KR20220112446A - Composition for improving skin, comprising Rosa wichuraiana Extract as effective components, Cosmetic composition and Food functional composition including the same - Google Patents
Composition for improving skin, comprising Rosa wichuraiana Extract as effective components, Cosmetic composition and Food functional composition including the same Download PDFInfo
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- KR20220112446A KR20220112446A KR1020210016015A KR20210016015A KR20220112446A KR 20220112446 A KR20220112446 A KR 20220112446A KR 1020210016015 A KR1020210016015 A KR 1020210016015A KR 20210016015 A KR20210016015 A KR 20210016015A KR 20220112446 A KR20220112446 A KR 20220112446A
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- 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]
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- A—HUMAN NECESSITIES
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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- A—HUMAN NECESSITIES
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- A23V2200/00—Function of food ingredients
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Abstract
Description
본 발명은 돌가시나무 추출물을 유효성분으로 함유하는 피부개선용 조성물, 이를 포함하는 피부 개선용 화장료 조성물 및 건강기능식품 조성물에 관한 것이다.The present invention relates to a composition for improving skin containing an extract of stony hornwort as an active ingredient, a cosmetic composition for skin improvement and a health functional food composition comprising the same.
피부는 노화 정도를 가장 먼저 판단할 수 있는 요소이므로 많은 사람들이 다양한 방법으로 피부 노화를 막기 위해 노력하고 있다. 특히 자외선, 스트레스, 환경 오염 물질 등을 통해 생성되는 활성산소 (Reactive Oxygen Species, ROS)는 피부노화에 가장 중요한 원인으로 꼽히며, 주름, 아토피, 피부염 등의 영향을 미친다. 따라서 기존의 화장료 조성물들은 항산화제를 활용하여 자유 라디칼과 병합하거나 중화하여 ROS에 의한 손상을 완화하는데 목적을 두고 있다.Since the skin is the first factor to determine the degree of aging, many people are trying to prevent skin aging in various ways. In particular, Reactive Oxygen Species (ROS) generated through ultraviolet rays, stress, and environmental pollutants is considered the most important cause of skin aging and has an effect on wrinkles, atopy, and dermatitis. Therefore, existing cosmetic compositions aim to alleviate damage caused by ROS by merging or neutralizing free radicals by utilizing antioxidants.
한편, 외부로부터 자극을 받은 피부 세포는 이를 방어하기 위해 높은 에너지를 필요로 한다. 진피의 경우 주로 미토콘드리아에서 산화적 인산화 과정을 통해 에너지를 공급받는 것으로 알려져 있다. 에너지 생성이 감소하면 교원섬유와 탄력섬유의 합성 및 항산화 효소 합성에 필요한 에너지를 충분히 공급받지 못해 교원섬유와 탄력섬유의 발현을 감소시켜 피부 주름을 생성시킬 수 있다.On the other hand, skin cells stimulated from the outside require high energy to defend them. In the case of the dermis, it is known that energy is supplied mainly through oxidative phosphorylation in mitochondria. When energy production is reduced, the energy required for the synthesis of collagen and elastic fibers and the synthesis of antioxidant enzymes is not sufficiently supplied, and the expression of collagen and elastic fibers is reduced, which can cause skin wrinkles.
또한, 미토콘드리아 기능 이상으로 인해 활성산소종이 증가하는 경우, 이들 물질이 멜라닌 생성을 촉진하고, 색소침착을 유발하며 피부 내 단백질, 지방, 세포막, 핵산 등을 산화시켜 피부 노화를 더욱 촉진할 수도 있다.In addition, when reactive oxygen species increase due to abnormal mitochondrial function, these substances promote melanin production, cause pigmentation, and oxidize proteins, fats, cell membranes, nucleic acids, etc. in the skin, thereby further promoting skin aging.
더욱이 미토콘드리아 DNA는 세포의 핵 DNA와는 달리 자체적인 수선 기작(repair mechanism)이 없으며, DNA를 보호하는 역할을 하는 히스톤 단백질이 없기 때문에 상대적으로 손상되기 쉽다. 따라서 노화 및 활성산소로 인해 미토콘드리아 DNA 손상 및 기능 이상이 생기면, 에너지 생성이 감소되고, 동시에 전자 유출이 증가하여 활성 산소를 더 많이 생성하게 된다. 증가한 활성 산소는 다시 미토콘드리아를 표적으로 해 미토콘드리아의 기능 이상을 초래하여 과도한 활성산소 발생으로 이어진다 (Prahl, S., et al., 2008, 32.1-4: 245-255). Moreover, mitochondrial DNA, unlike nuclear DNA of cells, does not have its own repair mechanism and is relatively susceptible to damage because there is no histone protein that protects the DNA. Therefore, when mitochondrial DNA damage and dysfunction occur due to aging and free radicals, energy production is reduced, and at the same time, electron flux is increased to generate more reactive oxygen species. The increased free radicals target the mitochondria again, resulting in mitochondrial dysfunction, leading to excessive generation of reactive oxygen species (Prahl, S., et al., 2008, 32.1-4: 245-255).
이러한 기술적 배경하에서, 미토콘드리아 보호 및 기능 활성화를 통해 미토콘드리아의 에너지 생성을 촉진하는 것이 산화적 스트레스를 저해하고, 피부 노화를 방지할 수 있는 방법으로 제시될 수 있다.Under this technical background, promoting mitochondrial energy generation through mitochondrial protection and functional activation can be suggested as a method to inhibit oxidative stress and prevent skin aging.
이에 본 발명의 목적은 돌가시나무천연 물질을 이용하여 친환경적이면서도, 산화적 스트레스로부터 세포 사멸을 억제하여 피부 세포를 보호하고, 피부 세포 내 미토콘드리아 DNA 합성 증가 및 기능 활성화를 통해 활성산소를 억제함으로써, 피부를 개선하는 조성물을 제공하는 데 있다.Accordingly, an object of the present invention is to protect skin cells by inhibiting apoptosis from oxidative stress while being eco-friendly by using a natural substance of rhododendron, and by inhibiting reactive oxygen species through increased mitochondrial DNA synthesis and functional activation in skin cells, An object of the present invention is to provide a composition for improving skin.
본 발명의 추가적인 목적은 인체에 해가 없는 친환경적인 피부개선용 화장료 조성물을 제공하는 것이다.An additional object of the present invention is to provide an eco-friendly cosmetic composition for skin improvement that is harmless to the human body.
본 발명의 또 다른 목적은 인체에 해가 없는 친환경적인 피부개선용 건강기능성식품 조성물을 제공하는 것이다.Another object of the present invention is to provide an eco-friendly functional health food composition for skin improvement that is harmless to the human body.
상기 목적을 달성하기 위하여, 본 발명은 돌가시나무 추출물을 유효성분으로 포함하는 피부개선용 조성물을 제공한다.In order to achieve the above object, the present invention provides a composition for improving the skin comprising an extract of oleander as an active ingredient.
본 발명의 일 실시예에 의하면, 상기 피부개선이 항산화 및 항노화인 것이 바람직하다.According to an embodiment of the present invention, it is preferable that the skin improvement is antioxidant and anti-aging.
본 발명의 일 실시예에 따르면, 상기 조성물은 산화스트레스에 대한 피부 세포 보호용인 것이 바람직하다.According to an embodiment of the present invention, the composition is preferably for protecting skin cells against oxidative stress.
본 발명의 일 실시예에 의하면, 상기 조성물은 미토콘드리아의 보호 및 기능 활성화에 의한 피부 개선용인 것이 바람직하다.According to an embodiment of the present invention, the composition is preferably for skin improvement by mitochondrial protection and functional activation.
본 발명의 일 실시예에 의하면, 상기 조성물은 피부 주름 개선용인 것이 바람직하다.According to an embodiment of the present invention, the composition is preferably for improving skin wrinkles.
본 발명의 일 실시예에 의하면, 상기 조성물은 피부 활력 증진용인 것이 바람직하다.According to one embodiment of the present invention, the composition is preferably for promoting skin vitality.
본 발명의 일 실시예에 의하면, 상기 조성물은 활성산소종(ROS)의 감소에 의한 피부 개선용인 것이 바람직하다.According to one embodiment of the present invention, the composition is preferably for skin improvement by reducing reactive oxygen species (ROS).
본 발명의 일 실시예에 의하면, 상기 조성물은 돌가시나무 추출물을 상기 조성물 총 중량에 대하여 0.0001~10% 중량%로 포함하는 것이 바람직하다.According to an embodiment of the present invention, the composition preferably contains stony hornwort extract in an amount of 0.0001 to 10% by weight based on the total weight of the composition.
본 발명의 일 실시예에 의하면, 상기 돌가시나무 추출물은 물, 유기용매 또는 이들의 혼합 용매로 추출되는 것이 바람직하다.According to an embodiment of the present invention, it is preferable that the extract of the rhododendron is extracted with water, an organic solvent, or a mixed solvent thereof.
본 발명의 일 실시예에 의하면, 상기 유기용매는 메탄올, 에탄올, 프로필알코올, 부틸알코올, 글리세린, 프로필렌글리콜 및 부틸렌글리콜 중에서 선택된 1종 이상인 것이 바람직하다.According to an embodiment of the present invention, the organic solvent is preferably at least one selected from methanol, ethanol, propyl alcohol, butyl alcohol, glycerin, propylene glycol and butylene glycol.
본 발명의 일 실시예에 의하면, 상기 조성물이 화장료 조성물로 사용되는 것이 바람직하다.According to an embodiment of the present invention, the composition is preferably used as a cosmetic composition.
본 발명의 일 실시예에 의하면, 상기 화장료 조성물이 수용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성 함유 클렌징, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이로 구성된 군으로부터 선택되는 어느 하나의 제형인 것이 바람직하다.According to an embodiment of the present invention, the cosmetic composition is an aqueous solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation and spray It is preferable that any one formulation selected from the group consisting of
본 발명의 일 실시예에 의하면, 상기 화장료 조성물은 수중유형 및 유중수형 메이크업베이스, 파운데이션, 스킨커버, 립스틱, 립그로스, 페이스파우더, 투웨이케익, 아이섀도, 치크칼라 및 아이브로우 펜슬류로 이루어진 색조화장료 제형 중에서 선택되는 어느 하나의 제형인 것이 바람직하다.According to an embodiment of the present invention, the cosmetic composition is a color made of oil-in-water and water-in-oil makeup base, foundation, skin cover, lipstick, lip gloss, face powder, two-way cake, eye shadow, cheek color and eyebrow pencils. It is preferable that any one formulation selected from cosmetic formulations.
본 발명의 일 실시예에 의하면, 상기 조성물이 건강기능성식품 조성물인 것이 바람직하다.According to an embodiment of the present invention, it is preferable that the composition is a health functional food composition.
본 발명의 일 실시예에 의하면, 상기 건강기능식품 조성물은 캡슐, 정제, 과립, 분말 또는 음료 형태인 것이 바람직하다. According to an embodiment of the present invention, the health functional food composition is preferably in the form of capsules, tablets, granules, powders or beverages.
본 발명에 따른 조성물은 산화적 스트레스로부터 세포 사멸을 억제하여 세포의 생존율을 증가시켜 세포 보호 효과를 나타내므로, 이에 따라서 피부 탄력, 주름 방지 및 노화 방지 효과를 나타낸다.The composition according to the present invention suppresses apoptosis from oxidative stress to increase cell viability, thereby exhibiting a cytoprotective effect, thus exhibiting skin elasticity, anti-wrinkle and anti-aging effects.
본 발명에 따른 조성물은 산화적 스트레스에 대하여 미토콘드리아 DNA 합성을 증가시키고, 주요 기능인 ATP 생성을 증가시켜 기능을 활성화시켜 미토콘드리아를 보호하고 기능을 활성화시켜 피부를 개선하는 효과가 있다.The composition according to the present invention increases mitochondrial DNA synthesis against oxidative stress and activates the function by increasing ATP production, which is the main function, thereby protecting mitochondria and activating the function to improve the skin.
또한 본 발명에 따른 조성물은 미토콘드리아의 주요 기능인 ATP 생성을 증가시킴으로서 피부 활력을 증가시키는 효과가 있다.In addition, the composition according to the present invention has an effect of increasing skin vitality by increasing ATP production, which is a major function of mitochondria.
또한, 본 발명에 따른 조성물은, ROS 생성량을 감소시켜, 주름, 아토피, 피부염 등의 피부 문제를 개선시키는 효과를 나타낸다.In addition, the composition according to the present invention exhibits an effect of improving skin problems such as wrinkles, atopy, and dermatitis by reducing the amount of ROS production.
본 발명에 따른 조성물은 피부 세포 노화를 방지하는 효과를 나타낸다.The composition according to the present invention exhibits an effect of preventing skin cell aging.
본 발명에 따른 조성물은 피부 세포에 악영향을 미치지 않는 안전성을 가지면서 항산화 효과 및 피부 세포 노화를 방지하므로 피부 외용제, 화장료 조성물 및 약학 조성물에 널리 적용될 수 있다.The composition according to the present invention can be widely applied to external preparations for skin, cosmetic compositions, and pharmaceutical compositions because it has safety that does not adversely affect skin cells and prevents antioxidant effects and skin cell aging.
도 1은 인간 정상 섬유아세포에 돌가시나무 지상부, 잎, 줄기 추출물을 처리하여 세포 독성 여부를 MTT assay를 통해 확인한 결과이다. (대조군으로는 어떠한 처리도 하지 않은 인간 정상 섬유아세포를, 음성대조군으로는 DMSO를 처리한 인간 정상 섬유아세포를 사용하였다.)
도 2는 돌가시나무 지상부, 잎, 줄기 추출물이 처리된 인간 정상 섬유아세포의 과산화수소에 대한 세포보호효과를 MTT assay를 통하여 확인한 결과이다. (대조군으로는 어떠한 처리도 하지 않은 인간 정상 섬유아세포를, 음성대조군으로는 과산화수소만을 처리한 인간 정상 섬유아세포를, 양성대조군으로는 과산화수소와 10μM의 Ascorbic acid를 처리한 인간 정상 섬유아세포를 사용하였다.)
도 3은 돌가시나무 지상부, 잎, 줄기 추출물이 처리된 인간 정상 섬유아세포의 미토콘드리아 DNA Copy 수를 real time PCR를 통하여 측정한 결과이다. (대조군으로는 어떠한 처리도 하지 않은 인간 정상 섬유아세포를, 음성대조군으로는 과산화수소만을 처리한 인간 정상 섬유아세포를, 양성대조군으로는 과산화수소와 10μM의 Ascorbic acid를 처리한 인간 정상 섬유아세포를 사용하였다.)
도 4는 돌가시나무 지상부, 잎, 줄기 추출물이 처리된 인간 정상 섬유아세포의 미토콘드리아 ATP 합성을 측정한 결과이다. (대조군으로는 어떠한 처리도 하지 않은 인간 정상 섬유아세포를, 음성대조군으로는 과산화수소만을 처리한 인간 정상 섬유아세포를, 양성대조군으로는 과산화수소와 10μM의 Ascorbic acid를 처리한 인간 정상 섬유아세포를 사용하였다.)
도 5는 돌가시나무 지상부, 잎, 줄기 추출물이 처리된 인간 정상 섬유아세포의 ROS 생성량을 DCF-DA를 통해 측정한 결과이다. (대조군으로는 어떠한 처리도 하지 않은 인간 정상 섬유아세포를, 음성대조군으로는 과산화수소만을 처리한 인간 정상 섬유아세포를, 양성대조군으로는 과산화수소와 10μM의 Ascorbic acid를 처리한 인간 정상 섬유아세포를 사용하였다.)1 is a result of confirming the cytotoxicity through MTT assay by treating normal human fibroblasts with extracts from the above-ground parts of brambles, leaves, and stems. (As a control group, normal human fibroblasts without any treatment were used, and as a negative control group, normal human fibroblasts treated with DMSO were used.)
Figure 2 is the result of confirming the cytoprotective effect on hydrogen peroxide in normal human fibroblasts treated with the above-ground part, leaves, and stem extracts of bramble by MTT assay. (As a control group, normal human fibroblasts without any treatment were used, as a negative control, human normal fibroblasts treated only with hydrogen peroxide, and as a positive control, normal human fibroblasts treated with hydrogen peroxide and 10 μM ascorbic acid were used. )
3 is a result of measuring the number of mitochondrial DNA copies of normal human fibroblasts treated with the above-ground part, leaf, and stem extracts of bramble by real-time PCR. (As a control group, normal human fibroblasts without any treatment were used, as a negative control, human normal fibroblasts treated only with hydrogen peroxide, and as a positive control, normal human fibroblasts treated with hydrogen peroxide and 10 μM ascorbic acid were used. )
4 is a result of measuring mitochondrial ATP synthesis in normal human fibroblasts treated with an above-ground part, leaves, and stem extracts of Ash tree. (As a control group, normal human fibroblasts without any treatment were used, as a negative control, human normal fibroblasts treated only with hydrogen peroxide, and as a positive control, normal human fibroblasts treated with hydrogen peroxide and 10 μM ascorbic acid were used. )
5 is a result of measuring the ROS production amount of normal human fibroblasts treated with the above-ground part, leaves, and stem extracts of stony hornwort through DCF-DA. (As a control group, normal human fibroblasts without any treatment were used, as a negative control, human normal fibroblasts treated only with hydrogen peroxide, and as a positive control, normal human fibroblasts treated with hydrogen peroxide and 10 μM ascorbic acid were used. )
이하, 본 발명의 내용을 하기 실시예를 통해 상세히 설명하고자 한다. 이들 실시 예는 오로지 본 발명을 예시하기 위한 것일 뿐, 본 발명의 범위가 이들 실시 예에 의해 한정되는 것은 아니다.Hereinafter, the contents of the present invention will be described in detail through the following examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.
본 발명에 따른 조성물은 돌가시나무 추출물을 유효성분으로 포함하는 피부개선용 조성물이다.The composition according to the present invention is a composition for improving the skin comprising an extract of oleracea as an active ingredient.
본 발명에 있어서 용어 "피부 개선용"이란, 피부 보호 및 피부 상태 개선, 피부 미백, 피부 노화 및 주름의 예방 또는 개선, 피부 보습, 피지분비억제, 여드름개선, 모공축소, 피부보호 및 피부의 염증 반응의 완화, 항염, 피부 장벽 기능, 보습 개선, 피부 활력 증진, 자외선으로부터의 피부 보호, 항산화 및 항노화 등을 모두 포함하는 개념이다. In the present invention, the term "for skin improvement" means skin protection and skin condition improvement, skin whitening, prevention or improvement of skin aging and wrinkles, skin moisturizing, sebum secretion suppression, acne improvement, pore reduction, skin protection and inflammation of the skin It is a concept that includes alleviation of reaction, anti-inflammatory, skin barrier function, moisture improvement, skin vitality enhancement, skin protection from UV rays, antioxidant and anti-aging.
본 발명에서 "피부"는 얼굴 뿐만 아니라, 두피, 전신도 포함되는 개념이다.In the present invention, "skin" is a concept that includes not only the face, but also the scalp and the whole body.
여기서 "유효성분으로 함유하는"은 피부 개선 효과를 나타낼 수 있는 정도의 돌가시나무 추출물을 포함한다는 것을 의미한다.Here, "contained as an active ingredient" means that it includes an extract of rhododendron to an extent capable of exhibiting a skin improvement effect.
돌가시나무(Rosa wichuraiana)는 장미과의 낙엽 활엽 관목. 줄기는 옆으로 누워 뻗으며, 잎은 우상 복엽이다. 여름에 흰 꽃이 총상(總狀) 화서로 피고 열매는 난상(卵狀) 원형으로 가을에 붉게 익는다. 산기슭 양지에서 나는데 한국, 일본, 대만 등지에 분포한다.Stone thorn (Rosa wichuraiana) is a deciduous broad-leaved shrub in the Rosaaceae family. The stem is stretched out lying on its side, and the leaves are bilobed to the right. In summer, white flowers bloom in raceme inflorescences, and the fruits are egg-shaped and ripen red in autumn. It grows in the sun at the foot of the mountain and is distributed in Korea, Japan, and Taiwan.
상기 돌가시나무는 잎, 꽃, 줄기, 뿌리 모두 다 사용가능하며, 바람직하게는 지상부, 잎, 줄기가 사용될 수 있고, 특히 건조한 후 사용하는 것이 바람직하다. All of the leaves, flowers, stems, and roots of the bramble can be used, preferably above-ground parts, leaves, and stems can be used, and it is particularly preferable to use them after drying.
본 발명의 일 실시예에 의하면 상기 피부개선은 항산화 및 항노화인 것이 바람직하다.According to an embodiment of the present invention, it is preferable that the skin improvement is antioxidant and anti-aging.
본 발명에서 용어 "항산화"란 대사 과정을 통해 생성된 활성 산소가 축적되면 조직 세포의 노화, 퇴행성 질환 등이 유발되는데 이러한 활성 산소의 생성을 억제하는 것을 말한다.In the present invention, the term "antioxidant" refers to inhibiting the generation of active oxygen, which is caused by the accumulation of active oxygen produced through metabolic processes, such as aging of tissue cells, degenerative diseases, and the like.
본 발명에서 용어 "노화"란 피부 내 또는 피부 외 요인으로 인하여 세포 기능이 저하되어 피부 주름 발생, 피부 탄력 저하, 피부 건조, 색소 침착, 또는 피부 재생력 저하 등을 말하며, "항노화"란 노화를 방지하는 것을 말한다.In the present invention, the term "aging" refers to the occurrence of skin wrinkles, decreased skin elasticity, dry skin, pigmentation, or decreased skin regeneration ability due to a decrease in cell function due to factors inside or outside the skin, and "anti-aging" refers to aging say to prevent
본 발명의 일 실시예에 의하면, 상기 조성물은 산화스트레스에 대한 피부 세포 보호용인 것이 바람직하다.According to an embodiment of the present invention, the composition is preferably for protecting skin cells against oxidative stress.
본 발명에서 용어 "산화스트레스"란 체내 활성산소가 많아져 생체 산화 균형이 무너진 상태를 말한다.As used herein, the term "oxidative stress" refers to a state in which the body's oxidative balance is disrupted due to an increase in free radicals in the body.
본 발명의 일 실시예에 의하면, 상기 조성물은 미토콘드리아의 보호 및 기능 활성화에 의한 피부 개선용인 것이 바람직하다.According to an embodiment of the present invention, the composition is preferably for skin improvement by mitochondrial protection and functional activation.
본 발명에서 용어 "미토콘드리아의 보호 및 기능 활성화"란 미토콘드리아의 DNA의 합성 및/또는 미토콘드리아의 ATP 합성을 통해 미토콘드리아를 보호하고 그 기능을 지속적으로 유지 및 활성화시키는 것을 말한다.In the present invention, the term "protection and function activation of mitochondria" refers to protecting mitochondria through the synthesis of mitochondrial DNA and/or mitochondrial ATP synthesis and continuously maintaining and activating its functions.
본 발명의 일 실시예에 의하면, 상기 조성물은 피부 주름 개선용인 것이 바람직하다.According to an embodiment of the present invention, the composition is preferably for improving skin wrinkles.
본 발명의 일 실시예에 의하면, 상기 조성물은 피부 활력 증진용인 것이 바람직하다.According to one embodiment of the present invention, the composition is preferably for promoting skin vitality.
본 발명의 일 실시예에 의하면, 상기 조성물은 활성산소종(ROS)의 감소에 의한 피부 개선용인 것이 바람직하다.According to an embodiment of the present invention, the composition is preferably for skin improvement by reducing reactive oxygen species (ROS).
본 발명에서 용어 "활성산소종(ROS)"란 화학적으로 활성화된, 즉 불안정한 산소를 포함하는 화학물질들을 말하며, 일반적으로 산소 대비 화학적 반응성이 높은 산소를 포함하는 분자들의 총칭으로, 생체 조직을 공격하고 세포를 손상시키는 산화력이 강한 산소이다.In the present invention, the term "reactive oxygen species (ROS)" refers to chemically activated, that is, chemical substances containing unstable oxygen, and is a generic term for molecules containing oxygen with high chemical reactivity compared to oxygen. It is oxygen with strong oxidizing power that damages cells.
본 발명의 일 실시예에 의하면, 상기 조성물은 돌가시나무 추출물을 상기 조성물 총 중량에 대하여 0.0001~10% 중량% 로 포함하는 것이 바람직하다.According to one embodiment of the present invention, the composition preferably contains stony hornwort extract in an amount of 0.0001 to 10% by weight based on the total weight of the composition.
돌가시나무 추출물이 0.0001중량% 미만으로 포함되면, 항산화 및 항노화 효능의 발휘가 나타나기 어려울 수 있으며, 10중량% 이상 포함되는 경우 변색, 변취 등 제형의 안정성이 저하되어 항산화 및 항노화 효과가 미미해질 수 있으며, 화장료 등으로 제조 시 향이 좋지 않을 수 있다.When the amount of rhododendron extract is less than 0.0001% by weight, it may be difficult to exhibit antioxidant and anti-aging effects. and may not smell good when manufactured with cosmetics.
본 발명의 일 실시예에 의하면, 본 발명에서 추출 방법은 특별히 제한되지 않고, 예를 들어 냉침 추출, 초음파 추출, 환류 추출, 열수 추출 등이 있다. 여기서, 열수추출의 경우, 열탕증류기에서 8~48시간동안, 80~ 100℃ 로 가열하여 열수 추출물을 얻는다. 냉수 추출의 경우, 예컨대, 냉수 (15-25℃)에 상기 조직 배양물 자체 또는 그것의 건조된 분말을 혼합하여 3일동안 추출하여 냉수추출물을 얻는다.According to an embodiment of the present invention, the extraction method in the present invention is not particularly limited, and for example, cold extraction, ultrasonic extraction, reflux extraction, hot water extraction, and the like. Here, in the case of hot water extraction, the hot water extract is obtained by heating at 80 to 100° C. for 8 to 48 hours in a hot water distiller. In the case of cold water extraction, for example, the tissue culture itself or its dried powder is mixed with cold water (15-25° C.) and extracted for 3 days to obtain a cold water extract.
또는, 물, 유기 용매, 또는 이의 혼합 용매를 사용하여 추출하는 방법으로 제조될 수 있다. 추출한 액은 바로 사용하거나 또는 농축 및/또는 건조하여 사용할 수 있다. 유기용매를 사용하여 추출하는 경우, 메탄올, 에탄올, 이소프로판올, 부탄올, 에틸렌, 아세톤, 헥산, 에테르, 클로로포름, 에틸아세테이트, 부틸아세테이트, 디클로로메탄, N, N-디메틸포름아미드(DMF), 디메틸설폭사이드(DMSO), 1,3-부틸렌글리콜, 프로필렌글리콜 또는 이들의 혼합용매인 유기용매를 사용하며 생약의 유효 성분이 파괴되지 않거나 최소화된 조건에서 실온 또는 가온하여 추출할 수 있다. Alternatively, it may be prepared by extraction using water, an organic solvent, or a mixed solvent thereof. The extracted liquid may be used directly or concentrated and/or dried. In case of extraction using an organic solvent, methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or a mixture thereof, an organic solvent, is used, and the active ingredient of the herbal medicine is not destroyed or can be extracted by heating at room temperature or under conditions in which it is minimized.
본 발명의 일 실시예에서, 상기 돌가시나무 추출물은 물, 유기용매 또는 이들의 혼합 용매로 추출되는 것이 바람직하다.In one embodiment of the present invention, it is preferable that the extract of the rhododendron is extracted with water, an organic solvent, or a mixed solvent thereof.
특히, 본 발명에 있어서는 에탄올 추출물, 바람직하게는 20-80% 에탄올 추출물, 일 구체예로써 80% 에탄올 추출물이 바람직하며, 추출 방법은 80% 에탄올에 섞은 후 3일동안 교반하여 얻는다. 본 발명에 있어서, 상기 추출물은 농축, 또는 희석하여 사용할 수 있고, 추출물의 증류액을 사용할 수도 있다.In particular, in the present invention, an ethanol extract, preferably a 20-80% ethanol extract, as an embodiment, an 80% ethanol extract is preferable, and the extraction method is obtained by mixing with 80% ethanol and stirring for 3 days. In the present invention, the extract may be used after concentration or dilution, or a distillate of the extract may be used.
본 발명의 일 실시예에 의하면, 상기 유기용매는 메탄올, 에탄올, 프로필알코올, 부틸알코올, 글리세린, 프로필렌글리콜 및 부틸렌글리콜 중에서 선택된 1종 이상인 것이 바람직하다.According to an embodiment of the present invention, the organic solvent is preferably at least one selected from methanol, ethanol, propyl alcohol, butyl alcohol, glycerin, propylene glycol and butylene glycol.
본 발명의 일 실시예에 의하면, 상기 조성물이 화장료 조성물로 사용되는 것이 바람직하다.According to an embodiment of the present invention, the composition is preferably used as a cosmetic composition.
본 발명의 일 실시예에 의하면, 상기 화장료 조성물이 수용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성 함유 클렌징, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이로 구성된 군으로부터 선택되는 어느 하나의 제형인 것이 바람직하다.According to an embodiment of the present invention, the cosmetic composition is an aqueous solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation and spray It is preferable that any one formulation selected from the group consisting of
본 발명의 일 실시예에 의하면, 상기 화장료 조성물은 수중유형 및 유중수형 메이크업베이스, 파운데이션, 스킨커버, 립스틱, 립그로스, 페이스파우더, 투웨이케익, 아이섀도, 치크칼라 및 아이브로우 펜슬류로 이루어진 색조화장료 제형 중에서 선택되는 어느 하나의 제형인 것이 바람직하다.According to an embodiment of the present invention, the cosmetic composition is a color made of oil-in-water and water-in-oil makeup base, foundation, skin cover, lipstick, lip gloss, face powder, two-way cake, eye shadow, cheek color and eyebrow pencils. It is preferable that any one formulation selected from cosmetic formulations.
본 발명의 일 실시예에 의하면, 상기 조성물이 건강기능성식품 조성물인 것이 바람직하다.According to an embodiment of the present invention, it is preferable that the composition is a health functional food composition.
본 발명의 일 실시예에 의하면, 상기 건강기능식품 조성물은 캡슐, 정제, 과립, 분말 또는 음료 형태인 것이 바람직하다. According to an embodiment of the present invention, the health functional food composition is preferably in the form of capsules, tablets, granules, powders or beverages.
상기 화장료 조성물에 있어서는, 화장품 제제에 있어서 수용가능한 담체를 포함할 수 있다. 여기서, "화장품 제제에 있어서 수용가능한 담체"란 화장품 제제에 포함될 수 있는 이미 공지되어 사용되고 있는 화합물 또는 조성물이거나 앞으로 개발될 화합물 또는 조성물로서 피부와의 접촉시 인체가 적응 가능한 이상의 독성, 불안정성 또는 자극성이 없는 것을 말한다.In the cosmetic composition, it may include a carrier acceptable in the cosmetic formulation. Here, "acceptable carrier in cosmetic preparations" refers to compounds or compositions that are already known and used that can be included in cosmetic preparations, or compounds or compositions to be developed in the future that have toxicity, instability, or irritation beyond what the human body can adapt when in contact with the skin. say nothing
싱기 담체는 본 발명의 화장료 조성물에 그것의 전체 중량에 대하여 약 1 중량% 내지 약 99.99 중량%, 바람직하게는 조성물의 중량의 약 90 중량% 내지 약 99.99 중량%로 포함될 수 있다. 그러나 상기 비율은 본 발명의 화장료 조성물이 제조되는 후술한 바의 제형에 따라 또 그것의 구체적인 적용 부위(얼굴, 목 등)나 그것의 바람직한 적용량 등에 따라 달라지는 것이기 때문에, 상기 비율은 어떠한 측면으로든 본 발명의 범위를 제한하는 것으로 이해되어서는 안 된다.The carrier may be included in the cosmetic composition of the present invention in an amount of from about 1% to about 99.99% by weight, preferably from about 90% to about 99.99% by weight of the total weight of the composition. However, since the above ratio varies depending on the formulation as described below in which the cosmetic composition of the present invention is prepared, its specific application site (face, neck, etc.) or its desired application amount, the ratio is the present invention in any aspect. should not be construed as limiting the scope of
상기 담체로서는 알코올, 오일, 계면활성제, 지방산, 실리콘 오일, 습윤제, 보습제, 점성 변형제, 유제, 안정제, 자외선산란제, 자외선흡수제, 발색제, 향료 등이 예시될 수 있다. 상기 알코올, 오일, 계면활성제, 지방산, 실리콘 오일, 습윤제, 보습제, 점성 변형제, 유제, 안정제, 자외선산란제, 자외선흡수제, 발색제, 향료로 사용될 수 있는 화합물/조성물 등은 이미 당업계에 공지되어 있기 때문에 당업자라면 적절한 해당 물질/조성물을 선택하여 사용할 수 있다.Examples of the carrier include alcohol, oil, surfactant, fatty acid, silicone oil, humectant, humectant, viscosity modifier, emulsion, stabilizer, UV scattering agent, UV absorber, color developer, fragrance, and the like. The alcohol, oil, surfactant, fatty acid, silicone oil, wetting agent, humectant, viscosity modifier, emulsion, stabilizer, UV scattering agent, UV absorber, coloring agent, perfume, etc. are already known in the art. Therefore, those skilled in the art can select and use an appropriate material/composition.
본 발명의 일 구현예로써, 본 발명에 따른 화장료 조성물은 상기 유효성분 이외에 글리세린, 부틸렌글리콜, 프로필렌글키롤, 폴리옥시에틸렌 경화피마자유, 에탄올, 트리에탄올아민 등을 포함할 수 있으며, 방부제, 항료, 착색료, 정제수 등을 필요에 따라 미량 포함할 수 있다.As an embodiment of the present invention, the cosmetic composition according to the present invention may include glycerin, butylene glycol, propylene glycol, polyoxyethylene hydrogenated castor oil, ethanol, triethanolamine, etc. in addition to the active ingredients, and preservatives, fragrances , a colorant, purified water, etc. may be included in a trace amount as needed.
또한, 종래에 잘 알려져 있는 생리활성성분이나 다른 태좌세포 배양물 또는 추출물을 더 포함할 수 있다.In addition, it may further include physiologically active ingredients or other placental cell cultures or extracts known in the prior art.
본 발명에 있어서의 피부는 얼굴뿐만 아니라, 두피, 전신도 포함되는 개념으로, 이러한 두피에 적용될 수 있는 화장료 조성물로써, 샴푸, 린스, 트리트먼트, 발모제 등이 있고, 전신에 적용될 수 있는 바디클렌져 등의 용도로써 다양한 형태로 제조될 수 있다.The skin in the present invention is a concept that includes not only the face, but also the scalp and the whole body. As a cosmetic composition that can be applied to the scalp, there are shampoo, conditioner, treatment, hair growth agent, etc., and body cleanser that can be applied to the whole body, etc. It can be prepared in various forms for the purpose of
본 발명에 따른 화장료 조성물의 제조방법은 전술한 제조방법에 한정되는 것은 아니며, 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자라면 상기 제조방법을 일부 변형시킨 방법으로도 제조할 수 있다.The manufacturing method of the cosmetic composition according to the present invention is not limited to the above-described manufacturing method, and those skilled in the art to which the present invention pertains may also prepare a method by partially modifying the above manufacturing method.
또한, 본 발명의 화장료 조성물은 추가로 지방 물질, 유기 용매, 용해제, 농축제 및 겔화제, 연화제, 항산화제, 현탁화제, 안정화제, 발포제(foaming agent), 방향제, 계면활성제, 물, 이온형 또는 비이온형 유화제, 충전제, 금속이온봉쇄제 및 킬레이트화제, 보존제, 비타민, 차단제, 습윤화제, 필수 오일,염료,안료, 친수성 또는 친유성 활성제, 지질 소낭 또는 화장품에 통상적으로 사용되는 임의의 다른 성분과 같은 화장품학 또는 피부과학 분야에서 통상적으로 사용되는 보조제를 함유할 수 있다. 그리고, 상기의 성분들은 피부과학 분야에서 일반적으로 사용되는 양으로 도입될 수 있다.In addition, the cosmetic composition of the present invention is a fatty substance, an organic solvent, a solubilizer, a thickening agent and a gelling agent, an emollient, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, a surfactant, water, an ionic type. or nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives, lipid vesicles or any other conventionally used in cosmetics. It may contain adjuvants commonly used in the field of cosmetology or dermatology, such as ingredients. And, the above ingredients may be introduced in an amount generally used in the field of dermatology.
본 발명의 또 다른 실시예에 따라 본 발명의 건강음료 조성물은 지시된 비율로 필수성분으로서 상기 분획물을 함유하는 외에는 액체성분에는 특별한 제한점은 없으며, 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상기 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리스리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진 등) 및 합성 향미제(사카린, 아스파탐 등)를 유리하게 사용할 수 있다. 또한, 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 ㎖ 당 일반적으로 약 1 ~ 20 g, 또는 약 5 ~ 12 g이다.According to another embodiment of the present invention, the health beverage composition of the present invention has no particular limitation on the liquid component except for containing the fraction as an essential component in the indicated ratio, and various flavoring agents or natural carbohydrates, etc. may be contained as an additional component. Examples of the natural carbohydrate include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides, for example, conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatine, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. , the proportion of the natural carbohydrate is generally about 1 to 20 g, or about 5 to 12 g per 100 ml of the composition of the present invention.
상기 외에 본 발명의 조성물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 증진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다.In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and enhancers (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof , organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like.
그밖에 본 발명의 조성물들은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 본 발명의 조성물 100중량부당 0 ~ 약 20 중량부의 범위에서 선택되는 것이 일반적이다.In addition, the compositions of the present invention may contain natural fruit juice and pulp for the production of fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The proportion of these additives is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.
이하 본 발명을 하기 실시예에 따라 상세히 설명한다. 단 하기 실시예는 본 발명을 보다 상세히 설명하기 위한 것일 뿐이며, 본 발명의 범위를 제한하는 방법으로 해석되어서는 안된다. Hereinafter, the present invention will be described in detail according to the following examples. However, the following examples are only for illustrating the present invention in more detail, and should not be construed as limiting the scope of the present invention.
제조예 1: 돌가시나무 추출물의 제조Preparation Example 1: Preparation of extract
건조된 돌가시나무를 분쇄하여 분말화 한 후, 2 배량의 80% 에탄올에 24시간 이상 침지하여 유효성분을 추출한다. 이렇게 추출된 추출물을 여과지에 거른 다음 회전감압농축기로 용매를 제거하여 추출물을 제조하였다.After pulverizing and pulverizing the dried bramble, the active ingredient is extracted by immersing it in twice the amount of 80% ethanol for more than 24 hours. The extract thus extracted was filtered through a filter paper and the solvent was removed with a rotary vacuum concentrator to prepare an extract.
[시험예 1: 돌가시나무 추출물의 세포 독성 평가][Test Example 1: Evaluation of Cytotoxicity of Stone Extracts]
본 시험예에서는 돌가시나무 추출물의 세포 독성 여부를 확인하고자 실시예 1에 따라 제조된 돌가시나무 추출물을 각각 0, 10, 50, 100, 200 μg/mL와 0, 100, 200, 300, 400 μg/mL로 희석하여, 농도가 다른 돌가시나무 지상부, 잎, 줄기 추출물을 인간 정상 섬유아세포인 CCD986sk에 처리하여 세포생존능력을 측정하였다.In this test example, 0, 10, 50, 100, 200 μg/mL and 0, 100, 200, 300, 400 Cell viability was measured by diluting to μg/mL and treating CCD986sk, which is a normal human fibroblast, with extracts of above-ground parts, leaves, and stems of rhododendrons of different concentrations.
인간 정상 피부 섬유아세포(CCD-986sk)를 1X104 cells/well 농도로 96-well plate에 분주한 후 37℃, 5% CO2 조건에서 24시간 동안 배양하였다. 배양 배지를 모두 제거 후, 무혈청배지에 돌가시나무 지상부 또는 잎, 줄기 추출물이 각각 0, 10, 50, 100, 200 μg/mL와 0, 100, 200, 300, 400 μg/mL로 희석된 시료를 각 웰 당 100 μL씩 세포에 처리하여 24시간 동안 추가 배양하였다. 그 후, 각 well 당 0.5 mg/ml 농도의 MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) 용액 100 ㎕를 처리하여 3시간동안 반응시킨 후 생성된 포마잔(formazan)을 DMSO 200 ㎕로 용해시켜 micro-plate reader를 이용하여 540 nm 파장에서 흡광도를 측정하였다. 아무것도 처리하지 않은 well의 세포 생존율을 100%으로 하여 정상 대조군 대비 시료 처리군의 세포 생존율을 계산하여 결과를 도 1에 나타내었다.Normal human skin fibroblasts (CCD-986sk) were aliquoted in a 96-well plate at a concentration of 1X10 4 cells/well, and then cultured at 37° C. and 5% CO 2 conditions for 24 hours. After removing all of the culture medium, extracts of the above-ground part or leaves and stems of Ash tree are diluted to 0, 10, 50, 100, 200 μg/mL and 0, 100, 200, 300, and 400 μg/mL, respectively, in serum-free medium. Cells were treated with 100 μL of the sample per well and further cultured for 24 hours. After that, 100 μl of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution at a concentration of 0.5 mg/ml per well was treated and reacted for 3 hours. The formazan was dissolved in 200 μl of DMSO and absorbance was measured at 540 nm wavelength using a micro-plate reader. The cell viability of the untreated well was calculated as 100%, and the cell viability of the sample treated group compared to the normal control group was calculated, and the results are shown in FIG. 1 .
실험결과, 돌가시나무 추출물을 처리한 인간정상섬유아세포 중, 돌가시나무 지상부 추출물 100 μg/mL, 돌가시나무 잎, 줄기 추출물 300 μg/mL에서 각각 유의적인 세포독성효과가 확인되지 않았다 (도 1). 따라서 100, 300 μg/mL 농도의 돌가시나무 지상부, 잎, 줄기 추출물을 선택하여 하기 실험에 사용하였다. As a result of the experiment, no significant cytotoxic effect was confirmed in 100 μg/mL of the above-ground extract of hornbill and 300 μg/mL of the extract of stony hornwort leaf and stem extract among normal human fibroblasts treated with the rhododendron extract (Fig. One). Therefore, extracts from above 100 and 300 μg/mL rhododendron, leaf, and stem extracts were selected and used in the following experiment.
[시험예 2: 돌가시나무 추출물의 세포 보호 효과 확인][Test Example 2: Confirmation of the cytoprotective effect of the Ash tree extract]
본 시험예에서는 돌가시나무 추출물의 산화 스트레스에 의한 세포 손상 보호 효과를 MTT법을 변형하여 측정하였다. 인간 정상 피부 섬유아세포(CCD-986sk)를 1X104 cells/well 농도로 96-well plate에 분주한 후 37℃, 5% CO2 조건에서 24시간 동안 배양하였다. 배양 배지를 제거 후 무혈청배지에 희석한 돌가시나무 지상부, 잎, 줄기 추출물을 각각 100, 300 μg/mL 농도의 시료를 세포에 처리하여 24시간 동안 추가 배양하였다. 배양이 끝난 후, 세포 내 산화스트레스를 유발하기 위하여 800 μM의 과산화수소 (hydrogen peroxide, H2O2)를 3시간 동안 처리하였다. 3시간 후, 각 well 당 0.5 mg/ml 농도의 MTT용액 100 ㎕를 처리하여 3시간동안 반응시킨 후 생성된 포마잔(formazan)을 DMSO 100 ㎕로 용해시켜 micro-plate reader를 이용하여 540 nm 파장에서 흡광도를 측정하였다. 정상 대조군으로는 시료와 과산화수소를 처리하지 않은 well을 사용하여 정상 대조군의 세포 생존율을 100%으로 하여 정상 대조군 대비 과산화수소만 처리군 (음성대조군)과 시료 및 과산화수소 처리군, Ascorbic acid 10 μM 처리군의 세포 생존율을 계산하여 결과를 도 2에 나타내었다.In this test example, the protective effect of oxidative stress-induced cellular damage of the oleander extract was measured by modifying the MTT method. Normal human skin fibroblasts (CCD-986sk) were aliquoted in a 96-well plate at a concentration of 1X10 4 cells/well, and then cultured at 37° C. and 5% CO 2 conditions for 24 hours. After removing the culture medium, the cells were treated with a sample of 100 and 300 μg/mL concentration of extracts of the above-ground part, leaf, and stem of rhododendron diluted in a serum-free medium, respectively, and further cultured for 24 hours. After incubation, 800 μM of hydrogen peroxide (H 2 O 2 ) was treated for 3 hours to induce intracellular oxidative stress. After 3 hours, 100 μl of MTT solution of 0.5 mg/ml concentration per well was treated and reacted for 3 hours, then the generated formazan was dissolved with 100 μl of DMSO using a micro-plate reader at a wavelength of 540 nm. absorbance was measured. As a normal control, using wells not treated with sample and hydrogen peroxide, the cell viability of the normal control was set to 100%, and compared to the normal control group, only hydrogen peroxide treatment group (negative control group), sample and hydrogen peroxide treatment group, and
실험결과, 음성대조군의 경우, 과산화수소 (H2O2)에 의한 산화스트레스 증가로 세포 생존율이 64.5%로 감소하였으나, Ascorbic acid(Vit C) 10 μM을 비롯한 돌가시나무 지상부, 잎, 줄기 추출물을 처리한 인간정상섬유아세포의 경우, 과산화수소 (H2O2)에 의해 감소한 세포 생존률 각각 105.6 %, 103.1%로 정상 대조군 수준으로 회복되어 세포 보호 효과를 나타내었다. 도 2는 돌가시나무 지상부 추출물 100 μg/mL, 돌가시나무 잎, 줄기 추출물 300 μg/mL 농도로 처리된 인간정상섬유아세포의 세포보호 효과를 확인한 결과이다.As a result of the experiment, in the case of the negative control group, the cell viability decreased to 64.5% due to the increase in oxidative stress caused by hydrogen peroxide (H 2 O 2 ). In the case of the treated normal human fibroblasts, the cell viability decreased by hydrogen peroxide (H 2 O 2 ) was recovered to the normal control level to 105.6% and 103.1%, respectively, indicating a cytoprotective effect. Figure 2 is the result of confirming the cytoprotective effect of human normal fibroblasts treated with 100 μg/mL of an above-ground extract of stony hornwort, and 300 μg/mL of a stony leaf, stem extract.
[시험예 3: 돌가시나무 추출물이 미토콘드리아의 DNA 합성에 미치는 영향 확인][Test Example 3: Confirmation of Effect of Ash Tree Extract on Mitochondrial DNA Synthesis]
본 시험예에서는 돌가시나무 추출물이 미토콘드리아의 DNA 합성에 어떠한 영향을 미치는지 확인하고자 하였다. 인간 정상 피부 섬유아세포(CCD-986sk)를 1X105 cells/well 농도로 6-well plate에 분주한 후 37℃, 5% CO2 조건에서 24시간 동안 배양하였다. 배양 배지를 제거 후 무혈청배지에 실시예 1에 따라 제조한 돌가시나무 지상부, 잎, 줄기 추출물을 각각 희석한 돌가시나무 지상부, 잎, 줄기 추출물 100, 300 μg/mL 농도의 시료를 세포에 처리하여 24시간 동안 추가 배양하였다. 배양이 끝난 후, 세포 내 산화스트레스를 유발하기 위하여 300 μM의 과산화수소 (hydrogen peroxide, H2O2)를 1시간 동안 처리하였다. 1시간 후, 1
세포 내 미토콘드리아 수는 미토콘드리아에서 합성되는 COX Ⅱ (Cytochrome oxidase subunit Ⅱ) 양을 핵에서 만들어지는 cyclophilin A의 양으로 나누어 미토콘드리아 DNA 카피 수를 계산함으로써 측정하였다. 정상 대조군으로는 시료와 과산화수소를 처리하지 않은 미토콘드리아 DNA 카피 수를 1로 하여 정상 대조군 대비 과산화수소만 처리군 (음성대조군)과, 시료(돌가시나무 추출물 및 아스코르브산) 및 과산화수소 처리군의 미토콘드리아 DNA Copy 수를 계산하여 결과를 도 3에 나타내었다. The number of mitochondria in cells was measured by dividing the amount of cytochrome oxidase subunit II (COX II) synthesized in mitochondria by the amount of cyclophilin A produced in the nucleus to calculate the mitochondrial DNA copy number. As a normal control, the mitochondrial DNA copy number of the sample and the non-hydrogen peroxide-treated mitochondrial DNA copy was set to 1, and the mitochondrial DNA Copy of the hydrogen peroxide-only group (negative control group), the sample (Salwood extract and ascorbic acid) and the hydrogen peroxide-treated group compared to the normal control group. The number was calculated and the results are shown in FIG. 3 .
실험 결과, 미토콘드리아 DNA(mtDNA)의 카피 수가 과산화수소를 처리한 음성대조군의 경우 정상 대조군 대비 0.49-fold 로 감소하였다. 또한, Ascorbic acid 10 μM을 처리한 실험군의 경우, 정상 대조군 대비 0.62-fold로 음성대조군과 비슷한 수준으로 감소하였다. 돌가시나무 지상부, 잎, 줄기 추출물 100, 300 μg/mL 농도의 시료를 각각 처리한 경우, 미토콘드리아 DNA(mtDNA) 카피 수는 정상 대조군과 비슷한 수준인 1.0-fold로 각각 증가하였다. 미토콘드리아 DNA(mtDNA) 카피 수의 증가는 미토콘드리아 생성이 증가되었음을 직접적으로 보여주는 지표로 10 μM의 Ascorbic acid(Vit C)는 미토콘드리아 DNA 합성을 유의미하게 증가시키지 못하였으나 돌가시나무 지상부, 잎, 줄기 추출물의 경우, 미토콘드리아의 생성을 증가시킨다는 것을 확인하였다.As a result of the experiment, the copy number of mitochondrial DNA (mtDNA) was reduced to 0.49-fold in the case of the negative control group treated with hydrogen peroxide compared to the normal control group. In addition, in the case of the experimental group treated with 10 μM of ascorbic acid, it was reduced to a level similar to that of the negative control group by 0.62-fold compared to the normal control group. When samples with concentrations of 100 and 300 μg/mL of extracts of the above-ground parts of the hornbeam tree, leaf, and stem were treated, respectively, the mitochondrial DNA (mtDNA) copy number increased to 1.0-fold, which is similar to that of the normal control group. The increase in mitochondrial DNA (mtDNA) copy number is a direct indicator of increased mitochondrial production. Ascorbic acid (Vit C) at 10 μM did not significantly increase mitochondrial DNA synthesis. In this case, it was confirmed that the production of mitochondria was increased.
[시험예 4: 돌가시나무 추출물이 미토콘드리아의 ATP 합성에 미치는 영향 확인][Test Example 4: Confirmation of Effect of Ash Tree Extract on Mitochondrial ATP Synthesis]
본 시험예에서는 미토콘드리아의 주 기능은 ATP합성으로, 돌가시나무 추출물이 미토콘드리아 내의 ATP 합성에 미치는 영향을 확인하고자 하였다. 사람의 정상섬유아세포인 CCD-986sk 세포를 12-웰 플레이트에 5X104 cells/well의 농도로 접종하고, 37℃, 5% CO2 조건에서 24시간 동안 배양하였다. 그 후, 배양 배지를 제거하고, 혈청이 포함되지 않은 배지에 실시예 1에 따라 제조된 돌가시나무 지상부, 잎, 줄기 추출물을 100, 300 μg/mL 의 농도로 희석된 시료를 well 당 1 ml씩 처리하여 24시간 동안 추가 배양하였다. 그 후, 300 μM의 과산화수소를 1시간 동안 처리한 세포를 PBS로 세척하고, 100 ℃의 증류수를 가해 여러 번 섞어주어 세포를 수거하였다. 수거한 세포를 4 ℃에서 5분간 12,000 xg로 원심분리하여 얻은 상층액 10 ㎕와 미리 준비한 ATP 어세이 혼합물 90 ㎕ 넣고 흡광값을 측정하였다. 정상 대조군으로는 시료와 과산화수소를 처리하지 않은 well을 사용하여 정상 대조군의 세포 생존율을 100%으로 하여 정상 대조군 대비 과산화수소만 처리군 (음성 대조군)과 시료 및 과산화수소 처리군의 ATP 양을 계산하여 결과를 도 4에 나타내었다.In this test example, the main function of mitochondria is ATP synthesis. CCD-986sk cells, which are normal human fibroblasts, were inoculated in a 12-well plate at a concentration of 5X10 4 cells/well, and cultured at 37° C., 5% CO 2 condition for 24 hours. After that, the culture medium is removed, and the sample diluted to a concentration of 100 or 300 μg/mL of the above-mentioned rhododendron, leaf, and stem extracts prepared according to Example 1 in a serum-free medium is diluted to 1 ml per well. Each treatment was further cultured for 24 hours. Thereafter, cells treated with 300 μM hydrogen peroxide for 1 hour were washed with PBS, and distilled water at 100° C. was added and mixed several times to collect cells. The collected cells were centrifuged at 12,000 x g for 5 minutes at 4°C, and 10 μl of the supernatant obtained and 90 μl of the ATP assay mixture prepared in advance were added, and the absorbance value was measured. As a normal control, using a sample and a well not treated with hydrogen peroxide, the cell viability of the normal control was set to 100%, and the amount of ATP of the hydrogen peroxide-treated group (negative control) and the sample and hydrogen peroxide-treated group compared to the normal control group was calculated and the result was calculated. 4 is shown.
실험결과, 인간정상섬유아세포에서 무첨가 대비 과산화수소 (H2O2)에 의해 65.8%로 감소되었던 ATP 합성 정도가 돌가시나무 지상부, 잎, 줄기 추출물을 처리한 결과, 양성 대조군 보다도 ATP 생성 정도가 증가한 것을 확인하였다. As a result of the experiment, the level of ATP synthesis, which was reduced to 65.8% by hydrogen peroxide (H 2 O 2 ) in human normal fibroblasts compared to no addition, was increased by the treatment of the above-ground part, leaf, and stem extracts of hornwort. confirmed that.
[시험예 5: 돌가시나무 추출물이 ROS 생성에 미치는 영향 확인][Test Example 5: Confirmation of Effect of Ash Tree Extract on ROS Generation]
세포 내에 생성되는 활성 산소는 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA)를 사용하여 측정하였다. H2DCF-DA는 지용성 물질로, 세포 안으로 쉽게 투과하여 esterase에 의해 가수분해되어 형광물질인 DCF (dichlorofluorescein)를 생성한다. 따라서 이를 측정함으로써 활성산소 생성량을 측정할 수 있다. 물질 처리 및 과산화수소(H2O2) 처리를 통해 얻어진 세포를 DPBS로 두 번 세척 후 25 μ M의 농도로 희석한 H2DCF-DA를 100μL 가하고 37 ℃, 5% CO2 조건에서 45 min 간 배양 후, 초기에 활성산소종으로 산화된 DCF의 형광 강도를 형광플레이트리더(Ex = 485 nm, Em = 530 nm)로 측정하였다. 과산화수소만 처리한 well (음성 대조군)의 ROS 생성 정도를 100%로 하여 시료와 과산화수소 모두 처리하지 않은 군과 시료 및 과산화수소 처리 군의 ROS 생성 양을 계산하여 도 5에 나타내었다.Active oxygen generated in cells was measured using 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA). H2DCF-DA is a fat-soluble substance that easily penetrates into cells and is hydrolyzed by esterase to generate DCF (dichlorofluorescein), a fluorescent substance. Therefore, it is possible to measure the amount of active oxygen production by measuring it. After washing the cells obtained through material treatment and hydrogen peroxide (H 2 O 2 ) treatment twice with DPBS, 100 μL of H2DCF-DA diluted to a concentration of 25 μM was added, and incubated for 45 min at 37 ° C., 5% CO 2 condition. , The fluorescence intensity of DCF initially oxidized with reactive oxygen species was measured with a fluorescence plate reader (Ex = 485 nm, Em = 530 nm). Assuming that the ROS generation level of the wells treated with only hydrogen peroxide (negative control) was 100%, the ROS generation amount of the sample and the hydrogen peroxide treatment group was calculated and shown in FIG. 5 .
실험 결과, 인간정상섬유아세포에서 과산화수소 (H2O2)에 의해 증가되었던 ROS 생성 정도가 아무것도 처리하지 않은 무 첨가 대조 군과 비슷한 수준인 65.0%, 70.3%로 감소한 것을 확인하였다.As a result of the experiment, it was confirmed that the level of ROS production increased by hydrogen peroxide (H 2 O 2 ) in human normal fibroblasts was reduced to 65.0% and 70.3%, which were similar to the control group that was not treated with anything.
시험예 1, 2, 3, 4 및 5의 실험 결과에 따르면, 본 발명에 따른 돌가시나무 추출물을 처리 시 과산화수소 (H2O2)로부터 세포를 보호, 미토콘드리아 DNA의 생합성 증가, 미토콘드리아의 주된 기능인 ATP 합성, 그리고 ROS 생성을 감소시키는 효과가 있음을 확인하였다. 따라서, 본 발명에 따른 조성물은 피부 내 산화스트레스로 유도되는 피부 항산화 및 노화를 효과적으로 개선시킬 수 있음을 기대할 수 있다.According to the experimental results of Test Examples 1, 2, 3, 4 and 5, when the bramble extract according to the present invention is treated, the cells are protected from hydrogen peroxide (H 2 O 2 ), the biosynthesis of mitochondrial DNA is increased, and the main function of mitochondria is It was confirmed that there is an effect of reducing ATP synthesis and ROS production. Therefore, it can be expected that the composition according to the present invention can effectively improve skin antioxidant and aging induced by oxidative stress in the skin.
이하, 상기한 실험예의 결과를 근거로 하여, 돌가시나무 추출물을 이용한 여러 제형 예를 조성하여 제시한다. 그러나 이들 제형예는 본 발명을 설명하기 위한 것으로, 본 발명의 제형이 이들 제형예에만 국한되지 않는 다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명한 것이다.Hereinafter, on the basis of the results of the above-described experimental examples, various formulation examples using an extract of stony hornwort are formulated and presented. However, these formulation examples are for illustrating the present invention, and it is obvious to those skilled in the art that the formulations of the present invention are not limited to these formulation examples.
Claims (15)
상기 피부개선이 항산화 및 항노화인 것을 특징으로 하는 조성물.According to claim 1,
The composition, characterized in that the skin improvement is antioxidant and anti-aging.
상기 조성물은 돌가시나무 산화스트레스에 대한 피부 세포 보호용인 것을 특징으로 하는 조성물.According to claim 1,
The composition is a composition, characterized in that for protecting skin cells against oxidative stress.
상기 조성물은 돌가시나무 미토콘드리아 기능 유지 및 기능 활성화에 의한 피부 개선용인 것을 특징으로 하는 조성물.According to claim 1,
The composition is characterized in that for skin improvement by maintaining and activating the function of the mitochondria of Ash tree.
상기 조성물은 피부 주름 개선용인 것을 특징으로 하는 조성물.The method of claim 1,
The composition is a composition, characterized in that for improving skin wrinkles.
상기 조성물은 피부 활력 증진용인 것을 특징으로 하는 조성물.The method of claim 1,
The composition is a composition, characterized in that for enhancing skin vitality.
상기 조성물은 활성산소종(ROS)의 감소에 의한 피부 개선용인 것을 특징으로 하는 조성물.The method of claim 1,
The composition is characterized in that for skin improvement by the reduction of reactive oxygen species (ROS).
상기 조성물은 돌가시나무 추출물을 상기 조성물 총 중량에 대하여 0.0001~10% 중량%로 포함하는 것을 특징으로 하는 조성물.The method of claim 1,
The composition is characterized in that it comprises 0.0001 to 10% by weight based on the total weight of the composition based on the total weight of the composition.
상기 돌가시나무 추출물은 물, 유기용매 또는 이들의 혼합 용매로 추출되는 것을 특징으로 하는 조성물.According to claim 1,
The composition, characterized in that the extract is extracted with water, an organic solvent, or a mixed solvent thereof.
상기 유기용매는 메탄올, 에탄올, 프로필알코올, 부틸알코올, 글리세린, 프로필렌글리콜 및 부틸렌글리콜 중에서 선택된 1종 이상인 것을 특징으로 하는 조성물.10. The method of claim 9,
The organic solvent is a composition, characterized in that at least one selected from methanol, ethanol, propyl alcohol, butyl alcohol, glycerin, propylene glycol and butylene glycol.
상기 화장료 조성물이 수용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성 함유 클렌징, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이로 구성된 군으로부터 선택되는 어느 하나의 제형인 것을 특징으로 하는 피부개선용 화장료 조성물.12. The method of claim 11,
Any one selected from the group consisting of an aqueous solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation and spray A cosmetic composition for skin improvement, characterized in that it is a formulation.
상기 화장료 조성물은 수중유형 및 유중수형 메이크업베이스, 파운데이션, 스킨커버, 립스틱, 립그로스, 페이스파우더, 투웨이케익, 아이섀도, 치크칼라 및 아이브로우 펜슬류로 이루어진 색조화장료 제형 중에서 선택되는 어느 하나의 제형인 것을 특징으로 하는 피부개선용 화장료 조성물.12. The method of claim 11,
The cosmetic composition is any one formulation selected from the color cosmetic formulations consisting of oil-in-water and water-in-oil makeup base, foundation, skin cover, lipstick, lip gloss, face powder, two-way cake, eye shadow, cheek color and eyebrow pencils. A cosmetic composition for skin improvement, characterized in that
상기 건강기능식품 조성물은 캡슐, 정제, 과립, 분말 또는 음료 형태인 것을 특징으로 하는 피부개선용 건강기능식품 조성물.15. The method of claim 14,
The health functional food composition is a health functional food composition for skin improvement, characterized in that it is in the form of a capsule, tablet, granule, powder or beverage.
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