WO2014058084A1 - Cosmetic composition containing gamma-oligopeptide or salts thereof for improving wrinkles - Google Patents

Cosmetic composition containing gamma-oligopeptide or salts thereof for improving wrinkles Download PDF

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WO2014058084A1
WO2014058084A1 PCT/KR2012/008159 KR2012008159W WO2014058084A1 WO 2014058084 A1 WO2014058084 A1 WO 2014058084A1 KR 2012008159 W KR2012008159 W KR 2012008159W WO 2014058084 A1 WO2014058084 A1 WO 2014058084A1
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cosmetic composition
salt
oligopeptide
gamma
skin
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PCT/KR2012/008159
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French (fr)
Korean (ko)
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성문희
최재철
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주식회사 바이오리더스
국민대학교산학협력단
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Priority to PCT/KR2012/008159 priority Critical patent/WO2014058084A1/en
Publication of WO2014058084A1 publication Critical patent/WO2014058084A1/en

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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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/74—Biological properties of particular ingredients
    • A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
    • A61K2800/782—Enzyme inhibitors; Enzyme antagonists

Definitions

  • the present invention relates to a cosmetic composition for improving skin wrinkles containing gamma oligopeptides or salts thereof, and more particularly to skin permeation, inhibiting collagen degrading enzymes in the skin, and increasing collagen production to improve skin wrinkles. It relates to a cosmetic composition for improving wrinkles containing gamma oligopeptide or a salt thereof.
  • Functional cosmetics which were mainly focused on product development for the purpose of skin protection, such as UV protection in the early years, have been developing products for cosmetic purposes such as wrinkle improvement in the past 10 years, and they can simultaneously whiten, wrinkle and color.
  • the main cause of wrinkles is skin aging. Aging can be classified into two types: natural aging and photo aging.
  • the wrinkles of the skin may be caused by a decrease in elastic fibers such as collagen or elastin due to the deterioration of cell regeneration ability, the generation of lipid peroxide by active oxygen, degeneration due to oxidation of biological components, and fatigue caused by excessive muscle movement.
  • functions such as promoting collagen synthesis, antioxidant activity, and preventing muscle fatigue are required.
  • Retinol (2500 IU / g), Retinyl Palmitate (10000 IU / g), Adenosine (0.04%), Polyethoxylatediretinamide (0.05-0.2) 4 types). They are also exempted from submitting safety validation data when using the indicated concentrations.
  • products approved as functional cosmetics through individual certification are hydroxyproline, 7-dihydrocholesterol, kainetin, peony extract, and betel nut. There are self extracts
  • Anti-wrinkle functional materials are mainly used to control the differentiation and regeneration of epidermal cells, substances to regulate extracellular matrix (ECM), antioxidants to eliminate free radicals, anti-inflammatory components, components to protect against UV damage Peptides with a similar Botox concept that slow muscle movement are the mainstream.
  • ECM extracellular matrix
  • mevalonic acid has been developed and used as an agent for controlling the differentiation and regeneration of epidermal cells, and ingredients such as paoniflorin extracted from peony or prangenidin extracted from white paper have been developed and used as ECM control ingredients.
  • Prangenidin collagen biosynthesis through PCIP enzyme immunoassay was found to be about 20 times better than vitamin C.
  • a component that protects or regenerates the DNA susceptible to UV damage has been developed, a typical example is a photolyase extracted from phytoplankton, known as an enzyme of DNA damage healing function.
  • Polygamma glutamic acid ([gamma] -PGA) is a mucous substance produced by microorganisms.
  • Poly-gamma-glutamic acid is produced from Bacillus isolates from Cheonggukjang, a traditional Korean fermented soybean meal using rice straw.
  • Polygamma glutamic acid is an edible, water-soluble, anionic and biodegradable polymer that can be used as a raw material and functional food material for cosmetics such as moisture absorbents and moisturizers.
  • the present inventors produce polygamma glutamic acid using a high-molecular weight polygamma glutamic acid and a material patent for the method of using the same (Korean Patent No. 399091), a salt-tolerant strain Bacillus subtilis Chungkukjang strain producing high molecular weight poly gamma glutamic acid.
  • a patent for a method (Korean Patent No. 500796), and a patent for anti-cancer composition containing polygamma glutamic acid, an immunoadjuvant, an immune enhancer, and a virus infection inhibition (Korean Patent No. 496606, Republic of Korea) Patent No. 517114, Republic of Korea Patent No. 475406, Republic of Korea Patent No. 0873179).
  • the cosmetic composition containing the conventional polygamma glutamic acid provides an application aspect as a moisturizing (Korean Patent Publication No. 2010-0101328), skin whitening (Korean Patent Publication No. 2010-0086728) or for enhancing the skin absorption of minerals (2009-0132372). However, it has not been applied for skin wrinkle improvement.
  • the present inventors have made efforts to confirm the effect on wrinkle improvement by developing gamma oligopeptide, a polygamma glutamic acid formulation capable of permeation of the skin, and thus successfully prepared a skin permeable poly gamma glutamic acid formulation to produce a collagen degrading enzyme. It was confirmed that to improve the skin wrinkles by inhibiting and promoting collagen synthesis, to complete the present invention.
  • An object of the present invention is to provide a cosmetic composition for improving skin wrinkles containing gamma oligopeptide or a salt thereof having an effect on skin wrinkle improvement.
  • the present invention provides a cosmetic composition for improving skin wrinkles containing gamma oligopeptide or a salt thereof as an active ingredient.
  • Figure 1 shows the collagenase enzyme inhibitory activity of the gamma oligopeptide.
  • Figure 2 shows the procollagen synthesis of gamma oligopeptides.
  • a gamma oligopeptide or a salt thereof having low molecular weight of polygamma glutamic acid was intended to determine whether it is effective in improving skin wrinkles.
  • a gamma oligopeptide or a salt thereof having low molecular weight of polygamma glutamic acid was prepared.
  • the molecular weight of the gamma oligopeptide or salt thereof prepared from the low molecular weight polygammaglutamic acid used in the present invention is characterized in that 600 ⁇ 5,000 Da, the salt is sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt and zinc It is characterized in that it is selected from the group consisting of salts.
  • UVA was irradiated to HDF-N cells, and then cultured by adding gamma oligopeptide (0.05, 0.1%, 0.5%) and analyzing the activity of collagenase.
  • the activity of collagenase and gamma oligopeptide in the control group were measured to be 177%, 154% and 133%, respectively, compared to the control group without UVA irradiation. Compared with the 183% of the experimental group did not significantly reduce the activity of collagenase enzymes (Fig. 1).
  • the concentration of procollagen was analyzed by adding gamma oligopeptide (0.01, 0.05%, 0.1%) to HDF-N cells to confirm collagen production ability, and compared with the control group. Increased to 129%, 168%, and 199%, respectively (FIG. 2). Through this, gamma oligopeptide was shown to increase the procollagen synthesis capacity in a concentration-dependent manner to show the effect on wrinkle improvement.
  • the cosmetic composition for improving skin wrinkles of the present invention may be added to wrinkle improvement functional cosmetics.
  • the content of gamma oligopeptide in the cosmetic composition for wrinkle improvement of the present invention is preferably 0.005% to 10%, when the content is less than 0.005%, anti-wrinkle effect cannot be expected, and when the content is more than 10%, the wrinkle improvement effect is increased. Not only can it not be expected, it is expensive, and it is not economical.
  • Cosmetics prepared from the compositions of the present invention can be prepared in the form of common emulsifying and solubilizing formulations.
  • Cosmetics of the emulsified formulations include nutrient cosmetics, creams, essences, etc., and cosmetics of the solubilized formulations are flexible cosmetics.
  • the compositions of the present invention may be prepared in adjuvant form for topical or systemic application commonly used in the field of dermatology by containing a dermatologically acceptable medium or base in addition to cosmetics.
  • Formulations of suitable cosmetics include, for example, emulsions, suspensions, emulsions, pastes, microemulsions, microcapsules, microgranules or ionic (liposomes) obtained by dispersing an oil phase in a solution, gel, solid or pasty anhydrous product, aqueous phase.
  • ionic vesicle dispersants creams, skins, lotions, powders, soaps, ointments, sprays, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations or conceal sticks Can be provided. It may also be prepared in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.
  • “Dermatologically acceptable carriers” that can be used according to the desired formulation include purified water, oils, waxes, fatty acids, fatty alcohols, fatty acid esters, surfactants, humectants, thickening agents, antioxidants, and viscosity stabilizers. Examples include, but are not limited to, topical stabilizers, chelating agents, buffers, preservatives, lower alcohols, and the like, and their types and concentrations are varied and modifications may be made by those skilled in the art without departing from the spirit and scope of the present invention. And parts that can be changed.
  • a whitening agent may be included, and these may be included in the cosmetic composition according to the present invention in an amount commonly used in the cosmetic field. May be included. Suitable from 0.001 to 50% by weight per total weight of the composition.
  • the oil may be hydrogenated vegetable oil, perm oil, cottonseed oil, olive oil, palm oil, jojoba oil, avocado oil, wax, beeswax, carnauba, candelilla, montan, ceresin, liquid paraffin Lanolin may be used.
  • As the fatty acid ester isopropyl myristate, isopropyl palmitate, butyl stearate and the like may be used, but is not limited thereto.
  • surfactant examples include anionic surfactants such as sodium stearate, sodium cetyl sulfate, polyoxyethylene laurylether phosphate, sodium N-acyl glutamate; Cationic surfactants such as stearyldimethylbenzylammonium chloride and stearyltrimethylammonium chloride; Amphoteric surfactants such as alkylaminoethylglycine hydrochloride and lecithin; Glycerine monostearate, sorbitan monostearate, propylene glycol monostearate, polyoxyethylene oleyl ether, polyethylene glycol monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene coconut fatty acid monoethanol arnide (monoethaolarnide ), Polyoxypropylene glycol, polyoxyethylene castor oil, nonionic surfactants such as polyoxyethylene lanolin and the like.
  • anionic surfactants such as sodium stearate, sodium cetyl sulfate
  • Glycerin, 1,3-butylene glycol, propylene glycol may be used as the moisture absorbent, and ethanol or isopropanol may be used as the lower alcohol.
  • thickeners include sodium alginate, sodium caseate, gelatin agar, xanthan gum, starch, cellulose ethers (e.g., hydroxyethyl cellulose, methyl cellulose, carboxymethyl cellulose, hydroxy propylmethyl cellulose), polyvinyl pyrrolidone, Polyvinyl alcohol, polyethylene glycol, sodium carboxymethyl cellulose, and the like, but are not limited thereto.
  • antioxidants butylated hydroxytoluene, butylated hydroxyanisole, propyl gallate, citric acid, ethoxyquin can be used, and chelating agents include disodium edetate and ethane hydroxy diphosphate.
  • Citric acid, sodium citrate, boric acid, borax, disodium hydrogen phosphate are available as buffers, and methyl parahydroxybenzoate, ethyl parahydroxybenzoate, dihydro as preservatives.
  • Acetic acid, salicylic acid, benzoic acid are available, but are not limited to these.
  • Basic medium for the production of polygamma glutamic acid (medium with 3% L-glutamic acid; glucose 3%, (NH 4 ) 2 SO 4 1%, KH 2 PO 4 0.27%, Na 2 HPO 4 / 12H 2 O 0.17% , NaCl 0.1%, sodium citrate 0.5%, Soypeptone 0.02%, MgSO 4 / 7H 2 O 0.7%, vitamin solution (10ml / l, pH 6.8) to sterilize, 5L fermenter ( Jar fermentor, working volume was fermented by inoculating 4% seed culture (LB medium) of Bacillus subtilis var chungkookjang (KCTC 0697BP) in 3L).
  • the stirring speed was 500rpm, the air injection speed was 1.0vvm, and fermented at 37 ° C. for 48 hours to remove the cells using a filter press (containing diatomaceous earth) to obtain a polygamma glutamic acid-containing sample liquid.
  • the polygamma glutamic acid-containing sample solution was adjusted to pH 2.0 using 2N sulfuric acid solution and then left at 10 ° C. for 15 hours to obtain a polygamma glutamic acid precipitate.
  • the obtained polygamma glutamic acid precipitate was washed with a sufficient amount of cold distilled water of 10 ° C. or less (pH 3.5 or more), and then obtained polygamma glutamic acid precipitate using a Licech filter, followed by lyophilization to prepare a high molecular weight polygamma glutamic acid.
  • this test was intended to evaluate the wrinkle improvement effect of the sample by measuring the activity of collagen degrading enzyme MMP-1 (Matrix metalloproteinases) by immuno ELISA assay (FIG. One).
  • MMP-1 collagen degrading enzyme MMP-1 (Matrix metalloproteinases)
  • HDF-N normal human primary dermal fibroblasts-Neonatal derived from human neonatal foreskin.
  • Fibroblast Basal Medium FBM, lonza, CC-3131
  • FBS Fibroblast Basal Medium
  • ATCC PCS-201-010 FBS after inoculating HDF-N (ATCC PCS-201-010) on the bottom of the culture dish was added and maintained at 37 ° C. and incubated in an incubator containing 5% carbon dioxide.
  • HDF-N cells were dispensed at 2.0 ⁇ 10 4 cells / well in 24 well plates and incubated for 48 hours under cell culture conditions. After 48 hours, the medium was discarded and washed with DPBS, and then UV-A 5J / cm 2 was irradiated by adding 200 ⁇ l of DPBS. Samples were diluted in medium at appropriate concentrations and then treated with cells and incubated in cell culture conditions for 24 hours. Retinoic acid (RA) was used as a positive control and the concentration was 1 uM. The culture medium was taken and the amount of MMP-1 was measured using a human total MMP-1 ELISA kit (R & D system, DY901). The amount of MMP-1 measured was corrected to the total protein amount.
  • RA Retinoic acid
  • HDF-N cells were dispensed at 1.0 ⁇ 10 4 cells / well in 48 well plate and incubated for 24 hours in the cell culture conditions. Thereafter, the medium was removed, and the cells were starved for 24 hours, after which the test substance was diluted in FBM (except for media supplement), which is a cell culture medium, and treated with the cells by concentration, followed by incubation for 24 hours. After 24 hours, the cultured cells were harvested and the amount of procollagen was measured using procollagen type I c-peptide (PIP) EIA kit (TAKARA, MK101) (FIG. 2). On the other hand, the cells attached to the bottom was washed with DPBS and lysed with 1N NaOH to measure the total protein amount to determine the amount of procollagen synthesis per constant protein. Procollagen and protein amounts were calculated according to the calibration curve.
  • PIP procollagen type I c-peptide
  • the procollagen synthesis increased by 195.23% when 10 ng / ml of TGF- ⁇ was used as a positive control.
  • BLS-PGA increased to 129.34%, 168.74%, and 199.60%, respectively, when treated with 0.01%, 0.05%, and 0.10% concentrations with 0.10% as the highest concentration.
  • This increased the procollagen synthesis capacity in a concentration-dependent manner, and the three concentrations showed statistically significant results.
  • gamma oligopeptides are thought to show efficacy in wrinkle improvement by increasing procollagen synthesis ability in a concentration-dependent manner at 0.01%, 0.05%, and 0.10%.
  • formulations comprising the compositions of the present invention are not limited in any way.
  • the cosmetic composition for improving skin wrinkles inhibits collagen degrading enzymes, enhances collagen synthesis, has little toxicity and side effects, and can be used for a long time, which is useful for improving skin wrinkles.

Abstract

The present invention relates to a cosmetic composition containing gamma-oligopeptide or salts thereof for improving wrinkles on the skin, and more specifically, to a cosmetic composition which can penetrate the skin, inhibit collagen-dissolving enzymes in the skin, and increase collagen generation to improve wrinkles on the skin. The cosmetic composition for improving wrinkles on the skin according to the present invention inhibits collagen-dissolving enzymes, increases collagen synthesis, is not toxic and has few side effects and thus can be used for a long time, and is useful in improving wrinkles on the skin.

Description

감마올리고펩타이드 또는 그 염을 함유하는 주름개선용 화장료 조성물Cosmetic composition for anti-wrinkle containing gamma oligopeptide or salt thereof
본 발명은 감마올리고펩타이드 또는 그 염을 함유하는 피부 주름개선용 화장료 조성물에 관한 것으로, 더욱 상세하게는 피부 투과가 가능하고, 피부 내 콜라겐 분해 효소를 저해하며, 콜라겐 생성을 증가시켜 피부 주름을 개선시키는 감마올리고펩타이드 또는 그 염을 함유하는 주름개선용 화장료 조성물에 관한 것이다.The present invention relates to a cosmetic composition for improving skin wrinkles containing gamma oligopeptides or salts thereof, and more particularly to skin permeation, inhibiting collagen degrading enzymes in the skin, and increasing collagen production to improve skin wrinkles. It relates to a cosmetic composition for improving wrinkles containing gamma oligopeptide or a salt thereof.
미래의 화장품 개발은 안정성을 강조한 일반 화장품에 비해 효능, 효과가 부각된 기능성 화장품의 중요성이 강조됨에 따라 앞으로 기능성 화장품 범위의 확대는 향후 화장품 산업발전을 위한 현안과제가 되고 있다. 즉 화장품의 개념이 피부 손질, 노화 방지와 세포부활, 미백 및 자외선 차단 등 기존의 개념에서 창상치유, 항아토피, 체형개선 등 현행 의약외품과 약용화장품의 성격의 제품으로 확대될 전망임. 화장품법의 시행과 기능성화장품의 범위 확대에 따라 화장품의 연구개발제조기술의 발전에 박차가 가해지고 있으며 기능성화장품 시대를 선점하기 위한 초석으로 기능성화장품의 특허출원 및 승인이 점차적으로 증가할 전망이다. 국내 뿐만 아니라 국외에서의 특허 획득을 통하여 기술력을 인정 받으려는 경향이 커지고 있다.The future development of cosmetics is emphasized the importance of functional cosmetics that have enhanced efficacy and effectiveness compared to general cosmetics that emphasize stability, and the expansion of the range of functional cosmetics in the future is becoming an issue for the future development of the cosmetics industry. In other words, the concept of cosmetics will be expanded from existing concepts such as skin care, anti-aging and cell regeneration, whitening and UV protection to products of current quasi-drugs and medicinal cosmetics such as wound healing, anti-atopy, and body improvement. The enforcement of the Cosmetic Act and the expansion of the range of functional cosmetics are accelerating the development of R & D manufacturing technology of cosmetics, and the patent application and approval of functional cosmetics is expected to gradually increase as a cornerstone for preoccupation of the era of functional cosmetics. There is a growing tendency to be recognized for technology through domestic and foreign patent acquisition.
초창기 자외선차단과 같이 피부보호의 목적으로 상품개발이 주를 이루었던 기능성화장품이 지난 10년 새 주름개선과 같은 미용 목적에 대한 상품개발이 늘어나고 있으며, 미백, 주름개선, 색조를 동시에 할 수 있는 다중기능성 화장품, 일명 비비크림에 대한 유행에 따라 이에 대한 시장 수요 또한 증가한 것으로 나타났다.Functional cosmetics, which were mainly focused on product development for the purpose of skin protection, such as UV protection in the early years, have been developing products for cosmetic purposes such as wrinkle improvement in the past 10 years, and they can simultaneously whiten, wrinkle and color. The market demand for functional cosmetics, also known as BB creams, has also increased.
미백과는 다르게 주름개선에 관한 정확한 메커니즘은 상대적으로 불명확하다. 주름의 주원인으로는 피부노화를 들 수 있는데, 노화는 크게 자연노화와 광노화의 두 가지 경우로 나눌 수 있다. 즉, 피부의 주름은 세포재생 능력의 저하로 인해 콜라겐이나 엘라스틴 같은 탄력섬유가 감소하거나 활성산소에 의해 과산화지질이 생성된다든지 생체 구성물질의 산화에 따른 변성, 과도한 근육운동에 의한 피로 등이 원인으로 알려져 있다. 그러므로 노화 방지 또는 주름을 개선하기 위해서는 콜라겐 합성 촉진, 항산화 작용, 근육 피로 방지 등의 기능이 요구되고 있다. Unlike whitening, the exact mechanism for wrinkle improvement is relatively unclear. The main cause of wrinkles is skin aging. Aging can be classified into two types: natural aging and photo aging. In other words, the wrinkles of the skin may be caused by a decrease in elastic fibers such as collagen or elastin due to the deterioration of cell regeneration ability, the generation of lipid peroxide by active oxygen, degeneration due to oxidation of biological components, and fatigue caused by excessive muscle movement. Known as Therefore, in order to prevent aging or to improve wrinkles, functions such as promoting collagen synthesis, antioxidant activity, and preventing muscle fatigue are required.
주름개선 기능성화장품을 만들기 위한 주성분으로 고시된 성분으로는 레티놀(2500 IU/g), 레티닐팔미테이트(10000 IU/g), 아데노신(0.04%), 폴리에톡실레이티드레틴아마이드(0.05∼0.2%)의 4종이 있다. 이들 역시 고시된 농도를 사용한 경우 안전성 유효성 심사 자료의 제출이 면제되며, 이외에도 개별 인증을 통해 기능성 화장품으로 승인된 제품 중에는 주성분으로 하이드록시프롤린, 7-디하이드로콜레스테롤, 카이네틴, 작약추출물, 빈랑자추출물 등이 있다Retinol (2500 IU / g), Retinyl Palmitate (10000 IU / g), Adenosine (0.04%), Polyethoxylatediretinamide (0.05-0.2) 4 types). They are also exempted from submitting safety validation data when using the indicated concentrations.In addition, products approved as functional cosmetics through individual certification are hydroxyproline, 7-dihydrocholesterol, kainetin, peony extract, and betel nut. There are self extracts
주름개선 기능성 소재는 주로 표피세포의 분화 및 재생을 조절하는 성분, extracellular matrix(ECM)을 조절하는 물질, 활성산소를 소거하는 항산화제, 항염증작용 성분, 자외선에 의한 손상을 방어하는 성분 및 기타 근육운동을 저하하는 유사 보톡스 개념의 펩타이드류들이 주류를 이루고 있다. 표피세포의 분화 및 재생을 조절하는 물질로는 기존의 retinoids와 AHAs 외에도 mevalonic acid 등이 개발 이용되고 있으며, ECM 조절 성분으로는 작약에서 추출한 paoniflorin 또는 백지에서 추출한 prangenidin과 같은 성분들이 개발되어 이용되고 있다. Prangenidin의 경우 PCIP enzyme immunoassay를 통한 콜라겐 생합성능을 조사한 결과 비타민 C에 비하여 약 20배 정도 우수한 것으로 확인되었다. 한편, 자외선에 의해 손상받기 쉬운 DNA를 보호하거나 재생하는 성분이 개발되기도 하였는데, 대표적인 예로 식물성 플랑크톤에서 추출한 것으로 DNA 손상치유 기능의 효소로 알려진 photolyase 가 있다.Anti-wrinkle functional materials are mainly used to control the differentiation and regeneration of epidermal cells, substances to regulate extracellular matrix (ECM), antioxidants to eliminate free radicals, anti-inflammatory components, components to protect against UV damage Peptides with a similar Botox concept that slow muscle movement are the mainstream. In addition to conventional retinoids and AHAs, mevalonic acid has been developed and used as an agent for controlling the differentiation and regeneration of epidermal cells, and ingredients such as paoniflorin extracted from peony or prangenidin extracted from white paper have been developed and used as ECM control ingredients. . In the case of Prangenidin, collagen biosynthesis through PCIP enzyme immunoassay was found to be about 20 times better than vitamin C. On the other hand, a component that protects or regenerates the DNA susceptible to UV damage has been developed, a typical example is a photolyase extracted from phytoplankton, known as an enzyme of DNA damage healing function.
폴리감마글루탐산(γ-PGA)은 미생물에 의해 생산되는 점액성 물질이다. 폴리감마글루탐산은 볏짚을 이용한 한국의 전통 콩 발효식품인 청국장 등에서 분리된 바실러스로부터 생산된다. 폴리감마글루탐산은 식용, 수용성, 음이온성 및 생분해성 고분자물질로 흡습제, 보습제 등의 화장품의 원료물질 및 기능성 식품 소재로 이용이 가능하다.Polygamma glutamic acid ([gamma] -PGA) is a mucous substance produced by microorganisms. Poly-gamma-glutamic acid is produced from Bacillus isolates from Cheonggukjang, a traditional Korean fermented soybean meal using rice straw. Polygamma glutamic acid is an edible, water-soluble, anionic and biodegradable polymer that can be used as a raw material and functional food material for cosmetics such as moisture absorbents and moisturizers.
본 발명자들은 고분자량 폴리감마글루탐산 및 그 이용방법에 대한 물질특허(대한민국 등록특허 제399091호), 고분자량의 폴리감마글루탐산을 생산하는 내염성 균주 바실러스 서브틸리스 청국장 균주를 사용하여 폴리감마글루탐산을 생산하는 방법에 관한 특허(대한민국 등록특허 제500796호)를 획득하였으며, 그 외에 폴리감마글루탐산을 함유하는 항암조성물, 면역보강제, 면역증강제 및 바이러스 감염 억제에 대한 특허(대한민국 등록특허 제496606호, 대한민국 등록특허 제517114호 및 대한민국 등록특허 제475406호, 대한민국 등록특허 제0873179호)를 획득한 바 있다. 또한, 폴리감마글루탐산의 면역증강을 통한 항암기능을 밝힘으로써 [Poo, H. R. et al., Journal of Immunology, 178:775, 2007, Poo, H.R. et al., Cancer Immunol Immunother, 59:11, 2010], 폴리감마글루탐산의 의약 용도에 대한 연구가 진행되는 등, 폴리감마글루탐산에 대한 지속적인 용도 개발을 수행하며 다양한 효능들을 연구하였다. The present inventors produce polygamma glutamic acid using a high-molecular weight polygamma glutamic acid and a material patent for the method of using the same (Korean Patent No. 399091), a salt-tolerant strain Bacillus subtilis Chungkukjang strain producing high molecular weight poly gamma glutamic acid. Acquired a patent for a method (Korean Patent No. 500796), and a patent for anti-cancer composition containing polygamma glutamic acid, an immunoadjuvant, an immune enhancer, and a virus infection inhibition (Korean Patent No. 496606, Republic of Korea) Patent No. 517114, Republic of Korea Patent No. 475406, Republic of Korea Patent No. 0873179). In addition, by revealing the anti-cancer function through immuno-enhancing polygamma glutamic acid [Poo, HR et al., Journal of Immunology , 178: 775, 2007, Poo, HR et al., Cancer Immunol Immunother , 59:11, 2010] In addition, research on the medical use of polygamma glutamic acid has been carried out, and various effects have been studied through continuous development of polygamma glutamic acid.
종래 폴리감마글루탐산을 함유하는 화장료 조성물은 보습용(대한민국공개특허 2010-0101328), 피부 미백용(대한민국공개특허 2010-0086728) 또는 미네랄의 피부흡수 증진용(2009-0132372)으로서의 응용적 측면을 제공하였지만, 피부 주름개선용으로 적용된 바 없다.The cosmetic composition containing the conventional polygamma glutamic acid provides an application aspect as a moisturizing (Korean Patent Publication No. 2010-0101328), skin whitening (Korean Patent Publication No. 2010-0086728) or for enhancing the skin absorption of minerals (2009-0132372). However, it has not been applied for skin wrinkle improvement.
이에, 본 발명자들은 피부 투과가 가능한 폴리감마글루탐산 제형인 감마올리고펩타이드를 개발하여 주름개선에 대한 효과를 확인하고자 예의 노력한 결과, 피부 투과 가능한 폴리감마글루탐산 제형을 성공적으로 제조하여 이 소재가 콜라겐 분해 효소를 저해하고 콜라겐 합성을 증진시켜 피부 주름을 개선시키는 것을 확인하고, 본 발명을 완성하게 되었다.Therefore, the present inventors have made efforts to confirm the effect on wrinkle improvement by developing gamma oligopeptide, a polygamma glutamic acid formulation capable of permeation of the skin, and thus successfully prepared a skin permeable poly gamma glutamic acid formulation to produce a collagen degrading enzyme. It was confirmed that to improve the skin wrinkles by inhibiting and promoting collagen synthesis, to complete the present invention.
발명의 요약Summary of the Invention
본 발명의 목적은 피부 주름개선에 대한 효과가 있는 감마올리고펩타이드 또는 그 염을 함유하는 피부 주름개선용 화장료 조성물을 제공하는데 있다.An object of the present invention is to provide a cosmetic composition for improving skin wrinkles containing gamma oligopeptide or a salt thereof having an effect on skin wrinkle improvement.
상기 목적을 달성하기 위하여, 본 발명은 감마올리고펩타이드 또는 그 염을 유효성분으로 함유하는 피부 주름개선용 화장료 조성물을 제공한다.In order to achieve the above object, the present invention provides a cosmetic composition for improving skin wrinkles containing gamma oligopeptide or a salt thereof as an active ingredient.
도 1은 감마올리고펩타이드의 콜라겐 분해 효소 활성 저해능을 나타낸 것이다.Figure 1 shows the collagenase enzyme inhibitory activity of the gamma oligopeptide.
도 2는 감마올리고펩타이드의 프로콜라겐 합성능을 나타낸 것이다.Figure 2 shows the procollagen synthesis of gamma oligopeptides.
발명의 상세한 설명 및 구체적인 구현예Detailed Description of the Invention and Specific Embodiments
본 발명에서는 폴리감마글루탐산을 저분자화한 감마올리고펩타이드 또는 그의 염이 피부 주름개선에 효과가 있는지를 확인하고자 하였다. In the present invention, a gamma oligopeptide or a salt thereof having low molecular weight of polygamma glutamic acid was intended to determine whether it is effective in improving skin wrinkles.
본 발명의 실시예에서는 폴리감마글루탐산을 저분자화한 감마올리고펩타이드 또는 그 염을 제조하였다. 본 발명에 사용된 저분자화한 폴리감마글루탐산으로 제조한 감마올리고펩타이드 또는 그 염의 분자량은 600~5,000Da인 것을 특징으로 하며, 상기 염은 나트륨염, 칼륨염, 칼슘염, 마그네슘염, 암모늄염 및 아연염으로 구성된 군에서 선택되는 것을 특징으로 하였다.In an embodiment of the present invention, a gamma oligopeptide or a salt thereof having low molecular weight of polygamma glutamic acid was prepared. The molecular weight of the gamma oligopeptide or salt thereof prepared from the low molecular weight polygammaglutamic acid used in the present invention is characterized in that 600 ~ 5,000 Da, the salt is sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt and zinc It is characterized in that it is selected from the group consisting of salts.
본 발명의 다른 실시예에서는 콜라겐 분해효소의 저해능을 확인하기 위해 HDF-N 세포에 UVA를 조사한 후 감마올리고펩타이드를 첨가(0.05, 0.1%, 0.5%)하여 배양한 후 콜라겐 분해효소의 활성을 분석한 결과, 대조군에서의 콜라겐 분해효소의 활성과 감마올리고펩타이드 처리군에서의 분해효소의 활성이 각각 UVA를 조사하지 않은 대조군에 비해 177%, 154%, 133%로 측정되어 감마올리고펩타이드를 처리하지 않은 실험군의 수치인 183%와 비교하여 유의적으로 콜라겐 분해 효소의 활성이 감소되는 것을 확인하였다 (도 1).In another embodiment of the present invention, to investigate the inhibitory ability of the collagenase, UVA was irradiated to HDF-N cells, and then cultured by adding gamma oligopeptide (0.05, 0.1%, 0.5%) and analyzing the activity of collagenase. As a result, the activity of collagenase and gamma oligopeptide in the control group were measured to be 177%, 154% and 133%, respectively, compared to the control group without UVA irradiation. Compared with the 183% of the experimental group did not significantly reduce the activity of collagenase enzymes (Fig. 1).
본 발명의 또다른 실시예에서는 콜라겐 생성능을 확인하기 위해 HDF-N 세포에 감마올리고펩타이드를 첨가(0.01, 0.05%, 0.1%)하여 배양한 후 프로콜라겐의 농도를 분석한 결과, 대조군과 비교하여 각각 129%, 168%, 199%로 증가하였다 (도 2). 이를 통해 감마올리고펩타이드는 프로콜라겐 합성능을 농도 의존적으로 증가시켜 주름개선에 효능을 나타내는 것으로 확인되었다.In another embodiment of the present invention, the concentration of procollagen was analyzed by adding gamma oligopeptide (0.01, 0.05%, 0.1%) to HDF-N cells to confirm collagen production ability, and compared with the control group. Increased to 129%, 168%, and 199%, respectively (FIG. 2). Through this, gamma oligopeptide was shown to increase the procollagen synthesis capacity in a concentration-dependent manner to show the effect on wrinkle improvement.
본 발명의 피부 주름 개선용 화장료조성물은 주름개선 기능성화장품에 첨가될 수 있다. 본 발명의 주름개선용 화장료조성물에서 감마올리고펩타이드의 함유량은 0.005%~10%가 바람직하며, 0.005% 이하일 경우, 피부 주름개선 효과를 기대할 수 없으며, 10% 이상일 경우 함량증가에 따른 주름개선 효과를 기대할 수 없을 뿐만 아니라 고비용을 초래하여, 경제적이지 못하다.The cosmetic composition for improving skin wrinkles of the present invention may be added to wrinkle improvement functional cosmetics. The content of gamma oligopeptide in the cosmetic composition for wrinkle improvement of the present invention is preferably 0.005% to 10%, when the content is less than 0.005%, anti-wrinkle effect cannot be expected, and when the content is more than 10%, the wrinkle improvement effect is increased. Not only can it not be expected, it is expensive, and it is not economical.
본 발명의 조성물로부터 제조되는 화장품은 일반적인 유화 제형 및 가용화 제형의 형태로 제조할 수 있다. 유화 제형의 화장품으로는 영양화장수, 크림, 에센스 등이 있으며, 가용화 제형의 화장품으로는 유연화장수가 있다. 또한, 본 발명의 조성물은 화장품 이외에도 피부학적으로 허용된 매질 또는 기제를 함유함으로써 피부학 분야에서 통상적으로 사용되는 국소 적용 또는 전신 적용할 수 있는 보조제 형태로 제조될 수도 있다.Cosmetics prepared from the compositions of the present invention can be prepared in the form of common emulsifying and solubilizing formulations. Cosmetics of the emulsified formulations include nutrient cosmetics, creams, essences, etc., and cosmetics of the solubilized formulations are flexible cosmetics. In addition, the compositions of the present invention may be prepared in adjuvant form for topical or systemic application commonly used in the field of dermatology by containing a dermatologically acceptable medium or base in addition to cosmetics.
적합한 화장품의 제형으로는 예를 들면, 용액, 겔, 고체 또는 반죽 무수 생성물, 수상에 유상을 분산시켜 얻은 에멀젼, 현탁액, 유탁액, 페이스트, 마이크로에멀젼, 마이크로캡슐, 미세과립구 또는 이온형 (리포좀), 비이온형의 소낭 분산제의 형태, 크림, 스킨, 로션, 파우더, 비누, 연고, 스프레이, 계면활성제 함유 클렌징, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 또는 콘실 스틱 (conceal stick)의 형태로 제공될 수 있다. 또한, 포말 (foam)의 형태 또는 압축된 추진제를 더 함유한 에어로졸 조성물의 형태로도 제조될 수 있다.Formulations of suitable cosmetics include, for example, emulsions, suspensions, emulsions, pastes, microemulsions, microcapsules, microgranules or ionic (liposomes) obtained by dispersing an oil phase in a solution, gel, solid or pasty anhydrous product, aqueous phase. , In the form of non-ionic vesicle dispersants, creams, skins, lotions, powders, soaps, ointments, sprays, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations or conceal sticks Can be provided. It may also be prepared in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.
목적한 제형에 따라 이용할 수 있는 "피부학적으로 허용된 담체"는 정제수, 오일, 왁스, 지방산, 지방산 알코올, 지방산 에스테르, 계면활성제, 흡습제 (humectant), 증점제 (thickening agent), 항산화제, 점도 안정화제 (viscosity stabilizer), 킬레이팅제, 완충제, 방부제, 저급 알코올 등이 포함되지만, 이에 제한되는 것은 아니며, 그 종류와 농도는 다양하고, 당업자가 본 발명의 사상 및 영역으로부터 벗어나지 않는 범위 내에서 수정 및 변경할 수 있는 부분을 포함한다. 필요에 따라 미백제, 보습제, 항염증제, 항박테리아제, 항진균제, 비타민, 자외선 차단제, 항생제, 여드름 방지제, 향수, 염료가 포함될 수도 있으며, 이들은 화장품 분야에서 통상적으로 사용되는 양으로 본 발명에 따른 화장료 조성물에 포함될 수 있다. 조성물 총 중량 당 0.001 내지 50 중량%가 적합하다. “Dermatologically acceptable carriers” that can be used according to the desired formulation include purified water, oils, waxes, fatty acids, fatty alcohols, fatty acid esters, surfactants, humectants, thickening agents, antioxidants, and viscosity stabilizers. Examples include, but are not limited to, topical stabilizers, chelating agents, buffers, preservatives, lower alcohols, and the like, and their types and concentrations are varied and modifications may be made by those skilled in the art without departing from the spirit and scope of the present invention. And parts that can be changed. If necessary, a whitening agent, a moisturizer, an anti-inflammatory agent, an antibacterial agent, an antifungal agent, vitamins, sunscreens, antibiotics, anti-acne agents, perfumes, dyes may be included, and these may be included in the cosmetic composition according to the present invention in an amount commonly used in the cosmetic field. May be included. Suitable from 0.001 to 50% by weight per total weight of the composition.
구체적으로 상기 오일로는 수소화 식물성유, 파마자유, 면실유, 올리브유, 야자인유, 호호바유, 아보카도유가 이용될 수 있고, 왁스로는 밀랍, 경랍, 카르나우바, 칸델릴라, 몬탄, 세레신, 액체 파라핀, 라놀린이 이용될 수 있다. 지방산으로는 스테아르산, 리놀레산, 리놀렌산, 올레산이 이용될 수 있고, 지방산 알코올로는 세틸 알코올, 옥틸 도데칸올, 올레일 알코올, 판텐올, 라놀린 알코올, 스테아릴 알코올, 헥사데칸올이 이용될 수 있으며, 지방산 에스테르로는 이소프로필 미리스테이트, 이소프로필 팔미테이트, 부틸 스테아레이트 등이 이용될 수 있지만, 이에 제한되는 것은 아니다. Specifically, the oil may be hydrogenated vegetable oil, perm oil, cottonseed oil, olive oil, palm oil, jojoba oil, avocado oil, wax, beeswax, carnauba, candelilla, montan, ceresin, liquid paraffin Lanolin may be used. Stearic acid, linoleic acid, linolenic acid, oleic acid may be used as a fatty acid, cetyl alcohol, octyl dodecanol, oleyl alcohol, pantenol, lanolin alcohol, stearyl alcohol, hexadecanol may be used as a fatty acid. As the fatty acid ester, isopropyl myristate, isopropyl palmitate, butyl stearate and the like may be used, but is not limited thereto.
계면활성제의 예로는 소듐 스테아레이트, 소듐 세틸설페이트, 폴리옥시에틸렌 라우릴에테르 포스페이트, 소듐 N-아실 글루타메이트와 같은 음이온 계면활성제; 스테아릴디메틸벤질암모늄 클로라이드 및 스테아릴트리메틸암모늄 클로라이드와 같은 양이온 계면활성제; 알킬아미노에틸글리신 하이드로클로라이드 및 레시틴과 같은 양성 계면활성제; 글리세린 모노스테아레이트, 소르비탄 모노스테아레이트, 프로필렌 글리콜 모노스테아레이트, 폴리옥시에틸렌 올레일에테르, 폴리에틸렌 글리콜 모노스테아레이트, 폴리옥시에틸렌 소르비탄 모노팔미테이트, 폴리옥시에틸렌 코코넛 지방산 모노에탄올아르니드(monoethaolarnide), 폴리옥시프로필렌 글리콜, 폴리옥시에틸렌 캐스터유, 폴리옥시에틸렌 라놀린과 같은 비이온성 계면활성제 등이 포함된다.Examples of the surfactant include anionic surfactants such as sodium stearate, sodium cetyl sulfate, polyoxyethylene laurylether phosphate, sodium N-acyl glutamate; Cationic surfactants such as stearyldimethylbenzylammonium chloride and stearyltrimethylammonium chloride; Amphoteric surfactants such as alkylaminoethylglycine hydrochloride and lecithin; Glycerine monostearate, sorbitan monostearate, propylene glycol monostearate, polyoxyethylene oleyl ether, polyethylene glycol monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene coconut fatty acid monoethanol arnide (monoethaolarnide ), Polyoxypropylene glycol, polyoxyethylene castor oil, nonionic surfactants such as polyoxyethylene lanolin and the like.
흡습제에는 글리세린, 1,3-부틸렌 글리콜, 프로필렌 글리콜이 이용될 수 있으며, 저급 알코올로는 에탄올, 이소프로판올이 이용 가능하다. 증점제의 예에는 알긴산 나트륨, 카제인산 나트륨, 젤라틴 한천, 크산탄 고무, 전분, 셀룰로오스 에테르 (예, 하이드록시에틸 셀룰로오스, 메틸 셀룰로오스, 카르복시메틸 셀룰로오스, 하이드록시 프로필메틸 셀룰로오스), 폴리비닐 피롤리돈, 폴리비닐 알코올, 폴리에틸렌 글리콜 및 소듐 카르복시메틸 셀룰로오스 등이 포함되지만, 이에 제한되는 것은 아니다. 항산화제로는 부틸레이티드 하이드록시톨루엔, 부틸레이티드 하이드록시아니솔, 프로필 갈레이트, 시트르산, 에톡시퀸(ethoxyquin)이 이용 가능하고, 킬레이팅제로는 디소듐 에데테이트, 에탄하이드록시 디포스페이트가 이용 가능하며, 완충제로는 시트르산, 소듐 시트레이트, 붕산, 보랙스(borax), 디소듐 하이드로젠 포스페이트가 이용 가능하고, 방부제로는 메틸 파라하이드록시벤조에이트, 에틸 파라하이드록시벤조에이트, 디하이드로아세트산, 살리실산, 벤조산이 이용 가능하지만, 이에 제한되는 것은 아니다.Glycerin, 1,3-butylene glycol, propylene glycol may be used as the moisture absorbent, and ethanol or isopropanol may be used as the lower alcohol. Examples of thickeners include sodium alginate, sodium caseate, gelatin agar, xanthan gum, starch, cellulose ethers (e.g., hydroxyethyl cellulose, methyl cellulose, carboxymethyl cellulose, hydroxy propylmethyl cellulose), polyvinyl pyrrolidone, Polyvinyl alcohol, polyethylene glycol, sodium carboxymethyl cellulose, and the like, but are not limited thereto. As antioxidants, butylated hydroxytoluene, butylated hydroxyanisole, propyl gallate, citric acid, ethoxyquin can be used, and chelating agents include disodium edetate and ethane hydroxy diphosphate. Citric acid, sodium citrate, boric acid, borax, disodium hydrogen phosphate are available as buffers, and methyl parahydroxybenzoate, ethyl parahydroxybenzoate, dihydro as preservatives. Acetic acid, salicylic acid, benzoic acid are available, but are not limited to these.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.
실시예 1 : 감마올리고펩타이드 및 그 염의 제조Example 1 Preparation of Gamma Oligopeptide and Its Salts
폴리감마글루탐산 생산용 기본배지(3%의 L-글루탐산이 첨가된 배지; 글루코오스 3%, (NH4)2SO4 1%, KH2PO4 0.27%, Na2HPO4/12H2O 0.17%, NaCl 0.1%, 시트르산나트륨(Sodium citrate) 0.5%, Soypeptone 0.02%, MgSO4/7H2O 0.7%, 비타민용액(Vitamin solution) 10㎖/ℓ, pH 6.8)을 멸균시켜 준비하고, 5ℓ 발효기(Jar fermentor, working volume은 3ℓ)에 바실러스 서브틸리스 청국장 균주(Bacillus subtilis var chungkookjang, KCTC 0697BP)의 종균 배양액(LB 배지)을 4% 접종하여 발효시켰다. 교반속도는 500rpm, 공기주입속도는 1.0vvm으로 하여 37℃에서 48시간 동안 발효시킨 후 필터프레스(규조토 함유)를 사용하여 균체를 제거하여 폴리감마글루탐산 함유 시료 액을 수득하였다.Basic medium for the production of polygamma glutamic acid (medium with 3% L-glutamic acid; glucose 3%, (NH 4 ) 2 SO 4 1%, KH 2 PO 4 0.27%, Na 2 HPO 4 / 12H 2 O 0.17% , NaCl 0.1%, sodium citrate 0.5%, Soypeptone 0.02%, MgSO 4 / 7H 2 O 0.7%, vitamin solution (10ml / ℓ, pH 6.8) to sterilize, 5L fermenter ( Jar fermentor, working volume was fermented by inoculating 4% seed culture (LB medium) of Bacillus subtilis var chungkookjang (KCTC 0697BP) in 3L). The stirring speed was 500rpm, the air injection speed was 1.0vvm, and fermented at 37 ° C. for 48 hours to remove the cells using a filter press (containing diatomaceous earth) to obtain a polygamma glutamic acid-containing sample liquid.
폴리감마글루탐산 함유 시료액에 2N 황산용액을 이용하여 pH 2.0으로 조정한 후 10℃에서 15시간 동안 정치시켜 폴리감마글루탐산 침전물을 수득하였다. 수득한 폴리감마글루탐산 침전물을 충분한 양의 10℃ 이하의 냉각 증류수로 세척한 후(pH 3.5 이상), 누체여과기를 이용하여 폴리감마글루탐산 침전물을 수득 후 동결건조하여 고분자량 폴리감마글루탐산을 제조하였다.The polygamma glutamic acid-containing sample solution was adjusted to pH 2.0 using 2N sulfuric acid solution and then left at 10 ° C. for 15 hours to obtain a polygamma glutamic acid precipitate. The obtained polygamma glutamic acid precipitate was washed with a sufficient amount of cold distilled water of 10 ° C. or less (pH 3.5 or more), and then obtained polygamma glutamic acid precipitate using a Licech filter, followed by lyophilization to prepare a high molecular weight polygamma glutamic acid.
실시예 2 : 콜라겐 분해 효소 저해 기능Example 2 Collagen Degrading Enzyme Inhibition Function
자연노화 및 광노화로 인하여 주름이 생성되는 여러 기전 중에 본 시험은 콜라겐 분해효소인 MMP-1(Matrix metalloproteinases)의 활성 정도를 immuno ELISA assay를 이용하여 측정함으로서 시료의 주름개선효능을 평가하고자 하였다 (도 1).Among various mechanisms in which wrinkles are formed due to natural aging and photo aging, this test was intended to evaluate the wrinkle improvement effect of the sample by measuring the activity of collagen degrading enzyme MMP-1 (Matrix metalloproteinases) by immuno ELISA assay (FIG. One).
세포주는 Human neonatal foreskin에서 유래한 Normal Human Primary Dermal fibroblasts-Neonatal(HDF-N)를 사용하였다. HDF-N(ATCC PCS-201-010)를 배양접시 바닥에 접종한 후 0.1% insulin, 0.1% rhFGF, 0.1% gentamicin, 2% FBS를 함유하는 Fibroblast Basal Medium(FBM, lonza, CC-3131) 배지를 넣고, 37℃를 유지하여 5% 이산화탄소를 포함하는 배양기내에서 배양하였다.Cell lines were normal human primary dermal fibroblasts-Neonatal (HDF-N) derived from human neonatal foreskin. Fibroblast Basal Medium (FBM, lonza, CC-3131) medium containing 0.1% insulin, 0.1% rhFGF, 0.1% gentamicin and 2% FBS after inoculating HDF-N (ATCC PCS-201-010) on the bottom of the culture dish Was added and maintained at 37 ° C. and incubated in an incubator containing 5% carbon dioxide.
감마올리고펩타이드의 콜라겐 분해 효소(MMP-1) 억제능을 평가하기 위해 HDF-N cell을 24 well plate에 2.0×104 cells/well로 분주한 다음 세포배양 조건에서 48시간 배양하였다. 48시간 이후 배지를 버리고 DPBS로 세척한 다음, DPBS 200㎕를 첨가하여 UV-A 5J/㎠를 조사하였다. 시료를 적당한 농도로 배지에 희석한 후 세포에 처리하여 세포 배양 조건에서 24시간 배양하였다. 양성대조군은 Retinoic acid(RA)을 사용하였으며, 농도는 1uM로 처리하였다. 배양액을 취하여 Human total MMP-1 ELISA kit(R＆D system, DY901)을 사용하여 MMP-1양을 측정하였다. 측정된 MMP-1양은 총 단백질 양으로 보정하였다.In order to evaluate the ability of gamma oligopeptide to inhibit collagen degrading enzyme (MMP-1), HDF-N cells were dispensed at 2.0 × 10 4 cells / well in 24 well plates and incubated for 48 hours under cell culture conditions. After 48 hours, the medium was discarded and washed with DPBS, and then UV-A 5J / cm 2 was irradiated by adding 200 µl of DPBS. Samples were diluted in medium at appropriate concentrations and then treated with cells and incubated in cell culture conditions for 24 hours. Retinoic acid (RA) was used as a positive control and the concentration was 1 uM. The culture medium was taken and the amount of MMP-1 was measured using a human total MMP-1 ELISA kit (R & D system, DY901). The amount of MMP-1 measured was corrected to the total protein amount.
먼저, HDF-N cell에 UVA 파장 5J/cm2을 조사한 결과, UVA를 조사한 실험군에서는 조사하지 않은 대조군에 비하여, MMP-1의 생성이 183.03% 증가하였다. 이로써 UVA 5J/cm2의 조사는 HDF-N 세포에서의 MMP-1생성을 효과적으로 유도하였음을 알 수 있었다. 한편, 감마올리고펩타이드를 0.05, 0.1, 0.5% 농도로 처리한 후 Human total MMP-1 ELISA kit(R&D system, DY901)로 측정한 결과, 대조군과 대비하여 각각 177.34%, 154.43%, 133.34%로 나타났다. 이를 통해 감마올리고펩타이드는 농도 의존적으로 MMP-1을 감소시키는 것을 확인하였다.First, as a result of irradiating the UVA wavelength 5J / cm 2 to the HDF-N cells, in the experimental group irradiated with UVA, the production of MMP-1 increased by 183.03% compared to the control group not irradiated. As a result, irradiation of UVA 5J / cm 2 was found to effectively induce MMP-1 production in HDF-N cells. On the other hand, after treating gamma oligopeptides at 0.05, 0.1, and 0.5% concentrations, human total MMP-1 ELISA kit (R & D system, DY901) measured 177.34%, 154.43%, and 133.34%, respectively. . Through this, gamma oligopeptide was found to decrease MMP-1 in a concentration dependent manner.
실시예 3 : 프로콜라겐 합성 기능Example 3 Procollagen Synthesis Function
콜라겐의 전구체인 프로콜라겐(procollagen) 합성능을 평가하기 위해 HDF-N 세포를 48 well plate에 1.0×104cells/well로 분주하고 세포배양 조건에서 24시간 배양하였다. 그 후, 배지를 제거하고, 24시간 세포의 기아상태를 유지하며 이 후 시험 물질을 세포 배양 배지인 FBM(media supplement 제외)에 희석하여 농도별로 세포에 처리한 후 24시간 배양하였다. 24시간 후에 배양된 세포의 배지를 수거하여 procollagen typeI c-peptide(PIP) EIA kit(TAKARA, MK101)를 사용하여 procollagen 양을 측정하였다 (도 2). 한편, 바닥에 부착되어 있는 세포는 DPBS로 세척한 후, 1N NaOH로 lysis 시켜 총 단백질 양을 측정하여 일정 단백질 당 procollagen 합성 양을 구하였다. 각각의 Procollagen 양과 단백질 양은 검량선에 따라 계산하였다.In order to evaluate the procollagen (procollagen) synthesis ability of the collagen precursor HDF-N cells were dispensed at 1.0 × 10 4 cells / well in 48 well plate and incubated for 24 hours in the cell culture conditions. Thereafter, the medium was removed, and the cells were starved for 24 hours, after which the test substance was diluted in FBM (except for media supplement), which is a cell culture medium, and treated with the cells by concentration, followed by incubation for 24 hours. After 24 hours, the cultured cells were harvested and the amount of procollagen was measured using procollagen type I c-peptide (PIP) EIA kit (TAKARA, MK101) (FIG. 2). On the other hand, the cells attached to the bottom was washed with DPBS and lysed with 1N NaOH to measure the total protein amount to determine the amount of procollagen synthesis per constant protein. Procollagen and protein amounts were calculated according to the calibration curve.
그 결과, 양성대조군으로 사용한 TGF-β는 10ng/㎖을 처리하였을 경우 대조군에 비해 procollagen 합성이 195.23% 증가하였다. 한편, BLS-PGA는 0.10%를 최고 농도로 하여 0.01%, 0.05%, 0.10% 세농도 처리하였을 때, 대조군과 대비하여 각각 129.34%, 168.74%, 199.60%로 증가하였다. 이를 통해 procollagen 합성능을 농도 의존적으로 증가시키며, 세농도 모두 통계적으로 유의한 결과를 나타내었다. 이에 따라 감마올리고펩타이드는 0.01%, 0.05%, 0.10%에서 농도의존적으로 procollagen 합성능을 증가시킴으로서 주름개선에 효능을 나타내는 것으로 판단된다.As a result, the procollagen synthesis increased by 195.23% when 10 ng / ml of TGF-β was used as a positive control. On the other hand, BLS-PGA increased to 129.34%, 168.74%, and 199.60%, respectively, when treated with 0.01%, 0.05%, and 0.10% concentrations with 0.10% as the highest concentration. This increased the procollagen synthesis capacity in a concentration-dependent manner, and the three concentrations showed statistically significant results. Accordingly, gamma oligopeptides are thought to show efficacy in wrinkle improvement by increasing procollagen synthesis ability in a concentration-dependent manner at 0.01%, 0.05%, and 0.10%.
이하, 본 발명의 조성물을 위한 제제예를 예시한다. 그러나 본 발명의 조성물을 포함하는 제형은 이제 한정되는 것은 아니다.Hereinafter, the formulation example for the composition of this invention is illustrated. However, formulations comprising the compositions of the present invention are not limited in any way.
제제예 1 : 유연 화장수 (스킨)Formulation Example 1 Flexible Lotion (Skin)
감마올리고펩타이드 5.00 (%)Gamma Oligopeptide 5.00 (%)
1,3-부틸렌글리콜 1.00 (%)1,3-butylene glycol 1.00 (%)
디소듐이디티에이 0.05 (%)Disodium ID 0.05 (%)
알란토인 0.10 (%)Allantoin 0.10 (%)
디포타슘글리시리제이트 0.05 (%)Dipotassium glycylizate 0.05 (%)
시트릭애씨드 0.01 (%)Citrix Acid 0.01 (%)
소듐시트레이트 0.02 (%)Sodium Citrate 0.02 (%)
글리세레스-26 1.00 (%)Glyceres-26 1.00 (%)
알부틴 2.00 (%)Arbutin 2.00 (%)
하이드로제네이티드캐스터오일 1.00 (%)Hydrogenated Castor Oil 1.00 (%)
에탄올 30.00 (%)Ethanol 30.00 (%)
보존제 미량Preservative Trace
착색제 미량Trace amount of colorant
착향제 미량Trace amount of flavor
정제수 잔량Purified water level
제제예 2 : 영양 크림Formulation Example 2: Nutrition Cream
감마올리고펩타이드 5.00 (%)Gamma Oligopeptide 5.00 (%)
1,3-부틸렌 글리콜 7.0 (%)1,3-butylene glycol 7.0 (%)
글리세린 1.0 (%)Glycerin 1.0 (%)
D-판테놀 0.1 (%)D-panthenol 0.1 (%)
식물 추출물 3.2 (%)Botanical Extracts 3.2 (%)
마그네슘알루미늄실리케이트 0.3 (%)Magnesium Aluminum Silicate 0.3 (%)
PEG-40 스테아레이트 1.2 (%)PEG-40 Stearate 1.2 (%)
스테아릭애씨드 2.0 (%)Stearic Acid 2.0 (%)
폴리소르베이트 60 1.5 (%)Polysorbate 60 1.5 (%)
친유형글리세릴스테아레이트 2.0 (%)Lipid Glyceryl Stearate 2.0 (%)
소르비탄세스퀴올리에이트 1.5 (%)Sorbitan Sesquioleate 1.5 (%)
세테아릴알코올 3.0 (%)Cetearyl Alcohol 3.0 (%)
미네랄오일 4.0 (%)Mineral oil 4.0 (%)
스쿠알란 3.8 (%)Squalane 3.8 (%)
카르릴릭/카프릭트리글리세라이드 2.8 (%)Carlylic / Capric Triglycerides 2.8 (%)
식물성 오일 1.8 (%)Vegetable Oil 1.8 (%)
디메치콘 0.4 (%)Dimethicone 0.4 (%)
디포타슘글리시리제이트 미량Dipotassium glycylizate traces
알란토인 미량Allantoin Trace
소듐 히아루로네이트 미량Sodium Hyaluronate Trace
토코페릴아세테이트 적량Tocopheryl acetate appropriate amount
트리에탄올아민 적량Triethanolamine appropriate amount
보존제 적량Preservative
착향제 적량Flavoring Agent
정제수 잔량Purified water level
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As described above in detail specific parts of the present invention, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
본 발명에 따른 피부 주름개선용 화장료 조성물은 콜라겐 분해 효소를 저해하고 콜라겐 합성을 증진시키고, 독성 및 부작용이 거의 없어 장기 사용이 가능하여, 피부 주름 개선에 유용하다.The cosmetic composition for improving skin wrinkles according to the present invention inhibits collagen degrading enzymes, enhances collagen synthesis, has little toxicity and side effects, and can be used for a long time, which is useful for improving skin wrinkles.

Claims (7)

  1. 감마올리고펩타이드 또는 그 염을 유효성분으로 함유하는 피부 주름개선용 화장료 조성물.A cosmetic composition for improving skin wrinkles comprising gamma oligopeptide or a salt thereof as an active ingredient.
  2. 제1항에 있어서, 상기 감마올리고펩타이드 또는 그 염은 폴리감마글루탐산을 저분자화하여 제조하는 것을 특징으로 하는 피부 주름개선용 화장료 조성물.The cosmetic composition for improving skin wrinkles according to claim 1, wherein the gamma oligopeptide or a salt thereof is prepared by lowering polygamma glutamic acid.
  3. 제1항에 있어서, 상기 감마올리고펩타이드 또는 그 염의 분자량은 600~5,000Da인 것을 특징으로 하는 피부 주름개선용 화장료 조성물.The cosmetic composition for improving skin wrinkles according to claim 1, wherein the gamma oligopeptide or its salt has a molecular weight of 600 to 5,000 Da.
  4. 제1항에 있어서, 상기 감마올리고펩타이드는 5~15개의 글루탐산으로 이루어진 펩타이드인 것을 특징으로 하는 피부 주름개선용 화장료 조성물.The cosmetic composition for improving skin wrinkles according to claim 1, wherein the gamma oligopeptide is a peptide consisting of 5 to 15 glutamic acids.
  5. 제1항에 있어서, 상기 염은 나트륨염, 칼륨염, 칼슘염, 마그네슘염, 암모늄염 및 아연염으로 구성된 군에서 선택되는 것을 특징으로 하는 피부 주름개선용 화장료 조성물.The cosmetic composition for improving skin wrinkles according to claim 1, wherein the salt is selected from the group consisting of sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt and zinc salt.
  6. 제1항에 있어서, 상기 감마올리고펩타이드 또는 그 염의 총 함량은 화장료 조성물 총 중량 대비 0.001 내지 10중량%인 것을 특징으로 하는 피부 주름개선용 화장료 조성물.The cosmetic composition for improving skin wrinkles according to claim 1, wherein the total content of the gamma oligopeptide or its salt is 0.001 to 10% by weight based on the total weight of the cosmetic composition.
  7. 제1항에 있어서, 상기 화장료 조성물은 화장수류, 에센스류, 스킨류, 로션류, 크림류, 팩류, 파운데이션류 및 메이크업베이스류로 이루어진 군에서 선택된 어느 하나의 제형인 것을 특징으로 하는 피부 주름개선용 화장료 조성물.According to claim 1, wherein the cosmetic composition is a skin wrinkle improvement, characterized in that any one selected from the group consisting of cosmetics, essences, skins, lotions, creams, packs, foundations and makeup bases Cosmetic composition.
PCT/KR2012/008159 2012-10-09 2012-10-09 Cosmetic composition containing gamma-oligopeptide or salts thereof for improving wrinkles WO2014058084A1 (en)

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