KR102053994B1 - Cosmetic composition for skin whitening and moisturizing comprising larva extract of Protaetia brevitarsis as effective component - Google Patents
Cosmetic composition for skin whitening and moisturizing comprising larva extract of Protaetia brevitarsis as effective component Download PDFInfo
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- KR102053994B1 KR102053994B1 KR1020170178625A KR20170178625A KR102053994B1 KR 102053994 B1 KR102053994 B1 KR 102053994B1 KR 1020170178625 A KR1020170178625 A KR 1020170178625A KR 20170178625 A KR20170178625 A KR 20170178625A KR 102053994 B1 KR102053994 B1 KR 102053994B1
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- moisturizing
- skin
- extract
- composition
- lotion
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- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
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Abstract
본 발명은 흰점박이꽃무지유충의 추출물을 유효성분으로 포함하는 피부 미백 및 보습용 화장료 조성물에 관한 것으로, 본 발명의 흰점박이꽃무지유충의 추출물은 멜라닌의 생성을 억제하며, 티로시나아제 활성을 저해하고, 보습관련 인자의 발현을 증진시키는 효과가 우수하므로, 미백 및 보습용 화장료의 신규한 소재로 유용하게 활용될 수 있을 것이다.The present invention relates to a skin whitening and moisturizing cosmetic composition comprising the extract of the white spotted flower larva as an active ingredient, the extract of the white spotted flower larvae of the present invention inhibits the production of melanin, tyrosinase activity Inhibiting and improving the expression of the moisturizing-related factors is excellent, it can be usefully used as a novel material of the whitening and moisturizing cosmetics.
Description
본 발명은 흰점박이꽃무지유충의 추출물을 유효성분으로 포함하는 피부 미백 및 보습용 화장료 조성물에 관한 것이다.The present invention relates to a skin whitening and moisturizing cosmetic composition comprising an extract of white spotted flower larva as an active ingredient.
피부는 외부를 덮고 있는 기관으로, 체온 조절과 다양한 외부 자극으로부터 몸을 보호함과 동시에 내측의 수분을 막는 장벽 기능이 있다. 피부의 표피층은 분화과정을 통하여 수분을 함유하면서 얇은 각질층(stratum corneum)으로 발달하게 된다. 각질층을 구성하고 있는 세포에는 수용성 성분인 고농도의 천연보습인자(natural moisturizing factor)가 존재하여 피부가 유연성을 나타내도록 작용함은 물론 적절한 수분을 유지할 수 있도록 하여 피부 보습에 중요한 역할을 담당하고 있다.The skin is an organ that covers the outside and has a barrier function that protects the body from body temperature control and various external stimuli while preventing moisture from the inside. The epidermal layer of the skin develops into a thin stratum corneum that contains water through differentiation. The cells constituting the stratum corneum have a high concentration of natural moisturizing factor, which is a water-soluble ingredient, which plays an important role in moisturizing the skin by maintaining flexibility and of maintaining proper moisture.
멜라닌은 피부의 진피와 표피 경계에 존재하는 멜라닌세포(melanocyte)로부터 합성되는 독특한 색소이다. 자외선에 노출되면, 멜라닌세포로부터 멜라닌이 합성되고, 합성된 멜라닌은 자외선에 의해 유발되는 피부 상해를 예방하는 역할을 한다. 티로시나아제(tyrosinase) 유전자 패밀리는 멜라닌 생합성(melanogenesis)을 조절하는데 중요한 역할을 한다. 티로시나아제는 이원기능성(bifunctional)의 효소로, 티로신(tyrosine)의 DOPA(3,4-dihydroxyphenylalanine)로의 수산화(hydroxylation) 및 DOPA의 DOPA 퀴논(quinone)으로의 산화를 촉매하여 멜라닌 생성을 조절한다.Melanin is a unique pigment synthesized from melanocytes that exist at the dermal and epidermal boundaries of the skin. When exposed to ultraviolet rays, melanin is synthesized from melanocytes, and the synthesized melanin serves to prevent skin injury caused by ultraviolet rays. The tyrosinase gene family plays an important role in regulating melanogenesis. Tyrosinase is a bifunctional enzyme that regulates melanin production by catalyzing the hydroxylation of tyrosine to DOPA (3,4-dihydroxyphenylalanine) and the oxidation of DOPA to DOPA quinone. .
흰점박이꽃무지는 민간 및 동의보감(東醫寶鑑) 등의 전통 한방의서에서 '제조' 또는 '굼벵이'라는 속명으로 불리어지고 있는 딱정벌레목(Coleoptera), 풍뎅이과(Scarabaeidae), 꽃무지아과(Cetoniinae)에 속하는 크기 17~24㎝의 식식성(phytophagous) 곤충으로, 우리나라를 비롯하여 중국, 일본 및 시베리아 동부 지역에 서식하는 것으로 알려져 있다. 흰점박이꽃무지는 3령의 성숙유충 상태로 월동하며, 성충은 주간에 활동하고 복숭아, 배 등의 성숙한 과일이나 옥수수, 상수리 나무 등의 즙액을 먹이로 하며, 항상 군집성이다. 또한, 이들의 유충은 퇴비나 건초더미 등의 유기물이 풍부한 부식성 토양속에서 서식한다.White spotted radish is known as `` manufacture '' or `` slugs '' in traditional Chinese medicine such as folk or Dongbogam, Coleoptera, Scarabaeidae and Cetoniinae. Phytophagous insects, 17-24 cm in size, are known to live in Korea, China, Japan, and Siberia. White spotted radish overwinters as a three-year-old mature larva. Adults are active during the day and feed on mature fruits such as peaches and pears, or juices such as corn and oak, and are always clustered. Their larvae also live in corrosive soils rich in organic matter such as compost and haystacks.
흰점박이꽃무지는 오래전부터 간질환 등의 치료를 위한 한방 약재로서 이용되어 왔다. 또한, 최근에 유용한 생체 활성물질의 탐색 및 개발을 위한 곤충자원으로 크게 주목을 받고 있으며, 항생물질의 생산, 흰쥐(mouse)를 이용한 실험에서 알코올 과량섭취에 의해 손상된 간지질대사의 회복작용 등이 알려져 있으며 혈전용해성 효소에 대한 연구와 집쥐(rat)에서 사염화탄소의 투여에 의해 유도된 간독성에 대한 간보호효과를 나타내는 등 유용성이 확인된 바 있다.White spotted radish has been used as a herbal medicine for the treatment of liver disease for a long time. In addition, it has recently attracted great attention as an insect resource for the exploration and development of useful bioactive substances, and the production of antibiotics, recovery of hepatic lipid metabolism damaged by alcohol intake in experiments using mice, etc. Known and useful, such as studies on thrombolytic enzymes and hepatoprotective effects on hepatotoxicity induced by administration of carbon tetrachloride in rats.
한편, 한국등록특허 제1758718호에는 '흰점박이꽃무지 유충으로부터 분리된 화합물을 함유하는 항혈전용 조성물'이 개시되어 있고, 한국등록특허 제1557788호는 '무당벌레 유충 유래 신규 펩타이드를 함유하는 항염증 화장료 및 피부외용제 조성물'이 개시되어 있으나, 본 발명의 흰점박이꽃무지유충의 추출물을 유효성분으로 포함하는 피부 미백 및 보습용 화장료 조성물에 대해서는 기재된 바 없다.Meanwhile, Korean Patent No. 1758718 discloses an 'anti-blood composition containing a compound isolated from white spotted radish larva', and Korean Patent No. 1557788 describes an anti-inflammatory containing a novel peptide derived from ladybug larvae. Cosmetics and external application composition for the skin 'is disclosed, but the skin whitening and moisturizing cosmetic composition comprising the extract of the white spotted flower larvae of the present invention as an active ingredient has not been described.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자들은 흰점박이꽃무지유충의 열풍건조 또는 동결건조 분말의 열수 및 에탄올 추출물을 제조하고 상기 각 추출물의 티로시나아제 활성 저해능, 멜라닌 생성 억제능 및 보습관련 인자의 발현 촉진능을 분석한 결과, 흰점박이꽃무지유충의 열풍건조 분말의 추출물이 티로시나아제 효소 활성 저해 및 멜라닌 생성 효과가 우수하고, 히알루론산 합성 효소의 발현을 증진시키는 효과가 있음을 확인함으로써, 본 발명을 완성하였다.The present invention was derived by the above requirements, the present inventors prepared hot water and ethanol extracts of hot air dried or lyophilized powder of white spotted radish larvae and inhibited tyrosinase activity, melanin production inhibitory activity of each extract and As a result of analyzing the expression promoting ability of moisturizing-related factors, the extract of the hot-air dried powder of white spotted radish larvae has the effect of inhibiting tyrosinase enzyme activity and melanin production, and enhancing the expression of hyaluronic acid synthase. By confirming that, this invention was completed.
상기 과제를 해결하기 위해, 본 발명은 흰점박이꽃무지유충의 추출물을 유효성분으로 포함하는 피부 미백 및 보습용 화장료 조성물을 제공한다.In order to solve the above problems, the present invention provides a skin whitening and moisturizing cosmetic composition comprising the extract of the white spotted flower larva as an active ingredient.
또한, 본 발명은 흰점박이꽃무지유충의 추출물을 유효성분으로 포함하는 피부 미백 및 보습용 건강기능식품 조성물을 제공한다.The present invention also provides a health functional food composition for skin whitening and moisturizing containing the extract of white spotted flower larva as an active ingredient.
또한, 본 발명은 흰점박이꽃무지유충의 추출물을 유효성분으로 함유하는 멜라닌 색소 과다 침착 질환의 예방 또는 치료용 약학 조성물을 제공한다.In another aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of melanin-pigmentation hyperpigmentation disease containing the extract of white spotted radish larvae as an active ingredient.
본 발명의 흰점박이꽃무지유충의 추출물은 멜라닌의 생성을 억제하며, 티로시나아제 활성을 저해하고, 보습관련 인자의 발현을 증진시키는 효과가 우수하므로, 미백 및 보습용 화장료의 신규한 소재로 유용하게 활용될 수 있을 것이다.The extract of the white spotted radish larvae of the present invention inhibits the production of melanin, inhibits tyrosinase activity, and enhances the expression of moisturizing-related factors, and thus is useful as a novel material for whitening and moisturizing cosmetics. Can be utilized.
도 1은 굼벵이(흰점박이꽃무지유충) 열풍건조 분말 추출물의 세포 독성을 분석한 결과이다.
도 2는 굼벵이 동결건조 분말 추출물의 세포 독성을 분석한 결과이다.
도 3은 코지산(kojic acid) 처리에 따른 버섯 티로시나아제 효소의 활성 저해 수준을 보여주는 그래프이다.
도 4는 굼벵이 열풍건조 분말 추출물 처리에 따른 버섯 티로시나아제 효소의 활성 수준을 보여주는 그래프이다.
도 5는 굼벵이 동결건조 분말 추출물 처리에 따른 버섯 티로시나아제 효소의 활성 수준을 보여주는 그래프이다.
도 6은 마우스 흑색종 세포에서 코지산 처리에 따른 멜라닌 함량을 분석한 결과이다.
도 7은 마우스 흑색종 세포에서 굼벵이 열풍건조 분말 추출물 처리에 따른 멜라닌 함량을 분석한 결과이다.
도 8은 각질형성세포주에서 1μM의 레티노산(retinoic acid) 처리에 따른 보습인자의 mRNA 발현 변화를 보여주는 결과이다. IVL: involucrin, FLG: filaggrin, HAS2; hyaluronic acid synthase-2, HAS3; hyaluronic acid synthase-3.
도 9는 각질형성세포주에서 굼벵이 열풍건조 분말의 열수 추출물 처리에 따른 보습인자의 mRNA 발현 변화를 보여주는 결과이다.
도 10은 각질형성세포주에서 굼벵이 열풍건조 분말의 에탄올 추출물 처리에 따른 보습인자의 mRNA 발현 변화를 보여주는 결과이다.1 is a result of analyzing the cytotoxicity of the slug (white spotted radish larva) hot air dried powder extract.
Figure 2 is the result of analyzing the cytotoxicity of the slug lyophilized powder extract.
Figure 3 is a graph showing the activity inhibition level of mushroom tyrosinase enzyme according to kojic acid treatment.
Figure 4 is a graph showing the activity level of mushroom tyrosinase enzyme according to the slug hot air dry powder extract treatment.
5 is a graph showing the activity level of mushroom tyrosinase enzyme according to the treatment of slug lyophilized powder extract.
6 is a result of analyzing the melanin content according to koji acid treatment in mouse melanoma cells.
Figure 7 is the result of analyzing the melanin content according to the treatment of slug hot air drying powder in mouse melanoma cells.
8 is a result showing the mRNA expression change of the moisturizing factor according to the retinoic acid treatment of 1μM in keratinocytes. IVL: involucrin, FLG: filaggrin, HAS2; hyaluronic acid synthase-2, HAS3; hyaluronic acid synthase-3.
9 is a result showing the mRNA expression change of the moisturizing factor according to the hydrothermal extract treatment of slug hot air drying powder in keratinocytes.
10 is a result showing the mRNA expression change of the moisturizing factor according to the ethanol extract treatment of slug hot air drying powder in keratinocytes.
본 발명의 목적을 달성하기 위하여, 본 발명은 흰점박이꽃무지유충의 추출물을 유효성분으로 포함하는 피부 미백 및 보습용 화장료 조성물을 제공한다.In order to achieve the object of the present invention, the present invention provides a skin whitening and moisturizing cosmetic composition comprising the extract of white spotted flower larva as an active ingredient.
본 발명의 피부 미백 및 보습용 화장료 조성물에 있어서, 상기 흰점박이꽃무지유충의 추출물은 물, 탄소수 1 내지 4의 저급 알코올 및 이들의 혼합용매로 구성되는 군으로부터 선택되는 용매의 추출물일 수 있고, 바람직하게는 물 또는 저급 알코올일 수 있고, 가장 바람직하게는 물 또는 에탄올일 수 있으나, 이에 제한되지 않는다.In the cosmetic composition for skin whitening and moisturizing of the present invention, the extract of the white spotted radish larva may be an extract of a solvent selected from the group consisting of water, a lower alcohol having 1 to 4 carbon atoms, and a mixed solvent thereof, Preferably it may be water or lower alcohol, most preferably water or ethanol, but is not limited thereto.
또한, 상기 흰점박이꽃무지유충의 추출물은 추출처리에 의해 얻어지는 추출액, 추출액의 희석액 또는 농축액, 추출액을 건조하여 얻어지는 건조물, 또는 조정제물 또는 정제물 중 어느 하나를 포함하는 것일 수 있다.In addition, the extract of the white spotted flower larva larva may include any one of an extract obtained by the extraction treatment, a diluent or concentrate of the extract, a dried product obtained by drying the extract, or a crude or purified product.
본 발명의 일 구현예에 따른 화장료 조성물에 있어서, 상기 흰점박이꽃무지유충의 추출물은 열풍건조된 흰점박이꽃무지유충을 분말화한 후, 80~90%(v/v) 에탄올로 추출하거나, 80~90℃에서 2.5~3.5시간 동안 열수 추출한 것일 수 있고, 바람직하게는 열풍건조한 흰점박이꽃무지유충의 분말을 70% 에탄올로 추출하거나, 70℃에서 3시간 동안 열수 추출한 것일 수 있으나, 이에 제한되지 않는다.In the cosmetic composition according to an embodiment of the present invention, the extract of the white spotted flower larva larva is powdered after hot-air dried white spotted flower larva larvae, extracted with 80 to 90% (v / v) ethanol, Hot water may be extracted at 80-90 ° C. for 2.5-3.5 hours, preferably, hot-air dried white spotted radish larvae are extracted with 70% ethanol, or hot water extracted at 70 ° C. for 3 hours, but not limited thereto. It doesn't work.
본 발명에 따른 상기 조성물은 멜라닌(melanin)의 생성을 저해하고, 티로시나아제(tyrosinase)의 활성을 저해하며, 히알루론산 합성효소와 같은 보습인자의 발현을 증가시키는 특징이 있다.The composition according to the present invention is characterized by inhibiting the production of melanin, inhibiting the activity of tyrosinase, and increasing the expression of moisturizing factors such as hyaluronic acid synthase.
한편, 미백 효과란, 자외선 노출, 호르몬 밸런스의 변화, 유전적 프로그램 등의 각종 요인에 의해 발생하는 거뭇한 피부나 기미, 주근깨, 다크서클을 개선 또는 방지하는 효과, 피부를 투명감 있는 아름다운 것으로 하는, 혹은 투명감 있는 아름다운 피부를 유지하는 효과, 피부의 칙칙함을 경감하여 윤기ㆍ팽팽함을 증가시키는 효과 등을 의미한다.On the other hand, the whitening effect is to improve or prevent dark skin, blemishes, freckles, dark circles caused by various factors such as exposure to ultraviolet rays, changes in hormonal balance, genetic programs, and make skin beautiful and transparent. Or it means the effect of maintaining the beautiful skin with transparency, the effect of reducing the dullness of the skin and increasing the luster and tension.
또한, 보습 효과란, 피부의 수분이 감소되는 것을 저해 또는 억제하거나 피부의 수분 함유량을 증가시켜 피부 표면을 매끄럽게 하며, 윤기를 부여하는 것을 말한다. 또는 수분 손실(수분 증발) 등을 적절히 조절하여 생체의 항상성을 유지하는 것을 의미한다.In addition, a moisturizing effect means inhibiting or suppressing the reduction of the moisture of skin, or increasing the moisture content of skin, to make skin surface smooth, and to give gloss. Or means to maintain the homeostasis of the living body by appropriately adjusting the water loss (moisture evaporation).
본 발명의 상기 피부 미백 및 보습용 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 있고, 보다 상세하게는, 스킨, 스킨 소프트너, 스킨토너, 아스트린젠트, 로션, 밀크로션, 모이스쳐로션, 영양로션, 마사지크림, 영양크림, 아이 크림, 모이스쳐 크림, 핸드크림, 에센스, 영양에센스, 팩, 클렌징폼, 클렌징 워터, 클렌징 로션, 클렌징 크림, 바디로션, 바디클렌져, 비누 또는 파우더의 화장품 제형으로 제조될 수 있으나, 이에 한정하는 것은 아니다. The skin whitening and moisturizing cosmetic composition of the present invention may be prepared in any formulation commonly prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, oils , Powder foundation, emulsion foundation, wax foundation, spray, and the like, and more specifically, skin, skin softener, skin toner, astringent, lotion, milk lotion, moisturizer, nutrition lotion, massage cream, It may be prepared as a cosmetic formulation of nutrition cream, eye cream, moisturizer cream, hand cream, essence, nutrition essence, pack, cleansing foam, cleansing water, cleansing lotion, cleansing cream, body lotion, body cleanser, soap or powder It is not limited.
본 발명의 화장료 조성물의 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.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. This can be used.
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 클로로플루오로 히드로카본, 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 chlorofluoro hydro 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, 프로필렌글리콜, 1,3-부틸글리콜 오일, 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.
본 발명의 화장료 조성물의 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 formulation of the cosmetic composition of the present invention is a suspension, a liquid diluent such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester as carrier components , Microcrystalline cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.
본 발명의 화장료 조성물은 상기 유효성분 이외에 화장품 조성물에 통상적으로 이용되는 성분들을 포함하며, 예컨대 지방 물질, 유기 용매, 용해제, 농축제 및 겔화제, 연화제, 항산화제, 현탁화제, 안정화제, 발포제(foaming agent), 방향제, 계면활성제, 물, 이온형 또는 비이온형 유화제, 충전제, 금속이온 봉쇄제 및 킬레이트화제, 보존제, 비타민, 차단제, 습윤화제, 필수 오일, 염료, 안료, 친수성 또는 친유성 활성제, 지질 소낭과 같은 통상적인 보조제, 그리고 담체를 포함한다.The cosmetic composition of the present invention includes components commonly used in cosmetic compositions in addition to the active ingredient, and include, for example, fatty substances, organic solvents, solubilizers, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents ( foaming agents), fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents , Conventional adjuvants such as lipid vesicles, and carriers.
본 발명은 또한, 흰점박이꽃무지유충의 추출물을 유효성분으로 포함하는 피부 미백 및 보습용 건강기능식품 조성물을 제공한다.The present invention also provides a health functional food composition for skin whitening and moisturizing containing the extract of white spotted flower larva as an active ingredient.
상기 건강기능식품 조성물은 분말, 과립, 환, 정제, 캡슐, 캔디, 시럽 또는 음료의 제형으로 제조할 수 있으나, 이에 제한되지 않는다. The dietary supplement composition may be prepared in the form of powder, granules, pills, tablets, capsules, candy, syrup or beverage, but is not limited thereto.
본 발명의 건강기능식품 조성물을 식품첨가물로 사용하는 경우, 상기 건강기능식품 조성물을 그대로 첨가하거나 다른 식품 또는 식품성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 양은 그의 사용 목적에 따라 적절하게 사용될 수 있다. 일반적으로, 식품 또는 음료의 제조시 본 발명의 건강기능식품 조성물은 원료에 대하여 15 중량부 이하, 바람직하게는 10 중량부 이하의 양으로 첨가된다. 그러나 건강을 목적으로 장기간 섭취하는 경우에는 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로 사용될 수 있다.When the health functional food composition of the present invention is used as a food additive, the health functional food composition may be added as it is or used with other foods or food ingredients, and may be appropriately used according to a conventional method. The amount of the active ingredient may be appropriately used depending on the purpose of use thereof. In general, in the preparation of food or beverages, the nutraceutical composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less with respect to the raw material. However, in the case of long-term intake for health purposes, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.
상기 건강기능식품의 종류에 특별한 제한은 없다. 상기 건강기능식품 조성물을 첨가할 수 있는 식품의 예로는 육류, 소시지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다.There is no particular limitation on the type of dietary supplement. Examples of foods to which the health functional food composition may be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products, including ice cream, various soups, drinks, tea Drinks, alcoholic beverages, vitamin complexes, and the like, and include all health foods in the conventional sense.
또한, 본 발명의 건강기능식품 조성물은 식품, 특히 기능성 식품으로 제조될 수 있다. 본 발명의 기능성 식품은 식품 제조 시에 통상적으로 첨가되는 성분을 포함하며, 예를 들어, 단백질, 탄수화물, 지방, 영양소 및 조미제를 포함한다. 예컨대, 드링크제로 제조되는 경우에는 유효성분 이외에 천연 탄수화물 또는 향미제를 추가 성분으로서 포함시킬 수 있다. 상기 천연 탄수화물은 모노사카라이드(예컨대, 글루코오스, 프럭토오스 등), 디사카라이드(예컨대, 말토스, 수크로오스 등), 올리고당, 폴리사카라이드(예컨대, 덱스트린, 시클로덱스트린 등) 또는 당알코올(예컨대, 자일리톨, 소르비톨, 에리쓰리톨 등)인 것이 바람직하다. 상기 향미제는 천연 향미제(예컨대, 타우마틴, 스테비아 추출물 등)와 합성 향미제(예컨대, 사카린, 아스파르탐 등)를 이용할 수 있다.In addition, the nutraceutical composition of the present invention may be prepared as a food, in particular a functional food. Functional foods of the present invention include ingredients that are commonly added in food production, and include, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when prepared with a drink, natural carbohydrates or flavors may be included as additional ingredients in addition to the active ingredient. The natural carbohydrates can be monosaccharides (e.g. glucose, fructose, etc.), disaccharides (e.g. maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g. dextrins, cyclodextrins, etc.) or sugar alcohols (e.g. , Xylitol, sorbitol, erythritol and the like). The flavourant may be a natural flavourant (eg, taumartin, stevia extract, etc.) and a synthetic flavourant (eg, saccharin, aspartame, etc.).
상기 건강기능식품 조성물 이외에 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산음료에 사용되는 탄산화제 등을 더 함유할 수 있다. 이러한 상기 첨가되는 성분의 비율은 크게 중요하진 않지만 본 발명의 건강기능식품 조성물 100 중량부에 대하여, 0.01 내지 0.1 중량부의 범위에서 선택되는 것이 일반적이다.Various nutritional supplements, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonic acid The carbonation agent etc. which are used for a drink can be contained further. Although the ratio of the above-mentioned ingredients is not critical, it is generally selected from 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food composition of the present invention.
본 발명은 또한, 흰점박이꽃무지유충의 추출물을 유효성분으로 함유하는 멜라닌 색소 과다 침착 질환의 예방 또는 치료용 약학 조성물을 제공한다.The present invention also provides a pharmaceutical composition for the prevention or treatment of melanin hyperpigmentation disease containing the extract of white spotted radish larvae as an active ingredient.
본 명세서에서 사용되는 용어 "멜라닌 색소 과다 침착(hyperpigmentation)"은 피부 또는 손·발톱의 특정 부위에서 멜라닌의 과도한 증가에 의해 다른 부위에 비해 검게 또는 어둡게 되는 것을 의미한다. 상기 멜라닌 색소 과다 침착 질환은 주근깨, 노인성 반점, 간반, 기미, 갈색 또는 흑점, 일광 색소반, 푸른흑피증(cyanic melasma), 약물 사용 후의 과다색소침착, 임신성 갈색반(gravidic chloasma), 또는 찰상 및 화상을 비롯한 상처 또는 피부염으로 인한 염증 후 과다 색소 침착 등을 포함하나 이에 한정되지 않는다.As used herein, the term "melanin hyperpigmentation" means blacking or darkening in comparison to other areas by excessive increase of melanin in the skin or in certain areas of the nails. The melanin hyperpigmentation disease is freckles, senile plaques, liver spots, blemishes, brown or sunspots, sunrays, cyanic melasma, hyperpigmentation after drug use, gravidic chloasma, or scratches and Hyperpigmentation after inflammation due to wounds or dermatitis, including burns, and the like.
본 발명의 약학 조성물은 유효성분 이외에 약학적으로 허용되는 담체를 포함할 수 있으며, 이러한 담체는 제제 시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient, which is commonly used in the preparation of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, Calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and minerals Oils and the like, but are not limited thereto.
본 발명의 약학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약학적으로 허용되는 담체 및 제제는 레밍턴의 약학적 과학(Remington's Pharmaceutical Sciences, 19th ed., 1995)에 상세히 기재되어 있다.In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences, 19th ed., 1995.
본 발명에 따른 약학 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다. 한편, 본 발명의 약학 조성물의 투여량은 바람직하게는 1일 당 0.0001~100mg/kg(체중)이다.Suitable dosages of the pharmaceutical compositions according to the present invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. On the other hand, the dosage of the pharmaceutical composition of the present invention is preferably 0.0001 ~ 100mg / kg (body weight) per day.
본 발명의 약학 조성물은 경구 또는 비경구로 투여할 수 있으며, 비경구 투여의 경우, 피부에 국소적으로 도포, 정맥 내 주입, 피하 주입, 근육 주입, 복강 주입, 경피 투여 등으로 투여할 수 있다. 본 발명의 약학 조성물이 멜라닌 색소 과다 침착 질환을 치료 또는 예방을 위해 적용되는 점을 감안하면, 피부에 국소적으로 도포되어 이루어지는 것이 바람직하다.The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered topically to the skin, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration and the like. In view of the fact that the pharmaceutical composition of the present invention is applied for treating or preventing melanin hyperpigmentation disease, it is preferable that the pharmaceutical composition is applied topically to the skin.
본 발명의 조성물에 포함되는 유효성분의 농도는 치료 목적, 환자의 상태, 필요기간 등을 고려하여 결정할 수 있으며 특정 범위의 농도로 한정되지 않는다.The concentration of the active ingredient included in the composition of the present invention can be determined in consideration of the purpose of treatment, the condition of the patient, the period of time, etc., and is not limited to a specific range of concentration.
본 발명의 약학 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약학적으로 허용되는 담체 또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 주사제, 크림, 패취, 분무제, 연고제, 경고제, 로션제, 리니멘트제, 파스타제 및 카타플라스마제 중에서 선택된 어느 하나의 제형으로 제조될 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The pharmaceutical compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier or excipient according to methods which can be easily carried out by those skilled in the art. It can be prepared by incorporation into a dose container. In this case, the formulation may be prepared in any one formulation selected from injections, creams, patches, sprays, ointments, warnings, lotions, linings, pastas, and cataplasmas, and may further include dispersants or stabilizers. have.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.
재료 및 방법Materials and methods
1. 세포독성 평가1. Cytotoxicity Assessment
약용 곤충의 유효성분 추출을 위해, 굼벵이(흰점박이꽃무지유충)를 울산소재 홍이농장에서 열풍건조 상태와 생물로 공급받아 전처리 하였다. 생물 굼벵이는 동결건조 후 분말화 하였고, 열풍건조 굼벵이도 분말화 하였다. 분말화한 동결건조 또는 열풍건조 굼벵이는 70% 에탄올 추출 및 70℃에서 3시간 동안 열수 추출하여 여과(filtration)한 후 실험에 사용하였다.For extracting the active ingredient of medicinal insects, slugs (white spotted radish larvae) were pre-treated with hot-air dried condition and organisms at Hongi Farm, Ulsan. Bio slugs were powdered after lyophilization, and hot air dried slugs were also powdered. The powdered lyophilized or hot air dried slugs were extracted by 70% ethanol extraction and hot water extraction for 3 hours at 70 ° C., and then used for experiments.
인간 정상 세포주인 293T 세포주(Human kidney epithelial cell line)는 한국세포주 은행(KCLB 21573)에서 분양 받았으며, 10% FBS(fetal bovine serum)를 함유한 DMEM(Dulbecco's Modified Eagle Medium) 배지를 사용하여 배양하였다. 세포의 상태와 생사여부는 위상차 현미경으로 관찰하였고, 합류(confluency) 80~90% 상태가 되면 세포를 계대배양하며 유지하였고, 5% CO2가 공급되는 37℃ 항온 세포 배양기에서 배양하였다.Human kidney epithelial cell line (Human kidney epithelial cell line) was obtained from Korea Cell Line Bank (KCLB 21573) and cultured using DMEM (Dulbecco's Modified Eagle Medium) medium containing 10% FBS (fetal bovine serum). The state and viability of the cells were observed under a phase contrast microscope. When the confluency reached 80-90%, the cells were passaged and maintained, and cultured in a 37 ° C. incubator with 5% CO 2 .
약용 곤충 추출물의 세포독성을 알아보기 위해 인간 293T 세포에서 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS, Promega, 미국) 어세이 방법을 통해 세포의 생존율을 측정하였다. 정상 293T 세포를 96 웰 플레이트에 2 x 10⁴세포/웰 씩 분주한 다음 약용 곤충 추출물을 농도별(0, 5, 10, 25, 50, 100, 500 및 1,000㎍/㎖)로 처리하고 37℃, 5% CO₂조건으로 24시간 동안 배양하였다. 배양 후 MTS 용액을 첨가하고 4시간 동안 다시 배양한 후 마이크로플레이트 리더기(microplate reader)로 490nm에서 흡광도를 측정하였다. 세포의 생존능력은 하기의 식에 따라 계산하였다.To investigate the cytotoxicity of medicinal insect extracts, 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium (MTS, Promega, USA) assay was used to determine the viability of the cells. Normal 293T cells were dispensed in 96 well plates at 2 × 10 cells / well, followed by treatment with medicinal insect extracts by concentration (0, 5, 10, 25, 50, 100, 500 and 1,000 μg / ml) and 37 ° C., Incubated for 24 hours under 5% CO₂. After incubation, the MTS solution was added and incubated again for 4 hours, and then the absorbance was measured at 490 nm using a microplate reader. The viability of the cells was calculated according to the following formula.
세포 생존율(%) = (시료 첨가군의 흡광도/대조군의 흡광도) X 100% Cell viability = (absorbance of sample added / absorbance of control)
2. 미백 효능 실험2. Whitening efficacy test
한국 세포주 은행에서 구입한 B16F10 마우스 흑색종(mouse melanoma) 세포를 10% FBS, 1% 페니실린-스트렙토마이신이 첨가된 DMEM에 배양하여 실험에 사용하였다. 각 세포는 10cm 세포 배양접시에 10㎖의 배지로 37℃, 5% CO2 배양기에서 세포가 70~80% 합류(confluency)가 되도록 배양하였다. 배지는 일주일에 두번씩 갈아주고, 합류에 도달한 세포는 계대 배양하여 유지하였다.B16F10 mouse melanoma cells purchased from the Bank of Korea Cell Line were cultured in DMEM with 10% FBS, 1% penicillin-streptomycin and used for the experiment. Each cell was incubated in a 10 cm cell culture dish so that the cells were 70-80% confluent in 37 ° C., 5% CO 2 incubator with 10 ml of medium. The medium was changed twice a week, and the cells reaching the confluence were passaged and maintained.
2-1. 버섯 티로시나아제(tyrosinase) 활성도 측정2-1. Determination of Mushroom Tyrosinase Activity
버섯 티로시나아제(Sigma, 미국)는 최종 1,000 U/㎖이 되도록 0.1M 인산염 완충액(phosphate buffer, pH 6.8)에 녹여 냉동고에 동결 보관한 후, 실험 수행 전에 녹여 효소원으로 사용하였다. L-DOPA(L-3,4-dihydroxyphenyl alanine)는 사용 직전에 제조하였는데, 차광병에서 0.1M 인산염 완충액에 녹여 사용하였다. 96 웰 플레이트에 대조군인 0.1M 인산염 완충액(pH 6.5)과 열풍 또는 동결건조한 굼벵이 분말의 각각의 추출물(100, 500 및 1,000㎍/㎖) 60㎕ 및 1mM의 L-DOPA 20㎕ 넣고 전배양(pre-incubation)시킨 후, 티로시나아제 20㎕ 섞은 다음 485nm에서 5분 간격으로 1시간 동안 흡광도를 측정하였다. 티로시나아제 활성 저해의 양성 대조군으로는 코지산(kojic acid)을 사용하였다. 각 실험군은 모두 3반복으로 수행하였으며, 측정값 중 흡광도가 최대 수치일 때 값을 선택하였다. 하기의 식을 활용하여 티로시나아제 효소 저해율(%)을 산출하였다.Mushroom tyrosinase (Sigma, USA) was dissolved in 0.1M phosphate buffer (pH 6.8) so that the final 1,000 U / ㎖, stored in the freezer and frozen before the experiment was used as an enzyme source. L-DOPA (L-3,4-dihydroxyphenyl alanine) was prepared immediately before use, and was dissolved in 0.1M phosphate buffer in a shading bottle. In a 96 well plate, add 60 μl of each control (100, 500 and 1,000 μg / ml) and 0.1 μl phosphate buffer (pH 6.5) and hot or lyophilized slug powder, and 20 μl of 1 mM L-DOPA to pre-culture (pre After incubation), 20 μl of tyrosinase was mixed and the absorbance was measured at 485 nm for 5 hours at 1 minute intervals. Kojic acid was used as a positive control of tyrosinase activity inhibition. Each experimental group was performed in three repetitions, and the value was selected when the absorbance was the maximum value among the measured values. Tyrosinase enzyme inhibition rate (%) was calculated using the following formula.
티로시나아제 효소 저해율(%) = (100 - (A - A') / (B - B')) X 100Tyrosinase enzyme inhibition rate (%) = (100-(A-A ') / (B-B'))
A : 시료 반응 후의 흡광도, B : 공시료 반응 후의 흡광도,A: absorbance after sample reaction, B: absorbance after blank sample reaction,
A' : 시료와 완충액의 흡광도, B' : 공시료와 완충액의 흡광도.A ': absorbance of the sample and the buffer, B': absorbance of the blank and the buffer.
2-2. 멜라닌 생성률 측정2-2. Melanin production rate measurement
B16F10 세포를 24 웰 플레이트에 각각 3 x 104 세포/웰로 세포를 분주하고 24 시간 배양하여 부착 및 안정화시킨 후, PBS로 2회 세척하고 5% FBS가 첨가된 phenol red free DMEM으로 배지를 바꿔준 다음 농도별로 희석한 굼벵이 추출물 시료와 10μM의 포스콜린(forskolin)을 세포에 처리하여 멜라닌이 합성되도록 37℃, 5% CO2 배양기에서 4일 동안 배양하였다. 4일 후 배양 상등액 200㎕를 96 웰 플레이트로 옮겨 생성된 멜라닌 양을 405nm에서 흡광도를 측정하여 분석하였다. 시료액 대신 용매를 사용한 블랭크(blank)의 흡광도를 기준으로 멜라닌 생성 억제율을 산출하였고, 대조군으로는 멜라닌 생성 저해 효과를 갖는 코지산을 사용하여 시료와 비교하였다.B16F10 cells were seeded at 3 x 10 4 cells / well in 24 well plates, incubated for 24 hours, attached and stabilized, washed twice with PBS and changed to medium with phenol red free DMEM with 5% FBS. Next, the slug extract samples diluted at the following concentrations and 10 μM of forskolin were treated to cells and incubated in 37 ° C., 5% CO 2 incubator for 4 days to synthesize melanin. Four days later, 200 μl of the culture supernatant was transferred to a 96 well plate, and the amount of melanin produced was analyzed by measuring absorbance at 405 nm. The melanin production inhibition rate was calculated based on the absorbance of the blank using a solvent instead of the sample solution, and compared with the sample using kojic acid having a melanin production inhibitory effect as a control.
멜라닌 생성 억제율(%) = (100 - (A - A') / (B - B')) X 100% Melanogenesis inhibition = (100-(A-A ') / (B-B'))
A : 시료 반응 후의 흡광도, B : 공시료 반응 후의 흡광도,A: absorbance after sample reaction, B: absorbance after blank sample reaction,
A' : 시료와 완충액의 흡광도, B' : 공시료와 완충액의 흡광도.A ': absorbance of the sample and the buffer, B': absorbance of the blank and the buffer.
3. 보습 효능 실험3. Moisturizing effect experiment
사람의 각질형성세포주(human keratinocyte cell)인 HaCaT 세포는 10% FBS와 1% 페니실린-스트렙토마이신이 함유된 DMEM 배지를 사용하여 37℃, 5% CO2 조건에서 배양하였다.HaCaT cells, which are human keratinocyte cells, were cultured at 37 ° C. and 5% CO 2 using DMEM medium containing 10% FBS and 1% penicillin-streptomycin.
피부장벽은 각질형성세포의 분화 말기과정에서 분비되는 분화 촉진인자들에 의해 각질세포막을 형성함으로써 물리적 장벽으로서의 정상적인 기능을 유지하게 된다. 열풍건조한 굼벵이 분말의 추출물 시료가 피부장벽기능에 미치는 영향을 살펴보기 위해, 각질형성세포의 분화 촉진 시에 분비되는 필라그린(filaggrin), 인보루크린(involucrin)과 표피의 수분 증발을 막아 피부의 탄력성을 유지하고, 영양성분의 저장과 확산, 세포 이동 등에 관여하는 보습인자인 히알루론산의 합성 효소인 HAS2(hyaluronic acid synthase)와 HAS3의 발현을 Real-time PCR을 이용하여 측정하였다. HaCaT 세포에 시료를 농도별로 처리하여 24시간 동안 배양한 후 mRNA의 발현을 확인하였다. 레티노산(retinoic acid)은 각질형성세포 분화의 촉진에 관여하는 생체 인자로서 피부장벽기능의 강화에 관련하는 인자들의 발현 관찰에서 양성 대조군으로 사용하였다. The skin barrier maintains its normal function as a physical barrier by forming keratinocyte membranes by differentiation facilitating factors secreted during the end stage of differentiation of keratinocytes. To examine the effects of hot air-dried slug powder extract on skin barrier function, it prevents the evaporation of filaggrin, involucrin and epidermis, which are secreted at the time of differentiation of keratinocytes. The expression of hyaluronic acid synthase (HAS2) and HAS3, a moisturizing factor that is involved in nutrient storage, diffusion, and cell migration, was measured by real-time PCR. Samples were treated in concentrations in HaCaT cells and cultured for 24 hours, and then mRNA expression was confirmed. Retinoic acid is a biological factor involved in the promotion of keratinocyte differentiation and was used as a positive control in the expression of factors related to the enhancement of skin barrier function.
실시예 1. 굼벵이 분말 추출물의 세포독성 분석Example 1. Cytotoxicity Analysis of Slugs Powder Extract
293T 세포에 굼벵이(흰점박이꽃무지유충) 분말 추출물을 농도별(0, 1, 10, 25, 50, 100, 500 및 1,000㎍/㎖)로 처리한 후 세포의 생존율을 조사한 결과, 건조 방법 및 추출물 종류에 관계없이 모든 농도 구간에서 도 1 및 2와 같이 최고 처리 농도인 1,000㎍/㎖까지 세포 독성이 나타나지 않아 약용 곤충 추출물 시료가 안전성이 우수함을 확인하였다. 특히, 굼벵이 동결건조 분말의 추출물은 최고 처리 농도에서 세포의 생존율이 무처리구 대비 현저히 증가되어, 세포 증식 효과가 있음을 알 수 있었다.After treatment of 293T cells with the extracts of slug powder (white spotted radish larvae) at different concentrations (0, 1, 10, 25, 50, 100, 500 and 1,000 µg / ml), the survival rate of the cells was examined. Regardless of the type of extract, the medicinal insect extract sample was confirmed to have excellent safety because it did not show cytotoxicity up to 1,000 µg / ml, the highest treatment concentration, as shown in FIGS. In particular, the extract of the slug freeze-dried powder was found to have a cell proliferation effect, the survival rate of the cells at the highest treatment concentration significantly increased compared to the untreated group.
실시예 2. 굼벵이 분말 추출물의 티로시나아제 활성 저해능 분석Example 2 Analysis of Inhibitory Activity of Tyrosinase Activity of Slugs Powder Extract
티로시나아제는 멜라닌 생성에 중요한 역할을 하는 효소로, 본 발명에서는 생성되는 멜라닌의 양을 흡광도로 측정함으로써 굼벵이 추출물의 티로시나아제 효소 활성 억제 효과를 검토하였다. 코지산을 티로시나아제 저해 활성의 양성 대조군으로 사용하였다(도 3). 열풍건조 또는 동결건조 굼벵이(흰점박이꽃무지유충) 분말의 열수 및 에탄올 추출물을 100, 500 및 1,000㎍/㎖ 농도로 처리한 결과, 굼벵이 분말의 열수 추출물에서는 티로시나아제 활성 저해능이 거의 확인되지 않았고, 열풍건조 및 동결건조 굼벵이 분말의 에탄올 추출물 처리 조건에서 시료 농도 의존적으로 티로시나아제 활성이 저해되는 것이 확인되었다(도 4 및 도 5). 상기 결과를 통해, 굼벵이 분말의 에탄올 추출물이 열수 추출물에 비해 티로시나아제 활성 저해능이 우수한 것을 알 수 있었다.Tyrosinase is an enzyme that plays an important role in melanin production. In the present invention, the effect of inhibiting tyrosinase enzyme activity of the slug extract was examined by measuring the amount of melanin produced by absorbance. Kojic acid was used as a positive control of tyrosinase inhibitory activity (FIG. 3). The hot water and ethanol extracts of hot-air dried or lyophilized slug powder (white spotted larvae) powder were treated at concentrations of 100, 500 and 1,000 ㎍ / ml, and the hydrothermal extract of slug powders showed little inhibition of tyrosinase activity. It was confirmed that tyrosinase activity was inhibited depending on the sample concentration under the ethanol extract treatment conditions of hot air drying and lyophilized slug powder (FIGS. 4 and 5). Through the above results, it was found that the ethanol extract of slug powder was superior to the tyrosinase activity inhibitory effect compared to the hydrothermal extract.
실시예 3. 굼벵이 분말 추출물의 멜라닌 합성 저해능 분석Example 3 Analysis of Melanin Synthesis of Slug Powder Extract
멜라닌은 자외선이 조사되면 멜라닌세포(melanocyte)의 멜라닌생성반응(melanogenesis)을 통해 생성되고, 피부색을 검게 변화시켜 자외선으로부터 피부를 보호하는 역할을 한다. 티로시나아제 활성 저해 효과가 있는 굼벵이(흰점박이꽃무지유충) 분말 추출물의 미백 개선능을 검증하기 위해 B16F10 마우스 흑색종 세포를 이용하여 멜라닌 생합성 억제능을 측정하였다. 코지산을 양성 대조군으로 사용하였고(도 6), 열풍건조 또는 동결건조 굼벵이 분말의 열수 및 에탄올 추출물을 최대 500㎍/㎖까지 처리한 후 생성된 멜라닌 양을 측정한 결과, 동결건조 굼벵이 분말 추출물에서는 멜라닌 합성이 저해되지 않았고(결과 미제시), 열풍건조 굼벵이 분말 추출물에서만 멜라닌 합성이 저해되었다. 열풍건조 굼벵이 분말의 열수 추출물은 500㎍/㎖에서 30.1%, 에탄올 추출물은 100㎍/㎖ 농도에서 60% 이상, 500㎍/㎖ 농도에서 90% 이상 멜라닌 합성이 저해된 것이 확인되었다(도 7). 이를 통해, 상기 티로시나아제 활성 저해에서와 유사하게, 열수 추출물보다 에탄올 추출물의 멜라닌 합성 저해능이 우수함을 알 수 있었다.Melanin is generated through melanogenesis of melanocytes when the ultraviolet rays are irradiated, and serves to protect the skin from ultraviolet rays by changing the skin color to black. In order to verify the whitening ability of the slug (white spotted radish larva) powder extract having an inhibitory effect on tyrosinase activity, melanin biosynthesis inhibition activity was measured using B16F10 mouse melanoma cells. Koji acid was used as a positive control (FIG. 6), and hot water and lyophilized slug powder were treated with hot water and ethanol extract up to 500 µg / ml, and the amount of melanin produced was measured. Melanin synthesis was not inhibited (results not shown), and melanin synthesis was inhibited only in hot air dried slug powder extracts. Hot-water-dried slug powder was found to inhibit melanin synthesis in hot water extracts of 500 μg / ml at 30.1%, ethanol extracts at 100 μg / ml and at least 60% at 500 μg / ml (90 μg) (FIG. 7). . Through this, similar to the inhibition of tyrosinase activity, it was found that the melanin synthesis inhibitory ability of the ethanol extract than the hot water extract.
실시예 4. 굼벵이 분말 추출물의 보습능 분석Example 4 Moisturizing Analysis of Slugs Powder Extract
상기 실시예 2 및 3을 통해, 티로시나아제 활성과 멜라닌 합성을 모두 저해하는 열풍건조 굼벵이(흰점박이꽃무지유충) 분말 추출물을 선택하고, 상기 추출물의 보습능을 평가하기 위해, 각질형성세포주(HaCaT)에서 보습관련 인자인 인보루크린(IVL), 필라그린(FLG) 및 히알루론산 합성 효소(HAS2, HAS3)의 mRNA 발현 수준을 분석하였다.Through Examples 2 and 3, to select the hot air dry slug (white spotted radish larva) powder extract that inhibits both tyrosinase activity and melanin synthesis, to evaluate the moisturizing ability of the extract, keratinocyte cell line ( MRNA levels of moisturizing-related factors, inboleuklin (IVL), filaggrin (FLG) and hyaluronic acid synthase (HAS2, HAS3) were analyzed in HaCaT).
먼저, 대표적인 피부 보습용 화장료 원료인 레티노산을 각질형성세포주에 처리하고 보습관련 인자의 발현 수준을 분석한 결과, 무처리 대조구에 비해 레티노산을 처리한 세포에서 인보루크린, HAS2 및 HAS3의 발현이 현저히 증가된 것이 확인되었다(도 8).First, retinoic acid, a representative skin moisturizing cosmetic raw material, was treated to keratinocytes and analyzed for expression levels of moisturizing-related factors. As a result, the expression of inborukrrin, HAS2, and HAS3 in retinoic acid-treated cells compared to the untreated control. This was found to be significantly increased (FIG. 8).
반면, 열풍건조 굼벵이 분말의 열수 추출물을 처리한 군에서는 50㎍/㎖ 이상의 농도에서 필라그린과 HAS3의 발현이 현저히 증가된 것을 확인할 수 있었다(도 9). 그리고 열풍건조 굼벵이 분말의 에탄올 추출물을 처리한 군에서는 HAS3에서만 시료 처리 농도 의존적으로 mRNA의 발현이 증가하는 양상을 보여주었다(도 10). 이를 통해, 열풍건조 굼벵이 분말의 추출물이 보습인자의 발현 증진을 유도하여 피부 보습 개선에 효과가 있을 것으로 유추되었다.On the other hand, in the group treated with hot water dried hot slug powder, the expression of filaggrin and HAS3 was significantly increased at a concentration of 50 μg / ml or more (FIG. 9). And in the group treated with ethanol extract of the hot air dry slug powder showed that the expression of mRNA was increased only in HAS3 sample concentration concentration (Fig. 10). Through this, it was inferred that the extract of hot air dry slug powder induces the enhancement of the expression of the moisturizing factor and is effective in improving skin moisturizing.
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