resin. Then, the reaction was carried out at a temperature of 24 to 32 ℃ for 5 hours or more using a reactor.

1-3 deprotection of resin fluorenylmethoxycarbonyl chloride

The synthetic process using the above chlorotrifluoroethylene resin or RINK amide resin includes a deprotection reaction process at fluorenylmethoxycarbonyl chloride (Fmoc). In the deprotection process of the resin fluorenylmethoxycarbonyl chloride, the solvent was removed by a filtration membrane under reduced pressure, and after washing for 5 minutes by adding 20% (v/v) piperidine in dimethylformamide, washing was performed again for 10 minutes. The reaction solution was removed by filtration under reduced pressure, and washed with dichloromethane or Dimethylformamide (DMF) for 5 minutes 6 times or more.

1-4 synthesis of fatty acid-amino acid compound and fatty acid oligopeptide compound

After 3 to 5 equivalents of amino acid was completely dissolved in dimethylformamide, the solvent-removed RINK amide resin was added. As a coupling reagent (coupling reagent), 2M hydroxybenzotriazole/diisopropylcarbodiimide (HOBt/DIC, hydroxybenzotriazole/diisopropylcarbodiimide) was placed in such amounts that the amino acid equivalent and the amount of RINK amide resin were matched. And then, synthesizing for more than 5 hours at the temperature of 24-32 ℃ by using a reactor. When the reaction was completed, the solvent was removed by filtration through a membrane under reduced pressure, and then washed 6 times with clean dimethylformamide for 5 minutes. After completion of washing, amino acids were coupled in order by the same method as the above-described amino acid binding method, respectively. Then, 3 equivalents of 2M hydroxybenzotriazole/diisopropylcarbodiimide were added to the resin in the state of peptide synthesis in such an amount that the amino acid equivalent and the resin amount were satisfied. And then, reacting for more than 5 hours at the temperature of 24-32 ℃ by using a reactor.

1-5 separation (clearage)

After the reaction was completed, the solvent was removed by filtration membrane under reduced pressure, washed 2 times for 2 minutes by clean dimethylformamide, washed 2 times for two minutes by dichloromethane, and then removed by filtration membrane under reduced pressure. Utilizing 70% (v/v) trichloroacetic acid/29% (v/v) dichloromethane/1% (v/v) H₂O solution, the dried peptide complex resin was isolated for 4 hours.

1-6 recrystallization (crystalize)

The crude product was recrystallized and extracted with diethyl ether (ethyl ether) against the solvent to complete the separation. The specific synthetic procedures are shown in table 2 below.

TABLE 2

Procedure for Synthesis of peptide Complex of the present invention

Resin substitution rate: -Rink amide MBHA resin (substitution rate: 0.54mmole/g) -Chlorotrityl chloride resin (substitution rate: 1.25mmol/g) amino acids: 3 to 10eq of coupling agent: diisopropylcarbodiimide, hydroxybenzotriazole, dimethylformamide coupling time: 6 hours coupling temperature: separating at 24-32 ℃: 70% trichloroacetic acid/29% dichloromethane/1% H₂O, 4 hours, 5% trichloroacetic acid/95% dichloromethane 5 minutes extraction: diethyl ether

1-7. Synthesis of products

Novel complexes in which fatty acids having 6 to 24 carbon atoms (table 3) are bound to glycine (G), glutamic acid (E), lysine (K) or oligopeptides having sequences 1 to 30 as amino acids were developed by Solid Phase Peptide Synthesis (SPPS). Specifically, an amino group (NH) present at the N-terminus of an amino acid or oligopeptide may be bound₂) And a carboxyl group (COOH) of a fatty acid (fig. 1A), and a carboxyl group of a fatty acid (fig. 1B) may be bound to a side chain of lysine (K) present in an oligopeptide, using glycol as a crosslinking agent (linker).

Finally, the complexes of synthetic amino acids or oligopeptides with fatty acids are presented in tables 4 and 10 below, in table 10, the upper case is the L-form amino acid and the lower case is the D-form amino acid. The fatty acid-bound complex at the N-terminus of an amino acid or oligopeptide is described in the order of fatty acid-peptide, the fatty acid-bound complex at the C-terminus of an amino acid or oligopeptide is described in the order of peptide-fatty acid, and the fatty acid-bound complex at the side chain of an oligopeptide is shown in parentheses after the amino acid attached to the side chain of the fatty acid is underlined (a), and is described in the following.

TABLE 3

Kind of fatty acid

TABLE 4

Synthetic fatty acid-amino acid complex and fatty acid-oligopeptide complex species

TABLE 5

Synthetic fatty acid oligopeptide complex species

TABLE 6

Synthetic fatty acid oligopeptide complex species

TABLE 7

Synthetic fatty acid oligopeptide complex species

TABLE 8

Synthetic fatty acid oligopeptide complex species

TABLE 9

Synthetic fatty acid oligopeptide complex species

Watch 10

Synthetic fatty acid oligopeptide complex species

Example 2: complex purification

The crude complex synthesized in the above example 1 was dissolved in distilled water to which 10% (v/v) acetonitrile (acetonitrile) was added, respectively, and then purified by High Performance Liquid Chromatography (HPLC) under gradient conditions (fig. 2 to 4), and freeze-dried to obtain the objective complex. The conditions for high performance liquid chromatography and the gradient conditions for the separation are shown in tables 11 and 12 below. Further, the molecular weight of the purified crude complex was measured by MALDI-TOF-MASS, and the molecular weight measurement method is shown in Table 13 and the results of the molecular weight measurement are shown in tables 14 to 23.

TABLE 11

High performance liquid chromatography conditions

TABLE 12

Gradient condition

Time (minutes)	H₂0(％)	Acetonitrile (acetonitrile) (%)
			0～12	89→77	11→23
12～13	77→5	23→95
			13～18	5→5	95→95
18～19	5→89	95→11
			19～27	89→99	11→11

Watch 13

Method for measuring molecular weight

TABLE 14

Molecular weight confirmation

K side chain: the ninth K amino acid (underlined) of the oligopeptide of sequence 30 (LLWIALRKK, llwilrkk) binds to the fatty acid.

Watch 15

Molecular weight confirmation

TABLE 16

Molecular weight confirmation

TABLE 17

Molecular weight confirmation

Watch 18

Molecular weight confirmation

Watch 19

Molecular weight confirmation

Watch 20

Molecular weight confirmation

TABLE 21

Molecular weight confirmation

TABLE 22

Molecular weight confirmation

TABLE 23

Molecular weight confirmation

Example 3: analysis of the cytotoxicity of ultra-fine dusts in human keratinocytes (HaCat)

In order to confirm the cytotoxicity of the fatty acid-amino acid complex and the fatty acid oligomer peptide (SEQ ID Nos. 1 to 30) complex, the number of cells reached 2X 10⁴The cells/ml were diluted in Darber modified eagle's medium (10% fetal bovine serum), and cultured for 16-20 hours after being seeded in 100. mu.l of a 6-well plate until the cells were attached to the plate surface. The medium was removed, and each complex substance was diluted to a concentration of 10 μm by Dulbecco's Modified Eagle's Medium (DMEM) and treated in cells at 100. mu.l, at 37 ℃ with 5% CO₂After 24 hours of incubation in the incubator, the medium was removed again. After a 10-fold diluted solution of thiazole blue (5mg/ml in Phosphate Buffered Saline (PBS)) reagent was inoculated into Darber's modified eagle's medium in 100. mu.l and reacted for 3 to 4 hours, the thiazole blue reagent was removed, dimethyl sulfoxide (DMSO) was inoculated in 100. mu.l and reacted in an incubator for 30 minutes, and then the Darber's modified eagle's medium-treated group, 0.2% dimethyl sulfoxide/Darber's modified eagle's medium-treated group (negative control group), 10% dimethyl sulfoxide/Darber's modified eagle's medium-treated group (positive control group) and the above-mentioned tables 4 to 10 were treated by measuring absorbance (540nm)Cell viability in the group of complexes of disclosed amino acids or oligopeptides bound to fatty acids.

As a result, the cell survival rate in the group treated with the complex was at a level of 100 ± 4% similar to that of the negative control group when compared with the negative control group having a cell survival rate of 100%, and the cell survival rate in the positive control group for cytotoxicity was 8-25% (retention result not revealed). Thus, the complex of the present invention, which binds a fatty acid at the N-terminus, C-terminus or side chain of an amino acid or oligopeptide, is not toxic to skin cells.

Example 4: cytoprotective effect of amino acid or oligopeptide and fatty acid complex of ultramicro dust (human keratinocyte)

In order to confirm whether the fatty acid-amino acid complex and the fatty acid oligomer peptide (seq. nos. 1 to 30) complex could reduce the cytotoxicity increased by the ultra-fine dust, ultra-fine dust (Diesel exhaust particles, DEP) and complex treatment were performed on human skin-derived keratinocytes (HaCaT cells), and the cytotoxicity was analyzed by the thiazole blue analysis method. The diesel exhaust particles (PM 2.5, which is a fine dust having a diameter of 2.5 μm or less) were purchased from National Institute for Environmental standards, Japan, and collected by a glass fiber filter by operating a 2740cc4 cylinder direct injection diesel engine (Isuzu Automobile co., Japan) model 4JB1 under a load condition of 1500rpm and a torque condition of 10 (10 kg/ml).

So as to make the number of cells reach 2X 10⁴The cells were diluted in Darber modified eagle's medium (10% fetal bovine serum) at a volume/ml, and cultured for 16 to 20 hours after being seeded in 100. mu.l in a 96-well plate until the cells were attached to the plate surface. After completion of the culture, the medium was taken out, and 100. mu.l of each of the solutions containing 10 μm of the complex and 50. mu.g/ml of ultra-fine dust was inoculated into each well at 37 ℃ with 5% CO₂After 24 hours of incubation in the incubator, the medium was removed again. Thereafter, the cell viability was confirmed by thiazole blue assay in the same manner as in example 3.

As a result, a significant increase in cell survival rate was confirmed in the group treated with the ultra fine dust and the complex of the amino acid or oligopeptide and the fatty acid simultaneously, compared to the group treated with the ultra fine dust alone (fig. 5 to 15).

Thus, the complex of the present invention, which binds fatty acids at amino acids or oligopeptides, can effectively protect skin cells by agglutinating ultra fine dust.

Example 5: analysis of ultra-fine dust cytotoxicity in Lung cancer epithelial cells (A549)

Cytotoxicity was analyzed by thiazole blue assay after ultramicro dust treatment in human lung cancer epithelial cell line (a 549). The thiazole blue assay was performed by the same method as in example 3 above, and ultramicrodusts were treated at concentrations of 50. mu.g/ml, 60. mu.g/m, 70. mu.g/ml, 80. mu.g/ml, 90. mu.g/ml, 100. mu.g/ml, 110. mu.g/ml, 120. mu.g/ml and 200. mu.g/ml in cultured lung cancer epithelial cells.

As a result, the cell viability decreased as the treatment concentration of the ultra-fine dust increased, and in particular, only about 20% of the cells survived in the 200. mu.g/ml dust-treated group at the highest concentration (FIG. 16).

Example 6: analysis of the cytoprotective Effect of peptide and fatty acid complexes on ultramicro dust (Lung cancer epithelial cells)

To confirm whether the fatty acid oligomer peptide (a saturated fatty acid bonded to the side chain of the amino acid sequence of SEQ ID NO: 30) complex can reduce cytotoxicity due to ultrafme dust, the cytotoxicity was analyzed by thiazole blue assay. The thiazole blue assay was performed by the same method as in example 3 above.

As a result, cell survival rate was increased in the group treated with the fatty acid oligopeptide complex, particularly, significantly increased in the peptide complex bound to the remaining saturated fatty acids of C8 to C16 except for the saturated fatty acid of C6, compared to the group treated with the ultra fine dust alone (about 40%), wherein the complex bound with Capric acid (C10) (P4, P11, P18, P25) exhibited a cell survival rate of 70% or more, thereby confirming a reduction in cytotoxicity against the ultra fine dust (fig. 17).

Example 7: analysis of the aggregation Effect of fatty acid oligopeptide complexes and ultrafine dust Using silver staining (silver staining)

Ultramicrodust and fatty acid oligomer peptide complex P11 (capric acid bonded to the side chain of the amino acid sequence of SEQ ID NO: 30) were treated simultaneously on a 20% SDS-PAGE gel, and the amount of the complex not aggregated with the ultramicrodust was confirmed by silver staining.

After treating 100. mu.g/ml concentration of ultrafme dust alone or together with 10 μm, 50 μm, 100 μm concentration of P11 complex in Dulbecco's modified eagle's medium at 37 ℃ with 5% CO₂The culture was carried out in an incubator for 24 hours, and a first-class liquid sample was prepared by centrifugal separation with a centrifugal force of 10000Xg for 1 hour. Then, the 20% SDS-PAGE gel was filled with 15. mu.l of the supernatant and subjected to electrophoresis. Silver staining procedure kit using ELPIS biotechnology (Peptigel)^TMELPISBIOTECH, EBA-1053) is performed as follows. The gel subjected to electrophoresis and a solution of 30% ethanol mixed with 10% acetic acid were reacted for 1 hour to solidify, and washed 2 times with distilled water. Then, the gel was placed in solution A1 minutes and washed 2 times with distilled water, where the gel was placed in solution B2 containing formaldehyde for 20 to 30 minutes. Finally, after washing the gel 2 times with distilled water for 1 minute, it was contained in solution C and waited for the appearance of bands, and the reaction was stopped using a fixative.

As a result, the intensity of the band was slightly decreased in the group in which the ultrafme and the complex P11 were treated simultaneously, as compared with the group treated with the complex P11 alone, and it was confirmed that the ultrafme and the fatty acid oligopeptide complex were aggregated and the amount of the peptide existing in the supernatant was decreased (FIG. 18). Thus, the oligopeptide-fatty acid-bound complex of the present invention is aggregated with ultrafine dust, whereby ultrafine dust can be removed.

Example 8: analysis of dust Capture (particle removal) efficiency of fatty acid oligopeptide composites Using comparison of micronic particle penetration of masks

In order to confirm whether or not the fatty acid oligopeptide compound adsorbs the moving ultrafine dust to improve the filtration efficiency of the mask, the dust collection (particle removal) efficiency was analyzed, and the evaluation method and calculation formula of the dust collection efficiency are shown in table 24 below.

Watch 24

Evaluation and calculation formula of dust trapping (particle removal) efficiency

Mask particle removal efficiency (%) was measured by quantifying the amount of 50nm to 300nm sized fine dust particles in the air passing through general masks (HIP's) and fine dust masks (KF84), and the fine dust masks had particle removal efficiency of about 89 to 100% for 50 to 300nm sized fine dust particles (dust capture), whereas the general masks had particle removal efficiency of about 39 to 57% for 50 to 300nm sized fine dust particles (fig. 19).

Further, as a result of measuring mask particle removal (separation and collection) efficiency (%) by spraying distilled water or a 250ppm solution of a fatty acid oligopeptide complex (diluted with distilled water) once onto a general mask and then quantifying the amount of fine dust particles having a size of 50 to 300nm in the air, the mask particle removal efficiency (%) was similar to that of a general mask not sprayed with any substance and a general mask sprayed with distilled water, and the mask treated with a solution of a fatty acid-peptide complex (Palmitic acid-KTTKS, Palmitic acid-rrrr, Palmitic acid-kkkkkkkkk, Capric acid-rrr, and Capric acid-RRRRRRRRR) had an increased dust collection efficiency compared to a general mask sprayed with any substance, and in particular, the smaller the size of the fine dust particles was excellent in the particle removal efficiency of the fatty acid-peptide complex (fig. 20 and 21).

Sequence listing

<110> Anjiajiajian Co Ltd

<120> cosmetic composition for removing or adsorbing fine dusts comprising peptide complex as an active ingredient

<130> P20117565WP

<150> 10-2018-0078889

<151> 2018-07-06

<160> 30

<170> PatentIn version 3.5

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Claims

1. A cosmetic composition for removing or adsorbing fine dust, comprising a complex in which a fatty acid is bound to the N-terminus, C-terminus or side chain of an amino acid or oligopeptide as an active ingredient.

2. The cosmetic composition for removing or adsorbing fine dust according to claim 1, wherein the oligopeptide is one or more selected from the group consisting of amino acid sequences of seq id nos. 1 to 30.

3. The cosmetic composition for removing or adsorbing fine dust according to claim 1, wherein said fatty acid is a saturated fatty acid or an unsaturated fatty acid.

4. The cosmetic composition for removing or adsorbing fine dusts according to claim 3, wherein the saturated fatty acid is at least one selected from the group consisting of caproic acid, caprylic acid, pelargonic acid, capric acid, undecanoic acid, lauric acid, myristic acid, pentadecanoic acid, palmitic acid, margaric acid, stearic acid, nonadecanoic acid, arachidic acid, heneicosanoic acid, behenic acid, tricosanic acid, tetracosanoic acid, pentacosanoic acid, cerotic acid, heptacosanoic acid, montanic acid, nonacosanoic acid, melissic acid, hentriacontanoic acid, laccerotic acid, tridecanoic acid, tetracosanoic acid, pentacosanoic acid and hexacosanoic acid.

5. The cosmetic composition for removing or adsorbing fine dusts according to claim 3, wherein the unsaturated fatty acid is at least one selected from the group consisting of palmitoleic acid, oleic acid, elaidic acid, petroselinic acid, vaccenic acid, macrocephalic acid, sinapic acid, nervonic acid, linoleic acid, linolenic acid, punicic acid, eleostearic acid, stearidonic acid, arachidonic acid, eicosapentaenoic acid, menhadenic acid, adrenic acid, docosahexaenoic acid and ricinoleic acid.