KR101283810B1 - A method of isolating quercetin-3-O-α-rhamnoside and kaempferol-3-O-α-rhamnoside from Lindera obtusiloba Blume and A use of the compound as an antioxidant or liver protection - Google Patents
A method of isolating quercetin-3-O-α-rhamnoside and kaempferol-3-O-α-rhamnoside from Lindera obtusiloba Blume and A use of the compound as an antioxidant or liver protection Download PDFInfo
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- KR101283810B1 KR101283810B1 KR1020110034142A KR20110034142A KR101283810B1 KR 101283810 B1 KR101283810 B1 KR 101283810B1 KR 1020110034142 A KR1020110034142 A KR 1020110034142A KR 20110034142 A KR20110034142 A KR 20110034142A KR 101283810 B1 KR101283810 B1 KR 101283810B1
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- South Korea
- Prior art keywords
- loe
- antioxidant
- rhamnoside
- hepatoprotective
- quercetin
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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
-
- 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
-
- 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
Abstract
본 발명은 생강나무로부터 쿼세틴-3-O-알파-람노사이드 및 캠페롤-3-O-알파-람노사이드의 분리 방법 및 그것의 항산화 및 간 보호 용도에 관한 것이다.The present invention relates to a process for the separation of quercetin-3-O-alpha-rhamnoside and camphorol-3-O-alpha-rhamnoside from ginger and its antioxidant and hepatoprotective use.
Description
본 발명은 생강나무로부터 쿼세틴-3-O-알파-람노사이드 및 캠페롤-3-O-알파-람노사이드의 분리 방법 및 그것의 항산화 및 간 보호 용도에 관한 것이다.The present invention relates to a process for the separation of quercetin-3-O-alpha-rhamnoside and camphorol-3-O-alpha-rhamnoside from ginger and its antioxidant and hepatoprotective use.
일반적으로 산화스트레스(oxidative stress)란 살아 있는 세포에서 항산화 방어기전 (antioxidant defense mechanism)에 대하여 산화 기전이 우세하여 세포 단백, 지질, 핵산의 산화 손상을 일으키는 상태라고 정의할 수 있다. 산화스트레스는 활성.산소 (reactive oxygen species)에 의해 발생되며, 산소이온, 유리 라디칼, 과산화물 등을 말한다. 활성산소는 세포전달체계에서 생성되어 축적되며 이온화방사능 등에 의해서도 생성된다. 인체에서는 산화스트레스가 흡연, 고혈압, 죽상경화증, 고지혈증, 당뇨병, 허혈-재관류, 암, 류마치스성 관절염, 베체트 질환, 신경손상성 질환 등에서 중요한 발병기전으로 추정되고 있을 뿐만 아니라 노.화 과정서도 주요한 인자로 보고 있다(김정호, 산화스트레스의 진단, 2008, 13~15, 대한임상노인학회 춘계학술대회).In general, oxidative stress can be defined as a condition in which oxidative mechanisms prevail over the antioxidant defense mechanism in living cells, causing oxidative damage of cellular proteins, lipids, and nucleic acids. Oxidative stress is caused by reactive oxygen species and refers to oxygen ions, free radicals, and peroxides. Free radicals are produced and accumulated in cell delivery systems, and are also produced by ionizing radioactivity. In humans, oxidative stress is considered to be an important pathogenesis in smoking, hypertension, atherosclerosis, hyperlipidemia, diabetes mellitus, ischemia-reperfusion, cancer, rheumatoid arthritis, Behcet's disease, neuroinjury disease, etc. (Jung Ho Kim, Diagnosis of Oxidative Stress, 2008, 13 ~ 15, Korean Society of Clinical Geriatrics Spring Conference).
활성산소에 대한 항산화물질의 작용기전을 보면 크게 다음의 3가지로 나뉠 수 있다. 즉 예방적인 측면, 활성산소를 제거하는 측면 그리고 조직회복 및 신생에 관여하는 기능으로 분류할 수 있다. 이 중 식물유래 항산화 물질의 작용기전은 주로 활성산소를 제거하는 항산화 물질로서의 기능을 담당한다. 즉 반응성이 크고 유해한 활성산소와 같은 유리기와 먼저 반응하여 자기 자신은 안정성이 있는 유리기로 되어 다른 중요한 화합물이 유리기가 되는 연쇄반응을 막아준다. 생체 내에서는 예방적 항산화물질의 예를 들면 SOD와 같은 효소작용에 의해 활성산소와의 반응은 어느 정도 억제되지만, 여전히 생체 내에서는 소량의 활성산소가 생성되며 외부에서도 활성산소(대기오염, 담배연기 등)가 체내로 들어오기도 한다. 식물에서의 항산화 기능을 갖는 물질의 분류는 비타민 E, 카로티노이드(carotenoids), 비타민 C 등이 있다.The mechanism of action of antioxidants on active oxygen can be largely divided into the following three. In other words, it can be classified into preventive aspects, removal of free radicals, and functions related to tissue recovery and angiogenesis. Of these, the mechanism of action of plant-derived antioxidants is primarily responsible for the function of antioxidants to remove free radicals. In other words, by reacting free radicals such as reactive and harmful free radicals first, they become stable free radicals to prevent chain reactions in which other important compounds are free radicals. In vivo, the reaction with prophylactic antioxidants such as SOD inhibits the reaction with free radicals to some extent, but a small amount of free radicals are still produced in vivo and free radicals (air pollution, tobacco smoke) Etc.) may enter the body. The class of substances that have antioxidant function in plants is vitamin E, carotenoids, vitamin C and the like.
이런 항산화와 관련하여 현재까지 생강나무의 항산화 기능은 아직 밝혀져 있지 않으며, 특히 쿼세틴-3-O-α-람노사이드 및 캠페롤-3-O-α-람노사이드의 항산화 기능에 대해서도 전무한 상황이다. The antioxidant function of ginger tree is not yet known with respect to such antioxidants, and there is no particular antioxidant activity of quercetin-3-O-α-rhamnoside and camphorol-3-O-α-rhamnoside.
본 발명은 상기의 필요성에 의하여 안출된 것으로서 발명의 목적은 천연식물인 생강나무(Lindera obtusiloba Blume)로부터 쿼세틴-3-O-α-람노사이드 및 캠페롤-3-O-α-람노사이드의 추출·분리·정제 방법을 제공하는 것이다.The present invention has been made by the necessity of the above, an object of the present invention is the extraction of quercetin-3-O-α-rhamnoside and camphorol-3-O-α-rhamnoside from natural plant Ginger (Lindera obtusiloba Blume) It is to provide a separation and purification method.
본 발명의 또 다른 목적은 생강나무(Lindera obtusiloba Blume)로부터 분리된 쿼세틴-3-O-α-람노사이드 및 캠페롤-3-O-α-람노사이드의 항산화 효과를 제공하는 것이다.Still another object of the present invention is to provide an antioxidant effect of quercetin-3-O-α-rhamnoside and camphorol-3-O-α-rhamnoside isolated from ginger tree (Lindera obtusiloba Blume).
본 발명의 또 다른 목적은 생강나무(Lindera obtusiloba Blume)로부터 분리된 쿼세틴-3-O-α-람노사이드 및 캠페롤-3-O-α-람노사이드의 간 보호 효과를 제공하는 것이다.Still another object of the present invention is to provide a hepatoprotective effect of quercetin-3-O-α-rhamnoside and camphorol-3-O-α-rhamnoside isolated from the ginger tree (Lindera obtusiloba Blume).
상기의 목적을 달성하기 위하여, 본 발명은 a) 생강나무 (Lindera obtusiloba Blume)로부터 물 또는 알콜을 이용하여 조추출물을 제조하는 단계; b) 상기 조 추출물을 헥산을 처리하여 수층을 얻는 단계; 및 c)상기 얻어진 수층에 에틸아세테이트를 처리하여 에틸아세테이트 층을 얻는 단계를 포함하는 퀘세틴-3-O-α-람노사이드 또는 캠페롤-3-O-α-람노사이드 추출ㆍ분리방법을 제공한다.In order to achieve the above object, the present invention is a) ginger tree ( Lindera obtusiloba Blume) to prepare a crude extract using water or alcohol; b) treating the crude extract with hexane to obtain an aqueous layer; And c) treating the obtained aqueous layer with ethyl acetate to obtain an ethylacetate layer. Quercetin-3- O- α-rhamnoside or camphorol-3- O- α-rhamnoside extraction and separation method are provided. do.
본 발명의 일 구체예에 있어서, 상기 방법은 c)단계 후 크로마토그래피 정제과정을 더욱 포함하는 것이 바람직하나 이에 한정되지 아니한다.In one embodiment of the invention, the method preferably further comprises a chromatographic purification step after step c), but is not limited thereto.
또한 본 발명의 일 구체예에 있어서, 상기 알콜은 메탄올, 에탄올, 프로판올 및 부탄올로 구성된 군으로부터 선택된 알콜이 바람직하고, 메탄올 또는 에탄올인 것이 더욱 바람직하나 이에 한정되지 아니한다.In addition, in one embodiment of the present invention, the alcohol is preferably an alcohol selected from the group consisting of methanol, ethanol, propanol and butanol, more preferably methanol or ethanol, but is not limited thereto.
또한 본 발명은 퀘세틴-3-O-α-람노사이드를 유효성분으로 함유하는 항산화 조성물을 제공한다.The present invention also provides an antioxidant composition containing quercetin-3- O- α-rhamnoside as an active ingredient.
또한 본 발명은 캠페롤-3-O-α-람노사이드를 유효성분으로 함유하는 항산화 조성물을 제공한다.The present invention also provides an antioxidant composition containing camphorol-3- O- α-rhamnoside as an active ingredient.
또 본 발명은 퀘세틴-3-O-α-람노사이드를 유효성분으로 함유하는 간 보호 조성물을 제공한다.The present invention also provides a hepatoprotective composition containing quercetin-3- O- α-rhamnoside as an active ingredient.
또한 본 발명은 캠페롤-3-O-α-람노사이드를 유효성분으로 함유하는 간 보호 조성물을 제공한다.The present invention also provides a hepatoprotective composition containing camphorol-3- O- α-rhamnoside as an active ingredient.
본 발명은 또한 항산화 효과로서의 피로, 만성질병이나 성인성질환 같이 산화스트레스로 유발 될 수 있는 고혈압, 죽상경화증, 고지혈증, 당뇨병, 허혈-재관류, 암, 류마치스성 관절염, Behcet 질환, 신경손상성 질환, 폐암 등의 예방 개선효과를 가진다.The present invention also provides anti-oxidant effects such as fatigue, chronic disease or adult disease such as hypertension, atherosclerosis, hyperlipidemia, diabetes, ischemia-reperfusion, cancer, rheumatoid arthritis, Behcet disease, neuroinjury disease, It has the effect of preventing and preventing lung cancer.
본 발명의 화합물을 의약으로서 사용하는 경우, 통상, 본 발명의 화합물과 적당한 첨가제를 혼화하여 제제화한 것을 사용한다. When using the compound of this invention as a medicine, the thing which mixed and formulated the compound of this invention and a suitable additive is used normally.
상기 첨가제로서는 일반적으로 의약에 사용되는 부형제, 결합제, 활택제, 붕괴제, 착색제, 교미 교취제, 유화제, 계면 활성제, 용해 보조제, 현탁화제, 등장화제, 완충제, 방부제, 항산화제, 안정화제, 흡수 촉진제 등을 들 수 있고, 소망에 따라 이들을 적절하게 조합하여 사용할 수도 있다.As the additives, excipients, binders, lubricants, disintegrants, coloring agents, copulation agents, emulsifiers, surfactants, dissolution aids, suspending agents, isotonic agents, buffers, preservatives, antioxidants, stabilizers, absorption accelerators, which are generally used in medicine These etc. can be mentioned, You can use combining these suitably as needed.
상기 부형제로서는 예컨대 젖당, 백당, 포도당, 콘스타치, 만니톨, 소르비톨, 전분, α화 전분, 덱스트린, 결정 셀룰로오스, 경질 무수 규산, 규산알루미늄, 규산칼슘, 메타규산알루민산마그네슘, 인산수소칼슘 등을 들 수 있고,Examples of the excipient include lactose, white sugar, glucose, corn starch, mannitol, sorbitol, starch, α-starch, dextrin, crystalline cellulose, hard anhydrous silicic acid, aluminum silicate, calcium silicate, magnesium aluminate silicate and calcium hydrogen phosphate. There is,
상기 결합제로서는 예컨대 폴리비닐알코올, 메틸셀룰로오스, 에틸셀룰로오스, 아라비아 고무, 트래거캔스, 젤라틴, 셸락, 히드록시프로필메틸셀룰로오스, 히드록시프로필셀룰로오스, 카르복시메틸셀룰로오스나트륨, 폴리비닐피롤리돈, 매크로골 등을 들 수 있으며,Examples of the binder include polyvinyl alcohol, methyl cellulose, ethyl cellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl methyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose sodium, polyvinylpyrrolidone, macrogol and the like. Can be
상기 활택제로서는 예컨대 스테아르산마그네슘, 스테아르산칼슘, 푸마르산스테아릴나트륨, 탈크, 폴리에틸렌글리콜, 콜로이드실리카 등을 들 수 있고,Examples of the lubricant include magnesium stearate, calcium stearate, sodium stearyl fumarate, talc, polyethylene glycol, colloidal silica, and the like.
상기 붕괴제로서는 예컨대 결정 셀룰로오스, 한천, 젤라틴, 탄산칼슘, 탄산수소나트륨, 시트르산칼슘, 덱스트린, 펙틴, 저치환도 히드록시프로필셀룰로오스, 카르복시메틸셀룰로오스, 카르복시메틸셀룰로오스칼슘, 크로스카멜로오스나트륨, 카르복시메틸스타치, 카르복시메틸스타치나트륨 등을 들 수 있다.Examples of the disintegrating agent include crystalline cellulose, agar, gelatin, calcium carbonate, sodium bicarbonate, calcium citrate, dextrin, pectin, low-substituted hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium, croscarmellose sodium, carboxymethyl Starch, carboxymethyl starch sodium, and the like.
상기 착색제로서는 예컨대 삼이산화철, 황색 삼이산화철, 카르민, 카라멜, β-카로틴, 산화티탄, 탈크, 인산리보플라빈나트륨, 황색 알루미늄레이크 등, 의약품에 첨가하는 것이 허가되어 있는 것을 들 수 있고,Examples of the colorant include those which are permitted to be added to medicines such as iron trioxide, yellow iron trioxide, carmine, caramel, β-carotene, titanium oxide, talc, sodium riboflavin sodium and yellow aluminum lake.
상기 교미 교취제로서는 코코아 분말, 박하뇌, 방향산, 박하유, 용뇌, 계피분말 등을 들 수 있으며,Cocoa powder, peppermint brain, aromatic acid, peppermint oil, cedar, cinnamon powder, etc. are mentioned as said copulation agent,
상기 유화제 또는 계면 활성제로서는 예컨대 스테아릴트리에탄올아민, 라우릴황산나트륨, 라우릴아미노프로피온산, 레시틴, 모노스테아르산글리세린, 자당 지방산 에스테르, 글리세린 지방산 에스테르 등을 들 수 있고,Examples of the emulsifiers or surfactants include stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, glycerin monostearate, sucrose fatty acid esters, glycerin fatty acid esters, and the like.
상기 용해 보조제로서는 예컨대 폴리에틸렌글리콜, 프로필렌글리콜, 벤조산벤질, 에탄올, 콜레스테롤, 트리에탄올아민, 탄산나트륨, 시트르산나트륨, 폴리솔베이트 80, 니코틴산아미드 등을 들 수 있으며,Examples of the dissolution aid include polyethylene glycol, propylene glycol, benzyl benzoate, ethanol, cholesterol, triethanolamine, sodium carbonate, sodium citrate,
상기 현탁화제로서는 상기 계면 활성제 이외에 예컨대 폴리비닐알코올, 폴리비닐피롤리돈, 메틸셀룰로오스, 히드록시메틸셀룰로오스, 히드록시에틸셀룰로오스, 히드록시프로필셀룰로오스 등의 친수성 고분자를들 수 있고,Examples of the suspending agent include hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, and hydroxypropyl cellulose, in addition to the surfactant.
상기 등장화제로서는 예컨대 포도당, 염화나트륨, 만니톨, 소르비톨 등을 들 수 있으며,Examples of the tonicity agent include glucose, sodium chloride, mannitol, sorbitol, and the like.
상기 완충제로서는 예컨대 인산염, 아세트산염, 탄산염, 시트르산염 등의 완충액을 들 수 있고,
Examples of the buffer include buffers such as phosphate, acetate, carbonate and citrate,
*상기 방부제로서는 예컨대 메틸파라벤, 프로필파라벤, 클로로부탄올, 벤질알코올, 페네틸알코올, 디히드로아세트산, 소르브산 등을 들 수 있으며,Examples of the preservative include methyl paraben, propyl paraben, chlorobutanol, benzyl alcohol, phenethyl alcohol, dihydroacetic acid, sorbic acid, and the like.
상기 항산화제로서는 예컨대 아황산염, 아스코르빈산, α-토코페롤 등을 들 수 있다.Examples of the antioxidant include sulfite, ascorbic acid and α-tocopherol.
상기 안정화제로서는 일반적으로 의약에 사용되는 것을 들 수 있다.As said stabilizer, what is generally used for medicine is mentioned.
상기 흡수 촉진제로서는 일반적으로 의약에 사용되는 것을 들 수 있다.As said absorption promoter, what is generally used for medicine is mentioned.
또한, 상기 제제로서는 예컨대 정제, 산제, 과립제, 캡슐제, 시럽제, 트로치제, 흡입제와 같은 경구제; 예컨대 좌제, 연고제, 안연고제, 테이프제, 점안제, 점비제, 점이제, 파프제, 로션제와 같은 외용제 또는 주사제를 들 수 있다.In addition, the preparations include, for example, oral preparations such as tablets, powders, granules, capsules, syrups, troches, inhalants; Examples include suppositories, ointments, eye ointments, tapes, eye drops, eye drops, ear drops, pape, lotions such as lotions, or injections.
상기 경구제는 상기 첨가제를 적절하게 조합하여 제제화한다. 또한, 필요에 따라 이들 표면을 코팅하여도 좋다.The oral formulation is formulated by combining the additives as appropriate. Moreover, you may coat these surfaces as needed.
상기 외용제는 상기 첨가제 중 특히 부형제, 결합제, 교미 교취제, 유화제, 계면 활성제, 용해 보조제, 현탁화제, 등장화제, 방부제, 항산화제, 안정화제 또는 흡수 촉진제를 적절하게 조합하여 제제화한다.The external preparations are formulated by appropriate combination of excipients, binders, copulation agents, emulsifiers, surfactants, dissolution aids, suspending agents, isotonic agents, preservatives, antioxidants, stabilizers or absorption accelerators, among others.
상기 주사제는 상기 첨가제 중 특히 유화제, 계면 활성제, 용해 보조제, 현탁화제, 등장화제, 완충제, 방부제, 항산화제, 안정화제 또는 흡수 촉진제를 적절하게 조합하여 제제화한다.The injectables are formulated in appropriate combination of the above additives, in particular emulsifiers, surfactants, dissolution aids, suspending agents, isotonic agents, buffers, preservatives, antioxidants, stabilizers or absorption promoters.
본 발명에 따른 화합물을 의약으로서 사용하는 경우, 그 사용량은 증상이나 연령에 따라 다르지만, 통상, 경구제의 경우에는. 0.1 ㎎ 내지 10 g(바람직하게는 1 ㎎ 내지 2 g), 외용제의 경우에는 0.01 ㎎ 내지 10 g(바람직하게는 0.1 ㎎ 내지 2 g), 주사제의 경우에는, 0.01 ㎎ 내지 10 g(바람직하게는 0.1 ㎎ 내지 2 g)를 1일 1회 투여 또는 2 내지 4회로 나누어 사용한다.When the compound according to the present invention is used as a medicament, its amount varies depending on symptoms and age, but usually in the case of oral preparations. 0.1 mg to 10 g (preferably 1 mg to 2 g), 0.01 mg to 10 g (preferably 0.1 mg to 2 g) for external preparations, 0.01 mg to 10 g (preferably for injections) 0.1 mg to 2 g) is used once daily or divided into 2 to 4 times.
본 발명은 간질환의 예방 및 개선효과를 갖는 상기한 본 발명의 화합물을 유효성분으로 함유하는 간질환의 예방 및 개선용 건강기능식품을 제공한다. 본 발명의 화합물을 첨가할 수 있는 식품으로는, 예를 들어, 각종 식품류, 음료, 껌, 차, 비타민 복합제, 건강 기능성 식품류 등이 있다.The present invention provides a health functional food for the prevention and improvement of liver disease containing the compound of the present invention as an active ingredient having an effect of preventing and improving liver disease. Examples of the food to which the compound of the present invention can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.
또한, 간질환의 예방 효과를 목적으로 식품 또는 음료에 첨가될 수 있다. 이 때, 식품 또는 음료 중의 상기 추출물의 양은 전체 식품 중량의 0.01 내지 15 중량%로 가할 수 있으며, 건강 음료 조성물은 100 ㎖를 기준으로 0.02 내지 5 g, 바람직하게는 0.3 내지 1g의 비율로 가할 수 있다. It may also be added to foods or beverages for the purpose of preventing the liver disease. At this time, the amount of the extract in the food or beverage may be added in 0.01 to 15% by weight of the total food weight, the health beverage composition may be added in a ratio of 0.02 to 5 g, preferably 0.3 to 1g based on 100 ml. have.
본 발명의 건강기능식품은 정제, 캡슐제, 환제, 액제등의 형태를 포함한다.The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids.
본 발명의 건강 기능성 음료 조성물은 지시된 비율로 필수 성분으로서 상기 화합물을 함유하는 외에는 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 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㎖당 일반적으로 약 1 내지 20g, 바람직하게는 약 5 내지 12g이다.The health functional beverage composition of the present invention is not particularly limited to the other ingredients other than the above-mentioned compounds as essential ingredients in the indicated ratios and may contain various flavors or natural carbohydrates as additional ingredients such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio 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 화합물은 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, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 본 발명의 화합물들은 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 화합물 100 중량부 당 0 내지 약 20 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above, the compounds of the present invention include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the compounds of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is usually selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the compound of the present invention.
이하 본 발명을 설명한다.Hereinafter, the present invention will be described.
본 발명은 천연식물인 생강나무(Lindera obtusiloba Blume)로부터 항산화 및 간 보호 기능이 있는 추출분리정제 분획물, 쿼세틴-3-O-α-람노사이드(쿼시트린) 및 캠페롤-3-O-α-람노사이드(아프젤린 유도체)의 추출분리정제방법과 추출분리정제 분획물, 쿼세틴-3-O-α-람노사이드(쿼시트린) 및 캠페롤-3-O-α-람노사이드(아프젤린 유도체)의 항산화 기능과 간보호 기능이 있음을 증거로 제공하여 항산화 기능 및 간 보호 기능 추출분리정제 분획물과 단일물질로 등재하고자 함. 또한 식품 소재, 건강기능식품 및 의약품의 소재로 활용함에 있다.The present invention is a natural plant ginger tree ( Lindera Extraction and Extraction of Quercetin-3- O- α-Rhamnoside (Quitcirine) and Camperol-3- O- α-Rhamnoside (Azegline Derivative) from Antitusks with Obtusiloba Blume Evidence of Antioxidant and Hepatoprotective Functions of Purification and Extraction and Purification Fractions, Quercetin-3- O- α-Rhamnoside (Quitcitrin) and Camperol-3- O- α-Rhamnoside (Apzeline Derivative) Antioxidant and hepatoprotective functions Extracted and purified as fractions and listed as a single substance. It is also used as a food material, health functional food and medicine.
본 발명에 사용된 천연식물인 생강나무(Lindera obtusiloba Blume) 알콜 추출물(LOE)이 항산화 효과가 있는지를 실험예 1 등을 통하여 실험한 결과 1000 μg/mL의 농도에서 94.9 ± 9.8 %, 500 μg/mL에서는 63.8 ± 2.6 %, 100 μg/mL에서 38.4 ± 0.8 %의 높은 항산화 활성 및 간 보호 활성을 보였다.Ginger tree ( lindera) is a natural plant used in the present invention Experimental results of obtusiloba Blume) alcohol extract (LOE) was tested by Experimental Example 1, etc., at 94.9 ± 9.8% at 1000 μg / mL, 63.8 ± 2.6% at 500 μg / mL, and 100 μg / mL It showed high antioxidant activity and hepatoprotective activity of 38.4 ± 0.8%.
이렇게 높은 항산화 활성 및 간 보호 활성이 확인된 생강나무(Lindera obtusiloba Blume)의 항산화 및 간 보호 기능물질을 포함하는 분리ㆍ정제 분획물 및 쿼세틴-3-O-α-람노사이드(쿼시트린), 캠페롤-3-O-α-람노사이드(아프젤린 유도체)는 실시예 1과 같은 방법으로 추출ㆍ분리ㆍ정제하여 얻었다. 생강나무(Lindera obtusiloba Blume) 70% 에탄올 추출물(LOE)로부터 분액깔대기를 이용한 극성별 유기용매 분획결과 높은 항산화 및 간 보호 활성을 보인 LOE-E (도 2 참고), 1000 μg/mL의 농도에서 141.0 ± 7.1 %, 500 μg/mL에서는 99.2 ± 6.3 %, 100 μg/mL에서 66.5 ± 5.0 %) 획분은 vacuum liquid chromatography를 이용하여 정제를 계속하였다. 이동상의 조건은 0% MeOH(100% D.W.) → 100% MeOH로, 단계별로 MeOH 농도 (25% 씩)를 증가시키면서 column 용량의 3배 이상의 이동상을 용출 시켰다(도 1). 각 단계 마다의 분리를 정확하게 하기 위하여 용출되어 나온 물질의 흡광도가 254nm에서 0.1 이하가 될 때까지 용출하였다. 용출되어 나온 각 획분을 농축 및 동결건조 후 항산화 활성을 측정한 결과 <50% MeOH + 50% D.W.>에서 용출된 LOE-E-2 획분의 활성이 500 μg/mL의 농도에서 97.3 ± 2.5 %, 100 μg/mL에서는 86.8 ± 2.4 %의 높은 항산화 활성을 보였다(도 3 참고). 이 획분을 Prep HPLC를 이용하여 이동상의 조건을 23 % acetonotrile(77% D.W)의 조건으로 LOE-E-2-Ⅰ와 LOE-E-2-Ⅱ의 2가지 획분을 얻었다(도 1 참고). 이 2 가지 획분을 HPLC로 단일물질임을 확인(도 4)하였다. 그리고 1D NMR의 1H과 13C NMR의 spectrum 분석을 통하여 최종적으로 쿼세틴-3-O-α-람노사이드(쿼시트린) 및 캠페롤-3-O-α-람노사이드(아프젤린 유도체)으로 동정(도 5~9) 하였다.Ginger tree ( Lindera) with high antioxidant activity and liver protection activity isolated and purified fractions containing antioxidant and hepatoprotective substances of obtusiloba Blume) and quercetin-3- O- α-rhamnoside (quacitrine), camphorol-3- O- α-rhamnoside (afzelin derivative) Obtained by extraction, separation and purification in the same manner as in Example 1. Ginger Tree ( Lindera obtusiloba Blume) LOE-E showing high antioxidant and hepatoprotective activity as a result of organic solvent fractionation by polarization funnel from 70% ethanol extract (LOE), 141.0 ± 7.1%, at a concentration of 1000 μg / mL, 99.2 ± 6.3% at 500 μg / mL and 66.5 ± 5.0% at 100 μg / mL) fractions were purified using vacuum liquid chromatography. Mobile phase conditions were from 0% MeOH (100% DW) to 100% MeOH, eluting the mobile phase more than three times the column capacity while increasing the MeOH concentration (25% increments) step by step (FIG. 1). In order to accurately separate each step, the eluted material was eluted until the absorbance of the eluted material became 0.1 or less at 254 nm. After concentration and freeze-drying of each eluted fraction, antioxidant activity was measured and the activity of LOE-E-2 fraction eluted at <50% MeOH + 50% DW> was 97.3 ± 2.5% at a concentration of 500 μg / mL, At 100 μg / mL showed a high antioxidant activity of 86.8 ± 2.4% (see Figure 3). This fraction was obtained using Prep HPLC to obtain two fractions of LOE-E-2-I and LOE-E-2-II under mobile phase conditions of 23% acetonotrile (77% DW) (see FIG. 1). These two fractions were confirmed to be homogeneous by HPLC (FIG. 4). And finally, through the spectrum analysis of 1 H and 13 C NMR of 1D NMR, finally identified as quercetin-3- O- α-lamnoside (quacitrine) and camphorol-3- O- α-rhamnoside (afzelin derivative) (FIGS. 5-9).
화합물 LOE-E-2-Ⅰ은 황색 분말로 FeCl3 시액과 Mg-HCl 반응에 양성이며, MeOH 용액의 UV spectrum에서 351, 257 nm의 흡수대가 나타나 flavonol 유도체로 추정할 수 있었다. 1H-NMR spectrum에서는 H-6'의 signal이 δH 7.41 (1H, dd, J = 8.5, 2.0 Hz)에서 관찰되었고, δH 7.01 (1H, d, J = 8.5 Hz)은 H-6'과 ortho coupling을 이루고 있는 H-5'로 확인하였다. 또한 H-2'는 δH 7.52에서 J = 2.0 Hz인 doublet으로 추정되어 H-6′와 meta coupling하고 있음을 알 수 있었고, δH 6.48과 6.28에서 flavonoid A환의 H-6 및 H-8의 전형적인 meta coupling이 관찰되었으며, 이상의 분광학적 데이터로 aglycone은 quercetin으로 추정할 수 있었다. 또한, δH 5.54에서 anomeric proton과 δH 0.93 (3H, d, J = 6.0 Hz)의 signal은 rhamnose에 기인하는 signal로 확인하였다. 13C-NMR spectrum에서는 21 개의 signal을 확인하였으며, ESI-MS spectrum에서는 m/z 447 [M - H]-에서 molecular ion peak를 관찰할 수 있었다. 이상의 기기분석 결과 및 문헌과의 비교로 화합물 LOE-E-2-Ⅰ은 quercetin C-3의 hydroxy에 rhamnose가 결합되어 있는 quercetin-3-O-α-L-rhamnopyranoside (quercitrin)로 동정하였다.Compound LOE-E-2-I was a yellow powder, positive for FeCl 3 solution and Mg-HCl reaction, and absorbed bands of 351 and 257 nm in the UV spectrum of MeOH solution. In the 1 H-NMR spectrum, H-6 'signal was observed at δ H 7.41 (1H, dd, J = 8.5, 2.0 Hz), and δ H 7.01 (1H, d, J = 8.5 Hz) was H-6'. H-5 'with ortho coupling. In addition, H-2 'was estimated to be a double coupling with J = 2.0 Hz at δ H 7.52, indicating that it is meta- coupling with H-6', and H-6 and H-8 of flavonoid A rings at δ H 6.48 and 6.28. Typical meta coupling was observed, and the spectroscopic data indicated that aglycone could be estimated as quercetin. In addition, the signals of anomeric protons and δ H 0.93 (3H, d, J = 6.0 Hz) at δ H 5.54 were identified as signals due to rhamnose. In the 13 C-NMR spectrum, 21 signals were identified, and in the ESI-MS spectrum, molecular ion peaks were observed at m / z 447 [M-H] - . Compound LOE-E-2-I was identified as quercetin-3- O- α-L-rhamnopyranoside (quercitrin) in which rhamnose is bound to hydroxy of quercetin C-3.
화합물 LOE-E-2-Ⅱ는 등황색의 분말상 결정으로 FeCl3 test에서 오록색, Mg-HCl 반응에서 홍색을 각각 나타냈으며, ESI-MS spectrum에서는 m/z 431 [M - H]-에서 molecular ion peak를 관찰할 수 있었다. UV spectrum에서는 342 및 264 nm에서 흡수극대를 나타내어 flavonol 유도체로 추정할 수 있었다. 1H-NMR 스펙트럼 중 aromatic field의 δH 6.49와 6.28에서 flavonoid A환의 H-6 및 H-8의 전형적인 meta coupling이 관찰되었으며, δH 7.87 (1H, d, J = 8.0 Hz)과 7.03 (1H, d, J = 8.0 Hz)에서 B환의 H-2', H-3', H-5', H-6'의 AA'BB' type이 관찰되어, aglycone은 kaempferol로 추정할 수 있었다. 또한, δH 5.57 (1H, brs)와 0.91 (3H, d, J = 5.5 Hz) 그리고 δC 101.9, 72.1, 71.2, 70.6, 70.5, 16.9에서 rhamnose에 기인하는 signal을 확인하였다. 이상의 결과로 화합물 LOE-E-2-Ⅱ는 afzelin (kaempferol-3-O-α-L-rhamnopyranoside)으로 추정하였으며, 보고된 문헌과의 비교로 일치함을 확인하였다.Compound LOE-E-2-Ⅱ is naeteumyeo respectively represent the red color from O Testing items, Mg-HCl reaction in the FeCl 3 test as powdery crystal of deunghwangsaek, the ESI-MS spectrum m / z 431 [M - H] - from molecular ion Peaks could be observed. UV spectra showed absorption peaks at 342 and 264 nm, suggesting flavonol derivatives. In the 1 H-NMR spectrum, typical meta coupling of H-6 and H-8 of the flavonoid A ring was observed at δ H 6.49 and 6.28 in the aromatic field, and δ H 7.87 (1H, d, J = 8.0 Hz) and 7.03 (1H). , d, J = 8.0 Hz), the AA'BB 'type of H-2', H-3 ', H-5', and H-6 'of the B ring was observed, and aglycone was assumed to be kaempferol. In addition, signals due to rhamnose were confirmed at δ H 5.57 (1H, brs) and 0.91 (3H, d, J = 5.5 Hz) and at δ C 101.9, 72.1, 71.2, 70.6, 70.5, and 16.9. As a result, the compound LOE-E-2-II was estimated to be afzelin (kaempferol-3- O- α-L-rhamnopyranoside), and confirmed by the comparison with the reported literature.
생강나무(Lindera obtusiloba Blume)로 부터 얻은 LOE-E-2-Ⅰ와 LOE-E-2-Ⅱ 획분의 항산화 및 간 보호 기능 물질인 쿼세틴-3-O-α-람노사이드(쿼시트린) 및 캠페롤-3-O-α-람노사이드(아프젤린 유도체)의 항산화 측정 결과(표 1) 두 물질 모두 높은 항산화 활성을 보였으며, 그 중 쿼세틴-3-O-α-람노사이드(쿼시트린)가 캠페롤-3-O-α-람노사이드(아프젤린 유도체) 보다 2배 정도 더 높은 항산화 기능을 나타내었다.Ginger Tree ( Lindera Quercetin -3- O- α-Ramnoside (Quetcitrin) and Camperol-3-, Antioxidant and Hepatoprotective Compounds of LOE-E-2-I and LOE-E-2-Ⅱ Fractions from obtusiloba Blume) Antioxidant measurement of O- α-rhamnoside (afzelin derivative) (Table 1) Both substances showed high antioxidant activity, of which quercetin-3- O- α-rhamnoside (quatcirine) was camphorol-3 It exhibited about 2 times higher antioxidant activity than O- α-rhamnoside (afzelin derivative).
표 1은 생강나무(Lindera obtusiloba Blume)에서 높은 항산화 및 간 보호 기능성을 따라 추출ㆍ분리ㆍ정제한 단일물질인 쿼시트린(quercitrin)과 아프젤린(afzelin) 유도체의 항산화 효과 측정 결과로써 DPPH radical 소거능과 FRAP을 측정한 결과이다. 두 결과에서 쿼시트린(quercitrin)이 아프젤린(afzelin)유도체 보다 2배 정도 더 높은 항산화 활성을 가지고 있다. Table 1 shows ginger tree ( Lindera The results of DPPH radical scavenging activity and FRAP were measured by the antioxidant activity of quercitrin and afzelin derivatives, which were extracted, isolated and purified according to high antioxidant and hepatoprotective properties in obtusiloba Blume. In both results, quercitrin has twice as much antioxidant activity as afzelin derivatives.
표 2는 LOE-E-2 획분을 Prep HPLC에 걸어 생강나무에서 가장 높은 항산화 및 간 보호 활성을 나타내는 단일물질인 LOE-E-2-Ⅰ(쿼시트린, quercitrin)과 LOE-E-2-Ⅱ(아프젤린, afzelin)를 얻기 위한 Prep HPLC 정제 조건을 보여준다.Table 2 shows LOE-E-2-I (Quercitrin) and LOE-E-2-Ⅱ, which are the single substances exhibiting the highest antioxidant and hepatoprotective activity in ginger tree by applying LOE-E-2 fraction to Prep HPLC. Prep HPLC purification conditions for obtaining (Afzelin) are shown.
표 3은 LOE-E-2 획분을 Prep HPLC에 걸어 얻은 LOE-E-2-Ⅰ(쿼시트린, quercitrin)과 LOE-E-2-Ⅱ(아프젤린 유도체)획분에 대하여 단일물질임을 보여주는 HPLC 분석을 위한 HPLC 분석 조건을 보여준다.Table 3 shows HPLC analyzes showing that the LOE-E-2 fractions were homogeneous for the LOE-E-2-I (qualercitrin) and LOE-E-2-II (afzelin derivative) fractions obtained by prep HPLC. HPLC analysis conditions are shown.
위 결과를 토대로 생강나무(Lindera obtusiloba Blume)에서 추출ㆍ분리ㆍ정제 분획물 및 단일물질인 쿼세틴-3-O-α-람노사이드(쿼시트린)와 캠페롤-3-O-α-람노사이드(아프젤린 유도체)가 높은 항산화 및 간 보호 기능을 보임을 확인 할 수 있으며, 이 결과로 추출ㆍ분리ㆍ정제 분획물 및 단일물질인 쿼세틴-3-O-α-람노사이드(쿼시트린)와 캠페롤-3-O-α-람노사이드(아프젤린 유도체)를 이용하여 피로, 만성질병이나 성인 성질환 같이 산화스트레스로 유발 될 수 있는 고혈압, 죽상경화증, 고지혈증, 당뇨병, 허혈-재관류, 암, 류마치스성 관절염, Behcet 질환, 신경손상성 질환, 또는 폐암 등과 간 질환의 예방 / 치료 효과를 높임으로써 식품소재, 건강기능 식품 소재, 의약품으로도 사용될 수 있다.
Based on the above results, the ginger tree ( Lindera obtusiloba Blume) extract, separation, and purification fractions and high antioxidants and hepatic antioxidants of Quercetin-3- O- α-Rhamnoside (Quetitrine) and Camperol-3- O- α-Rhamnoside (Aphzelin derivative) It can be seen that it shows the protective function, and as a result, it is extracted, separated and purified fractions and a single substance, quercetin-3- O- α-lamnoside (quacitrine) and camphorol-3- O- α-lamnoside ( Azelin derivatives), which can cause hypertension, atherosclerosis, hyperlipidemia, diabetes, ischemia-reperfusion, cancer, rheumatoid arthritis, Behcet disease, neuroinjury disease, Or it can be used as a food material, nutraceutical material, pharmaceuticals by increasing the prevention / treatment effect of lung cancer and liver disease.
이상에서 살펴 본 바와 같이, 본 발명은 천연식물인 생강나무(Lindera obtusiloba Blume)에서 추출ㆍ분리ㆍ정제한 분획물 및 쿼세틴-3-O-α-람노사이드(쿼시트린)와 캠페롤-3-O-α-람노사이드(아프젤린 유도체)를 단일물질로 추출ㆍ분리ㆍ정제 하여 얻었고, 본 발명의 추출ㆍ분리ㆍ정제 분획물 및 쿼세틴-3-O-α-람노사이드(쿼시트린)와 캠페롤-3-O-α-람노사이드(아프젤린 유도체)는 무독성의 천연식물소재에서 얻음으로서 종래의 의약품보다 부작용과 독성이 없고 안전성이 보강되었다. 또한 항산화 및 간 보호 기능물질로 활용될 수 있기에 피로, 만성질병이나 성인성질환 같이 산화스트레스로 유발 될 수 있는 간 보호, 고혈압, 죽상경화증, 고지혈증, 당뇨병, 허혈-재관류, 암, 류마치스성 관절염, Behcet 질환, 신경손상성 질환, 또는 폐암 등과 간 질환의 예방 / 치료 효과를 높임으로써 식품소재, 건강기능 식품 소재, 의약품으로도 사용될 수 있다.As described above, the present invention is a natural plant ginger tree ( lindera fractions extracted, separated and purified from obtusiloba Blume), and Quercetin-3- O- α-Rhamnoside (Quetitrine) and Camperol-3- O- α-Rhamnoside (Afzelin derivative) as a single substance Obtained by purification; the extract, separation and purification fractions of the present invention and quercetin-3- O- α-lamnoside (quacitrine) and camphorol-3- O- α-lamnoside (afzelin derivative) are non-toxic natural Obtained from plant materials, it has no side effects and toxicity compared to conventional medicines and has enhanced safety. It can also be used as an antioxidant and hepatoprotective agent to protect liver, which can be caused by oxidative stress such as fatigue, chronic disease or adult disease, hypertension, atherosclerosis, hyperlipidemia, diabetes, ischemia-reperfusion, cancer, rheumatoid arthritis, It can be used as a food material, nutraceutical material, and medicine by enhancing the prevention / treatment effect of Behcet disease, neuro-injury disease, or lung cancer.
도 1은 생강나무(Lindera obtusiloba Blume) 70% 에탄올 추출물(LOE)에서 간세포유래 셀인 HepG2 cell에 t-BHP(tert-butyl hydroperoxide)로 산화적 손상을 유발 한 후, 산화적 손상을 막아주는 항산화 및 간 보호 효과가 있는 추출물(LOE), 분리정제 획분(LOE-E, LOE-E-2)과 단일물질인 쿼시트린(quercitrin)과 아프젤린(afzelin) 유도체의 추출ㆍ분리ㆍ정제 과정을 보여주는 모식도이다.
도 2는 간세포유래 셀인 HepG2 cell에 t-BHP(tert-butyl hydroperoxide)로 산화적 손상을 유발 한 후, 이 산화적 손상을 막아줄거라 생각하는 항산화 및 간 보호 효과가 있는 생강나무 70% 에탄올 추출물(LOE)과 극성별 유기용매 분리획분(LOE-H, LOE-C, LOE-E, LOE-B, LOE-W)들의 항산화 및 간 보호 효과를 MTT 분석(cell viability, 셀 생존율)을 통하여 본 실험 결과이다. 결과는 t-BHP 단독으로 처리한 세포와 비교한 평균 ± 표준편차(n=3)로 나타내었다. ** P <0.01.
생강나무 70% 에탄올 추출물이 항산화 및 간 보호 효과가 있음을 확인 하였으며, 이 물질로 부터 출발한 극성별 유기용매 분리 획분들 역시 모두 항산화 및 간 보호 효과가 있는 것을 확인 하였다. 그러나 그 중 에틸아세테이트 분리 획분인 LOE-E가 극성별 유기용매 분리 획분 중에서 항산화 및 간 보호 활성이 가장 높음을 알 수 있다. 비교는 산화적 손상만을 준 t-BHP 그룹과 비교하여 이 그룹보다 셀 생존율이 높음을 기준으로 항산화 및 간 보호 효과가 있음을 확인하였다. 이는 항산화 및 간 보호 효과가 있는 물질이 들어가면 t-BHP에 의해 발생되는 산화적 손상을 막아 주므로 상대적으로 산화적 손상을 받은 그룹에 비하여 더 많은 셀 생존율을 나타내기 때문이다.
도 3은 간세포유래 셀인 HepG2 cell에 t-BHP(tert-butyl hydroperoxide)로 산화적 손상을 유발 한 후, 앞선 Fig. 1에서 항산화 및 간 보호 효과가 입증된 LOE-E를 다시 컬럼정제법을 통하여 얻은 5개의 정제 획분들(LOE-E-0, LOE-E-1, LOE-E-2, LOE-E-3, LOE-E-4)의 항산화 및 간 보호 효과를 MTT 분석(cell viability, 셀 생존율)을 통하여 본 실험 결과이다.정제획분들 역시 모두 항산화 및 간 보호 효과가 있는 것을 확인 하였다. 그러나 그 중 LOE-E-2가 항산화 및 간 보호 활성이 가장 높음을 알 수 있다.
비교는 산화적 손상만을 준 t-BHP 그룹과 비교하여 이 그룹보다 셀 생존율이 높음을 기준으로 항산화 및 간 보호 효과가 있음을 확인하였다. 이는 항산화 및 간 보호 효과가 있는 물질이 들어가면 t-BHP에 의해 발생되는 산화적 손상을 막아 주므로 상대적으로 산화적 손상을 받은 그룹에 비하여 더 많은 셀 생존율을 나타내기 때문이다. 결과는 t-BHP 단독으로 처리한 세포와 비교한 평균 ± 표준편차(n=3)로 나타내었다. ** P <0.01.
도 4는 LOE-E-2 획분을 Prep HPLC에 걸어 얻은 LOE-E-2-Ⅰ(쿼시트린, quercitrin, 4a)과 LOE-E-2-Ⅱ(아프젤린 유도체, 4b)획분에 대하여 HPLC 분석을 하여 단일 피크를 얻음으로써 단일물질임을 확인해주는 chromatogram을 보여준다.
도 5는 LOE-E-2-Ⅰ(쿼시트린, quercitrin) 획분에 대하여 구조분석을 하기 위하여 실시한 1D NMR의 1H-NMR 스펙트럼의 결과를 보여준다.
도 6은 LOE-E-2-Ⅰ(쿼시트린, quercitrin) 획분에 대하여 구조분석을 하기 위하여 실시한 1D NMR의 13C-NMR 스펙트럼의 결과를 보여준다.
도 7은 LOE-E-2-Ⅱ(아프젤린 유도체, afzelin) 획분에 대하여 구조분석을 하기 위하여 실시한 1D NMR의 1H-NMR 스펙트럼의 결과를 보여준다.
도 8은 LOE-E-2-Ⅱ(아프젤린 유도체, afzelin) 획분에 대하여 구조분석을 하기 위하여 실시한 1D NMR의 13C-NMR 스펙트럼의 결과를 보여준다.
도 9는 LOE-E-2-Ⅰ(쿼시트린, quercitrin, A)과 LOE-E-2-Ⅱ(아프젤린 유도체, B) 획분에 대하여 구조분석을 하기 위하여 실시한 1D NMR과 2D NMR의 결과를 종합하여 도출된 단일천연물질인 쿼시트린(LOE-E-2-Ⅰ, quercitrin)과 아프젤린 유도체(LOE-E-2-Ⅱ, afzelin)의 화학구조를 보여준다.
도 10은 생강나무(Lindera obtusiloba Blume) 100% 메탄올 추출물(LOE)에서 단일물질인 쿼시트린(quercitrin)과 아프젤린 유도체의 추출ㆍ분리ㆍ정제 과정을 보여주는 모식도이다.
도 11은 생강 추출물과 LOE의 배양된 간세포에서 t-BHP-유도된 산화적 스트레스에 대한 보호효과를 비교한 것이다. 세포는 30분 동안 여러 농도의 LOE 및 1.5 mM t-BHP를 처리하였다. 세포 생존률은 MTT 분석에 의하여 결정하였다. 결과는 t-BHP 단독으로 처리한 세포와 비교한 평균 ± 표준편차(n=3)로 나타내었다. ** P <0.01. LOE는 메탄올로 생강나무를 추출한 분획이고, 생강 추출물은 생강 분말을 메탄올로 추출한 분획이다.
도 12는 배양된 간세포에서 t-BHP-유도된 산화적 스트레스에 대한 LOE의 각 유기용매별 분획층의 보호 효과를 비교한 것이다. 세포는 30분 동안 500 μg/mL의 각층과 1.5 mM t-BHP를 처리하였다. 세포 생존률은 MTT 분석에 의하여 결정하였다. 결과는 t-BHP 단독으로 처리한 세포와 비교한 평균 ± 표준편차(n=3)로 나타내었다. ** P <0.01. H, n-헥산 층; C, 클로로포름 층; E, EtOAc 층; B, n-부탄올 층; W, 수층.
도 13은 배양된 간세포에서 t-BHP-유도된 산화적 스트레스에 대한 LOE-E의 각 분획의 보호효과를 비교한 것이다. 세포는 30분 동안 100 μg/mL의 각층과 1.5 mM t-BHP를 처리하였다. 결과는 t-BHP 단독으로 처리한 세포와 비교한 평균 ± 표준편차(n=3)로 나타내었다. **P<0.01,*P<0.05.
LOE-E-0, 100% EtOAc : 0% MeOH, LOE-E-10, 90% EtOAc : 10% MeOH, LOE-E-20, 80% EtOAc : 20% MeOH, LOE-E-30, 70% EtOAc : 30% MeOH, LOE-E-40, 60% EtOAc : 40% MeOH, LOE-E-50, 50% EtOAc : 50% MeOH, LOE-E-60, 40% EtOAc : 60% MeOH, LOE-E-70, 30% EtOAc : 70% MeOH, LOE-E-80, 20% EtOAc : 80% MeOH, LOE-E-90, 10% EtOAc : 90% MeOH, LOE-E-100, 0% EtOAc : 100% MeOH을 나타낸다.
도 14는 수퍼옥사이드 래디컬에 대한 소거 활성을 나타낸 그림.
수퍼옥사이드 래디컬은 NADH-페나진 메토설페이트 시스템에 의하여 생성되고, NBT의 환원에 의하여 분석되었다. 바는 평균 ±SD (n=3)를 나타낸다. 결과는 t-테스트(** P<0.01)에 의하여 분석하였다.
도 15는 LOE-E-0의 실리카겔 컬럼으로부터 얻은 서브분획의 세파덱스 LH-20크로마토그램을 나타낸다.
컬럼(1.5 X 40 cm)을 50 % 메탄올로 1 ml/분으로 용출하고, 10 ml/투브로 수집하였다.
도 16은 정제된 LOE-E-O-I 및 LOE-E-O-I II의 HPLC 크로마토그램을 나타낸다.
HPLC을 Waters SymmetryPrep C18 (7um) 컬럼(7.8 X 30 cm)을 사용하여 수행되었다. 용출은 1 ml/분 속도로 물과 메탄올의 선형구배를 사용하였다. 그 용출액은 340 nm에서 모니터하였다.
두 개의 분획층(LOE-E-0-Ⅰ, LOE-E-0-Ⅱ)을 HPLC로 단일물질로 확인하였다.
LOE-E-0 획분을 Sephadex LH-20 레진 컬럼에 걸어 얻은 LOE-E-2-Ⅰ(아프젤린 유도체, 16a)과 LOE-E-2-Ⅱ(쿼시트린, quercitrin, 16b)획분에 대하여 HPLC 분석을 하여 단일 피크를 얻음으로써 단일물질임을 확인해주는 크로마토그램을 보여준다.Figure 1 is an antioxidant that prevents oxidative damage after inducing oxidative damage with t-BHP (tert-butyl hydroperoxide) in HepG2 cells, which are hepatocyte-derived cells, from
Figure 2 is a ethanol extract of ginger tree with antioxidant and hepatoprotective effects, which is thought to prevent oxidative damage after inducing oxidative damage to t-BHP (tert-butyl hydroperoxide) in HepG2 cells, a hepatocyte-derived cell. Antioxidant and hepatoprotective effects of (LOE) and organic solvent separation fractions (LOE-H, LOE-C, LOE-E, LOE-B, LOE-W) by polarity were analyzed through MTT analysis (cell viability). Experimental results. The results are expressed as mean ± standard deviation (n = 3) compared to cells treated with t-BHP alone. ** P <0.01.
It was confirmed that 70% ethanol extract of ginger tree had antioxidant and hepatoprotective effects, and all the organic solvent separation fractions by polarity from this substance also had antioxidant and hepatoprotective effects. However, it can be seen that LOE-E, an ethyl acetate fraction, has the highest antioxidant and hepatoprotective activity among the organic solvent fractions. The comparison was confirmed to have antioxidant and hepatoprotective effects on the basis of higher cell viability than the t- BHP group which gave only oxidative damage. This prevents the oxidative damage caused by t- BHP when the substance having antioxidant and hepatoprotective effects enters, thus exhibiting more cell viability compared to the oxidatively damaged group.
Figure 3 is after inducing oxidative damage to t- BHP ( tert -butyl hydroperoxide) in HepG2 cells, hepatocyte-derived cells. Five purified fractions (LOE-E-0, LOE-E-1, LOE-E-2, LOE-E-3), obtained by column purification method, showed LOE-E, which showed antioxidant and hepatoprotective effects in 1 The antioxidative and hepatoprotective effects of LOE-E-4) are the results of this experiment through MTT analysis (cell viability). Purified fractions also have antioxidant and hepatoprotective effects. However, it can be seen that LOE-E-2 has the highest antioxidant and hepatoprotective activity.
The comparison was confirmed to have antioxidant and hepatoprotective effects on the basis of higher cell viability than the t- BHP group which gave only oxidative damage. This prevents the oxidative damage caused by t- BHP when the substance having antioxidant and hepatoprotective effects enters, thus exhibiting more cell viability compared to the oxidatively damaged group. The results are expressed as mean ± standard deviation (n = 3) compared to cells treated with t- BHP alone. ** P <0.01.
Figure 4 HPLC analysis of LOE-E-2-I (quatrine, quercitrin, 4a) and LOE-E-2-II (afzelin derivative, 4b) fractions obtained by preparative HPLC of LOE-E-2 fractions Shows a chromatogram confirming that it is a single substance by obtaining a single peak.
Figure 5 shows the results of the 1 H-NMR spectrum of the 1D NMR carried out for structural analysis on the LOE-E-2-I (Quercitrin) fraction.
FIG. 6 shows the results of 13 C-NMR spectra of 1D NMR performed for structural analysis on LOE-E-2-I (Quercitrin) fractions.
Figure 7 shows the results of the 1 H-NMR spectrum of 1D NMR performed for structural analysis on the LOE-E-2-II (afzelin derivative, afzelin) fraction.
FIG. 8 shows the results of 13 C-NMR spectra of 1D NMR conducted for structural analysis on the LOE-E-2-II (afzelin derivative, afzelin) fraction.
FIG. 9 shows the results of 1D NMR and 2D NMR performed for structural analysis on the fractions of LOE-E-2-I (quatrine, quercitrin, A) and LOE-E-2-II (afzelin derivative, B). The chemical structures of the quencitrin (LOE-E-2-I, quercitrin) and afzelin derivatives (LOE-E-2-II, afzelin) are shown.
10 is a ginger tree ( Lindera obtusiloba Blume is a schematic diagram showing the extraction, separation and purification of quercitrin and afzelin derivatives from 100% methanol extract (LOE).
Figure 11 compares the protective effect of t -BHP-induced oxidative stress in cultured hepatocytes of ginger extract and LOE. Cells were treated with various concentrations of LOE and 1.5 mM t- BHP for 30 minutes. Cell viability was determined by MTT assay. The results are expressed as mean ± standard deviation (n = 3) compared to cells treated with t- BHP alone. ** P <0.01. LOE is a fraction obtained by extracting a ginger tree with methanol, and a ginger extract is a fraction obtained by extracting ginger powder with methanol.
Figure 12 compares the protective effect of each organic solvent fractionation layer of LOE on t -BHP-induced oxidative stress in cultured hepatocytes. Cells were treated with 500 μg / mL of each layer and 1.5 mM t- BHP for 30 minutes. Cell viability was determined by MTT assay. The results are expressed as mean ± standard deviation (n = 3) compared to cells treated with t- BHP alone. ** P <0.01. H, n -hexane layer; C, chloroform layer; E, EtOAc layer; B, n -butanol layer; W, water column.
Figure 13 compares the protective effect of each fraction of LOE-E against t -BHP-induced oxidative stress in cultured hepatocytes. Cells were treated with 100 μg / mL of each layer and 1.5 mM t- BHP for 30 minutes. The results are expressed as mean ± standard deviation (n = 3) compared to cells treated with t- BHP alone. ** P <0.01, * P <0.05.
LOE-E-0, 100% EtOAc: 0% MeOH, LOE-E-10, 90% EtOAc: 10% MeOH, LOE-E-20, 80% EtOAc: 20% MeOH, LOE-E-30, 70% EtOAc: 30% MeOH, LOE-E-40, 60% EtOAc: 40% MeOH, LOE-E-50, 50% EtOAc: 50% MeOH, LOE-E-60, 40% EtOAc: 60% MeOH, LOE- E-70, 30% EtOAc: 70% MeOH, LOE-E-80, 20% EtOAc: 80% MeOH, LOE-E-90, 10% EtOAc: 90% MeOH, LOE-E-100, 0% EtOAc: 100% MeOH.
14 shows scavenging activity for superoxide radicals.
Superoxide radicals were generated by the NADH-phenazine methosulfate system and analyzed by reduction of NBT. Bars represent mean ± SD ( n = 3). The results were analyzed by t-test ( ** P <0.01).
FIG. 15 shows Sephadex LH-20 chromatogram of subfraction obtained from silica gel column of LOE-E-0.
The column (1.5 × 40 cm) was eluted with 50% methanol at 1 ml / min and collected at 10 ml / tub.
16 shows HPLC chromatograms of purified LOE-EOI and LOE-EOI II.
HPLC was performed using a Waters SymmetryPrep C18 (7um) column (7.8 × 30 cm). Elution was performed using a linear gradient of water and methanol at a rate of 1 ml / min. The eluate was monitored at 340 nm.
Two fractionation layers (LOE-E-0-I, LOE-E-0-II) were identified as single material by HPLC.
HPLC of LOE-E-2-I (afzelin derivative, 16a) and LOE-E-2-II (quatcitrin, quercitrin, 16b) fractions obtained by hanging LOE-E-0 fraction on Sephadex LH-20 resin column The analysis shows a chromatogram that identifies a single substance by obtaining a single peak.
이하, 비한정적인 실시예를 통하여 본 발명을 더욱 상세하게 설명한다. 단 하기 실시예는 본 발명을 설명하기 위한 목적으로 기재된 것으로, 본 발명의 범위는 하기 실시예에 의하여 제한되는 것으로 해석되지 아니한다.
Hereinafter, the present invention will be described in more detail with reference to non-limiting examples. However, the following examples are described for the purpose of illustrating the present invention, the scope of the present invention is not to be construed as limited by the following examples.
실시예 1: 생강나무( Lindera obtusiloba Blume )로부터 항산화 및 간 보호 기능물질의 추출·분리 및 정제-에탄올 용매 사용 (도 1 참고) Example 1 Ginger Tree ( Lindera obtusiloba Extraction, Separation and Purification of Antioxidant and Hepatoprotective Compounds from Blume ) -Use of Ethanol Solvent (See Figure 1)
건조된 생강나무(Lindera obtusiloba Blume) 500g을 70% 에탄올 5 L를 이용하여 3시간 동안 환류냉각 추출하여 얻은 후 7,500 rpm, 30 min, 4℃에서 원심분리하여 상등액을 얻었다. 원심분리된 상층액에 남아 있을지 모르는 불용성 물질을 제거하기 위하여 재차 상등액을 whatman No. 42 여과하였다. 여과된 상등액을 감압증류한 다음 동결건조를 통하여 분말 형태의 생강나무(Lindera obtusiloba Blume) 70% 에탄올 추출물(LOE)을 얻었다. 이렇게 얻어진 LOE를 물에 녹인 뒤 순차적으로 유기용매의 극성도를 달리하며 hexane(LOE-H), chloroform(LOE-C), ethylacetate (LOE-E), butanol(LOE-B), water(LOE-W) 획분으로 극성에 따라 일차적 분리를 하였다. 항산화 및 간 보호 활성이 가장 높은 LOE-E를 연속적으로 vacuum liquid chromatography에 흡착 시킨 후 <D.W. : methanol> 혼합용출액의 농도를 달리하여 5개의 획분을 얻은 뒤, 역시 항산화 활성이 가장 높은 <50% D.W. : 50% methanol> 획분(LOE-E-2)을 재차 Prep HPLC를 이용하여 표 2(가변적인 조건, 용매의 종류와 유속, %함량은 바뀔 수 있음)의 조건으로 생강나무(Lindera obtusiloba Blume)의 항산화 및 간 보호 기능물질인 쿼세틴-3-O-α-람노사이드(쿼시트린)와 캠페롤-3-O-α-람노사이드(아프젤린 유도체)를 분리하였다. 용출된 항산화 기능물질을 Table 3의 조건으로 순도를 확인하였다.Dried Ginger Tree ( Lindera Obtusiloba Blume) 500g was obtained by reflux extraction for 3 hours using 5 L of 70% ethanol and then centrifuged at 7,500 rpm, 30 min, 4 ℃ to obtain a supernatant. To remove insoluble matter that may remain in the centrifuged supernatant, replicate the supernatant again with whatman No. 42 < / RTI > The filtered supernatant was distilled under reduced pressure and then lyophilized to form a powdered ginger tree ( Lindera obtusiloba Blume) 70% ethanol extract (LOE) was obtained. After dissolving LOE in water, the polarity of organic solvent was sequentially changed, and hexane (LOE-H), chloroform (LOE-C), ethylacetate (LOE-E), butanol (LOE-B), water (LOE- W) The fractions were separated first by polarity. LOE-E, which has the highest antioxidant and hepatoprotective activity, was continuously adsorbed by vacuum liquid chromatography, and five fractions were obtained by varying the concentration of <DW: methanol> mixed eluent, and the highest antioxidant activity was <50% DW. : 50% methanol> fraction (LOE-E-2) the re-using the Prep HPLC under the conditions of Table 2, ginger tree (variable conditions, that the solvent, type and flow rate,% content is subject to change) (Lindera Quercetin- 3- O- α-rhamnoside (quacitrine) and camphorol-3- O- α-rhamnoside (apzelin derivatives), which are antioxidants and hepatoprotective agents of obtusiloba Blume), were isolated. The eluted antioxidant functional substances were checked for purity under the conditions of Table 3.
실시예 2: 생강나무( Lindera obtusiloba Blume )로부터 항산화 및 간 보호기능물질의 추출·분리 및 정제-메탄올 용매 사용 (도 10 참고) Example 2 Ginger Tree ( Lindera obtusiloba Extraction, Separation and Purification of Antioxidant and Hepatoprotective Compounds from Blume ) -Use of Methanol Solvent (see Figure 10)
건조된 생강나무(Lindera obtusiloba Blume) 500g을 100% 메탄올 5 L를 이용하여 3시간 동안 환류냉각 추출하여 얻은 후 7,500 rpm, 30 min, 4℃에서 원심분리하여 상등액을 얻었다. 원심분리된 상층액에 남아 있을지 모르는 불용성 물질을 제거하기 위하여 재차 상등액을 whatman No. 42 여과하였다. 여과된 상등액을 감압증류한 다음 동결건조를 통하여 분말 형태의 생강나무(Lindera obtusiloba Blume) 100% 메탄올 추출물(LOE)을 얻었다. 이렇게 얻어진 LOE를 물에 녹인 뒤 순차적으로 유기용매의 극성도를 달리하며 hexane(LOE-H), chloroform(LOE-C), ethylacetate (LOE-E), butanol(LOE-B), water(LOE-W) 획분으로 극성에 따라 일차적 분리를 하였다. 항산화 활성이 가장 높은 LOE-E를 연속적으로 silica gel column chromatography에 흡착 시킨 후 <ethyl acetate : methanol> 혼합용출액의 농도를 달리하여 11개의 획분을 얻은 뒤, 역시 항산화 및 간 보호 활성이 가장 높은 <100% ethyl acetate. : 0% methanol> 획분(LOE-E-0)을 재차 Sephadex LH-20 resin를 이용한 column 정제를(용매는 methanol과 D.W.를 사용)하여 생강나무(Lindera obtusiloba Blume)의 항산화 기능물질인 쿼세틴-3-O-α-람노사이드(쿼시트린, LOE-E-0-Ⅱ)와 캠페롤-3-O-α-람노사이드(아프젤린 유도체, LOE-E-0-Ⅰ)를 분리하였다.Dried Ginger Tree ( Lindera Obtusiloba Blume) 500g was obtained by reflux extraction for 3 hours using 5 L of 100% methanol and centrifuged at 7,500 rpm, 30 min, 4 ℃ to obtain a supernatant. To remove insoluble matter that may remain in the centrifuged supernatant, replicate the supernatant again with whatman No. 42 < / RTI > The filtered supernatant was distilled under reduced pressure and then lyophilized to form a powdered ginger tree ( Lindera obtusiloba Blume) 100% methanol extract (LOE) was obtained. After dissolving LOE in water, the polarity of organic solvent was sequentially changed, and hexane (LOE-H), chloroform (LOE-C), ethylacetate (LOE-E), butanol (LOE-B), water (LOE- W) The fractions were separated first by polarity. After LOE-E, which has the highest antioxidant activity, was continuously adsorbed on silica gel column chromatography, 11 fractions were obtained by varying the concentration of <ethyl acetate: methanol> mixed eluent, and also the highest antioxidant and hepatoprotective activity <100 % ethyl acetate. : Quercetin-3, an antioxidant functional substance of Lindera obtusiloba Blume, using 0% methanol> fraction (LOE-E-0) and column purification using Sephadex LH-20 resin (solvent using methanol and DW) O- α-rhamnoside (quacitrine, LOE-E-0-II) and camphorol-3- O- α-rhamnoside (afzelin derivative, LOE-E-0-I) were separated.
실험예 1: 생강나무( Lindera obtusiloba Blume ) 에탄올 추출ㆍ분리ㆍ정제 단계에서의 각 획분, 쿼세틴-3-O-α-람노사이드(쿼시트린)와 캠페롤-3-O-α-람노사이드(아프젤린 유도체)의 항산화 및 간 보호기능 (표 1, 도 2, 및 3 참고) Experimental Example 1 Ginger Tree ( Lindera obtusiloba Antioxidant and hepatoprotective of Blume) ethanol extraction and separation, each fraction, kwosetin -3- O -α- ramno side (quartz citrine) campaigns and roll -3- O -α- ramno side (ill jelrin derivative) in a purification step Functions (see Tables 1, 2, and 3)
간세포 유래 셀인 HepG2 cell에 t-BHP(tert-butyl hydroperoxide)로 산화적 손상을 유발 한 후, 산화적 손상을 막아줄거라 생각하는 항산화 및 간 보호 효과가 있는 생강나무 70% 에탄올 추출물(LOE)과 극성별 유기용매 분리획분(LOE-H, LOE-C, LOE-E, LOE-B, LOE-W)들의 항산화 및 간 보호 효과를 MTT 분석(cell viability, 셀 생존율)을 통하여 확인하였다. 생강나무 70% 에탄올 추출물(LOE)이 항산화 및 간 보호 효과가 있음을 확인하였으며, 이 물질로 부터 출발하여 극성별 유기용매 분리한 획분들 역시 모두 항산화 및 간 보호 효과가 있는 것을 확인 하였다. 그러나 그 중 에틸아세테이트 획분인 LOE-E가 유기용매별 분리 획분 중에서 항산화 및 간 보호 활성이 가장 높음을 알 수 있다. 항산화 및 간 보호 효과가 입증된 LOE-E를 다시 컬럼정제법을 통하여 얻은 5개의 정제획분들(LOE-E-0, LOE-E-1, LOE-E-2, LOE-E-3, LOE-E-4)의 항산화 및 간 보호 효과를 MTT 분석을 통하여 측정한 결과 정제획분들 역시 모두 항산화 및 간 보호 효과가 있는 것을 확인 하였다. 그러나 그 중 LOE-E-2가 항산화 및 간 보호 활성이 가장 높음을 알 수 있다. 그리고 이 LOE-E-2 획분에서 Prep HPLC를 통하여 얻은 두 개의 단일물질인 쿼세틴-3-O-α-람노사이드(쿼시트린, LOE-E-2-Ⅰ)와 캠페롤-3-O-α-람노사이드(아프젤린 유도체, LOE-E-2-Ⅱ)를 항산화 측정방법의 하나인 FRAP assay와 DPPH-radical scavenging activity로 측정한 결과 두 물질 모두 항산화 및 간 보호 활성이 높음을 확인 하였다.After inducing oxidative damage with t -BHP ( tert -butyl hydroperoxide) to HepG2 cells, hepatocyte-derived cells,
실험예 2: 쿼세틴 -3- O -α- 람노사이드(쿼시트린)와 캠페롤 -3- O -α-람노사이드( 아프젤린 유도체)의 단일물질 확인과 구조 동정 (표 3, 도 4~9 참고) Experimental Example 2: Quercetin- 3- O- α- rhamnoside (Quetcitrin) Campaign roll -3- O -α- ramno side single substance identification and the identification of structure (ill gel incorrect derivative) (Table 3, see Fig. 4-9)
실시예1에서 실시한 추출ㆍ분리ㆍ정제 방법을 통하여 얻은 두 획분(LOE-E-2-Ⅰ과 LOE-E-2-Ⅱ)을 우선 HPLC 분석을 통하여 단일물질임을 확인하였고, 이 물질이 정확히 어떤 물질인지를 알기위해 1D NMR의 1H과 13C NMR의 spectrum 분석을 통하여 그것이 최종적으로 쿼세틴-3-O-α-람노사이드(쿼시트린)와 캠페롤-3-O-α-람노사이드(아프젤린 유도체)로 동정하였다.Two fractions (LOE-E-2-I and LOE-E-2-II) obtained through the extraction, separation, and purification method described in Example 1 were first identified by HPLC analysis. Spectral analysis of 1 H and 13 C NMR of 1D NMR to determine if it is a substance, it was finally performed on quercetin-3- O- α-lamnoside (quacitrine) and camphorol-3- O- α-lamnoside (af Jellyline derivatives).
실험예 3: 생강나무 메탄올 추출물과 생강 메탄올 추출물의 MTT assay 를 통한 항산화 및 간보호 활성 비교 Experimental Example 3: MTT of ginger tree methanol extract and ginger methanol extract Comparison of Antioxidant and Hepatoprotective Activities by Assay
Rat의 간세포(hepatocyte)를 primary culture 기술을 이용하여 분리하여 t-BHP(tert-butyl hydroperoxide)로 산화적 손상을 유발 한 후, 산화적 손상을 막아줄거라 생각하는 항산화 및 간 보호 효과가 있는 생강나무 100% 메탄올 추출물(LOE)과 비교군으로 생강 메탄올 추출물의 항산화 및 간 보호 효과를 MTT 분석(cell viability, 셀 생존율)을 통하여 확인하였다. 생강나무 100% 메탄올 추출물(LOE)이 항산화 및 간 보호 효과가 있음을 확인 하였으며, 또한 이 생강나무 메탄올 추출물은 좋은 효과가 있다고 잘 알려진 생강보다 더 높은 항산화 및 간 보호 활성이 있음을 확인하였다.Hepatocytes from rats were isolated using primary culture technology to induce oxidative damage with tert- butyl hydroperoxide ( t -BHP), and ginger with antioxidant and liver protection effects thought to prevent oxidative damage. Antioxidant and hepatoprotective effects of ginger methanol extract as a comparison group with 100% methanol extract (LOE) were confirmed by MTT analysis (cell viability).
실험예 4: 생강나무 메탄올 층에서 분획한 각각의 분획층에 대해서 MTT assay 를 통한 항산화 및 간보호 활성 Experimental Example 4: each fraction from the ginger layer of methanol In the fractionation layer about MTT assay ThroughAntioxidant and hepatoprotective activity
Rat의 간세포(hepatocyte)를 primary culture 기술을 이용하여 분리하여 t-BHP(tert-butyl hydroperoxide)로 산화적 손상을 유발 한 후, 산화적 손상을 막아줄거라 생각하는 항산화 및 간 보호 효과가 있는 생강나무의 극성별 유기용매 분리획분(LOE-H, LOE-C, LOE-E, LOE-B, LOE-W)들의 항산화 및 간 보호 효과를 MTT 분석(cell viability, 셀 생존율)을 통하여 확인하였다. 모든 분획물에서 항산화 및 간 보호 효과가 있음을 확인 하였으며, 그 중 에틸아세테이트 획분인 LOE-E가 유기용매별 분리 획분 중에서 항산화 및 간 보호 활성이 가장 높음을 알 수 있다. Hepatocytes from rats were isolated using primary culture technology to induce oxidative damage with tert- butyl hydroperoxide ( t -BHP), and ginger with antioxidant and liver protection effects thought to prevent oxidative damage. The antioxidant and hepatoprotective effects of organic solvent separation fractions (LOE-H, LOE-C, LOE-E, LOE-B, LOE-W) of each polar tree were confirmed by MTT analysis (cell viability, cell viability). It was confirmed that all fractions had antioxidant and hepatoprotective effects. Among them, LOE-E, an ethyl acetate fraction, showed the highest antioxidant and hepatoprotective activity among the organic fractions.
항산화 및 간 보호 효과가 입증된 LOE-E를 다시 컬럼정제법을 통하여 얻은 11개의 정제획분들(LOE-E-0, LOE-E-1 ~ LOE-E-10)의 항산화 및 간 보호 효과를 MTT 분석을 통하여 측정한 결과 정제획분들 역시 모두 항산화 및 간 보호 효과가 있는 것을 확인 하였다. 그러나 그 중 LOE-E-0이 항산화 및 간 보호 활성이 가장 높음을 알 수 있다. 그래서 이 획분을 Sephadex LH-20 resin를 이용한 column 정제(용매는 methanol과 D.W.를 사용)를 하여 생강나무(Lindera obtusiloba Blume)의 항산화 및 간 보호 기능물질인 쿼세틴-3-O-α-람노사이드(쿼시트린, LOE-E-0-Ⅱ)와 캠페롤-3-O-α-람노사이드(아프젤린 유도체, LOE-E-0-Ⅰ)를 분리하였다.Antioxidant and hepatoprotective effects of 11 purified fractions (LOE-E-0, LOE-E-1 to LOE-E-10), which were obtained through the column purification method, were again proved LOE-E, which has been proved to be antioxidant and hepatoprotective. As a result of MTT analysis, it was confirmed that all the purified fractions also had antioxidant and liver protection effects. However, it can be seen that LOE-E-0 has the highest antioxidant and hepatoprotective activity. Thus, this fraction was subjected to column purification using Sephadex LH-20 resin (solvent using methanol and DW) and ginger tree ( Lindera Quercetin- 3- O- α-rhamnoside (quacitrine, LOE-E-0-II) and camphorol-3- O- α-rhamnoside (afzelin derivatives), which are antioxidant and hepatoprotective agents of obtusiloba Blume) LOE-E-0-I) was isolated.
실험예 5: 생강나무 메탄올 층에서 분획한 에틸아세테이트 분획층(LOE-E)에 대해서 superoxide radical scavenging assay를 통한 항산화 및 간 보호 활성 Experimental Example 5: ginger methanol wood layers through the superoxide radical scavenging assay for the ethyl acetate fraction layer (LOE-E) fraction antioxidant and protective activity on the liver
항산화 실험의 하나인 superoxide radical 제거 효과를 본 결과로 항산화 물질로 잘 알려진 Vit E와 비교한 결과 Vit E보다 더 높은 superoxide radical 제거 효과가 있음을 확인하였다.The superoxide radical removal effect, one of the antioxidant experiments, was compared with Vit E, which is known as an antioxidant.
Superoxide radical scavenging 분석은 산화로 인해 발생되는 superoxide radical을 NADH-phenazine methosulfate 시스템으로 발생하고 이에 시료를 처리하면 즉 항산화 활성이 있으면 superoxide radical을 소거하는 기능을 나타내는데 기존에 알려진 항산화 물질인 Vit E와 비교를 하였을 때 Vit E 보다 더 높은 superoxide radical 소거 기능을 보이는 것으로 보아 높은 항산화 활성이 알 수 있다.Superoxide radical scavenging analysis shows that superoxide radical generated by oxidation is generated by NADH-phenazine methosulfate system, and the sample is processed to scaveng superoxide radical when there is antioxidant activity, compared with the known antioxidant Vit E. Higher antioxidant activity was observed as it showed higher superoxide radical scavenging ability than Vit E.
Claims (3)
b) 상기 조추출물에 헥산을 처리하여 수층을 얻는 단계; 및
c) 상기 얻어진 수층에 에틸아세테이트를 처리하여 에틸아세테이트 층을 얻는 단계를 포함하여, 생강나무의 물 추출물, 에탄올 추출물 또는 메탄올 추출물에서 분리된 캠페롤-3-o-알파-람노사이드를 유효성분으로 포함하는 간세포의 산화적 손상을 회복시키는 간보호용 기능성 식품 조성물을 제조하는 방법. a) preparing a crude extract using water, ethanol or methanol from Lindera obtusiloba Blume;
b) treating the crude extract with hexane to obtain an aqueous layer; And
c) treating the obtained aqueous layer with ethyl acetate to obtain an ethyl acetate layer, wherein the camphorol-3-o-alpha-rhamnoside isolated from the water extract, ethanol extract or methanol extract of ginger tree as an active ingredient. Method for producing a functional food composition for liver protection to restore the oxidative damage of liver cells comprising.
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KR20090079584A (en) * | 2008-01-18 | 2009-07-22 | 양지화학 주식회사 | Composition Comprising the Extracts of Lindera obtusiloba for Prevention and Treatment of Cardiovascular Diseases |
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Non-Patent Citations (2)
Title |
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Jung, H.A. et al., Archives of Pharmacal Research (1999) Vol.22, No.2, pp.213-218 * |
이상준 외 3명, 한국식품과학회지 (1999) 제31권 제3호 pp.815-822 * |
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