KR102637563B1 - Cosmetic composition for improving skin condition comprising extract of silene acaulis callus - Google Patents
Cosmetic composition for improving skin condition comprising extract of silene acaulis callus Download PDFInfo
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- KR102637563B1 KR102637563B1 KR1020220122492A KR20220122492A KR102637563B1 KR 102637563 B1 KR102637563 B1 KR 102637563B1 KR 1020220122492 A KR1020220122492 A KR 1020220122492A KR 20220122492 A KR20220122492 A KR 20220122492A KR 102637563 B1 KR102637563 B1 KR 102637563B1
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- South Korea
- Prior art keywords
- moss
- arctic
- skin
- extract
- callus
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9789—Magnoliopsida [dicotyledons]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/005—Preparations for sensitive skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/52—Stabilizers
- A61K2800/522—Antioxidants; Radical scavengers
Abstract
본 발명은 북극이끼장구채 캘러스 배양 추출물을 포함하는 피부 개선용 화장료 조성물에 관한 것이다.
본 발명의 화장료 조성물은 피부 세포에 독성이 없으면서도, 피부 미백, 피부 염증 완화, 피부 자극 완화, 주름 개선, 항노화, 항산화 및 피부 보습 효과를 가지는 바, 북극이끼장구채 캘러스 배양 추출물을 포함하는 화장료 조성물은 피부 개선용으로 유용하게 사용될 수 있다.The present invention relates to a cosmetic composition for improving skin containing a callus culture extract of Arctic moss Janguchae.
The cosmetic composition of the present invention is non-toxic to skin cells and has skin whitening, skin inflammation relief, skin irritation relief, wrinkle improvement, anti-aging, antioxidant and skin moisturizing effects, and is a cosmetic composition containing Arctic moss callus culture extract. The composition can be usefully used for skin improvement.
Description
본 발명은 북극이끼장구채 캘러스 배양 추출물을 포함하는 피부 개선용 화장료 조성물에 관한 것이다.The present invention relates to a cosmetic composition for improving skin containing a callus culture extract of Arctic moss Janguchae.
북극 툰드라는 얼음과 추위, 척박한 토양 등으로 인해, 식물이 살아가기 어려운 극한 환경이지만, 이러한 극한 환경에서 자생하는 식물들은 춥고 건조하며 자외선이 강한 환경에서 자라기 때문에 다른 지역 식물과 차별화된 2차대사 물질을 보유하고 있어, 피부 생명력을 높일 수 있는 강인한 성분들이 다량 함유되어 있는 것으로 알려져 있다.The Arctic tundra is an extreme environment in which plants find it difficult to survive due to ice, cold, and barren soil. However, plants that grow in this extreme environment grow in a cold, dry, and strong ultraviolet environment, so they have a secondary metabolism that is differentiated from plants in other regions. It is known to contain a large amount of strong ingredients that can increase skin vitality.
남극에 서식하고 있는 관속식물은 2종에 불과하지만, 북극에는 1,800여종의 관속식물이 서식하고 있으며, 이러한 식물들은 대부분 극지와 고산지대에 분포하고 있으므로, 우리나라에 서식하는 식물과는 뚜렷하게 차별화되기 때문에, 이를 원료로 한 화장품 연구개발이 많이 이루어지고 있다.Although there are only two species of vascular plants living in Antarctica, there are over 1,800 species of vascular plants living in the Arctic. Most of these plants are distributed in polar regions and alpine areas, so they are clearly differentiated from plants living in Korea. , a lot of research and development is being conducted on cosmetics using this raw material.
노르웨이어로 ‘차가운 해변의 땅’이라는 뜻을 가진 스발바르 전역에서 널리 발견되는 가장 흔한 식물 중 하나인 북극이끼장구채(학명: Silene acaulis (L.) Jacq.)는 석죽과(Caryophyllaceae)에 속하는 다년생 식물로, 일반적으로 Moss Campion 또는 Cushion pink 라고 알려진 고산툰드라 식물이다. 꽃이 남쪽에서 먼저 피어나 살아있는 툰드라의 나침반으로도 알려져 있다. 수명이 긴 다년생 초본으로 한 개체가 방석 모양이며 높이는 2~8cm까지 자라고 곧은 뿌리가 있다. 잎은 밀집하여 마주 나고 모양이 가늘고 끝이 뾰족하다. 죽은 잎은 수년 간 남아 줄기 안쪽에서 잔가지를 보호한다. 꽃잎은 5개이며 줄기 끝에서 한 개씩 피는 꽃은 양성화 또는 암꽃이며, 열매는 삭과(열매 속이 여러 칸으로 나뉘고 칸 속에 종자가 든 구조)이다. 북극이끼장구채는 다양한 기질에서 자라며, 그린란드, 러시아, 미국 북서부, 스발바르, 아이슬란드, 알래스카, 캐나다 북부에 분포하는 것으로 알려져 있다.Silene acaulis (L.) Jacq., one of the most common plants widely found throughout Svalbard, which means 'cold seaside land' in Norwegian, is a perennial plant belonging to the Caryophyllaceae family. It is an alpine tundra plant commonly known as Moss Campion or Cushion pink. Since the flowers bloom first in the south, it is also known as the compass of the living tundra. It is a long-lived perennial herb. Each individual is cushion-shaped, grows up to 2~8cm in height, and has straight roots. The leaves grow densely opposite each other, are thin in shape, and have pointed ends. Dead leaves remain for several years, protecting twigs inside the stem. It has 5 petals, and the flowers that bloom one at a time at the end of the stem are either bisexual or female, and the fruit is a capsule (a structure in which the fruit is divided into several compartments and contains seeds). Arctic moss grows on a variety of substrates and is known to be distributed in Greenland, Russia, the northwestern United States, Svalbard, Iceland, Alaska, and northern Canada.
'캘러스(Callus)'는 식물체에 상처가 났을 때, 세포가 분열 능력을 회복하여 상처를 막고 비대하여 지는 유상조직으로, 식물체에서 잘라낸 조직을 옥신을 함유한 배지에서 배양하는 방법 등에 의해 생기는 특수한 조직덩어리를 의미한다. 식물은 구성요소인 잎, 줄기, 뿌리 등 그 일부만을 이식하여도 전체 개체가 다시 형성되는 전형성능(totipotency)이 뛰어나다. 캘러스는 식물세포 배양, 특히 조직배양을 통하여 식물의 어느 부분에서도 유도될 수 있으며, 계대 배양에 의해 계속해서 만들어 낼 수 있다는 특징을 가지고 있다. 이러한 캘러스의 특징을 이용하여 다양한 방면에 활용하고자 하는 관심이 고조되고 있으며, 관련 연구가 활발한 추세이다.'Callus' is a callus tissue that, when a plant is injured, cells regain their ability to divide, block the wound, and enlarge. It is a special tissue created by culturing tissue cut from the plant in a medium containing auxin. It means lump. Plants have excellent totipotency, allowing the entire plant to be re-formed by transplanting only part of its components, such as leaves, stems, and roots. Callus can be derived from any part of the plant through plant cell culture, especially tissue culture, and has the characteristic of being continuously produced through subculture. There is growing interest in utilizing these characteristics of callus in various fields, and related research is active.
이러한 배경하에, 본 발명자들은 북극이끼장구채 캘러스 배양 추출물이 피부 미백, 항염, 자극완화, 주름개선, 항산화 및 보습 효능이 있음을 확인하고, 본 발명을 완성하였다.Against this background, the present inventors confirmed that the Arctic moss Janguchae callus culture extract has skin whitening, anti-inflammatory, irritation relief, wrinkle improvement, antioxidant and moisturizing effects, and completed the present invention.
본 발명은 전술한 문제 및 이와 연관된 다른 문제를 해결하는 것을 목적으로 한다.The present invention aims to solve the above-described problems and other problems associated therewith.
본 발명의 일 예시적 목적은 북극이끼장구채 캘러스 배양 추출물을 포함하는 피부 개선용 화장료 조성물을 제공하는 것이다.An exemplary object of the present invention is to provide a cosmetic composition for improving skin containing a culture extract of Arctic moss Janguchae callus.
본 발명의 다른 예시적 목적은 북극이끼장구채 캘러스 배양 추출물을 포함하는 피부 미백용 화장료 조성물을 제공하는 것이다.Another exemplary object of the present invention is to provide a cosmetic composition for skin whitening containing a callus culture extract of Arctic moss Jangguchae.
본 발명의 또 다른 예시적 목적은 북극이끼장구채 캘러스 배양 추출물을 포함하는 피부 항염용 화장료 조성물을 제공하는 것이다.Another exemplary object of the present invention is to provide a cosmetic composition for anti-inflammatory skin containing a callus culture extract of Arctic moss Jangguchae.
본 발명의 또 다른 예시적 목적은 북극이끼장구채 캘러스 배양 추출물을 포함하는 피부 자극 완화용 화장료 조성물을 제공하는 것이다.Another exemplary object of the present invention is to provide a cosmetic composition for alleviating skin irritation containing a callus culture extract of Arctic moss Jangguchae.
본 발명의 또 다른 예시적 목적은 북극이끼장구채 캘러스 배양 추출물을 포함하는 피부 주름 개선용 화장료 조성물을 제공하는 것이다.Another exemplary object of the present invention is to provide a cosmetic composition for improving skin wrinkles containing Arctic moss Janguchae callus culture extract.
본 발명의 또 다른 예시적 목적은 북극이끼장구채 캘러스 배양 추출물을 포함하는 피부 항산화용 화장료 조성물을 제공하는 것이다.Another exemplary object of the present invention is to provide a cosmetic composition for skin antioxidant containing Arctic moss Janguchae callus culture extract.
본 발명의 또 다른 예시적 목적은 북극이끼장구채 캘러스 배양 추출물을 포함하는 피부 보습용 화장료 조성물을 제공하는 것이다.Another exemplary object of the present invention is to provide a cosmetic composition for moisturizing skin containing a callus culture extract of Arctic moss Jangguchae.
본 명세서에 개시된 발명의 기술적 사상에 따라 이루고자 하는 기술적 과제는 이상에서 언급한 문제점을 해결하기 위한 과제로 제한되지 않으며, 언급되지 않은 또 다른 과제는 아래의 기재로부터 통상의 기술자에게 명확하게 이해될 수 있을 것이다.The technical problem to be achieved according to the technical idea of the invention disclosed in this specification is not limited to the problem to solve the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.
이를 구체적으로 설명하면 다음과 같다. 한편, 본 출원에서 개시된 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본 출원에서 개시된 다양한 요소들의 모든 조합이 본 출원의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 출원의 범주가 제한된다고 볼 수 없다.This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Additionally, the scope of the present application cannot be considered limited by the specific description described below.
상기 목적을 달성하기 위한 일 양태로서, 본 발명은 북극이끼장구채 캘러스 배양 추출물을 포함하는 화장료 조성물을 제공한다.As an aspect for achieving the above object, the present invention provides a cosmetic composition containing a callus culture extract of Arctic moss Jangguchae.
본 발명의 용어 "북극이끼장구채(Silene acaulis)"는 유라시아와 북미의 높은 산 및 툰드라 전역에 서식하는 야생화로서, 석죽과(Caryophyllaceae)에 속하는 상록 다년생 꽃 식물을 의미한다.The term "Silene acaulis" of the present invention refers to an evergreen perennial flowering plant belonging to the Caryophyllaceae family, which is a wild flower that grows throughout the high mountains and tundra of Eurasia and North America.
본 발명의 용어 "캘러스(callus)"는 식물체에서 잘라낸 조직을 옥신을 함유한 배지에서 배양하거나, 어떤 종류의 식물에 상처를 내거나, 상처 부위를 옥신으로 처리하거나 할 때에 생기는 미분화 조직 또는 세포덩어리를 말하는 것으로서, 새로운 배지에 계대 배양함으로써 영구적으로 분열 및 증식할 수 있다.The term "callus" of the present invention refers to an undifferentiated tissue or cell mass that is formed when a tissue cut from a plant is cultured in a medium containing auxin, a certain type of plant is wounded, or the wounded area is treated with auxin. In other words, they can permanently divide and proliferate by subculturing them in new media.
본 발명에 있어서, 북극이끼장구채 캘러스는 이끼장구채 캘러스 자체, 이끼장구채의 일부, 예를 들어 잎, 줄기, 뿌리 등 또는 그 일부를 유도배지에서 배양을 통해 얻어지는 것으로서, 이끼장구채 식물체의 일부를 배양하여 유도한 캘러스, 또는 캘러스가 배양된 배양액을 포함한다.In the present invention, Arctic moss acacia callus is obtained by culturing the moss acacia callus itself, parts of the moss acacia, such as leaves, stems, roots, etc., or parts thereof, in an induction medium, and culturing a part of the moss acacia plant. Includes induced callus or culture medium in which callus is cultured.
본 발명의 용어 "배양 추출물"은 상기 북극이끼장구채 캘러스 배양물의 추출처리에 의하여 얻어지는 추출액, 상기 추출액의 희석액이나 농축액, 상기 추출물을 건조하여 얻어지는 건조물, 상기 추출액의 조정제물이나 정제물, 또는 이들의 혼합물 등, 추출액 자체 및 추출액을 이용하여 형성 가능한 모든 제형의 추출물을 포함하는 것으로, 상기 이끼장구채 캘러스 배양물을 분말화하거나, 감압추출법, 냉수추출법, 열수추출법, 에탄올 추출법 등 종래 알려진 다양한 추출법에 의해 수득한 추출물을 의미한다.The term "culture extract" of the present invention refers to an extract obtained by extraction treatment of the Arctic moss Janguchae callus culture, a diluted or concentrated liquid of the extract, a dried product obtained by drying the extract, a crude product or purified product of the extract, or a product thereof. It includes extracts of all formulations that can be formed using the extract itself and the extract, such as mixtures, by pulverizing the Moss Jangjichae callus culture or by various conventionally known extraction methods such as reduced pressure extraction, cold water extraction, hot water extraction, and ethanol extraction. It refers to the obtained extract.
본 발명에서 추출 방법은 특별히 제한되지 않고, 예를 들어 주정 추출, 냉침 추출, 초음파 추출, 환류 추출, 열수 추출 등이 있다.In the present invention, the extraction method is not particularly limited and includes, for example, alcohol extraction, cold extraction, ultrasonic extraction, reflux extraction, and hot water extraction.
상기 열수 추출의 경우, 열탕증류기에서 2~24시간동안, 80 내지 100℃ 로 가열하여 열수 추출물을 얻을 수 있다.In the case of the hot water extraction, the hot water extract can be obtained by heating at 80 to 100° C. for 2 to 24 hours in a hot water distiller.
상기 냉침 추출의 경우, 냉수(15-25℃)와 상기 캘러스 배양물 자체 또는 그것의 건조된 분말을 혼합하여 3일동안 추출하여 추출물을 얻을 수 있다.In the case of the cold immersion extraction, the extract can be obtained by mixing cold water (15-25°C) with the callus culture itself or its dried powder and extracting for 3 days.
추출 용매는 물, 유기 용매, 또는 이의 혼합 용매를 사용할 수 있으며, 상기 추출 방법을 이용하여 추출한 액은 바로 사용하거나 또는 농축 및/또는 건조하여 사용할 수 있다. The extraction solvent may be water, an organic solvent, or a mixed solvent thereof, and the liquid extracted using the above extraction method may be used directly or after being concentrated and/or dried.
상기 유기 용매는 메탄올, 에탄올, 이소프로판올, 부탄올, 에틸렌, 아세톤, 헥산, 에테르, 클로로포름, 에틸아세테이트, 부틸아세테이트, 디클로로메탄, N, N-디메틸포름아미드(DMF), 디메틸설폭사이드(DMSO), 1,3-부틸렌글리콜, 프로필렌글리콜 또는 이들의 혼합용매일 수 있으며, 이러한 유기용매를 이용하는 경우에는 활성 성분이 파괴되지 않거나 최소화된 조건에서 실온 또는 가온하여 추출물을 제조할 수 있다. 추출하는 유기용매에 따라 식물이 함유하고 있는 활성 성분의 추출정도와 손실정도가 차이가 날 수 있으므로, 알맞은 유기용매를 선택하여 사용하도록 한다.The organic solvents include 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 mixed solvent thereof. When using such an organic solvent, the extract can be prepared at room temperature or by heating under conditions in which the active ingredient is not destroyed or is minimized. Depending on the organic solvent being extracted, the degree of extraction and loss of the active ingredients contained in the plant may vary, so select and use an appropriate organic solvent.
본 발명에 있어서, 상기 추출물은 농축, 또는 희석하여 사용할 수 있고, 추출물의 증류액을 사용할 수도 있다.In the present invention, the extract can be used concentrated or diluted, and a distillate of the extract can also be used.
본 발명에 있어서, "피부 개선"이란 본 발명의 북극이끼장구채 캘러스 배양 추출물을 사용하여 피부가 호전되도록 하거나 이롭게 되도록 하는 모든 행위를 의미한다.In the present invention, “skin improvement” means any action that improves or benefits the skin using the Arctic moss callus culture extract of the present invention.
본 발명의 상기 피부 개선은 일 예시로, 피부 미백, 피부 염증 완화, 피부 자극 완화, 주름 개선, 항노화, 항산화 및/또는 피부 보습을 의미할 수 있으나, 이에 제한되는 것은 아니다.The skin improvement of the present invention may mean, as an example, skin whitening, skin inflammation relief, skin irritation relief, wrinkle improvement, anti-aging, antioxidant and/or skin moisturizing, but is not limited thereto.
본 발명에 있어서, "주름 개선"은 피부의 주름 및 탄력을 유지 또는 강화시키는 것을 의미한다.In the present invention, “wrinkle improvement” means maintaining or strengthening wrinkles and elasticity of the skin.
본 발명에 있어서, "항노화"는 피부 노화 방지를 의미하는 것으로, 시간이 흐름에 따라 신체의 생리적 변화가 발생하여 나타나는 자연적 노화인 연대학적 노화(내인성 노화)와 햇빛에 노출되는 부위에서 발생하는 광노화(광인성 노화)를 예방 또는 지연시키는 것 모두를 포함한다.In the present invention, "anti-aging" refers to preventing skin aging, and includes chronological aging (intrinsic aging), which is natural aging that occurs due to physiological changes in the body over time, and natural aging that occurs in areas exposed to sunlight. This includes everything that prevents or delays photoaging (photogenic aging).
본 발명에 있어서, "피부 보습"은 피부에 수분감을 증가시켜주고, 촉촉한 상태를 유지시키는 것을 의미한다. 피부 보습 효과를 높일 경우 피부의 주름 개선, 탄력도 증가에도 이로운 영향을 미칠 수 있다.In the present invention, “skin moisturizing” means increasing moisture in the skin and maintaining a moist state. Increasing the skin moisturizing effect can have a beneficial effect on improving skin wrinkles and increasing elasticity.
본 발명의 실험예에서는 북극이끼장구채 캘러스 배양 추출물이 피부 자극 없이 피부 개선 효과를 나타냄을 확인하였다. 구체적으로, 북극이끼장구채 캘러스 배양 추출물의 멜라닌 생성 억제를 통한 미백 효과를 확인하였으며, 북극이끼장구채 캘러스 배양 추출물에 의해 염증 관련 인자(일산화 질소, NO)의 생성량이 감소함을 확인하였다. 또한, 피부자극 유발물질(SDS)에 의한 세포 생존율 감소가 북극이끼장구채 캘러스 배양 추출물에 의해 완화됨을 확인하였고, 북극이끼장구채 캘러스 배양 추출물에 의해 PIP(Procollagen Type 1 C-Peptide)의 생성이 촉진되고, MMP-1(콜라겐분해효소) 생성이 감소됨을 확인하여 주름 개선 효과가 있음을 확인하였다. 또한, 북극이끼장구채 캘러스 배양 추출물의 ABTS 라디컬 소거능 및 활성산소(ROS) 제거능을 확인하였으며, 북극이끼장구채 캘러스 배양 추출물을 함유한 조성물을 도포한 경우, 북극이끼장구채 캘러스 배양 추출물을 함유하지 않은 조성물의 경우보다 우수한 보습력이 지속됨을 확인하고, 경피수분손실량 역시 개선되었음을 확인하였다.In an experimental example of the present invention, it was confirmed that the Arctic moss Janguchae callus culture extract showed a skin improvement effect without skin irritation. Specifically, the whitening effect of the Arctic moss callus culture extract was confirmed by inhibiting melanin production, and the production of inflammation-related factors (nitric oxide, NO) was confirmed to be reduced by the Arctic moss callus culture extract. In addition, it was confirmed that the decrease in cell survival rate caused by skin irritants (SDS) was alleviated by the Arctic Moss Janguchae callus culture extract, and the production of PIP (Procollagen Type 1 C-Peptide) was promoted by the Arctic Moss Janguchae callus culture extract. , the production of MMP-1 (collagen degrading enzyme) was confirmed to be reduced, confirming the wrinkle improvement effect. In addition, the ABTS radical scavenging ability and reactive oxygen species (ROS) removal ability of the Arctic Mossy callus culture extract were confirmed, and when a composition containing the Arctic Moss Janguchae callus culture extract was applied, a composition that did not contain the Arctic Moss Janguchae callus culture extract It was confirmed that excellent moisturizing power was maintained compared to the case of , and transepidermal water loss was also confirmed to be improved.
본 발명에 있어서, 상기 북극이끼장구채 캘러스 배양 추출물은 화장료 조성물의 총 중량에 대하여 0.1 내지 60 중량%로 함유될 수 있는 것으로, 본 발명에 따른 화장료 조성물은 상기 북극이끼장구채 캘러스 배양 추출물 외에도 필요에 따라 본 발명의 효과를 저하시키지 않는 범위 내에서 화장료 조성물에 일반적으로 사용하는 각종 성분, 예를 들면 수용성 성분, 분말성분, 유분, 계면활성제, 보습제, 점도조절제, 방부제, 산화방지제, 향료, 색소 등을 배합하여 구성될 수 있다.In the present invention, the Arctic Moss Janguchae callus culture extract may be contained in an amount of 0.1 to 60% by weight based on the total weight of the cosmetic composition, and the cosmetic composition according to the present invention may contain the Arctic Moss Janguchae callus culture extract as needed. Various ingredients commonly used in cosmetic compositions, such as water-soluble ingredients, powder ingredients, oils, surfactants, moisturizers, viscosity modifiers, preservatives, antioxidants, fragrances, colorants, etc., are added within the range that does not reduce the effect of the present invention. It can be composed by combining.
상기 사용 가능한 계면활성제의 비제한적인 예로는, 음이온계 계면활성제, 양이온성 계면활성제, 비이온성 계면활성제, 양쪽성 계면활성제 등이 있다. 보다 구체적으로, 상기 음이온계 계면활성제로는 알킬벤젠설폰산염, 폴리옥시알킬렌알킬황산 에스테르염, 알킬황산 에스테르염, 올레핀설폰산염, 알킬인산염, 폴리옥시알킬렌알킬에테르인산염, 디알킬설포석신산염, 지방산염 등을 들 수 있고, 비이온성 계면활성제로서, 폴리옥시에틸렌알킬에테르, 폴리옥시에틸렌지방산 에스테르, 다가 알콜지방산 부분 에스테르, 폴리옥시에틸렌 다가 알콜지방산 부분 에스테르, 폴리글리세린지방산 에스테르, 폴리옥시에틸렌 경화 피마자유 유도체, 지방산디에탄올아미드 등을 들 수 있다. 또한, 양이온성 계면활성제로서는, 3급 지방족 아민염, 알킬트리메틸암모늄 할라이드, 디알킬디메틸암모늄할라이드 등을 들 수 있고, 양쪽성 계면활성제로서는, 아미드베타인형, 이미다졸리늄베타인형, 설포베타인형 등을 들 수 있다.Non-limiting examples of the surfactants that can be used include anionic surfactants, cationic surfactants, nonionic surfactants, and amphoteric surfactants. More specifically, the anionic surfactant includes alkylbenzene sulfonate, polyoxyalkylene alkyl sulfuric acid ester salt, alkyl sulfuric acid ester salt, olefin sulfonate, alkyl phosphate, polyoxyalkylene alkyl ether phosphate, and dialkyl sulfosuccine. Salts, fatty acid salts, etc. can be mentioned, and nonionic surfactants include polyoxyethylene alkyl ether, polyoxyethylene fatty acid ester, polyhydric alcohol fatty acid partial ester, polyoxyethylene polyhydric alcohol fatty acid partial ester, polyglycerol fatty acid ester, and polyoxyethylene fatty acid ester. Examples include ethylene hydrogenated castor oil derivatives and fatty acid diethanolamide. In addition, cationic surfactants include tertiary aliphatic amine salts, alkyltrimethylammonium halides, dialkyldimethylammonium halides, etc., and examples of amphoteric surfactants include amide betaine type, imidazolinium betaine type, and sulfobetaine type. etc. can be mentioned.
상기 보습제로서는, 글리세린, 프로필렌글리콜, 1,3-부틸렌글리콜, 디프로필렌글리콜, 소르비톨 등을 들 수 있다. 상기 방부제로서는, 벤조산, 데하이드로아세트산, 파라옥시벤조산에스테르(파라옥시벤조산메틸, 파라옥시벤조산부틸 등), 페녹시에탄올 등을 들 수 있다. 또한, 상기 산화방지제로서는, 아스코르브산, BHA 등을 들 수 있으며, 이외에도, 자외선 흡수제, 소염제 및 청량제 등을 첨가할 수 있다.Examples of the moisturizing agent include glycerin, propylene glycol, 1,3-butylene glycol, dipropylene glycol, and sorbitol. Examples of the preservative include benzoic acid, dehydroacetic acid, paraoxybenzoic acid ester (methyl paraoxybenzoate, butyl paraoxybenzoate, etc.), phenoxyethanol, etc. In addition, the antioxidants include ascorbic acid, BHA, etc., and in addition, ultraviolet absorbers, anti-inflammatory agents, fresheners, etc. can be added.
본 발명의 화장료 조성물은 용액, 외용 연고, 크림, 폼, 영양 화장수, 유연 화장수, 팩, 유연수, 유액, 메이크업 베이스, 에센스, 비누, 액체 세정료, 입욕제, 선 스크린 크림, 선오일, 현탁액, 유탁액, 페이스트, 겔, 로션, 파우더, 비누, 계면 활성제-함유 클렌징, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션, 패취 및 스프레이로 이루어진 군으로부터 선택되는 제형으로 제조할 수 있으나, 이에 제한되는 것은 아니다.The cosmetic composition of the present invention includes solution, external ointment, cream, foam, nutritional lotion, softening lotion, pack, softening water, emulsion, makeup base, essence, soap, liquid cleanser, bath agent, sunscreen cream, sun oil, suspension, and emulsion. It can be prepared in a formulation selected from the group consisting of liquid, paste, gel, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, patch and spray. no.
본 발명의 화장료 조성물은 화장품 제제에 있어서 수용 가능한 담체를 1종 이상 추가로 포함할 수 있으며, 통상의 성분으로 예를 들면 유분, 물, 계면 활성제, 보습제, 저급 알코올, 증점제, 킬레이트제, 색소, 방부제, 향료 등을 적절히 배합할 수 있으나, 이에 제한되는 것은 아니다. 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. 상기 담체는 본 발명의 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 조성물이 제조되는 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 cosmetic composition of the present invention may further include one or more carriers acceptable in cosmetic formulations, and common ingredients include, for example, oil, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, pigments, Preservatives, fragrances, etc. may be appropriately mixed, but are not limited thereto. Here, “acceptable carrier in cosmetic preparations” refers to compounds or compositions 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 to upon contact with the skin. It says something that doesn't exist. The carrier may be included in the composition of the present invention in an amount of about 1% by weight to about 99.99% by weight based on the total weight thereof, preferably about 90% by weight to about 99.99% by weight of the weight of the composition. However, since the ratio varies depending on the formulation in which the composition of the present invention is manufactured, its specific application area (face, neck, etc.), and its preferred application amount, the ratio does not limit the scope of the present invention in any respect. It should not be understood as
본 발명의 화장료 조성물에 포함되는 화장품 제제에 있어서 수용 가능한 담체는 화장료 조성물의 제형에 따라 다양하다.In the cosmetic formulation included in the cosmetic composition of the present invention, acceptable carriers vary depending on the formulation of the cosmetic composition.
본 발명의 제형이 연고, 페이스트, 크림 또는 젤인 경우에는, 담체 성분으로서 동물성 유, 식물성 유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크, 산화 아연 등이 이용될 수 있으나, 이에 제한되는 것은 아니다. 이들은 단독으로 사용되거나 2종 이상 혼합되어 사용될 수 있다.When the formulation of the present invention is an ointment, paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. are used as carrier ingredients. may be used, but is not limited thereto. These may be used alone or in combination of two or more types.
본 발명의 제형이 파우더 또는 스프레이인 경우에는, 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄 히드록사이드, 칼슘 실케이트, 폴리아미드 파우더 등이 이용될 수 있고, 특히 스프레이인 경우에는 추가적으로 클로로플루오로하드로카본, 프로판/부탄 또는 디메틸 에테르와 같은 추진제를 포함할 수 있으나, 이에 제한되는 것은 아니며, 이들은 단독으로 사용되거나 2종 이상 혼합되어 사용될 수 있다.When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silcate, polyamide powder, etc. may be used as carrier ingredients, and especially in the case of a spray, chlorofluorohydride may be additionally used. It may include propellants such as locarbon, propane/butane, or dimethyl ether, but is not limited thereto, and these may be used alone or in a mixture of two or more types.
본 발명의 제형이 용액 또는 유탁액인 경우에는, 담체 성분으로서 용매, 용해화제 또는 유탁화제 등이 이용될 수 있으며, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌 글리콜, 1,3-부틸글리콜 오일 등이 이용될 수 있고, 특히, 목화씨 오일, 땅콩 오일, 옥수수 배종 오일, 올리브 오일, 피마자 오일 및 참깨 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르가 이용될 수 있으나, 이에 제한되는 것은 아니며, 이들은 단독으로 사용되거나 2종 이상 혼합되어 사용될 수 있다.When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent, or emulsifying agent may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, Propylene glycol, 1,3-butyl glycol oil, etc. can be used, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic esters, polyethylene glycol or fatty acid esters of sorbitan. may be used, but is not limited thereto, and may be used alone or in a mixture of two or more types.
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 메타하이드록시드, 벤토나이트, 아가 또는 트라칸트 등이 이용될 수 있으나, 이에 제한되는 것은 아니며, 이들은 단독으로 사용되거나 2종 이상 혼합되어 사용될 수 있다.When the formulation of the present invention is a suspension, the carrier components include water, liquid diluents such as ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, and miso. Crystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used, but are not limited thereto, and they may be used alone or in a mixture of two or more types.
본 발명의 제형이 비누인 경우에는, 담체 성분으로서 지방산의 알칼리 금속 염, 지방산 헤미에스테르 염, 지방산 단백질 히드롤리제이트, 이세티오네이트, 라놀린 유도체, 지방족 알코올, 식물성 유, 글리세롤, 당 등이 이용될 수 있으나, 이에 제한되는 것은 아니며, 이들은 단독으로 사용되거나 2종 이상 혼합되어 사용될 수 있다.When the formulation of the present invention is soap, alkali metal salts of fatty acids, hemiester salts of fatty acids, fatty acid protein hydrolysates, isethionates, lanolin derivatives, fatty alcohols, vegetable oils, glycerol, sugars, etc. are used as carrier components. It may be, but is not limited to, these may be used alone or in combination of two or more types.
상기 목적을 달성하기 위한 다른 양태로서, 본 발명은 (a) 북극이끼장구채 식물로부터 캘러스를 배양하는 단계; 및In another aspect for achieving the above object, the present invention includes the steps of (a) culturing callus from Arctic moss Janguchae plants; and
(b) 상기 (a)단계에서 얻은 캘러스 배양물 또는 상기 배양물의 추출물을 함유하는 조성물을 제조하는 단계.(b) Preparing a composition containing the callus culture obtained in step (a) or an extract of the culture.
본 발명의 상기 배양을 위하여 적절한 배지를 사용할 수 있으며, 본 발명의 기술분야에서 캘러스 배양에 일반적으로 사용되고 있는 배지가 있다면 제한 없이 사용가능하다.An appropriate medium can be used for the above-mentioned culture of the present invention, and any medium commonly used for callus culture in the technical field of the present invention can be used without limitation.
일 실시예로, 본 발명의 캘러스 배양을 위해 MS 배지를 사용할 수 있다. 상기 MS 배지는 제아틴, 수크로스 및 아가(agar)를 포함할 수 있으며, 바람직하게는 0.1 내지 10 mg/L 제아틴(Zeatin), 1 내지 10 % 수크로스(Sucrose) 및 아가(agar) 1 내지 15 g/L를 포함할 수 있고, 더 바람직하게는 0.5 내지 3 mg/L 제아틴(Zeatin), 2 내지 5 % 수크로스(Sucrose) 및 아가(agar) 5 내지 10 g/L를 포함할 수 있다.In one example, MS medium can be used for callus culture of the present invention. The MS medium may contain zeatin, sucrose, and agar, preferably 0.1 to 10 mg/L zeatin, 1 to 10% sucrose, and agar 1. to 15 g/L, more preferably 0.5 to 3 mg/L Zeatin, 2 to 5% sucrose, and 5 to 10 g/L agar. You can.
일 실시예로, 본 발명의 캘러스 배양은 북극이끼장구채 식물의 전부 또는 일부 조직 또는 세포를 상기 전술한 MS 배지에서 명배양 또는 암배양하는 것일 수 있고, 바람직하게는 암배양하는 것일 수 있다.In one embodiment, the callus culture of the present invention may be light or dark culture, preferably dark culture, of all or part of the tissues or cells of the Arctic moss plant in the above-mentioned MS medium.
본 발명에 있어서 상기 배양물의 추출은 특별히 제한되지 아니하며, 당해 기술 분야에서 통상적으로 사용하는 방법에 따라 추출할 수 있다. 상기 추출 방법의 비제한적인 예로는, 열수 추출법, 냉침 추출법, 용매 추출법, 수증기 증류법, 용출법, 압착법, 초음파 추출법, 여과법, 환류 추출법 등이 있으며, 이들은 단독으로 수행되거나 2종 이상의 방법을 병용하여 수행될 수 있다.In the present invention, extraction of the culture is not particularly limited and can be extracted according to methods commonly used in the art. Non-limiting examples of the extraction method include hot water extraction, cold immersion extraction, solvent extraction, steam distillation, elution, compression, ultrasonic extraction, filtration, and reflux extraction, which are performed alone or using a combination of two or more methods. It can be performed by doing this.
일 실시예로, 본 발명의 추출 단계는 열수 추출에 의한 것일 수 있다. 바람직하게는 70 ℃ 내지 140 ℃에서 1시간 내지 24시간 열수 추출하는 것일 수 있으며, 더욱 바람직하게는 90 ℃ 내지 120 ℃에서 4시간 열수 추출하는 것일 수 있다.In one embodiment, the extraction step of the present invention may be by hot water extraction. Preferably, hydrothermal extraction may be performed at 70°C to 140°C for 1 hour to 24 hours, and more preferably, hydrothermal extraction may be performed at 90°C to 120°C for 4 hours.
본 발명의 상기 추출물은 적절한 용매를 이용하여 북극이끼장구채 캘러스로부터 추출한 것이며, 예를 들어 조추출물, 극성용매 가용 추출물 또는 비극성 용매 가용 추출물을 모두 포함할 수 있다. 상기 추출물을 제조하기 위해 사용되는 추출 용매의 종류는 특별히 제한되지 아니하며, 당해 기술 분야에서 공지된 임의의 용매를 사용할 수 있다. 상기 추출 용매의 비제한적인 예로는 물; 메탄올, 에탄올, 프로판올, 이소프로판올, 부탄올, 프로필알콜, 부틸알콜 등의 탄소수 1 내지 4 저급 알콜; 글리세린, 부틸렌글리콜, 프로필렌글리콜 등의 다가 알코올; 메틸아세테이트, 에틸아세테이트, 아세톤, 벤젠, 헥산, 디에틸에테르, 디클로로메탄 등의 탄화수소계 용매; 또는 이들의 혼합물을 사용할 수 있다. 또한, 상기 용매를 사용하여 1회 이상 추출하여 용매 추출물을 제조할 수 있다.The extract of the present invention is extracted from Arctic moss Janguchae callus using an appropriate solvent, and may include, for example, a crude extract, a polar solvent-soluble extract, or a non-polar solvent-soluble extract. The type of extraction solvent used to prepare the extract is not particularly limited, and any solvent known in the art can be used. Non-limiting examples of the extraction solvent include water; lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, propanol, isopropanol, butanol, propyl alcohol, and butyl alcohol; Polyhydric alcohols such as glycerin, butylene glycol, and propylene glycol; Hydrocarbon solvents such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane; Or a mixture thereof can be used. Additionally, a solvent extract can be prepared by extracting the extract one or more times using the solvent.
본 발명의 상기 추출물은 부유하는 고체 입자를 제거하기 위해 여과, 예를 들어 메쉬(mesh)로 여과하여 고형분을 제거하고 사용하거나, 이를 동결건조, 열풍건조, 분무건조 등을 이용해 건조시켜 사용될 수 있다. 또한, 상기 추출물은 추가로 통상의 분획 공정을 거칠 수도 있으며, 통상의 정제 방법을 이용하여 정제될 수도 있다.The extract of the present invention can be used by filtering to remove floating solid particles, for example, by filtering with a mesh to remove solid content, or by drying it using freeze-drying, hot air drying, spray drying, etc. . Additionally, the extract may further undergo a conventional fractionation process or may be purified using a conventional purification method.
본 발명의 북극이끼장구채 캘러스 배양 추출물은 피부 세포에 독성이 없으면서도, 피부 미백, 피부 염증 완화, 피부 자극 완화, 주름 개선, 항노화, 항산화 및 피부 보습 등의 효능이 있어, 피부 개선용으로 유용하게 사용될 수 있다.The Arctic moss callus culture extract of the present invention is not toxic to skin cells, but has effects such as skin whitening, skin inflammation relief, skin irritation relief, wrinkle improvement, anti-aging, antioxidant and skin moisturizing, and is useful for skin improvement. It can be used effectively.
도 1은 북극 다산기지 근처에서 직접 채취한 이끼장구채 식물 및 이를 캘러스로 배양한 사진이다.
도 2는 북극이끼장구채 캘러스 배양 추출물의 멜라닌 생성 억제를 통해 미백 효과를 확인한 결과이다.
도 3은 북극이끼장구채 캘러스 배양 추출물의 NO(Nitric oxide, 염증 관련 인자)의 활성 변화를 통해 항염 효능을 평가한 결과이다.
도 4는 북극이끼장구채 캘러스 배양 추출물의 계면활성제(SDS)에 의해 유발된 피부자극(세포 생존율 감소) 완화 효과를 확인한 결과이다.
도 5는 북극이끼장구채 캘러스 배양 추출물의 프로콜라겐 1형(주름 개선 관련 단백질)의 함량 변화를 통해 항노화 효능을 확인한 결과이다.
도 6은 북극이끼장구채 캘러스 배양 추출물의 MMP-1(주름 개선 관련 단백질)의 함량 변화를 통해 항노화 효능을 확인한 결과이다.
도 7은 북극이끼장구채 캘러스 배양 추출물의 ABTS 라디컬 소거능을 통해 항산화 효능을 확인한 결과이다.
도 8은 북극이끼장구채 캘러스 배양 추출물의 활성산소(ROS) 제거능을 통해 항산화 효능을 확인한 결과이다.
도 9는 북극이끼장구채 캘러스 배양 추출물을 포함한 에센스 제형의 화장료 조성물의 도포에 따른 보습력 효능을 확인한 결과이다.
도 10은 북극이끼장구채 캘러스 배양 추출물을 포함한 크림 제형의 화장료 조성물의 도포에 따른 보습력 효능을 확인한 결과이다.
도 11은 북극이끼장구채 캘러스 배양 추출물을 포함한 에센스 제형의 화장료 조성물의 도포에 따른 경피수분손실량(TEWL)을 측정한 결과이다.
도 12는 북극이끼장구채 캘러스 배양 추출물을 포함한 크림 제형의 화장료 조성물의 도포에 따른 경피수분손실량(TEWL)을 측정한 결과이다.Figure 1 is a photograph of a Moss janguchae plant collected directly near the Dasan Station in the Arctic and cultured into callus.
Figure 2 shows the results of confirming the whitening effect of Arctic moss Janguchae callus culture extract through inhibition of melanin production.
Figure 3 shows the results of evaluating the anti-inflammatory efficacy through changes in the activity of NO (Nitric oxide, an inflammation-related factor) of the Arctic moss Janguchae callus culture extract.
Figure 4 shows the results of confirming the effect of alleviating skin irritation (reduced cell viability) caused by surfactant (SDS) of the Arctic moss Janguchae callus culture extract.
Figure 5 shows the results of confirming the anti-aging effect through changes in the content of procollagen type 1 (a protein related to wrinkle improvement) of the Arctic moss janguchae callus culture extract.
Figure 6 shows the results of confirming the anti-aging effect through changes in the content of MMP-1 (wrinkle improvement-related protein) of the Arctic moss janguchae callus culture extract.
Figure 7 shows the results of confirming the antioxidant efficacy of the Arctic moss Janguchae callus culture extract through the ABTS radical scavenging ability.
Figure 8 shows the results of confirming the antioxidant efficacy of the Arctic moss Janguchae callus culture extract through the reactive oxygen species (ROS) removal ability.
Figure 9 shows the results of confirming the moisturizing effect upon application of a cosmetic composition in an essence formulation containing a callus culture extract of Arctic moss Janguchae.
Figure 10 shows the results of confirming the moisturizing effect upon application of a cream-type cosmetic composition containing callus culture extract of Arctic moss Janguchae.
Figure 11 shows the results of measuring transepidermal water loss (TEWL) upon application of a cosmetic composition in an essence formulation containing a callus culture extract of Arctic moss Janguchae.
Figure 12 shows the results of measuring transepidermal water loss (TEWL) upon application of a cream-type cosmetic composition containing callus culture extract of Arctic moss Jangguchae.
이하, 본 발명을 하기 실시예를 통하여 보다 상세하게 설명한다. 그러나 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예만으로 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.
실시예 1. 북극이끼장구채 캘러스 배양 추출물 제조Example 1. Preparation of Arctic moss Janguchae callus culture extract
1-1. 북극이끼장구채 캘러스 유도1-1. Induction of arctic moss callus
이끼장구채 식물은 (주)바이오에프디엔씨 연구원이 북극 다산기지(Norway, Spitsbergen, Ny-Alesund, 노르웨이 스피츠베르겐섬 니알슨 과학기지촌(북위 78도)) 근처에서 직접 채취하였고, 70 % 에탄올에 30초 동안 침지한 후 멸균수로 세척하고 다시 소독액 (30 % 락스 + Tween 20)으로 20분 소독한 후 멸균수로 3회 수세한 뒤, 날카로운 칼을 이용하여 단번에 상처를 내어 1 mg/L 제아틴(Zeatin), 3% 수크로스(Sucrose) 및 아가(agar) 8g/L 가 포함된 기본 MS배지(Murashige and Skoog 1962, Duchefa 사, Cat No. M0221)에서 25℃, 습도 70%의 생장상 조건으로 암배양하여 초기 이끼장구채 캘러스를 유도하였다(도 1).The moss plant was collected directly by a researcher at BioFD&C Co., Ltd. near the Arctic Dasan Station (Norway, Spitsbergen, Ny-Alesund, Ny-Alesund Science Base Village on Spitsbergen Island, Norway (78 degrees north latitude)), and soaked in 70% ethanol for 30 seconds. After soaking for a while, wash with sterilized water, disinfect again with disinfectant solution (30% bleach + Tween 20) for 20 minutes, wash 3 times with sterilized water, then cut a wound with a sharp knife and add 1 mg/L zeatin ( Zeatin, 3% sucrose, and agar 8g/L under growth conditions of 25°C and 70% humidity on basic MS medium (Murashige and Skoog 1962, Duchefa, Cat No. M0221). Early Moss Janguchae calli were induced by culturing in the dark (Figure 1).
계대배양은 2주간격으로 진행하였고, 대량배양은 온도조건 23±2 ℃, 습도 70 % 배양실 조건에서 20 L 생물반응기(Bioreactor)(구입처:(주)삼성과학)에 공기공급량 0.1 vvm 정도로 5주간 배양하여 준비하였다.Subculture was carried out at two-week intervals, and mass culture was carried out in a 20 L bioreactor (purchased by Samsung Science Co., Ltd.) in a culture room with a temperature of 23 ± 2 ℃ and 70% humidity for 5 weeks with an air supply of 0.1 vvm. It was prepared by culturing.
1-2. 북극이끼장구채 캘러스 배양 추출물 제조1-2. Manufacture of Arctic moss janguchae callus culture extract
실시예 1-1에서 배양하여 얻은 북극이끼장구채 캘러스를 수확하여 깨끗한 티슈로 충분히 수분을 제거한 후 45 ℃로 1일 동안 건조기에서 건조하였다. 건조된 이끼장구채 캘러스 2 g에 정제수 1 L를 넣은 후 80 ~ 100 ℃에서 4시간 열수 추출하였다. 추출 후, 메쉬(mesh)로 여과하여 고형분을 제거하고, 여과한 북극이끼장구채 캘러스 배양 추출물을 본 발명에 사용하였다.The Arctic moss callus obtained by culturing in Example 1-1 was harvested, moisture was sufficiently removed with a clean tissue, and then dried in a dryer at 45° C. for 1 day. 1 L of purified water was added to 2 g of dried Moss Janguchae callus and then subjected to hot water extraction at 80 to 100°C for 4 hours. After extraction, solids were removed by filtering through a mesh, and the filtered Arctic moss janguchae callus culture extract was used in the present invention.
실시예 2. 본 발명에 따른 화장료 조성물 제조Example 2. Preparation of cosmetic composition according to the present invention
2-1. 북극이끼장구채 캘러스 배양 추출물 함량을 달리한 에센스 제형의 조성물 제조2-1. Manufacture of essence formulation compositions with different contents of Arctic moss janguchae callus culture extract
북극이끼장구채 캘러스 배양 추출물, 다이소듐이디티에이, 글리세린, 프로판다이올, 카보머를 정제수에 분산시켜 70℃ 내지 75℃로 가열한 수상에 용해된 유화계를 첨가한 후 아지믹서로 5분간 교반하였다. 60℃ 내지 65℃에서 트로메타민을 첨가한 후 아지믹서로 3분간 교반하여 중화하였다. 45℃에서 1,2-헥산다이올, 에틸헥실글리세린을 투입하고 3분간 교반한 후 30℃까지 냉각한 후 탈포하여 에센스 제형을 제조하였다(표 1).Arctic moss callus culture extract, disodium EDTA, glycerin, propanediol, and carbomer were dispersed in purified water and the dissolved emulsifying system was added to the water phase heated to 70°C to 75°C and stirred for 5 minutes with an azimixer. did. Tromethamine was added at 60°C to 65°C and then stirred for 3 minutes with an Azimixer to neutralize. 1,2-hexanediol and ethylhexylglycerin were added at 45°C, stirred for 3 minutes, cooled to 30°C, and degassed to prepare an essence formulation (Table 1).
2-2. 북극이끼장구채 캘러스 배양 추출물 함량을 달리한 크림 제형의 조성물 제조2-2. Manufacture of cream-type compositions with different contents of Arctic moss callus culture extract
세틸에틸헥사노에이트, 세테아릴알코올, 글리세릴스테아레이트를 75℃ 내지 80℃로 가열 용해하여 투명 유화계를 제조하였다. 북극이끼장구채 캘러스 배양 추출물, 다이소듐이디티에이, 글리세린, 프로판다이올, 세테아릴올리베이트, 솔비탄올리베이트, 카보머를 정제수에 분산시켜 70℃ 내지 75℃로 가열한 수상에 용해된 유화계를 첨가한 후 호모믹서로 3500 내지 5000 rpm 조건 하에서 5분간 유화하였다. 60℃ 내지 65℃에서 트로메타민을 첨가한 후 호모믹서로 3000 내지 3500 rpm 조건 하에서 3분간 교반하여 중화하였 다. 45℃에서 1,2-헥산다이올, 에틸헥실글리세린을 투입하고 3분간 교반한 후 30℃까지 냉각한 후 탈포하여 크림 제형을 제조하였다(표 2). A transparent emulsion system was prepared by heating and dissolving cetyl ethyl hexanoate, cetearyl alcohol, and glyceryl stearate at 75°C to 80°C. An emulsion system in which Arctic moss callus culture extract, disodium EDTA, glycerin, propanediol, cetearyl olivate, sorbitan olivate, and carbomer are dispersed in purified water and dissolved in water heated to 70°C to 75°C. After addition, it was emulsified for 5 minutes under conditions of 3500 to 5000 rpm using a homomixer. Tromethamine was added at 60°C to 65°C and then neutralized by stirring for 3 minutes using a homomixer at 3000 to 3500 rpm. 1,2-hexanediol and ethylhexylglycerin were added at 45°C, stirred for 3 minutes, cooled to 30°C, and defoamed to prepare a cream formulation (Table 2).
실험예 1. 안전성 평가(피부 자극 테스트)Experimental Example 1. Safety evaluation (skin irritation test)
본 발명에 따른 제형예 1 내지 10을 피부에 첩포한 뒤 육안 평가를 통해 시험 제품의 인체피부 일차 자극의 유무 평가 및 객관적 검증을 위해 피부 첩포 시험을 수행하였다. 건강한 성인 10명을 대상으로 실험을 실시하였고, IQ Ultra 챔버에 시료 25 mg 도포 후 이를 시험부위인 팔 하박부 안쪽에 24시간 폐쇄 첩포하였다. 첩포 제거 후 1시간, 24시간, 48시간 경과 후 피부반응을 관찰하였다. ICDRG(The International Contact Dermatitis Research Group)의 기준에 준하여 인체에 대한 피부 자극 시험을 평가한 결과, 하기 표 3과 같이 제형예 1 내지 10 모두 피부 자극을 유발하지 않으며, 피부 적용에 안전한 것을 확인할 수 있었다.After applying Formulation Examples 1 to 10 according to the present invention to the skin, a skin patch test was performed to evaluate and objectively verify the presence or absence of primary irritation of the test product to human skin through visual evaluation. An experiment was conducted on 10 healthy adults, and 25 mg of the sample was applied to the IQ Ultra chamber and applied as an occlusive patch on the inside of the lower arm, the test area, for 24 hours. Skin reactions were observed 1 hour, 24 hours, and 48 hours after removing the patch. As a result of evaluating skin irritation tests on humans according to the standards of ICDRG (The International Contact Dermatitis Research Group), it was confirmed that none of Formulation Examples 1 to 10 caused skin irritation and were safe for skin application, as shown in Table 3 below. .
자수embroidery
반응도Responsiveness
판정Judgment
실험예 2. 멜라닌 생성 억제능 평가Experimental Example 2. Evaluation of melanin production inhibition ability
미백 효과를 검증하기 위해서 멜라닌 생성능력이 뛰어난 것으로 알려진 B16F10 마우스 흑색종(melanoma) 세포에 α-MSH(melanocyte-Stimulating homone)를 처리하여 멜라닌 생성을 촉진하며, 이 때 시료의 멜라닌 생성 억제능을 확인하여 간접적으로 피부에 대한 미백 효능을 확인하였다. To verify the whitening effect, B16F10 mouse melanoma cells, known to have excellent melanin production ability, were treated with α-MSH (melanocyte-stimulating homone) to promote melanin production. At this time, the melanin production inhibition ability of the sample was confirmed. The skin whitening effect was indirectly confirmed.
24웰 플레이트에 웰당 2 × 104개의 B16F10 세포를 분주한 후, 세포 배양조건(37℃, 5% 이산화탄소)에서 배양하였다. 24시간 후, 멜라닌 생성 과정을 촉진하는 α-MSH 100 nM과 함께 일정 농도의 시험물질을 처리하여 72시간 배양하였다. 72시간 후, 세포 배양액을 면역플레이트(immunoplate)에 옮겨 세포 외 멜라닌(Release melanin)의 양을 405nm에서 흡광도를 측정하였다. 배지를 제거하고 남은 세포는 PBS로 세척 후, 1N의 수산화나트륨을 100 ul를 넣고 용해시켰다. 용해시켜 얻은 세포 내 멜라닌(Cellular melanin)의 양을 405nm에서 흡광도를 측정하였다. 세포 외 멜라닌 양과 세포 내 멜라닌 양을 일정 단백질 당 멜라닌 양으로 환산하여 음성대조군과 비교하였다. 세포독성이 나타나지 않는 농도 내에서 실험하였다.After dispensing 2 × 10 4 B16F10 cells per well in a 24-well plate, the cells were cultured under cell culture conditions (37°C, 5% carbon dioxide). After 24 hours, a certain concentration of test substance was treated with 100 nM of α-MSH, which promotes the melanin production process, and cultured for 72 hours. After 72 hours, the cell culture was transferred to an immunoplate, and the amount of extracellular melanin (release melanin) was measured by absorbance at 405 nm. After removing the medium, the remaining cells were washed with PBS and dissolved by adding 100 ul of 1N sodium hydroxide. The amount of intracellular melanin obtained by dissolution was measured by absorbance at 405 nm. The amount of extracellular melanin and intracellular melanin were converted to the amount of melanin per certain protein and compared with the negative control group. The experiment was conducted within a concentration that does not cause cytotoxicity.
본 발명에 따른 북극이끼장구채 캘러스 배양 추출물의 멜라닌 생성억제를 통한 미백 효능을 평가한 결과, α-MSH에 의해 207.3% 증가한 멜라닌 생성이 이끼장구채 캘러스 추출물에 의해 농도 의존적으로 감소하였으며, 최고 176.6% 감소하는 것으로 나타났다(도 2 및 표 4).As a result of evaluating the whitening efficacy of the Arctic moss callus culture extract according to the present invention by inhibiting melanin production, melanin production, which increased by 207.3% by α-MSH, was reduced in a concentration-dependent manner by the Moss japonicum callus extract, with a maximum decrease of 176.6%. It was found that (Figure 2 and Table 4).
실험예 3. NO(Nitric oxide) 생성 억제능 평가Experimental Example 3. Evaluation of NO (Nitric oxide) production inhibition ability
NO를 발현시키는 염증유발인자인 LPS(Lipopolysaccharide)로 자극된 뮤린(murine) RAW 264.7 대식세포에서 시험물질의 NO 생성 억제능을 통해 항염 효능을 확인하였다. The anti-inflammatory efficacy of the test substance was confirmed through its ability to inhibit NO production in murine RAW 264.7 macrophages stimulated with LPS (Lipopolysaccharide), an inflammatory factor that expresses NO.
96웰 플레이트에 웰당 6 × 104개의 RAW 264.7 세포를 분주한 후, 세포 배양조건(37℃, 5% 이산화탄소)에서 배양하였다. 24시간 후, 배양액을 버리고 PBS로 세척한 다음 FBS를 함유하지 않은 DMEM(Dulbecco's Modified Eagle's Medium) 배지를 사용하여 세포를 기아상태로 만들어주었다. 다음 날, 1 ug/ml의 LPS와 함께 일정 농도의 시험물질을 처리하여 배양하였다. 24시간 후, 세포 배양액과 griess 시약(reagent)을 동량 넣어 혼합 후, 15분간 상온에서 반응시켰다. 560nm에서 흡광도를 측정하였으며, NO의 양은 아질산나트륨(sodium nitrite)으로부터 얻은 표준곡선을 이용하여 결정하였다. 세포독성이 나타나지 않는 농도 내에서 실험하였다.After dispensing 6 × 10 4 RAW 264.7 cells per well in a 96-well plate, the cells were cultured under cell culture conditions (37°C, 5% carbon dioxide). After 24 hours, the culture medium was discarded, washed with PBS, and the cells were starved using DMEM (Dulbecco's Modified Eagle's Medium) medium without FBS. The next day, the test substances were treated with a certain concentration along with 1 ug/ml LPS and cultured. After 24 hours, equal amounts of cell culture medium and griess reagent were mixed and reacted at room temperature for 15 minutes. Absorbance was measured at 560 nm, and the amount of NO was determined using a standard curve obtained from sodium nitrite. The experiment was conducted within a concentration that does not cause cytotoxicity.
본 발명에 따른 북극이끼장구채 캘러스 배양 추출물에 의한 염증 관련 인자(NO)의 활성 변화를 통해 항염 효능을 평가한 결과, LPS에 의해 47.7% 증가한 NO 생성량이 북극이끼장구채 캘러스 추출물에 의해 농도 의존적으로 감소하였으며, 최고 70.3% 감소하는 것으로 나타났다(도 3 및 표 5).As a result of evaluating the anti-inflammatory efficacy through changes in the activity of inflammation-related factors (NO) by the Arctic moss callus culture extract according to the present invention, NO production, which was increased by 47.7% by LPS, was reduced in a concentration-dependent manner by the Arctic moss callus extract. It was found to decrease by up to 70.3% (Figure 3 and Table 5).
실험예 4. 자극 완화(세포 보호) 평가Experimental Example 4. Evaluation of irritation relief (cell protection)
96웰 플레이트에 웰당 1.5 × 104개의 HaCaT(Human keratinocyte) 세포를분주한 후, 세포 배양조건에서 배양하였다. 24시간 후, 배양액을 버리고 PBS로 세척한 다음 FBS를 함유하지 않은 DMEM 배지를 사용하여 세포를 기아상태로 만들어주었다. 다음 날, 피부자극 유발물질인 SDS(Sodium dodecyl sulfate)와 함께 일정 농도의 시험물질을 처리하여 24시간 배양하였다. 배지에 10배 희석시킨 WST-1 시약을 각 well에 100 ul씩 넣고 2시간 배양 후, 450nm에서 흡광도를 측정하였다. 세포독성이 나타나지 않는 농도 내에서 실험하였다.1.5 × 10 4 HaCaT (Human keratinocyte) cells per well were dispensed into a 96-well plate and then cultured under cell culture conditions. After 24 hours, the culture medium was discarded, washed with PBS, and the cells were starved using DMEM medium without FBS. The next day, the test substance was treated with a certain concentration along with SDS (Sodium dodecyl sulfate), a substance that causes skin irritation, and cultured for 24 hours. 100 ul of WST-1 reagent diluted 10 times in medium was added to each well, and after 2 hours of incubation, the absorbance was measured at 450 nm. The experiment was conducted within a concentration that does not cause cytotoxicity.
본 발명에 따른 북극이끼장구채 캘러스 배양 추출물의 세포 생존율을 통한 자극완화 효능을 평가한 결과, SDS에 의해 35.4% 감소한 세포 생존율이 북극이끼장구채 캘러스 배양 추출물에 의해 최고 14.4% 증가하는 것으로 나타났다(도 4 및 표 6).As a result of evaluating the irritation-relieving efficacy of the Arctic moss callus culture extract according to the present invention through cell viability, it was found that the cell survival rate, which was reduced by 35.4% by SDS, increased by up to 14.4% by the Arctic moss janguchae callus culture extract (Figure 4 and Table 6).
실험예 5. 주름 개선 효능 시험 (PIP 생성능 및 MMP-1 억제능 평가)Experimental Example 5. Wrinkle improvement efficacy test (evaluation of PIP generation ability and MMP-1 inhibition ability)
인체섬유아세포(Human dermal fibroblast)에서 PIP(Procollagen Type 1 C-Peptide)의 생성 촉진능과 MMP-1(콜라겐분해효소) 생성 억제능을 통해 간접적으로 항노화에 대한 피부 효능을 확인하였다.The skin anti-aging effect was confirmed indirectly through the ability to promote the production of PIP (Procollagen Type 1 C-Peptide) and the ability to inhibit the production of MMP-1 (collagenase) in human dermal fibroblasts.
5-1. 프로콜라겐 1형(Procollagen Type 1) 생성능 평가5-1. Evaluation of Procollagen Type 1 production ability
96웰 플레이트에 웰당 6 × 103개의 NHF(Human dermal fibroblast)를 분주한 후, 세포 배양조건(37℃, 5% 이산화탄소)에서 배양하였다. 24시간 후, 배양액을 버리고 PBS로 세척한 다음 supplement를 함유하지 않은 FBM(Fibroblast Basal Medium) 배지를 사용하여 세포를 기아상태로 만들어주었다. 다음 날, 일정 농도의 시험물질을 처리하여 24시간 배양하였다. 프로콜라겐 1형 Elisa kit를 이용하여 실험 후, 450nm에서 흡광도를 측정하였다. 최종 프로콜라겐(Procollagen)의 양은 일정 단백질 당 프로콜라겐의 양으로 환산하여 음성대조군과 비교하였다. 세포독성이 나타나지 않는 농도 내에서 실험하였다.6 × 10 3 NHF (Human dermal fibroblasts) per well were dispensed into a 96-well plate and then cultured under cell culture conditions (37°C, 5% carbon dioxide). After 24 hours, the culture medium was discarded, washed with PBS, and the cells were starved using supplement-free FBM (Fibroblast Basal Medium) medium. The next day, the test substances were treated with a certain concentration and cultured for 24 hours. After the experiment using the procollagen type 1 Elisa kit, the absorbance was measured at 450 nm. The final amount of procollagen was converted to the amount of procollagen per certain protein and compared with the negative control group. The experiment was conducted within a concentration that does not cause cytotoxicity.
본 발명에 따른 북극이끼장구채 캘러스 배양 추출물에 의한 프로콜라겐 1형의 변화를 통한 항노화 효능을 평가한 결과, 프로콜라겐 1형의 생성량이 북극이끼장구채 캘러스 배양 추출물에 의해 최고 49.1% 증가하는 것으로 나타났다(도 5 및 표 7).As a result of evaluating the anti-aging effect through changes in procollagen type 1 by the Arctic moss janguchae callus culture extract according to the present invention, it was found that the production of procollagen type 1 was increased by up to 49.1% by the Arctic moss janguchae callus culture extract. (Figure 5 and Table 7).
5-2. MMP-1 억제능 평가5-2. Evaluation of MMP-1 inhibition ability
6웰 플레이트에 웰당 2.5 × 105개의 HaCaT 세포를 분주한 후, 세포 배양조건(37℃, 5% 이산화탄소)에서 배양하였다. 24시간 후, 배지를 버리고 PBS로 세척한 다음 FBS를 포함하지 않은 DMEM 배지(serum free 배지)를 사용하여 세포를 기아상태로 만들어 준 후, 다음 날 UVB를 조사하여 배양하였다.After dispensing 2.5 × 10 5 HaCaT cells per well in a 6-well plate, they were cultured under cell culture conditions (37°C, 5% carbon dioxide). After 24 hours, the medium was discarded, washed with PBS, and the cells were starved using DMEM medium (serum free medium) that does not contain FBS, and then cultured under UVB irradiation the next day.
96웰 플레이트에 웰당 6 × 103개의 NHF를 분주한 후, 세포 배양조건에서 배양하였다. 24시간 후 보충물질(supplement)를 포함하지 않은 FBM 배지를 사용하여 세포를 기아상태로 만들어 준 후, 다음 날 UVB 자극을 받은 HaCaT의 배양액을 시료와 함께 인체섬유아세포에 처리하여 배양하였다. 24시간 배양 후, MMP-1 Elisa kit를 이용하여 실험 후, 450nm에서 흡광도를 측정하였다. 최종 MMP-1의 양은 일정 단백질 당 MMP-1의 양으로 환산하여 음성대조군과 비교하였다. 세포독성이 나타나지 않는 농도 내에서 실험하였다.After dispensing 6 × 10 3 NHF per well in a 96-well plate, the cells were cultured under cell culture conditions. After 24 hours, the cells were starved using FBM medium without supplements, and the next day, the culture medium of UVB-stimulated HaCaT was treated with the sample and cultured into human fibroblasts. After culturing for 24 hours, the absorbance was measured at 450 nm after experiment using the MMP-1 Elisa kit. The final amount of MMP-1 was converted to the amount of MMP-1 per certain protein and compared with the negative control group. The experiment was conducted within a concentration that does not cause cytotoxicity.
본 발명에 따른 북극이끼장구채 캘러스 배양 추출물에 의한 콜라겐 합성 저해 효소인 MMP-1의 억제를 통한 항노화 효능을 평가한 결과, UVB에 의해 25.3% 증가한 MMP-1 생성량이 북극이끼장구채 캘러스 배양 추출물에 의해 농도 의존적으로 감소하였으며, 최고 79.2% 감소하는 것으로 나타났다(도 6 및 표 8).As a result of evaluating the anti-aging effect through inhibition of MMP-1, an enzyme that inhibits collagen synthesis, by the Arctic moss callus culture extract according to the present invention, the MMP-1 production increased by 25.3% by UVB in the Arctic moss japonicum callus culture extract. It decreased in a concentration-dependent manner, with a maximum decrease of 79.2% (Figure 6 and Table 8).
실험예 6. 항산화 효능 시험 (ABTS Assay, DCF-DA Assay)Experimental Example 6. Antioxidant efficacy test (ABTS Assay, DCF-DA Assay)
6-1. ABTS 라디칼 소거능 평가6-1. ABTS radical scavenging ability evaluation
ABTS 라디칼을 이용한 항산화력 측정은 7.4 mM 2,2-azino-bis(3-ethyl-benthiazoline-6-sulfonic acid)와 2.6mM 과황산칼륨(potassium persulfate)를 혼합하여 실온에서 24시간 동안 보관하여 ABTS+를 형성시킨 후 에탄올로 희석시켜 사용하였으며, ABTS+ 100㎕에 대조군 및 농도별 시료 100㎕를 1:1로 가하여 6~7분 반응 후 700nm에서 흡광도를 측정하였다. ABTS 라디칼 소거능은 다음의 수학식 1의 방법으로 계산하였다.Measurement of antioxidant power using ABTS radicals was performed by mixing 7.4mM 2,2-azino-bis(3-ethyl-benthiazoline-6-sulfonic acid) and 2.6mM potassium persulfate and storing it at room temperature for 24 hours to obtain ABTS+. After forming, it was diluted with ethanol and used. 100 ㎕ of the control group and each concentration sample were added to 100 ㎕ of ABTS+ in a 1:1 ratio, and the absorbance was measured at 700 nm after reaction for 6 to 7 minutes. ABTS radical scavenging ability was calculated using Equation 1 below.
[수학식 1] [Equation 1]
Radical scavenging activity(%)=(1 - 반응구 OD700/대조구 OD700)×100Radical scavenging activity(%)=(1 - reaction group OD 700 / control group OD 700 )×100
본 발명에 따른 북극이끼장구채 캘러스 배양 추출물에 의한 ABTS 라디컬 소거능을 통한 항산화 효능을 평가한 결과, 북극이끼장구채 캘러스 배양 추출물에 의해 농도 의존적으로 라디컬 소거능이 증가하여 40%에서 최고 46.7%의 활성을 나타났다(도 7 및 표 9).As a result of evaluating the antioxidant efficacy through ABTS radical scavenging ability by the Arctic moss callus culture extract according to the present invention, the radical scavenging ability increased in a concentration-dependent manner by the Arctic moss callus culture extract, reaching an activity of 40% to a maximum of 46.7%. appeared (Figure 7 and Table 9).
6-2. 활성산소(ROS) 제거능 평가 (DCF-DA Assay)6-2. Evaluation of reactive oxygen species (ROS) removal ability (DCF-DA Assay)
96웰 플레이트에 웰당 1.5 × 104개의 HaCaT 세포를 분주한 후, 세포 배양조건(37℃, 5% 이산화탄소)에서 배양하였다. 24시간 후, 배지를 버리고 PBS로 세척한 다음 시료 또는 양성 대조군으로 처리한 후 인큐베이터에서 24시간 배양하였다. 차광조건에서 웰 내 배양액을 모두 제거한 후 37℃의 PBS 용액으로 세척하였다. PBS 용액을 제거하고 새로운 PBS와 함께 20 μM DCF-DA 용액을 처리한 후 호일로 싸서 인큐베이터에서 45분간 배양하였다. 차광조건에서 well 안의 배양액을 모두 제거한 후 37℃의 PBS로 1회 세척하였다. 산화스트레스 유발물질인 H2O2를 PBS(1% FBS 함유)에 200 μM 농도가 되도록 희석하여 각 웰에 200 ㎕씩 처리한 후 호일로 싸서 인큐베이터에서 30분간 배양한 후, 여기(excitation) 485 nm, 방출(emission) 535 nm에서 형광을 측정하였다. 세포독성이 나타나지 않는 농도 내에서 실험하였다.After dispensing 1.5 × 10 4 HaCaT cells per well in a 96-well plate, they were cultured under cell culture conditions (37°C, 5% carbon dioxide). After 24 hours, the medium was discarded, washed with PBS, treated with a sample or positive control, and cultured in an incubator for 24 hours. All culture medium in the wells was removed under light-blocking conditions, and then washed with 37°C PBS solution. The PBS solution was removed, and 20 μM DCF-DA solution was treated with new PBS, then wrapped in foil and incubated in an incubator for 45 minutes. All culture medium in the well was removed under light-blocking conditions, and then washed once with PBS at 37°C. H 2 O 2 , a substance that causes oxidative stress, was diluted in PBS (containing 1% FBS) to a concentration of 200 μM, and 200 μl was treated in each well, then wrapped in foil and incubated in an incubator for 30 minutes, followed by excitation 485 Fluorescence was measured at nm, emission 535 nm. The experiment was conducted within a concentration that does not cause cytotoxicity.
본 발명에 따른 북극이끼장구채 캘러스 배양 추출물에 의한 ROS 제거능을 통한 항산화 효능을 평가한 결과, 음성 대조군에서 100.4% 증가한 세포내 ROS 생성이 북극이끼장구채 캘러스 배양 추출물에 의해 농도 의존적으로 감소하여 최고 24.6% 감소하는 것으로 나타났다(도 8 및 표 10).As a result of evaluating the antioxidant efficacy through ROS removal ability by the Arctic Moss Janguchae callus culture extract according to the present invention, intracellular ROS production, which increased by 100.4% in the negative control group, was reduced in a concentration-dependent manner by the Arctic Moss Janguchae callus culture extract, reaching a maximum of 24.6%. It appeared to decrease (Figure 8 and Table 10).
실험예 7. 보습력 및 지속 보습력 평가Experimental Example 7. Evaluation of moisturizing power and continuous moisturizing power
준비 단계에서 피험자들의 측정 조건을 동일하게 하고자 시험 부위를 깨끗하고 마른 상태로 유지하였으며 최소 30분간 항온항습 (22±2℃, R.H. 40~60%)이 유지되는 곳에서 피부 안정을 취한 후 진행하였다. 측정 단계에서 시험 제품을 전완부의 선정된 시험부위(5cm X 4cm)에 마이크로 파이펫을 사용하여 2mg/cm2의 양으로 도포하였다. 측정은 제품 사용 전, 사용 후, 3시간 후로 총 3회 측정하며 3개의 값을 이용해 평균값을 구하였다. 피부 수분 측정은 아프로디테 수분 측정기(Aphrodite moisture checker, MC-1000)를 이용하여 측정하였다.In the preparation stage, to ensure that the measurement conditions were the same for the subjects, the test area was kept clean and dry, and the skin was stabilized in a place where constant temperature and humidity (22±2℃, RH 40-60%) was maintained for at least 30 minutes before proceeding. . In the measurement stage, the test product was applied in an amount of 2 mg/cm 2 to the selected test area (5cm Measurements were made three times: before using the product, after using the product, and 3 hours later, and the average value was calculated using the three values. Skin moisture was measured using an Aphrodite moisture checker (MC-1000).
그 결과, 본 발명의 북극이끼장구채 캘러스 배양 추출물을 함유한 제형예 1 내지 10은 북극이끼장구채 캘러스 배양 추출물을 함유하지 않은 비교제형예 1 및 2에 비하여 도포 직후 보습력 및 도포 3시간 후 보습력이 증가하여 지속되는 것으로 나타났다. 특히, 제형예 5 및 10의 도포 직후 보습력 및 도포 3시간 후 보습력이 가장 우수하였다(표 11, 도 9 및 도 10).As a result, Formulation Examples 1 to 10 containing the Arctic Moss Janguchae callus culture extract of the present invention increased the moisturizing power immediately after application and 3 hours after application compared to Comparative Formulation Examples 1 and 2 which did not contain the Arctic Moss Janguchae callus culture extract. It was shown to last. In particular, the moisturizing power immediately after application and the moisturizing power 3 hours after application of Formulation Examples 5 and 10 were the best (Table 11, Figures 9 and 10).
실험예 8. 경피수분손실량(TEWL) 평가Experimental Example 8. Transepidermal water loss (TEWL) evaluation
준비 단계에서 피험자들의 측정 조건을 동일하게 하고자 시험 부위를 깨끗하고 마른 상태로 유지하였으며 최소 30분간 항온항습 (22±2℃, R.H. 40~60%)이 유지되는 곳에서 피부 안정을 취한 후 진행하였다. 측정 단계에서 시험 제품을 전완부의 선정된 시험부위(5cm X 4cm)에 마이크로 파이펫을 사용하여 2mg/cm2의 양으로 도포하였다. 측정은 제품 사용 전, 3시간 후 총 3회 측정하며 3개의 값을 이용해 평균값을 구하였다. 경피수분손실량 측정은 Tewameter TM 300 기기를 이용하여 측정하였다.In the preparation stage, to ensure that the measurement conditions were the same for the subjects, the test area was kept clean and dry, and the skin was stabilized in a place where constant temperature and humidity (22±2℃, RH 40-60%) was maintained for at least 30 minutes before proceeding. . In the measurement stage, the test product was applied in an amount of 2 mg/cm 2 to the selected test area (5cm Measurements were made a total of three times, before using the product and three hours after, and the average value was calculated using the three values. Transepidermal water loss was measured using a Tewameter TM 300 device.
그 결과, 본 발명의 북극이끼장구채 캘러스 배양 추출물을 함유한 제형예 1 내지 10은 북극이끼장구채 캘러스 배양 추출물을 함유하지 않은 비교제형예 1 및 2에 비하여 도포 직후 및 도포 3시간 후 경피수분손실량이 개선되었음을 알 수 있다. 특히, 제형예 5 및 10의 도포 직후 및 도포 3시간 후 경피수분손실량 개선 효과가 가장 우수하였다(표 12, 도 11 및 도 12).As a result, Formulation Examples 1 to 10 containing the Arctic Moss Janguchae callus culture extract of the present invention had a transepidermal water loss amount immediately after and 3 hours after application compared to Comparative Formulation Examples 1 and 2 which did not contain the Arctic Moss Janguchae callus culture extract. You can see that it has improved. In particular, Formulation Examples 5 and 10 had the best effect on improving transepidermal water loss immediately after and 3 hours after application (Table 12, Figures 11 and 12).
이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the patent claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present invention.
Claims (8)
상기 피부 개선은 피부 염증 완화 또는 항산화 또는 피부 보습인, 조성물.
A cosmetic composition for improving skin containing Arctic moss janguchae callus culture extract,
The composition, wherein the skin improvement is alleviating skin inflammation or antioxidant or skin moisturizing.
상기 피부 개선은 피부 미백인 것인, 화장료 조성물.
According to paragraph 1,
A cosmetic composition wherein the skin improvement is skin whitening.
(b) 상기 (a)단계에서 얻은 캘러스 배양물 또는 상기 배양물의 추출물을 함유하는 조성물을 제조하는 단계;를 포함하는,
북극이끼장구채 캘러스 배양 추출물을 포함하는 제1항에 따른 피부 개선용 화장료 조성물의 제조방법.
(a) cultivating callus from Arctic moss plants; and
(b) preparing a composition containing the callus culture obtained in step (a) or an extract of the culture; comprising,
A method for producing a cosmetic composition for improving skin according to claim 1, comprising a callus culture extract of Arctic moss jangguchae.
상기 (a)단계는 상기 북극이끼장구채 식물을 제아틴, 수크로스 및 아가(agar)를 함유하는 MS 배지에서 암배양하여 북극이끼장구채 캘러스를 유도하는 단계를 포함하는 것인, 북극이끼장구채 캘러스 배양 추출물을 포함하는 피부 개선용 화장료 조성물의 제조방법.
According to clause 6,
Step (a) includes the step of inducing Arctic moss acae callus by culturing the Arctic moss acacia plant in the dark on MS medium containing zeatin, sucrose, and agar. Method for producing a cosmetic composition for skin improvement containing an extract.
상기 (b)단계는, 상기 북극이끼장구채 캘러스 배양물을 80 내지 100 ℃에서 2 내지 6시간동안 열수 추출하여 추출물을 얻는 단계를 포함하는 것인, 북극이끼장구채 캘러스 배양 추출물을 포함하는 피부 개선용 화장료 조성물의 제조방법.
According to clause 6,
The step (b) includes the step of obtaining an extract by extracting the Arctic moss Janguchae callus culture at 80 to 100° C. for 2 to 6 hours. A method for improving skin containing a callus culture extract. Method for producing a cosmetic composition.
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