KR20090018459A - Whitening cosmetic compositions containing extract of isodon inflexus - Google Patents
Whitening cosmetic compositions containing extract of isodon inflexus Download PDFInfo
- Publication number
- KR20090018459A KR20090018459A KR1020070082919A KR20070082919A KR20090018459A KR 20090018459 A KR20090018459 A KR 20090018459A KR 1020070082919 A KR1020070082919 A KR 1020070082919A KR 20070082919 A KR20070082919 A KR 20070082919A KR 20090018459 A KR20090018459 A KR 20090018459A
- Authority
- KR
- South Korea
- Prior art keywords
- extract
- acidic
- melanin
- whitening
- fraction
- Prior art date
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9789—Magnoliopsida [dicotyledons]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
- A61K36/534—Mentha (mint)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/31—Hydrocarbons
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Birds (AREA)
- Mycology (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Medicinal Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Medical Informatics (AREA)
- Alternative & Traditional Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Dermatology (AREA)
- Cosmetics (AREA)
- Medicines Containing Plant Substances (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
Description
본 발명은 산박하(Isodon inflexus) 추출물을 유효성분으로 함유하는 미백용 화장료조성물에 관한 것이다. 또한, 본 발명은 산박하 추출물로부터 멜라닌생성 억제작용을 갖는 화학식 1로 표시되는 excisanin A 화합물을 분리 정제하는 방법을 제공한다. 본 발명의 excisanin A 화합물 및 산박하 추출물은 멜라닌 생성 억제작용이 우수하여 과다한 색소 침착의 피부질환에 대한 천연 미백제로서의 그 이용이 기대된다.The present invention is an acid peppermint ( Isodon inflexus ) relates to a whitening cosmetic composition containing the extract as an active ingredient. The present invention also provides a method for separating and purifying an excisanin A compound represented by Chemical Formula 1 having melanogenesis inhibitory activity from an acidic peppermint extract. The excisanin A compound and the acidic peppermint extract of the present invention are excellent in inhibiting melanin production and are expected to be used as natural whitening agents for skin diseases of excessive pigmentation.
산박하(Isodon inflexus)는 쌍떡잎식물 통화식물목 꿀풀과의 여러해살이풀로 원산지는 한국, 일본, 중국 등지에 분포하고 산지에서 흔히 자란다. 높이는 40∼100cm로 자라며 줄기는 곧게 서고 모가 나며, 가지가 많이 갈라지고 전체에 잔털이 난다. 잎은 마주나고 삼각 달걀모양이며 길이 3∼6cm, 나비 2∼4cm이고, 밑은 잎자루의 날개같이 되고 가장자리에 둔한 톱니가 있다. 꽃은 6∼8월에 파란빛을 띤 자줏빛으로 피고 줄기 위에 취산꽃차례[聚揀花序]로 달린다. 꽃 지름 길이 8∼10mm이며, 전체가 커다란 꽃이삭이 된다. 꽃받침은 종 모양이며 털이 나고 5갈래로 갈라 지는데, 갈래조각은 좁은 삼각형이다. 화관은 입술 모양으로서 윗입술은 위를 향하며 5갈래로 갈라지고, 아랫입술은 밑동이 볼록 나온 모양이다. 4개의 수술 중 2개가 길고, 암술은 1개이며 암술대는 2갈래로 갈라진다. 열매는 작은 견과로서 꽃받침 속에 들어 있으며 9∼10월에 익는다. 어린순은 나물로 식용한다.Mountain pepper ( Isodon inflexus ) is a perennial herb of the dicotyledon and moss family. Its origin is distributed in Korea, Japan and China and grows in mountainous areas. It grows to 40-100cm in height, stems stand upright, gathered, many branches are split, and the whole hairs grow. The leaves are opposite each other, triangular egg-shaped, 3-6cm long, 2-4cm butterfly, and the bottom is like a wing of petiole with dull teeth at the edge. Flowers bloom in June-August with a blue-purple color, and hang on the stems with a pickled inflorescence. Flower diameter is 8-10mm in length, and the whole becomes a large flower ear. Calyxes are bell-shaped, hairy, and divided into 5 branches, the fragments being narrow triangles. Corollas are shaped like lips, upper lip facing up, split into 5 branches, and lower lip is convex. Two of the four stamens are long, with one pistil and the pistil split into two branches. Fruits are small nuts in the calyx and ripen in September to October. Young shoots are eaten as herbs.
산박하를 대상으로 미백에 관련한 활성을 갖는 활성물질의 규명에 대한 연구는 보고된 바가 없어 본 발명자들은 천연물에서의 천연 미백제 관련 활성물질에 대해 조사한 결과 산박하를 유기용매로 추출한 산박하 추출물의 좋은 미백효과를 멜라닌 세포에서의 멜라닌 생성억제를 통해 확인할 수 있었다. 이로부터 미백 효과를 갖는 물질을 분리하여 그 화학적 특성을 조사한 결과 excisanin A 임을 확인함으로써 본 발명을 완성하였다.There have been no studies on the identification of active substances having activity related to whitening in acidic peppermint. Therefore, the present inventors have investigated the active substances related to natural whitening agent in natural products. The whitening effect was confirmed through the inhibition of melanin production in melanocytes. From this, a material having a whitening effect was separated and examined for its chemical properties, thereby confirming that it was excisanin A, thereby completing the present invention.
산박하 유기용매 추출물에서부터 분리한 유효성분 excisanin A의 구조.Structure of the active ingredient excisanin A isolated from the extract of organic acidic peppermint.
인간이 미에 대한 욕구는 삶에 대한 여유를 갖고자 하는 현대인들에게 있어서 필수적이라 할 수 있다. 국내에서는 화장품법이 분리되어 시행된 이후로 기능성 화장품에 대한 많은 관심과 함께 연구 개발이 활발히 이루어지고 있다. 특히, 동양 인들은 희고 고운 피부에 대한 열망이 과거에서부터 미의 상징으로 여겨져 미백화장품에 대한 관심과 함께 그에 대한 연구가 집중되고 있다.Man's desire for beauty is essential for modern people who want to have time for life. In Korea, since the Cosmetic Act was implemented separately, research and development has been actively conducted with much interest in functional cosmetics. In particular, Asians have long been considered as a symbol of beauty because of their desire for white and fine skin.
인간의 피부 색깔은 환경, 인종, 성별 등의 요인과 멜라닌, 카로틴 및 헤모글로빈 양과 같은 여러 가지 요인에 의해 결정되지만 피부의 과색소 침착과 관련된 주요한 원인은 표피 내 멜라닌색소의 이상적 증가에 기인한다. 멜라닌은 표피층의 기저층에 있는 멜라닌형성세포(melanocyte)내의 멜라노좀(melanosome)에서 티로신(tyrosin)으로부터 생합성되며 주변 각질형성세포(kerotinocyte)로 전이되어 피부색을 나타낸다(V. J. Hearing and T. M. Ekel, Mammalian tyrosinase, Biochem. J., 157, 549, 1976). 멜라닌은 자외선으로부터 피부가 손상되는 것을 방지하는 중요한 역할을 하지만(Y. Mishima, S. Hatta, and Y. Ohyama, Induction of melanogenesis suppression : cellular pharmacology and mode of differential action, Pigment Cell Res., 1, 367, 1998, H. Matubara, Inhibitoryh effect of lichen metabolites and their synthetic analogues on melanin biosynthesis in cultered B-16 mouse melanoma cells, Nateral Product Sciences, 4, 3, 1998) 비정상적으로 과잉 생산되는 경우는 기미나 주근깨 등의 미용상 문제를 일으키고, 반대로 너무 적게 생산되면 백반증과 같은 피부 병변이 유발된다. Melanocyte는 자외선, 피부의 염증반응, 호르몬 이상, 유전질환, 화학적 자극 등에 의해 활성화되고, 증식 및 분화하여 멜라닌 색소를 과다하게 만들어 결국 피부 색소 침착을 일으킨다. 특히 피부의 멜라닌 색소를 증가시키는 대표적인 인자인 자외선에 대한 자극은 최근 환경오염에 따른 오존층의 파괴와 여가 활동에 대한 욕구가 많아지면서 더욱 많아지게 되었다. 자외선에 의한 피부색소증가에 대한 경로는 명확하게 밝혀지지는 않았지만 주로 각질형성세포(kerotinocyte)에서 분비되는 인자들에 기인한다. 자극된 각질형성세포가 분비하는 인자들로는 endothelin-1(ET-1), α-melanocyte stimulating hormone(α-MSH), adrenocorticotropic hormome(ACTH), nitric oxide(NO-)등이 있다. ET-1인 경우 멜라닌 세포의 protein kinase C(PKC) 경로와 cAMP 경로를 자극시켜 멜라닌색소형성을 증가시키고 α-MSH와 ACTH도 cAMP 경로를 통해 피부 색소형성을 촉진시킨다(Imokawa G, Kobayashi T, Miyagishi M, Higashi K, Yada Y: The role of endothelin-1 in epidermal hyperpigmentation and signaling mechanism of mitogenesis and melanogenesis. Pigment Cell Res 10:218-28, 1997, Thody AJ, Graham A : Does alpha-MSH have a role in regulating skin pigmentation in humans? Pigment Cell Res 11:265-74, 1998). α-MSH 의 전구체인 pro-opiomelano-cortin(POMC)도 α-MSH와 같이 멜라닌세포에 있는 melanocortin-1(MC-1) 수용체에 결합하여 cAMP활성을 증가시켜 멜라닌 생성을 촉진시킨다 (Sungbin Im, Moro O, Peng F, Medrano EE, Cnelius J, Babcokc G, Nordlund JJ, Abdel Malek ZA: Activation of the cyclic AMP pathway by α-melanotropin mediated the response of human melanocytes to ultraviolet B radiation. Cancer RES 58:47-54, 1998, Romero-Grailler C, Aberdam E, Clement M, Ortonne JP, Bailotti R: Cycle AMP a Key Messenger in the Regulation of Skin Pimentation. Pigment Cell Res. 13: 60-69. 2000).Human skin color is determined by factors such as the environment, race, and sex and various factors such as the amount of melanin, carotene and hemoglobin, but the main cause associated with hyperpigmentation of the skin is due to the ideal increase in melanin pigment in the epidermis. Melanin is biosynthesized from tyrosin in melanocytes in melanocytes in the basal layer of the epidermis and metastasizes to peripheral keratinocytes (VJ Hearing and TM Ekel, Mammalian tyrosinase, Biochem. J. , 157, 549, 1976). Melanin plays an important role in preventing skin damage from UV rays (Y. Mishima, S. Hatta, and Y. Ohyama, Induction of melanogenesis suppression: cellular pharmacology and mode of differential action, Pigment Cell Res ., 1, 367 , 1998, H. Matubara, Inhibitoryh effect of lichen metabolites and their synthetic analogues on melanin biosynthesis in cultered B-16 mouse melanoma cells, Nateral Product Sciences, 4, 3, 1998) It causes cosmetic problems and, on the contrary, too little production leads to skin lesions such as vitiligo. Melanocytes are activated by ultraviolet rays, inflammatory reactions of the skin, hormonal abnormalities, genetic diseases, chemical stimuli, etc., proliferate and differentiate, causing excessive melanin pigmentation, which in turn causes skin pigmentation. In particular, the stimulation for ultraviolet rays, which is a representative factor for increasing the melanin pigment in the skin, has been increased as the desire for destruction of the ozone layer and the desire for leisure activities have increased recently. The path to skin pigmentation caused by UV light is not clear, but is mainly due to factors secreted from keratinocytes. Factors secreted by stimulated keratinocytes include endothelin-1 (ET-1), α-melanocyte stimulating hormone (α-MSH), adrenocorticotropic hormome (ACTH), and nitric oxide (NO − ). ET-1 stimulates the protein kinase C (PKC) pathway and cAMP pathway in melanocytes to increase melanocyte formation and α-MSH and ACTH also promote skin pigmentation through cAMP pathway (Imokawa G, Kobayashi T, Miyagishi M, Higashi K, Yada Y: The role of endothelin-1 in epidermal hyperpigmentation and signaling mechanism of mitogenesis and melanogenesis.Pigment Cell Res 10: 218-28, 1997, Thody AJ, Graham A: Does alpha-MSH have a role in regulating skin pigmentation in humans? Pigment Cell Res 11: 265-74, 1998). Pro-opiomelano-cortin (POMC), a precursor of α-MSH, also binds to melanocortin-1 (MC-1) receptors in melanocytes, like α-MSH, to increase cAMP activity and promote melanin production (Sungbin Im, Moro O, Peng F, Medrano EE, Cnelius J, Babcokc G, Nordlund JJ, Abdel Malek ZA: Activation of the cyclic AMP pathway by α-melanotropin mediated the response of human melanocytes to ultraviolet B radiation.Cancer RES 58: 47-54 , 1998, Romero-Grailler C, Aberdam E, Clement M, Ortonne JP, Bailotti R: Cycle AMP a Key Messenger in the Regulation of Skin Pimentation.Pigment Cell Res. 13: 60-69. 2000).
기능성 미백화장품에서는 과다하게 생성되는 멜라닌 색소의 억제 및 자외선 차단을 통한 색소 침착 억제 그리고, 생성된 멜라닌 색소의 환원작용을 통하여 미백효과를 유도한다. 멜라닌 생성을 억제하는 물질로는 hydroquinone, resorcinol 등 페놀 유도체나, L-ascrobic acid와 그 유도체 및 arbutin, lactic acid, glucosamin, tunicamycin 등이 개발되었으나, 피부자극성이나 안전성에 문제가 있어 극히 제한된 양만 사용되는 것들이 많다(H. Matubara, Inhibitory effect of lichen metabolites and their synthetic analogues on melanin biosynthesis in cultured B-16 mouse melanoma cells, Natural Product Sciences, 4,3,1998).In functional whitening cosmetics, the whitening effect is induced through inhibition of excessively generated melanin pigment, inhibition of pigmentation through UV protection, and reduction of the produced melanin pigment. Phenolic derivatives such as hydroquinone and resorcinol, L-ascrobic acid and its derivatives, and arbutin, lactic acid, glucosamin, and tunicamycin have been developed as inhibitors of melanin production, but due to skin irritation and safety problems, only a limited amount is used. Many (H. Matubara, Inhibitory effect of lichen metabolites and their synthetic analogues on melanin biosynthesis in cultured B-16 mouse melanoma cells, Natural Product Sciences, 4,3,1998).
인간의 생활이 윤택해지면서 웰빙에 대한 욕구가 강해지고, 그에 따른 자아실현에 대한 욕구의 증가와 미에 대한 관심이 급증하면서 단순한 의미의 화장품 개념이 아닌, 유효성이 강조된 고기능성 화장품에 대한 구매 욕구가 많아지고 있다. 특히나, 그러한 효능이 인공적인 것보다는 인체에 친화적이고 무해한 자연적인 것으로부터 얻고자 하는 경향이 증가하고 있어 화장품 원료뿐만 아니라 식품, 의약계 등에서도 천연원료의 사용이 증가되고 있고, 그에 따르는 연구 역시 증가되고 있다.As people's lives get better, the desire for well-being grows stronger, and the demand for self-realization and the interest in beauty soar. As a result, the desire for purchasing high-functional cosmetics that emphasizes effectiveness rather than a simple concept of cosmetics It is getting bigger. In particular, there is an increasing tendency to obtain such effects from human-friendly and harmless natural ones rather than artificial ones, and thus the use of natural raw materials is increasing not only in cosmetic raw materials but also in food and pharmaceutical systems, and the researches that follow are increasing. have.
본 발명의 목적은 인체에 무해하고 친환경적인 소재의 활용을 통해 좀 더 안전하고 효능이 우수한 천연물질의 개발을 통해 새로운 미백용 화장료를 개발하며, 나아가 피부색소 변화에 대한 치유제를 제공함을 목적으로 한다. 또한 본 발명의 또 다른 목적은 산박하로부터 excisanin A를 분리하는 방법을 제공하고, 처음으로 이 물질이 미백기능이 있음을 밝힌다.The purpose of the present invention is to develop new whitening cosmetics through the development of safer and more effective natural materials through the use of harmless and environmentally friendly materials, and to provide a healing agent for skin pigment changes. do. It is another object of the present invention to provide a method for separating excisanin A from acidic peppermint, and for the first time reveals that this material has a whitening function.
상기 목적을 달성하기 위하여, 본 발명은 산박하를 메틸알코올과 기타 유기용매들로 추출하여 멜라닌 생성 억제활성을 갖는 산박하 추출물을 제공한다. 또한 본 발명은 산박하의 지상부를 80% 메탄올로 추출한 다음 유기용매인 헥산, 에틸아세테이트, 부탄올 등의 유기용매를 가하여 다시 추출한 미백 활성을 가지는 산박하 추출물의 용매분획물을 제공한다.In order to achieve the above object, the present invention provides an acidic peppermint extract having melanin production inhibitory activity by extracting the acidic peppermint with methyl alcohol and other organic solvents. In another aspect, the present invention provides a solvent fraction of the acidic peppermint extract having a whitening activity extracted by extracting the ground portion of acidic peppermint with 80% methanol, and then added again an organic solvent such as hexane, ethyl acetate, butanol, and the like.
본 발명의 추출용매로는 메탄올, 에탄올 등의 저급알코올, 헥산, 에틸아세테이트, DMF(Dimethyl foramide), THF(Tetrahydrofuran), 클로로포름, 디에틸에테르 등이 사용 가능하고 바람직하기로는 메탄올 또는 에탄올이 바람직하다.As the extraction solvent of the present invention, lower alcohols such as methanol and ethanol, hexane, ethyl acetate, DMF (dimethyl foramide), THF (Tetrahydrofuran), chloroform, diethyl ether and the like can be used, and preferably methanol or ethanol is preferable. .
또한, 본 발명은 산박하 전초를 80% 메탄올(물, 메탄올 혼합용액)로 침지시켜 반복 추출하고 농축하여 메탄올 추출물을 얻은 다음 이를 20% 메탄올로 현탁시키고 헥산, 에틸아세테이트 및 부탄올 등의 유기용매를 차례대로 가하여 그에 해당되는 용매 추출분획을 제조한다.In addition, the present invention is immersed in acidic peppermint starch with 80% methanol (water, methanol mixed solution) and repeatedly extracted and concentrated to obtain a methanol extract and then suspended in 20% methanol and organic solvents such as hexane, ethyl acetate and butanol In turn, a corresponding solvent extraction fraction is prepared.
또한 본 발명은 산박하 추출물로부터 실리카겔 크로마토그래피, prep-HPLC를 이용하여 excisanin A를 분리·정제하는 방법을 제공한다.The present invention also provides a method for separating and purifying excisanin A from acidic peppermint extract using silica gel chromatography and prep-HPLC.
본 발명자는 상기과정으로 분리·정제한 물질이 미백효과 등의 활성을 나타내는 활성 물질임을 밝히고, 화학적 구조를 수소 및 탄소 핵자기 공명 스펙트럼 방법으로 조사하여 excisanin A임을 확인하였다.The inventors found that the substance separated and purified by the above procedure was an active substance showing activity such as a whitening effect, and confirmed that the chemical structure was excisanin A by investigating hydrogen and carbon nuclear magnetic resonance spectra.
본 발명의 산박하추출물 또는 산박하추출물의 용매분획물을 유효성분으로 함유하는 미백용 화장료 조성물은 일반화장수, 크림제, 에센스, 마스크팩 등의 제형으로 제공될 수 있으며, 본 발명의 산박하추출물 또는 산박하추출물의 용매분획물은 화장료조성물 전체 중량의 1×10-4 내지 10 중량% 함유하는 것이 바람직하다. 0.0001 중량% 이하 함유시는 본 발명의 미백효과를 기대하기 어렵고, 10 중량% 이상 함유시는 제제화에 문제가 있다.The cosmetic composition for whitening containing the acidic peppermint extract of the present invention or the solvent fraction of the acidic peppermint extract as an active ingredient may be provided in the form of general cosmetic water, cream, essence, mask pack, or the like. The solvent fraction of the acidic peppermint extract preferably contains 1 × 10 −4 to 10 wt% of the total weight of the cosmetic composition. When it is contained 0.0001% by weight or less, the whitening effect of the present invention is hardly expected, and when it is contained by 10% by weight or more, there is a problem in formulation.
excisanin A로는 화장료조성물 전체 중량의 1×10-5 내지 1 중량% 함유하는 것이 바람직하다. 1×10-5 중량% 이하 함유시는 본 발명의 미백효과를 기대하기 어렵다.As excisanin A, it is preferable to contain 1 * 10 <-5> -1 weight% of the total weight of a cosmetic composition. It is difficult to expect the whitening effect of this invention when it contains 1 * 10 <-5> weight% or less.
본 발명의 산박하 추출물 및 산박하 추출물의 활성성분인 excisanin A 화합물은 탁월한 멜라닌 생성 억제 효과를 나타내므로 앞으로 천연식물에서 유래한 안전하고 효과적인 미백제로 널리 사용될 수 있다.The acid and peppermint extract of the present invention and the excisanin A compound, which is an active ingredient of the peppermint extract, exhibit excellent melanin production inhibitory effect and can be widely used as a safe and effective whitening agent derived from natural plants in the future.
또한, 본 발명에 의거 얻어진 산박하 추출물은 화장품은 물론 의약품 분야에도 널리 응용될 수 있다.In addition, the peppermint extract obtained according to the present invention can be widely applied to the cosmetics as well as the pharmaceutical field.
본 발명은 산박하의 지상부를 80% 메탄올에 침지시켜 반복 추출하여 미백 효능을 가지는 산박하 메탄올 추출물을 얻는다. 또한 산박하의 메탄올 추출물을 다시 유기용매인 헥산, 에틸아세테이트, 부탄올 등을 이용하여 추출한 후 미백 활성을 가지는 추출 분획을 제공한다.The present invention is extracted repeatedly by immersing the ground portion of acidic peppermint in 80% methanol to obtain an acidic peppermint methanol extract having a whitening effect. In addition, the methanol extract of acid and mint is extracted again using an organic solvent hexane, ethyl acetate, butanol and the like to provide an extraction fraction having a whitening activity.
본 발명은 산박하의 지상부를 80% 메탄올로 추출하여 농축한 메탄올 추출물을 이용하여 유기용매로 분획함으로서 미백활성 효과가 우수한 산박하 에틸아세테이트 분획을 제공한다.The present invention provides an acidic ethylacetate fraction with excellent whitening activity by fractionating the ground portion of acidic peppermint with 80% methanol and fractionating it with an organic solvent using a concentrated methanol extract.
본 발명은 상기 산박하 추출물을 유효성분으로 하는 미백활성이 있는 조성물을 제공한다. 구체적으로는 상기 추출물은 생체내의 멜라닌 생성을 억제하여 미백 향장품 및 의약품 등의 조성물로서 사용될 수 있다.The present invention provides a composition having a whitening activity using the acidic peppermint extract as an active ingredient. Specifically, the extract may be used as a composition for whitening cosmetics and pharmaceuticals by inhibiting melanin production in vivo.
상기 산박하 추출물의 미백 유효 성분은 산박하 메탄올 추출물을 이용하여 mouse의 melanocyte에서 멜라닌 생성 억제 효과를 측정한 결과, 산박하 조추출물에서 유효 효능이 확인 되었고, 유기용매 분획층에서 특히, 에틸아세테이트 추출분획은 멜라닌 생성 억제 효과를 탁월하게 나타내었다.The whitening active ingredient of the acidic peppermint extract was determined to be effective in extracting melanin from melanocytes of mice using an acidic peppermint methanol extract. The fraction showed the melanin production inhibitory effect outstanding.
본 발명은 상기의 우수한 미백 효능을 보이는 에틸아세테이트 분획추출에서 실리카겔 컬럼 크로마토그래피와 prep-HPLC를 이용하여 단일 물질을 얻어낼 수 있었다. 또한 수소 및 탄소 핵자기 공명 스펙트럼 등의 방법으로 조사하여 이 화합물이 excisanin A임을 확인하였다.The present invention was able to obtain a single material using silica gel column chromatography and prep-HPLC in the ethyl acetate fraction extraction showing the excellent whitening efficacy. In addition, hydrogen and carbon nuclear magnetic resonance spectroscopy was carried out to confirm that this compound is excisanin A.
본 발명은 상기 excisanin A를 포함하는 산박하 추출물의 미백효능을 조사하기 위하여 melanocyte인 melan-a 세포와 B16 세포를 이용하여 멜라닌 생성 억제 효과 및 생합성 과정에 관련된 유전자들의 발현을 조절하는 기능을 측정함으로써 효과를 확인하였다.The present invention by measuring the ability to control the expression of genes involved in the melanogenesis inhibitory effect and biosynthetic process using melanocytes melan-a cells and B16 cells to investigate the whitening efficacy of the extract of the acidic peppermint containing excisanin A The effect was confirmed.
<실시예 1 : 산박하의 지상부로부터 용매분획물의 제조>Example 1 Preparation of Solvent Fractions from the Ground Part of Acid Peppermint
제주도에 자생하는 산박하 전초 2240.7g을 채취하여 깨끗이 손질한 후 잘게 파쇄하여 80% 메탄올 10L로 3회 교반 추출 후 감압 농축하여 320g의 메탄올 조출물을 얻었다. 건조 조출물 10g을 20% 메탄올 1L로 현탁 시킨 후 헥산 1L를 첨가하여 헥산층을 분리하고, 이것을 3회 반복하여 헥산 분획층을 얻었다. 남은 물층에 에틸아세테이트 1L를 가하여 에틸아세테이트층을 분리하고, 이것을 3회 반복하여 에틸아세테이트 분획층을 얻었다. 최종적으로 물층에 부탄올 1L를 3회 가하여 부탄올층과 물층으로 분획하였고, 나머지 메탄올 조추출물들도 이와 같은 과정을 반복하여 모두 용매분획 하여 사용하였다.2240.7 g of acidic peppermint outpost native to Jeju Island were collected, trimmed, and finely crushed. After stirring and extracting three times with 10 L of 80% methanol, the mixture was concentrated under reduced pressure to obtain 320 g of methanol extract. 10 g of the dried crude product was suspended in 1 L of 20% methanol, and then, 1 L of hexane was added to separate the hexane layer. This was repeated three times to obtain a hexane fraction layer. Ethyl acetate 1L was added to the remaining water layer to separate the ethyl acetate layer, and this was repeated three times to obtain an ethyl acetate fraction layer. Finally, 1 L of butanol was added to the water layer three times, and the mixture was partitioned into a butanol layer and a water layer. The remaining methanol crude extracts were repeatedly used in a solvent fraction.
상기에서 얻은 각 분획물을 감압 농축한 결과 에틸아세테이트 분획 7.752g을 얻을 수 있었고, 이 분획층은 다시 셀라이트(celite)를 충진한 컬럼을 통해 용매들을 순차적으로 사용하여 분획을 얻었다. 그 결과 헥산 분획 0.168g, 디메틸클로라이드 분획 4.298g, 에틸아세테이트 분획 2.149g, 메탄올 분획 0.63g을 얻을 수 있었다.The obtained fractions were concentrated under reduced pressure to yield 7.752 g of ethyl acetate fraction, which was then fractionated using solvents sequentially through a column filled with celite. As a result, 0.168 g of hexane fraction, 4.298 g of dimethyl chloride fraction, 2.149 g of ethyl acetate fraction and 0.63 g of methanol fraction were obtained.
산박하 추출물의 미백 효과를 조사하기 위하여 상기 실시예 1에서 얻은 각 분획물의 멜라닌 생성 억제 효과를 두 가지 mouse melanocyte를 사용하여 조사하다.In order to investigate the whitening effect of the peppermint extract, the effect of inhibiting melanin production of each fraction obtained in Example 1 was investigated using two mouse melanocytes.
실험예 1 : 멜라닌생성 억제 실험Experimental Example 1: Melanogenesis Inhibition Experiment
melan-a cell은 non-tumorigrinc mouse melanocyte cell line으로써 정상 mouse melanocyte의 성질을 대부분 가지고 있으면서도 불멸화 되어 실험에 용이하게 사용할 수 있다. 따라서 시료 추출물의 멜라닌 생성 억제 정도를 melan-a cell 을 이용하여 최종 생성되는 멜라닌 생성 억제 정도를 측정하였다. Melan-a cells을 RPMI 1640배지(Gibco, USA) 배지를 이용하여 24 well plate에 1×105 cells/mL로 plating 한 후, 시료를 처리하여 37℃ 10% CO2 항온기에서 3일간 배양하였다. Plate의 배지 제거 후 세포를 수확하여 1N NaOH를 첨가하여 세포를 완전히 녹인 후 450 nm에서 enzyme linked immunosorbant assay(ELISA) reader로 측정하여 대조군과 비교하였다.The melan-a cell is a non-tumorigrinc mouse melanocyte cell line that has most of the properties of normal mouse melanocytes, but is immortalized and can be easily used in experiments. Therefore, the degree of inhibition of melanin production of the sample extract was measured by using melan-a cells. Melan-a cells were plated at 1 × 10 5 cells / mL in 24 well plates using RPMI 1640 medium (Gibco, USA) medium, and the samples were treated and incubated in a 37 ° C. 10% CO 2 incubator for 3 days. After removal of the plate medium, the cells were harvested, 1N NaOH was added to completely dissolve the cells, and measured by enzyme linked immunosorbant assay (ELISA) reader at 450 nm.
합성 멜라닌을 이용하여 standard soultion을 만들고 sample과 standard soultion을 96 well plate에 넣고 흡광도를 측정한다. 멜라닌 농도는 합성 멜라닌으로 작성된 표준 농도 곡선으로부터 결정하였다.Make standard soultion using synthetic melanin and measure the absorbance by putting sample and standard soultion in 96 well plate. Melanin concentrations were determined from standard concentration curves prepared with synthetic melanin.
시료 추출물의 멜라닌 암세포인 B16 mouse melanocyte에도 유의한 효과가 나타나는 지 확인하기 위하여 B16 cell을 사용하여 멜라닌 생성 억제 정도를 측정하였다. B16 melanocyte를 Dulbecco's modified eagle medium(DMEM)을 이용하여 6well plate에 1×105 cells/well로 plating 한 후, 시료를 처리하여 37℃ 5% CO2 항온기에서 3일간 배양하였다. Plate의 배지 제거 후 세포를 수확하여 1N NaOH를 첨가하여 세포를 완전히 녹인 후 450 nm에서 ELISA reader로 측정하여 대조군과 비교하였다.In order to determine whether a significant effect on the melanocyte B16 mouse melanocyte of the sample extract was measured, the degree of inhibition of melanogenesis was measured using B16 cells. B16 melanocytes were plated at 1 × 10 5 cells / well in 6well plates using Dulbecco's modified eagle medium (DMEM), and then the samples were treated and incubated in a 37 ° C. 5% CO 2 incubator for 3 days. After removal of the plate medium, the cells were harvested, 1N NaOH was added to completely dissolve the cells, and measured by ELISA reader at 450 nm.
세포의 Viability 측정(MTT assay)Viability measurement of cells (MTT assay)
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole (MTT) 정량은 Mosmann의 방법을 변형하여 실시하였다. 멜라닌 양 측정 시 plate를 duplicate로 하여 똑같은 방법으로 시료를 처리하여 배양 후 MTT (50 ㎎/mL) 시약을 처리 (0.1㎎)하여 형성된 포마잔(formazan)을 DMSO롤 녹인 후 540 nm에서 ELISA reader로 측정하여 대조군과 비교하였다.Determination of 3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, a tetrazole (MTT) was performed by modifying Mosmann's method. When measuring the amount of melanin, the sample was treated in the same manner with duplicate plates, and after incubation, formazan (formazan) formed by treating MTT (50 mg / mL) reagent (0.1 mg) was dissolved in DMSO, followed by ELISA reader at 540 nm. Measured and compared with control.
상기 표 1에 나타난 바와 같이 산박하 조출물인 상태에서 50㎍/mL의 농도에서 세포의 증식을 거의 억제하지 않은 수준에서 좋은 효과를 보였고, 분획층에서는 같은 농도에서 세포증식을 억제하는 결과에 의해 멜라닌 생성 억제 효과를 보여 세포 증식에 영향을 주지 않는 농도로 시료를 희석하여 melan-a cell과 B16 cell에 처리하여 멜라닌 생성 억제효과를 확인하였다. 상기 표에서 음의 부호를 가지는 결과는 멜라닌생성을 촉진한다는 의미이다.As shown in Table 1, it showed a good effect at the level of almost inhibiting the proliferation of cells at the concentration of 50 ㎍ / mL in the state of acidic extract, melanin by the result of inhibiting cell proliferation at the same concentration in the fractionation layer Inhibition of melanin production was confirmed by diluting the sample to a concentration that does not affect cell proliferation and treating it with melan-a cells and B16 cells. A negative sign in the table indicates that it promotes melanogenesis.
상기 표 2에서 나타난 바와 같이 에틸아세테이트 분획 추출물에서 세포 증식에 영향을 주지 않는 농도에서도 melan-a 및 B16 cell 모두에서 탁월한 멜라닌 생성 억제 효과를 나타내었다.As shown in Table 2, the ethyl acetate fraction extract showed excellent melanin production inhibitory effect in both melan-a and B16 cells even at concentrations that did not affect cell proliferation.
<실시예 2 : 산박하로부터 미백 물질의 분리 및 구조 결정>Example 2 Separation and Structure Determination of Whitening Materials from Acid Peppermint
실시예 1의 산박하 전초에서 얻은 용매 분획 중 미백 활성이 우수한 에틸아세테이트 분획을 대상으로 하기의 과정에 따라 활성물질을 분리하였다. 도 1, 도 2, 도 3은 분획하고 분리하는 과정을 표시하였다.In the solvent fraction obtained from the acidic starch outpost of Example 1, an ethyl acetate fraction having excellent whitening activity was isolated according to the following procedure. 1, 2, and 3 show the process of fractionation and separation.
에틸아세테이트 분획 7.752g중 4.4783g을 우선 celite를 통해 헥산 분획 0.04g 디메틸클로라이드 분획 3.29g, 에틸아세테이트 분획 1.03g, 메탄올 분획 0.24g 을 얻어 멜라닌 생성 억제 실험을 우선적으로 시행한 결과, CH2Cl2 분획층에서 좋은 효능을 보여 순상 실리카겔 (Merck사)을 유리 컬럼 (3×24 cm)에 전개 용매 CHCl3와 MeOH를 0~100~%의 gradient로 용출 분리하였다. 이중 10%에서 얻은 1.5307g을 다시 순상 실리카겔로 유리 컬럼 (3×30 cm)에 전개 용매 (CHCl3 : MeOH = 10:1)로 서스펜션시켜 충진 한 후 6개의 분획을 얻었다. 이 중 멜라닌 억제 효과가 좋게 나타나는 F4의 0.2343g을 순상 실리카겔로 유리 컬럼 (3×35 cm)에 전개 용매 (CHCl3 : MeOH = 9:1)로 서스펜션시켜 충진 한 후 5개의 분획을 얻었다. 그리고 그 중에서 멜라닌 생성 억제 효과가 좋게 나오는 F4-4 0.1405g을 순상 실리카겔로 유리 컬럼 (1.5×35 cm)에 전개 용매 (CH2Cl2 : CHCl3 : Acetone =1 :1 : 1)로 서스펜션시켜 충진 한 후 10개의 분획을 얻었다. 그리고 이 중 멜라닌 억제 활성을 보이는 f6~f8을 모아 prep-HPLC를 이용하여 H(excisanin A)를 분리하였다(도 4).Ethyl acetate-hexane fractions were fractionated over a 7.752g 4.4783g of the first celite dimethyl chloride 0.04g 3.29g fraction, ethyl acetate fraction 1.03g, 0.24g of a result of the methanol fraction obtained preferentially performed in the melanogenesis inhibition test, CH 2 Cl 2 Pure silica gel (Merck) was eluted with developing solvent CHCl 3 and MeOH with a gradient of 0-100% by weight on a glass column (3 × 24 cm). 1.5307 g of the obtained 10% was again charged with a silica gel (3 × 30 cm) by using a developing solvent (CHCl 3 : MeOH = 10: 1), followed by filling with silica gel to obtain 6 fractions. Of these, 0.2343 g of F4, which exhibits a good melanin inhibitory effect, was filled with pure silica gel in a glass column (3 × 35 cm) by suspension with a developing solvent (CHCl 3 : MeOH = 9: 1), and five fractions were obtained. Among them, 0.1405 g of F4-4, which has a good melanin inhibitory effect, was developed on a glass column (1.5 × 35 cm) with pure silica gel (CH 2 Cl 2 : CHCl 3). : 10 fractions were obtained after the suspension was filled with Acetone = 1: 1: 1). Among them, f6 to f8 showing melanin inhibitory activity were collected and H (excisanin A) was isolated using prep-HPLC (FIG. 4).
Prep-HPLC로 분리한 단일 물질 중 H는 수소 핵자기 공명스펙트럼과 탄소 핵자기 공명 스펙트럼에서 excisanin A임을 확인할 수 있었으며 문헌치(Nihon Kagakkai, Chemistry Letters, 1981, 6, 753-756, 1972)와 일치하였다.Among the single materials separated by Prep-HPLC, H was found to be excisanin A in the hydrogen nuclear magnetic resonance spectrum and carbon nuclear magnetic resonance spectrum, which is consistent with Nihon Kagakkai, Chemistry Letters, 1981, 6, 753-756, 1972. It was.
<실험예 2 : 순상 컬럼을 통과한 분획층들의 미백 효과>Experimental Example 2 Whitening Effect of Fraction Layers Passed Through a Normal Phase Column
실시예 2에서 각 컬럼을 통과하면서 얻은 분획들은 농축하여 melan-a 세포를 이용하여 멜라닌 생성 억제 효과를 측정하였다.The fractions obtained while passing through each column in Example 2 were concentrated to measure the melanin production inhibitory effect using melan-a cells.
상기 표 3에서 보는 바와 같이 디클로로메탄 분획층에서 세포독성의 없는 농도로 낮추어 세포에 처리한 결과 낮은 농도에서도 우수한 멜라닌 생성 억제 효능을 나타내었다.As shown in Table 3, the dichloromethane fractionation layer exhibited excellent melanin production inhibitory effect even at low concentrations as a result of treatment with cells lowered to no concentration of cytotoxicity.
도 5은 저해제가 없는 상태(C)의 멜라닌양과 CH2Cl2 와 EtOAc 분획과 Positive control인 알부틴(Ar)의 멜라닌 생성 저해 정도를 나타낸다. 알부틴 보다도 분획물들의 저해능력이 훨씬 우수함을 보여준다.Figure 5 shows the melanin amount in the absence of inhibitor (C), CH 2 Cl 2 and EtOAc fraction and the degree of inhibition of melanin production of arbutin (Ar) as a positive control. Fractions are much better than arbutin.
상기 표 4에서와 같이 10%, 20% 분획에서 낮은 농도에서도 우수한 멜라닌 생성 억제 효과가 나타남을 확인할 수 있었다.As shown in Table 4, it was confirmed that excellent melanin production inhibitory effect was observed even at low concentrations in the 10% and 20% fractions.
상기 표 5은 10%의 분획을 순상 실리카겔을 통해서 다시 분획해서 얻은 6개의 분획들의 멜라닌 생성 억제 효과를 melan-a 세포를 이용하여 농도별로 측정한 결과 F4가 낮은 농도에서도 가장 우수한 효과를 나타내었고, F2와 F3에서도 좋은 효과가 조사되었다.Table 5 shows the melanin production inhibitory effect of the 6 fractions obtained by re-fractionation of 10% fractions through normal silica gel, measured by concentration using melan-a cells. Good effects were also investigated in F2 and F3.
상기 표 6에서는 10%에서 얻은 분획 중 4번째 분획을 다시 컬럼을 통해 얻은 5개의 분획중에서 4번째 분획의 세포 증식에 영향을 미치지 않는 농도에서 melan-a 세포에서의 멜라닌 생성 억제 효과를 측정한 결과 대조군으로 사용된 알부틴에 비해 우수한 효과가 있음을 확인 하였다.In Table 6, the fourth fraction of the fraction obtained in 10% was again measured the melanin production inhibitory effect in melan-a cells at a concentration that does not affect the cell proliferation of the fourth fraction of the five fractions obtained through the column It was confirmed that there is an excellent effect compared to the arbutin used as a control.
상기 표 7에서 분리된 물질 H의 서로 다른 melanocyte에서의 멜라닌 생성 억제 효과를 측정한 결과 에틸아세테이트 분획이나 다음 단계의 분획에 비해 멜라닌 생성 억제 효과는 떨어지나 대조군에 비교하여 우수한 효능을 보이고 있고, 전단계의 분획이 멜라닌 생성 효과가 더 좋게 나타난 것은 excisanin A와 다른 화합물들의 시너지 효과에 의한 것으로 여겨진다.As a result of measuring the melanin production inhibitory effect on the different melanocytes of the substance H isolated in Table 7, the melanin production inhibitory effect is lower than that of the ethyl acetate fraction or the next step fraction, but shows superior efficacy compared to the control group, The better melanogenesis effect of the fraction is believed to be due to the synergistic effect of excisanin A and other compounds.
<실시예 3: UV B 조사에 대한 멜라닌 생성 억제 효과>Example 3: Melanin Production Inhibition Effect on UV B Irradiation
UV B 조사에 따른 멜라닌 생성 억제 효과 측정Measurement of melanin production inhibitory effect by UV B irradiation
Melan-a cells을 RPMI 배지를 이용하여 24 well plate에 1×105 cells/mL로 plating 한 후, 시료를 처리하여 37℃ 10% CO2 항온기에서 하루 동안 배양하였다. 다음 날부터 이틀 동안 UV B 10mJ/cm2로 조사한 후 시료가 들어있는 새배지로 교환하고 하루동안 더 배양한 후 Plate의 배지 제거 후 세포를 수확하여 1N NaOH를 첨가하여 세포를 완전히 녹인 후 450 nm에서 ELISA로 측정하여 대조군과 비교하였다.Melan-a cells were plated at 1 × 10 5 cells / mL in a 24 well plate using RPMI medium, and the samples were treated and incubated in a 37 ° C. 10% CO 2 incubator for one day. After irradiating with UV B 10mJ / cm 2 for 2 days from the next day, exchange it with a new medium containing the sample, incubate for one more day, remove the plate medium, harvest the cells, add 1N NaOH, and completely dissolve the cells 450 nm Measured by ELISA at and compared with control.
합성 멜라닌을 이용하여 standard soultion을 만들고 sample과 standard soultion을 96 well plate에 넣고 흡광도를 측정한다. 멜라닌 농도는 합성 멜라닌으로 작성된 표준 농도 곡선으로부터 결정하였다.Make standard soultion using synthetic melanin and measure the absorbance by putting sample and standard soultion in 96 well plate. Melanin concentrations were determined from standard concentration curves prepared with synthetic melanin.
세포의 Viability 측정(MTT assay)Viability measurement of cells (MTT assay)
MTT 정량은 Mosmann의 방법을 변형하여 실시하였다. 멜라닌 양 측정 시 plate를 duplicate로 하여 똑같은 방법으로 시료를 처리하여 배양 후 MTT (50 ㎎/mL) 시약을 처리 (0.1㎎)하여 형성된 formazan을 DMSO를 녹인 후 540 nm에서 ELISA롤 측정하여 대조군과 비교하였다.MTT quantification was performed by modifying Mosmann's method. When measuring the melanin amount, the plate was duplicated and the sample was treated in the same way. After incubation, formazan formed by treatment with MTT (50 mg / mL) reagent (0.1 mg) was dissolved in DMSO and measured by ELISA at 540 nm. It was.
RT-PCRRT-PCR
Melan-a cells을 RPMI 배지를 이용하여 60mm plate에 4.5×105 cells/well로 plating 한 후, 시료를 처리하여 37℃ 10% CO2 항온기에서 하루 동안 배양하였다. 다음 날부터 이틀 동안 UV B 10mJ/cm2로 조사한 후 시료가 들어있는 새배지로 교환하고 하루 동안 더 배양한 후 Plate의 배지를 제거하고 Tri-zol solution (Invitrogen, USA) 을 이용하여 RNA를 추출하였다. 1μL의 total RNA를 oligo (dT)18 primer, dNTP (0.5 μM), 1 unit RNase inhibitor, M-MuLV reverse transcriptase (2U)로 70℃ 5min, 4℃ 5min, 37℃ 60min, 그리고 70℃에서 10min heating시켜 cDNA를 합성하였다. Polymerase chain reaction (PCR)은 합성된 cDNA로부터 primer들을 증폭시키기 위하여 2μL cDNA, 10×buffer (10mM Tris-HCl, pH 8.3, 50mM KCl, 0.1% Triton X-100), 250μM dNTP, 1unit Taq polymerase(Promega, USA)를 증류수로 전체를 25μL로 맞춘 후 94℃에서 45s간 denaturation, 55~60℃에서 45s간 annealing, 72℃에서 90s간 extension을 25~30 cycles 수행하여 amplification 하였다. 사용된 primer는 다음과 같다.Melan-a cells were plated at 4.5 × 10 5 cells / well on a 60 mm plate using RPMI medium, and the samples were treated and incubated in a 37 ° C. 10% CO 2 incubator for one day. After irradiating with UV B 10mJ / cm 2 for 2 days from the next day, exchange with a new medium containing the sample, incubate for one more day, remove the plate medium, and extract RNA using Tri-zol solution (Invitrogen, USA). It was. 1 μL total RNA was heated at 70 ° C 5min, 4 ° C 5min, 37 ° C 60min, and 10min at 70 ° C with oligo (dT) 18 primer, dNTP (0.5 μM), 1 unit RNase inhibitor, M-MuLV reverse transcriptase (2U) CDNA was synthesized. Polymerase chain reaction (PCR) was performed with 2μL cDNA, 10 × buffer (10mM Tris-HCl, pH 8.3, 50mM KCl, 0.1% Triton X-100), 250μM dNTP, 1unit Taq polymerase (Promega) to amplify primers from synthesized cDNA. , USA) was amplified by distilled water at 25 μL, followed by denaturation at 94 ° C for 45s, annealing at 45s for 45s, and extension for 90s at 72 ° C for 25-30 cycles. The primers used are as follows.
도 6에서 산박하 에틸아세테이트 분획층이 농도 의존적으로 mitf, tyrosinase의 mRNA 발현을 현저히 억제하고 있음을 확인하였다. Excisanin A로 구조가 규명된 H인 경우 처리 농도 1 ㎍/mL에서 mitf, tyrosinase, c-kit의 mRNA 발현을 현저히 억제 시켰으며, MC1r, TRP-1은 미미하게 억제 하였으며, TRP-2에는 거의 영향을 미치지 않고 있음을 확인하였다. 전반적으로 대조군인 arbutin(Ar)과 hydroquinone(HQ)보다 우수한 억제활성을 지님을 알 수 있다.In Figure 6 it was confirmed that the acidic ethyl acetate fraction layer significantly inhibited mRNA expression of mitf, tyrosinase in a concentration-dependent manner. Excisanin A significantly inhibited the expression of mitf, tyrosinase and c-kit mRNA at 1 ㎍ / mL, and suppressed MC1r and TRP-1, but had little effect on TRP-2. It was confirmed that it does not have. In general, it can be seen that it has superior inhibitory activity than the control group arbutin (Ar) and hydroquinone (HQ).
도 7에서 산박하 EtOAc층과 분리한 excisanin A의 tyrosinase 발현 억제 정도를 western blotting을 통해 확인한 결과, 대조군인 arbutin과 hydroquinone에 비해 단백질 수준에서 강한 억제 효과를 나타내었다.In FIG. 7, Western blotting confirmed the degree of inhibition of tyrosinase expression of excisanin A isolated from the acid and mint EtOAc layers, and showed a strong inhibitory effect at the protein level compared to the control groups arbutin and hydroquinone.
표 9는 UV B 10mJ/cm2로 이틀간 조사 후 멜라닌 생성 억제 정도를 측정한 값으로 조사 전과 마찬가지로 대조군으로 사용된 hydroquinone과 비슷하거나 좋은 효과를 보이고 있었고, 알부틴에 비해서는 월등한 효과를 나타내고 있음을 확인 하였다. 그러나 여기에서도 전단계 분획보다는 멜라닌 생성 억제 효과가 비교적 낮게 나타남을 확인함으로써 전단계에서의 활성물질들의 시너지 효과가 작용하고 있음을 확인할 수 있었다.Table 9 is a measure of the inhibition of melanin production after two days of irradiation with UV B 10mJ / cm 2 , showing similar or better effects than hydroquinone used as a control as before the irradiation, and superior effect to arbutin. Confirmed. However, it was also confirmed that the synergistic effect of the active substances in the previous step was confirmed by confirming that the melanin production inhibitory effect was relatively lower than the previous step fraction.
도 1는 산박하 EtOAc 분획층의 추출분리 계통도1 is an extractive separation system diagram of an acid-mineral EtOAc fraction layer.
도 2은 F4의 분리도2 is an exploded view of F4
도 3는 F4-4로부터 excisanin A의 분리 계통도3 is a flow diagram of the separation of excisanin A from F4-4
도 4는 분획 F4-4의 f6의 HPLC 분석 결과4 shows the results of HPLC analysis of f6 of the fraction F4-4.
도 5은 디클로로메탄과 EtOAc 분획 추출물의 멜라닌생성저해도를 보여주는 사진Figure 5 is a photograph showing the melanogenesis inhibition of dichloromethane and EtOAc fraction extract
도 6은 산박하 용매분획물과 H의 멜라닌 생합성 관련 유전자 발현조절 결과FIG. 6 shows the results of regulation of gene expression related to melanin biosynthesis of acidic peppermint solvent fraction and H.
도 7은 산박하 EtOAc 분획층 및 compound들의 melan-a cell에서의 western blotting 결과FIG. 7 shows western blotting results in melan-a cells of acid and mint EtOAc fraction layers.
Claims (6)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020070082919A KR100888742B1 (en) | 2007-08-17 | 2007-08-17 | Whitening cosmetic compositions containing extract of Isodon inflexus |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020070082919A KR100888742B1 (en) | 2007-08-17 | 2007-08-17 | Whitening cosmetic compositions containing extract of Isodon inflexus |
Publications (2)
Publication Number | Publication Date |
---|---|
KR20090018459A true KR20090018459A (en) | 2009-02-20 |
KR100888742B1 KR100888742B1 (en) | 2009-03-17 |
Family
ID=40686733
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
KR1020070082919A KR100888742B1 (en) | 2007-08-17 | 2007-08-17 | Whitening cosmetic compositions containing extract of Isodon inflexus |
Country Status (1)
Country | Link |
---|---|
KR (1) | KR100888742B1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR100967617B1 (en) * | 2008-02-20 | 2010-07-05 | 재단법인 제주하이테크산업진흥원 | Antioxidation and whitening cosmetic compositions containing extract of Isodon inflexus var. canescens |
KR20180057227A (en) * | 2016-11-22 | 2018-05-30 | 신동선 | The composition comprising the extracts of Ramaria formosa(Fr.) Quel for prevention of skin whitening as an active ingredient |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20200114352A (en) | 2019-03-28 | 2020-10-07 | 주식회사 웰스킨 | Whitening cosmetic composition containing extract of Euonymus hamiltonianus |
-
2007
- 2007-08-17 KR KR1020070082919A patent/KR100888742B1/en active IP Right Grant
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR100967617B1 (en) * | 2008-02-20 | 2010-07-05 | 재단법인 제주하이테크산업진흥원 | Antioxidation and whitening cosmetic compositions containing extract of Isodon inflexus var. canescens |
KR20180057227A (en) * | 2016-11-22 | 2018-05-30 | 신동선 | The composition comprising the extracts of Ramaria formosa(Fr.) Quel for prevention of skin whitening as an active ingredient |
Also Published As
Publication number | Publication date |
---|---|
KR100888742B1 (en) | 2009-03-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
KR102126469B1 (en) | Cosmetic Compositions comprising extract of Dendrobium candidum flower extract | |
CN109820739A (en) | A kind of skin composition and its preparation method and application of Plantain skin lightening | |
WO2008147022A1 (en) | Anti-wrinkle composition for external applications to the skin containing biflavonoid derivatives | |
KR100888743B1 (en) | Cosmetic compositions containing extracts of Distylium racemosum for anti-oxidation, skin whitening and anti-wrinkles | |
KR20100111066A (en) | Skin whitening composition | |
KR100888742B1 (en) | Whitening cosmetic compositions containing extract of Isodon inflexus | |
TW201630589A (en) | Compositions including obacunone and methods of using the same in skin whitening applications | |
KR20130001123A (en) | Composition for regulating expression of pigmentation-related genes containing microrna | |
KR102066652B1 (en) | Cosmetic composition for skin whitening comprising the extract of Silene seoulensis Nakai as active ingredient | |
KR100967073B1 (en) | cosmetic compositions containing extract of Carpinus tschonoskii used for skin whitening | |
KR100959393B1 (en) | Skin whitening cosmetics composition containing an effective ingredient extracted from Acanthopanax sessiliflorum Seeman and manufacturing method thereof | |
KR20200064899A (en) | Cosmetic Composition Containing Fraction of Extract Derived from Passiflora edulis for Skin-Whitening | |
JP6143167B2 (en) | Microphthalmia-related transcription factor inhibitor, melanin production inhibitor, cosmetic composition and anticancer agent | |
KR100967617B1 (en) | Antioxidation and whitening cosmetic compositions containing extract of Isodon inflexus var. canescens | |
KR101781111B1 (en) | Cosmetic composition containing extracts of magnolia officinalis, buplerum falcatum and/or schizonepeta tenuifolia | |
KR102202554B1 (en) | Skin whitening cosmetic composition with the extract of Saururus chinensis leaf, the extract of Grape seed, the extract of Portulaca oleracea and rice fermented product | |
KR20150000719A (en) | Cosmetic Composition Comprising Persicaria thunbergii Extract for Skin Whitening | |
KR101618180B1 (en) | Cosmetic composition comprising an extract of the tissue-cultured raoulia australis shoot | |
KR102021263B1 (en) | PHARMACEUTICAL COMPOSITION Porphyra dentata Kjellman EXTRACT FOR SKIN REGENERATION. | |
KR101959811B1 (en) | Method for preparing fermented extract of Sargassum thunbergii and Colpomenia expansa and cosmetic composition containing the same | |
KR102124698B1 (en) | Cosmetic composition for improving skin wrinkle or skin aging comprising mixture extract of Chrysanthemum morfiolium, Aloe vera, and Hedera Helix | |
KR101317433B1 (en) | The cosmetic composition for skin whitening comprising the mixture of extract of Hovenia dulcia, Platycodon grandiflorum, wheat bud and sage | |
KR20100102439A (en) | A cosmetic composition comprising an extract of tissue cultured anoectochilus formosanus and a preparation method of the extract of tissue cultured anoectochilus formosanus | |
KR102491296B1 (en) | Skin whitening cosmetic composition using pear peel | |
KR102688902B1 (en) | Cosmetic composition using unripe citrus unshiu extract containing citrus bio-gel |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A201 | Request for examination | ||
E902 | Notification of reason for refusal | ||
E701 | Decision to grant or registration of patent right | ||
GRNT | Written decision to grant | ||
FPAY | Annual fee payment |
Payment date: 20130311 Year of fee payment: 5 |
|
FPAY | Annual fee payment |
Payment date: 20140304 Year of fee payment: 6 |
|
FPAY | Annual fee payment |
Payment date: 20150306 Year of fee payment: 7 |
|
FPAY | Annual fee payment |
Payment date: 20160322 Year of fee payment: 8 |
|
FPAY | Annual fee payment |
Payment date: 20170324 Year of fee payment: 9 |
|
FPAY | Annual fee payment |
Payment date: 20180102 Year of fee payment: 10 |
|
FPAY | Annual fee payment |
Payment date: 20190311 Year of fee payment: 11 |