KR20230170629A - Compound extracspted from the leave of Viburnum erosum Thunb and tyrosinase activity inhibitory ability composition comprising the same - Google Patents
Compound extracspted from the leave of Viburnum erosum Thunb and tyrosinase activity inhibitory ability composition comprising the same Download PDFInfo
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- KR20230170629A KR20230170629A KR1020230173986A KR20230173986A KR20230170629A KR 20230170629 A KR20230170629 A KR 20230170629A KR 1020230173986 A KR1020230173986 A KR 1020230173986A KR 20230173986 A KR20230173986 A KR 20230173986A KR 20230170629 A KR20230170629 A KR 20230170629A
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- compound
- tyrosinase
- vee2
- meoh
- skin
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
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- A—HUMAN NECESSITIES
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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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
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/35—Caprifoliaceae (Honeysuckle family)
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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
- A61K8/60—Sugars; Derivatives thereof
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- A—HUMAN NECESSITIES
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Abstract
덜꿩나무 잎 유래 신규 화합물, 상기 화합물을 포함하는 타이로시나제 활성 저해능력을 갖는 조성물, 덜꿩나무 잎 추출물 또는 이의 분획물을 포함하는 타이로시나제 활성 저해능력을 갖는 조성물에 관한 것이다. 일 양상에 따른 덜꿩나무 잎 유래 신규 화합물은 낮은 세포 독성을 나타내고, 타이로시나제 저해 활성화능을 가지므로, 타이로시나제 억제 기능성을 갖는 식품, 의약품, 화장품 등에 안전하게 사용될 수 있다. 다른 양상에 따른 타이로시나제 활성 저해능력을 갖는 조성물은 덜꿩나무 잎 유래 신규 화합물이나 덜꿩나무 잎 추출물 또는 이의 분획물을 포함함으로써 타이로시나제에 의한 질환을 개선하거나 피부 미용 목적으로 사용될 수 있다It relates to a novel compound derived from Lesser Pheasant tree leaves, a composition containing the compound having the ability to inhibit tyrosinase activity, and a composition containing Lesser Pheasant tree leaf extract or a fraction thereof having the ability to inhibit tyrosinase activity. According to one aspect, the novel compound derived from the leaves of the pheasant tree exhibits low cytotoxicity and has tyrosinase inhibitory activation ability, so it can be safely used in foods, medicines, cosmetics, etc. with tyrosinase inhibitory functionality. Compositions having the ability to inhibit tyrosinase activity according to different aspects can be used to improve diseases caused by tyrosinase or for skin beauty purposes by containing novel compounds derived from Lesser Pheasant tree leaves, Lesser Pheasant leaf extract, or fractions thereof.
Description
본 발명은 덜꿩나무 잎 유래 화학물, 상기 화합물을 포함하는 타이로시나제 활성 저해능력 조성물, 덜꿩나무 잎 추출물 또는 이의 분획물을 포함하는 타이로시나제 활성 저해능력 조성물에 관한 것이다.The present invention relates to a chemical derived from Lesser Pheasant tree leaves, a composition with the ability to inhibit tyrosinase activity containing the above compounds, and a composition with the ability to inhibit Tyrosinase activity containing a Lesser Pheasant tree leaf extract or a fraction thereof.
멜라닌은 동물체내에서 페놀화합물의 산화중합에 의하여 생성되는 흑갈색 색소이며 인간의 피부, 털, 눈 등의 색을 결정짓고, 자외선에 의한 피부 손상으로부터 보호해 주는 역할을 한다. Melanin is a dark brown pigment produced by oxidative polymerization of phenol compounds in the animal body. It determines the color of human skin, fur, and eyes, and plays a role in protecting the skin from damage caused by ultraviolet rays.
그러나 멜라닌이 과도하게 생성되면 피부침착, 주근깨, 기미 등의 원인이 되기도 한다. 타이로시나제(tyrosinase)는 멜라닌 생성에 관여하는 효소로 melanocyte내의 L-tyrosine을 순차적으로 산화시켜 멜라닌을 생성한다. However, if melanin is produced excessively, it can cause skin darkening, freckles, and freckles. Tyrosinase is an enzyme involved in melanin production and produces melanin by sequentially oxidizing L-tyrosine in melanocytes.
타이로시나제(tyrosinase) 억제제는 화장품 산업에서 피부 미백제로 개발 될 수 있으며, 피부암, 기미, 주근깨 등에서 과다 색소 침착을 억제하거나 치료할 수 있는 핵심요소로 간주된다. 천연물에서 발견되는 대표적인 타이로시나제(tyrosinase) 억제제로는 arbutin, kojic acid, niacinamide 등이 있다. Tyrosinase inhibitors can be developed as skin whitening agents in the cosmetics industry and are considered a key element in suppressing or treating hyperpigmentation in skin cancer, spots, freckles, etc. Representative tyrosinase inhibitors found in natural products include arbutin, kojic acid, and niacinamide.
덜꿩나무(Viburnum erosum Thunb.)는 산토끼꽃목 산분꽃나무과에 속하며, 동아시아국가, 한국, 중국, 및 일본에 서식하는 낙엽 활엽 관목이다. 덜꿩나무의 줄기에서 신경보호 및 항 염증성 리그난이 보고된 바 있지만, 덜꿩나무의 다양한 화학적 구성물은 아직 광범위하게 연구되지 않고 있다.Viburnum erosum Thunb. belongs to the Viburnum erosum Thunb. family and is a deciduous broad-leaved shrub native to East Asian countries, Korea, China, and Japan. Although neuroprotective and anti-inflammatory lignans have been reported in the stems of Lesser Pheasant tree, the diverse chemical composition of Lesser Pheasant tree has not yet been extensively studied.
상술한 기술적 배경을 바탕으로 안출된 것으로 본 발명의 일 실시예는 덜꿩나무 잎으로부터 추출된 6개의 신규 화합물을 제공한다. One embodiment of the present invention, which was devised based on the above-described technical background, provides six new compounds extracted from Lesser Pheasant tree leaves.
다른 일시예로, 상기 화합물 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 타이로시나제 활성에 의한 질환을 예방 또는 치료하기 위한 약학적 조성물을 제공한다. In another example, a pharmaceutical composition for preventing or treating diseases caused by tyrosinase activity is provided, comprising the compound or a pharmaceutically acceptable salt thereof as an active ingredient.
또 다른 실시예로 상기 화합물을 유효성분으로 포함하는 타이로시나제에 의한 질환을 예방 또는 개선하기 위한 건강기능식품 조성물을 제공한다.In another embodiment, a health functional food composition for preventing or improving diseases caused by tyrosinase containing the above compound as an active ingredient is provided.
또 다른 실시예로 상기 화합물을 유효성분으로 포함하는 피부 미용 개선용 화장료 조성물을 제공한다.In another embodiment, a cosmetic composition for improving skin beauty containing the above compound as an active ingredient is provided.
또 다른 실시예로 덜꿩나무 잎 추출물 또는 이의 분획물을 유효성분으로 포함하는 타이로시나제에 의한 질환을 예방 또는 치료하기 위한 약학적 조성물을 제공한다.In another embodiment, a pharmaceutical composition for preventing or treating a disease caused by tyrosinase is provided, which contains a leaf extract or a fraction thereof as an active ingredient.
또 다른 실시예로 덜꿩나무 잎 추출물 또는 이의 분획물을 유효성분으로 포함하는 타이로시나제에 의한 질환을 예방 또는 개선하기 위한 건강기능식품 조성물을 제공한다.In another embodiment, a health functional food composition for preventing or improving diseases caused by tyrosinase is provided, which contains a leaf extract or a fraction thereof as an active ingredient.
또 다른 실시예로 덜꿩나무 잎 추출물 또는 이의 분획물을 유효성분으로 포함하는 피부 미용 개선용 화장료 조성물을 제공한다.In another embodiment, a cosmetic composition for improving skin beauty is provided, which includes a Lesser Pheasant tree leaf extract or a fraction thereof as an active ingredient.
본 발명의 일 실시예는 하기 화학식 1 내지 9로 표시되는 화합물, 또는 이의 약학적으로 허용가능한 염, 또는 이의 이성질체, 또는 이의 수화물, 또는 이의 용매화물을 제공한다.One embodiment of the present invention provides a compound represented by the following formulas 1 to 9, or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a hydrate thereof, or a solvate thereof.
[화학식][Chemical formula]
용어 "약학적으로 허용 가능"은 통상의 의약적 복용량으로 이용할 때 상당한 독성 효과를 피함으로써, 동물, 보다 구체적으로는 인간에게 사용할 수 있다는 정부 또는 이에 준하는 규제 기관의 승인을 받을 수 있거나 승인 받거나, 또는 약전에 열거되거나 기타 일반적인 약전에 기재된 것으로 인지되는 것을 의미한다.The term "pharmaceutically acceptable" means capable of or has been approved by a government or equivalent regulatory agency for use in animals, and more specifically in humans, by avoiding significant toxic effects when used in usual medicinal doses; or is listed in a pharmacopoeia or is recognized as being listed in another general pharmacopoeia.
용어 "약학적으로 허용 가능한 염"은 약학적으로 허용 가능하고 모 화합물(parent compound)의 바람직한 약리 활성을 갖는 염을 의미한다. 상기 염은 염산염, 나트륨염, 칼륨염, 칼슘염, 마그네슘염, 암모늄염 등을 포함할 수 있으나, 이에 한정되지 않는다.The term “pharmaceutically acceptable salt” means a salt that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. The salt may include, but is not limited to, hydrochloride, sodium, potassium, calcium, magnesium, and ammonium salts.
용어 "이성질체"는 광학 이성질체(optical isomers)(예를 들면, 본래 순수한 거울상 이성질체(essentially pure enantiomers), 본래 순수한 부분 입체 이성질체(essentially pure diastereomers) 또는 이들의 혼합물)뿐 만 아니라, 형태 이성질체(conformation isomers)(즉, 하나 이상의 화학 결합의 그 각도만 다른 이성질체), 위 치 이성질체(position isomers)(특히, 호변이성체(tautomers)) 또는 기하 이성질체(geometric isomers)(예컨대, 시스-트랜스 이성질체)를 포함할 수 있다.The term “isomer” refers to optical isomers (e.g., essentially pure enantiomers, essentially pure diastereomers, or mixtures thereof), as well as conformational isomers. ) (i.e., isomers that differ only in the angle of one or more chemical bonds), positional isomers (particularly tautomers), or geometric isomers (e.g., cis-trans isomers). You can.
용어 "본래 순수(essentially pure)"란, 예컨대 거울상 이성질체 또는 부분 이성질체와 관련하여 사용한 경우, 거울상 이성질체 또는 부분 이성질체를 예로 들 수 있는 구체적인 화합물이 약 90% 이상, 바람직하게는 약 95% 이상, 보다 바람직하게는 약 97% 이상 또는 약 98% 이상, 보다 더 바람직하게는 약 99% 이상, 보다 더욱 더 바 람직하게는 약 99.5% 이상(w/w) 존재하는 것을 의미할 수 있다.The term "essentially pure", when used in relation to, for example, enantiomers or partial isomers, means that the specific compound exemplified by the enantiomer or partial isomer is at least about 90%, preferably at least about 95%, or more. Preferably, it may mean that it is present in about 97% or more or about 98% or more, more preferably about 99% or more, and even more preferably about 99.5% or more (w/w).
용어 “수화물(hydrate)”은 물이 결합되어 있는 화합물을 의미하며, 물과 화합물 사이에 화학적인 결합력이 없 는 내포 화합물을 포함하는 광범위한 개념을 의미할 수 있다.The term “hydrate” refers to a compound to which water is bound, and can refer to a broad concept including embedded compounds in which there is no chemical bond between water and the compound.
용어 “용매화물”은 용질의 분자나 이온과 용매의 분자나 이온 사이에 생긴 고차의 화합물을 의미할 수 있다.The term “solvate” can refer to a higher-order compound formed between a molecule or ion of a solute and a molecule or ion of a solvent.
상기 화합물은 덜꿩나무 잎로부터 추출된 것일 수 있다. 상기 화합물은 덜꿩나무 잎 추출물 또는 이의 분획물로부터 추출 또는 분리된 것일 수 있다.The compound may be extracted from pheasant tree leaves. The compound may be extracted or isolated from Lesser Pheasant tree leaf extract or fractions thereof.
덜꿩나무(Viburnum erosum Thunb.)는 산토끼꽃목 산분꽃나무과에 속하는 식물로, 동아시아국가, 한국, 중국, 및 일본에 서식하는 낙엽 활엽 관목이다. 잎은 마주나고 장란형이며 잎자루에 털이 있고 뾰족한 턱잎이 있다.Viburnum erosum Thunb. is a plant belonging to the Viburnum erosum Thunb. family and is a deciduous broad-leaved shrub native to East Asian countries, Korea, China, and Japan. The leaves are opposite, oval-shaped, have hairs on the petioles, and have pointed stipules.
상기 화합물은 타이로시나제 활성 저해능력을 갖는 것일 수 있다. The compound may have the ability to inhibit tyrosinase activity.
용어 "활성 저해능력"이란 유전자 또는 단백질의 발현 수준을 증가시키는 것을 억제하거나 유전자 또는 단백질을 활성화시키는 것을 억제하는 능력을 의미 할 수 있다. 구체적으로, 상기 화합물은 타이로시나제 활성을 억제 또는 저해할 수 있다. The term “activity inhibition ability” may mean the ability to inhibit increasing the expression level of a gene or protein or to inhibit activating a gene or protein. Specifically, the compound may inhibit or inhibit tyrosinase activity.
다른 양상은 상기 화합물 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 타이로시나제 활성 저해능력을 갖는 조성물을 제공 한다. Another aspect provides a composition having the ability to inhibit tyrosinase activity, comprising the compound or a pharmaceutically acceptable salt thereof as an active ingredient.
상기 타이로시나제 활성 저해능력 조성물은 타이로시나제 활성 저해능력이 요구되는 다양한 목적 및 용도로 사용될 수 있으며, 구체적으로는 의약품, 화장품, 식품 및 동물 사료 등 다양한 산업분야에서 적용되는 물품에 타이로시나제 활성 저해능력화능을 부여할 수 있는 기능성 소재로 사용할 수 있다. The composition capable of inhibiting tyrosinase activity can be used for various purposes and uses that require the ability to inhibit tyrosinase activity. Specifically, it can be used in products applied in various industrial fields such as pharmaceuticals, cosmetics, food, and animal feed. It can be used as a functional material that can provide the ability to inhibit rosinase activity.
또한 의약품 보존제, 화장품 보존제, 식품 보존제, 의약품 첨가제, 화장품 첨가제, 식품첨가제 및 사료첨가제 등의 소재로도 사용될 수 있다. 따라서, 상기 타이로시나제 저해 활성화능 조성물은 화장료, 식품, 또는 약학 조성물일 수 있다.It can also be used as a material for pharmaceutical preservatives, cosmetic preservatives, food preservatives, pharmaceutical additives, cosmetic additives, food additives, and feed additives. Accordingly, the composition with tyrosinase inhibitory activation ability may be a cosmetic, food, or pharmaceutical composition.
또 다른 양상은 상기 화합물 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 타이로시나제에 의한 질환을 예방 또는 치료하기 위한 약학적 조성물을 제공한다.Another aspect provides a pharmaceutical composition for preventing or treating diseases caused by tyrosinase, comprising the compound or a pharmaceutically acceptable salt thereof as an active ingredient.
상기 약학적 조성물에 있어서, 화합물, 이의 약학적으로 허용 가능한 염에 대해서는 상기한 바와 같다.In the pharmaceutical composition, the compound and its pharmaceutically acceptable salt are as described above.
용어, "예방"은 질병의 발생을 억제하는 것을 포함할 수 있다. 용어, "치료"는 질병의 발전의 억제, 경감, 또는 제거를 포함할 수 있다.The term “prevention” may include inhibiting the occurrence of a disease. The term “treatment” may include inhibiting, alleviating, or eliminating the development of a disease.
또 다른 양상은 상기 화합물을 유효성분으로 포함하는 타이로시나제에 의한 질환을 예방 또는 개선하기 위한 건강기능식품 조성물을 제공한다.Another aspect provides a health functional food composition for preventing or improving diseases caused by tyrosinase, including the above compound as an active ingredient.
상기 건강기능식품 조성물에 있어서, 화합물, 타이로시나제에 의한 질환, 예방에 대해서는 상기한 바와 같다.In the health functional food composition, the compounds, diseases caused by tyrosinase, and prevention are as described above.
용어 “개선”은 상태의 완화 또는 치료와 관련된 파라미터, 예를 들어, 증상의 정도를 적어도 감소시키는 모든 행위를 의미할 수 있다.The term “improvement” can refer to any action that results in at least reducing the severity of a parameter, such as a symptom, associated with the alleviation or treatment of a condition.
또 다른 양상은 상기 화합물을 유효성분으로 포함하는 피부 미용 개선용 화장료 조성물을 제공한다.Another aspect provides a cosmetic composition for improving skin beauty containing the above compound as an active ingredient.
상기 화장료 조성물에 있어서, 화합물에 대해서는 상기한 바와 같다.In the cosmetic composition, the compounds are as described above.
상기 피부 미용 개선은 피부 주름 개선 또는 미백제일 수 있다.The skin beauty improvement agent may be skin wrinkle improvement or whitening agent.
또 다른 양상은 덜꿩나무 잎 추출물 또는 이의 분획물을 유효성분으로 포함하는 타이로시나제 저해 활성화능 조성물을 제공한다. 상기 타이로시나제 저해 활성화능 조성물은 타이로시나제 저해 활성화능이 요구되는 다양한 목적 및 용도로 사용될 수 있으며, 구체적으로는 의약품, 화장품, 식품 및 동물 사료 등 다양한 산업분야에서 적용되는 물품에 타이로시나제 저해 활성화능 활성을 부여 할 수 있는 기능성 소재로 사용할 수 있다. 또한 의약품 보존제, 화장품 보존제, 식품 보존제, 의약품 첨가제, 화장품 첨가제, 식품첨가제 및 사료첨가제 등의 소재로도 사용될 수 있다. 따라서, 상기 타이로시나제 저해 활성화능 조성물은 화장료, 식품, 또는 약학 조성물일 수 있다.Another aspect provides a tyrosinase inhibitory activation activity composition comprising a Lesser Pheasant tree leaf extract or a fraction thereof as an active ingredient. The composition with tyrosinase inhibitory activation ability can be used for various purposes and uses that require tyrosinase inhibition activation ability, and specifically, it can be used as a tyrosinase agent for products applied in various industrial fields such as pharmaceuticals, cosmetics, food, and animal feed. It can be used as a functional material that can impart synthase inhibition activation activity. It can also be used as a material for pharmaceutical preservatives, cosmetic preservatives, food preservatives, pharmaceutical additives, cosmetic additives, food additives, and feed additives. Accordingly, the composition with tyrosinase inhibitory activation ability may be a cosmetic, food, or pharmaceutical composition.
또 다른 양상은 덜꿩나무 잎 추출물 또는 이의 분획물을 유효성분으로 포함하는 타이로시나제에 의한 질환을 예방 또는 치료하기 위한 약학적 조성물을 제공한다.Another aspect provides a pharmaceutical composition for preventing or treating diseases caused by tyrosinase, comprising a Lesser Pheasant tree leaf extract or a fraction thereof as an active ingredient.
상기 약학적 조성물에 있어서, 타이로시나제에 의한 질환, 예방, 치료에 대해서는 상기한 바와 같다.In the pharmaceutical composition, diseases caused by tyrosinase, prevention, and treatment are as described above.
상기 덜꿩나무 잎 추출물 또는 이의 분획물은 상기 화합물을 포함하는 것일 수 있다. 예를 들어, 상기 화합물 1 내지 화합물 6 중 하나 이상을 포함하는 것일 수 있다. 상기 화합물 1 내지 화합물 6에 대한 설명은 상술한 바와 같다.The Lesser Pheasant tree leaf extract or fraction thereof may contain the above compound. For example, it may include one or more of compounds 1 to 6 above. The description of Compound 1 to Compound 6 is as described above.
상기 덜꿩나무 잎은 전체 또는 그 일부분일 수 있다. 추출에 사용된 덜꿩나무 잎은 전체 또는 그 일부 분을 분쇄 또는 세절하거나 적당하게 건조한 것일 수 있다.The leaf of the pheasant tree may be whole or a part thereof. The leaves of the pheasant tree used in the extraction may be whole or part of them crushed, shredded, or suitably dried.
상기 추출물은 가온 추출, 가압 추출, 초음파 추출, 열수 추출, 환류 냉각 추출, 아임계 추출, 또는 초임계 추 출 등 당업 기술분야에서 통상적인 방법으로 추출된 것일 수 있다.The extract may be extracted by a method common in the art, such as heating extraction, pressure extraction, ultrasonic extraction, hot water extraction, reflux cooling extraction, subcritical extraction, or supercritical extraction.
일 구체예에 따른 조성물은 상기 덜꿩나무 잎 추출물의 분획물을 유효성분으로서 포함할 수 있다.The composition according to one embodiment may include a fraction of the Lesser Pheasant tree leaf extract as an active ingredient.
용어, "분획물(fraction)"은 상기 덜꿩나무 잎의 추출물이 그 일부의 성분으로 나누어진 물질 즉, 분획되어진 물질을 나타낸다. 상기 분획물은 용매 분획화 (fractionation)에 의하여 얻어진 것일 수 있다. 상기 용매 분획 화는 덜꿩나무 잎 추출물을 용매와 혼합하고 상기 용매에 존재하는 물질을 분리하는 것일 수 있다. 상기 분획물은 The term “fraction” refers to a substance in which the extract of the leaf of the pheasant tree is divided into its parts, that is, a substance in which the extract is fractionated. The fraction may be obtained by solvent fractionation. The solvent fractionation may be mixing the Lesser pheasant leaf extract with a solvent and separating substances present in the solvent. The fraction is
상기 약학적 조성물은 상기 추출물 또는 이의 분획물을 유효한 양, 또는 유효 성분으로서 포함할 수 있다. 상기 유효한 양은 개체에 따라 적절하게 선택할 수 있다. 질환 내지 상태의 중증도, 개체의 연령, 체중, 건강, 성별, 개체의 추출물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 투여 기간, 상기 조성물과 배합 또는 동시 사용되는 다른 조성물을 포함한 요소 및 기타 생리 내지 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다.The pharmaceutical composition may contain the extract or fraction thereof in an effective amount or as an active ingredient. The effective amount can be appropriately selected depending on the individual. The severity of the disease or condition, the subject's age, weight, health, gender, the subject's sensitivity to the extract, administration time, administration route and excretion rate, administration period, factors including other compositions combined or used simultaneously with the composition, and other factors. It can be determined according to factors well known in the field of physiology or medicine.
상기 약학적 조성물은 약학적으로 허용가능한 부형제 또는 담체를 더 포함할 수 있다. 상기 약학적으로 허용가 능한 부형제 또는 담체에 관한 설명은 상기한 바와 같다.The pharmaceutical composition may further include a pharmaceutically acceptable excipient or carrier. The description of the pharmaceutically acceptable excipient or carrier is the same as above.
또 다른 양상은 덜꿩나무 잎 추출물 또는 이의 분획물을 유효성분으로 포함하는 타이로시나제에 의한 질환을 예방 또는 개선하기 위한 건강기능식품 조성물을 제공한다.Another aspect provides a health functional food composition for preventing or ameliorating diseases caused by tyrosinase, which contains a leaf extract or a fraction thereof as an active ingredient.
상기 건강기능식품 조성물에 있어서, 덜꿩나무 잎 추출물, 분획물, 타이로시나제에 의한 질환, 예방, 개선에 대해서는 상기한 바와 같다.In the above-mentioned health functional food composition, the disease, prevention, and improvement caused by Lesser Pheasant tree leaf extract, fraction, and tyrosinase are as described above.
또 다른 양상은 덜꿩나무 잎 추출물 또는 이의 분획물을 유효성분으로 포함하는 피부 미용 개선용 화장료 조성물을 제공한다.Another aspect provides a cosmetic composition for improving skin beauty comprising a Lesser Pheasant tree leaf extract or a fraction thereof as an active ingredient.
상기 화장료 조성물에 있어서, 덜꿩나무 잎 추출물, 분획물, 피부 미용 개선에 대해서는 상기한 바와 같다.In the cosmetic composition, the leaf extract, fraction, and improvement of skin beauty are as described above.
또 다른 양상은 상기 약학적 조성물을 개체에게 투여하는 단계를 포함하는 개체의 타이로시나제에 의한 질환 을 예방, 개선 또는 치료하는 방법을 제공한다. 상기 약학적 조성물, 타이로시나제에 의한 질환, 예방, 개선, 치료에 대해서는 상기한 바와 같다.Another aspect provides a method for preventing, improving, or treating a disease caused by tyrosinase in a subject, comprising administering the pharmaceutical composition to the subject. Diseases, prevention, improvement, and treatment caused by the pharmaceutical composition and tyrosinase are as described above.
용어 "투여하는", "도입하는" 및 "이식하는"은 상호교환적으로 사용되고 일 구체예에 따른 조성물의 원하는 부위로의 적어도 부분적 국소화를 초래하는 방법 또는 경로에 의한 개체 내로의 일 구체예에 따른 조성물의 배치 를 의미할 수 있다. 일 구체예에 따른 조성물의 화합물, 추출물, 또는 이의 분획물 성분의 적어도 일부를 생존 하는 개체 내에서 원하는 위치로 전달하는 임의의 적절한 경로에 의해 투여될 수 있다.The terms “administering,” “introducing,” and “implanting” are used interchangeably and, in one embodiment, are administered to an individual by a method or route that results in at least partial localization of the composition according to one embodiment to the desired site. It may refer to the arrangement of the composition according to the composition. It can be administered by any suitable route to deliver at least a portion of the components of the compound, extract, or fraction thereof of the composition according to one embodiment to a desired location within a living individual.
본 일 양상에 따른 덜꿩나무 잎 유래 신규 화합물은 낮은 세포 독성을 나타내고, 타이로시나제 저해 활성화능 활성을 가지므로, 타이로시나제 저해 활성화능 기능성을 갖는 식품, 의약품, 화장품 등에 안전하게 사용될 수 있다.The novel compound derived from Lesser pheasant leaves according to this aspect exhibits low cytotoxicity and has tyrosinase inhibitory activation activity, so it can be safely used in foods, medicines, cosmetics, etc. with tyrosinase inhibition activation activity. .
다른 양상에 따른 타이로시나제 저해 활성화능 조성물은 덜꿩나무 잎 유래 신규 화합물이나 덜꿩나무 잎 추출물 또는 이의 분획 물을 포함함으로써 타이로시나제에 의한 질환을 개선하거나 피부 미용 목적으로 사용될 수 있다.A composition with tyrosinase inhibition and activation ability according to another aspect may be used to improve diseases caused by tyrosinase or for skin care purposes by containing a novel compound derived from the leaves of the tyrosinase tree, leaf extract of the pheasant tree, or a fraction thereof.
도 1 은 본 발명의 일 실시에 따른 덜꿩나무 추출 및 분획을 나타낸 도면이다.
도 2는 도 1에 나타낸 아틸아세테이트로 부터 분획을 나타낸 도면이다.
도 3은 본 발명의 화합물 1 내지 9의 화학구조를 나타낸 도면이다.
도 4는 덜꿩나무 잎으로부터 분획된 분획물의 세포독성을 비교한 결과를 나타낸 그래프이다.
도 5는 덜꿩나무 잎으로부터 분획된 분획물의 타이로시나제 활성도를 비교한 결과를 나타낸 그래프이다.
도 6은 도 3에 나타낸 화합물의 타이로시나제 활성도를 비교한 결과를 나타낸 그래프이다.
도 7은 도 3에 나타낸 화합물 9의 함량을 나타낸 표이다.
도 8은 도 7에 나타낸 화합물 9의 크로마토그래피 결과를 나타낸 그래프이다.Figure 1 is a diagram showing extraction and fractionation of Lesser pheasant tree according to one embodiment of the present invention.
Figure 2 is a diagram showing the fraction from acylacetate shown in Figure 1.
Figure 3 is a diagram showing the chemical structures of compounds 1 to 9 of the present invention.
Figure 4 is a graph showing the results of comparing the cytotoxicity of fractions fractionated from Lesser pheasant leaves.
Figure 5 is a graph showing the results of comparing the tyrosinase activity of fractions fractionated from Lesser pheasant leaves.
Figure 6 is a graph showing the results of comparing the tyrosinase activities of the compounds shown in Figure 3.
Figure 7 is a table showing the content of compound 9 shown in Figure 3.
Figure 8 is a graph showing the chromatography results of compound 9 shown in Figure 7.
이하, 첨부한 도면을 참고로 하여 본 발명의 실시예에 대하여 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있도록 상세히 설명한다. Hereinafter, with reference to the attached drawings, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement the present invention.
덜꿩나무 잎으로부터 화합물 1 ~ 9의 추출 및 분리Extraction and isolation of compounds 1 to 9 from Lesser Pheasant leaves
(1) 추출 및 분획 (1) Extraction and fractionation
도 1 내지 도 3을 참고하면, 건조된 덜꿩나무의 잎 (V.erosum) 2kg을 80% MeOH로 3시간 3회 반복 추출하였다. 추출액을 필터 후 감압 농축기로 농축을 진행하여 추출물 356.0g을 얻었다. 추출물을 증류수에 현탁하여 n-Hexane (0.5 g), EtOAc (66.4 g), n-BuOH (123.7 g), 수용성 (165.0 g) 순으로 분획물을 획득하였다. Referring to Figures 1 to 3, 2 kg of dried V. erosum leaves were extracted three times for 3 hours with 80% MeOH. The extract was filtered and concentrated using a reduced pressure concentrator to obtain 356.0 g of extract. The extract was suspended in distilled water to obtain fractions in the following order: n-Hexane (0.5 g), EtOAc (66.4 g), n-BuOH (123.7 g), and water-soluble (165.0 g).
덜꿩나무 잎의 EtOAc 분획물(66.4g) 중 23.0g 을 MPLC와 RediSep® Rf RP-C18 column을 사용하여 MeOH : H2O (5:5→6:4→7:3→8:2→9:1→MeOH)의 용매 조건으로 분리하였고, 8개의 분획(VEE1 ~ VEE8)으로 나누었다. 이중 VEE2로부터 화합물 1-8을 얻었으며, VEE3으로부터 화합물 9를 얻었다. 23.0g of the EtOAc fraction (66.4g) of Lesser pheasant leaves was analyzed using MPLC and RediSep® Rf RP-C18 column to MeOH: H2O (5:5→6:4→7:3→8:2→9:1→ It was separated under solvent conditions (MeOH) and divided into eight fractions (VEE1 to VEE8). Among them, compound 1-8 was obtained from VEE2, and compound 9 was obtained from VEE3.
화합물 1의 경우, VEE2 분획을 MPLC와 silica gel column 을 사용하여 DCM : MeOH (9:1→8:2→7:3→6:4→5:5→3:7→MeOH)의 용매 조건으로 분리하였고 9개의 소분획(VEE2-1 ~ VEE2-9)으로 나누었다. 이중 VEE2-6을 Sephadex LH-20 column을 사용하여 MeOH : H2O (6:4→MeOH)의 용매 조건으로 분리하였고 11개의 소분획(VEE2-6-1 ~ VEE2-6-11)으로 나누었다. VEE2-6-4를 MPLC와 RediSep® Rf RP-C18 column을 사용하여 MeOH : H2O (5:5→6:4→7:3→8:2→9:1→MeOH)의 용매 조건으로 분리하였고 10개의 소분획(VEE2-6-4-1 ~ VEE2-6-4-10)으로 나누었다. 마지막으로 VEE2-6-4-7을 MPLC 와 YMC-Actus Triart C18 column 을 사용하여 MeOH : H2O (65:35)의 용매조건으로 분리하였다. 7개의 소분획(VEE2-6-4-7-1 ~ VEE2-6-4-7-7)을 Thin Layer Chromatography(TLC) 확인시험법을 이용하여 순도를 확인하였고, VEE2-6-4-7-5에서 단일한 화합물 1 (11.7mg)을 얻었다. In the case of compound 1, the VEE2 fraction was analyzed using MPLC and a silica gel column under solvent conditions of DCM: MeOH (9:1→8:2→7:3→6:4→5:5→3:7→MeOH). It was separated and divided into 9 subfractions (VEE2-1 to VEE2-9). Among them, VEE2-6 was separated using a Sephadex LH-20 column under solvent conditions of MeOH: H2O (6:4→MeOH) and divided into 11 small fractions (VEE2-6-1 to VEE2-6-11). VEE2-6-4 was separated using MPLC and RediSep® Rf RP-C18 column under solvent conditions of MeOH: H2O (5:5→6:4→7:3→8:2→9:1→MeOH) It was divided into 10 subfractions (VEE2-6-4-1 to VEE2-6-4-10). Finally, VEE2-6-4-7 was separated using MPLC and YMC-Actus Triart C18 column under solvent conditions of MeOH: H2O (65:35). The purity of 7 small fractions (VEE2-6-4-7-1 to VEE2-6-4-7-7) was confirmed using the Thin Layer Chromatography (TLC) confirmation test, and VEE2-6-4-7 A single compound 1 (11.7 mg) was obtained at -5.
화합물 2의 경우, VEE2-6을 Sephadex LH-20 column을 사용하여 MeOH : H2O (6:4→MeOH)의 용매 조건으로 분리하였고 11개의 소분획(VEE2-6-1 ~ VEE2-6-11)으로 나누었다. VEE2-6-4를 MPLC와 RediSep® Rf RP-C18 column을 사용하여 MeOH : H2O (5:5→6:4→7:3→8:2→9:1→MeOH)의 용매 조건으로 분리하였고 10개의 소분획(VEE2-6-4-1 ~ VEE2-6-4-10) 으로 나누었다. 이중 VEE2-6-4-9를 silica gel column을 사용하여 DCM : MeOH (9:1→8:2→MeOH)의 용매 조건으로 분리하였고 9개의 소분획(VEE2-6-4-9-1 ~ VEE2-6-4-9-9)으로 나누었다. 마지막으로 VEE2-6-4-9-5를 Preparative HPLC와 RP-C18 column을 사용하여 MeOH : H2O (7:3)의 용매 조건으로 정제하였다. Thin Layer Chromatography(TLC) 확인시험법을 이용하여 순도를 확인하였고, VEE2-6-4-9-5-1에서 단일한 화합물 2 (0.9mg)을 얻었다.In the case of compound 2, VEE2-6 was separated using a Sephadex LH-20 column under solvent conditions of MeOH: H2O (6:4→MeOH), and 11 small fractions (VEE2-6-1 to VEE2-6-11) were obtained. divided into VEE2-6-4 was separated using MPLC and RediSep® Rf RP-C18 column under solvent conditions of MeOH: H2O (5:5→6:4→7:3→8:2→9:1→MeOH) It was divided into 10 subfractions (VEE2-6-4-1 to VEE2-6-4-10). Among them, VEE2-6-4-9 was separated using a silica gel column under solvent conditions of DCM: MeOH (9:1→8:2→MeOH), and nine subfractions (VEE2-6-4-9-1 ~ VEE2-6-4-9-9). Finally, VEE2-6-4-9-5 was purified using Preparative HPLC and RP-C18 column under solvent conditions of MeOH: H2O (7:3). Purity was confirmed using the Thin Layer Chromatography (TLC) identification test, and single compound 2 (0.9 mg) was obtained from VEE2-6-4-9-5-1.
화합물 3의 경우, VEE2 분획을 MPLC와 silica gel column 을 사용하여 DCM : MeOH (9:1→8:2→7:3→6:4→5:5→3:7→MeOH)의 용매 조건으로 분리하였고 9개의 소분획(VEE2-1 ~ VEE2-9)으로 나누었다. 이중 VEE2-6을 Sephadex LH-20 column을 사용하여 MeOH : H2O (6:4→MeOH)의 용매 조건으로 분리하였고 11개의 소분획(VEE2-6-1 ~ VEE2-6-11)으로 나누었다. VEE2-6-4를 MPLC와 RediSep® Rf RP-C18 column을 사용하여 MeOH : H2O (5:5→6:4→7:3→ 8:2→9:1→MeOH)의 용매 조건으로 분리하였고 10개의 소분획(VEE2-6-4-1 ~ VEE2-6-4-10) 으로 나누었다. 마지막으로 VEE2-6-4-7을 MPLC 와 YMC-Actus Triart C18 column 을 사용하여 MeOH : H2O (65:35)의 용매조건으로 분리하였다. 7개의 소분획(VEE2-6-4-7-1 ~ VEE2-6-4-7-7)을 Thin Layer Chromatography(TLC) 확인시험법을 이용하여 순도를 확인하였고, VEE2-6-4-7-2에서 단일한 화합물 3 (19.4mg)을 얻었다. In the case of compound 3, the VEE2 fraction was analyzed using MPLC and a silica gel column under solvent conditions of DCM: MeOH (9:1→8:2→7:3→6:4→5:5→3:7→MeOH). It was separated and divided into 9 subfractions (VEE2-1 to VEE2-9). Among them, VEE2-6 was separated using a Sephadex LH-20 column under solvent conditions of MeOH: H2O (6:4→MeOH) and divided into 11 small fractions (VEE2-6-1 to VEE2-6-11). VEE2-6-4 was separated using MPLC and RediSep® Rf RP-C18 column under solvent conditions of MeOH: H2O (5:5→6:4→7:3→ 8:2→9:1→MeOH) It was divided into 10 subfractions (VEE2-6-4-1 to VEE2-6-4-10). Finally, VEE2-6-4-7 was separated using MPLC and YMC-Actus Triart C18 column under solvent conditions of MeOH: H2O (65:35). The purity of 7 small fractions (VEE2-6-4-7-1 to VEE2-6-4-7-7) was confirmed using the Thin Layer Chromatography (TLC) confirmation test, and VEE2-6-4-7 A single compound 3 (19.4 mg) was obtained from -2.
화합물 4의 경우, VEE2 분획을 MPLC와 silica gel column 을 사용하여 DCM : MeOH (9:1→8:2→7:3→6:4→5:5→3:7→MeOH)의 용매 조건으로 분리하였고 9개의 소분획(VEE2-1 ~ VEE2-9)으로 나누었다. 이중 VEE2-6을 Sephadex LH-20 column을 사용하여 MeOH : H2O (6:4→MeOH)의 용매 조건으로 분리하였고 11개의 소분획(VEE2-6-1 ~ VEE2-6-11)으로 나누었다. VEE2-6-4를 MPLC와 RediSep® Rf RP-C18 column을 사용하여 MeOH : H2O (5:5→6:4→7:3→ 8:2→9:1→MeOH)의 용매 조건으로 분리하였고 10개의 소분획(VEE2-6-4-1 ~ VEE2-6-4-10) 으로 나누었다. 마지막으로 VEE2-6-4-7을 MPLC 와 YMC-Actus Triart C18 column 을 사용하여 MeOH : H2O (65:35)의 용매조건으로 분리하였다. 7개의 소분획(VEE2-6-4-7-1 ~ VEE2-6-4-7-7)을 Thin Layer Chromatography(TLC) 확인시험법을 이용하여 순도를 확인하였고, VEE2-6-4-7-3에서 단일한 화합물 4 (11.1mg)을 얻었다. In the case of compound 4, the VEE2 fraction was purified using MPLC and a silica gel column under solvent conditions of DCM: MeOH (9:1→8:2→7:3→6:4→5:5→3:7→MeOH). It was separated and divided into 9 subfractions (VEE2-1 to VEE2-9). Among them, VEE2-6 was separated using a Sephadex LH-20 column under solvent conditions of MeOH: H2O (6:4→MeOH) and divided into 11 small fractions (VEE2-6-1 to VEE2-6-11). VEE2-6-4 was separated using MPLC and RediSep® Rf RP-C18 column under solvent conditions of MeOH: H2O (5:5→6:4→7:3→ 8:2→9:1→MeOH) It was divided into 10 subfractions (VEE2-6-4-1 to VEE2-6-4-10). Finally, VEE2-6-4-7 was separated using MPLC and YMC-Actus Triart C18 column under solvent conditions of MeOH: H2O (65:35). The purity of 7 small fractions (VEE2-6-4-7-1 to VEE2-6-4-7-7) was confirmed using the Thin Layer Chromatography (TLC) confirmation test, and VEE2-6-4-7 A single compound 4 (11.1 mg) was obtained from -3.
화합물 5의 경우, VEE2 분획을 MPLC와 silica gel column 을 사용하여 DCM : MeOH (9:1→8:2→7:3→6:4→5:5→3:7→MeOH)의 용매 조건으로 분리하였고 9개의 소분획(VEE2-1 ~ VEE2-9)으로 나누었다. 이중 VEE2-6을 Sephadex LH-20 column을 사용하여 MeOH : H2O (6:4→MeOH)의 용매 조건으로 분리하였고 11개의 소분획(VEE2-6-1 ~ VEE2-6-11)으로 나누었다. VEE2-6-4를 MPLC와 RediSep® Rf RP-C18 column을 사용하여 MeOH : H2O (5:5→6:4→7:3→ 8:2→9:1→MeOH)의 용매 조건으로 분리하였고 10개의 소분획(VEE2-6-4-1 ~ VEE2-6-4-10) 으로 나누었다. 마지막으로 VEE2-6-4-2를 Preparative HPLC와 RP-C18 column을 사용하여 MeOH : H2O (35:65)의 용매 조건으로 정제하였다. 2개의 소분획(VEE2-6-4-2-1, VEE2-6-4-2-2)을 Thin Layer Chromatography(TLC) 확인시험법을 이용하여 순도를 확인하였고, VEE2-6-4-2-1에서 단일한 화합물 5 (6.3mg)을 얻었다In the case of compound 5, the VEE2 fraction was purified using MPLC and a silica gel column under solvent conditions of DCM: MeOH (9:1→8:2→7:3→6:4→5:5→3:7→MeOH). It was separated and divided into 9 subfractions (VEE2-1 to VEE2-9). Among them, VEE2-6 was separated using a Sephadex LH-20 column under solvent conditions of MeOH: H2O (6:4→MeOH) and divided into 11 small fractions (VEE2-6-1 to VEE2-6-11). VEE2-6-4 was separated using MPLC and RediSep® Rf RP-C18 column under solvent conditions of MeOH: H2O (5:5→6:4→7:3→ 8:2→9:1→MeOH) It was divided into 10 subfractions (VEE2-6-4-1 to VEE2-6-4-10). Finally, VEE2-6-4-2 was purified using Preparative HPLC and RP-C18 column under solvent conditions of MeOH: H2O (35:65). The purity of two small fractions (VEE2-6-4-2-1, VEE2-6-4-2-2) was confirmed using the Thin Layer Chromatography (TLC) confirmation test, and VEE2-6-4-2 A single compound 5 (6.3mg) was obtained from -1
화합물6의 경우, VEE2 분획을 MPLC와 silica gel column 을 사용하여 DCM : MeOH (9:1→8:2→7:3→6:4→5:5→3:7→MeOH)의 용매 조건으로 분리하였고 9개의 소분획(VEE2-1 ~ VEE2-9)으로 나누었다. 이중 VEE2-6을 Sephadex LH-20 column을 사용하여 MeOH : H2O (6:4→MeOH)의 용매 조건으로 분리하였고 11개의 소분획(VEE2-6-1 ~ VEE2-6-11)으로 나누었다. VEE2-6-4를 MPLC와 RediSep® Rf RP-C18 column을 사용하여 MeOH : H2O (5:5→6:4→7:3→ 8:2→9:1→MeOH)의 용매 조건으로 분리하였고 10개의 소분획(VEE2-6-4-1 ~ VEE2-6-4-10)으로 나누었다. 마지막으로 VEE2-6-4-2를 Preparative HPLC와 RP-C18 column을 사용하여 MeOH : H2O (35:65)의 용매 조건으로 정제하였다. 2개의 소분획(VEE2-6-4-2-1, VEE2-6-4-2-2)을 Thin Layer Chromatography(TLC) 확인시험법을 이용하여 순도를 확인하였고, VEE2-6-4-2-2에서 단일한 화합물 6 (5.2mg)을 얻었다In the case of compound 6, the VEE2 fraction was analyzed using MPLC and a silica gel column under solvent conditions of DCM: MeOH (9:1→8:2→7:3→6:4→5:5→3:7→MeOH). It was separated and divided into 9 subfractions (VEE2-1 to VEE2-9). Among them, VEE2-6 was separated using a Sephadex LH-20 column under solvent conditions of MeOH: H2O (6:4→MeOH) and divided into 11 small fractions (VEE2-6-1 to VEE2-6-11). VEE2-6-4 was separated using MPLC and RediSep® Rf RP-C18 column under solvent conditions of MeOH: H2O (5:5→6:4→7:3→ 8:2→9:1→MeOH) It was divided into 10 subfractions (VEE2-6-4-1 to VEE2-6-4-10). Finally, VEE2-6-4-2 was purified using Preparative HPLC and RP-C18 column under solvent conditions of MeOH: H2O (35:65). The purity of two small fractions (VEE2-6-4-2-1, VEE2-6-4-2-2) was confirmed using the Thin Layer Chromatography (TLC) confirmation test, and VEE2-6-4-2 A single compound 6 (5.2 mg) was obtained from -2.
화합물7의 경우, VEE2 분획을 MPLC와 silica gel column 을 사용하여 DCM : MeOH (9:1→8:2→7:3→6:4→5:5→3:7→MeOH)의 용매 조건으로 분리하였고 9개의 소분획(VEE2-1 ~ VEE2-9)으로 나누었다. 이중 VEE2-6을 Sephadex LH-20 column을 사용하여 MeOH : H2O (6:4→MeOH)의 용매 조건으로 분리하였고 11개의 소분획(VEE2-6-1 ~ VEE2-6-11)으로 나누었다. VEE2-6-4를 MPLC와 RediSep® Rf RP-C18 column을 사용하여 MeOH : H2O (5:5→6:4→7:3→ 8:2→9:1→MeOH)의 용매 조건으로 분리하였고 10개의 소분획(VEE2-6-4-1 ~ VEE2-6-4-10) 으로 나누었다. VEE2-6-4-4를 다시한번 MPLC와 RediSep® Rf RP-C18 column을 사용하여 MeOH : H2O (4:6→5:5→6:4→MeOH)의 용매 조건으로 분리하였고 3개의 소분획(VEE2-6-4-4-1 ~ VEE2-6-4-4-3) 으로 나누었다. 마지막으로 VEE2-6-4-4-2를 MPLC 와 YMC-Actus Triart C18 column 을 사용하여 MeOH : H2O (4:6)의 용매조건으로 분리하였다. 3개의 소분획(VEE2-6-4-4-2-1 ~ VEE2-6-4-4-2-3)을 Thin Layer Chromatography(TLC) 확인 시험법을 이용하여 순도를 확인하였고, VEE2-6-4-4-2-3에서 단일한 화합물 7 (114mg)을 얻었다. In the case of compound 7, the VEE2 fraction was analyzed using MPLC and a silica gel column under solvent conditions of DCM: MeOH (9:1→8:2→7:3→6:4→5:5→3:7→MeOH). It was separated and divided into 9 subfractions (VEE2-1 to VEE2-9). Among them, VEE2-6 was separated using a Sephadex LH-20 column under solvent conditions of MeOH: H2O (6:4→MeOH) and divided into 11 small fractions (VEE2-6-1 to VEE2-6-11). VEE2-6-4 was separated using MPLC and RediSep® Rf RP-C18 column under solvent conditions of MeOH: H2O (5:5→6:4→7:3→ 8:2→9:1→MeOH) It was divided into 10 subfractions (VEE2-6-4-1 to VEE2-6-4-10). VEE2-6-4-4 was separated once again using MPLC and RediSep® Rf RP-C18 column under solvent conditions of MeOH: H2O (4:6→5:5→6:4→MeOH) and divided into three subfractions. Divided into (VEE2-6-4-4-1 ~ VEE2-6-4-4-3). Finally, VEE2-6-4-4-2 was separated using MPLC and YMC-Actus Triart C18 column under solvent conditions of MeOH: H2O (4:6). The purity of three small fractions (VEE2-6-4-4-2-1 to VEE2-6-4-4-2-3) was confirmed using the Thin Layer Chromatography (TLC) confirmation test, and VEE2-6 A single compound 7 (114 mg) was obtained from -4-4-2-3.
화합물8의 경우, VEE2 분획을 MPLC와 silica gel column 을 사용하여 DCM : MeOH (9:1→8:2→7:3→6:4→5:5→3:7→MeOH)의 용매 조건으로 분리하였고 9개의 소분획(VEE2-1 ~ VEE2-9)으로 나누었다. 이중 VEE2-2를 MPLC와 RediSep® Rf RP-C18 column을 사용하여 MeOH : H2O (5:5→6:4→7:3→8:2→9:1→MeOH)의 용매 조건으로 분리하였고 7개의 소분획(VEE2-2-1 ~ VEE2-2-7) 으로 나누었다. 마지막으로VEE2-2-5를 Preparative HPLC와 RP-C18 column을 사용하여 MeOH : H2O (1:1)의 용매 조건으로 정제하였다. 4개의 소분획(VEE2-2-5-1 ~ VEE2-2-5-4)을 Thin Layer Chromatography(TLC) 확인시험법을 이용하여 순도를 확인하였고, VEE2-2-5-2에서 단일한 화합물 8 (7.3mg)을 얻었다.In the case of compound 8, the VEE2 fraction was analyzed using MPLC and a silica gel column under solvent conditions of DCM: MeOH (9:1→8:2→7:3→6:4→5:5→3:7→MeOH). It was separated and divided into 9 subfractions (VEE2-1 to VEE2-9). Among them, VEE2-2 was separated using MPLC and RediSep® Rf RP-C18 column under solvent conditions of MeOH: H2O (5:5→6:4→7:3→8:2→9:1→MeOH) and 7 It was divided into two small fractions (VEE2-2-1 to VEE2-2-7). Finally, VEE2-2-5 was purified using Preparative HPLC and RP-C18 column under solvent conditions of MeOH: H2O (1:1). The purity of four small fractions (VEE2-2-5-1 to VEE2-2-5-4) was confirmed using Thin Layer Chromatography (TLC), and a single compound was identified in VEE2-2-5-2. 8 (7.3 mg) was obtained.
화합물8의 경우, 덜꿩나무 잎의 EtOAc 분획물(66.4g) 중 23.0g 을 MPLC와 RediSep® Rf RP-C18 column을 사용하여 MeOH : H2O (5:5→6:4→7:3→8:2→9:1→MeOH)의 용매 조건으로 분리하였고 8개의 분획(VEE1 ~ VEE8) 으로 나누었다.In the case of compound 8, 23.0g of the EtOAc fraction (66.4g) of Lesser pheasant leaves was analyzed using MPLC and RediSep® Rf RP-C18 column to MeOH: H2O (5:5→6:4→7:3→8:2 It was separated under solvent conditions of (→9:1→MeOH) and divided into eight fractions (VEE1 to VEE8).
화합물9의 경우, 8개의 분획(VEE1 ~ VEE8) 중 VEE3로부터 crystallization 되어 석출된 소분획 VEE3-C를 얻었다. Thin Layer Chromatography(TLC) 확인시험법을 이용하여 순도를 확인하였고, VEE3-C에서 단일한 화합물 9 (198mg)을 얻었다. In the case of compound 9, among the eight fractions (VEE1 to VEE8), a small fraction VEE3-C was obtained by crystallization from VEE3 and precipitated. Purity was confirmed using the Thin Layer Chromatography (TLC) identification test, and a single compound 9 (198 mg) was obtained from VEE3-C.
(2) 덜꿩나무 잎(V.erosum) 에서 분리한 화합물의 구조(2) Structure of compounds isolated from V. erosum leaves
화합물 1 내지 9에 대해 NMR 분석 및 MS 분석을 수행하였다. 피리딘-d5를 용매로 사용하여 Bruker Avance II 600 (Bruker, Germany) 분광계에서 H는 600 MHz, C는 150 MHz에서 얻었다. 하기 표 1에 화합물 1 내지 4의 NMR 결과를 나타내었으며, 하기 표 2에 화합물 5 내지 7의 NMR 결과를 나타내었다.NMR analysis and MS analysis were performed on compounds 1 to 9. H was obtained at 600 MHz and C at 150 MHz on a Bruker Avance II 600 (Bruker, Germany) spectrometer using pyridine-d5 as the solvent. The NMR results of compounds 1 to 4 are shown in Table 1 below, and the NMR results of compounds 5 to 7 are shown in Table 2 below.
[표 1][Table 1]
[표 2][Table 2]
화합물 1은 하얀색 무정형 분말로서 얻어졌으며, HRESI-MS (m/z 419.1690 [M-H]- calcd for 419.1706)측정값을 통해 분자식 C22H28O8 로 결정되었다.Compound 1 was obtained as a white amorphous powder, and its molecular formula was determined to be C22H28O8 through HRESI-MS (m/z 419.1690 [M-H]- calcd for 419.1706) measurements.
화합물 1의 1D NMR 과 HSQC 데이터에서 전형적인 β-D-glucosyl group으로 예상되는 신호인 δH/δC 4.71 (1H, d, J = 7.4 Hz, H-1) / 103.6 (C-1), 4.51 (1H, dd, J = 2.1, 11.8 Hz, H-6) / 65.1 (C-6), 4.24 (1H, dd, J = 7.2, 11.8 Hz, H-6) / 65.1 (C-6), 3.62 (1H, ddd, J = 2.1, 7.2, 9.5 Hz, H-5) / 75.4 (C-5), 3.43 (H, m, H-3) / 77.9 (C-3), 3.43 (H, m, H-2) / 74.9 (C-2), 3.36 (1H, dd, J = 6.5, 11.7 Hz, H-4) / 71.9 (C-4) 신호를 확인 했으며, 153.9 (C-6′), 152.3 (C-1′), 6.93 (2H, d, J = 8.9 Hz, H-2′, 6′) / 119.5 (C-4′, 6′), 6.66 (2H, d, J = 8.9 Hz, H-3′, 5′) / 116.6 (C-3′, 5′) 의 신호를 통해 para위치에 두개의 hydroxyl group을 갖는 hydroquinone group이 존재함을 확인하였다. HMBC데이터에서 β-D-glucosyl group 의δH 4.71 (1H, d, J = 7.4 Hz, H-1)와 hydroquinone group의 δC 152.3 (C-1′)가 상관 피크를 나타낸 것을 토대로 화합물 1이 arbutin의 유도체임을 예상하였다.In the 1D NMR and HSQC data of compound 1, the signal expected to be a typical β-D-glucosyl group, δH/δC 4.71 (1H, d, J = 7.4 Hz, H-1) / 103.6 (C-1), 4.51 (1H) , dd, J = 2.1, 11.8 Hz, H-6) / 65.1 (C-6), 4.24 (1H, dd, J = 7.2, 11.8 Hz, H-6) / 65.1 (C-6), 3.62 (1H) , ddd, J = 2.1, 7.2, 9.5 Hz, H-5) / 75.4 (C-5), 3.43 (H, m, H-3) / 77.9 (C-3), 3.43 (H, m, H- 2) / 74.9 (C-2), 3.36 (1H, dd, J = 6.5, 11.7 Hz, H-4) / 71.9 (C-4) signals were confirmed, and 153.9 (C-6′), 152.3 (C) -1′), 6.93 (2H, d, J = 8.9 Hz, H-2′, 6′) / 119.5 (C-4′, 6′), 6.66 (2H, d, J = 8.9 Hz, H-3 Through the signal of ′, 5′) / 116.6 (C-3′, 5′), it was confirmed that a hydroquinone group with two hydroxyl groups exists at the para position. Based on the correlation peak of δH 4.71 (1H, d, J = 7.4 Hz, H-1) of the β-D-glucosyl group and δC 152.3 (C-1′) of the hydroquinone group in the HMBC data, it was found that compound 1 is a It was expected to be a derivative.
나아가, 화합물 1의 1D NMR 과 HSQC 데이터에서 두 개의 olefinic groups:δH/δC 6.82 (1H, td, J = 1.4, 7.3, 7.3 Hz, H-3″) / 143.7 (C-3″), 6.40 (1H, dd, J = 10.9, 17.7 Hz, H-7″) / 139.7 (C-7″), 두 개의 exomethylene: 5.28 (1H, d, J = 14.3 Hz, H-8″), 5.09 (1H, d, J = 14.3 Hz, H-8″) / 113.9 (C-8″), 5.03 (2H, d, J = 16.2 Hz, H-7″) / 116.9 (C-7″), 두 개의 methylene: 2.43 (2H, dd, J = 7.3, 14.5 Hz, H-4″) / 28.4 (C-4″), 2.37 (2H, dd, J = 6.8, 7.9 Hz, H-5″) / 31.1 (C-5″), 하나의 methyl group: 1.84 (3H, s, H-9″) / 12.6 (C-9″) 신호가 확인되었다. 13C NMR 데이터에서 하나의 carboxyl group: δC 169.4 (C-1″), 두 개의 olefinic quaternary carbons: 146.76 (C-6″) and 128.9 (C-2″) 신호가 확인되었다. 이 신호들은 COSY 데이터에서 H-3″과 H-4″, H-4″과 H-5″, H-7″과 H-8″의 상관 피크를 나타내었으며, HMBC 데이터에서 H-3″과 C-1″, C-9″사이, H-4″과 C-2″, C-3″, C-6″사이, H-5″과 C-3″, C-6″, C-7″, C-10″사이, H-7″과 C-5″, C-6″, C-10″사이, H-8″과 C-6″, C-7″사이, H-9″과 C-1″, C-2″ 사이, H-10″과 C-5″, C-7″사이의 상관피크를 나타내었다. 이를 토대로 화합물 1 이 2-methyl-6-methylene-2,7-octadienoyl group을 포함하는 것을 예상하였다.Furthermore, the 1D NMR and HSQC data of compound 1 show two olefinic groups: δH/δC 6.82 (1H, td, J = 1.4, 7.3, 7.3 Hz, H-3″) / 143.7 (C-3″), 6.40 ( 1H, dd, J = 10.9, 17.7 Hz, H-7″) / 139.7 (C-7″), two exomethylene: 5.28 (1H, d, J = 14.3 Hz, H-8″), 5.09 (1H, d, J = 14.3 Hz, H-8″) / 113.9 (C-8″), 5.03 (2H, d, J = 16.2 Hz, H-7″) / 116.9 (C-7″), two methylenes: 2.43 (2H, dd, J = 7.3, 14.5 Hz, H-4″) / 28.4 (C-4″), 2.37 (2H, dd, J = 6.8, 7.9 Hz, H-5″) / 31.1 (C- 5″), one methyl group: 1.84 (3H, s, H-9″) / 12.6 (C-9″) signal was confirmed. In the 13C NMR data, one carboxyl group: δC 169.4 (C-1″) and two olefinic quaternary carbons: 146.76 (C-6″) and 128.9 (C-2″) signals were confirmed. These signals showed correlation peaks of H-3″ and H-4″, H-4″ and H-5″, and H-7″ and H-8″ in the COZY data, and H-3″ and H-8″ in the HMBC data. Between C-1″ and C-9″, between H-4″ and C-2″, C-3″, C-6″, between H-5″ and C-3″, C-6″, C-7 ″, between C-10″, between H-7″ and C-5″, C-6″, C-10″, between H-8″ and C-6″, between C-7″, between H-9″ and Correlation peaks were shown between C-1″, C-2″, H-10″, C-5″, and C-7″. Based on this, it was predicted that Compound 1 contains a 2-methyl-6-methylene-2,7-octadienoyl group.
마지막으로, HMBC 데이터에서 arbutin 분자의 H-6과 monoterpene 분자의 C-1″사이의 상관피크를 확인함으로써 화합물 1은 6-O-(2-methyl-6-methylene-2,7-octadienoyl)-arbutin 으로 결정되었다.Finally, by confirming the correlation peak between H-6 of the arbutin molecule and C-1″ of the monoterpene molecule in the HMBC data, compound 1 is 6-O-(2-methyl-6-methylene-2,7-octadienoyl)- It was decided to be arbutin.
화합물 2는 하얀색 무정형 분말로서 얻어졌으며, HRESI-MS (m/z 437.1834 [M-H]- calcd for 437.1812)측정값을 통해 분자식 C22H30O9 로 결정되었다. Compound 2 was obtained as a white amorphous powder, and its molecular formula was determined to be C22H30O9 through HRESI-MS (m/z 437.1834 [M-H]-calcd for 437.1812) measurements.
1D 및 2D NMR 데이터는 glucopyranosyl group의 C-6에 결합된 monoterpene 부분을 제외하고는 화합물 1과 유사한 패턴을 보여주었다. 1D NMR 과 HSQC 데이터에서 두 개의 olefinic groups: δH/δC 6.79 (1H, td, J = 1.4, 7.3 Hz, H-3″) / 143.7 (C-3″), 5.39 (1H, t, J = 6.1 Hz, H-7″) / 125.7 (C-7″), 하나의 oxymethylene: 4.08 (2H, d, J = 6.7 Hz, H-8″) / 59.4 (C-8″), 두 개의 methylene: 2.36 (2H, dd, J = 7.4, 14.9 Hz, H-4″) / 28.0 (C-4″), 2.16 (2H, t, J = 7.6, 7.6 Hz, H-5″) / 39.1 (C-5″), 두 개의 methyl groups 1.85 (3H, s, H-9″) / 12.5 (C-9″), 1.70 (3H, s, H-10″) / 16.2 (C-10″) 신호가 확인되었다. 13C NMR 데이터에서 하나의 carboxyl group: δC 169.4 (C-1″), 두 개의 olefinic quaternary carbons: 138.5 (C-6″), 128.9 (C-2″) 신호가 확인되었다. 이 신호들은 COSY 데이터에서 H-3″과 H-4″, H-4″과 H-5″, H-7″과 H-8″의 상관 피크를 나타내었으며, HMBC 데이터에서 H-5″과 C-3″, C-6″, C-7″, C-10″사이, H-7″과 C-5″, C-10″사이, H-8″과 C-6″, C-7″사이, H-10″과 C-6″, C-7″사이의 상관피크를 나타내었다. 이를 토대로 화합물 2가 2,6-dimethyl-8-hydroxy-2,6-octadienoyl group을 포함하는 것을 예상하였다.1D and 2D NMR data showed a similar pattern to compound 1 except for the monoterpene moiety bound to C-6 of the glucopyranosyl group. Two olefinic groups in 1D NMR and HSQC data: δH/δC 6.79 (1H, td, J = 1.4, 7.3 Hz, H-3″) / 143.7 (C-3″), 5.39 (1H, t, J = 6.1 Hz, H-7″) / 125.7 (C-7″), one oxymethylene: 4.08 (2H, d, J = 6.7 Hz, H-8″) / 59.4 (C-8″), two methylene: 2.36 (2H, dd, J = 7.4, 14.9 Hz, H-4″) / 28.0 (C-4″), 2.16 (2H, t, J = 7.6, 7.6 Hz, H-5″) / 39.1 (C-5) ″), two methyl groups 1.85 (3H, s, H-9″) / 12.5 (C-9″), 1.70 (3H, s, H-10″) / 16.2 (C-10″) signals were confirmed. . In the 13C NMR data, one carboxyl group: δC 169.4 (C-1″) and two olefinic quaternary carbons: 138.5 (C-6″), 128.9 (C-2″) signals were confirmed. These signals showed correlation peaks of H-3″ and H-4″, H-4″ and H-5″, and H-7″ and H-8″ in the COZY data, and H-5″ and H-8″ in the HMBC data. Between C-3″, C-6″, C-7″, C-10″, between H-7″ and C-5″, C-10″, between H-8″ and C-6″, C-7 Correlation peaks were shown between ″, H-10″, C-6″, and C-7″. Based on this, it was predicted that compound 2 contains a 2,6-dimethyl-8-hydroxy-2,6-octadienoyl group.
마지막으로, HMBC 데이터에서 arbutin 분자의 H-6과 monoterpene 분자의 C-1″사이의 상관피크를 확인함으로써 화합물 2는 6-O-(2,6-dimethyl-8-hydroxy-2,6-octadienoyl)-arbutin 으로 결정되었다.Finally, by confirming the correlation peak between H-6 of the arbutin molecule and C-1″ of the monoterpene molecule in the HMBC data, compound 2 was identified as 6-O-(2,6-dimethyl-8-hydroxy-2,6-octadienoyl )-arbutin.
화합물 3은 하얀색 무정형 분말로서 얻어졌으며, HRESI-MS (m/z 451.1975[M-H]- calcd for 451.1968)측정값을 통해 분자식 C23H32O9 로 결정되었다. Compound 3 was obtained as a white amorphous powder, and its molecular formula was determined to be C23H32O9 through HRESI-MS (m/z 451.1975[M-H]- calcd for 451.1968) measurements.
1D 및 2D NMR 데이터는 C-8″의 hydroxyl group이 methoxy group으로 치환된 것을 제외하고는 화합물 2와 유사한 패턴을 보여주었다. 화합물 2에 비하여 C-6″ (δC 140.8, +2.3 ppm), C-7″ (δC 122.6, -3.1 ppm), C-8″ (δC 69.7, +10.3 ppm)의 chemical shift값의 유의한 변화가 있었다. HMBC 데이터에서 δH 3.28 (3H, s, H-11″) 과 δC 69.7 (C-8″)의 상관피크를 확인함으로써 C-8″에 methoxy group이 위치함을 확인했다.1D and 2D NMR data showed a similar pattern to compound 2 except that the hydroxyl group at C-8″ was replaced with a methoxy group. Significant changes in chemical shift values of C-6″ (δC 140.8, +2.3 ppm), C-7″ (δC 122.6, -3.1 ppm), and C-8″ (δC 69.7, +10.3 ppm) compared to compound 2. There was. By confirming the correlation peak of δH 3.28 (3H, s, H-11″) and δC 69.7 (C-8″) in the HMBC data, it was confirmed that the methoxy group is located at C-8″.
이를 토대로 화합물 3이 2,6-dimethyl-8-methoxy-2,6-octadienoyl group을 포함하는 것을 예상하였다. 따라서, 화합물 3은 6-O-(2,6-dimethyl-8-methoxy-2,6-octadienoyl)-arbutin 으로 결정되었다.Based on this, it was predicted that compound 3 contains a 2,6-dimethyl-8-methoxy-2,6-octadienoyl group. Therefore, compound 3 was determined to be 6-O-(2,6-dimethyl-8-methoxy-2,6-octadienoyl)-arbutin.
화합물 4는 하얀색 무정형 분말로서 얻어졌으며, HRESI-MS (m/z 451.1975[M-H]- calcd for 451.1968)측정값을 통해 분자식 C23H32O9 로 결정되었다. 1D 및 2D NMR 데이터는 C-6″의 hydroxyl group이 methoxy group으로 치환된 것을 제외하고는 화합물 7과 유사한 패턴을 보여주었다. 화합물 7에 비하여 C-5″ (δC 39.2, -2.5 ppm), C-6″ (δC 78.5, +4.8 ppm), C-7″ (δC 144.3, -1.6 ppm), C-8″ (δC 115.8, +3.3 ppm) and C-10″ (δC 21.9, -6.0 ppm)의 chemical shift값의 유의한 변화가 있었다. HMBC 데이터에서 δH 3.17 (3H, s, H-11″) 와 δC 78.5 (C-6″)의 상관피크를 확인함으로써 C-6″에 methoxy group이 위치함을 확인했다. 화합물 4의 methoxy group 절대구조는 화합물 4와 7의 acid hydrolysis에 의해 확인되었다. 이들의 acid hydrolysis 생성물은 TLC 분석에서 동일한 Rf값을 가지며, H NMR 스펙트럼이 일치한다. 이를 토대로 화합물 4가 (6S)-2,6-methyl-6-methoxy-2,7-octadienoyl group을 포함하는 것을 예상하였다. 따라서, 화합물 4는 6-O-[(6S)-2,6-dimethyl-6-methoxy-2,7-octadienoyl]-arbutin 으로 결정되었다.Compound 4 was obtained as a white amorphous powder, and its molecular formula was determined to be C23H32O9 through HRESI-MS (m/z 451.1975[M-H]- calcd for 451.1968) measurements. 1D and 2D NMR data showed a similar pattern to compound 7 except that the hydroxyl group at C-6″ was replaced with a methoxy group. Compared to compound 7, C-5″ (δC 39.2, -2.5 ppm), C-6″ (δC 78.5, +4.8 ppm), C-7″ (δC 144.3, -1.6 ppm), C-8″ (δC 115.8) , +3.3 ppm) and C-10″ (δC 21.9, -6.0 ppm), there was a significant change in the chemical shift values. By confirming the correlation peak of δH 3.17 (3H, s, H-11″) and δC 78.5 (C-6″) in the HMBC data, it was confirmed that the methoxy group is located at C-6″. The absolute structure of the methoxy group of compound 4 was confirmed by acid hydrolysis of compounds 4 and 7. These acid hydrolysis products have the same Rf value in TLC analysis, and their H NMR spectra are consistent. Based on this, it was predicted that compound 4 contains a (6S)-2,6-methyl-6-methoxy-2,7-octadienoyl group. Therefore, compound 4 was determined to be 6-O-[(6S)-2,6-dimethyl-6-methoxy-2,7-octadienoyl]-arbutin.
화합물 5는 하얀색 무정형 분말로서 얻어졌으며, HRESI-MS (m/z 471.1868[M-H]- calcd for 471.1866)측정값을 통해 분자식 C22H32O11 로 결정되었다. 1D 및 2D NMR 데이터는 monoterpene의 말단부분을 제외하고는 화합물 7과 유사한 패턴을 보여주었다. 화합물 7에 비하여 C-5″ (δC 38.6, -3.1 ppm), C-7″ (δC 78.2, -67.7 ppm), C-8″ (δC 64.0, -48.5 ppm) and C-10″ (δC 22.1, -5.8 ppm)의 chemical shift값의 유의한 변화가 있었다. 화합물 7과 상이하게, 1D NMR 과 HSQC 데이터에서 하나의 oxymethine: δH/δC 3.50 (1H, dd, J = 3.2, 7.9 Hz, H-7″) / 78.2 (C-7″), 하나의 oxymethylene: 3.55 (1H, m, H-8″), 3.80 (1H, m, H-8″) / 64.0 (C-8″) 신호가 확인되었다. HMBC데이터에서 H-7″과 C-6″, C-8″, C-10″사이, H-8″과 C-7″사이의 상관피크를 나타내었다. 이는 화합물 7 의 C-7″,C-8″double bond가 single bond 로 변경되고, 각각의 C-7″,C-8″자리에 hydroxyl group이 결합된 것을 의미한다. Compound 5 was obtained as a white amorphous powder, and its molecular formula was determined to be C22H32O11 through HRESI-MS (m/z 471.1868[M-H]- calcd for 471.1866) measurements. 1D and 2D NMR data showed a similar pattern to compound 7 except for the terminal portion of the monoterpene. Compared to compound 7, C-5″ (δC 38.6, -3.1 ppm), C-7″ (δC 78.2, -67.7 ppm), C-8″ (δC 64.0, -48.5 ppm) and C-10″ (δC 22.1 There was a significant change in the chemical shift value of -5.8 ppm). Different from compound 7, 1D NMR and HSQC data show one oxymethine: δH/δC 3.50 (1H, dd, J = 3.2, 7.9 Hz, H-7″) / 78.2 (C-7″), one oxymethylene: Signals 3.55 (1H, m, H-8″), 3.80 (1H, m, H-8″) / 64.0 (C-8″) were confirmed. The HMBC data showed correlation peaks between H-7″ and C-6″, C-8″ and C-10″, and between H-8″ and C-7″. This means that the C-7″, C-8″ double bond of compound 7 is changed to a single bond, and a hydroxyl group is attached to each C-7″ and C-8″ site.
이를 토대로 화합물 5가 2,6-dimethyl-6,7,8-trihydroxy-2-octenoyl group 을 포함하는 것을 예상하였다. 따라서, 화합물 5는 6-O-(2,6-dimethyl-6,7,8-trihydroxy-2-octenoyl)-arbutin 으로 결정 되었다. 화합물 5의 C-7″,C-8″ hydroxyl group 절대구조는 밝히지 못하였다.Based on this, it was predicted that compound 5 contains a 2,6-dimethyl-6,7,8-trihydroxy-2-octenoyl group. Therefore, compound 5 was determined to be 6-O-(2,6-dimethyl-6,7,8-trihydroxy-2-octenoyl)-arbutin. The absolute structures of the C-7″ and C-8″ hydroxyl groups of compound 5 were not revealed.
화합물 6은 하얀색 무정형 분말로서 얻어졌으며, HRESI-MS (m/z 471.1868[M-H]- calcd for 471.1866)측정값을 통해 분자식 C22H32O11 로 결정되었다. 1D 및 2D NMR 데이터는 화합물 5와 매우 유사한 패턴을 보여주었다. 화합물 5에 비하여 C-4″ (δC 24.0, -0.3 ppm), C-5″ (δC 37.9, -0.7 ppm), C-7″ (δC 78.5, -0.3 ppm), H-8″(δH 3.75, -0.05 ppm) 의 미세한 chemical shift값의 변화를 제외하고는 1D 및 2D NMR 스펙트럼이 일치하였다. 이는 화합물 5와 6이 서로 stereoisomer 관계임을 의미한다. Compound 6 was obtained as a white amorphous powder, and its molecular formula was determined to be C22H32O11 through HRESI-MS (m/z 471.1868[M-H]-calcd for 471.1866) measurements. 1D and 2D NMR data showed a very similar pattern to compound 5. Compared to compound 5, C-4″ (δC 24.0, -0.3 ppm), C-5″ (δC 37.9, -0.7 ppm), C-7″ (δC 78.5, -0.3 ppm), H-8″ (δH 3.75 , -0.05 ppm), the 1D and 2D NMR spectra were consistent except for a slight change in chemical shift value. This means that compounds 5 and 6 have a stereoisomer relationship with each other.
따라서, 화합물 6은 6-O-(2,6-dimethyl-6,7,8-trihydroxy-2-octenoyl)-arbutin 으로 결정되었다. 화합물 6의 C-7″,C-8″ hydroxyl group 절대구조는 밝히지 못하였다.Therefore, compound 6 was determined to be 6-O-(2,6-dimethyl-6,7,8-trihydroxy-2-octenoyl)-arbutin. The absolute structures of the C-7″ and C-8″ hydroxyl groups of compound 6 were not revealed.
화합물 7은 하얀색 무정형 분말로서 얻어졌으며, HRESI-MS (m/z 437.1834 [M-H]- calcd for 437.1812)측정값을 통해 분자식 C22H30O9 로 결정되었다. 1D 및 2D NMR 데이터는 glucopyranosyl group의 C-6에 결합된 monoterpene 부분을 제외하고는 화합물 1과 유사한 패턴을 보여주었다. 1D NMR 과 HSQC 데이터에서 두 개의 olefinic groups: δH/δC 6.80 (1H, td, J = 1.4, 7.6 Hz, H-3″) / 144.4 (C-3″), 5.93 (1H, ddd, J = 2.9, 10.8, 17.4 Hz, H-7″) / 145.9 (C-7″), 하나의 exomethylene: 5.07 (H, d, J = 14.1 Hz, H-8″), 5.23 (H, d, J = 14.1 Hz, H-8″) / 112.48 (C-8″), 두 개의 methylene: 2.25 (2H, m, H-4″) / 24.6 (C-4″), 1.60 (2H, m, H-5″) / 41.7 (C-5″), 두 개의 methyl groups 1.84 (3H, s, H-9″) / 12.4 (C-9″), 1.29 (3H, s, H-10″) / 27.9 (C-10″) 신호가 확인되었다. 13C NMR 데이터에서 하나의 carboxyl group: δC 169.4 (C-1″), 두 개의 olefinic quaternary carbons: 73.7 (C-6″), 128.5 (C-2″) 신호가 확인되었다. 위의 결과를 참고문헌의 NMR shift값과 비교하여 화합물 7은 Phlebotrichin, 6-O-[(6S)-2,6-dimethyl-6-hydroxy-2,7-octadienoyl]-arbutin 으로 결정되었다. Compound 7 was obtained as a white amorphous powder, and its molecular formula was determined to be C22H30O9 through HRESI-MS (m/z 437.1834 [M-H]-calcd for 437.1812) measurements. 1D and 2D NMR data showed a similar pattern to compound 1 except for the monoterpene moiety bound to C-6 of the glucopyranosyl group. Two olefinic groups in 1D NMR and HSQC data: δH/δC 6.80 (1H, td, J = 1.4, 7.6 Hz, H-3″) / 144.4 (C-3″), 5.93 (1H, ddd, J = 2.9) , 10.8, 17.4 Hz, H-7″) / 145.9 (C-7″), one exomethylene: 5.07 (H, d, J = 14.1 Hz, H-8″), 5.23 (H, d, J = 14.1 Hz, H-8″) / 112.48 (C-8″), two methylene: 2.25 (2H, m, H-4″) / 24.6 (C-4″), 1.60 (2H, m, H-5″) ) / 41.7 (C-5″), two methyl groups 1.84 (3H, s, H-9″) / 12.4 (C-9″), 1.29 (3H, s, H-10″) / 27.9 (C- 10″) signal was confirmed. In the 13C NMR data, one carboxyl group: δC 169.4 (C-1″) and two olefinic quaternary carbons: 73.7 (C-6″), 128.5 (C-2″) signals were confirmed. By comparing the above results with the NMR shift values in the reference, compound 7 was determined to be Phlebotrichin, 6-O-[(6S)-2,6-dimethyl-6-hydroxy-2,7-octadienoyl]-arbutin.
화합물 8은 갈색 무정형 분말로서 얻어졌으며, ESI-MS (m/z 357.18[M-H]-)측정값을 통해 분자식 C20H22O6 로 결정되었다. 13C NMR 데이터에서 20개의 탄소를 확인했으며 furofuran lignan에 나타나는 non-oxidized furofuran carbon δC 89.2 (C-7), 83.3 (C-7′), 72.0 (C-9), 70.6 (C-9′), 55.7 (C-8), 51.2(C-8′)을 확인하였다. signal 1H NMR 데이터에서 pinoresinol-type lignan의 benzene ring δH 6.89 (2H, t, J = 7.9 Hz, H-2, 2′), 6.84(2H, dd, J = 5.8, 1.7 Hz, H-5, 5′), 6.81(2H, dd, J = 8.2, 1.8 Hz, H-6, 6′) 신호를 확인했다. 이어서 3.85 (6H, s, H-10, 10′)신호를 확인하여 lignan의 두 benzene ring에 methoxy group이 존재함을 확인하였다. Compound 8 was obtained as a brown amorphous powder, and its molecular formula was determined to be C20H22O6 through ESI-MS (m/z 357.18[M-H]-) measurements. Twenty carbons were identified in the 13C NMR data, and non-oxidized furofuran carbon δC 89.2 (C-7), 83.3 (C-7′), 72.0 (C-9), 70.6 (C-9′), which appears in furofuran lignan. 55.7 (C-8) and 51.2 (C-8′) were confirmed. In the signal 1H NMR data, the benzene ring δH of pinoresinol-type lignan was 6.89 (2H, t, J = 7.9 Hz, H-2, 2′), 6.84 (2H, dd, J = 5.8, 1.7 Hz, H-5, 5). ′), 6.81(2H, dd, J = 8.2, 1.8 Hz, H-6, 6′) signals were confirmed. Subsequently, the 3.85 (6H, s, H-10, 10′) signal was confirmed to confirm the presence of methoxy groups in the two benzene rings of lignan.
위의 결과를 참고문헌의 NMR shift값과 비교하여 화합물 8은 (-)-epipinoresinol 으로 결정되었다. By comparing the above results with the NMR shift values in the reference, compound 8 was determined to be (-)-epipinoresinol.
화합물 9는 노란색 분말로서 얻어졌으며, ESI-MS (m/z 537.18[M-H]- )측정값을 통해 분자식 C30H18O10 로 결정되었다. 13C NMR 데이터에서 30개의 탄소를 확인했으며 flavonoid에 나타나는 conjugated carbonyl carbon δC 181.8 (C-4), 182.2 (C-4″)를 확인하였다. signal 1H NMR 데이터에서 전형적인 flavonoid로 예상되는 신호인 δH 6.16 (1H, d, J = 2.1 Hz, H-6), 6.44 (1H, d, J = 2.1 Hz, H-8) 신호를 확인했다. 두 doublet peaks는 aromatic ring의 A ring H-6, H-8이다. 이어서, 6.38 (1H, s, H-6″)신호를 확인하여 flavonoid A ring의 H-6위치가 추가로 존재함을 확인하였고, H-6″신호가 singlet인 점과 또 다른 flavonoid A ring의 H-8위치 신호의 부재를 통해 화합물 9가 biflavonoid 인 것을 예상하였다. 6.77 (1H, s, H-3), 6.81(1H, s, H-3″)의 신호는 flavonoid C ring의 H-3위치의 신호이며, 이웃한 위치에 proton이 존재하지 않기 때문에 singlet peak를 나타낸다. Compound 9 was obtained as a yellow powder, and its molecular formula was determined to be C30H18O10 through ESI-MS (m/z 537.18[M-H]-) measurements. 30 carbons were identified in the 13C NMR data, and conjugated carbonyl carbon δC 181.8 (C-4), 182.2 (C-4″) appearing in the flavonoid was confirmed. In signal 1H NMR data, δH 6.16 (1H, d, J = 2.1 Hz, H-6) and 6.44 (1H, d, J = 2.1 Hz, H-8) signals, which are signals expected to be typical flavonoids, were confirmed. The two doublet peaks are the A ring H-6 and H-8 of the aromatic ring. Next, by checking the 6.38 (1H, s, H-6″) signal, it was confirmed that the H-6 position of the flavonoid A ring additionally exists, and the H-6″ signal is a singlet and the presence of another flavonoid A ring Compound 9 was predicted to be a biflavonoid through the absence of the H-8 position signal. The signals of 6.77 (1H, s, H-3) and 6.81 (1H, s, H-3″) are signals from the H-3 position of the flavonoid C ring, and since there is no proton at the adjacent position, a singlet peak is produced. indicates.
위의 결과를 참고문헌의 NMR shift값과 비교하여 화합물 9는 amentoflavone 으로 결정되었다.By comparing the above results with the NMR shift values in the reference, compound 9 was determined to be amentoflavone.
실험1: 덜꿩나무 잎 분획물의 세포독성확인Experiment 1: Confirmation of cytotoxicity of Lesser Pheasant leaf fraction
(1) B16F10, HEP-2, PC3 세포주 배양(1) B16F10, HEP-2, PC3 cell line culture
B16F10, HEP-2, PC3 암세포주는 KCLB (Korean Cell Line Bank, Seoul, Republic of Korea)에서 분양받았으며 배지는 RPMI 1640 medium (added 1% Penicillin and 10% FBS)과 DMEM medium (added 1% Penicillin and 10% FBS)를 사용하였다 (WELGENE, Republic of Korea). 각 세포주는 5% CO2가 지속적으로 공급되는 배양기에서 온도 조건 37℃ 하에 배양하였다. B16F10, HEP-2, and PC3 cancer cell lines were purchased from KCLB (Korean Cell Line Bank, Seoul, Republic of Korea), and the medium was RPMI 1640 medium (added 1% Penicillin and 10% FBS) and DMEM medium (added 1% Penicillin and 10% FBS). % FBS) was used (WELGENE, Republic of Korea). Each cell line was cultured under temperature conditions of 37°C in an incubator continuously supplied with 5% CO2.
(2) 세포독성 확인(2) Confirmation of cytotoxicity
B16F10, HEP-2, PC3 세포주를 96 well plate에 5.0 x 103 cells/well로 희석하여 분주한 후 24시간 배양하였다. DMSO에 녹인 덜꿩나무잎 소분획을 최종농도 100νg/mL로 처리하여 72시간 배양한 후 MTT assay를 통해 흡광도 540nm에서 확인하였다. 음성대조군으로 DMSO를 사용하였으며 시료당 3반복 실험하였다. B16F10, HEP-2, and PC3 cell lines were diluted and dispensed at 5.0 x 103 cells/well in a 96 well plate and cultured for 24 hours. A small fraction of Lesser pheasant leaves dissolved in DMSO was treated at a final concentration of 100 νg/mL, cultured for 72 hours, and the absorbance was confirmed at 540 nm through MTT assay. DMSO was used as a negative control, and the experiment was repeated 3 times per sample.
(3) 통계처리(3) Statistical processing
덜꿩나무잎 소분획에 대한 세포독성을 비교하기위해 음성대조군으로 DMSO를 사용하였으며 생존율을 100%로 설정하였다. 시료 처리 후 생존율을 %값으로 계산하여 비교하였다.. To compare the cytotoxicity of small fractions of Lesser Pheasant tree leaves, DMSO was used as a negative control and the survival rate was set at 100%. After sample processing, the survival rate was calculated and compared as a percentage value.
(4) 덜꿩나무 잎 분획물의 세포독성 결과(4) Cytotoxicity results of leaf fractions of Lesser Pheasant tree
덜꿩나무 잎의 EtOA 분획물 VEE1~VEE8의 세포독성을 확인하고자 시료농도 100νg/mL 로 확인하였고, 세포주로는 B16F10(피부암세포), HEp-2(후두암세포), PC-3(전립선암세포)를 사용하였다. 음성대조군인 DMSO를 통해 세포생존율을 100%로 계산하였으며, 각 분획물 시료를 세 번 반복 처리 후 평균값을 산출하여 비교하였다. To confirm the cytotoxicity of EtOA fractions VEE1~VEE8 from Lesser Pheasant tree leaves, the sample concentration was confirmed to be 100νg/mL, and B16F10 (skin cancer cells), HEp-2 (laryngeal cancer cells), and PC-3 (prostate cancer cells) were used as cell lines. did. Cell viability was calculated as 100% using DMSO as a negative control, and each fraction sample was treated three times and the average value was calculated and compared.
도 4를 참고하면, 피부암세포인 B16F10에서 세포생존율은 각 분획마다 눈에띄는 차이를 보였다. VEE1~3, VEE8에서 세포생존율은 103%~128%로, 세포독성이 존재하지 않음을 확인하였다. 대조적으로 VEE4~7에서는 세포생존율이 19%~22%로 강한 세포독성이 존재하였다. Referring to Figure 4, the cell viability of B16F10 skin cancer cells showed a noticeable difference in each fraction. The cell viability in VEE1~3 and VEE8 was 103%~128%, confirming that there was no cytotoxicity. In contrast, VEE4~7 showed strong cytotoxicity with a cell survival rate of 19%~22%.
* 후두암세포인 HEp-2에서 세포생존율은 B16F10의 세포생존율과 비슷한 경향을 보였다. VEE1~2에서 세포생존율이 각각 112%, 100로, 세포독성이 존재하지않았다. VEE3, VEE8에서는 세포생존율이 각각 86%, 83%로, 매우 약한 세포독성이 존재하였다. VEE4~7에서는 세포생존율이 15%~36%로 강한 세포독성이 존재하였다. * The cell survival rate of HEp-2, a laryngeal cancer cell, showed a similar trend to that of B16F10. In VEE1~2, the cell viability was 112% and 100, respectively, and there was no cytotoxicity. In VEE3 and VEE8, cell survival rates were 86% and 83%, respectively, and very weak cytotoxicity existed. In VEE4~7, there was strong cytotoxicity with a cell survival rate of 15%~36%.
전립선암세포인 PC-3에서 세포생존율은 VEE2를 제외한 모든분획에서 강한 세포독성을 보였다. VEE2에서 세포생존률은 85%로 매우 약한 세포독성이 존재하였지만, 나머지 모든 분획에서는 14%~39%의 세포생존률을 보여 강한 세포독성이 존재함을 확인하였다. Cell viability in PC-3, a prostate cancer cell, showed strong cytotoxicity in all fractions except VEE2. In VEE2, the cell viability was 85%, indicating very weak cytotoxicity, but all remaining fractions showed cell viability of 14% to 39%, confirming the presence of strong cytotoxicity.
분획물 VEE1~VEE8 에서 상대적으로 극성이 높은 fraction인 VEE1~VEE3에서 비교적 높은 세포생존률을 보였으며, 이 중 모든 세포주에서 높은 생존률을 보이는 VEE2분획물을 분리에 사용하였다. VEE4~VEE7은 실험에사용된 모든 세포주에서 강한 세포독성이 있었다. 해당 분획은 항암활성을 가지는 활성성분 분리에 유용하게 연구될 수 있을것으로 사료된다. Fractions VEE1 to VEE8 showed relatively high cell survival rates in VEE1 to VEE3, which are relatively highly polar fractions, and among these, the VEE2 fraction, which showed high survival rates in all cell lines, was used for separation. VEE4~VEE7 had strong cytotoxicity in all cell lines used in the experiment. It is believed that this fraction can be usefully studied for the isolation of active ingredients with anticancer activity.
실험2: 덜꿩나무 잎 화합물의 Tyrosinase 활성 저해시험Experiment 2: Tyrosinase activity inhibition test of Lesser Pheasant leaf compounds
*(1) 시험 방법*(1) Test method
시료를 에탄올에 녹여 Tyrosinase 활성 저해를 확인할 수 있는 농도로 설정하였다. 96 well plate에 0.1M 인산염완충액 (pH 6.5) 220νM, 시료액 20νL, Mushroom tyrosinase액 (2000U/mL) 20νL을 순서대로 처리한 후 37℃ 배양기에서 10분간 배양하였다. 이 액에 1.5mM 타이로신액 40νL를 처리하고 37℃에서 15분동안 반응시킨 다음 490nm에서 흡광도를 측정하였다. The sample was dissolved in ethanol and set to a concentration that could confirm inhibition of Tyrosinase activity. A 96 well plate was treated with 220νM of 0.1M phosphate buffer (pH 6.5), 20νL of sample solution, and 20νL of mushroom tyrosinase solution (2000U/mL) in that order and incubated in an incubator at 37°C for 10 minutes. This solution was treated with 40νL of 1.5mM tyrosine solution, reacted at 37°C for 15 minutes, and absorbance was measured at 490nm.
(2) 통계 처리(2) Statistical processing
덜꿩나무 잎 소분획과 덜꿩나무 잎에서 분리한 화합물 1-9에대한 tyrosinase 활성저해능을 비교하기위해 음성대조군으로 EtOH, 양성대조군으로 arbutin을 사용하였다. tyrosinase 활성능력은 아래와 같은 방법으로 계산되었다. To compare the tyrosinase activity inhibition ability of compound 1-9 isolated from the Lesser Pheasant tree leaf fraction and the Lesser Pheasant tree leaf, EtOH was used as a negative control and arbutin was used as a positive control. Tyrosinase activity capacity was calculated as follows.
Tyrosinase activity(%): [(A1-A0)/(B1-B0)] ×100Tyrosinase activity (%): [(A1-A0)/(B1-B0)] ×100
A0은 반응 전 샘플의 흡광도, A1은 반응 후 샘플의 흡광도이다. B0 및 B1은 음성대조군의 반응 전후의 흡광도이다.A0 is the absorbance of the sample before reaction, and A1 is the absorbance of the sample after reaction. B0 and B1 are the absorbance before and after the reaction of the negative control group.
(3) 덜꿩나무 잎 화합물의 타이로시나제 활성 저해능력(3) Tyrosinase activity inhibition ability of Lesser Pheasant leaf compounds
덜꿩나무 잎의 EtOA 분획물 VEE1~VEE8의 타이로시나제 저해능을 확인하고자 시료농도 0.2mg/mL 로 확인하였다. 효소는 mushroom tyrosinase, 기질은 L-tyrosine을 사용하였고 음성대조군으로 EtOH을, 양성대조군으로 arbutin을 사용하였다. 음성대조군을 통해 타이로시나제 활성을 100%로 계산하였고, 각 분획물 시료를 세번 반복 처리 후 평균값을 산출하여 비교하였다. To confirm the tyrosinase inhibitory ability of EtOA fractions VEE1 to VEE8 from Lesser pheasant leaves, a sample concentration of 0.2 mg/mL was confirmed. Mushroom tyrosinase was used as an enzyme, L-tyrosine was used as a substrate, EtOH was used as a negative control, and arbutin was used as a positive control. Tyrosinase activity was calculated as 100% using the negative control group, and each fraction sample was processed three times and the average value was calculated and compared.
도 5를 참고하면, 양성대조군인 arbutin은 농도 0.2mg/mL에서 타이로시나제 활성을 21%로 감소시켜 강한 타이로시나제 저해능력을 보였다. VEE1 과 VEE2 는 타이로시나제 활성이 각각 29%, 28%로, 양성대조군인 arbutin과 비슷한 저해능력을 보여주었다. VEE3의 타이로시나제 활성은 52%로, 앞선 분획물의 저해능력에 비해 약한 효능이지만 저해능력이 있음을 확인하였다. Referring to Figure 5, arbutin, a positive control, showed strong tyrosinase inhibition ability by reducing tyrosinase activity by 21% at a concentration of 0.2 mg/mL. VEE1 and VEE2 had tyrosinase activities of 29% and 28%, respectively, showing inhibitory abilities similar to those of arbutin, a positive control. The tyrosinase activity of VEE3 was 52%, and it was confirmed that it had inhibitory ability, although its efficacy was weaker than that of the previous fraction.
VEE4 ~ VEE8 분획물의 경우에는 타이로시나제 활성이 94%~124%의 수치로 저해능력이 없거나 역효과가 있음을 확인했다. 분획물 VEE1~VEE8 에서 상대적으로 극성이 높은 fraction인 VEE1~VEE3에서 비교적 높은 타이로시나제 활성 저해능력을 확인하였다. In the case of VEE4 to VEE8 fractions, tyrosinase activity was confirmed to be 94% to 124%, indicating no inhibition ability or adverse effect. Among fractions VEE1 to VEE8, relatively high tyrosinase activity inhibition ability was confirmed in VEE1 to VEE3, which are relatively highly polar fractions.
이 중 가장 높은 저해능력을 보인 VEE2분획물에서 화합물 1-8을 얻었다. Among these, compound 1-8 was obtained from the VEE2 fraction, which showed the highest inhibitory ability.
(4) 덜꿩나무 잎에서 분리한 화합물 1-9의 타이로시나제 활성 저해능력(4) Tyrosinase activity inhibition ability of compound 1-9 isolated from Lesser Pheasant tree leaves
덜꿩나무 잎에서 분리한 화합물 1-9의 타이로시나제 저해능을 확인하고자 시료농도 200μM 로 확인하였다. 효소는 mushroom tyrosinase, 기질은 L-tyrosine을 사용하였고 음성대조군으로 EtOH을, 양성대조군으로 arbutin을 사용하였다. 음성대조군을 통해 타이로시나제 활성을 100%로 계산하였고, 각 분획물 시료를 세 번 반복 처리 후 평균값을 산출하여 비교하였다. To confirm the tyrosinase inhibitory ability of compound 1-9 isolated from Lesser Pheasant tree leaves, a sample concentration of 200 μM was confirmed. Mushroom tyrosinase was used as an enzyme, L-tyrosine was used as a substrate, EtOH was used as a negative control, and arbutin was used as a positive control. Tyrosinase activity was calculated as 100% using the negative control group, and each fraction sample was processed three times and the average value was calculated and compared.
도 6을 참고하면, 양성대조군인 arbutin은 농도 200μM 에서 타이로시나제 활성을 30%로 감소시켜 강한 타이로시나제 저해능력을 보였다. 화합물 1-6 및 화합물 9는 타이로시나제 활성이 91%~112%의 수치로 저해능력이 없음을 확인하였다. Referring to Figure 6, arbutin, a positive control, showed strong tyrosinase inhibition ability by reducing tyrosinase activity by 30% at a concentration of 200 μM. Compound 1-6 and Compound 9 were confirmed to have no inhibitory ability with tyrosinase activity ranging from 91% to 112%.
화합물 7은 타이로시나제 활성이 75%로, 매우 약한 저해능력이 있음을 확인하였다. 화합물 8의 경우에는 타이로시나제 활성이 27%의 수치로 양성대조군인 arbutin과함께 강한 저해능력을 보여주었다. 화합물 8의 tyrosinase 활성 저해능력의 IC50값은 37.9μM 이다. 화합물1-7의 경우 분자구조내에 arbutin 구조를 포함하고있지만, C-6자리에 결합된 monoterpen에 의해 저해능력이 상실되는 것을 보여주었다. Compound 7 was confirmed to have a very weak inhibitory ability with a tyrosinase activity of 75%. In the case of compound 8, tyrosinase activity was 27%, showing strong inhibitory ability along with arbutin, a positive control. The IC50 value of compound 8's ability to inhibit tyrosinase activity is 37.9μM. Compound 1-7 contained an arbutin structure in its molecular structure, but showed that its inhibitory ability was lost due to the monoterpene bound to the C-6 position.
실험3: 덜꿩나무 잎 추출물의 분획별 화합물 9 함량 비교Experiment 3: Comparison of Compound 9 content in each fraction of Lesser Pheasant tree leaf extract
(1) 분석용 시료 제조(1) Preparation of samples for analysis
덜꿩나무 잎 추출물(VET)과 n-Hexane(VEH), EtOAc(VEE), n-BuOH(VEB) 및 수용성(VEW) fraction을 각각 동결 건조한 뒤, 각각 10mg/mL 농도로 용해 시킨 후 0.45μm syringe filter로 여과하여 HPLC 분석용 시료로 제조하였다. 용매는 100% MeOH을 사용하였다. VET leaf extract (VET), n-Hexane (VEH), EtOAc (VEE), n-BuOH (VEB), and water-soluble (VEW) fractions were freeze-dried, dissolved at a concentration of 10 mg/mL, and then syringed with 0.45 μm. It was filtered using a filter and prepared as a sample for HPLC analysis. The solvent was 100% MeOH.
(2) 검량선 시료 제조(2) Preparation of calibration curve samples
화합물 9을 0.02~1mg/mL의 범위에서 5개의 농도를 선택하여 검량선을 작성하였다. 용매는 100% MeOH을 사용하였다. A calibration curve was created by selecting five concentrations of compound 9 in the range of 0.02 to 1 mg/mL. The solvent was 100% MeOH.
(3) HPLC-UV 분석 방법(3) HPLC-UV analysis method
VET, VEH, VEE, VEB, VEW 및 화합물9 에 대한 HPLC mobile phase로 0.1F.A in water(A)와 MeOH(B)을 사용하였으며, B용매 5%를 시작으로 0분에서 10분까지 5%(B), 10분에서 20분까지 20%(B), 20분에서 30분까지 70%(B), 30분에에서 40분까지70%(B)의 gradient condition으로 분석하였다. (flow rate: 1mL/min, column temperature: 30℃, wavelength: 260nm) 0.1 F.A in water (A) and MeOH (B) were used as HPLC mobile phases for VET, VEH, VEE, VEB, VEW, and compound 9, starting with 5% of solvent B, 5% from 0 to 10 minutes ( B), 20% from 10 to 20 minutes (B), 70% from 20 to 30 minutes (B), and 70% from 30 to 40 minutes (B). (flow rate: 1mL/min, column temperature: 30℃, wavelength: 260nm)
(4) 덜꿩나무 잎 추출물의 분획별 화합물 9 함량비교 (4) Comparison of compound 9 content in each fraction of Lesser Pheasant tree leaf extract
덜꿩나무 잎 추출물의 EtOAc 분획물에서 다량으로 추출된 화합물 9를 덜꿩나무 잎 total extract (VET)와 n-Hexane(VEH), EtOAc(VEE), n-BuOH(VEB) 및 수용성(VEW) fraction 에서의 함량을 비교하였다. Compound 9, which was extracted in large quantities from the EtOAc fraction of the Lesser Pheasant leaf extract, was analyzed in the Lesser Pheasant tree leaf total extract (VET), n-Hexane (VEH), EtOAc (VEE), n-BuOH (VEB), and water-soluble (VEW) fractions. The contents were compared.
도 7 및 도 8을 참고하면, 화합물 9는 MeOH : Water 70:30 의 농도구간에서 38.572min 에 용출되었으며, 검출파장은 260nm였다. 화합물 9은 VET에서 7.39mg/g, VEE 에서 31.48mg/g. VEB에서 4.61mg/g의 함량을 보였으며, VEH와 VEW에서는 검출되지 않았다. Referring to Figures 7 and 8, compound 9 was eluted in 38.572 min in the concentration range of MeOH: Water 70:30, and the detection wavelength was 260 nm. Compound 9 was 7.39 mg/g in VET and 31.48 mg/g in VEE. The content was 4.61 mg/g in VEB, and was not detected in VEH and VEW.
화합물 9는 항산화, 항노화, 항염효과가 있는 화합물로 연구되어왔으며, 화장품산업에서 주름 개선 효과를위한 대표적인 천연성분으로 알려져있다. 7.39mg/g의 높은 함량을 가진 덜꿩나무 잎 추출물이 뛰어난 피부개선 소재로 사용될 수 있을것으로 사료된다. Compound 9 has been studied as a compound with antioxidant, anti-aging, and anti-inflammatory effects, and is known as a representative natural ingredient for wrinkle improvement in the cosmetics industry. It is believed that pheasant tree leaf extract with a high content of 7.39 mg/g can be used as an excellent skin improvement material.
이상에서 본 발명의 실시예에 대하여 설명하였으나, 본 발명의 사상은 본 명세서에 제시되는 실시 예에 제한되지 아니하며, 본 발명의 사상을 이해하는 당업자는 동일한 사상의 범위 내에서, 구성요소의 부가, 변경, 삭제, 추가 등에 의해서 다른 실시 예를 용이하게 제안할 수 있을 것이나, 이 또한 본 발명의 사상범위 내에 든다고 할 것이다. Although the embodiments of the present invention have been described above, the spirit of the present invention is not limited to the embodiments presented in the present specification, and those skilled in the art who understand the spirit of the present invention will understand the spirit of the present invention within the scope of the same spirit, including addition of components, Other embodiments can be easily proposed by changes, deletions, additions, etc., but this will also be said to fall within the scope of the present invention.
Claims (14)
[화학식1]
A compound represented by the following formula (1), or a pharmaceutically acceptable salt thereof, or a hydrate thereof, or a solvate thereof:
[Formula 1]
[화학식2]
A compound represented by the following formula (2), or a pharmaceutically acceptable salt thereof, or a hydrate thereof, or a solvate thereof:
[Formula 2]
[화학식3]
A compound represented by the following formula (3), or a pharmaceutically acceptable salt thereof, or a hydrate thereof, or a solvate thereof:
[Formula 3]
[화학식4]
A compound represented by the following formula (4), or a pharmaceutically acceptable salt thereof, or a hydrate thereof, or a solvate thereof:
[Formula 4]
상기 화합물은 덜꿩나무 (Viburnum erosum Thunb.) 잎으로부터 추출된 것인 화합물.According to any one of claims 1 to 4,
The compound is a compound extracted from the leaves of Viburnum erosum Thunb.
상기 화합물은 타이로시나제 활성 저해능력을 갖는 것을 특징으로 하는 화합물.According to any one of claims 1 to 4,
The compound is characterized in that it has the ability to inhibit tyrosinase activity.
상기 타이로시나제에 의한 질환은 피부암, 피부침착, 기미, 주근깨 등에서 과다 색소 침착 질환으로 이루어진 군에서 선택되는 어느 하나인 것인 약학적 조성물.A pharmaceutical composition for preventing or treating diseases caused by tyrosinase, comprising the compound of any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof as an active ingredient,
A pharmaceutical composition wherein the disease caused by tyrosinase is any one selected from the group consisting of hyperpigmentation diseases such as skin cancer, skin deposition, freckles, and freckles.
상기 타이로시나제에 의한 질환은 피부암, 피부침착, 기미, 주근깨 등에서 과다 색소 침착 질환으로 이루어진 군에서 선택되는 어느 하나인 것인 건강기능식품 조성물.A health functional food composition for preventing or improving diseases caused by tyrosinase, comprising the compound of any one of claims 1 to 4 as an active ingredient,
A health functional food composition wherein the disease caused by tyrosinase is any one selected from the group consisting of hyperpigmentation diseases such as skin cancer, skin deposition, freckles, and freckles.
상기 피부 미용 개선은 피부 주름 개선 또는 미백제인 것인 화장료 조성물.
A cosmetic composition for improving skin beauty comprising the compound of any one of claims 1 to 4 as an active ingredient,
A cosmetic composition wherein the skin beauty improvement is a skin wrinkle improvement or whitening agent.
상기 화합물은 덜꿩나무 (Viburnum erosum Thunb.) 잎으로부터 추출된 것인 화합물.
[화학식5]
A compound represented by the following formula (5), or a pharmaceutically acceptable salt thereof, or a hydrate thereof, or a solvate thereof,
The compound is a compound extracted from the leaves of Viburnum erosum Thunb.
[Formula 5]
상기 화합물은 타이로시나제 활성 저해능력을 갖는 것을 특징으로 하는 화합물.
[화학식5]
A compound represented by the following formula (5), or a pharmaceutically acceptable salt thereof, or a hydrate thereof, or a solvate thereof,
The compound is characterized in that it has the ability to inhibit tyrosinase activity.
[Formula 5]
상기 타이로시나제에 의한 질환은 피부암, 피부침착, 기미, 주근깨 등에서 과다 색소 침착 질환으로 이루어진 군에서 선택되는 어느 하나인 것인 약학적 조성물.
[화학식5]
A pharmaceutical composition for preventing or treating diseases caused by tyrosinase, comprising a compound represented by the following formula (5), or a pharmaceutically acceptable salt thereof, as an active ingredient,
A pharmaceutical composition wherein the disease caused by tyrosinase is any one selected from the group consisting of hyperpigmentation diseases such as skin cancer, skin deposition, freckles, and freckles.
[Formula 5]
상기 타이로시나제에 의한 질환은 피부암, 피부침착, 기미, 주근깨 등에서 과다 색소 침착 질환으로 이루어진 군에서 선택되는 어느 하나인 것인 건강기능식품 조성물.
[화학식5]
A health functional food composition for preventing or improving diseases caused by tyrosinase, comprising a compound represented by the following formula 5 as an active ingredient,
A health functional food composition wherein the disease caused by tyrosinase is any one selected from the group consisting of hyperpigmentation diseases such as skin cancer, skin deposition, freckles, and freckles.
[Formula 5]
상기 피부 미용 개선은 피부 주름 개선 또는 미백제인 것인 화장료 조성물.
[화학식5]
A cosmetic composition for improving skin beauty containing a compound represented by the following formula (5) as an active ingredient,
A cosmetic composition wherein the skin beauty improvement is a skin wrinkle improvement or whitening agent.
[Formula 5]
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020230173986A KR20230170629A (en) | 2021-04-07 | 2023-12-05 | Compound extracspted from the leave of Viburnum erosum Thunb and tyrosinase activity inhibitory ability composition comprising the same |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020210045495A KR102612269B1 (en) | 2021-04-07 | 2021-04-07 | Compound extracspted from the leave of Viburnum erosum Thunb and tyrosinase activity inhibitory ability composition comprising the same |
| KR1020230173986A KR20230170629A (en) | 2021-04-07 | 2023-12-05 | Compound extracspted from the leave of Viburnum erosum Thunb and tyrosinase activity inhibitory ability composition comprising the same |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020210045495A Division KR102612269B1 (en) | 2021-04-07 | 2021-04-07 | Compound extracspted from the leave of Viburnum erosum Thunb and tyrosinase activity inhibitory ability composition comprising the same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| KR20230170629A true KR20230170629A (en) | 2023-12-19 |
Family
ID=83809984
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020210045495A KR102612269B1 (en) | 2021-04-07 | 2021-04-07 | Compound extracspted from the leave of Viburnum erosum Thunb and tyrosinase activity inhibitory ability composition comprising the same |
| KR1020230173986A KR20230170629A (en) | 2021-04-07 | 2023-12-05 | Compound extracspted from the leave of Viburnum erosum Thunb and tyrosinase activity inhibitory ability composition comprising the same |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020210045495A KR102612269B1 (en) | 2021-04-07 | 2021-04-07 | Compound extracspted from the leave of Viburnum erosum Thunb and tyrosinase activity inhibitory ability composition comprising the same |
Country Status (1)
| Country | Link |
|---|---|
| KR (2) | KR102612269B1 (en) |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102176710B1 (en) * | 2017-12-27 | 2020-11-10 | 경희대학교 산학협력단 | A cosmetic composition comprising fractions of extracts of flower of Forsythia koreana as an active ingredient |
-
2021
- 2021-04-07 KR KR1020210045495A patent/KR102612269B1/en active IP Right Grant
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2023
- 2023-12-05 KR KR1020230173986A patent/KR20230170629A/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| KR20220139512A (en) | 2022-10-17 |
| KR102612269B1 (en) | 2023-12-12 |
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