KR20210050316A - Cosmetic Composition Comprising Phytochelatin - Google Patents

Abstract

Provided are a cosmetic composition comprising phytochelatin, a product comprising the same, and a method for preparing the composition and/or product. The composition for external application for skin and/or cosmetic composition exhibits excellent antioxidant activity by containing phytochelatin, does not show skin cytotoxicity, is water-soluble, has excellent stability even when stored for a long time, and is excellent in protecting the skin from harmful substances.

Description

Cosmetic Composition Comprising Phytochelatin}

A cosmetic composition comprising phytochelatin, a product comprising the same, and a method of manufacturing the composition and/or product are provided.

The skin is the primary barrier of the human body and protects the internal organs from stimuli such as temperature and humidity changes, ultraviolet rays, and pollutants, and plays an important role in maintaining biological homeostasis such as body temperature control.

External foreign substances such as rapid industrialization, automobile exhaust gas, heavy metals and fine dust originating from China pollute the skin, and are considered one of the main causes of skin aging and troubles.

Although removing heavy metals and fine dust from the skin through cleansing is an effective method of preventing skin troubles, there is a problem that heavy metals and fine dust are not easily desorbed by adsorbing into cells.

Accordingly, studies on various materials to remove foreign substances in the skin and to realize skin improvement effects have been conducted, but cosmetic ingredients developed until recently have various problems such as insufficient efficacy or causing skin side effects.

In addition, even when the efficacy is excellent, current cosmetic ingredients have a limited range of action on the skin, and most of them have similar effects on the skin, such as promoting collagen synthesis, inhibiting collagen degradation, and removing active oxygen.

Therefore, in solving these problems, there is a need to develop a material that can be safely used while effectively having complex functions such as heavy metal chelating, antioxidant, and anti-inflammatory by using human-compatible raw materials.

The present specification provides a composition for external application for skin and a method of manufacturing the same, which protects the skin from environmental pollutants, has antioxidant and/or anti-inflammatory effects, and has excellent skin improvement ability.

One example provides a composition for external application for skin containing phytochelatin.

Another example provides a cosmetic composition containing phytocheletin.

The external composition for skin and cosmetic composition may have one or more effects selected from the group consisting of antioxidant, anti-inflammatory, skin contaminant removal (anti-pollution; for example, removal of heavy metals), skin improvement, and the like.

Another example provides a product comprising the cosmetic composition. The product may be a product for application to the skin, hair, and/or scalp.

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

Phytochelatin is an oligopeptide containing glutathione, a tripeptide containing glutamic acid, cysteine, and glycine. In one example, the phytocheletin may be represented by the following Formula 1:

[Formula 1]

In the above formula, n is the repetition number of the dipeptide consisting of [N-glutamic acid-cysteine-C], and is 1 or 2, and R is -OH or -NH ₂ . For example, n may be 1. The phytochelatin may include a carboxylic acid at the C-terminus (R is -OH in Formula 1) or an amidated carboxylic acid (R is -NH in Formula 1). The phytocheletin may be plant-derived, but is not limited thereto.

The phytochelatin has the advantage of not only having excellent antioxidant activity, but also exhibiting no skin cytotoxicity (excellent skin safety), water-soluble, and excellent stability even when stored for a long time. In addition, phytochelatin is excellent in removing harmful factors (eg, free radicals, inflammation, etc.) and/or environmental harmful factors (eg, heavy metals, fine dust, cigarette smoke, multinuclear aromatic hydrocarbons, etc.) in the body, When applied to, it is possible to protect the skin from the harmful factors and prevent skin damage (eg, skin damage due to the harmful factors).

Accordingly, the composition for external application for skin and the cosmetic composition including phytochelatin provided in the present specification are anti-oxidation, anti-inflammatory, skin contamination prevention or removal of skin contaminants (anti-pollution; for example, removal of heavy metals and/or fine dust) , And/or skin harmful factors (e.g., free radicals, inflammation, heavy metals, and/or fine dust) can prevent skin damage, and due to these effects, skin tone improvement, skin texture improvement, etc. It can exert an improvement effect.

In one example, the composition for external application for skin including phytochelatin provided herein may be a composition for external application for skin for antioxidant, anti-inflammatory, prevention of skin contamination, and/or removal of skin contaminants. In another example, the composition for external application for skin may be a composition for external application for skin for preventing, treating, and/or improving skin damage caused by one or more harmful substances selected from the group consisting of free radicals, inflammation, heavy metals, fine dust, and the like. In the present specification, the composition for external application for skin may refer to a composition, such as a pharmaceutical composition, for application to the skin epidermis, such as the outer surface (stratum corneum) of the skin epidermis in all parts of the body.

In another example, the cosmetic composition including phytochelatin provided herein may be an antioxidant, anti-inflammatory, anti-skin contamination, and/or a cosmetic composition for removing skin contaminants. In another example, the cosmetic composition may be a cosmetic composition for preventing and/or improving skin damage caused by one or more harmful substances selected from the group consisting of free radicals, inflammation, heavy metals, fine dust, and the like. In the present specification, the cosmetic composition may be applied to all skin, scalp, and/or hair of the body including the face, hands, and feet.

The free radical is a generic term for an atom or complex compound having very high reactivity since it has unpaired electrons, and may be active oxygen in one example. The composition provided herein may have an antioxidant effect by removing active oxygen when applied to the skin. The heavy metal is a generic term for a metal having a specific gravity of 4 to 5 or more, and in one example, Ag, Al, B, Ba, Bi, Ca, Cd, Co, Cr, Cu, Fe, Ga, In, K, Li, Mg , Mn, Na, Ni, Pb, Sr, Tl, Zn　, etc. at least one selected from the group consisting of (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), 2 It may be a species or more, or three or more, but is not limited thereto. In one embodiment, the heavy metal may be lead (Pb) or cadmium (Cd), but is not limited thereto.

Another example provides a composition for antioxidant, anti-inflammatory, or anti-pollution (eg, removal of heavy metals and/or fine dust) comprising phytochelatin.

The content of the phytochelatin peptide contained in the composition for external application for skin or cosmetic composition provided herein is from 0.00000001 to 3% by weight, 0.00000001 to 2.5% by weight, 0.00000001 to 2% by weight, 0.00000001 to 1.5% by weight, 0.00000001 to 1% by weight, 0.00000001 to 0.5% by weight, 0.00000001 to 0.1% by weight, 0.0000001 to 3% by weight, 0.0000001 to 2.5% by weight, 0.0000001 to 2% by weight, 0.0000001 to 1.5% by weight, 0.0000001 to 1% by weight %, 0.0000001 to 0.5% by weight, 0.0000001 to 0.1% by weight, 0.000001 to 3% by weight, 0.000001 to 2.5% by weight, 0.000001 to 2% by weight, 0.000001 to 1.5% by weight, 0.000001 to 1% by weight, 0.000001 to 0.5% by weight, 0.000001 to 0.1 wt%, 0.00001 to 3 wt%, 0.00001 to 2.5 wt%, 0.00001 to 2 wt%, 0.00001 to 1.5 wt%, 0.00001 to 1 wt%, 0.00001 to 0.5 wt%, or 0.00001 to 0.1 wt% have.

There is no particular limitation on the formulation of the composition for external application for skin or cosmetic composition provided herein, and for example, formulation as a solution, suspension, emulsion (emulsion), paste, gel, cream, powder, ointment, patch or spray, etc. Can be mad.

In one example, the composition comprises a conventional surfactant (emulsifier), O / W (oil in water; oil in water), W / O (water in oil; water in oil), W / O / W, or O It may have the properties of an emulsified or solubilized form of the /W/O formulation. The surfactant may be at least one selected from all surfactants commonly used in the preparation of an external skin composition and/or cosmetic composition, for example, at least one selected from the group consisting of nonionic surfactants and anionic surfactants. However, it is not limited thereto. The nonionic surfactant may be one or more selected from the group consisting of polyoxyethylene surfactants, polyglycerin surfactants, sugar ester surfactants, etc., for example, polyglyceryl-methylglucose distearate, polyglyceride Reel-methylglucose stearate, polyglyceryl-distearate (e.g., polyglyceryl-10 distearate, etc.), polyglyceryl-stearate, hydrogenated lecithin, cetearylolibate, sorbitanolibate ,　PEG (poly(ethylene glycol))-stearates (eg, PEG-100 stearate, PEG-40 stearate, etc.), glyceryl stearate, glyceryl stearate SE, decylglucoside, coco-glucoside, lauryl Glucoside, caprylyl/caprylglucoside, arachidyl glucoside, C12-20 (straight or branched) alkyl glucoside, cetearyl glucoside, ceteares (e.g., ceteares-20, etc.), steares (e.g., Steareth-2, steareth-21, etc.), polyethylene glycol ether of oleyl alcohol (e.g., oleth-20, etc.), methylglucose sesquistearate, sorbitan stearate, sucrosecoate, polysorb Baits (eg, polysorbate 60, etc.), sorbitan sesquioleate, sorbitan oleate, oil palm oil, oil palm kernel oil, etc. may be one or more selected from the group consisting of, but is not limited thereto. Anionic surfactants are sodium lauryl sulfate, ammonium lauryl sulfate, magnesium lauryl sulfate, triethanolamine lauryl sulfate, sodium polyoxyethylene lauryl sulfate, polyoxyethylene lauryl ammonium sulfate, sodium cocoamphoacetate, ammonium laureth sulfate, ammonium Lauryl Sulfate, Cocamide MI, Disodium Laureth Sulfosuccinate, TEA-Cocoyl Glutamate, Sodium Cocoyl Apple Amino Acid, Cocamide DIA, Sodium Laureth Sulfate, Sodium Lauryl Sulfate, Potassium Cocoyl Gly It may be one or more selected from the group consisting of sinate, sodium stearoyl glutamate, potassium cetyl phosphate, and the like, but is not limited thereto.

The composition for external application for skin and/or the cosmetic composition is one containing at least one selected from the group consisting of ingredients, oils, polymers, thickeners, additives, etc. that are commonly contained for application to the skin, in addition to phytochelatin. I can. The commonly contained components and their content may be appropriately adjusted according to the function, properties, and the like of the desired composition.

For example, the oil is a silicone oil such as cyclopentasiloxane, dimethicone, dimethicone, cyclomethicone, trisiloxane, amodimethicone, phenyltrimethicone, disiloxane, cyclohexasiloxane, PEG-dimethicone. (E.g., PEG-12 dimethicone, PEG-10 dimethicone, PEG-7 dimethicone, etc.), polysilicons (e.g., polysilicon-11, etc.), simethicone, diphenyl dimethicone, PEG-9 polydimethyl At least one selected from the group consisting of siloxyethyldimethicone, etc.; Non-silicone oils such as caprylic/capric triglyceride, butylene glycol dicaprylate/dicaprate, hydrogenated polyisobutene, isopropyl myristate, cetylethylhexanoate, hydrogenated polydecene, At least one selected from the group consisting of diethoxyethylsuccinate, hexyllaurate, triethylhexanoine, isononylisononanoate, polyisobutene, and the like; Triglycerides; It may be one or more selected from the group consisting of wax and the like. The polyol is butylene glycol, glycerin, glyceres (eg, glyceryl-26, etc.), methylpropanediol, PEG (poly(ethylene glycol)) / PPG (poly(propylene glycol)) copolymers (eg, PEG /PPG-17/6 copolymer, etc.), methylgluces (e.g., methylgluces-20, etc.), dipropylene glycol, propylene glycol, propanediol, caprylyl glycol, pentylene glycol It may be one or more selected from the group consisting of. The thickener is carbomer, ammonium acryloyldimethyltaurate/Vpicopolymer, xanthan gum, sodium polyacrylate, hydroxyethylcellulose, acrylate/C10-30 alkyl acrylate crosspolymer, sodium polyacrylate, sodium It may be at least one selected from polymer compounds consisting of acrylate/sodium acryloyldimethyltaurate copolymer, magnesium aluminum silicate, sodium carbomer, and the like. The additives are commonly used in the preparation of skin external preparations and/or cosmetics, ion sequestering agents, emulsion stabilizers, pH adjusters, skin conditioning agents, fragrances, preservatives, disinfectants, oxidation stabilizers, antioxidants, free radical destroying agents, opacity. It may contain one or more selected from the group consisting of an agent, a stabilizer, an emollient, a vitamin, an insect repellent, a preservative, an anti-inflammatory agent, a basification or acidifying agent, a colorant (pigment), a solubilizing agent, a carrier, etc. Not limited.

The composition may include a solvent commonly used for preparing skin external preparations and/or cosmetic compositions, and the available solvents are selected from the group consisting of purified water, bedrock water, hot spring water, glacial water, sea water, deep sea water, plant extract water, etc. It may be one or more, but is not limited thereto.

In addition to the above-described ingredients, the composition may further contain an active ingredient that exerts a beneficial effect on skin (skin) care and/or hair/scalp care. For example, the advantageous effects are skin whitening, skin elasticity improvement, skin wrinkle improvement, skin aging prevention, skin hydration, skin moisturizing, skin nutrition supply, pore shrinking, skin texture improvement, dead skin cell removal, skin volume improvement, scalp protection, scalp Alternatively, it may mean preventing sebum of the skin, preventing hair loss, supplying hair moisture, supplying hair nutrition, and improving hair volume, but is not limited thereto, and may be any effect desired in the skin/hair/scalp to be applied. For example, the active ingredient is a variety of plant extracts (e.g., Jimo extract, green tea extract, grape extract, lotus extract, tomato extract, gold and silver extract, camellia extract, seaweed extract, etc.), plant callus culture or culture extract, yeast extract, cells Containing or cell-free yeast cultures or culture extracts, vitamins, various peptides, lipids, or fatty acids (e.g., lipids derived from various fish and shellfish, fatty acids, peptides, etc.) hyaluronic acid, glycosides (e.g., arbutin, etc.), and other types of skin It may be one or more selected from the group consisting of compounds, etc., but is not limited thereto, and an appropriate active ingredient may be selected and used according to a desired effect. In one example, the active ingredient may be a mixture of hydrogenated polyisobutene and Zymo extract, but is not limited thereto.

Another example provides a product comprising the composition for external application for skin or a cosmetic composition. The product may be a product for skin, hair, and/or scalp care, for example, for all forms of cosmetics and/or washing (cleansing ) It can be a product. In one example, the product is skin, lotion, cream, essence, pack, foundation, color cosmetics, sun cream (sunscreen), BB cream, two-way cake, face powder, compact, makeup base, skin cover, eye shadow, lipstick, Cosmetics such as lip gloss, lip fix, eyebrow pencil, and lotion; And it may be one or more selected from the group consisting of cleaning products such as shampoo, soap, cleansing foam, and body cleanser.

In one example, the phytocheletin of Formula 1 may be synthesized by combining each amino acid by a solid-phase peptide synthesis method, synthesized through a converging reaction in which small peptide fragments are combined, or synthesized by a chemical synthesis method using a peptide synthesizer or the like. .

In another example, phytochelatin of Formula 1 can be prepared by performing the following steps:

(1) obtaining a protected peptide-resin by conventional solid phase peptide synthesis (SPPS);

(2) It can be prepared including the step of removing the resin from the obtained protected peptide-resin.

When a functional group is present in the side chain of the amino acid residue constituting the desired phytocheletin compound, a peptide can be synthesized using the amino acid whose functional group is protected in step (1), and the protecting group bonded to the functional group is It is removed from (2).

One specific example of the process for preparing phytochelatin according to the present invention is shown in Scheme 1:

[Scheme 1]

Peptide Synthesis and purification

Phytochelatin of Formula 1 can be prepared by using the solid phase peptide synthesis method using Fmoc (Solid Phase Peptide Synthesis).

In the solid-phase peptide synthesis method, the first amino acid in the sequence (ie, glycine, the first amino acid on the C-terminal side) is attached to the resin, and then coupling-deprotection is repeated according to the sequence of the next sequence. The carboxyl group at the C-terminus of the next amino acid whose N-terminus is protected reacts with the unprotected (Fmoc) N-terminal amide group of the peptide bound to the solid support. It is then deprotected to expose the N-terminus so that the next amino acid can react.

In the solid-phase peptide synthesis method, Fmoc and tert-butoxycarbonyl (Boc) protecting groups are mainly used for the amino group, and the synthesis proceeds from the C-terminus to the N-terminus. The N-terminus of the amino acid is protected by this protecting group. At the N-terminal end of the amino acid, a Fmoc protecting group that is easily removed from the base and tert-butyl (tBu) and/or Trityl protecting groups that are easily removed from an acid protect the side chain. The first Fmoc-amino acid is bonded to the resin linker and then piperidine is used to remove the Fmoc. After confirming that the Fmoc protecting group has been removed by Kaiser test, the second reaction is carried out by activating the carboxyl group of the Fmoc-amino acid. Through the Kaiser test, free amines are checked in the resin to confirm the completion of the reaction. Synthesis is synthesized according to the peptide sequence by removing the Fmoc protecting group and then reacting with the Fmoc-amino acid in one cycle. The synthesized peptide is treated with TFA (Trifluoroacetic acid) to separate the peptide from the resin, and the protective side chain is also removed at this time.

The composition for external application for skin and/or the cosmetic composition provided in the present specification not only exhibits excellent antioxidant activity by including phytochelatin, but also does not show skin cytotoxicity, is water-soluble, and has excellent stability even when stored for a long time, and harmful substances It features an excellent function to protect the skin from.

1 is a graph showing the results of a cytotoxicity test of phytocheletin and glutathione prepared in Examples.
Figure 2 is a graph showing the results of testing the antioxidant effects of phytocheletin and glutathione prepared in Examples.
Figure 3 is a graph showing the results of testing the anti-inflammatory effects of phytocheletin and glutathione prepared in Examples.

Hereinafter, the present invention will be described in more detail through Examples and Test Examples. However, these Examples and Test Examples are for illustrating the present invention and should not be construed as limiting the present invention.

Example One: Phytochelatin Preparation of the compound

A phytocheletin compound having the following formula was prepared:

[Formula 1]

(In the above formula, n is 1 or 2, R is -OH or -NH ₂ )

1.1. gamma- Glutamyl - Cysteinyl -gamma- Glutamyl - Cysteinyl -Glycine ( γECγECG ) (n=1, R= -OH)

1.1.1 Gamma- Glutamyl (O- t butyl )- Cysteinyl ( Trityl )-gamma- Glutamyl (O- t butyl )-Cysteinyl(trityl)-glycine (protected Peptide ) Manufacturing of resin

The peptide was synthesized by a conventional solid phase peptide synthesis (SPPS) using 9-fluorenylmethoxycarbonyl (Fmoc) as an amino acid protecting group, and N-hydroxybenzotriazole (N-hydroxybenzotriazole; HOBt) and N,N-diisopropylcarbodiimide (DIC) were used as activators to extend amino acid residues [Reference: Wang C. Chan, Perter D. White,'Fmoc-solid phase peptide synthesis', Oxford].

Specifically, in a glass solid phase reactor, 0.04 g of an amino group 2-chlorotrityl chloride resin (Bead Tech, Inc) was swelled in 3 ml of dichloromethane as a solvent for 10 minutes, and then the solvent was removed, and Fmoc-Gly Fmoc-Gly-O-Resin was synthesized by reacting -OH/DIEA (N,N-Diisopropylethylamine) and dichloromethane, and then treated twice with 3 ml of 20% piperidine to remove Fmoc. The resin from which Fmoc was removed was treated 6 times with DMF (N,N'-Dimethylformamide), and reacted with a solution of Fmoc-Cys(Trt)-OH activated with HOBt-DIC at room temperature for about 4 hours. Afterwards, NH2-protected peptide (gamma-glucose) obtained by successively reacting with Fmoc-γ-Glu-O(tBu), Fmoc-Cys(Trt)-OH, Fmoc-γ-Glu-O(tBu), etc. Tamil (OtBu)-cysteinyl (trityl)-gamma-glutamyl (OtBu)-cysteinyl (trityl)-glycine) resin 3 times with DMF (N,N'-Dimethylformamide), 3 times with DCM Washed.

1.1.2. gamma- Glutamyl - Cysteinyl -gamma- Glutamyl - Cysteinyl -Glycine ( γECγECG ) Of the manufacture

The above synthesized NH2-protected peptide (gamma-glutamyl-cysteinyl-gamma-glutamyl-cysteinyl-glycine)-resin is trifluoroacetic acid: triisopropylsilane: water (95: 2.5: 2.5) (v/v)) reacted at room temperature for 2 to 3 hours at room temperature to remove tert-Bu and Trt groups, which are the protecting groups of the functional groups present in the side chains of the amino acid residues constituting the peptide, and gamma-glutamyl from the resin. -Cysteinyl-gamma-glutamyl-cysteinyl-glycine was separated, and then the peptide was crystallized with cooled diethyl ether. The obtained dried gamma-glutamyl-cysteinyl-gamma-glutamyl-cysteinyl-glycine Crude peptide was used in water containing 0.1% trifluoroacetic acid and acetonitrile as a solvent. After purification in High Performance Liquid Chromatography; Column: C18, 20x250mm), as a result of freeze-drying, a peptide with a purity of 95% or more was obtained.

Yield: 48%

1.2. gamma- Glutamyl - Cysteinyl -gamma- Glutamyl - Cysteinyl -gamma- Glutamyl -Cysteinyl-glycine ( γECγECγECG ) (n=2, R= -OH)

1.2.1. Gamma-glutamyl (Otbutyl)-cysteinyl (trityl)-gamma-glutamyl (Otbutyl)-cysteinyl (trityl)-gamma-glutamyl (Otbutyl)-cysteinyl (trityl) -Glycine (protected Peptide ) Manufacturing of resin

The peptide was synthesized by a conventional solid phase peptide synthesis (SPPS) using 9-fluorenylmethoxycarbonyl (Fmoc) as an amino acid protecting group, and N-hydroxybenzotriazole (N-hydroxybenzotriazole; HOBt) and N,N-diisopropylcarbodiimide (DIC) were used as activators to extend amino acid residues [Reference: Wang C. Chan, Perter D. White,'Fmoc-solid phase peptide synthesis', Oxford].

Specifically, in a glass solid phase reactor, 0.04 g of an amino group 2-chlorotrityl chloride resin (Bead Tech, Inc) was swelled in 3 ml of dichloromethane as a solvent for 10 minutes, and then the solvent was removed, and Fmoc-Gly Fmoc-Gly-O-Resin was synthesized by reacting -OH/DIEA (N,N-Diisopropylethylamine) and dichloromethane, and then treated twice with 3 ml of 20% piperidine to remove Fmoc. The resin from which Fmoc was removed was treated with DMF 6 times, and reacted with a solution of Fmoc-Cys(Trt)-OH activated with HOBt-DIC at room temperature for about 4 hours. Afterwards, NH2-protected peptide (gamma-glucose) obtained by successively reacting with Fmoc-γ-Glu-O(tBu), Fmoc-Cys(Trt)-OH, Fmoc-γ-Glu-O(tBu), etc. Tamil (OtBu)-cysteinyl (trityl)-gamma-glutamyl (OtBu)-cysteinyl (trityl)-gamma-glutamyl (OtBu)-cysteinyl (trityl)-glycine) resin was added to DMF. Washed 3 times with and 3 times with DCM.

1.2.2. gamma- Glutamyl - Cysteinyl -gamma- Glutamyl - Cysteinyl -gamma- Glutamyl -Cysteinyl-glycine ( γECγECγECG ) Of the manufacture

NH2-protected peptide synthesized above (gamma-glutamyl-cysteinyl-gamma-glutamyl-cysteinyl-gamma-glutamyl-cysteinyl-glycine)-resin trifluoroacetic acid: triisopropyl Silane: reacted for 2 to 3 hours at room temperature with a mixed solution of water (95: 2.5: 2.5 (v/v)), tert-Bu and Trt groups, which are the protecting groups of the functional groups present in the side chains of the amino acid residues constituting the peptide. After removal, gamma-glutamyl-cysteinyl-gamma-glutamyl-cysteinyl-glycine was separated from the resin, and the peptide was crystallized with cooled diethyl ether. The obtained dried gamma-glutamyl-cysteinyl-gamma-glutamyl-cysteinyl-gamma-glutamyl-cysteinyl-glycine Crude peptide was used as a solvent in water containing 0.1% trifluoroacetic acid and acetonitrile. After purification by Reverse Phase High Performance Liquid Chromatography (Column: C18, 20x250mm), peptides with a purity of 95% or more were obtained as a result of freeze-drying.

Yield: 39%

1.3. gamma- Glutamyl - Cysteinyl -gamma- Glutamyl - Cysteinyl -Glycine- Amide (γECγECG-NH ₂ ) (n=1, R= -NH ₂ )

1.3.1. gamma- Glutamyl (O- t butyl )- Cysteinyl ( Trityl )-gamma- Glutamyl (O- t butyl )-Cysteinyl(trityl)-glycine-amide (protected Peptide ) Manufacturing of resin

The peptide was synthesized by a conventional solid phase peptide synthesis (SPPS) using 9-fluorenylmethoxycarbonyl (Fmoc) as an amino acid protecting group, and N-hydroxybenzotriazole (N-hydroxybenzotriazole; HOBt) and N,N-diisopropylcarbodiimide (DIC) were used as activators to extend amino acid residues [Reference: Wang C. Chan, Perter D. White,'Fmoc-solid phase peptide synthesis', Oxford].

Specifically, in a glass solid phase reactor, 0.04 g of amide resin (Bead Tech, Inc) was swelled in 3 ml of dichloromethane as a solvent for 10 minutes, and then the solvent was removed, and Fmoc-Gly- activated with HOBt-DIC. It was reacted with OH solution at room temperature for about 4 hours. Subsequently, obtained by continuously reacting with Fmoc-Cys(Trt)-OH, Fmoc-γ-Glu-O(tBu), Fmoc-Cys(Trt)-OH, Fmoc-γ-Glu-O(tBu) and the like in the same process. NH2-protected peptide (gamma-glutamyl (OtBu)-cysteinyl (trityl)-gamma-glutamyl (OtBu)-cysteinyl (trityl)-glycine-amide resin 3 times with DMF, with DCM. Washed 3 times.

1.3.2. gamma- Glutamyl - Cysteinyl -gamma- Glutamyl - Cysteinyl -Glycine- Amide (γECγECG-NH ₂ )of Produce

The NH2-protected peptide synthesized above (gamma-glutamyl-cysteinyl-gamma-glutamyl-cysteinyl-glycine) amide-resin is trifluoroacetic acid: triisopropylsilane: water (95: 2.5: 2.5 (v/v)) reacted at room temperature for 2 to 3 hours at room temperature to remove tert-Bu and Trt groups, which are the protecting groups of the functional groups present in the side chains of the amino acid residues constituting the peptide, and gamma-glucose from the resin. Tamil-cysteinyl-gamma-glutamyl-cysteinyl-glycine amide was separated, and then the peptide was crystallized with cooled diethyl ether. The obtained dried gamma-glutamyl-cysteinyl-gamma-glutamyl-cysteinyl-glycine-amide Crude peptide was used in water containing 0.1% trifluoroacetic acid and acetonitrile as a solvent in reverse phase high performance liquid chromatography ( After purification in Reverse Phase High Performance Liquid Chromatography; Column: C18, 20x250mm), peptides with a purity of 95% or more were obtained as a result of freeze-drying.

Yield: 38%

1.4. gamma- Glutamyl - Cysteinyl -gamma- Glutamyl - Cysteinyl -gamma- Glutamyl -Cysteinyl-glycine-amide (γECγECγECG-NH ₂ ) (n=2, R= -NH ₂ )

1.4.1. Gamma-glutamyl (Otbutyl)-cysteinyl (trityl)-gamma-glutamyl (Otbutyl)-cysteinyl (trityl)-gamma-glutamyl (Otbutyl)-cysteinyl (trityl) -Glycine-amide (protected Peptide ) Manufacturing of resin

The peptide was synthesized by a conventional solid phase peptide synthesis (SPPS) using 9-fluorenylmethoxycarbonyl (Fmoc) as an amino acid protecting group, and N-hydroxybenzotriazole (N-hydroxybenzotriazole; HOBt) and N,N-diisopropylcarbodiimide (DIC) were used as activators to extend amino acid residues [Reference: Wang C. Chan, Perter D. White,'Fmoc-solid phase peptide synthesis', Oxford].

Specifically, 0.04 g of amide resin (Bead Tech, Inc) was swelled for 10 minutes in 3 ml of dichloromethane as a solvent in a glass solid-phase reactor, and then the solvent was removed, and Fmoc-Gly- activated with HOBt-DIC. The OH solution was reacted at room temperature for about 4 hours. Subsequently, obtained by continuously reacting with Fmoc-Cys(Trt)-OH, Fmoc-γ-Glu-O(tBu), Fmoc-Cys(Trt)-OH, Fmoc-γ-Glu-O(tBu) and the like in the same process. NH2- protected peptide (gamma-glutamyl (OtBu) - cis table carbonyl (trityl) - gamma-glutamyl (Ot-butyl) - cis table carbonyl (trityl) - gamma-glutamyl (OtBu) - cis table carbonyl (Trityl)-glycine-amide resin was washed 3 times with DMF and 3 times with DCM.

1.4.2. gamma- Glutamyl - Cysteinyl -gamma- Glutamyl - Cysteinyl -gamma- Glutamyl -Cysteinyl-glycine-amide (γECγECγECG-NH ₂ ) Of the manufacture

The NH2-protected peptide synthesized above (gamma-glutamyl-cysteinyl-gamma-glutamyl-cysteinyl-gamma-glutamyl-cysteinyl-glycine) amide-resin is trifluoroacetic acid: triiso Propylsilane: reacted for 2 to 3 hours at room temperature with a mixed solution of water (95: 2.5: 2.5 (v/v)), tert-Bu, Trt, which are protecting groups of functional groups present in the side chains of amino acid residues constituting the peptide After removing the group and separating gamma-glutamyl-cysteinyl-gamma-glutamyl-cysteinyl-gamma-glutamyl-cysteinyl-glycine amide from the resin, the peptide was crystallized with cooled diethyl ether. Made it. The obtained dried gamma-glutamyl-cysteinyl-gamma-glutamyl-cysteinyl-gamma-glutamyl-cysteinyl-glycine-amide Crude peptide was used in water containing 0.1% trifluoroacetic acid and acetonitrile. Was purified by Reverse Phase High Performance Liquid Chromatography (Column: C18, 20x250mm) with a solvent, and then freeze-dried to obtain a peptide with a purity of 95% or more.

Yield: 32%

Comparative example 1. Gamma- Glutamyl - Cysteinyl -Glycine ( γECG ) Of the manufacture

According to the preparation method described in Example 1, the title compound, gamma-glutamyl-cysteinyl-glycine (γECG; glutathione) was prepared.

Yield: 41%

Example 2. Cytotoxicity test using cell culture

In order to examine the toxicity of the phytocheletin compound prepared in Example 1 to skin cells, the effect on the cell proliferation of human fibroblasts was tested. Specifically, human fibroblasts were inoculated into a 24-well culture plate at a concentration of 1 ^{×10 4 cells/ml.} As a medium, DMEM (Dubelcco's Modified Eagle's Medium, BRL, USA) containing 10% (v/v) fetal bovine serum (FBS) was used. When incubated in DMEM containing 10% (v/v) FBS for 48 hours and cultured for 25 to 30% of the surface area of the culture vessel, the phytocheletin compounds prepared in Examples 1.1 to 1.4 and the glutathione prepared in Comparative Example 1 were respectively It was replaced with FBS-free DMEM contained in an amount of 0.5, 1, 10, or 30 ppm, and incubated for 24 hours. Thereafter, 50 µl of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma M5655, USA) solution (2.5 mg/ml) was added, After incubating for an additional 3 hours, the supernatant was removed, and 200 µl of dimethyl sulfoxide (DMSO, Sigma D2650, USA) solution was added to each well, followed by stirring for 20 minutes to dissolve the formed formazan crystals, 100 µl of 96 wells were taken, and absorbance was measured at 570 nm by the lesion infection diagnostic test (ELISA).

The degree of cytotoxicity was measured as the degree of cell proliferation, and the degree of cell proliferation was calculated as a percentage by the following calculation formula 1 based on the absorbance of the control group using pure water, and the results are shown in FIG.

[Calculation 1]

Cell proliferation effect (%) = [(absorbance at the time of extract treatment-absorbance of control group)/absorbance of control group]x100

As shown in FIG. 1, all kinds of phytocheletin compounds used in the test did not show cytotoxicity at all tested concentrations, and these results indicate that the phytocheletin compound can be safely used as a material for external skin preparations such as cosmetics Show that you can.

Example 3. DPPH Checking the antioxidant effect used

In order to confirm the antioxidant efficacy of the phytocheletin compounds, a conventional DPPH assay was used, and as a positive control, vitamin C (Ascorbic acid; Sigma) was used as a positive control. Specifically, ascorbic acid, the phytocheletin compound prepared in Examples 1.1 to 1.4, and glutathione prepared in Comparative Example 1 were dissolved in purified water, respectively, and 0.3 mL of the solution was added to 0.2 mL ethanol, followed by 0.1 mM DPPH. (1,1-diphenyl-2-picrylhydrazyl: DPPH, Sigma D9132-1G, USA) 0.5 ml of the solution was added, so that the final concentration of each was 0.5, 1, 10 or 30 ppm. Thereafter, in order to check how much the amount of free radicals generated in each decreases, vortexing was performed for 10 seconds and stored for 30 minutes in a cool and dark place, and the absorbance was measured at 517 nm. The degree of antioxidant activity (free radical scavenging effect) was calculated as a percentage through the following calculation formula 2 based on the absorbance of the control group using pure water, and the results are shown in FIG. 2.

[Calculation 2]

As shown in Figure 2, the phytocheletin compounds of Examples 1.1 to 1.4 show superior antioxidant activity than the compound of Comparative Example 1, and have similar or better antioxidant activity compared to ascorbic acid, which has proven antioxidant efficacy. I can confirm.

Example 4. Anti-inflammatory effect check

In order to confirm the anti-inflammatory effect of the phytocheletin compounds, the degree of inhibition of iNOS (Inducible nitric oxide synthase) activity was confirmed. Specifically, after activating the HaCaT cell line (keratinocytes) with 1 g of LPS, L-NMMA (CAS 53308-83-1; positive control), which is well known as an NOS inhibitor, phytocheletin compounds prepared in Examples 1.1 to 1.4 And glutathione prepared in Comparative Example 1 was treated in a medium at a concentration of 0.5, 1, 10 or 30 ppm, and then cultured for 24 hours. After incubation, 100 μl of cell culture supernatant and 100 μl of Griess reagent (1%(w/v) sulfanilamide in 5%(w/v) phosphoric acid and 1%(w/v)-naphthylamide in H ₂ O) were mixed Then, the reaction was carried out in a 96-well plate for 10 minutes, and the absorbance was measured at 540 nm, and the amount of NO (NO ₂ form) was measured. The concentration of NO ₂ was obtained by measuring the absorbance by diluting sodium nitrate.

Fig. 3 shows the obtained results. As shown in FIG. 3, it can be seen that the phytocheletin compounds of Examples 1.1 to 1.4 exhibit an anti-inflammatory effect in a concentration-dependent manner, and have an anti-inflammatory effect superior to that of glutathione of Comparative Example 1.

Example 5. Heavy metal Chelating Check the effect

The effects of removing lead and cadmium, which are representative heavy metals, of the phytochelatin compounds of Examples 1 to 4 and the glutathione of Comparative Example 1 were confirmed.

Lead (60 ppm) and cadmium (30 ppm) were mixed with the phytochelatin compounds of Examples 1 to 4 and the glutathione of Comparative Example 1 at a concentration of 0.5, 1, 10, or 30 ppm, respectively, and reacted three times.

After the reaction three times, the content of the heavy metal remaining in the reaction solution was measured using inductively coupled plasma (ICP-MS). As a control, a solution containing 10% (w/v) MCT (caprylyl/capric triglyceride) was used.

The adsorption rate was calculated by dividing the concentration of the heavy metal and the sample after adsorption by the concentration of the control sample, and the higher the adsorption rate, the more effectively the heavy metal can be adsorbed and removed.

The heavy metal removal rate was calculated as (1-adsorption rate) x 100, and the evaluation results are shown in Table 1 below:

division Concentration (ppm) Lead adsorption rate (%) Cadmium adsorption rate (%) Example 1.1 0.5 4.5 5.2 One 8.4 10.2 10 18.9 22.7 30 35.2 40.1 Example 1.2 0.5 6.4 4.9 One 9.6 9.2 10 19.2 18.3 30 36.5 39.8 Example 1.3 0.5 4.1 4.5 One 7.4 7.8 10 16.5 18.2 30 29.5 30.1 Example 1.4 0.5 4.3 4.8 One 7.5 7.7 10 17.4 18.2 30 30.6 33.9 Comparative Example 1 0.5 3.9 4.1 One 6.4 7.1 10 15.5 17.3 30 27.8 29.2

As shown in Table 1, it can be seen that the phytocheletin compounds of Examples 1.1 to 1.4 exhibited a heavy metal removal effect in a concentration-dependent manner, and had an excellent heavy metal removal effect than glutathione of Comparative Example 1 at the same concentration.

Example 6: manufacture of cosmetics

6.1. Phytochelatin Preparation of containing lotion

A lotion (skin lotion) containing phytochelatin peptide prepared in Example 1.1 was prepared in the composition and content shown in Table 2 below.

Specifically, ingredients 2, 3, 4, 5, and 6 were added in order to component No. 12, and stirred to dissolve (dissolved product 1). On the other hand, component No. 7 was heated to about 50° C. to dissolve, and then No. 8, 9, and 11 were added thereto to dissolve (dissolved product 2). Then, the lysate 2 was added to the lysate 1, and finally, ingredients 1 and 10 were added, sufficiently stirred, and aged to prepare a lotion.

number ingredient Content (% by weight) One Aqueous solution of phytocheletin (100 ppm) of Example 1 1.0 2 Disodium EDTA 0.02 3 Butylene Glycol 4.0 4 glycerin 2.0 5 Citric acid 0.04 6 Sodium citrate 0.1 7 Polyoxyethylene hydrogenated castor oil 1.0 8 ethanol 3.0 9 antiseptic a very small amount 10 Pigment a very small amount 11 Spices a very small amount 12 Purified water Balance

6.2. Phytochelatin Inclusive Nutritional lotion Produce

A nutritional lotion containing the phytochelatin peptide prepared in Example 1.1 was prepared in the composition and content shown in Table 3 below.

Specifically, components 2, 3, 4, 5, 6, and 18 were added to an aqueous phase dissolving bath, heated to 80°C, and stirred to prepare an aqueous agitating bath. Components 7, 8, 9, 10, 11, 12, and 13 were added to the oil phase dissolving tank, heated to 80°C, and completely dissolved to prepare an oil phase stirring tank. At 80° C., an oily raw material mixture in an oily agitation tank was added to an aqueous agitation tank, followed by uniform emulsification for 5 minutes. After cooling the obtained emulsion to 65° C., component 14 was added and stirred for 3 minutes to neutralize. After cooling the obtained resultant to 40° C., components 1, 15, 16, and 17 were added and uniformly stirred for 3 minutes. The resulting product was cooled to 25° C. and then aged to prepare a nourishing lotion.

number ingredient Content (% by weight) One Aqueous solution of phytochelatin peptide (100 ppm) of Example 1.1 1.0 2 Disodium EDTA 0.02 3 Butylene Glycol 4.0 4 glycerin 2.0 5 Carbomer 0.1 6 Xanthan gum 0.05 7 Glyceryl stearate 1.5 8 PEG-100 Stearate 0.5 9 Beeswax 0.5 10 Polysorbate 60 1.0 11 Stearic acid 1.0 12 Caprylic/Capric Triglyceride 6.0 13 Squalane 2.0 14 Triethanolamine 0.1 15 antiseptic a very small amount 16 Pigment a very small amount 17 Spices a very small amount 18 Purified water Balance

Claims (15)

A cosmetic composition comprising phytochelatin. The cosmetic composition of claim 1, wherein the phytocheletin is represented by Chemical Formula 1:
[Formula 1]

In the above formula, n is 1 or 2, and R is -OH or -NH ₂ . According to claim 1, Based on the total weight of the composition, containing 0.00000001 to 3% by weight of phytocheletin, cosmetic composition. The cosmetic composition according to claim 1, for applying to one or more selected from the group consisting of skin, scalp, and hair. The cosmetic composition according to any one of claims 1 to 4, having at least one effect selected from the group consisting of antioxidant, anti-inflammatory, skin contamination prevention, skin contamination removal, and skin damage prevention. The cosmetic composition of claim 5, wherein the skin contaminants are heavy metals, fine dust, or both. A composition for external application for skin comprising phytochelatin. The composition for external application for skin according to claim 7, wherein the phytocheletin is represented by Chemical Formula 1:
[Formula 1]

In the above formula, n is 1 or 2, and R is -OH or -NH ₂ . According to claim 7, Based on the total weight of the composition, containing 0.00000001 to 3% by weight of phytocheletin, composition for external application for skin. The composition for external application for skin according to any one of claims 7 to 9, having at least one effect selected from the group consisting of antioxidant, anti-inflammatory, skin contamination prevention, skin contamination removal, and skin damage prevention. The composition for external application for skin according to claim 10, wherein the skin contaminants are heavy metals, fine dust, or both. A product comprising the cosmetic composition of any one of claims 1 to 4. The product of claim 12, wherein the product is a cosmetic or cleansing product for application to the skin, scalp, or hair. The product according to claim 13, having at least one effect selected from the group consisting of antioxidant, anti-inflammatory, skin contamination prevention, skin contamination removal, and skin damage prevention. The product of claim 14, wherein the skin contaminant is a heavy metal, fine dust, or both.

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