KR102236022B1 - Composition for skin whitening or improving winkle comprising sonchus brachyotus extract - Google Patents
Composition for skin whitening or improving winkle comprising sonchus brachyotus extract Download PDFInfo
- Publication number
- KR102236022B1 KR102236022B1 KR1020190075004A KR20190075004A KR102236022B1 KR 102236022 B1 KR102236022 B1 KR 102236022B1 KR 1020190075004 A KR1020190075004 A KR 1020190075004A KR 20190075004 A KR20190075004 A KR 20190075004A KR 102236022 B1 KR102236022 B1 KR 102236022B1
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- extract
- sadepul
- activity
- methanol extract
- skin whitening
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Abstract
본 발명은 사데풀 추출물을 유효성분으로 함유하는 피부 미백 및 주름개선용 조성물에 관한 것으로, 상기 사데풀 추출물은 콜라겐과 엘라스틴을 분해시켜 주름을 생성시키는 콜라게나아제 및 엘라스타아제의 활성을 억제하고, 색소침착을 유도하는 티로시나아제 활성을 억제하는 효과를 나타내는 것으로 확인됨에 따라, 상기 사데풀 추출물을 유효성분으로 함유하는 조성물은 피부미백 및 주름개선용 화장료 조성물 또는 건강식품으로 제공될 수 있다.The present invention relates to a composition for skin whitening and wrinkle improvement comprising a sadepul extract as an active ingredient, wherein the sadepul extract inhibits the activity of collagenase and elastase to generate wrinkles by decomposing collagen and elastin, and , As it was confirmed to exhibit an effect of inhibiting tyrosinase activity that induces pigmentation, the composition containing the sadepul extract as an active ingredient may be provided as a cosmetic composition or health food for skin whitening and wrinkle improvement.
Description
본 발명은 사데풀 추출물을 유효성분으로 함유하는 피부 미백 또는 주름개선용 조성물에 관한 것이다.The present invention relates to a composition for skin whitening or wrinkle improvement, containing sadepul extract as an active ingredient.
오늘날 삶의 질이 향상되면서 취미 생활을 위한 야외활동 인구가 증가하고 있으나 환경오염으로 인해 지표면에 도달하는 자외선의 양은 해마다 증가하고 있다. 또한, 산업화된 사회에서는 서구화된 생활습관 및 스트레스와 같이 피부노화 요소들도 증가하고 있어 피부건강을 증진시킬 수 있는 다양한 방법들이 연구되고 있다. 피부건강에 대한 관심과 욕구가 증가함에 따라 나이가 들어감에 따라 생리적 기능 저하로 인한 내인성 피부 노화뿐만 아니라 자외선과 환경에 의한 외인성 피부 노화를 지연시키고 건강한 피부를 유지하기 위해 천연소재로서의 화장품에 대한 연구가 활발히 진행되고 있다. Today, as the quality of life improves, the number of outdoor activities for hobbies is increasing. However, due to environmental pollution, the amount of ultraviolet rays reaching the surface is increasing year by year. In addition, in the industrialized society, skin aging factors such as westernized lifestyle and stress are also increasing, and various methods to improve skin health are being studied. As interest and desire for skin health increase, research on cosmetics as natural materials to delay extrinsic skin aging due to ultraviolet rays and the environment as well as endogenous skin aging due to physiological decline with age and to maintain healthy skin Is actively progressing.
피부는 표피(epidermis), 진피(dermis), 피하층(hypodermis)으로 구성된 기관으로써 수분 유지 및 병원균을 포함한 외부 감염원의 침입을 방어하는 장벽기능(barrier function)을 수행한다. 주로 각질형성세포(keratinocyte)로 구성된 표피는 두께가 0.05-1.5 mm정도 밖에 되지 않지만 낮은 투과성으로 외부 환경에서 인체를 보호하는 물리적 장벽은 물론, 체내 수분을 보호하는 보습장벽, 항균 펩타이드(antimicrobial peptide)로 구성된 화학적 장벽 등 대부분의 피부장벽 기능을 수행하고 있다. The skin is an organ composed of the epidermis, the dermis, and the hypodermis, and performs a barrier function that maintains moisture and prevents the invasion of external infectious agents including pathogens. The epidermis, mainly composed of keratinocytes, is only about 0.05-1.5 mm thick, but its low permeability provides a physical barrier that protects the human body from the external environment, as well as a moisturizing barrier that protects the body's moisture, antimicrobial peptide. It performs most of the skin barrier functions, such as a chemical barrier composed of.
피부노화 요인 중 자외선(ultraviolet radiation, UV)의 반복적인 노출에 의한 광노화가 대표적이며, 자외선은 피부세포 내에 유해한 활성산소종(reactive oxygen species, ROS)을 생성시켜 여러 가지 신호전달 체계를 활성화함으로써 피부세포를 구성하는 불포화 지방산, 단백질, DNA 등의 고분자 물질과 반응하여 피부결합조직인 콜라겐 (collagen), 엘라스틴 (elastin) 등과 같은 세포외 기질 단백질의 합성을 줄이고, 구조를 변형시켜 탄력을 감소시키는 것으로 알려져 있다. 자외선 및 활성산소 등에 의해 유발되는 피부 진피층에 존재하는 기질금속단백질분해효소(matrix-metalloproteinases, MMPs)는 피부노화, 특히 주름생성과 밀접한 관계가 있다. MMPs를 이루는 주요성분으로 콜라게나아제 (collagenase), 젤라티나제 (gelatinase) 및 엘라스타제 (elastase) 등이 있으며, 피부의 탄력감소 및 주름생성에 있어서 엘라스타제 및 콜라게나아제의 활성 감소는 매우 중요하다.Among the factors of skin aging, photoaging due to repeated exposure to ultraviolet radiation (UV) is a representative, and ultraviolet rays generate harmful reactive oxygen species (ROS) in the skin cells, thereby activating various signal transmission systems. It is known to reduce the synthesis of extracellular matrix proteins such as collagen and elastin, which are skin connective tissues by reacting with polymeric substances such as unsaturated fatty acids, proteins, and DNA that make up cells, and to reduce elasticity by modifying the structure. have. Matrix-metalloproteinases (MMPs) present in the dermal layer of the skin caused by ultraviolet rays and free radicals are closely related to skin aging, especially wrinkle formation. Major components of MMPs include collagenase, gelatinase, and elastase, and the decrease in the activity of elastase and collagenase in reducing skin elasticity and wrinkle formation very important.
또한, 자외선을 받으면 피부에서 색소침착이 증가되는데, 색소침착이 증가하는 것은 자외선 흡수에 의한 피부 세포의 손상을 억제할 목적으로 멜라닌 (melanin) 생성이 증가된 결과이다. 멜라닌 합성은 아미노산의 하나인 티로신(tyrosine)을 기질로 티로시나제(tyrosinase), TRP-1(tyrosinase related protein-1), TRP-2에 의해 DOPA(3,4-dihydroxyphenylalanine)를 거쳐 DOPAquinone으로 전환되고 자동산화반응과 효소반응으로 DOPAchrome을 거쳐 흑갈색의 공동합체인 멜라닌을 생성하게 된다. 또한 멜라닌 형성의 조절인자로 잘 알려진 MITF(microphthalmia-asociated transcription factor)는 티로시나제 및 TRPs의 M-box sequences에 결합하여 멜라닌 형성에 관여하는 효소들의 발현을 조절하는 것으로 잘 알려져 있다. 피부 미백제의 개발에 있어서 티로시나제, TRP-1 및 TRP-2 활성 억제 실험은 유용한 일차 평가법으로 인정되고 있다. 젊고 아름다운 피부를 유지하기 위해서는 피부세포 및 조직을 보호할 수 있는 항산화제 개발과 함께 새로운 미백제 개발이 절실히 요구되고 있는 실정이다.In addition, when UV rays are applied, pigmentation increases in the skin. The increase in pigmentation is a result of increased melanin production for the purpose of suppressing damage to skin cells caused by absorption of UV rays. Melanin synthesis is converted to DOPAquinone through DOPA (3,4-dihydroxyphenylalanine) by tyrosinase, TRP-1 (tyrosinase related protein-1), and TRP-2 as a substrate using tyrosine, one of the amino acids. Through an oxidation reaction and an enzymatic reaction, melanin, a dark brown co-polymer, is produced through DOPAchrome. In addition, MITF (microphthalmia-asociated transcription factor), which is well known as a modulator of melanin formation, is well known to regulate the expression of enzymes involved in melanin formation by binding to M-box sequences of tyrosinase and TRPs. In the development of skin whitening agents, tyrosinase, TRP-1, and TRP-2 activity inhibition experiments are recognized as useful primary evaluation methods. In order to maintain youthful and beautiful skin, the development of antioxidants that can protect skin cells and tissues and development of new whitening agents is urgently required.
본 발명은 피부 주름 생성 및 피부색소 침착을 억제시켜 건강한 피부 유지하기 위한 천연소재로 사데풀 추출물을 유효성분으로 함유하는 조성물을 제공하고자 한다. An object of the present invention is to provide a composition containing sadepul extract as an active ingredient as a natural material for maintaining healthy skin by inhibiting the formation of skin wrinkles and skin pigmentation.
본 발명은 사데풀 추출물을 유효성분으로 함유하는 피부 미백 또는 주름개선용 화장료 조성물을 제공한다.The present invention provides a cosmetic composition for skin whitening or wrinkle improvement containing the sadepul extract as an active ingredient.
또한, 본 발명은 사데풀 추출물을 유효성분으로 함유하는 피부 미백 또는 주름개선용 건강식품을 제공한다.In addition, the present invention provides a health food for skin whitening or wrinkle improvement containing the sadepul extract as an active ingredient.
본 발명에 따르면, 사데풀 추출물은 콜라겐과 엘라스틴을 분해시켜 주름을 생성시키는 콜라게나아제 및 엘라스타아제의 활성을 억제하고, 색소침착을 유도하는 티로시나아제 활성을 억제하는 효과를 나타내는 것으로 확인됨에 따라, 상기 사데풀 추출물을 유효성분으로 함유하는 조성물은 피부미백 및 주름개선용 화장료 조성물 또는 건강식품으로 제공될 수 있다.According to the present invention, it was confirmed that the sadeful extract inhibits the activity of collagenase and elastase, which degrades collagen and elastin to produce wrinkles, and inhibits the tyrosinase activity that induces pigmentation. Accordingly, the composition containing the sadepul extract as an active ingredient may be provided as a cosmetic composition or health food for skin whitening and wrinkle improvement.
도 1 사데풀 메탄올추출물 (SBME)의 전체 폴리페놀 및 플라보노이드 함량을 확인한 결과로, 3번 반복 실험 후 값을 평균 ± SD로 나타내었으며, 전체 폴리페놀 및 플라보노이드 함량의 정량을 위해 각각 탄닌산 및 루틴을 이용하여 검량곡선을 나타내었다.
도 2는 사데풀 메탄올추출물 (SBME)의 전자공여능을 확인한 결과로, 3번 반복 실험 후 값을 평균 ± SD로 나타내었으며, AA는 아스코르빅산이다.
도 3은 사데풀 메탄올추출물 (SBME)의 콜라게나아제 활성 억제 효과를 확인한 결과로, 3번 반복 실험 후 값을 평균 ± SD로 나타내었으며, AA는 아스코르빅산이다.
도 4는 사데풀 메탄올추출물 (SBME)의 엘라스타아제 활성 억제 효과를 확인한 결과로, 3번 반복 실험 후 값을 평균 ± SD로 나타내었으며, AA는 아스코르빅산이다.
도 5는 사데풀 메탄올추출물 (SBME)의 버섯 티로시나아제 활성 억제 효과를 확인한 결과로, 3번 반복 실험 후 값을 평균 ± SD로 나타내었다.1 As a result of checking the total polyphenol and flavonoid contents of the methanol extract (SBME) of Sadeful, the values after repeated experiments were expressed as mean ± SD, and tannic acid and rutin were respectively used for quantification of the total polyphenol and flavonoid contents. Was used to show the calibration curve.
Figure 2 is a result of confirming the electron donating ability of the methanol extract (SBME) of sadepul, the value after repeated experiments 3 times was shown as mean ± SD, AA is ascorbic acid.
Figure 3 is a result of confirming the inhibitory effect of the collagenase activity of the methanol extract (SBME) of sadepul, the value after repeated experiments 3 times was shown as mean ± SD, AA is ascorbic acid.
4 is a result of confirming the effect of inhibiting elastase activity of the methanol extract (SBME) of sadeful, and the value after repeated experiments 3 times was expressed as mean ± SD, and AA is ascorbic acid.
5 is a result of confirming the inhibitory effect of mushroom tyrosinase activity of the methanol extract of Sardeful (SBME), and the values after repeated experiments 3 times are shown as mean ± SD.
이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명은 사데풀 추출물을 유효성분으로 함유하는 피부 미백 또는 주름개선용 화장료 조성물을 제공할 수 있다.The present invention can provide a cosmetic composition for skin whitening or wrinkle improvement containing sadepul extract as an active ingredient.
상기 사데풀 추출물은 사데풀의 잎, 줄기 또는 이들의 혼합물을 추출한 것일 수 있다.The sadepul extract may be obtained by extracting leaves, stems, or mixtures of sadepul.
또한, 상기 사데풀 추출물은 물, C1 내지 C4의 알콜, 또는 이들의 혼합용매로 추출되는 것일 수 있으며, 보다 바람직하게는 메탄올 추출물일 수 있으나, 이에 한정되는 것은 아니다.In addition, the sadepul extract may be extracted with water, C1 to C4 alcohol, or a mixed solvent thereof, and more preferably a methanol extract, but is not limited thereto.
상기 사데풀 추출물은 티로시나아제, 콜라게나아제 또는 엘라스타아제의 활성을 억제하는 것일 수 있다.The sadepul extract may be one that inhibits the activity of tyrosinase, collagenase, or elastase.
본 발명의 실시예에 따르면, 사데풀 추출물의 주름개선 효과를 확인하기 위해, 양성대조군인 아스코르빅산과 탄력 섬유인 콜라겐과 엘라스틴을 분해시켜 피부 주름생성을 유도하는 콜라게나아제 및 엘라스타아제에 처리하여 각각의 활성 저해 효과를 확인한 결과, 도 3과 같이 아스코르빅산와 사데풀 메탄올추출의 콜라게나아제 활성저해능은 1000 μg/mL에서 각각 71.7% 및 73.3%로 사데풀 메탄올추출물의 콜라게나아제 활성저해능이 양성대조군인 아스코르빅산과 유사한 수준으로 확인되었다. 또한, 도 4와 같이 아스코르빅산와 사데풀 메탄올추출의 엘라스타아제 활성저해능은 1000 μg/mL에서 각각 45.6% 및 62.6%로 나타남에 따라, 엘라스타아제 활성 억제효과는 사데풀 메탄올추출물이 양성대조군인 아스코르빅산 보다 우수한 것으로 확인되었다.According to an embodiment of the present invention, in order to confirm the wrinkle improvement effect of the sadepul extract, collagenase and elastase that induces skin wrinkle formation by decomposing ascorbic acid as a positive control and collagen and elastin, which are elastic fibers, are used. As a result of confirming the inhibitory effect of each activity by treatment, as shown in FIG. 3, collagenase activity inhibitory activity of ascorbic acid and sadeful methanol extract was 71.7% and 73.3%, respectively, at 1000 μg/mL. The activity inhibitory activity was confirmed to be similar to the positive control, ascorbic acid. In addition, as shown in Figure 4, ascorbic acid and sadeful methanol extracts showed 45.6% and 62.6% of elastase activity inhibition at 1000 μg/mL, respectively, so that the inhibitory effect of elastase activity was positive for the sadeful methanol extract. It was found to be superior to the control ascorbic acid.
또한, 본 발명의 다른 실시예에 따르면, 사데풀 추출물의 미백효과를 확인하기 위해, 색소 침착을 유도하는 티로시나아제에 사데풀 메탄올추출물을 처리하여 티로시나아제 활성 억제효과를 확인한 결과, 도 5와 같이 양성대조군인 알부틴과 사데풀 메탄올추출물의 티로시나아제 활성저해능은 1000 μg/mL 농도에서 각각 70.8% 및 57.1%로 나타남에 따라, 사데풀 메탄올추출물의 티로시나아제 활성저해능은 양성대조군인 알부틴과 유사한 수준으로 확인되었다.In addition, according to another embodiment of the present invention, in order to confirm the whitening effect of the extract of sadepul, the effect of inhibiting tyrosinase activity was confirmed by treating the methanol extract of sadeful with tyrosinase that induces pigmentation, FIG. 5 As shown, the inhibitory activity of tyrosinase activity of the positive controls, arbutin and sadeful methanol extract, was 70.8% and 57.1%, respectively, at a concentration of 1000 μg/mL. It was confirmed at a level similar to that of.
상기 결과로부터 사데풀 메탄올추출물은 피부미백 또는 주름개선용 기능성 천연소재로 활용가능성이 높은 것으로 확인되었다.From the above results, it was confirmed that the methanol extract of Sadepul has a high possibility of application as a functional natural material for skin whitening or wrinkle improvement.
상기 화장료 조성물은 조성물 총 100 중량부에 대하여, 사데풀 추출물이 0.1 내지 90 중량부로 포함되는 것일 수 있다.The cosmetic composition may be one containing 0.1 to 90 parts by weight of sadepul extract based on a total of 100 parts by weight of the composition.
상기 화장료 조성물은 유연화장수, 영양화장수, 영양크림, 엣센스 또는 팩의 제형을 갖는 것일 수 있다.The cosmetic composition may have a formulation of a flexible lotion, a nutritional lotion, a nutritional cream, an essence, or a pack.
상기 화장료 조성물은 유효성분인 사데풀 추출물 외에 안정화제, 용해화제, 비타민, 안료 및 향료와 같은 통상적인 보조제, 그리고 담체를 포함할 수 있다.The cosmetic composition may include a stabilizer, a solubilizer, a conventional auxiliary such as a vitamin, a pigment and a fragrance, and a carrier, in addition to the sadepul extract as an active ingredient.
상기 화장료 조성물은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 예를 들어, 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이 등으로 제형화될 수 있으나, 이에 한정되는 것은 아니다. 보다 상세하게는, 썬 크림, 유연 화장수, 수렴 화장수, 영양 화장수, 영양 크림, 마사지 크림, 에센스, 아이 크림, 팩, 스프레이 또는 파우더의 제형으로 제조될 수 있다.The cosmetic composition may be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, oil, powder foundation, emulsion foundation, It may be formulated as a wax foundation or spray, but is not limited thereto. In more detail, it may be prepared in the form of a sun cream, a flexible lotion, a converging lotion, a nourishing lotion, a nourishing cream, a massage cream, an essence, an eye cream, a pack, a spray, or a powder.
상기 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물성유, 식물성유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜,실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 이용될 수 있다.When the formulation is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicon, bentonite, silica, talc, or zinc oxide may be used as a carrier component. .
상기 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 is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additional chlorofluorohydrocarbon, propane/butane Or a propellant such as dimethyl ether.
상기 제형이 용액 또는 유탁액인 경우에는 담체 성분으로서 용매, 용해 화제 또는 유탁화제가 이용되고, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌 글리콜, 1,3-부틸글리콜 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르가 있다.When the formulation is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -Butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.
상기 제형이 현탁액인 경우에는 담체 성분으로서 물, 에탄올 또는 프로필렌글리콜과 같은 액상의 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가 또는 트라칸트 등이 이용될 수 있다.When the above formulation is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose , Aluminum metahydroxide, bentonite, agar or tracant, and the like may be used.
또한, 본 발명은 사데풀 추출물을 유효성분으로 함유하는 피부 미백 또는 주름개선용 건강식품을 제공할 수 있다.In addition, the present invention can provide a health food for skin whitening or wrinkle improvement containing the sadepul extract as an active ingredient.
상기 건강식품은 상기 사데풀 추출물 이외에 다른 식품 또는 식품 첨가물과 함께 사용되고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 혼합양은 그의 사용 목적 예를 들어 예방, 건강 또는 치료적 처치에 따라 적합하게 결정될 수 있다.The health food is used together with other foods or food additives in addition to the sadepul extract, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use, for example, prevention, health or therapeutic treatment.
상기 건강식품에 함유된 화합물의 유효용량은 상기 치료제의 유효용량에 준해서 사용할 수 있으나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 범위 이하일 수 있으며, 유효성분은 안전성 면에서 아무런 문제가 없기 때문에 상기 범위 이상의 양으로도 사용될 수 있음은 확실하다.The effective dose of the compound contained in the health food may be used in accordance with the effective dose of the therapeutic agent, but in the case of long-term intake for the purpose of health and hygiene or health control, it may be less than the above range, and is effective. It is clear that the ingredient can be used in an amount beyond the above range because there is no problem in terms of safety.
상기 건강식품의 종류에는 특별한 제한이 없고, 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제등을 들 수 있다.There is no particular limitation on the kind of health food, for example, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, Drinks, alcoholic beverages, and vitamin complexes.
또한, 본 발명은 사데풀 추출물을 유효성분으로 함유는 티로시나아제, 콜라게나아제 또는 엘라스타아제의 활성 억제용 시약조성물로 제공될 수 있다.In addition, the present invention may be provided as a reagent composition for inhibiting the activity of tyrosinase, collagenase, or elastase containing the sadepul extract as an active ingredient.
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely describe the present invention to those of ordinary skill in the art.
<참고예><Reference Example>
Folin & Ciocalteu's 페놀 시약, DMSO (dimethyl sulfoxide), 디에틸렌 글리콜 (diethylene glycol), 포타슘 페록소디설페이트 (potassium peroxodisulfate), 1,1-디페닐-2-폴릴 하이드라질 (1,1-diphenyl-2-polryl hydrazyl; DPPH), 탄닌산 (tannic acid), 루틴 (rutin), 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg, N-succinyl-(L-Ala)3-p-(nitroanilide), 콜라게나아제 (from Clostridium histolyticum), 엘라스타아제 ( from porcine pancreas), 타이로신 (tyrosine), 티로시나아제 (from mushroom)은 Sigma 사(St. Louis, MO, USA)의 제품을 사용하였으며, 그 외 일반시약들은 특급품을 사용하였다. Folin & Ciocalteu's phenol reagent, DMSO (dimethyl sulfoxide), diethylene glycol, potassium peroxodisulfate, 1,1-diphenyl-2-polyl hydrazyl (1,1-diphenyl-2- polryl hydrazyl; DPPH), tannic acid, rutin, 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg, N-succinyl-(L-Ala)3-p-(nitroanilide), Collagenase (from Clostridium histolyticum), elastase (from porcine pancreas), tyrosine, and tyrosinase (from mushroom) were manufactured by Sigma (St. Louis, MO, USA), and others. General reagents used special products.
<실시예 1> 사데풀 추출물 준비<Example 1> Preparation of sadepul extract
사데풀(Sonchus brachyotus) 메탄올 추출물은 한국식물추출물은행에서 분양받아 사용하였다. 사데풀 잎과 줄기는 2015년 9월 강원도 평창군 봉평면 흥정리 태기산 일원에서 채집하였다. 플라스크에 분쇄된 시료 100 g과 1 L의 100% 메탄올을 넣고 45℃에서 1시간 동안 추출하였고, 이를 3회 반복 추출하였다. 추출액을 여과하고, 농축 및 건조시켰다. 사데풀 메탄올추출물의 수율은 12.0%였으며, 시료를 사용하기 전에 DMSO에 용해시켰다.The methanol extract of Sadepul (Sonchus brachyotus) was sold and used by the Korea Plant Extract Bank. The leaves and stems of sadae grass were collected in the area of Taegisan, Heungjeong-ri, Bongpyeong-myeon, Pyeongchang-gun, Gangwon-do in September 2015. 100 g of the pulverized sample and 1 L of 100% methanol were added to the flask, followed by extraction at 45° C. for 1 hour, and this was repeatedly extracted three times. The extract was filtered, concentrated and dried. The yield of the methanol extract of Sadeful was 12.0%, and the sample was dissolved in DMSO before use.
<실시예 2> 항산화능 확인<Example 2> Confirmation of antioxidant activity
2-1. 총 폴리페놀 및 총 플라보노이드 함량 확인2-1. Determination of total polyphenol and total flavonoid content
총 폴리페놀 함량은 Folin-Denis법(Folin & Denis, 1912)으로 확인하였다. 200 μg/mL 농도로 희석한 시료 1 mL에 folin-reagent 1 mL를 가하여 3분간 실온에서 반응시킨 후 10% Na2CO3 1 mL를 혼합하고 1시간 실온에서 방치하여 725 nm에서 흡광도를 측정하였다. 검량곡선은 탄닌산(tannic acid)을 이용하여 작성하였다.The total polyphenol content was confirmed by the Folin-Denis method (Folin & Denis, 1912). 1 mL of folin-reagent was added to 1 mL of the sample diluted to 200 μg/mL, reacted at room temperature for 3 minutes, and then 1 mL of 10% Na 2 CO 3 was mixed and left at room temperature for 1 hour to measure absorbance at 725 nm. . The calibration curve was prepared using tannic acid.
총 플라보노이드 함량은 Davies et al.(1980)의 변법으로 확인하였다. 200 μg/mL 농도로 희석한 시료 100 μL에 디에틸렌 글리콜 1 mL를 혼합하여 5분간 실온에서 반응시킨 후 1 N NaOH 용액 100 μL를 첨가하여 37℃에서 1시간 방치 후 420 nm에서 흡광도를 측정하였다. 검량곡선은 루틴을 사용하여 작성하였다.The total flavonoid content was confirmed by a variant of Davies et al. (1980). 1 mL of diethylene glycol was mixed with 100 μL of a sample diluted to a concentration of 200 μg/mL, reacted at room temperature for 5 minutes, and then 100 μL of 1 N NaOH solution was added and left at 37° C. for 1 hour, and the absorbance was measured at 420 nm. . The calibration curve was prepared using a routine.
그 결과, 도 1과 같이 사데풀 메탄올추출물의 총 폴리페놀 및 총 플라보노이드 함량이 각각 350.6 TAE(tannic acid equivalent) mg/g 및 242.0 RE(rutin equivalent) mg/g으로 나타났다.As a result, as shown in FIG. 1, the total polyphenol and total flavonoid contents of the methanol extract of Sadeful were 350.6 TAE (tannic acid equivalent) mg/g and 242.0 RE (rutin equivalent) mg/g, respectively.
2-2. 전자공여능 확인2-2. Confirmation of e-donation capability
전자공여능은 Perez et al.(2004)의 방법으로 확인하였다. 96-웰 플레이트에 에탄올에 녹인 0.2 mM DPPH 용액 50 μL와 사데풀 메탄올추출물을 62.5, 125, 250, 500, 1000 μg/mL 농도로 각각 100 μL씩 가하고 차광 상태에서 30분 동안 방치한 후에 517 nm에서 흡광도를 측정하였다. GraphPrism 소프트웨어 Ver. 7(Graphpad Software, lnc., La Jolla, CA, USA)을 사용하여 SC50(50% scavenging concentration, 자유라디칼을 50% 소거하는 농도)을 산출하였다.The electron donating ability was confirmed by the method of Perez et al. (2004). To a 96-well plate, 50 μL of 0.2 mM DPPH solution dissolved in ethanol and 100 μL of Sadeful methanol extract at 62.5, 125, 250, 500, and 1000 μg/mL concentrations were added each, and allowed to stand for 30 minutes in a light-shielding state, and then 517 nm. The absorbance was measured at. GraphPrism software Ver. 7 (Graphpad Software, lnc., La Jolla, CA, USA) was used to calculate SC 50 (50% scavenging concentration, 50% scavenging concentration).
그 결과, 도 2와 같이 사데풀 메탄올추출물은 농도 의존적으로 정적(+)인 용량-반응 상관관계를 보였으며, 아스코르빅산(ascorbic acid)과 사데풀 메탄올추출물의 전자공여능은 1000 μg/mL에서 각각 97.7% 및 87.2%로 확인되다. 또한 사데풀 메탄올추출물의 SC50은 293.9 μg/mL로 산출되었으며, 아스코르빅산의 SC50은 산출되지 않았으나 62.5 μg/mL 보다 작은 것으로 확인되었다.As a result, as shown in Fig. 2, the methanol extract of Sadeful showed a positive (+) dose-response correlation in a concentration-dependent manner, and the electron donating ability of the ascorbic acid and the methanol extract of Sadeful was at 1000 μg/mL. It was identified as 97.7% and 87.2%, respectively. In addition, the SC 50 of the methanol extract of Sadepul was calculated to be 293.9 μg/mL, and the SC 50 of ascorbic acid was not calculated, but it was found to be less than 62.5 μg/mL.
<실시예 3> 효소활성 확인<Example 3> Enzyme activity confirmation
3-1. 콜라게나아제 (Collagenase) 활성 저해능 확인3-1. Confirmation of the ability to inhibit collagenase activity
62.5, 125, 250, 500 및 1000 μg/mL 농도로 희석한 시료 100 μL와 4 mM의 CaCl2를 첨가한 0.1 M의 Tris-HCl buffer(pH 7.5)에 4-phenylazobenzyloxycarbonyl- Pro-Leu-Gly-Pro-D-Arg(0.3 mg/mL)를 녹인 기질액 250 μL를 혼합한 후 콜라게나아제 (0.2 mg/mL) 150 μL를 가하여 실온에서 20분간 반응시켰다. 4-phenylazobenzyloxycarbonyl- Pro-Leu-Gly- in 0.1 M Tris-HCl buffer (pH 7.5) containing 100 μL of the sample diluted to 62.5, 125, 250, 500 and 1000 μg/mL and 4 mM CaCl 2 After mixing 250 μL of a substrate solution in which Pro-D-Arg (0.3 mg/mL) was dissolved, 150 μL of collagenase (0.2 mg/mL) was added and reacted at room temperature for 20 minutes.
이후 6% 시트르산 (citric acid) 1 mL를 혼합하고 반응을 정지시키기 위하여 에틸 아세테이트 (ethyl acetate) 1.5 mL를 혼합하였다. 그 후 상층액을 취해 320 nm에서 흡광도를 측정하였다. 양성대조군으로 아스코르빅산(ascorbic acid)을 사용하였으며, GraphPrism 소프트웨어를 사용하여 IC50(50% inhibitory concentration, collagenase 활성을 50% 저해하는 농도)을 산출하였다.Thereafter, 1 mL of 6% citric acid was mixed, and 1.5 mL of ethyl acetate was mixed to stop the reaction. Then, the supernatant was taken and absorbance was measured at 320 nm. Ascorbic acid was used as a positive control, and IC 50 (50% inhibitory concentration, concentration that inhibits collagenase activity by 50%) was calculated using GraphPrism software.
그 결과, 도 3과 같이 사데풀 메탄올추출물은 농도 의존적으로 정적(+)인 용량-반응 상관관계를 보였으며, 아스코르빅산과 사데풀 메탄올추출물의 콜라게나아제 활성저해능은 1000 μg/mL에서 각각 71.7% 및 73.3%로 확인되었다. 또한, 아스코르빅산과 사데풀 메탄올추출물의 IC50은 각각 360.3 μg/mL 및 351.3 μg/mL로 산출되었다.As a result, as shown in FIG. 3, the methanol extract of Sadeful showed a positive (+) dose-response correlation in a concentration-dependent manner, and the inhibitory activity of collagenase activity of the ascorbic acid and the methanol extract of Sadeful was 1000 μg/mL, respectively. It was found to be 71.7% and 73.3%. In addition, the IC 50 of the methanol extract of ascorbic acid and sadeful was calculated to be 360.3 μg/mL and 351.3 μg/mL, respectively.
3-2. 엘라스타아제 (Elastase) 활성 저해능 확인3-2. Confirmation of inhibitory ability of elastase activity
62.5, 125, 250, 500 및 1000 μg/mL 농도로 희석한 시료 15 μL에 100 mM의 Tris-HCl buffer(pH 8)에 녹인 돼지 췌장 엘라스타아제 (porcine pancrease elastase) 0.2 unit 5 μL와 N-succcinyl-(L-Ala)3-p-nitroa- nilide(0.5 mg/mL)를 녹인 기질액 20 μL를 첨가하여 37℃에서 10분간 반응시켰다. 5 μL of 0.2 unit of porcine pancrease elastase dissolved in 100 mM Tris-HCl buffer (pH 8) in 15 μL of samples diluted to 62.5, 125, 250, 500 and 1000 μg/mL and N- 20 μL of a substrate solution in which succcinyl-(L-Ala)3-p-nitroa-nilide (0.5 mg/mL) was dissolved was added and reacted at 37° C. for 10 minutes.
그 후 얼음에서 5분간 냉각 시킨 후 410 nm에서 흡광도를 측정하였다. 양성대조군으로 아스코르빅산을 사용하였으며, GraphPrism 소프트웨어를 사용하여 IC50(50% inhibitory concentration, elastase 활성을 50%로 저해하는 농도)을 산출하였다.After cooling for 5 minutes on ice, the absorbance was measured at 410 nm. Ascorbic acid was used as a positive control, and IC 50 (50% inhibitory concentration, concentration inhibiting elastase activity by 50%) was calculated using GraphPrism software.
그 결과, 도 4와 같이 사데풀 메탄올추출물은 농도 의존적으로 정적(+)인 용량-반응 상관관계를 보였으며, 아스코르빅산과 사데풀 메탄올추출물의 엘라스타아제 활성저해능은 1000 μg/mL에서 각각 45.6% 및 62.6%로 확인되었으며, 사데풀 메탄올추출물의 IC50은 531.0 μg/mL로 산출되었다. 아스코르빅산의 IC50은 산출되지 않았으나 1000 μg/mL 보다 큰 것으로 확인되었다.As a result, as shown in Fig. 4, the sadeful methanol extract showed a positive (+) dose-response correlation in a concentration-dependent manner, and the inhibitory activity of the elastase activity of the ascorbic acid and the sadeful methanol extract was at 1000 μg/mL, respectively. It was found to be 45.6% and 62.6%, and the IC 50 of the methanol extract of Sadeful was calculated to be 531.0 μg/mL. IC 50 of ascorbic acid was not calculated, but was found to be greater than 1000 μg/mL.
3-3. 티로시나아제 (Mushroom tyrosinase) 활성 저해능 확인3-3. Confirmation of tyrosinase (Mushroom tyrosinase) activity inhibition
62.5, 125, 250, 500 및 1000 μg/mL 농도로 희석한 시료 5 μL에 sodium phosphate buffer(pH 6.8) 50 μL, 0.03% 티로신 50 μL, 버섯 티로시나아제 (mushroom tyrosinase) 10 μL를 넣은 후 37℃에서 10분간 반응시키고 얼음에서 5분간 냉각 시킨 후 475 nm에서 흡광도를 측정하였다. 양성대조군으로 알부틴(arbutin)을 사용하였으며, GraphPrism 소프트웨어를 사용하여 IC50(50% inhibitory concentration, tyrosinase 활성을 50% 저해하는 농도)을 산출하였다.Add 50 μL of sodium phosphate buffer (pH 6.8), 50 μL of 0.03% tyrosine, and 10 μL of mushroom tyrosinase to 5 μL of the sample diluted to 62.5, 125, 250, 500 and 1000 μg/mL. After reacting at °C for 10 minutes, cooling on ice for 5 minutes, absorbance was measured at 475 nm. Arbutin was used as a positive control, and IC 50 (50% inhibitory concentration, 50% inhibitory concentration of tyrosinase activity) was calculated using GraphPrism software.
그 결과, 도 5와 같이 사데풀 메탄올추출물은 농도 의존적으로 정적(+)인 용량-반응 상관관계를 보였으며, 알부틴과 사데풀 메탄올추출물의 버섯 티로시나아제 활성저해능은 1000 μg/mL 농도에서 각각 70.8% 및 57.1%으로 확인되었으며, 알부틴과 사데풀 메탄올추출물의 IC50은 각각 240.0 μg/mL 및 608.1 μg/mL로 산출되었다.As a result, as shown in FIG. 5, the methanol extract of Sardeful showed a positive (+) dose-response correlation in a concentration-dependent manner, and the inhibitory activity of the mushroom tyrosinase activity of arbutin and the methanol extract of Sardeful was at 1000 μg/mL concentration, respectively. It was found to be 70.8% and 57.1%, and the IC 50 of the methanol extract of arbutin and sadeful was calculated to be 240.0 μg/mL and 608.1 μg/mL, respectively.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 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, a specific part of the present invention has been described in detail, and for those of ordinary skill in the art, it is obvious that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.
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