KR101971254B1 - Composition for skin whitening comprising Pavetta indica extract as effective component - Google Patents
Composition for skin whitening comprising Pavetta indica extract as effective component Download PDFInfo
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- KR101971254B1 KR101971254B1 KR1020170096708A KR20170096708A KR101971254B1 KR 101971254 B1 KR101971254 B1 KR 101971254B1 KR 1020170096708 A KR1020170096708 A KR 1020170096708A KR 20170096708 A KR20170096708 A KR 20170096708A KR 101971254 B1 KR101971254 B1 KR 101971254B1
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- South Korea
- Prior art keywords
- indica
- composition
- extract
- active ingredient
- present
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Abstract
본 발명은 파베타 인디카 추출물을 유효성분으로 함유하는 미백용 조성물에 관한 것으로, 보다 상세하게는 본 발명의 파베타 인디카 추출물은 멜라노마 세포(B16-F10 mouse melanoma cell)에서 멜라닌의 생성 및 티로시나아제의 활성을 저해하는 효과가 현저하게 우수하므로, 파베타 인디카 추출물을 유효성분으로 함유하는 본 발명의 조성물은 피부에 자극을 일으키지 않는 피부 미백을 위한 기능성 소재로 유용하게 사용될 수 있다. The present invention relates to a whitening composition comprising Pavela Indica extract as an active ingredient. More particularly, the Pavela indica extract of the present invention is useful for the production of melanin and tyrosine in a melanoma cell (B16-F10 mouse melanoma cell) Since the effect of inhibiting the activity of the azela is remarkably excellent, the composition of the present invention containing the pravastatin indica extract as an active ingredient can be usefully used as a functional material for skin whitening which does not cause skin irritation.
Description
본 발명은 파베타 인디카 추출물을 유효성분으로 함유하는 피부 미백용 조성물에 관한 것이다.The present invention relates to a composition for whitening skin, which contains Pavela Indica extract as an active ingredient.
멜라닌은 머리카락 및 피부의 색상을 결정하는 주요 인자이며, 자외선으로부터 피부세포를 보호하는 역할을 한다. 그러나 과도한 멜라닌의 축적은 주근깨 및 기미와 같은 과다 색소 침착 현상을 유발한다. 표피층의 기저층(basal layer)에 존재하는 멜라닌 형성 세포는 멜라노솜이라고 불리는 특정한 구조를 가지고 있으며, 이곳에서 멜라닌형성 효소(melanogenic enzyme)의 작용으로 멜라닌이 만들어진다. 멜라닌은 검은색 및 갈색계열의 유멜라닌(eumelanin)과 노란색 및 붉은계열의 페오멜라닌(pheomelanin)이 있으며, 멜라닌의 합성은 티로시나아제(tyrosinase)에 의해 티로신(tyrosine)이 도파(DOPA) 및 도파 퀴논(DOPA quinone)으로 전환되면서 시작된다. 또한, TRP-2(tyrosinase-related protein 2)는 도파 크롬(DOPA chrome)을 5,6-디히드록시인돌-2-카복실산(5,6-dihydroxyindole-2-carboxylic acid)으로, TRP-1(tyrosinase-related protein 1)은 인돌-5,6-퀴논-2-카복실산(indole-5,6-quinone-2-carboxylic acid)으로 산화반응을 촉매하며, 유멜라닌(eumelanin) 합성에 티로시나아제(tyrosinase)와 TRP-1 및 TRP-2가 관여하고 있다. 또한, 멜라닌 형성의 조절인자로 잘 알려진 MITF(microphthalmia-associated transcription factor)는 나선-고리-나선-루신 지퍼(helix-loop-helix-leucine zipper) 구조를 가진 전사인자(transcription factor)로서 멜라닌 형성을 통해 흑화과정과 세포의 증식 및 생존을 조절하는 것으로 잘 알려져 있다. 멜라닌 생합성에 관여하는 인자로는 α-MSH(α-melanocyte stimulating hormone), 부신피질자극호르몬(adrenocorticotropic hormone), PGE2(prostaglandine E2) 등이 있으며, cAMP를 통해 멜라닌 합성을 조절하는 것으로 보고되어 있다.Melanin is a key determinant of hair and skin color, and it protects skin cells from ultraviolet light. However, excessive accumulation of melanin causes hyperpigmentation such as freckles and spots. The melanin-forming cells in the basal layer of the epidermis have a specific structure called melanosomes, in which melanin is produced by the action of a melanogenic enzyme. Melanin has black and brown series of eumelanin and yellow and red series of pheomelanin. The synthesis of melanin is caused by tyrosinase, tyrosine is doped (DOPA) and dopa It begins with conversion to quinone (DOPA quinone). In addition, TRP-2 (tyrosinase-related protein 2) can be produced by converting DOPA chrome into 5,6-dihydroxyindole-2-carboxylic acid, TRP-1 tyrosinase-
현재 합성 항산화제로 사용되는 코직산, 비타민 유도체, 알부틴, BHT(dibutylated hydroxytoluene) 등은 가격도 저렴하고 항산화 활성으로 인하여 우수한 미백 효과를 나타내지만, 과다 사용하게 되면 질병을 유발하는 등 안전성에 문제점이 있는 것으로 보고되고 있어 사용량을 규제하고 있다. 현재 국내외적으로 천연물로부터 기능성 성분의 소재를 발굴하기 위한 연구가 활발히 진행되고 있다.Currently, kojic acid, vitamin derivatives, arbutin and dibutylated hydroxytoluene (BHT), which are used as synthetic antioxidants, are inexpensive and exhibit excellent whitening effect due to their antioxidative activity. However, It is reported and regulates usage. Currently, studies are being actively carried out to find functional materials from natural products both domestically and externally.
한편, 파베타 인디카(Pavetta indica L.)는 꼭두서니과(Rubiaceae)에 속하는 관목성 식물로 인도차이나반도(베트남, 라오스, 캄보디아), 인도네시아, 방글라데시 및 인도 등에 분포한다. 파베타 인디카는 전통적으로 이뇨제, 염증, 류마티스, 황달, 궤양 및 치질에 사용되었으며, 이화학적 활성에 대한 연구는 항균, 항바이러스, 항말라리아 및 진통 활성에 대한 보고된 바가 있다.Meanwhile, the Pavetta indica L.) is a shrubby plant belonging to the Rubiaceae, distributed in the Indochina Peninsula (Vietnam, Laos, Cambodia), Indonesia, Bangladesh and India. Pavera Indica has traditionally been used for diuretics, inflammation, rheumatism, jaundice, ulcers, and hemorrhoids. Studies on physicochemical activities have been reported on antibacterial, antiviral, anti-malarial and analgesic activities.
한편, 한국등록특허 제0724168호는 멜라닌 생성 억제 활성을 갖는 식물 추출물을 함유하는 피부미백용 화장료를 개시하고 있으나, 본 발명의 파베타 인디카 추출물을 유효성분으로 함유하는 미백용 조성물에 대해 아직까지 개시된 바가 없다.Meanwhile, Korean Patent No. 0724168 discloses a skin whitening cosmetic composition containing a plant extract having an activity of inhibiting melanin production. However, the whitening composition containing the perveter indica extract of the present invention as an active ingredient has not yet been disclosed There is no bar.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자들은 파베타 인디카(Pavetta indica) 추출물을 멜라노마 세포(B16F10 mouse melanoma cell)에 처리한 결과, 세포독성이 없으며, 멜라닌의 함량을 현저하게 감소시킬 뿐만 아니라 티로시나아제의 활성을 효과적으로 저해하는 것을 확인함으로써, 본 발명을 완성하였다.DISCLOSURE OF THE INVENTION The present invention has been made in view of the above-mentioned needs. The present inventors have found that treatment of Pavetta indica extract with melanoma cells (B16F10 mouse melanoma cell) results in no cytotoxicity and a remarkable content of melanin But also effectively inhibited the activity of tyrosinase, thereby completing the present invention.
상기 과제를 해결하기 위하여, 본 발명은 파베타 인디카(Pavetta indica) 추출물을 유효성분으로 함유하는 피부 미백용 화장료 조성물을 제공한다.In order to achieve the foregoing object, the present invention is a beta wave indica (Pavetta indica ) as an active ingredient.
또한, 본 발명은 파베타 인디카(Pavetta indica) 추출물을 유효성분으로 함유하는 멜라닌 색소 과다 침착 질환의 예방 또는 치료용 약학 조성물을 제공한다.In addition, the present invention relates to a method for producing a protein, indica ) as an active ingredient. The present invention also provides a pharmaceutical composition for preventing or treating melanin pigment hyperpigmentation disease.
또한, 본 발명은 파베타 인디카(Pavetta indica) 추출물을 유효성분으로 함유하는 멜라닌 색소 과다 침착 질환의 예방 또는 개선용 건강기능식품 조성물을 제공한다.In addition, the present invention relates to a method for producing a protein, indica ) as an active ingredient. The present invention also provides a health functional food composition for prevention or amelioration of a melanin pigment hyperpigmentation disease.
본 발명의 파베타 인디카 추출물은 멜라노마 세포(B16-F10 mouse melanoma cell)에서 멜라닌의 생성 및 티로시나아제의 활성을 저해하는 효과가 현저하게 우수하므로, 파베타 인디카 추출물을 유효성분으로 함유하는 본 발명의 조성물은 피부에 자극을 일으키지 않는 피부 미백을 위한 기능성 소재로 유용하게 사용될 수 있다. Since the Pavela Indica extract of the present invention is remarkably excellent in the effect of inhibiting melanin production and tyrosinase activity in melanoma cells (B16-F10 mouse melanoma cells), the pavela indica extract The composition of the present invention can be usefully used as a functional material for skin whitening which does not cause skin irritation.
도 1은 본 발명의 일 실시예에 따른 농도별로 제조한 파베타 인디카 추출물(1.25, 2.5, 5, 10 및 20㎍/㎖)을 B16F10 마우스 멜라노 세포(A) 및 인간 피부섬유아세포(human dermal fibroblast cell)(B)에 처리하였을 때, 세포생존율(cell viability)을 확인한 결과이다.
도 2는 본 발명의 일 실시예에 따른 α-MSH(α-melanocyte stimulating hormone) 및 농도별로 제조한 파베타 인디카 추출물(2.5, 5 및 10㎍/㎖)을 B16F10 마우스 멜라노 세포에 처리하였을 때, B16F10 마우스 멜라노 세포의 펠렛(pellet) 형상(A) 및 멜라닌의 함량을 측정한 결과이다. α-MSH은 멜라닌 생성 유도물질이며, 레스베라트롤(Resveratrol)은 양성 대조군이다.
도 3은 본 발명의 일 실시예에 따른 α-MSH(α-melanocyte stimulating hormone) 및 농도별로 제조한 파베타 인디카 추출물(2.5, 5 및 10㎍/㎖)을 B16F10 마우스 멜라노 세포에 처리하였을 때, in situ 세포 내 티로시나아제의 활성을 나타낸 현미경 사진(A) 및 그래프(B)이다.
도 4는 본 발명의 일 실시예에 따른 α-MSH(α-melanocyte stimulating hormone) 및 농도별로 제조한 파베타 인디카 추출물(2.5, 5 및 10㎍/㎖)을 B16F10 마우스 멜라노 세포에 처리하였을 때, 티로시나아제, TRP1(tyrosinase related protenin 1) 및 MITF(Microphthalmia-associated transcription factor)의 단백질 발현 양상을 나타낸 결과이다. 베타-액틴(β-actin)은 로딩 컨트롤이다.FIG. 1 is a graph showing the effect of the extract of Pavela indica (1.25, 2.5, 5, 10 and 20 / / ml) prepared according to the concentration of the present invention on B16F10 mouse melanocyte (A) and human dermal fibroblast cell (B), the cell viability was confirmed.
FIG. 2 shows the results of treatment of B16F10 mouse melanocyte cells with α-MSH (α-melanocyte stimulating hormone) and Pavela Indica extract (2.5, 5 and 10 μg / B16F10 mouse Melanocyte pellet shape (A) and melanin content. α-MSH is a melanogenesis inducer, and Resveratrol is a positive control.
FIG. 3 is a graph showing the results of treatment of B16F10 mouse melanocytes with P-beta-Indica extracts (2.5, 5 and 10 μg / ml) prepared according to α-MSH (α-melanocyte stimulating hormone) (A) and graph (B) showing the activity of in situ intracellular tyrosinase.
FIG. 4 is a graph showing the results of treatment of α-MSH (α-melanocyte stimulating hormone) and Pavela Indica extract (2.5, 5 and 10 μg / ml) prepared according to the concentration of B16F10 mouse melanocytes, Tyrosinase, TRP1 (tyrosinase related protenin 1) and MITF (Microphthalmia-associated transcription factor). Beta-actin is a loading control.
본 발명의 목적을 달성하기 위하여, 본 발명은 파베타 인디카 추출물을 유효성분으로 함유하는 피부 미백용 화장료 조성물을 제공한다.In order to accomplish the object of the present invention, the present invention provides a skin whitening cosmetic composition comprising Pavela Indica extract as an active ingredient.
본 발명의 일 구현 예에 따른 화장료 조성물에서, 상기 파베타 인디카 추출물은 물, 탄소수 1 내지 4의 저급 알코올 및 이들의 혼합용매로 구성되는 군으로부터 선택되는 용매, 바람직하게는 메탄올을 용매로 이용하여 추출한 것이다.In the cosmetic composition according to one embodiment of the present invention, the Pava betaine extract may be prepared by using a solvent selected from the group consisting of water, a lower alcohol having 1 to 4 carbon atoms and a mixed solvent thereof, preferably methanol as a solvent It is extracted.
예를 들면, 상기 파베타 인디카 추출물은For example, the < RTI ID = 0.0 >
1) 파베타 인디카의 잎 및 가지의 혼합물 중량 대비 12.5배의 부피부로 메탄올을 첨가하고, 상온에서 5~30분 동안 초음파(sonication)를 처리하여 2~4일간 20~40회 반복하여 추출하는 단계; 및 1) The mixture of leaves and branches of Pavera Indica is added 12.5 times as much as the weight of methanol, and the mixture is sonicated at room temperature for 5 to 30 minutes and then extracted 20 to 40 times for 2 to 4 days. step; And
2) 상기 단계 1)에서 추출하여 획득한 파베타 인디카 추출물을 여과 및 감압농축한 후 동결건조하는 단계;를 포함하여 제조한 것일 수 있으며, 2) filtration and concentration under reduced pressure of the P. indica extract obtained in step 1), followed by lyophilization,
바람직하게는, 1) 파베타 인디카의 잎 및 가지를 1:1의 중량비로 혼합한 혼합물 중량 대비 58배의 부피부로 99.9% 메탄올 1ℓ를 첨가하고, 상온에서 15분 동안 초음파(sonication)를 처리하여 3일간 30회 반복하여 추출하는 단계; 및Preferably 1) 1 liter of 99.9% methanol is added to the mixture of the mixture of the leaves and branches of the pavetainindica at a weight ratio of 1: 1 by 58 times the weight of the mixture, and sonication is performed at room temperature for 15 minutes And repeating extraction for 30 days for 3 days; And
2) 상기 단계 1)에서 추출하여 획득한 파베타 인디카 추출물을 여과 및 감압농축한 후 동결건조하는 단계;를 포함하여 제조한 것일 수 있으나, 이에 제한되지 않는다.2) The step of extracting and extracting the Porphyria indica extract obtained in step 1), followed by filtration and concentration under reduced pressure, followed by lyophilization, but the present invention is not limited thereto.
또한, 상기 파베타 인디카 추출물은 파베타 인디카의 잎 및 가지의 혼합물을 추출한 것일 수 있으나, 이에 제한되지 않는다.In addition, the P. indica extract may be a mixture of leaves and branches of P. indica, but is not limited thereto.
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 의도하고 있다. 일반적으로, 거뭇한 피부나 기미, 주근깨, 다크서클은 자외선에 의한 자극이나 호르몬 밸런스의 변화 등에 의해 멜라노사이트(melanocyte)가 자극되고, 그래서 생합성된 멜라닌 색소가 피부에 침착하여 발생한다고 알려져 있다. 따라서, 멜라닌의 생성을 억제할 수 있다면, 거뭇한 피부나 기미, 주근깨, 다크서클을 예방ㆍ개선하는 것이 가능하다. On the other hand, the whitening effect refers to an effect to improve or prevent glaring skin, spots, freckles, and dark circles caused by various factors such as exposure to ultraviolet rays, change in hormone balance, genetic program, Or an effect of maintaining a beautiful skin with a sense of transparency and an effect of reducing the dullness of the skin to increase gloss and tautness. In general, it is known that the skin, spots, freckles, and dark circles of the skin, stamens, freckles and dark circles are stimulated by melanocytes due to stimulation by ultraviolet rays or changes in hormone balance, and melanin pigment is deposited on the skin. Therefore, if the production of melanin can be inhibited, it is possible to prevent or improve gross skin, spots, freckles and dark circles.
또한, 상기 피부 미백용 화장료 조성물은 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성제-함유 클린징, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이로 이루어지는 군으로부터 선택된 어느 하나의 제형으로 제조되는 것일 수 있으나, 이에 제한되지 않는다. 보다 구체적으로는, 유연 화장수, 영양 화장수, 영양 크림, 마사지 크림, 에센스, 아이 크림, 클렌징 크림, 클렌징 폼, 클렌징 워터, 팩, 스프레이, 파우더, 팩트, 립글로즈, 립스틱, 섀도우 등의 제형으로 제조될 수 있다. 이들 각 제형으로 이루어진 화장료 조성물은 그 제형의 제제화에 필요하고 적절한 각종의 기제와 첨가물을 함유할 수 있으며, 이들 성분의 종류와 양은 당업자에 의해 용이하게 선정될 수 있다.The cosmetic composition for skin whitening may be in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing oil, powder foundation, emulsion foundation, wax foundation and spray But the present invention is not limited thereto. More specifically, it is made up of formulations such as soft lotion, nutritional lotion, nutritional cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray, powder, fact, lip gloss, lipstick and shadow . The cosmetic composition comprising each of these formulations may contain various bases and additives necessary for formulation of the formulation, and the kinds and amounts of these ingredients can be easily selected by those skilled in the art.
본 발명의 화장료 조성물은 유효성분 이외에 추가로 동일 또는 유사한 기능을 나타내는 피부 미백 활성 성분을 1종 이상 함유할 수 있다. 피부 미백 활성 성분으로는 코지산 및 이의 유도체, 알부틴, 아스코르브산 및 이의 유도체, 하이드로퀴논 및 이의 유도체, 레조르시놀, 사이클로알카논, 메틸렌디옥시페닐 알칸올, 2,7-디니트로인다졸 또는 덩굴귤 추출물, 쌀 추출물, 감초 추출물과 같은 식물 추출물 등이 있으나, 이에 제한되는 것은 아니다.The cosmetic composition of the present invention may contain, in addition to the active ingredient, at least one skin whitening active ingredient exhibiting the same or similar functions. Skin whitening active ingredients include kojic acid and its derivatives, arbutin, ascorbic acid and its derivatives, hydroquinone and its derivatives, resorcinol, cycloalkanone, methylenedioxyphenylalkanol, 2,7-dinitroindazole or Plant extracts such as mandarin orange extract, rice extract, licorice extract, and the like, but are not limited thereto.
본 발명의 화장료 조성물의 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물섬유, 식물섬유, 왁스, 파라핀, 전분, 트라가칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 이용될 수 있다.When the formulation of the cosmetic composition of the present invention is a paste, a cream or a gel, an animal fiber, a plant fiber, a wax, a paraffin, a starch, a tragacanth, a cellulose derivative, a polyethylene glycol, a silicone, a bentonite, Etc. may 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 클로로플루오로히드로카본, 프로판-부탄 또는 디메틸 에테르와 같은 추진체를 포함할 수 있다.When the formulation of the cosmetic composition of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. Especially, in the case of a spray, Propellants such as carbon, propane-butane or dimethyl ether.
본 발명의 화장료 조성물의 제형이 용액 또는 유탁액의 경우에는 담체 성분으로서 용매, 용매화제 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 an emulsion, a solvent, a solvent or an emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, Glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid esters of sorbitan.
본 발명의 화장료 조성물의 제형이 현탁액인 경우에는 담체 성분으로서 물, 에탄올 또는 프로필렌 글리콜과 같은 액상 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 carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspension such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.
본 발명의 화장료 조성물의 제형이 계면-활성제 함유 클렌징인 경우에는 담체 성분으로서 지방족 알코올 설페이트, 지방족 알코올 에테르설페이트, 설포숙신산 모노에스테르, 아세티오네이트, 이미다졸리늄 유도체, 메틸타우레이트, 사르코시네이트, 지방산 아미드 에테르 설페이트, 알킬아미도베타인, 지방족 알코올, 지방산 글리세리드, 지방산 디에탄올아미드, 식물성 유, 리놀린 유도체 또는 에톡실화 글리세롤 지방산 에스테르 등이 이용될 수 있다.When the formulation of the cosmetic composition of the present invention is an interfacial-active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, acethionate, imidazolinium derivative, methyltaurate, sarcosinate , Fatty acid amide ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linolenic derivative or ethoxylated glycerol fatty acid ester.
본 발명의 화장료 조성물은 형광물질, 살진균제, 굴수성 유발물질, 보습제, 방향제, 방향제 담체, 단백질, 용해화제, 당 유도체, 일광차단제, 비타민, 식물 추출물 등을 포함하는 부형제를 추가로 함유할 수 있다.The cosmetic composition of the present invention may further contain an excipient including a fluorescent substance, a fungicide, a hygroscopic substance, a moisturizer, a fragrance, a fragrance carrier, a protein, a solubilizer, a sugar derivative, a sunscreen, a vitamin, .
또한, 본 발명은 파베타 인디카 추출물을 유효성분으로 함유하는 멜라닌 색소 과다 침착 질환의 예방 또는 치료용 약학 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating a melanin pigment hyperproliferative disorder containing Pavela indica extract 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 색소 과다 침착, 임신성 갈색반(gravidic chloasma), 또는 찰상 및 화상을 비롯한 상처 또는 피부염으로 인한 염증 후 과다 색소 침착 등을 포함하나 이에 한정되지 않는다.The term " melanin pigment hyperpigmentation " as used herein means darker or darker than other areas due to excessive increase of melanin in specific areas of skin or hands and toenails. The melanin pigment hyperpigmentation disorder may be selected from the group consisting of freckles, senile spots, liver, spots, brown or black spots, daylight pigmentation, cyanic melasma, pigment overdose after drug use, gravidic chloasma, Post-inflammatory hyperpigmentation due to burns 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. 본 발명의 약학 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.The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier in addition to the active ingredient. Such a carrier is usually used at the time of formulation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, Calcium carbonate, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and minerals Oils, and the like, but are not limited thereto. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc., in addition to the above components. 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. The appropriate dosage of the pharmaceutical composition according to the present invention may vary depending on such factors as the formulation method, the administration method, the patient's age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, .
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 may be administered orally or parenterally. In the case of parenteral administration, the composition may be administered topically to the skin, intravenously, subcutaneously, intramuscularly, intraperitoneally, or transdermally. In view of the fact that the pharmaceutical composition of the present invention is applied for the treatment or prevention of a melanin pigment hyperpigmentation disease, it is preferable that the composition is topically applied 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 contained 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 required, and the like, 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 composition of the present invention may be manufactured in a unit dosage form by formulating it using a pharmaceutically acceptable carrier or excipient according to a method which can be easily carried out by those having ordinary skill in the art to which the present invention belongs Into a capacity container. The formulations may be formulated into any one of the formulations selected from injectables, creams, patches, sprays, ointments, alerts, lotions, liniments, pastes and cataplasms, and may additionally contain dispersing or stabilizing agents have.
또한, 본 발명은 파베타 인디카 추출물을 유효성분으로 함유하는 멜라닌 색소 과다 침착 질환의 예방 또는 개선용 건강기능식품 조성물을 제공한다.In addition, the present invention provides a health functional food composition for preventing or ameliorating melanin pigment hyperproliferative disorder containing pravastatin indica extract as an active ingredient.
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 목적(예방 또는 개선)에 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 하는 장기간의 섭취한 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 may be used together with other food or food ingredients, and suitably used according to a conventional method. The amount of the active ingredient can be suitably used depending on its use purpose (prevention or improvement). Generally, the health functional food composition of the present invention is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight based on the raw material, when the food or beverage is produced. However, in case of long-term ingestion for health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding 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 kind of the health functional food. Examples of the foods to which the health functional food composition can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, soups, Drinks, alcoholic beverages, and vitamin complexes, all of which include health foods in a 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 health functional food composition of the present invention can be produced as a food, particularly a functional food. The functional food of the present invention includes components that are ordinarily added in food production, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, in the case of a drink, a natural carbohydrate or a flavoring agent may be included as an additional ingredient in addition to the active ingredient. The natural carbohydrate may be selected from the group consisting of monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose etc.), oligosaccharides, polysaccharides (e.g., dextrin, cyclodextrin, , Xylitol, sorbitol, erythritol, etc.). The flavoring agent may be a natural flavoring agent (e.g., tau Martin, stevia extract, etc.) and a synthetic flavoring agent (e.g., 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. In addition to the above health functional food composition, it is also possible to use various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, A carbonating agent used in beverages, and the like.
이하, 실시예를 이용하여 본 발명을 더욱 상세하게 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로 본 발명의 범위가 이들에 의해 제한되지 않는다는 것은 당해 기술분야에서 통상의 지식을 가진 자에게 있어 자명한 것이다. Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited thereto.
제조예Manufacturing example 1. One. 파베타Parbetta 인디카 추출물의 제조 Manufacture of Indica Extract
파베타 인디카(Pavetta indica)는 생명공학연구원 해외생물소재센터로부터 제공받아 본 발명에 사용하였다. 베트남에서 확보한 파베타 인디카의 잎 및 가지를 1:1의 중량비로 혼합한 혼합물 17.2g에 99.9%(v/v) 메탄올 1ℓ를 첨가하여 상온에서 15분 동안 초음파(sonication)를 처리하여 3일간 30회 반복하여 추출한 후, 무형광 솜을 사용하여 여과한 다음 45℃에서 감압농축한 후, -70℃에서 건조시켜 제조하였다. Pavetta indica ) was obtained from the Center for Biological and Material Research, Biotechnology Research Institute and used in the present invention. To the mixture (17.2 g) of a mixture of leaves and branches of Paeta Indica obtained in Vietnam at a weight ratio of 1: 1, 1 liter of 99.9% (v / v) methanol was added and the mixture was sonicated for 15 minutes at room temperature. The mixture was extracted 30 times, filtered, then concentrated under reduced pressure at 45 ° C, and dried at -70 ° C.
실시예Example 1. 세포생존율(cell viability) 측정 1. Measurement of cell viability
멜라닌 생성 억제 효과를 갖는 유효성분에 대하여 미백 화장료 조성물로 사용할 수 있는지 여부를 판단하기 위해 세포생존율 실험을 실시하였다. B16F10 마우스 멜라노 세포(mouse melanoma cell) 및 인간 피부섬유아세포(human dermal fibroblast cell)를 96-웰 플레이트(well plate)에 4.0×103 세포/웰의 농도로 각각 분주하여 24시간 동안 37℃ 및 5% CO2 조건의 배양기에서 배양하였다. 배양한 후 농도별로 제조한 파베타 인디카 추출물(1.25, 2.5, 5, 10 및 20㎍/㎖)을 각각 20㎕씩 처리하여 48시간 배양하였다. 배지를 제거한 다음 5㎎/㎖의 MTT(3-(4,5-디메틸티아졸-2yl)-2,5-디페닐-2H-테트라졸륨 브로마이드) 용액 20㎕를 B16F10 마우스 멜라노 세포 및 인간 피부섬유아세포에 각각 첨가하여 실온에서 4시간 동안 반응시켰다. 반응이 끝난 후, 배양액을 제거하고 각 웰(well)당 디메틸 설폭사이드(dimethyl sulfoxide, DMSO) 150㎕를 가하여 실온에서 30분간 반응시킨 뒤 570nm에서 흡광도를 측정하였다. 세포생존율(cell viability)은 파베타 인디카 추출물의 첨가군과 무첨가군의 흡광도 감소율을 나타내었다.To determine whether the active ingredient having melanin production inhibitory effect can be used as a whitening cosmetic composition, a cell survival rate experiment was conducted. B16F10 mouse melanoma cells (mouse melanoma cell) and human skin fibroblast (human dermal fibroblast cell) the 96-well plate (well plate) to 4.0 × 10 3 cells, 37 ℃ and 5 for a / 24 hours, respectively, divided by the concentration of the well and incubated in an incubator of% CO 2 conditions. After culturing, 20 μl of each of the P. indica extracts (1.25, 2.5, 5, 10 and 20 μg / ml) prepared for each concentration was treated and cultured for 48 hours. After the medium was removed, 20 μl of a solution of 5 mg / ml MTT (3- (4,5-dimethylthiazol-2yl) -2,5-diphenyl-2H-tetrazolium bromide) was added to B16F10 mouse melanocytes and human skin fibroblasts And reacted at room temperature for 4 hours. After completion of the reaction, the culture medium was removed, 150 μl of dimethyl sulfoxide (DMSO) was added to each well, reacted at room temperature for 30 minutes, and absorbance was measured at 570 nm. Cell viability showed the decrease of the absorbance of the Pravastatin Indica extract group and the no additive group.
그 결과, 도 1에 개시한 바와 같이 B16F10 마우스 멜라노 세포의 경우, 20㎍/㎖의 파베타 인디카 추출물을 제외한 모든 농도에서 90%~100%의 세포생존율을 나타내었으며, 인간 피부섬유아세포(human dermal fibroblast cell)의 경우, 모든 농도에서 세포생존율이 잘 유지되고 있는 것을 확인할 수 있었다. 따라서 상기 결과를 토대로 1.25, 2.5, 5 및 10㎍/㎖의 파베타 인디카 추출물을 하기 실시예 2 내지 4에 사용하였다.As a result, as shown in Fig. 1, in the case of B16F10 mouse melanocyte cells, cell survival rate was 90% to 100% at all concentrations except 20 μg / ml of pavetaindica extract, and human dermal fibroblast fibroblast cell), cell viability was maintained well at all concentrations. Based on the above results, 1.25, 2.5, 5 and 10 μg / ml of pavetaindica extract were used in the following Examples 2 to 4.
실시예Example 2. 멜라닌 함량(melanin contents) 측정 2. Measurement of melanin contents
B16F10 마우스 멜라노 세포(mouse melanoma cell)를 6-웰 플레이트(well plate)에 4.0×104 세포/웰의 농도로 분주하여 24시간 동안 37℃, 5% CO2 조건의 배양기에서 배양시킨 후, 각 웰(well)에 α-MSH(500nM, melanocyte stimulating hormone)와 농도별로 제조한 파베타 인디카 추출물(2.5, 5 및 10㎍/㎖)을 처리하였다. 48시간 동안 배양한 후 배지를 제거하고 인산완충액(phosphate buffered saline, PBS)으로 세척한 다음, 부착된 세포를 마이크로 테스트 튜브(micro test tube)에 옮기고 원심분리하여 펠릿(pellet)을 얻었다. 침전된 B16F10 마우스 멜라노 세포의 형상을 촬영하고, 1N NaOH에 디메틸 설폭사이드(dimethyl sulfoxide, DMSO)가 10%가 되도록 만든 용액을 펠릿(pellet)에 가하고 30분 동안 80℃ 히팅블럭(heating block)에서 펠렛을 녹인 후 470nm에서 흡광도를 측정하였다.B16F10 mouse melanoma cells were seeded in a 6-well plate at a concentration of 4.0 × 10 4 cells / well and cultured in an incubator at 37 ° C. and 5% CO 2 for 24 hours. (500 nM, melanocyte stimulating hormone) and pervbeta Indica extract (2.5, 5 and 10 μg / ml) prepared by concentration were treated in wells. After 48 hours of culture, the medium was removed and washed with phosphate buffered saline (PBS). The adhered cells were transferred to a micro test tube and centrifuged to obtain a pellet. The shape of the precipitated B16F10 mouse melanocytes was photographed, and a solution of 10% dimethyl sulfoxide (DMSO) in 1 N NaOH was added to the pellet and the mixture was heated for 30 minutes in an 80 ° C heating block The absorbance of the pellet was measured at 470 nm.
그 결과, 도 2에 개시한 바와 같이 α-MSH(500nM, melanocyte stimulating hormone) 처리로 인해 증가하였던 멜라닌 함량이 α-MSH와 농도별로 제조한 파베타 인디카 추출물(2.5, 5 및 10㎍/㎖)을 함께 처리하였을 때, 농도 의존적으로 멜라닌 함량이 현저하게 감소되었고, B16F10 세포의 펠렛(pellet) 색이 확연하게 탈색된 것을 확인할 수 있었으며, 특히, 10㎍/㎖의 파베타 인디카 추출물의 경우, 양성대조군인 레스베라트롤(20μM, resveratrol)보다 멜라닌 함량 감소효과가 더 우수한 것을 확인할 수 있었다.As a result, as shown in FIG. 2, the melanin content increased due to α-MSH (500 nM, melanocyte stimulating hormone) treatment was significantly lower than that of the Pervata Indica extract (2.5, 5 and 10 μg / , The melanin content was significantly decreased in a concentration dependent manner and the pellet color of B16F10 cells was markedly discolored. In particular, in the case of 10 μg / ml of pavetaindica extract, positive It was confirmed that the melanin content reduction effect was better than that of the control group, resveratrol (20 μM, resveratrol).
실시예Example 3. 세포 내 티로시나아제(Intracellular 3. Intracellular tyrosinase tyrosinasetyrosinase ) 활성 측정) Active measurement
B16F10 마우스 멜라노 세포(mouse melanoma cell)를 60mm 디쉬(dish)에 8.0×104 세포/웰의 농도로 분주하여 24시간 동안 37℃, 5% CO2 조건의 배양기에서 배양시킨 후, 각 웰(well)에 α-MSH(500nM)와 농도별로 제조한 파베타 인디카 추출물(2.5, 5 및 10㎍/㎖)을 처리하였다. 48시간 후 배지를 제거하고 인산완충액(phosphate buffer saline, PBS)으로 세척하였다. 각 디쉬(dish)에 분해완충용액(lysis buffer)을 첨가한 후 초음파처리(ultra-sonication)하였다. 이를 30분 동안 방치한 후 원심분리하여 얻어진 상층액을 효소용액으로 사용하였다. 이를 PBS에 녹인 L-DOPA(L-β-3,4-dihydroxyphenylalanine(2mM)) 160㎕와 파베타 인디카 추출물 40㎕을 96-웰 플레이트(well plate)에 넣은 후, 37℃에서 10, 20 및 30분간 배양한 후, 생성된 도파 크롬(DOPA chrome)의 490nm에서 흡광도를 측정하였다.B16F10 mouse melanoma cells were seeded at a concentration of 8.0 × 10 4 cells / well in a 60 mm dish and cultured in an incubator at 37 ° C. and 5% CO 2 for 24 hours. Then, each well ) Were treated with? -MSH (500 nM) and Pavela Indica extract (2.5, 5 and 10 μg / ml) prepared by concentration. After 48 hours, the medium was removed and washed with phosphate buffered saline (PBS). Each dish was added with lysis buffer and subjected to ultra-sonication. The supernatant obtained by centrifugation after being left for 30 minutes was used as an enzyme solution. After adding 160 μl of L-DOPA (L-β-3,4-dihydroxyphenylalanine (2mM)) dissolved in PBS and 40 μl of pravastatin Indica extract into a 96-well plate, After incubation for 30 minutes, the absorbance of the generated DOPA chrome was measured at 490 nm.
또한, in situ 세포 내 티로시나아제 활성 측정 방법은 다음과 같다. B16F10 마우스 멜라노 세포(mouse melanoma cell)를 60mm 디쉬(dish)에 8.0×104 세포/웰의 농도로 분주하여 24시간 동안 37℃, 5% CO2 조건의 배양기에서 배양시킨 후, 각 웰(well)에 α-MSH(500nM)와 농도별로 제조한 파베타 인디카 추출물(2.5, 5 및 10㎍/㎖)을 처리하였다. 48시간 후 배지를 제거하고 인산완충액(phosphate buffer saline, PBS)으로 세척하였다. 각 디쉬에 4% 파라포름알데히드(paraformaldehyde)를 이용하여 40분 동안 세포를 고정시킨 후, PBS로 세척하였다. 0.1% Triton X-100을 이용하여 2분간 세포를 투과(permeabilization) 시킨 후, PBS로 세척한 뒤, 2mM의 L-DOPA로 2시간 동안 세포를 염색시켜 현미경으로 촬영하였다.The in situ intracellular tyrosinase activity measurement method is as follows. B16F10 mouse melanoma cells were seeded at a concentration of 8.0 × 10 4 cells / well in a 60 mm dish and cultured in an incubator at 37 ° C. and 5% CO 2 for 24 hours. Then, each well ) Were treated with? -MSH (500 nM) and Pavela Indica extract (2.5, 5 and 10 μg / ml) prepared by concentration. After 48 hours, the medium was removed and washed with phosphate buffered saline (PBS). Each dish was fixed with 4% paraformaldehyde for 40 minutes and then washed with PBS. Cells were permeabilized with 0.1% Triton X-100 for 2 minutes, washed with PBS, and stained with 2 mM L-DOPA for 2 hours.
그 결과, 도 3에 개시한 바와 같이 α-MSH(500nM) 처리로 인해 증가하였던 세포 내 티로시나아제 활성이 α-MSH와 파베타 인디카 추출물을 농도별로 동시에 처리하였을 때, 세포 내 티로시나아제의 활성 또한 마찬가지로 농도 의존적으로 감소하였으며, 특히 10㎍/㎖의 파베타 인디카 추출물의 경우, 양성대조군인 레스베라트롤(20μM, resveratrol)보다 더 우수한 세포 내 티로시나아제 억제 활성을 나타내었다. As a result, the intracellular tyrosinase activity, which was increased due to the treatment with α-MSH (500 nM) as shown in FIG. 3, when the α-MSH and pravastatin indica extracts were simultaneously treated at different concentrations, the intracellular tyrosinase activity The activity also decreased in a dose dependent manner. Particularly, the 10 μg / ㎖ of pervbetaindica extract showed a better inhibitory activity of intracellular tyrosinase than the positive control, resveratrol (20 μM, resveratrol).
실시예Example 4. 4. 웨스턴Western 블럿(Western blot)을Western blot 통해 미백 관련 단백질 활성 검증 Verification of whitening-related protein activity through
B16F10 마우스 멜라노 세포(mouse melanoma cell)를 100mm 조직배양접시(tissue culture dish)에 2.0×105 세포/웰의 농도가 되도록 분주하여 24시간 동안 배양시킨 후, 각 웰(well)에 α-MSH(500nM)와 농도별로 제조한 파베타 인디카 추출물(2.5, 5 및 10㎍/㎖)을 처리하고 48시간 동안 배양하였다. 배양한 후, 분해완충용액(Lysis buffer)을 첨가하여 세포를 용해시키고 원심분리하여 세포막 성분들을 제거한 다음, 원심분리하여 얻은 단백질은 BSA(bovine serum albumin)로 정량하였으며, 단백질을 SDS-PAGE(Sodiumdodecylsulfate-polyacrylamide gel)를 이용하여 전기영동한 후, 항체(antibody)의 비특이적 결합을 억제시키기 위해 PVDF 멤브레인(polyvinyl difluoride membrane)에 옮긴 다음 트랜스퍼(transfer)하였다. 트랜스퍼(transfer)가 끝난 후, 5% 탈지분유(skim milk)로 밤새(overnight) 반응시켜 백그라운드(background)를 제거시켰다. 멤브레인을 TTBS로 3회 세척 후, 1차 항체를 밤새(overnight) 동안 붙인 후 다시 2차 항체를 붙이고 ECL 키트를 이용하여 엑스-레이 필름(X-ray film)에 감광시켜 밴드를 확인하였다.B16F10 mouse melanoma cells (mouse melanoma cell) after incubation for 100mm tissue culture plate with 2.0 × 10 5 cells / seeded to a concentration of the well for 24 hours (tissue culture dish), each well (well) α-MSH ( (2.5, 5 and 10 μg / ml) were cultured and cultured for 48 hours. After incubation, the cells were lysed by adding lysis buffer, centrifuged to remove cell membrane components, and the proteins obtained by centrifugation were quantitated with bovine serum albumin (BSA). Proteins were analyzed by SDS-PAGE (sodium dodecylsulfate -polyacrylamide gel), and transferred to a PVDF membrane (polyvinyl difluoride membrane) to inhibit non-specific binding of the antibody. After the transfer was completed, the background was removed by overnight reaction with 5% skim milk. After the membrane was washed three times with TTBS, the primary antibody was stuck overnight, then the secondary antibody was attached to the membrane, and the band was detected by exposing the membrane to an X-ray film using an ECL kit.
그 결과, 도 4에 개시한 바와 같이 α-MSH(500nM) 처리로 인해 증가된 티로시나아제 및 TRP1(tyrosinase related protenin 1)의 단백질 발현량이 파베타 인디카 추출물에 의해 농도 의존적으로 감소하였으며, 티로시나아제 및 TRP1의 전사인자인 MITF(Microphthalmia-associated transcription factor)의 발현량도 감소되는 것을 확인할 수 있었다. 이러한 결과는, 파베타 인디카 추출물이 MITF의 발현을 감소시킴으로써, 티로시나아제 및 TRP1의 생합성에 영향을 주어 멜라닌 생성을 저해하여 미백효과를 나타내는 것으로 사료된다.As a result, as shown in FIG. 4, protein expression of tyrosinase and TRP1 (tyrosinase related protenin 1) increased due to α-MSH (500 nM) treatment was decreased in a concentration-dependent manner by pravastatin Indica extract, The expression level of MITF (Microphthalmia-associated transcription factor), which is a transcription factor of azo and TRP1, was also decreased. These results suggest that the pervbeta Indica extract reduces the expression of MITF, thereby affecting the biosynthesis of tyrosinase and TRP1, thereby inhibiting melanogenesis and thus exhibiting a whitening effect.
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