KR102540485B1 - Cosmetic compositions for skin improvement comprising exosome derived from leontopodium alpinum, adenium obesum, centella asiatica or polygonum multiflorum as an active ingredient - Google Patents
Cosmetic compositions for skin improvement comprising exosome derived from leontopodium alpinum, adenium obesum, centella asiatica or polygonum multiflorum as an active ingredient Download PDFInfo
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- KR102540485B1 KR102540485B1 KR1020220137839A KR20220137839A KR102540485B1 KR 102540485 B1 KR102540485 B1 KR 102540485B1 KR 1020220137839 A KR1020220137839 A KR 1020220137839A KR 20220137839 A KR20220137839 A KR 20220137839A KR 102540485 B1 KR102540485 B1 KR 102540485B1
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- exosomes
- skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
Description
본 발명은 식물 유래 엑소좀을 유효성분으로 포함하는 피부 개선용 화장료 조성물에 관한 것이다.The present invention relates to a cosmetic composition for skin improvement comprising plant-derived exosomes as an active ingredient.
사람의 피부는 표피(Epidermis), 진피(Dermis) 및 피하조직(Subcutis)의 3층 구조로 이루어져 있다. 주름과 가장 밀접하게 연관되어 있는 것은 피부 세포의 노화이다. 일반적으로 피부 노화의 원인은 고령화에 따라 나타나는 자연 노화와 외부환경에 의해 나타나는 내적인 노화로 분류될 수 있다. 자연 노화는 유전자적 요인 등이 많이 관여되기 때문에 제어가 어렵다. 그렇기 때문에 자외선，산화，건조 등과 같은 환경적 요인을 제어하기 위해 많은 연구가 진행되고 있다. 환경적 요인은 주로 진피층에 포함된 콜라겐(Collagen) 단백질, 엘라스틴(Elastin) 단백질 등을 파괴하거나 변성을 일으켜 주름을 유발한다. 특히, 태양의 자외선은 피부세포(섬유아세포)를 산화시키고 엘라스틴의 생성저하 및 파괴를 일으키는데, 자외선 등에 의해 피부 내에 엘라스틴 분해효소(Elastase)가 발현되며 이 효소가 피부 내의 정상적인 엘라스틴을 분해하여 피부 탄력 저하를 가져 온다. 또한, 환경적 요인에 의해 양산된 활성 산소는 산화 작용에 의해 피부 세포 내의 단백질 파괴, 당의 산화, 유전자 변형 등을 일으켜 피부에 노화 및 주름을 유발할 수 있다. 이러한 산화력과 활성 산소는 피부 세포 내의 단백질 파괴, 당의 산화, 유전자 변형 등을 일으켜 피부에 노화 및 주름을 유발할 수 있다. 이에 따라, 상기 부작용은 최소화하면서도 피부 미백과 산화 방지 및 주름 방지 효과를 모두 극대화할 수 있는 물질의 개발이 요구되고 있다. 또한, 피부 자극, 세포 독성 등의 위해 요소가 적어 함량의 제한 없이 사용할 수 있는 화장료 조성물에 대한 요구도 증가하고 있다.Human skin consists of a three-layer structure of epidermis, dermis, and subcutaneous tissue. What is most closely related to wrinkles is the aging of skin cells. In general, the causes of skin aging can be classified into natural aging caused by aging and internal aging caused by the external environment. Natural aging is difficult to control because many genetic factors are involved. Therefore, many studies are being conducted to control environmental factors such as ultraviolet rays, oxidation, and drying. Environmental factors mainly cause wrinkles by destroying or denaturing collagen and elastin proteins contained in the dermal layer. In particular, ultraviolet rays from the sun oxidize skin cells (fibroblasts) and cause reduction and destruction of elastin production. brings down In addition, active oxygen mass-produced by environmental factors may cause protein destruction in skin cells, oxidation of sugars, genetic modification, etc. by oxidation to cause aging and wrinkles in the skin. Such oxidizing power and active oxygen can cause protein destruction in skin cells, oxidation of sugars, genetic modification, etc., which can cause aging and wrinkles in the skin. Accordingly, there is a demand for the development of a material capable of maximizing both skin whitening, anti-oxidation and anti-wrinkle effects while minimizing the side effects. In addition, there is an increasing demand for a cosmetic composition that can be used without limiting the content due to low risk factors such as skin irritation and cytotoxicity.
피부는 환경의 변화나 내부의 요인에 의한 스트레스 및 외부 요인에 의한 자극 등으로 민감해지며 수분 보유력이 저하될 수 있으며, 노화가 진행됨에 따라 각질층의 수분이 감소하여 피부가 건조해지고 윤기를 잃어 칙칙해 보이는 등의 현상이 발생한다. 피부의 수분이 감소하는 경우 피부는 건조하게 되며 탄력이 떨어지고 주름 생성이 이루어지게 된다. 이와 더불어 피부 톤도 어두워지며 피부 보호막 기능이 저하되어 외부의 자극에도 더욱 민감한 상태가 되어 각종 트러블이 증가할 수 있다. 이처럼 각질층의 보습 상태는 피부의 외형(피부의 유연성 및 거친 정도)과 직접적으로 연관되어 있으며, 각질층의 피부 수분 함량은 피부의 컨디션 및 투과성과 관련되는 피부 막 항상성에 영향을 미치므로, 화장품 또는 바르는 의약품의 흡습도가 달라질 수 있어 피부 보습이 중요한 요소로 작용하고 있다. 따라서 피부가 보습 및 탄력을 잃지 않도록 피부의 보습력을 유지해주고, 그 효과를 지속시키는 화장료 조성물에 대한 요구가 증가하는 추세이다.The skin becomes sensitive due to changes in the environment, stress caused by internal factors, and irritation caused by external factors, and its ability to retain moisture may decrease. phenomena such as appearing. When the moisture of the skin decreases, the skin becomes dry, loses elasticity, and wrinkles are formed. In addition, the skin tone becomes darker and the skin barrier function is lowered, making it more sensitive to external stimuli, which can increase various troubles. As such, the moisturizing state of the stratum corneum is directly related to the appearance of the skin (skin flexibility and roughness), and the skin moisture content of the stratum corneum affects the skin membrane homeostasis related to the condition and permeability of the skin. The moisture absorption of medicines can change, so skin moisturizing is an important factor. Therefore, there is an increasing demand for a cosmetic composition that maintains the moisturizing power of the skin so that the skin does not lose moisture and elasticity and maintains the effect.
한편, 에델바이스(Leontopodium alpinum)는 국화과의 다년생 고산 식물로써 잎 표면에 솜털이 약간 있고, 잎의 뒷면은 회백색이다. 에델바이스란 ‘고귀한 흰 빛’이란 뜻으로 유럽에서는 흔히 ‘알프스의 별’이라고도 부르며 스위스와 오스트리아의 국화이다. 에델바이스 꽃 추출물은 천연 안티 에이징의 효능을 내는 천연 노화방지제이며 혈액순환 증진을 통해 피부가 세포 생성을 활성화 할 수 있게 한다.On the other hand, edelweiss ( Leontopodium alpinum ) is a perennial alpine plant of the Asteraceae family, and has some downy hairs on the leaf surface, and the back side of the leaf is grayish white. Edelweiss means 'noble white light' and is often called 'Star of the Alps' in Europe and is the national flower of Switzerland and Austria. Edelweiss flower extract is a natural anti-aging agent that has the effect of natural anti-aging, and enables the skin to activate cell production by enhancing blood circulation.
사막장미(Adenium obesum)는 용담목 협죽도과에 속하며 줄기가 비대하는 특징을 가지며 동아프리카 전역에 넓게 자생한다. 석화는 사막과 같은 건조한 날씨에 살아남기 위해 다량의 수분을 땅 위의 줄기나 잎에 저장하는 다육식물의 하나로 피부에 수분을 공급하는 화장품의 원료로써 중요한 공급원으로 여겨진다.Desert rose ( Adenium obesum ) belongs to the Gentian oleander family and has a characteristic of having a swollen stem and grows widely throughout East Africa. Petrification is one of the succulents that store a large amount of moisture in the stems or leaves on the ground to survive in dry weather such as deserts.
병풀(Centella asiatica)은 쌍떡잎식물로 미나리과의 여러해살이 식물로 덩굴이나 뿌리가 땅 위로 길게 뻗으며 자라나는 포복성 식물이다. 우리나라에서는 ‘병을 치료하는 식물’이라 해서 병풀로 불리우며, 중국, 인도 등에서는 수명 연장 및 에너지 약초로 널리 사용되어지고 있다. 병풀의 효능으로 피부 상처, 만성 궤양, 관절염, 나병의 치료, 해열제, 이뇨제, 강장제 등이 보고되어 있고, 혈관 강화 및 혈액 순환 촉진 기능을 갖고 있으며, 체내 노폐물을 원화하게 배설해 준다. 병풀이 가지고 있는 대표적인 유효 성분은 아시아틱산(Asiatic acid), 아시아티코사이드(Asiaticoside), 마데카식산 (Madecassic acid), 마데카소사이드(Madecassocide)의 총 4가지 성분으로 이들은 피부 재생과 콜라겐 합성 촉진 능력을 지닌다. 특히, 아시아티코사이드(Asiaticoside)는 항균 및 항진균 활성에 있어 탁월한 효과를 지닌다. Centella asiatica is a dicotyledonous perennial plant of the Apiaceae family. It is a creeping plant with vines or roots extending long above the ground. In Korea, it is called Centella Asiatica because it is a 'plant that treats disease', and in China and India, it is widely used as a life extension and energy herb. The efficacy of centella asiatica has been reported to treat skin wounds, chronic ulcers, arthritis, leprosy, antipyretic, diuretic, tonic, etc. The representative active ingredients of Centella asiatica are four ingredients: Asiatic acid, Asiaticoside, Madecassic acid, and Madecassocide, which have the ability to promote skin regeneration and collagen synthesis. has In particular, Asiaticoside has an excellent effect on antibacterial and antifungal activity.
하수오(Polygonum multiflorum)는 마디풀과 여러해살이 덩굴식물로 옛날부터 산삼과 견줄만한 영약으로 알려져 왔으며, 머리를 검게하는 강장제 한약재로 쓰인다. 이와 유사한 백수오는 사실 과(科)수준에서 차이가 나는데, 백수오는 마디풀과가 아닌 박주가리과에 속하며, 하수오는 덩어리진 뿌리를 지니는 반면, 백수오는 길쭉한 모양의 뿌리를 지닌다. 멜라닌 억제 성분으로 알려진 알부틴보다 멜라닌 억제율이 좋아 피부 미백 등에 탁월한 효능이 알려져 있다.Hasuo ( Polygonum multiflorum ) is a knotweed and perennial vine plant that has been known since ancient times as an elixir comparable to wild ginseng, and is used as a tonic herbal medicine to blacken hair. Baeksuo, which is similar to this one, is actually different at the family level. Baeksuo belongs to the family Pacaceae, not to the Knobaceae family, and Hasuo has lumpy roots, while Baeksuo has elongated roots. It has a better melanin inhibition rate than arbutin, known as a melanin inhibitor, and is known for its excellent efficacy in skin whitening.
세포로 구성된 모든 생명체는 세포들간의 정보 교환 또는 외부 환경과의 정보 및 물질 교환을 하며, 이를 위해 세포밖으로 작은 소낭의 소포체인 세포밖소포체(Extracellular vesicles)를 내보낸다. 엑소좀(Exosome)은 이런 세포와 세포사이의 신호 전달에 있어 중요한 메신저로 50 ~ 200 nm 정도의 크기를 지니는 나노사이즈의 소포체이다. 구조를 보면 세포막과 동일한 인지질이중막(phospholipid bilayer)을 지니고 있어 세포와 세포간의 이동 및 침투가 용이하고, 면역반응, 신호 전달과 같은 다양한 생리적, 병리적 기능을 수행한다. 엑소좀은 그 내부에는 단백질, 지질, 핵산 등의 기원세포 유래 생리활성 물질을 포함하는데, 유래된 세포의 성질 및 상태에 따라 특이적인 유전물질과 생체활성 인자들이 포함된다. 동물 줄기세포에서 분리되는 엑소좀은 포함된 인자가 목적 이외의 효과를 나타낼 수 있으며, 엑소좀의 분리에 있어 줄기세포가 포함될 수 있다는 점에서 안전성의 문제가 될 수도 있다. 이런 동물 세포 유래 엑소좀의 안전성 문제는 식물 세포의 엑소좀을 이용하는 것으로 해결될 수 있다.All organisms composed of cells exchange information between cells or exchange information and materials with the external environment. Exosomes are nano-sized endoplasmic reticulum having a size of about 50 to 200 nm as important messengers in signal transmission between cells. Looking at the structure, it has the same phospholipid bilayer as the cell membrane, so it is easy to move and penetrate between cells, and it performs various physiological and pathological functions such as immune response and signal transmission. Exosomes contain physiologically active substances derived from the cells of origin, such as proteins, lipids, and nucleic acids, and include specific genetic substances and bioactive factors depending on the nature and state of the derived cells. Exosomes separated from animal stem cells may have effects other than the intended ones, and may be a safety issue in that stem cells may be included in the isolation of exosomes. The safety problem of animal cell-derived exosomes can be solved by using plant cell exosomes.
최근 식물에서 유래한 엑소좀의 다양한 효능에 관한 연구가 이루어지고 있다. 식물은 동물 세포와 달리 세포막(Cell membrane) 뿐만이 아닌 세포벽(Cell wall)을 지니고 있고, 이런 세포벽과 세포막 사이에는 공간이 형성되는데, 이런 공간을 전세포벽(Apoplast)로 명한다. 전세포벽에서는 양분 및 수분의 이동뿐만 아닌 수많은 세포외 소포체(Extracellular vesicles) 또는 소립자(Nanovesicles)인 엑소좀(Exosomes)이 축적된다. 식물 유래 엑소좀도 동물 유래 엑소좀과 마찬가지로 다양한 유전 정보를 지니는 핵산(DNAs, messenger RNAs, micro RNAs), 단백질(Proteins), 지질(Lipids) 등이 주로 존재하며, 식물만이 지니는 다양한 특이적 이차대사물질(Secondary metabolites)도 존재한다. 또한, 식물체는 다양한 지질(Lipids)을 지니는데, 식물 유래 엑소좀의 경우에도 동물 유래 엑소좀에 비해 지질의 구성이 다양하고, 함유량이 높아 이에 따른 다양한 생리 활성을 기대할 수 있다. 식물 유래 엑소좀은 천연물 또는 비건 유도체로 동물 세포가 능동적으로 흡수 할 수 있고, 이는 합성 전달체(Synthetic carrier)보다 생체 적합성(Biocompatibility)이 높다.Recently, studies on various effects of plant-derived exosomes have been conducted. Unlike animal cells, plants have a cell wall as well as a cell membrane, and a space is formed between the cell wall and the cell membrane, and this space is called an apoplast. Numerous extracellular vesicles or nanovesicles, exosomes, as well as movement of nutrients and water are accumulated in the whole cell wall. Plant-derived exosomes, like animal-derived exosomes, mainly contain nucleic acids (DNAs, messenger RNAs, micro RNAs), proteins, and lipids with various genetic information, and various specific secondary Secondary metabolites also exist. In addition, plants have various lipids, and even in the case of plant-derived exosomes, the composition of lipids is diverse and high in content compared to animal-derived exosomes, so various physiological activities can be expected accordingly. Plant-derived exosomes are natural products or vegan derivatives that can be actively absorbed by animal cells, and have higher biocompatibility than synthetic carriers.
식물 유래 천연물 원료의 수요는 나날이 증가하고 있지만, 현실적으로 지구 온난화에 따른 기후 변화 및 생육에 있어 계절적, 공간적 제약을 받는 식물은 원활한 공급을 위한 개선책이 필요하다. 그 일환으로 식물 세포?조직배양 기술력을 이용한 식물 줄기세포인 캘러스 배양은 이런 문제점을 극복 할 수 있을 뿐만 아니라 식물 자원의 고갈 및 환경 파괴로부터 자연환경 보존 등 이용가치가 높은 기술력이다. 고기능성 물질로 다양한 이차대사물질인 Phytochemicals을 지니고 있는 캘러스 유래 식물 추출물은 이미 고가의 핵심원료로 화장품에 적용이 되고 있지만, 이들 배양체로부터 분리된 식물 줄기세포 유래 엑소좀 개발은 취약한 현실이다.Demand for plant-derived natural raw materials is increasing day by day, but in reality, climate change due to global warming and plants subject to seasonal and spatial restrictions in growth require improvement measures for smooth supply. As part of this, callus cultivation, which is a plant stem cell using plant cell/tissue culture technology, can not only overcome these problems, but also is a technology with high use value such as preservation of the natural environment from depletion of plant resources and environmental destruction. Callus-derived plant extracts, which are highly functional substances and have various secondary metabolites, phytochemicals, are already applied to cosmetics as an expensive core raw material, but the development of plant stem cell-derived exosomes isolated from these cultures is a weak reality.
엑소좀을 이용하는 피부 개선용 화장료 조성물 기술로는, 국내의 경우 대한민국 등록특허공보 제10-2192317호「천연 추출물 유래 엑소좀을 유효성분으로 포함하는 피부 진정용 조성물」, 대한민국 등록특허공보 제10-2159394호「바다제비집에서 유래된 엑소좀을 유효성분으로 포함하는 피부 상태 개선용 조성물」, 대한민국 등록특허공보 제10-2058444호「장미 줄기세포 유래의 엑소좀을 유효성분으로 포함하는 화장료 조성물」 등이 개시되어 있다.As for cosmetic composition technology for skin improvement using exosomes, in Korea, Korean Patent Registration No. 10-2192317 "Composition for skin soothing containing exosomes derived from natural extracts as an active ingredient", Korean Patent Registration No. 10-2192317 No. 2159394 "Composition for improving skin conditions containing exosomes derived from bird's nest as an active ingredient", Korean Patent Registration No. 10-2058444 "Cosmetic composition containing exosomes derived from rose stem cells as an active ingredient", etc. This is disclosed.
그러나, 아직까지 에델바이스(Leontopodium alpinum) 유래 엑소좀, 사막장미(Adenium obesum) 유래 엑소좀, 병풀(Centella asiatica) 유래 엑소좀, 하수오(Polygonum multiflorum) 유래 엑소좀 및 이들의 혼합 엑소좀으로 이루어진 군으로부터 선택되는 식물 유래 엑소좀을 이용하여 피부 탄력, 주름 방지, 피부 장벽 강화 및 보습 효과를 동시에 부여할 수 있는 화장료 조성물에 대한 연구는 존재하지 않은 실정이다.However, from the group consisting of exosomes derived from edelweiss ( Leontopodium alpinum ), exosomes derived from desert rose ( Adenium obesum ), exosomes derived from Centella asiatica , exosomes derived from Polygonum multiflorum , and mixed exosomes thereof There is no research on a cosmetic composition capable of simultaneously imparting skin elasticity, wrinkle prevention, skin barrier strengthening, and moisturizing effects using selected plant-derived exosomes.
이에, 본 발명자들은 에델바이스(Leontopodium alpinum) 유래 엑소좀, 사막장미(Adenium obesum) 유래 엑소좀, 병풀(Centella asiatica) 유래 엑소좀, 하수오(Polygonum multiflorum) 유래 엑소좀 및 이들의 혼합 엑소좀으로 이루어진 군으로부터 선택되는 식물 유래 엑소좀이 MMP-1 mRNA 발현 저해 활성 및 COL1A1 mRNA 발현 활성이 우수할 뿐만 아니라, 아쿠아포린-3(AQP3) 유전자 및 Filaggrin 유전자의 발현을 증가시키기 때문에, 피부 탄력, 주름 방지, 피부 장벽 강화 및 보습 효과를 동시에 부여할 수 있다는 사실을 발견하고 본 발명을 완성하게 되었다.Accordingly, the present inventors have proposed a group consisting of exosomes derived from edelweiss ( Leontopodium alpinum ), exosomes derived from desert rose ( Adenium obesum ), exosomes derived from Centella asiatica , exosomes derived from Polygonum multiflorum , and mixed exosomes thereof. Since plant-derived exosomes selected from are not only excellent in MMP-1 mRNA expression inhibitory activity and COL1A1 mRNA expression activity, but also increase the expression of aquaporin-3 (AQP3) gene and filaggrin gene, skin elasticity, anti-wrinkle, The present invention was completed by discovering that skin barrier strengthening and moisturizing effects can be imparted at the same time.
본 발명의 목적은 식물 유래 엑소좀의 피부 탄력, 주름 방지, 피부 장벽 강화 및 보습용 화장료 조성물을 제공하는 데 있다.An object of the present invention is to provide a cosmetic composition for skin elasticity, wrinkle prevention, skin barrier enhancement and moisturizing of plant-derived exosomes.
본 발명의 다른 목적은 상기 조성물을 포함하는 화장품을 제공하는 데 있다.Another object of the present invention is to provide a cosmetic comprising the composition.
상기와 같은 목적을 달성하기 위하여, 본 발명은 에델바이스(Leontopodium alpinum) 유래 엑소좀, 사막장미(Adenium obesum) 유래 엑소좀, 병풀(Centella asiatica) 유래 엑소좀, 하수오(Polygonum multiflorum) 유래 엑소좀 및 이들의 혼합 엑소좀으로 이루어진 군으로부터 선택되는 식물 유래 엑소좀을 유효성분으로 포함하는 피부 탄력, 주름 방지, 피부 장벽 강화 및 보습용 화장료 조성물을 제공한다.In order to achieve the above object, the present invention provides exosomes derived from edelweiss ( Leontopodium alpinum ), exosomes derived from desert rose ( Adenium obesum ), exosomes derived from Centella asiatica , exosomes derived from Polygonum multiflorum and these Provided is a cosmetic composition for skin elasticity, anti-wrinkle, strengthening of skin barrier and moisturizing, comprising plant-derived exosomes selected from the group consisting of mixed exosomes as an active ingredient.
본 발명에 있어서, 상기 에델바이스 유래 엑소좀은 에델바이스 캘러스 유래 엑소좀인 것을 특징으로 한다.In the present invention, the edelweiss-derived exosome is characterized in that it is an edelweiss callus-derived exosome.
본 발명에 있어서, 상기 사막장미 유래 엑소좀은 사막장미 캘러스 유래 엑소좀인 것을 특징으로 한다.In the present invention, the desert rose-derived exosomes are characterized in that they are desert rose callus-derived exosomes.
본 발명에 있어서, 상기 병풀 유래 엑소좀은 생병풀의 잎 유래 엑소좀인 것을 특징으로 한다.In the present invention, the exosomes derived from Centella asiatica are characterized in that they are exosomes derived from leaves of Centella asiatica.
본 발명에 있어서, 상기 하수오 유래 엑소좀은 하수오의 전초 유래 엑소좀인 것을 특징으로 한다.In the present invention, the exosomes derived from sewage are characterized in that they are exosomes derived from the plant of sewage.
본 발명에 있어서, 상기 혼합 엑소좀은 에델바이스 유래 엑소좀, 사막장미 유래 엑소좀, 병풀 유래 엑소좀 및 하수오 유래 엑소좀이 1:0.5~2:0.5~2:0.5~2의 중량비로 혼합된 것을 특징으로 한다.In the present invention, the mixed exosome is a mixture of edelweiss-derived exosomes, desert rose-derived exosomes, centella-derived exosomes, and sewage-derived exosomes in a weight ratio of 1:0.5 to 2:0.5 to 2:0.5 to 2 to be characterized
본 발명에 있어서, 상기 엑소좀은 평균 입자 크기가 50 ~ 200 nm인 세포외소포체(extracellular vesicles)인 것을 특징으로 한다.In the present invention, the exosome is characterized in that the average particle size is 50 ~ 200 nm of extracellular vesicles (extracellular vesicles).
본 발명에 있어서, 상기 엑소좀은 1x107 ~ 1x109 particles/mL의 입자수를 포함하는 것을 특징으로 한다.In the present invention, the exosome is characterized in that it comprises a particle number of 1x10 7 ~ 1x10 9 particles/mL.
본 발명에 있어서, 상기 엑소좀은 100 ~ 300ug/mL의 단백질을 포함하는 것을 특징으로 한다.In the present invention, the exosome is characterized in that it contains a protein of 100 ~ 300ug / mL.
본 발명에 있어서, 상기 식물 유래 엑소좀은 조성물 총 중량에 대하여 1 내지 40 중량% 포함되는 것을 특징으로 한다.In the present invention, the plant-derived exosome is characterized in that it is included in 1 to 40% by weight based on the total weight of the composition.
또한, 본 발명은 상기 조성물을 포함하는 화장품을 제공한다.In addition, the present invention provides a cosmetic comprising the composition.
본 발명으로부터 제공되는 식물 유래 엑소좀은 MMP-1 mRNA 발현 저해 활성 및 COL1A1 mRNA 발현 활성이 우수할 뿐만 아니라, 아쿠아포린-3(AQP3) 유전자 및 Filaggrin 유전자의 발현을 증가시키기 때문에, 피부 탄력, 주름 방지, 피부 장벽 강화 및 보습용 화장료 조성물로 활용 가능하다.The plant-derived exosomes provided by the present invention not only have excellent MMP-1 mRNA expression inhibitory activity and COL1A1 mRNA expression activity, but also increase the expression of aquaporin-3 (AQP3) gene and filaggrin gene, so skin elasticity and wrinkles It can be used as a cosmetic composition for prevention, skin barrier strengthening, and moisturizing.
도 1은 에델바이스 캘러스 유래 엑소좀을 NTA 장비(ZetaView, Particle Metrix, German)를 이용하여 엑소좀의 크기 및 농도를 측정한 것이다.
도 2는 사막장미 캘러스 유래 엑소좀을 NTA 장비(ZetaView, Particle Metrix, German)를 이용하여 엑소좀의 크기 및 농도를 측정한 것이다.
도 3은 병풀 유래 엑소좀을 NTA 장비(ZetaView, Particle Metrix, German)를 이용하여 엑소좀의 크기 및 농도를 측정한 것이다.
도 4는 하수오 유래 엑소좀을 NTA 장비(ZetaView, Particle Metrix, German)를 이용하여 엑소좀의 크기 및 농도를 측정한 것이다.
도 5는 에델바이스 캘러스 유래 엑소좀, 사막장미 캘러스 유래 엑소좀, 병풀 유래 엑소좀 및 하수오 유래 엑소좀의 단백질 함량을 나타낸 그래프이다.
1 shows the measurement of exosome size and concentration of Edelweiss callus-derived exosomes using NTA equipment (ZetaView, Particle Metrix, German).
2 shows the measurement of exosome size and concentration of desert rose callus-derived exosomes using NTA equipment (ZetaView, Particle Metrix, German).
3 is a measurement of the size and concentration of exosomes derived from Centella asiatica using NTA equipment (ZetaView, Particle Metrix, German).
4 is a measurement of the size and concentration of exosomes derived from sewage using NTA equipment (ZetaView, Particle Metrix, German).
5 is a graph showing the protein content of exosomes derived from edelweiss callus, exosomes derived from desert rose callus, exosomes derived from centella asiatica, and exosomes derived from S. aureus.
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 가진다. 일반적으로, 본 명세서에서 사용된 명명법 은 본 기술분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclatures used herein are those well known and commonly used in the art.
본원 명세서 전체에서, 어떤 부분이 어떤 구성 요소를 "포함" 한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성 요소를 제외하는 것이 아니라 다른 구성요소를 더 포함할 수 있는 것을 의미한다.Throughout the present specification, when a certain component is said to "include", it means that it may further include other components without excluding other components unless otherwise stated.
본 발명의 일 구현예에 따르면, 본 발명은 에델바이스(Leontopodium alpinum) 유래 엑소좀, 사막장미(Adenium obesum) 유래 엑소좀, 병풀(Centella asiatica) 유래 엑소좀, 하수오(Polygonum multiflorum) 유래 엑소좀 및 이들의 혼합 엑소좀으로 이루어진 군으로부터 선택되는 식물 유래 엑소좀을 유효성분으로 포함하는 피부 탄력, 주름 방지, 피부 장벽 강화 및 보습용 화장료 조성물에 관한 것이다.According to one embodiment of the present invention, the present invention provides exosomes derived from edelweiss ( Leontopodium alpinum ), exosomes derived from desert rose ( Adenium obesum ), exosomes derived from Centella asiatica , exosomes derived from Polygonum multiflorum and these It relates to a cosmetic composition for skin elasticity, anti-wrinkle, skin barrier strengthening and moisturizing, comprising plant-derived exosomes selected from the group consisting of mixed exosomes as an active ingredient.
본 발명의 일 구현예에 따르면, 상기 에델바이스 유래 엑소좀은 에델바이스 캘러스 유래 엑소좀이며, 더욱 상세하게는 에델바이스 줄기세포인 캘러스의 현탁 액체배양을 통해 수득된 세포와 배양된 세포를 여과지에 걸러낸 배양액을 통해 수득된 것을 특징으로 한다.According to one embodiment of the present invention, the edelweiss-derived exosomes are exosomes derived from edelweiss callus, and more specifically, cells obtained through suspension liquid culture of callus, which is an edelweiss stem cell, and a culture medium obtained by filtering the cultured cells through filter paper It is characterized in that obtained through.
또한, 상기 사막장미 유래 엑소좀은 사막장미 캘러스 유래 엑소좀이며, 더욱 상세하게는 사막장미 줄기세포인 캘러스의 현탁 액체배양을 통해 수득된 세포와 배양된 세포를 여과지에 걸러낸 배양액을 통해 수득된 것을 특징으로 한다.In addition, the desert rose-derived exosomes are desert rose callus-derived exosomes, and more specifically, the cells obtained through suspension liquid culture of desert rose stem cells, callus, and the culture medium obtained by filtering the cultured cells through filter paper characterized by
또한, 상기 병풀 유래 엑소좀은 생병풀의 잎 착즙액을 통해 수득된 엑소좀인 것일 수 있으며, 하수오 유래 엑소좀은 하수오의 잎, 줄기, 뿌리 등으로 이루어진 하수오 전초의 착즙액을 통해 수득된 엑소좀인 것일 수 있다.In addition, the exosomes derived from Centella asiatica may be exosomes obtained through the juice of the leaves of Centella Asiatica, and the exosomes derived from S. aureus are exosomes obtained through the juice of S. It could be a little bit.
본 발명의 일 구현예에 따르면, 상기 혼합 엑소좀은 에델바이스 유래 엑소좀, 사막장미 유래 엑소좀, 병풀 유래 엑소좀 및 하수오 유래 엑소좀이 1:0.5~2:0.5~2:0.5~2의 중량비로 혼합된 것이 바람직하고, 그 중에서도 에델바이스 유래 엑소좀, 사막장미 유래 엑소좀, 병풀 유래 엑소좀 및 하수오 유래 엑소좀이 1:1:1:1 중량비로 혼합된 것이 피부 탄력, 주름 방지, 피부 장벽 강화 및 보습 효과가 우수하다는 측면에서 바람직하다.According to one embodiment of the present invention, the mixed exosomes include edelweiss-derived exosomes, desert rose-derived exosomes, centella-derived exosomes, and sewage-derived exosomes in a weight ratio of 1:0.5 to 2:0.5 to 2:0.5 to 2. is preferably mixed with, among others, a mixture of edelweiss-derived exosomes, desert rose-derived exosomes, centella-derived exosomes, and sewage-derived exosomes in a weight ratio of 1:1:1:1 for skin elasticity, wrinkle prevention, and skin barrier It is preferable in terms of excellent strengthening and moisturizing effects.
본 발명의 일 구현예에 따르면, 상기 엑소좀은 평균 입자 크기가 50 ~ 200 nm인 세포외소포체(extracellular vesicles)인 것일 수 있으며, 엑소좀이 상기 범위를 포함할 경우 모공 보다도 작은 크기의 나노소포체로 인하여 피부 내 흡수능이 증대되기 때문에 피부의 탄성이 증진될 수 있는 측면에서 바람직하다.According to one embodiment of the present invention, the exosomes may be extracellular vesicles having an average particle size of 50 to 200 nm, and when the exosomes contain the above range, nanovesicles smaller than pores It is preferable in terms of improving the elasticity of the skin because the absorption capacity in the skin is increased due to.
또한, 상기 엑소좀은 1x107 ~ 1x109 particles/mL의 입자수를 포함하는 것일 수 있으며, 엑소좀이 상기 범위를 포함할 경우 피부 탄력, 주름 방지, 피부 장벽 강화 및 보습 효과가 우수하다는 측면에서 바람직하다.In addition, the exosome may include a particle number of 1x10 7 to 1x10 9 particles / mL, and when the exosome contains the above range, skin elasticity, wrinkle prevention, skin barrier strengthening and moisturizing effect are excellent. desirable.
또한, 상기 엑소좀은 100 ~ 300ug/mL의 단백질을 포함하는 것일 수 있으며, 엑소좀이 상기 범위를 포함할 경우 피부 탄력, 주름 방지, 피부 장벽 강화 및 보습 효과가 우수하다는 측면에서 바람직하다.In addition, the exosome may contain 100 ~ 300ug / mL of protein, and when the exosome contains the above range, it is preferable in terms of excellent skin elasticity, wrinkle prevention, skin barrier strengthening, and moisturizing effect.
본 발명의 일 구현예에 따르면, 상기 식물 유래 엑소좀은 조성물 총 중량에 대하여 1 내지 40 중량% 포함될 수 있고, 더욱 바람직하게는 1 내지 20 중량%포함될 수 있다. 상기 식물 유래 엑소좀이 조성물의 총 중량에 대하여 1 중량% 미만일 경우에는 피부 탄력, 주름 방지, 피부 장벽 강화 및 보습 효과를 충분히 얻을 수 없기 때문에 조성물로서의 기능이 떨어지고, 40 중량%를 초과하는 경우에는 그 이상을 함유하지 않아도 충분한 효능을 나타낼 수 있기 때문에 비경제적일 뿐만 아니라, 제형의 안정성 및 발림성 등이 저하되어 화장품으로 사용에 어려움이 있기 때문에 바람직하지 않다.According to one embodiment of the present invention, the plant-derived exosomes may be included in an amount of 1 to 40% by weight, more preferably 1 to 20% by weight, based on the total weight of the composition. If the plant-derived exosome is less than 1% by weight relative to the total weight of the composition, skin elasticity, wrinkle prevention, skin barrier strengthening and moisturizing effects cannot be sufficiently obtained, so the function as a composition is deteriorated, and if it exceeds 40% by weight, It is undesirable because it is uneconomical because it can show sufficient efficacy without containing more than that, and it is difficult to use it as a cosmetic because the stability and spreadability of the formulation are lowered.
전술된 바와 같이, 식물 유래 엑소좀은 MMP-1 mRNA 발현 저해 활성 및 COL1A1 mRNA 발현 활성이 우수할 뿐만 아니라, 아쿠아포린-3(AQP3) 유전자 및 Filaggrin 유전자의 발현을 증가시키기 때문에, 피부 탄력, 주름 방지, 피부 장벽 강화 및 보습용 화장료 조성물로 사용될 수 있다.As described above, plant-derived exosomes not only have excellent MMP-1 mRNA expression inhibitory activity and COL1A1 mRNA expression activity, but also increase the expression of the aquaporin-3 (AQP3) gene and the filaggrin gene, thereby improving skin elasticity and wrinkles. It can be used as a cosmetic composition for prevention, skin barrier strengthening and moisturizing.
본 발명의 일 구현예에 따르면, 본 발명은 상기 조성물을 포함하는 화장품 에 관한 것이다.According to one embodiment of the present invention, the present invention relates to cosmetics comprising the composition.
상기 화장품은 피부 탄력, 주름 방지, 피부 장벽 강화 및 보습용으로 적용이 가능하다.The cosmetics can be applied for skin elasticity, wrinkle prevention, skin barrier strengthening, and moisturizing.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명 하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 요지 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it is to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. It will be self-explanatory.
<실시예 1> 에델바이스 캘러스 유래 엑소좀 제조<Example 1> Preparation of exosomes derived from Edelweiss callus
(1) 에델바이스 식물 세포 유도(1) Edelweiss plant cell induction
에델바이스 종자(Chilternseeds, UK)를 70% 에탄올 60초 침지한 후, 소독액(50% 락스 + 0.1% Tween-20)으로 20분 소독한 후, 멸균수로 3회 세척하였다. 종자의 휴면타파 및 발아를 촉진하기 위해 지베렐린계 식물생장 조절제인 GA3 (Gibberellic acid)를 10 uM 농도가 포함된 MS배지를 사용하였다. 종자 발아 후, 생육을 위해서 호르몬 free 배지로 옮겨 발근 유도 및 생육을 유지하였다. 발아 후, 8주 정도 된 식물체의 잎을 날카로운 칼로 가로*세로 4*4mm 크기의 절편체를 만들어 0.5 mg/L 6-BA(6-Benzylaminopurine), 0.3 mg/L 2,4-D(2,4-dichlorophenoxyacetic acid), 3% Sucrose 및 0.4% gelrite가 포함된 기본 MS배지에서 25 ℃, 습도 70% 의 생장상 조건으로 암배양하여 캘러스를 유도한 다음, 캘러스 유도 상태를 현미경으로 관찰하였다. 고체배지 상태에서 4주 간격으로 3 ~ 4회 계대배양을 통해 우량 세포주를 선발하였다. 선발된 우량 세포주는 적정 호르몬이 포함된 MS 액체배지에서 온도조건 23±2℃에서 10 L 생물반응기(Bioreactor)에 공기공급량 0.2 vvm으로 6주간 액상 현탁배양을 통해 에델바이스 캘러스 배양체를 준비하였다 .Edelweiss seeds (Chilternseeds, UK) were immersed in 70% ethanol for 60 seconds, disinfected with a disinfectant solution (50% lacquer + 0.1% Tween-20) for 20 minutes, and washed three times with sterile water. MS medium containing GA3 (Gibberellic acid), a gibberellin-based plant growth regulator, at a concentration of 10 uM was used to promote seed dormancy breaking and germination. After seed germination, they were transferred to a hormone-free medium for growth, and rooting induction and growth were maintained. After germination, 8-week-old plant leaves were cut with a sharp knife, measuring 4*4 mm in length and width, and 0.5 mg/L 6-BA (6-Benzylaminopurine), 0.3 mg/L 2,4-D (2,4-D) 4-dichlorophenoxyacetic acid), 3% sucrose, and 0.4% gelrite in the basic MS medium containing 25 ℃, 70% humidity in the dark culture to induce callus, and then callus induction was observed under a microscope. Excellent cell lines were selected through subcultures 3 to 4 times at intervals of 4 weeks in a solid medium. Edelweiss callus cultures were prepared through liquid suspension culture for 6 weeks in a 10 L bioreactor with an air supply of 0.2 vvm in a 10 L bioreactor at a temperature of 23 ± 2 ° C in MS liquid medium containing appropriate hormones.
(2) 에델바이스 캘러스 유래 엑소좀 제조(2) Preparation of exosomes derived from Edelweiss callus
에델바이스 캘러스 유래 엑소좀 제조를 위해 바이오리액터에서 배양된 에델바이스 캘러스 배양체를 여과하여 배양체와 배양액을 분리한 다음, 캘러스 배양체 10% 및 캘러스 배양액 90%(w/w%) 중량비로 취하여 homogenizer를 이용하여 배양된 세포를 배양액과 함께 50~100 rpm 속도로 분쇄하였다. 분쇄액은 400 um mesh로 거른 다음, 원심분리기를 이용하여 1,500 ~ 2,000 xg로 20 ~ 30분 동안 원심분리 후, 상등액만을 취하여 세포의 잔여물을 분리 및 제거하였다. 상등 추출액을 모아 초고속 원심분리기를 이용하여 100,000 ~ 150,000 xg로 2시간 동안 원심분리 후, 펠렛층에 엑소좀을 가라앉힌 후 재분산하는 단계를 거쳐 엑소좀을 분리하였다. 상기에서 얻어진 에델바이스 캘러스 배양액 유래 엑소좀을 DNase/RNase free water로 녹인 다음, 0.22 ㎛ 필터로 여과하여 제균 작업을 실시하였다. 최종적으로 수득된 에델바이스 캘러스 배양액 유래 엑소좀을 NTA 장비(ZetaView, Particle Metrix, German)를 이용하여 크기와 농도를 확인하였다.For the production of Edelweiss callus-derived exosomes, the cultured Edelweiss callus cultured in a bioreactor was filtered to separate the cultured body and the culture medium, and then the callus cultured 10% and the callus cultured medium were taken at a weight ratio of 90% (w/w%) and cultured using a homogenizer. The cells were ground together with the culture medium at a speed of 50-100 rpm. The pulverized solution was filtered through a 400 um mesh, and then centrifuged at 1,500 to 2,000 xg for 20 to 30 minutes using a centrifuge, and only the supernatant was taken to separate and remove cell residues. After collecting the supernatant extract and centrifuging at 100,000 to 150,000 xg for 2 hours using an ultra-high-speed centrifuge, the exosomes were settled in the pellet layer and then redispersed to separate the exosomes. The exosomes derived from the edelweiss callus culture medium obtained above were dissolved in DNase/RNase free water, and then filtered through a 0.22 μm filter to remove bacteria. Finally, the size and concentration of the exosomes derived from the Edelweiss callus culture medium were confirmed using NTA equipment (ZetaView, Particle Metrix, German).
그 결과, 도 1에서 확인할 수 있듯이, 에델바이스 캘러스 배양액 유래 엑소좀은 엑소좀의 평균 입자 크기가 136.8 nm인 세포외소포체인 것을 확인할 수 있었고, 입자수는 2.9x109 particles/mL 인 것을 확인하였다.As a result, as can be seen in FIG. 1, it was confirmed that the exosomes derived from the culture medium of Edelweiss callus were extracellular vesicles having an average particle size of 136.8 nm, and the number of particles was 2.9x10 9 particles/mL.
<실시예 2> 사막장미 캘러스 유래 엑소좀 제조 <Example 2> Preparation of exosomes derived from desert rose callus
(1) 사막장미 잎세포 유도(1) Induction of desert rose leaf cells
사막장미 어린잎(서울화훼)을 70% 에탄올 60초 침지한 후, 소독액(50% 락스 + 0.1% Tween-20)으로 20분 소독한 후, 멸균수로 3회 세척하였다. 소독된 사막장미의 잎을 날카로운 칼로 가로*세로 4*4 mm 크기의 절편체를 만들어 2.0 mg/L NAA(1-naphthaleneacetic acid), 1.0 mg/L 6-BA(6-Benzylaminopurine), 3% Sucrose 및 0.4% gelrite가 포함된 기본 MS배지에서 25 ℃, 습도 70% 의 생장상 조건으로 암배양하여 캘러스를 유도한 후 캘러스 유도 상태를 현미경으로 관찰하였다. 고체배지 상태에서 4주 간격으로 3~4회 계대배양을 통해 우량 세포주를 선발하였다. 선발된 우량 세포주는 적정 호르몬(NAA 2 mg/L, 6-BA 1 mg/L)이 포함된 MS 액체배지에서 온도조건 23±2 ℃에서 10 L 생물반응기(Bioreactor)에 공기공급량 0.2 vvm로 6주간 액체현탁배양하여 사막장미 캘러스 배양체를 준비하였다.Young leaves of desert rose (Seoul Flower) were immersed in 70% ethanol for 60 seconds, disinfected with a disinfectant solution (50% bleach + 0.1% Tween-20) for 20 minutes, and washed three times with sterile water. The leaves of the sterilized desert rose were cut with a sharp knife to a size of 4 x 4 mm in length and width, and 2.0 mg/L NAA (1-naphthaleneacetic acid), 1.0 mg/L 6-BA (6-Benzylaminopurine), 3% Sucrose and 0.4% gelrite in a basic MS medium containing 25 ° C., 70% humidity in the dark to induce callus, and then observe the state of callus induction under a microscope. Excellent cell lines were selected through subcultures 3 to 4 times at 4-week intervals in a solid medium. Selected excellent cell lines were cultured in MS liquid medium containing appropriate hormones (NAA 2 mg/L, 6-
(2) 사막장미 캘러스 유래 엑소좀 제조(2) Preparation of exosomes derived from desert rose callus
사막장미 캘러스 유래 엑소좀 제조를 위해, 바이오리액터에서 배양된 사막장미 캘러스 배양체를 여과하여 배양체와 배양액을 분리한 다음, 캘러스 배양체 10%와 캘러스 배양액 90%(w/w%) 중량비로 취하여 homogenizer를 이용하여 배양된 세포를 배양액과 함께 50~100 rpm 속도로 분쇄하였다. 분쇄액은 400 um mesh로 거른 다음, 원심분리기를 이용하여 1,500 ~ 2,000 xg로 20 ~ 30분 동안 원심분리 후, 상등액만을 취하여 세포의 잔여물을 분리 및 제거하였다. 상등 추출액을 모아 초고속 원심분리기를 이용하여 100,000 ~ 150,000 xg로 2시간 동안 원심분리 후, 엑소좀을 펠렛층에 가라앉힌 후 재분산하는 단계를 거쳐 엑소좀을 분리하였다. 상기에서 얻어진 사막장미 캘러스 유래 엑소좀을 DNase/RNase free water로 녹인 다음, 0.22 ㎛ 필터로 여과하여 제균 작업을 실시하였다. 최종적으로 수득된 사막장미 캘러스 유래 엑소좀을 NTA 장비(ZetaView, Particle Metrix, German)를 이용하여 크기와 농도를 확인하였다.For the production of desert rose callus-derived exosomes, the desert rose callus cultures cultured in the bioreactor were filtered to separate the cultures and the culture medium, and then the callus cultures were taken at a weight ratio of 10% and the callus culture medium 90% (w/w%), and a homogenizer was used. The cultured cells were ground together with the culture medium at a speed of 50 to 100 rpm. The pulverized solution was filtered through a 400 um mesh, and then centrifuged at 1,500 to 2,000 xg for 20 to 30 minutes using a centrifuge, and only the supernatant was taken to separate and remove cell residues. After the supernatant extract was collected and centrifuged at 100,000 to 150,000 xg for 2 hours using an ultra-high-speed centrifuge, the exosomes were settled in a pellet layer and then redispersed to separate the exosomes. The above-obtained desert rose callus-derived exosomes were dissolved in DNase/RNase free water, and then filtered through a 0.22 μm filter to remove bacteria. The size and concentration of the finally obtained exosomes derived from desert rose callus were confirmed using NTA equipment (ZetaView, Particle Metrix, German).
그 결과, 도 2에서 확인할 수 있듯이, 사막장미 캘러스 유래 엑소좀은 엑소좀의 평균 입자 크기가 159.1 nm인 세포외소포체인 것을 확인할 수 있었고, 입자수는 1.3x1010 particles/mL 인 것을 확인하였다.As a result, as can be seen in FIG. 2, it was confirmed that the exosomes derived from desert rose callus were extracellular vesicles with an average particle size of 159.1 nm, and the number of particles was 1.3x10 10 particles/mL.
<실시예 3> 병풀 유래 엑소좀 제조 <Example 3> Preparation of centella asiatica-derived exosomes
(1) 생병풀 착즙(1) Centella asiatica juice
생병풀 잎(센텔라농원, 충북 충주)을 깨끗이 씻어 물기를 제거한 다음, 일반 분쇄기에 세척된 병풀잎에 10~20배 중량의 정제수를 넣어 30 ~ 50 rpm의 속도로 분쇄를 수행하였으며, 수득된 병풀 잎 분쇄액은 400 um mesh를 통해 부유물을 제거하였다. 회수된 분쇄액은 엑소좀 분석을 위한 시료로 사용하였다.Centella asiatica leaves (Centella Farm, Chungju, Chungcheongbuk-do) were washed and dried, and then 10 to 20 times the weight of purified water was added to the washed centella leaves in a general grinder to pulverize at a speed of 30 to 50 rpm. Centella asiatica leaf pulverized liquid was removed from the suspension through a 400 um mesh. The recovered pulverized liquid was used as a sample for exosome analysis.
(2) 병풀 유래 엑소좀 제조(2) Centella asiatica-derived exosome preparation
병풀 유래 엑소좀 제조를 위해 생병풀 분쇄액에 10~20배 중량의 정제수를 첨가하였다. 원심분리기를 이용하여 3,000 ~ 4,000 xg로 20분 동안 원심분리 후, 상등액만을 취하여 세포의 잔여물을 분리 및 제거하였다. 상기 단계를 한번 더 수행하여 불순물 제거하였다. 상등 추출액을 초고속 원심분리기를 이용하여 100,000 ~ 150,000 xg로 2시간 동안 원심분리 후, 엑소좀을 펠렛층에 가라앉힌 후, 멸균수로 재분산하는 단계를 거쳐 엑소좀을 분리하였다. 상기에서 얻어진 병풀 유래 엑소좀을 0.22 ㎛ 필터로 여과하여 제균 작업을 실시하였다. 최종적으로 수득된 병풀 유래 엑소좀을 NTA 장비(ZetaView, Particle Metrix, German)를 이용하여 크기와 농도를 확인하였다.To prepare centella asiatica-derived exosomes, 10 to 20 times the weight of purified water was added to centella asiatica pulverized liquid. After centrifugation at 3,000 to 4,000 xg for 20 minutes using a centrifuge, only the supernatant was taken to separate and remove cell residues. The above step was performed once more to remove impurities. After the supernatant extract was centrifuged at 100,000 to 150,000 xg for 2 hours using an ultra-high-speed centrifuge, the exosomes were settled in a pellet layer and redispersed in sterile water to separate the exosomes. The exosomes derived from centella asiatica obtained above were filtered through a 0.22 μm filter to remove bacteria. The size and concentration of the finally obtained exosomes derived from centella asiatica were confirmed using NTA equipment (ZetaView, Particle Metrix, German).
그 결과, 도 3에서 확인할 수 있듯이, 병풀 유래 엑소좀은 엑소좀의 평균 입자 크기가 139.5 nm인 세포외소포체인 것을 확인할 수 있었고, 입자수는 3.3x1011 particles/mL 인 것을 확인하였다.As a result, as can be seen in FIG. 3, it was confirmed that the exosomes derived from centella asiatica were extracellular vesicles having an average particle size of 139.5 nm, and the number of particles was 3.3x10 11 particles/mL.
<실시예 4> 하수오 유래 엑소좀 제조 <Example 4> Preparation of sewage-derived exosomes
(1) 하수오 착즙(1) Hasuo Juice
하수오(원진약초농원, 울산)를 깨끗이 씻어 물기를 제거한 다음, 일반 분쇄기에 세척된 하수오에 10~20배 중량의 정제수를 넣어 30 ~ 50 rpm의 속도로 분쇄를 수행하였으며, 수득된 하수오 분쇄액은 400 um mesh를 통해 부유물을 제거하였다. 회수된 분쇄액은 엑소좀 분석을 위한 시료로 사용하였다.After washing sewage (Wonjin Herb Farm, Ulsan) cleanly and removing water, 10 to 20 times the weight of purified water was added to the washed sewage in a general grinder, and pulverization was performed at a speed of 30 to 50 rpm. Suspended matter was removed through a 400 um mesh. The recovered pulverized liquid was used as a sample for exosome analysis.
(2) 하수오 유래 엑소좀 제조(2) Manufacture of sewage-derived exosomes
하수오 유래 엑소좀 제조를 위해 하수오 분쇄액에 10~20배 중량의 정제수를 첨가하였다. 원심분리기를 이용하여 3,000 ~ 4,000 xg로 20분 동안 원심분리 후, 상등액만을 취하여 세포의 잔여물을 분리 및 제거하였다. 상기 단계를 한번 더 수행하여 불순물 제거하였다. 상등 추출액을 초고속 원심분리기를 이용하여 100,000 ~ 150,000 xg로 2시간 동안 원심분리 후, 엑소좀을 펠렛층에 가라앉힌 후, 멸균수로 재분산하는 단계를 거쳐 엑소좀을 분리하였다. 상기에서 얻어진 하수오 유래 엑소좀을 0.22 ㎛ 필터로 여과하여 제균 작업을 실시하였다. 최종적으로 수득된 하수오 유래 엑소좀을 NTA 장비(ZetaView, Particle Metrix, German)를 이용하여 크기와 농도를 확인하였다.To prepare sewageo-derived exosomes, 10 to 20 times the weight of purified water was added to the sewage pulverized liquid. After centrifugation at 3,000 to 4,000 xg for 20 minutes using a centrifuge, only the supernatant was taken to separate and remove cell residues. The above step was performed once more to remove impurities. After the supernatant extract was centrifuged at 100,000 to 150,000 xg for 2 hours using an ultra-high-speed centrifuge, the exosomes were settled in a pellet layer and redispersed in sterile water to separate the exosomes. The sewage-derived exosomes obtained above were filtered through a 0.22 μm filter to remove bacteria. The size and concentration of the finally obtained exosomes derived from sewage were confirmed using NTA equipment (ZetaView, Particle Metrix, German).
그 결과, 도 4에서 확인할 수 있듯이, 하수오 유래 엑소좀은 엑소좀의 평균 입자 크기가 139.0 nm인 세포외소포체인 것을 확인할 수 있었고, 입자수는 9.8x1010 particles/mL 인 것을 확인하였다.As a result, as can be seen in Figure 4, it was confirmed that the exosomes derived from sewage were extracellular vesicles having an average particle size of 139.0 nm, and the number of particles was 9.8x10 10 particles / mL.
<실험예 1> 엑소좀의 수율 증대 효과 분석<Experimental Example 1> Analysis of the yield increase effect of exosomes
실시예 1~4에서 수득된 식물 유래 엑소좀의 정량(입자 크기, 입자 수) 분석은 나노 입자 추적 분석기(Nano Tracking Analyzer; NTA, ZetaView TWIN, Particle Metrix, German)를 이용하여 측정하였으며, 본 기기의 분석 프로그램인 ZetaView software를 이용하여 분석을 수행하였다.Quantitative analysis (particle size, particle number) of the plant-derived exosomes obtained in Examples 1 to 4 was measured using a nano tracking analyzer (Nano Tracking Analyzer; NTA, ZetaView TWIN, Particle Metrix, German), and this device Analysis was performed using ZetaView software, an analysis program of .
실시예 1~4에서 수득된 식물 유래 엑소좀은 정량적 및 분자 생물학적 평가를 위해 일반적으로 사용되고 있는 BCA(Bicinchoninic Acid) assay 법을 통해 단백질 농도를 측정하였다. 상기 실시예 1~4에서 수득된 식물 유래 엑소좀 시료 20 ㎕와 BCA 시료 180 ㎕을 96-well plate에서 혼합한 후, 호일로 빛을 차단한 상태에서 37℃에서 1시간 반응시켰다. 이후, Microplate reader 장비(BioTek)를 이용하여 562 nm의 파장에서 흡광도를 측정하였다. 측정된 흡광도를 통한 단백질의 농도는 시약과 함께 제공된 표준 단백질 물질인 소혈청알부민단백질 BSA(Bovine serum albumin)를 이용한 흡광도의 표준 곡선(Standard curve)을 통해 분석하였다. 각 엑소좀을 이용한 시료 단백질의 농도는 입자수를 십억개(1.0x109 particles/mL) 로 맞춘 다음 측정하였다.The protein concentration of the plant-derived exosomes obtained in Examples 1 to 4 was measured through Bicinchoninic Acid (BCA) assay, which is generally used for quantitative and molecular biological evaluation. After mixing 20 μl of plant-derived exosome samples obtained in Examples 1 to 4 and 180 μl of BCA sample in a 96-well plate, they were reacted at 37° C. for 1 hour while blocking light with foil. Then, absorbance was measured at a wavelength of 562 nm using a Microplate reader (BioTek). The protein concentration through the measured absorbance was analyzed through a standard curve of absorbance using bovine serum albumin (BSA), a standard protein material provided with the reagent. The concentration of sample protein using each exosome was measured after setting the number of particles to one billion (1.0x10 9 particles/mL).
그 결과, 도 5에서 확인할 수 있듯이, 에델바이스 캘러스 유래 엑소좀은 단백질의 농도가 149.8ug/mL, 사막장미 캘러스 유래 엑소좀은 단백질의 농도가 193.1ug/mL 병풀 유래 엑소좀은 단백질의 농도가 170.6ug/mL 및 하수오 유래 엑소좀은 단백질의 농도가 134.2ug/mL 인 것을 확인하였다.As a result, as can be seen in FIG. 5, the exosomes derived from edelweiss callus had a protein concentration of 149.8 ug/mL, the exosomes derived from desert rose callus had a protein concentration of 193.1 ug/mL, and the exosomes derived from centella asiatica had a protein concentration of 170.6 It was confirmed that the protein concentration of ug/mL and sewage-derived exosomes was 134.2ug/mL.
<실험예 2> 피부 세포 배양을 통한 세포 활성도 평가 <Experimental Example 2> Evaluation of cell activity through skin cell culture
HaCaT 세포(Human epidermal keratinocyte, 각질형성세포), Detroit 551 세포(Human skin fibroblast, 섬유아세포)를 각각 10% FBS(Fetal Bovine Serum), 1% penicillin/streptomycin(GIBCO, 10,000U/mL) 함유된 DMEM(Dubelcco's Modified Eagle's Medium) 배지를 이용하여 37℃, 5% CO2의 조건인 항온기에서 24시간 배양하였다. 이렇게 배양된 세포를 96 well plate에 한 well 당 0.5 X 104개의 농도로 분주하고, 37℃, 5% CO2 조건인 항온기에서 배양하여 plate에 부착시켰다. 24시간 뒤, 정제수를 첨가한 대조군과 시험물질인 실시예 1~4에서 수득된 식물 유래 엑소좀을 세포에 처리하고, 24시간 동안 추가 배양하였고, 10% WST-1 시약을 각 well에 처리하였다. 이때. 각 엑소좀의 처리 농도는 십억개(1.0x109 particles/mL)를 100% 농도 기준으로 정하고, 각각의 엑소좀을 10%(1.0x108 particles/mL), 5%(5.0x107 particles/mL), 1%(1.0x107 particles/mL) 농도로 처리하였다. 2시간 동안 배양기에서 반응시킨 후, Microplate reader기(BioTeck)를 이용하여 450nm에서 흡광도 값을 측정한 후, 하기 수식에 따라 세포 생존율(%)을 측정하였다.HaCaT cells (Human epidermal keratinocyte, keratinocyte) and Detroit 551 cells (Human skin fibroblast, fibroblast) were mixed in DMEM containing 10% FBS (Fetal Bovine Serum) and 1% penicillin/streptomycin (GIBCO, 10,000 U/mL), respectively. (Dubelcco's Modified Eagle's Medium) medium was used and cultured for 24 hours in an incubator under conditions of 37°C and 5% CO 2 . The cultured cells were dispensed in a 96 well plate at a concentration of 0.5 X 10 4 cells per well, cultured in an incubator at 37°C and 5% CO 2 , and adhered to the plate. After 24 hours, the cells were treated with the plant-derived exosomes obtained in Examples 1 to 4, which were the test substances and the control group to which purified water was added, and further cultured for 24 hours. 10% WST-1 reagent was treated in each well . At this time. The processing concentration of each exosome is determined by billions (1.0x10 9 particles/mL) based on 100% concentration, and each exosome is 10% (1.0x10 8 particles/mL) and 5% (5.0x10 7 particles/mL). ), treated at a concentration of 1% (1.0x10 7 particles/mL). After reacting in an incubator for 2 hours, the absorbance value was measured at 450 nm using a Microplate reader (BioTeck), and cell viability (%) was measured according to the following formula.
세포생존율(%)=[(시험물질의 흡광도)/(대조군의 흡광도)] X 100Cell viability (%) = [(absorbance of test substance)/(absorbance of control group)]
(%)(%)
생존율(%)survival rate (%)
생존율(%)survival rate (%)
그 결과, 상기 표 1에서 확인할 수 있듯이 에델바이스 캘러스 유래 엑소좀, 사막장미 캘러스 유래 엑소좀, 병풀 유래 엑소좀 및 하수오 유래 엑소좀은 1~10% 처리 농도에서 각질형성세포인 HaCaT 세포(Human epidermal keratinocyte), 섬유아세포인 Detroit 551 세포(Human skin fibroblast)에서 세포 내 독성을 보이지 않을 뿐만 아니라 세포생존율(%)이 농도 의존적으로 증가함을 확인하였다. 따라서, 에델바이스 캘러스 유래 엑소좀, 사막장미 캘러스 유래 엑소좀, 병풀 유래 엑소좀 및 하수오 유래 엑소좀은 세포에 대한 자극이 없어 피부 자극 유발 가능성이 낮은 안전한 물질임을 확인하였다.As a result, as can be seen in Table 1 above, exosomes derived from edelweiss callus, exosomes derived from desert rose callus, exosomes derived from centella asiatica, and exosomes derived from S. aureus were keratinocytes, HaCaT cells (Human epidermal keratinocytes) at 1-10% treatment concentration. ) and Detroit 551 cells (Human skin fibroblast), which are fibroblasts, showed no intracellular toxicity and increased cell viability (%) in a concentration-dependent manner. Therefore, it was confirmed that the exosomes derived from edelweiss callus, exosomes derived from desert rose callus, exosomes derived from Centella asiatica, and exosomes derived from S. aureus were safe materials with low possibility of skin irritation because they did not stimulate cells.
<실험예 3> 항주름 효능 시험: Type I collagen (COL1A1), Matrix metalloproteinase-1 (MMP-1)<Experimental Example 3> Anti-wrinkle efficacy test: Type I collagen (COL1A1), Matrix metalloproteinase-1 (MMP-1)
실시예 1~4에서 수득된 식물 유래 엑소좀의 피부 주름 개선 효과를 알아보고자, Type I collagen(COL1A1), Matrix metalloproteinase-1(MMP-1)의 mRNA 발현량을 측정하였다. 노화된 피부에서 나타나는 주름살과 피부 탄력의 감소, 피부 처짐 , 건조 현상 등은 대부분 진피에서 존재하는 기질 단백질의 변화가 주원인이다. 이런 기질 단백질을 분해하는 효소로서 대표적인 것이 Matrix metalloproteinase(MMP)이다. MMP-1은 MMP-2, MMP-9와 함께 collagen I, II, III, VII, X, 젤라틴을 분해하여 피부 노화 및 손상을 주는 효소로 알려져 있는데, 가장 근본적으로 작용하는 효소는 collagenase로 알려진 MMP-1이다. 따라서 MMP-1을 억제하는 활성 성분은 피부 노화 방지에 좋은 효과를 지니는 active compound로, 대표적인 TGF-β(transforming growth factor-β)는 MMP-1의 생성을 억제하여 결국 콜라겐 합성을 촉진 물질로 항주름 효능 시험을 위한 지표 물질로 사용되어진다. Type I collagen alpha1(COL1A1)은 아형인 Type I collagen alpha2(COL1A2)와 함께 콜라겐 생합성을 증가시킨다.In order to investigate the skin wrinkle improvement effect of the plant-derived exosomes obtained in Examples 1 to 4, the mRNA expression levels of Type I collagen (COL1A1) and Matrix metalloproteinase-1 (MMP-1) were measured. Changes in matrix proteins present in the dermis are the main causes of wrinkles, loss of skin elasticity, skin sagging, and dryness in aged skin. Matrix metalloproteinase (MMP) is a representative enzyme that degrades these matrix proteins. MMP-1, along with MMP-2 and MMP-9, is known as an enzyme that breaks down collagen I, II, III, VII, X, and gelatin to cause skin aging and damage. The most fundamental enzyme is MMP, known as collagenase. is -1. Therefore, the active ingredient that inhibits MMP-1 is an active compound that has a good effect on preventing skin aging. Representative TGF-β (transforming growth factor-β) inhibits the production of MMP-1 and eventually acts as a collagen synthesis promoting substance. It is used as an indicator material for wrinkle efficacy test. Type I collagen alpha1 (COL1A1) increases collagen biosynthesis together with its subtype, Type I collagen alpha2 (COL1A2).
인체 유래 섬유아세포인 CCD-986sk를 6 well plate에 4 X 105의 수만큼 분주한 후, 37℃, 5% CO2 조건의 배양기에서 24시간 동안 배양하였다. 이후 MMP-1 mRNA 발현량 측정 실험군은 배지를 제거하고 DPBS를 넣어준 후 UVB 비조사군을 제외한 나머지 세포군에 20 mJ/cm2의 UVB를 조사하였다. COL1A1 mRNA 발현량 측정 실험군은 UV 조사하지 않았다. 각 실시예 1~4에서 수득된 식물 유래 엑소좀의 처리 농도는 십억개(1.0x109 particles/mL)를 100% 농도 기준으로 정하고 각각의 실시예 1~4에서 수득된 식물 유래 엑소좀 및 이의 혼합 엑소좀(1:1:1:1 비율)을 10%(1.0x108 particles/mL)로 처리하였다. 24시간 배양 후 세포에서 RNA preparation kit를 이용하여 RNA를 분리한 뒤 나노드롭(nanodrop)을 이용하여 260 nm에서 RNA를 정량한 후, 각각 2 ㎍의 RNA를 사용하여 증폭기에서 cDNA를 합성하였다(C1000 Thermal Cycler, Bio-Rad, 미국). 합성된 cDNA에 타겟 유전자 특이적 프라이머와 시아닌 염료인 사이버그린(SYBR Green supermis, Applied Biosystems, 미국)을 첨가한 혼합물을 이용하여 실시간(real-time) PCR 기계에서 실시간 연쇄중합효소반응을 실시함으로써 최종적으로 타겟 유전자의 발현 정도를 평가하였다. 프라이머의 서열은 하기 표 2에 나타내었으며, 유전자의 발현량은 β-actin 유전자에 대한 보정을 통해 최종적으로 분석하였다.Human-derived fibroblasts, CCD-986sk, were dispensed by the number of 4 X 10 5 in a 6 well plate, and then cultured for 24 hours in an incubator at 37°C and 5% CO 2 conditions. Thereafter, in the MMP-1 mRNA expression level measurement experimental group, the medium was removed, DPBS was added, and 20 mJ/cm 2 of UVB was irradiated to the remaining cell groups except for the non-UVB irradiated group. The COL1A1 mRNA expression level measurement experimental group was not irradiated with UV. The treatment concentration of the plant-derived exosomes obtained in each of Examples 1 to 4 was set at 1 billion (1.0x10 9 particles/mL) based on the 100% concentration, and the plant-derived exosomes obtained in each of Examples 1 to 4 and their Mixed exosomes (1:1:1:1 ratio) were treated with 10% (1.0x10 8 particles/mL). After culturing for 24 hours, RNA was isolated from the cells using an RNA preparation kit, RNA was quantified at 260 nm using a nanodrop, and cDNA was synthesized in an amplifier using 2 μg of RNA each (C1000 Thermal Cycler, Bio-Rad, USA). Finally, real-time chain polymerase reaction is performed in a real-time PCR machine using a mixture in which target gene-specific primers and cyanine dye Cybergreen (SYBR Green supermis, Applied Biosystems, USA) are added to the synthesized cDNA. The expression level of the target gene was evaluated. The sequences of the primers are shown in Table 2 below, and the expression levels of the genes were finally analyzed through correction for the β-actin gene.
5'-CGAATTGCCGACAGAGATGA-3' SEQ ID NO: 1:
5′-CGAATTGCCGACAGAGATGA-3′
5'-GTCCCTGAACAGCCCAGTACTT-3'SEQ ID NO: 2:
5′-GTCCCTGAACAGCCCAGTACTT-3′
5'-TGACGTTCCCATTAGACAACTG-3'SEQ ID NO: 3:
5′-TGACGTTCCCATTAGACAACTG-3′
5'-CCGTCTTTCATTACACAGGACA-3'SEQ ID NO: 4:
5′-CCGTCTTTCATTACACAGGACA-3′
5'-GGCCATCTCTTGCTCGAAGT-3'SEQ ID NO: 5:
5′-GGCCATCTCTTGCTCGAAGT-3′
5'-GAGACCTTCAACACCCCAGC-3'SEQ ID NO: 6:
5′-GAGACCTTCAACACCCCAGC-3′
(% of control)Suppression of MMP-1 expression
(% of control)
(% of control)COL1A1 expression activity
(% of control)
그 결과, 상기 표 3 및 표 4에서 확인할 수 있듯이 무처리군(control)에 비해 실시예 1~4에서 전반적으로 MMP-1 mRNA 발현 저해 활성 및 COL1A1 mRNA 발현 활성이 우수한 것을 확인하였다. 특히, 혼합 엑소좀인 실시예 5의 경우, 단독 엑소좀 처리 뿐만 아닌 같은 농도 조건에서 콜라겐 생합성 관련 유전자 발현이 약간 높았다. TGF-β는 양성 대조군으로 5 ng/mL을 처리하였을 때, MMP1은 44.0% 발현율로 66% 감소하였고, COL1A1은 62% 증가하였다.As a result, as can be seen in Tables 3 and 4, it was confirmed that the MMP-1 mRNA expression inhibitory activity and the COL1A1 mRNA expression activity were generally excellent in Examples 1 to 4 compared to the control group. In particular, in the case of Example 5, which is a mixed exosome, the expression of genes related to collagen biosynthesis was slightly higher under the same concentration conditions as well as the single exosome treatment. When 5 ng/mL of TGF-β was treated as a positive control, MMP1 expression was reduced by 66% to 44.0%, and COL1A1 was increased by 62%.
따라서, 에델바이스 캘러스 유래 엑소좀, 사막장미 캘러스 유래 엑소좀, 병풀 유래 엑소좀, 하수오 유래 엑소좀 및 이들의 혼합 엑소좀은 피부 탄력 및 주름 방지 효능이 있음을 확인하였다.Accordingly, it was confirmed that exosomes derived from edelweiss callus, exosomes derived from desert rose callus, exosomes derived from centella asiatica, exosomes derived from sage, and mixed exosomes had skin elasticity and anti-wrinkle effects.
<실험예 4> 피부장벽 강화 및 보습효능 시험 : Filaggrin 및 AQP3 발현<Experimental Example 4> Skin barrier strengthening and moisturizing effect test: Filaggrin and AQP3 expression
실시예 1~4에서 수득된 식물 유래 엑소좀의 피부 장벽 강화 및 보습 효능 시험을 알아보고자, Filaggrin 및 AQP3 발현을 확인하였다. Filaggrin은 분해 과정을 통해 Pyrrocarboxylic acid나 Trans-urocanic acid 등과 같은 천연보습인자(natural moisturizing factors, NMF)를 형성하여 피부 보습에 중요한 역할을 수행함은 물론, 각질층 pH의 정상화, 항염 작용 등도 수행한다. 피부 노화는 각질층의 수분 부족이나 진피층의 세포외 기질 단백질 이상으로 인해 발생하며, 노화가 진행되면서 피부를 구성하는 물질인 콜라겐, 엘라스틴, 히알루론산 및 당단백질 등의 함량 및 배열이 변하거나 감소하면서 전반적으로 장벽이 무너진다. 아쿠아포린(Aquaporin)은 물분자만을 선택적으로 통과시키며 모든 세포막에 존재하는 막단백질로 세포 내외의 물과 글리세롤의 이동에 관여한다고 알려져 있다. 포유동물에서 발견되는 아쿠아포린은 13개가 존재하며, 이 중 아쿠아포린-3(Aquaporin 3, AQP3)은 사람의 표피에 풍부하게 존재한다. AQP3는 피부의 각질형성세포에서 발현하며, 이를 통한 물 및 글리세롤의 흡수 및 이동은 세포의 수분 손실을 방지해 피부의 탄력 및 보습력을 증가시킨다.In order to investigate the skin barrier strengthening and moisturizing efficacy test of the plant-derived exosomes obtained in Examples 1 to 4, the expression of Filaggrin and AQP3 was confirmed. Filaggrin plays an important role in skin moisturizing by forming natural moisturizing factors (NMF) such as Pyrrocarboxylic acid or Trans-urocanic acid through the decomposition process, as well as normalizing the pH of the stratum corneum and performing anti-inflammatory actions. Skin aging is caused by a lack of moisture in the stratum corneum or an abnormality in extracellular matrix proteins in the dermis layer. As aging progresses, the content and arrangement of collagen, elastin, hyaluronic acid, and glycoprotein, etc. barriers are broken down by Aquaporin is a membrane protein that selectively passes only water molecules and is present in all cell membranes and is known to be involved in the movement of water and glycerol in and out of cells. There are 13 aquaporins found in mammals, and among them, aquaporin-3 (AQP3) is abundant in human epidermis. AQP3 is expressed in keratinocytes of the skin, and the absorption and movement of water and glycerol through it prevents the loss of water in the cells and increases the elasticity and moisturizing power of the skin.
인체 피부 각질형성 세포주인 HaCaT 세포를 6 well plate에 4 X 105의 수만큼 분주한 후, 37℃, 5% CO2 조건의 배양기에서 24시간 동안 배양하였다. 이후 DPBS로 2번 washing 후, FBS가 없는 DMEM/High glucose 배지로 교체하고, 각각의 실시예 1~4에서 수득된 식물 유래 엑소좀 및 이의 혼합 엑소좀(1:1:1:1 비율)을 10% 농도로 처리하고, 24시간 동안 배양하였다. 24시간 배양된 세포에서 RNA prep kit를 이용하여 RNA를 분리한 뒤 나노드롭(nanodrop)을 이용하여 260 nm에서 RNA를 정량한 후, 각각 1 ㎍의 RNA를 사용하여 증폭기에서 cDNA를 합성하였다(C1000 Thermal Cycler, Bio-Rad, 미국). 합성된 cDNA에 타겟 유전자 특이적 프라이머와 시아닌 염료인 사이버그린(SYBR Green supermis, Applied Biosystems, 미국)을 첨가한 혼합물을 이용하여 실시간(real-time) PCR 기계에서 실시간 연쇄중합효소반응을 실시함으로써 최종적으로 타겟 유전자의 발현 정도를 평가하였다. 유전자의 발현량은 β-actin 유전자에 대한 보정을 통해 최종적으로 분석하였다. 프라이머의 서열은 하기 표 5에 나타내었다. 유전자의 발현량은 β-actin 유전자에 대한 보정을 통해 최종적으로 분석하였다.HaCaT cells, a human skin keratinocyte cell line, were dispensed by the number of 4 X 10 5 in a 6 well plate, and then cultured for 24 hours in an incubator at 37°C and 5% CO 2 conditions. Then, after washing twice with DPBS, the medium was replaced with DMEM/High glucose medium without FBS, and the plant-derived exosomes obtained in each of Examples 1 to 4 and their mixed exosomes (1:1:1:1 ratio) It was treated with 10% concentration and cultured for 24 hours. RNA was isolated using an RNA prep kit from cells cultured for 24 hours, RNA was quantified at 260 nm using a nanodrop, and cDNA was synthesized using an amplifier using 1 μg of RNA each (C1000 Thermal Cycler, Bio-Rad, USA). Finally, real-time chain polymerase reaction is performed in a real-time PCR machine using a mixture in which target gene-specific primers and cyanine dye Cybergreen (SYBR Green supermis, Applied Biosystems, USA) are added to the synthesized cDNA. The expression level of the target gene was evaluated. The expression level of the gene was finally analyzed through correction for the β-actin gene. The sequences of the primers are shown in Table 5 below. The expression level of the gene was finally analyzed through correction for the β-actin gene.
이때, Filaggrin의 발현 분석을 위해 무처리군인 음성 대조군으로 DPBS를 처리하고, 양성 대조군으로 CaCl2 20mM 처리하였으며, AQP3 발현 분석을 위해 무처리군인 음성 대조군으로 DPBS를 처리하고, 양성 대조군으로는 Hyaluronic acid 0.005%를 처리하였다.At this time, DPBS was treated as an untreated negative control group for the expression analysis of filaggrin, CaCl 2 20mM was treated as a positive control group, and DPBS was treated as a negative control group, an untreated group for AQP3 expression analysis, and Hyaluronic acid as a positive control group. 0.005% was treated.
5'-TGCAATCTGGCACTTCGC-3' SEQ ID NO: 7:
5′-TGCAATCTGGCACTTCGC-3′
5'-GCCAGCACACACACGATAA-3'SEQ ID NO: 8:
5′-GCCAGCACACACACGATAA-3′
5'-GGCTAAGTGAAAGACTTGAAGAGA-3'SEQ ID NO: 9:
5′-GGCTAAGTGAAAGACTTGAAGAGA-3′
5'-AATAGACTATCAGTGGTGTCATAGG-3'SEQ ID NO: 10:
5′-AATAGACTATCAGTGGTGTCATAGG-3′
5'-GGCCATCTCTTGCTCGAAGT-3'SEQ ID NO: 11:
5′-GGCCATCTCTTGCTCGAAGT-3′
5'-GAGACCTTCAACACCCCAGC-3'SEQ ID NO: 12:
5′-GAGACCTTCAACACCCCAGC-3′
(% or mM)treatment concentration
(% or mM)
(%)treatment concentration
(%)
그 결과, 상기 표 6 및 표 7에서 확인할 수 있듯이 무처리군(control)에 비해 실시예 1~5에서 수득된 식물 유래 엑소좀이 세포에서 Filaggrin 및 AQP3 유전자 발현이 증가됨을 확인하였고, 이를 통해 전반적인 피부 보습 효과가 있음을 확인하였다.As a result, as can be seen in Tables 6 and 7, it was confirmed that the plant-derived exosomes obtained in Examples 1 to 5 increased the expression of Filaggrin and AQP3 genes in the cells compared to the control group. It was confirmed that there was a skin moisturizing effect.
더욱 상세하게는, 각각의 실시예 1~4를 10% 처리하였을 때, Filaggrin의 발현이 음성 대조군 대비 1.18~1.25배 증가하였으며, 실시예 5를 10% 처리하였을 때, 음성대조군 대비 1.29배 높은 발현율을 보여 혼합 엑소좀이 더 높은 보습 효과가 있음을 확인하였다.More specifically, when each of Examples 1 to 4 was treated with 10%, the expression of filaggrin increased 1.18 to 1.25 times compared to the negative control group, and when Example 5 was treated with 10%, the expression rate was 1.29 times higher than that of the negative control group. It was confirmed that the mixed exosomes had a higher moisturizing effect.
또한, 각각의 실시예 1~4를 10% 처리하였을 때, AQP3의 발현이 음성 대조군 대비 1.9~2.5배 증가하였으며, 실시예 5를 10% 처리하였을 때, 음성대조군 대비 3.0배 높은 발현율을 보여 혼합 엑소좀이 더 높은 보습 효과가 있음을 확인하였다.In addition, when each of Examples 1 to 4 was treated with 10%, the expression of AQP3 increased 1.9 to 2.5 times compared to the negative control group, and when Example 5 was treated with 10%, the expression rate was 3.0 times higher than that of the negative control group. It was confirmed that exosomes have a higher moisturizing effect.
그 결과, 본 발명의 조성물인 에델바이스 캘러스 및 배양액 유래 엑소좀, 사막장미 잎세포 및 배양액 유래 엑소좀, 병풀 유래 엑소좀 및 하수오 유래 엑소좀, 또는 이들의 일정 비율로 혼합한 혼합 엑소좀 모두에서 피부세포 내 AQP3과 Filaggrin의 발현 증가를 통해 보습과 피부장벽 강화에 기여하는 것으로 사료된다.As a result, the composition of the present invention, edelweiss callus and exosomes derived from the culture medium, desert rose leaf cells and exosomes derived from the culture medium, exosomes derived from centella asiatica and exosomes derived from S. aureus, or mixed exosomes mixed at a certain ratio It is thought to contribute to moisturizing and strengthening the skin barrier through the increase in the expression of AQP3 and Filaggrin in cells.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시 양태일 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.Having described specific parts of the present invention in detail above, it will be clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
Claims (11)
Edelweiss ( Leontopodium alpinum ) derived exosomes, desert rose ( Adenium obesum ) derived exosomes, centella asiatica ( Centella asiatica ) derived exosomes and sewage ( Polygonum multiflorum ) derived exosomes are 1:0.5~2:0.5~2:0.5~2 A cosmetic composition for skin elasticity, wrinkle prevention, skin barrier strengthening and moisturizing comprising plant-derived exosomes as an active ingredient, characterized in that they are mixed in a weight ratio of.
According to claim 1, wherein the edelweiss-derived exosomes are edelweiss callus-derived exosomes characterized in that the plant-derived exosomes as an active ingredient Skin elasticity, wrinkle prevention, skin barrier strengthening and moisturizing cosmetic composition for moisturizing.
The cosmetic composition for skin elasticity, anti-wrinkle, skin barrier strengthening and moisturizing according to claim 1, wherein the desert rose-derived exosomes are exosomes derived from desert rose callus as an active ingredient.
According to claim 1, wherein the exosomes derived from Centella Asiatica are leaf-derived exosomes of Centella asiatica Plant-derived exosomes as an active ingredient Skin elasticity, wrinkle prevention, skin barrier strengthening and moisturizing cosmetic composition.
The cosmetic composition for skin elasticity, anti-wrinkle, skin barrier strengthening and moisturizing, comprising plant-derived exosomes as an active ingredient according to claim 1, wherein the exosomes derived from the sewage plant are exosomes derived from the outpouring of the sewage plant.
The method of claim 1, wherein the exosomes are extracellular vesicles having an average particle size of 50 to 200 nm, comprising plant-derived exosomes as an active ingredient for skin elasticity, wrinkle prevention, and skin barrier enhancement and a cosmetic composition for moisturizing.
The method of claim 1, wherein the exosomes contain plant-derived exosomes as an active ingredient, characterized in that the number of particles is 1x10 7 to 1x10 9 particles/mL, for skin elasticity, wrinkle prevention, skin barrier strengthening and moisturizing cosmetic composition.
According to claim 1, wherein the exosome is 100 ~ 300ug / mL of protein, characterized in that it contains plant-derived exosomes as an active ingredient skin elasticity, wrinkle prevention, skin barrier strengthening and moisturizing cosmetic composition.
The method of claim 1, wherein the plant-derived exosome is contained in an amount of 1 to 40% by weight based on the total weight of the composition. cosmetic composition.
A cosmetic comprising the composition of any one of claims 1, 2, 3, 4, 5, 7, 8, 9 and 10.
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