KR20140097718A - A health food composition for preventing inflammatory comprising the extracts or fractions of Amaranth as effective component - Google Patents
A health food composition for preventing inflammatory comprising the extracts or fractions of Amaranth as effective component Download PDFInfo
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- KR20140097718A KR20140097718A KR1020130009931A KR20130009931A KR20140097718A KR 20140097718 A KR20140097718 A KR 20140097718A KR 1020130009931 A KR1020130009931 A KR 1020130009931A KR 20130009931 A KR20130009931 A KR 20130009931A KR 20140097718 A KR20140097718 A KR 20140097718A
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- South Korea
- Prior art keywords
- amaranth
- extract
- fraction
- composition
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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)
- A61K36/21—Amaranthaceae (Amaranth family), e.g. pigweed, rockwort or globe amaranth
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9789—Magnoliopsida [dicotyledons]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
Abstract
Description
본 발명은 아마란스(Mastixia arborea C.B.Clarke) 추출물 또는 이의 분획물을 유효성분으로 함유하는 염증방지용 조성물에 관한 것으로, 보다 구체적으로 본 발명의 아마란스 추출물 또는 이의 분획물은 농도의존적으로 NO 소거 활성 및 iNOS 생성 억제 효과를 나타내어 탁월한 항염증 효과를 가짐으로써, 건강식품의 유효성분, 약학적 조성물 및 노화방지용 화장료 조성물로 유용하게 사용될 수 있다.The present invention relates to an anti-inflammatory composition comprising an extract of Mastixia arborea CBClarke or a fraction thereof as an active ingredient. More specifically, the extract of Amaranth or the fraction thereof according to the present invention has NO-scavenging activity and iNOS production inhibitory effect And has an excellent anti-inflammatory effect, it can be effectively used as an active ingredient, a pharmaceutical composition and a cosmetic composition for preventing aging of health food.
아마란스는 비름과에 속하는 일년생 유사화곡류로서 식물학적으로 옥수수, 귀리, 벼, 밀과 같은 단자엽 식물이 아니고 쌍자엽 식물이다. 해발이 높고 고온건조한 중앙 남아메리키가 원산지로 페루의 잉카시대와 멕시코의 아즈텍 시대에 옥수수, 완두콩과 함께 주식품원이었다. 크기는 모래알보다 약간 작은 사이즈로 매우 작으며 종자의 형태를 보면 배가 고리처럼 배유를 감싸고 있는 모양을 하고 있는데, 주로 고리 모양의 배에 단백질이나 지방이 축적되어있고 배로 감싸져 있는 외배우에 전분이 축저되어있다. 이삭길이는 보통 20 ~ 60cm 이고 종피색은 황색, 연한살색, 자주색이다. 보통 4월 하순부터 5월 중순사이에 파종하고, 개화기는 보통 6월 하순에서 8월 중하순까지도 품종에 따라 개화기의 다양한 변이를 보이며 초장은 토양비옥도에 따라 달라지나 작은 품종은 120cm정도 이고 큰 품종은 250cm정도이다. Amaranth is a biennial-like grains belonging to the family Amaranthus and is a biplot plant, not a monocotyledon such as corn, oats, rice, and wheat. It was the main food source along with corn and peas in the Inca era of Peru and the Aztec era of Mexico. The size is slightly smaller than the size of a grain of sand, and it is very small in size. The shape of a seed encircles the endosperm like a ring. It has a protein and fat accumulation in an annular pear, and starch It is compassion. The length of the ears is usually 20 to 60cm, and the color of the seed coat is yellow, light flesh, purple. It is usually planted from late April to mid May, and the flowering period usually varies from late June to mid-August depending on the variety of flowering period. The plant length varies depending on the soil fertility, but small varieties are about 120 cm. It is about 250cm.
아마란스를 대상으로 항염 및 미백에 관련한 활성 규명에 대한 연구는 보고된 바가 없어 본 발명자들은 제주도에서 재배된 아마란스추출물을 이용하여 염증에 관여하는 NO 생성 억제 및 iNOS와 Cytokine 억제 활성을 측정하여, 아마란스 종실과 자주색 전초에서 우수한 NO 억제 효과와 iNOS 단백질 발현 억제 및 염증관련 TNF-α, IL-6 등의 cytokine 억제 효과가 뛰어남을 확인하였다. 뿐만 아니라 아마란스의 영양성분에 대해 조사한 결과, 타지방에서 재배된 아마란스에 비해 우수한 영양성분을 확인함으로써 본 발명을 완성하였다. The present inventors measured the inhibition of NO production and iNOS and cytokine inhibitory activity involved in inflammation by using the extract of Amaranth cultivated in Jeju Island and found that the amaranth seeds And purple outpost showed excellent NO inhibitory effect, inhibition of iNOS protein expression, and cytokine inhibitory effects such as inflammation-related TNF-α and IL-6. In addition, the present inventors completed the present invention by examining the nutritional components of the amaranth, and confirming the superior nutritional components compared to the amaranth grown in the other lipid.
최근 사회가 급변하고, 여성의 사회진출 등의 여러 가지 요인들에 의해 바쁜 현대인을 위한 패스트푸드 형태의 식품이 넘쳐나고 있다. 이에 따라 소비자들은 충분한 영양성분이 가미되지 않은 식단을 일상적으로 접하게 되고, 그로 인한 각종 불균형 신체 발달 및 질병들이 발병하고 있어 사회적 문제화 되고 있는 실정이다.Recently, society is rapidly changing, and various factors such as the advancement of women into society are overflowing with fast food foods for busy modern people. As a result, consumers are routinely exposed to diets that do not have sufficient nutritional components, and various unbalanced body developments and diseases arise, resulting in social problems.
인체 질병 및 노화를 유도하는 물질 중에 하나인 nitric oxide(NO)는 대기오염물질이며 담배연기에 포함된 수많은 화학물질중의 하나로서 독성을 가진 매우 불안한 기체이며 고농도에는 세포의 기질적 손상을 초래하는 free radical의 하나이다. Nitric oxide synthase (NOS)는 물리화학적 성질에 따라 Type Ⅰ, Ⅱ, Ⅲ 등 세종류의 동종 효소로 분류된다. 즉, Type I(neuronal NOS, nNOS)과 Type Ⅲ(endothelial NOS, eNOS)는 세포 속에 계속적으로 존재하기 때문에 지속적 NOS(constitutive NOS)로 분류되며, 상대적으로 일부 세포에서 lipopolysaccharide(LPS), cytokines 및 박테리아 독소 같은 특수한 자극제들에 노출되는 경우에만 발현되는 Type Ⅱ인 inducible NOS(iNOS)이 있다. 이러한 NOS에 의해 L-arginine이 L-citrulline으로 전환되면서 NO가 형성된다. Nitric oxide (NO), one of the substances that induce human diseases and aging, is an air pollutant. It is one of the many chemicals contained in tobacco smoke, which is a very unstable gas with toxicity. At high concentration, It is one of the free radicals. Nitric oxide synthase (NOS) is classified into three types of isoenzymes, Type I, II, and III, depending on their physicochemical properties. In other words, Type I (neuronal NOS, nNOS) and Type Ⅲ (endothelial NOS, eNOS) are classified as constitutive NOS because they are continuously present in the cells. In some cells, lipopolysaccharide (LPS), cytokines and bacteria There is an inducible NOS (iNOS), Type II, which is expressed only when exposed to specific stimuli such as toxins. This NOS converts NO to L-arginine into L-citrulline.
자극에 유도된 iNOS인 경우 오랜 기간 동안 다량의 NO를 생성하게 되고, 생성된 NO는 guanylyl cyclase의 활성과 동시에 주위 조직에 세포독성을 나타내는 것이 특징이다. NO는 매우 작으면서도 반응성이 있고 전기적으로 중성이기 때문에 합성된 곳에서 곧바로 확산되어 사방으로 퍼져 혈관 투과성, 부종 등의 염증반응을 촉진시킬 뿐만 아니라 염증매개체의 생합성을 촉진하여 염증을 심화시켜 종양을 유발하는 것으로 알려져 있다(Lee, H. J., Jeong, Y. S., Ryu, S. Y., and Ryu. J. H., “Inhibition of nitric oxide synthesis by 8-epi-xanthatin in activated RAW 264.7 cell”, Yakhak Hoeji ., 1998, 42:5.). 현재 NO에 대한 많은 연구 보고서들에 의해 생체에서 여러 가지 생리생화학적 현상의 messenger molecule로서 mediator, neurotransmitter 또는 regulator 등으로 다양하게 작용함이 밝혀지고 있다(Kuo, P. C. and Schroeder, R. A. “The cmerging multifaceted roles of nitric oxide. Ann Surg”, 1995, 221(3):220-235.). 일반적인 NO의 형성은 박테리아를 죽이거나 종양을 제거시키는 중요한 역할을 하지만, 병리적인 원인에 의한 과도한 NO의 형성은 정상세포의 손상을 초래하여 염증을 유발시키며 조직의 손상, 유전자 변이 및 신경 손상 등을 유발한다(Weisz, A., C Icatiello, I., and Esumi, H., “Regulation of the mouse inducible type nitric oxide synthase gene promoter by interferon- gamma, bacterial lipopolysaccharide and NG-monomethyl-L- arginine”, Biochem. J., 1996, 316: 209.).Stimulated iNOS produces a large amount of NO over a long period of time, and the produced NO is characterized by the activity of guanylyl cyclase and cytotoxicity to surrounding tissues. NO is very small, but reactive and electrically neutral, so it diffuses straight from the synthesis site to spread in all directions, promoting inflammatory reactions such as vascular permeability and edema, promoting the biosynthesis of inflammatory mediators, JH, "Inhibition of nitric oxide synthesis by 8-epi-xanthatin in activated RAW 264.7 cell", Yakhak, Hoeji . , 1998 , 42 : 5.). Many research reports on NO have been shown to act as mediators, neurotransmitters or regulators as messenger molecules of various physiological biochemical phenomena in living organisms (Kuo, PC and Schroeder, RA "The cmerging multifaceted roles of nitric oxide. Ann Surg ", 1995 , 221 (3): 220-235.). Although normal NO formation plays an important role in killing bacteria or eliminating tumors, excessive NO formation due to pathological causes causes damage to normal cells, causing inflammation, and damages tissue, gene mutation, and nerve damage The cause (Weisz, A., C Icatiello, I., and Esumi, H., "Regulation of the mouse inducible type nitric oxide synthase gene promoter by interferon- gamma, bacterial lipopolysaccharide and NG-monomethyl-L- arginine", Biochem J., 1996 , 316 : 209).
염증이 발생하게 되면 림프구(lymphocyte), 대식세포(macrophage), 내피세포 (endothelial cell), 섬유아세포(fibroblast)등 염증반응에 관여하는 세포들이 관여하게 되고 이 세포들이 lipopolysaccharide(LPS), interferon-γ(INF-γ) 및 tumor necrosis factor-α(TNF-α)등의 자극제들에 노출되게 되면 iNOS의 발현이 증가되고 이로 인한 NO 생성이 증가되어 염증반응을 촉진시킨다.When inflammation occurs, cells involved in inflammatory reactions such as lymphocytes, macrophages, endothelial cells, and fibroblasts are involved When these cells are exposed to stimulants such as lipopolysaccharide (LPS), interferon-γ (INF-γ) and tumor necrosis factor-α (TNF-α), the expression of iNOS is increased, .
염증반응 시 생겨나는 염증매개 물질들과, cytokine들이 c-Jun의 활성화를 통해 AP-1 전사인자를 활성화시켜 진피내의 무코다당류와 결합섬유들을 분해하는 matrix metalloprotease(MMPs)의 발현을 촉진시키고, 이것의 저해제인 tissue inhibitor of matrix metalloproteases(TIMPs)의 합성을 억제한다고 알려져 있다(Burger D, Rezzonico R, Li JM, Modoux C, Pierce RA, Welgus HG, et al. “Imbalance between interstitial collagenase and tissue inhibitor of metalloproteinases 1 in synoviocytes and fibroblasts upon direct contact with stimlated T lymphocytes: involvement of membrane- associated cytokines”, Arthritis Rheum., 1998, 41: 1748-59.) . Activation of AP-1 transcription factors through the activation of c-Jun by inflammatory mediators and cytokines during inflammatory reactions promotes the expression of matrix metalloproteases (MMPs) that break down mucopolysaccharides and binding fibers in the dermis, (Burger D, Rezzonico R, Li JM, Modoux C, Pierce RA, Welgus HG, et al., &Quot; Imbalance between interstitial collagenase and tissue inhibitor of metalloproteinases (TIMPs) 1 in synoviocytes and fibroblasts upon direct contact with stimulated T lymphocytes: involvement of membrane-associated cytokines ", Arthritis Rheum ., 1998 , 41 : 1748-59.).
무기질은 신체의 구성과 일부 신체기능을 조절하는 데 필수적인 요소이다. 특히, 칼슘은 뼈와 신경세포를 구성하며, 철분은 적혈구의 혈색소를 구성하는 등 인체의 뼈와 피를 형성함으로써 아동기에서부터 노년기에 이르기까지 인체 성장발육에 아주 중요한 무기질 성분이다. Minerals are an essential element in controlling body composition and some body functions. In particular, calcium constitutes bones and nerve cells, and iron constitutes the hemoglobin of red blood cells, forming the bones and blood of the human body, which is an important mineral component for human growth and development from childhood to old age.
칼슘은 체내 무기질 중 가장 많은 원소로 인체 내 총 칼슘의 양은 체중의 약 2% 정도로 대부분이 인산칼슘의 형태로 뼈와 치아의 성분을 이루고, 이밖에 혈장 중에 약간 존재하며 근육 및 신경의 기능조절, 혈액응고에 필요하다. 칼슘은 전 생애를 통해 필요하며, 특히 성장기, 임신기, 수유기에는 더욱 많이 필요하고, 성장이나 골다공증 및 관절염과 관련이 있는 중요한 성분이다(이성현, 황보영숙, 김지연, 이연숙, 칼슘급원식품의 체내이용성 연구, 한국영양학회지, 30(5): 499-505, 1997)Calcium is the most abundant element in the body. The total amount of calcium in the body is about 2% of body weight. Most of calcium is calcium phosphate, which is a component of bone and teeth. It is necessary for blood coagulation. Calcium is an essential ingredient throughout life, especially in growing, pregnancy, and lactation, and is an important component of growth, osteoporosis and arthritis (Lee Sung Hyun, Hwangbo Youngsook, Kim Ji Yeon, Lee Yun Sook, , Korean Nutrition Society, 30 (5): 499-505, 1997)
철은 미량원소로 헤모글로빈의 성분으로 산소 운반에 관여한다. 따라서 출혈성 질환, 월경개시시기, 임신, 출산, 성장기에는 수요가 높아져 음식의 종류에 주의하지 않으면 결핍되기 쉬운 성분이다(김천수, 홍희옥, 김정윤, 맹원재, 이정숙, 서울지역 여고생들의 식이 철분밀도에 따른 영양섭취상태 및 철분 급원식품에 관한 연구, 한국영양학회지, 40(4): 371-384, 2007).최근에는 염증성 질환 등 다양한 질병을 예방 및 치료하기 위해 기능성 식품, 기능성 화장료 및 치료제제 등 각 분야에서 인공물질이 아닌 천연물질을 이용한 연구가 활발히 진행되어지고 있다. 본 발명자들은 철과 칼슘과 같은 무기질 함량이 우수할 뿐만 아니라, 다른 곡물에 비해 단백질 함량이 우수하고, 스쿠알렌 및 토코페롤과 같은 우수한 성분이 함유되어 있는 아마란스의 항염 효과를 측정하였고 우수한 억제 효능이 있음을 확인하였다. Iron is a trace element that is involved in oxygen transport as a component of hemoglobin. Therefore, it is a component which is easily deficient if there is a high demand for hemorrhagic disease, menstrual onset period, pregnancy, childbirth, and growing period, (4): 371-384, 2007). In recent years, in order to prevent and treat various diseases such as inflammatory diseases, various foods such as functional foods, functional cosmetics and therapeutic agents Researches using natural materials that are not artificial materials are being actively carried out. The present inventors have measured the anti-inflammatory effect of amaranth which is excellent in the content of minerals such as iron and calcium, has an excellent protein content as compared with other grains, and contains excellent components such as squalene and tocopherol. Respectively.
본 발명의 목적은 인체에 무해하고 친환경적인 소재의 활용을 통해 좀 더 안전하고 효능이 우수한 천연물질의 개발을 통해 염증성 질환에 따른 다양한 질병을 예방하고, 건강한 영양성분을 함유한 제품을 개발하여 환자 및 성장기 어린이와 노약자들에게 우수한 영양조성식을 제공함을 목적으로 한다. 또한 본 발명의 또 다른 목적은 아마란스에서는 처음으로 항염효과 및 미백 효능에 대해서도 처음으로 그 기능을 밝힌다.The object of the present invention is to prevent the various diseases caused by inflammatory diseases through development of safer and more effective natural substances through the use of harmless and environmentally friendly materials to the human body and to develop products containing healthy nutrients, And to provide excellent nutritional formula to children and the elderly. Another object of the present invention is also the first to disclose its anti-inflammatory and whitening effects in Amaranth for the first time.
상기 목적을 달성하기 위하여 본 발명은 아마란스를 에탄올과 기타 유기용매들로 추출하여NO 억제 효과 및 iNOS 와 COX-2 단백질 억제 효과, 그리고 cytokine 발현 억제 효과를 갖는 아마란스 추출물을 제공한다. 또한 본 발명은 아마란스의 지상부와 종실 부분을 각각 70% 에탄올로 추출한 다음 유기용매인 헥산, 디클로로메탄, 에틸아세테이트, 부탄올 등의 유기용매를 가하여 다시 추출한 용매분획물을 제공한다. 본 발명의 추출용매로는 에탄올, 헥산, 디콜로로메탄, 에틸아세테이트, DMF(Dimethyl foramide), THF(Tetrahydrofuran), 클로로포름, 디에틸에테르 등이 사용가능하고 바람직하기로는 에탄올이 바람직하다.
To achieve the above object, the present invention provides an extract of Amaranth which has an NO inhibitory effect, an iNOS and COX-2 protein inhibitory effect, and an inhibitory effect on cytokine expression by extracting amaranth with ethanol and other organic solvents. Also, the present invention provides a solvent fraction obtained by extracting the above ground part and seedling part of amaranth with 70% ethanol, and then extracting again with an organic solvent such as an organic solvent such as hexane, dichloromethane, ethyl acetate, butanol and the like. As the extraction solvent of the present invention, ethanol, hexane, dichloromethane, ethyl acetate, dimethylformamide (DMF), tetrahydrofuran (THF), chloroform, diethyl ether and the like can be used.
또한, 본 발명은 아마란스 전초와 종실을 70% 에탄올로 침지시켜 반복 추출하고 농축하여 에탄올 추출물을 얻은 다음 이를 20% 에탄올로 현탁시키고 헥산, 디클로로메탄, 에틸아세테이트 및 부탄올 등의 유기용매를 차례대로 가하여 그에 해당되는 용매 추출분획을 제조한다.
In the present invention, the amaranth seeds and the seedling are dipped in 70% ethanol and repeatedly extracted and concentrated to obtain an ethanol extract, which is then suspended in 20% ethanol and an organic solvent such as hexane, dichloromethane, ethyl acetate and butanol is added successively And a corresponding solvent-extracted fraction is prepared.
본 발명자는 상기 과정으로 추출 및 분획한 혼합물질이 항염효과 등의 활성을 나타냄을 밝히고, 영양성분 분석을 위해 식품공전법에 의해 아미노산과 무기질 성분을 확인하였고, HPLC 분석 방법으로 스쿠알렌과 토코페롤 등의 함량을 확인하였다.
The inventors of the present invention have found that a mixed substance extracted and fractioned by the above process exhibits activity such as anti-inflammatory effect, and amino acid and inorganic components are identified by a food or drink method in order to analyze nutritional components, and squalene and tocopherol Respectively.
본 발명의 아마란스 추출물 또는 아마란스 추출물의 용매분획물을 유효성분으로 함유하는 항염효과를 가지는 건강보조식품 조성물은 죽, 선식, 씨리얼, 음료 등의 제형으로 제공될 수 있으며, 본 발명의 아마란스 추출물 또는 아마란스 용매분획물은 건강보조식품 조성물 전체 중량의 0.01 내지 10 중량 % 함유하는 것이 바람직하다. 0.01 중량% 이하 함유시는 본 발명의 염증효과를 기대하기 어렵고, 10 중량% 이상 함유시는 제제화에 문제가 있다.The anti-inflammatory health supplement composition containing the amaranth extract of the present invention or the solvent fraction of the amaranth extract as an active ingredient may be provided in a form such as a porridge, a wire, a cereal, a drink, and the like. The amaranth extract or the amaranth solvent It is preferred that the fraction contains 0.01 to 10% by weight of the total weight of the composition. When it is 0.01% by weight or less, it is difficult to expect the inflammatory effect of the present invention, and when it is contained by 10% by weight or more, there is a problem in formulation.
본 발명의 아마란스 추출물은 항염 효과가 우수하고 특히 아마란스 전초부분은 에탄올 추출물로도 그 효능이 우수하여 앞으로 종실과 전초부분이 다양하게 식품으로 사용할 수 있어 천연식물에서 유래한 안전하고 효과적인 항염효과를 갖는 식품 조성물로 널리 사용될 수 있다.The amaranth extract of the present invention has an excellent anti-inflammatory effect. Especially, the amaranth outermost portion has excellent efficacy as an ethanol extract, and can be used as a food in a variety of seeds and outpost portions. Thus, it has a safe and effective anti- It can be widely used as a food composition.
또한, 본 발명에 의거 얻어진 아마란스 추출물은 식품은 물론 화장품과 의약품 분야에도 널리 응용될 수 있다.The amaranth extract obtained according to the present invention can be widely applied not only to foods but also to cosmetics and pharmaceuticals.
도 1은 Western blot analysis를 이용하여 종실에서 분리한 에틸아세테이트층의 NO 생성에 관여하는 유전자들의 억제 효과를 확인한 결과 iNOS를 현저하게 억제하고, COX-2도 농도 의존적으로 억제 효과가 있음을 확인할 수 있었다.
도2는 도2에서 자주색 아마란스 상층부의 에탄올 조출물의 iNOS와 COX-2 단백질 발현 억제를 확인한 결과, 두 단백질 모두 농도의존적으로 현저하게 발현 억제함을 확인하였다.
도3은 아마란스 추출 방법을 나타냄.
도4는 아마란스 지상부 조추출물의 NO 억제효과와 세포 독성
도5는 아마란스 종실 조추출물의 NO 억제효과와 세포 독성
도6은 아마란스 종실 에틸아세테이트층의 NO 억제효과와 세포 독성
도7은 아마란스 지상부 조추출물의 TNF-α 억제 효과
A: 자주색 아마란스 70% EtOH 추출(100ug/mL),
B: 자주색 아마란스 70% EtOH 추출(200 ug/mL).
C: 자주색 아마란스 70% EtOH 추출(400 ug/mL).
D: 아마란스 혼합물 70% EtOH 추출(100ug/mL),
E: 아마란스 혼합물 70% EtOH 추출(200 ug/mL),
F: 아마란스 혼합물 70% EtOH 추출(400 ug/mL)
도8은 아마란스 지상부 조추출물의 IL-6 억제 효과
A: 자주색 아마란스 70% EtOH 추출(100ug/mL),
B: 자주색 아마란스 70% EtOH 추출(200 ug/mL).
C: 자주색 아마란스 70% EtOH 추출(400 ug/mL).
D: 아마란스 혼합물 70% EtOH 추출(100ug/mL),
E: 아마란스 혼합물 70% EtOH 추출(200 ug/mL),
F: 아마란스 혼합물 70% EtOH 추출(400 ug/mL)
도9는 아마란스 종실 에틸아세트층의 TNF-α 억제 효과
도10은 아마란스 자주색 전초 에탄올 추출물의 PGE2 억제 효과
도11은 아마란스 종실 에?아세테이트 추출물의 PGE2 억제 효과
도12는 아마란스 잎과 종실의 단백질 함량
* A : Raw seeds(grinding)
B : Roasted seeds
C : Raw Seeds(do not grind)
D : leaf
도13은 아마란스 잎과 종실의 철 함량
도14는 아마란스 잎과 종실의 칼슘 함량
도15는 아마란스와 다른 곡물의 단백질 함량 비교
도16은 아마란스와 다른 곡물의 철 함량 비교
도17은 아마란스와 다른 곡물의 칼슘 함량 비교
도18은 아마란스 종실의 스쿠알렌 함량
도19는 생 아마란스 헥산추출물의 스쿠알렌 함량
도20은 볶은 아마란스 헥산추출물의 스쿠알렌 함량
도21은 아마란스 종실의 α-토코페롤 함량
도22는 생 아마란스 70% 에탄올 추출물의 α-토코페롤 함량
도23은 볶은 아마란스 헥산 추출물의 α-토코페롤
도24는 아마란스 종실의 β-토코페롤 함량FIG. 1 shows that the inhibitory effect of genes involved in NO production in the ethyl acetate layer isolated from the seeds using Western blot analysis showed that iNOS was significantly suppressed and COX-2 was also inhibited in a concentration-dependent manner there was.
FIG. 2 shows that the inhibition of iNOS and COX-2 protein expression of the ethanol extract of purple amaranth upper layer was remarkably inhibited by both proteins in a concentration-dependent manner.
Figure 3 shows the amaranth extraction method.
Fig. 4 shows the NO inhibitory effect and cytotoxicity
Figure 5 shows the NO inhibitory effect and cytotoxicity
Figure 6 shows the NO inhibitory effect and cytotoxicity of the amaranth seed ethyl acetate layer
Figure 7 shows the effect of TNF-alpha inhibition
A:
B: Purple Amaranth 70% EtOH Extraction (200 ug / mL).
C:
D:
E:
F:
FIG. 8 shows the effect of the extract of the above-ground Amaranth extract on the IL-6 inhibitory effect
A:
B:
C:
D:
E:
F:
Figure 9 shows the effect of the amaranth seed ethyl acetate layer on the TNF-
10 shows the PGE2 inhibitory effect of the amaranth purple pre-plant ethanol extract
11 shows the PGE2 inhibitory effect of? Acetate extract on amaranth seeds
Figure 12 shows the protein content of amaranth leaves and seeds
* A: Raw seeds (grinding)
B: Roasted seeds
C: Raw Seeds (do not grind)
D: leaf
Figure 13 shows the iron content of amaranth leaves and seeds
14 shows the calcium content of amaranth leaves and seeds
15 shows the comparison of the protein content of amaranth and other grains
Figure 16 compares the iron content of amaranth and other grains
17 shows the comparison of the calcium content of amaranth and other grains
18 shows the squalene content of amaranth seeds
19 shows the squalene content of raw amaranth hexane extract
20 shows the squalene content of roasted amaranth hexane extract
Figure 21 shows the alpha-tocopherol content of the amaranth seeds
22 is a graph showing the change in the? -Tocopherol content
23 is a graph showing the effect of the roasted amaranth hexane extract on? -Tocopherol
Figure 24 shows the < RTI ID = 0.0 > β-tocopherol content
본 발명은 아마란스 지상부 및 종실부분을 각각 70% 에탄올에 침지시켜 반복 추출하여 항염효능을 가지는 아마란스 에탄올 추출물을 얻는다. 또한 아마란스의 에탄올 추출물을 다시 유기용매인 헥산, 디클로로메탄, 에틸아세테이트, 부탄올 등을 이용하여 추출한 후 항염효과를 가지는 추출 분획을 제공한다.
The present invention provides amaranth ethanol extract having anti-inflammatory activity by repeatedly extracting amaranth surface portion and seed portion by immersing in 70% ethanol, respectively. In addition, the ethanol extract of amaranth is further extracted with an organic solvent such as hexane, dichloromethane, ethyl acetate, butanol, etc., to provide an extract fraction having anti-inflammatory effect.
본 발명은 아마란스 상층부 및 종실부를 70% 에탄올로 추출하여 농축한 에탄올 추출물을 이용하여 유기용매로 분획함으로서 항염 효과가 우수한 아마란스 에틸아세테이트 분획을 제공한다.
The present invention provides an amaranth ethyl acetate fraction having an excellent anti-inflammatory effect by fractionating the upper part of amaranth and the seedling part with 70% ethanol and fractionating the concentrated ethanol extract with an organic solvent.
본 발명은 상기 아마란스 에탄올 추출물을 유효성분으로 하는 항염활성이 있는 조성물을 제공한다. 구체적으로는 상기 추출물은 염증 생성을 억제하여 염증 예방 식품 및 향장품과 의약품 등의 조성물로서 사용될 수 있다.
The present invention provides a composition having anti-inflammatory activity using the above-described amaranth ethanol extract as an active ingredient. Specifically, the extract can be used as a composition for preventing inflammation and inhibiting the production of inflammation, as well as for an anti-inflammatory food, an aromatic product, and a medicine.
상기 아마란스 상층부 추출물은 에탄올 조추출물에서 NO 억제 효과 및 iNOS와 COX-2 단백질 억제 효과가 우수하고, IL-6와 PEG2 역시 농도의존적으로 억제해 좋은 염증 억제효과를 보였다. 종실 추출물의 항염 효과는 NO 억제 활성을 이용하여 측정한 결과, 유기 용매 분획 중 에틸아세테이트 분획층에서 가장 좋은 효과를 나타내었다. 또한 iNOS와 COX-2 단백질 발현 억제 효과를 확인하기 위하여 Western blotting을 통해 측정한 결과 에틸아세트층에서 iNOS와 COX-2 단백질 발현을 농도 의존적으로 억제하고 있음을 확인하였고, 특히 iNOS는 억제 효과가 탁월하게 나타내었다.The above extract of Amaranth showed excellent NO inhibitory effect and inhibitory effect on iNOS and COX-2 protein in ethanol crude extract, and IL-6 and PEG2 also showed a dose-dependent inhibitory effect on inflammation. The anti - inflammatory effect of seed extract was best measured by the ethyl acetate fraction in the organic solvent fraction. In order to confirm the inhibitory effect of iNOS and COX-2 protein expression, Western blotting assay showed that iNOS and COX-2 protein expression was suppressed in a concentration-dependent manner in the ethyl acetate layer. In particular, Respectively.
또한 본 발명에서는 제주도에서 자란 아마란스의 영양성분을 타지역에 비해 우수함을 확인하고자 식품공전법과 HPLC를 이용하여 측정한 결과 칼슘과 철분에서 월등히 우수한 함량 보유를 확인하였고, 단백질 함량도 비교적 높게 확인되었다. 특히 스쿠알렌과 토코페롤 함량은 타지역에서 자란 아마란스에 비해 월등히 높은 수치를 보여, 제주산 아마란스가 영양적으로 우수하게 식품 및 화장품류나 약품에 다양하게 활용할 수 있음을 확인하였다.
In addition, in the present invention, it was confirmed that the nutritional content of amaranth grown in Jeju Island was superior to other regions, and the content of calcium and iron was higher than that of other regions, and the protein content was also confirmed to be relatively high. Especially, the content of squalene and tocopherol was significantly higher than that of other areas, indicating that Jeju amaranth can be used in various foods, cosmetics and pharmaceuticals.
<실시 예1 : ≪ Example 1: 아마란스의Amaranth 지상부로부터 From above 용매분획물의Of the solvent fraction 제조> Manufacturing>
본 실험에 사용된 아마란스 전초와 종실은 제주도 어음리에서 재배하여 수확한 것을 사용하였다. 아마란스 전초는 3가지 색상의 아마란스 혼합물과 자주색 아마란스 전초를 가지고 실험하였으며 종실은 분쇄기를 이용하여 분쇄한 것을 사용 하였다.Amaranth plantlets and seedlings used in this experiment were grown and harvested from Jeju Island. The amaranth outpost was experimented with amaranth mixture of three colors and purple amaranth outpost. The seedlings were pulverized using a pulverizer.
제주도 애월읍 어음2리에서 재배한 아마란스 전초와 종실을 채취하여 깨끗이 손질한 후 잘게 파쇄 하여 전초는 80% 에탄올로 3L 3회 교반 추출 후 감압 농축하여 15g의 에탄올 추출물을 얻었다. 종실은 80% 에탄올로 5L를 추출 후 감압 농축하여 12g의 에탄올 조추출물을 얻었다. 건조 조추출물 12g을 물 1L로 현탁 시킨 후 헥산 1L를 첨가하여 헥산층을 분리하고, 이것을 2회 반복하여 헥산 분획층을 얻었다. 남은 물층에 디클로로메탄 1L를 가하여 디클로로메탄층을 분리하고, 이것을 2회 반복하여 디클로로메탄 분획층을 얻었다. 남은 물층에 에틸아세테이트 1L를 가하여 에틸아세테이트층을 분리하고, 이것을 2회 반복하여 에틸아세테이트 분획층을 얻었다. 최종적으로 물층에 부탄올 1L를 2회 가하여 부탄올층과 물층으로 분획하여 사용하였다.Amaranth outposts and seedlings cultivated on Aewol-eup berry cultivated in Jeju Island were picked and finely crushed. After finely crushing, the outpastes were extracted with
아마란스 추출물의 항염 효과를 조사하기 위하여 상기 실시예 1에서 얻은 각 분획물의 항염효과를 NO 억제 효과와 RAW 264.7 세포를 통해 조사하였다.
To investigate the anti-inflammatory effect of the extract of Amaranth, the anti-inflammatory effect of each fraction obtained in Example 1 was investigated through NO inhibitory effect and RAW 264.7 cells.
<실험 예1 : Experimental Example 1: NONO 억제 활성과 And 세포생존률Cell survival rate (( LDHLDH ) 측정>) Measurement>
Raw264.7 세포를 10% FBS가 포함된 배지를 사용하여 24 well plate에 1.8×105cells/mL로 분주하고 18시간 뒤, 10% FBS가 포함된 배지에 LPS(1ug/mL)가 포함되지 않은 것 또는 포함한 배지에 추출물을 100,200,400 ug/mL로 처리하였고, 아마란스 종실 분획물의 NO생성 억제 능력을 조사하기 위해 위와 같은 조건의 배지에 각각 n-hexan, MC, EA, BuOH, H2O로 분획한 아마란스 종실 분획물을 200ug/ml로 처리하거나 또는 농도를 세분화(25, 50, 100, 200 ug/ml)한 후에 37℃, 5% CO2에서 24시간 배양하였다. Raw264.7 세포로부터 생성된 NO의 양은 세포 배양액 중에 존재하는 NO2의 형태로서 Griess reagentsystem(Promega, Madison, WI, USA)을 이용하여 측정하였다. 상층액을 96well plate에 각각 분주한 후 제조사에서 제시한 방법대로 Griess 시약을 첨가하여 반응시킨 뒤 540 nm 파장에서 microplate reader(BIO-RAD, Hercules, CA, USA)를 이용하여 nitrite 농도를 측정하였다. Raw264.7 cells were plated at 1.8 × 10 5 cells / mL in a 24-well plate using a medium containing 10% FBS. After 18 hours, LPS (1 ug / mL) was included in the medium containing 10% FBS The extracts were treated with 100, 200, and 400 ug / mL of the extracts in the medium containing or without aminobutyric acid and fractionated with n-hexane, MC, EA, BuOH and H 2 O An amaranth seed fraction was treated with 200 ug / ml or fractionated (25, 50, 100, 200 ug / ml) and cultured at 37 ° C and 5% CO 2 for 24 hours. The amount of NO produced from Raw264.7 cells was measured using a Griess reagent system (Promega, Madison, WI, USA) as the form of NO 2 present in the cell culture. The supernatant was dispensed into 96-well plates, and the Griess reagent was added and reacted at the wavelength of 540 nm using a microplate reader (BIO-RAD, Hercules, CA, USA).
세포생존율을 측정하기 위해 nitrite를 측정하였던 동일한 배지의 상측액을 96well plate에 각각 50ul 씩 분주한 후 LDH 시약 50ul를 분주 후 30분 동안 방치한다. 30분 후에 stop solution을 50ul 넣어 반응을 stop 시킨 후 490 nm 파장에서 microplate reader(BIO-RAD, Hercules, CA, USA)를 이용하여 측정한다.In order to measure the cell viability, 50ul of the upper liquid of the same medium in which the nitrite was measured was dispensed into each well of a 96-well plate, and 50ul of the LDH reagent was dispensed and left for 30 minutes. After 30 minutes, stop the reaction by adding 50 μl of stop solution, and measure with a microplate reader (BIO-RAD, Hercules, CA, USA) at a wavelength of 490 nm.
아마란스 3종 혼합
A mixture of three kinds of amaranth
자주색 아마란스
Purple amaranth
상기 표 1에 나타난 바와 같이 아마란스 조추출물인 상태에서 유효간 NO 억제 효과As shown in Table 1, in the case of Amaranth crude extract,
를 보였다. 특히, 지상부 3종 혼합보다 자주색 단일 추출물이 세포독성이 없는 상태에서 농도 의존적으로 좋은 NO 억제 효과를 확인할 수 있었다.Respectively. Especially, it was possible to confirm a good NO inhibitory effect in a concentration - dependent manner in the absence of cytotoxicity of the purple single extract from the mixture of three kinds of the above - ground parts.
100ug/mL
100 ug / mL
상기 표 2에 나타난 바와 같이 종실인 경우 에틸아세트층에서 가장 우수한 NO 억제 효과가 나타남을 확인하였다.As shown in Table 2, it was confirmed that the best inhibitory effect of NO on the ethyl acetate layer was obtained when the seeds were seeded.
<실험 예2 : Experimental Example 2: CytokineCytokine ( ( TNFTNF -a, -a, ILIL -6) 억제 실험 -6) inhibition experiment
Raw 264.7 cell을 1.8 x 105 cell/mL의 밀도로 배양하였다. LPS(1ug/mL)로 RAW 264.7 cell을 자극하고 추출물을 여러 농도로 처리하여 37℃ 10% CO2 항온기에서 24시간 배양하였다. 24시간 뒤 세포 상층액을 얻어 ELISA법으로 cytokine의 발현을 측정하였다. ELISA는 BD pharmingen (San Diego, CA, USA)에서 Mouse ELISA kit for IL-6,IL-1b, TNF-를 구입하여 시행하였다.
Raw 264.7 cells were cultured to cell density of 1.8 x 10 5 cell / mL. RAW 264.7 cells were stimulated with LPS (1 ug / mL) and the extracts were treated at various concentrations and cultured in a 37 ° C, 10% CO 2 incubator for 24 hours. Twenty-four hours later, cell supernatants were obtained and cytokine expression was measured by ELISA. ELISA was performed by purchasing a mouse ELISA kit for IL-6, IL-1b and TNF- from BD pharmingen (San Diego, CA, USA).
<실험 예3 : Experimental Example 3: WesternWestern blotblot 를 통한 Through iNOSiNOS 및 And COXCOX -2 억제 효과 실험>-2 inhibitory effect experiment>
Raw264.7 세포를 1.2×106 cells/ml의 밀도로 60 mm dish에 분주하고 18시간 뒤 10% FBS가 함유된 배지에 LPS(1 ug/ml)가 포함되지 않은 것 또는 포함한 배지에 아마란스 추출물 50, 100, 200, 400 ug/ml의 농도로 처리한 뒤 24시간 37℃, 5% CO2 배양기에서 배양하였다. 24시간 후에 배양액을 제거, 2~3회 PBS로 세척 후 100 μl의 lysis buffer를 첨가하고, 30분 동안 4℃에서 lysis 시킨다. 30분 후 15,000 rpm에서 15분간 원심분리 하였다. 단백질 농도는 BSA (Bovine serum albumin)을 표준화하여 Protein Assay Kit (Bio-Rad, USA)를 사용하여 정량하였다. Total cell lysate를 4-12% Bis -Tris Gel 1.0mm X 15well로 크기에 따라 분리한 후, Polyvinylidene difluoride (PVDF) membrane (Bio-Rad, USA)에 200mA로 120분 동안 transfer하였다. 그리고 membrane의 blocking은 5% skim milk가 함유된 TTBS (TBS (Tris-buffered saline) + 0.1% Tween 20) 용액에서 상온에서 3시간동안 실시하였다. 반응이 끝난 뒤에 측정하고자 하는 항체(iNOS 1:2000, COX-2 1:2000, β-actin 1:2000)를 각각 첨가하여 상온에서 2시간 또는 3시간 동안 교반 후 4℃에서 24시간 반응시켰다. 반응 시킨 후 TTBS와 D.W로 3회 세척하고 2차 항체 (1: 5000)와 상온에서 3시간 반응시킨 뒤에 TTBS와 D.W로 4~5회 세척하여 ECL 기질(Amersham Biosciences, Piscataway, Nj, USA)과 1~3분간 반응 후 X-ray 필름에 감광하였다.
Raw 264.7 cells were seeded in a 60 mm dish at a density of 1.2 × 10 6 cells / ml. After 18 hours, the culture medium containing 10% FBS or without LPS (1 μg / ml) 50, 100, 200, and 400 ug / ml, and cultured in a 5% CO 2 incubator at 37 ° C for 24 hours. After 24 hours, remove the culture medium, wash with PBS 2-3 times, add 100 μl of lysis buffer, and lysis at 4 ° C for 30 minutes. After 30 minutes, the cells were centrifuged at 15,000 rpm for 15 minutes. Protein concentration was determined by standardizing BSA (Bovine serum albumin) using Protein Assay Kit (Bio-Rad, USA). Total cell lysate was separated by size from 4-12% Bis-Tris Gel 1.0
<실시 예2 : Example 2: 아마란스amaranth 종실부터From seedling HPLCHPLC 분석시료 제조> Analytical sample preparation>
본 분석에 사용된 아마란스 종실은 제주도 어음리에서 재배하여 수확한 것을 사용하였다. Amaranth seeds used in this analysis were harvested from Jeju Island.
각각의 종실은 가공하지 않은 Raw상태의 시료와 고온에서 볶아낸 것을 사용한 Popping상태의 시료를 사용하였으며, 각각은 Raw, Popping이라 하였다.
Raw and popping samples were used for each seed, and popping samples were used for roasting at high temperatures.
추출extraction
○ 70%EtOH 추출 : 분쇄한 두 가지 상태의 식물시료를 Raw상태의 것은 500g을 Popping상태의 것은 120g을 70%에탄올 각각 5L, 1.2L에 넣고 6일간 추출하였다. 이를 여과한 다음 감압농축기를 사용하여 농축하고 동결건조한 다음 사용하였다.○ 70% EtOH Extraction: 500 g of the plant sample in the Raw state and 120 g of the plant sample in the popped state were extracted in 6 L of 5 L and 1.2 L of 70% ethanol, respectively. It was filtered, concentrated using a vacuum concentrator, lyophilized and then used.
○ Hexane추출 ○ Hexane extraction
- Squalene 분석 : 분쇄한 두 가지 상태의 시료를 각각 5g을 헥산 40ml에 넣고 24시간 동안 상온에서 교반시키며 추출하였다.Squalene analysis: Five grams of each of the two milled samples were added to 40 ml of hexane and extracted with stirring at room temperature for 24 hours.
- Tocopherol 분석 : 분쇄한 두 가지 상태의 시료를 각각 20g을 헥산 1L에 넣고 혼합하여 30분씩 3회 sonication하여 추출하였다.- Tocopherol analysis: Twenty grams of each of the two milled samples were mixed with 1 L of hexane, and the mixture was sonicated three times for 30 minutes each.
위의 두 가지 추출물은 여과한 다음 감압농축기를 사용하여 농축하고 동결건조한 다음 사용하였다.
The two extracts were filtered, concentrated using a vacuum concentrator, and lyophilized before use.
시료전처리Sample preparation
70%EtOH 추출물은 동결건조한 추출물에 ACN용매를 넣어 100mg/ml의 농도로 제조하여 충분히 섞일 수 있도록 sonication하며, 0.45㎛의 실린지 필터를 이용하여 여과 후 바이알에 담아 준비하였다. The 70% EtOH extract was prepared by adding ACN solvent to the lyophilized extract at a concentration of 100 mg / ml and sonication so that the mixture could be mixed well. The syringe was filtered using a syringe filter of 0.45 μm and prepared in a vial.
Hexane추출물은 동결건조한 추출물에 ACN용매를 넣어 10mg/ml의 농도로 제조하여 충분히 섞일 수 있도록 sonication하며, 0.45㎛의 실린지 필터를 이용하여 여과 후 바이알에 담아 준비하였다.
Hexane extract was prepared by adding ACN solvent to the lyophilized extract at a concentration of 10 mg / ml and sonication so as to be mixed thoroughly. The extract was filtered and prepared in a vial using a 0.45 μm syringe filter.
표준시료의 제조Preparation of standard samples
Squalene : ACN용매에 녹여 0.1, 0.5, 1mg/ml의 농도로 제조하였다.Squalene was dissolved in ACN solvent and prepared at concentrations of 0.1, 0.5, and 1 mg / ml.
α-Tocopherol : ACN용매에 녹여 0.05, 0.1, 0.5, 1mg/ml의 농도로 제조하였다.α-Tocopherol was dissolved in ACN solvent and prepared at concentrations of 0.05, 0.1, 0.5 and 1 mg / ml.
β-Tocopherol : ACN용매에 녹여 0.1, 0.5, 1mg/ml의 농도로 제조하였다.
β-Tocopherol was dissolved in ACN solvent and prepared at concentrations of 0.1, 0.5 and 1 mg / ml.
<실험 예4 : Experimental Example 4: SqualeneSqualene 및 And TocopherolTocopherol 류 분석> Flow analysis>
SqualeneSqualene 분석analysis
스쿠알렌 분석을 위한 PDA detector의 파장은 208nm이었다. 이동상으로 사용된 ACN용매는 각각 solvent A와 solvent B에 나눠 담아 50:50의 비율을 사용하였다. 흐름속도는 0.4ml/min이었으며 각각 10분간 running하였다.
The wavelength of the PDA detector for squalene analysis was 208 nm. The ACN solvent used as the mobile phase was divided into solvent A and solvent B, and a ratio of 50:50 was used. The flow rate was 0.4 ml / min and each run for 10 minutes.
TocopherolTocopherol 류 분석Flow analysis
토코페놀 분석을 위한 PDA detector의 파장은 284nm이었다. 이동상으로 사용된 ACN용매는 각각 solvent A와 solvent B에 나눠 담아 50:50의 비율을 사용하였다. 흐름속도는 0.4ml/min이었으며 각각 10분간 running하였다.The wavelength of the PDA detector for tocophenol analysis was 284 nm. The ACN solvent used as the mobile phase was divided into solvent A and solvent B, and a ratio of 50:50 was used. The flow rate was 0.4 ml / min and each run for 10 minutes.
(mean)3 species
(mean)
제주 어음리
Jeju Compilation
* A : Raw seeds(70%EtOH추출)* A: Raw seeds (70% EtOH extraction)
B : Roasted seeds(70%EtOH추출) B: Roasted seeds (70% EtOH extraction)
A' : Raw seeds(Hexans추출) A ': Raw seeds (Hexans extraction)
B' : Roasted seeds(Hexans추출) B ': Roasted seeds (Hexans extraction)
상기 표 3에서와 같이 제주 어음리에서 재배 생산된 아마란스는 기존 농림부연구보고서(고영양 곡물자원 아마란스의 유효성분 활용 및 가공제품 개발, 2002년)에서 제시된 경기 지역에서 재배된 아마란스에 비해 스쿠알렌이나 토코페롤 함량이 월등하게 높은 것을 확인할 수 있었다. As shown in Table 3, amaranths grown in Jeju harvesting showed higher content of squalene and tocopherol than the amaranth grown in the Gyeonggi province, which was presented in the previous report of the Ministry of Agriculture and Forestry (high-nutrient grain resource amaranth application of active ingredients and processing product development, 2002) It was able to confirm that it was remarkably high.
<실시 예3 : 아미노산 분석 용액 제조>≪ Example 3: Preparation of amino acid analysis solution >
단백질구성 아미노산Protein-constituting amino acids
○ 시스틴, 트립토판 이외의 아미노산 정량용 시험용액○ Test solution for quantification of amino acids except cystine and tryptophan
단백질로서 약 10 mg을 함유하는 검체를 가수분해 시험관에 정밀히 달아 넣는다. 0.05%(w/v) 2-머캅토에탄올(C2H6SO)을 함유한 6 N 염산을 단백질양에 대하여 약 1,000배량 즉, 10 mL를 가한다.A sample containing about 10 mg of protein is precisely weighed into a hydrolysis test tube. Add about 1,000 times, that is, 10 mL of 6 N hydrochloric acid containing 0.05% (w / v) 2-mercaptoethanol (C 2 H 6 SO) to the amount of protein.
드라이아이스에탄올로 동결한 후 탈기장치에 장착하여 융해, 동결을 수회 반복하며 충분히 탈기한다. 봉관하여, 정온가열로에서 110℃±1℃, 22～24시간 가수분해한다. 가수분해 종료 후 봉관을 절단하여 즉시 감압하여 40℃에서 농축 건조하여 염산을 제거한다. 염산을 최대한 제거하기 위하여 잔사에 물을 가해 다시 농축 건조한다. 0.2 N 구연산나트륨 완충액(pH 2.2) 또는 0.02 N 염산용액으로 일정량으로 만들어 시험용액으로 한다. 침전이 있는 경우에는 멤브레인 필터를 사용하여 여과한다.After freezing with dry ice ethanol, install it in the degasser, melt and freeze repeatedly several times and thoroughly degas. With respect to the rods, hydrolyze in a constant temperature furnace at 110 ° C ± 1 ° C for 22-24 hours. After completion of the hydrolysis, the shaft tube is cut, and immediately decompressed, and concentrated and dried at 40 ° C to remove hydrochloric acid. To remove the hydrochloric acid as much as possible, water is added to the residue, and it is concentrated and dried again. Prepare a test solution with a certain amount of 0.2 N sodium citrate buffer (pH 2.2) or 0.02 N hydrochloric acid solution. If there is a precipitate, filter using a membrane filter.
○ 시스틴 정량용 시험용액○ Test solution for cystine determination
단백질 약 10 mg 함유하는 검체를 정밀히 달아 미리 0℃로 냉각시켜 놓은 과개미산 용액 10 mL를가하여 단백질이 가용성인 경우 0℃에서 4시간, 불용성인 경우 0℃에서 하루 밤 방치한다. 방치 후 공기를 흡입하고 소량의 물을 가해 동결 건조한다. 잔사를 0.05%(v/v) 2-머캅토에탄올 함유의 6 N 염산 10 mL를 사용하여 가수분해 시험관으로 옮긴다. 동결한 후, 탈기장치에 장착하여 충분히 탈기한 후 봉관하여 110±1℃에서 18시간 분해한다. 이하 방법은 (1) 시스틴, 트립토판 이외의 아미노산 정량용 시험용액에 따라 시험용액을 조제한다.10 mL of protein and 10 mL of a solution of formic acid, which had been cooled to 0 ° C beforehand, were precisely weighed and stored at 0 ° C for 4 hours when the protein was soluble and at 0 ° C overnight if insoluble. After left standing, air is sucked in and a small amount of water is added to freeze-dry. Transfer the residue to a hydrolysis test tube using 10 mL of 6 N hydrochloric acid containing 0.05% (v / v) 2-mercaptoethanol. After freezing, it is mounted on a degasser, thoroughly degassed, and then disassembled at 110 ± 1 ° C for 18 hours. In the following method, (1) Prepare the test solution according to the test solution for quantification of amino acids other than cystine and tryptophan.
○ 트립토판 정량용 시험용액○ Test solution for quantitation of tryptophan
단백질 10 mg을 함유하는 검체를 가수분해 시험관에 정밀히 달아 넣고, 가용성전분 100 mg을 한 다음 4.2 N 수산화나트륨용액 3 mL를 가한다. 동결시킨 후, 탈기장치에 장착하여 충분히 탈기하고 밀봉하여 135±1℃에서 22시간 가수분해한다. 가수분해 후 봉관을 잘라 열고 6 N 염산으로 중화하여 0.2 N 구연산나트륨완충액(pH 4.25)으로 일정량을 만들어 시험용액으로 한다. 용액이 탁한 경우 30,000～40,000 rpm에서 30분간 원심분리하여 상등액을 취한다.
A sample containing 10 mg of protein is precisely weighed into a hydrolysis test tube, 100 mg of soluble starch is added, and then 3 mL of 4.2 N sodium hydroxide solution is added. After freezing, it is mounted on a degasser, thoroughly degassed, sealed, and hydrolyzed at 135 ± 1 ° C for 22 hours. After hydrolysis, cut off the capillary tube, neutralize with 6 N hydrochloric acid and make a test solution with 0.2 N sodium citrate buffer (pH 4.25). If the solution is cloudy, centrifuge at 30,000 to 40,000 rpm for 30 minutes to take the supernatant.
유리 아미노산Free amino acid
○ 물에 의한 추출○ Extraction by water
고체 또는 페이스트상의 검체에 적용된다.It is applied to solid or paste-like specimens.
균질한 검체 약 1 g을 정밀히 달아 약 10배량을 물을 가해 비등 수욕상에서 가열하여 응고시킨 다음 여과하여 물층을 취한다. 잔사는 2～3회 소량의 물로 세정하고, 세액은 앞서의 물과 합한다. 지방이 있는 경우에는 에테르로 추출하여 제거한다.Approximately 1 g of a homogeneous sample is precisely weighed, and about ten times the amount of water is added. The mixture is heated in a boiling water bath to coagulate and then filtered to take the water layer. The residue is washed with a small amount of water two to three times, and the washing liquid is combined with the above-mentioned water. If there is fat, remove with ether.
물층을 감압하에 농축 건조하여 얻은 잔사를 0.2 N 구연산나트륨 완충액(pH 2.2) 또는 0.02 N 염산으로 용해하여 일정량으로 하여 시험용액으로 한다. 불용물질이 있는 경우에는 멤브레인 필터를 사용하여 여과한다.The water layer is concentrated and dried under reduced pressure, and the residue is dissolved in 0.2 N sodium citrate buffer (pH 2.2) or 0.02 N hydrochloric acid to obtain a test solution. When insoluble substances are present, they are filtered using a membrane filter.
○ 트리클로로초산법○ trichloroacetic acid method
액체상의 검체에 적용한다.Apply to liquid specimens.
검체 1.0～3.0 mL를 시험관에 달아 16% 트리클로로초산용액을 동용량 가하여 15분간 진탕한다. 3,000 rpm에서 15분간 원심분리하여 상등액을 시험용액으로 한다.
1.0 to 3.0 mL of the specimen is placed in a test tube, and 16% trichloroacetic acid solution is added to the same volume and shaken for 15 minutes. Centrifuge at 3,000 rpm for 15 minutes and use the supernatant as the test solution.
<실험 예5 : 아미노산 분석 > Experimental Example 5: Amino acid analysis >
아미노산 자동분석기 조건Amino Acid Analyzer Condition
완충액, 발색시약의 유무, 칼럼오븐의 온도를 점검하고, 자동완충액 전환기구 및 정유량 펌프, 유량을 소정의 조건에 따라 설정하고 측정을 실시한다.
Check the presence of buffers, color reagents, temperature of the column oven, set the automatic buffer switching mechanism, constant flow pump, and flow rate according to predetermined conditions and perform measurement.
정성시험Qualitative examination
정성분석을 위하여 표준물질과 시험용액을 동일한 조건에서 분석하여 머무름 시간이 일치하는 성분을 비교 동정한다.
For qualitative analysis, the standard material and the test solution are analyzed under the same conditions and the components with the same retention time are compared and identified.
정량시험Quantitative test
정량분석의 경우에는 미리 구해 놓은 농도의 아미노산표준의 피크 면적과 함량으로부터 검량 곡선을작성한 다음 시험용액으로부터 얻어진 피크면적으로 시험용액중의 아미노산 함량을 계산한다.For quantitative analysis, prepare a calibration curve from the peak area and content of the amino acid standard of the previously obtained concentration, and calculate the amino acid content in the test solution at the peak area obtained from the test solution.
<실험 예6 : 칼슘 분석 > Experimental Example 6: Calcium analysis [
과망간산칼륨 용량법은 Ca를 함유하는 용액에 수산염을 첨가해두고, 물에 매우 난용성인 수산칼슘 CaC2O4H2O로서 침전시키고 이 침전을 H2SO4에 녹여 용액내의 수산을 KMnO4 용액으로 적정하여 정량하는 방법이다.In the potassium permanganate capacity method, a hydroxide is added to a solution containing Ca, precipitated as calcium hydroxide CaC 2 O 4 H 2 O which is very hardly soluble in water, the precipitate is dissolved in H 2 SO 4 , and the aqueous solution in the solution is dissolved in KMnO 4 solution .
무기성분 시험용액 조제에서 얻은 시험용액 중에 40 mL(Ca 1～12 mg에 대응하는 양)를 200 mL의 비커에 취하여 메틸레드시액 수방울 및 수산암모늄용액 10 mL를 가한 다음 요소 2～5 g을 가해 녹인다. 비커를 시계접시로 덮고 약하게 가열을 계속시킨다. 메틸레드시액을 색이 적색에서 등황색으로 (pH 약 5.6) 되었을 때 가열을 중지하고 방냉한다. 이를 2시간 이상(침전이 적을 때는 하룻밤) 실온으로 방치한다. 이때의 액량은 약 20～30 mL정도이다. 충분히 침전을 형성시킨 후 석출전 수산칼슘 결정을 유리여과기(15AG-4)로 흡입여과하고 세척용 암모니아수 30～40 mL를 수회 나누어 침전과 여과지를 잘 씻는다. 세척이 끝나면 침전 생성에 사용한 비커를 앞의 유리여과기의 밑에 놓고 여과에 사용한 유리봉을 유리여과기에 넣어 미리 70℃이상으로 가온한 황산용액(1→25)을 유리여과기에 주입한다. 유리봉으로 교반하여 잠시 방치하고 수산칼슘을 용해한 다음 흡입한다. 다음 흡입을 그치고 가온한 황산용액(1→25) 약 5～7 mL를 유리여과기에 주입하고 유리봉으로 교반하여 내벽을 씻고 흡입한다. 이 조작을 2회 반복한다. 비커를 여과장치에서 들어내고 65～80℃로 가열하여 0.02 N 과망간산칼륨용액으로 적정한다. 공시험에 대하여도 같은 조작을 한다.
a：공시험에 대한 0.02 N 과망간산칼륨용액의 소비 mL 수a: consumption of 0.02 N potassium permanganate solution for blank test
b：검액에 대한 0.02 N 과망간산칼륨용액의 소비 mL 수b: consumption of 0.02 N potassium permanganate solution to the sample solution
F：0.02 N 과망간산칼륨용액의 역가F: 0.02 N Potency of potassium permanganate solution
V：시험용액의 희석배수V: Dilution of test solution
S：검체의 채취량(g)S: Amount of sample (g)
<실험 예6 : 철분 분석 > Experimental Example 6: Analysis of iron powder [
Ortho-phenanthroline(C12H8n2)은 일정 범위의 pH하에서 검체용액 중에 있는 Fe2+1원자와 O-phenanthroline 3분자가 정량적으로 반응하여 적색의 착화합물 [(C12H8n2)3Fe2+]을 생성한다. 이 적색의 농도는 Fe 100~200 γ, pH 2.9~9.0 사이에서 Fe의 농도와 비례하므로 비색정량에 의해 검체 중의 Fe 함량을 구할 수 있다.Ortho-phenanthroline (C 12 H 8n 2 ) is a red complex [(C 12 H 8n 2 ) 3 Fe 2+ ] reacting quantitatively with
시험용액의 조제에서 얻은 시험용액중의 10 mL를 피펫으로 25 mL의 메스플라스크에 취하고 같은 10 mL를 시험관(혹은 작은 삼각플라스크)에 취한다.
후자는 pH조절용 대조액으로 쓴다. 시험관쪽에는 브롬페놀블루시액 4방울을 가하고 25 mL 메스플라스크 쪽에는 하이드로퀴논용액 1 mL와 또 페난트로린용액 2 mL를 피펫으로 가한다. 구연산나트륨용액을 뷰렛 또는 5 mL 메스피펫으로 취하여 시험관의 액중에 적가하고 액의 pH가 3.5가 되면 지시액을 가한 액의 색이 황색에서 황록색으로 변하는 점에서 적가를 그치고 이때까지 소요된 구연산나트륨용액의mL수를 기록한다. The latter is used as a control solution for pH control. Add 4 drops of bromophenol blue TS to the test tube,
이와 같은 용량의 구연산나트륨용액을 메스플라스크 쪽에 가하여 물로 25 mL의 표선까지 희석하여 20℃이상의 온도에서 1시간이상 방치한다. 하룻밤 방치해도 무방하다. 일단, 완전히 발색된 액은 적어도 48시간은 퇴색하지 않는다. Add the sodium citrate solution of this volume to the measuring flask, dilute to 25 mL of water with water, and allow to stand at a temperature of 20 ° C or more for 1 hour or more. It is safe to leave overnight. Once fully developed, the solution does not fade for at least 48 hours.
분광광도계(Spectrophotometer)에서는 510 nm의 파장에서, 광전비색계에서는 녹색휠타를 이용해서 발색액의 흡광도를 측정하여 비흡광계수를 구한다. 이것을 미리 작성하여 놓은 검량선과 대조하여 철의 양을 구한다. The absorbance of the coloring liquid is measured at a wavelength of 510 nm in a spectrophotometer and in a photoelectric colorimeter using a green filter to obtain a non-absorbance coefficient. This is compared with the calibration curve prepared in advance to obtain the amount of iron.
검량선은 다음과 같이 하여 만든다. 즉, 철 표준액을 물로 정확히 10배로 희석한다. 1, 2, 5, 10, 15 및 20 mL를 각각 1조씩 25 mL의 메스플라스크와 시험관 또는 작은 삼각플라스크에 취한다. 10 mL가 안되는 것은 물을 추가하여 거의 10 mL로 만든다. 상기 발색조작과 동일하게 pH 3.5에서 각 메스플라스크에서 발색시켜 흡광도를 측정하며, 농도 흡광곡선을 작성한다.
The calibration curve is made as follows. That is, iron standard solution is diluted with water exactly 10 times. 1, 2, 5, 10, 15, and 20 mL each into a 25 mL volumetric flask and test tube or small Erlenmeyer flask. Add 10 mL of water to make approximately 10 mL. Similarly to the above-mentioned coloring operation, color development is performed in each of the measuring flasks at a pH of 3.5, absorbance is measured, and a concentration absorption curve is prepared.
철(mg/100 g) ＝×V×100Iron (mg / 100 g) = x V x 100
A：흡광곡선에서 구한 철의 양(mg) A: Amount of iron (mg) determined from the extinction curve
S：검체채취량(g) S: Sample weight (g)
V：시험용액의 희석배수
V: Dilution of test solution
항목
Item
제주어음리
Jeju Compilation
단위
unit
연구보고서Ministry of Agriculture and Forestry
Research Report
100gg /
100g
100gmg /
100g
100gmg /
100g
* A : Raw seeds(grinding)* A: Raw seeds (grinding)
B : Roasted seeds B: Roasted seeds
C : Raw Seeds(do not grind) C: Raw Seeds (do not grind)
D : leaf D: leaf
상기 표 4에서 제주산 아마란스가 전체적으로 단백질 함량도 우수하고 특히 철분과 칼슘은 타지방에서 분석된 양보다 월등하게 우수한 함량을 보유하고 있음을 확인할 수 있었다.
In Table 4, it can be seen that the amaranth juice from Jeju has excellent protein contents as a whole, and iron and calcium particularly have superior contents than the amounts analyzed in the other lipids.
(g/100g) Protein
(g / 100 g)
(mg/100g) Ca
(mg / 100g)
(mg/100g) Fe
(mg / 100g)
제주 어음리
Jeju Compilation
상기 표 5에서 제주산 아마란스인 경우 타 곡물에 비해서 단백질, 칼슘, 철분이 모두 우수하게 나타남을 확인하였다. In Table 5, it was confirmed that protein, calcium and iron were superior to those of other grains in Jeju amaranth.
Claims (6)
Composition of supplements for the amaranth and top seeds
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20150050995A (en) * | 2013-11-01 | 2015-05-11 | 가천대학교 산학협력단 | Composition for skin whitening comprising amaranthus spp. l. extract or fraction thereof |
| KR20160139328A (en) * | 2015-05-27 | 2016-12-07 | 주식회사 효성 | Polyketone blend vehicle head lamp bezel |
-
2013
- 2013-01-29 KR KR1020130009931A patent/KR20140097718A/en not_active Application Discontinuation
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20150050995A (en) * | 2013-11-01 | 2015-05-11 | 가천대학교 산학협력단 | Composition for skin whitening comprising amaranthus spp. l. extract or fraction thereof |
| KR20160139328A (en) * | 2015-05-27 | 2016-12-07 | 주식회사 효성 | Polyketone blend vehicle head lamp bezel |
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