WO2016093461A1 - Cosmetic composition, containing peptide of astragalus membranaceus, for preventing skin aging - Google Patents

Cosmetic composition, containing peptide of astragalus membranaceus, for preventing skin aging Download PDF

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WO2016093461A1
WO2016093461A1 PCT/KR2015/007388 KR2015007388W WO2016093461A1 WO 2016093461 A1 WO2016093461 A1 WO 2016093461A1 KR 2015007388 W KR2015007388 W KR 2015007388W WO 2016093461 A1 WO2016093461 A1 WO 2016093461A1
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peptide
cosmetic composition
astragalus
present
mmp
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PCT/KR2015/007388
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French (fr)
Korean (ko)
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박선기
차영권
최순호
노현하
히엔 팜반
곽병문
변상요
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주식회사 코스메카코리아
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/645—Proteins of vegetable origin; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations

Definitions

  • the present invention relates to a cosmetic composition for preventing skin aging comprising a yellow peptide, and more particularly, a matrix metalloproteinase related to collagen degeneration in human fibroblasts, characterized in that it comprises a brown peptide as an active ingredient.
  • a matrix metalloproteinase related to collagen degeneration in human fibroblasts, characterized in that it comprises a brown peptide as an active ingredient.
  • Collagen is one of the major components of mammalian connective tissue. In humans, collagen makes up about 40% of human protein, providing skin cells with firmness, flexibility, recovery and elasticity. Skin aging, expressed as wrinkles, is the final result due to the incongruity of collagen degradation. During aging of the skin there is an imbalance in the balance between collagen synthesis and decline, causing excessive collagen decline. Degeneration occurs through matrix metalloproteinases (MMPs), calcium-dependent zinc containing endopeptidases, in particular through MMP-1, MMP-3, MMP-8 and MMP-13. Many active ingredients have been reported that inhibit MMP and collagen degeneration and stimulate collagen synthesis, which are used in the manufacture of cosmetics. Active ingredients derived from plant peptides are not toxic at recommended doses and are being studied for their outstanding effectiveness.
  • MMPs matrix metalloproteinases
  • Astragalus root one of the health-promoting plants popular in the Orient, has been used to treat common colds, diarrhea, fatigue, anorexia and heart disease for more than 2,000 years.
  • the roots contain more than 100 compounds, including triterpenoids saponins and flavonoids.
  • Astragalus peptides have been developed by developing cosmetic materials that can prevent skin aging by inhibiting collagen degeneration, while the peptides of Astragalus reduce MMP expression, increase the synthesis of procollagen in fibroblasts, and ultimately The present invention was completed by discovering that collagen degradation can be prevented and improved by reducing MMPs and increased procollagen, thereby preventing skin aging.
  • the technical problem to be solved in the present invention is to provide a cosmetic composition for preventing skin aging comprising a natural material.
  • the present invention provides a cosmetic composition for preventing skin aging comprising a sulfur peptide as an active ingredient.
  • one embodiment of the present invention provides a cosmetic composition for preventing collagen deterioration comprising an astragalus peptide as an active ingredient.
  • the cosmetic composition comprising the Astragalus peptide of the present invention reduces the expression of matrix metalloproteinases (MMPs), increases the synthesis of procollagen in fibroblasts, and ultimately by reduced MMPs and increased procollagen It can be used as a cosmetic for preventing skin aging by reducing collagen decomposition and preventing and improving skin aging.
  • MMPs matrix metalloproteinases
  • the present invention provides a cosmetic composition for preventing collagen deterioration comprising a sulfur-based peptide.
  • Figure 1 compares the expression of MMP-1 in human skin fibroblasts treated with several peptide moieties of Astragalus isolated by electrophoresis.
  • Figure 2 compares the expression of MMP-3 in human dermal fibroblasts treated with several peptide moieties of Astragalus isolated by electrophoresis.
  • Figure 3 compares the expression of MMP-8 in human dermal fibroblasts treated with several peptide moieties of Astragalus isolated by electrophoresis.
  • Figure 4 compares the expression of MMP-13 in human dermal fibroblasts treated with several peptide moieties of Astragalus isolated by electrophoresis.
  • the present invention provides a cosmetic composition for preventing skin aging comprising a sulfur peptide as an active ingredient.
  • one embodiment of the present invention provides a cosmetic composition for preventing collagen deterioration comprising an astragalus peptide as an active ingredient.
  • Cosmetic composition for preventing skin aging of the present invention is characterized in that it comprises a sulfur peptide as an active ingredient.
  • the sulfuric peptides of the present invention can be obtained by hydrolyzing sulfuric acid with a mixture of trypsin and chymotrypsin, followed by centrifugation.
  • peptides can be separated using only 40% polyacrylamide gel without SDS to separate small peptides according to molecular weight.
  • the peptide partially purified from Astragalus is characterized by having a molecular weight of less than 1500 Da as a result of mass spectrum analysis.
  • the cosmetic composition comprising the Astragalus peptide of the present invention reduces the expression of matrix metalloproteinases (MMPs), increases the synthesis of procollagen in fibroblasts, and ultimately reduces collagen degradation by reduced MMPs and increased procollagen, resulting in skin aging. It can be used as a cosmetic for preventing and improving the skin to prevent skin aging.
  • MMPs matrix metalloproteinases
  • the present invention provides a cosmetic composition for preventing collagen deterioration including a sulfur peptide.
  • the cosmetic composition of the present invention is preferably contained in the amount of the Astragalus peptide in an amount of 0.001-50% by weight relative to the total weight of the cosmetic composition, more preferably in an amount of 0.01-20% by weight, even more preferably Is included in the amount of 0.1-10% by weight. If the content of the Astragalus peptide is less than a certain amount can not expect a distinct effect, if the content exceeds a certain amount, there is a difficulty in the safety of cosmetics or formulation formulation.
  • the formulation of the cosmetic composition of the present invention as described above is not particularly limited and may be appropriately selected as desired.
  • the cosmetic composition of the present invention may be prepared in the form of external skin ointment, softening longevity, nourishing longevity, nutrition cream, massage cream, essence, pack, emulsion, oil gel and the like.
  • the external skin ointment may be prepared to contain 50 to 97% by weight of petrolatum and 0.1 to 5% by weight of polyoxyethylene oleyl-ether phosphate in addition to the sulfuric peptide of the present invention, the flexible cosmetics in addition to the sulfuric peptide of the present invention It can be manufactured to contain 1-10 weight% of polyhydric alcohols (propylene glycol, glycerin etc.) and 0.05-2 weight% of surfactant (polyethylene oleyl ether, polyoxyethylene hardened castor oil, etc.).
  • polyhydric alcohols propylene glycol, glycerin etc.
  • surfactant polyethylene oleyl ether, polyoxyethylene hardened castor oil, etc.
  • the nutritional longevity and nourishing cream is 5 to 20% by weight of oils (squalane, petrolatum, octyldodecanol, etc.) and wax components (cetanol, stearyl alcohol, beeswax, etc.) in addition to the sulfuric peptide of the present invention
  • oils squalane, petrolatum, octyldodecanol, etc.
  • wax components cetanol, stearyl alcohol, beeswax, etc.
  • the essence can be prepared by containing 5 to 30% by weight of polyhydric alcohols, such as glycerin, propylene glycol in addition to the sulfuric peptide of the present invention.
  • Massage cream is prepared by containing 30 to 70% by weight of oil, such as liquid paraffin, petrolatum, isononyl isononanoate in addition to the sulfuric peptide of the present invention, the pack is 5 to 20% by weight of polyvinyl alcohol in addition to the sulfuric peptide of the present invention Peel off pack or general emulsified cosmetics in addition to the sulfur-based peptide of the present invention in the wash off pack containing 5 to 30% by weight of pigments such as kaolin, talc, zinc oxide, titanium dioxide, etc. It can manufacture.
  • oil such as liquid paraffin, petrolatum, isononyl isononanoate
  • the pack is 5 to 20% by weight of polyvinyl alcohol in addition to the sulfuric peptide of the present invention
  • Peel off pack or general emulsified cosmetics in addition to the sulfur-based peptide of the present invention in the wash off pack containing 5 to 30% by weight of pigments such as kaolin, talc, zinc oxide, titanium dioxide, etc.
  • the cosmetic composition of the present invention contains components conventionally used in cosmetic compositions in addition to the above-mentioned Astragalus peptides, and common auxiliaries such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavors, and carriers. It may include, but is not limited thereto.
  • the carrier component is animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide. May be used, but is not limited thereto.
  • the formulation of the cosmetic composition of the present invention is a powder or a spray
  • lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as the carrier component, and in particular, in the case of a spray, additionally chlorofluorohydro Propellant such as carbon, propane / butane or dimethyl ether.
  • a solvent, solubilizing agent or emulsifying agent is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene.
  • Astragalus was purchased from the market using roots. Astragalus protein was digested by mixing trypsin and chymotrypsin (10: 1, w / w). Specifically, the enzyme was dissolved in 10 mM CaCl 2 and 2 mM HCl and stored 20 times concentrated. Substrate-enzyme ratio was set to 25: 1 (w / w) for digestion. Hydrolysis was performed in 50 mM ammonia bicarbonate solution at pH 8-9 for 12-18 hours. The reaction was terminated by heating the sample to 80 ° C. for 5 minutes and then the hydrolyzed suspension was centrifuged at 10,000 rpm for 10 minutes. The obtained supernatant was used as the sulfuric peptide solution.
  • Astragalus peptides were separated on the basis of molecular weight by electrophoresis on 40% polyacrylamide (acrylamide: bisacrylamide, 19: 1, w / w) gel. Specifically, the peptide mixture was placed on the gel with agar stacking gel solution. The electrophoresis process was performed at 10 mA / gel for 1 hour and then at 40 mA / gel until the trace line passed half of the gel, where the gel was divided into 10 parts from the top to the bottom of the gel according to the dimensions ( From PEP1 to PEP10, each part is 15 mm long). Peptides in each portion were extracted with 50% acetonitrile (ACN) and 1% acetic acid.
  • ACN acetonitrile
  • Human skin fibroblasts were 5 ⁇ 10 in 10 ml medium (DMEM + 10% fetal bovine serum (FBS) + 1% antibiotic antimicrobial (penicillin 10,000 units / mL, streptomycin 10,000 ⁇ g / mL, amphotericin B 25 ⁇ g / mL)). Inoculated at 5 cells / plate. The cells were incubated for 3 days at 37 ° C. under 5% CO 2 . The peptide isolated in Example 1 was added to the culture medium at 100 ppm. In addition, cells were cultured in a medium free of peptide as a control. After 3 days, human skin fibroblasts were obtained by scraping and washed in diluted 1/10 concentration of DPBS.
  • DMEM + 10% fetal bovine serum (FBS) + 1% antibiotic antimicrobial penicillin 10,000 units / mL, streptomycin 10,000 ⁇ g / mL, amphotericin B 25 ⁇ g / mL
  • antibiotic antimicrobial
  • Proteins of human dermal fibroblasts were extracted for 5 hours with elution buffer (7M urea, 2M thiourea, 4% CHAPS, 1% DTT, 0.002% bromophenol blue, 2% carrier positive electrolyte, 4% protease inhibitor mixture) , Centrifuged at 13,500 rpm for 10 minutes. The supernatant was then quantified by Bradford assay on an ELISA microreader at 595 nm using bovine albumin as the standard solution.
  • Isoelectric Focusing The extracted protein was in 600 ⁇ l of a rehydration solution (7 M urea, 2 M thiurea, 4% CHAPS, 1% DTT, 2% carrier amphoteric electrolyte, 10% glycerol, 0.002% bromophenol Blue) and placed on immobiline DryStrips (pH 3-10 linear, 24-cm long) rehydrated for 12 hours. Each strip was placed with 100 ⁇ g protein, 2% DNAse, and 5% protein markers. Proteins are equipotentially focused at 500V for 1.5 hours using an Ettan IPGphor 3 (US GE Healthcare) system, then ramped to 1000V for 1.0 hour, ramped to 10,000V for 3.5 hours, and for 11 hours.
  • Ettan IPGphor 3 US GE Healthcare
  • Quantification of Protein Expression The colored gels were scanned using a Umax Powerlook 2100XL scanner and analyzed using the Image Master 2-D Platinum program. Protein spots on the gels were identified based on the pi and molecular weight (MW) of the reference protein. The individual spot volume in the test sample was compared with the volume of the relevant standard spot.
  • MMP-1 known as collagenase-1
  • collagenase-1 is one of the most influential enzymes in skin collagen decline.
  • MMP-1 substrates include collagen types I, II, III, VII, VIII, X and gelatin.
  • MMP-1 preferentially cleaves type III collagen. Active ingredients that inhibit MMP-1 can inhibit skin decline and have a good effect on skin aging.
  • CTR refers to a control group without reagents
  • PEP1 to PEP10 are human dermal fibroblasts treated with peptides 1 to 10, respectively, separated by electrophoresis with 40% polyacrylamide gel.
  • MMP-1 was reduced in all human dermal fibroblasts treated with Astragal peptides, except for samples treated with Astragal peptides isolated from the first portion of a 40% acrylamide gel.
  • the test sample treated with Astragalus peptide isolated from the fifth portion of 40% acrylamide gel showed the greatest reduction. In this case, MMP-1 was reduced by 43.2% compared to the control.
  • MMP-3 shows the effect of Astragalus peptides on MMP-3 regulation.
  • MMP-3 of human dermal fibroblasts treated with Astragalus peptide isolated from 2-7 parts of 40% polyacrylamide gel was present at lower levels compared to untreated control cells. The greatest reduction was seen in human dermal fibroblasts treated with Astragalus peptides isolated from the second (7.6%) and third (5%) of the 40% acrylamide gel.
  • MMP-3 known as stromelysin-1, activates some pro-MMPs during extracellular matrix conversion.
  • MMP-3 substrates include gelatin and collagen III, IV, V, IX, X, and XI.
  • MMP-3 efficiently activates collagenase, matrilysine and gelatinase B, and the action of MMP-3 on partially treated pro-MMP-1 is very critical for the production of fully activated MMP-1. Element.
  • MMP-3 activates TGF-a, which in turn stimulates expression of MMP-9 by macrophages in a COX-2 dependent manner.
  • the active ingredients inhibit MMP-3 expression, which makes it possible to inhibit skin decay.
  • the Astragalus peptide inhibits the expression of MMP-3, indicating that it inhibits skin decay and has a good anti-aging effect.
  • MMP-8 also known as collagenase-2 or neutrophil collagenase, is one of the major collagens responsible for the catalysis of the decline of skin collagen. MMP-8 has the greatest effect on collagen I, a major component of the skin.
  • MMP-8 was reduced in human skin fibroblasts treated with Astragalus peptide.
  • MMP-8 of human dermal fibroblasts treated with Astragal peptides was reduced compared to control cells, up to 22 compared to control cells in human dermal fibroblasts treated with Astragal peptides isolated from the fifth portion of 40% polyacrylamide gel. A decrease of% was observed.
  • MMP-13 also known as collagenase-3, preferentially hydrolyses type II collagen, a major form of collagen found in cartilage. It is noted that the gelatin soluble activity of MMP-13 is about 44 times greater than that of MMP-1 and has a significant effect on the extracellular matrix decline.
  • peptides In order to separate small peptides (ultra peptides), in particular, to separate those having a molecular weight of less than 1500 Da, techniques such as ultracentrifugation, molecular cutoff amount filtration, ultrafiltration, and chromatography are required. In addition, these peptides could be separated using urea-dodecyl sodium sulfate (SDS) -polyacrylamide gel or tricin-SDS-polyacrylamide gel.
  • SDS urea-dodecyl sodium sulfate
  • the Astragalus peptide inhibits MMP expression in human skin fibroblasts associated with skin decline.
  • the inhibitory effect was increased by the electrophoresis clarified peptide.
  • MMP-1, MMP-3, MMP-8 and MMP-13 expressions are reduced to 43%, 5%, 22% and 57%, respectively, compared to controls not treated with Astragalus peptides to inhibit skin aging by inhibiting collagen degeneration. It can be seen that it can prevent.
  • the cytotoxicity of the Astragalus peptide of the present invention was examined.
  • Cytotoxicity was carried out by culturing fibroblasts and adding each of the Astragalus peptides of the present invention, and then examining the survival rate of the cells. Cell viability was determined by MTT assay. First, fibroblasts were laid at 10 5 cells / ml in a 24-well plate, and 0.1%, 0.5%, 2.5% (w / v) of Astragal peptides (PEP4, PEP5, PEP6) were added thereto, and then cultured for 48 hours. Survival was compared. The results are shown in Table 1 below.
  • composition of the present invention was confirmed that the safety does not exhibit cytotoxicity.
  • SIRCOL collgen assay was performed by measuring collagen production promoting effect using human epidermal cell line, CSK323. Cell lines were aliquoted into 24well plates at a concentration of 10 5 cells / ml and incubated for 24 hours in 10% FBS / DMEM medium.
  • the composition of the present invention was confirmed to have an excellent collagen production promoting effect.
  • the HaCaT cell line was incubated with DMEM / 10% FBS, aliquoted at a concentration of 10 5 cells / ml in a 24well plate, stabilized, and then the samples were added at a concentration of 1 mg / ml, respectively, and 25 ug / ml of hesperidin as a control. Then, the amount of TNF- ⁇ secretion was compared by treating hydrogen peroxide at a concentration of 100 uM and then separating and quantifying the supernatant after 24 hours. The smaller the secretion amount of TNF- ⁇ , the better the anti-aging action by the radical. Analysis of TNF- ⁇ was performed by ELISA method. The results are shown in Table 3 below.
  • composition of the present invention can be said to have a very excellent anti-aging effect.
  • a cream containing the Astragalus peptide (PEP5) of the present invention was prepared.
  • Sample "PEP5" of the extract of the present invention 5%, argan oil 5%, shea butter 5%, green tea seed oil 3%, avocado oil 2%, isoamy laurate (5% isoamy laurate), cetiaryl alcohol (Cetearyl alcohol ) 5%, hyaluronic acid 10% solution 10%, beta glucan 10% solution 10%, the remainder 3% of the remainder was added to the purified water in a volume ratio and mixed to prepare a cream.
  • a lotion containing the Astragalus peptide (PEP5) of the present invention was prepared.
  • Sample of the extract of the present invention "PEP5" 5%, argan oil 2%, shea butter 2%, green tea seed oil 1%, jojoba oil 1%, isoamy laurate (4%), cetiaryl alcohol (Cetearyl alcohol ) 3%, sorbitan olivate (Sorbitan Olivate) 1%, hyaluronic acid 5% solution 10%, beta glucan 10% solution 5%, the fragrance 3% was added to the purified water in a volume ratio and mixed to prepare a lotion.
  • composition of the present invention can be used as a raw material in the manufacture of cosmetics, cosmetics containing the composition of the present invention has a very excellent skin anti-aging effect can be developed as a high value-added product.

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Abstract

A cosmetic composition containing a peptide of Astragalus membranaceus, of the present invention, reduces the expression of matrix metalloproteinases (MMPs), increases the synthesis of procollagen in fibroblasts, and ultimately reduces the collagen degradation through the reduced MMPs and the increased procollagen, thereby preventing and alleviating skin aging, and thus can be used as a cosmetic agent for preventing skin aging. In addition, the present invention provides a cosmetic composition, containing a peptide of Astragalus membranaceus, for preventing collagen degeneration.

Description

황기 펩타이드를 포함하는 피부 노화 방지를 위한 화장료 조성물Cosmetic composition for preventing skin aging containing astragalus peptide
본 발명은 황기 펩타이드를 포함하는 피부 노화 방지를 위한 화장료 조성물에 관한 것으로, 보다 구체적으로는 황기 펩타이드를 유효성분으로 포함하는 것을 특징으로 하는, 인간 섬유아세포에서 콜라겐 퇴화와 관련된 매트릭스 메탈로프로티나아제(matrix metalloproteinase) 효소의 발현을 감소시키고 콜라겐 퇴화를 억제함으로써 피부 노화를 방지하는 화장료 조성물에 관한 것이다.The present invention relates to a cosmetic composition for preventing skin aging comprising a yellow peptide, and more particularly, a matrix metalloproteinase related to collagen degeneration in human fibroblasts, characterized in that it comprises a brown peptide as an active ingredient. (Matrix metalloproteinase) relates to a cosmetic composition that prevents skin aging by reducing the expression of enzymes and inhibiting collagen degeneration.
콜라겐은 포유류의 연결 조직의 주요 성분중 하나이다. 사람의 경우 콜라겐은 인체 단백질의 약 40%를 차지하므로 피부 세포의 견고성, 유연성, 회복성, 탄성을 공급한다. 주름으로 표현되는 피부 노화는 콜라겐의 합성 저하의 부조화로 인한 최종 결과이다. 피부의 노화가 진행되는 동안에는 콜라겐의 합성과 쇠퇴사이의 조화에 불균형이 일어나서, 과도한 콜라겐 쇠퇴를 야기한다. 퇴화는 매트릭스 메탈로프로티나아제(matrix metalloproteinase, MMP), 칼슘 의존성 아연 함유의 엔도펩티다아제(endopeptinase)를 통해 일어나며, 특히 MMP-1, MMP-3, MMP-8 및 MMP-13을 통해 일어난다. MMP 및 콜라겐 퇴화를 억제하고 콜라겐 합성을 자극하는 많은 활성 성분들이 보고되었는데, 이들은 화장품 제조에 이용되고 있다. 식물 펩타이드로부터 유도된 활성 성분들은 권장 사용량 복용시 독성이 없고 그 뛰어난 효과로 이에 대한 연구가 계속 진행되고 있다.Collagen is one of the major components of mammalian connective tissue. In humans, collagen makes up about 40% of human protein, providing skin cells with firmness, flexibility, recovery and elasticity. Skin aging, expressed as wrinkles, is the final result due to the incongruity of collagen degradation. During aging of the skin there is an imbalance in the balance between collagen synthesis and decline, causing excessive collagen decline. Degeneration occurs through matrix metalloproteinases (MMPs), calcium-dependent zinc containing endopeptidases, in particular through MMP-1, MMP-3, MMP-8 and MMP-13. Many active ingredients have been reported that inhibit MMP and collagen degeneration and stimulate collagen synthesis, which are used in the manufacture of cosmetics. Active ingredients derived from plant peptides are not toxic at recommended doses and are being studied for their outstanding effectiveness.
동양에서 많이 보급된 건강 증진 식물중 하나인 황기 뿌리는 2천년이상동안 통상의 감기, 설사, 피로, 식욕부진, 심장질환 등을 치료하는 데에 이용되어 왔다. 트리테르페노이드 사포닌 및 플라보노이드를 포함하여 뿌리에는 100개 이상의 합성물이 함유되어 있다. Astragalus root, one of the health-promoting plants popular in the Orient, has been used to treat common colds, diarrhea, fatigue, anorexia and heart disease for more than 2,000 years. The roots contain more than 100 compounds, including triterpenoids saponins and flavonoids.
그러나, 황기의 펩타이드에 대한 연구는 현재까지 거의 행해지지 않았다. 이에 본 발명자들은 콜라겐 퇴화를 억제하여 피부 노화를 방지할 수 있는 화장료 재료를 개발하기 위해 계속 연구를 진행하던 중 황기의 펩타이드가 MMP 발현을 감소시키고, 섬유아세포에서의 procollagen의 합성을 증가시키며, 궁극적으로 감소된 MMPs와 증가된 procollagen에 의하여 collagen분해를 감소시켜 피부노화를 예방하고 개선할 수 있다는 사실을 발견하여 본 발명을 완성하였다.However, little research has been done on Astragalus peptides. The inventors of the present invention continue to develop cosmetic materials that can prevent skin aging by inhibiting collagen degeneration, while the peptides of Astragalus reduce MMP expression, increase the synthesis of procollagen in fibroblasts, and ultimately The present invention was completed by discovering that collagen degradation can be prevented and improved by reducing MMPs and increased procollagen, thereby preventing skin aging.
따라서, 본 발명에서 해결하고자 하는 기술적 과제는 천연재료를 포함하는 피부 노화 방지를 위한 화장료 조성물을 제공하기 위한 것이다.Therefore, the technical problem to be solved in the present invention is to provide a cosmetic composition for preventing skin aging comprising a natural material.
상기한 기술적 과제를 해결하기 위하여, 본 발명에서는 황기 펩타이드를 유효성분으로 포함하는 피부 노화 방지를 위한 화장료 조성물을 제공한다. In order to solve the above technical problem, the present invention provides a cosmetic composition for preventing skin aging comprising a sulfur peptide as an active ingredient.
또한, 본 발명의 일 실시양태에서는 황기 펩타이드를 유효성분으로 포함하는 콜라겐 퇴화 방지를 위한 화장료 조성물을 제공한다.In addition, one embodiment of the present invention provides a cosmetic composition for preventing collagen deterioration comprising an astragalus peptide as an active ingredient.
이상에서 상술한 바와 같이, 본 발명의 황기 펩타이드를 포함하는 화장료 조성물은 MMPs(matrix metalloproteinases) 발현을 감소시키고, 섬유아세포에서의 procollagen의 합성을 증가시키며, 궁극적으로 감소된 MMPs와 증가된 procollagen에 의하여 collagen분해를 감소시켜 피부노화를 예방하고 개선하여 피부 노화를 방지를 위한 화장료로서 이용할 수 있다. 또한, 본 발명에서는 황기 펩타이드를 포함하는 콜라겐 퇴화 방지를 위한 화장료 조성물을 제공한다.As described above, the cosmetic composition comprising the Astragalus peptide of the present invention reduces the expression of matrix metalloproteinases (MMPs), increases the synthesis of procollagen in fibroblasts, and ultimately by reduced MMPs and increased procollagen It can be used as a cosmetic for preventing skin aging by reducing collagen decomposition and preventing and improving skin aging. In addition, the present invention provides a cosmetic composition for preventing collagen deterioration comprising a sulfur-based peptide.
본 명세서에 첨부되는 다음의 도면들은 본 발명의 바람직한 실시예를 예시하는 것이며, 전술한 발명의 내용과 함께 본 발명의 기술사상을 더욱 이해시키는 역할을 하는 것이므로, 본 발명은 그러한 도면에 기재된 사항에만 한정되어 해석되어서는 아니 된다.The following drawings, which are attached to this specification, illustrate preferred embodiments of the present invention, and together with the contents of the present invention serve to further understand the technical spirit of the present invention, the present invention is limited to the matters described in such drawings. It should not be construed as limited.
도 1은 전기영동에 의해 분리된 황기의 여러 펩타이드 부분으로 처리한 인체 피부 섬유아세포에서의 MMP-1의 발현을 비교한 것이다. Figure 1 compares the expression of MMP-1 in human skin fibroblasts treated with several peptide moieties of Astragalus isolated by electrophoresis.
도 2는 전기영동에 의해 분리된 황기의 여러 펩타이드 부분으로 처리한 인체 피부섬유아세포에서의 MMP-3의 발현을 비교한 것이다.  Figure 2 compares the expression of MMP-3 in human dermal fibroblasts treated with several peptide moieties of Astragalus isolated by electrophoresis.
도 3은 전기영동에 의해 분리된 황기의 여러 펩타이드 부분으로 처리한 인체 피부섬유아세포에서의 MMP-8의 발현을 비교한 것이다. Figure 3 compares the expression of MMP-8 in human dermal fibroblasts treated with several peptide moieties of Astragalus isolated by electrophoresis.
도4는 전기영동에 의해 분리된 황기의 여러 펩타이드 부분으로 처리한 인체 피부섬유아세포에서의 MMP-13의 발현을 비교한 것이다. Figure 4 compares the expression of MMP-13 in human dermal fibroblasts treated with several peptide moieties of Astragalus isolated by electrophoresis.
도5는 40% 폴리아크릴아미드 젤로 전기영동처리하여 분리한 제5부분으로부터의 황기 펩타이드의 질량 스펙트럼 분석이다. 5 is a mass spectral analysis of the Astragalus peptide from the fifth portion separated by electrophoresis with 40% polyacrylamide gel.
상기한 기술적 과제를 해결하기 위하여, 본 발명에서는 황기 펩타이드를 유효성분으로 포함하는 피부 노화 방지를 위한 화장료 조성물을 제공한다. In order to solve the above technical problem, the present invention provides a cosmetic composition for preventing skin aging comprising a sulfur peptide as an active ingredient.
또한, 본 발명의 일 실시양태에서는 황기 펩타이드를 유효성분으로 포함하는 콜라겐 퇴화 방지를 위한 화장료 조성물을 제공한다.In addition, one embodiment of the present invention provides a cosmetic composition for preventing collagen deterioration comprising an astragalus peptide as an active ingredient.
본 발명의 피부 노화 방지를 위한 화장료 조성물은 황기 펩타이드를 유효성분으로 포함하는 것을 특징으로 한다.Cosmetic composition for preventing skin aging of the present invention is characterized in that it comprises a sulfur peptide as an active ingredient.
본 발명의 황기 펩타이드는 황기를 트립신과 키모트립신의 혼합물로 가수분해시킨 다음, 원심분리하여 수득할 수 있다. The sulfuric peptides of the present invention can be obtained by hydrolyzing sulfuric acid with a mixture of trypsin and chymotrypsin, followed by centrifugation.
본 발명에서는 분자량에 따라 소형 펩타이드를 분리하기 위해 SDS 없이 40%폴리아크릴아미드 젤만을 이용하여 펩타이드를 분리할 수 있다. In the present invention, peptides can be separated using only 40% polyacrylamide gel without SDS to separate small peptides according to molecular weight.
본 발명에서 황기로부터 부분 정제된 펩타이드는 질량 스펙트럼 분석 결과 1500Da미만의 분자량을 가지는 것이 특징이다.In the present invention, the peptide partially purified from Astragalus is characterized by having a molecular weight of less than 1500 Da as a result of mass spectrum analysis.
본 발명의 황기 펩타이드를 포함하는 화장료 조성물은 MMPs(matrix metalloproteinases) 발현을 감소시키고, 섬유아세포에서의 procollagen의 합성을 증가시키며, 궁극적으로 감소된 MMPs와 증가된 procollagen에 의하여 collagen분해를 감소시켜 피부노화를 예방하고 개선하여 피부 노화를 방지를 위한 화장료로서 이용할 수 있다. The cosmetic composition comprising the Astragalus peptide of the present invention reduces the expression of matrix metalloproteinases (MMPs), increases the synthesis of procollagen in fibroblasts, and ultimately reduces collagen degradation by reduced MMPs and increased procollagen, resulting in skin aging. It can be used as a cosmetic for preventing and improving the skin to prevent skin aging.
한편, 본 발명에서는 황기 펩타이드를 포함하는 콜라겐 퇴화 방지를 위한 화장료 조성물을 제공한다.On the other hand, the present invention provides a cosmetic composition for preventing collagen deterioration including a sulfur peptide.
본 발명의 화장료 조성물에는 상기 황기 펩타이드가 화장료 조성물의 총 중량에 대하여 0.001-50 중량%의 양으로 포함되는 것이 바람직하며, 더욱 바람직하게는 0.01-20 중량%의 양으로 포함되고, 더욱 더 바람직하게는 0.1-10 중량%로 포함된다. 황기 펩타이드의 함량이 일정 함량 미만일 경우에는 뚜렷한 효과를 기대할 수 없으며, 일정 함량을 초과일 경우에는 화장료의 안전성 또는 제형상의 제조에 어려움이 있다.The cosmetic composition of the present invention is preferably contained in the amount of the Astragalus peptide in an amount of 0.001-50% by weight relative to the total weight of the cosmetic composition, more preferably in an amount of 0.01-20% by weight, even more preferably Is included in the amount of 0.1-10% by weight. If the content of the Astragalus peptide is less than a certain amount can not expect a distinct effect, if the content exceeds a certain amount, there is a difficulty in the safety of cosmetics or formulation formulation.
상기와 같은 본 발명의 화장료 조성물의 제형은 특별히 제한되지 않으며, 목적하는 바에 따라 적절히 선택할 수 있다. 예를 들어, 본 발명의 화장료 조성물은 피부외용연고, 유연화장수, 영양화장수, 영양크림, 마사지크림, 에센스, 팩, 에멀젼, 오일젤 등의 제형으로 제조될 수 있다. 이때, 상기 피부외용연고는 본 발명의 황기 펩타이드 이외에 바셀린 50∼97 중량% 및 폴리옥시에틸렌올레일-에테르 포스페이트 0.1∼5중량%를 함유하도록 제조할 수 있으며, 유연화장수는 본 발명의 황기 펩타이드 이외에 다가알콜류(프로필렌글리콜, 글리세린 등) 1∼10 중량% 및 계면활성제(폴리에틸렌올레일에테르, 폴리옥시에틸렌 경화피마자유 등) 0.05∼2 중량%를 함유하도록 제조할 수 있다. 또한, 영양화장수 및 영양크림은 본 발명의 황기 펩타이드 이외에 오일류(스쿠알란, 바셀린, 옥틸도데칸올 등) 5∼20 중량% 및 왁스성분(세탄올, 스테아릴알콜, 밀납 등) 3∼15 중량%를 함유하도록 제조할 수 있으며, 에센스는 본 발명의 황기 펩타이드 이외에 글리세린, 프로필렌글리콜 등의 다가알콜류 5∼30 중량%를 함유하여 제조할 수 있다. 마사지 크림은 본 발명의 황기 펩타이드 이외에 유동파라핀, 바셀린, 이소노닐이소노나노에이트 등의 오일 30∼70 중량%를 함유하여 제조되며, 팩은 본 발명의 황기 펩타이드 이외에 폴리비닐알콜 5∼20 중량%를 함유하는 필 오프(peel off) 팩 또는 일반유화형화장료에 본 발명의 황기 펩타이드 이외에 카올린, 탈크, 산화아연, 이산화티탄 등의 안료가 5∼30 중량% 함유된 워시오프(wash off) 팩으로 제조할 수 있다.The formulation of the cosmetic composition of the present invention as described above is not particularly limited and may be appropriately selected as desired. For example, the cosmetic composition of the present invention may be prepared in the form of external skin ointment, softening longevity, nourishing longevity, nutrition cream, massage cream, essence, pack, emulsion, oil gel and the like. At this time, the external skin ointment may be prepared to contain 50 to 97% by weight of petrolatum and 0.1 to 5% by weight of polyoxyethylene oleyl-ether phosphate in addition to the sulfuric peptide of the present invention, the flexible cosmetics in addition to the sulfuric peptide of the present invention It can be manufactured to contain 1-10 weight% of polyhydric alcohols (propylene glycol, glycerin etc.) and 0.05-2 weight% of surfactant (polyethylene oleyl ether, polyoxyethylene hardened castor oil, etc.). In addition, the nutritional longevity and nourishing cream is 5 to 20% by weight of oils (squalane, petrolatum, octyldodecanol, etc.) and wax components (cetanol, stearyl alcohol, beeswax, etc.) in addition to the sulfuric peptide of the present invention It can be prepared to contain, the essence can be prepared by containing 5 to 30% by weight of polyhydric alcohols, such as glycerin, propylene glycol in addition to the sulfuric peptide of the present invention. Massage cream is prepared by containing 30 to 70% by weight of oil, such as liquid paraffin, petrolatum, isononyl isononanoate in addition to the sulfuric peptide of the present invention, the pack is 5 to 20% by weight of polyvinyl alcohol in addition to the sulfuric peptide of the present invention Peel off pack or general emulsified cosmetics in addition to the sulfur-based peptide of the present invention in the wash off pack containing 5 to 30% by weight of pigments such as kaolin, talc, zinc oxide, titanium dioxide, etc. It can manufacture.
또한, 본 발명의 화장료 조성물은 유효 성분으로서 상기 황기 펩타이드 이외에 화장료 조성물에 통상적으로 이용되는 성분들을 포함하며, 예컨대 항산화제, 안정화제, 용해화제, 비타민, 안료 및 향료와 같은 통상적인 보조제, 그리고 담체를 포함할 수 있으나, 이에 한정되는 것은 아니다.In addition, the cosmetic composition of the present invention, as an active ingredient, contains components conventionally used in cosmetic compositions in addition to the above-mentioned Astragalus peptides, and common auxiliaries such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavors, and carriers. It may include, but is not limited thereto.
본 발명의 화장료 조성물의 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물성유, 식물성유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 이용될 수 있으나, 이에 한정되는 것은 아니다.In case the formulation of the cosmetic composition of the present invention is a paste, cream or gel, the carrier component is animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide. May be used, but is not limited thereto.
본 발명의 화장료 조성물의 제형이 파우더 또는 스프레이인 경우에는 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄 히드록시드, 칼슘 실리케이트 또는 폴리아미드 파우더가 이용될 수 있고, 특히 스프레이인 경우에는 추가적으로 클로로플루오로히드로카본, 프로판/부탄 또는 디메틸 에테르와 같은 추진체를 포함할 수 있다.When the formulation of the cosmetic composition of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as the carrier component, and in particular, in the case of a spray, additionally chlorofluorohydro Propellant such as carbon, propane / butane or dimethyl ether.
본 발명의 화장료 조성물의 제형이 용액 또는 유탁액인 경우에는 담체 성분으로서 용매, 용해화제 또는 유탁화제가 이용되고, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌 글리콜, 1,3-부틸글리콜오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르가 있다.When the formulation of the cosmetic composition of the present invention is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene. Fatty acid esters of glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan.
이하, 본 발명의 이해를 돕기 위하여 실시예 등을 들어 상세하게 설명하기로 한다. 그러나, 본 발명에 따른 실시예들은 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 하기 실시예들에 한정되는 것으로 해석되어서는 안 된다. 본 발명의 실시예들은 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다. Hereinafter, examples and the like will be described in detail to help understand the present invention. However, embodiments according to the present invention can be modified in many different forms, the scope of the invention should not be construed as limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.
<실시예 1> 황기 펩타이드 제조Example 1 Preparation of Astragalus Peptides
황기는 뿌리를 이용하며 시장에서 구입하였다. 황기 단백질은 트립신과 키모트립신(10:1, w/w)의 혼합에 의해 분해하였다. 구체적으로, 효소를 10mM CaCl2 and 2mM HCl에서 용해시켜 20배 저장 농축하였다. 분해를 위해 기질-효소 비율은 25:1 (w/w)로 설정하였다. 가수분해과정은 pH 8-9에서 12-18 시간동안 50mM 암모니아 중탄산염 용액에서 수행하였다. 시료를 5분동안 80℃로 가열시킴으로써 반응은 종료시킨 다음 가수분해된 현탁액을 10분동안 10,000 rpm 에서 원심분리하였다. 수득된 상층액은 황기 펩타이드 용액으로 사용하였다. Astragalus was purchased from the market using roots. Astragalus protein was digested by mixing trypsin and chymotrypsin (10: 1, w / w). Specifically, the enzyme was dissolved in 10 mM CaCl 2 and 2 mM HCl and stored 20 times concentrated. Substrate-enzyme ratio was set to 25: 1 (w / w) for digestion. Hydrolysis was performed in 50 mM ammonia bicarbonate solution at pH 8-9 for 12-18 hours. The reaction was terminated by heating the sample to 80 ° C. for 5 minutes and then the hydrolyzed suspension was centrifuged at 10,000 rpm for 10 minutes. The obtained supernatant was used as the sulfuric peptide solution.
황기 펩타이드를 40% 폴리아크릴아미드 (아크릴아미드: 비스아크릴아미드, 19:1, w/w) 젤 상에서 전기영동을 통해 분자량에 기초하여 분리하였다. 구체적으로, 펩타이드 혼합물을 아가 스태킹 젤 용액과 함께 상기 젤 상에 위치시켰다. 전기영동 과정은 1시간동안은 10mA/gel로 수행한 후, 추적 라인이 젤의 절반을 지날 때까지는 40mA/gel로 수행해졌는데, 이때에 젤은 치수에 따라 젤 상단에서 하단까지 10파트로 나누었다(PEP1로부터 PEP10까지, 각 부분은 15mm 길이임). 각 부분에서 펩타이드를 50% 아세토니트릴 (ACN) 및 1% 아세트산으로 추출하였다. 이어서, 추출물을 질소하에서 증발시켜 고농도로 수득하였다. 펩타이드 추출물을 사용전 50% ACN 용액 및 0.1% 포름산에서 활성화된 단백질/펩타이드 ToptipsTMTT3CAR를 이용하여 탈염시켰다. 0.1% 포름산을 첨가하여 오염 염분을 제거하고, 펩타이드를 50% ACN용액에서 용리하였다. 추출물을 진공에서 증발시킨 후 나머지 부분들은 인체 피부 섬유아세포에 대한 테스트를 위해 DMEM에 용해시켰다. Astragalus peptides were separated on the basis of molecular weight by electrophoresis on 40% polyacrylamide (acrylamide: bisacrylamide, 19: 1, w / w) gel. Specifically, the peptide mixture was placed on the gel with agar stacking gel solution. The electrophoresis process was performed at 10 mA / gel for 1 hour and then at 40 mA / gel until the trace line passed half of the gel, where the gel was divided into 10 parts from the top to the bottom of the gel according to the dimensions ( From PEP1 to PEP10, each part is 15 mm long). Peptides in each portion were extracted with 50% acetonitrile (ACN) and 1% acetic acid. The extract was then evaporated under nitrogen to give a high concentration. Peptide extracts were desalted using protein / peptide Toptips ™ TT3CAR activated in 50% ACN solution and 0.1% formic acid prior to use. Contaminant salts were removed by adding 0.1% formic acid and the peptide eluted in 50% ACN solution. After evaporation of the extract in vacuo, the remaining portions were dissolved in DMEM for testing on human skin fibroblasts.
*<실시예 2> 인체 피부 섬유아세포 배양Example 2 Human Skin Fibroblast Culture
인체 피부 섬유아세포를 10ml 배지 (DMEM + 10% 우태아혈청(FBS) + 1% 항생 항균제(페니실린 10,000 units/mL, 스트렙토마이신 10,000μg/mL, 암포테리신 B 25μg/mL))에서 5 × 105 cells/plate로 접종하였다. 상기 세포들을 5% CO2하 37℃에서 3일동안 배양하였다. 상기 실시예 1에서 분리된 펩타이드를 100 ppm에서 배양 배지에 가하였다. 추가로, 대조군으로서 펩타이드를 포함하지 않는 배지에서 세포를 배양하였다. 3일 후, 인체 피부 섬유아세포들을 스크래핑하여 얻었으며, 희석된 1/10 농도의 DPBS에서 세척하였다. 인체 피부 섬유아세포의 단백질을 용리 완충액(7M 우레아, 2M 티오우레아, 4% CHAPS, 1% DTT, 0.002% 브로모페놀 블루, 2% 담체양성전해질, 4% 프로테아제 억제제 혼합물)으로 5시간동안 추출한 후, 13,500 rpm에서 10분 동안 원심분리하였다. 이어서, 표준액으로 소 알부민을 사용하여 상청액을 595nm에서 ELISA 마이크로리더에서 Bradford분석에 의해 정량하였다.Human skin fibroblasts were 5 × 10 in 10 ml medium (DMEM + 10% fetal bovine serum (FBS) + 1% antibiotic antimicrobial (penicillin 10,000 units / mL, streptomycin 10,000 μg / mL, amphotericin B 25 μg / mL)). Inoculated at 5 cells / plate. The cells were incubated for 3 days at 37 ° C. under 5% CO 2 . The peptide isolated in Example 1 was added to the culture medium at 100 ppm. In addition, cells were cultured in a medium free of peptide as a control. After 3 days, human skin fibroblasts were obtained by scraping and washed in diluted 1/10 concentration of DPBS. Proteins of human dermal fibroblasts were extracted for 5 hours with elution buffer (7M urea, 2M thiourea, 4% CHAPS, 1% DTT, 0.002% bromophenol blue, 2% carrier positive electrolyte, 4% protease inhibitor mixture) , Centrifuged at 13,500 rpm for 10 minutes. The supernatant was then quantified by Bradford assay on an ELISA microreader at 595 nm using bovine albumin as the standard solution.
<실시예 3> 2-D PAGE (폴리아크릴아미드 젤 전기영동)Example 3 2-D PAGE (Polyacrylamide Gel Electrophoresis)
등전 초점화 (IEF): 상기 추출된 단백질을 in 600 μl의 재수화 용액(7 M 우레아, 2M 티우레아, 4% CHAPS, 1% DTT, 2% 담체 양성전해질, 10% 글리세롤, 0.002% 브로모페놀 블루)에서 12시간동안 재수화된 immobiline DryStrips(pH 3-10 선형, 길이 24-cm)위에 배치하였다. 각각의 스트립을 100μg 단백질, 2% DNAse, 및 5% 단백질 마커와 함께 배치하였다. 단백질은 Ettan IPGphor 3 (미국 GE 헬스케어) 시스템을 이용하여 500V에서 1.5시간동안 등전위적으로 포커싱된 후, 1.0시간동안 1000V까지 램핑(ramping)시키고, 3.5시간동안 10,000V까지 램핑시키고, 11시간동안 10,000 V로 유지시켰다. 2차 분리 전에, 평형 완충액(7M 우레아, 2M 티오우레아, 2% SDS, 50 mM Tris-HCl, 30% 글리세롤, 0.002% 브로모페놀 블루)에서 1% DTT와 고정화된 DryStrips를 15분동안 배양하여 다이설파이드 결합을 감소시켰고, 이후에 유리 SH-기를 평형 완충액 중에서 2.5% 아이오도아세트아미드로 처리하여 알킬화시켰다. 이후, 스트립은 12.5% 폴리아크릴아미드 젤(25 x 20 cm)로 옮기고, 제2차 전기영동(1단계: 1시간동안 10mA/gel 수행, 2단계: 5시간동안 40 mV/gel 수행)하였다. 이어서, 젤을 고정하고 질산염 실버 착색 방법에 따라 착색하였다. Isoelectric Focusing (IEF) : The extracted protein was in 600 μl of a rehydration solution (7 M urea, 2 M thiurea, 4% CHAPS, 1% DTT, 2% carrier amphoteric electrolyte, 10% glycerol, 0.002% bromophenol Blue) and placed on immobiline DryStrips (pH 3-10 linear, 24-cm long) rehydrated for 12 hours. Each strip was placed with 100 μg protein, 2% DNAse, and 5% protein markers. Proteins are equipotentially focused at 500V for 1.5 hours using an Ettan IPGphor 3 (US GE Healthcare) system, then ramped to 1000V for 1.0 hour, ramped to 10,000V for 3.5 hours, and for 11 hours. Maintained at 10,000 V. Before secondary separation, incubate 1% DTT and immobilized DryStrips for 15 minutes in equilibration buffer (7M urea, 2M thiourea, 2% SDS, 50 mM Tris-HCl, 30% glycerol, 0.002% bromophenol blue) Disulfide bonds were reduced and then alkylated by treating the free SH-group with 2.5% iodoacetamide in equilibration buffer. The strip was then transferred to 12.5% polyacrylamide gel (25 x 20 cm) and subjected to secondary electrophoresis (step 1: 10 mA / gel for 1 h, step 2: 40 mV / gel for 5 h). The gel was then fixed and colored according to the nitrate silver coloring method.
단백질 발현의 정량화: 상기 착색된 젤을 Umax Powerlook 2100XL 스캐너를 이용하여 스캔하고, Image Master 2-D Platinum 프로그램을 이용하여 분석하였다. 젤 위의 단백질 반점은 기준 단백질의 pI 및 분자량(MW)에 기초하여 식별하였다. 테스트 시료에서의 개별 반점 부피는 관련 표준 반점의 부피와 비교하였다. Quantification of Protein Expression : The colored gels were scanned using a Umax Powerlook 2100XL scanner and analyzed using the Image Master 2-D Platinum program. Protein spots on the gels were identified based on the pi and molecular weight (MW) of the reference protein. The individual spot volume in the test sample was compared with the volume of the relevant standard spot.
콜라게나제-1로 알려진 MMP-1은 피부 콜라겐 쇠퇴에서 가장 영향을 끼치는 효소중 하나이다. MMP-1 기질은 콜라겐 타입 I, II, III, VII, VIII, X 및 젤라틴을 포함한다. MMP-1은 우선적으로 타입 III 콜라겐을 절단한다. MMP-1을 억제하는 활성 성분은 피부 쇠퇴를 억제할 수 있으며, 피부 노화방지에 좋은 효과를 가진다. MMP-1, known as collagenase-1, is one of the most influential enzymes in skin collagen decline. MMP-1 substrates include collagen types I, II, III, VII, VIII, X and gelatin. MMP-1 preferentially cleaves type III collagen. Active ingredients that inhibit MMP-1 can inhibit skin decline and have a good effect on skin aging.
그 결과는 도 1에 나타내었다. 도 1에서 CTR은 시약처리하지 않은 대조군을 의미하며, PEP1 내지 PEP10은 각각 40% 폴리아크릴아미드 젤로 전기영동법에 의해 분리한 펩타이드 제1내지 제10부분으로 처리한 인체 피부 섬유아세포들이다. The results are shown in FIG. In FIG. 1, CTR refers to a control group without reagents, and PEP1 to PEP10 are human dermal fibroblasts treated with peptides 1 to 10, respectively, separated by electrophoresis with 40% polyacrylamide gel.
40% 아크릴아미드 젤의 제1부분으로부터 분리된 황기 펩타이드로 처리된 샘플을 제외하고는 황기 펩타이드로 처리된 모든 인체 피부 섬유아세포들에서 MMP-1은 대조군에 비하여 감소되었다. 40% 아크릴아미드 젤의 제5부분으로부터 분리된 황기 펩타이드로 처리된 테스트 시료가 가장 큰 감소를 보였다. 이 경우, MMP-1은 대조군에 비하여 비해 43.2%까지 감소한 것이다. 이러한 결과는 황기 펩타이드가, 특히 40% 아크릴아미드 젤의 제5부분으로부터 분리된 황기 펩타이드가 MMP-1의 저하를 유도하는 것을 나타낸다.MMP-1 was reduced in all human dermal fibroblasts treated with Astragal peptides, except for samples treated with Astragal peptides isolated from the first portion of a 40% acrylamide gel. The test sample treated with Astragalus peptide isolated from the fifth portion of 40% acrylamide gel showed the greatest reduction. In this case, MMP-1 was reduced by 43.2% compared to the control. These results indicate that the Astragalus peptide, particularly the Astragal peptide isolated from the fifth part of the 40% acrylamide gel, induces a decrease in MMP-1.
도 2에는 MMP-3 조절에 대한 황기 펩타이드의 효과를 나타내었다. 여기에서 보듯이, 40% 폴리아크릴아미드 젤의 제2-7부분에서 분리한 황기 펩타이드로 처리한 인체 피부 섬유아세포의 MMP-3는 미처리된 대조군 세포와 비교할 때 더 낮은 수준으로 존재하였다. 40% 아크릴아미드 젤의 제2부분(7.6%) 및 제3부분(5%)으로부터 분리한 황기 펩타이드로 처리한 인체 피부 섬유아세포에서 가장 큰 감소를 나타내었다. 스트로멜리신-1로 알려진 MMP-3는 세포외 기질 전환동안에 일부 pro-MMP들을 활성화시킨다. MMP-3 기질은 젤라틴 및 콜라겐 III, IV, V, IX, X, 및 XI을 포함한다. MMP-3는 콜라게나제, 마트릴리신 및 젤라티나제 B를 효율적으로 활성화시키며, 부분적으로 처리된 pro-MMP-1에 대한 MMP-3의 작용은 완전 활성화된 MMP-1의 생성에 매우 결정적인 요소이다. 또한, MMP-3는 TGF-a를 활성화시키며, 다음에는 COX-2 의존 방식으로 대식세포에 의해 MMP-9의 발현을 자극한다. 다음으로, 활성성분들은 MMP-3 발현을 억제하며, 이는 피부의 쇠퇴를 억제할 수 있게 한다. 결과적으로, 황기 펩타이드는 MMP-3의 발현을 억제하는데, 이는 피부 쇠퇴를 억제하고 양호한 노화방지효과를 가지는 것을 나타낸다. 2 shows the effect of Astragalus peptides on MMP-3 regulation. As shown here, MMP-3 of human dermal fibroblasts treated with Astragalus peptide isolated from 2-7 parts of 40% polyacrylamide gel was present at lower levels compared to untreated control cells. The greatest reduction was seen in human dermal fibroblasts treated with Astragalus peptides isolated from the second (7.6%) and third (5%) of the 40% acrylamide gel. MMP-3, known as stromelysin-1, activates some pro-MMPs during extracellular matrix conversion. MMP-3 substrates include gelatin and collagen III, IV, V, IX, X, and XI. MMP-3 efficiently activates collagenase, matrilysine and gelatinase B, and the action of MMP-3 on partially treated pro-MMP-1 is very critical for the production of fully activated MMP-1. Element. In addition, MMP-3 activates TGF-a, which in turn stimulates expression of MMP-9 by macrophages in a COX-2 dependent manner. Next, the active ingredients inhibit MMP-3 expression, which makes it possible to inhibit skin decay. As a result, the Astragalus peptide inhibits the expression of MMP-3, indicating that it inhibits skin decay and has a good anti-aging effect.
콜라게나제-2 또는 호중구 콜라게나제라고도 알려진 MMP-8은 피부 콜라겐의 쇠퇴의 촉매작용의 원인이 되는 주요 콜라겐아제중의 하나이다. MMP-8은 피부의 주요 성분인 콜라겐 I에 대해 가장 큰 효과를 나타낸다. MMP-8, also known as collagenase-2 or neutrophil collagenase, is one of the major collagens responsible for the catalysis of the decline of skin collagen. MMP-8 has the greatest effect on collagen I, a major component of the skin.
도 3에서 보듯이, 황기 펩타이드로 처리된 인체 피부 섬유아세포에서 MMP-8은 감소되었다. 황기 펩타이드로 처리된 인체 피부 섬유아세포의 MMP-8은 대조군 세포에 비해 감소되었는데, 40% 폴리아크릴아미드 젤의 제5부분으로부터 분리된 황기 펩타이드로 처리된 인체 피부 섬유아세포에서 대조군 세포에 비하여 최대 22%의 감소가 관찰되었다. 이러한 결과는 황기 펩타이드가 MMP-8의 발현을 현격하게 억제하며, 이는 피부 쇠퇴를 효율적으로 억제하고 피부 노화방지를 하는 것을 나타낸다. As shown in Figure 3, MMP-8 was reduced in human skin fibroblasts treated with Astragalus peptide. MMP-8 of human dermal fibroblasts treated with Astragal peptides was reduced compared to control cells, up to 22 compared to control cells in human dermal fibroblasts treated with Astragal peptides isolated from the fifth portion of 40% polyacrylamide gel. A decrease of% was observed. These results indicate that Astragalus peptides significantly inhibit the expression of MMP-8, which effectively inhibits skin decline and prevents skin aging.
또한, MMP-13 발현에 대한 황기 펩타이드의 효과는 도 4에 나타내었다.In addition, the effect of Astragalus peptides on MMP-13 expression is shown in FIG. 4.
도 4에서 보듯이, 40% 폴리아크릴아미드 젤의 제3 내지 제9부분으로 분리된 황기 펩타이드가 MMP-13의 발현을 억제한 것을 나타내었다. 최고 57%, 58% 및 59% 감소는 젤의 제3부분, 제4부분, 제5부분으로부터 분리된 황기 펩타이드로 처리된 인체 피부섬유아세포에서 각각 관찰되었다. 이러한 결과는 황기 펩타이드가 MMP-13 발현을 억제하는 것을 나타낸다. 콜라게나제-3으로도 알려진 MMP-13은 연골에서 발견된 콜라겐의 주요 형태인 타입 II 콜라겐을 우선적으로 가수분해한다. MMP-13의 젤라틴용해성 활동은 MMP-1보다 약 44배 더 크며, 세포외 기질 쇠퇴에 많은 영향을 끼침에 유의한다. 작은 펩타이드(울트라 펩타이드)를 분리하기 위해서 특히 분자량 1500 Da미만의 것을 분리하기 위해서는 초원심분리법, 분자 컷오프량 여과, 한외여과 및 크로마토그래피 등의 기술이 필요하다. 또한, 이들 펩타이드는 우레아-황산도데실나트륨 (SDS)-폴리아크릴아미드 젤 또는 트리신- SDS-폴리아크릴아미드 젤을 이용하여 분리될 수 있었다. As shown in FIG. 4, it was shown that the sulfuric peptide separated into the third to ninth portions of the 40% polyacrylamide gel inhibited the expression of MMP-13. Up to 57%, 58% and 59% reductions were observed in human dermal fibroblasts treated with Astragal peptides isolated from the third, fourth and fifth portions of the gel, respectively. These results indicate that Astragalus peptides inhibit MMP-13 expression. MMP-13, also known as collagenase-3, preferentially hydrolyses type II collagen, a major form of collagen found in cartilage. It is noted that the gelatin soluble activity of MMP-13 is about 44 times greater than that of MMP-1 and has a significant effect on the extracellular matrix decline. In order to separate small peptides (ultra peptides), in particular, to separate those having a molecular weight of less than 1500 Da, techniques such as ultracentrifugation, molecular cutoff amount filtration, ultrafiltration, and chromatography are required. In addition, these peptides could be separated using urea-dodecyl sodium sulfate (SDS) -polyacrylamide gel or tricin-SDS-polyacrylamide gel.
이상과 같이, 황기 펩타이드는 피부 쇠퇴와 관련된 인체 피부 섬유아세포에서의 MMP발현을 억제하는 것을 알 수 있다. 억제 효과는 전기영동으로 정화된 펩타이드에 의해 증가되었다. MMP-1, MMP-3, MMP-8 and MMP-13 발현은 황기 펩타이드로 처리하지 않은 대조군에 비해 각각 43%, 5%, 22% 및 57%로 감소되어 콜라겐의 퇴화를 억제함으로써 피부 노화를 방지할 수 있음을 알 수 있다. As described above, it can be seen that the Astragalus peptide inhibits MMP expression in human skin fibroblasts associated with skin decline. The inhibitory effect was increased by the electrophoresis clarified peptide. MMP-1, MMP-3, MMP-8 and MMP-13 expressions are reduced to 43%, 5%, 22% and 57%, respectively, compared to controls not treated with Astragalus peptides to inhibit skin aging by inhibiting collagen degeneration. It can be seen that it can prevent.
<실시예 4> 세포독성 여부에 대한 검증Example 4 Validation of Cytotoxicity
본 발명의 황기 펩타이드에 대한 세포독성 여부를 검토하였다.The cytotoxicity of the Astragalus peptide of the present invention was examined.
세포독성은 섬유아세포(Fibroblast)를 배양하고 여기에 본 발명의 황기 펩타이드를 각각 가한 다음 세포의 생존율을 조사하는 방식으로 진행하였다. 세포의 생존율은 MTT asssay를 통하여 결정하였다. 먼저 섬유아세포를 24-well plate에 105 cells/ml로 깔고, 여기에 황기 펩타이드(PEP4, PEP5, PEP6)를 각각 0.1%, 0.5%, 2.5%(w/v)로 투입하고 48시간 배양 후 생존율을 비교하였다. 그 결과를 하기 표 1에 나타내었다.Cytotoxicity was carried out by culturing fibroblasts and adding each of the Astragalus peptides of the present invention, and then examining the survival rate of the cells. Cell viability was determined by MTT assay. First, fibroblasts were laid at 10 5 cells / ml in a 24-well plate, and 0.1%, 0.5%, 2.5% (w / v) of Astragal peptides (PEP4, PEP5, PEP6) were added thereto, and then cultured for 48 hours. Survival was compared. The results are shown in Table 1 below.
표 1
PEP4 PEP5 PEP6
0.1% 98% 100% 99%
0.5% 100% 100% 100%
2.5% 99% 99% 99%
Table 1
PEP4 PEP5 PEP6
0.1% 98% 100% 99%
0.5% 100% 100% 100%
2.5% 99% 99% 99%
상기의 결과에서 알 수 있듯이 본 발명의 조성물은 세포독성을 나타내지 않는 안전성이 있음을 확인할 수 있었다.As can be seen from the above results, the composition of the present invention was confirmed that the safety does not exhibit cytotoxicity.
<실시예 5> 콜라겐 생합성 촉진 효과Example 5 Collagen Biosynthesis Promoting Effect
인체 상피세포주, CSK323를 이용한 콜라겐 생성 촉진 효과를 측정하기 하여 SIRCOL collgen assay를 실시하였다. 세포주를 105 cells/ml의 농도로 24well plate에 분주하고, 10% FBS / DMEM 배지에서 24시간 배양하였다. 배지를 제거하고, 10% FBS/DMEM 배지를 각각 0.8ml 씩 투입하고, 여기에 테스트용 시료를 각각 0.8ml 투입한 다음 1일간 추가 배양하고, 상등액을 0.5ml 취하여 염색시약을 1ml 첨가하고 상온에서 30분간 교반한 다음, 원심분리하여 상등액을 제거하고 다시 알칼리 agent를 0.1ml 첨가하고 5분간 섞어 준 다음, 96-well plate에 옮기고 micro reader로 흡광도를 측정하였다. 그 결과를 하기 표 2에 나타내었다. 양성대조군의 경우 ascorbic acid, 100ppm을 이용하였고, 음성대조군은 생리심염수 첨가군이다.SIRCOL collgen assay was performed by measuring collagen production promoting effect using human epidermal cell line, CSK323. Cell lines were aliquoted into 24well plates at a concentration of 10 5 cells / ml and incubated for 24 hours in 10% FBS / DMEM medium. Remove the medium, add 0.8 ml of 10% FBS / DMEM medium, add 0.8 ml of test sample to each sample, incubate for 1 day, and add 0.5 ml of the supernatant, add 1 ml of dyeing reagent, and at room temperature After stirring for 30 minutes, the supernatant was removed by centrifugation, and 0.1 ml of alkali agent was added again and mixed for 5 minutes, and then transferred to a 96-well plate and the absorbance was measured by a micro reader. The results are shown in Table 2 below. Ascorbic acid, 100ppm was used in the positive control group and the physiological saline added group was negative.
표 2
음성 대조군 양성 대조군 본 발명의 시료
콜라겐 생성 촉진(%) 100% 205% 165%
TABLE 2
Negative control Positive control Sample of the Invention
Promote collagen production (%) 100% 205% 165%
상기 표 2에 나타난 바와 같이 본 발명의 조성물은 뛰어난 콜라겐 생성 촉진효과가 있음을 확인할 수 있었다.As shown in Table 2, the composition of the present invention was confirmed to have an excellent collagen production promoting effect.
<실시예 6> 피부 항노화 효과의 비교Example 6 Comparison of Skin Anti-Aging Effects
과산화물에 의한 세포의 산화적 스트레스에 의하여 초래되는 노화를 억제하는 효과를 비교하기 위하여 사이토카인 TNF-α의 분비량을 비교하는 시험을 행하였다. 실험은 다음과 같이 진행하였다.In order to compare the effect of inhibiting the aging caused by the oxidative stress of the cells by the peroxide, a test was performed to compare the secretion amount of the cytokine TNF-α. The experiment proceeded as follows.
HaCaT 세포주를 DMEM/10% FBS 로 배양하고, 24well plate에 105 cells/ml의 농도로 분주하고, 안정화시킨 다음, 시료를 각각 1mg/ml의 농도로 가하고, 대조군으로는 헤스페리딘을 25ug/ml 처리하고, 여기에 과산화수소를 100uM 농도로 처리한 다음 24시간 후 상층액을 분리하여 정량하는 방법으로 TNF-α의 분비량을 비교하였다. TNF-α의 분비량이 적을수록 라디칼에 의한 항노화 작용이 우수한 것으로 볼 수 있다. TNF-α의 분석은 ELISA 법을 이용하였다. 그 결과를 하기 표 3에 나타내었다.The HaCaT cell line was incubated with DMEM / 10% FBS, aliquoted at a concentration of 10 5 cells / ml in a 24well plate, stabilized, and then the samples were added at a concentration of 1 mg / ml, respectively, and 25 ug / ml of hesperidin as a control. Then, the amount of TNF-α secretion was compared by treating hydrogen peroxide at a concentration of 100 uM and then separating and quantifying the supernatant after 24 hours. The smaller the secretion amount of TNF-α, the better the anti-aging action by the radical. Analysis of TNF-α was performed by ELISA method. The results are shown in Table 3 below.
표 3
음성 대조군 과산화수소 본 발명의 시료 헤스페리딘
TNF-α 상대량(%) 0% 100% 65% 55%
TABLE 3
Negative control Hydrogen peroxide Sample of the Invention Hesperidin
TNF-α relative amount (%) 0% 100% 65% 55%
따라서 본 발명의 조성물은 매우 뛰어난 항노화 효과가 있다고 할 수 있다.Therefore, the composition of the present invention can be said to have a very excellent anti-aging effect.
<제조예 1> 화장료로서 크림의 제조Production Example 1 Preparation of Cream as Cosmetic
본 발명의 황기 펩타이드(PEP5)를 함유하는 크림을 제조하였다. 본 발명의 추출물인 시료 "PEP5" 5%, 아르간오일 5%, 쉐어버터 5%, 녹차씨유 3%, 아보카도오일 2%, 이소아미 라우레이트(isoamy laurate) 5%, 세티아릴 알콜 (Cetearyl alcohol) 5%, 히알루론산 10% 솔루션 10%, 베타글루칸 10% 솔루션 10%, 향료 3% 나머지는 정제수를 각각 부피비로 가하고 혼합하여 크림을 제조하였다.A cream containing the Astragalus peptide (PEP5) of the present invention was prepared. Sample "PEP5" of the extract of the present invention 5%, argan oil 5%, shea butter 5%, green tea seed oil 3%, avocado oil 2%, isoamy laurate (5% isoamy laurate), cetiaryl alcohol (Cetearyl alcohol ) 5%, hyaluronic acid 10% solution 10%, beta glucan 10% solution 10%, the remainder 3% of the remainder was added to the purified water in a volume ratio and mixed to prepare a cream.
<제조예 2> 화장료로서 로션의 제조Production Example 2 Preparation of Lotion as Cosmetic
본 발명의 황기 펩타이드(PEP5)를 함유하는 로션을 제조하였다. 본 발명의 추출물인 시료 "PEP5" 5%, 아르간오일 2%, 쉐어버터 2%, 녹차씨유 1%, 호호바오일 1%, 이소아미 라우레이트(isoamy laurate) 4%, 세티아릴 알콜 (Cetearyl alcohol) 3%, 소르비탄 올리베이트(Sorbitan Olivate) 1%, 히알루론산 5% 솔루션 10%, 베타글루칸 10% 솔루션 5%, 향료 3% 나머지는 정제수를 각각 부피비로 가하고 혼합하여 로션을 제조하였다.A lotion containing the Astragalus peptide (PEP5) of the present invention was prepared. Sample of the extract of the present invention "PEP5" 5%, argan oil 2%, shea butter 2%, green tea seed oil 1%, jojoba oil 1%, isoamy laurate (4%), cetiaryl alcohol (Cetearyl alcohol ) 3%, sorbitan olivate (Sorbitan Olivate) 1%, hyaluronic acid 5% solution 10%, beta glucan 10% solution 5%, the fragrance 3% was added to the purified water in a volume ratio and mixed to prepare a lotion.
<제조예 3> 화장료로서 에센스의 제조Production Example 3 Production of Essence as Cosmetic
본 발명의 황기 펩타이드(PEP5)를 함유하는 에센스를 제조하였다. 본 발명의 추출물인 시료 "PEP5 2%, 아르간오일 2%, 드럼스틱씨오일 2%, 호호바오일 1%, 이소아미 라우레이트(isoamy laurate) 2%, 세티아릴 알콜(Cetearyl alcohol) 1.5%, 소르비탄 올리베이트(Sorbitan Olivate) 1%, 글리세린 올리에이트(Glyceryl oleate) 1% 히알루론산 10% 솔루션 5%, 베타글루칸 10% 솔루션 5%, 향료 3% 나머지는 정제수를 각각 부피비로 가하고 혼합하여 에센스를 제조하였다.Essences containing the Astragalus peptide (PEP5) of the present invention were prepared. Samples of the extract of the present invention "PEP5 2%, argan oil 2%, drumstick sea oil 2%, jojoba oil 1%, isoamy laurate 2%, cetiaryl alcohol (Cetearyl alcohol) 1.5%, sorb Sorbitan Olivate 1%, Glyceryl oleate 1% Hyaluronic Acid 10% Solution 5%, Beta Glucan 10% Solution 5%, Perfume 3% Prepared.
본 발명의 조성물은 화장품의 제조시 원료로서 사용가능하며, 본 발명의 조성물을 함유한 화장품은 매우 뛰어난 피부 노화 방지효과를 가지고 있어 고부가가치의 제품으로 개발될 수 있다.The composition of the present invention can be used as a raw material in the manufacture of cosmetics, cosmetics containing the composition of the present invention has a very excellent skin anti-aging effect can be developed as a high value-added product.

Claims (6)

  1. 황기 펩타이드를 유효성분으로 포함하는 것을 특징으로 하는 피부 노화를 방지를 위한 화장료 조성물.Cosmetic composition for preventing skin aging, characterized in that containing an amino acid peptide as an active ingredient.
  2. 상기 제 1 항에 있어서,The method of claim 1,
    상기 황기 펩타이드가 황기를 트립신과 키모트립신의 혼합물로 가수분해시킨 다음, 원심분리하여 수득된 것임을 특징으로 하는 화장료 조성물.Cosmetic composition, characterized in that the sulfuric peptide is obtained by hydrolyzing the sulfur group with a mixture of trypsin and chymotrypsin, followed by centrifugation.
  3. 상기 제 1 항에 있어서,The method of claim 1,
    상기 황기 펩타이드가 40% 폴리아크릴아미드 젤을 이용하여 분리된 것임을 특징으로 하는 화장료 조성물.Cosmetic composition, characterized in that the Astragalus peptide is separated using a 40% polyacrylamide gel.
  4. 제 1 항에 있어서,The method of claim 1,
    상기 황기 펩타이드가 1500Da 미만의 분자량을 가지는 것을 특징으로 하는 화장료 조성물.Cosmetic composition, characterized in that the Astragalus peptide has a molecular weight of less than 1500 Da.
  5. 제 1 항에 있어서,The method of claim 1,
    상기 황기 펩타이드가 화장료 조성물의 총 중량에 대하여 0.001-50 중량%의 양으로 포함되는 것을 특징으로 하는 화장료 조성물.Cosmetic composition, characterized in that the Astragalus peptide is contained in an amount of 0.001-50% by weight relative to the total weight of the cosmetic composition.
  6. 황기 펩타이드를 유효성분으로 포함하는 것을 특징으로 하는 콜라겐 퇴화 방지를 위한 화장료 조성물.Cosmetic composition for preventing collagen degeneration, characterized in that it comprises an amino acid peptide as an active ingredient.
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