KR102451162B1 - Composition for Anti-Inflammatory, Anti-Wrinkling, Moisturizing, and Skin Cell Regeneration Property Comprising Enzyme Treated Extract of Krill Oil as Active Ingredient - Google Patents
Composition for Anti-Inflammatory, Anti-Wrinkling, Moisturizing, and Skin Cell Regeneration Property Comprising Enzyme Treated Extract of Krill Oil as Active Ingredient Download PDFInfo
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- KR102451162B1 KR102451162B1 KR1020200113859A KR20200113859A KR102451162B1 KR 102451162 B1 KR102451162 B1 KR 102451162B1 KR 1020200113859 A KR1020200113859 A KR 1020200113859A KR 20200113859 A KR20200113859 A KR 20200113859A KR 102451162 B1 KR102451162 B1 KR 102451162B1
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- skin
- treated
- krill oil
- enzyme
- inflammatory
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Abstract
본 발명은 단백분해효소 처리 크릴 오일 추출물을 유효성분으로 포함하는 항염증, 피부 주름개선, 보습 및 피부 재생 촉진용 조성물에 관한 것이다.
본 발명의 효소처리 크릴 오일 추출물은 우수한 항산화 활성 및 항염증 활성을 갖는 동시에, 피부 주름을 억제하고, 피부 보습에 효과적이며, 손상된 피부의 재생을 촉진하여 종합적으로 피부상태를 개선하는 우수한 효과가 있으므로 화장품, 식품, 약제학 분야에서 유용하게 이용 가능하다. 또한, 본 발명의 조성물은 식용 가능한 크릴에서 분리된 물질로, 인체에 매우 안전할 뿐만 아니라, 안정성도 매우 탁월하다.The present invention relates to a composition for anti-inflammatory, skin wrinkle improvement, moisturizing and promoting skin regeneration comprising a protease-treated krill oil extract as an active ingredient.
The enzyme-treated krill oil extract of the present invention has excellent antioxidant activity and anti-inflammatory activity, and at the same time suppresses skin wrinkles, is effective in moisturizing the skin, and promotes the regeneration of damaged skin to improve the skin condition comprehensively. It can be usefully used in cosmetics, food, and pharmaceutical fields. In addition, the composition of the present invention is a material separated from edible krill, and is not only very safe for the human body, but also has excellent stability.
Description
본 발명은 효소처리 크릴 오일 추출물을 유효성분으로 화장료 조성물에 관한 것으로, 더욱 상세히는 단백분해효소 처리 크릴 오일 추출물을 유효성분으로 포함하는 항염증, 피부 주름개선, 보습 및 피부 재생 촉진용 조성물에 관한 것이다.The present invention relates to a cosmetic composition comprising an enzyme-treated krill oil extract as an active ingredient, and more particularly, to a composition for anti-inflammatory, skin wrinkle improvement, moisturizing and skin regeneration promotion comprising a protease-treated krill oil extract as an active ingredient will be.
생물체의 항상성(homeostasis)을 유지하는 과정에서 중요한 역할을 담당하고 있는 물질 중 일부가 각종 생물체 유래의 생리 활성 물질이다. 지금까지 수많은 생리 활성 물질에 관해 많은 연구가 진행되고 있으며, 그 중, 미생물, 식물, 또는 동물체 등에서 분리된 생리 활성을 갖는 물질에 관한 연구는 생명과학 및 의학 분야에서 매우 중요한 영역이다.Some of the substances that play an important role in the process of maintaining homeostasis of living organisms are physiologically active substances derived from various living organisms. So far, many studies have been conducted on numerous physiologically active substances, and among them, research on substances having physiological activity isolated from microorganisms, plants, or animals, etc. is a very important field in life science and medicine.
한편, 콜라겐은 피부 진피층에 존재하는데, 피부 전체 건조 중량의 약 70%에서 80%를 차지하는 엘라스틴과 함께 피부에 탄력을 부여하는 주요 성분으로 알려져 있다. 콜라겐은 자연 노화에 따른 세포 활성 저하와 같은 내부 요인에 의해 감소되고, 여러 유해 환경에서의 스트레스 증가나 태양 광선에 의한 활성산소종의 증가와 같은 외부 요인에 의하여 생합성이 감소하거나, 분해가 촉진되고 있다.On the other hand, collagen is present in the dermal layer of the skin, and is known as a major component that gives elasticity to the skin together with elastin, which accounts for about 70% to 80% of the total dry weight of the skin. Collagen is reduced by internal factors such as decreased cell activity due to natural aging, and biosynthesis is reduced or decomposition is accelerated by external factors such as increased stress in various harmful environments or increased reactive oxygen species due to sunlight. have.
또한, 활성산소종(reactive oxygen species, ROS)의 과도한 생산은 UVB 노출 환경에서 보고된 바 있고, UVB 노출과 연관된 신호전달 체계는 MAPKs(mitogen activated protein kinases), ERK(extracellular signal-regulated kinase), p38 키나아제, 및 JNK(c-Jun amino-terminal kinase) 등의 단백질과 관련되어 있는데, NF-ĸB 및 AP-1 전사인자의 발현증가를 유도한다. 또한, AP-1 단백질은 MMP(matrix metalloproteinase) 유전자의 활성과 관련되어 있고, NF-ĸB 단백질은 인터루킨과 같은 전염증 사이토카인(proinflammatory cytokines)의 발현에 영향을 미친다. 노화된 피부에서 콜라겐 및 엘라스틴의 붕괴는 주로 MMP 단백질과 같은 분해효소의 증가된 발현에 의해 야기된다. 또한, 전염증 사이토카인은 콜라겐의 합성을 방해하고, 콜라겐의 붕괴를 촉진한다. 타입 I 콜라겐은 연결 조직의 세포외 기질에서 가장 풍부한 단백질이다. 세포외 기질에는 타입 III, V, 및 VII 콜라겐, 엘라스틴, 프로테오글라이칸, 파이브로넥틴과 같은 다른 유형의 단백질을 포함한다. 콜라겐 감소를 저해하여 피부 주름개선에 효과가 있는 천연물질을 탐색하고자 하는 여러 다양한 시도가 있었다. 그러나 종래 개발된 비타민C, 레티노익산, 형질전환 생장인자(transforming growth factor, TGF), 동물태반 유래의 단백질(JP8-231370), 베툴린산(betulinic acid, JP8-208424), 클로렐라 추출물(JP9-40523, JP10-36283, 섬유아세포 증식 촉진작용) 등의 소재들은 피부에 적용시 자극과 발적 등 안전성 문제로 사용량에 제한이 있거나, 그 효과가 미미하여 실질적으로 피부 탄력 및 주름의 개선에 효과를 기대할 수 없다는 문제점이 있었다. In addition, excessive production of reactive oxygen species (ROS) has been reported in a UVB exposure environment, and the signaling systems associated with UVB exposure include mitogen activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), It is related to proteins such as p38 kinase and JNK (c-Jun amino-terminal kinase), and induces increased expression of NF-ĸB and AP-1 transcription factors. In addition, AP-1 protein is associated with the activity of matrix metalloproteinase (MMP) gene, and NF-ĸB protein affects the expression of proinflammatory cytokines such as interleukin. The breakdown of collagen and elastin in aged skin is mainly caused by increased expression of degrading enzymes such as MMP protein. In addition, proinflammatory cytokines interfere with the synthesis of collagen and promote the breakdown of collagen. Type I collagen is the most abundant protein in the extracellular matrix of connective tissue. The extracellular matrix includes other types of proteins such as type III, V, and VII collagen, elastin, proteoglycans, and fibronectin. There have been various attempts to search for natural substances that are effective in improving skin wrinkles by inhibiting collagen reduction. However, conventionally developed vitamin C, retinoic acid, transforming growth factor (TGF), animal placenta-derived protein (JP8-231370), betulinic acid (JP8-208424), chlorella extract (JP9-40523) , JP10-36283, fibroblast proliferation promoting action), etc., have limited usage due to safety issues such as irritation and redness when applied to the skin, or their effect is insignificant, so it is not possible to expect an effect on improvement of skin elasticity and wrinkles. There was a problem.
또한, 일반적으로 피부에 사용되는 보습용 화장료 조성물은 사람의 모발이나 피부 등에 수분을 일정량 유지함으로써 부드럽고 생기있게 하며, 갈라짐, 건조함 등의 손상을 방지해주는 역할을 한다. 즉, 피부 보습용 화장료 조성물은 피부나 모발에 일정량 이상의 수분을 공급하거나 수분이 유지함으로써 피부나 모발을 미화하고, 건강하게 유지하기 위한 목적으로 사용된다. 피부는 수분 유지에 필수적인 기능을 하는 기관으로, 수분 조절에 대략 2/3 정도의 비중을 담당하는 것으로 알려져 있다. 따라서, 화장품 분야에서는 수분의 공급, 유지, 즉 보습과 관련된 기능성 연구가 활발하게 진행되고 있다. 또한, 최근에는 천연물 등으로부터 얻은 생리 활성 물질을 화장료에 도입하여 피부 자체의 고유 방어 기능을 더욱 강화함으로써 피부의 보습력을 증가시키는 화장품의 개발이 활발하게 진행되는 추세이다.In addition, the cosmetic composition for moisturizing generally used for the skin functions to keep a certain amount of moisture in human hair or skin, thereby making it soft and lively, and preventing damage such as cracking and dryness. That is, the cosmetic composition for moisturizing the skin is used for the purpose of beautifying the skin or hair by supplying a certain amount of moisture to the skin or hair or maintaining the moisture, and to keep the skin or hair healthy. The skin is an organ that has an essential function to maintain moisture, and it is known that it accounts for about 2/3 of the weight in moisture control. Therefore, in the cosmetic field, functional research related to supply and maintenance of moisture, that is, moisturizing, is being actively conducted. In addition, in recent years, the development of cosmetics that increase the moisturizing power of the skin by introducing physiologically active substances obtained from natural products into cosmetics to further strengthen the inherent defense function of the skin itself is being actively developed.
따라서, 생체에 안전하고, 유효성분이 안정하며, 무엇보다도 기존의 항염증, 피부 주름 개선, 항산화, 보습, 및 피부 재생 효과가 있는 물질보다 효과가 우수한 천연소재의 개발이 절실히 요망되고 있다.Therefore, there is an urgent need for the development of natural materials that are safe to the body, stable in active ingredients, and more effective than substances having anti-inflammatory, skin wrinkle improvement, antioxidant, moisturizing, and skin regeneration effects above all else.
이러한 배경 아래에서, 본 발명자들은 항염증, 피부 주름 개선, 보습, 및 피부 재생 효과가 우수한 물질을 찾기 위하여 예의 연구노력한 결과, 효소처리된 크릴 오일 추출물을 피부에 적용하는 경우, 우수한 항산화 활성 및 항염증 활성을 갖는 동시에, 피부 주름을 억제하고, 피부 보습에 효과적이며, 손상된 피부의 재생을 촉진하여 종합적으로 피부상태를 개선시키는데 우수한 효과가 있음을 확인하고, 본 발명을 완성하게 되었다.Under this background, the present inventors made intensive research efforts to find substances with excellent anti-inflammatory, skin wrinkle improvement, moisturizing, and skin regeneration effects. As a result, when enzyme-treated krill oil extract is applied to the skin, excellent antioxidant activity and anti It has been confirmed that it has an inflammatory activity, suppresses skin wrinkles, is effective in moisturizing the skin, and promotes the regeneration of damaged skin, thereby having an excellent effect in improving the skin condition comprehensively, and completed the present invention.
따라서, 본 발명의 주된 목적은 우수한 항산화 활성 및 항염증 활성을 갖는 동시에, 피부 주름을 억제하고, 피부 보습에 효과적이며, 손상된 피부의 재생을 촉진하여 종합적으로 피부상태를 개선시키는데 우수한 효과가 있는 효소처리된 크릴 오일 추출물을 유효성분으로 하는 조성물을 제공하는 데 있다.Therefore, the main object of the present invention is an enzyme that has excellent antioxidant activity and anti-inflammatory activity, and at the same time suppresses skin wrinkles, is effective in moisturizing the skin, and promotes the regeneration of damaged skin to comprehensively improve the skin condition. To provide a composition comprising the treated krill oil extract as an active ingredient.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.
본 발명의 한 양태에 따르면, 본 발명은 단백분해효소 처리 크릴 오일 추출물을 유효성분으로 포함하는 항염증, 피부 주름개선, 보습 및 피부 재생 촉진용 화장료 조성물을 제공한다.According to one aspect of the present invention, the present invention provides a cosmetic composition for anti-inflammatory, skin wrinkle improvement, moisturizing and promoting skin regeneration comprising a protease-treated krill oil extract as an active ingredient.
본 발명자들은 피부상태 개선에 유용하게 사용될 수 있는 천연유래 성분을 발굴하기 위하여 예의 연구노력하였다. 그 결과, 단백분해효소 처리된 크릴 오일 추출물이 항염증, 피부 주름개선, 보습 및 피부 재생 촉진 활성을 나타내어 피부상태 개선에 효과적으로 적용할 수 있다는 사실을 확인하였다.The present inventors made intensive research efforts to discover naturally-derived ingredients that can be usefully used to improve skin condition. As a result, it was confirmed that the proteolytic enzyme-treated krill oil extract exhibits anti-inflammatory, skin wrinkle improvement, moisturizing and skin regeneration promoting activities and can be effectively applied to skin condition improvement.
본 발명에서 크릴새우(Eupausia superba)는 절지동물 난바다곤쟁이목 난바다곤쟁이과의 갑각류를 의미한다. 또한, ‘크릴 오일’은 크릴새우에서 분리된 오일 성분을 의미하며, ‘크릴 오일 추출물’은 크릴 오일에서 수용성 성분을 제거한 추출물을 의미한다. 본 발명에서 사용되는 용어, "추출물"은 크릴 오일의 추출 처리에 의하여 얻어지는 추출액, 상기 추출액의 희석액이나 농축액, 상기 추출액을 건조하여 얻어지는 건조물, 상기 추출액의 조정제물이나 정제물, 또는 이들의 혼합물 등, 추출액 자체 및 추출액을 이용하여 형성 가능한 모든 제형의 추출물을 포함한다. 본 발명의 상기 추출물은 바람직하게 추출 후, 액상 또는 건조 분말 형태로 제조되어 사용될 수 있다. In the present invention, krill ( Eupausia superba ) refers to a crustacean of the order Arthropod Apidae. In addition, 'krill oil' refers to an oil component separated from krill, and 'krill oil extract' refers to an extract obtained by removing water-soluble components from krill oil. As used herein, the term "extract" refers to an extract obtained by extraction treatment of krill oil, a diluted or concentrated liquid of the extract, a dried product obtained by drying the extract, a prepared or purified product of the extract, or a mixture thereof, etc. , including extracts of all formulations that can be formed using the extract itself and the extract. The extract of the present invention may be prepared and used in the form of a liquid or dry powder, preferably after extraction.
본 발명의 용어 "효소처리 크릴 오일"은 단백분해효소(protease) 처리된 크릴 분쇄물에서 분리한 오일을 의미한다. 또한, 상기 단백분해효소는 프로테아제와 동일한 의미로 사용되며, 단백질과 펩티드결합을 가수분해하는 효소를 의미하는데, 알칼라제(Alkalase), 에스페라제(Esperase), 프로테아제(Protease) NL, 프로테아제(Protease) A, 프로테아제(Protease) P, 프로테아제(Protease) S, 에버라제(Evelase) 6.0 등이 효소가 사용 가능하다. 본 발명의 바람직한 실시예에서는 알칼라제(Alkalase)를 사용하여 효소처리된 크릴 분쇄물로부터 오일 성분을 분리하여 크릴 오일 추출물을 수득하였다. As used herein, the term "enzyme-treated krill oil" refers to an oil separated from pulverized krill treated with protease. In addition, the protease is used in the same sense as protease, and refers to an enzyme that hydrolyzes protein and peptide bonds, alkalase, esperase, protease NL, protease ( Protease A, Protease P, Protease S, Evelase 6.0 and the like can be used as enzymes. In a preferred embodiment of the present invention, a krill oil extract was obtained by separating the oil component from the enzymatically-treated krill pulverized product using Alkalase.
본 발명의 상기 크릴 오일의 추출에 있어서, 상기 추출물을 추출하는 방법은 특별히 제한되지 아니하며, 당해 기술 분야에서 통상적으로 사용하는 방법에 따라 추출할 수 있다. 상기 추출 방법의 비제한적인 예로는, 용매분획법, 원심분리법 등을 들 수 있으며, 이들은 단독으로 수행되거나 2종 이상의 방법을 병용하여 수행될 수 있다. In the extraction of the krill oil of the present invention, the method of extracting the extract is not particularly limited, and may be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method include a solvent fractionation method, a centrifugation method, and the like, and these may be performed alone or in combination of two or more methods.
본 발명에서 용어, "피부 상태 개선"은 항산화 활성을 나타내고, 염증성 피부 질환 증상을 개선시키며, 피부 주름을 감소시키거나 예방하여 항주름 효과를 나타내고, 피부에 수분을 보충해주며, 피부 혈행개선에 도움을 주고, 피부조직 또는 피부세포의 재생을 촉진하는 것을 모두 포함하는 의미로 사용된다.As used herein, the term "improving skin condition" refers to antioxidant activity, improvement of inflammatory skin disease symptoms, anti-wrinkle effect by reducing or preventing skin wrinkles, replenishing moisture to the skin, and improving skin blood circulation. It is used in the meaning of helping and promoting the regeneration of skin tissue or skin cells.
본 발명의 화장료 조성물은 항산화 활성을 갖는 것을 특징으로 한다. 본 발명의 바람직한 실시예에서는 본 발명에 따른 효소처리된 크릴 오일 추출물이 농도 의존적으로 항산화 활성이 증가하는 것을 확인한 바 있으며, 대조군인 바이오틴과 비교하여 동등한 수준의 우수한 항산화능을 나타내는 것을 확인하였다(도 1 참조).The cosmetic composition of the present invention is characterized in that it has antioxidant activity. In a preferred embodiment of the present invention, it was confirmed that the antioxidant activity of the enzyme-treated krill oil extract according to the present invention increased in a concentration-dependent manner, and it was confirmed that it exhibited an excellent antioxidant activity at the same level as compared to the control biotin (Fig. see 1).
또한, 본 발명의 화장료 조성물은 산화질소 생성을 억제하여 항염증 활성을 나타내는 것을 특징으로 한다. 본 발명의 바람직한 실시예에서는 효소처리된 크릴 오일 추출물이 산화질소 생성 억제에 우수한 효과를 나타내는 것으로 확인할 수 있었다(도 2 및 3 참조).In addition, the cosmetic composition of the present invention is characterized in that it exhibits anti-inflammatory activity by inhibiting the production of nitric oxide. In a preferred embodiment of the present invention, it was confirmed that the enzyme-treated krill oil extract exhibited an excellent effect in inhibiting nitric oxide production (see FIGS. 2 and 3).
또한, 본 발명의 화장료 조성물은 MMP-1 유전자 발현을 억제하고, 타입 1 프로콜라겐 단백질 발현을 증진하여 피부 주름 개선 활성을 나타내는 것을 특징으로 한다. 피부섬유아세포의 콜라겐은 피부 재생, 피부 탄력, 피부 주름 형성 및 피부 손상 시 조직의 수복 또는 재생과 직접적인 관련이 있다. 즉, 피부 섬유아세포의 콜라겐 또는 프로콜라겐의 합성이 촉진되거나, 콜라겐을 분해하는 기질 단백질 분해효소(matrix metallo-proteinase, 이하 'MMP'라 함)의 합성이 저해되면, 피부 탄력 개선, 피부 재생, 피부 주름 개선, 상처 치유, 손상된 피부 조직의 수복 및 재생, 및 피부 노화 방지 등의 효과를 얻을 수 있다. 본 발명의 바람직한 실시예에서는 상기 효소처리된 크릴 오일 추출물의 처리로 MMP-1 단백질의 발현이 감소하고 타입 1 프로콜라겐 단백질 발현이 증가하는 현상을 확인하였고, 양성대조군인 바이오틴 대비 현저하게 개선된 효과가 있음을 확인할 수 있었다(도 5 및 도 6 참조). In addition, the cosmetic composition of the present invention is characterized in that it suppresses MMP-1 gene expression and promotes
또한, 본 발명의 화장료 조성물은 히알루론산 합성을 촉진하여 피부 보습 활성을 나타내는 것을 특징으로 한다. 본 발명의 바람직한 실시예에서는 상기 효소처리된 크릴 오일 추출물의 처리로 히알루론산(Hyaluronan) 생성량이 농도 의존적으로 증가하며, 모든 농도 구간에서는 바이오틴 처리 양성대조군에 비해 더욱 증가된 히알루론산 생성 촉진 효과를 나타내는 것을 확인한 바 있다(도 7 참조). In addition, the cosmetic composition of the present invention is characterized in that it promotes the synthesis of hyaluronic acid to exhibit skin moisturizing activity. In a preferred embodiment of the present invention, the amount of hyaluronic acid production is increased in a concentration-dependent manner by the treatment of the enzyme-treated krill oil extract, and in all concentration ranges, the hyaluronic acid production promoting effect is further increased compared to the biotin-treated positive control group. It has been confirmed that (see FIG. 7).
또한, 본 발명의 화장료 조성물은 VEGF(vascular endothelial growth factor) 인자의 발현을 촉진하여 피부 혈행 개선 활성을 나타내는 것을 특징으로 한다. 본 발명의 바람직한 실시예에서는 상기 효소처리된 크릴 오일 추출물을 처리한 실험군은 스크레치만 낸 대조군(Con)과 비교하여 모든 농도 구간에서 VEGF 인자의 발현이 현저하게 증가하여 피부 혈행 개선에 효과가 있음을 확인한 바 있다(도 8 참조). In addition, the cosmetic composition of the present invention is characterized in that it promotes the expression of vascular endothelial growth factor (VEGF) factor to exhibit skin blood circulation improvement activity. In a preferred embodiment of the present invention, the experimental group treated with the enzyme-treated krill oil extract significantly increased the expression of the VEGF factor in all concentration sections compared to the control group (Con) only scratched, so that it is effective in improving skin blood circulation. It has been confirmed (see FIG. 8).
본 발명의 화장료 조성물은 식용 가능한 크릴에서 유래한 오일 소재를 사용하여 인체에 해가 없으며, 그 첨가로 인해 화장품의 물질의 품질을 손상하지 않으므로 화장료 조성물에 포함되어 사용되는데 적합하다. 특히, 항산화, 항염증, 주름개선, 보습, 혈행 개선, 및 피부 재생 활성과 관련된 기능이 있으므로, 기능성 화장료 조성물에 포함되어 사용되는 것이 바람직하다.The cosmetic composition of the present invention does not harm the human body by using an edible krill-derived oil material, and does not impair the quality of the cosmetic material due to its addition, so it is suitable for being included in the cosmetic composition. In particular, since it has functions related to antioxidant, anti-inflammatory, wrinkle improvement, moisturizing, blood circulation improvement, and skin regeneration activity, it is preferably included in a functional cosmetic composition.
본 발명의 화장료 조성물은 스킨로션, 스킨소프너, 스킨토너, 아스트린젠트, 로션, 밀크로션, 모이스쳐 로션, 영양로션, 맛사지크림, 영양크림, 모이스처크림, 핸드크림, 파운데이션, 에센스, 영양에센스, 마스크팩, 비누, 클렌징폼, 클렌징로션, 클렌징크림, 바디로션 및 바디클린저에서 선택된 어느 하나의 제형으로 제조할 수 있으나, 이에 제한되는 것은 아니다.The cosmetic composition of the present invention includes skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, foundation, essence, nourishing essence, mask pack, It may be prepared in any one formulation selected from soap, cleansing foam, cleansing lotion, cleansing cream, body lotion and body cleanser, but is not limited thereto.
본 발명의 상기 화장료 조성물은 일반 피부 화장료에 배합되는 화장품학적으로 허용 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.The cosmetic composition of the present invention may further include one or more cosmetically acceptable carriers to be formulated in general skin cosmetics, and common ingredients include, for example, oil, water, surfactant, humectant, lower alcohol, and thickener. , a chelating agent, a colorant, a preservative, a fragrance, and the like may be appropriately mixed, but the present invention is not limited thereto.
본 발명의 화장료 조성물에 포함되는 화장품학적으로 허용 가능한 담체는 화장료 조성물의 제형에 따라 다양하다.The cosmetically acceptable carrier included in the cosmetic composition of the present invention varies 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, 산화아연 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, as a carrier component, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. may be used, but is not limited thereto. These may be used alone or in combination of two or more.
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 아니다. 이들은 단독으로 사용되거나 2종 이상 혼합되어 사용될 수 있다.When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohard propellants such as, but not limited to, locarbon, propane/butane or dimethyl ether. These may be used alone or in combination of two or more.
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 아니다. 이들은 단독으로 사용되거나 2종 이상 혼합되어 사용될 수 있다.When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier may be used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, Propylene glycol, 1,3-butyl glycol oil, and the like can be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic esters, fatty acid esters of polyethylene glycol or sorbitan may be used, but is not limited thereto. These may be used alone or in combination of two or more.
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 아니다. 이들은 단독으로 사용되거나 2종 이상 혼합되어 사용될 수 있다.When the formulation of the present invention is a suspension, as a carrier component a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters; Crystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth may be used, but is not limited thereto. These may be used alone or in combination of two or more.
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 아니다. 이들은 단독으로 사용되거나 2종 이상 혼합되어 사용될 수 있다.When the formulation of the present invention is soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolysates, isethionate, lanolin derivatives, aliphatic alcohols, vegetable oils, glycerol, sugar, etc. are used as carrier components. may be, but is not limited thereto. These may be used alone or in combination of two or more.
본 발명의 화장료 조성물에서, 상기 효소처리된 크릴 오일 추출물은 구체적으로는 건조 중량으로, 화장료 조성물 총 중량의 0.0001 중량% 내지 50 중량%로 포함될 수 있으며, 보다 구체적으로는 0.0005 중량% 내지 10 중량%로 포함될 수 있다. 상기 범위 내에서 항산화, 항염증, 피부 주름 및 혈행 개선, 보습, 및 피부 재생 활성에 대한 우수한 효능을 나타내는 이점이 있으며, 조성물의 제형이 안정화되는 이점이 있다.In the cosmetic composition of the present invention, the enzyme-treated krill oil extract is specifically, by dry weight, 0.0001% to 50% by weight of the total weight of the cosmetic composition, more specifically 0.0005% to 10% by weight may be included as Within the above range, there is an advantage of exhibiting excellent efficacy for antioxidant, anti-inflammatory, skin wrinkle and blood circulation improvement, moisturizing, and skin regeneration activity, and there is an advantage in that the formulation of the composition is stabilized.
본 발명의 다른 양태에 따르면, 본 발명은 효소처리 크릴 오일 추출물을 유효성분으로 포함하는 항염증, 피부 주름개선, 보습 및 피부 재생 촉진용 식품 조성물을 제공한다.According to another aspect of the present invention, the present invention provides a food composition for anti-inflammatory, skin wrinkle improvement, moisturizing and promoting skin regeneration comprising an enzyme-treated krill oil extract as an active ingredient.
본 발명의 효소처리 크릴 오일 추출물은 항산화, 항염증, 피부 주름 및 혈행 개선, 보습, 및 피부 재생에 우수한 효과를 나타내어 피부 상태 개선을 위한 식품 조성물로 유용하게 사용될 수 있다. 본 발명의 효소처리 크릴 오일 추출물과 항산화, 항염증, 보습, 주름 개선, 혈행 개선, 및 피부 재생에 대한 효과는 상기에서 설명한 바와 같다. 상기 식품 조성물은 건강기능식품의 형태로 사용될 수 있으나, 이에 제한되는 것은 아니다. The enzyme-treated krill oil extract of the present invention exhibits excellent effects in antioxidant, anti-inflammatory, skin wrinkle and blood circulation improvement, moisturizing, and skin regeneration, and thus can be usefully used as a food composition for improving skin condition. The enzyme-treated krill oil extract of the present invention and the effects on antioxidant, anti-inflammatory, moisturizing, wrinkle improvement, blood circulation improvement, and skin regeneration are as described above. The food composition may be used in the form of a health functional food, but is not limited thereto.
본 발명의 식품 조성물은 효소처리 크릴 오일 추출물과, 이들의 가공물의 형태로 포함될 수 있다. 또한, 상기 조성물은 유효성분 이외에 식품학적으로 허용 가능한 식품 보조첨가제를 포함할 수 있다.The food composition of the present invention may be included in the form of an enzyme-treated krill oil extract and a processed product thereof. In addition, the composition may include a food pharmaceutically acceptable food additive in addition to the active ingredient.
본 발명에 있어서, "식품 보조첨가제"란 식품에 보조적으로 첨가될 수 있는 구성요소를 의미하며, 각 제형의 건강기능식품을 제조하는데 첨가되는 것으로서 당업자가 적절히 선택하여 사용할 수 있다. 식품 보조첨가제의 예로는 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 충진제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산음료에 사용되는 탄산화제 등이 포함되지만, 상기 예들에 의해 본 발명의 식품 보조첨가제의 종류가 제한되는 것은 아니다.In the present invention, "food supplementary additive" means a component that can be added auxiliary to food, and is added to the manufacture of health functional food of each formulation, and those skilled in the art can appropriately select and use it. Examples of food supplement additives include various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners , pH adjuster, stabilizer, preservative, glycerin, alcohol, carbonation agent used in carbonated beverages, etc., but the above examples are not limited to the type of food supplementary additive of the present invention.
본 발명의 식품 조성물에는 건강기능식품이 포함될 수 있다. 본 발명에 있어서, "건강기능식품"이란 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 정제, 캅셀, 분말, 과립, 액상 및 환 등의 형태로 제조 및 가공한 식품을 말한다. 여기서 “기능성”이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻는 것을 의미한다. 본 발명의 건강기능식품은 당업계에서 통상적으로 사용되는 방법으로 제조 가능하며, 상기 제조시에는 당업계에서 통상적으로 첨가하는 원료 및 성분을 첨가하여 제조할 수 있다. The food composition of the present invention may include a health functional food. In the present invention, "health functional food" refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients useful for the human body. Here, the term “functionality” refers to obtaining useful effects for health purposes such as regulating nutrients or physiological effects on the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art.
또한, 상기 건강기능식품의 제형 또한 건강기능식품으로 인정되는 제형이면 제한 없이 제조될 수 있다. 본 발명의 식품 조성물은 다양한 형태의 제형으로 제조될 수 있으며, 일반 약품과는 달리 식용 크릴 오일을 원료로 하여 식품의 장기 복용 시 발생할 수 있는 부작용 등이 없는 장점이 있고, 휴대성이 뛰어나며, 항산화, 항염증, 주름 개선, 보습, 혈행 개선, 및 피부 재생 활성에 대한 효과를 증진시키기 위한 보조제로 섭취할 수 있다.In addition, if the formulation of the health functional food is also recognized as a health functional food, it can be prepared without limitation. The food composition of the present invention can be prepared in various forms, and unlike general drugs, it uses edible krill oil as a raw material and has the advantage of not having side effects that may occur when taking food for a long time, excellent portability, and antioxidant , anti-inflammatory, wrinkle improvement, moisturizing, blood circulation improvement, and can be taken as an adjuvant to enhance the effect on skin regeneration activity.
본 발명의 건강기능식품이 취할 수 있는 형태에는 제한이 없으며, 통상적인 의미의 식품을 모두 포함할 수 있고, 기능성 식품 등 당업계에 알려진 용어와 혼용하여 사용할 수 있다. 아울러 본 발명의 건강기능식품은 당업자의 선택에 따라 식품에 포함될 수 있는 적절한 기타 보조 성분과 공지의 첨가제를 혼합하여 제조할 수 있다. 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올 음료 및 비타민 복합제 등이 있으며, 본 발명에 따른 효소처리 크릴 오일 추출물을 주성분으로 하여 제조한 즙, 차, 젤리 및 주스 등에 첨가하여 제조할 수 있다. 또한, 동물을 위한 사료로 이용되는 식품도 포함된다.There is no limitation in the form that the health functional food of the present invention can take, and it may include any food in a conventional sense, and may be used in combination with terms known in the art, such as functional food. In addition, the health functional food of the present invention can be prepared by mixing known additives with other suitable auxiliary ingredients that may be included in the food according to the selection of those skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and There is a vitamin complex, and it can be prepared by adding the enzyme-treated krill oil extract according to the present invention as a main component to juice, tea, jelly and juice. Also included are foods used as feed for animals.
본 발명의 다른 양태에 따르면, 본 발명은 효소처리 크릴 오일 추출물을 유효성분으로 포함하는 항염증용 약학적 조성물을 제공한다.According to another aspect of the present invention, the present invention provides an anti-inflammatory pharmaceutical composition comprising an enzyme-treated krill oil extract as an active ingredient.
본 발명의 항염증용 약학적 조성물에서, 상기 항염증 활성은 일산화질소(NO) 생성 억제를 통해 수행되는데, 본 발명자들은 효소처리 크릴 오일 추출물이 염증 유발물질인 LPS에 의해 매개되는 신호전달체계에서 일산화질소(Nitric Oxide; NO)의 생성 억제를 통해 염증 관련 세포 내 신호전달체계를 효과적으로 차단함으로써 염증성 질환의 예방 또는 치료에 사용될 수 있음을 확인한 바 있다. In the anti-inflammatory pharmaceutical composition of the present invention, the anti-inflammatory activity is performed through inhibition of nitric oxide (NO) production. It has been confirmed that it can be used for the prevention or treatment of inflammatory diseases by effectively blocking the intracellular signaling system related to inflammation by inhibiting the production of nitric oxide (NO).
본 발명의 조성물이 약제학적 조성물로 제조되는 경우, 본 발명의 약제학적 조성물은 약제학적으로 허용되는 담체를 포함한다. 본 발명의 약제학적 조성물에 포함되는 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약제학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약제학적으로 허용되는 담체 및 제제는 Remington's Pharmaceutical Sciences(19th ed., 1995)에 상세히 기재되어 있다.When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. it's not going to be The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
본 발명의 약제학적 조성물은 경구 또는 비경구 투여할 수 있으며, 본 발명의 일 구현예에 따르면 비경구 방식으로 투여되고, 본 발명의 다른 구현예에 따르면 경피투여방식으로 투여된다. The pharmaceutical composition of the present invention may be administered orally or parenterally. According to one embodiment of the present invention, it is administered by a parenteral method, and according to another embodiment of the present invention, it is administered by a transdermal administration method.
본 발명의 약제학적 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다. 본 발명의 약제학적 조성물의 바람직한 투여량은 성인 기준으로 0.0001-100㎎/kg 범위 내이다.A suitable dosage of the pharmaceutical composition of the present invention is variously prescribed according to factors such as formulation method, administration method, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and reaction sensitivity of the patient. can be A preferred dosage of the pharmaceutical composition of the present invention is within the range of 0.0001-100 mg/kg for adults.
본 발명의 약제학적 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액, 시럽제 또는 유화액 형태이거나 엑스제, 산제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. or may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, syrup, or emulsion in oil or aqueous medium, or may be in the form of an extract, powder, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.
본 발명의 특징 및 이점을 요약하면 다음과 같다:The features and advantages of the present invention are summarized as follows:
(a) 본 발명은 효소처리 크릴 오일 추출물을 유효성분으로 포함하는 항염증, 피부 주름개선, 피부 보습 및 피부 혈행과 피부 재생 촉진용 조성물을 제공한다.(a) The present invention provides a composition for anti-inflammatory, skin wrinkle improvement, skin moisturizing, and skin circulation and skin regeneration promotion composition comprising an enzyme-treated krill oil extract as an active ingredient.
(b) 본 발명의 효소처리 크릴 오일 추출물은 우수한 항산화 활성 및 항염증 활성을 갖는 동시에, 피부 주름을 억제하고, 피부 보습에 효과적이며, 손상된 피부의 재생을 촉진하여 종합적으로 피부상태를 개선하는 우수한 효과가 있으므로 화장품, 식품, 약제학 분야에서 유용하게 이용 가능하다. (b) The enzyme-treated krill oil extract of the present invention has excellent antioxidant activity and anti-inflammatory activity, and at the same time suppresses skin wrinkles, is effective in moisturizing the skin, and promotes the regeneration of damaged skin, thereby improving the skin condition comprehensively. Because it is effective, it can be usefully used in cosmetics, food, and pharmaceutical fields.
(c) 또한, 본 발명의 조성물은 식용 가능한 크릴에서 분리된 물질로, 인체에 매우 안전할 뿐만 아니라, 안정성도 매우 탁월하다.(c) In addition, the composition of the present invention is a material isolated from edible krill, and is not only very safe for the human body, but also has excellent stability.
도 1은 효소처리 크일 오일 추출물 처리에 따른 항산화 활성 변화를 측정하여 나타낸 그래프이다.
도 2는 효소처리 크릴 오일 추출물 처리에 따른 RAW 264.7의 세포 생존능을 측정하여 나타낸 그래프이다.
도 3은 효소처리 크릴 오일 추출물을 RAW 264.7 세포에 처리한 후, NO 생성량의 변화를 측정하여 나타낸 그래프이다.
도 4는 효소처리 크릴 오일 추출물 처리에 따른 인간 피부섬유아세포(NHDF, normal human dermal fibroblasts)의 세포 생존능을 측정하여 나타낸 그래프이다.
도 5는 효소처리 크릴 오일 추출물을 인간 피부섬유아세포에 처리한 후, ELISA 방법에 의해 MMP-1 단백질의 발현량 변화를 측정하여 나타낸 그래프이다.
도 6은 효소처리 크릴 오일 추출물을 인간 피부섬유아세포에 처리한 후, ELISA 방법에 의해 타입 1 프로콜라겐 단백질의 발현량 변화를 측정하여 나타낸 그래프이다.
도 7은 효소처리 크릴 오일 추출물을 인간각질형성세포에 처리한 후, ELISA 방법에 의해 히알루론산(Hyaluronan) 합성량 변화를 측정하여 나타낸 그래프이다.
도 8은 효소처리 크릴 오일 추출물을 인간각질형성세포에 처리한 후, ELISA 방법에 의해 VEGF 인자의 발현량 변화를 측정하여 나타낸 그래프이다.
도 9는 효소처리 크릴 오일 추출물을 인간각질형성세포에 처리한 후, cell stain 용액을 이용하여 플레이트 내에서 세포 염색을 한 결과를 촬영한 사진이다.1 is a graph showing the change in antioxidant activity according to the enzyme-treated kile oil extract treatment.
Figure 2 is a graph showing the measurement of the cell viability of RAW 264.7 according to the enzyme-treated krill oil extract treatment.
3 is a graph showing the change in NO production after the enzyme-treated krill oil extract was treated in RAW 264.7 cells.
Figure 4 is a graph showing the measurement of the cell viability of human dermal fibroblasts (NHDF, normal human dermal fibroblasts) according to the enzyme-treated krill oil extract treatment.
5 is a graph showing the change in the expression level of MMP-1 protein by ELISA after the enzyme-treated krill oil extract was treated on human skin fibroblasts.
6 is a graph showing the change in the expression level of
7 is a graph showing the change in the amount of hyaluronic acid (Hyaluronan) synthesized by ELISA after the enzyme-treated krill oil extract was treated on human keratinocytes.
8 is a graph showing changes in the expression level of VEGF factors by ELISA after the enzyme-treated krill oil extract was treated on human keratinocytes.
9 is a photograph of the results of cell staining in a plate using a cell stain solution after the enzyme-treated krill oil extract was treated on human keratinocytes.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention by these examples.
실시예 1. 효소처리 크릴 오일 추출물의 제조Example 1. Preparation of enzyme-treated krill oil extract
냉동 크릴 2kg을 해동하고 물로 세척하여 염분 및 불순물을 제거한 다음 분쇄하였다. 상기 분쇄된 크릴을 중탄산나트륨 200g으로 처리하고 80℃에서 2시간 자숙하였다. 상기 분쇄된 크릴의 pH는 중탄산나트륨 처리를 통한 상기 자숙과정에 의해 pH 7 내지 pH 9로 조절되었다.2 kg of frozen krill was thawed, washed with water to remove salts and impurities, and then pulverized. The pulverized krill was treated with 200 g of sodium bicarbonate and self-retained at 80° C. for 2 hours. The pH of the pulverized krill was adjusted to pH 7 to pH 9 by the self-sustaining process through sodium bicarbonate treatment.
상기 자숙한 크릴에 정제수 1,500g을 첨가하고 단백질 분해 효소인 알칼라제(Alkalase) 100g을 첨가하여 단백질 분해 효소 반응을 수행하였다. 상기 효소 반응을 약 55℃에서 약 2시간 동안 수행하여 크릴의 단백질을 가수분해하였다.A proteolytic enzyme reaction was performed by adding 1,500 g of purified water to the mature krill and adding 100 g of a proteolytic enzyme Alkalase. The enzymatic reaction was performed at about 55° C. for about 2 hours to hydrolyze the krill protein.
이후, 효소 반응 후에 효소 반응을 멈추는 효소 실활을 수행하였다. 효소 실활의 온도는 90℃이었고, 상기 크릴의 가수분해물에 구연산을 100g 첨가하고 30분 동안 정치하여 pH 4.0 ~ 5.0으로 조절하였다.Thereafter, enzyme inactivation was performed to stop the enzymatic reaction after the enzymatic reaction. The temperature of the enzyme inactivation was 90° C., and 100 g of citric acid was added to the hydrolyzate of the krill and left still for 30 minutes to adjust the pH to 4.0 to 5.0.
상기 효소 실활 후에 상기 가수분해물을 데칸터(Decanter)로 여과하고 여과액을 얻었다. 상기 데칸터를 이용하여 70℃ ~ 99℃에서 2시간 동안 10,000rpm으로 여과하였다.After inactivation of the enzyme, the hydrolyzate was filtered through a decanter to obtain a filtrate. Using the decanter, it was filtered at 10,000 rpm at 70° C. to 99° C. for 2 hours.
상기 여과액을 상기 여과액을 10,000rpm의 속도로 70℃ ~ 99℃에서 2시간 동안 원심분리하여 라이트(light) 오일, 헤비(heavy) 오일, 및 단백질로 분리하였다. 상기 라이트 오일은 원심분리하여 얻은 오일의 상등액이고, 상기 헤비 오일은 원심분리하여 얻은 오일의 하등액이다. 상기 라이트 오일과 상기 헤비 오일은 모두 EPA, DHA, 인지질, 및 아스타잔틴을 포함한다. 상기 아스타잔틴이 포함된 크릴 오일을 TFE(Thin Film Evaporator)를 이용하여 건조 수분 함량 2% 미만으로 농축하였다.The filtrate was centrifuged at a speed of 10,000 rpm at 70° C. to 99° C. for 2 hours to separate the filtrate into light oil, heavy oil, and protein. The light oil is a supernatant of the oil obtained by centrifugation, and the heavy oil is a supernatant of the oil obtained by centrifugation. The light oil and the heavy oil both contain EPA, DHA, phospholipids, and astaxanthin. The astaxanthin-containing krill oil was concentrated to a dry moisture content of less than 2% using a TFE (Thin Film Evaporator).
상기 농축된 아스타잔틴을 포함하는 크릴 오일을 발효 주정으로 정제한 후 가압여과하여 효소처리 크일 오일 추출물을 제조하였다.The krill oil containing the concentrated astaxanthin was purified with fermented alcohol and then filtered under pressure to prepare an enzyme-treated krill oil extract.
비교예. 크릴 오일 추출물의 제조comparative example. Preparation of Krill Oil Extract
단백질 분해 효소인 알카라제를 이용하여 단백질 분해 효소 반응을 수행하는 과정과, 효소 실활과정을 제외하고, 상기 실시예 1의 방법과 동일한 방법으로 크릴 오일 추출물을 제조하였다. A krill oil extract was prepared in the same manner as in Example 1, except for the process of performing the proteolytic enzyme reaction using the proteolytic enzyme alkalase and the enzyme inactivation process.
실험예 1. 항산화 활성 Experimental Example 1. Antioxidant activity
DPPH(1,1-diphenyl-2-picrylhydrazyl, Sigma, USA)의 환원력을 이용하는 상 등의 문헌(Sang S et al., J.Agric. Food Chem. 50(8), p2459-2463, 2002)에 기재된 방법에 따라, 본 발명에 따른 효소처리 크일 오일 추출물의 항산화 활성을 측정하였다. DPPH는 항산화 활성이 있는 물질과 반응하면 라디칼이 소거되어 짙은 자색으로 탈색되기 때문에, 흡광도 측정을 통하여 항산화 물질의 수소 공여능을 측정하는데 널리 이용되고 있다. Sang S et al. using the reducing power of DPPH (1,1-diphenyl-2-picrylhydrazyl, Sigma, USA) (Sang S et al., J. Agric. Food Chem. 50(8), p2459-2463, 2002) According to the described method, the antioxidant activity of the enzyme-treated kile oil extract according to the present invention was measured. DPPH is widely used to measure the hydrogen donating ability of antioxidants through absorbance measurement because DPPH is decolorized to dark purple by removing radicals when reacting with substances with antioxidant activity.
메탄올에 녹인 0.2mM DPPH 용액 160μL와 DMSO 40μL에 녹인 효소처리 크일 오일 추출물(시험예) 및 효소처리 하지 않은 크릴 오일 추출물(비교예)을 각각 1, 10, 및 50μg/mL 농도로 96-웰 마이크로플레이트에 넣고 37℃에서 30분간 배양한 후, 환원된 DPPH의 양을 분광 광도계(Molecular Devices FilterMax F5; San Francisco, CA, USA)를 이용하여 520nm에서 흡광도를 측정하여 항산화 활성을 비교하였다. 비교실험을 위해 아스코르브산(ascorbic acid)과 바이오틴(Biotin)을 양성대조군으로 사용하였다. 또한, 모든 실험은 3회 반복하여 측정되었다.Enzyme-treated krill oil extract (Test Example) and non-enzyme-treated krill oil extract (Comparative Example) dissolved in 160 μL of 0.2 mM DPPH solution in methanol and 40 μL of DMSO were added to a 96-well microstructure at concentrations of 1, 10, and 50 μg/mL, respectively. After being put on a plate and incubated at 37° C. for 30 minutes, the amount of reduced DPPH was measured by measuring absorbance at 520 nm using a spectrophotometer (Molecular Devices FilterMax F5; San Francisco, CA, USA) to compare antioxidant activity. For comparative experiments, ascorbic acid and biotin were used as positive controls. In addition, all experiments were repeated three times.
측정 결과, 효소처리 하지 않은 크릴 오일 추출물의 항산화 활성은 미약한 것으로 나타났으나, 본 발명에 따른 효소처리 크릴 오일 추출물은 농도 의존적으로 항산화 활성이 증가하는 것으로 나타났으며, 대조군인 바이오틴과 비교하여도 동등한 수준으로 우수한 항산화능을 나타내는 것으로 관찰되었다(도 1 참조).As a result of the measurement, it was found that the antioxidant activity of the krill oil extract not treated with enzyme was weak, but the antioxidant activity of the enzyme-treated krill oil extract according to the present invention was increased in a concentration-dependent manner. It was also observed to exhibit excellent antioxidant activity at the same level (see FIG. 1).
실험예 2. 항염 활성 Experimental Example 2. Anti-inflammatory activity
2-1. 세포 보호 효과2-1. cytoprotective effect
효소처리 크릴 오일 추출물이 LPS 처리된 대식세포주인 RAW 264.7 세포의 생장에 미치는 영향을 통해 세포 보호 효과를 아래와 같은 방법으로 확인하였다. Through the effect of the enzyme-treated krill oil extract on the growth of the LPS-treated macrophage, RAW 264.7 cells, the cytoprotective effect was confirmed in the following way.
대식세포주인 RAW 264.7 세포를 한국세포주은행(KTCC, Seoul, Korea)에서 분양받아 사용하였고, 10% FBS(Fetal bovine serum)를 함유한 DMEM(Dulbecco’s modified Eagle’s medium)에 10% FBS, 100μg/mL 페니실린 및 100μg/mL 스트렙토마이신을 첨가한 배지에 상기 세포를 접종하여 37℃, 5% CO2 조건에서 배양하였다.RAW 264.7 cells, a macrophage cell line, were purchased from the Korea Cell Line Bank (KTCC, Seoul, Korea), and 10% FBS, 100 μg/mL penicillin in DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS (Fetal bovine serum). And 100 μg/mL streptomycin was inoculated with the cells in a medium, and cultured at 37° C., 5% CO 2 condition.
상기 RAW 264.7 세포를 1×106 cells/mL의 농도(in DMEM)로 96웰 플레이트에 접종하고 24시간 배양 후, 실시예 1에서 제조된 효소처리 크릴 오일 추출물을 각각 1, 10, 및 50μg/mL 농도로 희석한 시료와 내독소로 알려진 LPS(1μg/mL)를 함유한 새로운 배지를 동시에 처리하여 24시간 배양하였다. 이후, MTT 시약의 농도가 0.1 mg/mL이 되도록 처리하고 3시간 후, 세포에 MTT 처리로 생성된 포마잔(formazan)을 DMSO로 녹여 595nm에서 흡광도를 측정하였다. 비교실험을 위해 덱사메타손(Dexamethasone) 1μg/mL 처리군과 바이오틴(Biotin) 1, 10, 및 50μg/mL 처리군을 양성대조군으로 사용하였다. 또한, 모든 실험은 3회 반복하여 측정되었다.The RAW 264.7 cells were inoculated in a 96-well plate at a concentration of 1×10 6 cells/mL (in DMEM) and incubated for 24 hours, each of the enzyme-treated krill oil extract prepared in Example 1 was 1, 10, and 50 μg/ The sample diluted to a concentration of mL and a fresh medium containing LPS (1 μg/mL) known as endotoxin were simultaneously treated and cultured for 24 hours. Thereafter, the concentration of the MTT reagent was treated to be 0.1 mg/mL, and after 3 hours, the formazan generated by the MTT treatment was dissolved in DMSO in the cells to measure the absorbance at 595 nm. For the comparative experiment,
실험 결과, 도 2를 참조하면, 효소처리 크릴 오일 추출물 처리군 모두에서 효소처리하지 않은 크릴 오일 추출물에 비해 세포 보호 효과가 더욱 우수한 것으로 나타났다. 또한, 본 발명의 효소처리 크릴 오일 추출물은 모든 구간에서 바이오틴(Biotin)을 처리한 양성대조군에 비해 세포 보호 효과가 더욱 우수한 것으로 나타났으며, 효소처리 크릴 오일 추출물 50μg/mL 처리군은 덱사메타손(Dexamethasone) 처리군과 비교하여 동등 이상의 세포 보호 효과를 나타내는 것으로 관찰되었다.As a result of the experiment, referring to FIG. 2 , it was found that in all of the enzyme-treated krill oil extract treatment groups, the cytoprotective effect was more excellent than that of the krill oil extract that was not enzymatically treated. In addition, the enzyme-treated krill oil extract of the present invention showed more excellent cytoprotective effect than the positive control group treated with biotin in all sections, and the enzyme-treated
5-2. 산화질소 생성 억제능5-2. Inhibition of nitric oxide production
효소처리 크릴 오일 추출물의 항염증 효과를 확인하기 위해, 염증 유발에 관여하는 대표적인 세포 독성물질인 산화질소(nitric oxide, NO)의 생성 억제율을 측정하였다.In order to confirm the anti-inflammatory effect of the enzyme-treated krill oil extract, the inhibition rate of the production of nitric oxide (NO), a representative cytotoxic substance involved in inducing inflammation, was measured.
먼저, 대식세포주인 RAW 264.7 세포를 한국세포주은행(KTCC, Seoul, Korea)에서 분양받아 사용하였고, 10% FBS(Fetal bovine serum)를 함유한 DMEM(Dulbecco’s modified Eagle’s medium)에 10% FBS, 100μg/mL 페니실린 및 100μg/mL 스트렙토마이신을 첨가한 배지에 상기 세포를 접종하여 37℃, 5% CO2 조건에서 배양하였다.First, RAW 264.7 cells, a macrophage cell line, were purchased from the Korea Cell Line Bank (KTCC, Seoul, Korea), and 10% FBS, 100 μg/ The cells were inoculated into a medium supplemented with mL penicillin and 100 μg/mL streptomycin, and cultured at 37° C., 5% CO 2 conditions.
구체적으로, 상기 RAW 264.7 세포를 1×106 cells/mL의 농도(in DMEM)로 96 웰 플레이트에 접종하고 24시간 배양 후, 실시예 1에서 제조된 효소처리 크릴 오일 추출물을 각각 1, 10, 및 50μg/mL 농도로 희석한 시료와 내독소로 알려진 LPS(1μg/mL)를 함유한 새로운 배지를 동시에 처리하여 24시간 배양하였다. 그 후, 세포 배양 상등액 100μL와 Griess reagent[1%(w/v) sulfanilamide, 0.1%(w/v) naphtylethylenediamine in 2.5%(v/v) phosphoric acid] 100μL를 혼합하여 96 웰 플레이트에서 10분 동안 반응시키고, ELISA 리더를 사용하여 540nm에서 흡광도를 측정하여 생성된 산화질소의 양을 측정하였다. 생성된 아질산염(nitrite)의 농도는 아질산나트륨(sodium nitrite)을 DMEM 배지에 용해한 표준 곡선을 이용하여 계산하였다. LPS를 처리한 대조군과 LPS를 처리하지 않은 대조군에서 생성된 아질산염의 생성량 차이를 기준으로 각 시료의 일산화질소 생성 저해 활성을 확인하였다. 비교실험을 위해 덱사메타손(Dexamethasone) 1μg/mL 처리군과 바이오틴(Biotin) 1, 10, 및 50μg/mL 처리군을 양성대조군으로 사용하였다. 또한, 모든 실험은 3회 반복하여 측정되었다.Specifically, the RAW 264.7 cells were inoculated in a 96-well plate at a concentration (in DMEM) of 1 × 10 6 cells/mL and cultured for 24 hours, followed by the enzyme-treated krill oil extract prepared in Example 1 of 1, 10, and a sample diluted to a concentration of 50 μg/mL and a fresh medium containing LPS (1 μg/mL), known as an endotoxin, were simultaneously treated and cultured for 24 hours. After that, 100 μL of cell culture supernatant and 100 μL of Griess reagent [1% (w/v) sulfanilamide, 0.1% (w/v) naphtylethylenediamine in 2.5% (v/v) phosphoric acid] were mixed and in a 96-well plate for 10 minutes. After reacting, the amount of nitric oxide produced was measured by measuring absorbance at 540 nm using an ELISA reader. The concentration of the produced nitrite (nitrite) was calculated using a standard curve in which sodium nitrite (sodium nitrite) was dissolved in DMEM medium. The nitric oxide production inhibitory activity of each sample was confirmed based on the difference in the amount of nitrite produced in the control group treated with LPS and the control group not treated with LPS. For the comparative experiment,
실험 결과, 도 3을 참조하면, 효소처리 하지 않은 크릴 오일 추출물에 비해 본 발명의 효소처리 크릴 오일 추출물의 산화질소 생성 억제 효과가 더 우수한 것을 확인할 수 있었다. As a result of the experiment, referring to FIG. 3 , it was confirmed that the nitric oxide production inhibitory effect of the enzyme-treated krill oil extract of the present invention was better than that of the enzyme-treated krill oil extract.
구체적으로, LPS(1μL/mL) 단독 처리군 대비 덱사메타손 처리 양성대조군은 산화질소 생성이 51.8% 감소하였다. 또한, 효소 처리하지 않은 비교예의 크릴 오일 추출물 처리군은 각각 1, 및 10μg/mL 농도에서는 LPS 단독처리군 대비 산화질소의 생성이 증가하였으나, 50μg/mL 농도에서 산화질소 생성량이 19.4% 감소한 것으로 나타났다. 이에 반해 본 발명의 효소처리 크릴 오일 추출물 처리군은 1, 10, 및 50μg/mL 농도에서 산화질소 생성량이 12.2%, 37%, 및 40.7% 감소하는 것으로 관찰되었고, 바이오틴 처리 양성대조군과 비교하여도 전반적으로 산화질소 생성 억제 효과가 우수한 것으로 나타났다. Specifically, nitric oxide production was reduced by 51.8% in the dexamethasone-treated positive control group compared to the LPS (1 μL/mL) alone treatment group. In addition, the krill oil extract treatment group of the comparative example without enzyme treatment increased the production of nitric oxide compared to the LPS alone treatment group at the concentrations of 1 and 10 μg/mL, respectively, but at the concentration of 50 μg/mL, the amount of nitric oxide was reduced by 19.4%. . In contrast, the enzyme-treated krill oil extract treatment group of the present invention was observed to decrease the amount of nitric oxide production by 12.2%, 37%, and 40.7% at the concentrations of 1, 10, and 50 μg/mL, even compared to the biotin-treated positive control group Overall, it was found that the nitric oxide production inhibitory effect was excellent.
이러한 결과를 통해 본 발명의 효소처리 크릴 오일 추출물이 산화질소 생성 억제에 우수한 효과를 나타내는 것으로 확인할 수 있었다(도 3 참조). Through these results, it was confirmed that the enzyme-treated krill oil extract of the present invention exhibits an excellent effect in inhibiting nitric oxide production (see FIG. 3 ).
실험예 3. 항주름 활성 Experimental Example 3. Anti-wrinkle activity
3-1. 세포 독성3-1. cytotoxicity
상기 실시예 1에서 제조한 효소처리 크릴 오일 추출물이 인간 피부섬유아세포(NHDF, normal human dermal fibroblasts)의 생장에 미치는 효과를 아래와 같은 방법으로 확인하였다. The effect of the enzyme-treated krill oil extract prepared in Example 1 on the growth of normal human dermal fibroblasts (NHDF) was confirmed by the following method.
우선, 10% FBS(fetal bovine serum, Cambrex)가 포함된 전용 배지인 DMEM(Dulbeccos modified Eagles medium) 배지에 피부섬유아세포를 40mm 크기의 세포 배양 접시에 1.25 농도로 접종한 후 24시간 동안, 37℃, 5% CO2 습윤 조건에서 배양하였다. 이후, 배지를 제거하고 300㎕ 용량의 PBS를 가하고, 무혈청 DMEM 배지로 희석한 효소처리 크릴 오일 추출물을 농도별(1, 10, 및 50㎍/㎕)로 처리하고 72시간 동안 배양하였다. 72시간 후, 1mg/mL의 MTT를 처리하고 2시간 후, 세포에 MTT 처리로 인해 생성된 포마잔(formazan)을 DMSO로 녹여 570nm에서 흡광도를 측정하였다.First, the skin fibroblasts were inoculated at a concentration of 1.25 in a 40mm-sized cell culture dish in DMEM (Dulbeccos modified Eagles medium) medium, which is a dedicated medium containing 10% FBS (fetal bovine serum, Cambrex), for 24 hours at 37°C. , 5% CO 2 Incubated under humid conditions. Thereafter, the medium was removed, 300 μl of PBS was added, and the enzyme-treated krill oil extract diluted with serum-free DMEM medium was treated with different concentrations (1, 10, and 50 μg/μl) and cultured for 72 hours. After 72 hours, 1 mg/mL of MTT was treated and 2 hours later, the cells were dissolved in DMSO and absorbance was measured at 570 nm.
그 결과, 효소처리 크릴 오일 추출물을 인간 피부섬유아세포에 처리한 경우, 효소처리 크릴 오일 추출물이 인간 피부섬유아세포의 세포 생존율에 크게 영향을 미치지 않는 것으로 확인하였다(도 4 참조). As a result, when the enzyme-treated krill oil extract was treated on human dermal fibroblasts, it was confirmed that the enzyme-treated krill oil extract did not significantly affect the cell viability of human dermal fibroblasts (see FIG. 4 ).
3-2. MMP-1 단백질 합성 저해3-2. Inhibition of MMP-1 protein synthesis
40mm 세포 배양 접시에 2㎖의 DMEM 배양액을 넣고, 인간섬유아세포를 약 1.2x105 농도로 접종한 후, 37℃, 5% CO2 환경에서 24시간 동안 배양하였다. 그 후, 실시예 1에서 제조된 효소처리 크릴 오일 추출물(각각 1, 10, 및 50㎍/㎖)을 포함한 배지로 교체하여 3일간 배양하였다. 이후 배양액을 회수(harvest)하여 4℃, 7,500rpm 환경에서 5분간 원심분리(centrifuge)하여 ELISA 방법을 이용하여 MMP-1(Human Total MMP-1 kit, R&D Systems, Inc., Minneapolis, MN, USA)의 단백질 발현량 변화를 확인하였다. 비교실험을 위해 TGF-β1 10ng/mL 처리군과 바이오틴(Biotin) 1, 10, 및 50μg/mL 처리군을 양성대조군으로 사용하였다. 또한, 모든 실험은 3회 반복하여 측정되었다.2ml of DMEM culture solution was put in a 40mm cell culture dish, and human fibroblasts were inoculated at a concentration of about 1.2x10 5 , and then cultured at 37° C., 5% CO 2 environment for 24 hours. Thereafter, the medium was replaced with the medium containing the enzyme-treated krill oil extract (1, 10, and 50 μg/ml, respectively) prepared in Example 1 and cultured for 3 days. Afterwards, the culture medium was harvested and centrifuged for 5 minutes at 4 ° C., 7,500 rpm environment, and MMP-1 (Human Total MMP-1 kit, R&D Systems, Inc., Minneapolis, MN, USA) using ELISA method. ) of the protein expression level was confirmed. For the comparative experiment, TGF-β1 10ng/mL treatment group and
그 결과, 효소처리 크릴 오일 추출물 처리군은 MMP-1 단백질의 생성을 농도 의존적으로 억제하고 있음을 확인할 수 있었다. As a result, it was confirmed that the enzyme-treated krill oil extract treatment group inhibited the production of MMP-1 protein in a concentration-dependent manner.
이 때, 효소처리 크릴 오일 추출물 처리군은 효소처리하지 않은 비교예의 크릴 오일 처리군과 비교하여 현저하게 감소된 MMP-1 단백질 생성량이 측정되었는다. At this time, the enzyme-treated krill oil extract treatment group, compared with the krill oil treatment group of the comparative example not treated with the enzyme, a significantly reduced amount of MMP-1 protein production was measured.
구체적으로 무처리 정상대조군(Nor)과 비교하여 비교예의 효소처리하지 않은 크릴 오일 처리군의 경우, 1, 10㎍/㎖ 농도에서 오히려 MMP-1 단백질의 생성을 증가되는 것으로 관찰되었고, 50㎍/㎖ 농도에서만 MMP-1 단백질의 생성이 7.6% 감소되는 것으로 관찰되었다. 이에 반해, 본 발명의 효소처리된 크릴 오일 추출물 처리군은 정상대조군(Nor)과 비교하여 1, 10, 및 50㎍/㎖ 용량에서 MMP-1 단백질 생성이 각각 20.2%, 31.8% 및 45.4% 감소된 것으로 관찰되었다. 효소처리하지 않은 비교예의 크릴 오일 처리군과 비교하였을 경우 본 발명의 효소처리 크릴 오일은 1, 10, 및 50㎍/㎖ 용량에서 MMP-1 단백질 생성이 각각 33.6%, 35.9% 및 40.8% 감소된 것으로 나타나 효소처리로 인해 MMP-1 단백질의 생성 저해에 더욱 효과적임을 확인할 수 있었다(도 5 참조). Specifically, compared to the untreated normal control group (Nor), in the case of the krill oil-treated group that was not treated with the enzyme of Comparative Example, it was observed that the production of MMP-1 protein was rather increased at concentrations of 1 and 10 μg/ml, and 50 μg/m A 7.6% reduction in the production of MMP-1 protein was observed only at the ml concentration. In contrast, the enzyme-treated krill oil extract treatment group of the present invention reduced MMP-1 protein production by 20.2%, 31.8% and 45.4%, respectively, at 1, 10, and 50 μg/ml doses compared to the normal control group (Nor). was observed to have been When compared with the krill oil-treated group of the comparative example not treated with the enzyme, the enzyme-treated krill oil of the present invention exhibited 33.6%, 35.9% and 40.8% reduction in MMP-1 protein production at 1, 10, and 50 μg/ml doses, respectively. It could be confirmed that the enzyme treatment was more effective in inhibiting the production of MMP-1 protein (see FIG. 5).
또한, 양성대조군으로 사용한 바이오틴 처리군에 비하여 본 발명의 효소처리 크릴 오일 추출물 처리군의 효과가 모든 농도 구간에서 월등한 것으로 나타났으며, 효소처리 크릴 오일 추출물 50㎍/㎖ 용량 처리군에서는 10,536.29pg/ml의 MMP-1 단백질이 측정되어 TGF-β1 10ng/mL 처리군의 MMP-1 단백질 측정값 9,219.86 pg/ml와 비교하여 동등한 수준의 효과를 나타내는 것으로 관찰되었다. In addition, compared to the biotin-treated group used as a positive control group, the effect of the enzyme-treated krill oil extract treatment group of the present invention was found to be superior in all concentration sections, and 10,536.29 pg in the enzyme-treated
이러한 결과를 통해, 본 발명의 효소처리 크릴 오일 추출물은 MMP-1 단백질의 생성 저해에 효과적으로 사용할 수 있음을 확인할 수 있었다(도 5 참조). Through these results, it was confirmed that the enzyme-treated krill oil extract of the present invention can be effectively used to inhibit the production of MMP-1 protein (see FIG. 5 ).
3-3. 타입 1 프로콜라겐 단백질의 합성 촉진3-3. Promotes the synthesis of
40mm 세포 배양 접시에 2㎖의 DMEM 배양액을 넣고, 인간섬유아세포를 약 1.2x105 농도로 접종한 후, 37℃, 5% CO2 환경에서 24시간 동안 배양하였다. 그 후, 실시예 1에서 제조한 효소처리 크릴 오일 추출물(각각 1, 10, 및 50㎍/㎖)을 포함한 배지로 교체하여 배양하였다. 이후 배양액을 회수(harvest)하여 4℃, 7,500rpm 환경에서 5분간 원심분리(centrifuge)하고 ELISA 방법을 이용하여 타입 1 프로콜라겐(Procollagen Type I C Peptide EIA Kit, Takara, Shiga, Japan)의 단백질 발현량 변화를 확인하였다.2ml of DMEM culture solution was put in a 40mm cell culture dish, and human fibroblasts were inoculated at a concentration of about 1.2x10 5 , and then cultured at 37° C., 5% CO 2 environment for 24 hours. Thereafter, the culture medium was replaced with a medium containing the enzyme-treated krill oil extract (1, 10, and 50 μg/ml, respectively) prepared in Example 1. Thereafter, the culture medium was harvested, centrifuged for 5 minutes at 4°C and 7,500 rpm, and protein expression level of
도 6을 참조하면, 바이오틴 또는 효소처리하지 않은 비교예의 크릴 오일 추출물을 처리한 인간섬유아세포는 타입 1 프로콜라겐 단백질의 생성량에 크게 변화를 보이지 않았으며, 무처리 정상 대조군에 비해 오히려 타입 1 프로콜라겐 단백질 생성량이 감소하는 현상이 관찰되었다. Referring to FIG. 6 , human fibroblasts treated with the krill oil extract of Comparative Example without biotin or enzyme treatment did not show a significant change in the amount of
이와 달리, 본 발명의 효소처리 크릴 오일 추출물 처리군은 무처리 정상대조군과 비교할 때 뚜렷한 타입 1 프로콜라겐 단백질 발현량 증가가 관찰되었는데, 1, 10, 및 50㎍/㎖ 용량에서 각각 95.8%, 123%, 138% 개선된 타입 1 프로콜라겐 단백질 발현량 증가를 나타내었다.In contrast, in the enzyme-treated krill oil extract treatment group of the present invention, a distinct increase in
상기와 같은 결과를 통해, 본 발명의 효소처리 크릴 오일 추출물은 주름 생성을 억제하는 타입 1 프로콜라겐 단백질의 생성을 효과적으로 증진하여 피부 주름의 예방 및 개선에 효과적으로 사용될 수 있음을 확인할 수 있었다. Through the above results, it was confirmed that the enzyme-treated krill oil extract of the present invention can be effectively used to prevent and improve skin wrinkles by effectively promoting the production of
실험예 4. 피부 보습 활성 Experimental Example 4. Skin moisturizing activity
본 발명의 효소처리 크릴 오일 추출물의 피부 보습 효과를 검증하기 위해 히알루론산(Hyaluronan) 합성 촉진에 미치는 영향을 실험하였다.In order to verify the skin moisturizing effect of the enzyme-treated krill oil extract of the present invention, the effect on the synthesis of hyaluronan was tested.
히알루론산(HA, Hyaluronan, Hyaluronic acid)은 주로 표피 각질형성세포와 진피 섬유아세포에 의해 합성되며, 수분과 결합하여 세포가 정상적으로 활동할 수 있는 환경을 조성하는 중요한 역할을 하는 물질이다. 피부에서 히알루론산의 감소는 피부 탄력 저하 및 수분 함유량 감소의 직접적인 원인 중 하나이다. 따라서 본 실험예에서는 인간각질형성세포 내에서 히알루론산의 생성량을 확인하였다. Hyaluronic acid (HA, Hyaluronan, Hyaluronic acid) is mainly synthesized by epidermal keratinocytes and dermal fibroblasts, and is a substance that plays an important role in creating an environment in which cells can function normally by combining with water. A decrease in hyaluronic acid in the skin is one of the direct causes of a decrease in skin elasticity and a decrease in moisture content. Therefore, in this experimental example, the amount of hyaluronic acid produced in human keratinocytes was confirmed.
먼저, 인간각질형성세포를 24 well 세포배양 접시에 1mL의 배양액을 넣고 약 1×105 cells/mL의 농도로 세포를 접종한 후, 37℃, 5% CO2 환경에서 24시간 동안 배양하였다. 그 후, 실시예 1에서 제조한 효소처리 크릴 오일 추출물과 효소 처리하지 않은 비교예의 크릴 오일 추출물(각각 1, 10, 및 50㎍/㎖)을 포함한 배지로 교체하여 24시간 배양하였다. 이후 배양액을 회수(harvest)하여 4℃, 7,500rpm 환경에서 5분간 원심분리(centrifuge)한 뒤, ELISA 방법을 이용하여 Hyaluronan(Hyaluronan kit, R&D Systems, Inc., Minneapolis, MN, USA)의 발현량을 확인하였다. 비교실험을 위해, 정상대조군(Nor, 100% DMEM)과 대조군(Con, 용매 + 시료 무첨가 배지)을 사용하였고, 바이오틴(Biotin) 처리군을 양성대조군으로 사용하였다. 또한, 모든 실험은 3회 반복하여 측정되었다.First, human keratinocytes were inoculated with 1 mL of a culture solution in a 24-well cell culture dish at a concentration of about 1×10 5 cells/mL, and then cultured at 37° C., 5% CO 2 environment for 24 hours. Thereafter, the medium containing the enzyme-treated krill oil extract prepared in Example 1 and the krill oil extract of Comparative Example without enzyme treatment (1, 10, and 50 μg/ml, respectively) was replaced and cultured for 24 hours. Thereafter, the culture medium was harvested and centrifuged for 5 minutes at 4° C., 7,500 rpm, and the expression level of Hyaluronan (Hyaluronan kit, R&D Systems, Inc., Minneapolis, MN, USA) was performed using an ELISA method. was confirmed. For the comparative experiment, a normal control group (Nor, 100% DMEM) and a control group (Con, solvent + sample-free medium) were used, and a biotin-treated group was used as a positive control group. In addition, all experiments were repeated three times.
실험 결과, 도 7을 참조하면, 본 발명의 효소처리 크릴 오일 추출물을 처리한 시험군에서는 히알루론산(Hyaluronan)의 생성량이 10㎍/㎖ 농도까지 농도 의존적으로 증가하는 경향을 확인하였고, 정상대조군에 비해 히알루론산의 생성량이 1, 10, 및 50㎍/㎖ 농도에서 각각 178.4%, 229%, 및 222.9% 증가하는 것으로 관찰되었다. 또한, 모든 구간에서 효소처리된 크릴오일 추출물을 처리한 모든 구간에서 바이오틴(Biotin) 처리군 대비 더욱 증가된 히알루론산 생성량이 측정되었다.As a result of the experiment, referring to FIG. 7 , in the test group treated with the enzyme-treated krill oil extract of the present invention, it was confirmed that the production amount of hyaluronan increased in a concentration-dependent manner up to a concentration of 10 μg/ml, and in the normal control group In comparison, it was observed that the production amount of hyaluronic acid increased by 178.4%, 229%, and 222.9%, respectively, at the concentrations of 1, 10, and 50 μg/ml. In addition, in all sections treated with the enzyme-treated krill oil extract in all sections, an increased amount of hyaluronic acid production was measured compared to the biotin treatment group.
상기와 같은 결과를 통해, 본 발명의 효소처리 크릴 오일 추출물은 피부 세포에서 히알루론산 생성을 효과적으로 증가시켜 피부 보습과 관련된 용도로 매우 효과적으로 사용될 수 있음을 확인할 수 있었다. Through the above results, it was confirmed that the enzyme-treated krill oil extract of the present invention effectively increases the production of hyaluronic acid in skin cells and can be very effectively used for skin moisturizing purposes.
실험예 5. 피부 혈행 개선 및 재생 활성 Experimental Example 5. Skin blood circulation improvement and regeneration activity
혈행 개선 인자인 VEGF(vascular endothelial growth factor) 인자의 발현 조절과 관련하여 본 발명의 효소처리 크릴 오일 추출물의 효과를 검증하였다.The effect of the enzyme-treated krill oil extract of the present invention was verified in relation to the regulation of expression of vascular endothelial growth factor (VEGF) factor, which is a blood circulation improving factor.
먼저, 24 well 세포배양 접시에 1 mL 용량의 DMEM 배양액을 넣고, 인간각질형성세포(HaCaT) 세포를 약 1.2x105 cells/mL 농도로 접종한 후, 37℃, 5% CO2 환경에서 24시간 동안 배양하였다. 그 후, 실시예 1에서 제조된 효소처리 크릴 오일 추출물을 각각 1, 10, 및 50㎍/㎖ 농도로 희석한 시료를 포함한 배지로 교체하여 24시간 배양하였다. 이후 배양액을 회수(harvest)하여 4℃, 7,500rpm 환경에서 5분간 원심분리(centrifuge)하고, VEGF 항체(Human VEGF kit, R&D Systems, Inc., Minneapolis, MN, USA)를 사용하여 ELISA 방법으로 VEGF 단백질의 발현량 변화를 확인하였다. 이때, 무처리 정상대조군(Nor)을 제외한 모든 군은 스크레치를 낸 후 시료를 처리하였다. 또한, 모든 실험은 3회 반복하여 측정되었다.First, put 1 mL of DMEM culture medium in a 24-well cell culture dish, and inoculate human keratinocytes (HaCaT) cells at a concentration of about 1.2x10 5 cells/mL, 37°C, 5% CO 2 environment for 24 hours. incubated during Thereafter, the enzyme-treated krill oil extract prepared in Example 1 was replaced with a medium containing a sample diluted to a concentration of 1, 10, and 50 μg/ml, respectively, and cultured for 24 hours. Thereafter, the culture medium was harvested and centrifuged for 5 minutes at 4° C., 7,500 rpm, and VEGF antibody (Human VEGF kit, R&D Systems, Inc., Minneapolis, MN, USA) was used to ELISA method. A change in the expression level of the protein was confirmed. At this time, all groups except for the untreated normal control group (Nor) were treated with the sample after scratching. In addition, all experiments were repeated three times.
실험 결과, 도 8을 참조하면, 스크레치만 낸 처리군(Con)에서는 무처리 정상대조군(Nor)에 비해 VEGF 인자의 발현이 소폭 감소하였고, 효소 처리하지 않은 크릴 오일 추출물을 처리한 군에서도 VEGF 인자의 발현량에 변화가 없는 것으로 관찰되었다. As a result of the experiment, referring to FIG. 8 , the expression of VEGF factor was slightly reduced in the treated group (Con) with only scratch compared to the untreated normal control group (Nor), and even in the group treated with the krill oil extract without the enzyme treatment, the VEGF factor It was observed that there was no change in the expression level of
이와 달리, 본 발명의 효소처리 크릴 오일 추출물을 처리한 실험군에서는 10㎍/㎖ 농도까지 농도 의존적으로 VEGF 인자의 발현이 증가하였으며, 스크레치만 낸 대조군(Con)과 비교하여 1, 10, 및 50㎍/㎖ 농도에서 VEGF 인자의 발현이 각각 106.4%, 155.5%, 및 152.4% 증가하는 것으로 관찰되었다. In contrast, in the experimental group treated with the enzyme-treated krill oil extract of the present invention, the expression of VEGF factor was increased in a concentration-dependent manner up to a concentration of 10 μg/ml, and 1, 10, and 50 μg compared to the control group (Con) with only scratches. At the concentration /ml, the expression of VEGF factor was observed to increase by 106.4%, 155.5%, and 152.4%, respectively.
또한, 스크레치가 재생되는 과정의 피부세포의 형태적인 모양에 있어서도 무처리군의 피부 세포보다 세포 증식(proliferation) 및 분화(differentiation)가 원활하며 상처(wound) 부위의 세포 밀도가 더욱 높은 것으로 관찰되었다(도 9 참조).In addition, in the morphological shape of the skin cells in the process of regenerating the scratch, cell proliferation and differentiation were smoother than the skin cells of the untreated group, and it was observed that the cell density in the wound area was higher. (See Fig. 9).
상기와 같은 결과를 통해, 본 발명의 효소처리 크릴 오일 추출물은 혈관 형성을 촉진하고 미세 혈관 투과성을 증가시키는 VEGF 인자의 발현 촉진을 통해 피부 혈행 개선에 효과적으로 사용될 수 있음을 확인할 수 있었다. Through the above results, it was confirmed that the enzyme-treated krill oil extract of the present invention can be effectively used to improve skin blood circulation by promoting the expression of VEGF factors that promote blood vessel formation and increase microvascular permeability.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
Claims (8)
VEGF(vascular endothelial growth factor) 인자의 발현을 촉진하여 피부 혈행 개선 활성을 나타내는 것을 특징으로 하는,
항염증, 피부 주름개선, 보습 및 피부 재생 촉진용 화장료 조성물.Contains protease-treated krill oil extract as an active ingredient,
Characterized in that it promotes the expression of vascular endothelial growth factor (VEGF) factor to exhibit skin blood circulation improvement activity,
A cosmetic composition for anti-inflammatory, skin wrinkle improvement, moisturizing and skin regeneration promotion.
상기 단백분해효소는 알칼라제(Alkalase), 에스페라제(Esperase), 프로테아제(Protease) NL, 프로테아제(Protease) A, 프로테아제(Protease) P, 프로테아제(Protease) S, 및 에버라제(Evelase) 6.0에서 선택된 어느 하나인 것을 특징으로 하는 항염증, 피부 주름개선, 보습 및 피부 재생 촉진용 화장료 조성물.According to claim 1,
The protease is Alkalase, Esperase, Protease NL, Protease A, Protease P, Protease S, and Everase 6.0 Anti-inflammatory, skin wrinkle improvement, moisturizing and skin regeneration promotion cosmetic composition, characterized in that any one selected from.
상기 조성물은 산화질소 생성을 억제하여 항염증 활성을 나타내는 것을 특징으로 하는 항염증, 피부 주름개선, 보습 및 피부 재생 촉진용 화장료 조성물.According to claim 1,
The composition is a cosmetic composition for anti-inflammatory, skin wrinkle improvement, moisturizing and promoting skin regeneration, characterized in that it exhibits anti-inflammatory activity by inhibiting nitric oxide production.
상기 조성물은 MMP-1 유전자 발현을 억제하고, 타입 1 프로콜라겐 단백질 발현을 증진하여 피부 주름 개선 활성을 나타내는 것을 특징으로 하는 항염증, 피부 주름개선, 보습 및 피부 재생 촉진용 화장료 조성물.According to claim 1,
The composition suppresses MMP-1 gene expression and promotes type 1 procollagen protein expression to exhibit anti-inflammatory, skin wrinkle improvement, moisturizing and skin regeneration promotion cosmetic composition, characterized in that it exhibits skin wrinkle improvement activity.
상기 조성물은 히알루론산 합성을 촉진하는 것을 특징으로 하는 항염증, 피부 주름개선, 보습 및 피부 재생 촉진용 화장료 조성물.According to claim 1,
The composition is a cosmetic composition for promoting anti-inflammatory, skin wrinkle improvement, moisturizing and skin regeneration, characterized in that it promotes hyaluronic acid synthesis.
VEGF(vascular endothelial growth factor) 인자의 발현을 촉진하여 피부 혈행 개선 활성을 나타내는 것을 특징으로 하는,
항염증, 피부 주름개선, 보습 및 피부 재생 촉진용 식품 조성물.Contains protease-treated krill oil extract as an active ingredient,
Characterized in that it promotes the expression of vascular endothelial growth factor (VEGF) factor to exhibit skin blood circulation improvement activity,
Food composition for anti-inflammatory, skin wrinkle improvement, moisturizing and promoting skin regeneration.
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