KR101656561B1 - Anti-inflammation Composition Using Phacelocarpus japonicus Extracts - Google Patents
Anti-inflammation Composition Using Phacelocarpus japonicus Extracts Download PDFInfo
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- KR101656561B1 KR101656561B1 KR1020140058688A KR20140058688A KR101656561B1 KR 101656561 B1 KR101656561 B1 KR 101656561B1 KR 1020140058688 A KR1020140058688 A KR 1020140058688A KR 20140058688 A KR20140058688 A KR 20140058688A KR 101656561 B1 KR101656561 B1 KR 101656561B1
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Abstract
본 발명은 꿩꼬리풀 추출물을 이용한 항염증용 조성물을 개시한다.
구체적으로 LPS(lipopolysaccharide)로 자극된 대식세포에서 NO 생성 억제 활성, PGE2 생성 억제 활성, 염증성 사이토카인(TNF-α, IL-6 및 IL-1β) 생성 억제 활성 그리고 iNOS 및 COX-2 생성 억제 활성을 보이고, LPS로 자극된 골수 유래 수지상세포에서 염증성 사이토카인인 IL-12p40의 생성 억제 활성을 보이며, 나아가 IFN-γ로 자극된 각질형성세포인 HaCaT keratinocytes에서 염증성 케모카인인 MDC(macrophage-derived chemokine)의 생성 억제 활성을 보이는 꿩꼬리풀 추출물을 이용한 항염증용 조성물을 개시한다.The present invention discloses a composition for anti-inflammation using a pheasant herring extract.
Specifically, inhibition of NO production, inhibition of PGE 2 production, inhibition of inflammatory cytokine production (TNF-α, IL-6 and IL-1β) and inhibition of iNOS and COX-2 production in macrophages stimulated by lipopolysaccharide (LPS) 12p40, which is a proinflammatory cytokine, in the bone marrow-derived dendritic cells stimulated by LPS. In addition, IFN-gamma-stimulated keratinocytes HaCaT keratinocytes stimulate macrophage-derived chemokine The present invention relates to a composition for anti-inflammation using a pheasant extract of pheasant,
Description
본 발명은 꿩꼬리풀(Phacelocarpus japonicus) 추출물을 이용한 항염증용 조성물에 관한 것이다.
The present invention relates to the use of Phacelocarpus japonicus extract of the present invention.
염증은 외부의 물리·화학적 자극, 박테리아, 곰팡이, 바이러스, 각종 알레르기 유발 물질 등 외부 감염원의 감염에 대한 생체의 방어 반응이다. Inflammation is the defensive response of a living body to external infectious agents such as external 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.The inflammatory response is part of the innate immune response, and as in other animals, the innate immune response of humans begins by recognizing and attacking macrophages as non-self through a pattern of cell surfaces that are specific to the pathogen. During the inflammation reaction, plasma accumulates on the inflamed area, diluting the toxicities secreted by the bacteria, increasing the 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 reactions involve a variety of biochemical events, in particular, cyclooxygenase (COX), which is associated with the nitric oxide synthase (NOS) and the biosynthesis of various prostaglandins, .
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: endotoxin of bacteria such as calcium or camodanol-dependent eNOS (endothelial NOS), nNOS (neurotic NOS), and lipopolysaccharide (IL-1β, TNF-α, IL There are iNOS (inducible NOS) induced by various inflammatory cytokines such as IL-6, IL-8 and IL-12 and produce 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 maintenance of homeostasis by performing various physiological responses related to blood pressure control, neurotransmission, learning, memory, etc. However, NO produced by iNOS is associated with arthritis, sepsis, (Moncade S. et al, Pharmacol. Rev., 1991, 43, 109, Nature Medicine, 2001, 7, 1138; Mu, S., et al., Immunoprecipitation, , 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 synthesizes PGG 2 and PGH 2 from arachidonic acid with hydroperoxidase (HOX) activity together with the function of COX. These compounds include PGE 2 , PGF 2 , PGD 2 , prostacyclin and create a duplex thromboxane a 2 (thromboxane A2, TxA2) . Among the functions of COX, the function of PGH synthase is involved in the pain and inflammation reaction through 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 NO, PGE2, 염증성 사이토카인 등의 억제제나 iNOS 억제제 그리고 COX-2 억제제는 염증질환 치료제로서 활용될 수 있다.Therefore, NO, PGE 2 , Inhibitors such as inflammatory cytokines, iNOS inhibitors and COX-2 inhibitors may be used as therapeutic agents for inflammatory diseases.
본 발명은 NO 생성 억제 활성, PGE2의 생성 억제 활성, 염증성 사이토카인의 생성 억제 활성 등을 가지는 꿩꼬리풀 추출물을 개시한다.
The present invention discloses a pheasant extract of Pheasant having a NO production inhibitory activity, a PGE 2 production inhibitory activity, an inflammatory cytokine production inhibitory activity and the like.
본 발명의 목적은 꿩꼬리풀 추출물을 이용한 항염증용 조성물을 제공하는 데 있다.It is an object of the present invention to provide a composition for anti-inflammation using an extract of pheasant corn borer.
본 발명의 다른 목적이나 구체적인 목적은 이하에서 제시될 것이다.
Other and further objects of the present invention will be described below.
본 발명은 아래의 실시예 및 실험예에서 확인되는 바와 같이, 꿩꼬리풀 80% 에탄올 추출물과 그 분획물 그리고 초임계 추출물이 LPS(lipopolysaccharide)로 자극된 대식세포에서 NO 생성 억제 활성, PGE2 생성 억제 활성, 염증성 사이토카인(TNF-α, IL-6 및 IL-1β) 생성 억제 활성 그리고 iNOS 및 COX-2 생성 억제 활성을 보이고, LPS로 자극된 골수 유래 수지상세포에서 염증성 사이토카인인 IL-12p40의 생성 억제 활성을 보이며, 나아가 IFN-γ로 자극된 각질형성세포인 HaCaT keratinocytes에서 염증성 케모카인인 MDC(macrophage-derived chemokine)의 생성 억제 활성을 보임을 확인함으로써 완성된 것이다. 상기에서 그람 음성균의 외막성분인 LPS는 대식세포를 자극하여 NO, PGE2, 염증성 사이토카인(TNF-α, IL-1β, IL-6) 등의 염증 매개 물질의 분비를 유발하는 것으로 알려져 있고(Proc Soc Exp Biol Med. 1996;211 : 32-24; Kor J Herbol. 2009;24 : 47-39), 염증성 케모카인인 MDC의 경우 아토피성 피부염 환자에서 그 혈청 농도가 현저히 증가한다는 보고가 있으며(Hijnen et. al., J. Allergy Clin. Immunol. 113(2) 334-340), 아토피성 피부염의 치료 물질로 사용되는 사이크로스포린 A나 코르티코스테로이드를 아토피성 피부염 환자에게 투여하였을 때는 MDC의 혈청 농도가 감소한다는 보고가 있고(Y. Shimada et al., J. Dermatol. Sci., 34, 201-208, 2004), 또 시험관내 실험에서 HaCaT 세포에 INF-γ나 TNF-α를 처리하였을 때 MDC가 다량 발현되는데 이러한 발현을 억제할 수 있는 물질은 아토피성 피부염 치료제의 후보물질이 될 수 있음이 제시된 바 있다(Horikawa et. al., Int. Immunol., 14(7) 767-773, 2002).The present invention is based on the finding that the 80% ethanol extract and its fractions and the supercritical extract of Pheasant lupine have inhibited NO production, LPS (lipopolysaccharide) stimulated macrophages, PGE 2 production inhibitory activity , IL-6 and IL-1β) and inhibitory activity on iNOS and COX-2 production, and the production of IL-12p40, an inflammatory cytokine, in bone marrow-derived dendritic cells stimulated by LPS Inhibited the activity of IFN-y, and further confirmed the activity of inhibiting the production of MDC (macrophage-derived chemokine), an inflammatory chemokine, in HaCaT keratinocytes, keratinocytes stimulated with IFN-y. LPS, which is an outer membrane component of Gram-negative bacteria, is known to stimulate macrophages to induce the secretion of inflammatory mediators such as NO, PGE 2 , and inflammatory cytokines (TNF-α, IL-1β, IL-6) MDC, an inflammatory chemokine, has been reported to significantly increase its serum concentration in patients with atopic dermatitis (Hijnen et al., 1996; (2) 334-340). When ciclosporin A or corticosteroids, which are used as therapeutic agents for atopic dermatitis, were administered to patients with atopic dermatitis, serum concentrations of MDC (Y. Shimada et al., J. Dermatol. Sci., 34, 201-208, 2004), and in vitro experiments revealed that when HaCaT cells were treated with INF-? Or TNF- ?, MDC A substance capable of inhibiting such expression can be used as a therapeutic agent for the treatment of atopic dermatitis There is shown a bar could be a material (Horikawa et. Al., Int. Immunol., 14 (7) 767-773, 2002).
전술한 바를 고려할 때, 본 발명의 항염증용 조성물은 꿩꼬리풀 추출물을 유효성분으로 포함함을 특징으로 한다.In view of the foregoing, the composition for anti-inflammation of the present invention is characterized by containing an extract of Pheasant corn borer as an active ingredient.
본 명세서에서, "추출물"이란 추출 대상을 물, 메탄올, 에탄올, 부탄올 등의 탄소수 1 내지 4의 저급 알콜, 메틸렌클로라이드, 에틸렌, 아세톤, 헥산, 에테르, 클로로포름, 에틸아세테이트, 부틸아세테이트, N, N-디메틸포름아미드(DMF), 디메틸설폭사이드(DMSO), 1,3-부틸렌글리콜, 프로필렌글리콜 또는 이들의 혼합 용매를 사용하여 침출하여 얻어진 추출물, 이산화탄소, 펜탄 등 초임계 추출 용매를 사용하여 얻어진 추출물 또는 그 추출물을 분획하여 얻어진 분획물을 의미하며, 추출 방법은 활성물질의 극성, 추출 정도, 보존 정도를 고려하여 냉침, 환류, 가온, 초음파 방사, 초임계 추출 등 임의의 방식을 적용할 수 있다. 분획된 추출물의 경우 상기 조추출물을 특정 용매에 현탁시킨 후 극성이 다른 용매와 혼합·정치시켜 얻은 분획물을 포함하고, 상기 조추출물을 상기 용매들을 극성이 증가 또는 감소하는 순으로 사용하여 순차적으로 분획하여 얻어진 분획물을 포함하며, 나아가 크기, 전하, 소수성, 친화성 등의 성질을 이용한 크로마토그래피에 의하여 얻어진 분획물을 포함한다. 또한 상기 추출물의 의미에는 동결건조, 진공건조, 열풍건조, 분무건조 등의 방식으로 추출 용매가 제거된 농축된 액상 또는 고형상의 추출물이 포함된다. 아래의 실시예를 참조할 때, 바람직하게는 추출용매로서 물, 에탄올 또는 이들의 혼합 용매를 사용하여 침출하여 얻어진 추출물(특히 물과 에탄올의 혼합 용매), 그 추출물에서 추출용매를 제거하여 얻은 고형상의 추출물을 물에 현탁하고 이를 헥산, 에틸아세테이트 및 부탄올로 순차적으로 분획하였을 때 얻어지는 각층의 분획물, 또는 이산화탄소를 용매로 사용하여 초임계 추출하여 얻어진 추출물을 의미한다. 특히 상기 분획물 중에서도 아래의 실시예 및 실험예가 보여주듯이 NO 생성 억제 활성, PGE2 생성 억제 활성, 염증성 사이토카인(TNF-α, IL-6) 생성 억제 활성, iNOS 및 COX-2 생성 억제 활성이 우수한 헥산 분획물 또는 에틸아세테이트 분획물이 바람직하다. 여기서 '순차적으로 분획한다'는 의미는 분획 후의 잔여 물층을 계속적으로 사용하여 상기 열거된 순서대로의 분획 용매로 분획한다는 의미이다.As used herein, the term "extract" refers to a substance which is extracted with water, a lower alcohol having 1 to 4 carbon atoms such as methanol, ethanol, and butanol, methylene chloride, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, - Extracts obtained by leaching using dimethylformamide (DMF), dimethylsulfoxide (DMSO), 1,3-butylene glycol, propylene glycol or a mixed solvent thereof, and extracts obtained using supercritical extraction solvents such as carbon dioxide and pentane Extract means the fraction obtained by fractionating the extract or the extract. The extraction method can be applied by any method such as cold rolling, refluxing, heating, ultrasonic irradiation, supercritical extraction, etc. in consideration of the polarity, extraction degree, . The fraction extracted is a fraction obtained by suspending the crude extract in a specific solvent and mixing and leaving with a solvent having a different polarity. The crude extract is used in the order of increasing or decreasing the polarity, , And further includes fractions obtained by chromatography using properties such as size, charge, hydrophobicity, affinity, and the like. Also, the meaning of the extract includes concentrated liquid or solid extract from which the extraction solvent has been removed by freeze drying, vacuum drying, hot air drying, spray drying and the like. The following examples are intended to illustrate the present invention, but it is not intended to limit the scope of the present invention. EXAMPLES The following examples are intended to illustrate, but are not to be construed to limit the scope of the present invention. Means an extract obtained by suspending the extract in water in water and fractionating it with hexane, ethyl acetate and butanol sequentially, or extract obtained by supercritical extraction using carbon dioxide as a solvent. Particularly, as shown in Examples and Experimental Examples below, it was found that, among the above-mentioned fractions, the inhibitory activity against NO production, the inhibitory activity against PGE 2 formation, the inhibitory activity against inflammatory cytokine (TNF-α, IL-6) production, and the inhibitory activity against iNOS and COX- Hexane fractions or ethyl acetate fractions are preferred. Here, 'sequential fractionation' means that the residue layer after fractionation is continuously used to fractionate into fraction solvents in the order listed above.
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 the present specification, the above-mentioned "active ingredient" means an ingredient that exhibits the desired activity alone or can exhibit activity together with a carrier that is itself inactive.
또 본 명세서에서, "항염증"은 아래에서 정의되는 염증성 질환의 개선, 치료, 그러한 질환의 발병 억제 또는 지연을 포함하는 의미이다.As used herein, "anti-inflammatory" is meant to include the improvement, treatment, inhibition or delay of the onset of an inflammatory disease 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)의 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(아토피성 피부염 포함), 습진, 다발성 경화증 등이 포함될 것이다. 특히 아래의 실시예 및 실험예에서 꿩꼬리풀 추출물이 IFN-γ로 자극된 각질형성세포인 HaCaT keratinocytes에서 염증성 케모카인인 MDC(macrophage-derived chemokine)의 생성 억제 활성을 보인다는 점에서 상기 염증성 질환은 아토피성 피부염인 것이 바람직하다.In the present specification, the above-mentioned "inflammatory disease" is defined as any state specified by a local or systemic bio-defense reaction against external physical or chemical stimulation or infection of an external infectious agent such as bacteria, fungi, viruses, . This response is secretion of activated, inflammatory mediators of the enzyme (for example, iNOS, COX-2, and so on) associated with various inflammatory mediators and the immune cells (e. G., NO, of TNF-α, IL-6, IL-1β,
본 발명의 조성물은 그 유효성분을 용도, 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 15 중량 % 범위 내에서 결정될 것이다. 여기서 "유효량"이란 그 적용 대상인 포유동물 바람직하게는 사람에게서, 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.The composition of the present invention may be contained in any amount (effective amount) as long as it can exhibit the improving activity of an inflammatory disease intended for treatment depending on the purpose of use, formulation, blending purpose and the like. By weight based on the total weight of the composition. The term "effective amount" as used herein refers to the amount of active ingredient capable of inducing the improvement, treatment, or inhibition / delay of the onset of such pathological conditions in a mammal, preferably a human, to which it is applied. Such effective amounts can be determined experimentally within the ordinary skill of those skilled in the art.
본 발명의 조성물이 적용(처방)될 수 있는 대상은 포유동물 및 사람이며, 특히 사람인 경우가 바람직하다.The subject to which the composition of the present invention can be applied (prescription) is preferably a mammal and a person, particularly a human.
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 그 유효성분을 포함하는 이외에 약제학적으로 허용되는 담체, 부형제 등을 포함하여, 경구용 제형(정제, 현탁액, 과립, 에멀젼, 캡슐, 시럽 등), 비경구형 제형(멸균 주사용 수성 또는 유성 현탁액), 국소형 제형(용액, 크림, 연고, 겔, 로션, 패치) 등으로 제조될 수 있다.The pharmaceutical composition of the present invention may be in the form of oral dosage forms (tablets, suspensions, granules, emulsions, capsules, syrups, etc.), parenteral formulations (including sterile injectable preparations (E.g., aqueous or oily suspensions in the form of injectable solutions), and small formulations (solutions, creams, ointments, gels, lotions, patches).
상기에서 "약제학적으로 허용되는" 의미는 유효성분의 활성을 억제하지 않으면서 적용(처방) 대상이 적응가능한 이상의 독성(충분히 낮은 독성)을 지니지 않는다 의미이다.The term "pharmaceutically acceptable" as used herein means that the application (prescribing) subject does not have the above-mentioned toxicity (sufficiently low toxicity) without inhibiting the activity of the active ingredient.
약제학적으로 허용되는 담체의 예로서는 락토스, 글루코스, 슈크로스, 전분(예컨대 옥수수 전분, 감자 전분 등), 셀룰로오스, 그것의 유도체(예컨대 나트륨 카르복시메틸 셀룰로오스, 에틸셀룰로오스, 등) 맥아, 젤라틴, 탈크, 고체 윤활제(예컨대 스테아르산, 스테아르산 마그네슘 등), 황산 칼슘, 식물성 기름(예컨대 땅콩 기름, 면실유, 참기름, 올리브유 등), 폴리올(예컨대 프로필렌 글리콜, 글리세린 등), 알긴산, 유화제(예컨대 TWEENS), 습윤제(예컨대 라우릴 황산 나트륨), 착색제, 풍미제, 정제화제, 안정화제, 항산화제, 보존제, 물, 식염수, 인산염 완충 용액 등을 들 수 있다. 이러한 담체는 본 발명의 약제학적 조성물의 제형에 따라 적당한 것을 하나 이상 선택하여 사용할 수 있다.Examples of pharmaceutically acceptable carriers include lactose, glucose, sucrose, starch (e.g. corn starch, potato starch, etc.), cellulose, derivatives thereof (e.g. sodium carboxymethylcellulose, ethylcellulose, etc.) malt, gelatin, talc, (E.g., peanut oil, cottonseed oil, sesame oil, olive oil, etc.), polyol (e.g., propylene glycol, glycerin and the like), alginic acid, emulsifiers (e.g., TWEENS), humectants Such as sodium lauryl sulfate, a coloring agent, a flavoring agent, a tableting agent, a stabilizer, an antioxidant, a preservative, water, a saline solution, and a phosphate buffer solution. The carrier may be selected from one or more of suitable pharmaceutical formulations according to the formulation of the pharmaceutical composition of the present invention.
부형제도 본 발명의 약제학적 조성물의 제형에 따라 적합한 것을 선택하여 사용할 수 있는데, 예컨대 본 발명의 약제학적 조성물이 수성 현탁제로 제조될 경우에 적합한 부형제로서는 나트륨 카르복시메틸 셀룰로오스, 메틸 셀룰로오스, 히드로프로필메틸셀룰로오스, 알긴산 나트륨, 폴리비닐피롤리돈 등의 현탁제나 분산제 등을 들 수 있다. 주사액으로 제조되는 경우 적합한 부형제로서는 링거액, 등장 염화나트륨 등을 들 수 있다.The excipient may be selected according to the formulation of the pharmaceutical composition of the present invention. For example, when the pharmaceutical composition of the present invention is prepared by an aqueous suspension, suitable excipients include sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose , Sodium alginate, polyvinylpyrrolidone, and the like. Suitable excipients when prepared from injection solutions include Ringer's solution, isotonic sodium chloride, and the like.
본 발명의 약제학적 조성물은 경구 또는 비경구로 투여될 수 있고, 아토피성 피부염 조성물처럼 경우에 따라서는 국소적으로 투여될 수 있다.The pharmaceutical compositions of the present invention may be administered orally or parenterally and may be administered topically, as the case may be, such as an atopic dermatitis composition.
본 발명의 약제학적 조성물은 그 1일 투여량이 통상 0.001 ~ 150 mg/kg 체중 범위이고, 1회 또는 수회로 나누어 투여할 수 있다. 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 아니 된다. The daily dose of the pharmaceutical composition of the present invention is usually 0.001 to 150 mg / kg body weight, and may be administered once or several times. However, since the dosage of the pharmaceutical composition of the present invention is determined in view of various related factors such as route of administration, age, sex, weight, and patient's severity of the patient, the dose is limited in any aspect to the scope of the present invention Should not be understood to be.
본 발명의 조성물은 구체적인 양태에 있어서, 식품 조성물로 파악할 수 있다. In a specific embodiment, the composition of the present invention can be identified as a food composition.
본 발명의 식품 조성물에는 그 유효성분 이외에 감미제, 풍미제, 생리활성 성분, 미네랄 등이 포함될 수 있다.The food composition of the present invention may contain sweetening agents, flavoring agents, physiologically active ingredients, minerals and the like in addition to the active ingredients thereof.
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. Sweetening agents may be used in an amount that sweetens the food in a suitable manner, and may be natural or synthetic. Preferably, natural sweeteners are used. Examples of natural sweeteners 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 등이 이용될 수 있다. Flavors may be used to enhance taste or flavor, 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. Examples of natural flavoring agents include those obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, and the like, or those obtained from green tea leaves, Asiatica, Daegu, Cinnamon, Chrysanthemum leaves and Jasmine. 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. Synthetic flavors may be used depending on the case, and synthetic flavors such as esters, alcohols, aldehydes, terpenes and the like may be used.
생리 활성 물질로서는 카테킨, 에피카테킨, 갈로가테킨, 에피갈로카테킨 등의 카테킨류나, 레티놀, 아스코르브산, 토코페롤, 칼시페롤, 티아민, 리보플라빈 등의 비타민류 등이 사용될 수 있다.Examples of the physiologically active substance include catechins such as catechin, epicatechin, gallocatechin and epigallocatechin, and vitamins such as retinol, ascorbic acid, tocopherol, calciferol, thiamine and riboflavin.
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 the mineral, calcium, magnesium, chromium, cobalt, copper, fluoride, germanium, iodine, iron, lithium, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, silicon, sodium, sulfur, vanadium and zinc 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 preservatives, emulsifiers, acidifiers, thickeners and the like as needed in addition to the above sweeteners.
이러한 보존제, 유화제 등은 그것이 첨가되는 용도를 달성할 수 있는 한 극미량으로 첨가되어 사용되는 것이 바람직하다. 극미량이란 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 in a very small amount as long as they can attain an application to which they are added. The term " trace amount " means, when expressed numerically, in the range of 0.0005% by weight 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 the preservative which can be used include calcium sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate and EDTA (ethylenediaminetetraacetic acid).
사용될 수 있는 유화제로서는 아카시아검, 카르복시메틸셀룰로스, 잔탄검, 펙틴 등을 들 수 있다.Examples of the emulsifier which can be used include acacia gum, carboxymethyl cellulose, 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 the acidulant that can be used include acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, and phosphoric acid. Such an acidulant may be added so that the food composition has a proper acidity for the purpose of inhibiting the growth of microorganisms other than the purpose of enhancing the taste.
사용될 수 있는 점증제로서는 현탁화 구현제, 침강제, 겔형성제, 팽화제 등을 들 수 있다. Agents that may be used include suspending agents, sedimentation agents, gel formers, bulking agents and the like.
또한 향미나 기호성을 향상시키고 다른 기능성을 추가하기 위하여 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, herbal medicines may be added to improve flavor and palatability and to add other functionalities. Examples of medicinal herbs that can be added include mulberry extract, early-stage extract, antler extract, safflower extract, tosaja extract, , Gugija extract, licorice extract, Angelica gigantosa extract, Puerariae Radix extract, Gangjin extract, Manganese extract, Mountain beet root extract, Bulgogi extract, Radix extract.
본 발명은 또 다른 측면에 있어서, 화장품 조성물로 파악할 수 있다.In another aspect, the present invention can be identified as a cosmetic composition.
본 발명의 조성물이 화장품 조성물로서 파악될 경우, 그 화장품 조성물은 다양한 형태로 제조될 수 있는데, 예컨대, 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성제-함유 클린싱, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션, 스프레이 등으로 제형화될 수 있으나, 이에 한정되는 것은 아니다. 바람직하게는 에멀젼, 로션, 크림(수중유적형, 유중수적형, 다중상), 용액, 현탁액(무수 및 수계), 무수 생성물(오일 및 글리콜계), 젤, 마스크, 팩 또는 분말 등의 제형으로 제조될 수 있다.When the composition of the present invention is recognized as a cosmetic composition, the cosmetic composition may be prepared in various forms, for example, as a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, , Oil, powder foundation, emulsion foundation, wax foundation, spray, and the like, but is not limited thereto. It is preferably formulated into emulsions, lotions, creams (water-in-oil type, water-in-water type, multiphase), solutions, suspensions (anhydrous and aquatic), anhydrous products (oil and glycol), gels, masks, packs or powders .
본 발명의 조성물은 그 유효성분 이외에 화장품 제제에 있어서 수용가능한 담체를 포함할 수 있다. The composition of the present invention may contain an acceptable carrier in cosmetic preparations in addition to its active ingredients.
여기서 "화장품 제제에 있어서 수용가능한 담체"란 화장품 제제에 포함될 수 있는 이미 공지되어 사용되고 있는 화합물 또는 조성물이거나 앞으로 개발될 화합물 또는 조성물로서 피부와의 접촉시 인체가 적응 가능한 독성 이상의 독성이 없는 것을 말한다.As used herein, the term " acceptable carrier for a cosmetic preparation "refers to a compound or composition which is already known and used in the cosmetic preparation, or which is a compound or composition to be developed in the future, and which is not toxic to the human body.
상기 담체는 본 발명의 조성물에 그것의 전체 중량에 대하여 약 1 중량 % 내지 약 99.99 중량 %, 바람직하게는 조성물의 중량의 약 50 중량% 내지 약 99 중량 %로 포함될 수 있다. The carrier may be included in the composition of the present invention in an amount of from about 1% by weight to about 99.99% by weight, preferably from about 50% by weight to about 99% by weight of the composition, based on the total weight thereof.
그러나 상기 비율은 화장품의 전술한 바의 제형에 따라 또 그것의 구체적인 적용 부위(얼굴이나 손)나 그것의 바람직한 적용량 등에 따라 달라지는 것이기 때문에, 상기 비율은 어떠한 측면으로든 본 발명의 범위를 제한하는 것으로 이해되어서는 안 된다. However, since the ratio depends on the above-mentioned formulation of the cosmetic product and its specific application site (face or hands) or the desired amount of application thereof, the ratio is to limit the scope of the present invention in any aspect It should not be.
한편, 상기 담체로서는 알코올, 오일, 계면활성제, 지방산, 실리콘 오일, 습윤제, 보습제, 점성 변형제, 유제, 안정제, 자외선 차단제, 발색제, 향료 등이 예시될 수 있다. Examples of the carrier include alcohols, oils, surfactants, fatty acids, silicone oils, humectants, moisturizers, viscosifiers, emulsifiers, stabilizers, sunscreens, coloring agents and perfumes.
상기 담체로서 사용될 수 있는 알코올, 오일, 계면활성제, 지방산, 실리콘 오일, 습윤제, 보습제, 점성 변형제, 유제, 안정제, 자외선 차단제, 발색제, 향료 로 사용될 수 있는 화합물/조성물 등은 이미 당업계에 공지되어 있기 때문에 당업자라면 적절한 해당 물질/조성물을 선택하여 사용할 수 있다.
The compounds / compositions which can be used as the carrier and which can be used as alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, moisturizers, viscosifiers, emulsions, stabilizers, sunscreens, A person skilled in the art can select and use appropriate substances / compositions.
전술한 바와 같이, 본 발명에 따르면 꿩꼬리풀 추출물을 이용한 항염증용 조성물을 제공할 수 있다. 본 발명의 항염증용 조성물은 약제학적 조성물, 식품 조성물 또는 화장품 조성물로 제품화될 수 있다.
As described above, according to the present invention, it is possible to provide a composition for anti-inflammation using an extract of pheasant corn borer. The anti-inflammatory composition of the present invention can be produced into pharmaceutical compositions, food compositions or cosmetic compositions.
도 1 내지 도 3은 LPS로 자극된 대식세포에서 꿩꼬리풀 추출물과 분획물의 NO 생성 억제 활성을 나타낸 결과이다.
도 4 및 도 5는 LPS로 자극된 대식세포에서 꿩꼬리풀 추출물의 헥산 분획물과 에틸아세테이트 분획물 그리고 초임계 추출물의 PGE2 생성 억제 활성을 나타낸 결과이다.
도 6 내지 도 11은 LPS로 자극된 대식세포에서 꿩꼬리풀 추출물의 헥산 분획물과 에틸아세테이트 분획물 그리고 초임계 추출물의 염증성 사이토카인의 생성 억제 활성을 나타낸 결과이다.
도 12 및 도 13은 LPS로 자극된 대식세포에서 꿩꼬리풀 추출물과 분획물의 iNOS 와 COX-2 발현 억제 활성을 나타낸 결과이다.
도 14는 LPS로 자극된 골수 유래 수지상세포에서 염증성 사이토카인인 IL-12 p40의 생성 억제 활성을 나타낸 결과이다.
도 15는 IFN-γ로 자극된 각질형성세포에서 염증성 케모카인인 MDC의 생성 억제 활성을 나타낸 결과이다.FIGS. 1 to 3 show the results of inhibiting the NO production of the extracts and fractions of pheasant corn borer on the LPS-stimulated macrophages.
FIG. 4 and FIG. 5 show the results of inhibiting the formation of PGE 2 by hexane fraction, ethyl acetate fraction and supercritical extract of the pheasant extract from LPS-stimulated macrophages.
FIGS. 6 to 11 show the results of inhibiting the production of inflammatory cytokines by the hexane fraction, the ethyl acetate fraction and the supercritical extract of the pheasant extract from the macrophages stimulated by LPS.
FIGS. 12 and 13 show the inhibitory activity of iNOS and COX-2 on the LPS-stimulated macrophages.
14 shows the results of inhibiting the production of IL-12 p40, an inflammatory cytokine, in bone marrow-derived dendritic cells stimulated by LPS.
Fig. 15 shows the results of inhibiting the production of inflammatory chemokine MDC in keratinocytes stimulated with IFN-y.
이하 본 발명을 실시예 및 실험예를 참조하여 설명한다. 그러나 본 발명의 범위가 이러한 실시예 및 실험예에 한정되는 것은 아니다.
Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these examples and experimental examples.
<< 실시예Example > > 꿩꼬리풀Pheasant 추출물의 제조 Preparation of extract
<실시예 1> 꿩꼬리풀의 용매 추출물의 제조 Example 1 Preparation of Solvent Extract of Pheasant < RTI ID = 0.0 >
꿩꼬리풀 전초 분말 1 kg을 5 L의 80% 에탄올에 24시간 동안 침지시켜 3회 반복 추출한 후 여과하고, 그 여액을 감압 농축하여 용매를 제거하고 분말상의 추출물을 얻었다. 이렇게 얻어진 꿩꼬리풀 80% 에탄올 추출물을 10 배 중량의 증류수에 현탁시킨 후 헥산(n-Hexane), 에틸아세테이트(EtOAc) 및 부탄올(butanol)을 이용하여 순차적으로 분획하여 헥산 분획물, 에틸아세테이트 분획물, 부탄올 분획물 및 잔여 물층을 얻었다.1 kg of pheasant sprout outpowder powder was immersed in 5 L of 80% ethanol for 24 hours, repeatedly extracted three times, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent to obtain a powdery extract. The 80% ethanol extract of pheasant horsetail obtained in this manner was suspended in distilled water having a weight of 10 times and then fractionated successively using n-hexane, ethyl acetate (EtOAc) and butanol to obtain hexane fraction, ethyl acetate fraction, Fractions and a residue layer were obtained.
<실시예 2> 꿩꼬리풀의 초임계 추출물의 제조 ≪ Example 2 & gt ; Preparation of Supercritical Extracts
꿩꼬리풀의 초임계 추출물은 초임계추출장치(SFE500R1, EYESEL CO, Ltd.)를 이용하여 제조하였다.The supercritical extract of Pheasant lupine was prepared using supercritical extraction system (SFE500R1, EYESEL CO, Ltd.).
구체적으로 꿩꼬리풀 전초 분말 100g를 초임계 추출조에 투입하고 추출조에 이산화탄소를 공급하면서 승온시켜 300bar 압력과 60℃의 온도를 유지하여 초임계 상태를 만든 후 3시간 동안 추출과정을 진행하여 꿩꼬리풀 초임계 추출물을 얻었다( 추출 방법 전체적으로 확인 바랍니다 ).
Specifically, 100 g of pheasant phyllotaxis powder was added to the supercritical extraction tank, and the temperature was elevated while supplying carbon dioxide to the extraction tank to maintain a supercritical state at a pressure of 300 bar and a temperature of 60 ° C., followed by extraction for 3 hours. The extract was obtained ( please check the extraction method as a whole ).
<< 실험예Experimental Example > > 항염증 활성 실험Anti-inflammatory activity experiment
<실험예 1> 세포 배양 ≪ Experimental Example 1 > Cell culture
American Type Culture Collection (ATCC, Rockville, MD, USA)으로부터 생쥐(murine) 대식세포주인 RAW 264.7을 구입하여, 10% fetal bovine serum (FBS), penicillin (100 units/mL), 및 streptomycin (100 g/mL)을 첨가하여 Dulbeccos Modified Eagles Medium (DMEM; GIBCO Inc.)에서 보관하였다. 이들 세포들은 37℃ 에서 95% air, 5% CO2 가습 공기 조건 하 포화 상태(subconfluence)에서 배양하였으며, 3일마다 계대배양하였다. RAW 264.7 was purchased from the American Type Culture Collection (ATCC, Rockville, Md., USA), and 10% fetal bovine serum (FBS), penicillin (100 units / mL), and streptomycin (100 g / mL) was added and stored in Dulbeccos Modified Eagles Medium (DMEM; GIBCO Inc.). These cells were cultured at 37 ° C in a subconfluence condition of 95% air, 5% CO 2 humidified air, and subcultured every 3 days.
<실험예 2> NO ( Nitric oxide ) 생성 억제 효능 평가 <Experimental Example 2> NO ( Nitric oxide production inhibition
RAW 264.7 세포를 10% FBS가 첨가된 DMEM 배지를 이용하여 1.5×105cells/mL로 조절한 후 24 well plate에 접종하고, 실시예의 시료와 LPS (1 ㎍/mL)를 동시에 처리하여 24시간 배양하였다. 생성된 NO의 양은 Griess 시약 [1% (w/v) sulfanilamide, 0.1% (w/v) naphylethylenediamine in 2.5% (v/v) phosphoric acid]을 이용하여 세포배양액 중에 존재하는 NO2 -의 형태로 측정하였다. 세포배양 상등액 100 L와 Griess 시약 100 L를 혼합하여 96 well plates에서 10분 동안 반응시킨 후 540 nm에서 흡광도를 측정하였다. 생성된 NO의 양은 sodium nitrite (NaNO2)를 standard로 비교하였다.RAW 264.7 cells were adjusted to 1.5 × 10 5 cells / mL using DMEM supplemented with 10% FBS, and then inoculated into a 24-well plate. LPS (1 μg / mL) Lt; / RTI > The amount of produced NO Griess reagent [1% (w / v) sulfanilamide, 0.1% (w / v) naphylethylenediamine in 2.5% (v / v) phosphoric acid] by using the NO 2 present in the cell culture - in the form of Respectively. 100 L of cell culture supernatant and 100 L of Griess reagent were mixed and reacted on 96-well plates for 10 minutes, and the absorbance was measured at 540 nm. The amount of NO produced was compared with sodium nitrite (NaNO 2 ) as standard.
결과를 [도 1] 내지 [도 3]에 나타내었다.The results are shown in [Figure 1] to [Figure 3].
[도 1]은 상기 실시예의 80% 에탄올 추출물 및 그 분획물을 50 ㎍/㎖로 처리하였을 때의 결과이고, [도 2]는 [도 1]에서 NO 생성 억제 활성이 우수한 헥산 분획물과 에틸아세테이트 분획물을 각각 12.5, 25 및 50 ㎍/㎖로 처리하였을 때의 결과이며, [도 3]은 25, 50 및 100 ㎍/㎖로 처리하였을 때의 결과이다. 결과는 3번의 독립된 실험의 평균±SD를 나타내었다. [Figure 1] shows the results obtained when the 80% ethanol extract of the above example and the fraction thereof were treated at 50 占 퐂 / ml. Fig. 2 shows the results of the hexane fraction and the
[도 1] 내지 [도 3]을 참조하여 보면, 꿩꼬리풀 용매 추출물과 초임계 추출물이 모두 활성을 보이고 또 헥산 분획물과 에틸아세테이트 분획물이 농도 의존적으로 활성을 보임을 알 수 있다. Referring to FIGS. 1 to 3, it can be seen that both the solvent extract of Pheasant and the supercritical extract show activity, and the hexane fraction and the ethyl acetate fraction are active in a concentration-dependent manner.
<실험예 3> 세포독성 평가 - LDH assay <Experimental Example 3> Cytotoxicity evaluation - LDH assay
RAW 264.7 세포 (1.5×105cells/mL)를 DMEM 배지에 실시예의 시료와 LPS (1 ㎍/mL)를 동시 처리하여 24시간 배양 한 후 배양 배지를 얻어 3,000 rpm에서 5분간 원심분리 하였다. LDH (lactate dehydrogenase) assay는 non-radioactive cytotoxicity assay kit (Promega)를 이용하여 측정했으며, 96 well plate에 원심 분리하여 얻은 배양 배지 50 L와 reconstituted substrate mix를 50 L를 넣고, 실온에서 30분 반응시킨 후 50 L의 stop solution을 넣은 후 microplate reader (Bio-TEK Instruments Inc., Vermont, WI, USA)를 사용하여 490 nm에서 흡광도를 측정하였다. 각 시료군에 대한 평균 흡광도 값을 구하였으며, 대조군 (LDH control, 1:5000)의 흡광도 값과 비교하여 세포독성을 평가하였다. 결과는 3번의 독립된 실험의 평균±SD를 나타내었다. RAW 264.7 cells (1.5 × 10 5 cells / mL) were co-treated with DMEM medium and LPS (1 μg / mL) in a DMEM medium for 24 hours, followed by culture at 3,000 rpm for 5 minutes. LDH (lactate dehydrogenase) assay was performed using a non-radioactive cytotoxicity assay kit (Promega). 50 L of the culture medium obtained by centrifugation on a 96-well plate and 50 L of the reconstituted substrate mix were added and reacted at room temperature for 30 minutes After the addition of 50 L stop solution, the absorbance was measured at 490 nm using a microplate reader (Bio-TEK Instruments Inc., Vermont, WI, USA). The average absorbance values for each sample group were determined and compared with the absorbance values of the control (LDH control, 1: 5000) to evaluate cytotoxicity. The results showed the mean ± SD of three independent experiments.
결과를 [도 1] 내지 [도 3]에 함께 나타내었는데, 헥산 분획물이 50 ㎍/㎖의 처리 농도에서 세포독성을 나타내는 이외에 나머지 실시예의 시료는 모두 특별한 세포독성을 보이지 않았다. The results are shown together in FIGS. 1 to 3, except that the hexane fraction showed cytotoxicity at a treatment concentration of 50 占 퐂 / ml, all of the other examples showed no cytotoxicity.
<실험예 4> PGE 2 ( Prostaglandin E 2 )생성 억제 효능 평가 Experimental Example 4: Experimental Example 4 PGE 2 ( Prostaglandin E 2 ) Evaluation of production inhibition efficacy
RAW 264.7 세포를 DMEM 배지를 이용하여 1.5×105cells/mL로 조절한 후 24 well plate 에 접종하고, 5% CO2항온기에서 18시간 전 배양 하였다. 이후 배지를 제거하고 실시예의 시료와 LPS (1 ㎍/mL)를 동시 함유한 새로운 배지를 처리하여 전배양과 동일 조건에서 배양하였다. 24시간 후 PGE2를 측정하기 위해 배양 배지를 원심분리 (12,000 rpm, 3 min)하여 상층액을 얻었다. PGE2의 측정은 PGE2 ELISA kit(R&DSystemsInc.,Minneapolis,MN,USA)를 이용하여 정량하였으며 standard 에 대한 표준곡선의 r2값은 0.99 이상이었다. 결과는 3번의 독립된 실험의 평균±SD를 나타내었다. RAW 264.7 cells were adjusted to 1.5 × 10 5 cells / mL using DMEM medium, inoculated into 24-well plates, and incubated 18 hours before in a 5% CO 2 incubator. Then, the medium was removed and a new medium containing the sample of Example and LPS (1 / / mL) was treated and cultured under the same conditions as the pre-culture. After 24 hours, the supernatant was obtained by centrifuging the culture medium (12,000 rpm, 3 min) to measure PGE 2 . PGE 2 was quantified using a PGE 2 ELISA kit (R & DS Systems Inc., Minneapolis, MN, USA). The r 2 value of the standard curve for the standard was 0.99 or more. The results showed the mean ± SD of three independent experiments.
헥산 분획물과 에틸아세테이트 분획물을 12.5, 25 및 50 ㎍/㎖로 처리하였을 때의 결과를 [도 4]에 나타내었고, 초임계 추출물을 25, 50 및 100 ㎍/㎖로 처리하였을 때의 결과를 [도 5]에 나타내었다. 상기 분획물과 초임계 추출물 모두 농도 의존적으로 PGE2 생성 억제 활성을 보였다.The results obtained when the hexane fraction and the ethyl acetate fraction were treated at 12.5, 25 and 50 μg / ml are shown in FIG. 4, and the results when the supercritical extract was treated at 25, 50 and 100 μg / 5]. Both the fractions and the supercritical extracts contained PGE 2 Respectively.
<실험예 5> 사이토카인( TNF -α, IL -6 및 IL-1β) 생성 억제 효능 평가 <Experimental Example 5> Evaluation of cytotoxicity ( TNF- α, IL- 6 and IL-1β)
RAW 264.7 세포 (1.5×105cells/mL)를 DMEM 배지를 이용하여 24 well plate 에 접종하고, 5% CO2 항온기에서 18 시간 전 배양하였다. 이후 배지를 제거하고 실시예 시료와 LPS (1 ㎍/mL)를 동시 함유한 새로운 배지를 처리하여 전 배양과 동일 조건에서 배양하였다. 24 시간 후 배양 배지를 원심분리 (12,000 rpm, 3 분)하여 얻어진 상층액의 pro-inflammatory cytokines 생성 함량을 측정하였다. 모든 시료는 정량 전까지 -20℃ 이하에 보관하였다. pro-inflammatory cytokines 정량은 mouse enzyme-linked immnunosorbent assay (ELISA) kit (R&D Systems Inc., Minneapolis, MN, USA)를 이용하여 정량하였으며 standard 에 대한 표준곡선의 r2값은 0.99 이상이었다. 결과는 3번의 독립된 실험의 평균±SD를 나타내었다. RAW 264.7 cells (1.5 × 10 5 cells / mL) were inoculated into 24-well plates using DMEM medium and cultured for 18 hours in a 5% CO 2 incubator. Then, the medium was removed and a new medium containing both the sample and LPS (1 / / mL) was treated and cultured under the same conditions as the pre-culture. After 24 hours, the amount of pro-inflammatory cytokines produced in the supernatant obtained by centrifuging the culture medium (12,000 rpm, 3 minutes) was measured. All samples were stored at -20 ° C or lower before quantification. Quantification of pro-inflammatory cytokines was quantitated using a mouse enzyme-linked immunosorbent assay (ELISA) kit (R & D Systems Inc., Minneapolis, MN, USA). The results showed the mean ± SD of three independent experiments.
헥산 분획물과 에틸아세테이트 분획물을 12.5, 25 및 50 ㎍/㎖로 처리하였을 때의 결과를 [도 6] 내지 [도 8]에 나타내었고, 초임계 추출물을 25, 50 및 100 ㎍/㎖로 처리하였을 때의 결과를 [도 9] 내지 [도 11]에 나타내었다. 상기 분획물과 초임계 추출물 모두 농도 의존적으로 염증성 사이토카인의 생성 억제 활성을 보였다.The results obtained when the hexane fraction and the ethyl acetate fraction were treated at 12.5, 25 and 50 μg / ml were shown in FIGS. 6 to 8, and supercritical extracts were treated at 25, 50 and 100 μg / Are shown in [Figure 9] to [Figure 11]. Both the fractions and supercritical extracts showed an inhibitory activity on the production of inflammatory cytokines in a concentration-dependent manner.
<실험예 6> iNOS 및 COX -2 발현 억제 효능 평가( western - blotting ) Experimental Example 6 Evaluation of iNOS and COX- 2 expression inhibition ( western - blotting )
배양이 끝난 세포를 수집하여 2~3회 phosphate buffered saline (PBS)로 세척 한 후 세포 용해 버퍼 [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 2 mM EDTA, 1 mM EGTA, 1 mM NaVO3, 10 mM NaF, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 25 ㎍/mL aprotinin, 25 ㎍/mL leupeptin]를 첨가하여 30분간 4℃에서 용해시킨 후 4, 15,000 rpm에서 15분간 원심 분리하여 세포막 성분 등을 제거하였다. 단백질 농도는 bovine serum albumin (BSA)를 표준화하여 Bio-Rad Protein Assay Kit를 사용하여 정량하였다. 분리된 단백질 20~30 ㎍를 4~12% NuPAGE Tris-Acetate mini gel 분리하여, 이를 PVDF (polyvinylidene difluoride) membrane iBlot gel transfer(Invitrogen, La Jolla, CA, USA)를 이용하여 7분 동안 transfer하였다. 그리고 membrane의 blocking은 5% skim milk가 함유된 TTBS (0.1% Tween 20 + TBS) 용액에서 상온에서 2시간 동안 실시하였다. iNOS의 발현 양을 검토하기 위한 항체로는 anti-rabbit iNOS (Calbiochem, La Jolla, CA, USA)를 COX-2의 발현 양을 검토하기 위한 항체로는 anti-mouse COX-2 (BD Biosciences Pharmingen, San Jose, CA, USA)를 TTBS 용액에서 1:2000으로 희석하여 상온에서 2시간 반응시킨 후 TTBS로 3회 세정하였다. 2차 항체로는 HRP (horse radish peroxidase)가 결합된 anti-rabbit IgG(Cell Signaling Technology, Inc., Danvers, MA), anti-mouse IgG (Cell Signaling Technology, Inc., Danvers, MA)를 1:5000으로 희석하여 상온에서 30분 간 반응시킨 후, TTBS로 3회 세척하여 West-Zol Plus(iNtRON, Gyeong, Seongnam, Kora)과 1~3분 간 반응 후 X-ray 필름에 감광하였다. 결과는 3번의 독립된 실험의 평균±SD를 나타내었다. After incubation, cells were collected and washed twice with phosphate buffered saline (PBS). Cell lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P- After incubation for 30 min at 4 ° C, the cells were incubated at 4, 15,000 rpm for 1 h at 37 ° C. The cells were incubated at 37 ° C for 1 h. And the cell membrane components were removed by centrifugation for 15 minutes. Protein concentrations were quantified by standardizing bovine serum albumin (BSA) using the Bio-Rad Protein Assay Kit. 20 ~ 30 ㎍ of the separated proteins were separated by 4-12% NuPAGE Tris-Acetate mini gel and transferred to PVDF (polyvinylidene difluoride) membrane iBlot gel transfer (Invitrogen, La Jolla, CA, USA) for 7 minutes. Blocking of the membranes was performed in TTBS (0.1
실시예의 80% 에탄올 추출물 및 그 분획물을 50 ㎍/㎖로 처리하였을 때의 결과와 초임계 추출물을 25, 50 및 100 ㎍/㎖로 처리하였을 때의 결과를 [도 12]에 나타내었고, 헥산 분획물과 에틸아세테이트 분획물을 12.5, 25 및 50 ㎍/㎖로 처리하였을 때의 결과를 [도 13]에 나타내었다. The results obtained when the 80% ethanol extract and the fraction thereof of the Example were treated with 50 μg / ml and the supercritical extract was treated with 25, 50 and 100 μg / ml were shown in FIG. 12, and the hexane fraction And ethyl acetate fraction were treated with 12.5, 25 and 50 占 퐂 / ml, respectively. The results are shown in Fig.
<실험예 7> IL -12 p40 의 생성 억제 효과 측정 실험 <Experimental Example 7> Experiment to measure the inhibitory effect of IL- 12 p40
<실험예 7-1> 실험동물 <Experimental Example 7-1> Animals
야생형 C57BL/6 마우스를 오리엔트 바이오 사(Orient Bio Inc,: Seongnam, South Korea)에서 구입하였다. 국립보건원(National Institutes of Health)의 지침에 따라 무균 장소에서 사육시켰으며, 마우스를 사용한 모든 실험은 제주대학교 실험동물윤리위원회의 규정(#2010-0028)에 따라 수행되었다.Wild type C57BL / 6 mice were purchased from Orient Bio Inc (Seongnam, South Korea). It was raised in an aseptic place according to the guidelines of the National Institutes of Health. All experiments using mice were carried out according to the regulations of the Experimental Animals Ethics Committee of Jeju University (# 2010-0028).
<실험예 7-2> 마우스 골수 유래 대식세포 및 수지상세포 준비. Experimental Example 7-2 Preparation of mouse bone marrow-derived macrophages and dendritic cells .
골수 유래 수지상세포(BMDC)를 야생형 C57BL/6 마우스로부터 수득하였다. 마우스의 정강이뼈 및 대퇴골을 DMEM(Dulbecco’s modified Eagles medium) 배지에서 플러싱(flushing)함으로써 골수 세포를 수득하였다. 수득한 골수 세포들은, 그래뉼로사이트-마크로파지 콜로니-자극인자(GM-CSF)를 함유하고 3% J558L 하이브리도마 세포 배양 상청액으로 보충되어 있으며, 10%의 열-비활성화된 FBS(Gibco,NY, USA), 50uM β-머캅토에탄올, 2mM 글루타민을 함유하는 RPMI 1640 배지에서 배양하였다.Bone marrow-derived dendritic cells (BMDC) were obtained from wild-type C57BL / 6 mice. Bone marrow cells were obtained by flushing mouse shinbone and femur in DMEM (Dulbecco's modified Eagles medium) medium. The obtained bone marrow cells were supplemented with 3% J558L hybridoma cell culture supernatant containing granulocyte-macrophage colony-stimulating factor (GM-CSF), supplemented with 10% heat-inactivated FBS (Gibco, NY, USA), 50 uM [beta] -mercaptoethanol, 2 mM glutamine.
<실험예 7-3> IL -12 p40 의 생성 억제 효과 측정 실험 <Experimental Example 7-3> Experiment to measure the inhibitory effect of IL- 12 p40
상기 <실시예 1>의 꿩꼬리풀 80% 에탄올 추출물이 염증을 유발시키는 염증성 사이토카인의 생성을 억제하는 활성이 있는지 확인하기 위하여, 다음과 같은 실험을 수행하였다. The following experiment was carried out to confirm that the 80% ethanol extract of Pheasant lupine from Example 1 had an activity of inhibiting the inflammatory cytokine production which induces inflammation.
위 실험방법에서 준비한 마우스의 골수로부터 유래한 수지상세포를 1 x 105 cells/0.5ml의 세포수가 되도록 48웰 플레이트에 분주하고 꿩꼬리풀 추출물을 2, 10, 25, 50 ㎍/ml의 농도별로 처리하였다. 이때 대조군으로는 꿩꼬리풀 추출물을 처리하지 않은 군을 사용하였다. 1시간 후 LPS 10 ng/ml을 처리하고 18시간 후, 세포 배양액을 수득하여 IL-12 p40의 발현 양을 ELISA(BD PharMingen, CA, USA, R&D system, MN, USA)를 이용하여 측정하였다. 결과는 3번의 독립된 실험의 평균±SD를 나타내었다. The dendritic cells derived from the bone marrow prepared in the above test method were dispensed into a 48-well plate so as to have a cell number of 1 x 10 5 cells / 0.5 ml, and the pheasant extract was treated at a concentration of 2, 10, 25 and 50 μg / ml Respectively. As a control group, a group not treated with pheasant extract was used. After 1 hour, 10 ng / ml of LPS was treated and 18 hours later, cell culture was obtained and the expression level of IL-12 p40 was measured by ELISA (BD PharMingen, CA, USA, R & D system, MN, USA). The results showed the mean ± SD of three independent experiments.
<실험예 7-4> 실험 결과 <Experimental Example 7-4> Experimental results
결과를 [도 14]에 나타내었다. [도 14]를 참조하여 보면 꿩꼬리풀 80% 에탄올 추출물이 농도 의존적으로 IL-12 p40의 발현을 억제함을 보여준다. IL-12 p40에 대한 IC50은 13.62㎍/ml로 나타났다.The results are shown in Fig. Referring to FIG. 14, it is shown that the 80% ethanol extract of Pheasant lupine inhibits IL-12 p40 expression in a concentration-dependent manner. The IC 50 for IL-12 p40 was 13.62 μg / ml.
<실험예 8> MDC ( macrophage - derived chemokine ) 생성 억제 활성 실험 Experimental Example 8 Experimental Example 8 Experiments were conducted on MDC ( macrophage - derived chemokine) produced inhibition experiment
HaCaT 각질형성세포는 10%의 우태아 혈청 (fetal bovine serum; FBS, 6 Gibco)과 1%의 antibiotics (100X antibiotic antimycotic; Gibco)를 첨가한 RPMI 1640 1X (cellgro, USA) 배지를 이용하여 37℃, 5% CO2 농도가 유지되는 항온배양기에서 배양했다.HaCaT keratinocytes were cultured in RPMI 1640 1X (cellgro, USA) supplemented with 10% fetal bovine serum (FBS, 6 Gibco) and 1% antibiotics (100 × antibiotic antimycotic; Gibco) , And incubated in a constant temperature incubator maintained at a concentration of 5% CO 2 .
각질형성세포인 HaCaT keratinocytes에서 꿩꼬리풀 추출물의 MDC 활성 억제 활성을 확인하기 위해, 계수된 세포 (2.0×105cells/mL)를 96well plate에 접종하고 18시간 동안 전배양 했다. 전배양 후 배양액을 serum free RPMI 배지로 교체하고 IFN-γ (10ng/mL)와 시료(12.5, 25, 50 ㎍/mL)을 배지에 희석해 처리한 뒤, 다시 24시간 동안 추가적으로 배양했다. 발생된 MDC를 얻고자, 각 well의 상층액을 취해 human MDC/CCL22 ELISA kit를 이용해 정량 했다. 표준곡선 (standard curve)은 human MDC standard를 이용하여 500 pg/mL에서부터 각각 1/2씩 순차적으로 희석 (serial dilution)했으며 MDC 생성량은 450nm의 파장에서 VersaMax ELISA microplate reader를 이용해 분석했다. 결과는 3번의 독립된 실험의 평균±SD를 나타내었다. In order to confirm the MDC activity inhibitory activity of the extract of pheasant corn borer on HaCaT keratinocytes, keratinocytes, counted cells (2.0 × 10 5 cells / mL) were inoculated on a 96-well plate and pre-cultured for 18 hours. After preincubation, the medium was replaced with serum-free RPMI medium, IFN-γ (10 ng / mL) and samples (12.5, 25, 50 μg / mL) were diluted in the medium and further cultured for another 24 hours. To obtain the generated MDC, the supernatant of each well was taken and quantified using a human MDC / CCL22 ELISA kit. Standard curves were serially diluted by 1/2 each from 500 pg / mL using the human MDC standard and MDC production was analyzed using a VersaMax ELISA microplate reader at a wavelength of 450 nm. The results showed the mean ± SD of three independent experiments.
결과를 [도 15]에 나타내었다. [도 15]의 결과는 꿩꼬리풀 추출물이 농도 의존적으로 MDC의 생성을 억제함을 보여준다. IC50은 19.18㎍/ml로 나타났다.The results are shown in Fig. The results of [Fig. 15] show that the extract of Pheasant myrtle extract inhibits MDC production in a concentration-dependent manner. The IC 50 was 19.18 / / ml.
Claims (7)
(Ⅰ) 꿩꼬리풀을 물, 에탄올 또는 이들의 혼합 용매로 추출하여 얻어진 추출물;
(Ⅱ) 꿩꼬리풀의 물과 에탄올 혼합 용매 추출물을 증류수에 현탁시키고 헥산, 에틸아세테이트 및 부탄올로 순차적으로 분획하였을 때 얻어지는 헥산 분획물;
(Ⅲ) 꿩꼬리풀의 물과 에탄올 혼합 용매 추출물을 증류수에 현탁시키고 헥산, 에틸아세테이트 및 부탄올로 순차적으로 분획하였을 때 얻어지는 에틸아세테이트 분획물; 및
(Ⅳ) 꿩꼬리풀을 이산화탄소를 용매로 사용하여 초임계 추출하여 얻어진 추출물
An antiinflammatory composition comprising any one of the following (I) to (IV) as an active ingredient.
(I) An extract obtained by extracting pheasant tailpid with water, ethanol or a mixed solvent thereof;
(II) a hexane fraction obtained by suspending a mixture of water and ethanol in a pheasant crab pooh suspension in distilled water and sequentially fractionating the mixture with hexane, ethyl acetate and butanol;
(III) Ethyl acetate fractions obtained by suspending a mixture of water and ethanol in a pheasant flask with distilled water and sequentially fractionating the mixture with hexane, ethyl acetate and butanol; And
(Ⅳ) Extracts obtained by supercritical extraction of pheasant cormorant using carbon dioxide as a solvent
상기 항염증용은 아토피성 피부염에 대한 항염증용인 것을 특징으로 하는 항염증용 조성물.
The method according to claim 1,
Wherein said anti-inflammatory agent is anti-inflammatory for atopic dermatitis.
상기 조성물은 약제학적 조성물인 것을 특징으로 하는 항염증용 조성물.
3. The method according to claim 1 or 2,
Wherein the composition is a pharmaceutical composition.
상기 조성물은 식품 조성물인 것을 특징으로 하는 항염증용 조성물.
3. The method according to claim 1 or 2,
Wherein the composition is a food composition.
상기 조성물은 화장품 조성물인 것을 특징으로 하는 항염증용 조성물.
3. The method according to claim 1 or 2,
Wherein the composition is a cosmetic composition.
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JP2004035431A (en) | 2002-07-01 | 2004-02-05 | Noevir Co Ltd | Skin care preparation for externa use |
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US20060165636A1 (en) | 2003-03-10 | 2006-07-27 | Kouhei Hasebe | Hair treatment composition and hair cosmetic for damaged hair |
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JP2004035431A (en) | 2002-07-01 | 2004-02-05 | Noevir Co Ltd | Skin care preparation for externa use |
US20060165636A1 (en) | 2003-03-10 | 2006-07-27 | Kouhei Hasebe | Hair treatment composition and hair cosmetic for damaged hair |
US20060018867A1 (en) | 2004-05-12 | 2006-01-26 | Ichimaru Pharcos Co., Ltd | Cosmetic composition and production thereof |
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