KR102628089B1 - Cosmetic composition containing exosomes derived from Centella asiatica stem cell culture medium as an active ingredient and having cell regeneration and skin elastic effects - Google Patents
Cosmetic composition containing exosomes derived from Centella asiatica stem cell culture medium as an active ingredient and having cell regeneration and skin elastic effects Download PDFInfo
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- KR102628089B1 KR102628089B1 KR1020230034538A KR20230034538A KR102628089B1 KR 102628089 B1 KR102628089 B1 KR 102628089B1 KR 1020230034538 A KR1020230034538 A KR 1020230034538A KR 20230034538 A KR20230034538 A KR 20230034538A KR 102628089 B1 KR102628089 B1 KR 102628089B1
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- centella asiatica
- exosomes
- cosmetic composition
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- stem cell
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/04—Plant cells or tissues
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
- A61K8/14—Liposomes; Vesicles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9789—Magnoliopsida [dicotyledons]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
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- Cell Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Mycology (AREA)
- Dermatology (AREA)
- Cosmetics (AREA)
Abstract
본 발명은 병풀 줄기세포 배양액 유래 엑소좀을 유효성분으로 함유하는 세포재생 및 피부탄력 효능을 갖는 화장료 조성물 및 이의 제조방법에 관한 것이다. 본 발명을 통해 병풀 줄기세포 배양액에서 분리한 엑소좀은 이를 화장료로 활용할 경우, 피부세포의 증식능이 우수하여 피부 세포 재생에 효과가 있고, Collagen Type I과 III의 발현을 증가시켜 피부 탄력에 효과가 있다. The present invention relates to a cosmetic composition having cell regeneration and skin elasticity effects containing exosomes derived from Centella asiatica stem cell culture medium as an active ingredient, and a method for producing the same. When used as a cosmetic, the exosomes isolated from Centella asiatica stem cell culture medium through the present invention are effective in skin cell regeneration due to their excellent proliferative ability of skin cells, and are effective in skin elasticity by increasing the expression of Collagen Type I and III. there is.
Description
본 발명은 병풀 줄기세포 배양액 유래 엑소좀을 유효성분으로 함유하는 세포재생 및 피부탄력 효능을 갖는 화장료 조성물 및 이의 제조방법에 관한 것이다. The present invention relates to a cosmetic composition having cell regeneration and skin elasticity effects containing exosomes derived from Centella asiatica stem cell culture medium as an active ingredient, and a method for producing the same.
피부는 인체 내에서 외부 환경과 가장 밀접하게 접하고 있는 신체 기관으로 인체 내부를 보호하는 기능을 가지며, 표피(epidermis), 진피(dermis) 및 피하지방(hypodermis) 3가지로 크게 나눌 수 있다. 피부는 매우 다양한 기능의 세포와 그에 맞는 물성의 물질들로 구성된 복잡한 기관으로 진피층은 탄력성이 있는 고형의 물질(섬유)들과 점성이 있는 액상의 물질들이 결합되어 있어 고유의 피부 탄력을 유지하게 된다. 그리하여 피부는 기기적 장력에 대해 독특한 물성으로 인한 반응을 나타내게 되는데 이를 점탄성이라고 한다. 피부가 이러한 점탄성을 갖게 되는 것은 프로테오글리칸류의 기질에 콜라겐 섬유와 엘라스틴 섬유가 특유의 삼차원 구조를 형성하고 있기 때문이다. 일반적으로 나이를 먹어감에 따라 자외선이나, 공해, 스트레스 등에 의해 피부의 기능저하와 위축성 변화가 나타나며 피부 세포수가 감소하거나 피부의 두께가 감소하는 현상이 나타난다. 이러한 진행에 피부의 탄성 섬유는 변형되어 피부의 탄력이 감소하거나 피부가 늘어지게 된다. The skin is the body organ in closest contact with the external environment within the human body and has the function of protecting the inside of the human body. It can be broadly divided into three types: epidermis, dermis, and hypodermis. The skin is a complex organ composed of cells with a wide variety of functions and materials with appropriate physical properties. The dermal layer is a combination of elastic solid substances (fibers) and viscous liquid substances to maintain the skin's inherent elasticity. . Thus, the skin responds to mechanical tension due to its unique physical properties, which are called viscoelasticity. The reason skin has such viscoelasticity is because collagen fibers and elastin fibers form a unique three-dimensional structure in a proteoglycan matrix. In general, as we age, skin function declines and atrophic changes occur due to ultraviolet rays, pollution, stress, etc., and the number of skin cells decreases or the thickness of the skin decreases. As this progresses, the elastic fibers of the skin are deformed, causing the skin's elasticity to decrease or the skin to become saggy.
콜라겐은 피부를 구성하고 있는 주요한 성분으로 이 중 type I 콜라겐이 80%, type Ⅲ 콜라겐이 15%를 차지하고 있다. 자외선에 피부가 장시간 노출될 경우 피부 내 활성산소가 다량 생성되고, 이로 인해 기질 금속단백질분해 효소(matrix metalloproteinases, MMPs)들 중에서 콜라겐을 분해하는 효소인 MMP-1(matrix metalloproteinase-1)의 발현 또는 활성이 촉진되어 콜라겐 합성이 저하되며, 결국 이로 인해 피부탄력이 감소하고 주름이 생성된다.Collagen is a major component of skin, of which type I collagen accounts for 80% and type III collagen accounts for 15%. When the skin is exposed to ultraviolet rays for a long time, a large amount of active oxygen is generated in the skin, which causes the expression of MMP-1 (matrix metalloproteinase-1), an enzyme that decomposes collagen, among matrix metalloproteinases (MMPs). Activation is promoted and collagen synthesis decreases, which ultimately reduces skin elasticity and creates wrinkles.
Wound healing 과정 중 proliferative phase가 시작될 때 제일 먼저 진피의 fibroblast가 나타난다. Fibroblast가 활성화되어 새로운 ECM(extracellular matrix) 성분들, collagen I, II, III, IV, glycoprotein, hyaluronic acid 등을 만들어낸다. 손상된 부위가 채워지고 reepithelialization을 거쳐 피부는 원래의 모습을 회복하게 된다 (Journal of Wound Care 22(8), 407-408, 410-412, 2013).When the proliferative phase begins during the wound healing process, dermal fibroblasts appear first. Fibroblasts are activated and produce new ECM (extracellular matrix) components, collagen I, II, III, IV, glycoprotein, and hyaluronic acid. Damaged areas are filled and the skin regains its original appearance through reepithelialization (Journal of Wound Care 22(8), 407-408, 410-412, 2013).
따라서, 피부 노화 예방 및 회복을 위한 신소재를 발굴하는데 있어 콜라겐 합성을 촉진시키고 wound healing에 효과가 있는 물질을 찾는 것이 좋은 전략이 될 수 있다 (Journal of Investigative Dermatology, 107(3), 404-411, 1996; Science, 276, 75-81, 1997)Therefore, in discovering new materials for preventing and recovering skin aging, finding substances that promote collagen synthesis and are effective in wound healing can be a good strategy (Journal of Investigative Dermatology, 107(3), 404-411, 1996; Science, 276, 75-81, 1997)
병풀은 산형과의 여러해살이풀로서, 옆으로 뻗어가면서 마디에서 뿌리가 내리고 비늘 같은 잎이 있다. 주로 고온 다습한 인도 남방 지역, 인도양의 해안지역, 아프리카 마다가스카르 섬에서 자생한다. 병풀은 아시아티코사이드, 마데카소사이드, 아시아틱애시드, 마데가식애시드 등을 함유하고 있어 상처 치료에 탁월하다 알려져 있다.Centella asiatica is a perennial plant of the Umbrella family. It spreads out laterally, roots from the nodes, and has scale-like leaves. It grows mainly in the hot and humid southern region of India, coastal areas of the Indian Ocean, and the island of Madagascar in Africa. Centella asiatica contains asiaticoside, madecassoside, asiatic acid, and madegasic acid, and is known to be excellent for treating wounds.
캘러스는 식물체에 상처가 났을 때, 상처부위를 재생시키기 위해 형성하는 유상조직이다. 식물체에 상처를 낸 뒤, 적절한 식물 호르몬 농도를 함유한 배지에서 배양하면 캘러스 세포를 얻을 수 있다. 캘러스는 식물 생장 조절 물질 등의 처리를 통해 전체 식물로 분화할 수 있는 전형성능을 가지기 때문에 식물 줄기세포로도 불리고 있다. 특히 식물 줄기세포는 동물 줄기세포에 비해 증식속도가 빠르고 대량 배양이 쉬운 편이며, 윤리적 문제가 없기 때문에 관심이 높아지고 있다. 또한, 다양한 식물 유래 대사산물을 생산할 수 있기 때문에 다방면의 연구가 이루어지고 있다.Callus is a callus tissue that forms when a plant is injured to regenerate the injured area. Callus cells can be obtained by wounding the plant and culturing it in a medium containing an appropriate concentration of plant hormones. Callus is also called a plant stem cell because it has the ability to differentiate into a whole plant through treatment with plant growth regulators. In particular, interest in plant stem cells is increasing because they proliferate faster than animal stem cells, are easier to mass culture, and do not have ethical issues. In addition, because various plant-derived metabolites can be produced, research in various fields is being conducted.
한편, 엑소좀은 세포가 세포 외부로 방출하는 소낭인 extra cellular vesicles의 일종으로, 진핵생물에서 세포 간 정보 교환을 위해 분비하는 나노미터 크기의 물질이다. 세포막의 구조와 동일한 인지질 이중막으로 이루어지며, 모체가 되는 세포 내부의 단백질, 지질, 핵산 등을 유사하게 포함하고 있다. 초기 연구에서 엑소좀은 특정 기능을 가지는 것이 아닌 세포의 노폐물 배설과 관계되어 이해되었지만, 최근 많은 연구에서 엑소좀의 다양한 역할들이 새롭게 규명되었다. 예를 들어, 세포 유래 호르몬이나 사이토카인의 역할인 세포 대사의 자극 및 억제 기능을 엑소좀이 비슷하게 한다는 것이 밝혀졌다.Meanwhile, exosomes are a type of extra cellular vesicles, which are vesicles released by cells to the outside of the cell. They are nanometer-sized substances secreted by eukaryotes to exchange information between cells. It is composed of a phospholipid bilayer that is identical to the structure of a cell membrane, and contains proteins, lipids, and nucleic acids similar to those inside the parent cell. In early studies, exosomes were understood to be related to the excretion of cellular waste rather than having a specific function, but many recent studies have newly identified various roles of exosomes. For example, it has been revealed that exosomes have similar stimulating and suppressing functions of cell metabolism, which are the functions of cell-derived hormones or cytokines.
이에 본 발명자들은 Elicitor 처리를 통한 병풀 줄기세포 배양액에서 엑소좀의 발현량을 증가시키고 이를 분리하는 공정을 발명하였다. 또한 세포재생 및 피부 탄력 효능을 나타내는 것을 확인함으로써 본 발명을 완성하였다.Accordingly, the present inventors invented a process to increase the expression level of exosomes in Centella asiatica stem cell culture medium through elicitor treatment and to isolate them. In addition, the present invention was completed by confirming that it exhibits cell regeneration and skin elasticity effects.
본 발명의 목적은 병풀 줄기세포 배양액 유래 엑소좀을 유효성분으로 함유하는 세포재생 및 피부탄력 효능을 갖는 화장료 조성물 및 이의 제조방법을 제공하는 데에 있다. The purpose of the present invention is to provide a cosmetic composition having cell regeneration and skin elasticity effects containing exosomes derived from Centella asiatica stem cell culture medium as an active ingredient, and a method for producing the same.
(제1단계) 병풀 잎으로부터 MS 고체배지 및 액체배지를 이용하여 병풀 식물세포를 배양하고, 유인제(Elicitor)를 첨가하여 병풀 줄기세포 배양액을 얻는 단계; (Step 1) Culturing Centella asiatica plant cells from Centella asiatica leaves using MS solid medium and liquid medium, and adding an attractor to obtain Centella asiatica stem cell culture medium;
(제2단계) 상기 병풀 줄기세포 배양액과 세포용해버퍼를 혼합하고, 교반 및 유화하여 세포를 파쇄하여 세포 파쇄액을 얻는 단계; (Second step) mixing the Centella asiatica stem cell culture medium and cell lysis buffer, stirring and emulsifying to disrupt the cells to obtain a cell disruption solution;
(제3단계) 상기 세포 파쇄액을 원심분리하여 상층액을 얻는 단계; 및 (Third step) centrifuging the cell lysate to obtain a supernatant; and
(제4단계) 접선흐름여과법(PTFF; PERFORMANCE TANGENTIAL FLOW FILTRATION)을 이용하여 제3단계에서 얻은 상층액으로부터 50 ~ 150 kDa 사이의 분자량인 물질만 회수하여 병풀 엑소좀을 얻는 단계; (Step 4) Obtaining Centella asiatica exosomes by recovering only substances with a molecular weight between 50 and 150 kDa from the supernatant obtained in the third step using tangential flow filtration (PTFF; PERFORMANCE TANGENTIAL FLOW FILTRATION);
를 포함하는 것을 특징으로 하는 병풀 엑소좀의 제조방법에 관한 것이다. It relates to a method for producing Centella asiatica exosomes, characterized in that it comprises.
상기 제1단계에서 병풀줄기세포 배양액은, 병풀 잎으로부터 MS 고체배지 및 액체배지를 이용하여 병풀 식물세포를 배양하고, 유인제(Elicitor)를 첨가 후 추가배양하여 얻을 수 있다. In the first step, the Centella asiatica stem cell culture medium can be obtained by culturing Centella asiatica plant cells from Centella asiatica leaves using MS solid medium and liquid medium, adding an attractor, and further culturing.
상기 제1단계에서 유인제로서 TNF-α(tumor necrosis factor-α) 및 살리실산(salicylic acid)을 이용할 수 있다. In the first step, tumor necrosis factor-α (TNF-α) and salicylic acid can be used as attractants.
상기 제3단계의 원심분리는 3~5℃의 1,000 ~ 10,000 x g에서 원심분리하고 상층액만을 회수함으로써 수행하는 것이 특징이다. The third step of centrifugation is characterized by centrifuging at 1,000 to 10,000 x g at 3 to 5°C and recovering only the supernatant.
상기 제4단계의 접선흐름여과법은 통상의 방법을 적용할 수 있으며, 40~60℃, 10~15ml/min, 2.5~6.0 bar 조건의 한외여과/초미세여과(Ultrafiltration)를 수행할 수 있다. The tangential flow filtration method of the fourth step can be applied as a conventional method, and ultrafiltration/ultrafiltration can be performed under the conditions of 40 to 60°C, 10 to 15 ml/min, and 2.5 to 6.0 bar.
이에 본 발명은 상기 제조방법으로 얻은 병풀 엑소좀을 제공할 수 있고, 이를 포함하는 화장료 조성물을 제공한다. Accordingly, the present invention can provide Centella asiatica exosomes obtained by the above production method, and provides a cosmetic composition containing the same.
상기 병풀 엑소좀은 세포 재생 효능을 갖는 것을 특징으로 한다. The Centella asiatica exosomes are characterized by having a cell regenerative effect.
또한 상기 병풀 엑소좀은 피부 탄력 효능을 가지며, 제1형 콜라겐(Collagen Type I)과 제3형 콜라겐(Collagen Type III)의 발현을 증강시키는 효능이 있다. In addition, the Centella asiatica exosomes have skin elasticity effects and are effective in enhancing the expression of type 1 collagen (Collagen Type I) and type 3 collagen (Collagen Type III).
또 다른 양태에서 본 발명은 유인제가 처리된 병풀 줄기세포 유래의 엑소좀을 함유하는 화장료 조성물에 관한 것일 수 있다. In another aspect, the present invention may relate to a cosmetic composition containing exosomes derived from Centella asiatica stem cells treated with an attractant.
이하 본 발명을 상세히 설명한다. Hereinafter, the present invention will be described in detail.
본 발명의 제1단계에서 사용하는 유인제는 TNF-α(tumor necrosis factor-α) 50~100㎍/ℓ 및 살리실산(salicylic acid) 0.1~5 mM이 포함된 것일 수 있다. The attractant used in the first step of the present invention may contain 50 to 100 μg/l of tumor necrosis factor-α (TNF-α) and 0.1 to 5 mM of salicylic acid.
상기 유인제 중, TNF-α가 50㎍/ℓ 미만으로 처리되거나 100㎍/ℓ를 초과하여 처리될 경우, 또는 살리실산이 0.1 mM 미만의 농도로 처리되거나 5 mM을 초과하여 처리되어도 병풀 줄기세포 내의 엑소좀 형성이 잘 되지 않거나 세포의 상태가 좋지 않아 배양이 잘 되지 않을 수 있어 바람직하지 않다. Among the above attractants, even if TNF-α is treated at a concentration of less than 50 ㎍/ℓ or more than 100 ㎍/ℓ, or salicylic acid is treated at a concentration of less than 0.1 mM or more than 5 mM, This is undesirable because exosomes may not form well or the cells may not be in good condition, which may result in poor culture.
상기 제1단계의 병풀 줄기세포 배양액은 살균한 병풀 잎을 습도 60~70% 및 온도 28~32℃ 조건에서 MS 고체배지(pH 5.7~5.9)에서 3~4주간 암배양하고, 습도 60~70% 및 온도 23~27℃ 조건에서 6~8주간 MS 액체배지에서 액체 배양한 후, 유인제를 첨가하여 5~10일간 더 추가 배양하여 얻은 것일 수 있다. The Centella asiatica stem cell culture medium in the first step is obtained by culturing sterilized Centella asiatica leaves in the dark for 3 to 4 weeks on MS solid medium (pH 5.7 to 5.9) at a humidity of 60 to 70% and a temperature of 28 to 32°C, and at a humidity of 60 to 70. It may be obtained by culturing liquid in MS liquid medium for 6 to 8 weeks under conditions of % and temperature of 23 to 27°C, then adding an attractant and culturing for an additional 5 to 10 days.
상기 MS 고체배지에서 암배양 상태로 고체배양 할 때의 시간이 3주 미만이거나 4주를 초과할 경우, 액체배양 시, 병풀 식물세포의 성장이 저하될 수 있어 바람직하지 않으며, 액체배양 시간이 6주 미만이거나 8주를 초과하게 될 경우 유인제를 충분히 처리해도 병풀 줄기세포 내의 엑소좀 형성이 잘 되지 않을 수 있다. 또한 유인제를 첨가하여 액체배양을 추가로 수행할 때에도 5일 미만 또는 10일을 초과하더라도 역시 병풀 줄기세포 내의 엑소좀 형성이 잘 되지 않을 수 있어 바람직하지 않다. If the time for solid culture in dark culture on the MS solid medium is less than 3 weeks or more than 4 weeks, it is not desirable because the growth of centella asiatica plant cells may be reduced during liquid culture, and the liquid culture time is 6. If it is less than a week or more than 8 weeks, exosome formation in Centella asiatica stem cells may not work well even if the attractant is sufficiently treated. In addition, even when additional liquid culture is performed by adding an attractant, even if it is less than 5 days or more than 10 days, exosome formation in the centella asiatica stem cells may not occur well, which is not desirable.
상기 제2단계의 교반은 3~5℃에서 300~1,000 x g에서 1~3시간 동안 교반할 수 있고, 이 때, 세포벽이 파쇄될 수 있다. 교반 속도가 300 x g 미만이거나 교반시간이 1시간 미만일 경우, 세포벽 파쇄가 잘 되지 않을 수 있다. 교반 속도가 1,000 x g를 초과하게 되거나 3시간을 초과하게 되는 것은 본 발명에서 크게 제한되거나 구애되지는 않지만, 이 이상의 조건을 넘기는 것은 효율적인 면에서 바람직하지 않다. The second step of stirring may be performed at 300 to 1,000 x g at 3 to 5°C for 1 to 3 hours, and at this time, the cell wall may be broken. If the stirring speed is less than 300 x g or the stirring time is less than 1 hour, cell wall disruption may not be successful. The stirring speed exceeding 1,000
상기 제2단계의 분산화는 100~1,000 bar로 1~5 Cycle 하에 수행할 수 있으며, 이 때, 추가적으로 남아있는 세포벽까지 대부분 파쇄될 수 있다. 상기 분산화를 100bar 미만에서 수행하게 될 경우, 세포벽 찌꺼기가 남아있을 수 있어 엑소좀을 보다 고순도로 정제하기에 좋지 않으며, 1000bar를 초과하게 되면 엑소좀 입자 상태가 손상될 수 있어 바람직하지 않다. 또한 분산화를 5 cycle을 초과해서 수행하는 것은 본 발명에서 크게 제한되거나 구애되지는 않지만, 이 이상의 조건을 넘기는 것은 효율적인 면에서 바람직하지 않다. The second stage of dispersion can be performed under 1 to 5 cycles at 100 to 1,000 bar, and at this time, most of the remaining cell walls can be additionally broken. If the dispersion is performed at less than 100 bar, cell wall debris may remain, which is not good for purifying exosomes to a higher purity, and if it exceeds 1000 bar, the state of the exosome particles may be damaged, which is not desirable. In addition, performing decentralization beyond 5 cycles is not greatly limited or restricted in the present invention, but exceeding this condition is not desirable from an efficiency standpoint.
상기 제2단계에서 세포용해버퍼는 통상의 식물세포 용해용 버퍼를 사용할 수 있다. 바람직하게는 Tris-HCl(tris(hydroxymethyl)aminomethane hydrochloride; pH 7.0~7.5), NaCl, SDS 0.05~0.15, EDTA(ethylenediaminetetraacetic acid), 폴리에틸렌글리콜 3차-옥틸페닐에스테르(Polyethylene glycol tert-octylphenyl ether), CaCl2 0.05~0.15 (w/v)% 및 물 잔량을 포함하는 것을 사용할 수 있고, 더 바람직하게는 상기 세포용해버퍼는 Tris-HCl(tris(hydroxymethyl)aminomethane hydrochloride; pH 7.0~7.5) 50~100 mM, NaCl 100~100 mM, SDS(sodium dodecyl sulfate) 0.05~0.15 (w/v)%, EDTA(ethylenediaminetetraacetic acid) 1~5 mM, 폴리에틸렌글리콜 3차-옥틸페닐에스테르(Polyethylene glycol tert-octylphenyl ether) 0.5~1.5 (v/v)%, CaCl2 0.05~0.15 (w/v)% 및 물 잔량을 포함하는 것을 특징으로 한다. In the second step, the cell lysis buffer may be a typical plant cell lysis buffer. Preferably, Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride; pH 7.0-7.5), NaCl, SDS 0.05-0.15, EDTA (ethylenediaminetetraacetic acid), polyethylene glycol tert-octylphenyl ether, A buffer containing 0.05-0.15 (w/v)% of CaCl 2 and the remaining amount of water may be used, and more preferably, the cell lysis buffer is Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride; pH 7.0-7.5) 50-100. mM, NaCl 100~100 mM, SDS (sodium dodecyl sulfate) 0.05~0.15 (w/v)%, EDTA (ethylenediaminetetraacetic acid) 1~5 mM, polyethylene glycol tert-octylphenyl ether It is characterized in that it contains 0.5 to 1.5 (v/v)%, CaCl 2 0.05 to 0.15 (w/v)%, and the remaining amount of water.
상기 제3단계의 원심분리는 바람직하게는 3~5℃의 1,000 ~ 10,000 x g에서 60~180분간 원심분리하여 펠릿(Pellet) 형태의 찌꺼기 세포(Debris cell)나 죽은 세포(Dead Cell)를 제거하고 상층액만을 회수함으로써 수행할 수 있다. 더 바람직하게는 3~5℃의 1,000~3,000 x g에서 30~120분간 원심분리하여 수행하여 1차 상층액만을 회수하고, 상기 1차 상층액을 다시 3~5℃의 5,000~1,000 x g에서 30~60분간 초고속 원심분리를 수행하여 1차 상층액만을 회수할 수 있다. The third step of centrifugation is preferably centrifuged at 1,000 to 10,000 x g at 3 to 5°C for 60 to 180 minutes to remove debris cells or dead cells in the form of pellets. This can be performed by recovering only the supernatant. More preferably, centrifugation is performed at 1,000-3,000 Only the primary supernatant can be recovered by performing ultra-high-speed centrifugation for 60 minutes.
본 발명은 상기 병풀 엑소좀의 제조방법으로 얻은 병풀 엑소좀에 관한 것이다.The present invention relates to Centella asiatica exosomes obtained by the method for producing Centella asiatica exosomes.
상기 병풀 엑소좀은 총 단백질 함량이 700~800 μg/ml인 것을 특징으로 한다. The Centella asiatica exosomes are characterized by a total protein content of 700-800 μg/ml.
또한 본 발명은 상기 병풀 엑소좀을 함유하는 화장료 조성물에 관한 것일 수 있다. 상기 화장료 조성물은 세포 재생 또는 피부 탄력 효능을 갖는 것을 특징으로 한다.Additionally, the present invention may relate to a cosmetic composition containing the Centella asiatica exosomes. The cosmetic composition is characterized by having cell regeneration or skin elasticity effects.
상기 병풀 엑소좀은 제1형 콜라겐(Collagen Type I) 또는 제3형 콜라겐(Collagen Type III)의 발현을 증강시키는 것을 특징으로 한다. The Centella asiatica exosome is characterized by enhancing the expression of type 1 collagen (Collagen Type I) or type 3 collagen (Collagen Type III).
상기 병풀 엑소좀 내 엑소좀 입자가 1.5×106 particle/ml일 때, 인간 섬유아세포(fibroblast)에서 제1형 콜라겐(Collagen Type I)의 유전자 발현이 200~300% 증강되고, 제3형 콜라겐(Collagen Type III)은 유전자 발현이 140~160% 증가되는 특징이 있다. When the exosome particles in the Centella asiatica exosomes are 1.5×10 6 particles/ml, gene expression of collagen type I in human fibroblasts is enhanced by 200 to 300%, and type 3 collagen (Collagen Type III) is characterized by a 140-160% increase in gene expression.
상기 화장료 조성물에는 병풀 엑소좀이 화장료 조성물 총 중량 대비 0.01~20 중량%를 함유할 수 있다. The cosmetic composition may contain 0.01 to 20% by weight of Centella asiatica exosomes based on the total weight of the cosmetic composition.
상기 화장료 조성물의 제형으로는 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 에센스, 로션, 에멀젼, 팩, 핸드크림, 풋크림, 립밤, 립스틱, 아이섀도우, 아이라이너, 아이브로우 펜슬, 블러셔, 하이라이터, 일반화장수, 스킨, 크림, 세럼, 미용비누, 유연화장수, 약용화장수, 전신세정제, 클렌징폼, 클렌징로션, 겔, 클렌징 오일, 클렌징 크림, 샴푸, 린스, 헤어트리트먼트, 헤어로션, 클렌징 티슈 및 클렌징 워터에서 선택되는 것을 제공할 수 있다. The formulation of the cosmetic composition may be any formulation commonly manufactured in the art, including essence, lotion, emulsion, pack, hand cream, foot cream, lip balm, lipstick, eye shadow, eyeliner, and eyebrow pencil. , blusher, highlighter, general lotion, skin, cream, serum, beauty soap, softening lotion, medicated lotion, body cleanser, cleansing foam, cleansing lotion, gel, cleansing oil, cleansing cream, shampoo, conditioner, hair treatment, hair You can provide a selection from lotions, cleansing tissues and cleansing water.
보다 더 자세하게는, 본 발명의 화장료 조성물의 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물섬유, 식물섬유, 왁스, 파라핀, 전분, 트라가칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 이용될 수 있다. 본 발명의 화장료 조성물의 제형이 파우더 또는 스프레이인 경우에는 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄 히드록시드, 칼슘 실리케이트 또는 폴리아미드 파우더가 이용될 수 있고, 특히 스프레이인 경우에는 추가적으로 클로로플루오로히드로카본, 프로판-부탄 또는 디메틸 에테르와 같은 추진체를 포함할 수 있다. 본 발명의 화장료 조성물의 제형이 용액 또는 유탁액의 경우에는 담체 성분으로서 용매, 용매화제 또는 유탁화제가 이용되고, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌글리콜, 1,3-부틸글리콜 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르가 있다. 본 발명의 화장료 조성물의 제형이 현탁액인 경우에는 담체 성분으로서 물, 에탄올 또는 프로필렌 글리콜과 같은 액상 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가 또는 트라칸트 등이 이용될 수 있다. 본 발명의 화장료 조성물의 제형이 계면-활성제 함유 클렌징인 경우에는 담체 성분으로서 지방족 알코올 설페이트, 지방족 알코올 에테르설페이트, 설포숙신산 모노에스테르, 아세티오네이트, 이미다졸리늄 유도체, 메틸타우레이트, 사르코시네이트, 지방산 아미드 에테르 설페이트, 알킬아미도베타인, 지방족 알코올, 지방산 글리세리드, 지방산 디에탄올아미드, 식물성 유, 리놀린 유도체 또는 에톡실화 글리세롤 지방산 에스테르 등이 이용될 수 있다. 본 발명의 화장료 조성물은 형광물질, 살진균제, 굴수성 유발물질, 보습제, 방향제, 방향제 담체, 단백질, 용해화제, 당 유도체, 일광차단제, 비타민, 식물 추출물 등을 포함하는 부형제를 추가로 함유할 수 있다. 상기 성분들은 제형 또는 사용목적에 따라 그 첨가량을 화장료 조성물 고유의 효과를 손상시키지 않는 범위 내에서 선택할 수 있다. 상기 성분들의 첨가량은 예를 들어 조성물 총 중량에 대하여 0.1~10 중량%, 바람직하게는 0.1~6 중량%일 수 있으나 이에 제한되는 것은 아니다.More specifically, when the formulation of the cosmetic composition of the present invention is a paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, and silica are used as carrier ingredients. , talc or zinc oxide can be used. When the formulation of the cosmetic composition of the present invention is powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as the carrier ingredient. In particular, when the cosmetic composition is a spray, chlorofluorohydride may be used as a carrier ingredient. It may contain propellants such as carbon, propane-butane or dimethyl ether. When the formulation of the cosmetic composition of the present invention is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene. These include fatty acid esters of glycol, 1,3-butylglycol oil, glycerol aliphatic esters, polyethylene glycol or sorbitan. When the formulation of the cosmetic composition of the present invention is a suspension, the carrier component includes water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used. When the formulation of the cosmetic composition of the present invention is a surfactant-containing cleansing agent, the carrier ingredients include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, acethionate, imidazolinium derivative, methyl taurate, and sarcosinate. , fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linoline derivative, or ethoxylated glycerol fatty acid ester can be used. The cosmetic composition of the present invention may further contain excipients including fluorescent substances, fungicides, hydrotropes-inducing substances, moisturizers, fragrances, fragrance carriers, proteins, solubilizers, sugar derivatives, sunscreens, vitamins, plant extracts, etc. . The amount of the above ingredients can be selected depending on the formulation or purpose of use within a range that does not impair the inherent effect of the cosmetic composition. The amount of the ingredients added may be, for example, 0.1 to 10% by weight, preferably 0.1 to 6% by weight, based on the total weight of the composition, but is not limited thereto.
본 발명은 병풀 줄기세포 배양액 유래 엑소좀을 유효성분으로 함유하는 세포재생 및 피부탄력 효능을 갖는 화장료 조성물 및 이의 제조방법에 관한 것이다. 본 발명을 통해 병풀 줄기세포 배양액에서 분리한 엑소좀은 이를 화장료로 활용할 경우, 피부세포의 증식능이 우수하여 피부 세포 재생에 효과가 있고, Collagen Type I과 III의 발현을 증가시켜 피부 탄력에 효과가 있다. The present invention relates to a cosmetic composition having cell regeneration and skin elasticity effects containing exosomes derived from Centella asiatica stem cell culture medium as an active ingredient, and a method for producing the same. When used as a cosmetic, the exosomes isolated from Centella asiatica stem cell culture medium through the present invention are effective in skin cell regeneration due to their excellent proliferative ability of skin cells, and are effective in skin elasticity by increasing the expression of Collagen Type I and III. there is.
도 1은 본 발명의 실시예 1의 엑소좀 입자 형태를 확인한 투과 전자 현미경 (TEM) 사진이다.
도 2는 본 발명의 실시예 1 엑소좀 분산액의 제타전위 측정 결과를 나타내는 그래프이다. Figure 1 is a transmission electron microscope (TEM) photograph confirming the shape of exosome particles in Example 1 of the present invention.
Figure 2 is a graph showing the zeta potential measurement results of the exosome dispersion in Example 1 of the present invention.
이하 본 발명의 바람직한 실시예를 상세히 설명하기로 한다. 그러나, 본 발명은 여기서 설명되는 실시예에 한정되지 않고 다른 형태로 구체화될 수도 있다. 오히려, 여기서 소개되는 내용이 철저하고 완전해지도록, 당업자에게 본 발명의 사상을 충분히 전달하기 위해 제공하는 것이다. Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, it is provided to ensure that the content introduced here is thorough and complete, and to sufficiently convey the spirit of the present invention to those skilled in the art.
<실시예 1. Elicitor (TNF-α 및 salicylic acid)를 첨가한 병풀 줄기세포 배양액 유래 엑소좀 제조><Example 1. Preparation of exosomes derived from centella asiatica stem cell culture medium with added elicitors (TNF-α and salicylic acid)>
병풀(제주자원식물연구소에서 구입)을 70(v/v)% 에탄올 수용액에 몇초간 침지하여 살균한 후 멸균수로 세척하였다. 세척한 병풀 잎을 절삭하여 상처를 내어 1 ㎎/ℓ 2,4-D, 3(w/v)% Sucrose 및 agar 8g/ℓ 가 포함된 기본 MS배지(Murashige and Skoog 1962, Duchefa 사)에서 30℃, 습도 65%의 조건으로 암배양하며 초기 병풀 식물세포를 유도하였다. 이와 같은 고체 배양 중에는 지속적으로 1N 수산화나트륨(NaOH)을 이용하여 배지의 pH를 5.8 로 조정하였다. 또한 병풀 식물세포가 배양되고 있는 배지에 대해, 고체상태에서의 배양은 3주간으로 진행하였다. Centella asiatica (purchased from Jeju Resource Plant Research Institute) was sterilized by immersing it in a 70 (v/v) % ethanol aqueous solution for a few seconds and then washed with sterilized water. Washed Centella asiatica leaves were cut, wounded, and grown on basic MS medium (Murashige and Skoog 1962, Duchefa) containing 1 mg/l 2,4-D, 3(w/v)% sucrose, and 8g/l agar. Early Centella asiatica plant cells were induced by culturing in the dark under conditions of ℃ and 65% humidity. During such solid culture, the pH of the medium was continuously adjusted to 5.8 using 1N sodium hydroxide (NaOH). In addition, for the medium in which Centella asiatica plant cells were cultured, culture in solid state was carried out for 3 weeks.
이 후, 식물세포를 대량의 액체배지로 옮겨 액체배양을 실시하였다. 이와같은 대량 배양(MS 배지 액체 배지 이용)은 온도조건 25±2 ℃, 습도 65 % 배양실 조건에서 배지 추가 없이 20 ℓ 생물반응기에 총 8주간 배양하였다. Afterwards, the plant cells were transferred to a large amount of liquid medium and liquid culture was performed. This mass culture (using MS medium liquid medium) was cultured in a 20 L bioreactor for a total of 8 weeks without adding any medium at a temperature of 25±2°C and a humidity of 65% in a culture room.
이 때, 배양 8주차 시작일(7주 액체 배양 후)에 최종농도가 TNF-α 50㎍/ℓ 및 salicylic acid 1mM가 되도록, TNF-α와 salicylic acid를 첨가하여 1주간 배양을 더 진행하여 TNF-α와 salicylic acid를 첨가한 병풀 줄기세포 배양액을 얻었다. (이 줄기세포 배양액에서 엑소좀이 확인된다.) At this time, at the beginning of the 8th week of culture (after 7 weeks of liquid culture), TNF-α and salicylic acid were added so that the final concentration was 50㎍/ℓ of TNF-α and salicylic acid 1mM, and culture was continued for another week to increase TNF-α. Centella asiatica stem cell culture medium containing α and salicylic acid was obtained. (Exosomes are identified in this stem cell culture medium.)
상기 병풀 줄기세포 배양액 1ℓ와 표 1의 Lysis buffer 1ℓ를 혼합하여 Homo mixer(또는 Homogenizer)로 4℃에서 1,000 x g에서 2시간 동안 교반하였고, 교반한 용액을 Microfluidizer를 이용하여 800 bar의 고압 조건에서 분산하는 과정을 3회 반복하여 세포벽을 파쇄하였다. 1 liter of the Centella Asiatica stem cell culture medium and 1 liter of Lysis buffer in Table 1 were mixed and stirred at 4°C at 1,000 This process was repeated three times to crush the cell walls.
그 후, 원심분리기를 이용하여 4℃에서 3,000 x g로 60분 동안 원심분리하고 Pellet 층을 제거한 후 상층액을 회수하였다. 다시 추가적으로 초고속 원심분리기를 이용하여 4℃에서 10,000 x g로 60분 동안 원심분리하고 Pellet 층을 제거하고 상층액을 회수하였다. Afterwards, centrifugation was performed at 3,000 x g at 4°C for 60 minutes using a centrifuge, the pellet layer was removed, and the supernatant was recovered. Additionally, centrifugation was performed at 10,000 x g at 4°C for 60 minutes using an ultra-high-speed centrifuge, the pellet layer was removed, and the supernatant was recovered.
회수한 상층액을 접선흐름여과법(TFF; TANGENTIAL FLOW FILTRATION)을 이용하여 여과 및 농축을 수행하였다. 필터는 Pellicon XL 필터(Biomax membrane, 10kDa, 13ml/min)를 사용하였으며, 필터를 반복적으로 통과하게 하여 원하는 분자량을 갖는 용출액이 농축될 수 있게 하였다. 이를 통해 50 ~ 150 kDa사이의 분자량을 갖는 병풀 엑소좀을 분리하였다. The recovered supernatant was filtered and concentrated using tangential flow filtration (TFF). The filter used was a Pellicon XL filter (Biomax membrane, 10kDa, 13ml/min), and the eluate with the desired molecular weight was concentrated by repeatedly passing through the filter. Through this, Centella asiatica exosomes with a molecular weight between 50 and 150 kDa were isolated.
<비교예 1. TNF-α만을 첨가한 병풀 줄기세포 배양액 유래 엑소좀 제조><Comparative Example 1. Preparation of exosomes derived from Centella asiatica stem cell culture medium with the addition of only TNF-α>
실시예 1과 동일한 방법으로 병풀 줄기세포 배양액 유래 엑소좀을 얻되, salicylic acid는 첨가하지 않고 TNF-α만 처리하였다. Exosomes derived from Centella asiatica stem cell culture medium were obtained in the same manner as in Example 1, but only TNF-α was treated without adding salicylic acid.
<비교예 2. Salicylic acid를 첨가한 병풀 줄기세포 배양액 유래 엑소좀 제조><Comparative Example 2. Preparation of exosomes derived from Centella asiatica stem cell culture medium with added salicylic acid>
실시예 1과 동일한 방법으로 병풀 줄기세포 배양액 유래 엑소좀을 얻되, TNF-α는 첨가하지 않고 salicylic acid만 처리하였다. Exosomes derived from Centella asiatica stem cell culture were obtained in the same manner as in Example 1, but only salicylic acid was treated without the addition of TNF-α.
<비교예 3. TNF-α 및 salicylic acid를 모두 처리하지 않은 병풀 줄기세포 배양액 유래 엑소좀 제조><Comparative Example 3. Preparation of exosomes derived from Centella asiatica stem cell culture medium without treatment with either TNF-α or salicylic acid>
실시예 1과 동일한 방법으로 병풀 줄기세포 배양액 유래 엑소좀을 얻되, salicylic acid와 TNF-α를 모두 처리하지 않았다. Exosomes derived from Centella asiatica stem cell culture medium were obtained in the same manner as in Example 1, but neither salicylic acid nor TNF-α were treated.
<비교예 4. 병풀 줄기세포 배양 단계를 생략한 병풀 유래 엑소좀 제조><Comparative Example 4. Preparation of Centella asiatica-derived exosomes omitting the Centella asiatica stem cell culture step>
실시예 1의 방법에서 병풀 줄기세포 배양 단계를 모두 생략하고, 병풀 잎50g을 바로 Lysis buffer 1ℓ와 혼합하고, 이후의 단계는 실시예 1과 동일한 방법으로 엑소좀을 얻었다. In the method of Example 1, the entire Centella asiatica stem cell culture step was omitted, and 50 g of Centella asiatica leaves were immediately mixed with 1 liter of Lysis buffer, and subsequent steps were performed in the same manner as in Example 1 to obtain exosomes.
<비교예 5. 병풀 줄기세포 배양액 유래 엑소좀 제조 (초원심분리법)> <Comparative Example 5. Preparation of exosomes derived from Centella asiatica stem cell culture medium (ultracentrifugation method)>
실시예 1의 방법에서 병풀 줄기세포 배양액을 얻는 단계(TNF-α와 salicylic acid를 첨가후 1주일 배양)까지는 동일하게 진행하고, 상기 병풀 줄기세포 배양액을 이용하여 Ultracentrifugation을 이용하여 엑소좀을 추출하였다. The method of Example 1 was carried out in the same manner up to the step of obtaining a centella asiatica stem cell culture medium (culturing for 1 week after adding TNF-α and salicylic acid), and exosomes were extracted using the centella asiatica stem cell culture medium using ultracentrifugation. .
구체적으로, 병풀 줄기세포 배양액을 1,000 x g로 20분간 원심분리 후 pellet (dead cell)을 제거한 상층액을 4℃에서 2,000 x g로 20분간 원심분리 후 다시 상층액만을 모아, 상기 상층액을 4℃에서 3,000 x g로 30분간 원심분리하였다. 3,000 x g로 분리된 상층액을 추가적으로 4℃에서 10,000 x g로 60분간 원심분리하여 pellet 형태의 debris cell을 제거하고 상층액을 회수하였다. Specifically, after centrifuging the Centella asiatica stem cell culture medium at 1,000 Centrifuged at 3,000 x g for 30 minutes. The supernatant separated at 3,000
다시 10,000 x g로 분리된 상층액을 초원심분리기를 이용하여 4℃에서 100,000 x g로 60분간 초원심분리하여 이번에는 Pellet 형태를 회수하여 엑소좀을 분리하였다.The supernatant was again ultracentrifuged at 10,000
<비교예 6. 일반 접선흐름여과법만을 적용한 병풀 줄기세포 배양액 유래 엑소좀 제조> <Comparative Example 6. Preparation of exosomes derived from Centella asiatica stem cell culture fluid using only general tangential flow filtration method>
실시예 1의 방법에서 병풀 줄기세포 배양액을 얻는 단계(TNF-α와 salicylic acid를 첨가후 1주일 배양)까지는 동일하게 진행하고, 이를 에멀전화하고, 원심분리하는 단계는 진행하지 않은 채, 바로, 상기 병풀 줄기세포 배양액을 1.0 ㎛ 기공크기 필터로 여과하여 액상을 얻고, 상기 액상을 접선흐름여과법을 이용하여 50 ~ 150 kDa 사이의 분자량만 회수하여 엑소좀을 분리하였다.In the method of Example 1, the step of obtaining a centella asiatica stem cell culture medium (culturing for 1 week after adding TNF-α and salicylic acid) was carried out in the same manner, without proceeding with the steps of emulsifying and centrifuging, The centella asiatica stem cell culture medium was filtered through a 1.0 ㎛ pore size filter to obtain a liquid phase, and exosomes were isolated by recovering only the molecular weight between 50 and 150 kDa from the liquid phase using tangential flow filtration.
<비교예 7. 수성 2상계법을 적용한 병풀 줄기세포 배양액 유래 엑소좀 제조 ()> <Comparative Example 7. Preparation of exosomes derived from Centella asiatica stem cell culture using aqueous two-phase method ()>
실시예 1의 방법에서 병풀 줄기세포 배양액을 얻는 단계(TNF-α와 salicylic acid를 첨가후 1주일 배양)까지는 동일하게 진행하고, 이를 에멀전화하고, 원심분리하는 단계는 진행하지 않은 채, 바로, 상기 병풀 줄기세포 배양액을 1.0 ㎛ 기공크기 필터로 여과하여 액상을 얻고, 상기 액상을 PEG(Polyethylene glycol)/Dextran을 사용하여 수성 2상계를 형성하였다. 그 후, 3,000 x g, 60분, 4℃에서 원심분리를 하였다. 원심분리 후 뚜렷하게 분리된 2개의 층 중에서, 상층액을 제거하고 하층액을 회수하여 엑소좀을 분리하였다.In the method of Example 1, the step of obtaining a centella asiatica stem cell culture medium (culturing for 1 week after adding TNF-α and salicylic acid) was carried out in the same manner, without proceeding with the steps of emulsifying and centrifuging, The centella asiatica stem cell culture medium was filtered through a 1.0 ㎛ pore size filter to obtain a liquid phase, and the liquid phase was formed into an aqueous two-phase system using PEG (Polyethylene glycol)/Dextran. Afterwards, centrifugation was performed at 3,000 x g, 60 minutes, 4°C. Among the two clearly separated layers after centrifugation, the supernatant was removed, the lower layer was recovered, and exosomes were separated.
<실험예 1. 병풀 엑소좀 내 총 단백질 함량 확인> <Experimental Example 1. Confirmation of total protein content in Centella asiatica exosomes>
실시예 1, 비교예 1 내지 7에서 제조한 엑소좀의 분리가 잘 되었는지는 총 단백질 함량과 입자 수를 통해 확인하였고, 먼저, 총 단백질 함량을 확인하였다. Whether the exosomes prepared in Example 1 and Comparative Examples 1 to 7 were well separated was confirmed through the total protein content and number of particles. First, the total protein content was confirmed.
각 엑소좀 내 단백질 함량은 BCA assay(Bicinchoninic acid assay)를 진행하여 확인하였고, 실험방법은 다음과 같다 The protein content in each exosome was confirmed by performing BCA assay (Bicinchoninic acid assay), and the experimental method was as follows.
(1) Standard Solution 제조 : BSA (Bovine Serum Albumin, 98%) 23 ㎎을 취해 정제수 10 ml를 채운다. 이를 0 (Blank), 20 ㎕, 40 ㎕, 60 ㎕ 취해 각 1,000 ㎕, 980 ㎕, 960 ㎕, 940 ㎕의 정제수를 첨가한다. (1) Preparation of Standard Solution: Take 23 mg of BSA (Bovine Serum Albumin, 98%) and fill with 10 ml of purified water. Take 0 (Blank), 20 ㎕, 40 ㎕, and 60 ㎕ and add 1,000 ㎕, 980 ㎕, 960 ㎕, and 940 ㎕ of purified water respectively.
(2) Test Solution 제조 (2) Test Solution Manufacturing
: 각 엑소좀 샘플 20 ㎕을 취해 정제수 980 ㎕를 채운다. : Take 20 ㎕ of each exosome sample and fill with 980 ㎕ of purified water.
(3) BCA Procedure (3) BCA Procedure
: Standard Solution 및 Test Solution 각 0.1 ml 취해 BCA working Solution 1 ml를 첨가한 후, 37℃ Water bath에서 30분간 반응하고 562 nm에서 흡광도를 측정한다 (Working Solution : Bicinchoninic acid : 4% CuSO4·5H2O Sol = 49 : 1): Take 0.1 ml each of Standard Solution and Test Solution, add 1 ml of BCA Working Solution, react for 30 minutes in a 37℃ water bath, and measure absorbance at 562 nm (Working Solution: Bicinchoninic acid: 4% CuSO 4 5H 2 O Sol = 49:1)
실험 결과는 표 2에 나타내었는데, 하기 표 2의 결과와 같이, 본 발명에서 병풀 줄기세포 배양액 제조시 TNF-α 및 salicylic acid를 첨가한 경우가 최종 회수된 엑소좀에서의 총 단백질 함량이 가장 높은 것으로 확인되었다. The experimental results are shown in Table 2. As shown in Table 2 below, the total protein content in the final recovered exosomes was highest when TNF-α and salicylic acid were added when preparing the centella asiatica stem cell culture medium in the present invention. It was confirmed that
<실험예 2. 각 분산액 내 엑소좀 입자의 특징 확인> <Experimental Example 2. Confirmation of characteristics of exosome particles in each dispersion>
엑소좀 분리 수율 확인을 하기 위한 두번째 방법으로서, 실시예 1, 비교예 1 내지 7에서 제조한 분산액 내 엑소좀 입자 크기 분포와 단위 부피당 입자 수를 나노입자추적분석 (Nanoparticle Tracking Analysis, NTA)으로 분석하여 확인하였다. 이렇게 확인된 결과는 하기의 표 3에 기재하였다. As a second method to confirm the exosome isolation yield, the size distribution of exosome particles and the number of particles per unit volume in the dispersions prepared in Example 1 and Comparative Examples 1 to 7 were analyzed using Nanoparticle Tracking Analysis (NTA). It was confirmed. The results confirmed in this way are listed in Table 3 below.
(엑소좀 입자 미검출)Unable to confirm
(Exosome particles not detected)
(엑소좀 입자 미검출)Unable to confirm
(Exosome particles not detected)
표 3과 같이 각 입자크기와 단위 부피당 입자 수를 확인한 결과, 실시예 1의 엑소좀 입자크기가 작은 편이면서도, 단위 부피당 입자 수가 현저하게 많은 것을 알 수 있다. As a result of checking each particle size and the number of particles per unit volume as shown in Table 3, it can be seen that the exosome particle size of Example 1 is relatively small, but the number of particles per unit volume is significantly large.
따라서 실시예 1이 엑소좀의 정제 수율도 가장 좋은 것을 알 수 있다. Therefore, it can be seen that Example 1 has the best purification yield of exosomes.
비교예 4는 병풀 잎을 바로 세포 용해 버퍼에 용해한 것이기에, 단백질 총 함량은 높게 나타났지만 나노입자가 전혀 확인되지 않아, 엑소좀 형성 및 분리가 될 수 없는 것이었고, 예상한 바와 같은 결과가 나타났다. 이에 이후의 효능 실험에서는 비교예 4는 제외하였다. In Comparative Example 4, because the centella asiatica leaves were immediately dissolved in cell lysis buffer, the total protein content was high, but no nanoparticles were identified, so exosomes could not be formed and separated, and the results were as expected. Accordingly, Comparative Example 4 was excluded from subsequent efficacy experiments.
또한, 수득된 엑조솜 분산액 중에서, 실시예 1 엑소좀 입자에 대한 투과 전자 현미경 (TEM) 사진은 도 1에 나타내었고, 제타 전위(zeta potential) 결과 그래프는 도 2에 나타내었다. In addition, among the obtained exosome dispersions, a transmission electron microscopy (TEM) photograph of the exosome particles of Example 1 is shown in Figure 1, and a graph of the zeta potential results is shown in Figure 2.
도 1을 살펴보면, 실시예 1 분산액 내 엑소좀 나노입자의 크기가 균일하게 제조되었음을 확인할 수 있다. Looking at Figure 1, it can be seen that the size of the exosome nanoparticles in the dispersion of Example 1 was produced uniformly.
한편, Zeta potential은 입자들간의 반발력 정도로 콜로이드의 안정성을 평가한다. 값이 클수록 입자간의 응집력이 높음을 나타내기 때문에 엑소좀이 안정적으로 형성되었는지 확인할 수 있다. Meanwhile, Zeta potential evaluates the stability of colloids by the degree of repulsion between particles. The larger the value, the higher the cohesion between particles, so it can be confirmed whether the exosomes were formed stably.
이에 도 2에서와 같이 실시예 1 엑소좀 분산액에 대한 제타전위 측정 결과, -13mV의 값을 나타냈으며 이는 본 발명의 엑소좀 입자들이 서로 반발하는 힘이 없어 응집력이 높음을 나타낸다.Accordingly, as shown in Figure 2, the zeta potential measurement result for the exosome dispersion of Example 1 showed a value of -13 mV, which indicates that the exosome particles of the present invention have high cohesion since there is no force to repel each other.
<실험예 3. 세포 독성 확인 - Fibroblast MTT assay><Experimental Example 3. Confirmation of cytotoxicity - Fibroblast MTT assay>
각 엑소좀을 이용하여 Fibroblast 에 대한 세포독성 실험을 실시하였다. 실험방법은 다음과 같다. Cytotoxicity tests on Fibroblast were conducted using each exosome. The experimental method is as follows.
24 well plate에 세포를 접종하여 24시간 배양한 뒤, 배양된 세포의 배지를 무혈청(serum-free) 배지로 교체하고 시료를 농도 별로 처리하여 24시간 배양하였다. 배양이 끝난 세포에 2.5 mg/ml의 MTT 용액을 10배 희석시킨 배지로 교체하여 반응시킨 후 상등액을 제거하고 DMSO 1 ml을 첨가하여 생성된 MTT-formazan 결정체를 용해시켜 ELISA reader로 570 nm에서 흡광도를 측정하였다. 이때 사용된 대조군은 용해 용매로 DMSO (Dimethyl Sulfoxide)를 사용하였다. 세포 독성은 대조군과 비교하여 흡광도의 백분율로 나타냈다. Cells were inoculated into a 24 well plate and cultured for 24 hours. The medium of the cultured cells was replaced with serum-free medium, and the samples were treated according to concentration and cultured for 24 hours. After culturing the cells, replace them with a medium containing 2.5 mg/ml MTT solution diluted 10 times, remove the supernatant, add 1 ml of DMSO to dissolve the resulting MTT-formazan crystals, and measure the absorbance at 570 nm using an ELISA reader. was measured. The control used at this time used DMSO (Dimethyl Sulfoxide) as a dissolution solvent. Cytotoxicity was expressed as a percentage of absorbance compared to control.
그 결과, 각 엑소좀이 모두 7.5×106 particle/ml까지는 독성이 없음을 확인하였다. 이 결과 중, 실시예 1에 대한 것을 대표적으로 하기 표 4에 나타내었고, 이후에는 1.5×106 particle/ml의 농도인 것을 적용하여 실험하였다. As a result, it was confirmed that each exosome was not toxic up to 7.5 × 10 6 particle/ml. Among these results, those for Example 1 are representatively shown in Table 4 below, and the subsequent experiments were conducted using a concentration of 1.5×10 6 particles/ml.
실시예 1
Example 1
한편, 기존에 세포 재생용 조성물로 사용되던 병풀 추출물, 병풀 추출물로부터 분리된 TECA(센텔라 정량 추출물)와 본 발명의 실시예 1 엑소좀의 효능을 비교하기 위해, 0.1 중량%의 병풀 에탄올 추출물, 0.1 중량%의 TECA 용액을 준비하였다. 실시예 1 엑소좀은 이를 동결건조했을 때의 조성물을 이용하여 농도 환산을 하였는데, 고형분 0.1 중량%가 되도록 엑소좀이 포함된 액상을 환산하여 시료로 준비하였다. Meanwhile, in order to compare the efficacy of Centella asiatica extract, which was previously used as a composition for cell regeneration, and TECA (Centella quantitative extract) isolated from Centella asiatica extract, and the exosome of Example 1 of the present invention, 0.1% by weight of Centella asiatica ethanol extract, A 0.1 wt% TECA solution was prepared. Example 1 The concentration of exosomes was converted using the composition obtained when freeze-dried. The liquid phase containing exosomes was converted to a solid content of 0.1% by weight and prepared as a sample.
이들을 각각 위의 세포 재생 실험에 사용하였는데, 실시예 1 조성물만에 세포 증식 효과가 나타나며, 병풀 추출물과 TECA의 처리 세포에서는 오히려 세포사멸이 일어나는 것이 확인되었다. 이는 용매 추출된 병풀 추출물과 TECA는 고농도의 병풀 유래 화합물이 포함되어 있기에, 이를 고용량으로 세포에 투여하는 것이 오히려 세포 독성을 일으키는 것임을 알 수 있다. 이에 반해, 병풀 유래 줄기세포에서 분리된 엑소좀은 세포가 세포 외부로 방출하는 소낭인 extra cellular vesicles의 일종으로, 진핵생물에서 세포 간 정보 교환을 위해 분비하는 나노미터 크기의 물질로서, 세포막의 구조와 동일한 인지질 이중막으로 이루어지며, 모체가 되는 세포 내부의 단백질, 지질, 핵산 등을 유사하게 포함하고 있기에, 세포 독성을 나타내지 않고, 기존 화합물이 대부분 포함된 추출물 유래 조성물에 비해 체내에 보다 더 효과적으로 적용 가능한 유용성 물질임을 확인할 수 있다. These were each used in the above cell regeneration experiment, and it was confirmed that only the composition of Example 1 showed a cell proliferation effect, while cell death occurred in cells treated with Centella asiatica extract and TECA. This shows that solvent-extracted Centella asiatica extract and TECA contain high concentrations of Centella asiatica-derived compounds, so administering them to cells in high doses actually causes cytotoxicity. On the other hand, exosomes isolated from centella asiatica-derived stem cells are a type of extra cellular vesicles, which are vesicles released by cells to the outside of the cell. They are nanometer-sized substances secreted by eukaryotes for information exchange between cells, and are related to the structure of the cell membrane. It is composed of the same phospholipid bilayer and contains similar proteins, lipids, and nucleic acids inside the parent cell, so it does not exhibit cytotoxicity and is more effective in the body than extract-derived compositions containing most of the existing compounds. It can be confirmed that it is an applicable useful substance.
<실험예 4. Wound healing efficacy - scratch assay><Experimental Example 4. Wound healing efficacy - scratch assay>
24 well plate (corning)에 인간섬유아세포(human dermal fibroblast)를 1일 (37℃, CO2 5%) 배양한 뒤 Scar™ Scratcher(SPL)로 긁은 후 HBSS (Hank's Balanced Salt Solution, Gibco)로 washing한다. Serum-free 배지(IMDM, welgene)로 교체하고 시료를 농도 별로 처리하여 26시간 동안 37℃, CO2 5% incubator에 배양하였다. Human dermal fibroblasts were cultured in a 24 well plate (corning) for 1 day (37°C, CO 2 5%), then scratched with Scar™ Scratcher (SPL) and washed with HBSS (Hank's Balanced Salt Solution, Gibco). do. Serum-free medium (IMDM, welgene) was replaced, samples were treated according to concentration, and cultured in an incubator at 37°C and CO 2 5% for 26 hours.
세포 사진은 scratcher로 긁은 직후와 26시간 배양한 후 현미경(Leica)을 사용하여 촬영하고 ImageJ를 이용하여 wound closure 값을 산출하였다. Cell photos were taken using a microscope (Leica) immediately after scratching with a scratcher and after culturing for 26 hours, and wound closure values were calculated using ImageJ.
실험 결과는 표 5에 나타내었다. The experimental results are shown in Table 5.
표 5의 결과와 같이, 실시예 1 엑소좀(1.5×106 particle/ml)이 세포 재생에 가장 효과적임을 확인하였다.As shown in Table 5, it was confirmed that Example 1 exosomes (1.5×10 6 particles/ml) were most effective in cell regeneration.
<실험예 5. Collagen type I 발현 증가 효과 (Skin Elasticity)><Experimental Example 5. Effect of increasing Collagen type I expression (Skin Elasticity)>
탄력 증가 효능을 확인하기 위해 Collagen type I 유전자 발현 실험을 수행하였다. 60 mm culture dish (corning) 에 인간 섬유아세포(fibroblast)를 1일 (37℃, CO2 5%) 배양한 뒤 Serum-free 배지(IMDM, welgene)로 교체하고, 시료를 농도 별로 처리하여 24시간 동안 37℃, CO2 5% incubator에 배양하였다. 배양 후 세포를 QIAzol™ Lysis Reagent (Qiagen) 로 모아 제조사의 방법에 따라 RNA를 분리한. 분리된 RNA를 정량한 뒤 1 μg의 RNA로 cDNA를 합성하여 Real-time PCR을 실시한다. PCR에 사용된 Collagen type I, β-Actin primer 는 코스모진텍사 (Korea)에서 합성하여 사용하였으며 primer sequence 는 표 6에, 실험 결과는 표 7에 나타내었다.Collagen type I gene expression experiment was performed to confirm the effectiveness of increasing elasticity. Human fibroblasts were cultured in a 60 mm culture dish (corning) for 1 day (37°C, CO 2 5%), then replaced with serum-free medium (IMDM, Welgene), and the samples were treated according to concentration and incubated for 24 hours. Cultured in an incubator at 37°C and CO 2 5%. After culturing, cells were collected with QIAzol™ Lysis Reagent (Qiagen) and RNA was isolated according to the manufacturer's method. After quantifying the separated RNA, cDNA is synthesized from 1 μg of RNA and real-time PCR is performed. Collagen type I and β-Actin primers used in PCR were synthesized by Cosmogenetec (Korea). The primer sequences are shown in Table 6 and the experimental results are shown in Table 7.
상기 표 7의 결과와 같이 실시예 1 엑소좀 (1.5×106 particle/ml) 처리시 가장 높은 Collagen type Ⅰ 합성율을 나타내었다. As shown in Table 7, the highest Collagen type I synthesis rate was observed when exosomes (1.5×10 6 particles/ml) were treated in Example 1.
<실험예 6. Collagen type Ⅲ의 발현 증가 효과 (Skin Elasticity)><Experimental Example 6. Effect of increasing expression of Collagen type Ⅲ (Skin Elasticity)>
실험예 5와 같은 조건으로 인간 섬유아세포(fibroblast)에 시료를 처리하여 실험을 진행하되, Collagen type Ⅰ 대신 Collagen type Ⅲ의 발현을 확인하였다. 이를 위해 하기 표 8의 프라이머를 이용하였고, 그 결과를 표 9에 나타내었다. The experiment was conducted by treating samples with human fibroblasts under the same conditions as in Experimental Example 5, but the expression of Collagen type Ⅲ instead of Collagen type Ⅰ was confirmed. For this purpose, the primers shown in Table 8 below were used, and the results are shown in Table 9.
표 9의 결과와 같이 실시예 1 엑소좀(1.5×106 particle/ml)을 처리했을 때, 가장 높은 Collagen type Ⅲ 합성율을 나타내었다. As shown in the results in Table 9, when exosomes (1.5×10 6 particles/ml) of Example 1 were treated, the highest Collagen type III synthesis rate was observed.
이상의 결과를 통해, 본 발명의 방법으로 제조된 병풀 줄기세포 배양액 유래 엑소좀이 총 엑소좀 입자의 수율이 높을 뿐만 아니라, 입자 상태가 균일하고 안정성이 있는 것으로 확인되며, 이 엑소좀이 갖는 세포 재생과 피부탄력 효능이 매우 우수함을 알 수 있다. Through the above results, it was confirmed that exosomes derived from Centella asiatica stem cell culture medium prepared by the method of the present invention not only have a high yield of total exosome particles, but also have a uniform and stable particle state, and the cell regeneration properties of these exosomes. It can be seen that the skin elasticity effect is very excellent.
<화장료 제형예 1. 유연 화장수의 제조><Cosmetic formulation example 1. Preparation of flexible lotion>
1.5×106 particle/ml의 실시예 1 엑소좀을 시료로서 함유한 유연 화장수(스킨, 100g)를 통상의 방법에 따라 표 10과 같이 제조하였다. 이 후의 화장료 제형에서는 상기 1.5×106 particle/ml의 실시예 1 엑소좀을 이용하였다. A soft lotion (skin, 100g) containing 1.5×10 6 particle/ml of Example 1 exosomes as a sample was prepared as shown in Table 10 according to a conventional method. In the subsequent cosmetic formulation, the 1.5×10 6 particle/ml exosome of Example 1 was used.
<화장료 제형예 2. 영양 화장수의 제조><Cosmetic formulation example 2. Preparation of nutritious lotion>
하기 표 11과 같이 실시예 1 엑소좀을 함유한 영양 화장수(로션, 100g)를 통상의 방법에 따라 제조하였다. As shown in Table 11 below, Example 1 nutritional lotion (lotion, 100g) containing exosomes was prepared according to a conventional method.
<화장료 제형예 3. 영양크림의 제조><Cosmetic formulation example 3. Preparation of nutritional cream>
하기 표 12의 조성과 같이 실시예 1 엑소좀을 함유한 영양크림(100g)을 통상의 방법에 따라 제조하였다. As shown in Table 12 below, nutritional cream (100 g) containing exosomes in Example 1 was prepared according to a conventional method.
<화장료 제형예 4. 에센스의 제조><Cosmetic formulation example 4. Preparation of essence>
하기 표 13의 조성과 같이 실시예 1 엑소좀을 함유한 에센스(100g)를 통상의 방법에 따라 제조하였다. Exosome-containing essence (100 g) of Example 1 was prepared according to a conventional method, as shown in Table 13 below.
<화장료 제형예 5. 파운데이션의 제조><Cosmetic formulation example 5. Preparation of foundation>
하기 표 14의 조성과 같이 실시예 1 엑소좀을 함유한 파운데이션(100g)을 통상의 방법에 따라 제조하였다. Example 1 Exosome-containing foundation (100 g) was prepared according to a conventional method, as shown in Table 14 below.
<화장료 제형예 6. 헤어샴푸의 제조><Cosmetic formulation example 6. Production of hair shampoo>
하기 표 15의 조성과 같이 실시예 1 엑소좀을 함유한 헤어샴푸(100g)를 통상의 방법에 따라 제조하였다. Example 1 Hair shampoo (100 g) containing exosomes was prepared according to a conventional method, as shown in Table 15 below.
Claims (15)
(제2단계) 상기 병풀 줄기세포 배양액과 세포용해버퍼를 혼합하고, 교반 및 유화하여 세포를 파쇄하여 세포 파쇄액을 얻는 단계;
(제3단계) 상기 세포 파쇄액을 원심분리하여 상층액을 얻는 단계; 및
(제4단계) 접선흐름여과법(PTFF; PERFORMANCE TANGENTIAL FLOW FILTRATION)을 이용하여 제3단계에서 얻은 상층액으로부터 50 ~ 150 kDa 사이의 분자량인 물질만 회수하여 병풀 엑소좀을 얻는 단계;
를 포함하는 것을 특징으로 하는 병풀 엑소좀의 제조방법.(Step 1) Obtaining Centella asiatica stem cell culture medium from Centella asiatica leaves using tumor necrosis factor-α (TNF-α) and salicylic acid as attractors;
(Second step) mixing the Centella asiatica stem cell culture medium and cell lysis buffer, stirring and emulsifying to disrupt the cells to obtain a cell disruption solution;
(Third step) centrifuging the cell lysate to obtain a supernatant; and
(Step 4) Obtaining Centella asiatica exosomes by recovering only substances with a molecular weight between 50 and 150 kDa from the supernatant obtained in the third step using tangential flow filtration (PTFF; PERFORMANCE TANGENTIAL FLOW FILTRATION);
A method for producing Centella asiatica exosomes, comprising:
제1단계에서 병풀줄기세포 배양액은, 병풀 잎으로부터 MS 고체배지 및 액체배지를 이용하여 병풀 식물세포를 배양하고, 유인제(Elicitor)를 첨가 후 추가배양하여 얻는 것을 특징으로 하는 병풀 엑소좀의 제조방법.According to paragraph 1,
In the first step, the Centella asiatica stem cell culture medium is obtained by culturing Centella asiatica plant cells from Centella asiatica leaves using MS solid medium and liquid medium, adding an attractor, and then further culturing. Preparation of Centella asiatica exosomes. method.
상기 병풀 엑소좀은 세포 재생 효능을 갖는 것을 특징으로 하는 화장료 조성물.According to clause 5,
A cosmetic composition characterized in that the Centella asiatica exosomes have a cell regenerative effect.
상기 병풀 엑소좀은 피부 탄력 효능을 갖는 것을 특징으로 하는 화장료 조성물.According to clause 5,
A cosmetic composition characterized in that the Centella asiatica exosomes have a skin elasticity effect.
상기 병풀 엑소좀은 제1형 콜라겐(Collagen Type I)의 발현을 증강시키는 것을 특징으로 하는 화장료 조성물.According to clause 5,
A cosmetic composition characterized in that the Centella asiatica exosomes enhance the expression of collagen type I.
상기 병풀 엑소좀은 제3형 콜라겐(Collagen Type III)의 발현을 증강시키는 것을 특징으로 하는 화장료 조성물.According to clause 5,
A cosmetic composition characterized in that the Centella asiatica exosomes enhance the expression of type 3 collagen (Collagen Type III).
상기 유인제는 TNF-α(tumor necrosis factor-α) 및 살리실산(salicylic acid)이고,
상기 엑소좀은 유인제가 처리된 병풀 줄기세포 배양액의 세포파쇄액으로부터 50 ~ 150 kDa 사이의 분자량인 물질을 회수한 것을 특징으로 하는 화장료 조성물.A cosmetic composition containing exosomes derived from Centella asiatica stem cell culture medium treated with an attractant,
The attractants are tumor necrosis factor-α (TNF-α) and salicylic acid,
The exosome is a cosmetic composition characterized in that a substance with a molecular weight of between 50 and 150 kDa is recovered from the cell lysate of Centella asiatica stem cell culture medium treated with an attractant.
상기 엑소좀은 세포 재생 효능이 있는 것을 특징으로 하는 화장료 조성물. According to clause 10,
The exosome is a cosmetic composition characterized in that it has a cell regenerative effect.
상기 엑소좀은 피부 탄력 효능이 있는 것을 특징으로 하는 화장료 조성물. According to clause 10,
The exosome is a cosmetic composition characterized in that it has a skin elasticity effect.
상기 엑소좀은 제1형 콜라겐(Collagen Type I)의 증강 효능이 있는 것을 특징으로 하는 화장료 조성물.According to clause 10,
The exosome is a cosmetic composition characterized in that it has an enhancing effect on type 1 collagen (Collagen Type I).
상기 엑소좀은 제3형 콜라겐(Collagen Type III)의 증강 효능이 있는 것을 특징으로 하는 화장료 조성물. According to clause 10,
The exosome is a cosmetic composition characterized in that it has an enhancing effect on type 3 collagen (Collagen Type III).
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