KR20210101572A - Composition for preventing or improving skin damage by ultraviolet comprising compounds derived from Sargassum horneri extracts - Google Patents
Composition for preventing or improving skin damage by ultraviolet comprising compounds derived from Sargassum horneri extracts Download PDFInfo
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- KR20210101572A KR20210101572A KR1020200015689A KR20200015689A KR20210101572A KR 20210101572 A KR20210101572 A KR 20210101572A KR 1020200015689 A KR1020200015689 A KR 1020200015689A KR 20200015689 A KR20200015689 A KR 20200015689A KR 20210101572 A KR20210101572 A KR 20210101572A
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- preventing
- skin damage
- ultraviolet rays
- damage caused
- extract
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Images
Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
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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
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
- A61K8/4973—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/16—Emollients or protectives, e.g. against radiation
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/318—Foods, ingredients or supplements having a functional effect on health having an effect on skin health and hair or coat
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- 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
Description
본 발명은 괭생이모자반 추출물 유래 화합물을 포함하는 자외선에 의한 피부손상 예방 또는 개선용 조성물에 관한 것이다.The present invention relates to a composition for preventing or improving skin damage caused by ultraviolet rays, which includes a compound derived from the extract derived from A.
피부는 사람의 장기 중 가장 바깥에 위치해 있으며, 세균, 바이러스, 물리적 손상, 자외선 등과 같은 외부환경에 대한 1차적인 보호장벽의 역할을 한다. 피부는 지속적인 외부 스트레스가 가해지는 경우 손상을 받기 쉽다. 이러한 외부 스트레스의 주요 원인으로는 자외선 노출, 배기가스, 황사, 환경오염물, 중금속, 온도변화, 미생물을 대표적인 예로 들 수 있으며, 이들은 주로 산화적 스트레스 (oxidative stress)를 통하여 활성산소종 (reactive oxygen species, ROS)을 생성함으로써, 항산화 방어체계를 붕괴시켜 세포 손상 및 사멸을 야기하게 된다.The skin is located at the outermost part of the human organ and serves as a primary protective barrier against the external environment such as bacteria, viruses, physical damage, and UV rays. The skin is susceptible to damage when subjected to constant external stress. The main causes of such external stress include UV exposure, exhaust gas, yellow dust, environmental pollutants, heavy metals, temperature changes, and microorganisms, which are mainly reactive oxygen species through oxidative stress. , ROS), disrupts the antioxidant defense system, causing cell damage and death.
외부 스트레스 중 태양광선 특히 자외선 (ultraviolet, UV)은 활성산소종의 생성을 통하여 피부 세포 및 조직에 손상을 일으키며, 지속적인 스트레스는 생체내 거대분자인 지질의 과산화, 단백질 산화, DNA 산화, 단백 분해효소 활성화, 탄력섬유 손상, 멜라닌 생성반응, 염증반응, 면역기능 저하 등을 유발하며 궁극적으로 피부 노화 과정을 가속화하는 것으로 알려져 있다. Among external stresses, sunlight, especially ultraviolet (ultraviolet, UV), causes damage to skin cells and tissues through the generation of reactive oxygen species, and continuous stress causes in vivo macromolecular lipid peroxidation, protein oxidation, DNA oxidation, and proteolytic enzymes. It is known to cause activation, damage to elastic fibers, melanin production, inflammatory response, and decrease in immune function, and ultimately accelerate the skin aging process.
특히, 자외선은 파장의 길이에 따라 장파장 (UV-A, 320-400 nm), 중파장 (UV-B, 290-320 nm), 단파장 (UV-C, 200-290 nm)로 구분된다. 자외선 A는 피부 내 진피까지 도달하며 탄력 및 교원섬유의 변성으로 인한 피부탄력 감소, 피부위축, 주름형성, 색소침착, 광독성, 광알러지, 피부암 등을 유발할 수 있다. 자외선 B는 표피의 기저층 또는 진피의 상부까지 도달하며 다량으로 노출되는 경우 면역저하, 염증 악화, 피부각화, 홍반, 일광화상, 색소침착, DNA 손상, 피부암 등을 일으키게 된다. 자외선 A 및 자외선 B 조사 후 생체 내 활성산소의 증가 및 내인성 항산화 효소 및 항산화제들의 감소가 보고되고 있으며, 그 결과로 인하여 광노화 과정이 촉진되게 된다.In particular, ultraviolet rays are classified into long wavelengths (UV-A, 320-400 nm), medium wavelengths (UV-B, 290-320 nm), and short wavelengths (UV-C, 200-290 nm) according to the length of the wavelength. Ultraviolet A reaches the dermis in the skin and can cause skin elasticity reduction due to elasticity and degeneration of collagen fibers, skin atrophy, wrinkle formation, pigmentation, phototoxicity, photoallergy, and skin cancer. Ultraviolet B rays reach the basal layer of the epidermis or the upper part of the dermis, and when exposed in large amounts, cause immunosuppression, exacerbation of inflammation, skin keratinization, erythema, sunburn, pigmentation, DNA damage, and skin cancer. After UV A and UV B irradiation, an increase in free radicals in vivo and a decrease in endogenous antioxidant enzymes and antioxidants have been reported, and as a result, the photoaging process is promoted.
따라서, 외부 스트레스로부터 피부 세포를 보호하고 노화를 예방하기 위해서는 활성산소로 인한 산화적 스트레스를 억제하고, 활성산소를 효율적으로 제거할 수 있는 항산화 방어 강화가 중요하며, 최근에는 인체에 무해하고 보다 강력한 항산화력을 가진 천연 물질에 대한 연구가 활발하게 진행되고 있다.Therefore, in order to protect skin cells from external stress and prevent aging, it is important to suppress oxidative stress caused by free radicals and to strengthen antioxidant defenses that can effectively remove free radicals, and recently, it is harmless to the human body and has more powerful Research on natural substances with antioxidant power is being actively conducted.
본 발명자들은 자외선에 의한 피부손상 억제 및 항산화 효과를 가지면서 인체 부작용이 적은 새로운 천연물질을 개발하기 위해 노력한 결과, 괭생이모자반 추출물 유래 화합물이 항산화 효과뿐만 아니라 자외선에 의한 피부손상을 억제하는 효과를 나타낸다는 것을 규명함으로써 본 발명을 완성하였다.As a result of the present inventors' efforts to develop a new natural substance with fewer side effects to the human body while inhibiting skin damage caused by UV rays and having an antioxidant effect, the compound derived from the extract derived from A. The present invention was completed by clarifying that
이에, 본 발명의 목적은 괭생이모자반 추출물 유래 화합물을 포함하는 자외선에 의한 피부손상 예방 또는 개선용 식품 조성물을 제공하는 것이다.Accordingly, it is an object of the present invention to provide a food composition for preventing or improving skin damage caused by ultraviolet rays, which includes a compound derived from the extract derived from hoesaengnimojabanum extract.
본 발명의 또 다른 목적은 괭생이모자반 추출물 유래 화합물을 포함하는 자외선에 의한 피부손상 예방 또는 개선용 화장료 조성물을 제공하는 것이다.Another object of the present invention is to provide a cosmetic composition for preventing or improving skin damage caused by ultraviolet rays, which includes a compound derived from the extract derived from hoesaengnimojabanum extract.
본 발명의 또 다른 목적은 괭생이모자반 추출물 유래 화합물을 포함하는 항산화용 화장료 조성물을 제공하는 것이다.Another object of the present invention is to provide a cosmetic composition for antioxidants comprising a compound derived from the extract derived from hoesaengnimojaban extract.
본 발명의 다른 목적은 괭생이모자반 추출물 유래 화합물을 포함하는 자외선에 의한 피부손상 예방 또는 치료용 약제학적 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating skin damage caused by ultraviolet rays, which includes a compound derived from the extract derived from hoesaengi caprifolia.
본 발명은 괭생이모자반 추출물 유래 화합물을 포함하는 피부손상 예방 또는 개선용 식품 조성물 및 화장료 조성물, 항산화용 화장료 조성물 및 자외선에 의한 피부손상 예방 또는 치료용 약제학적 조성물에 관한 것으로, 본 발명에 따른 괭생이모자반 추출물 유래 화합물은 항산화 효과뿐만 아니라 자외선에 의한 피부손상을 억제하는 효과를 나타낸다.The present invention relates to a food composition and a cosmetic composition for preventing or improving skin damage, a cosmetic composition for antioxidant, and a pharmaceutical composition for preventing or treating skin damage caused by ultraviolet rays, which include a compound derived from the extract derived from hoesaengifolia extract. The compound derived from Saengimozan extract exhibits an antioxidant effect as well as an effect of inhibiting skin damage caused by UV rays.
이하 본 발명을 더욱 자세히 설명하고자 한다. Hereinafter, the present invention will be described in more detail.
본 발명자들은 자외선에 의한 피부손상 억제 및 항산화 효과를 가지면서 인체 부작용이 적은 새로운 천연물질을 개발하기 위해 노력한 결과, 괭생이모자반 추출물 유래 화합물이 항산화 효과뿐만 아니라 자외선에 의한 피부손상을 억제하는 효과를 나타낸다는 것을 규명하였다. As a result of the present inventors' efforts to develop a new natural substance with fewer side effects to the human body while inhibiting skin damage caused by UV rays and having an antioxidant effect, the compound derived from the extract derived from A. It has been shown to indicate
괭생이모자반 (Sargassum horneri)은 한국, 일본 및 중국 연안에 폭넓게 분포하고 있으며, 주요 서식 수심층은 3-5 m 내외 암반으로 1-5 m까지 성장한다. 배우체 수명은 1년이며 고갈·유실을 반복하는 다년생 해조류로 주 성장 시기는 2-5월까지이다. 괭생이모자반은 5개월 동안 부유 가능한 모자반으로써 엽체 기질에서 떨어져 나와 본래 서식지로부터 해상 수백킬로(km)까지 이동이 가능하다. Sargassum horneri is widely distributed along the coasts of Korea, Japan, and China, and its main habitat is a bedrock of about 3-5 m and grows to 1-5 m. Gametophyte lifespan is one year, and it is a perennial seaweed that repeats exhaustion and loss, and the main growth period is from February to May. As a fertility fern for 5 months, it is detached from the thallus matrix and can migrate hundreds of kilometers (km) from its original habitat to the sea.
본 발명의 일 양태는 하기 화학식 1로 표시되는 화합물을 포함하는 자외선에 의한 피부손상 예방 또는 개선용 식품 조성물에 관한 것이다:One aspect of the present invention relates to a food composition for preventing or improving skin damage caused by ultraviolet rays comprising a compound represented by the following formula (1):
[화학식 1][Formula 1]
본 발명에 따르면, 상기 화학식 1로 표시되는 화합물은 괭생이모자반 추출물로부터 분리한 것일 수 있다. 본 발명의 발명자들은 괭생이모자반으로부터 상기 화합물을 추출하여 구조를 규명하였고, 상기 화합물이 피부손상에 대한 우수한 보호효과 및 ROS 생성 억제 효과를 가짐을 확인하였다. 상기 화학식 1의 화합물은 괭생이모자반 이외에 다른 식물로부터 추출할 수 있으며, 또한 일반적인 화학적 합성 방법에 의해서 합성할 수도 있다.According to the present invention, the compound represented by the formula (1) may be isolated from the extract The inventors of the present invention identified the structure by extracting the compound from the ferns, and confirmed that the compound has an excellent protective effect against skin damage and an inhibitory effect on ROS generation. The compound of Chemical Formula 1 may be extracted from other plants other than the ferns, and may also be synthesized by a general chemical synthesis method.
상기 화학식 1로 표시되는 화합물은 HTT (6-hydroxy-4,4,7a-trimethyl-5,6,7,7a-tetrahydrobenzofuran-2(4H)-one)으로 명명할 수 있다. The compound represented by
본 발명에 따르면, 상기 화학식 1로 표시되는 화합물은 괭생이모자반을 물, 탄소수 1 내지 4개의 직쇄 또는 분지형 알코올, 아세톤, 에틸아세테이트, 부틸아세테이트, 1,3-부틸렌 글리콜, 헥산 및 디에틸에테르로 이루어진 군으로부터 선택되는 1 이상의 용매로 추출하여 얻어진 추출물로부터 분리할 수 있다. According to the present invention, the compound represented by the formula (1) is obtained by mixing hoesaeng hat with water, a linear or branched alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, butyl acetate, 1,3-butylene glycol, hexane and diethyl. It can be separated from the extract obtained by extraction with one or more solvents selected from the group consisting of ethers.
본 발명의 일 구현예에 따르면, 건조한 괭생이모자반을 에탄올에 침지시킨 다음 원심분리 및 여과하여 얻은 괭생이모자반 추출물로부터 상기 화학식 1로 표시되는 화합물을 수득할 수 있다. 상기 괭생이모자반 추출물로부터 상기 화학식 1로 표시되는 화합물을 수득하는 방법은 당업계에 알려진 다양한 화합물 분리 방법을 이용할 수 있고, 예를 들어, 컬럼 크로마토그래피를 이용하여 수득할 수 있다. According to one embodiment of the present invention, the compound represented by the above formula (1) can be obtained from the extract obtained by immersing the dried hoesaengni capan in ethanol, followed by centrifugation and filtration. A method of obtaining the compound represented by
본 발명에 있어서, 피부의 "손상"이란 자외선에 의한 인간 피부세포의 사멸, 피부 세포 DNA 손상, 활성산소종 증가, 지질과산화 증가 등을 포함한다. 또한, 손상의"예방"은 상기 자외선에 의한 피부세포의 손상을 억제시키거나 지연시키는 모든 행위를 의미한다. 또한, 손상의 "개선"은 상기 자외선에 의한 피부세포의 손상 상태의 완화 또는 증상의 정도를 감소시키는 모든 행위를 의미한다.In the present invention, "damage" of the skin includes apoptosis of human skin cells by ultraviolet rays, DNA damage of skin cells, increase in reactive oxygen species, increase in lipid peroxidation, and the like. In addition, "prevention" of damage refers to any action that inhibits or delays the damage of the skin cells by the ultraviolet rays. In addition, "improvement" of damage refers to any action of alleviating the damage state of the skin cells by the ultraviolet rays or reducing the severity of the symptoms.
상기 자외선에 의한 피부손상은 피부염, 피부암, 피부건조, 알레르기, 습진, 홍반반응, 색소침착, 일광화상, 광노화, 만성광선피부염, 일광두드러기, 광독성 접촉피부염, 광알레르기성 접촉피부염, 포르피린증, 홍반성낭창 및 단순포진으로 이루어진 군으로부터 선택되는 1 이상의 피부손상일 수 있다. The skin damage caused by ultraviolet rays is dermatitis, skin cancer, dry skin, allergy, eczema, erythema reaction, pigmentation, sunburn, photoaging, chronic photodermatitis, sun urticaria, phototoxic contact dermatitis, photoallergic contact dermatitis, porphyria, erythema It may be at least one skin injury selected from the group consisting of lupus and herpes simplex.
본 발명에 따르면, 상기 화학식 1로 표시되는 화합물은 자외선에 의한 피부세포 사멸 및 피부세포 내 ROS(reactive oxygen species) 생성을 억제함으로써 자외선으로부터 피부를 보호할 수 있다. 또한, 상기 화합물은 NFκB 신호전달 관련 단백질의 발현을 억제하여 NFκB 신호전달 활성화를 억제함으로써 자외선에 의한 피부세포 사멸을 억제할 수 있다. 상기 NFκB 신호전달 관련 단백질은 인산화된 IκBα(p-IκBα) 및 NFκB p65(p-NFκB p65)와 핵 내 NFκB p65를 포함하나, 이에 제한되는 것은 아니다. According to the present invention, the compound represented by Formula 1 can protect the skin from UV rays by inhibiting the apoptosis of skin cells by UV rays and the generation of reactive oxygen species (ROS) in skin cells. In addition, the compound can suppress the apoptosis of skin cells by ultraviolet rays by inhibiting the expression of NFκB signaling-related protein to inhibit the activation of NFκB signaling. The NFκB signaling-related proteins include, but are not limited to, phosphorylated IκBα (p-IκBα) and NFκB p65 (p-NFκB p65) and nuclear NFκB p65.
상기 식품 조성물은 피부손상 예방 또는 개선을 위한 식품 등에 다양하게 이용될 수 있다. The food composition can be used in various ways, such as food for preventing or improving skin damage.
본 발명의 추출물을 첨가할 수 있는 식품으로는, 예를 들어, 음료, 껌, 차, 비타민 복합제, 침출차, 건강 식품류 등의 각종 식품류 및 분말, 과립, 정제, 캡슐 등의 건강기능식품의 형태로 사용할 수 있다.Foods to which the extract of the present invention can be added include, for example, beverages, chewing gum, tea, vitamin complexes, leached tea, health foods, etc., and in the form of health functional foods such as powders, granules, tablets, and capsules. Can be used.
상기 식품 조성물에 함유된 유효성분으로서 괭생이모자반 추출물 유래 화합물의 함량은 식품의 형태, 소망하는 용도 등에 따라 적절하게 특별한 제한이 없으며, 예를 들어, 전체 식품 중량의 0.01 내지 15 중량%로 가할 수 있으며, 건강 음료 조성물은 100 mL를 기준으로 0.02 내지 10 g, 바람직하게는 0.3 내지 1 g의 비율로 가할 수 있다.As an active ingredient contained in the food composition, the content of the compound derived from the extract of oleracea extract is not particularly limited appropriately depending on the type of food, desired use, etc. For example, it can be added as 0.01 to 15% by weight of the total food weight. In addition, the health beverage composition may be added at a ratio of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 mL.
본 발명의 식품 조성물은 지시된 비율로 필수 성분으로서 상기 괭생이모자반 추출물 유래 화합물을 함유하는 것 외에 액체성분에는 특별한 제한점은 없으며, 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. The food composition of the present invention does not have any special limitations on the liquid component other than containing the compound derived from the extract derived from the extract as an essential component in the indicated ratio, and as an additional component, various flavoring agents or natural carbohydrates, such as conventional beverages, are added. may contain.
상기 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등의 디사카라이드, 예를 들어 말토스, 슈크로스 등의 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제 (타우마틴, 스테비아 추출물 (예를 들어, 레바우디오시드 A, 글리시르히진등) 및 합성 향미제 (사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 mL당 일반적으로 약 1 내지 20 g, 바람직하게는 약 5 내지 12 g이다. Examples of the natural carbohydrate include monosaccharides, such as disaccharides such as glucose, fructose, and the like, such as maltose, sucrose, and the like, and polysaccharides such as dextrin, cyclodextrin, and the like. and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g, per 100 mL of the composition of the present invention.
상기 외에 본 발명의 식품 조성물은 여러 가지 영양제, 비타민, 광물 (전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제 (치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명의 조성물들은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 그렇게 중요하진 않지만 본 발명의 조성물 100 중량부 당 0 내지 약 20 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above, the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, colorants and thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the compositions of the present invention may contain natural fruit juice and pulp for the production of fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The proportion of these additives is not critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.
예를 들어, 본 발명의 식품 조성물이 드링크제로 제조되는 경우에는 본 발명의 블루베리 추출물 이외에 구연산, 액상과당, 설탕, 포도당, 초산, 사과산, 과즙, 두충 추출액, 대추 추출액, 감초 추출액 등을 추가로 포함시킬 수 있다.For example, when the food composition of the present invention is prepared as a drink, in addition to the blueberry extract of the present invention, citric acid, high fructose, sugar, glucose, acetic acid, malic acid, fruit juice, cephalothorax extract, jujube extract, licorice extract, etc. are additionally added. can be included
본 발명의 다른 일 양태에 따르면, 본 발명은 괭생이모자반 추출물 유래의 상기 화학식 1로 표시되는 화합물을 포함하는 자외선에 의한 피부손상 예방 또는 개선용 화장료 조성물을 제공한다.According to another aspect of the present invention, the present invention provides a cosmetic composition for preventing or improving skin damage caused by ultraviolet rays, comprising the compound represented by the above formula (1) derived from the extract of hoesaengi hat.
본 발명에 있어서, 화장료 조성물은 상기 괭생이모자반 추출물 유래 화합물 이외에 화장료 조성물에 통상적으로 이용되는 성분들을 포함할 수 있다. 예를 들어, 항산화제, 안정화제, 용해화제, 비타민, 안료 및 향료와 같은 화장료로서 허용되는 통상적인 보조제, 그리고 담체를 포함한다.In the present invention, the cosmetic composition may include ingredients commonly used in the cosmetic composition in addition to the compound derived from the extract derived from the extract. For example, conventional adjuvants acceptable as cosmetics such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, and carriers are included.
본 발명에 있어서, 화장료 조성물은 상기 괭생이모자반 추출물 유래 화합물 이외에 화장료 조성물에 통상적으로 이용되는 성분들을 포함할 수 있다. 예를 들어, 항산화제, 안정화제, 용해화제, 비타민, 안료 및 향료와 같은 화장료로서 허용되는 통상적인 보조제, 그리고 담체를 포함한다.In the present invention, the cosmetic composition may include ingredients commonly used in the cosmetic composition in addition to the compound derived from the extract derived from the extract. For example, conventional adjuvants acceptable as cosmetics such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, and carriers are included.
상기 화장료 조성물은 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성제-함유 클렌징, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이 등으로 제형화될 수 있으나, 이에 한정되는 것은 아니다. 보다 상세하게는, 영양 크림, 수렴 화장수, 유연 화장수, 로션, 에센스, 샴푸, 영양젤 또는 마사지 크림의 제형으로 제조될 수 있다.The cosmetic composition may be formulated as a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation and spray, etc. The present invention is not limited thereto. More specifically, it may be prepared in the form of a nourishing cream, astringent lotion, softening lotion, lotion, essence, shampoo, nutrition gel or massage cream.
상기 화장료 조성물의 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물성유, 식물성유, 왁스, 파라핀, 전분, 트라가칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 이용될 수 있다.When the formulation of the cosmetic composition is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide as a carrier component can be used
상기 화장료 조성물의 제형이 파우더 또는 스프레이인 경우에는 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄 히드록시드, 칼슘 실리케이트 또는 폴리아미드 파우더가 이용될 수 있고, 특히 스프레이인 경우에는 추가적으로 클로로플루오로히드로카본, 프로판/부탄 또는 디메틸 에테르와 같은 추진체를 포함할 수 있다.When the formulation of the cosmetic composition is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydrocarbon, propellants such as propane/butane or dimethyl ether.
상기 화장료 조성물이 용액 또는 유탁액인 경우에는 담체 성분으로서 용매, 용해화제 또는 유탁화제가 이용되고, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌 글리콜, 1,3-부틸글리콜 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르 등이 이용될 수 있다.When the cosmetic composition is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol, or fatty acid ester of sorbitan may be used.
상기 화장료 조성물의 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, 미결정 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 한천 또는 트라가칸트 등이 이용될 수 있다.When the formulation of the cosmetic composition 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 ester and polyoxyethylene sorbitan ester, microcrystalline Cellulose, aluminum metahydroxide, bentonite, agar or tragacanth may be used.
상기 화장료 조성물의 제형이 계면-활성제 함유 클렌징인 경우에는 담체 성분으로서 지방족 알코올 설페이트, 지방족 알코올 에테르 설페이트, 설포숙신산 모노에스테르, 이세티오네이트, 이미다졸리늄 유도체, 메틸타우레이트, 사르코시네이트, 지방산 아미드 에테르 설페이트, 알킬아미도베타인, 지방족 알코올, 지방산 글리세리드, 지방산 디에탄올아미드, 식물성유, 라놀린 유도체 또는 에톡실화 글리세롤 지방산 에스테르 등이 이용될 수 있다.When the formulation of the cosmetic composition is surfactant-containing cleansing, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid as carrier components Amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester and the like can be used.
본 발명의 다른 일 양태에 따르면, 본 발명은 괭생이모자반 추출물 유래의 상기 화학식 1로 표시되는 화합물을 포함하는 항산화용 화장료 조성물에 관한 것이다.According to another aspect of the present invention, the present invention relates to an antioxidant cosmetic composition comprising the compound represented by
상기 괭생이모자반 추출물 유래 화합물은 우수한 활성산소종 (Reactive oxygen species, ROS) 생성 저해 활성을 나타내므로 항산화 용도로 이용할 수 있다. The compound derived from the extract of hoesaengnimojaban extract exhibits excellent reactive oxygen species (ROS) production inhibitory activity, and thus can be used for antioxidant purposes.
본 발명의 항산화용 화장료 조성물은 피부의 산화 상태를 억제 또는 제거하여 피부노화 억제, 피부결 개선 및 피부 탄력 증진 효과를 나타낼 수 있다. The cosmetic composition for antioxidants of the present invention can suppress or remove the oxidative state of the skin, thereby inhibiting skin aging, improving skin texture, and enhancing skin elasticity.
본 발명의 다른 일 양태에 따르면, 본 발명은 괭생이모자반 추출물 유래의 상기 화학식 1로 표시되는 화합물을 포함하는 자외선에 의한 피부손상 예방 또는 치료용 약제학적 조성물에 관한 것이다. According to another aspect of the present invention, the present invention relates to a pharmaceutical composition for preventing or treating skin damage caused by ultraviolet rays, comprising the compound represented by the above formula (1) derived from the extract
본 명세서에서, 용어 "예방"은 질환 또는 질병을 보유하고 있다고 진단된 적은 없으나, 이러한 질환 또는 질병에 걸리기 쉬운 경향이 있는 개체에서 질환 또는 질병의 발생을 억제하는 것을 의미한다. As used herein, the term “prevention” refers to inhibiting the occurrence of a disease or disease in an individual who has never been diagnosed as possessing a disease or disease, but is prone to the disease or disease.
본 명세서에서, 용어 "치료"는 개체에서 (a) 질환 또는 질병의 발전의 억제 (b) 질환 또는 질병의 경감 및 (c) 질환 또는 질환의 제거를 의미한다. As used herein, the term “treatment” refers to (a) inhibiting the development of a disease or disorder in a subject, (b) alleviating the disease or disorder, and (c) eliminating the disease or disorder.
상기 자외선에 의한 피부손상은 피부염, 피부암, 피부건조, 알레르기, 습진, 홍반반응, 색소침착, 일광화상, 광노화, 만성광선피부염, 일광두드러기, 광독성 접촉피부염, 광알레르기성 접촉피부염, 포르피린증, 홍반성낭창 또는 단순포진일 수 있다. The skin damage caused by ultraviolet rays is dermatitis, skin cancer, dry skin, allergy, eczema, erythema reaction, pigmentation, sunburn, photoaging, chronic photodermatitis, sun urticaria, phototoxic contact dermatitis, photoallergic contact dermatitis, porphyria, erythema It can be lupus or herpes simplex.
본 발명에 있어서, 약제학적 조성물은 상기 괭생이모자반 추출물 유래 화합물 이외에 약제학적으로 허용되는 담체를 포함할 수 있다. 본 발명의 약제학적 조성물에 포함되는 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 탄수화물류 화합물 (예: 락토스, 아밀로스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 셀룰로스, 등), 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 염 용액, 알코올, 아라비아 고무, 식물성 기름 (예: 옥수수 기름, 목화 종자유, 두유, 올리브유, 코코넛유), 폴리에틸렌 글리콜, 메틸 셀룰로스, 메틸히드록시 벤조에이트, 프로필히드록시 벤조에이트, 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.In the present invention, the pharmaceutical composition may include a pharmaceutically acceptable carrier in addition to the compound derived from the extract derived from the extract. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include carbohydrate compounds (eg, lactose, amylose, dextrose, sucrose, sorbitol, mannitol, starch, cellulose, etc.). ), gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, salt solution, alcohol, gum arabic, vegetable oil (e.g. corn oil, cotton seed oil, soy milk, olive oil, coconut oil), polyethylene glycol, methyl cellulose, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate, and mineral oil. 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 비경구로 투여할 수 있으며, 바람직하게는 비경구 투여, 보다 바람직하게는 도포에 의한 국부 투여 방식으로 적용될 수 있다.The pharmaceutical composition of the present invention may be administered orally or parenterally, preferably by parenteral administration, more preferably by topical administration by application.
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. 한편, 본 발명의 약제학적 조성물의 투여량은 바람직하게는 1일 당 0.001-100 ㎎/kg(체중)이다. 또한, 외용제인 경우에는 성인 기준으로 1.0 내지 3.0 ml의 양으로 1일 1회 내지 5회 도포하여 1개월 이상 계속 하는 것이 좋다.A suitable dosage of the pharmaceutical composition of the present invention is variously prescribed depending on 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 Meanwhile, the dosage of the pharmaceutical composition of the present invention is preferably 0.001-100 mg/kg (body weight) per day. In addition, in the case of an external preparation, it is recommended to apply it once to 5 times a day in an amount of 1.0 to 3.0 ml based on an adult and continue it for at least 1 month.
다만, 상기 투여량은 본 발명의 범위를 한정하는 것이 아니다.However, the dosage does not limit the scope of the present invention.
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 it 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 괭생이모자반 추출물 유래 화합물은 강력한 항산화 작용을 통해서 자외선에 의한 산화적 스트레스에 대응하여 세포 보호 효과를 나타내 자외선에 의한 피부 손상을 효과적으로 예방 및 개선할 수 있으므로, 과도한 자외선 조사에 의해 유발가능한 각종 피부 질환을 예방 또는 개선에 유용하게 사용될 수 있다.The compound derived from the extract derived from the extract of the present invention can effectively prevent and improve skin damage caused by ultraviolet rays by showing a cellular protective effect in response to oxidative stress caused by ultraviolet rays through a strong antioxidant action. It can be usefully used for preventing or improving various skin diseases.
도 1은 HTT (6-hydroxy-4,4,7a-trimethyl-5,6,7,7a-tetrahydrobenzofuran-2(4H)-one) 화합물에 대한 구조식이다.
도 2a는 농도별 HTT 처리에 따른 HaCaT 세포의 세포생존율 변화를 측정한 결과이다.
도 2b는 UVB를 조사한 HaCaT 세포에 농도별로 HTT를 처리하고 세포생존율 변화를 측정한 결과이다.
도 3은 UVB를 조사한 HaCaT 세포에 농도별로 HTT를 처리하고 ROS 생성 정도를 측정한 결과이다.
도 4a는 UVB를 조사한 HaCaT 세포에 농도별로 HTT를 처리하고 세포사멸과 관련된 단백질 Bcl-2, Bax, p53, Cleaved-caspase 3 및 PARP의 발현 정도를 웨스턴 블롯으로 측정한 결과이다.
도 4b는 도 4a의 Bcl-2에 대한 결과를 그래프화한 것이다.
도 4c는 도 4a의 Bax에 대한 결과를 그래프화한 것이다.
도 4d는 도 4a의 p53에 대한 결과를 그래프화한 것이다.
도 4e는 도 4a의 Cleaved-caspase 3에 대한 결과를 그래프화한 것이다.
도 4f는 도 4a의 PARP에 대한 결과를 그래프화한 것이다.
도 5a는 UVB를 조사한 HaCaT 세포에 농도별로 HTT를 처리하고 NFκB 신호전달과 관련된 세포질 단백질 IκBα, p-IκBα, NFκB p65 및 p-NFκB p65의 발현 정도를 웨스턴 블롯으로 측정한 결과이다.
도 5b는 UVB를 조사한 HaCaT 세포에 농도별로 HTT를 처리하고 NFκB 신호전달과 관련된 핵 단백질 NFκB p65의 발현 정도를 웨스턴 블롯으로 측정한 결과이다.
도 5c는 도 5a의 IκBα에 대한 결과를 그래프화한 것이다.
도 5d는 도 5a의 p-IκBα에 대한 결과를 그래프화한 것이다.
도 5e는 도 5a의 NFκB p65에 대한 결과를 그래프화한 것이다.
도 5f는 도 5a의 p-NFκB p65에 대한 결과를 그래프화한 것이다.
도 5g는 도 5b의 NFκB p65에 대한 결과를 그래프화한 것이다. 1 is a structural formula for HTT (6-hydroxy-4,4,7a-trimethyl-5,6,7,7a-tetrahydrobenzofuran-2(4H)-one) compound.
Figure 2a is a result of measuring the change in the cell viability of HaCaT cells according to the HTT treatment for each concentration.
Figure 2b is a result of measuring the change in cell viability by treating the UVB-irradiated HaCaT cells with HTT for each concentration.
3 is a result of HTT treatment for each concentration in HaCaT cells irradiated with UVB and measuring the degree of ROS generation.
4a is a result of HTT treatment by concentration in HaCaT cells irradiated with UVB, and the expression levels of Bcl-2, Bax, p53, Cleaved-
Figure 4b is a graph of the results for Bcl-2 of Figure 4a.
Figure 4c is a graph of the result for Bax of Figure 4a.
Figure 4d is a graph of the result for p53 of Figure 4a.
Figure 4e is a graph of the results for Cleaved-
Figure 4f is a graph of the results for the PARP of Figure 4a.
FIG. 5a shows the results of processing HTT by concentration in UVB-irradiated HaCaT cells and measuring the expression levels of cytoplasmic proteins IκBα, p-IκBα, NFκB p65 and p-NFκB p65 related to NFκB signaling by Western blot.
Figure 5b is a result of HTT treatment for each concentration in HaCaT cells irradiated with UVB, and the expression level of the nuclear protein NFκB p65 related to NFκB signaling was measured by Western blot.
FIG. 5c is a graph showing the results for IκBα of FIG. 5a.
FIG. 5D is a graph showing the results for p-IκBα of FIG. 5A.
Figure 5e is a graph of the results for NFκB p65 of Figure 5a.
Figure 5f is a graph of the results for p-NFκB p65 of Figure 5a.
Figure 5g is a graph of the results for NFκB p65 of Figure 5b.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시 예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시 예에 의해 제한되지 않는다는 것은 당 업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .
본 명세서 전체에 거쳐, 특정 물질의 농도를 나타내기 위하여 사용되는 “%"는 별도의 언급이 없는 한 고체/고체는 (중량/중량) %, 고체/액체는 (중량/부피) %, 그리고 액체/액체는 (부피/부피) %이다.Throughout this specification, "%" used to indicate the concentration of a specific substance is (weight/weight) % for solids/solids, (weight/volume) % for solids/liquids, and liquids unless otherwise specified. /liquid is (volume/volume) %.
실시예 1. 괭생이모자반 유용성분 분리 및 세포 배양Example 1. Isolation and cell culture of useful components of the ferns
1-1. 괭생이모자반 유용성분 분리1-1. Separation of useful ingredients for hoesaengi hat
괭생이모자반(Sargassμm horneri)을 건조시킨 후, 70% 에탄올에 침지하여 12시간동안 교반하였고, 원심분리 및 여과지(Whatman No.6)로 여과하여 추출물을 획득하였다. 얻어진 괭생이모자반 에탄올 추출물로부터 유용성분인 6-hydroxy-4,4,7a-trimethyl-5,6,7,7a-tetrahydrobenzofuran-2(4H)-one (이하, HTT)를 분리하여 본 실험에 사용하였다. HTT의 구조식은 도 1에 제시된 것과 같다. After drying, Sargass μm horneri was immersed in 70% ethanol and stirred for 12 hours, followed by centrifugation and filtration with filter paper (Whatman No. 6) to obtain an extract. 6-hydroxy-4,4,7a-trimethyl-5,6,7,7a-tetrahydrobenzofuran-2(4H)-one (hereinafter, HTT), an useful component, was isolated from the obtained ethanol extract did. The structural formula of HTT is as shown in FIG. 1 .
1-2. 피부각질형성세포(HaCaT) 배양1-2. Skin keratinocyte (HaCaT) culture
실험에 사용한 피부각질형성세포(HaCaT)는 DMEM(HyCloneTM)에 10% FBS (fetal bovine serum)과 페니실린 (100 unit/ml)을 첨가한 것을 사용하였으며, 배양기를 이용하여 37 ℃와 5% CO2를 유지하였다.Skin keratin used in the experiment forming cells (HaCaT) was 10% in DMEM (HyCloneTM) FBS (fetal bovine serum) and penicillin (100 unit / ml) was used as the addition of, using the incubator 37 ℃ and 5% CO 2 was maintained.
실시예 2. HaCaT세포에서 HTT의 세포독성 및 UVB 처리에 대한 보호효과 Example 2. Protective effect on cytotoxicity and UVB treatment of HTT in HaCaT cells
HaCaT 세포에 대해 HTT의 세포독성과 UVB처리에 대한 보호효과를 측정하기 위해 MTT (thiazolyl blue tetrazolium bromide) 어세이를 실시하였다. HaCaT 세포를 24웰 플레이트에 1x105 세포로 분주하고 각 웰에 10% FBS와 1% 스트렙토마이신과 페니실린이 포함되어 있는 DMEM 500 μL와 함께 37℃ 5% CO2 조건하에서 24시간 배양하였다. 그 후 샘플을 농도별 (15.6-62.5 μg/mL)로 처리하여 48시간 동안 배양한 후, 상층액을 제거한 다음 DMSO (dimethylsulfoxide) 500 μL를 가하여 MTT의 환원에 의해 생성된 포르마잔 침전물을 용해시킨 후 마이크로플레이트 리더를 사용하여 570 nm에서 흡광도를 측정하였다. 또한, UVB 처리에 의한 HTT의 보호효과를 측정하기 위해 동일한 농도로 세포를 시딩한 후 24시간 뒤, HTT를 농도별로 처리하였으며, 2시간 뒤 UVB 40 mJ/cm2로 자극하고 FBS가 없는 배지로 교체하였다. 48시간 배양 후, 위에 제시된 방법과 동일하게 흡광도를 측정하여 세포생존율을 측정하였다.MTT (thiazolyl blue tetrazolium bromide) assay was performed to measure the cytotoxicity of HTT and the protective effect against UVB treatment on HaCaT cells. HaCaT cells were seeded in a 24-well plate at 1x10 5 cells, and each well was incubated with 500 μL of DMEM containing 10% FBS, 1% streptomycin, and penicillin at 37° C. under 5% CO 2 conditions for 24 hours. After that, the samples were treated at different concentrations (15.6-62.5 μg/mL) and incubated for 48 hours, the supernatant was removed, and then 500 μL of DMSO (dimethylsulfoxide) was added to dissolve the formazan precipitate generated by the reduction of MTT. Then, the absorbance was measured at 570 nm using a microplate reader. In addition, in order to measure the protective effect of HTT by UVB treatment, 24 hours after seeding cells at the same concentration, HTT was treated by concentration, 2 hours later,
[계산식][formula]
생존율(%) = UVB 처리군 또는 UVB + 샘플 처리군의 흡광도/대조군의 흡광도*100Viability (%) = absorbance of UVB treatment group or UVB + sample treatment group/absorbance of control group*100
HaCaT세포에 대해 HTT의 세포독성과 UVB 처리에 대한 보호효과를 확인하기 위해 MTT 어세이를 수행한 결과, HTT만 처리한 경우, HaCaT세포의 생존율이 아무것도 처리하지 않은 대조군과 유의적인 차이를 보이지 않는 것으로 보아 사용된 HTT는 세포독성을 보이지 않았다(도 2a). 또한, HaCaT세포에 UVB를 처리하였을 때, 아무것도 처리하지 않은 대조군에 비해 세포 생존율이 55%로 감소하였으나, HTT를 처리한 경우, UVB처리에 의해 감소된 세포생존율을 향상시켰다(도 2b).As a result of performing an MTT assay to confirm the cytotoxicity of HTT and the protective effect against UVB treatment on HaCaT cells, when only HTT was treated, the survival rate of HaCaT cells did not show a significant difference from that of the control untreated. As it can be seen, the HTT used did not show cytotoxicity (FIG. 2a). In addition, when UVB was treated to HaCaT cells, the cell viability was reduced to 55% compared to the control untreated, but when treated with HTT, the cell viability decreased by UVB treatment was improved ( FIG. 2b ).
실시예 3. UVB가 조사된 HaCaT 세포에서 HTT의 세포 내 ROS 생성 저해 활성 효과Example 3. Inhibitory effect on intracellular ROS production of HTT in UVB-irradiated HaCaT cells
UVB 조사에 의해 활성화된 HaCaT 세포에서 HTT 처리에 의한 세포 내 ROS(reactive oxygen species) 생성량을 평가하기 위해 DCFH-DA(2',7'- Dichlorofluorescin diacetate) 어세이를 수행하였다. HaCaT 세포를 24웰 플레이트에 1x105 세포로 분주하고 각 웰에 10% FBS와 1% 스트렙토마이신과 페니실린이 포함되어 있는 DMEM 500 μL와 함께 37℃ 5% CO2 조건하에서 24시간 배양하였다. 그 후 HTT를 농도별 (15.6-62.5 μg/mL)로 처리하여 2시간 동안 배양한 후, UVB 40 mJ/cm2로 자극하고 FBS가 없는 배지로 교체하여 1시간동안 추가로 배양하였다. 그런 다음 0.5 mg/mL의 DCFH-DA 용액을 처리하여 30분 동안 배양 후, 마이크로플레이트 리더를 이용하여 Excitation 485 nm/Emission 528 nm에서 측정하여 세포 내 ROS 생성량을 계산하였다. DCFH-DA (2',7'-Dichlorofluorescin diacetate) assay was performed to evaluate the amount of intracellular reactive oxygen species (ROS) production by HTT treatment in HaCaT cells activated by UVB irradiation. HaCaT cells were seeded in a 24-well plate at 1x10 5 cells, and each well was incubated with 500 μL of DMEM containing 10% FBS, 1% streptomycin, and penicillin at 37° C. under 5% CO 2 conditions for 24 hours. After that, HTT was treated with each concentration (15.6-62.5 μg/mL) and cultured for 2 hours , stimulated with UVB 40 mJ/cm 2 , and replaced with a medium without FBS, and further cultured for 1 hour. Then, 0.5 mg/mL of DCFH-DA solution was treated, incubated for 30 minutes, and the amount of intracellular ROS production was calculated by measuring at Excitation 485 nm/Emission 528 nm using a microplate reader.
[계산식][formula]
ROS 생성량 (%) = 대조군 또는 SHM 처리군의 흡광도/UVB만 처리한 군의 흡광도*100ROS production (%) = Absorbance of control or SHM treatment group / Absorbance of UVB-only treatment group * 100
HaCaT 세포에 UVB를 처리하여 생성된 세포 내 ROS에 대한 HTT의 억제효능을 평가한 결과, 세포에 UVB를 처리하였을 때 아무것도 처리하지 않은 대조군과 비교 시, 세포 내 ROS가 유의적으로 증가되었으나, HTT를 처리한 경우 감소되었다(도 3).As a result of evaluating the inhibitory effect of HTT on intracellular ROS generated by treating HaCaT cells with UVB, when cells were treated with UVB, intracellular ROS was significantly increased when compared to the control group not treated with anything, but HTT was reduced when treated (Fig. 3).
실시예 4. UVB가 조사된 HaCaT 세포에서 HTT가 세포사멸에 미치는 영향 평가Example 4. Evaluation of the effect of HTT on apoptosis in UVB-irradiated HaCaT cells
SHM이 UVB가 조사된 HaCaT 세포에서 세포사멸에 미치는 영향을 확인하기 위해, 웨스턴 블롯을 수행하였다. 먼저, HaCaT 세포를 10 cm 디쉬에 1x106 세포로 분주하고 각 디쉬에 10% FBS와 1% 스트렙토마이신과 페니실린이 포함되어 있는 DMEM과 함께 37℃ 5% CO2 조건하에서 24시간 배양하였다. 그 후 SHM을 농도별(31.3-125 μg/mL)로 처리하여 2시간 배양하고 UVB 40 mJ/cm2로 자극한 후, 세포에 용해 버퍼를 넣고 볼텍싱을 10분씩 6회 실시하여 4℃ 14,000 rpm에서 10분 동안 원심을 돌려 얻은 상층액을 가지고 BCA 키트를 이용하여 단백질을 정량하였다. 동일한 농도의 단백질과 샘플 버퍼를 혼합한 후, 100℃에서 3분간 끓여서 단백질의 변성을 유도하였다. 10% SDS-PAGE 겔에서 전기영동 된 후, PVDF 멤브레인으로 단백질을 전이하였다. 비특이적인 단백질 결합 부분은 0.1% Tween 20과 블로킹 버퍼를 함유한 TBS (Tris-buffered saline)에 2시간 동안 반응시켜 블로킹하였다. 이후에 1차 항체 Bcl-2 (1:1000), Bax (1:1000), p53 (1:1000), cleaved PARP (1:1000), β-actin (1:10000), Lamin B (1:1000)가 첨가된 용액을 12시간동안 혼합한 후 TBS-T로 10분간 3번 세척하였다. 그런 다음 멤브레인을 2차 항체 고트 항-마우스 IgG, 고트 항-토끼 IgG가 첨가된 용액을 상온에서 1시간 30분 동안 결합시켜 반응한 후 TBS-T로 10분간 3번 세척하였고, ECL detection reagents (Thermo, USA)를 이용하여 효소 반응에 의한 발광을 통해 표적 단백질의 발현을 측정하였다.To determine the effect of SHM on apoptosis in UVB-irradiated HaCaT cells, Western blot was performed. First, HaCaT cells were aliquoted into 1x10 6 cells in a 10 cm dish, and each dish was incubated with DMEM containing 10% FBS, 1% streptomycin and penicillin at 37° C. under 5% CO 2 conditions for 24 hours. After that, SHM was treated with different concentrations (31.3-125 μg/mL), incubated for 2 hours, and stimulated with UVB 40 mJ/cm 2 , lysis buffer was added to the cells, and vortexing was performed 6 times for 10 minutes at 4°C 14,000. The protein was quantified using the BCA kit with the supernatant obtained by centrifugation at rpm for 10 minutes. After mixing the same concentration of protein and sample buffer, it was boiled at 100° C. for 3 minutes to induce protein denaturation. After electrophoresis on a 10% SDS-PAGE gel, the protein was transferred to a PVDF membrane. Non-specific protein-binding portions were blocked by reacting for 2 hours in TBS (Tris-buffered saline) containing 0.1
UVB가 처리된 HaCaT 세포의 세포 사멸 관련 단백질 발현에 대한 HTT의 효과를 확인하기 위해 웨스턴 블롯을 수행하였다. 도 4에 제시된 것처럼 UVB를 처리한 세포에서 아무것도 처리하지 않은 세포에 비해 항-세포사멸 단백질인 Bcl-2의 발현은 감소하였고, 전-세포사멸 단백질인 Bax, p53 및 cleaved PARP의 발현은 증가한 것을 확인할 수 있었다. 흥미롭게도 HTT의 농도별 (31.3-125 μg/mL) 처리는 UVB가 감소시킨 Bcl-2 단백질의 발현을 농도 의존적으로 증가시켰으나, UVB가 증가시킨 Bax, p53 및 cleaved PARP의 단백질 발현을 농도 의존적으로 감소시켰다.Western blot was performed to confirm the effect of HTT on the expression of apoptosis-related proteins in UVB-treated HaCaT cells. As shown in Figure 4, the expression of the anti-apoptotic protein Bcl-2 was decreased, and the expression of the pro-apoptotic proteins Bax, p53 and cleaved PARP was increased in UVB-treated cells compared to cells not treated with anything. could check Interestingly, the concentration-dependent treatment of HTT (31.3-125 μg/mL) increased the expression of Bcl-2 protein, which was reduced by UVB, in a concentration-dependent manner, but the protein expression of Bax, p53 and cleaved PARP increased by UVB was increased in a concentration-dependent manner. decreased.
실시예 5. UVB가 조사된 HaCaT 세포에서 HTT가 NFκB 신호전달에 미치는 영향 평가Example 5. Evaluation of the effect of HTT on NFκB signaling in UVB-irradiated HaCaT cells
UVB가 조사된 HaCaT 세포에서 NFκB 신호전달 경로와 관련된 단백질의 발현은 웨스턴 블롯으로 확인하였다. 먼저, HaCaT 세포를 10 cm 디쉬에 1x106 세포로 분주하고 각 디쉬에 10% FBS와 1% 스트렙토마이신과 페니실린이 포함되어 있는 DMEM과 함께 37℃ 5% CO2 조건하에서 24시간 배양하였다. 그 후 HTT를 농도별 (31.3-125 μg/mL)로 처리하여 2시간 배양하고 UVB 40 mJ/cm2로 자극한 후, 세포에 용해 버퍼를 넣고 볼텍싱을 10분씩 6회 실시하여 4℃ 14,000 rpm에서 10분 동안 원심을 돌려 얻은 상층액을 가지고 BCA 키트를 이용하여 단백질을 정량하였다. 동일한 농도의 단백질과 샘플 버퍼를 혼합한 후, 100℃에서 3분간 끓여서 단백질의 변성을 유도하였다. 10% SDS-PAGE 겔에서 전기영동 된 후, PVDF 멤브레인으로 단백질을 전이하였다. 비특이적인 단백질 결합 부분은 0.1% Tween 20과 블로킹 버퍼를 함유한 TBS에 2시간 동안 반응시켜 블로킹하였다. 이후에 1차 항체 IκBα (1:1000), p65 (1:1000), phospho-IκBα (1:1000), phospho-p65 (1:1000), β-actin (1:10000), Lamin B (1:1000)가 첨가된 용액을 12시간동안 혼합한 후 TBS-T로 10분간 3번 세척하였다. 그런 다음 멤브레인을 2차 항체 고트 항-마우스 IgG, 고트 항-토끼 IgG가 첨가된 용액을 상온에서 1시간 30분 동안 결합시켜 반응한 후 TBS-T로 10분간 3번 세척하였고, ECL detection reagents (Thermo, USA)를 이용하여 효소 반응에 의한 발광을 통해 표적 단백질의 발현을 측정하였다.The expression of proteins related to the NFκB signaling pathway in UVB-irradiated HaCaT cells was confirmed by Western blot. First, HaCaT cells were aliquoted into 1x10 6 cells in a 10 cm dish, and each dish was incubated with DMEM containing 10% FBS, 1% streptomycin and penicillin at 37° C. under 5% CO 2 conditions for 24 hours. After that, HTT was treated at different concentrations (31.3-125 μg/mL), incubated for 2 hours, and stimulated with UVB 40 mJ/cm 2 , lysis buffer was added to the cells, and vortexing was performed 6 times for 10 minutes at 4°C 14,000. The protein was quantified using the BCA kit with the supernatant obtained by centrifugation at rpm for 10 minutes. After mixing the same concentration of protein and sample buffer, it was boiled at 100° C. for 3 minutes to induce protein denaturation. After electrophoresis on a 10% SDS-PAGE gel, the protein was transferred to a PVDF membrane. Non-specific protein-binding portions were blocked by reacting them in TBS containing 0.1
NFκB 신호전달 관련 단백질을 확인한 결과(도 5), UVB를 처리한 세포에서는 아무것도 처리하지 않은 세포에 비해 인산화된 IκBα (p-IκBα)와 NFκB p65(p-NFκB p65)의 발현이 증가하였으나 농도별 (31.3-125 μg/mL)로 처리된 HTT에 의해 농도 의존적으로 p-IκBα 및 p-NFκB p65의 발현이 감소되었다. 또한 NFκB의 핵 내 발현을 확인한 결과 UVB를 처리한 세포에서 NFκB p65의 발현이 증가되었으나 HTT의 처리에 의해 농도 의존적으로 핵 내 NFκB p65의 발현이 감소되었다. 일반적으로 NFκB는 면역체계 조절(immune modulation), 세포사멸, 세포증식 등에 관여하는 단백질군(protein family)으로 이러한 NFκB 활성화 경로의 조절장애가 발생하면 미토콘드리아 의존성과정(mitochondria dependent pathway)이 억제될 뿐만 아니라, 종양의 생성에도 영향을 미치는 것으로 알려져 있다. 따라서, 이러한 결과로부터 UVB가 조사된 HaCaT 세포에서 HTT처리는 NFκB 신호전달의 활성화를 억제하여 세포사멸을 억제하는 것으로 볼 수 있다.As a result of confirming the NFκB signaling-related protein (FIG. 5), the expression of phosphorylated IκBα (p-IκBα) and NFκB p65 (p-NFκB p65) was increased in the cells treated with UVB compared to the cells untreated, but by concentration The expression of p-IκBα and p-NFκB p65 was decreased in a concentration-dependent manner by HTT treated with (31.3-125 μg/mL). In addition, as a result of confirming the nuclear expression of NFκB, the expression of NFκB p65 was increased in UVB-treated cells, but the expression of NFκB p65 in the nucleus was decreased in a concentration-dependent manner by the treatment of HTT. In general, NFκB is a protein family involved in immune modulation, apoptosis, and cell proliferation. It is also known to affect the formation of tumors. Therefore, from these results, it can be seen that HTT treatment in UVB-irradiated HaCaT cells inhibits apoptosis by inhibiting the activation of NFκB signaling.
상기 실험결과는 PASW Statistics 19.0 software (SPSS, Chicago, IL, USA)를 사용하여 통계적 유의성에 대하여 평가하였으며, 각 실험군 간의 평균치의 유의성을 P < 0.05 수준에서 Duncan's test을 사용하여 비교하였다.The experimental results were evaluated for statistical significance using PASW Statistics 19.0 software (SPSS, Chicago, IL, USA), and the significance of the mean values between each experimental group was compared using Duncan's test at P < 0.05 level.
Claims (10)
[화학식 1]
[Formula 1]
[화학식 1]
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[화학식 1]
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