KR20110036317A - Anti-inflammatory composition - Google Patents
Anti-inflammatory compositionInfo
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- KR20110036317A KR20110036317A KR1020090093911A KR20090093911A KR20110036317A KR 20110036317 A KR20110036317 A KR 20110036317A KR 1020090093911 A KR1020090093911 A KR 1020090093911A KR 20090093911 A KR20090093911 A KR 20090093911A KR 20110036317 A KR20110036317 A KR 20110036317A
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- leaf extract
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- outpost
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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)
- A61K36/77—Sapindaceae (Soapberry family), e.g. lychee or soapberry
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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 OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- 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)
- A61K36/35—Caprifoliaceae (Honeysuckle family)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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)
- A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation or decoction
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
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- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
본 발명은 항염증성 조성물에 관한 것으로, 구체적으로는 고로쇠나무 잎 추출물, 가막살나무 잎 추출물, 아그배나무 잎 추출물, 멀구슬나무 잎 추출물, 인동 전초 추출물, 고비 전초 추출물, 사방오리 잎 추출물, 비목나무 잎 추출물 등 식물 추출물을 유효성분으로 포함하는 항염증성 조성물에 관한 것이다.The present invention relates to an anti-inflammatory composition, specifically, Cypress leaf extract, Viburnum leaf extract, Ag pear leaf extract, Mulberry leaf extract, Honeysuckle outpost, Fern outpost extract, Rhododendron leaf extract, Leaf tree leaf It relates to an anti-inflammatory composition comprising a plant extract such as extract as an active ingredient.
염증은 외부의 물리·화학적 자극, 박테리아, 곰팡이, 바이러스, 각종 알레르기 유발 물질 등 외부 감염원의 감염에 대한 생체의 방어 반응이다. Inflammation is the body's defense against external infections such as physical and chemical stimuli, bacteria, fungi, viruses, and allergens.
염증 반응은 선천성 면역 반응의 일부이며, 다른 동물에서처럼 인간의 선천성 면역 반응은 대식세포가 병원체에 특이적으로 존재하는 세포 표면의 패턴을 통해 비자기(non-self)로 인식하고 공격함으로써 시작된다. 염증 반응 시에는 염증 부위에 혈장이 축적되어 세균이 분비한 독성을 희석시키며, 혈류가 증가하고, 홍반, 통증, 부종, 발열 등의 증상이 수반되게 된다.Inflammatory responses are part of the innate immune response, and as in other animals, the innate immune response in humans begins by recognizing and attacking non-self through patterns of cell surfaces where macrophages are specifically present in pathogens. During the inflammatory reaction, plasma accumulates at the site of inflammation, diluting the toxicity secreted by bacteria, increasing blood flow, and accompanied by symptoms such as erythema, pain, edema, and fever.
이러한 염증 반응에는 다양한 생화학적 현상이 관여하는데, 특히 산화질소 합성효소(nitric oxide synthase, NOS)와 다양한 프로스타글란딘(prostaglandins)의 생합성과 관련되는 사이클로옥시제나제(cyclooxygenase, COX)가 염증 반응의 중요한 매개체로 알려져 있다. These inflammatory responses involve a variety of biochemical phenomena, particularly cyclooxygenase (COX), which is associated with the biosynthesis of nitric oxide synthase (NOS) and various prostaglandins. Known as
NOS는 세 가지 이성질체가 존재하는데, 칼슘이나 카모듈린 의존성인 eNOS(내피성 NOS)와 nNOS(신경성 NOS), 그리고 LPS(lipopolysaccharide)와 같은 세균의 내독소나 IL-1β, TNF-α, IL-6, IL-8, IL-12과 같은 여러 염증성 사이토카인에 의해 유도되는 iNOS(유도성 NOS)가 있으며, L-아르기닌(L-arginine)으로부터 산화질소(NO)를 생성한다. There are three isomers of NOS, which are endotoxins of bacteria such as calcium- or capmodin-dependent eNOS (endothelial NOS), nNOS (neurotrophic NOS), and LPS (lipopolysaccharide) or IL-1β, TNF-α, IL There are iNOS (inducible NOS) induced by several inflammatory cytokines such as -6, IL-8 and IL-12, which produce nitric oxide (NO) from L-arginine.
eNOS나 nNOS에 의해 생성되는 NO는 혈압 조절 작용, 신경 전달 작용, 학습, 기억 등과 관련된 다양한 생리 반응을 수행함으로써 인체의 항상성 유지에 중요한 역할을 하지만, iNOS에 의해 생성되는 NO는 관절염, 패혈증, 조직이식거부반응, 자가면역질환, 신경세포의 사멸 등 다양한 염증성 질환에 관여하는 것으로 알려져 있다(Moncade S. et al, Pharmacol. Rev., 1991, 43, 109; Nature Medicine, 2001, 7, 1138; Mu, M. M., J. Endotoxic Res. 7, p341, 2001).NO produced by eNOS or nNOS plays an important role in maintaining homeostasis by performing various physiological reactions related to blood pressure control, neurotransmitter, learning, memory, etc. It is known to be involved in various inflammatory diseases such as transplant rejection, autoimmune diseases and neuronal death (Moncade S. et al, Pharmacol. Rev., 1991, 43, 109; Nature Medicine, 2001, 7, 1138; Mu , MM, J. Endotoxic Res. 7, p341, 2001).
COX 효소는 COX의 기능과 함께 하이드로퍼옥시다제(hydroperoxidase, HOX) 활성을 가지고 아라키돈산으로부터 중간체인 PGG2와 PGH2를 합성하며, 이들 화합물로 PGE2, PGF2, PGD2, 프로스타시클린 및 트롬복신A2(thromboxane A2, TxA2)를 만든다. COX의 기능 중 PGH 합성효소의 기능은 PGE2의 합성을 통해 통증과 염증 반응에 관여한다. The COX enzyme has hydroperoxidase (HOX) activity with COX function and synthesizes PGG 2 and PGH 2 intermediates from arachidonic acid, and these compounds include PGE 2 , PGF 2 , PGD 2 , and prostacyclin. And thromboxin A 2 (thromboxane A2, TxA2). Among the functions of COX, the function of PGH synthase is involved in pain and inflammatory responses through the synthesis of PGE 2 .
COX에는 두 가지 아형이 있고 COX-1은 대부분의 조직에 항시 발현되는데 비해, COX-2는 염증성 사이토카인에 의해 신속히 발현이 유도되어 염증 반응에서 중요한 역할을 한다. There are two subtypes of COX and COX-1 is always expressed in most tissues, whereas COX-2 is rapidly induced by inflammatory cytokines and plays an important role in the inflammatory response.
따라서 iNOX 억제 활성이나 COX-2 억제 활성, NO, PGE2, TNF-α, IL-6 등 염증 매개 인자의 생성 억제 활성을 갖는 물질은 염증질환 치료제로서 활용될 수 있다.Therefore, a substance having inhibitory activity for the production of inflammatory mediators such as iNOX inhibitory activity, COX-2 inhibitory activity, NO, PGE 2 , TNF-α, IL-6 can be used as a therapeutic agent for inflammatory diseases.
본 발명은 항염증성 조성물을 제공하는 데 있다.The present invention is to provide an anti-inflammatory composition.
본 발명의 구체적인 양태는 이하에서 제시될 것이다.Specific embodiments of the present invention will be presented below.
본 발명자는 260여 종의 식물 추출물의 항염증 활성을 평가하기 위해서 아래의 실시예 및 실험예에서 확인되는 바와 같은 방법으로 NO 생성 억제 활성, PGE2 생성 억제 활성, IL-6 생성 억제 활성 및/또는 TNF-α 생성 억제 활성을 살펴본 결과, 아래의 <표 1>에 나열된 122종의 식물 추출물이 NO 생성 억제 활성, PGE2 생성 억제 활성, IL-6 생성 억제 활성 및/또는 TNF-α 생성 억제 활성을 가짐을 확인할 수 있었다. In order to evaluate the anti-inflammatory activity of over 260 plant extracts, the present inventors identified NO production inhibitory activity, PGE 2 production inhibitory activity, IL-6 production inhibitory activity and / or the same method as confirmed in Examples and Experimental Examples below. Or TNF-α production inhibitory activity, as a result, 122 plant extracts listed in Table 1 below, NO production inhibitory activity, PGE 2 production inhibitory activity, IL-6 production inhibitory activity and / or TNF-α production inhibition It was confirmed that it has activity.
본 발명은 이러한 실험 결과에 기초하여 완성된 것이다.The present invention has been completed based on these experimental results.
따라서 본 발명의 항염증성 조성물은, 고로쇠나무 잎 추출물, 가막살나무 잎 추출물, 덜꿩나무 잎 추출물, 으름덩굴 잎 추출물, 박새 전초 추출물, 개족도리풀 전초 추출물, 화살나무 잎 추출물, 아그배나무 잎 추출물, 분단나무 잎 추출물, 붉나무 잎 추출물, 화살나무 잎 추출물, 실거리나무 잎 추출물, 구릿대 전초 추출물, 긴잎달맞이꽃 전초 추출물, 층층나무 잎 추출물, 멀구슬나무 잎 추출물, 인동 전초 추출물, 고비 전초 추출물, 사방오리 잎 추출물, 멍석딸기 잎 추출물, 뽕나무 잎 추출물, 해국 잎 추출물, 예덕나무 잎 추출물, 나문재 전초 추출물, 개다래 전초 추출물, 비목나무 잎 추출물, 쥐똥나무 잎 추출물, 정금나무 잎 추출물, 섬오갈피나무 가지 추출물, 등 잎 추출물, 굴피나무 잎 추출물, 떡갈나무 잎 추출물, 풍게나무 잎 추출물, 소사나무 잎 추출물, 병개암나무 잎 추출물, 서어나무 잎 추출물, 참꽃나무 잎 추출물, 작살나무 잎 추출물, 덧나무 잎 추출물, 삼색병꽃나무 잎 추출물, 소태나무 잎 추출물, 병꽃나무 잎 추출물, 때죽나무 잎 추출물, 버드나무 잎 추출물, 좀참빗살나무 잎 추출물, 붉나무 잎 추출물, 목련 잎 추출물, 참개암나무 잎 추출물, 이나무 잎 추출물, 말채나무 잎 추출물, 후피향나무 잎 추출물, 꽃댕강나무 잎 추출물, 참느릅나무 잎 추출물, 산뽕나무 잎 추출물, 아그배나무 잎 추출물, 마가목 잎 추출물, 털팥배나무 잎 추출물, 팥배나무 잎 추출물, 사옥 잎 추출물, 쪽동백나무 잎 추출물, 고추나무 잎 추출물, 머귀나무 잎 추출물, 사람주나무 잎 추출물, 솔비나무 잎 추출물, 육박나무 잎 추출물, 뽕잎피나무 잎 추출물, 산개벚지나무 잎 추출물, 꾸지뽕나무 잎 추출물, 꾸지나무 잎 추출물, 오구나무 잎 추출물, 광대싸리 잎 추출물, 들메나무 잎 추출물, 칠엽수 잎 추출물, 회화나무 잎 추출물, 붉은토끼풀 잎 추출물, 섬오갈피나무 잎 추출물, 애기땅빈대 전초 추출물, 은행나무 잎 추출물, 개옻나무 잎 추출물, 보리수나무 잎 추출물, 검양옻나무 잎 추출물, 두충 잎 추출물, 고추나무 잎 추출물, 고욤나무 잎 추출물, 큰달맞이꽃 잎 추출물, 큰달맞이꽃 줄기 추출물, 구릿대 줄기 추출물, 개머루 전초 추출물, 갯길경 전초 추출물, 질경이 전초 추출물, 붓순나무 잎 추출물, 좁은잎천선과나무 줄기 추출물, 모람 열매 추출물, 누리장나무 잎 추출물, 산유자나무 잎 추출물, 비쑥 전 초 추출물, 제비쑥 전초 추출물, 참나무겨우살이 잎 추출물, 등대풀 전초 추출물, 소귀나무 잎 추출물, 겨울딸기 잎 추출물, 유럽점나도나물 전초 추출물, 자주광대나물 전초 추출물, 무환자나무 잎 추출물, 당광나무 잎 추출물, 꾸지나무 잎 추출물, 참골무꽃 잎 추출물, 꽃향유 잎 추출물, 남오미자 지상부 추출물, 계요등 지상부 추출물, 모새나무 지상부 추출물, 센달나무 잎 추출물, 개속새 지상부 추출물, 동백나무 잎 추출물, 상산 잎 추출물, 작살나무 잎 추출물, 갯사상자 전초 추출물, 말오줌때 전초 추출물, 갯강활 전초 추출물, 수송나물 전초 추출물, 말똥비름 전초, 및 파초 줄기 추출물로 구성된 군에서 선택된 어느 하나 이상의 식물 추출물을 유효성분으로 포함함을 특징으로 한다. Thus, the anti-inflammatory composition of the present invention, Cypress leaf extract, Viburnum leaf extract, Pheasant leaf extract, Creeper leaf extract, Spearweed outpost extract, Forbidden tree outpost extract, Arrow leaf leaf extract, Ag pear leaf extract, Segmented Tree Leaf Extract, Rhododendron Leaf Extract, Arrowwood Leaf Extract, Stingray Leaf Extract, Copper Leaf Outpost Extract, Longleaf Evening Primrose Extract, Dogwood Leaf Extract, Mulberry Leaf Extract, Honeysuckle Outpost, Fern Outpost Extract, Rhododendron Leaf Extract Leaf, locust leaf extract, mulberry leaf extract, hydrangea leaf extract, viburnum leaf extract, namunjae outpost extract, forbidden tree outpost extract, birch leaf extract, Privet leaf extract, cedar leaf extract, gingko leaf branch extract, etc leaf Extract, Oysterwood Leaf Extract, Oak Tree Leaf Extract, Taraxacum Leaf Extract, Cypress Extract, Cranberry Leaf Extract, Rhododendron Leaf Extract, Oak Tree Leaf Extract, Harpoon Leaf Extract, Mulberry Leaf Extract, Tricolor Frond Leaf Extract, Pine Tree Leaf Extract, Cauliflower Leaf Extract, Bamboo Stick Leaf Extract, Bud Tree Leaf Extract, Black Oak Leaf Extract, Rhododendron Leaf Extract, Magnolia Leaf Extract, Hazel Leaf Extract, Alder Leaf Extract, Dogwood Leaf Extract, Epidermis Leaf Extract, Anthurium Leaf Extract, Elm Leaf Extract, Mountain Mulberry Leaf Extract, Agberry Leaf Extract, Rowan Leaf Extract, Red Bean Leaf Extract, Red Bean Leaf Extract, Office Building Leaf Extract, Camellia Leaf Extract, Chilli Leaf Extract, Mucus Leaf Extract, Mandarin Leaf Extract Pine Cone, Leaf Extract, Mulberry Leaf Extract, Mulberry Leaf Extract, Cabbage Leaf Extract, Cucumber Leaf Extract, Bud Radish Leaf Extract, Fuchsia Leaf Extract, Bumblebee Leaf Extract, Wildwood Leaf Extract, Horse Chestnut Leaf Extract, Panax Leaf Extract, Red Rabbit Leaf Extract, Iridaceae Leaf Extract, Celandine Leaf Extract, Ginkgo Leaf Extract, Red Oak Leaf Extract, Lime Tree Leaf Extract, Beethoven Leaf Extract, Beetle Leaf Extract, Chilli Leaf Extract, Horseradish Leaf Extract, Evening Primrose Leaf Extract, Evening Primrose Stem Extract, Copperberry Stem Extract, Kaleidoscope Outpost Extract, Algae Border Outpost Extract, Plantain Outpost Extract, Iridaceae Leaf Extract, Narrow Leaf Cedar Leaf Extract, Maraam Fruit Extract, Nutassium Leaf Extract, Citron Leaf Extract, Wormwood Sprout Extract, Sageweed Outpost Extract, Oak Mistletoe Leaf Extract, Lighthouse Grass Outpost Extract, Pine Tree Leaf Extract, Winter Berry Leaf Extract, European Sprout Leaf Extract Mandarin Leaf Extract, Solanum Leaf Extract, Lactobacillus Leaf Extract, Coconut Leaf Extract, Chamomile Leaf Extract, Flower Balm Leaf Extract, South Schisandra chinensis Extract, Lantern Et. Consisting of groundweed extract, Camellia leaf extract, locust leaf extract, harpoon leaf extract, sandbox outpost extract, urinary outpost extract, seaweed bow outpost extract, transport herb outpost extract, horseweed outpost, and leek stem extract At least one plant extract selected from the group is characterized in that it comprises as an active ingredient.
바람직하게는 NO 생성 억제 활성, PGE2 생성 억제 활성, IL-6 생성 억제 활성 및/또는 TNF-α 생성 억제 활성이 높은, 고로쇠나무 잎 추출물, 가막살나무 잎 추출물, 아그배나무 잎 추출물, 멀구슬나무 잎 추출물, 인동 전초 추출물, 고비 전초 추출물, 사방오리 잎 추출물, 비목나무 잎 추출물, 쥐똥나무 잎 추출물, 정금나무 잎 추출물, 굴피나무 잎 추출물, 소사나무 잎 추출물, 병개암나무 잎 추출물, 서어나무 잎 추출물, 참꽃나무 잎 추출물, 버드나무 잎 추출물, 목련 잎 추출물, 참개암나무 잎 추출물, 이나무 잎 추출물, 말채나무 잎 추출물, 후피향나무 잎 추출물, 참느릅나무 잎 추출물, 산뽕나무 잎 추출물, 아그배나무 잎 추출물, 마가목 잎 추출물, 털팥배나무 잎 추출물, 팥배나무 잎 추출물, 쪽동백나무 잎 추출물, 사람주나무 잎 추출물, 육박나무 잎 추출물, 꾸지뽕나무 잎 추출물, 오구나무 잎 추 출물, 광대싸리 잎 추출물, 들메나무 잎 추출물, 보리수나무 잎 추출물, 검양옻나무 잎 추출물, 큰달맞이꽃 잎 추출물, 좁은잎천선과나무 줄기 추출물, 비쑥 전초 추출물, 꽃향유 잎 추출물, 모새나무 지상부 추출물, 상산 잎 추출물, 갯사상자 전초 추출물, 및 말오줌때 전초 추출물로 구성된 군에서 선택된 어느 하나 이상의 식물 추출물을 유효성분으로 포함함을 특징으로 한다. Preferably, Cypress leaf extract, Viburnum leaf extract, Ag pear leaf extract, Mulberry tree having high NO production inhibitory activity, PGE 2 production inhibitory activity, IL-6 production inhibitory activity and / or TNF-α production inhibitory activity Leaf Extract, Honeysuckle Outpost Extract, Fern Outpost Extract, Rhododendron Leaf Extract, Rainforest Leaf Extract, Privet Leaf Extract, Gemini Leaf Extract, Humidified Leaf Extract, Pine Cone Leaf Extract, Hazel Leaf Extract, West Pine Leaf Extract, Oak Tree Leaf Extract, Willow Leaf Extract, Magnolia Leaf Extract, Hazel Leaf Extract, Birch Leaf Extract, Dogwood Leaf Extract, Epidermis Leaf Extract, Elm Leaf Extract, Mulberry Leaf Extract, Agberry Leaf Extract, Rowan Leaf Extract, Red Bean Leaf Extract, Red Bean Leaf Extract, Camellia Leaf Extract, Mandarin Leaf Extract, Cactus Leaf Extract Water, Cucumber Leaf Extract, Fuchsia Leaf Extract, Bumblebee Leaf Extract, Wildwood Leaf Extract, Bodhi Tree Leaf Extract, Glacier Leaf Extract, Evening Primrose Leaf Extract, Narrow Leaf Thymus Leaf Extract, Wormwood Outpost Extract, At least one plant extract selected from the group consisting of flowering balm leaf extract, ground bark extract, sangsan leaf extract, saecho outpost extract, and horse urinary outpost extract as an active ingredient.
본 명세서에서 "추출물"은 추출 방법을 불문하고 상기 각 추출 대상을 메탄올, 에탄올, 아세톤, 에틸아세테이트, 포화노말부탄올, 클로로포름, 메틸렌클로라이드, 물, 또는 이들의 혼합 용매로 추출하여 얻어진 추출물과 그 추출물에서 상기 열거된 용매로 더 정제된 분획을 포함하는 의미로서 이해된다. 추출 방법을 불문하므로, 추출 대상을 추출 용매에 침지시키는 단계를 통하여 추출되는 한, 추출 방법은 냉침, 환류, 가온, 초음파 등 임의의 방식이 모두 적용될 수 있는 것으로 이해되어야 한다. 그럼에도 상기 "추출물"은 바람직하게는 그 추출 대상을 물, 에탄올 또는 이들의 혼합 용매로 추출하여 얻어진 것을 의미하며, 더 바람직하게는 물과 에탄올의 혼합 용매로 추출하여 얻어진 것을 의미한다. 가장 바람직하게는 에탄올 함량이 70% 내지 90%의 에탄올 수용액을 추출 용매로 사용하는 경우이다. 여기서 %는 부피 백분율(v/v)을 의미한다. 본 명세서의 "추출물"의 의미에는 여과 등을 통하여 정제된 형태의 추출물, 추출 용매가 제거된 액상의 농축된 추출물 그리고 추출 용매가 제거된 고형상의 추출물이 포함된다. As used herein, the term "extract" refers to extracts obtained by extracting the extracts with methanol, ethanol, acetone, ethyl acetate, saturated normal butanol, chloroform, methylene chloride, water, or a mixed solvent thereof regardless of the extraction method. Is understood to mean fractions further purified with the solvents enumerated above. Regardless of the extraction method, it is to be understood that the extraction method may be applied to any method such as cooling, refluxing, heating, and ultrasonic wave, as long as the extraction object is extracted through immersion in the extraction solvent. Nevertheless, the term "extract" preferably means that the extraction target is obtained by extraction with water, ethanol or a mixed solvent thereof, and more preferably, by extraction with a mixed solvent of water and ethanol. Most preferably, an aqueous solution of ethanol having an ethanol content of 70% to 90% is used as the extraction solvent. Where% means volume percentage (v / v). As used herein, the term "extract" includes extracts in a purified form through filtration, concentrated liquid extracts from which the extraction solvent is removed, and solid extracts from which the extraction solvent is removed.
또 본 명세서에서, 상기 "유효성분"의 의미는 단독으로 목적하는 활성을 나타내거나 또는 그 자체는 활성이 없는 담체와 함께 활성을 나타낼 수 있는 성분을 의미한다.In addition, in the present specification, the meaning of "active ingredient" means a component that can exhibit activity alone or in combination with a carrier having no activity in itself.
또 본 명세서에서, "항염증"은 아래에서 정의되는 염증성 질환의 개선, 치료 및 그러한 질환의 발병 억제/지연을 포함하는 의미이다.In addition, in the present specification, "anti-inflammatory" is meant to include amelioration, treatment of inflammatory diseases and inhibition / delay of onset of such diseases as defined below.
또 본 명세서에서, 상기 "염증성 질환"이란 외부의 물리·화학적 자극 또는 박테리아, 곰팡이, 바이러스, 각종 알레르기 유발 물질 등 외부 감염원의 감염에 대한 국부적 또는 전신적 생체 방어 반응으로 특정되는 어떠한 상태로서 정의될 있다. 이러한 반응은 각종 염증 매개 인자와 면역세포와 관련된 효소(예컨대 iNOS, COX-2 등) 활성화, 염증 매개 물질의 분비(예컨대, NO, TNF-α, IL-6, IL-1β, PGE2의 분비), 체액 침윤, 세포 이동, 조직 파괴 등의 일련의 복합적인 생리적 반응을 수반하며, 홍반, 통증, 부종, 발열, 신체의 특정 기능의 저하 또는 상실 등의 증상에 의해 외적으로 나타난다. 상기 염증성 질환은 급성, 만성, 궤양성, 알레르기성 또는 괴사성을 띨 수 있으므로, 어떠한 질환이 상기와 같은 염증성 질환의 정의에 포함되는 한 그것이 급성이든지, 만성이든지, 궤양성이든지, 알레르기성이든지 또는 괴사성이든지를 불문한다. 구체적으로 상기 염증성 질환에는 천식, 알레르기성 및 비-알레르기성 비염, 만성 및 급성 비염, 만성 및 급성 위염 또는 장염, 궤양성 위염, 급성 및 만성 신장염, 급성 및 만성 간염, 만성 폐쇄성 폐질환, 폐섬유증, 과민성 대장 증후군, 염증성 통증, 편두퐁, 두통, 허리 통증, 섬유 근육통, 근막 질환, 바이러스 감염(예컨대, C형 감염), 박테리아 감염, 곰팡이 감염, 화상, 외과적 또는 치과적 수술에 의한 상처, 프로스타글라딘 E 과다 증후군, 아테롬성 동맥 경화증, 통풍, 관절염, 류머티스성 관절염, 강직성 척추염, 호지킨병, 췌장염, 결막염, 홍채염, 공막염, 포도막염, 피부염, 습진, 다발성 경화증 등이 포함될 것이다. In the present specification, the "inflammatory disease" may be defined as any condition specified by a local or systemic biological defense response against external physical and chemical stimuli or infection of an external infectious agent such as bacteria, fungi, viruses, and various allergens. . This response activates various inflammatory mediators and enzymes associated with immune cells (eg iNOS, COX-2, etc.), secretion of inflammatory mediators (eg NO, TNF-α, IL-6, IL-1β, PGE 2) . ), Accompanied by a series of complex physiological reactions such as fluid infiltration, cell migration, tissue destruction, etc., and are manifested externally by symptoms such as erythema, pain, edema, fever, deterioration or loss of certain functions of the body. The inflammatory disease may be acute, chronic, ulcerative, allergic or necrotic, so as long as any disease is included in the definition of an inflammatory disease as above, whether it is acute, chronic, ulcerative, allergic or Irrespective of necrosis Specifically, the inflammatory diseases include asthma, allergic and non-allergic rhinitis, chronic and acute rhinitis, chronic and acute gastritis or enteritis, ulcerative gastritis, acute and chronic nephritis, acute and chronic hepatitis, chronic obstructive pulmonary disease, pulmonary fibrosis Irritable bowel syndrome, inflammatory pain, migraines, headache, back pain, fibromyalgia, fascia disease, viral infections (eg, type C infections), bacterial infections, fungal infections, burns, surgical or dental wounds, Prostaglandin E excess syndrome, atherosclerosis, gout, arthritis, rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, irisitis, scleritis, uveitis, dermatitis, eczema, multiple sclerosis, and the like.
한편 본 발명의 조성물은 그 유효성분인 상기 식물 추출물을 제형, 배합 목적 등에 따라 치료를 의도하는 염증성 질환의 개선 활성을 나타낼 수 있는 한 임의의 양(유효량)으로 포함할 수 있는데, 통상적인 유효량은 조성물 전체 중량을 기준으로 할 때 0.001 중량 % 내지 99.999 중량 % 범위 내에서 결정될 것이다. 여기서 "유효량"이란 염증성 질환의 개선, 치료, 또는 이러한 병리적 증상의 발병 억제/지연을 유도할 수 있는 유효성분의 양을 말한다. 이러한 유효량은 당업자의 통상의 능력 범위 내에서 실험적으로 결정될 수 있다.On the other hand, the composition of the present invention may include the plant extract as an active ingredient in any amount (effective amount) as long as it can exhibit the improvement activity of the inflammatory disease intended to be treated according to the formulation, formulation purpose, etc. It will be determined within the range of 0.001% to 99.999% by weight based on the total weight of the composition. The term “effective amount” herein refers to an amount of an active ingredient that can induce improvement, treatment of an inflammatory disease, or inhibition / delay of the development of such pathological symptoms. Such effective amounts can be determined experimentally within the range of ordinary skill in the art.
본 발명의 조성물은 구체적인 양태에 있어서는 약제학적 조성물로 이용될 수 있다.The composition of the present invention can be used as a pharmaceutical composition in a specific embodiment.
본 발명의 약제학적 조성물은, 유효성분인 상기 식물 추출물을 제약상 허용가능한 담체, 부형제, 첨가제 등과 혼합하여 경구투여용 또는 비경구투여용으로 제제화하게 된다.The pharmaceutical composition of the present invention is formulated for oral or parenteral administration by mixing the plant extract as an active ingredient with a pharmaceutically acceptable carrier, excipient, additives and the like.
제약상 허용되는 담체 또는 부형제의 예는 락토스, 스테아린산 마그네슘, 전분, 탈크, 젤라틴, 한천, 펙틴, 아라비아고무, 올리브유, 참기름, 카카오버터, 에틸렌글리콜, 기타 상용되는 것을 포함한다.Examples of pharmaceutically acceptable carriers or excipients include lactose, magnesium stearate, starch, talc, gelatin, agar, pectin, gum arabic, olive oil, sesame oil, cacao butter, ethylene glycol, and the like.
경구투여용 고체 조성물의 예는 정제, 환제, 캡슐제, 분말제, 과립제 등을 포함한다. 이와 같은 고체 조성물에서는 유효성분인 식물 추출물이 적어도 하나의 불활성 희석제, 예를 들면, 락토스, 만니톨, 포도당, 히드록시 프로필 셀룰로오스, 미결정성 셀룰로오스, 전분, 폴리비닐 피롤리돈, 마그네슘 알루미노메타실리케이트 등과 혼합된다. 고체 조성물은, 통상적인 방법에 따라 불활성 희석제 이외의 첨가물, 예를들면, 마그네슘 스테아레이트와 같은 윤활제, 섬유소 글리콜산 칼슘과 같은 붕해제, 글루타민산 또는 아스파라긴산과 같은 용해보조제를 함유할 수 있다. 정제 또는 환제는, 필요에 따라 슈크로스, 젤라틴, 히드록시프로필 메틸셀룰로오스 프탈레이트 등의 물질로 이루어진 필름으로 피복될 수 있고, 경우에 따라서는 2개 이상의 층으로 피복될 수 있다. Examples of solid compositions for oral administration include tablets, pills, capsules, powders, granules and the like. In such solid compositions, the plant extract as an active ingredient may contain at least one inert diluent such as lactose, mannitol, glucose, hydroxypropyl cellulose, microcrystalline cellulose, starch, polyvinyl pyrrolidone, magnesium aluminometasilicate, and the like. Are mixed. The solid composition may contain additives other than inert diluents, for example, lubricants such as magnesium stearate, disintegrants such as calcium cellulose glycolate, dissolution aids such as glutamic acid or aspartic acid according to conventional methods. The tablets or pills may be coated with a film made of a material such as sucrose, gelatin, hydroxypropyl methylcellulose phthalate and the like, if desired, and may be coated with two or more layers.
경구투여용 액체조성물은, 약제학적으로 허용되는 유탁제, 용액제, 현탁제, 시럽제, 엘릭서제 등을 포함하고, 일반적으로 사용되는 불활성 희석제, 예를 들면, 정제수, 에탄올 등을 포함할 수 있다. 이 조성물은, 불활성 희석제 이외에 습윤제, 현탁제와 같은 보조제, 감미제, 풍미제, 방향제, 방부제 등을 포함할 수 있다.Liquid compositions for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs, and the like, and may include generally used inert diluents such as purified water and ethanol. This composition may contain, in addition to inert diluents, wetting agents, auxiliaries such as suspending agents, sweetening agents, flavoring agents, fragrances, preservatives and the like.
비경구투여용 주사제로는, 무균의 수성 또는 비수성의 용액제, 현탁제, 에멀젼화제가 포함된다. 수성의 용액제·현탁제로는, 예를 들면 주사용 물 및 주사용 생리식염수가 포함된다. 비수성의 용액제·현탁제로는, 예를 들면 프로필렌 글리콜, 폴리에틸렌글리콜, 올리브유와 같은 식물성유, 에탄올과 같은 알코올류, 폴리솔베이트 80® 등이 포함된다. 이와 같은 조성물은, 다시 방부제, 습윤제, 유화제, 분산제, 안정화제(예를 들면, 유당), 용해보조제(예를 들면, 글루타민산, 아스파르트산)와 같은 보조제를 포함할 수 있다. 이들은, 예를 들면, 정밀여과막에 의한 여과멸균, 고압증기멸균과 같은 가열멸균, 또는 살균제 배합 등의 통상적인 멸균 방 법으로 무균화될 수 있다. 주사제는 용액제제, 또는 사용 시에 용해시켜 사용할 수 있는 동결-건조 제제일 수 있다. 동결-건조를 위한 부형제로는 예를 들면, 만니톨, 글루코스 등의 당알콜이나 당류를 사용할 수 있다.Parenteral administration injections include sterile aqueous or nonaqueous solutions, suspensions, and emulsifiers. Aqueous solutions and suspensions include water for injection and physiological saline for injection. Examples of the non-aqueous solution and suspending agent include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, polysorbate 80, and the like. Such compositions may, in turn, include adjuvants such as preservatives, wetting agents, emulsifiers, dispersants, stabilizers (e.g. lactose), dissolution aids (e.g. glutamic acid, aspartic acid). These can be sterilized by conventional sterilization methods, such as, for example, filter sterilization by a microfiltration membrane, heat sterilization such as autoclaving, or a sterilizing agent. Injectables can be solution solutions or freeze-dried preparations which can be dissolved and used in use. As an excipient for freeze-drying, for example, sugar alcohols or sugars such as mannitol and glucose can be used.
본 발명의 약제학적 조성물은 경구투여방법 또는 비경구 투여방법으로 투여된다.The pharmaceutical composition of the present invention is administered by oral administration or parenteral administration.
바람직하기는 비경구 투여방법, 예를 들면, 주사에 의한 투여(피하주사, 정맥주사, 근육주사, 복강 내에의 주사 등으로 투여), 경피투여, 경점막투여(경직장투여 등), 경폐투여 등이다. 물론 경구투여방법도 사용될 수 있다.Preferably, parenteral administration method, for example, administration by injection (subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, etc.), transdermal administration, transmucosal administration (rectal administration, etc.), transpulmonary administration, etc. to be. Oral administration methods may also be used.
본 발명의 약제학적 조성물의 유효성분의 투여량은 질환의 중증도, 환자의 연령 등에 따라 결정할 수 있지만, 일반적으로는 0.005 ㎍/kg ~ l00 mg/kg의 범위이고, 바람직하기는 0.02㎍/kg ~ 5mg/kg의 범위이다. 그러나 본 발명의 약제학적 조성물의 투여량은 투여 경로, 환자의 연령, 성별, 체중, 환자의 중증도 등의 여러 관련 인자에 비추어 결정되는 것이므로 상기 투여량은 어떠한 측면으로든 본 발명의 범위를 제한하는 것으로 이해되어서는 아니된다. The dosage of the active ingredient of the pharmaceutical composition of the present invention can be determined depending on the severity of the disease, the age of the patient, etc., but is generally in the range of 0.005 µg / kg to l00 mg / kg, preferably 0.02 µg / kg to 5 mg / kg. However, since the dosage of the pharmaceutical composition of the present invention is determined in view of various related factors such as the route of administration, the age, sex, weight of the patient, and the severity of the patient, the dosage limits the scope of the present invention in any aspect. It should not be understood.
본 발명의 항염증성 조성물은 다른 구체적인 양태에 있어서, 식품 조성물 특히 기능성 음료로 파악할 수 있다.In another specific embodiment, the anti-inflammatory composition of the present invention can be regarded as a food composition, in particular a functional beverage.
본 발명의 식품 조성물에는 그 유효성분 이외에 감미제, 풍미제, 생리활성 성분, 미네랄 등이 포함될 수 있다.The food composition of the present invention may include sweeteners, flavoring agents, bioactive ingredients, minerals, etc. in addition to the active ingredients.
감미제는 식품이 적당한 단맛을 나게 하는 양으로 사용될 수 있으며, 천연의 것이거나 합성된 것일 수 있다. 바람직하게는 천연 감미제를 사용하는 경우인데, 천연 감미제로서는 옥수수 시럽 고형물, 꿀, 수크로오스, 프룩토오스, 락토오스, 말토오스 등의 당 감미제를 들 수 있다. Sweeteners may be used in amounts that give the food a suitable sweet taste, and may be natural or synthetic. Preferably, a natural sweetener is used. Examples of the natural sweetener include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose and maltose.
풍미제는 맛이나 향을 좋게 하기 위하여 사용될 수 있는데, 천연의 것과 합성된 것 모두 사용될 수 있다. 바람직하게는 천연의 것을 사용하는 경우이다. 천연의 것을 사용할 경우에 풍미 이외에 영양 강화의 목적도 병행할 수 있다. 천연 풍미제로서는 사과, 레몬, 감귤, 포도, 딸기, 복숭아 등에서 얻어진 것이거나 녹차잎, 둥굴레, 대잎, 계피, 국화 잎, 자스민 등에서 얻어진 것일 수 있다. 또 인삼(홍삼), 죽순, 알로에 베라, 은행 등에서 얻어진 것을 사용할 수 있다. 천연 풍미제는 액상의 농축액이나 고형상의 추출물일 수 있다. 경우에 따라서 합성 풍미제가 사용될 수 있는데, 합성 풍미제는 에스테르, 알콜, 알데하이드, 테르펜 등의 형태의 것들이 이용될 수 있다. Flavoring agents can be used to enhance the taste or aroma, both natural and synthetic. Preferably, a natural one is used. When using natural ones, the purpose of nutritional fortification can be performed in addition to the flavor. The natural flavor may be obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, and the like, or may be obtained from green tea leaves, round leaves, jujube leaves, cinnamon, chrysanthemum leaves, jasmine and the like. Also, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, banks and the like can be used. The natural flavoring agent may be a liquid concentrate or a solid form of extract. In some cases, synthetic flavoring agents may be used, and synthetic flavoring agents may be used in the form of esters, alcohols, aldehydes, terpenes and the like.
생리 활성 물질로서는 카테킨, 에피카테킨, 갈로가테킨, 에피갈로카테킨 등의 카테킨류나, 레티놀, 아스코르브산, 토코페롤, 칼시페롤, 티아민, 리보플라빈 등의 비타민류 등이 사용될 수 있다.As the physiologically active substance, catechins such as catechin, epicatechin, gallocatechin, epigallocatechin, vitamins such as retinol, ascorbic acid, tocopherol, calciferol, thiamine, riboflavin, and the like can be used.
미네랄로서는 칼슘, 마그네슘, 크롬, 코발트, 구리, 불소화물, 게르마늄, 요오드, 철, 리튬, 마그네슘, 망간, 몰리브덴, 인, 칼륨, 셀레늄, 규소, 나트륨, 황, 바나듐, 아연 등이 사용될 수 있다.As minerals, calcium, magnesium, chromium, cobalt, copper, fluoride, germanium, iodine, iron, lithium, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, silicon, sodium, sulfur, vanadium, zinc and the like can be used.
또한 본 발명의 식품 조성물은 상기 감미제 등 이외에도 필요에 따라 보존제, 유화제, 산미료, 점증제 등을 포함할 수 있다. In addition, the food composition of the present invention may contain a preservative, an emulsifier, an acidulant, a thickener, and the like, in addition to the sweetener.
이러한 보존제, 유화제 등은 그것이 첨가되는 용도를 달성할 수 있는 한 극 미량으로 첨가되어 사용되는 것이 바람직하다. "극미량"이란 수치적으로 표현할 때 식품 조성물 전체 중량을 기준으로 할 때 0.0005중량％ 내지 약 0.5중량％ 범위를 의미한다.Such preservatives, emulsifiers and the like are preferably added and used in an extremely small amount as long as the use to which they are added can be achieved. "Ultra trace" means numerically expressed in the range from 0.0005% to about 0.5% by weight based on the total weight of the food composition.
사용될 수 있는 보존제로서는 소듐 소르브산칼슘, 소르브산나트륨, 소르브산칼륨, 벤조산칼슘, 벤조산나트륨, 벤조산칼륨, EDTA(에틸렌디아민테트라아세트산) 등을 들 수 있다. Examples of preservatives that can be used include sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), and the like.
사용될 수 있는 유화제로서는 아카시아검, 카르복시메틸셀룰로스, 잔탄검, 펙틴 등을 들 수 있다.Emulsifiers that can be used include acacia gum, carboxymethylcellulose, xanthan gum, pectin and the like.
사용될 수 있는 산미료로서는 연산, 말산, 푸마르산, 아디프산, 인산, 글루콘산, 타르타르산, 아스코르브산, 아세트산, 인산 등을 들 수 있다. 이러한 산미료는 맛을 증진시키는 목적 이외에 미생물의 증식을 억제할 목적으로 식품 조성물이 적정 산도로 되도록 첨가될 수 있다.Examples of acidulants that may be used include lead acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, and the like. Such acidulant may be added so that the food composition is at an appropriate acidity for the purpose of inhibiting the growth of microorganisms in addition to the purpose of enhancing taste.
사용될 수 있는 점증제로서는 현탁화 구현제, 침강제, 겔형성제, 팽화제 등을 들 수 있다. Thickeners that can be used include suspending implements, sedimenters, gel formers, swelling agents and the like.
또한 본 발명의 식품 조성물은 향미나 기호성을 향상시키고 다른 기능성(예컨대 관절염 또는 골다공증 예방)을 부가하여 가지도록 한약재가 추가될 수 있는데, 추가될 수 있는 한약재로서는 두충 추출물, 속단 추출물, 녹용 추출물, 홍화인 추출물, 토사자 추출물, 숙지황 추출물, 별갑 추출물, 산수유 추출물, 구기자 추출물, 감초 추출물, 당귀 추출물, 갈근 추출물, 강진향 추출물, 합환피 추출물, 산두근 추출물, 괴화 추출물, 고삼 추출물 등이 예시될 수 있다.In addition, the herbal composition may be added to the food composition of the present invention to improve flavor and palatability and to add other functionalities (such as prevention of arthritis or osteoporosis), which may be added as a medicinal herb extract, soybean extract, antler extract, safflower Phosphorus extract, Tosa extract, Sukjihwang extract, Tortoiseshell extract, Cornus extract, Goji berry extract, Licorice extract, Angelica extract, Root extract, Gangjinhyang extract, Haphwanpi extract, Sandugeun extract, Cultivated extract, Red ginseng extract may be exemplified. .
전술한 바와 같이, 본 발명에 따르면 항염증성 조성물을 제공할 수 있다. 본 발명의 항염증성 조성물은 약제학적 조성물 또는 식품 조성물로 응용되어 사용될 수 있다.As described above, the present invention can provide an anti-inflammatory composition. The anti-inflammatory composition of the present invention can be applied and used as a pharmaceutical composition or a food composition.
이하 본 발명을 실시예를 참조하여 설명한다. 그러나 본 발명의 범위가 이러한 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described with reference to Examples. However, the scope of the present invention is not limited to these examples.
<실시예> <Examples> 각종 식물 추출물 제조Manufacture of various plant extracts
아래 <표 1>의 122종의 각종 식물의 잎, 줄기, 지상부, 가지 또는 전초(全草)를 80% 에탄올에 침지시켜 상온에서 24시간 동안 추출한 후 추출 잔사를 제거하고 감암농축하여 고형상의 추출물을 얻었다. 각각의 시료는 분주하여 DMSO에 녹여 사용하였다. The leaves, stems, ground parts, branches, or outposts of the 122 species of plants listed in Table 1 below were immersed in 80% ethanol and extracted at room temperature for 24 hours. An extract was obtained. Each sample was aliquoted and dissolved in DMSO.
[표 1]TABLE 1
추출에 사용한 식물 및 추출 부위Plants and Extraction Sites Used for Extraction
<실험예> 항염 활성 평가Experimental Example Evaluation of Anti-inflammatory Activity
<1> <1> 세포 배양 및 시약Cell Culture and Reagents
Murine macrophage cell line인 RAW 264.7 세포를 KCLB (Korean Cell Line Bank)로부터 분양 받아 100 units/ml penicillin-streptomycin과 10% fetal bovine serum (FBS)이 함유된 DMEM배지를 사용하여 37 ℃, 5% CORAW 264.7 cells, a Murine macrophage cell line, were distributed from KCLB (Korean Cell Line Bank) and were treated with DMEM medium containing 100 units / ml penicillin-streptomycin and 10% fetal bovine serum (FBS) at 37 ° C and 5% CO. 22 항온기 (incubator)에서 배양하였으며, 2~3일에 한번씩 계대 배양을 실시하였다. The cells were cultured in an incubator and passaged once every 2 to 3 days.
세포 계대 배양에 사용된 DMEM (Dulbecco’s Modified Eagle’s Medium) 배 지와 fetal bovine serum (FBS)은 Hyclone (Logan, Utah, USA)에서 구입하였고, lipopolysaccharide (LPS, E. coli serotype 0111:B4)는 sigma (St. Louis, MO)로부터 구입하였다. TNF-α와 IL-6 정량을 위한 enzyme-linked immunosorbent assay (ELISA) kit는 BD Biosciences (San Diego, USA)로부터 구입하였으며, PGE2 정량을 위한 ELISA kit는 R&D systems (Minneapolis, USA)으로부터 구입하였다. DMEM (Dulbecco's Modified Eagle's Medium) media and fetal bovine serum (FBS) used for cell passage were purchased from Hyclone (Logan, Utah, USA), and lipopolysaccharide (LPS, E. coli serotype 0111: B4) was used for sigma ( St. Louis, MO). An enzyme-linked immunosorbent assay (ELISA) kit for quantifying TNF-α and IL-6 was purchased from BD Biosciences (San Diego, USA) and an ELISA kit for quantifying PGE 2 was obtained from R & D systems (Minneapolis, USA). .
<2> Nitric oxide (NO) 생성 억제 활성 측정 <2> Determination of Nitric oxide (NO) production inhibitory activity
RAW 264.7 세포 (1.5×105 cells/㎖)를 18시간 전 배양 후, 실시예의 각 시료(100 ㎍/㎖)와 LPS(1 ㎍/㎖)를 동시 처리하여 24시간 배양하였고, 생성된 NO는 Griess 시약을 이용하여 세포 배양액 중에 존재하는 NO2 - 의 형태로 측정하였다. 세포배양 상층액 100㎕와 Griess시약[1% (w/v) sulfanilamide, 0.1% (w/v) naphtylethylenediamine in 2.5% (v/v) phosphoric acid]을 동량 혼합하여 10분간 실온 암소에서 반응시킨 후 ELISA reader를 이용하여 540 nm에서 흡광도를 측정하였다. 표준농도 곡선은 sodium nitrite (NaNO2)를 serial dilution하여 얻었다 (10-100μM).After culturing RAW 264.7 cells (1.5 × 10 5 cells / ml) 18 hours ago, each sample (100 μg / ml) and LPS (1 μg / ml) of the Example were incubated for 24 hours. The Griess reagent was used to measure NO 2 − present in the cell culture. 100 μl of cell culture supernatant and Griess reagent [1% (w / v) sulfanilamide, 0.1% (w / v) naphtylethylenediamine in 2.5% (v / v) phosphoric acid] Absorbance was measured at 540 nm using an ELISA reader. Standard concentration curves were obtained by serial dilution of sodium nitrite (NaNO 2 ) (10-100 μM).
결과를 <표 2>에 나타내었다.The results are shown in Table 2.
[표 2]TABLE 2
NO 생성 억제 활성NO production inhibitory activity
상기 <표 2>의 결과는 상기 각 시료가 모두 NO 생성 억제 활성을 가짐을 보여준다.The results in Table 2 show that each of the samples had NO production inhibitory activity.
참고로 상기 각 시료의 처리 농도에서의 세포 독성은 아래의 <표 4>에 나타내었는데, 상기 각 시료는 모두 특별한 세포독성을 보이지 않았다. 약간의 세포독성을 보이는 추출물(예컨대 으름덩굴 잎 추출물, 쥐똥나무 잎 추출물, 후피향나무 잎 추출물, 꾸지뽕나무 잎 추출물 등)이 있었지만, 그것의 세포독성 이상으로 NO 생성 억제 활성을 보였다.For reference, the cytotoxicity at the treatment concentration of each sample is shown in Table 4 below, and each of the samples did not show any particular cytotoxicity. There were some cytotoxic extracts (for example, ivy leaf extract, rodent leaf extract, cedar leaf extract, cucurbita leaf extract, etc.), but more than its cytotoxicity showed NO production inhibitory activity.
<3> 염증 매개 인자인 TNF-α, IL-6 및 PGE 2 의 정량 <3> Quantitation of inflammatory mediators TNF-α, IL-6 and PGE 2
Murine macrophage cell line인 RAW 264.7 세포 (4.0×105 cells/㎖)를 18시간 전 배양하고 상기 각 시료(100 ㎍/㎖)를 LPS(1 ㎍/㎖)와 동시처리 하여 24시간 배양하였다. 24시간 후 배양 배지를 원심분리(12,000 rpm, 3 min) 하여 얻어진 상층액의 PGE2, TNF-α와 IL-6 함량을 측정하였다. 정량은 murine enzyme-linked immunosorbent assay (ELISA) kit를 이용하여 정량하였으며 standard에 대한 표준 곡선의 r2 값은 0.99 이상이었다 (Cho et al., 2000). RAW 264.7 cells (4.0 × 10 5 cells / mL), a Murine macrophage cell line, were incubated 18 hours ago and each sample (100 μg / mL) was incubated with LPS (1 μg / mL) for 24 hours. After 24 hours, the culture medium was centrifuged (12,000 rpm, 3 min) to determine the PGE 2 , TNF-α and IL-6 contents of the supernatant. Quantification was performed using a murine enzyme-linked immunosorbent assay (ELISA) kit. The r 2 value of the standard curve for the standard was above 0.99 (Cho et al ., 2000).
결과를 아래의 <표 3>에 나타내었다.The results are shown in Table 3 below.
[표 3][Table 3]
염증 매개 인자의 억제율Inhibition of Inflammation Parameters
상기 <표 3>의 결과는 상기 각 시료를 LPS와 함께 처리했을 때의 결과이다. <표 3>에서 상기 각 시료가 염증 매개 인자 즉 PGEThe result of Table 3 is the result when each said sample was processed with LPS. In Table 3, each sample is an inflammation parameter or PGE. 22 , TNF-α및 IL-6의 생성 억제 활성을 가짐을 알 수 있다. 참고로 상기 각 시료의 세포 독성은 아래의 <표 4>에서 확인할 수 있다., TNF-α and IL-6 production inhibitory activity. For reference, the cytotoxicity of each sample can be confirmed in Table 4 below.
<4> <4> 세포독성 평가Cytotoxicity Assessment
세포독성은 MTT assay를 이용하여 실험하였다. 생존하면서 대사가 왕성한 세포는, 세포 내 mitochondria의 탈수소효소 작용에 의하여 수용성의 노란색 MTT [3-(4,5-dimethylthiaxol-2-yl)-2,5-diphenyl tetrazolium bromide]를 환원시켜 자주색을 띠는 비 수용성의 formazan을 형성한다 (Gerlier Cytotoxicity was tested using MTT assay. Cells that are alive and metabolized have a purple color by reducing water-soluble yellow MTT [3- (4,5-dimethylthiaxol-2-yl) -2,5-diphenyl tetrazolium bromide] by the dehydrogenase action of mitochondria in the cell. Forms a water-insoluble formazan (Gerlier et alet al ., 1986; Liu, 1999). RAW 264.7 세포 (1.5×10., 1986; Liu, 1999). RAW 264.7 cells (1.5 × 10 5 5 cells/㎖)에 각 시료(100 ㎍/㎖)를 LPS (1 ㎍/㎖)와 동시 처리하고 24시간 배양한 후, MTT (Sigma, MO, USA) 용액 50 ㎕ (2 ㎖/㎎)를 첨가하여 4시간 동안 반응시킨다. 상층액을 완전히 제거하고 dimethylsulfoxide (DMSO : Sigma, MO, USA)를 200 ㎕를 가하여 침전물을 완전히 용해시킨 후, microplate reader (Amersham Pharmacia Biotech, NY, USA)를 사용하여 540 nm에서 흡광도를 측정하였다. 각 시료군에 대한 평균 흡광도 값을 구하였으며, 대조군의 흡광도 값과 비교하여 독성 정도를 평가하였다.cells / ml) were treated with LPS (1 µg / ml) simultaneously with LPS (1 µg / ml) and incubated for 24 hours, and then 50 µl (2 ml / mg) of MTT (Sigma, MO, USA) solution was added. To react for 4 hours. The supernatant was completely removed, 200 μl of dimethylsulfoxide (DMSO: Sigma, MO, USA) was added to completely dissolve the precipitate, and the absorbance was measured at 540 nm using a microplate reader (Amersham Pharmacia Biotech, NY, USA). The average absorbance value for each sample group was obtained, and the degree of toxicity was evaluated by comparing with the absorbance value of the control group.
결과는 아래와 같다.The result is shown below.
[표 4][Table 4]
세포 독성Cytotoxicity
상기 <표 4>의 결과는 상기 각 실시예 시료의 NO 생성 억제 활성 및 염증 매개 인자의 생성 억제 활성이 세포 독성에 의한 결과가 아님을 보여준다.The results in Table 4 show that the NO production inhibitory activity and the production inhibitory activity of inflammatory mediators of the respective sample samples are not the result of cytotoxicity.
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