KR101354915B1 - Galloyl-peptide and cosmetic external preparation composition for skin using the same - Google Patents

Abstract

The present invention relates to galloyl-peptide which has excellent stability at alternating temperatures, antioxidant activity, whitening activity, and anti-inflammatory activity and a cosmetic external preparation composition for skins using the same. According to the present invention, a manufacturing method for galloyl-peptide which is stable at alternating temperatures and a cosmetic external preparation composition for skins using the same are provided. The galloyl-peptide of the present invention has excellent stability at alternating temperatures when compared with peptide used in the manufacturing of the galloyl-peptide. The cosmetic external preparation composition for skins using the galloyl-peptide can be manufactured into cosmetics, chemicals, and soaps.

Description

Carloyl-Peptide and Cosmetic External Preparation Composition for Skin Using the Same}

The present invention relates to a carloyl-peptide and an external composition for skin using the same.

People have a desire to stay young as they age. But the reality is that biological aging cannot be prevented. Therefore, efforts have been made to predict the progress of skin aging in advance and to prevent it. In general, skin aging can be divided into intrinsic aging and extrinsic aging. Endogenous aging refers to the aging phenomenon that occurs naturally as the physiological function of the skin gradually decreases with age. Clinically, skin elasticity is reduced, skin texture is rough, deep wrinkles and pigmentation is deposited (Archi Dermatol., 130: 87-95, 1994).

On the other hand, exogenous aging refers to the aging phenomenon caused by exogenous factors such as reactive oxygen species (ROS) and stress, the aging of the skin is rapidly progressed by these factors, as a result Coarse wrinkles are created and elasticity is significantly reduced.

As a general theory of skin aging, various theories are known, such as cell damage caused by gene damage, cell damage caused by free radicals, accumulation of toxic substances, and immune system abnormalities. Among these theories, free free radical theory is known to be a major cause of skin aging. In particular, free radicals in free radicals flow out of cells, causing inflammation of surrounding tissues, thereby promoting aging of the cells. Inflammation is a series of defenses aimed at minimizing the response and restoring damaged areas when cells or tissues are damaged by any cause.Inflammation causes physical factors such as trauma, burns, frostbite, and radiation. There are chemical factors caused by chemicals such as acids and bases, immunological factors caused by antibody reactions, and other causes of blood vessels and hormonal imbalances. Recently, various compounds have been developed for controlling such wrinkles, wrinkles and undesirable forms of skin surface tissue.

Among them, plant-derived phytochemicals, especially gallate and shikimic acid, are antioxidants that control free wrinkles and other uneven skin conditions caused by age or damage caused by UV rays. It is known to be important. However, despite its excellent efficacy, due to the instability (unstability) in the aqueous solution state of phytochemical was not widely used as an anti-aging material for cosmetics. Therefore, there is an urgent need for the development of derivative derivatives that can have stability and high solubility in aqueous solution of phytochemicals.

The present invention provides a galloyl-peptide having excellent stability by combining gallic acid with a peptide.

An object of the present invention is to provide a galloyl-peptide as a novel substance.

Another object of the present invention to provide an external composition for skin using the galloyl-peptide.

In one aspect, the invention relates to a galloyl-peptide having the formula

[Formula 1]

In the above R is arginine (Arginine), G is glycine (Glycine), D refers to aspartic acid (aspartic acid).

The galloyl-peptide of the present invention shows that the peptide, RGD, is about 66% stable after 60 days, but shows about 87% stability.

The galloyl-peptide of the present invention can be prepared by synthesizing the peptide and then reacting with gallic acid. Specifically, the galloyl-peptide of the present invention first obtains an NH2-protected peptide-resin by solid phase peptide synthesis as described in Scheme 1 below, and then obtains the NH2-protected peptide. It can be obtained by reacting resin with gallic acid and then removing the protecting group and resin. For more specific manufacturing process, reaction conditions and the like can refer to the following examples.

[Reaction Scheme 1]

In another aspect, the present invention relates to an anti-oxidant skin external composition, a whitening skin external composition and an anti-inflammatory skin external composition comprising the galloyl-peptide as an active ingredient.

Experimental example below shows that the galloyl-peptide shows DPPH scavenging activity in a concentration-dependent manner, and inhibits tyrosinase and inhibits oxidation of topa by tyrosinase in a concentration-dependent manner, and produces inflammatory factors PGE2 and NO. Shows inhibitory activity of COX-2 and iNOS. In particular, the galloyl-peptide does not show cytotoxicity to skin keratinocytes.

The external preparation composition for skin of the present invention may include galloyl peptide as an active ingredient in any amount (effective amount) according to the use, formulation, formulation purpose, etc. as long as it can exhibit antioxidant activity, whitening activity or anti-inflammatory activity. An effective amount of phosphorus will be determined in the range of 0.001% to 20.000% by weight, based on the total weight of the composition. As used herein, "effective amount" refers to an amount of an active ingredient that can exhibit antioxidant activity, whitening activity or anti-inflammatory activity. Such effective amounts can be determined experimentally within the ordinary skill of those skilled in the art.

The composition of the present invention can be identified as a cosmetic composition in a specific embodiment.

When the composition of the present invention is recognized as a cosmetic composition, the cosmetic composition may be prepared in various forms, for example, emulsion, lotion, cream (oil-in-water type, oil water type, multiphase), solution, suspension (Water and water), anhydrous products (oil and glycol), gel, mask, pack, powder and the like.

The composition of the present invention may contain an acceptable carrier in cosmetic preparations other than those containing the active ingredient.

As used herein, the term " acceptable carrier for cosmetic preparation "refers to a compound or composition which is already known and used in cosmetics, or which is a compound or composition to be developed in the future, and which has no toxicity that the human body can adapt to when contacted with skin.

The carrier may be included in the composition of the present invention in an amount of from about 1% by weight to about 99.99% by weight, preferably from about 50% by weight to about 99% by weight of the composition, based on the total weight thereof.

However, since the ratio depends on the above-mentioned formulation of the cosmetic product and its specific application site (face or hands) or the desired amount of application thereof, the ratio is to limit the scope of the present invention in any aspect It should not be.

Examples of the carrier include alcohols, oils, surfactants, fatty acids, silicone oils, humectants, moisturizers, viscosifiers, emulsifiers, stabilizers, sunscreens, coloring agents and perfumes.

The compounds / compositions which can be used as the carrier and which can be used as alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, moisturizers, viscosifiers, emulsions, stabilizers, sunscreens, A person skilled in the art can select and use appropriate substances / compositions.

The composition of the present invention may be regarded as a soap composition in another specific embodiment.

When the composition of the present invention is identified as a soap composition, the soap composition of the present invention can be prepared by incorporating an active ingredient into a soap base, and can be prepared as an additive including a skin moisturizer, an emulsifier, a water softener and the like.

Examples of the soap base material include vegetable oils such as palm oil, palm oil, soybean oil, perm free oil, olive oil and palm kernel oil or animal fats such as tallow, lard, sunshine and fish oil. Examples of the skin moisturizers include glycerin, erythritol, polyethylene glycol , Propylene glycol, butylene glycol, pentylene glycol, hexyl glycol, isopropyl myristate, silicone derivatives, aloe vera, sorbitol and the like. Examples of the emulsifier include natural oils, wax fatty alcohols, hydrocarbons, May be used. As the water softening agent, tetrasodium ethylenediate and the like may be used.

The soap composition of the present invention may further contain, as an additive, an antibacterial agent, a dispersant, a foam inhibitor, a solvent, a scouring inhibitor, a corrosion inhibitor, a fragrance, a dye, a sequestering agent, an antioxidant and an antiseptic.

In the soap composition of the present invention, the soap base or additive may be included in a content commonly used in the art, wherein the soap base generally comprises from 99.999% to 50% by weight, based on the total weight of the soap composition And the additive may be added in an amount of 1 wt% to 20 wt%.

The composition of the present invention may be regarded as a pharmaceutical composition in another specific embodiment.

The pharmaceutical composition of the present invention may be formulated into a wide variety of dosage forms, including pharmaceutically acceptable carriers, excipients and the like, in addition to the active ingredient, and may be formulated into a wide variety of preparations such as creams, lotions, ointments (semi-solid external medicine) (TTS) (e.g., patches, bandages, etc.), and the like.

The term "pharmaceutically acceptable" as used herein means that the application (prescribing) subject does not have the above-mentioned toxicity that is adaptable without inhibiting the activity of the active ingredient.

Examples of pharmaceutically acceptable carriers include lactose, glucose, sucrose, starch (e.g. corn starch, potato starch, etc.), cellulose, derivatives thereof (e.g. sodium carboxymethylcellulose, ethylcellulose, etc.), malt, gelatin, talc, (E.g., peanut oil, cottonseed oil, sesame oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, etc.), alginic acid, emulsifiers (e.g., TWEENS), wetting agents (For example, sodium lauryl sulfate), a coloring agent, a flavoring agent, a stabilizer, an antioxidant, a preservative, water, a saline solution, and a phosphate buffer solution. The carrier may be selected from one or more of suitable pharmaceutical formulations according to the formulation of the pharmaceutical composition of the present invention.

The excipient may be selected according to the formulation of the pharmaceutical composition of the present invention. For example, suspending agents such as sodium carboxymethyl cellulose, methyl cellulose, hydropropyl methyl cellulose, sodium alginate, polyvinyl pyrrolidone, .

The pharmaceutical compositions of the present invention may be administered orally or parenterally, and preferably topically.

The daily dose of the pharmaceutical composition of the present invention is usually 0.001 to 150 mg / kg body weight, and may be administered once or several times. However, since the dosage of the pharmaceutical composition of the present invention is determined in view of various related factors such as route of administration, age, sex, weight, and patient's severity of the patient, the dose is limited in any aspect to the scope of the present invention Should not be understood to be.

As described above, according to the present invention, it is possible to provide a method for preparing a galloyl peptide, which is stable even at room temperature, and an external composition for skin using the galloyl peptide.

The galloyl peptide of the present invention shows superior stability over the peptide used in its preparation at ambient temperature conditions.

The external preparation composition for skin using the galloyl peptide of the present invention may be commercialized into cosmetics, drugs, soaps, and the like.

1 is a MALDI-TOF MS analysis to confirm the molecular weight of galloyl-RGD.
Figure 2 shows the results showing that galloyl-RGD is stable for keratinocytes.
3 is a result showing the antioxidant activity of galloyl-RGD.
4 is a result showing the tyrosinase inhibitory activity of galloyl-RGD.
5 is a result showing the waveguide antioxidant activity of galloyl-RGD.
6 is a result showing the COX-2 production inhibitory activity of galloyl-RGD.
7 shows the iNOS production inhibitory activity of galloyl-RGD.

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these examples and experimental examples.

< Example > Galois - RGD Manufacture, purification and MS  analysis

<Example 1> galloyl-Preparation of RGD

The peptide was synthesized by a conventional solid phase peptide synthesis (SPPS) using 9-fluorenylmethoxycarbonyl (Fmoc) as a protecting group of an amino acid, and N-hydroxybenzotriazole N-hydroxybenzotriazole (HOBt) and N, N'-dicyclohexylcarbodiimide (DCC) were used as activating agents to extend the amino acid residues (Wang C. Chan, Perter D. White, 'Fmoc solid phase peptide synthesis', Oxford].

Specifically, 0.04 g of resin (Nova Biochem, Inc.) was swollen in 3 ml of N-methyl-2-pyrrolidone (NMP) for 20 minutes in a glass reactor, Aspartic acid (D), the first amino acid protected by Fmoc, was added to the resin and reacted at room temperature for 3 hours. The next solvent was removed and Fmoc was removed by treatment with 2 ml of 20% piperidine (3 ml). Next, the reaction was carried out twice with dichloromethane (DCM) and twice with NMP. Glycine (G), which is a second amino acid activated by HOBt-DCC, was added to the reactor and reacted at room temperature for about 3 hours to bind the second amino acid. The resulting NH ₂ -protected peptide (arginine-glycine-aspartic acid) -resin was washed twice with DCM, twice with NMP and twice with DCM, followed by coupling of the third amino acid, arginine (R) And then completely dried.

20% piperidine / NMP solution was added to the NH ₂ -protected peptide (arginine-glycine-aspartic acid) -resin synthesized above to remove Fmoc bound to the amino group, washed with NMP and DCM, and then gallic acid. (gallic acid, Lancaster Co., Ltd.) 5 equivalents and reacted overnight at room temperature. After the reaction was completed, the reaction was washed several times with NMP and DCM, and then dried.

The dried galloyl-peptide-resin was mixed with trifluoroacetic acid: phenol: thioanisole: water: ethanedithiol (82.5: 5: 5: 5: 2.5 (v / v)) at room temperature for 3 hours. Reaction to remove t-butyloxycarbonyl (Boc) and trityl (triphenylmethyl), the protecting groups of the functional groups present on the side chains of the amino acid residues constituting the peptide, to separate the galloyl-peptide from the resin Precipitation with ether gave galloyl-RGD.

<Example 2> galloyl - RGD of C18 Prep Column purification

For high purity purification of the synthesized galloyl-RGD, a widely used HPLC assay was used for peptide purification. In particular, Gradient analysis and purification conditions for varying the concentrations of solvent A and solvent B in the fluidized bed were established and performed to increase the resolution. By using these purification conditions, it was possible to secure a degree of purification of around 96% in repeated purification.

HPLC instrument type: Waters 650E Advanced Protein Purification System

Column Type: Gemini 5u C18 110 A (Phenomenex sk)

Column Specification: 250 * 4.60 mm, 5 micron

Solvent conditions: Solvent A: Water (containing 0.1% TFA (Trifluoro acetic acid))

          Solvent B: Acetonitrile (containing 0.1% TFA (Trifluoro acetic acid))

Wavelength: 230 nm (UV lamp)

Flow rate: 1 ml / min

Solvent slope conditions (Gradient): [Table 1]

Solvent slope conditions Time (min) Solvent A (%) Solvent B (%) 0 100 0 30 35 65 31 35 65 36 100 0 41 100 0

&Lt; Example 3 > MALDI - TOF Mass Spectrometry analysis

In order to confirm the molecular weight of the galloyl-RGD, MALDI-TOF MS analysis was performed under the following analytical conditions, and the results are shown in FIG. 1. From these results, it was confirmed that the synthesized galloyl-RGD is exactly the expected molecular weight.

- Device name: Kratos PC AximaCFR V2.3.3

- Mode: Linear (Positive)

- Laser power: 63

Matrix: -Cyano-4-hydroxy-cinnamic acid (CHCA)

Example 4 Physical Stability Test of Galloyl - RGD ( Ambient Stability)

In order to confirm the increased physical stability of galloyl-RGD, an appropriate concentration of Galloyl-RGD aqueous solution was applied at 8 ° C. in low temperature (4 ° C), room temperature (20 ° C) and high temperature (40 ° C). The stability was assessed by HPLC analysis at 20, 40 and 60 day intervals to assess physical stability. The evaluation showed that the initial 100% content of galloyl-RGD was 99 ± 2%, 94 ± 4%, 92 ± 8%, and 87 ± 29% after 0, 20, 40, and 60 days of treatment, respectively. The stability was shown to be very stable physically for a long time even under severe temperature conditions. Relatively, RGD showed a very poor physical stability of about 66 ± 23% after 60 days of temperature treatment.

Thermosafety of Galloyl-RGD term RGD Galloyl-rgd 0 days 99 ± 2%, 99 ± 2%, 20 days 81 ± 4%, 94 ± 4%, 40 days 74 ± 6%, 92 ± 8%, 60 days 66 ± 23% 87 ± 29%

< Experimental Example > Galois - RGD of Skin cell  Safety, antioxidant, whitening and anti-inflammatory activity experiment

< Experimental Example  1> Galloyl-RGD Skin cell  Stability experiment

The MTT assay was used to test the cellular safety of galloyl-RGD human dermal constituent cells, ie, the HaCaT cell line, a keratinocyte cell line. To this end, human keratinocyte cell line HaCaT cells Count the cells in a 24-well plate using 5 × 10 ³ cells / well and 5 × 10 ⁴ cells / well using a heamacytometer. When cultured in DMEM containing 10% FBS for 48 hours and cultured to 50% of the culture vessel surface area, the sample was treated for each concentration and cultured for another 48 hours. After incubation, 50 μl of a solution of 2.5 mg / ml 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT, Sigma M5655, USA) was added and cultured for additional 3 hours. 100 μl aliquots were transferred into 96 wells and eluted with Enzyme-Linked Immunosorbent Assay (ELISA) at 570 cells / well. The cell lysates were discarded and 200 μl of dimethyl sulfoxide (DMSO, Sigma D2650, USA) nm absorbance was measured. The extent of promoting toxicity or proliferation to cells was expressed as a percentage based on the absorbance intensity of the control using pure water.

The results are shown in Fig. Referring to Figure 2, it can be seen that galloyl-RGD is safe for particular keratinocytes.

< Experimental Example  2> Galois - RGD Antioxidant Activity Test of- DPPH Free radical  Scavenging activity experiment

Antioxidant activity of DPPH was performed to analyze the physical properties of galloyl-RGD. In particular, the gallic acid moiety, which enables various skin soothing effects, has a strong antioxidant power in itself, and the analysis of antioxidant capacity as galloyl-RGD after formation of a compound with peptide RGD is an important physical confirmation of structural integrity. Will be characteristic. Compound 1,1-diphenyl-2-picryl hydrazyl (DPPH, Sigma D9132-1G, USA) generates free radicals in ethanol. Mixing the prepared galloyl-RGD in a proportion to determine the amount of free radicals produced. Briefly, 0.2 ml of ethanol is added 0.5 ml of 0.1 mM DPPH solution made with ethanol and 0.3 ml of galloyl-RGD to a final concentration of 0.1 ppm to 1,000 ppm. Vortex strongly for 10 seconds and store in a cool dark place for 30 minutes. Absorbance is measured at 517 nm using ELISA. The degree of antioxidant activity was expressed as a percentage based on the absorbance intensity of the control group using pure water.

The results are shown in Fig. Referring to FIG. 3, it can be seen that galloyl-RGD shows antioxidant activity in a concentration-dependent manner.

< Experimental Example  3> Galois - RGD Skin whitening active experiment

< Experimental Example  3-1> Tyrosinase Inhibitory Activity Experiment

This test is performed in vitro ( in in vitro ) to determine the degree of inhibition of mushroom tyrosinase enzyme activity in a sample. The sample was dissolved in an appropriate solvent, and 220 µl of 0.1 M phosphate buffer (pH 6.5), 20 µl of sample solution and 20 µl of mushroom tyrosinase (1500 U / ml-2000 U / ml) were added sequentially. 40 μl of 1.5 mM tyrosine solution was added to the solution and reacted at 37 ° C. for 10-15 minutes. The absorbance was measured at 490 nm using an ELISA reader. 0.1 M phosphate buffer (pH 6.5) was used instead of the sample solution for the analysis.

The results are shown in FIG. 4 as a percentage compared to the negative control (sample untreated group). It can be seen that the concentration-dependent tyrosinase inhibitory activity in general.

< Experimental Example  3-2> Tyrosinase Topa  Oxidation Inhibition Experiment

This test is performed in vitro ( in In vitro ) to determine the degree of inhibition of enzyme activity against mushroom tyrosinase DOPA oxidation. The sample was dissolved in an appropriate solvent and 850 μl of 0.1M phosphate buffer (pH 7.0), 50 μl of sample solution and 50 μl of mushroom tyrosinase (1500 U / ml-2000 U / ml) were added in this order and reacted at 37 ° C. for 6 minutes. 50 μl of a 0.06 mM L-DOPA (L-3,4-dihydroxyphenylalaine) solution was added to the solution, followed by further reaction at 37 ° C. for 1 minute. This was measured for absorbance at 475 nm using an ELISA reader. As a blank sample, 0.1M phosphate buffer (pH 7.0) was used instead of the sample solution for analysis.

The results are shown in FIG. 5 as a percentage compared to the negative control (sample untreated group). Similar to the tyrosinase inhibitory activity of FIG.

< Experimental Example  4> Galois - RGD Anti-inflammatory activity of

< Example  4-1> COX -2 expression inhibition activity test

<1> cell culture

In this study, the expression level of COX-2, an enzyme that causes inflammation in human keratinocyte line (HaCaT), was determined at the mRNA level. Specifically, the human keratinocyte line (HaCaT) was mixed with Dulbecco's Modified Eagle's Medium (DMEM), 10% Fatal bovine serum (FBS), and 1% Antibiotic-Antimycotic (GIBCO, Cat No. 15240-062) in a 75 cm ² flask. The cells were cultured at 37 ° C. and 5% CO ₂ . When the human keratinocytes were confluenced at 80% or more, 110 ⁵ cells / well were dispensed into 6 well plates and incubated until the confluence reached at least 80% under cell culture conditions. Thereafter, the medium was removed, and then further cultured under cell culture conditions for 48 hours after exchange with fresh medium containing galloyl-RGD at a concentration of 100 ppm.

<2> RNA Isolation

RNA was isolated by total RNA isolation using QiAzol Lysis Reagent (QIAGEN). The cells from which the medium was removed were washed with PBS (phosphated buffer saline), and 1 ml of QIAzol Lysis Reagent was added thereto and treated at room temperature for 10 minutes. After moving to a new E-tube, 200ul chloroform (Amresco) was added and mixed well, and then reacted at room temperature for 5 minutes, followed by centrifugation at 4 ℃ and 12,000rpm for 15 minutes to separate the mRNA and protein DNA layers. 500ul of the supernatant containing mRNA was transferred to a new E-tube, and 500ul of Isopropanol (MERCK KGaA) was added thereto, inverted 4 to 5 times at room temperature, and further reacted at room temperature for 7 minutes. This was again centrifuged at 4, 13,000 rpm for 30 minutes to precipitate the mRNA pellet. Remove the supernatant, wash the pellet with 1ml of 75% Ethanol, dry it, dissolve in 20ul of RNA Free Water (WelGENE Inc.), and use the NanoDrop ^R Spectrophotometer (ND-1000, UV / VIS) to add 5g of total RNA. Quantified in ml.

<3> cDNA synthesis

cDNA synthesis was synthesized using PrimeScript ^™ reagent kit (TaKaRa, RR037A). Specifically, cDNA was synthesized by mixing 50 μM oligo-dT primer 1 μl, 100 μM Random Primer 4 μl, 5X primeScript Buffer 4 μl, PrimeScript RT enzyme Mix 1 μl with 15 μL of total RNA, and reacting at 37 ° C. for 15 minutes to inactivate RTase. For 5 seconds at 85 ° C.

<4> Real-time PCR

Real-time PCR was performed with the synthesized cDNA to analyze each gene. cDNA, a probe (GAPDH, COX-2), and TaqMan Gene expression master mix (AB, Pro. No. 4369016) were mixed and performed using a 7500 Real time PCR system (applied biosystems). All genes were performed once at 50 ° C. for 2 minutes, at 95 ° C. for 10 minutes, and at 95 ° C. for 15 seconds at 60 times. Inventoried primers from Applied Biosystems (USA) were used. Expression rates are shown by calibrating each GAPDH.

<5> results

The results are shown in FIG. 6 as a percentage compared to the negative control (sample untreated group). Referring to Figure 6 it can be seen that when treated with a concentration of 100 ppm concentration of galloyl-RGD compared to the sample untreated group it can be seen that inhibits the expression of about 43% COX-2.

Experimental Example 4-2 iNOS Expression Influence Test

<1> cell culture

In this study, the expression level of iNOS, an anti-inflammatory gene, in human keratinocyte line (HaCaT) was measured at the mRNA level. Specifically, the human keratinocyte line (HaCaT) was mixed with Dulbecco's Modified Eagle's Medium (DMEM), 10% Fatal bovine serum (FBS), and 1% Antibiotic-Antimycotic (GIBCO, Cat No. 15240-062) in a 75 cm ² flask. The cells were cultured at 37 ° C. and 5% CO ₂ . When the human keratinocytes were confluenced at 80% or more, 110 ⁵ cells / well were dispensed on 6well plates and incubated until the confluence reached at least 80% under cell culture conditions. Thereafter, the culture medium was further cultured under cell culture conditions for 48 hours after exchange with fresh medium containing galloyl-RGD at a concentration of 100 ppm.

<2> RNA Isolation

RNA was isolated by total RNA isolation using QiAzol Lysis Reagent (QIAGEN). The cells from which the medium was removed were washed with PBS (phosphated buffer saline), and 1 ml of QIAzol Lysis Reagent was added thereto and treated at room temperature for 10 minutes. After moving to a new E-tube, 200ul chloroform (Amresco) was added and mixed well, and then reacted at room temperature for 5 minutes, followed by centrifugation at 4 ℃ and 12,000rpm for 15 minutes to separate the mRNA and protein DNA layers. 500ul of the supernatant containing mRNA was transferred to a new E-tube, and 500ul of Isopropanol (MERCK KGaA) was added thereto, inverted 4 to 5 times at room temperature, and further reacted at room temperature for 7 minutes. The pellet was further centrifuged at 4 ° C. and 13,000 rpm for 30 minutes to precipitate mRNA pellets. Remove the supernatant, wash the pellet with 1ml of 75% Ethanol, dry it, dissolve in 20ul of RNA Free Water (WelGENE Inc.), and use the NanoDrop ^R Spectrophotometer (ND-1000, UV / VIS) to add 5g of total RNA. Quantified in ml.

<3> cDNA synthesis

cDNA synthesis was synthesized using PrimeScript ^™ reagent kit (TaKaRa, RR037A). Specifically, cDNA was synthesized by mixing 50 μM oligo-dT primer 1 μl, 100 μM Random Primer 4 μl, 5X primeScript Buffer 4 μl, PrimeScript RT enzyme Mix 1 μl with 15 μL of total RNA, and reacting at 37 ° C. for 15 minutes to inactivate RTase. For 5 seconds at 85 ° C.

<4> Real-time PCR

  Real-time PCR was performed with the synthesized cDNA to analyze each gene. cDNA was mixed with a probe (GAPDH, iNOS) and TaqMan Gene expression master mix (AB, Pro. All genes were performed once at 50 ° C. for 2 minutes, at 95 ° C. for 10 minutes, and at 95 ° C. for 15 seconds at 60 times. Inventoried primers from Applied Biosystems (USA) were used. Expression rates are shown by calibrating each GAPDH.

<5> results

The results are shown in FIG. 7 as a percentage compared to the negative control (sample untreated group). Referring to Figure 7 it can be seen that when treated with a concentration of 100 ppm concentration of galloyl-RGD compared to the sample untreated group, it can be seen that inhibits about 18% iNOS expression.

Claims (7)

delete delete delete Anti-inflammatory skin external composition comprising a galloyl-peptide having the formula as an active ingredient.

5. The method of claim 4,
Wherein the composition is a cosmetic composition.
5. The method of claim 4,
Wherein the composition is a pharmaceutical composition.
5. The method of claim 4,
Wherein the composition is a soap composition.

Images