KR20150142287A - Culture media compostion for promoting stem cell proliferation comprising plant extracts - Google Patents
Culture media compostion for promoting stem cell proliferation comprising plant extracts Download PDFInfo
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- KR20150142287A KR20150142287A KR1020140070804A KR20140070804A KR20150142287A KR 20150142287 A KR20150142287 A KR 20150142287A KR 1020140070804 A KR1020140070804 A KR 1020140070804A KR 20140070804 A KR20140070804 A KR 20140070804A KR 20150142287 A KR20150142287 A KR 20150142287A
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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Abstract
본 발명은 식물추출물을 포함하는 줄기세포 증식 촉진용 조성물에 관한 것이다. 보다 상세하게는, 아르니카꽃추출물, 겐티아나추출물, 서양톱풀추출물, 쓴쑥추출물, 캐모마일꽃수, 은행잎추출물, 병풀추출물, 마치현추출물 및 상백피추출물, 상기 9종의 식물추출물을 유효성분으로 포함하는 줄기세포 증식 촉진용 배지 조성물, 상기 조성물의 제조방법, 상기 조성물을 이용한 줄기세포의 배양방법 및 상기 방법에 의해 배양되어 수득된 배양물을 유효성분으로 포함하는 피부 상태 개선용 조성물에 관한 것이다. 본 발명의 식물추출물을 유효성분으로 포함하는 줄기세포 배양용 조성물은 고가의 동물성 혈청을 사용하지 않으므로, 제조비용을 현저히 낮출 수 있고, 동물성 혈청을 배지에 사용함으로써 발생되는 동물성 물질의 오염을 원천적으로 차단함으로써 보다 안전한 줄기세포를 제공할 수 있다. 또한, 본 발명의 식물추출물을 유효성분으로 포함하는 조성물을 이용하여 줄기세포를 배양한 배양액은 줄기세포의 증식촉진능 및 활성화능을 가지므로, 피부 주름의 개선, 피부노화 억제, 피부탄력의 개선, 상처 재생 및 피부 재생 효능 등의 피부 상태를 개선할 수 있는 화장료 조성물로 유용하게 사용될 수 있다. The present invention relates to a composition for promoting stem cell proliferation comprising a plant extract. More particularly, the present invention relates to a method for producing stem cell proliferation including arnica flower extract, Gentiana extract, wormwood extract, wortfly extract, chamomile flower, ginkgo biloba extract, centilla extract, A method for producing the composition, a method for culturing stem cells using the composition, and a composition for improving the skin condition comprising the culture obtained by culturing the composition as an active ingredient. Since the composition for culturing stem cells containing the plant extract of the present invention as an active ingredient does not use an expensive animal serum, the production cost can be remarkably lowered and the contamination of the animal material generated by using the animal serum in the medium can be fundamentally Thereby providing safer stem cells. In addition, since the culture solution in which the stem cells are cultured using the composition comprising the plant extract of the present invention as an active ingredient has the proliferation promoting activity and the activating ability of the stem cells, the improvement of the skin wrinkles, the prevention of skin aging, , Skin regeneration, wound regeneration, and skin regeneration efficacy.
Description
본 발명은 식물추출물을 포함하는 줄기세포 증식 촉진용 조성물에 관한 것이다. 보다 상세하게는, 아르니카꽃추출물, 겐티아나추출물, 서양톱풀추출물, 쓴쑥추출물, 캐모마일꽃수, 은행잎추출물, 병풀추출물, 마치현추출물 및 상백피추출물, 상기 9종의 식물추출물을 유효성분으로 포함하는 줄기세포 증식 촉진용 배지 조성물, 상기 조성물의 제조방법, 상기 조성물을 이용한 줄기세포의 배양방법 및 상기 방법에 의해 배양되어 수득된 배양물을 유효성분으로 포함하는 피부 상태 개선용 조성물에 관한 것이다.
The present invention relates to a composition for promoting stem cell proliferation comprising a plant extract. More particularly, the present invention relates to a method for producing stem cell proliferation including arnica flower extract, Gentiana extract, wormwood extract, wortfly extract, chamomile flower, ginkgo biloba extract, centilla extract, A method for producing the composition, a method for culturing stem cells using the composition, and a composition for improving the skin condition comprising the culture obtained by culturing the composition as an active ingredient.
생물의약품의 여러 생산기술 가운데 하나인 동물세포 산업화 기술은 유전자가 조작된 인간 및 동물세포를 대량으로 배양하여 고부가가치의 생물의약품을 생산하는 기술로 각광받고 있으며, 최근 유전자 치료(gene therapy) 및 세포 치료(cell therapy) 기법의 개발로 인해 산업화 기술로서의 중요성이 더욱 강조되고 있다. 일반적으로 동물세포 유래 의약품 생산 공정 개발은 생산 제품의 안전성 확보와 세포 생산성에 의한 높은 생산비의 절감 등에 역점을 두고 있다. Animal cell industrialization technology, which is one of the various production technologies of biologics, is attracting attention as a technology to produce high value-added biologics by cultivating large quantities of genetically engineered human and animal cells. Recently, gene therapy and cell The importance of industrial technology has been emphasized by the development of cell therapy techniques. Generally, the development of pharmaceutical production process based on animal cells is focused on securing the safety of production products and reducing the high production cost by cell productivity.
현재까지 사용되고 있는 동물세포 배양법은 성장을 위하여 반드시 세포배양배지에 혈청을 공급해 주어야 하며 가장 널리 사용되는 혈청으로 FBS(Fetal Bovine Serum)나 FCS(Fetal Calf Serum)가 있다. 이들 첨가 성분의 기능은 명확하진 않지만 성장 증진 작용, 영양분의 공급, 산화나 독소로부터 세포를 보호하는 역할을 하는 것으로 알려져 있으며, 이들 성분이 배제된 경우에는 세포배양 자체가 거의 안 되는 필수 성분이다. In animal cell culture methods used up to now, serum must be supplied to the cell culture medium for growth. FBS (Fetal Bovine Serum) or FCS (Fetal Calf Serum) are the most widely used serum. Although the function of these added components is not clear, it is known that it plays a role of growth promoting action, supply of nutrients, protection of cells from oxidation or toxin, and when these components are excluded, it is an essential ingredient which cell culture itself hardly occurs.
그러나 고가의 혈청은 배지가격을 상승시키고 또한 그 복잡한 성분으로 인해 재조합 단백질의 정제를 어렵게 할 뿐 아니라, 생산되는 과정마다 성분 차이가 있어서 실험을 할 때마다 다른 결과를 초래할 우려가 있다. 또한, 혈청은 동물 유래이기 때문에 배양과정 중에 세포들이 바이러스나 감염성 프리온 등에 오염될 가능성이 상존한다. 소혈청에 감염된 프리온은 사람에게 그대로 전이될 수 있으므로 동물세포 배양은 여러 가지 안전장치를 하고 있음에도 여전히 감염가능성은 존재한다고 해야 할 것이다. However, expensive sera not only raises the price of the medium but also makes it difficult to purify the recombinant protein due to the complex components thereof, and there is a possibility that different results are produced every time the experiment is performed because of the difference in the components in each process. Since the serum is derived from an animal, there is a possibility that the cells are contaminated with viruses and infectious prions during the culturing process. Prions infected with bovine serum may be transferred to humans, so animal cell culture should be said to be susceptible to infection even though it has several safeguards.
통상 혈청 사용시 다음과 같은 문제점을 수반한다: ⅰ) 혈청의 조성 변이 때문에 세포에 대한 성장촉진 능력에 있어서 변이가 크다; ⅱ) 인간에게 사용될 세포 생산물의 제조 시, 혈청이 감염물질에 의한 오염 잠재성을 가지고 있다; ⅲ) 상업적으로 제조된 FBS나 FCS에서 박테리오파아지(bacteriophage)와 보바인 바이러스(bovine virus)가 검출된다; ⅳ) 박테리아 오염 때문에 생긴 각종 독소(toxin)로 인해 세포배양 억제 물질도 함유된다; ⅴ) 여과(filteration) 전에는 오염으로 인한 세균성 독소(bacterial toxin)가 존재한다; ⅵ) 세포배양에 있어 생물학적 오염원(mycoplasmas, bacteriophages, viruses)이 되기도 한다; ⅶ) 감마-글로불린 분획에 배양액과 교차반응(cross reaction)하는 항체 및 세균이 분비한 독소를 함유한다; ⅷ) 열 불활성화(heat inactivation)는 면역글로불린의 세포독성(cytotoxin) 작용을 감소시키지만 불안정한 구성물질까지 파괴되어 열처리하지 않은 혈청보다 만족한 결과가 항상 나타나는 것은 아니다; ⅸ) 혈청 단백질의 흡착/결합에 의한 세포표면의 변형이 유도된다; ⅹ) 세포생산물의 분리 시 방해의 원인이 된다; xi) 1차 배양시에 섬유아세포의 과도한 성장이 조장된다.
The usual use of serum is accompanied by the following problems: i) the variability of the composition of the serum is highly variable in its ability to stimulate growth of cells; Ii) In the production of cell products for use in humans, the sera have a potential for contamination by infectious agents; Iii) Bacteriophage and bovine virus are detected in commercially produced FBS or FCS; Iv) cell culture inhibitors are also contained due to various toxins caused by bacterial contamination; V) There is a bacterial toxin due to contamination prior to filtration; Vi) may be biological contaminants (mycoplasmas, bacteriophages, viruses) in cell culture; Ⅶ) Gamma-globulin fractions contain antibodies and bacterial secreted toxins cross-reacting with the culture medium; Heat inactivation reduces the cytotoxic effects of immunoglobulin, but it does not always result in satisfactory results compared to untreated serum, even to unstable constituents; (Iii) induction of cell surface deformation by adsorption / binding of serum proteins; X) causes disturbances in the separation of cell products; xi) Over-growth of fibroblasts is promoted during primary culture.
1950년대에 합성배지로 자연배지를 부분적으로 대치할 수 있다고 보고된 이후로 혈청 없이 세포배양을 하려는 시도를 하게 되었고, 현재는 특정목적을 위하여 일부의 경우에 혈청없이 세포배양을 하고 있다. 그러나, 이들 무혈청배지는 특정세포만 잘 자라게 하고 혈청 대신에 사용하는 단백질성 세포성장인자 등이 너무 고가인데다 혈청을 포함한 배지에서보다 성장 및 산물생산이 안정적이지 못하다고 보고되고 있다. 또한 무혈청배지는 줄기세포의 배양에는 적용이 불가능한 것으로 알려져 있다. 줄기세포를 상업적으로 이용한 분야에서 가장 중요한 분야가 세포치료제로써 인체에서 얻어진 줄기세포를 시험관에서 배양한 후 다시 환자에게 투입하는 방식의 치료기술이다. 이러한 분야에서는 반드시 줄기세포 배양과정을 거치게 되는데 가능한한 동물성배지나 혈청을 피하는 것이 중요하다. 질병 치료 목적으로 줄기세포를 이식하는 경우 얻는 치료 이득을 고려할 때 이에 따르는 감염 위험은 감수될 수 있으나, 미용목적의 시술인 경우에는 줄기세포 등의 이식으로 얻어지는 이득보다 감염위험에 노출되는 위험이 더 크게 부각될 수 있다. 따라서 줄기세포 배양분야에서 동물성 혈청대체제를 찾는 일은 상업적으로 매우 의미있다.
In the 1950s, it was reported that the natural medium could be partially replaced with the synthetic medium. Since then, it has been attempted to cultivate cells without serum, and now, for certain purposes, cell culture is carried out without serum in some cases. However, these serum-free media have been reported to grow only well on specific cells, and the proteinaceous cell growth factors used in place of serum are too expensive and the growth and production of the products are not stable compared with the medium containing serum. It is also known that serum-free medium can not be applied to the culture of stem cells. The most important field in the field of commercial use of stem cells is a cell therapy agent, which is a therapeutic technique in which a stem cell obtained from a human is cultured in a test tube and then injected into a patient. In such a field, stem cell culturing is necessarily performed, and it is important to avoid animal sex slaughter or serum as much as possible. Considering the therapeutic benefit of transplanting stem cells for the purpose of treating diseases, the risk of infection can be reduced, but in the case of cosmetic procedures, there is a greater risk of exposure to the risk of infection than stem cell transplantation. Can be greatly highlighted. Therefore, finding an animal serum substitute in the field of stem cell culture is commercially meaningful.
한편, 피부에 존재하는 줄기세포는 피부의 건강과 직결된 전체적인 역할을 담당하고 있어 피부건강과 관련하여 가장 중요한 세포군이라 할 수 있다. 줄기세포들은 살아있는 동안 끊임없는 세포분열을 통해 증식과 여러 가지 세포 활성화물질을 분비하여 주위 세포들을 활성화시킴으로써 유해한 환경에 따른 세포들의 노화를 억제하고 문제가 있는 부위를 재생하거나 복구한다고 알려져 있다. 그러나 줄기세포 역시 세포군이기 때문에 나이에 따라 점차 그 기능을 서서히 소실하게 된다. 즉 피부의 줄기세포도 노화에 따라 그 활성이 점차 감소하기 때문에 세포분열능력이 떨어지고 점차 비활성화됨으로써 노화에 따른 주름, 검버섯, 더딘 상처치유, 더딘 복구 등의 여러 문제들이 발생하게 된다.
On the other hand, the stem cells present in the skin are the most important cell groups related to the skin health because they play an overall role directly linked to the health of the skin. Stem cells are known to regenerate and regenerate troubled areas by inhibiting aging of cells due to harmful environment by stimulating proliferation and various cell activating substances by activating the surrounding cells through continuous cell division during living. However, because stem cells are also a cell group, their functions gradually disappear with age. In other words, stem cells of the skin gradually decrease in activity due to aging, so that the cell division ability is inferior and gradually becomes inactive, thereby causing various problems such as wrinkles, black spots, slow wound healing and slow repair due to aging.
이러한 배경 하에서, 본 발명자들은 동물성 혈청을 대신하여 무혈청 배지에서도 줄기세포의 증식을 촉진할 수 있는 물질을 식물성 소재로부터 찾기 위하여 예의 연구 노력한 결과, 아르니카꽃추출물, 겐티아나추출물, 서양톱풀추출물, 쓴쑥추출물, 캐모마일꽃수, 은행잎추출물, 병풀추출물, 마치현추출물 및 상백피추출물, 상기 9종의 식물추출물이 동물성 혈청을 대체 가능한 정도로 줄기세포의 증식을 촉진할 수 있는 효과가 있음을 밝혀냄으로써, 이를 줄기세포 증식 촉진용 배지 조성물에 사용할 수 있음을 확인하여 본 발명을 완성하였다.
Under these circumstances, the present inventors have made intensive researches in order to find a substance capable of promoting the proliferation of stem cells even in a serum-free medium instead of an animal serum from plant materials. As a result, the present inventors have found that the extracts of Arginia flower, Gentiana extract, The present inventors have found that the 9 kinds of plant extracts have an effect of promoting the proliferation of stem cells to the extent that they can replace animal serum, The present invention has been completed upon confirming that it can be used in a promoting medium composition.
본 발명의 하나의 목적은 줄기세포의 증식 촉진 효과를 갖는 식물추출물을 유효성분으로 포함하는 줄기세포 배양용 배지 조성물을 제공하는 것이다.It is an object of the present invention to provide a culture medium for stem cell culture comprising a plant extract having an effect of promoting proliferation of stem cells as an active ingredient.
본 발명의 다른 하나의 목적은 상기 줄기세포 배양용 배지 조성물을 제조하는 방법을 제공하는 것이다.It is another object of the present invention to provide a method for producing the culture medium for culture of stem cells.
본 발명의 또 다른 하나의 목적은 상기 줄기세포 배양용 배지 조성물 내에서 줄기세포를 배양하는 방법을 제공하는 것이다.It is still another object of the present invention to provide a method for culturing stem cells in the culture medium for culturing stem cells.
본 발명의 또 다른 하나의 목적은 상기 방법에 의해 배양되어 수득된 배양물을 유효성분으로 포함하는 피부 상태 개선용 조성물을 제공하는 것이다.
It is still another object of the present invention to provide a composition for improving skin condition comprising the culture obtained by culturing by the above method as an active ingredient.
상기의 목적을 달성하기 위한 하나의 양태로서, 본 발명은 줄기세포의 증식 촉진 효과를 갖는 식물추출물을 유효성분으로 포함하는 줄기세포 배양용 배지 조성물을 제공한다.
In one aspect of the present invention, there is provided a culture medium for stem cell culture comprising a plant extract having an effect of stimulating proliferation of stem cells as an active ingredient.
구체적으로, 상기 식물추출물은 아르니카꽃추출물, 겐티아나추출물, 서양톱풀추출물, 쓴쑥추출물, 캐모마일꽃수, 은행잎추출물, 병풀추출물, 마치현추출물 및 상백피추출물, 총 9종의 식물추출물 중 하나 이상일 수 있으며, 바람직하게는 2종 이상, 3종 이상, 4종 이상, 5종 이상, 6종 이상, 7종 이상, 8종 이상을 포함할 수 있으며, 보다 바람직하게는 9종 모두를 포함할 수 있다.
Specifically, the plant extract may be at least one of the following nine plant extracts: arnica flower extract, gentiana extract, wormwood extract, wortley extract, chamomile flower, ginkgo leaf extract, centipede extract, May include two or more species, three or more species, four or more species, five or more species, six or more species, seven or more species, and eight or more species, and more preferably all nine species.
본 발명에서 용어, "아르니카(Arnica Motanna L.)"는 국화과의 여러해살이풀로, 알프스 고원, 독일 북서부, 북극 등지에 분포하며, 유럽의 민간에서는 옛날부터 꽃과 뿌리줄기를 만능약으로 사용하여 왔다. 한방에서도 협심증의 자극, 혈관확장, 혈관경련 완화, 타박상, 치질 등의 지혈제로 사용되어 온 것으로 알려져 있다. As used herein, the term "Arnica (Arnica Motanna L.) is a perennial plant of the Asteraceae family, distributed on the Alps Plateau, the northwestern part of Germany, the Arctic Circle , etc. In Europe, civilizations have been using flowers and rootstocks as universal drugs. Expansion, vasospasm relief, bruises, hemorrhoids and has been used as a hemostatic agent is known.
본 발명에서 용어, "겐티아나(Gentiana Lutea)"는 용담과에 속하는 다년생의 풀로, 스페인, 남프랑스, 독일, 스위스 등지에 분포하며, 겐티아나의 뿌리는 위의 기능을 촉진하고 또는 이를 조절하는 약제인 건위제로 사용되어 온 것으로 알려져 있다.As used herein, the term "gen tiahna (Gentiana Lutea "is a perennial herb that belongs to the family Yongdam, and is distributed in Spain, South France, Germany, Switzerland, etc. It is known that the roots of Gentiana have been used as a medicine for promoting or controlling the above functions .
본 발명에서 용어, "서양톱풀(Achillea Millefolium L.)"은 국화과의 여러해살이풀로, 유럽 등지에 분포하며, 지친 피부를 진정시키는 효과가 있으며, 소염 작용, 살균 작용 및 지혈 작용이 탁월한 것으로 알려져 있다. In the present invention, the term " Achillea Millefolium L.) "is a perennial plant of Asteraceae, distributed in Europe, has the effect of soothing tired skin, has excellent anti-inflammatory, bactericidal and hemostatic functions.
본 발명에서 용어, "쓴쑥(Artemisia Vulgaris L.)"은 국화과에 속하는 다년생 초본식물로, 한국, 일본 중국 등 동아시아 등지에 분포하며, 진정작용, 혈압강하작용 및 혈액응고작용이 있는 것으로 알려져 있다.In the present invention, the term " Artemisia & Vulgaris L.) "is a perennial herbaceous plant belonging to Asteraceae. It is distributed in Korea, Japan, China, and East Asia, and is known to have sedation, hypotensive effect and blood coagulation.
본 발명에서 용어, "캐모마일(Chamomilla Recutita)"은 국화과의 한해살이풀로, 유럽, 북아프리카, 북아시아 등지에 분포하며, 목욕, 미용, 습포 등에 이용되어져 왔으며, 방충, 진정, 진경, 진통, 발한, 소화촉진, 피로회복 등에 효과가 있는 것으로 알려져 있다.As used herein, the term "Chamomile (Chamomilla Recutita "is an annual plant of Asteraceae. It is distributed in Europe, North Africa and North Asia. It has been used for bath, beauty, and puffing . It is effective for insect, sedation, swallowing, analgesic, sweating, digestion, It is known.
본 발명에서 용어, "은행(Ginkgo Biloba)"은 은행나무과에 속하며, 한국, 일본, 중국 등지에 주로 분포하고 있다. 은행잎에는 플라보노이드, 징코라이트, 피로 피라이트, 엽록소, 비타민E, 비타민 B1, B2, 식물섬유 등의 성분을 함유하고 있는 것으로 알려져 있으며, 은행잎의 유효성분들은 혈관순환개선제로 사용하고 있으며, 말초순환 및 뇌 순환의 혈관 장해를 치료하는데 주로 사용되어져 오고 있다.In the present invention, the term "bank ( Ginkgo Biloba "belongs to Ginkgo biloba and is mainly distributed in Korea, Japan, China etc. Ginkgo leaf contains ingredients such as flavonoid, ginkgo light, pyrophyllite, chlorophyll, vitamin E, vitamin B1, B2, , And the active ingredients of Ginkgo biloba are used as an agent for improving blood circulation and have been mainly used for treating vascular disorders of peripheral circulation and brain circulation.
본 발명에서 용어, "병풀(Centella Asjatica)"는 미나리과에 속하는 여러해살이풀로, 한국, 일본, 중국, 인도, 남아프리카 및 남태평양 등지에 주로 분포하고 있으며, 병풀의 잎과 줄기에 있는 마데카식산이란 성분이 염증을 낫게 하고, 종양과 궤양 등의 상처 치유 효과가 있는 것으로 알려져 있어, 연고나 치약, 화장품 등의 원료로도 많이 쓰이고 있다.In the present invention, the term " Centella Asjatica "is a perennial herb that belongs to the family Mythaceae, and is distributed mainly in Korea, Japan, China, India, South Africa and the South Pacific. The compound of Madeca in the leaves and stalks of the herb remedies inflammation, It is known to have a healing effect on wounds such as ulcers, and is also widely used as a raw material for ointments, toothpaste, and cosmetics.
본 발명에서 용어, "마치현(Portulaca Oleracea L.)"은 쇠비름과의 한해살이풀로, 전 세계적으로 분포하고 있으며, 해열, 해독, 지혈효과가 있어 세균성이질, 종기, 치질, 경부림프절염, 습진, 대하, 자궁출혈, 소변불리 등에 사용하는 것으로 알려져 있으며, 약리작용으로 항균작용, 자궁평활근 수축력 증강으로 인한 장관 연동작용, 이뇨작용 등이 보고되어 있다.As used herein, the term "Portulaca Oleracea (Portulaca Oleracea L.) is an annual plant of the genus Porphyra . It is distributed throughout the world and has a fever, detoxification and hemostatic effect. It is used for bacterial dysentery, swelling, hemorrhoids, cervical lymphadenitis, eczema, subarachnoid hemorrhage, , Antimicrobial activity by pharmacological action, intestinal intercalation activity by diuretic effect of uterine smooth muscle contraction, and diuretic action have been reported.
본 발명에서 용어, "상백피(Morus Alba L.)"는 뽕나무과 식물 뽕나무의 뿌리껍질로, 뽕나무는 한국 및 중국의 대부분의 지역에 분포하고 있으며, 상기 상백피의 약리작용으로는 진해, 이뇨, 혈압강하, 진정, 진통, 해열, 진경, 항균작용 등이 보고되어 있다.
In the present invention, the term " Morus Alba L. "is a root bark of the mulberry tree, and mulberry is distributed in most parts of Korea and China. The pharmacological actions of the mulberry are as follows: Jinhae, diuretic, hypotension, sedation, analgesia, And antimicrobial action have been reported.
본 발명의 줄기세포 배양용 배지 조성물은 상기와 같은 9종의 식물 추출물 중 하나 이상의 식물 추출물을 유효 성분으로 포함함으로써, 줄기세포의 증식을 촉진시키는 효과를 달성할 수 있다.
The culture medium for stem cell culture of the present invention can achieve an effect of promoting the proliferation of stem cells by containing at least one plant extract of the above nine plant extracts as an effective ingredient.
바람직하게는, 본 발명의 줄기세포 배양용 배지 조성물에는 상기 9종의 식물 추출물이 모두 포함될 수 있다. 이 때, 각 식물 추출물 간의 비율은 줄기세포 증식 촉진 활성을 유지할 수 있는 한 별다른 제한은 없다. 그러나, 바람직하게는 아르니카꽃추출물 1 중량부에 대하여, 겐티아나추출물 0.3 중량부 내지 3 중량부, 서양톱풀추출물 0.3 중량부 내지 3 중량부, 쓴쑥추출물 0.3 중량부 내지 3 중량부, 캐모마일꽃수 1 중량부 내지 10 중량부, 은행잎추출물 0.4 중량부 내지 4 중량부, 병풀추출물 1 중량부 내지 10 중량부, 마치현추출물 0.4 중량부 내지 4 중량부, 및 상백피추출물 0.5 중량부 내지 5 중량부의 비율로 포함될 수 있다. 보다 바람직하게는, 아르니카꽃추출물 1 중량부에 대하여, 겐티아나추출물 1 중량부, 서양톱풀추출물 1 중량부, 쓴쑥추출물 1 중량부, 캐모마일꽃수 6 중량부, 은행잎추출물 1.2 중량부, 병풀추출물 6 중량부, 마치현추출물 1.2 중량부, 및 상백피추출물 1.6 중량부의 비율로 혼합할 수 있다. Preferably, the culture medium for stem cell culture of the present invention may contain all of the above nine plant extracts. At this time, the ratio between the plant extracts is not limited as long as it can maintain the stem cell proliferation promoting activity. Preferably, 0.3 part by weight to 3 parts by weight of Gentiana extract, 0.3 part by weight to 3 parts by weight of wormwood extract, 0.3 to 3 parts by weight of wortfly extract, 3 parts by weight of Chamomile flower, 1 part by weight , 0.4 to 4 parts by weight of Ginkgo biloba extract, 1 to 10 parts by weight of Centella asiatica extract, 0.4 to 4 parts by weight of Aspergillus oryzae extract, and 0.5 to 5 parts by weight of Eucalyptus root extract have. More preferably, 1 part by weight of ganoderma extract, 1 part by weight of wormwood extract, 1 part by weight of wortfly extract, 6 parts by weight of chamomile flower, 1.2 parts by weight of ginkgo leaf extract, 1.2 parts by weight of the extract, 1.2% by weight of the extract, and 1.6% by weight of the extract of the extract of Mulberry bark.
또한, 상기와 같이 제조된 혼합 식물추출물이 배양용 배지 전체 조성물에 첨가되는 비율은 이에 제한되지는 않으나, 바람직하게는 상기 배양용 배지 전체 조성물에 대해 0.1 내지 10 중량%로 첨가할 수 있으며, 보다 바람직하게는 1 내지 3 중량%로 첨가하는 것일 수 있으며, 보다 더 바람직하게는 2 중량%로 첨가하는 것일 수 있다. In addition, the proportion of the mixed plant extracts prepared as described above added to the entire culture medium composition is not limited thereto, but it is preferably 0.1 to 10% by weight based on the total composition of the culture medium. Preferably 1 to 3% by weight, and even more preferably 2% by weight.
아울러, 본 발명의 혼합 식물추출물의 농도는 바람직하게는 5% 내지 15 중량%, 보다 바람직하게는 10중량% 일 수 있으며, 그에 따라 최종적으로 유효성분인 식물추출물은 바람직하게는 배양용 배지 전체 조성물 성분 중 0.01 내지 1 중량%로 함유될 수 있으며, 보다 바람직하게는 0.1 내지 0.3 중량%, 보다 더 바람직하게는 0.2 중량%로 함유되는 것일 수 있다. In addition, the concentration of the mixed plant extract of the present invention may be preferably 5% to 15% by weight, more preferably 10% by weight, so that the plant extract as the final effective ingredient is preferably the whole composition of the culture medium May be contained in an amount of 0.01 to 1% by weight, more preferably 0.1 to 0.3% by weight, and even more preferably 0.2% by weight.
한편, 전술한 바와 같이 혼합 식물추출물을 제조한 후 배양용 배지 조성물에 첨가하는 방법 외에, 각각의 식물 추출물을 배양용 배지 조성물에 개별적으로 동시에 첨가할 수 있다.
On the other hand, in addition to the method of preparing a mixed plant extract as described above and then adding it to the culture medium composition, each plant extract may be added to the culture medium composition separately at the same time.
본 발명에서 용어, "추출물(extract)"은 생약을 적절한 침출액으로 짜내고 침출액을 증발시켜 농축한 제제를 의미하는 것으로, 이에 제한되지는 않으나, 추출처리에 의해 얻어지는 추출액, 추출액의 희석액 또는 농축액, 추출액을 건조하여 얻어지는 건조물, 이들의 조정제물 또는 정제물일 수 있다. 상기 혼합 약재 추출물은 당업계에 공지된 일반적인 추출방법, 분리 및 정제방법을 이용하여 제조할 수 있다. 상기 추출방법으로는, 이에 제한되지는 않으나, 바람직하게 열탕 추출, 열수 추출, 냉침 추출, 환류 냉각 추출 또는 초음파 추출 등의 방법을 사용할 수 있다.The term "extract" in the present invention means a preparation which is prepared by squeezing a herbal medicine with an appropriate extract solution and evaporating the extract solution to obtain a concentrate. The extract solution obtained by the extraction treatment, the diluent or concentrate of the extract, Or a dried product obtained by drying the above, a controlled preparation thereof or a purified product thereof. The mixed medicinal plant extract can be prepared by using common extraction, isolation and purification methods known in the art. The extraction method may be, but not limited to, hot water extraction, hot water extraction, cold extraction, reflux cooling extraction, or ultrasonic extraction.
본 발명에 있어서, 상기 추출물은 추출용매로 추출하거나 추출용매로 추출하여 제조한 추출물에 분획용매를 가하여 분획함으로써 제조할 수 있다. 상기 추출용매는 이에 제한되지 않으나, 물, 유기용매 또는 이들의 혼합용매 등을 사용할 수 있으며, 상기 유기용매는 탄소수 1 내지 4의 알코올이나, 에틸아세테이트 또는 아세톤 등의 극성용매, 헥산 또는 디크로로메탄의 비극성용매 또는 이들의 혼합용매를 사용할 수 있다.
In the present invention, the extract may be prepared by extracting with an extraction solvent or extracting with an extraction solvent, followed by fractionation with a fraction solvent. The organic solvent may be an alcohol having 1 to 4 carbon atoms, a polar solvent such as ethyl acetate or acetone, a solvent such as hexane or dichloro, or an organic solvent such as dichloromethane, Methane, or a mixed solvent thereof may be used.
또한, 상기 조성물은 양수 내 태아 유래 중간엽 줄기세포를 배양한 컨디션드 배지를 추가로 포함할 수 있다. 상기 컨디션드 배지 역시 첨가 비율은 제한이 없으나, 상기 배양용 배지 전체 조성물에 대해 상기 컨디션드 배지를 0.1 내지 10 중량%로 첨가할 수 있으며, 보다 바람직하게는 0.5 내지 5 중량%로 첨가하는 것일 수 있으며, 보다 더 바람직하게는 1 내지 3 중량%로 첨가하는 것일 수 있으며, 가장 바람직하게는 2 중량%로 첨가하는 것일 수 있다. In addition, the composition may further comprise conditioned medium in which fetal-derived mesenchymal stem cells in an amniotic fluid are cultured. The conditional medium may also be added in an amount of 0.1 to 10% by weight, more preferably 0.5 to 5% by weight, based on the total composition of the culture medium. More preferably 1 to 3% by weight, and most preferably 2% by weight.
또한, 전술한 바와 같이 상기 조성물에 추가로 포함될 수 있는 컨디션드 배지의 농도는 1 내지 10% 일 수 있으며, 바람직하게는 4 내지 6% 일 수 있으며, 보다 바람직하게는 5% 일 수 있다. In addition, as described above, the concentration of the conditional medium which can additionally be contained in the composition may be 1 to 10%, preferably 4 to 6%, and more preferably 5%.
그에 따라, 배양용 배지 전체 조성물에 첨가되어, 바람직하게는 배양용 배지 전체 조성물 성분 중 0.01 내지 1 중량%로 함유될 수 있으며, 보다 바람직하게는 0.05 내지 0.25 중량%, 보다 더 바람직하게는 0.1 중량%로 함유되는 것일 수 있다.
Therefore, the whole culture medium can be added to the whole composition, preferably 0.01 to 1% by weight, more preferably 0.05 to 0.25% by weight, and even more preferably 0.1% by weight %. ≪ / RTI >
본 발명의 "양수 내 태아 유래 중간엽 줄기세포를 배양한 컨디션드 배지(Amniotic Fluid Stem Cell Conditioned Media, AFSCM)"는, 인 비트로(in vitro) 상에서 양수 내 태아 유래 세포들의 성장 및 생존을 지지할 수 있게 하는 배지로서, Apo-1/Fas, 표피세포 성장인자(EGF), IP-10, 렙틴(Leptin), MIP4, MMP3, 란테스(Rantes), 인터페론-감마(IFN-γ), 인간형질전환 성장인자-베타(TGF-β), 종양괴사인자-알파(TNF-α), 종양괴사인자 수용체 Ⅰ(TNFRⅠ), 종양괴사인자 수용체Ⅱ(TNFRⅡ), 세포부착인자-1(ICAM-1), 혈관세포 부착인자-1(VCAM-1), 맥관내피세포 성장인자(VEGF), 인터루킨-1베타(IL-1β), 인터루킨-1 수용체 알파(IL-1Rα), IL-2, IL-3, IL-4, IL-5, IL-6, IL-6R, IL-7, IL-8, IL-12 및 IL-15를 포함하는 것일 수 있다.
"Condition cultured amniotic fetal-derived MSCs de medium (Amniotic Fluid Stem Cell Conditioned Media, AFSCM)" of the present invention, in vitro (in vitro) on a culture medium able to support the growth and survival of amniotic fetal derived cells, Apo-1 / Fas, epidermal growth factor (EGF), IP-10, leptin (Leptin), MIP4, MMP3, is tested (RANTES), interferon-gamma (IFN-y), human transforming growth factor-beta (TGF-beta), tumor necrosis factor- alpha (TNF- alpha), tumor necrosis factor receptor I (TNFR I), tumor necrosis factor receptor (ICAM-1), vascular cell adhesion factor-1 (VCAM-1), vascular endothelial growth factor (VEGF), interleukin-1 beta (IL- (IL-1R), IL-2, IL-3, IL-4, IL-5, IL-6, IL-6R, IL-7, IL-8, IL-12 and IL- Lt; / RTI >
본 발명에서 용어, "줄기세포(stem cell)"란 적합한 환경 및 자극을 통해 각종 세포로 분화할 수 있는 능력을 갖추고 있으며, 자가증식 능력을 갖추고 있는 세포이다. In the present invention, the term "stem cell" is a cell having an ability to differentiate into various cells through suitable environment and stimulation, and having a self-propagating ability.
본 발명의 조성물에 의해 증식이 촉진되어진 대상 줄기세포는 특정 줄기세포로 한정되지 않으며, 바람직하게는 중간엽 줄기세포일 수 있다. 보다 바람직하게는 상기 중간엽 줄기세포는 양수 내 태아 유래 중간엽 줄기세포일 수 있으나, 이에 제한되지는 않는다. Stem cells to which proliferation is promoted by the composition of the present invention are not limited to specific stem cells, and may be preferably mesenchymal stem cells. More preferably, the mesenchymal stem cells may be amniotic mesenchymal stem cells in amniotic fluid, but are not limited thereto.
본 발명에서 용어, "중간엽 줄기세포(mesenchymal stem cell; MSCs)"란 연골, 뼈, 지방, 골수간질, 근육, 신경 등을 만드는데 원조가 되는 세포로서, 성인에서는 일반적으로 골수에 머물러 있지만 제대혈, 말초혈액, 기타 조직 등에도 존재하며, 이들로부터 수득할 수 있는 세포이며, 본 발명의 "양수 내 태아 유래 중간엽줄기세포"는 산모로부터 얻은 양수에서 분리한 중간엽 줄기세포를 의미한다.The term "mesenchymal stem cells (MSCs)" in the present invention refers to cells that help to produce cartilage, bone, fat, bone marrow stroma, muscles and nerves. Peripheral blood, other tissues, etc., and can be obtained from them. "Amniotic fluid fetal-derived mesenchymal stem cells" of the present invention means mesenchymal stem cells isolated from amniotic fluid obtained from a mother.
본 발명에서 용어, "배지(culture media)"란 인 비트로(in vitro) 상에서 줄기세포들의 성장 및 생존을 지지할 수 있게 하는 배지로서, 줄기세포 배양에 적절한 당 분야에서 사용되는 통상의 배지를 모두 포함한다. 세포의 종류에 따라 배지와 배양 조건을 선택할 수 있다. 배양에 사용되는 배지는 바람직하게는 세포 배양 최소 배지(cell culture minimum medium; CCMM)로, 일반적으로 탄소원, 질소원 및 미량원소 성분을 포함한다. 이런 세포 배양 최소 배지에는 예를 들어, DMEM(Dulbecco's Modified Eagle's Medium), MEM(Minimal essential Medium), BME(Basal Medium Eagle), RPMI1640, F-10, F-12, αMEM(α Minimal essential Medium), GMEM(Glasgow's Minimal essential Medium) 및 IMEM(Iscove's Modified Dulbecco's Medium) 등이 있으나, 이에 제한되지는 않는다. 또한 상기 배지는 페니실린(penicillin), 스트렙토마이신(streptomycin) 또는 겐타마이신(gentamicin) 등의 항생제를 포함할 수 있다.
The term "culture medium (culture media)" in the present invention is in vitro (in The present invention also relates to a culture medium for supporting the growth and survival of stem cells on a medium, such as a human, Depending on the type of cells, medium and culture conditions can be selected. The medium used for the culture is preferably a cell culture minimum medium (CCMM), and generally includes a carbon source, a nitrogen source and a trace element component. For example, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, But are not limited to, GMEM (Glasgow's Minimal Essential Medium) and IMEM (Iscove's Modified Dulbecco's Medium). The medium may also contain antibiotics such as penicillin, streptomycin or gentamicin.
본 발명의 일 실시예에 의하면, 상기 식물추출물을 유효성분으로 함유하는 배지 조성물을 양수 내 태아 유래 중간엽 줄기세포에 처리하고 세포 증식력에 미치는 영향을 확인하였다.According to one embodiment of the present invention, the medium composition containing the plant extract as an active ingredient was treated with fetal-derived mesenchymal stem cells in amniotic fluid and the effect on cell proliferation was examined.
먼저 상기 조성물의 세포 증식력에 대한 효과를 확인하기 위해, 양수 내 태아 유래 중간엽 줄기세포에 처리한 결과, 성장인자로서 소태아혈청(Fetal Bovine Serum; FBS)을 첨가 또는 미첨가한 대조군에 비해, 줄기 세포의 증식을 촉진시킴을 확인하였다(도 1). 또한, 컨디션드 배지와 식물추출물을 동시에 유효성분으로 함유한 배지 조성물을 상기 줄기세포에 처리하여 세포 증식 정도를 분석해 본 결과, 성장인자로서 소태아혈청(Fetal Bovine Serum; FBS)을 첨가 또는 미첨가한 대조군과, 컨디션드 배지만을 유효성분으로 함유한 배지 조성물을 처리한 실험군에 비해, 컨드션드 배지와 식물추출물을 동시에 첨가한 실험군의 세포 증식이 현저하게 증가함을 확인하였다(도 2). 아울러, 컨디션드 배지와 식물추출물을 동시에 유효성분으로 함유한 배지 조성물을 상기 줄기세포에 처리하고, 배양 시간을 각각 24시간 및 72시간으로 달리하여, 세포 증식 정도를 분석해 본 결과, 24시간 동안 배양한 실험군에 비해 72시간 동안 배양한 실험군의 세포 증식이 현저하게 활성화되었음을 알 수 있었다(도 3).In order to confirm the effect of the composition on the cell proliferation ability, as compared with the control group in which fetal-derived mesenchymal stem cells in amniotic fluid were treated with Fetal Bovine Serum (FBS) as a growth factor, Promoting the proliferation of stem cells (Fig. 1). In addition, when the stem cell was treated with a medium composition containing the conditioned medium and the plant extract as an active ingredient, the degree of cell proliferation was analyzed. As a result, Fetal Bovine Serum (FBS) The cell proliferation of the experimental group to which the conditioned medium and the plant extract were simultaneously added was remarkably increased (FIG. 2), as compared to the control group and the medium group containing the conditioned medium containing the active ingredient. In addition, the stem cell was treated with a medium composition containing the conditioned medium and the plant extract as active ingredients, and the degree of cell proliferation was analyzed by varying the culture time for 24 hours and 72 hours, respectively. As a result, The cell proliferation of the experimental group cultured for 72 hours was remarkably activated compared to that of one experimental group (FIG. 3).
이를 통해, 본 발명의 조성물은 상기 9종의 식물추출물을 유효성분으로 함유함에 의해, 세포 증식 촉진에 있어서 우수한 효과를 가질 뿐만 아니라, 상기 9종의 식물추출물과 컨드션드 배지가 동시에 첨가될 경우, 세포 증식 촉진에 있어서 시너지 효과를 나타냄을 알 수 있었다.
Accordingly, the composition of the present invention has excellent effects in promoting cell proliferation by containing the above-mentioned 9 kinds of plant extracts as active ingredients, and when the 9 kinds of plant extracts and the conditional medium are simultaneously added, Indicating synergistic effects in promoting cell proliferation.
다른 하나의 양태로서, 본 발명은 상기 식물추출물을 유효성분으로 포함하는 줄기세포 배양용 배지 조성물을 제조하는 방법을 제공한다.
In another aspect, the present invention provides a method for producing a culture medium for stem cell culture comprising the plant extract as an active ingredient.
구체적으로, 본 발명의 제조 방법은 아르니카꽃추출물, 겐티아나추출물, 서양톱풀추출물, 쓴쑥추출물, 캐모마일꽃수, 은행잎추출물, 병풀추출물, 마치현추출물 및 상백피추출물로 구성된 군으로부터 선택된 하나 이상의 식물추출물을 배지에 첨가하는 단계를 포함하는, 줄기세포 배양용 배지 조성물의 제조방법일 수 있다.
Specifically, the production method of the present invention is a method for producing a plant extract comprising at least one plant extract selected from the group consisting of arnica flower extract, gentian extract, wormwood extract, wortley extract, chamomile flower, ginkgo biloba extract, To a culture medium for stem cell culture.
본 발명의 일 실시예에 의하면, 아르니카꽃추출물 1 중량부에 대하여, 겐티아나추출물 1 중량부, 서양톱풀추출물 1 중량부, 쓴쑥추출물 1 중량부, 캐모마일꽃수 6 중량부, 은행잎추출물 1.2 중량부, 병풀추출물 6 중량부, 마치현추출물 1.2 중량부, 및 상백피추출물 1.6 중량부의 비율로 혼합한 혼합 식물추출물을 제조하는 단계; 및 상기 혼합 식물추출물을 전체 배양용 배지 조성물에 대해 0.1 내지 10 중량%로 첨가하여 줄기세포 배양용 배지 조성물을 제조하였다.According to one embodiment of the present invention, 1 part by weight of gentiana extract, 1 part by weight of wormwood extract, 1 part by weight of wortfly extract, 6 parts by weight of chamomile flower, 1.2 parts by weight of ginkgo leaf extract, 6 parts by weight of a centilla sp. Extract, 1.2 parts by weight of a horseradish extract, and 1.6 parts by weight of an extract of a perennial herb extract. And 0.1 to 10% by weight of the mixed plant extract to the whole culture medium composition was added to prepare a culture medium for stem cell culture.
또한, 본 발명의 제조 방법은 전술한 바와 같이 혼합 식물추출물을 제조하는 단계; 상기 복합 식물추출물 1 중량부에 대하여 양수 내 태아 유래 중간엽 줄기세포를 배양한 컨디션드 배지를 0.5 중량부의 비율로 혼합하여 혼합조성물을 제조하는 단계; 및 상기 혼합조성물을 배양용 배지 전체 조성물에 대해 0.1 내지 10 중량%로 첨가하는 단계를 포함하는, 줄기세포 배양용 배지 조성물의 제조방법일 수 있다.
In addition, the method of the present invention comprises the steps of: preparing a mixed plant extract as described above; Preparing a mixed composition by mixing 0.5 part by weight of a conditioned medium prepared by culturing fetal-derived mesenchymal stem cells in an amniotic fluid on 1 part by weight of the complex plant extract; And adding the mixed composition to the entire culture medium for culture at 0.1 to 10% by weight.
또 다른 하나의 양태로서, 본 발명은 상기 식물추출물을 유효성분으로 포함하는 줄기세포 배양용 배지 조성물 내에서 줄기세포를 배양하는 방법을 제공한다.
In another aspect, the present invention provides a method for culturing a stem cell in a culture medium for culturing a stem cell comprising the plant extract as an active ingredient.
본 발명의 일 실시예에 의하면, 본 발명에서 제조된 상기 줄기세포 배양용 배지에 줄기세포를 분주하여 37, 5% CO2 인큐베이터에서 호기성(aerobic) 또는 저산소(hypoxice) 배양하여 줄기세포를 배양하였다.According to one embodiment of the present invention, stem cells are cultured in an aerobic or hypoxic culture medium in a 37, 5% CO 2 incubator in the medium for culturing stem cells prepared in the present invention .
또한, 본 발명의 제조 방법은 전술한 바와 같이 혼합 식물추출물을 제조하는 단계; 상기 혼합 식물추출물 1 중량부에 대하여 양수 내 태아 유래 중간엽 줄기세포를 배양한 컨디션드 배지를 0.5 중량부의 비율로 혼합하여 혼합조성물을 제조하는 단계; 상기 혼합조성물을 배양용 배지 전체 조성물에 대해 0.1 내지 10 중량%로 첨가하여 줄기세포 배양용 배지를 제조하는 단계; 및 상기와 같이 제조된 배양용 배지에 줄기세포를 분주하여 37℃, 5% CO2 인큐베이터에서 호기성(aerobic) 또는 저산소(hypoxice) 배양하는 단계를 포함하는, 줄기세포를 배양하는 방법일 수 있다.
In addition, the method of the present invention comprises the steps of: preparing a mixed plant extract as described above; Preparing a mixed composition by mixing 0.5 part by weight of conditioned medium prepared by culturing fetal-derived mesenchymal stem cells in amniotic fluid for 1 part by weight of the mixed plant extract; Preparing a culture medium for stem cell culture by adding the mixed composition to the whole composition for culture medium in an amount of 0.1 to 10% by weight; And the stem cells were dispensed into the culture medium prepared as described above and cultured at 37 ° C in 5% CO 2 Culturing an embryonic stem cell in an incubator in an aerobic or hypoxic culture.
또 다른 하나의 양태로서, 본 발명은 상기 식물추출물을 포함하는 조성물 내에서 줄기세포를 배양하는 방법에 의해 배양되어 수득된 배양물을 유효성분으로 포함하는 피부 상태 개선용 조성물을 제공한다.
In another aspect, the present invention provides a composition for improving skin condition comprising a culture obtained by culturing stem cells in a composition containing the plant extract as an active ingredient.
본 발명에서 용어, "피부 상태"는 주름, 자외선에 의한 피부 손상, 피부 탄력, 모공 확장 또는 피부 흉터 등을 포함한다.
The term "skin condition" in the present invention includes wrinkles, skin damage due to ultraviolet rays, skin elasticity, pore enlargement or skin scarring.
본 발명의 상기 피부 상태 개선용 조성물은 피부에 직접 적용되는 화장료 조성물로 제공될 수 있다. The skin condition improving composition of the present invention can be provided as a cosmetic composition directly applied to the skin.
본 발명의 화장료 조성물은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 예를 들어, 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성제-함유 클린싱, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이 등으로 제형화될 수 있으나, 이에 한정되는 것은 아니다. 보다 상세하게는, 유연 화장수, 영양 화장수, 영양 크림, 마사지 크림, 에센스, 아이 크림, 클렌징 크림, 클렌징 포옴, 클렌징 워터, 팩, 스프레이 또는 파우더의 제형으로 제조될 수 있다.
The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used as a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.
본 발명의 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물성유, 식물성유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 이용될 수 있다.
When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracer, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .
본 발명의 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.
When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, / Propane or dimethyl ether.
본 발명의 제형이 용액 또는 유탁액인 경우에는 담체 성분으로서 용매, 용해화제 또는 유탁화제가 이용되고, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌 글리콜, 1,3-부틸글리콜 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르가 있다.
When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.
본 발명의 제형이 현탁액인 경우에는 담체 성분으로서 물, 에탄올 또는 프로필렌 글리콜과 같은 액상의 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가 또는 트라칸트 등이 이용될 수 있다.
In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.
본 발명의 제형이 계면-활성제 함유 클린징인 경우에는 담체 성분으로서 지방족 알코올 설페이트, 지방족 알코올 에테르 설페이트, 설포숙신산 모노에스테르, 이세티오네이트, 이미다졸리늄 유도체, 메틸타우레이트, 사르코시네이트, 지방산 아미드 에테르 설페이트, 알킬아미도베타인, 지방족 알코올, 지방산 글리세리드, 지방산 디에탄올아미드, 식물성 유, 라놀린 유도체 또는 에톡실화 글리세롤 지방산 에스테르 등이 이용될 수 있다.
When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.
본 발명의 화장품 조성물에 포함되는 성분은 유효 성분과 담체 성분 이외에, 화장품 조성물에 통상적으로 이용되는 성분들을 포함하며, 예컨대 항산화제, 안정화제, 용해화제, 비타민, 안료 및 향료와 같은 통상적인 보조제를 포함할 수 있다.
The ingredients contained in the cosmetic composition of the present invention include, in addition to the active ingredient and the carrier ingredient, the ingredients conventionally used in cosmetic compositions and include conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, .
본 발명의 식물추출물을 유효성분으로 포함하는 줄기세포 배양용 조성물은 고가의 동물성 혈청을 사용하지 않으므로, 제조비용을 현저히 낮출 수 있고, 동물성 혈청을 배지에 사용함으로써 발생되는 동물성 물질의 오염을 원천적으로 차단함으로써 보다 안전한 줄기세포를 제공할 수 있다. 또한, 본 발명의 식물추출물을 유효성분으로 포함하는 조성물을 이용하여 줄기세포를 배양한 배양액은 줄기세포의 증식촉진능 및 활성화능을 가지므로, 피부 주름의 개선, 피부노화 억제, 피부탄력의 개선, 상처 재생 및 피부 재생 효능 등의 피부 상태를 개선할 수 있는 화장료 조성물로 유용하게 사용될 수 있다.
Since the composition for culturing stem cells containing the plant extract of the present invention as an active ingredient does not use an expensive animal serum, the production cost can be remarkably lowered and the contamination of the animal material generated by using the animal serum in the medium can be fundamentally Thereby providing safer stem cells. In addition, since the culture solution in which the stem cells are cultured using the composition comprising the plant extract of the present invention as an active ingredient has the proliferation promoting activity and the activating ability of the stem cells, the improvement of the skin wrinkles, the prevention of skin aging, , Skin regeneration, wound regeneration, and skin regeneration efficacy.
도 1은, 식물추출물의 처리에 따른, 양수 내 태아 유래 중간엽 줄기세포의 세포증식에 미치는 영향을 분석한 결과를 나타낸 그래프이다.
도 2는, 양수 내 태아 유래 중간엽 줄기세포의 컨디션드 배지(Amniotic Fluid Stem Cell Conditioned Media; AFSCM)와 식물추출물의 혼합조성물 처리에 따른, 양수 내 태아 유래 중간엽 줄기세포의 세포증식에 미치는 영향을 분석한 결과를 나타낸 그래프이다.
도 3은, 양수 내 태아 유래 중간엽 줄기세포의 컨디션드 배지(Amniotic Fluid Stem Cell Conditioned Media; AFSCM)와 식물추출물의 혼합조성물 처리에 따른, 양수 내 태아 유래 중간엽 줄기세포의 세포증식에 미치는 영향을, 1일(24시간) 및 3일(72시간)에 걸쳐 분석한 결과를 나타낸 그래프이다.Fig. 1 is a graph showing the results of analysis of the effect of amniotic fluid on the cell proliferation of embryo-derived mesenchymal stem cells after treatment with plant extracts.
FIG. 2 shows the effect of amniotic fluid stem cell conditioned media (AFSCM) and plant extract on the cell proliferation of embryo-derived mesenchymal stem cells in amniotic fluid after amniotic fluid embryo-derived mesenchymal stem cells FIG.
Figure 3 shows the effect of amniotic fluid stem cell conditioned media (AFSCM) and plant extracts on the cell proliferation of embryo-derived mesenchymal stem cells in amniotic fluid after treatment of embryo-derived mesenchymal stem cells in amniotic fluid (24 hours) and 3 days (72 hours), respectively.
이하, 실시예를 통하여 본 발명의 구성 및 효과를 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것일 뿐, 본 발명의 범위가 이들 실시예에 의해 한정되는 것은 아니다.
Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.
실시예Example 1: 줄기세포 증식 촉진용 혼합 식물추출물 및 혼합조성물의 제조 1: Preparation of mixed plant extracts and mixed compositions for promoting stem cell proliferation
줄기세포의 증식을 촉진하는 배양용 배지 조성물을 제조하기 위하여, 9종의 식물추출물을 혼합한 혼합 식물추출물, 그리고 상기 혼합 식물추출물과 양수 내 태아 유래 중간엽 줄기세포를 배양한 컨디션드 배지(Amniotic Fluid Stem Cell Conditioned Media, AFSCM)를 일정 비율로 혼합하여 혼합조성물을 제조하였다.
In order to prepare a culture medium composition for promoting the proliferation of stem cells, mixed plant extracts obtained by mixing 9 kinds of plant extracts, and conditioned mediums obtained by culturing the mixed plant extracts and amniotic fluid fetal-derived mesenchymal stem cells (Amniotic Fluid Stem Cell Conditioned Media, AFSCM) were mixed at a certain ratio to prepare a mixed composition.
실시예Example 1-1: 줄기세포 증식 촉진용 혼합 식물추출물의 제조 1-1: Preparation of mixed plant extracts for promoting stem cell proliferation
줄기세포의 증식을 촉진하는 배양용 배지 조성물을 제조하기 위하여, 9종의 식물추출물을 일정 비율로 혼합하여 혼합 식물추출물을 제조하였다.In order to prepare a culture medium composition for promoting the proliferation of stem cells, 9 kinds of plant extracts were mixed at a certain ratio to prepare a mixed plant extract.
구체적으로, 하기 표 1에 기재된 바와 같이, 아르니카꽃추출물 0.5 중량%, 겐티아나추출물 0.5 중량%, 서양톱풀추출물 0.5 중량%, 쓴쑥추출물 0.5 중량%, 캐모마일꽃수 3.0 중량%, 은행잎추출물 0.6 중량%, 병풀추출물 3.0 중량%, 마치현추출물 0.6 중량%, 및 상백피추출물 0.8 중량%로 상기 9종의 식물추출물을 혼합하여 최종 농도 10 중량%의 혼합 식물추출물을 제조하였다.
Specifically, as shown in the following Table 1, 0.5% by weight of arnica flower extract, 0.5% by weight of Gentiana extract, 0.5% by weight of wormwood extract, 0.5% by weight of wortfly extract, 3.0% by weight of chamomile flower, The nine plant extracts were mixed with 3.0% by weight of Centella asiatica extract, 0.6% by weight of Aspergillus oryzae and 0.8% by weight of Aspergillus oryzae as an extract to prepare a mixed plant extract having a final concentration of 10% by weight.
실시예Example 1-2: 양수 내 태아 유래 1-2: Amniotic fluid fetal birth 중간엽Intermediate lobe 줄기세포에서 In stem cells 컨디션드Conditioned 배지( badge( AmnioticAmniotic FluidFluid StemStem CellCell ConditionedConditioned MediaMedia , , AFSCMAFSCM )의 제조)
줄기세포의 증식을 촉진하는 배양용 배지 조성물을 제조하기 위하여, 양수 내 태아 유래 중간엽 줄기세포를 배양하여, 표피세포 성장인자(EGF), 인터페론-감마(IFN-γ), 인간형질전환 성장인자-베타(TGF-β), 맥관내피세포 성장인자(VEGF), 세포부착인자-1(ICAM-1) 등 인간 성장촉진인자를 포함하고 있는 컨디션드 배지를 수득하였다.In order to prepare a culture medium composition for promoting the proliferation of stem cells, embryo-derived mesenchymal stem cells in amniotic fluid were cultured to prepare epidermal growth factor (EGF), interferon-gamma (IFN-γ) (TGF-beta), vascular endothelial growth factor (VEGF), and cell adhesion factor-1 (ICAM-1).
구체적으로, 세포은행에 보관중인, 1 × 106개의 양수 내 태아 유래 중간엽 줄기세포(Amniotic Fluid Stem Cell, AFSCs)를 해동하여 T75 플라스크에 시딩하고, 10% 소태아혈청(fetal bovine serum, FBS)과 100 units/ml 농도의 페니실린 및 100 ug/ml 농도의 스트렙토마이신 항생제가 함유된 DMEM(low glucose) 배지를 사용하여 배양하였다. 상기 세포는 37, 5% CO2 인큐베이터에서 배양하였으며, 시딩하고 24시간 후 배지를 교환하였다. 이후 24시간 내지 48시간 간격으로 배지를 교환하였으며, 70 내지 90%의 컨플루언시(confluency)에 도달하면 계대배양(subculture)을 진행하였다. 각 계대(passage)당 3 내지 4일이 소요되었으며, 4번째 계대까지 증식 배양을 진행하였다. 이때 배양 용기는 T75 플라스크에서 T175 플라스크로 바꾸었으며, 이후 트리플(triple) 플라스크를 사용하여 배양하였다. 상기와 같이 4번째 계대 배양이 진행된 세포의 컨플루언시가 70 내지 90%에 도달하면 0.05% 트립신-EDTA(trypsin-EDTA)를 처리하여 부착세포를 떼어내고, 인산염완충액(Phosphate-buffered saline, PBS)으로 3회 세척하여 혈청 배지 성분 및 항생제 성분을 제거하였다. 이후, 5번째 계대 단계에서는 트리플 플라스크의 cm2 면적당 8,000 내지 20,000개의 세포수로 시딩하고, 무혈청배지 DMEM/F12를 용기당 90 내지 200 ml을 첨가하여 배양하였다. 배양 후, 5일 간격으로 컨디션드 배지(conditioned media)를 3회 수거하였으며, 매 수거때마다 새로운 DMEM/F12 배지를 플라스크에 첨가하여 주었다. 상기 수거된 컨디션드 배지는 원심분리하고 0.22 um 기공 크기(pore size)의 필터를 이용하여 여과(filtration)하여 세포 파편(cell debris)을 제거하였다. 상기와 같이 3회에 걸쳐 생산한 컨디션드 배지는 혼합하여 보관하였으며, 이후 5% 농도로 희석하여 사용하였다.
Specifically, that is stored in a cell bank, thawing the 1 × 10 6 of amniotic fetal-derived MSCs (Amniotic Fluid Stem Cell, AFSCs) and seeded in T75 flasks, 10% fetal calf serum (fetal bovine serum, and FBS ) And 100 μl / ml penicillin and 100 μg / ml streptomycin antibiotic in DMEM (low glucose) medium. The cells were cultured in a 37, 5% CO 2 incubator, seeded and the medium was changed after 24 hours. Subsequently, the medium was changed at intervals of 24 hours to 48 hours, and when the confluency of 70 to 90% was reached, subculture was carried out. It took 3 to 4 days for each passage, and the culture was proliferated to the 4th passage. At this time, the culture container was changed from a T75 flask to a T175 flask, and then cultured using a triple flask. When the confluency of the cells that have undergone the fourth subculture as described above reaches 70 to 90%, the adherent cells are removed by treatment with 0.05% trypsin-EDTA, and the cells are washed with phosphate buffered saline (PBS) PBS) to remove serum medium and antibiotic components. Then, in the 5th passage, seeds were seeded at 8,000 to 20,000 cells per cm 2 area of a triple flask and cultured in the serum-free medium DMEM / F12 supplemented with 90 to 200 ml per vessel. After the culture, conditioned media was collected three times at 5-day intervals, and fresh DMEM / F12 medium was added to the flask at each collection. The collected conditioned medium was centrifuged and filtered to remove cell debris using a 0.22-μm pore size filter. The conditioned medium produced three times as described above was mixed and stored, and then diluted to a concentration of 5%.
실시예Example 1-3: 줄기세포 증식 촉진용 혼합조성물의 제조 1-3: Preparation of a mixed composition for promoting stem cell proliferation
줄기세포의 증식을 촉진하는 배양용 배지 조성물을 제조하기 위하여, 상기 실시예 1-1에서 제조한 혼합 식물추출물과 상기 실시예 1-2에서 생산한 컨디션드 배지(Amniotic Fluid Stem Cell Conditioned Media, AFSCM)를 일정 비율로 혼합하여 혼합조성물을 제조하였다.In order to prepare a culture medium composition for promoting the proliferation of stem cells, the mixed plant extract prepared in Example 1-1 and the conditioned medium prepared in Example 1-2 (Amniotic Fluid Stem Cell Conditioned Media, AFSCM ) Were mixed at a certain ratio to prepare a mixed composition.
구체적으로, 양수 내 태아 유래 중간엽 줄기세포를 배양한 컨디션드 배지(Amniotic Fluid Stem Cell Conditioned Media, AFSCM) 5 중량% 및 상기 실시예 1-1의 혼합 식물추출물을 10 중량% 함유하도록 혼합조성물을 제조하였다.
Specifically, 5% by weight of Amniotic Fluid Stem Cell Conditioned Media (AFSCM) in which amniotic fluid fetal-derived mesenchymal stem cells were cultured, and 10% by weight of the mixed plant extract of Example 1-1, .
실시예Example 2: 혼합 식물추출물의 줄기세포 증식 촉진 효과 검증 2: Promoting stem cell proliferation of mixed plant extracts
혼합 식물추출물의 줄기세포 증식 촉진능을 검증하기 위하여, MTT assay를 수행하여 세포 증식 정도를 측정하였다.
To examine the ability of mixed plant extracts to stimulate stem cell proliferation, cell proliferation was measured by MTT assay.
구체적으로, 양수 내 태아 유래 중간엽 줄기세포에, 상기 실시예 1-1에서 제조한 혼합 식물추출물을, 배양용 배지로서 사용한, DMEM(Dulbecco's Modified Eagle's Medeium) 배지의 전체 조성물에 대해 각각 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0 중량%(최종 농도 0.025, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 중량%)로 첨가하고, 24시간 동안 배양한 후, 줄기세포의 증식 정도를 분석하였다. 이때, 성장촉진인자인 소태아혈청(Fetal Bovine Serum; FBS)을 10 중량% 첨가(양성대조군) 또는 미첨가(음성대조군)한 군을 대조군으로 하였다.Specifically, the amniotic fluid embryo-derived mesenchymal stem cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) medium in which the mixed plant extract prepared in Example 1-1 was used as a medium for culture, 0.25, 0.5 (Final concentration: 0.025, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 wt%) and incubated for 24 hours. The proliferation of stem cells Respectively. At this time, a group in which 10% by weight of Fetal Bovine Serum (FBS), which is a growth promoting factor, was added (positive control group) or not added (negative control group) was used as a control group.
실험 결과, FBS를 첨가 또는 미첨가한 대조군에 비해, 혼합 식물추출물을 처리한 모든 실험군에서, 줄기 세포의 증식을 촉진시킴을 확인하였으며, 특히 양성대조군 대비 상기 식물추출물을 2 중량%로 첨가한 경우, 최대치의 증식촉진효과를 나타내었으며, 대조군 대비 약 60% 정도 증가된 줄기세포 증식능 촉진효과를 보임을 알 수 있었다(도 1).
As a result, it was confirmed that stem cell proliferation was promoted in all the experimental groups treated with the mixed plant extracts, compared to the control group with or without FBS. In particular, when the plant extract was added at 2 wt% , Showing the maximum promoting effect on the proliferation and promoting the proliferation of stem cells by about 60% as compared with the control group (FIG. 1).
이러한 결과는 본 발명의 식물추출물이 배양을 위한 배지에 첨가되었을 때, 줄기세포 증식에 대해 우수한 효과를 가짐을 입증하는 결과이다.
These results show that when the plant extract of the present invention is added to a culture medium for culturing, it has an excellent effect on stem cell proliferation.
실시예Example 3: 혼합조성물의 줄기세포 증식 촉진 효과 검증 3: Promoting effect of mixed composition on stem cell proliferation
양수 내 태아 유래 중간엽 줄기세포를 배양한 컨디션드 배지(Amniotic Fluid Stem Cell Conditioned Media, AFSCM)와 혼합 식물추출물을 혼합하여 제조한, 혼합조성물이 줄기세포 증식력에 미치는 영향을 확인하기 위하여, MTT assay를 수행하여 세포 증식 정도를 측정하였다.
In order to investigate the effect of the mixed composition prepared by mixing amniotic fluid stem cell medium (AFSCM) and mixed plant extracts on embryonic stem cell proliferation, embryos were cultured in MTT assay And the degree of cell proliferation was measured.
구체적으로, 양수 내 태아 유래 중간엽 줄기세포에, 상기 실시예 1-3에서 제조한 혼합조성물을 배양용 배지 전체 조성물에 대해 각각 0.5, 1.0, 2.0 중량%로 첨가하고, 24시간 동안 배양한 후, 줄기세포의 증식 정도를 분석하였다. 이때, 성장촉진인자인 소태아혈청(Fetal Bovine Serum; FBS)을 10 중량% 첨가(양성대조군) 또는 미첨가(음성대조군)한 군과 컨디션드 배지만을 첨가(0.5, 1.0, 2.0 중량%)한 군을 대조군으로 하였다.Specifically, the mixed composition prepared in Example 1-3 was added to amniotic fluid fetal-derived mesenchymal stem cells at 0.5, 1.0, and 2.0 wt%, respectively, for the total culture medium for culture, followed by culturing for 24 hours , And the degree of proliferation of stem cells was analyzed. At this time, 10% by weight (positive control) or no addition (negative control) of growth promoting factor Fetal Bovine Serum (FBS) was added and the conditioned seeds were added (0.5, 1.0, 2.0% by weight) And the control group.
실험 결과, FBS를 첨가 또는 미첨가한 대조군 및 컨디션드 배지만을 첨가한 대조군에 비해, 컨디션드 배지와 식물추출물을 혼합하여 제조한 혼합조성물을 처리한 경우, 줄기 세포의 증식을 두드러지게 촉진시킴을 확인하였다. 또한, 양성대조군 대비 컨디션드 배지만을 첨가한 경우, 약 68% 정도 증가된 줄기세포 증식촉진효과를 보인데 반해, 혼합조성물을 첨가한 실험군의 경우, 약 120% 정도 증가된, 현저하게 두드러지는 줄기세포 증식촉진효과를 나타내었다(도 2).
As a result of the experiment, when the mixed composition prepared by mixing the conditional medium and the plant extract was treated, the proliferation of the stem cells was remarkably promoted as compared to the control group to which the FBS was added or not and the control group to which the conditioned seeds were added Respectively. In addition, the addition of conditioned seeds to the positive control showed an increase of about 68% in promoting stem cell proliferation, whereas in the case of the experimental group to which the mixed composition was added, the stem was remarkably prominent, And exhibited cell proliferation promoting effect (Fig. 2).
이러한 결과를 통하여, 본 발명의 식물추출물이 포함된 배지에 추가로 컨디션드 배지를 첨가하는 경우, 시너지 효과를 발휘하여 더욱 우수한 줄기세포 증식촉진이 이루어짐을 확인할 수 있었다.
From these results, it was confirmed that when the conditional medium was further added to the medium containing the plant extract of the present invention, the synergistic effect was exerted to further promote the proliferation of stem cells.
실시예Example 4: 배양시간에 따른, 혼합조성물의 줄기세포 증식 활성 효과 검증 4: Verification of stem cell proliferation activity of mixed composition according to incubation time
추가로, 상기 실시예 1-3에서 제조한 혼합조성물의 농도에 따른 증식능 차이를 확인하기 위하여, 배양시간을 달리하여 혼합조성물의 줄기세포 증식 촉진능을 MTT assay를 수행하여 평가하였다.
In addition, in order to confirm the difference in the growth potency according to the concentration of the mixed composition prepared in Example 1-3, MTT assay was performed to evaluate the stem cell proliferation promoting ability of the mixed composition with different incubation times.
구체적으로, 양수 내 태아 유래 중간엽 줄기세포에, 컨디션드 배지와 식물추출물을 동시에 유효성분으로 함유한 혼합조성물을 배양용 배지 전체 조성물에 대해 각각 0.25, 0.5, 0.75, 1.0, 2.0, 5.0 중량%로 첨가하고, 24시간 및 72시간 동안 배양한 후, 줄기세포의 증식 정도를 분석하였다. 이때, 성장촉진인자인 소태아혈청(Fetal Bovine Serum; FBS)을 10 중량% 첨가(양성대조군) 또는 미첨가(음성대조군)한 군을 대조군으로 하였다. Specifically, a mixed composition containing the conditioned medium and the plant extract simultaneously as active ingredients was added to the amniotic fluid fetal-derived mesenchymal stem cells at 0.25, 0.5, 0.75, 1.0, 2.0 and 5.0 wt%, respectively, , And cultured for 24 hours and 72 hours. Then, the degree of proliferation of the stem cells was analyzed. At this time, a group in which 10% by weight of Fetal Bovine Serum (FBS), which is a growth promoting factor, was added (positive control group) or not added (negative control group) was used as a control group.
실험 결과, 배양시간에 관계없이, FBS를 첨가 또는 미첨가한 대조군에 비해, 컨디션드 배지와 식물추출물을 혼합하여 제조한 혼합조성물을 처리한 경우, 줄기 세포의 증식을 두드러지게 촉진시킴을 확인하였으며, 배양시간에 따라 세포 증식이 현저하게 활성화되었음을 알 수 있었다(도 3). As a result of the experiment, it was confirmed that, when the mixed composition prepared by mixing the conditional medium and the plant extract was treated, the proliferation of the stem cells was remarkably promoted, compared to the control group with or without FBS, regardless of the culture time , And cell proliferation was markedly activated according to the incubation time (FIG. 3).
보다 구체적으로, 2 중량%의 혼합조성물을 첨가하고 24시간 동안 배양한 실험군의 경우, 24시간 동안 배양한 양성대조군에 비해 약 88% 정도 증가된 줄기세포 증식촉진 효과를 나타내었으며, 72시간 동안 배양한 실험군의 경우, 72시간 동안 배양한 양성대조군에 비해 약 184% 정도 증가된, 현저하게 두드러지는 줄기세포 증식촉진효과를 나타내었다.
More specifically, in the experimental group in which 2% by weight of the mixed composition was added and incubated for 24 hours, the stem cell proliferation promoting effect was enhanced by about 88% as compared with the positive control group cultured for 24 hours, One experimental group showed remarkable prominent stem cell proliferation promoting effect, which was increased by about 184% as compared with the positive control group cultured for 72 hours.
따라서, 상기 9종의 식물추출물을 유효성분으로 함유함에 의해, 세포 증식 촉진에 있어서 시너지 효과를 가짐을 확인할 수 있었으며, 성장촉진인자인 동물성 혈청 대체제로서의 활용이 가능함을 알 수 있었다.Therefore, it was confirmed that synergistic effect in promoting cell proliferation was obtained by containing the above-mentioned nine kinds of plant extracts as an active ingredient, and it could be utilized as an alternative agent for animal serum which is a growth promoting factor.
Claims (12)
A culture medium for stem cell culture comprising at least one plant extract selected from the group consisting of Arnica flower extract, Gentiana extract, Western blotch extract, wortfly extract, chamomile flower, Ginkgo biloba extract, Centella asiatica extract, .
The composition according to claim 1, wherein the plant extract is composed of arnica flower extract, gentian extract, wormwood extract, wortberry extract, chamomile flower, ginkgo biloba extract, centipede extract, horsetail extract and mulberry bark extract.
[Claim 3] The composition according to claim 2, wherein the composition comprises 0.3 part by weight to 3 parts by weight of Gentiana extract, 0.3 to 3 parts by weight of an extract of wormwood extract, 0.3 to 3 parts by weight of a wortfly extract, 1 to 10 parts by weight of chamomile flower, 0.4 to 4 parts by weight of Ginkgo biloba extract, 1 to 10 parts by weight of Centella asiatica extract, 0.4 to 4 parts by weight of the extract of Marchia and 0.5 to 5 parts by weight of extract ≪ / RTI > wherein the composition comprises plant extracts.
The composition according to claim 1, wherein the plant extract is contained in an amount of 0.01 to 1% by weight based on the total composition.
The composition of claim 1, wherein the composition further comprises conditioned medium in which fetal-derived mesenchymal stem cells in amniotic fluid are cultured.
The composition according to claim 1, wherein the composition has a stem cell proliferation promoting effect.
The composition according to claim 1, wherein the stem cells are mesenchymal stem cells.
8. The composition according to claim 7, wherein the mesenchymal stem cells are amniotic derived mesenchymal stem cells.
Comprising adding to the medium at least one plant extract selected from the group consisting of arnica flower extract, gentian extract, wormwood extract, wortley extract, chamomile flower, ginkgo leaf extract, centipede extract, Lt; RTI ID = 0.0 > 8. ≪ / RTI >
8. A method for culturing a stem cell, comprising culturing the stem cell in the medium composition of any one of claims 1 to 8.
A composition for improving skin condition comprising the culture obtained by culturing by the method of claim 10 as an active ingredient.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101707622B1 (en) | 2016-11-18 | 2017-02-20 | 주식회사 디지레이 | serum-free medium composition for proliferation of mesenchymal stem cell |
| WO2020013719A1 (en) * | 2018-07-10 | 2020-01-16 | Uniwersytet Jagielloński | Stem cell culture method using plant secretion |
| WO2022055034A1 (en) * | 2020-09-14 | 2022-03-17 | 주식회사 시리아이앤티 | Functional cosmetic composition using nordenau water |
| CN118308298A (en) * | 2024-06-11 | 2024-07-09 | 吉林省藏舍生物科技有限公司 | Method for improving proliferation capacity of adipose-derived mesenchymal stem cells |
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2014
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101707622B1 (en) | 2016-11-18 | 2017-02-20 | 주식회사 디지레이 | serum-free medium composition for proliferation of mesenchymal stem cell |
| WO2020013719A1 (en) * | 2018-07-10 | 2020-01-16 | Uniwersytet Jagielloński | Stem cell culture method using plant secretion |
| WO2022055034A1 (en) * | 2020-09-14 | 2022-03-17 | 주식회사 시리아이앤티 | Functional cosmetic composition using nordenau water |
| CN118308298A (en) * | 2024-06-11 | 2024-07-09 | 吉林省藏舍生物科技有限公司 | Method for improving proliferation capacity of adipose-derived mesenchymal stem cells |
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