KR102132009B1 - Composition for promoting hair growth and preventing hair loss lichen extract - Google Patents
Composition for promoting hair growth and preventing hair loss lichen extract Download PDFInfo
- Publication number
- KR102132009B1 KR102132009B1 KR1020180126893A KR20180126893A KR102132009B1 KR 102132009 B1 KR102132009 B1 KR 102132009B1 KR 1020180126893 A KR1020180126893 A KR 1020180126893A KR 20180126893 A KR20180126893 A KR 20180126893A KR 102132009 B1 KR102132009 B1 KR 102132009B1
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- South Korea
- Prior art keywords
- hair
- lichen
- extract
- composition
- hair growth
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- Medicines Containing Plant Substances (AREA)
Abstract
본 발명은 지의류 추출물을 함유하는 발모 촉진 및 탈모 방지용 조성물에 관한 것으로, 본 발명의 지의류 스테레오카룰론 자포니쿰(Stereocaulon japonicum) 추출물을 함유하는 조성물은 모발 성장 관련 유전자인 Shh, Krt25, Dlx2, Prom1, S100a9, Vegfc, Ptgfr, Pdgfrl, Igfbp4 및 Gli2의 유전자를 모두 발현시킴으로써 발모 촉진 및 탈모 방지 효과를 나타내며, 천연 물질로서 세포독성이 거의 없다. 또한, 본 발명의 지의류 추출물은 세포 독성 및 피부 부작용이 없어 화장료, 약학적 및 식품 조성물에 안전하게 사용할 수 있다. The present invention relates to a composition for promoting hair growth and preventing hair loss containing an extract of lichen, the composition containing an extract of lichen stereocarulon japonicum of the present invention is Shh, Krt25, Dlx2, Prom1 which are hair growth related genes. , S100a9, Vegfc, Ptgfr, Pdgfrl, Igfbp4 and Gli2 express all of the genes to promote hair growth and prevent hair loss, and have little cytotoxicity as a natural substance. In addition, the lichen extract of the present invention has no cytotoxicity and skin side effects and can be safely used in cosmetic, pharmaceutical, and food compositions.
Description
본 발명은 지의류 추출물을 함유하는 발모 촉진 또는 탈모 방지용 조성물에 관한 것으로, 보다 구체적으로는 지의류 스테레오카룰론 자포니쿰(Stereocaulon japonicum)의 메탄올 추출물을 유효성분으로 포함하는 발모 촉진 또는 탈모 방지용 화장료, 약학적 및 식품 조성물에 관한 것이다.The present invention relates to a composition for promoting hair growth or preventing hair loss containing an extract of lichen, more specifically, a cosmetic for stimulating hair loss or preventing hair loss, which comprises methanol extract of lichen stereocarulon japonicum as an active ingredient. Red and food composition.
비정상적으로 털이 많이 빠지는 탈모증은 화학적 요인, 유전학적인 요인, 오염원, 모근의 영양결핍, 내분비계의 장애, 노화 등 다양한 요인에 의하여 일어난다. 탈모증은 종래에는 남성형 탈모가 주종을 이루었으나, 최근에는 여성 및 젊은 층으로 확산되고 있다. 또한, 최근에는 경제 위기가 심화되고 이에 따른 실직이나 구직의 어려움으로 인해 불안감과 스트레스에 노출되면서 머리카락이 빠지는 증상을 호소하고 있는 탈모환자가 급속도로 증가하고 있다. Alopecia, which is unusually hairy, is caused by various factors such as chemical factors, genetic factors, pollutants, malnutrition of hair roots, endocrine system disorders, and aging. Alopecia has been predominantly male-type hair loss in the past, but has recently spread to women and younger people. In addition, in recent years, the economic crisis is intensifying and the number of patients with hair loss complaining of the symptoms of hair loss as they are exposed to anxiety and stress due to unemployment or job search difficulties is rapidly increasing.
정상인의 경우 머리카락이 하루에 50~70개가 빠지지만, 스트레스에 의한 탈모는 하루에 100개 이상 빠지는 현상을 동반하는 것으로 알려져 있다. 대한모발학회에 따르면 서양인은 성인의 50% 이상이 대머리가 될 가능성이 있으며 한국 사람은 성인의 15~20% 정도가 될 만큼 흔한 일이다. 국내 탈모인구는 약 900만명으로 추정된다. In normal people, 50 to 70 hairs are removed per day, but hair loss due to stress is known to accompany more than 100 hairs per day. According to the Korean Society of Hair Sciences, over 50% of adults are likely to become bald, and it is common for Koreans to be 15 to 20% of adults. The domestic population is estimated to be 9 million.
심한 탈모현상이 생기는 가장 중요한 원인은 유전적인 배경과 남성 호르몬인 안드로겐이 주원인으로 알려져 있으나 최근에는 스트레스가 가장 두드러진 탈모 촉진 원인으로 간주되고 있다. 그러나 스트레스에 의한 탈모현상에 관한 명확한 기전은 충분히 이해되고 있지 않다. 다만 쥐를 통한 연구 결과에 따르면, 스트레스는 털집(모낭) 주위에 염증을 일으켜 털의 퇴행기를 앞당기고, 결과적으로 털이 자라는 것을 억제하도록 유도하는 것으로 알려져 있다. 일반적으로 스트레스를 계속 받으면 뇌와 두피에 열이 발생하고 혈액공급이 부족해져 모발은 영양불량 상태가 된다. 스트레스성 열기운이 얼굴로 지나치게 몰리게 되면 두피의 피지선이 자극돼 피지가 모공을 막아 탈모를 유발하거나 두피의 모세혈관을 자극해 두피를 민감하게 만드는 요인이 된다. 따라서 스트레스에 의한 탈모는 스트레스를 예방해 주거나 조절해 주는 약물 사용시 매우 좋은 효과를 얻을 수 있는 것으로 나타났다. 게다가 빠르게 진행되는 탈모 예방 외에도 머리카락 성장의 촉진 기능을 나타내는 약물사용을 통해 어느 정도 스트레스성 탈모의 진행을 예방할 수 있을 것으로 알려져 있다. The most important causes of severe hair loss are genetic background and androgen, the male hormone, is the main cause, but recently, stress is considered to be the most prominent cause of hair loss. However, the clear mechanism of hair loss caused by stress is not fully understood. However, according to the research results through rats, stress is known to induce inflammation around the hair follicle (hair follicle), leading to the regression of the hair, and consequently to induce hair growth to be suppressed. In general, if the stress continues, the brain and scalp generate heat and the blood supply is insufficient, resulting in malnutrition of the hair. When the stressful heat cloud is overly concentrated on the face, the sebaceous glands of the scalp are stimulated, and the sebum becomes clogged pores to cause hair loss or to stimulate the capillaries of the scalp to make the scalp sensitive. Therefore, it has been found that hair loss caused by stress can have a very good effect when using drugs that prevent or control stress. In addition, it is known that, in addition to the prevention of rapidly progressing hair loss, it is possible to prevent the progression of stressful hair loss to some extent through the use of drugs that exhibit the function of promoting hair growth.
탈모치료 또는 발모효과를 나타내는 것으로 많은 물질들이 보고되어 있기는 하지만 현재까지 탈모된 모발의 성장을 효과적으로 촉진시킬 수 있는 제품의 개발은 미진한 실정이다. 따라서, 생체에 안전하고 효과가 좋을 뿐 만 아니라, 안정적이고 제조 시 경제성이 있는 발모촉진, 양모 또는 탈모의 예방 또는 치료용 제품의 개발이 절실하게 요구되고 있다.Although many substances have been reported as exhibiting hair loss treatment or hair growth effect, the development of products that can effectively promote the growth of hair loss has been insufficient. Accordingly, there is an urgent need to develop a product for promoting or preventing hair growth, promoting hair growth, and preventing hair loss, which is not only safe and effective in living body, but also stable and economical in manufacturing.
따라서, 본 발명에서 해결하고자 하는 기술적 과제는 발모 촉진 및 탈모 방지용 조성물을 제공하기 위한 것이다.Therefore, the technical problem to be solved in the present invention is to provide a composition for promoting hair growth and preventing hair loss.
상기한 기술적 과제를 해결하기 위하여, 본 발명에서는 지의류 스테레오카룰론 자포니쿰(Stereocaulon japonicum) 추출물을 유효성분으로 포함하는 발모 촉진 및 탈모 방지용 조성물을 제공한다.In order to solve the above technical problem, the present invention provides a composition for promoting hair growth and preventing hair loss, which includes an extract of lichen stereocarulon japonicum as an active ingredient.
바람직하게, 본 발명에서 지의류 스테레오카룰론 자포니쿰(Stereocaulon japonicum) 메탄올 추출물을 포함하는 조성물은 화장료 조성물, 약학적 조성물 또는 식품 조성물일 수 있다.Preferably, in the present invention, the composition comprising the methanol extract of lichen stereocarulon japonicum may be a cosmetic composition, a pharmaceutical composition, or a food composition.
본 발명에 있어서, "추출물"은 지의류로부터 통상의 방법에 의하여 추출하거나 이로부터 분리된 발모 촉진 및 탈모 방지 활성을 가지는 지의류의 대사산물을 말한다. 또한, 본 발명에 있어서 "추출물"은 상기 추출액뿐만 아니라 이의 건조 분말 또는 이를 이용하여 제형화된 모든 형태를 포함하는 의미로 사용된다.In the present invention, "extract" refers to metabolites of lichens that have the activity of promoting hair growth and preventing hair loss separated from or extracted from lichens by conventional methods. In addition, in the present invention, "extract" is used to mean not only the extract but also dry powder thereof or all forms formulated using the same.
본 발명에서 지의류 추출물은 유기 용매를 사용하여 추출할 수 있는데, 추출한 액은 액체 형태로 사용하거나 또는 농축 및/또는 건조하여 사용할 수 있다. 상기 유기용매는 메탄올, 에탄올, 이소프로판올, 부탄올, 에틸렌, 아세톤, 헥산, 에테르, 클로로포름, 에틸아세테이트, 부틸아세테이트, 디클로로메탄, N, N-디메틸포름아미드(DMF), 디메틸설폭사이드(DMSO), 1,3-부틸렌글리콜, 프로필렌글리콜 또는 이들의 혼합용매이며, 추출물의 유효성분이 파괴되지 않거나 최소화된 조건에서 실온 또는 가온하여 추출할 수 있다. 추출하는 유기용매에 따라 추출물의 유효성분의 추출정도와 손실정도가 차이가 날 수 있으므로, 알맞은 유기용매를 선택하여 사용하도록 한다. 추출 방법은 특별히 제한되지 않고, 예를 들어 냉침 추출, 초음파 추출, 환류 냉각 추출 등이 있다.In the present invention, the lichen extract may be extracted using an organic solvent, and the extracted liquid may be used in a liquid form or concentrated and/or dried. The organic solvent is methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1 ,3-butylene glycol, propylene glycol, or a mixed solvent thereof, and can be extracted at room temperature or by heating in a condition where the active ingredient of the extract is not destroyed or minimized. Depending on the organic solvent to be extracted, the extraction degree and loss degree of the active ingredient of the extract may differ, so select and use the appropriate organic solvent. The extraction method is not particularly limited, for example, cold immersion extraction, ultrasonic extraction, reflux cooling extraction, and the like.
상기 여과는 추출액으로부터 부유하는 고체 입자를 제거하는 과정으로, 면, 나일론 등을 이용하여 입자를 걸러 내거나 한외여과, 냉동여과법, 원심분리법 등을 사용할 수 있으나, 이에 제한되지 않는다. 또한, 다양한 크로마토그래피(크기, 전하, 소수성 또는 친화성에 따른 크로마토그래피)에 의한 분리 과정을 추가로 포함할 수 있다.The filtration is a process of removing the suspended solid particles from the extract, it may be used to filter the particles using cotton, nylon, etc., ultrafiltration, freezing filtration, centrifugation, etc., but is not limited thereto. In addition, the separation process by various chromatography (chromatography according to size, charge, hydrophobicity or affinity) may be further included.
상기 여액을 건조하는 단계는 동결건조, 진공건조, 열풍건조, 분무건조, 감압건조, 포말건조, 고주파건조, 적외선건조 등을 포함하나 이에 제한되지 않는다. 경우에 따라, 최종 건조된 추출물을 분쇄하는 공정을 추가할 수 있다.The step of drying the filtrate includes, but is not limited to, freeze drying, vacuum drying, hot air drying, spray drying, vacuum drying, foam drying, high frequency drying, infrared drying, and the like. In some cases, a process of grinding the final dried extract may be added.
본 발명에 따른 하나의 구체적 실시예에서, 본 발명의 지의류 추출물은 지의류 스테레오카룰론 자포니쿰(Stereocaulon japonicum)의 지의체를 무게(g)의 0.1배 내지 5배, 바람직하게는 0.1배 내지 3배의 메탄올 또는 아세톤을 사용하여 20 내지 50℃, 바람직하게는 20 내지 30 ℃의 추출 온도에서 1 내지 5시간, 바람직하게는 2 내지 3시간 동안 추출한 후 수득된 추출액을 여과, 감압 농축함으로써 수득하였다.In one specific embodiment according to the present invention, the lichen extract of the present invention is lichen stereocarulon japonicum ( stereocaulon japonicum ) of the lichen body 0.1 to 5 times the weight (g), preferably 0.1 to 3 times After extraction for 1 to 5 hours, preferably 2 to 3 hours at an extraction temperature of 20 to 50°C, preferably 20 to 30°C, using pear methanol or acetone, the obtained extract was filtered and concentrated under reduced pressure. .
본 발명의 조성물에는 지의류 추출물의 총 중량을 기준으로 하여 0.1 내지 90 중량%, 보다 바람직하게는 10 내지 80 중량%로 포함할 수 있다. 이 때 지의류 추출물의 함량이 0.1 중량% 미만일 경우 본 발명의 발모 촉진 및 탈모 방지 효과를 수득할 수 없으며, 90 중량%를 초과할 경우 함량의 증가에 따라 효과가 비례적이지 않아 비효율적일 수 있으며 제형상의 안정성이 확보되지 않는 문제점이 있다.The composition of the present invention may contain 0.1 to 90% by weight, more preferably 10 to 80% by weight based on the total weight of the lichen extract. At this time, when the content of the lichen extract is less than 0.1% by weight, the effect of promoting hair growth and preventing hair loss of the present invention cannot be obtained, and when it exceeds 90% by weight, the effect may not be proportional to the increase in content and thus may be inefficient. There is a problem that the shape stability is not secured.
본 발명의 구현예에 따르면, 본 발명에서는 지의류 스테레오카룰론 자포니쿰(Stereocaulon japonicum)의 메탄올 추출물을 유효성분으로 포함하는 발모 촉진 및 탈모 방지용 조성물을 제공한다.According to an embodiment of the present invention, the present invention provides a composition for promoting hair growth and preventing hair loss, which includes a methanol extract of lichen stereocarulon japonicum as an active ingredient.
본 발명의 지의류 스테레오카룰론 자포니쿰(Stereocaulon japonicum) 추출물을 함유하는 조성물은 모발 성장 관련 유전자인 Shh, Krt25, Dlx2, Prom1, S100a9, Vegfc, Ptgfr, Pdgfrl, Igfbp4 및 Gli2의 유전자를 모두 발현시킴으로써 발모 촉진 및 탈모 방지 효과를 나타내며, 천연 물질로서 세포독성이 거의 없다. The composition containing the lichen stereocarulon japonicum extract of the present invention expresses all of the genes of hair growth related genes Shh, Krt25, Dlx2, Prom1, S100a9, Vegfc, Ptgfr, Pdgfrl, Igfbp4 and Gli2. It has the effect of promoting hair growth and preventing hair loss and has little cytotoxicity as a natural substance.
상기 모발 성장 유전자의 알려진 기능은 다음과 같다. 고도로 발현된 유전자 중 하나인 Shh는 모발 모낭 플라코데에서 생성된 중요한 모낭 유도 신호이며 초기 모낭 상피의 상피 세포 증식을 유도한다. Krt25는 모발 내부 뿌리 덮개의 유형 I 케라틴이다. Dlx2는 모발 축 형성에 중요하며, Prom1은 일차 및 이차 모낭에서 HDP의 형성에 기여하며 출생 후의 모발 성장에서 HDP 세포의 성장 발병을 유지함으로써 중요한 역할을 하며 모발 팽창 영역에서 신호 교차 결합을 매개한다. S100a9는 성장기 모낭에서 강하고 케라틴 세포 분화에 관여한다. Vegfc는 모낭 성장 및 순환의 주요 중재자이다. Ptgfr은 모발 성장 조절에 관여한다. Pdgfrl은 모발주기에서 모낭 재생의 발전에 관여한다. Igfbp4는 인간 모낭의 HDP와 모발 사이의 경계에서 강력하게 발현하고 세포 성장을 조절한다. Gli2는 모낭 발달에 필수적이며 발달 모낭에서 Shh의 분열 촉진 효과를 중재한다(문헌 [Fuchs E, et al., Genes & Development. 2008;22(8):976-85]; [Huntzicker EG, et al., Genes & Development. 2006;20(3):276-81]; Hesse M, et al., European Journal of Cell Biology. 2004;83(1); [Lin KK, et al., Proceedings of the National Academy of Sciences of the United States of America. 2004;101(45):15955-60]; [Zhou L, Xu M, Yang Y, Yang K, Wickett RR, Andl T, et al. Activation of β-Catenin Signaling in CD133-Positive Dermal Papilla Cells Drives Postnatal Hair Growth. PLOS ONE. 2016;11(7)]; [J. Z, N. L, K. L, J. H, J. Y, R. B, et al. Identification of genes and proteins associated with anagen wool growth. Animal Genetics. 2017;48(1):67-79]; 및 [Amㅹlie R, Rachel S, Manon T, Carlos C, Michael R. PDGF signalling in the dermis and in dermal condensates is dispensable for hair follicle induction and formation. Experimental Dermatology. 2015;24(6):468-70].Known functions of the hair growth gene are as follows. One of the highly expressed genes, Shh, is an important hair follicle-inducing signal produced by hair follicle placode and induces epithelial cell proliferation of the initial follicular epithelium. Krt25 is a type I keratin of hair inner mulch. Dlx2 is important for hair axis formation, Prom1 contributes to the formation of HDP in primary and secondary hair follicles, plays an important role by maintaining the growth onset of HDP cells in postnatal hair growth, and mediates signal cross-linking in the hair expansion region. S100a9 is strong in growing follicles and is involved in keratinocyte differentiation. Vegfc is a major mediator of hair follicle growth and circulation. Ptgfr is involved in the regulation of hair growth. Pdgfrl is involved in the development of hair follicle regeneration in the hair cycle. Igfbp4 is strongly expressed at the boundary between HDP and hair in human hair follicles and regulates cell growth. Gli2 is essential for hair follicle development and mediates the effect of Shh on the development of hair follicles (Fuchs E, et al., Genes & Development. 2008;22(8):976-85); [Huntzicker EG, et al ., Genes & Development. 2006;20(3):276-81]; Hesse M, et al., European Journal of Cell Biology. 2004;83(1); [Lin KK, et al., Proceedings of the National Academy of Sciences of the United States of America. 2004;101(45):15955-60]; [Zhou L, Xu M, Yang Y, Yang K, Wickett RR, Andl T, et al. Activation of β-Catenin Signaling in CD133-Positive Dermal Papilla Cells Drives Postnatal Hair Growth.PLOS ONE. 2016;11(7)]; [J. Z, N. L, K. L, J. H, J. Y, R. B, et al Identification of genes and proteins associated with anagen wool growth.Animal Genetics. 2017;48(1):67-79]; and [Amㅹlie R, Rachel S, Manon T, Carlos C, Michael R. PDGF signaling in the dermis and in dermal condensates is dispensable for hair follicle induction and formation.Experimental Dermatology. 2015;24(6):468-70].
또한, 상기 추출물은 통상의 방법에 의해 추출한 추출액뿐만 아니라 이의 건조 분말 또는 이를 이용하여 제형화된 모든 형태를 말한다.In addition, the extract refers to not only the extract liquid extracted by a conventional method, but also a dry powder thereof or all forms formulated using the same.
본 발명에 있어서, 상기 "유효성분"이란 단독으로 목적하는 활성을 나타내거나 또는 그 자체는 활성이 없는 담체와 함께 활성을 나타낼 수 있는 성분을 의미한다. In the present invention, the "active ingredient" refers to a component that can exhibit the desired activity alone or itself can exhibit activity with an inactive carrier.
본 발명에서 "두피"는 머리 부위를 덮고 있는 피부를 의미한다. In the present invention, "scalp" refers to the skin covering the head area.
본 발명의 하나의 실시양태에 따르면, 지의류 추출물을 유효성분으로 포함하는 화장료 조성물을 제공한다.According to one embodiment of the present invention, there is provided a cosmetic composition comprising a lichen extract as an active ingredient.
본 발명의 하나의 실시양태에 따른 화장료 조성물에는 유효성분으로서의 지의류 추출물 이외에 화장품 조성물에 통상적으로 첨가되는 성분, 예컨대 항산화제, 안정화제, 가용화제, 비타민, 안료 및 향료와 같은 통상적인 보조제, 및 담체를 추가로 첨가할 수 있다. In the cosmetic composition according to one embodiment of the present invention, in addition to the lichen extract as an active ingredient, ingredients commonly added to cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavors, conventional adjuvants, and carriers Can be additionally added.
본 발명의 화장료 조성물은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 예를 들어, 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성제-함유 클린싱, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이 등으로 제형화될 수 있으나, 이에 한정되는 것은 아니다. 보다 상세하게는, 영양 크림, 수렴 화장수, 유연 화장수, 로션, 에센스, 영양젤 또는 마사지 크림의 제형으로 제조될 수 있다.The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleaning , May be formulated as an oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be prepared in the form of a nutrition cream, astringent lotion, soft lotion, lotion, essence, nutrition gel or massage cream.
본 발명의 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물성유, 식물성유, 왁스, 파라핀, 전분, 트라칸트 검, 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 oil, vegetable oil, wax, paraffin, starch, trakant gum, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide are used as carrier components. Can be.
본 발명의 하나의 구현예에 따르면, 상기 화장료는 헤어토닉, 모발영양 화장수, 스칼프 트리트먼트, 헤어 트리트먼트, 헤어린스, 헤어샴푸 또는 헤어로션을 조성하는 것일 수 있다.According to one embodiment of the invention, the cosmetic may be to create a hair tonic, hair nutrition lotion, scalp treatment, hair treatment, hair rinse, hair shampoo or hair lotion.
본 발명의 제형이 파우더 또는 스프레이인 경우에는 담체 성분으로서 토스, 탈크, 실리카, 알루미늄 히드록시드, 칼슘 실리케이트 또는 폴리아미드 파우더가 이용될 수 있고, 특히 스프레이인 경우에는 추가적으로 클로로플루오로히드로카본, 프로판/부탄 또는 디메틸에테르와 같은 추진체를 포함할 수 있다.When the formulation of the present invention is a powder or a spray, toss, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in the case of a spray, additionally chlorofluorohydrocarbon, propane /Propellant such as butane or dimethyl ether.
본 발명의 제형이 용액 또는 유탁액인 경우에는 담체 성분으로서 용매, 가용화제 또는 유탁화제가 이용되고, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌글리콜, 1,3-부틸글리콜 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르가 있다.When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 Fatty acid esters of ,3-butyl glycol oil, glycerol aliphatic esters, polyethylene glycol or sorbitan.
본 발명의 제형이 현탁액인 경우에는 담체 성분으로서 물, 에탄올 또는 프로필렌 글리콜과 같은 액상의 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가 또는 트라칸트 검등이 이용될 수 있다.When the formulation of the present invention is a suspension, liquid diluents such as water, ethanol or propylene glycol as carrier components, ethoxylated isostearyl alcohol, suspensions such as polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, microcrystals Sex cellulose, aluminum metahydroxide, bentonite, agar or trakant gum and the like can 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, 사르코시네이트, 지방산 아미드 에테르 설페이트, 알킬아미도베타인, 지방족 알코올, 지방산 글리세리드, 지방산 디에탄올아미드, 식물성 유, 라놀린 유도체 또는 에톡실화 글리세롤 지방산 에스테르 등이 이용될 수 있다.When the formulation of the present invention is a surfactant-containing cleansing, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivatives, methyltaurate, sarcosinate, fatty acid amide as a carrier component Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.
본 발명의 하나의 구체적 용도로서, 본 발명의 조성물은 발모 촉진 및 탈모 방지를 위한 약학적 조성물로 사용될 수 있다.As one specific use of the present invention, the composition of the present invention can be used as a pharmaceutical composition for promoting hair growth and preventing hair loss.
본 발명의 조성물이 약학적 조성물로 사용되는 경우, 유효성분으로서 지의류 추출물 이외에 약학적으로 허용되는 담체를 추가로 포함할 수 있다. 본 발명의 약학적 조성물에 포함되는 약학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 탄수화물류 화합물(예: 락토스, 아밀로스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 셀룰로스 등), 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 염 용액, 알코올, 아라비아 고무, 식물성 기름(예: 옥수수 기름, 목화 종자유, 두유, 올리브유, 코코넛유), 폴리에틸렌 글리콜, 메틸 셀룰로스, 메틸히드록시 벤조에이트, 프로필히드록시 벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약학적으로 허용되는 담체 및 제제는 Remington's Pharmaceutical Sciences (19th ed., 1995)에 상세히 기재되어 있다.When the composition of the present invention is used as a pharmaceutical composition, it may further include a pharmaceutically acceptable carrier in addition to the lichen extract as an active ingredient. The pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention is commonly used in the preparation, and carbohydrate compounds (eg, lactose, amylose, dextrose, sucrose, sorbitol, mannitol, starch, cellulose, etc.) , Acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, salt solution, alcohol, gum arabic, vegetable oils (eg corn oil, cotton seed oil , Soymilk, olive oil, coconut oil), polyethylene glycol, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil, and the like. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
상기 약학적 조성물은 통상의 방법에 따라 스프레이형, 액상형, 젤형, 분말, 고형의 비누, 거품타입 등으로 제형화된 것일 수 있다.The pharmaceutical composition may be formulated as a spray type, liquid type, gel type, powder, solid soap, foam type, or the like according to a conventional method.
본 발명의 다른 하나의 구체적 용도로서, 본 발명의 조성물은 발모 촉진 및 탈모 방지를 위한 식품 조성물로 사용될 수 있다.As another specific use of the present invention, the composition of the present invention may be used as a food composition for promoting hair growth and preventing hair loss.
본 발명의 조성물이 식품 조성물로 사용되는 경우, 유효성분으로서 지의류 추출물 이외에 식품 제조 시에 통상적으로 첨가되는 성분, 예를 들어, 단백질, 탄수화물, 지방, 영양소, 조미제 및 향미제를 추가로 포함할 수 있다.When the composition of the present invention is used as a food composition, as an active ingredient, in addition to the lichen extract, ingredients commonly added at the time of food preparation, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents may be further included. Can.
상기 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스, 올리고당 등; 및 폴리사카라이드, 예를 들어 덱스트린, 사이클로덱스트린 등과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 향미제로서 천연 향미제 [타우마틴, 스테비아 추출물 (예를 들어 레바우디오시드 A, 글리시르히진 등)] 및 합성 향미제(사카린, 아스파르탐 등)를 사용할 수 있다.Examples of the carbohydrate include monosaccharides such as glucose and fructose; Disaccharides such as maltose, sucrose, oligosaccharides, etc.; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents (tauumatin, stevia extract (eg, rebaudioside A, glycyrrhizine, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used.
상기 식품의 종류에는 특별한 제한이 없다. 본 발명의 지의류 추출물을 첨가할 수 있는 식품의 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 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다.There are no particular restrictions on the type of food. Examples of foods to which the lichen extract of the present invention can be added are meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea , Drinks, alcoholic beverages, and vitamin complexes, and includes all healthy foods in the ordinary sense.
본 발명의 식품 조성물이 드링크제로 제조되는 경우에는 본 발명의 지의류추출물 이외에 구연산, 액상과당, 설탕, 포도당, 초산, 사과산, 과즙 등을 추가로 포함시킬 수 있다.When the food composition of the present invention is prepared as a drink agent, in addition to the lichen extract of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, etc. may be further included.
또한, 상기 식품 조성물은 상술한 성분 외에 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명의 식품 조성물은 천연 과일쥬스, 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다.In addition, the food composition is a variety of nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin in addition to the above-mentioned ingredients , Alcohol, carbonic acid used in carbonated beverages, and the like. In addition, the food composition of the present invention may contain flesh for the production of natural fruit juices, beverages and vegetable beverages. These ingredients can be used independently or in combination.
한편, 본 발명의 지의류 추출물은 독성이 없어 장기간 사용시에도 안심하고 사용할 수 있으므로, 화장품, 약품이나 식품에 안전하게 사용할 수 있다.On the other hand, since the lichen extract of the present invention is non-toxic and can be safely used even for long-term use, it can be safely used in cosmetics, drugs, or food.
이와 같이, 본 발명의 지의류 스테레오카룰론 자포니쿰(Stereocaulon japonicum) 추출물을 함유하는 조성물은 모발 성장 관련 유전자인 Shh, Krt25, Dlx2, Prom1, S100a9, Vegfc, Ptgfr, Pdgfrl, Igfbp4 및 Gli2의 유전자를 모두 발현시킴으로써 발모 촉진 및 탈모 방지 효과를 나타내며, 천연 물질로서 세포독성이 거의 없다. 또한, 본 발명의 지의류 추출물은 세포 독성 및 피부 부작용이 없어 화장료, 약학적 및 식품 조성물에 안전하게 사용할 수 있다. As described above, the composition containing the lichen stereocarulon japonicum extract of the present invention contains the genes of the hair growth related genes Shh, Krt25, Dlx2, Prom1, S100a9, Vegfc, Ptgfr, Pdgfrl, Igfbp4 and Gli2. By expressing it all, it has the effect of promoting hair growth and preventing hair loss, and is a natural substance with little cytotoxicity. In addition, the lichen extract of the present invention has no cytotoxicity and skin side effects and can be safely used in cosmetic, pharmaceutical, and food compositions.
도 1은 양성 대조군 (tofacitinib) 처리 후 시간에 따른 모발 유전자 발현을 나타낸 것이다.
도 2은 지의류 메탄올 추출물 처리 후 시간에 따른 모발 유전자 발현을 나타낸 것이다.
도 3은 지의류 스테레오카룰론 자포니쿰(Stereocaulon japonicum)의 메탄올 추출물의 박막 크로마토그래피 분석결과이다.
도 4는 지의류 스테레오카룰론 자포니쿰(Stereocaulon japonicum)의 메탄올 추출물의 모발 유전자 발현 강도를 나타낸 것이다.
도 5는 지의류 스테레오카룰론 자포니쿰(Stereocaulon japonicum)의 메탄올 추출물의 세포 생존율을 나타낸 그래프이다.1 shows hair gene expression over time after treatment with a positive control (tofacitinib).
Figure 2 shows the expression of hair gene over time after treatment with lichen methanol extract.
Figure 3 is a thin-film chromatography analysis of the methanol extract of lichen stereocarulon japonicum ( Stereocaulon japonicum ).
Figure 4 shows the hair gene expression intensity of the methanol extract of lichen stereocarulon japonicum ( Stereocaulon japonicum ).
5 is a graph showing the cell viability of methanol extracts of lichen stereocarulon japonicum .
이하, 본 발명의 이해를 돕기 위하여 실시예 등을 들어 상세하게 설명하기로 한다. 그러나, 본 발명에 따른 실시예들은 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 하기 실시예들에 한정되는 것으로 해석되어서는 안 된다. 본 발명의 실시예들은 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다. Hereinafter, examples and the like will be described in detail to help understanding of the present invention. However, the embodiments according to the present invention can be modified in many different forms, and the scope of the present invention should not be interpreted as being limited to the following examples. The embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.
실험방법Experiment method
모발 성장 유전자 발현 검사에서 양성 대조군으로 tofacitinib을 사용하였다. tofacitinib은 JAK-STAT 신호 전달 경로를 방해하고, 세포 외 정보를 세포 핵으로 전달하며, DNA 전사에 영향을 미치는 janus kinase 1 (JAK1)과 janus kinase 3 (JAK 3)의 효소 억제제이다. 최근 Harel 등 문헌 (Harel S, Higgins CA, Cerise JE, Dai Z, Chen JC, Clynes R, et al. Pharmacologic inhibition of JAK-STAT signaling promotes hair growth. Science Advances. 2015;1(9))에서는 tofacitinib이 모낭의 성장, 피부 유두의 유도 성장 및 모발 성장을 촉진한다고 보고했다. 하기 표 1과 같은 총 10 개의 모발 성장 관련 유전자를 선택하였다. Tofacitinib was used as a positive control in the hair growth gene expression test. tofacitinib is an enzyme inhibitor of janus kinase 1 (JAK1) and janus kinase 3 (JAK 3) that interferes with the JAK-STAT signaling pathway, delivers extracellular information to the cell nucleus, and affects DNA transcription. Tofacitinib was recently reported in Harel et al. (Harel S, Higgins CA, Cerise JE, Dai Z, Chen JC, Clynes R, et al. Pharmacologic inhibition of JAK-STAT signaling promotes hair growth.Science Advances. 2015; 1(9)). It has been reported to promote hair follicle growth, induced growth of skin nipples and hair growth. A total of 10 hair growth-related genes were selected as shown in Table 1 below.
<실시예 1> 지의류 추출물 제조<Example 1> Preparation of lichen extract
지의류 표본은 하기 표 2에 나타낸 바와 같이 2016 년에 한국에서 수집되었으며, 지의류 식별은 한국 지의류 연구소에서 수행하였다. 건조된 지의류 엽상체(talli)를 초음파 분쇄기를 사용하여 35 ℃에서 2 시간 동안 99.8 % 메탄올 또는 아세톤으로 추출한 후 여과하였다. 지의류 추출물을 회전 진공 증발기에서 45 ℃로 농축시켰다. 추출물을 동결건조시키고 추가 실험을 위해 디메틸설폭사이드 (DMSO)에 용해시킨 후 사용하였다. Lichen samples were collected in Korea in 2016 as shown in Table 2 below, and lichen identification was performed at the Korea Lichen Research Institute. The dried lichen frond (talli) was extracted with 99.8% methanol or acetone for 2 hours at 35° C. using an ultrasonic grinder and filtered. The lichen extract was concentrated in a rotary vacuum evaporator to 45°C. The extract was lyophilized and dissolved in dimethylsulfoxide (DMSO) for further experiments before use.
각각 메탄올 및 아세톤 추출된 추출물을 지의류 순번에 따라 1M, 1A, 2M, 2A, 3M, 3A, 4M, 4A, 5M, 5A, 6M, 6A, 7M, 7A, 8M, 8A, 9M, 9A, 10M, 11A, 12M, 12A, 13M, 14A, 15M, 15A, 16M, 16A, 17M, 17A, 18M, 18A, 19M, 19A, 20M, 21M, 21A, 22M, 22A, 23M, 23A, 24M, 24A, 25M, 25A, 26M, 26A으로 명명하였다.The extracted extracts of methanol and acetone are 1M, 1A, 2M, 2A, 3M, 3A, 4M, 4A, 5M, 5A, 6M, 6A, 7M, 7A, 8M, 8A, 9M, 9A, 10M, respectively 11A, 12M, 12A, 13M, 14A, 15M, 15A, 16M, 16A, 17M, 17A, 18M, 18A, 19M, 19A, 20M, 21M, 21A, 22M, 22A, 23M, 23A, 24M, 24A, 25M, 25A, 26M, and 26A.
<< 실시예Example 2> 지의류 추출물에 의한 2> by lichen extract HDPHDP 세포의 유전자 발현 측정 Measuring gene expression in cells
(1) 세포 배양(1) Cell culture
HDP 일차 세포는 CEFO Co., Ltd.에서 구입하였다. 세포를 10 % FBS (fetal bovine serum)과 1 % 페니실린-스트렙토마이신 용액이 보충된 DMEM 배지 (Gen Depot, TX, USA)에서 배양하였다. 세포를 가습 분위기에서 5 % CO2로 37 ℃에서 배양하고 3 일마다 배지를 교체하였다. 세포의 80% 합류에서 0.5 % 트립신-EDTA (Gibco, Grand Island, New York)를 사용하여 분리하고 매 3-4 일마다 1 : 4의 비율로 통과시켰다. 모든 실험은 5 개의 계대 배양된 세포를 사용하였다.HDP primary cells were purchased from CEFO Co., Ltd. Cells were cultured in DMEM medium (Gen Depot, TX, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution. Cells were cultured in a humidified atmosphere with 5% CO 2 at 37° C. and the medium was changed every 3 days. Cells were isolated from 80% confluence using 0.5% trypsin-EDTA (Gibco, Grand Island, New York) and passed at a ratio of 1:4 every 3-4 days. All experiments used 5 passage cultured cells.
(2) 지의류 추출물 처리 및 배양(2) Treatment and culture of lichen extract
6-웰 플레이트 (Corning, New York, USA)에 DMEM (FBS과 항생제가 첨가되지 않은)중 HDP 세포의 5 번 구획 (2.0x104 cells/well)을 접종하고 37 ℃의 습윤 분위기에서 10 시간 동안 5 % CO2에서 배양하였다. 그런 다음 DMSO에 용해된 5mg/mL의 지의류 추출물 또는 양성 대조군 토파시티니브(tofacitinib)을 최종 농도 5㎍/mL로 처리하고 6 시간 동안 배양하였다.6-well plates (Corning, New York, USA) were inoculated with 5 compartments (2.0x10 4 cells/well) of HDP cells in DMEM (without FBS and antibiotics added) and in a humidified atmosphere at 37° C. for 10 hours. Cultured in 5% CO 2 . Then, a 5 mg/mL lichen extract dissolved in DMSO or a positive control tofacitinib was treated with a final concentration of 5 μg/mL and cultured for 6 hours.
(3) 양성 대조군(tofacitinib)에 의한 HDP 세포의 유전자 발현 측정(3) Gene expression measurement of HDP cells by positive control (tofacitinib)
모발 성장 유전자 발현의 시간을 결정하기 위해, 지의류 추출물을 처리 전에 tofacitinib이 HDP 세포에 우선적으로 처리되었다. HDP 세포를 18 시간 배양하는 동안 매 6 시간마다 3 회 수확한 다음 qRT-PCR을 분석하였다. To determine the time of hair growth gene expression, tofacitinib was preferentially treated to HDP cells prior to treatment of the lichen extract. HDP cells were harvested three times every 6 hours during 18 hour incubation and then qRT-PCR analyzed.
구체적으로, 제조사의 지시에 따라 easy-spinTM (iNtRON Biotechnology, Korea, Korea)을 이용하여 HDF 세포로부터 총 RNA를 분리하였다. 처리 된 세포의 각 샘플로부터 총 RNA (1μg)를 ImProm-IITM 역전사 효소 키트 (Promega, WI, USA) 및 SYBR green (Bio-Rad, CA, USA)을 사용하여 cDNA로 전환시켰다. 이 배열에는 모발 성장과 관련된 10 개의 유전자가 포함되어 있으며, 유전자의 상대적인 발현 수준은 내인성 항존 유전자(housekeeping gene) 글리세르알데히드 3-포스페이트 탈수소효소(glyceraldehyde 3-phosphate dehydrogenase, GAPDH)에 대해 정상화되었다. qRT-PCR은 CFX (Bio-Rad, Hercules, USA)에서 수행되었다.Specifically, according to the manufacturer's instructions, total RNA was isolated from HDF cells using easy-spinTM (iNtRON Biotechnology, Korea, Korea). Total RNA (1 μg) from each sample of treated cells was converted to cDNA using ImProm-IITM reverse transcriptase kit (Promega, WI, USA) and SYBR green (Bio-Rad, CA, USA). This arrangement contains 10 genes involved in hair growth, and the relative expression levels of the genes were normalized to the endogenous housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). qRT-PCR was performed on CFX (Bio-Rad, Hercules, USA).
도 1은 양성 대조군 (tofacitinib) 처리 후 시간에 따른 모발 유전자 발현을 나타낸 것으로, 여기에서 보듯이 12 시간 및 18 시간 배양 이외의 6 시간의 tofacininib 처리 후에 10 개의 유전자 중 8 개가 강력하게 발현된다는 것이 확인되었다.FIG. 1 shows hair gene expression over time after treatment with a positive control (tofacitinib), as shown here, confirming that 8 of 10 genes are strongly expressed after 6 hours of tofacininib treatment other than 12 and 18 hour culture Became.
(4) 지의류 추출물에 의한 HDP 세포의 유전자 발현 측정(4) Measurement of gene expression of HDP cells by lichen extract
상기 실시예 1에서 수득된 지의류 메탄올 및 아세톤 추출물 52 종에 대한 발모 유전자 발현 프로파일을 분석하기 위해 HDP 세포에 처리하였다. HDP cells were treated to analyze the hair growth gene expression profile for 52 species of lichen methanol and acetone extract obtained in Example 1 above.
도 2은 지의류 메탄올 추출물 처리 후 시간에 따른 모발 유전자 발현을 나타낸 것이다. 여기에서 보듯이, 가장 두드러진 결과는 메탄올 추출물 처리된 세포가 아세톤 추출물 처리된 세포보다 유전자 발현 강도가 높다는 것이었다. 또한, Shh, S100a9, Ptgfr 및 Gli2와 같은 발모 유전자는 일부 아세톤 추출물에서는 낮게 나타났으나 대부분의 메탄올 추출물에서는 특히 지의류 스테레오카룰론 자포니쿰(Stereocaulon japonicum)의 메탄올 추출물인 M25에서 매우 높게 나타났다. M25 처리 세포의 고도로 발현된 유전자는 Prom1, Ptgfr, Dlx2, Shh, S100a9 및 Krt25이었다. 한편, tofacitinib 처리된 세포는 M25 처리된 세포보다 상대적으로 낮은 유전자 발현 강도를 보였다.Figure 2 shows the expression of hair gene over time after treatment with lichen methanol extract. As can be seen here, the most prominent result was that the cells treated with methanol extract had higher gene expression intensity than the cells treated with acetone extract. In addition, hair growth genes such as Shh, S100a9, Ptgfr, and Gli2 were found to be low in some acetone extracts, but in most methanol extracts, they were particularly high in M25, a methanol extract of lichen stereocarulon japonicum . The highly expressed genes of M25 treated cells were Prom1, Ptgfr, Dlx2, Shh, S100a9 and Krt25. On the other hand, tofacitinib-treated cells showed a relatively low gene expression intensity than M25-treated cells.
<실시예 3> 박막크로마토그래피 분석<Example 3> Thin-film chromatography analysis
M25 물질을 스크리닝하기 위해 TLC 분획 화를 수행 하였다. TLC 플레이트를 분리한 후 발모 유전자 발현 프로파일을 가진 물질을 측정하였다. TLC fractionation was performed to screen the M25 material. After the TLC plate was separated, a substance having a hair growth gene expression profile was measured.
TLC와 분취용 TLC (PTLC) 플레이트에서 메탄올 지의류 추출물을 검출하였다. 전개 용매로는 톨루엔 : 클로로포름 : 아세톤 (7:5:8, v/v)을 사용하였다. 결과는 254 및 350nm에서 UV 광으로 표시하여 시각화하였다. 각각의 분리된 스팟을 긁어내어 메탄올로 물질을 용출시켰다. 용출된 시료는 45℃에서 회전식 진공 증발기로 농축시키고 동결 건조시켰다. 분말화된 샘플은 HDP (hair dermal papilla, 모유두) 세포 실험을 위해 디메틸설폭사이드 (DMSO)에 용해시킨 후 사용하였다.Methanol lichen extract was detected on TLC and preparative TLC (PTLC) plates. Toluene: chloroform: acetone (7:5:8, v/v) was used as the developing solvent. The results were visualized by marking with UV light at 254 and 350 nm. Each isolated spot was scraped off to elute the material with methanol. The eluted sample was concentrated in a rotary vacuum evaporator at 45°C and freeze dried. The powdered sample was used after dissolving in dimethyl sulfoxide (DMSO) for HDP (hair dermal papilla) treatment.
도 3은 지의류 스테레오카룰론 자포니쿰(Stereocaulon japonicum)의 메탄올 추출물의 박막 크로마토그래피 분석결과이다. 여기에서 보듯이, 흑색 배경상의 7 개의 상이한 컬러 밴드 중 하나 이상이 발모 유전자를 발현하는 것으로 간주되었다. Figure 3 is a thin-film chromatography analysis of the methanol extract of lichen stereocarulon japonicum ( Stereocaulon japonicum ). As can be seen, one or more of the 7 different color bands on a black background was considered to express the hair growth gene.
상기 7 개의 분획 중 주요 물질을 확인하기 위해, 각 물질 샘플을 HDP 세포로 처리하였다. To identify the main material among the 7 fractions, each material sample was treated with HDP cells.
도 4는 지의류 스테레오카룰론 자포니쿰(Stereocaulon japonicum)의 메탄올 추출물의 모발 유전자 발현 강도를 나타낸 것이다. 여기에서 보듯이, M25-1 및 M25-4 분획물은 다른 것보다 현저하게 높은 유전자 발현 강도를 나타내었다. M25-1과 M25-4 모두에서 10 개의 모든 유전자가 발현되었으며, 이들 중 9 개는 tofacitinib보다 매우 높은 발현 강도를 보였다.Figure 4 shows the hair gene expression intensity of the methanol extract of lichen stereocarulon japonicum ( Stereocaulon japonicum ). As can be seen here, the M25-1 and M25-4 fractions showed significantly higher gene expression intensity than others. All 10 genes were expressed in both M25-1 and M25-4, and 9 of them showed much higher expression intensity than tofacitinib.
<실시예 4> 세포 생존율 분석<Example 4> Cell viability analysis
세포 생존력은 비색 MTT 대사 활성 분석을 사용하여 평가되었다. 지의류 스테레오카룰론 자포니쿰(Stereocaulon japonicum)의 메탄올 추출물의 TLC 분획 (M25-1, M25-4)을 DMSO에 용해시키고 DMEM으로 연속 희석하여 156, 313, 625, 1250, 2500 및 5000㎍/mL의 농도를 얻었다. HDP 세포 (2.0 X 104 cells/well)를 96-웰 플레이트에 접종하고 밤새도록 부착시킨 다음 각 물질 10μL (156μg/mL 내지 5000㎍/mL)로 처리하였다. 37 ℃에서 48 시간 배양 한 후, 10μL의 MTT 용액을 첨가하고 4 시간 동안 배양하였다. 이어서, MTT를 제거하고, 세포를 50 % DMSO 및 20 % SDS를 함유하는 용해 완충액으로 용해시켰다. 흡광도는 마이크로 플레이트 판독기 (Epoch, BioTekㄾ, Winooski, VT, USA)를 사용하여 570 nm에서 판독 하였다. 세포 생존력은 하기 수학식 1을 사용하여 계산하였다:Cell viability was assessed using a colorimetric MTT metabolic activity assay. The TLC fraction (M25-1, M25-4) of the methanol extract of lichen stereocarulon japonicum was dissolved in DMSO and serially diluted with DMEM to 156, 313, 625, 1250, 2500 and 5000 μg/mL The concentration of was obtained. HDP cells (2.0 X 10 4 cells/well) were seeded in 96-well plates and attached overnight, and then treated with 10 μL of each material (156 μg/mL to 5000 μg/mL). After incubation at 37°C for 48 hours, 10 μL of MTT solution was added and incubated for 4 hours. MTT was then removed and cells were lysed with lysis buffer containing 50% DMSO and 20% SDS. Absorbance was read at 570 nm using a microplate reader (Epoch, BioTek®, Winooski, VT, USA). Cell viability was calculated using Equation 1 below:
도 5는 지의류 스테레오카룰론 자포니쿰(Stereocaulon japonicum)의 메탄올 추출물의 세포 생존율을 나타낸 그래프이다.5 is a graph showing the cell viability of methanol extracts of lichen stereocarulon japonicum .
하기에 본 발명의 조성물을 위한 제제예를 예시한다.Examples of formulations for the compositions of the present invention are illustrated below.
<제제예 1> 지의류 추출물을 포함하는 화장료 제제 제조<Formulation Example 1> Preparation of cosmetic preparations containing lichen extract
1-1. 유연화장수1-1. Flexible Cosmetics
1-2. 샴푸1-2. shampoo
1-3. 린스1-3. Rinse
1-5. 팩1-5. pack
Claims (7)
상기 화장료가 헤어토닉, 모발영양 화장수, 스칼프 트리트먼트, 헤어 트리트먼트, 헤어린스, 헤어샴푸 또는 헤어로션인 것을 특징으로 하는 발모 촉진 및 탈모 방지용 화장료 조성물.According to claim 1,
The cosmetic composition for promoting hair growth and preventing hair loss, wherein the cosmetic material is hair tonic, hair nutrition lotion, scalp treatment, hair treatment, hair rinse, hair shampoo or hair lotion.
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| JP2003212789A (en) | 2002-01-11 | 2003-07-30 | Maruzen Pharmaceut Co Ltd | Hair or skin cosmetic and food/drink |
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| JP3830960B1 (en) * | 2005-08-12 | 2006-10-11 | 建仁 河乃 | Hair growth substance |
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