KR20170080548A - Cosmetic composition for promoting the stemness of adipose-derived stem cell and proliferating skin cell containing hypericum extracts - Google Patents
Cosmetic composition for promoting the stemness of adipose-derived stem cell and proliferating skin cell containing hypericum extracts Download PDFInfo
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- KR20170080548A KR20170080548A KR1020170080225A KR20170080225A KR20170080548A KR 20170080548 A KR20170080548 A KR 20170080548A KR 1020170080225 A KR1020170080225 A KR 1020170080225A KR 20170080225 A KR20170080225 A KR 20170080225A KR 20170080548 A KR20170080548 A KR 20170080548A
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Abstract
본 발명은 하이페리쿰 추출물을 유효성분으로 함유하는 지방유래 줄기세포의 줄기세포성 증진용 조성물 및 피부세포 증식용 조성물을 제공한다. 본 발명의 하이페리쿰 추출물을 유효성분으로 함유하는 조성물은 지방유래 줄기세포 재생을 촉진시키고 표피의 재생에 관여하는 피부 표피 기저층의 전이-증폭 세포의 증식을 촉진시켜, 피부 볼륨감을 증진시키고 손상된 피하지방의 기능을 회복시켜 주므로, 피부 볼륨감 증대용 피부 미용 및 성형용 조성물 및 피부 볼륨감 증대용 피부외용제 조성물에 적용 가능하다.The present invention provides a composition for promoting stem cell function and a composition for skin cell proliferation of an adipose-derived stem cell containing Hypericum extract as an active ingredient. The composition containing the hypericum extract of the present invention as an active ingredient promotes the regeneration of adipose stem cells and promotes the proliferation of metastasis-amplified cells of the skin epidermal basal layer involved in the regeneration of the epidermis, It is possible to apply the composition for skin aesthetic and molding for increasing skin volume and the composition for external application for skin for increasing skin volume.
Description
본 발명은 하이페리쿰 추출물을 함유하는 지방유래 줄기세포의 줄기세포성 증진용 및 피부세포 증식용 조성물에 관한 것이다.The present invention relates to a composition for promoting stem cell growth and skin cell proliferation of adipose derived stem cells containing Hypericum extract.
피부는 인체를 구성하고 있는 기관 중 가장 큰 기관으로, 우리 몸 부피의 약 16%를 차지하고, 면적이 1.8 m2, 두께가 2~4mm, 무게가 약 3kg이며, 표피, 진피 및 피하지방층으로 구성되어 있다. 이러한 피부는 외부 환경과 직접 접하고 있으면서 인체 유해 미생물과 같은 다양한 유해인자들과 자외선 등과 같은 외부 자극으로부터 인체를 보호하는 중요한 보호막 역할을 담당하는 동시에 전신의 대사에 필요한 생화학적 기능을 영위하는 생명 유지에 필수 불가결한 기관이다. 특히, 진피 아래에 존재하는 피하지방층은 지방세포(adipocyte)의 소엽으로 구성되어 있는데, 외부의 열에 대해 피부 내부 기관의 보호 및 충격을 흡수하는 쿠션 역할을 하며 영양성분을 트리글리세라이드(Triglyceride)의 형태로 지방체(Lipid droplet)라는 세포 소기관에 저장하여 필요시 에너지원으로 사용하기도 한다. 그러나 나이가 들어감에 따라 자연적인 요인과 각종 외부 자극에 의해 피부는 손상을 입게 되는데, 특히 진피에서 탄력을 담당하는 교원섬유(콜라겐), 탄력섬유(엘라스틴)의 활성 및 발현이 저하되며 탄력이 줄어듦과 동시에, 피하지방에서도 지방 생성이 감소함으로써 결국에는 피부 전반적으로 각 구성 세포들의 본래 기능이 제대로 이루어지지 않게 된다. 그래서, 현재 널리 사용되고 있는 방법이 지방이식수술로, 이는 튤립 니들(tulip needle) 모양의 끝이 날카롭지 않은 바늘을 갖는 주사기를 이용하여 자신의 지방을 채취해 탄력이 떨어져 보이는 피부의 각 부위에 이식하는 수술이다. 그러나 지방이식수술 이후에 이식 부피의 40~60%가 다시 인체 내에 흡수된다는 것이 보고된 바 있고, 피부가 얇고 피하지방이 적은 부위에서의 지방이식수술은 세포 덩어리가 만져지는 등의 문제가 제기되며, 실제로 이식한 지방의 생존율과 생존기간이 기대에 미치지 못해 재이식이 필요한 경우가 발생한다.The skin is the largest organ of the human body, accounting for about 16% of our body volume, 1.8 m 2 in area, 2 ~ 4 mm in thickness, weighing about 3 kg and composed of epidermis, dermis and subcutaneous fat layer . Such skin is in direct contact with the external environment and plays an important protective layer for protecting the human body from various harmful factors such as harmful microorganisms and ultraviolet rays, and at the same time, it has a biochemical function necessary for metabolism of the whole body. It is an indispensable institution. In particular, the subcutaneous fat layer below the dermis is composed of adipocyte lobules. It serves as a cushion for absorbing the protection of internal organs of the skin against external heat, and the nutrient component is formed in the form of triglyceride (Lipid droplet), which is stored in the organelle and used as an energy source if necessary. However, as the age increases, the skin becomes damaged due to natural factors and various external stimuli. In particular, the activity and expression of collagen (collagen) and elastic fiber (elastin) At the same time, the fat production in the subcutaneous fat is reduced, so that the original function of each constituent cell in the skin is not achieved properly. Therefore, the widely used method is fat graft surgery, which uses a syringe with a tulip needle-shaped tip to remove its own fat and transplant it to each part of the skin that is not elastic It is an operation. However, it has been reported that 40-60% of the transplant volume is resorbed into the human body after the fat transplant operation, and fat grafting at the site where the skin is thin and the subcutaneous fat is low causes problems such as cell mass being touched , The survival rate of the transplanted fat and the survival period are less than expected and re-transplantation is necessary.
이에, 이러한 문제점들이 있는 지방이식수술을 대체하면서 피하지방층을 충분히 증가시킬 수 있는 조성물에 대한 요구가 높아지고 있다. 최근 피부 탄력 감소의 원인인 콜라겐과 엘라스틴의 감소를 억제하려는 목적으로 개발되어 사용되고 있는 레티놀과 레티노익산 등의 레티노이드는 탁월한 피부 탄력 개선 효과를 나타내지만, 소량만을 피부에 적용하여도 자극이 나타난다는 단점을 가진다. 따라서 최근 들어 이러한 원료들의 피부 자극 문제를 해소하기 위해 보다 안전하고 피부에 자극이 없는 원료를 이용한 화장품 개발에 대한 요구가 높아지고 있다.Accordingly, there is an increasing demand for a composition capable of sufficiently increasing the subcutaneous fat layer while replacing the fat transplant surgery with these problems. Recently, retinoids such as retinol and retinoic acid, which have been developed and used for the purpose of inhibiting the reduction of collagen and elastin, cause skin elasticity reduction, exhibit excellent skin elasticity improvement effect. However, . In recent years, there has been a growing demand for the development of cosmetics using safer and less irritating ingredients in order to solve the skin irritation problem of such raw materials.
피부의 기본적인 구조는 피하지방조직에 의해서 유지되고, 이러한 피하지방조직은 피부의 볼륨감과 강도를 좌우하는 역할을 하므로, 지방조직의 부피를 증가시키는 것이 피부의 볼륨감 유지에 좋은 해결책이 될 수 있으므로, 피부의 볼륨감 유지 및 증진을 위해 피부 자극이 없으면서 지방조직의 부피를 증가시킬 수 있는 조성물의 개발이 필요한 실정이었다.Since the basic structure of the skin is maintained by subcutaneous fat tissue, and this subcutaneous fat tissue plays a role in the volume sense and strength of the skin, increasing the volume of the adipose tissue may be a good solution for maintaining the volume of the skin , It is necessary to develop a composition capable of increasing the volume of adipose tissue without skin irritation in order to maintain and enhance the volume of the skin.
한편, 피부의 가장 바깥에 위치하는 표피(epidermis)에서 재생능력을 보이는 것으로 알려진 것은, 기저층에 존재하는 피부의 각질 줄기세포(Keratinocyte stem cells, KSC)이다. 각질 줄기세포가 지속적으로 분열하여 크게 두 가지 세포를 생성하는데, 하나는 자기 복제(self-renewal)를 하여 만드는 똑같은 줄기세포이고, 다른 하나는 전이-증폭 세포(Transit-Amplifying Cells, TA cells)이다. 전이-증폭 세포들은 더욱 증폭하고, 초기 분화하여 각질 세포(Keratinocyte cells)가 되고, 계속 분화가 진행되어 이들은 피부 표면으로 밀려 올라간다. 이 과정에서 각질 세포는 분화(Differentiation)와 각화(Keratinazation)를 일으키면서 각질층을 이루게 되며, 3~4주 후에는 각질층에서 떨어져 나간다.On the other hand, keratinocyte stem cells (KSC) of the skin present in the basal layer are known to regenerate in the epidermis located at the outermost part of the skin. The keratinocyte stem cells continue to divide and produce two large cells: the same stem cells that are made by self-renewal, and the other is Transit-Amplifying Cells (TA cells) . Metastasis-amplified cells are further amplified and differentiated early to become keratinocyte cells, which continue to differentiate and push them up to the surface of the skin. In this process, the keratinocytes are differentiated and keratinized, forming the stratum corneum, and after 3 ~ 4 weeks they are separated from the stratum corneum.
상기 각질 줄기세포 및 전이-증폭세포는 보통 기저층 세포들의 약 4-7% 정도로 극히 적은 수로 분포되며, 각질 줄기세포의 수를 증가시키는 것은 전이-증폭 세포의 수를 증가시키는 것보다 쉽지 않으며, 상대적으로 더 넓은 지역에 분포되어 있지 않아 연구에 많은 어려움이 따른다. 그래서 각질 줄기세포 및 전이-증폭 세포의 증식 능력을 보이는 조성물의 개발이 필요한 실정이었다.The keratinocyte and metastasis-amplified cells are usually distributed in an extremely small number of about 4-7% of the basal layer cells, and increasing the number of keratinocyte cells is not as easy as increasing the number of metastasis-amplifying cells, And it is not distributed in a wider area. Therefore, it was necessary to develop a composition showing the proliferative capacity of keratinocyte stem cells and metastasis-amplified cells.
상기 문제점을 해결하기 위해, 본 발명은 피부에 자극이 없으면서 지방조직을 증가시킬 수 있는 하이페리쿰 추출물을 함유하는 조성물을 제공하는 것을 목적으로 한다. 또한 본 발명은 지방유래 줄기세포의 줄기세포성을 증진시키고 피부 볼륨감을 증가시킬 뿐 아니라, 각질 세포의 전이-증폭 세포를 증식시키고 피부세포 재생 능력을 나타내는 조성물을 제공하는 것을 목적으로 한다.In order to solve the above problems, it is an object of the present invention to provide a composition containing a hypericum extract capable of increasing adipose tissue without irritation to the skin. It is another object of the present invention to provide a composition for promoting stem cell function of adipose derived stem cells and for increasing skin volume, as well as proliferating metaplasia cells of keratinocytes and regenerating skin cells.
상기 목적을 달성하기 위해, 본 발명의 일실시예는 하이페리쿰 추출물을 유효성분으로 함유하는 지방유래 줄기세포의 줄기세포성 증진용 조성물을 제공할 수 있다.In order to accomplish the above object, an embodiment of the present invention can provide a composition for promoting stem cellability of adipose derived stem cells containing Hypericum extract as an active ingredient.
본 발명의 일실시예는 하이페리쿰 추출물을 유효성분으로 함유하는 피부세포 증식용 조성물을 제공할 수 있다.One embodiment of the present invention can provide a composition for skin cell proliferation comprising Hyperpericum extract as an active ingredient.
본 발명의 일실시예는 하이페리쿰 추출물을 유효성분으로 함유하는 피부 볼륨감 증대용 피부 미용 및 성형용 조성물을 제공할 수 있다.An embodiment of the present invention can provide a skin cosmetic and molding composition for enhancing skin volume reduction containing Hypericum extract as an active ingredient.
본 발명의 일실시예는 하이페리쿰 추출물을 유효성분으로 함유하는 피부 볼륨감 증대용 피부외용제 조성물을 제공할 수 있다.One embodiment of the present invention can provide a composition for external application for skin for increasing skin volume, which comprises Hyperpericum extract as an active ingredient.
본 발명의 일실시예는 피부에 하이페리쿰 추출물을 처리하는 것을 포함하는 피부세포 증식 촉진에 의한 피부 미용 방법을 제공할 수 있다.An embodiment of the present invention can provide a skin cosmetic method by promoting skin cell proliferation, which comprises treating Hypericum extract on the skin.
본 발명의 하이페리쿰 추출물을 유효성분으로 함유하는 조성물은 지방유래 줄기세포 재생을 촉진시키고 표피의 재생에 관여하는 피부 표피 기저층의 전이-증폭 세포의 증식을 촉진시켜, 피부 볼륨감을 증진시키고 손상된 피하지방의 기능을 회복시켜 주므로, 피부 볼륨감 증대용 피부 미용, 성형용 조성물 및 피부 볼륨감 증대용 피부외용제 조성물에 적용 가능하다.The composition containing the hypericum extract of the present invention as an active ingredient promotes the regeneration of adipose stem cells and promotes the proliferation of metastasis-amplified cells of the skin epidermal basal layer involved in the regeneration of the epidermis, The composition of the present invention can be applied to cosmetic compositions for increasing skin volume, composition for molding, and compositions for external application for skin volume increase.
도 1은 본 발명의 일실시예에 따른 조성물의 농도에 따른 지방유래 줄기세포의 세포독성을 나타내는 그래프이다.
도 2는 본 발명의 일실시예에 따른 조성물의 농도에 따른 피부 각질 세포의 세포독성을 나타내는 그래프이다.
도 3은 본 발명의 일실시예에 따른 조성물을 산화 스트레스를 가한 지방유래 줄기세포에 처리했을 때, CD29 및 CD44 유전자의 발현양상을 나타내는 그래프이다.
도 4는 본 발명의 일실시예에 따른 조성물을 자외선을 가한 피부 각질 세포에 처리했을 때, CD71 유전자의 발현양상을 나타낸 그래프이다.
도 5는 본 발명의 일실시예에 따른 조성물을 산화 스트레스를 가한 지방유래 줄기세포에 처리했을 때, (a) CD29, (b) CD44 단백질의 발현을 비교하는 그래프이다.1 is a graph showing cytotoxicity of adipose-derived stem cells according to concentration of a composition according to an embodiment of the present invention.
2 is a graph showing cytotoxicity of keratinocytes according to the concentration of the composition according to an embodiment of the present invention.
FIG. 3 is a graph showing expression patterns of CD29 and CD44 genes when oxidative stressed adipose stem cells were treated with a composition according to an embodiment of the present invention.
FIG. 4 is a graph showing the expression pattern of the CD71 gene when the composition according to an embodiment of the present invention is treated with ultraviolet irradiated keratinocytes.
FIG. 5 is a graph comparing the expression of (a) CD29, (b) CD44 protein when the composition according to an embodiment of the present invention is treated with oxidative stress-induced adipose stem cells.
이하에서는, 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 본 발명을 용이하게 실시할 수 있도록 하기 위하여, 본 발명의 바람직한 실시예에 관하여 첨부된 도면을 참조하여 상세히 설명하기로 한다.Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings, so that those skilled in the art can easily carry out the present invention.
본 발명은 하이페리쿰 추출물을 유효성분으로 함유하는 지방유래 줄기세포의 줄기세포성 증진용 조성물을 제공한다.The present invention provides a composition for enhancing stem cell characteristics of adipose derived stem cells containing Hypericum extract as an active ingredient.
하이페리쿰(Hypericum)은 물레나물과의 한 속으로, 세인트존스워트, 물레나물, 망종화, 갈퀴망종화, 고추나물 등 약 400종의 현화식물을 포함하는 속이다. 과(family)는 Clusiaceae, 아과(subfamily)는 Hypericoideae로 알려져 있다. 하이페리쿰은 열대 저지방, 사막, 극지방을 제외한 전세계에 분포되어 있다.Hypericum is a genus containing about 400 flowering plants, such as St John's Wort, Spinach, Chrysanthemum spp. The family is known as Clusiaceae, and the subfamily is known as Hypericoideae. Hypericum is distributed all over the world except tropical low-fat, desert, and polar regions.
본 발명에서 하이페리쿰은 하이페리쿰 속에 속하는 어떠한 식물이라도 사용될 수 있으며, 바람직하게는 세인트존스워트(Hypericum perforatum)를 사용할 수 있으나 이에 한정되는 것은 아니다.In the present invention, hypericum may be any plant belonging to the genus Hypericum, preferably, but not limited to, St John's Wort (Hypericum perforatum).
하이페리쿰 추출물은 하이페리쿰을 열수 추출 또는 에탄올 추출하여 얻을 수 있으나, 이에 한정되는 것은 아니다. 또한, 하이페리쿰 추출물은 상업적으로 구입하여 사용할 수도 있다. 예컨대, 한국의 바이오 에프디엔씨(Bio-FD&C)에서 구입하여 사용할 수 있다.The hypericum extract can be obtained by subjecting hypericum to hot water extraction or ethanol extraction, but is not limited thereto. In addition, the hypericum extract may be purchased and used commercially. For example, it can be purchased from Bio-FD & C of Korea.
상기 '줄기세포성 (stemness)'이란 줄기세포의 자기재생능력과 항상성을 지니며 줄기세포의 활성이 있는 상태를 말하며 줄기세포성 증진이란 줄기세포의 활성이 증가하는 것을 의미한다.The term 'stemness' refers to a state of stem cells having the self-renewal ability and homeostasis of stem cells. The term 'stem cell growth' refers to an increase in stem cell activity.
상기 지방유래 줄기세포(Adipose derived stem cell: ADSC)는 인간의 지방유래 줄기세포(human adipose derived stem cells, hADSCs)일 수 있으나 이에 한정되는 것은 아니다.The adipose derived stem cell (ADSC) may be human adipose derived stem cells (hADSCs), but is not limited thereto.
상기 지방유래 줄기세포의 줄기세포성 증진용 조성물은 지방유래 줄기세포 표지마커인 분화 클러스터 29, 인테그린 베타 1(cluster of differentiation 29,integrin beta 1: 이하 CD29) 및 분화 클러스터 44(이하 CD44)의 발현량을 증가시킬 수 있다. CD29와 CD44의 발현량 증가는 지방유래 줄기세포의 활성이 증가된다는 것을 의미한다. 따라서 상기 지방유래 줄기세포의 줄기세포성 증진용 조성물은 지방조직을 증가시켜서 피부에 볼륨감을 증가시키는 효능을 가질 수 있다.The composition for promoting stem cell proliferation of the adipose-derived stem cells includes the expression of the differentiation cluster 29, integrin beta 1 (hereinafter referred to as CD29) and the differentiation cluster 44 (hereinafter referred to as CD44), which are adipose-derived stem cell markers Can be increased. The increased expression of CD29 and CD44 means that the activity of adipose derived stem cells is increased. Therefore, the composition for promoting stem cell growth of the adipose derived stem cells may have an effect of increasing the volume of the skin by increasing the adipose tissue.
상기 지방유래 줄기세포의 줄기세포성 증진용 조성물은 조성물 총 중량에 대하여 하이페리쿰 추출물 0.0001 내지 10중량% 함유할 수 있고, 상세하게는 0.0001 내지 0.01중량%을 함유할 수 있다. 0.0001중량% 미만이면 효과가 미미하고, 10중량% 이상이면 세포독성을 나타내는 문제가 있다.The composition for promoting stem cell proliferation of adipose derived stem cells may contain 0.0001 to 10% by weight, more specifically 0.0001 to 0.01% by weight of Hypericum extract, based on the total weight of the composition. If the amount is less than 0.0001% by weight, the effect is insignificant. If the amount is 10% by weight or more, there is a problem that the composition exhibits cytotoxicity.
또한, 본 발명의 일실시예는 하이페리쿰 추출물을 유효성분으로 함유하는 피부세포 증식용 조성물을 제공한다.In addition, one embodiment of the present invention provides a composition for skin cell proliferation comprising Hyperpericum extract as an active ingredient.
상기 피부세포는 전이-증폭 세포(Transit-Amplifying cell, 이하 TA cell)일 수 있으나 이에 한정되는 것은 아니다.The skin cell may be a transit-amplifying cell (TA cell), but is not limited thereto.
상기 피부세포 증식용 조성물에 의하면 전이-증폭 세포의 증식 촉진에 의해 피부 표피를 재생시킬 수 있으며, 전이-증폭 세포는 각질 줄기세포와 함께, 끊임없는 세포 분열을 통해 각질세포 탈락에 의한 표피 손실을 극복하고, 표피를 재생시키는 역할을 할 수 있다. 따라서 상기 피부세포 증식용 조성물은 피부를 재생시키거나 피부 노화를 방지할 수 있다.According to the composition for skin cell proliferation, the skin epidermis can be regenerated by promoting the proliferation of metastasis-amplified cells, and the metastasis-amplifying cells together with the keratinocyte cells can cause epidermal loss due to the elimination of keratinocytes through continuous cell division Overcome, and can play a role in regenerating the epidermis. Therefore, the composition for skin cell proliferation can regenerate the skin or prevent skin aging.
본 명세서에 사용된 용어 '피부 재생'은 전반적인 피부 노화 및 피부 표피 등에 유발된 상처 등에 대응하여 새로운 각질세포를 생성하는 능력을 포함하는 광의의 개념이다. 구체적으로, 피부 재생은 피부 표피의 기저층에 존재하는 각질 줄기세포의 분열에 의해 생성된 전이-증폭 세포들이 더욱 증폭하고, 초기 분화하여 각질 세포가 되고, 피부 표면으로 밀려 올라가 분화(Differentiation)와 각화(Keratinazation)를 일으키면서 각질층을 이루게 되는 모든 과정을 의미한다. 그 중에서 피부 재생의 핵심 과정은 기저층에 존재하는 각질 줄기세포와 이 세포의 딸세포인 전이-증폭 세포의 증식에 있다. 피부의 겉부분만이 소실되는 찰과상, 긁혔을 때 나타나는 열상, 불에 의한 화상 등 외부의 물리적, 화학적 환경에 의해 일어나는 피부 손상이 일어났을 때, 이를 빠른 시간에 회복하기 위해서는 피부 기저층에 존재하며 피부 재생을 담당하고 있는 각질 줄기세포와 전이-증폭세포들의 활발한 증식이 필요하다. 보통 어릴 때는 피부 손상이 일어나더라도 흉터가 남지 않는 반면, 나이가 들어 피부 손상이 일어나게 되면 흉터가 남는 이유도 피부 재생에 필요한 각질 줄기세포와 전이-증폭 세포의 증식의 수준 정도가 나이 정도에 따라 현저하게 낮아지기 때문이다. 즉, 노화가 진행하게 되면, 이러한 세포들의 증식 정도가 낮아지게 되며, 이는 피부 재생 능력이 감퇴하는 것으로, 상처 등의 회복이 지연되거나, 피부 탄력 감소가 심화될 수 있다.As used herein, the term " skin regeneration " is a broad concept including the ability to generate new keratinocytes in response to an overall skin aging, a wound caused by skin epidermis, and the like. Specifically, skin regeneration further amplifies the metastasis-amplifying cells generated by the division of keratinocyte stem cells existing in the basal layer of skin epidermis, and early differentiation becomes keratinocyte, which is pushed up to the surface of the skin, (Keratinization) and the formation of the stratum corneum means all the process. Among them, the key process of skin regeneration is the proliferation of keratinocyte stem cells in the basal layer and metastasis - amplification cells which are daughter cells of the cells. It is present in the basal layer of the skin in order to restore the skin damage caused by external physical and chemical environment such as abrasion which is only the outer surface of the skin, heat which appears when it is scratched, Active replication of keratinocyte stem cells and metastasis-amplifying cells responsible for regeneration is necessary. The reason for the scar remains when the skin is damaged due to age, while the scar is not left even when the skin damage occurs at a normal age. The level of proliferation of keratinocyte and metastasis-amplification cells necessary for skin regeneration is remarkable depending on the age . That is, as the aging progresses, the degree of proliferation of these cells becomes low, which leads to a decrease in skin regeneration ability, which may delay the recovery of wounds and the like and reduce skin elasticity.
상기 피부세포 증식용 조성물을 이용해 증식된 피부세포를, 각질 줄기세포를 필요로 하는 개체에 이식할 수 있다.The skin cell proliferated using the composition for skin cell proliferation can be transplanted into an individual in need of keratinocyte stem cells.
본 발명자들은 하기의 시험예를 통해 입증된 바와 같이, 본 발명에 따른 하이페리쿰 추출물을 유효 성분으로 함유하는 피부세포 증식용 조성물을 피부 각질 세포에 적용하여, 전이-증폭세포 표지 마커의 발현이 유효하게 증가함을 밝혀내었다.As demonstrated by the following test examples, the inventors of the present invention have applied skin cell proliferation composition containing hyperpericum extract of the present invention as an active ingredient to dermal keratinocytes, and found that the expression of metastasis-amplified cell markers Effective increase.
상기 각질 줄기세포는 앞서 언급한 바와 같이 표피를 재생시키는 데 중요한 역할을 담당한다. 따라서, 본 발명에 따른 조성물에 의한 각질 줄기세포수의 증가를 통해, 상처 회복을 위한 피부 재생 능력을 향상시키고, 피부 재생 능력 감퇴로 인한 피부 탄력 감소 및 피부 노화의 심화를 방지할 수 있다. As mentioned above, the keratinocyte cells play an important role in regenerating the epidermis. Therefore, through the increase of the number of keratinocyte stem cells by the composition according to the present invention, it is possible to improve skin regeneration ability for wound healing and to prevent skin elasticity and skin aging from deepening due to decline in skin regeneration ability.
상기 조성물은 분화 클러스터 71(Cluster of Differentiation 71: 이하 CD71)을 발현하는 전이-증폭 세포의 표지 마커의 발현을 증가시킬 수 있으며, 상기 CD71은 전이-증폭 세포에 특이적인 표지 마커 단백질들이다.The composition may increase the expression of the marker of the metastasis-amplification cell expressing Cluster of Differentiation 71 (hereinafter referred to as CD71), and CD71 is a marker marker protein specific for metastasis-amplified cells.
상기 CD71 단백질은 트랜스페린(Transferrin) 수용체라고도 하며, 기저층에만 존재한다. 구체적으로, 상기 CD71 단백질은 세포 혈류를 타고 흐르는 철분과 결합되는 트랜스페린(transferring) 단백질과 상호작용을 하여, 수용체 매개 트랜스페린 함입(receptor mediated transferrin endocytosis)이라는 과정을 거쳐, 세포 내에 철분을 공급하는 역할을 한다. 활성화되고 있는 세포나, 증식하는 세포는 세포 내에 철분을 많이 필요로 하는데, 이때 CD71 단백질의 발현이 증가할 수 있다.The CD71 protein, also referred to as the transferrin receptor, is present only in the basal layer. Specifically, the CD71 protein interacts with a transferring protein that binds to iron flowing in the cell blood stream, and acts as a receptor mediated transferrin endocytosis to supply iron in the cells do. Activated or proliferating cells require a large amount of iron in the cells, which can increase the expression of the CD71 protein.
상기 피부세포 증식용 조성물은 조성물 총 중량에 대하여 하이페리쿰 추출물을 0.0001 내지 10중량% 함유할 수 있고, 상세하게는 0.0001 내지 0.01 중량%을 함유할 수 있다. 0.0001중량% 미만이면 효과가 미미하고, 10중량% 이상이면 세포독성을 나타내는 문제가 있다.The composition for skin cell proliferation may contain 0.0001 to 10% by weight, more specifically 0.0001 to 0.01% by weight of Hypericum extract, based on the total weight of the composition. If the amount is less than 0.0001% by weight, the effect is insignificant. If the amount is 10% by weight or more, there is a problem that the composition exhibits cytotoxicity.
또한, 본 발명의 일실시예는 하이페리쿰 추출물을 유효성분으로 함유하는 피부 볼륨감 증대용 피부 미용 및 성형용 조성물을 제공한다.In addition, one embodiment of the present invention provides a skin cosmetic and molding composition for enhancing skin volume containing an extract of Hypericum as an active ingredient.
상기 조성물은 비제한적인 예로 유방 조직 형성을 통한 가슴 확대, 미세지방 이식술, 자가지방 이식술 등 얼굴에 피부 볼륨감을 증진시켜 팔자 주름 및 미간주름 제거 및 탄력 유지를 목적으로 사용할 수도 있다.The composition may be used, for example, for the purpose of removing wrinkles and wrinkles and maintaining elasticity by enhancing the volume feeling on the face such as breast enlargement through breast tissue formation, micro fat grafting, autologous fat transplantation.
상기 조성물은 약학적 분야에서 통상의 방법에 따라 환자의 신체 내 투여에 적합한 단위투여형의 제제로 제형화시켜 투여할 수 있다. 이러한 목적에 적합한 제형으로는 비경구투여 제제로서 주사제 또는 국소 투여용 제제 등이 바람직하다. 주사용 제형은 등장성 수용액 또는 현탁액이 바람직하다. 그러나, 이때, 일반적으로 사용되는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 함께 사용할 수 있다.The composition may be formulated into a unit dosage form suitable for administration to a patient in the body according to conventional methods in the pharmaceutical field. As a formulation suitable for such purpose, an injectable preparation or a preparation for topical administration is preferably used as the parenteral administration preparation. The injectable formulations are preferably isotonic aqueous solutions or suspensions. However, at this time, diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like can be used together.
이렇게 제조된 본 발명의 제제는 통상의 방법에 따라 비경구적으로, 예를 들면 특정 부위에 직접 투여할 수 있다. 이때, 조성물의 주입은 주사기를 이용하여 이루어진다. 상기 조성물의 1일 투여량은 1×105 내지 1×107세포/kg 체중, 바람직하게는 5×105 내지 5×106세포/kg 체중이며, 1회 또는 수회로 나누어 투여할 수 있다. 그러나, 유효성분의 실제 투여량은 분화 및 증식시키고자 하는 지방세포의 양, 투여경로, 환자의 체중, 연령 및 성별 등 여러 관련 인자에 비추어 결정되어야 하는 것으로 이해되어야 하며, 따라서, 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.The thus prepared preparation of the present invention can be administered parenterally according to a conventional method, for example, directly to a specific site. At this time, the composition is injected using a syringe. The daily dose of the composition is 1 × 10 5 to 1 × 10 7 cells / kg body weight, preferably 5 × 10 5 to 5 × 10 6 cells / kg body weight, and can be administered once or several times . It should be understood, however, that the actual dosage of the active ingredient should be determined in light of various relevant factors such as the amount of fat cells to differentiate and proliferate, the route of administration, the weight of the patient, age and sex, And are not intended to limit the scope of the invention in any way.
또한, 본 발명의 일실시예는 하이페리쿰 추출물을 유효성분으로 함유하는 피부 볼륨감 증진용 피부 외용제 조성물을 제공한다.In addition, one embodiment of the present invention provides a composition for external application of skin for enhancing skin volume feeling containing Hyperpericum extract as an active ingredient.
상기 피부 외용제 조성물은 화장료 조성물 또는 약학 조성물일 수 있다.The composition for external application for skin may be a cosmetic composition or a pharmaceutical composition.
상기 화장료 조성물의 제형은 특별히 제한되지 않으며, 목적하는 바에 따라 적절히 선택할 수 있다. 예를 들어 유연 화장수, 영양화장수, 마사지 크림, 에센스, 영양크림, 팩 등의 제형으로 제조할 수 있으나, 이에 제한되지 않는다.The formulation of the cosmetic composition is not particularly limited and may be appropriately selected according to the purpose. For example, it can be manufactured by a formulation such as a soft lotion, a nutritional lotion, a massage cream, an essence, a nutrition cream, a pack, and the like, but is not limited thereto.
상기 약학 조성물은 로션, 연고, 겔, 크림, 패취 또는 분무제와 같은 경피 투여형 제형일 수 있으나, 이에 제한되지 않는다.The pharmaceutical compositions may be in a transdermal dosage form such as, but not limited to, lotions, ointments, gels, creams, patches or sprays.
각 제형에 의한 피부 외용제 조성물에 있어서, 상기한 본 발명의 조성물 이외의 다른 성분들을 기타 피부 외용제의 제형 또는 사용 목적 등에 따라 당업자가 어려움 없이 적의 선정하여 배합할 수 있으며, 이 경우 다른 원료와 동시에 적용할 경우 상승효과가 일어날 수 있다.In the composition for external application for skin according to each formulation, components other than the composition of the present invention may be mixed and selected without difficulty by those skilled in the art depending on the formulation or use purpose of other external preparation for skin, etc. In this case, A synergistic effect can occur.
또한, 본 발명에 의한 조성물이 약학 조성물로 사용될 경우에, 방부제, 안정화제, 수화제 또는 유화 촉진제, 삼투압 조절을 위한 염 또는 완충제 등의 약학적 보조제 및 기타 치료적으로 유용한 물질을 추가로 함유할 수 있으며, 통상적인 방법에 따라 다양한 경구 또는 비경구 투여 형태로 제형화할 수 있다. In addition, when the composition according to the present invention is used as a pharmaceutical composition, it may further contain a preservative, a stabilizer, a wetting agent or an emulsifying accelerator, a pharmaceutical adjuvant such as a salt or buffer for controlling osmotic pressure and other therapeutically useful substances And can be formulated into various oral or parenteral dosage forms according to conventional methods.
유효성분의 실제 투여량은 증상의 중증도, 선택된 투여 경로, 대상의 연령, 성별, 체중 및 건강상태 등의 여러 관련 인자에 비추어 결정되어야 하는 것으로 이해되어야 한다.It should be understood that the actual dosage of the active ingredient should be determined in light of various relevant factors such as the severity of the symptoms, the route of administration selected, the age, sex, weight and health status of the subject.
일반적으로 유효성분의 투여량은 0.001mg/kg/일 내지 대략 2000mg/kg/일의 범위이다. 더 바람직한 투여량은 0.5mg/kg/일 내지2.5mg/kg/일이다.Generally, the dosage of active ingredient is in the range of 0.001 mg / kg / day to about 2000 mg / kg / day. A more preferred dosage is 0.5 mg / kg / day to 2.5 mg / kg / day.
또한, 본 발명의 일실시예는 피부에 하이페리쿰 추출물을 함유하는 조성물을 처리하는 단계 포함하는 피부세포 증식 촉진에 의한 피부 미용 방법을 제공한다. 상기 하이페리쿰 추출물은 하이페리쿰을 끓는 물에 녹여 필터링 하여 얻은 추출물인 것일 수 있다.In addition, one embodiment of the present invention provides a skin cosmetic method by promoting skin cell proliferation, comprising the step of treating a composition containing Hypericum extract on skin. The hyperpericum extract may be an extract obtained by dissolving hyperpericum in boiling water and filtering.
본 발명에 의한 조성물의 적용 부위는 신체의 모든 지방 조직을 포함하며, 특정 부위에 한정하지는 않지만, 예를 들면, 지방층이 적은 얼굴 부위를 포함할 수 있다. The application site of the composition according to the present invention includes all the adipose tissues of the body and may include, for example, a face region having fewer fat layers, though it is not limited to a specific region.
본 발명에 따른 조성물에 의한 피부세포 증식은 생체 외에서 수행될 수 있고, 증식된 피부세포는 각질 줄기세포를 필요로 하는 개체에 이식될 수 있다.The skin cell proliferation by the composition according to the present invention can be carried out in vitro, and the proliferated skin cells can be transplanted into an individual in need of corneal stem cells.
본 발명에 따른 조성물에 의한 지방조직의 증가 효과로, 피부 볼륨감 증가 효과 외에도 피부 탄력 증대 효과, 피부 주름 개선 효과, 피부 재생 효과, 피부 노화 방지 효과 등이 있으나, 이에 한정되지는 않는다.The effect of increasing the fat tissue by the composition according to the present invention includes, but is not limited to, skin elasticity increasing effect, skin wrinkle improving effect, skin regeneration effect, skin aging prevention effect,
이하 하기 실시예 및 시험예를 들어 본 발명을 보다 구체적으로 설명하지만, 이는 본 발명을 예시하기 위한 것일 뿐 본 발명의 범위가 이들만으로 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to the following Examples and Test Examples, which should not be construed as limiting the scope of the present invention.
[실시예 1] 하이페리쿰 추출물을 함유하는 조성물의 제조[Example 1] Preparation of a composition containing a hypericum extract
바이오 에프디엔씨(Bio-FD&C)에서 세인트존스워트(Hypericum perforatum)를 구입하였다. 이 세인트존스워트(Hypericum perforatum)의 분말을 끓는 물에 녹인 후, 0.2㎛ 시린지 필터로 필터링하였고, 필터링한 액체를 하이페리쿰 추출물로 사용하였다.I purchased St. John's Wort (Hypericum perforatum) from Bio-FD & C. The powder of St John's Wort (Hypericum perforatum) was dissolved in boiling water, filtered through a 0.2 μm syringe filter, and the filtered liquid was used as a hypericum extract.
[시험예 1] 하이페리쿰 추출물의 세포독성[Test Example 1] Cytotoxicity of Hypericum extract
상기 실시예 1에 따른 하이페리쿰 추출물 함유 조성물의 세포독성을 파악하기 위하여 인간 지방유래 줄기세포 및 피부 각질 세포에 대한 세포독성 실험을 수행하였다.To examine the cytotoxicity of the composition containing Hypericum extract according to Example 1, a cytotoxicity test on human adipose-derived stem cells and dermal keratinocytes was performed.
(1) 인간 지방유래 줄기세포에서의 세포독성(1) cytotoxicity in human adipose-derived stem cells
인간의 지방유래 줄기세포(human adipose derived stem cells, hADSCs)를 론자社(Lonza, Inc. Walkersville, MD, USA)에서 구입하였다. 상기 인간 지방유래 줄기세포를 75cm2 T-flask에 계대배양(passage)하여, CO2 배양기(CO2 Incubator)에서 37℃, 5% CO2 조건 하에서 배양을 하였다. 통상적으로 두 번째 또는 세 번째에서 세포의 계대배양을 진행하였다.Human adipose derived stem cells (hADSCs) were purchased from Lonza, Inc. (Walkersville, MD, USA). The human adipose-derived stem cells were subcultured in a 75 cm 2 T-flask and cultured in a CO 2 incubator (CO 2 Incubator) at 37 ° C and 5% CO 2 . Typically, subculture of the cells was performed in the second or third cycle.
상기 하이페리쿰 추출물을 0.0001, 0.001, 0.01, 0.1, 1, 5, 10%의 농도로 상기 지방유래 줄기세포에 처리하고, 지방유래 줄기세포에서의 세포독성 정도를 CCK-8키트(Dojindo)를 이용하여 확인하였다.Derived stem cells at a concentration of 0.0001, 0.001, 0.01, 0.1, 1, 5 and 10%, and the degree of cytotoxicity in adipose derived stem cells was measured by CCK-8 kit (Dojindo) Respectively.
상기 실험 결과를 도 1에 나타내었다. 도 1에서 볼 수 있는 바와 같이, 하이페리쿰 추출물이 상기 농도에서 세포독성이 없거나 극히 미미함을 확인할 수 있었다.The experimental results are shown in Fig. As can be seen from Fig. 1, it was confirmed that the hypericum extract had no or very little cytotoxicity at the above concentrations.
(2) 피부 각질 세포에서의 세포독성(2) Cytotoxicity in dermal keratinocytes
인간의 피부 각질 세포(keratinocyte)를 론자社(Lonza, Inc. Walkersville, MD, USA)에서 구입하였다. 상기 피부 각질 세포를 75cm2 T-flask에 계대배양(passage)을 하여, CO2 배양기(CO2 Incubator)에서 37℃, 5% CO2 조건하에서 배양을 하였다. 통상적으로 두 번째 또는 세 번째에서 세포의 계대배양을 진행하였다. Human keratinocytes were purchased from Lonza, Inc. (Walkersville, MD, USA). The keratinocytes were subcultured in a 75 cm 2 T-flask and cultured in a CO 2 incubator (CO 2 Incubator) at 37 ° C and 5% CO 2 . Typically, subculture of the cells was performed in the second or third cycle.
상기 실시예 1에 따른 하이페리쿰 추출물을 0.0001, 0.001, 0.01, 0.1, 1, 5, 10%의 농도로 피부 각질세포에 처리하고, 피부 각질세포에서의 세포독성 정도를 CCK-8키트(Dojindo)를 이용하여 확인하였다.The skin keratinocytes were treated with the hypericum extract according to Example 1 at concentrations of 0.0001, 0.001, 0.01, 0.1, 1, 5, and 10%, and the degree of cytotoxicity in keratinocytes was measured using a CCK-8 kit (Dojindo ).
상기 실험 결과를 도 2에 나타내었다. 도 2에서 볼 수 있는 바와 같이, 상기 농도에서 세포독성이 없거나 극히 미미함을 확인할 수 있었다.The experimental results are shown in Fig. As can be seen from FIG. 2, it was confirmed that there was no or very little cytotoxicity at the above concentrations.
[시험예 2] 하이페리쿰 추출물의 세포 표지 마커 유전자의 발현량 측정[Test Example 2] Measurement of expression amount of cell marker marker gene of Hypericum extract
상기 실시예 1에 따른 하이페리쿰 추출물 함유 조성물의 효과를 파악하기 위하여 인간 지방유래 줄기세포 및 전이-증폭세포 각각의 표지 마커 유전자의 발현 수준을 측정하였다. 지방유래 줄기세포(human adipose derived stem cells, hADSCs) 및 피부 각질세포(keratinocyte)는 론자社(Lonza, Inc. Walkersville, MD, USA)에서 구입하였다.In order to understand the effect of the composition containing Hypericum extract according to Example 1, expression levels of marker marker genes of human adipose-derived stem cells and metastasis-amplified cells were measured. Human adipose stem cells (hADSCs) and keratinocytes were purchased from Lonza, Inc. (Walkersville, MD, USA).
(1) 지방유래 줄기세포의 CD29, CD44 발현량 측정(1) Measurement of expression of CD29 and CD44 in adipose-derived stem cells
지방유래 줄기세포의 줄기세포성 증진효과를 파악하기 위해서 하이페리쿰 추출물을 0.01%로 배지에 첨가하였다. 지방유래 줄기세포의 산화스트레스는 과산화수소수(H2O2)를 200μM 농도로 24시간 처리하였다.Hyperpericum extract was added to the medium at 0.01% in order to evaluate the stem cell proliferation effect of adipose derived stem cells. Oxidative stress of adipose derived stem cells was treated with hydrogen peroxide (H 2 O 2 ) at a concentration of 200 μM for 24 hours.
2ml의 인산염 완충액(Phosphate Buffered Saline, PBS)으로 세포를 세척한 다음, 트리졸 시약(Trizol reagent, Invitrogen, Carlsbad, CA, USA)을 사용하여 세포 내의 RNA를 분리하였다. 분리된 RNA를 키아젠사의 RNA 키트(QIAGEN RNeasy kt, QIAGEN, Valencia, CA)로 한번 더 정제한 후, 애질런트社의 바이오어낼라이저 2100 모델 기기(Agilent 2100 BioAnalyzer, Agilent Technologies, Santa Clara, CA, USA)를 사용하여 RNA의 질(quality)을 확인하였다. 인비트로젠社의 역전사키트(Superscript Reverse Transcriptase (RT) kit, Invitrogen, Carlsbad, CA)를 이용하여 상기 RNA로부터 cDNA를 합성하였고, 이를 실시간 역전사 중합 효소 연쇄반응(real time-reverse transcription polymerase chain reaction, Q-RT-PCR)을 통해 정량적으로 분석하였다.Cells were washed with 2 ml of phosphate buffered saline (PBS), and then RNA was isolated from the cells using a Trizol reagent (Invitrogen, Carlsbad, Calif., USA). The separated RNA was further purified with an RNA kit (QIAGEN RNeasykt, QIAGEN, Valencia, Calif.) From Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) ) Was used to confirm the quality of the RNA. Reverse transcription polymerase chain reaction (RT-PCR) was performed using the reverse transcription kit (Invitrogen, Carlsbad, Calif.) Of Invitrogen. Q-RT-PCR).
지방유래 줄기세포에서 CD29, CD44 유전자의 발현 패턴 변화를 어플라이드바이오시스템社의 택맨 유전자 발현 시스템(TaqMan gene expression assay kit, Applied Biosystems, Foster City, CA) (CD29 유전자 프라이머: Hs00559595_m1, CD44 유전자 프라이머: Hs01075861_m1)을 이용하여 평가하였으며, 그 결과를 도 3((a)CD29, (b)CD44)에 나타내었다.(CD29 gene primer: Hs00559595_m1, CD44 gene primer: Hs01075861_m1) in adipide biosystems, TaqMan gene expression assay kit (Applied Biosystems, Foster City, CA) ), And the results are shown in Fig. 3 ((a) CD29, (b) CD44).
(2) 피부 각질 세포의 CD71 발현량 측정(2) Measurement of CD71 expression level of keratinocytes
피부 각질 세포에 UVB를 100mJ/㎠로 조사한 후 하이페리쿰 추출물 0.001%을 처리하였다.The skin keratinocytes were treated with UVB at a dose of 100 mJ / cm 2 and treated with 0.001% of hypericum extract.
2ml의 인산염 완충액(Phosphate Buffered Saline, PBS)으로 세포를 세척한 다음, 트리졸 시약(Trizol reagent, Invitrogen, Carlsbad, CA, USA)을 사용하여 세포 내의 RNA를 분리하였다. 분리된 RNA를 키아젠사의 RNA 키트(QIAGEN RNeasy kt, QIAGEN, Valencia, CA)로 한번 더 정제한 후, 애질런트社의 바이오어낼라이저 2100 모델 기기(Agilent 2100 BioAnalyzer, Agilent Technologies, Santa Clara, CA, USA)를 사용하여 RNA의 질(quality)을 확인하였다. 인비트로젠社의 역전사키트(Superscript Reverse Transcriptase (RT) kit, Invitrogen, Carlsbad, CA)를 이용하여 상기 RNA로부터 cDNA를 합성하였고, 이를 실시간 역전사 중합 효소 연쇄반응(real time-reverse transcription polymerase chain reaction, Q-RT-PCR)을 통해 정량적으로 분석하였다.Cells were washed with 2 ml of phosphate buffered saline (PBS), and then RNA was isolated from the cells using a Trizol reagent (Invitrogen, Carlsbad, Calif., USA). The separated RNA was further purified with an RNA kit (QIAGEN RNeasykt, QIAGEN, Valencia, Calif.) From Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) ) Was used to confirm the quality of the RNA. Reverse transcription polymerase chain reaction (RT-PCR) was performed using the reverse transcription kit (Invitrogen, Carlsbad, Calif.) Of Invitrogen. Q-RT-PCR).
피부 각질 세포에서 유전자의 발현 패턴 변화를 어플라이드바이오시스템社의 택맨 유전자 발현 시스템(TaqMan gene expression assay kit, Applied Biosystems, Foster City, CA) (CD71 유전자 프라이며: Hs00951083_m1)을 이용하여 평가하였으며, 그 결과를 도 4에 나타내었다.Changes in expression patterns of genes in keratinocytes were measured using Applied Biosystems' TaqMan gene expression system (TaqMan gene expression assay kit, Applied Biosystems, Foster City, CA) (CD71 gene: Hs00951083_m1). The results are shown in FIG.
통계는 양측 student t 테스트로 검증하였으며, p 값이 0.05 이하일 때 유의적인 차이가 있다고 표시하였다(*로 표시함).Statistics were verified by bilateral student t test and indicated significant difference when p value was less than 0.05 (marked with *).
도 3 및 도 4에서 볼 수 있는 바와 같이, 지방유래 줄기세포 및 피부 각질 세포의 각 유전자(CD29, CD44, CD71)의 발현양이 음성대조군 대비 약 1.35배, 1.38배, 1.67배 증가함을 확인하였다. 이러한 증가는 통계적으로 유의한 것이었다.As can be seen from FIGS. 3 and 4, the expression levels of the respective genes (CD29, CD44, CD71) in adipose derived stem cells and keratinocytes were increased by about 1.35 times, 1.38 times, and 1.67 times Respectively. This increase was statistically significant.
[시험예 3] 지방유래 줄기세포의 유세포 분석[Test Example 3] Flow cytometry analysis of adipose derived stem cells
인간 지방유래 줄기세포를 0.25% 트립신(Trypsin-EDTA)으로 75cm2 Ti 플라스크로부터 분리한 뒤 1500rpm에서 5분간 세포를 가라앉힌 다음, 배양액을 제거한 뒤에 3ml의 차단버퍼(Blocking buffer), 2% 우태아 혈청(Fetal calf serum: FCS), 2% 소의 혈청 알부민(Bovine Serum Albumin(BSA) KGM-2)을 넣어주어 세포를 현탁하였다.Human adipose-derived stem cells were isolated from a 75 cm 2 Ti flask with 0.25% trypsin-EDTA, and then the cells were allowed to settle at 1500 rpm for 5 minutes. After removing the culture medium, 3 ml of blocking buffer, 2% fetal bovine serum The cells were suspended by adding serum (Fetal calf serum: FCS) and 2% serum albumin (Bovine Serum Albumin (BSA) KGM-2).
4℃에서 15분 동안 세포 (약 106 개의 세포)를 적용시킨 다음, FITC (Fluorescein isothiocyanate)가 부착된 CD44 항체(Cat No. 560977)(BD Pharmingen, CA, US)와 PE-Cy(phycoerythrin-cyanine)가 부착된 CD29 항체(Cat No. 559882) (BD Pharmingen, CA, US)을 각각 100: l로 넣어주어, 4℃ 하에서 45분 동안 반응시켰다. 한편, 음성대조군으로 사용되는 세포에는 FITC Rat IgG2a, k 아이소타이프(isotype)(Cat No. 555742)와 PE-Cy5 mouse IgG1 k 아이소타이프(Cat No. 555750)를 역시 100: l로 넣어주어, 4℃ 하에서 45분 동안 반응시켰다.Cells (approximately 10 6 cells) were applied for 15 min at 4 ° C followed by incubation with a CD44 antibody (Cat No. 560977) (BD Pharmingen, CA, US) with FITC (Fluorescein isothiocyanate) and a phycoerythrin- (Cat No. 559882) (BD Pharmingen, CA, US) with cyanine attached thereto was added at a ratio of 100: 1, followed by reaction at 4 ° C for 45 minutes. FITC Rat IgG2a, k isotype (Cat No. 555742) and PE-Cy5 mouse IgG1 k isotype (Cat No. 555750) were also added to the cells used as the negative control, Lt; 0 > C for 45 minutes.
반응이 끝난 후 3ml의 워싱 버퍼(2% Bovine Serum Albumin (BSA) KGM-2)로 두 번 세척한 후, 세포를 현탁하였다. 이후 FACSCalibur(BD Bioscience, San Jose, CA, U.S)로 지방유래 줄기세포에서 CD29, 44을 발현하는 세포들의 활성의 변화를 관찰하고, 이를 도 5((a)CD29, (b)CD44) 및 표 1에 나타내었다.After the reaction was completed, the cells were washed twice with 3 ml of washing buffer (2% Bovine Serum Albumin (BSA) KGM-2), and then the cells were suspended. (A) CD29, (b) CD44, and (b) CD44, and (b) CD44, respectively, in FACSCalibur (BD Bioscience, San Jose, CA, US) Respectively.
줄기세포(세포의 수)Derived from CD29
Stem cells (number of cells)
줄기세포(세포의 수)Derived from CD44
Stem cells (number of cells)
도 5 및 표 1에서 볼 수 있듯이, 음성대조군에 비해서 CD29는 24%, CD44는 14% 증가하였음을 확인할 수 있었다. As can be seen from FIG. 5 and Table 1, CD29 and CD44 were increased by 24% and 14%, respectively, as compared with the negative control group .
하기에 본 발명의 조성물을 위한 제형예 및 제제예를 예시한다.Formulation Examples and Formulation Examples for the composition of the present invention are illustrated below.
[제형예 1] 유연화장수[Formulation Example 1] Flexible longevity
상기 하이페리쿰 추출물을 함유하는 유연화장수 제형을 하기 표 2에 나타낸 조성으로 통상의 제조 방법에 따라 제조하였다.The softening water-containing formulation containing the hypericum extract was prepared according to a conventional preparation method with the composition shown in Table 2 below.
[제형예 2] 영양화장수[Formulation Example 2] Nutritional lotion
상기 하이페리쿰 추출물을 함유하는 영양화장수 제형을 하기 표 3에 나타낸 조성으로 통상의 제조 방법에 따라 제조하였다.The nutritional lotion formulation containing the hypericum extract was prepared according to a conventional preparation method with the composition shown in Table 3 below.
[제형예 3] 크림[Formulation Example 3] Cream
상기 하이페리쿰 추출물을 함유하는 크림 제형을 하기 표 4에 나타낸 조성으로 통상의 제조 방법에 따라 제조하였다.The cream formulations containing the hypericum extract were prepared according to the usual preparation methods with the compositions shown in Table 4 below.
[제제예 1] 정제[Formulation Example 1] Tablets
본 발명에 따른 하이페리쿰 추출물 100mg, 락토오스 400mg, 옥수수 전분 400mg 및 스테아린산 마그네슘 2mg을 혼합한 후, 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조하였다.100 mg of Hypericum extract according to the present invention, 400 mg of lactose, 400 mg of corn starch and 2 mg of magnesium stearate were mixed and tableted according to a conventional preparation method.
[제제예 2] 캡슐제[Formulation Example 2]
본 발명에 따른 하이페리쿰 추출물 100mg, 락토오스 400mg, 옥수수 전분 400mg 및 스테아린산 마그네슘 2mg을 혼합한 후, 통상의 캡슐제의 제조 방법에 따라서 젤라틴 캡슐에 충전하여 캡슐제를 제조하였다.100 mg of Hypericum extract according to the present invention, 400 mg of lactose, 400 mg of corn starch and 2 mg of magnesium stearate were mixed and filled in gelatin capsules according to the conventional preparation method of capsules to prepare capsules.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
Claims (7)
상기 조성물은 조성물 총 중량에 대하여 상기 하이페리쿰 추출물을 0.0001 내지 10중량% 함유하는 것인, 미용 또는 성형용 조성물.7. The method according to any one of claims 1 to 6,
Wherein the composition contains 0. < RTI ID = 0.0 > 1001 < / RTI > to 10% by weight of the hypericum extract relative to the total weight of the composition.
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