KR101669359B1 - composition for promoting melanin synthesis - Google Patents
composition for promoting melanin synthesis Download PDFInfo
- Publication number
- KR101669359B1 KR101669359B1 KR1020150033718A KR20150033718A KR101669359B1 KR 101669359 B1 KR101669359 B1 KR 101669359B1 KR 1020150033718 A KR1020150033718 A KR 1020150033718A KR 20150033718 A KR20150033718 A KR 20150033718A KR 101669359 B1 KR101669359 B1 KR 101669359B1
- Authority
- KR
- South Korea
- Prior art keywords
- extract
- oak
- ethanol
- azalea
- melanin
- Prior art date
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Abstract
본 발명은 철쭉 추출물, 싸리 추출물, 굴참나무 목부 추출물 또는 이들의 혼합물을 포함하는 조성물에 관한 것으로써, 상기 조성물을 처리 시 멜라닌 합성을 유도하여 멜라닌 합성 장애에 의한 백반증 및 백모 개선 효과를 보이며, 이는 화학약품을 사용하지 않고 천연물만을 유효성분으로 한 백반증 또는 백모 개선용 조성물이므로, 백반증 또는 백모 개선을 위한 화장품, 약학 조성물 또는 건강기능식품으로써 유용하게 활용할 수 있다.The present invention relates to a composition comprising azalea extract, sari extract, oakwood extract or mixtures thereof, wherein melanin synthesis is induced upon treatment of the composition, thereby exhibiting an effect of improving vitiligo and white moth by melanin synthesis disorder, Since it is a composition for alleviating vitiligo or white moth, which contains only natural substances as an active ingredient without using chemicals, it can be usefully used as cosmetic, pharmaceutical composition or health functional food for alleviating vitiligo or white moth.
Description
본 발명은 멜라닌 합성을 촉진하는 조성물에 관한 것으로써, 보다 상세하게는 멜라닌 합성을 유도하여 백반증 또는 백모에 치료 효과가 있는 조성물에 관한 것이다.The present invention relates to a composition for promoting melanin synthesis, and more particularly, to a composition which induces melanin synthesis and has a therapeutic effect on vitiligo or white moth.
백반증은 피부 표피에 존재하는 멜라닌 세포에서 멜라닌이 오랫동안 생성되지 않아 하얀색 패치가 유발되는 색소 결핍증이다. 백반증이 유발되는 원인은 아직까지 정확하게 알려진 것은 없지만 유전적인 요인, 산화 활성 등의 자극에 의한 신경세포의 비정상적인 작용, 자가면역 파괴 등 멜라닌 세포의 기능을 감소시키는 것에 대한 가설들이 제시되고 있다. Vitiligo is a pigment deficiency in which melanin is not produced for a long time in melanocytes in skin epidermis, leading to white patches. Although the cause of vitiligo is not known precisely yet, hypotheses have been suggested for reducing the function of melanocytes such as genetic factors, abnormal action of nerve cells by stimulation of oxidative activity, and autoimmune destruction.
최근에는 이러한 가설들이 복합적으로 작용하여 백반증이 유발된다는 설이 주목받고 있다. 백반증을 가진 많은 환자들에 있어서 하얀 반점은 삶의 질에 큰 영향을 미치는 것으로 알려져 있으며 최근 신문기사에 의하면 백반증 환자의 취업문제, 대인관계 문제가 심각한 것으로 보도되었다. 이는 백반증이 작은 장애로 보일 수 있지만 정도가 심한 색소 부족 질병을 가진 사람에게는 심리학적 자아 존중감 및 사회 활동에 영향을 줄 수 있다는 것을 의미한다. Recently, it has been noticed that these hypotheses are combined to cause vitiligo. In many patients with vitiligo, white spots are known to have a significant impact on quality of life, and recent reports have reported serious problems in employment and interpersonal problems in patients with vitiligo. This means that vitiligo can be seen as a small disorder, but it can affect psychological self-esteem and social activity for people with severe pigmented disease.
백반증과 유사한 질병인 백모도 최근 경제 성장에 따른 문화 수준향상으로 외모에 대한 관심이 높아지면서 주목받고 있다. 백모는 모발색이 밝은 백인에 비해 흑인 모발을 가진 동양인에게 더욱 뚜렷이 나타나기 때문에 보건미용, 심리적인 면에서 연구대상이 되고 있다. 백모가 많을수록 백반증과 마찬가지로 심리적으로 우울하거나 스트레스가 증가하며 대인관계나 사회활동에서 불편함을 느끼고 있다. Baekmu, a disease similar to vitiligo, is also attracting attention due to the growing interest in appearance due to the recent improvement in the cultural level due to economic growth. White moths are more likely to be studied in health, beauty, and psychology because they appear more clearly in Asian people with black hair than in white ones. The more white moths are, the more psychological depression and stress increase like vitiligo, and feel discomfort in interpersonal relations and social activities.
이러한 백반증 또는 백모를 치료하기 위하여 색소 세포인 멜라닌 세포를 활성화 하는 것이 중요하며 멜라닌 세포의 활성화로 인한 멜라닌의 합성이 색소 재침착(repigmentation)에 큰 역할을 한다. 따라서 백반증 및 백모 치료의 목표는 주변 정상부 또는 병변 내부의 모낭에 존재하는 비활동성 멜라닌 세포 또는 색소 모세포를 자극하여 분화, 증식 및 이동을 촉진시키고 멜라닌을 생성하게 하는 것이다. It is important to activate melanocytes, which are pigment cells, in order to treat such vitiligo or white moth, and melanin synthesis due to activation of melanocytes plays a major role in pigment repigmentation. Therefore, the goal of treatment of vitiligo and white hair is to stimulate inactive melanocytes or pigment cells present in the hair follicles in the periphery or inside the lesion to promote differentiation, proliferation and migration and to produce melanin.
멜라닌 합성은 멜라닌 세포에서 멜라닌의 합성을 촉매 하는데 중요한 효소로 알려진 티로시나아제(tyrosinase)가 티로신(tyrosine)과 반응하여 도파(dopa)로, dopa가 다시 tyrosinase와 반응하여 도파-퀴논(dopa-quinone)으로 변환함으로써 최종적으로 멜라닌이 생성된다. 멜라닌은 검은색과 갈색을 띄는 유멜라닌(eumelanin)과 빨간색과 노란색을 띄는 페오멜라닌(pheomelanin)으로 나뉘며 pheomelanin의 경우 아미노산인 시스테인과 반응하면 생성되는 것으로 알려져 있다. 백반증 치료의 경우 검은색을 띄는 eumelanin의 생성이 높을수록 효과적이다. Melanin synthesis is a key enzyme in the synthesis of melanin in melanocytes. Tyrosinase, which is known to be an important enzyme, catalyzes the synthesis of melanin. It reacts with tyrosine to form dopa, while dopa reacts with tyrosinase to form dopa-quinone ) To finally produce melanin. Melanin is divided into black and brown eumelanin and pheomelanin with red and yellow. In the case of pheomelanin, melanin is known to be formed by the reaction with amino acid cysteine. The higher the production of black eumelanin in the treatment of vitiligo, the more effective it is.
현재, 백반증의 치료에 있어서 국부의 코르티코스테로이드, 칼시뉴린 억제제, 비타민 D 파생물, 광선치료(UVA, narrow band UVB, 광화학치료), 정상적인 멜라닌 세포를 가지고 있는 표피를 이식하는 수술적인 치료, 국부적인 치료와 광선치료의 조합을 포함한 다양한 치료들이 사용되고 있지만 비흑색종 피부암과 흑색종의 발생정도를 증가시키는 등 부작용을 가지고 있다.Currently, treatment of vitiligo includes local corticosteroids, calcineurin inhibitors, vitamin D derivatives, UVA (narrow band UVB, photochemical treatment), surgical treatment to transplant epidermis with normal melanocytes, local treatment And phototherapy, but they have side effects such as increased incidence of non-melanoma skin cancer and melanoma.
더불어 백모 억제에 관한 연구에 사용되는 아이소뷰틸 메틸잔틴(isobutyl-methylxanthine,IBMX)은 멜라닌 생성을 촉진시키고 작은 안구증 연관 전사 요소(Microphthalmia-associated transcription factor, MITF)와 tyrosinase의 단백질 수준을 증가시켜 백모를 억제하는 효능을 가지고 있는 것으로 보고된 바 있다. 현재 백모 해결방안으로 다양한 모발 염모제가 사용되고 있는 실정이다. 그러나 이런 영구 염모제는 식물성 염모제, 금속성 염모제, 합성 염모제 등으로 이루어지기 때문에 화학작용으로 인하여 모발과 두피에 손상, 자극 등의 부작용을 가지고 있다. In addition, isobutyl-methylxanthine (IBMX), which is used in studies on whitefly inhibition, promotes melanogenesis and increases protein levels of microphthalmia-associated transcription factors (MITF) and tyrosinase, Of the patients. Currently, various hair dyeing agents are being used as a solution to white hair. However, since this permanent hair dye is composed of a vegetable hair dye, a metallic hair dye, a synthetic hair dye and the like, it has side effects such as damage and stimulation to hair and scalp due to chemical action.
따라서 부작용이 적으며 멜라닌 생성에 효과적인 천연물에 대한 연구와 개발이 요구되고 있다.Therefore, there is a demand for research and development of natural products which have few side effects and are effective for the production of melanin.
따라서 본 발명은 부작용이 적은 천연물질을 포함하는 멜라닌 합성 촉진용 조성물을 제공하는 데 그 목적이 있다.Accordingly, it is an object of the present invention to provide a composition for promoting melanin synthesis, which comprises a natural substance having few side effects.
상기 목적을 달성하기 위하여, 본 발명은 철쭉 추출물을 포함하는 멜라닌 합성 촉진용 조성물을 제공한다.In order to attain the above object, the present invention provides a composition for promoting melanin synthesis comprising azalea extract.
상기 목적을 달성하기 위하여, 본 발명은 철쭉 추출물, 싸리 추출물, 굴참나무 추출물 또는 이들의 혼합물을 포함하는 멜라닌 합성 촉진용 조성물을 제공한다.In order to achieve the above object, the present invention provides a composition for promoting melanin synthesis, which comprises azalea extract, sari extract, oak oak extract or a mixture thereof.
본 발명은 철쭉 추출물, 싸리 추출물, 굴참나무 목부 추출물 또는 이들의 혼합물을 포함하는 조성물에 관한 것으로써, 상기 조성물을 처리 시 멜라닌 합성을 유도하여 멜라닌 합성 장애에 의한 백반증 및 백모 개선 효과를 보이며, 이는 화학약품을 사용하지 않고 천연물만을 유효성분으로 한 백반증 또는 백모 개선용 조성물이므로, 백반증 또는 백모 개선을 위한 화장품, 약학 조성물 또는 건강기능식품으로써 유용하게 활용할 수 있다. The present invention relates to a composition comprising azalea extract, sari extract, oakwood extract or mixtures thereof, wherein melanin synthesis is induced upon treatment of the composition, thereby exhibiting an effect of improving vitiligo and white moth by melanin synthesis disorder, Since it is a composition for alleviating vitiligo or white moth, which contains only natural substances as an active ingredient without using chemicals, it can be usefully used as cosmetic, pharmaceutical composition or health functional food for alleviating vitiligo or white moth.
도 1은 본 발명에 따른 철쭉, 싸리와 굴참나무 목부 에탄올 추출물 추출물의 버섯형 티로시나아제 활성을 분석한 것이고,
도 2 및 도 3은 본 발명에 따른 철쭉, 싸리와 굴참나무 목부 에탄올 추출물의 B16 흑색종(melanoma) 세포에 대한 세포독성을 분석한 것이고,
도 4는 본 발명에 따른 철쭉, 싸리와 굴참나무 목부 에탄올 추출물의 B16 melanoma 세포에서 티로시나아제(tyrosinase) 활성에 대한 영향을 분석한 것이고,
도 5는 본 발명에 따른 철쭉, 싸리와 굴참나무 목부 에탄올 추출물의 B16 melanoma 세포에서 멜라닌 생성에 대한 영향을 분석한 것이고,
도 6은 (C57/BL6×BALB)F1 마우스의 털에서 본 발명에 따른 철쭉, 싸리와 굴참나무 목부 에탄올 추출물 처리군의 멜라닌 색소 침착에 대한 육안적인 영향을 나타낸 것이고,
도 7은 (C57/BL6×BALB)F1 마우스의 털에서 본 발명에 따른 철쭉, 싸리와 굴참나무 목부 에탄올 추출물 처리군의 멜라닌 생성에 대한 영향을 분석한 것이고,
도 8은 (C57/BL6×BALB)F1 마우스의 피부에서 본 발명에 따른 철쭉, 싸리와 굴참나무 목부 에탄올 추출물 처리군의 멜라닌 세포 변화에 대한 영향을 육안적으로 나타낸 것이다. 1 is an analysis of the mushroom-type tyrosinase activity of ethanol extracts of azaleas, sari and oak wood of the present invention,
FIGS. 2 and 3 are graphs showing cytotoxicity of B16 melanoma cells of ethanol extracts of azaleas, sari and oak wood of the present invention,
FIG. 4 is a graph showing the effect of ethanol extracts of azaleas, sari and oakwood on the tyrosinase activity of B16 melanoma cells according to the present invention,
FIG. 5 is a graph showing the effect of ethanol extracts of azaleas, sari and oak oak wood on melanin production in B16 melanoma cells according to the present invention,
Fig. 6 shows the visual effects of melanin pigment deposition on the hair of the (C57 / BL6 x BALB) F1 mouse treated with the ethanol extract of azalea, sari and oak wood of the present invention,
FIG. 7 is a graph showing the effect of ethanolic extracts of Azalea, Sirius and Quercus serrata according to the present invention on melanin production in hair of (C57 / BL6xBALB) F1 mice,
FIG. 8 is a graphical representation of the effect of the ethanol extract of Azalea, Sari and Quercus serrata according to the present invention on the skin of (C57 / BL6 × BALB) F1 mice.
본 발명의 발명자들은 내성이나 안전성에 대한 문제가 대두되어지지 아니한 천연물 추출물의 기능성에 대하여 연구하던 중, 철쭉, 싸리와 굴참나무 목부 에탄올 추출물을 마우스에 처리할 시 멜라닌 합성이 유도되어 백반증과 백모에 개선 효과를 보이는 것을 확인하여 본 발명을 완성하였다. The inventors of the present invention have investigated the functionality of natural extracts which have not suffered from resistance or safety problems, and melanin synthesis was induced when ethanol extract of azaleas, And thus the present invention has been completed.
상기 철쭉(Rhododendron schlippenbachii)은 진달래과(Ericaceae) 진달래속(Rhododendron)에 속하는 식물로써 화경에 뚜렷한 검은 점들을 가지고 있으며, 꽃받침에 끈적끈적한 액이 있다. 철쭉의 꽃에는 그레이아노톡신(grayanotoxin) 등의 독성성분으로 인한 호흡마비 등의 위험성이 알려져 있으나, 철쭉의 잎과 목부는 고혈압의 치료 목적이나 당뇨와 관련하여 당대사 억제에 관련된 연구 소재로 사용되어 왔다. 국내에서는 주로 관상용으로 재배되는 특성상 철쭉 목부의 다양한 생리활성에 대한 연구는 아직까지 미흡한 상태이며, 일부 독성에 대한 증례만 보고되고 있다. Rhododendron schlippenbachii ( Rhododendron schlippenbachii ) is a plant belonging to the genus Rhododendron (Ericaceae). It has distinctive black spots on the flower bud and has a sticky solution on the calyx. Azaleas are known to have respiratory paralysis caused by toxic components such as grayanotoxin. However, leaf and throat of azaleas are used as research materials for the treatment of hypertension and inhibition of glucose metabolism related to diabetes come. Studies on various physiological activities of azalea spp. Are still insufficient, and only some cases of toxicity have been reported.
상기 싸리(Lespedeza bicolor)는 콩과 식물로 줄기가 매우 곧고 꽃대가 길게 나오는 것을 특징으로 하고 있다. 싸리 목부에는 항산화 활성 및 항균활성을 가지고 있으며 구성성분으로는 여러 가지 알칼로이드, 플라보노이드, 아스코르빈산, 사포닌, 탄닌 등을 포함하고 있다. 싸리나무 추출물은 예로부터 타박상, 두통, 심장병, 알레르기, 아토피 등 다양한 질병을 완화시키는데 효과가 있다고 알려져 있다. 또한 싸리나무 추출물을 이용한 전자공여능, 과산화물제거효소(Superoxide dismutase,SOD) 유사 활성, 아질산염 소거능에 대한 연구가 수행되어 왔다. 하지만 싸리나무의 목부를 이용한 생리활성에 대한 연구 결과가 미비하며 싸리의 뿌리 및 꽃을 이용하여 의학 분야에 응용한 연구 결과가 다수인 것으로 보고되고 있다. Lespedeza bicolor is a legume plant characterized by very straight stems and long peduncle. It has antioxidant activity and antimicrobial activity and contains various alkaloids, flavonoids, ascorbic acid, saponin, and tannin. Sri Lanka extract has been known to be effective in alleviating various diseases such as bruises, headache, heart disease, allergy, and atopy. Studies on electron donating ability, superoxide dismutase (SOD) - like activity, and nitrite scavenging ability using Sri Lanka extract have been carried out. However, the results of studies on the physiological activity using the thrips of the sorghum have not been reported, and many research results have been reported that have been applied to the medical field using sari roots and flowers.
상기 굴참나무(Quercus variabilis)는 쌍떡잎식물 참나무목 참나무과의 낙엽교목으로 나무껍질이 노란빛을 띠는 것을 특징으로 하고 있다. 굴참나무는 예로부터 종실을 식용으로 사용하였고 탄닌(tannin), 유지방, 퀘세틴(quercetin) 등 여러 성분이 함유되어 있어 식품으로는 물론 약용 식물로도 아주 유용하여 장 및 혈관을 수축시키는 작용을 한다. 더불어 민간요법에서 잎과 껍질을 수렴성 지혈, 설사, 탈황, 치질을 비롯하여 거담, 진통 등에 사용되어 왔다. 참나무류에 대한 물리적 특성, 생육특성, 분포 및 화학적 특성에 대한 연구는 그 동안 많이 진행되어 왔지만 이를 활용한 의학 분야의 개발은 미비한 실정이다. The oak oak ( Quercus variabilis ) is a deciduous arboreous tree with oak tree oak and oak tree, and the bark is yellowish. Quercus mongolica has been used for food for a long time and it is very useful as a food as well as a medicinal plant because it contains various ingredients such as tannin, milk fat, quercetin, . In folk remedies, leaves and bark have been used for astringent hemostasis, diarrhea, desulfurization, hemorrhoids, genomes, and painkillers. Studies on the physical properties, growth characteristics, distribution and chemical properties of oak have been carried out for a long time, but development of the medical field using them has been insufficient.
이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명은 철쭉 추출물을 포함하는 멜라닌 합성 촉진용 조성물을 제공한다.The present invention provides a composition for promoting melanin synthesis, which comprises azalea extract.
더불어 상기 조성물은 싸리 추출물, 굴참나무 추출물 또는 이들의 혼합물을 더 포함할 수 있다.In addition, the composition may further comprise sari extract, oakwood extract or a mixture thereof.
상기 조성물은 철쭉 추출물 5 내지 90 중량%, 싸리 추출물 5 내지 90 중량% 및 굴참나무 추출물 5 내지 90 중량%를 포함한다.The composition comprises 5 to 90% by weight of azalea extract, 5 to 90% by weight of sari extract and 5 to 90% by weight of oak oak extract.
상기 범위를 벗어날 시, 각 성분의 효능이 제대로 발휘되지 않는 문제가 발생할 수 있다.When the concentration is out of the above range, the effect of each component may not be exhibited properly.
상기 추출물은 에탄올, 메탄올, 증류수 및 이들의 혼합물로 이루어진 군에서 선택된 용매로 추출하며, 에탄올을 사용하는 것이 바람직하지만 이제 제한되지는 않는다.The extract is extracted with a solvent selected from the group consisting of ethanol, methanol, distilled water and a mixture thereof, and ethanol is preferably used, but is not limited thereto.
상기 조성물은 멜라닌 형성 장애에 의하여 발생하는 백반증 또는 백모의 예방 또는 치료용 조성물로 사용될 수 있다.The composition may be used as a composition for preventing or treating vitiligo or white moth caused by melanin-forming disorder.
상기 조성물은 화장품 조성물, 약학 조성물 또는 건강기능식품으로써 제공될 수 있다.The composition may be provided as a cosmetic composition, a pharmaceutical composition or a health functional food.
상기 화장품 조성물은 피부 외용연고, 기초화장품, 메이크업 화장품, 바디 화장품 또는 면도용 화장품 등의 용도로 제공될 수 있으며, 상기 기초화장품의 예로는 에센스, 로션, 크림, 젤, 화장수, 팩, 마사지 크림, 유액 등이 있을 수 있고, 상기 메이크업 화장품의 예로는 파운데이션, 립스틱, 아이섀도, 아이라이너, 마스카라, 아이브로우 펜슬, 메이크업 베이스 등이 있을 수 있으며, 바디 화장품으로는 비누, 액체 세정제, 입욕제, 썬 스크린 크림, 썬 오일 등이 있을 수 있고, 면도용 화장품으로는 애프터셰이브로션, 셰이빙 크림 등이 있다.The cosmetic composition may be used for external ointment for skin, foundation cosmetic, make-up cosmetic, body cosmetic or shaving cosmetic. Examples of the basic cosmetic include essence, lotion, cream, gel, lotion, Such as foundation, lipstick, eye shadow, eye liner, mascara, eyebrow pencil, make-up base, etc. The body cosmetics may include soap, liquid cleanser, bath powder, sunscreen Cream, sun oil, etc., and shaving cosmetics include aftershave lotion and shaving cream.
상기 유효성분인 철쭉 추출물, 싸리 추출물, 굴참나무 목부 추출물 또는 이들의 혼합물 외에 추가로 기능성 물질이나 물성 개선을 위한 부형제, 희석제 등의 물질이 포함될 수 있다.In addition to the above-mentioned active ingredients, such as azalea extract, sari extract, oak oak wood extract, and mixtures thereof, functional materials and excipients such as diluents for improving physical properties may be included.
일 예로, 상기 화장품 조성물에는 물성 개선을 위하여 향료, 색소, 살균제, 산화방지제, 방부제, 보습제, 점증제, 무기염류 또는 합성 고분자 물질 등이 추가로 첨가될 수 있다. 이외에 첨가해도 되는 배합 성분으로 유지 성분, 에몰리엔트제, 계면 활성제, 유기 안료, 무기 안료, 유기 분체, 자외선 흡수제, pH 조정제, 알코올, 혈행 촉진제, 냉감제, 제한제 또는 정제수 등일 수 있다.For example, the cosmetic composition may further contain a perfume, a pigment, a bactericide, an antioxidant, an antiseptic, a moisturizer, an increasing agent, an inorganic salt or a synthetic high molecular substance to improve physical properties. An organic pigment, an organic pigment, an ultraviolet absorber, a pH adjuster, an alcohol, a blood circulation accelerator, a cold agent, a limiting agent, or purified water may be used as a blending component that may be added.
상기 유효성분 이외의 첨가해도 되는 배합 성분은 한정되지 않으며, 상기 어느 성분도 본 발명의 목적 및 효과를 손상시키지 않는 범위 내에서 배합 가능하다.The compounding ingredient to be added other than the above-mentioned effective ingredient is not limited, and any of the above-mentioned ingredients can be compounded within a range that does not impair the purpose and effect of the present invention.
또한, 상기 화장품 조성물은 용액, 유화물, 점성형 혼합물 등의 형상을 취할 수 있으며, 당업계에서 통상적으로 제조되는 어떠한 제형일 수 있고 일 예로 스프레이, 유액, 크림, 화장수, 팩, 파운데이션, 로션, 미용액, 모발화장료 등의 제형일 수 있다.The cosmetic composition may be in the form of a solution, an emulsion, a viscous mixture, and the like, and may be any of the formulations conventionally produced in the art. Examples thereof include a spray, a milky lotion, a cream, a lotion, a pack, a foundation, , Hair cosmetics, and the like.
보다 구체적으로 본 발명의 화장품 조성물은 스프레이, 비누, 클렌징폼, 클렌징로션, 클렌징크림, 바디로션, 바디클린저, 스킨로션, 스킨소프너, 스킨토너, 아스트리젠트, 로션, 밀크로션, 모이스쳐 로션, 영양로션, 맛사지 크림, 영양크림, 모이스처크림, 핸드크림, 파운데이션, 에센스, 영양에센스 및 팩의 제형을 포함할 수 있다.More particularly, the cosmetic composition of the present invention can be used as a cosmetic composition in the form of a spray, a soap, a cleansing foam, a cleansing lotion, a cleansing cream, a body lotion, a body cleanser, a skin lotion, a skin softener, a skin toner, an astigmant, a lotion, , Massage cream, nutritional cream, moisturizing cream, hand cream, foundation, essence, nutritional essence and pack.
일 예로, 본 발명의 제형이 스프레이 또는 파우더인 경우에는 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄히드록시드, 칼슘 실리케이트 또는 폴리아미드 파우더가 이용될 수 있으며, 특히 본 발명의 제형이 스프레이인 경우에는 추가적으로 클로로플루오로히드로카본, 프로판/부탄 또는 디메틸 에테르와 같은 추진체를 포함할 수 있다.For example, when the formulation of the present invention is spray or powder, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, particularly when the formulation of the present invention is a spray In addition, propellants such as chlorofluorohydrocarbons, propane / butane or dimethyl ether may be included.
본 발명의 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물섬유, 식물섬유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 이용될 수 있다.When the formulation of the present invention is a paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .
본 발명의 제형이 용액 또는 유탁액의 경우에는 담체 성분으로서 용매, 용매화제, 또는 유탁화제가 이용되고, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 1,3-부틸글리콜 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르일 수 있다.When the formulation 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, -Butyl glycol oil, glycerol aliphatic esters, polyethylene glycols or fatty acid esters of sorbitan.
본 발명의 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 present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.
본 발명의 제형이 계면-활성제 함유 클린징인 경우에는 담체 성분으로서 지방족 알코올 설페이트, 지방족 알코올 에테르 설페이트, 설포숙신산 모노에스테르, 이세티오네이트, 이미다졸리늄 유도체, 메틸타우레이트, 사르코 시네이트, 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 present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linolenic derivatives or ethoxylated glycerol fatty acid esters.
상기 약학조성물은 약학조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 또는 희석제를 더 포함할 수 있다.The pharmaceutical compositions may further comprise suitable carriers, excipients or diluents conventionally used in the manufacture of pharmaceutical compositions.
본 발명에서 사용가능한 담체, 부형제 또는 희석제로는, 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 또는 광물유 등을 들 수 있다.Examples of the carrier, excipient or diluent which can be used in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.
본 발명에 따른 약학조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다.The pharmaceutical composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method .
제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 화합물은 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제할 수 있다. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose sucrose), lactose, gelatin, and the like.
또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .
비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.
본 발명에 따른 약학 조성물의 유효성분인 철쭉 추출물, 싸리 추출물, 굴참나무 목부 추출물 또는 이들의 혼합물의 사용량은 환자의 나이, 성별, 체중 또는 질환에 따라 달라질 수 있으나, 0.001 내지 100mg/kg으로, 바람직하게는 0.01 내지 10mg/kg을 일일 1회 내지 수회 투여할 수 있다. The use amount of the azalea extract, the sari extract, the oakwood extract or the mixture thereof, which is an effective ingredient of the pharmaceutical composition according to the present invention, may vary depending on the age, sex, body weight or disease of the patient but is preferably 0.001 to 100 mg / kg, 0.01 to 10 mg / kg may be administered once to several times per day.
또한, 상기 약학 조성물의 투여량은 투여경로, 질병의 정도, 성별, 체중, 나이 등에 따라서 증감될 수 있다. 따라서, 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.In addition, the dosage of the pharmaceutical composition may be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.
상기 약학 조성물은 쥐, 생쥐, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 기관지 내 흡입, 자궁 내 경막 또는 뇌혈관내(intracerebroventricular) 주사에 의해 투여될 수 있다.The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intratracheal, intrauterine or intracerebroventricular injections.
상기 건강식품은 분말, 과립, 정제, 캡슐, 시럽 또는 음료의 형태로 제공될 수 있으며, 상기 건강식품은 유효성분인 철쭉 추출물, 싸리 추출물, 굴참나무 목부 추출물 또는 이들의 혼합물 이외에 다른 식품 또는 식품 첨가물과 함께 사용되고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 혼합양은 그의 사용 목적 예를 들어 예방, 건강 또는 치료적 처치에 따라 적합하게 결정될 수 있다. The health food may be provided in the form of powder, granules, tablets, capsules, syrups or beverages. The health food may contain other ingredients such as azalea extract, sari extract, oakwood extract or mixtures thereof, And can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its use purpose, for example, prevention, health or therapeutic treatment.
상기 건강식품에 함유된 철쭉 추출물, 싸리 추출물, 굴참나무 목부 추출물 또는 이들의 혼합물의 유효용량은 상기 약학 조성물의 유효용량에 준해서 사용할 수 있으나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 범위 이하일 수 있으며, 유효성분은 안전성 면에서 아무런 문제가 없기 때문에 상기 범위 이상의 양으로도 사용될 수 있음은 확실하다.The effective dose of the azalea extract, the sari extract, the oakwood extract or the mixture thereof contained in the above health food can be used in accordance with the effective dose of the above pharmaceutical composition, but may be appropriately selected for the purpose of health and hygiene, It can be used in an amount of more than the above range because there is no problem in terms of safety of the active ingredient.
상기 건강식품의 종류에는 특별한 제한이 없고, 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등을 들 수 있다.There is no particular limitation on the type of the health food, and examples thereof include meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, Drinks, alcoholic beverages and vitamin complexes.
이하, 하기 실시예를 통해 본 발명을 보다 상세하게 설명한다. 다만, 이러한 실시예에 의해 본 발명이 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.
<< 실시예Example 1> 철쭉, 싸리와 굴참나무 목부 에탄올 추출물 제조 1> Production of ethanol extracts of azaleas, sari and oak wood
본 발명에 사용한 철쭉, 싸리와 굴참나무 목부는 경남 진주시 진주대로 경상대학교 학술림에서 채취한 후 -75℃ 저온 저장고에 보관하여 사용하였으며 이를 실온에서 음건시킨 후 사용하였다. The azaleas, sari and quercus serrata used in the present invention were collected from the scientific forests of Gyeongsang National University, Jinju, Gyeongnam, and stored in a low temperature storage at -75 ° C.
건조한 철쭉, 싸리와 굴참나무 목부를 각각 작은 조각(약 3cm)으로 자른 후 200g에 95% 에탄올 500mL를 혼합하여 실온에서 7일간 추출했다. 추출물은 여과지 (No.2, Whatman)로 여과한 후 동결건조기(freeze dryer)를 이용하여 여과된 추출액을 분말로 제조하였다. Dried azaleas, sari and oak wood were cut into small pieces (about 3 cm), and then 200 g of 95% ethanol (500 mL) was mixed and extracted at room temperature for 7 days. The extract was filtered with a filter paper (No. 2, Whatman), and then the filtered extract was prepared as a powder using a freeze dryer.
<< 실시예Example 2> 항산화 활성 분석 ( 2> Antioxidant Activity Analysis DPPHDPPH 라디칼 Radical 소거능Scatters 검토) Review)
추출물의 항산화 활성은 2,2-디페닐-1-피크릴히드라질(2,2-diphenyl-1-picrylhydrazyl, DPPH, Sigma, USA)을 이용한 DPPH 법에 의거하여 실시하였다(Monica et al., 2007 참고). 상기 실시예 1에서 제조한 추출물을 100ppm의 농도로 희석하여 이를 96 웰 플레이트(Falconⓡ, USA)의 각 웰에 50㎕씩 넣었다. 그리고 0.2 mM DPPH 용액을 200㎕ 첨가하고 실온에서 30분간 반응시킨 후 효소면역측정법(enzyme-linked immunosorbent assay, ELISA, Anthosⓡ, Austria) 리더기로 517nm에서 흡광도를 측정하였다. 대조군으로 뷰틸레이트하이드록시톨루엔(Butylated Hydroxy Toluene, BHT, Sigma, USA) 및 95% 에탄올을 사용하여 동일한 방법으로 측정하였다.The antioxidant activity of the extract was determined by the DPPH method using 2,2-diphenyl-1-picrylhydrazyl (DPPH, Sigma, USA) (Monica et al. 2007). The extract prepared in Example 1 was diluted to a concentration of 100 ppm, and 50 쨉 l of the extract was added to each well of a 96-well plate (Falcon ⓡ , USA). Then, 200 μl of 0.2 mM DPPH solution was added, reacted at room temperature for 30 minutes, and the absorbance was measured at 517 nm using an enzyme-linked immunosorbent assay (ELISA, Anthos ⓡ , Austria) reader. Control group was determined by the same method using butylated hydroxytoluene (BHT, Sigma, USA) and 95% ethanol.
그 결과, 하기 표 1과 같이 철쭉, 싸리, 굴참나무, 혼합(철쭉 에탄올 추출물 : 싸리 에탄올 추출물 : 굴참나무 에탄올 추출물, 1:1:1 (w/w/w)) 에탄올 추출물 각각 약 87, 91, 82 또는 90%로 양성 대조군인 BHT와 비슷한 활성(95%)을 가지는 것으로 확인되었다. 항산화 활성은 폴리페놀 함량과 비례적인 관계로, 높은 폴리페놀 함량 및 생리활성 효과를 기대할 수 있을 것으로 판단된다. As a result, as shown in Table 1, the ethanol extracts of rhodopsin, sari, oak, and ethanol extracts of mixed (rhodamine ethanol extract: sorly ethanol extract: oak oak ethanol extract, 1: 1: 1 (w / w / w) , 82 or 90%, similar to the positive control BHT (95%). Antioxidant activity was proportional to polyphenol contents, so it could be expected that high polyphenol content and physiological activity effect could be expected.
함량(mg/g)**Content (mg / g) **
추출물extract
*: 파우더의 갈산 등가의 mg/g*: Mg / g of galvanic equivalent of powder
**: 파우더의 등가의 퀘세틴 등가의 mg/g**: Equivalent quercetin equivalent of powder mg / g
<< 실시예Example 3> 폴리페놀 함량 분석 3> Analysis of polyphenol contents
추출물의 폴리페놀 함량은 폴린-시오칼토(Folin-Ciocalteu)법에 의거하여 실시하였다(Luis et al., 2007 참고). 갈산(Sigma, USA) 30 ㎕ (1 mg/mL)를 증류수 3 mL에 혼합하고 100 ㎕의 Folin-Ciocalteu(Sigma, USA)시약을 첨가하여 7분간 방치한 다음 300㎕의 탄산나트륨(sodium carbonate)를 혼합하고 30분간 반응시켰다. 그 후 UV-분광광도계(UV-spectrophotometer, U-3000ⓡ, Hitachi, Japan)를 사용하여 765nm에서 흡광도를 측정하여 검량선을 작성하였다. 동일한 방법으로 상기 실시예 1에서 제조한 추출물 분말의 흡광도를 측정한 후 검량선과 비교하여 추출물의 폴리페놀함량을 측정하였다.The polyphenol content of the extract was determined according to the Folin-Ciocalteu method (see Luis et al., 2007). (Sigma, USA) was added to each well. After the addition of 100 μl of Folin-Ciocalteu (Sigma, USA) reagent, the mixture was allowed to stand for 7 minutes and 300 μl of sodium carbonate Mixed and allowed to react for 30 minutes. Then using a UV- spectrophotometer (UV-spectrophotometer, U-3000 ⓡ, Hitachi, Japan) to prepare a calibration curve by measuring the absorbance at 765nm. The absorbance of the extract powder prepared in Example 1 was measured in the same manner and then the polyphenol content of the extract was measured by comparing with the calibration curve.
그 결과, 상기 실시예 2 중 표 1과 같이 싸리 에탄올 추출물, 철쭉 에탄올 추출물과 굴참나무 에탄올 추출물의 총 폴리페놀 함량은 각각 106.9mg/g, 252.1mg/g 또는 116.4mg/g이었고 싸리, 철쭉, 굴참나무 목부 에탄올 추출물의 1:1:1 (w/w/w) 혼합물은 200.2 mg/g 이었다. As a result, the total polyphenol contents of the cari ethanol extract, the azalea ethanol extract and the oak oak ethanol extract were 106.9 mg / g, 252.1 mg / g or 116.4 mg / g, respectively, as shown in Table 1 of Example 2, The 1: 1: 1 (w / w / w) mixture of oak woody ethanol extract was 200.2 mg / g.
<< 실시예Example 4> 플라보노이드 함량 분석 4> Analysis of flavonoid content
추출물의 플라보노이드 함량은 데이비스(Davis's) 법에 의거하여 실시하였다(Monica et al., 2007 참고). 상기 실시예 1에서 제조한 추출물 분말 0.5g을 메탄올 1ml와 혼합하고 디에틸렌 글리콜(diethylene glycol)을 10ml씩 가하여 혼합한 후 1N NaOH를 1ml 첨가하였다. 이를 37℃ 워터 배스(water bath)에서 1시간 반응시키고 UV-spectrophotometer (U-3000ⓡ, Hitachi, Japan) 420nm에서 흡광도를 측정하였다. 표준물질은 퀘세틴을 사용하였다.The flavonoid content of the extracts was determined according to the Davis's method (see Monica et al., 2007). 0.5 g of the extract powder prepared in Example 1 was mixed with 1 ml of methanol, mixed with 10 ml of diethylene glycol, and 1 ml of 1N NaOH was added. This was reacted for 1 hour in a 37 ° C water bath and absorbance was measured at 420 nm on a UV-spectrophotometer (U-3000 ⓡ , Hitachi, Japan). Quercetin was used as a reference material.
그 결과, 상기 실시예 2 중 표 1과 같이 싸리 에탄올 추출물과 철쭉 에탄올 추출물, 굴참나무 에탄올 추출물의 플라보노이드 함량은 각각 33.5mg/g, 74.4mg/g 또는 27.7mg/g이었고 싸리 에탄올 추출물과 철쭉 에탄올 추출물, 굴참나무 에탄올 추출물의 1:1:1 (w/w/w) 혼합물은 51.4mg/g을 나타냈다. As a result, the content of flavonoids of the cari ethanol extract, azalea ethanol extract and oak oak ethanol extract were 33.5 mg / g, 74.4 mg / g or 27.7 mg / g, respectively, as shown in Table 1 of Example 2, 1: 1: 1 (w / w / w) mixture of the extract and oyster oak ethanol extract showed 51.4 mg / g.
<< 실시예Example 5> L- 5> L- 도파(L-DOPA)를Dopa (L-DOPA) 이용한 Used 버섯형Mushroom type 티로시나아제(mushroom tyrosinase) Mushroom tyrosinase 활성능Bow performance 분석 analysis
싸리, 철쭉, 굴참나무 에탄올 추출물의 멜라닌 생성능을 확인하기 위하여 멜라닌 생성에 중요한 작용을 하는 티로시나아제(tyrosinase)에 직접적인 작용을 관찰하였다. Tyrosinase는 L-티로신(L-tyrosine)을 L-DOPA로 전환시키고 이어서 L-도파(L-DOPA)를 L-도파 퀴논(L-DOPA quinone)으로 산화시킨다. 그러므로 tyrosinase의 기질로서 L-DOPA를 이용하여 정제한 mushroom tyrosinase의 활성을 측정하였다(Chang et al., 2007 참고). 0.1M의 인산완충액(phosphate buffer, pH 6) 100㎕와 추출물 20㎕(1mg/ml, 2mg/ml)를 혼합한 다음 mushroom tyrosinase (2000U/ml in phosphate buffer, pH 6) 20㎕를 첨가하고 37℃에서 10분 동안 반응시켰다. 반응물은 UV-spectrophotometer (U-3000ⓡ, Hitachi, Japan)를 사용하여 475nm에서 흡광도를 측정하였다. Tyrosinase 활성은 하기 수학식 1에 의거하여 도출하였다. In order to confirm the melanin production ability of ethanol extract of sari, azalea, and oak oak, direct action was observed on tyrosinase, which plays an important role in the production of melanin. Tyrosinase converts L-tyrosine to L-DOPA and then L-DOPA to L-DOPA quinone. Therefore, the activity of mushroom tyrosinase purified using L-DOPA as a tyrosinase substrate was measured (see Chang et al., 2007). After adding 100 μl of 0.1 M phosphate buffer (pH 6) and 20 μl of extract (1 mg / ml, 2 mg / ml), add 20 μl of mushroom tyrosinase (2000 U / ml in phosphate buffer, pH 6) Lt; 0 > C for 10 minutes. The reactants and the absorbance at 475nm was measured using a UV-spectrophotometer (U-3000 ⓡ , Hitachi, Japan). The tyrosinase activity was derived from the following equation (1).
그 결과, 도 1과 같이 L-DOPA를 기질로 사용한 경우 싸리와 철쭉, 굴참나무 에탄올 추출물을 2mg/mL 반응시켰을 때 대조군에 비해 각각 약 8%, 20% 또는 8% 증가하였다. 싸리, 철쭉, 굴참나무 에탄올추출물의 1:1:1 (w/w/w) 혼합물에 대한 mushroom tyrosinase 활성은 대조군에 비해 12% 증가한 것으로 확인되었으며 싸리 에탄올 추출물 및 굴참나무 에탄올 추출물의 단독 처리보다 비교적 높은 활성을 보였다.As a result, when L-DOPA was used as a substrate as shown in Fig. 1, the ethanol extract of sari, azalea and oak oak was increased by about 8%, 20%, or 8%, respectively, The mushroom tyrosinase activity of the mixture of 1: 1: 1 (w / w / w) ethanol extract of sari, azalea and oak oak was 12% higher than that of the control. High activity.
<< 실시예Example 6> B166> B16 흑색종(melanoma) 세포에서의 활성 분석 Activity analysis in melanoma cells
1. B16 흑색종(melanoma) 세포 배양1. B16 melanoma cell culture
C57BL/6J 마우스에서 유래한 B16 melanoma 세포는 아주대학교병원으로부터 분양받아 10% 소태아혈청(fetal bovine serum, PBS)가 함유된 DMEM 배지로 37℃, 0.5% CO2 조건의 배양기에서 배양하였다. B16 melanoma cells derived from C57BL / 6J mice were purchased from Ajou University Hospital and cultured in DMEM medium containing 10% fetal bovine serum (PBS) at 37 ° C and 0.5% CO 2 .
2. B16 melanoma를 이용한 세포 독성 분석2. Cytotoxicity analysis using B16 melanoma
MTT 분석법은 3-(4,5-디메틸티아졸-2-일)-2,5-디페닐 테트라졸리움 브로마이드(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT) 시약이 세포 내로 흡수된 후 미토콘드리아의 숙신산 탈수소효소(succinate dehydrogenase)에 의해 포르말잔(formazan)을 형성하는데 이 물질의 세포 내 축적은 미토콘드리아의 활성, 넓게는 세포의 활성을 의미하는 것으로써 세포의 생장율을 측정하는 대표적인 방법이다.MTT assay was performed using 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide , MTT) After the reagent is absorbed into cells, the succinate dehydrogenase of mitochondria forms formazan. The intracellular accumulation of this substance means the activity of mitochondria, broadly, the activity of cells It is a representative method of measuring the growth rate of cells.
B16 melanoma 세포를 24 웰 플레이트에 적정 세포수 (7×103 cells/well)로 분주하고 37℃, 0.5% CO2 배양기에서 24시간 배양한 다음 싸리, 철쭉 및 굴참나무 에탄올 추출물을 각각 농도별(5, 10, 20 mg/ml)로 희석시켜 이를 1㎕씩 넣은 후 0.5% CO2 배양기에서 72시간 배양하였다. 배양 후 플레이트의 배지를 제거한 다음 PBS로 1회 세척하고 MTT(5mg/ml)를 배지 500㎕ 당 50㎕씩 넣은 후 37℃, 0.5% CO2 배양기에서 1시간 배양하였다. 반응 후 배지를 제거하고 디메틸설폭시화물(dimethyl sulfoxide, DMSO) 500㎕씩 넣고 플레이트 쉐이커(plate shaker)에서 15분간 세포를 융해한 다음 ELISA 리더기로 540nm 파장에서 흡광도를 측정하였다. 세포 생장률은 무처리구인 대조군과 비교하여 나타내었다. B16 melanoma cells were plated on 24-well plates (7 × 10 3 cells / well) and cultured in a 0.5% CO 2 incubator at 37 ° C. for 24 hours. The ethanol extracts of Sari, Azalea and
그 결과, 도 2와 같이 B16 melanoma 세포의 활성은 10, 20㎍/mL 철쭉 에탄올 추출물, 10, 20㎍/mL 혼합 처리군을 제외하고 세포 증식에 대한 억제를 일으키지 않는 것으로 확인되었다. 20㎍/mL 철쭉 에탄올 추출물 처리군의 경우 B16 melanoma 세포에 72시간 처리했을 때 최대 49%까지 세포 생존율이 감소하는 것으로 나타났다. 하지만 통상적으로 세포의 분화 상태는 수상돌기 유사(dendrite-like) 형상 돌기세포의 형성에 의해 모니터링된다. 도 3은 싸리와 철쭉, 굴참나무 에탄올 추출물이 처리된 세포와 대조구의 육안적인 세포 변화를 나타냈다. 도 2에서 세포 증식 억제가 나타난 철쭉의 경우 대조구 세포와 비교했을 때 전형적인 수지 돌기 세포상을 보였다. 이러한 dendrite-like 형상은 싸리, 철쭉과 굴참나무 에탄올 추출물 처리군에서 모두 농도가 증가함에 따라 점진적으로 확대하였다. 또한 기존 논문에서 dendrite-like 형상이 세포 분화 유도와 관계있는지 확인하기 위해 양성 대조군으로 삼산화 비소를 세포에 처리한 결과 dendrite-like 형상이 분명하게 형성되는 것을 확인하였다. As a result, as shown in FIG. 2, the activity of B16 melanoma cells was found not to inhibit cell proliferation except 10, 20 μg / mL azelaic ethanol extract and 10, 20 μg / mL mixed treatment group. The cell viability of B16 melanoma cells treated with 20 ㎍ / mL of azelaic ethanol extract decreased to 49% at 72 hours. However, the differentiation status of cells is usually monitored by the formation of dendrite-like shaped dendritic cells. FIG. 3 shows gross cell changes in cells treated with ethanol extracts of sari, azalea and oak oak and control. In Fig. 2, azodiazoles exhibiting cell proliferation inhibition showed a typical dendritic cell phase as compared with control cells. These dendrite-like morphologies were gradually enlarged as the concentration increased in the sari, azalea and oak oak ethanol extract-treated groups. In order to confirm whether dendrite-like morphology is related to induction of cell differentiation in the existing articles, it was confirmed that dendritic-like morphology was clearly formed when cells were treated with arsenic trioxide as a positive control.
따라서 싸리와 철쭉, 굴참나무 에탄올 추출물이 효과적으로 B16 melanoma 세포 분화를 유도하는 것으로 판단된다. Therefore, the ethanol extract of sari, azalea, and oak oak extract effectively induced B16 melanoma cell differentiation.
3. B16 melanoma를 이용한 3. Using B16 melanoma tyrosinasetyrosinase 활성능Bow performance 분석 analysis
Tyrosinase는 멜라닌 생합성 중 세 단계를 촉진하므로 tyrosinase 활성의 측정은 매우 중요하다. 따라서 B16 melanoma 세포에서 싸리와 철쭉, 굴참나무 에탄올 추출물의 tyrosinase 활성에 미치는 영향에 대해 측정하였다. B16 melanoma 세포를 60ø 플레이트에 적정 세포수 (8×104 cells/well)로 분주하고 37℃, 0.5% CO2, 배양기에서 24시간 배양한 다음 싸리, 철쭉과 굴참나무 에탄올 추출물을 각각 농도별 (5, 10, 20 mg/ml)로 희석시켜 1㎕씩 넣은 후 0.5% CO2, 37℃ 배양기에서 72시간 배양하였다. 배양 후 배지를 제거하고 트립신으로 세포를 e-튜브에 옮겨 담고 1% 트립톤 X-100 용액과 pH 6.8 PBS가 혼합된 용해 버퍼(lysis buffer)를 100㎕씩 넣어 용해한 후 아이스박스에서 1시간 반응시켰다. 4℃, 10000rpm에서 30분간 원심분리한 상층액을 tyrosinase 활성 측정 용액으로 사용하였고 단백질 분석 용액(Bio rad, USA)으로 흡광도를 측정, 동량의 단백질 양을 계산하였다. 단백질 20㎍을 포함한 세포 추출물과 시험 물질을 총 20㎕가 되게끔 섞은 후 L-DOPA를 180㎕씩 넣고 0.5% CO2, 37℃ 배양기에서 1시간 반응하였다. 반응 후 490nm에서 흡광도의 변화를 측정하였다.Because tyrosinase promotes three stages of melanin biosynthesis, measurement of tyrosinase activity is very important. Therefore, the effect of tyrosinase activity on ethanol extracts of sari, azalea and oak oak on B16 melanoma cells was evaluated. B16 melanoma cells were cultured in 60ø plate at a concentration of 8 × 10 4 cells / well and incubated at 37 ° C and 0.5% CO 2 for 24 hours. The ethanol extracts of sari, 5, 10, and 20 mg / ml), and the cells were incubated for 72 hours at 37 ° C in a 0.5% CO 2 incubator. After incubation, the medium was removed and the cells were transferred into trypticin and dissolved in 100 μl of a lysis buffer containing 1% tryptone X-100 solution and pH 6.8 PBS. . The supernatant was centrifuged at 10,000 rpm for 30 minutes at 4 ° C. The supernatant was used as the tyrosinase activity measurement solution. The absorbance was measured with protein analysis solution (Bio rad, USA) and the amount of protein was calculated. The cell extracts containing 20 μg of the protein and the test substance were mixed to make a total of 20 μl, and then 180 μl of L-DOPA was added thereto, followed by reaction at 37 ° C. for 1 hour in a 0.5% CO 2 incubator. After the reaction, the change in absorbance at 490 nm was measured.
그 결과, 도 4와 같이 싸리 목부 에탄올 추출물 처리군에서 20㎍/ml 처리했을 때 대조군에 비해 최대 14% 증가한 것으로 나타났고 철쭉 목부 에탄올 추출물의 경우 10㎍/ml 처리했을 때 13% 증가한 것으로 나타났으며 굴참나무 목부 에탄올 추출물은 20 ㎍/ml 처리했을 때 대조군에 비해 최대 14% 증가한 것으로 나타났다. 싸리, 철쭉과 굴참나무 목부 에탄올 추출물의 혼합물 (1:1:1(w/w/w))은 10㎍/ml 처리했을 때 7% 증가한 것으로 나타났고 분산분석(analysis of variance, ANOVA) 결과 혼합물을 제외한 싸리, 철쭉, 굴참나무 각각의 추출물의 단독 처리군은 P<0.05의 유의성을 나타냈다. 따라서 싸리, 철쭉과 굴참나무 목부 에탄올 추출물은 B16 melanoma 세포 내 tyrosinase 활성화에 영향을 미치는 것으로 판단되었다. As a result, as shown in Fig. 4, when treated with 20 μg / ml of the ethanol extract of Chenxianum, the maximum increase was 14% as compared with that of the control, and the ethanol extract of Azalea japonica increased by 13% when treated with 10 μg / ml The ethanol extract of Quercus mongolica showed a maximum increase of 14% at 20 ㎍ / ml compared to the control. (1: 1: 1 (w / w / w)) of ethanolic extracts of Silla, Azalea and Quercus variabilis were increased by 7% when treated with 10 μg / ml and the analysis of variance (ANOVA) Except for the extracts of Saffi, Azalea, and Quercus variabilis showed a significant P <0.05 significance. Therefore, the ethanol extracts of sari, azalea and oak wood were determined to affect tyrosinase activation in B16 melanoma cells.
4. B16 melanoma를 이용한 멜라닌 4. Melanin using B16 melanoma 생성능Generation 분석 analysis
B16 melanoma 세포를 60ø 플레이트에 적정 세포수 (8×104 cells/well)로 분주하고 37℃, 0.5% CO2, 배양기에서 24시간 배양한 다음 싸리, 철쭉, 굴참나무 에탄올 추출물을 각각 농도별 (5, 10, 20 mg/ml)로 희석시켜 1㎕씩 넣은 후 0.5% CO2, 배양기에서 72시간 배양하였다. 배양 후 배지를 제거하고 트립신으로 세포를 떼어 e-튜브에 옮겨 담고 1% triton X-100 용액과 pH 6.8 PBS가 혼합된 lysis buffer를 100㎕씩 넣어 용해한 후 얼음에 박아 1시간 반응시켰다. 4℃, 10000rpm에서 30분간 원심분리한 상층액을 제거한 후 남은 펠렛을 멜라닌 함량 측정에 사용하였다. 1N NaOH 용액으로 멜라닌을 용해시켜 490nm에서 흡광도를 측정하였고, 멜라닌 검량선(Y=0.00075X+0.0348)을 이용해 함량을 계산하였다. B16 melanoma cells were plated on 60ø plate at a density of 8 × 10 4 cells / well and incubated for 24 hours at 37 ° C and 0.5% CO 2 in an incubator. The ethanol extracts of sari, azalea and
그 결과, 도 5와 같이 B16 melanoma 세포에서 싸리와 철쭉, 굴참나무 목부 에탄올 추출물의 멜라닌 생성 효과에 대해 평가한 결과, 싸리, 철쭉과 굴참나무 목부 에탄올 추출물 모두 B16 melanoma 세포의 멜라닌 생성에 효과가 있었다. 대조군과 비교할 때 싸리 에탄올 추출물은 5, 10, 20㎍/ml에서 각각 39, 81 또는 89% 증가한 것으로 나타나 멜라닌 생성능이 우수한 것으로 확인되었으며 철쭉 에탄올 추출물은 5, 10, 20㎍/ml에서 각각 61, 78 또는 98% 증가한 것으로 나타났다. 굴참나무 에탄올 추출물은 5, 10 또는 20㎍/ml에서 각각 14, 58 또는 61% 증가한 것으로 나타났다. 싸리와 철쭉, 굴참나무 에탄올 추출물의 멜라닌 생성능은 추출물 처리 농도 의존적으로 증가했고 대조군과 비교하여 ANOVA 통계 분석한 결과 굴참나무 목부 에탄올 추출물 단독 처리군과 싸리, 철쭉, 굴참나무 목부 에탄올추출 혼합물 (1:1:1 (w/w/w)) 5㎍/ml를 제외하고 유의성 있게 증가한 것을 확인했다. As a result, as shown in FIG. 5, the melanin production of the ethanol extracts of Sacri, Azalea and Quercus variabilis from B16 melanoma cells was evaluated. As a result, ethanol extracts of Sari, Azalea and Quercus variabilis were effective in the melanin production of B16 melanoma cells . The ethanol extracts of 5, 10 and 20 μg / ml were increased by 39, 81 and 89%, respectively, and the melanin production was higher than that of the control. 78 or 98%, respectively. The extracts of oak oak ethanol increased by 14, 58 or 61% at 5, 10 or 20 ㎍ / ml, respectively. The melanin production of ethanol extracts of Silla, Azalea and Quercus variabilis increased depending on the concentration of extracts. The ANOVA statistical analysis showed that the ethanol extract of Quercus variabilis and the ethanol extract of Quercus, Azalea and Quercus variabilis (1: 1: 1 (w / w / w)) of 5 / / ml.
<< 실시예Example 7> 마우스 털 및 표피의 멜라닌 생성 효과 분석 7> Analysis of melanin production effect of mouse hair and epidermis
1. One. 실험 동물Experimental animal 준비 Ready
양성 대조군인 C57/BL6 마우스와 시험구인 (C57/BL6×BALB)F1 마우스는 6주령으로 중앙실험동물에서 구입하였으며, 경상대학교 실험동물관리실에서 사육하였다. 동물 사육실 온도 23±2℃, 상대습도 35 내지 60%, 12시간 조명 주기 조건하에서 식이와 식수는 자유롭게 섭취토록 하였으며 7일간의 적응기간을 거친 후 7 주령 마우스를 이용하여 실험을 수행하였다.Positive control C57 / BL6 mice and test mice (C57 / BL6 × BALB) F1 mice were purchased from a central laboratory animal at 6 weeks of age and raised in an experimental animal control room of Gyeongsang National University. Animals were fed diets and drinking water freely at a temperature of 23 ± 2 ° C and relative humidity of 35 to 60% for 12 hours. The animals were fed a 7-day adaptation period and then were tested using a 7-week-old mouse.
2. 싸리, 철쭉, 굴참나무 에탄올 추출물 또는 이들의 혼합물 처리2. Treatment of sari, azalea, oak oak ethanol extract or mixture thereof
마우스의 피부에 선택된 부분의 털을 먼저 제거한 후 휴지기의 분홍빛이 도는 피부에, 에탄올 추출물을 면봉을 사용하여 부드럽게 발라주었다(0.2 mL/cm2). 대조군은 동일한 마우스의 반대측의 피부에 60% 에탄올을 발라주어 유지하였다. 추출물 처리는 6달 동안 진행하였고 하루에 1회 처리해주었다. 피부 색깔과 관련된 마우스 털의 색상 변화는 정기적으로 관찰했다.The hair of the selected part of the mouse was first removed, and the ethanol extract was gently applied (0.2 mL / cm 2 ) to the pinkish skin of the resting paw using a cotton swab. The control group was maintained with 60% ethanol applied to the skin on the opposite side of the same mouse. The extract treatment was carried out for 6 months and treated once a day. Changes in color of mouse fur associated with skin color were regularly observed.
3. 마우스 털3. Mouse hair 의of 육안적 관찰 및 멜라닌 함량 측정 Gross observation and measurement of melanin content
복잡한 전처리 과정과 고가의 장비를 구비하여야 분석 가능한 HPLC 분석에 비해 모발 내 멜라닌 수준을 파악하는데 보다 편리하게 사용할 수 있는 분광 광도법 분석을 사용하여 멜라닌 함량을 측정하였다(Lee et al., 2007 참고). 추출물을 도포 후 자라난 모발 내의 멜라닌 함량을 분광 광도법으로 측정하여 물질의 백반 방지 효능을 정량적으로 평가하였다. Melanin content was measured using a spectrophotometric assay, which can be conveniently used to determine the level of melanin in the hair compared to analytical HPLC analysis with a complex pretreatment process and expensive equipment (Lee et al., 2007). The melanin content in the hair after the application of the extract was measured spectrophotometrically to quantitatively evaluate the antioxidant effect of the material.
양성 대조군인 C57/BL6와 시험군의 (C57/BL6×BALB)F1 마우스의 털을 면도기를 이용하여 수집한 다음 털 20mg당 1mL의 1M NaOH와 24시간 실온에서 반응시켰다. 대조군과 시험군 모두 파장 A500(total melanin)과 A650(eumelanin)에서 흡광도를 측정하였다. Melanin과 eumelanin 각각 1M의 NaOH에 농도 범위 0.05 내지 0.4 ㎎/㎖의 멜라닌을 용해하여 작성한 검량선에 대입하여 멜라닌 함량을 계산하였다.The hair of the positive control group C57 / BL6 and the test group (C57 / BL6 × BALB) F1 mice were collected using a razor and then reacted with 1 mL of 1 M NaOH per 20 mg of hair for 24 hours at room temperature. Absorbance was measured at wavelengths A500 (total melanin) and A650 (eumelanin) in both the control and test groups. Melanin and eumelanin were dissolved in 1 M NaOH in concentrations ranging from 0.05 to 0.4 mg / ml, respectively, and melanin was added to the calibration curve to calculate the melanin content.
그 결과, 도 6과 같이 60% 에탄올을 사용한 대조군과 비교했을 때 싸리 에탄올 추출물, 철쭉 에탄올 추출물, 굴참나무 에탄올 추출물, 혼합물(싸리 에탄올추출물: 철쭉 에탄올 추출물 : 굴참나무 에탄올 추출물, 1:1:1 (w/w/w))을 각각 처리 한 부분에서 검은색 털이 자란 것을 확인할 수 있다. As a result, as shown in FIG. 6, when compared with the control group using 60% ethanol, the extracts of Cari ethanol, Azalea ethanol extract, Quercus oak ethanol extract and mixture (Cari ethanol extract: Azalea ethanol extract: Quercy oak ethanol extract, 1: 1: (w / w / w)), respectively.
또한 추출물 도포 후 자라난 모발 내의 멜라닌 함량을 분광 광도법으로 측정하여 물질의 백반 방지 효능을 정량적으로 평가한 결과, 도 7과 같이 마우스 털의 멜라닌 함량 측정 결과 60% 에탄올 처리구인 대조군과 비교했을 때 Melanin과 eumelanin 함량이 증가하는 것으로 나타났다. 싸리 에탄올 추출물 처리군의 경우 멜라닌 함량이 40%, 철쭉 에탄올 추출물 처리군은 47%, 굴참나무 에탄올 추출물 처리군은 36%, 싸리와 철쭉, 굴참나무 에탄올 추출 혼합물(1:1:1(w/w/w))은 39% 증가했으며 검은색 색소를 나타내는 유멜라닌 함량도 마찬가지로 싸리 에탄올 추출물, 철쭉 에탄올 추출물, 혼합물 (싸리 에탄올 추출물: 철쭉 에탄올 추출물 : 굴참나무 에탄올 추출물 (1:1:1(w/w/w))이 각각 29%, 29%, 21%, 27% 증가한 것으로 나타났다. 따라서 싸리와 철쭉, 굴참나무 에탄올 추출물이 멜라닌 생성에 긍정적인 역할을 한다고 판단할 수 있다.The melanin content of the hair was measured by spectrophotometric method and quantitatively evaluated the anti-alopecia effect of the material. As a result, the melanin content of the mouse hair was measured as shown in Fig. 7. As a result, Melanin And eumelanin content were increased. The content of melanin was 40%, that of azalea ethanol extract was 47%, that of oak oak ethanol extract was 36%, that of sorghum azalea and oak oak ethanol was 1: 1: 1 (w / w / w)) was increased by 39%, and the content of yelmelinone showing black pigment was also the same as the content of ethanol extract, azalea ethanol extract and mixture of ethanol extract: azalea ethanol extract: oak oak ethanol extract (1: 1: 1 (w 29%, 29%, 21% and 27%, respectively), indicating that the ethanol extracts of sari, azalea and quercetin play a positive role in the production of melanin.
4. 마우스 피부의 L-4. L- DOPADOPA 염색을 통한 멜라닌 세포 관찰 Observation of melanocytes through staining
멜라닌 세포는 DOPA에 의해 양성반응을 일으켜 검은색을 나타냄으로써 멜라닌 세포의 위치와 추출물 처리에 따른 변화를 육안적으로 파악할 수 있다. 마우스 피부의 깨끗한 조각이나 프로말린으로 고정한 조직을 pH 7.4의 3: 4: 디히드록시페닐알라닌(dihydroxyphenylalanine, Dopa) 용액에서 배양했을 때 세포 내에서 Dopa-melanin과의 산화를 일으켜 검은색으로 염색되게 된다. 이러한 세포의 변화를 도파-양성반응(dopa-positive)이라고 한다. 마우스 표피에서 이 반응은 일반적으로 색소 침착활동을 하는 멜라닌 세포에서 일어난다. Melanocytes are positive by DOPA and show black color, so that the position of melanocytes and the changes of the treatment of extracts can be glanced. When a clean piece of mouse skin or pro-malignant tissue is cultured in a solution of 3: 4: dihydroxyphenylalanine (Dopa) at pH 7.4, it is oxidized with Dopa-melanin in the cells to be stained black . These changes in cells are called dopa-positive. In the mouse epidermis, this reaction usually occurs in melanocytes that undergo pigmentation activity.
이를 위하여 레이드로&블랙버그(Laidlaw & Blackberg)의 표준절차를 사용했고 반응을 수행하기 위해 깨끗한 시트와 마우스 피부를 준비했다. 우선 마우스 등에서 분리한 피부의 표피와 진피를 분리하기 위한 방법으로 에틸렌디아민사아세트산(ethylenediaminetetraacetic acid, EDTA) 용액을 사용했다. 6 웰 플레이트에서 2시간 동안 EDTA 용액과 반응시킨 후 마이크로포셉(microforcep)을 이용하여 표피를 분리하였다. 분리된 표피를 약 1분 동안 식염수로 세척하고 37℃, 1시간 동안 L-DOPA 용액에 넣어 배양하였다. 1시간 후 L-DOPA 용액을 교체하고 37℃, 8시간 동안 다시 배양하였다. 배양 후 약 1분 동안 식염수에 세척하고 10% 포르말린으로 20분 동안 고정시켰다. 고정 후 3분간 증류수를 이용하여 깨끗이 세척하고 이를 95% 에탄올(ethanol)과 100% ethanol에 20분 동안 순차적으로 반응시켜 표피 속의 공기를 제거하였다. 마지막으로 자일렌(xylene)에 2분간 반응시킨 후 카다나 블라썸(cadana blasm) 용액을 이용하여 기포가 생기지 않도록 조심스럽게 커버 글라스를 덮었다. 그런 후 광학 현미경을 이용하여 염색된 멜라닌 세포를 관찰하였다. To this end, we used the standard procedure of Laidlaw & Blackberg and prepared clean sheet and mouse skin to perform the reaction. First, ethylenediaminetetraacetic acid (EDTA) solution was used as a method for separating the epidermis and dermis of the skin isolated from mice and the like. After reacting with EDTA solution in a 6-well plate for 2 hours, the epidermis was separated using a microforcep. Separated epidermis was washed with saline for about 1 minute and incubated in L-DOPA solution for 1 hour at 37 ° C. After 1 hour, the L-DOPA solution was replaced and incubated at 37 ° C for 8 hours. After incubation, the cells were washed with saline for about 1 minute and fixed with 10% formalin for 20 minutes. After the fixation, it was cleaned using distilled water for 3 minutes, and the air in the epidermis was removed by sequentially reacting with 95% ethanol and 100% ethanol for 20 minutes. Finally, xylene was reacted for 2 minutes, and a cover glass was carefully covered with a cadana blasm solution so as to prevent air bubbles from forming. Then, stained melanocytes were observed using an optical microscope.
싸리와 철쭉, 굴참나무 에탄올 추출물을 마우스 등에 5개월 동안 처리 후 표피를 분리해 관찰한 결과 도 8과 같이, 대조군과 비교했을 때 멜라닌 세포가 모낭 주위에서 더 많이 관찰되는 것을 확인할 수 있었다. 싸리와 철쭉, 굴참나무 에탄올 추출물 처리한 후, 60% 에탄올 처리군(vehicle) 피부에서 관찰되지 않았던 멜라닌 세포들이 모낭 주위에서 더 많이 관찰되었다. 멜라닌 세포의 활성화로 인해 마우스 모낭의 뿌리 주변에 멜라닌 세포가 증가하는 것은 이미 보고된 바 있다. 따라서 이 결과는 싸리와 철쭉, 굴참나무 에탄올 추출물이 백반증의 착색을 유도하는 것으로 판단된다. After 5 months of treatment with ethanol extract of sari, azalea and oak oak for 5 months, epidermis was separated and observed. As shown in Fig. 8, it was confirmed that melanocytes were observed around the hair follicles as compared with the control group. Melanocytes, which were not observed in the skin of 60% ethanol treated group, were observed around the hair follicles after ethanol treatment of sari, azalea and oak oak. It has been previously reported that the activation of melanocytes causes an increase in melanocytes around the roots of mouse hair follicles. Therefore, it is concluded that the ethanol extracts of sari, azalea and oak oak induce the pigmentation of vitiligo.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시예일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다. While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims. will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
Claims (8)
상기 추출물은 에탄올, 메탄올, 증류수 및 이들의 혼합물로 이루어진 군에서 선택된 용매로 추출하는 것을 특징으로 하는 백반증 또는 백모 예방 또는 치료용 약학 조성물.The method according to claim 1,
Wherein the extract is extracted with a solvent selected from the group consisting of ethanol, methanol, distilled water, and mixtures thereof.
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| WO2018174453A1 (en) | 2017-03-21 | 2018-09-27 | 바이오스펙트럼 주식회사 | Composition containing cirsium japonicum extract as active ingredient for stimulating melanogenesis |
| KR20210012649A (en) * | 2019-07-26 | 2021-02-03 | 한국 한의학 연구원 | Composition for skin whitening comprising Rhododendron schippenbachii root extract as effective component |
| KR102240026B1 (en) | 2019-07-26 | 2021-04-14 | 한국 한의학 연구원 | Composition for skin whitening comprising Rhododendron schippenbachii root extract as effective component |
| KR20220059214A (en) | 2020-11-02 | 2022-05-10 | 제주대학교 산학협력단 | A composition for promoting melanin synthesis comprising syringetin |
| KR20220078294A (en) | 2020-12-03 | 2022-06-10 | 제주대학교 산학협력단 | A composition for promoting melanin synthesis comprising didymin |
| KR20220078293A (en) | 2020-12-03 | 2022-06-10 | 제주대학교 산학협력단 | A composition for promoting melanin synthesis comprising chrysoeriol |
| KR20220078295A (en) | 2020-12-03 | 2022-06-10 | 제주대학교 산학협력단 | A composition for promoting melanin synthesis comprising linarin |
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