KR20180057599A - Compositions for Treatment and Prevention of Inflammatory Disease Comprising Extract of Patrinia scabiosaefolia as Active Ingredient - Google Patents
Compositions for Treatment and Prevention of Inflammatory Disease Comprising Extract of Patrinia scabiosaefolia as Active Ingredient Download PDFInfo
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- KR20180057599A KR20180057599A KR1020180058379A KR20180058379A KR20180057599A KR 20180057599 A KR20180057599 A KR 20180057599A KR 1020180058379 A KR1020180058379 A KR 1020180058379A KR 20180058379 A KR20180058379 A KR 20180058379A KR 20180057599 A KR20180057599 A KR 20180057599A
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- A61Q19/00—Preparations for care of the skin
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Abstract
Description
본 발명은 패장 추출물을 유효성분으로 포함하는 염증성 질환의 예방 및 치료용 조성물에 관한 것이다. The present invention relates to a composition for the prevention and treatment of inflammatory diseases comprising the extract as an active ingredient.
패장(Patrinia scabiosaefolia)은 마타리과(Valerianaceae family)에 속하며, 아시아에서 한방약으로 주로 사용된다. 전통적으로 패장은 염증성 질환의 치료에 사용되며, 특히 장내 종기, 급성 맹장염 및 이질에 사용된다. 지금까지 대부분의 연구들은 패장 추출물에 초점을 맞추고 있으며, 패장 추출물은 간염, 자궁염(M.K. Shin et al., Clinical Traditional Herbalogy, 564-565(1998)) 및 급성 췌장염(S.W. Seo et al., World J Gastroenterol. 12:1110-1114(2006))을 포함하는 바이러스 감염 치료에 효과적인 것으로 알려져 있다. 올레아노닉산(oleanonic acid) 및 우르솔산(ursolic acid)을 포함하는 패장의 몇몇 구성 성분들에 대해서는 항염증 효과가 보고되었다(E.M. Giner-Larza et al., Eur J Pharmacol. 428:137-143(2001), T. Nakanishi et al., Chem Pharm Bull. 41:183-186(1993), L.A. Tapondjou et al., Arch Pharm Res. 26:143-146(2003), B. Yang et al., Zhong Yao Cai. 22:23-24(1999)). 그러나, 패장의 항염증 활성에 대한 인 비트로 및 인 비보 결과에 대해서는 발표된 바가 없으며, 어떠한 과정을 통해 패장이 항-염증 효과를 매개하는지에 대해서는 전혀 알려져 있지 않다. NF-κB(nuclear factor-κB) 활성 경로가 염증반응과 연계되어 있기 때문에 패장이 NF-κB 신호전달 경로를 조절하여 항염증 효과를 매개하는 것이 가능하다. iNOS, COX-2, IL-6 및 TNF-α 유전자의 상류 프로모터 부분에서 NF-κB 콘센서스 위치가 억제되기 때문에 NF-κB 활성은 iNOS, COX-2, IL-6 및 TNF-α 발현을 포함한다(K.S. Ahn et al., Ann N Y Acad Sci. 1056:218-233(2005), K.S. Ahn et al., Curr Mol Med. 7:619-637(2007)). 이러한 유전자의 생성 산물은 염증 반응에 주요한 요소이며, 몇몇 염증 질환의 발병과 연관되어 있다(P.J. Barnes et al., N Engl J Med. 336:1066-1071(1997)). 본 발명자 및 다른 연구자들은 다양한 피토케미칼(phytochemical)이 NF-κB 신호 전달 경로를 억제함으로써 항염증 활성을 나타낸다는 것을 규명하였다. Patrinia scabiosaefolia ) belongs to the Valerianaceae family, and is mainly used as herbal medicine in Asia. Traditionally, the shell is used for the treatment of inflammatory diseases, especially for intestinal boils, acute appendicitis and dysentery. Most of the studies to date have focused on shellfish extract, and shellfish extract includes hepatitis, uterine inflammation (MK Shin et al., Clinical Traditional Herbalogy , 564-565 (1998)) and acute pancreatitis (SW Seo et al., World J. Gastroenterol . 12:1110-1114 (2006)) is known to be effective in the treatment of viral infections. Anti-inflammatory effects have been reported on several components of the shell including oleanonic acid and ursolic acid (EM Giner-Larza et al., Eur J Pharmacol . 428:137-143 ( 2001), T. Nakanishi et al., Chem Pharm Bull . 41:183-186 (1993), LA Tapondjou et al., Arch Pharm Res . 26:143-146 (2003), B. Yang et al., Zhong Yao Cai . 22:23-24 (1999)). However, the in vitro and in vivo results on the anti-inflammatory activity of the shell have not been published, and it is not known how the shell media mediates the anti-inflammatory effect. Since the NF-κB (nuclear factor-κB) active pathway is linked to the inflammatory response, it is possible for the shell to mediate the anti-inflammatory effect by regulating the NF-κB signaling pathway. NF-κB activity includes iNOS, COX-2, IL-6 and TNF-α expression because the NF-κB consensus position is inhibited in the upstream promoter portion of iNOS, COX-2, IL-6 and TNF-α genes. (KS Ahn et al., Ann NY Acad Sci . 1056:218-233 (2005), KS Ahn et al., Curr Mol Med . 7:619-637 (2007)). The product of these genes is a major factor in the inflammatory response and is associated with the development of several inflammatory diseases (PJ Barnes et al., N Engl J Med . 336:1066-1071 (1997)). The present inventors and other researchers have identified that various phytochemicals exhibit anti-inflammatory activity by inhibiting the NF-κB signaling pathway.
본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.Throughout this specification, a number of papers and patent documents are referenced and citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated by reference in this specification as a whole, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.
본 발명자들은 여러 가지 천연물 중에서 보다 개선된 항염증 작용을 갖는 유효성분을 추출하고자 연구 노력한 결과, 패장(Patrinia scabiosaefolia) 추출물이 항염증 활성을 가짐을 실험적으로 규명함으로써 본 발명을 완성하였다. As a result of research efforts to extract an active ingredient having an improved anti-inflammatory action from various natural products, the present inventors, Patrinia scabiosaefolia ) The present invention was completed by experimentally identifying that the extract has anti-inflammatory activity.
따라서, 본 발명의 목적은 패장 추출물을 유효성분으로 포함하는 염증성 질환(inflammatory disease)의 예방 또는 치료용 조성물을 제공하는 데 있다. Accordingly, an object of the present invention is to provide a composition for the prevention or treatment of inflammatory diseases, comprising a shellfish extract as an active ingredient.
본 발명의 다른 목적은 패장 추출물의 약제학적 유효량 및 약제학적으로 허용되는 담체를 포함하는 염증성 질환의 치료 또는 예방용 약제학적 조성물을 제공하는 데 있다. Another object of the present invention is to provide a pharmaceutical composition for the treatment or prevention of an inflammatory disease, comprising a pharmaceutically effective amount of a shellfish extract and a pharmaceutically acceptable carrier.
본 발명의 다른 목적은 패장 추출물을 유효성분으로 포함하는 염증성 질환의 개선 또는 완화용 기능성 식품 조성물을 제공하는 데 있다.Another object of the present invention is to provide a functional food composition for improving or alleviating inflammatory diseases, comprising a shellfish extract as an active ingredient.
본 발명의 또 다른 목적은 패장 추출물을 유효성분으로 포함하는 염증성 질환의 예방 또는 개선용 화장료 조성물을 제공하는 데 있다.Another object of the present invention is to provide a cosmetic composition for the prevention or improvement of inflammatory diseases, comprising a shellfish extract as an active ingredient.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명 및 청구범위에 의해 보다 명확하게 된다.Other objects and advantages of the present invention will become more apparent by the following detailed description and claims.
본 발명의 일 양태에 따르면, 본 발명은 패장(Patrinia scabiosaefolia) 추출물을 유효성분으로 포함하는 염증성 질환(inflammatory disease)의 예방 또는 치료용 조성물을 제공한다. According to one aspect of the present invention, the present invention is Patrinia scabiosaefolia ) provides a composition for the prevention or treatment of inflammatory diseases comprising the extract as an active ingredient.
본 발명자들은 여러 가지 천연물 중에서 보다 개선된 항염증 작용을 갖는 유효성분을 추출하고자 연구 노력한 결과, 패장 추출물이 항염증 활성을 가짐을 실험적으로 규명함으로써 본 발명을 완성하였다. The present inventors completed the present invention by experimentally finding out that the shellfish extract has anti-inflammatory activity as a result of research efforts to extract an active ingredient having an improved anti-inflammatory action from among various natural products.
본 명세서에서 용어 “패장(Patrinia scabiosaefolia)”이란 강양취, 가얌취, 가양취, 미역취, 마타리뿌리 등으로 불리는 마타리과(Valerianaceae family)의 여러해살이 풀이다. 전국 산야지의 양지에서 흔히 자라는 풀로 1.0- 1.5 미터의 높이로 자라며 8월-10월에 황색의 꽃이 피고 꽃대 줄기도 황색이 돈다. 11월에 종자가 익으며 식용, 관상용, 약용으로 쓰이며, 어린순을 산채 나물로 먹는다. 한방 및 민간에서 안질, 화상, 단독, 정혈, 부종, 종창, 소염, 대하 등의 약재로 쓰인다. In the present specification, the term “ Parinia scabiosaefolia )” is a perennial plant of the Valerianaceae family called gangyangchwi, gayamchwi, gayangchwi, seaweed, and matari roots. It is a grass commonly grown in the sunny areas of the national mountains and grows to a height of 1.0-1.5 meters, and yellow flowers bloom in August-October, and the stalks of the stalks turn yellow. Seeds ripen in November and are used for edible, ornamental and medicinal purposes, and young shoots are eaten as wild vegetables. It is used in oriental medicine and in the private sector as a medicinal material for eye disease, burns, alone, blood loss, edema, swelling, anti-inflammatory, and lobster.
본 발명의 명세서에서 용어 “패장 추출물” 이란 상기 패장으로부터 적합한 용매를 사용하여 추출하여 얻는 혼합물을 의미한다. In the specification of the present invention, the term "shell paste extract" refers to a mixture obtained by extracting from the shell paste using a suitable solvent.
본 발명의 조성물에서의 유효성분인 패장 추출물을 분리하는 방법은 천연물로부터 추출물을 추출하는 당업계에 공지된 통상적인 방법에 따라, 즉, 통상적인 온도, 압력의 조건하에서 통상적인 용매를 사용하여 분리할 수 있다.The method of separating the shellfish extract, which is an active ingredient in the composition of the present invention, is performed according to a conventional method known in the art for extracting the extract from a natural product, that is, using a conventional solvent under conditions of normal temperature and pressure. can do.
본 발명의 조성물에서 이용되는 패장 추출물을 패장에 추출용매를 처리하여 얻는 경우에는, 다양한 추출용매가 이용될 수 있다. 바람직하게는, 극성 용매 또는 비극성 용매를 이용할 수 있다. 극성 용매로서 적합한 것은, (i) 물, (ii) 알코올(바람직하게는, 메탄올, 에탄올, 프로판올, 부탄올, 노말-프로판올, 이소-프로판올, 노말-부탄올, 1-펜탄올, 2-부톡시에탄올 또는 에틸렌글리콜), (iii) 아세트산, (iv) DMFO(dimethyl-formamide) 및 (v) DMSO(dimethyl sulfoxide)를 포함한다. 비극성 용매로서 적합한 것은, 아세톤, 아세토나이트릴, 에틸 아세테이트, 메틸 아세테이트, 플루오로알칸, 펜탄, 헥산, 2,2,4-트리메틸펜탄, 데칸, 사이클로헥산, 사이클로펜탄, 디이소부틸렌, 1-펜텐, 1-클로로부탄, 1-클로로펜탄, o-자일렌, 디이소프로필 에테르, 2-클로로프로판, 톨루엔, 1-클로로프로판, 클로로벤젠, 벤젠, 디에틸 에테르, 디에틸 설파이드, 클로로포름, 디클로로메탄, 1,2-디클로로에탄, 어닐린, 디에틸아민, 에테르, 사염화탄소 및 THF를 포함한다.When the shellfish extract used in the composition of the present invention is obtained by treating the shellfish with an extraction solvent, various extraction solvents may be used. Preferably, a polar solvent or a non-polar solvent can be used. Suitable as polar solvents are (i) water, (ii) alcohols (preferably methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal-butanol, 1-pentanol, 2-butoxyethanol. Or ethylene glycol), (iii) acetic acid, (iv) dimethyl-formamide (DMFO), and (v) dimethyl sulfoxide (DMSO). Suitable non-polar solvents include acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1- Pentene, 1-chlorobutane, 1-chloropentane, o -xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene, diethyl ether, diethyl sulfide, chloroform, dichloro Methane, 1,2-dichloroethane, aniline, diethylamine, ether, carbon tetrachloride and THF.
보다 바람직하게는, 본 발명에서 이용되는 추출용매는 (a) 물, (b) 탄소수 1-4의 무수 또는 함수 저급 알코올(메탄올, 에탄올, 프로판올, 부탄올 등), (c) 상기 저급 알코올과 물과의 혼합용매, (d) 아세톤, (e) 에틸 아세테이트, (f) 클로로포름, (g) 부틸아세테이트, (h) 1,3-부틸렌글리콜, (i) 헥산 및 (j) 디에틸에테르를 포함한다. 보다 더 바람직하게는, 본 발명의 추출물은 물, 에탄올, 부탄올, 에틸 아세테이트 또는 이의 조합을 패장에 처리하여 수득한 것이고, 가장 바람직하게는 에틸 아세테이트를 패장에 처리하여 수득한 것이다. More preferably, the extraction solvent used in the present invention is (a) water, (b) anhydrous or hydrated lower alcohol having 1-4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), (c) the lower alcohol and water Mixed solvent with, (d) acetone, (e) ethyl acetate, (f) chloroform, (g) butyl acetate, (h) 1,3-butylene glycol, (i) hexane and (j) diethyl ether. Includes. Even more preferably, the extract of the present invention is obtained by treating the shell with water, ethanol, butanol, ethyl acetate or a combination thereof, and most preferably, the extract of the present invention is obtained by treating the shell with ethyl acetate.
본 발명에서 상기 패장 추출물은, 패장의 전체 부분 즉, 잎, 줄기, 뿌리, 열매, 꽃 및 어린순의 추출물을 모두 포함하는 의미이며, 가장 바람직하게는, 패장의 뿌리 추출물이다.In the present invention, the shellfish extract is meant to include all parts of the shellfish, that is, leaves, stems, roots, fruits, flowers, and extracts of young shoots, and most preferably, it is a root extract of shellfish.
본 명세서에서 사용되는 용어 “추출물”은 상술한 바와 같이 당업계에서 조추출물(crude extract)로 통용되는 의미를 갖지만, 광의적으로는 추출물을 추가적으로 분획(fractionation)한 분획물도 포함한다. 즉, 패장 추출물은 상술한 추출용매를 이용하여 얻은 것뿐만 아니라, 여기에 정제과정을 추가적으로 적용하여 얻은 것도 포함한다. 예컨대, 상기 추출물을 일정한 분자량 컷-오프 값을 갖는 한외 여과막을 통과시켜 얻은 분획, 다양한 크로마토그래피(크기, 전하, 소수성 또는 친화성에 따른 분리를 위해 제작된 것)에 의한 분리 등, 추가적으로 실시된 다양한 정제 방법을 통해 얻어진 분획도 본 발명의 패장 추출물에 포함되는 것이다.As used herein, the term "extract" has the meaning commonly used as a crude extract in the art as described above, but broadly includes a fraction obtained by further fractionating the extract. That is, the shellfish extract includes not only those obtained by using the above-described extraction solvent, but also those obtained by additionally applying a purification process thereto. For example, fractions obtained by passing the extract through an ultrafiltration membrane having a certain molecular weight cut-off value, separation by various chromatography (one prepared for separation according to size, charge, hydrophobicity or affinity), etc. Fractions obtained through the purification method are also included in the shellfish extract of the present invention.
본 발명에 따르면, 본 발명의 조성물은 NF-κB의 활성화에 관여하는 IL-6 및 TNF-α의 분비를 억제하고, 이로부터 iNOS 및 COX-2 유전자 발현을 억제함으로써 결과적으로 산화질소의 생성을 저해하여 염증성 질환의 예방 및 치료에 뛰어난 효과를 나타낸다. According to the present invention, the composition of the present invention inhibits the secretion of IL-6 and TNF-α, which are involved in the activation of NF-κB, and thereby suppresses the expression of iNOS and COX-2 genes, resulting in the production of nitric oxide. By inhibiting, it exhibits excellent effects in the prevention and treatment of inflammatory diseases.
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 반응으로서 정의될 수 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, 조직파괴 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 수 있다. The "inflammatory disease" may be defined as a local or systemic biological defense response against external physical and chemical stimuli or infection by external infectious agents such as bacteria, fungi, viruses, and various allergens. These reactions include activation of various inflammatory mediators and enzymes related to immune cells (eg iNOS, COX-2, etc.), secretion of inflammatory mediators (eg, secretion of NO, TNF-α, IL-6, IL-1β, PGE2). , Body fluid infiltration, cell migration, tissue destruction, etc., accompanied by a series of complex physiological reactions, and externally manifested by symptoms such as erythema, pain, swelling, fever, deterioration or loss of specific functions of the body. Since the inflammatory disease may be acute, chronic, ulcerative, allergic or necrotic, so long as any disease is included in the definition of such an inflammatory disease, whether it is acute, chronic, ulcerative, allergic, or Regardless of whether it is necrotic or not. 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, migraine, headache, back pain, fibromyalgia, myofascial disease, viral infection (e.g., type C infection), bacterial infection, fungal infection, burns, wounds from surgical or dental surgery, Prostagladin E excess syndrome, atherosclerosis, gout, arthritis, rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, iritis, scleritis, uveitis, dermatitis, eczema, and multiple sclerosis.
본 발명의 다른 양태에 따르면, 본 발명은 패장 추출물을 유효성분으로 포함하는 조성물의 약제학적 유효량; 및 (b) 약제학적으로 허용되는 담체를 포함하는 염증성 질환의 예방 또는 치료용 약제학적 조성물을 제공한다.According to another aspect of the present invention, the present invention provides a pharmaceutically effective amount of a composition comprising a shellfish extract as an active ingredient; And (b) provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising a pharmaceutically acceptable carrier.
본 발명의 조성물은 약제학적 조성물로 제조될 수 있다. The composition of the present invention can be prepared as a pharmaceutical composition.
본 발명의 바람직한 구현예에 따르면, 본 발명의 조성물은 (a) 상술한 본 발명의 패장 추출물의 약제학적 유효량; 및 (b) 약제학적으로 허용되는 담체를 포함하는 약제학적 조성물이다. According to a preferred embodiment of the present invention, the composition of the present invention comprises (a) a pharmaceutically effective amount of the extract of the present invention described above; And (b) a pharmaceutically acceptable carrier.
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 수 있다. 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. The composition of the present invention may contain the plant extract, which is the active ingredient, in an arbitrary amount (effective amount) as long as it can exhibit the improvement activity of the inflammatory disease intended for treatment according to the formulation, combination purpose, etc. Based on the total weight, it may be determined within the range of 0.001% by weight to 99.999% by weight. Here, “effective amount” refers to the amount of an active ingredient capable of inducing improvement or treatment of inflammatory diseases, or suppression/delay of the onset of such pathological symptoms. Such effective amounts can be determined empirically within the range of ordinary skill in the art.
본 발명의 조성물이 약제학적 조성물로 제조되는 경우, 본 발명의 약제학적 조성물은 약제학적으로 허용되는 담체를 포함한다. 본 발명의 약제학적 조성물에 포함되는 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약제학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약제학적으로 허용되는 담체 및 제제는 Remington's Pharmaceutical Sciences (19th ed., 1995)에 상세히 기재되어 있다.When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used at the time of formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. It does not become. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
본 발명의 약제학적 조성물은 경구 또는 비경구 투여할 수 있으며, 바람직하게는 경구 투여 방식으로 적용된다.The pharmaceutical composition of the present invention can be administered orally or parenterally, and is preferably applied by way of oral administration.
본 발명의 약제학적 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다. 본 발명의 약제학적 조성물의 일반적인 투여량은 성인 기준으로 0.001-100 ㎎/kg 범위 내이다. A suitable dosage of the pharmaceutical composition of the present invention is formulated in various ways depending on factors such as the formulation method, the mode of administration, the patient's age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity. Can be. A typical dosage of the pharmaceutical composition of the present invention is in the range of 0.001-100 mg/kg on an adult basis.
본 발명의 약제학적 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액, 시럽제 또는 유화액 형태이거나 엑스제, 산제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다. The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the art. Or it can be prepared by incorporating it into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.
본 발명의 또 다른 양태에 따르면, 본 발명은 패장 추출물을 유효성분으로 포함하는 조성물의 식품학적 유효량; 및 (b) 식품학적으로 허용되는 담체를 포함하는 염증성 질환의 예방 또는 개선용 식품 조성물을 제공한다. According to another aspect of the present invention, the present invention is a food effective amount of a composition comprising a shellfish extract as an active ingredient; And (b) it provides a food composition for preventing or improving inflammatory diseases comprising a food physiologically acceptable carrier.
본 발명의 조성물은 식품 조성물로 제공될 수 있다. The composition of the present invention may be provided as a food composition.
본 발명의 조성물이 식품 조성물로 제조되는 경우, 유효성분으로서 패장 추출물뿐만 아니라, 식품 제조 시에 통상적으로 첨가되는 성분을 포함하며, 예를 들어, 단백질, 탄수화물, 지방, 영양소, 조미제 및 향미제를 포함한다. 상술한 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스, 올리고당 등; 및 폴리사카라이드, 예를 들어 덱스트린, 사이클로덱스트린 등과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 향미제로서 천연 향미제 [타우마틴, 스테비아 추출물 (예를 들어 레바우디오시드 A, 글리시르히진 등]) 및 합성 향미제(사카린, 아스파르탐 등)를 사용할 수 있다. 예컨대, 본 발명의 식품 조성물이 드링크제로 제조되는 경우에는 본 발명의 패장 추출물 이외에 구연산, 액상과당, 설탕, 포도당, 초산, 사과산, 과즙, 두충 추출액, 대추 추출액, 감초 추출액 등을 추가로 포함시킬 수 있다.When the composition of the present invention is prepared as a food composition, it includes not only the shellfish extract as an active ingredient, but also ingredients commonly added during food production, for example, proteins, carbohydrates, fats, nutrients, flavoring agents and flavoring agents. Includes. Examples of the aforementioned carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents [taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is prepared as a drink, it may further include citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, headworm extract, jujube extract, licorice extract, etc. in addition to the shellfish extract of the present invention. have.
본 발명의 또 다른 양태에 따르면, 본 발명은 패장 추출물을 유효성분으로 포함하는 조성물의 화장품학적 유효량; 및 (b) 화장품학적으로 허용되는 담체를 포함하는 염증성 질환의 예방 또는 개선용 화장료 조성물을 제공한다. According to another aspect of the present invention, the present invention provides a cosmetically effective amount of a composition comprising a shellfish extract as an active ingredient; And (b) it provides a cosmetic composition for preventing or improving inflammatory diseases comprising a cosmetically acceptable carrier.
본 발명의 조성물은 화장료 조성물로 제공될 수 있다. The composition of the present invention may be provided as a cosmetic composition.
본 발명의 화장료 조성물에 포함되는 성분은 유효 성분으로서 패장 추출물 이외에 화장료 조성물에 통상적으로 이용되는 성분들을 포함하며, 예컨대 안정화제, 용해화제, 비타민, 안료 및 향료와 같은 통상적인 보조제, 그리고 담체를 포함한다.Ingredients included in the cosmetic composition of the present invention include ingredients commonly used in cosmetic compositions in addition to the shellfish extract as an active ingredient, and include conventional adjuvants such as stabilizers, solubilizers, vitamins, pigments and fragrances, and carriers. do.
본 발명의 화장료 조성물은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 예를 들어, 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성제-함유 클린싱, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이 등으로 제형화될 수 있으나, 이에 한정되는 것은 아니다. 보다 상세하게는, 유연 화장수, 영양 화장수, 영양 크림, 마사지 크림, 에센스, 아이 크림, 클렌징 크림, 클렌징 포옴, 클렌징 워터, 팩, 스프레이 또는 파우더의 제형으로 제조될 수 있다.The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing , Oil, powder foundation, emulsion foundation, wax foundation, and may be formulated as a spray, but is not limited thereto. In more detail, it may be prepared in the form of a flexible lotion, nutritional lotion, nutritional cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.
본 발명의 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물성유, 식물성유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 이용될 수 있다.When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component. I can.
본 발명의 제형이 파우더 또는 스프레이인 경우에는 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄 히드록시드, 칼슘 실리케이트 또는 폴리아미드 파우더가 이용될 수 있고, 특히 스프레이인 경우에는 추가적으로 클로로플루오로히드로카본, 프로판/부탄 또는 디메틸 에테르와 같은 추진체를 포함할 수 있다.When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additionally chlorofluorohydrocarbon, propane / May contain propellants such as butane or dimethyl ether.
본 발명의 제형이 용액 또는 유탁액인 경우에는 담체 성분으로서 용매, 용해화제 또는 유탁화제가 이용되고, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌 글리콜, 1,3-부틸글리콜 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르가 있다.When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.
본 발명의 제형이 현탁액인 경우에는 담체 성분으로서 물, 에탄올 또는 프로필렌 글리콜과 같은 액상의 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가 또는 트라칸트 등이 이용될 수 있다.When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, and crystallites Sex cellulose, aluminum metahydroxide, bentonite, agar or tracanth, and the like can be used.
본 발명의 제형이 계면-활성제 함유 클린징인 경우에는 담체 성분으로서 지방족 알코올 설페이트, 지방족 알코올 에테르 설페이트, 설포숙신산 모노에스테르, 이세티오네이트, 이미다졸리늄 유도체, 메틸타우레이트, 사르코시네이트, 지방산 아미드 에테르 설페이트, 알킬아미도베타인, 지방족 알코올, 지방산 글리세리드, 지방산 디에탄올아미드, 식물성 유, 라놀린 유도체 또는 에톡실화 글리세롤 지방산 에스테르 등이 이용될 수 있다.When the formulation of the present invention is a surfactant containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters may be used.
본 발명의 특징 및 이점을 요약하면 다음과 같다:The features and advantages of the present invention are summarized as follows:
(ⅰ) 본 발명은 패장 추출물을 유효성분으로 포함하는 염증성 질환의 예방 및 치료용 조성물을 제공한다.(I) The present invention provides a composition for the prevention and treatment of inflammatory diseases comprising the extract as an active ingredient.
(ⅱ) 본 발명의 조성물은 NF-κB 신호전달 과정을 억제함으로써 염증성 질환의 예방 및 치료에 뛰어난 효과를 나타낸다. (Ii) The composition of the present invention exhibits excellent effects in the prevention and treatment of inflammatory diseases by inhibiting the NF-κB signaling process.
(ⅲ) 본 발명은 패장 추출물을 유효성분으로 포함하는 염증성 질환의 예방 및 치료용 약제학적 조성물을 제공한다. (Iii) The present invention provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases comprising a shellfish extract as an active ingredient.
(ⅳ) 본 발명은 패장 추출물을 유효성분으로 포함하는 염증성 질환의 예방 또는 개선용 식품 조성물을 제공한다.(Iv) The present invention provides a food composition for preventing or improving inflammatory diseases, comprising a shellfish extract as an active ingredient.
(v) 본 발명은 패장 추출물을 유효성분으로 포함하는 염증성 질환의 예방 또는 개선용 화장료 조성물을 제공한다.(v) The present invention provides a cosmetic composition for the prevention or improvement of inflammatory diseases comprising the extract as an active ingredient.
본 발명은 패장 추출물을 유효성분으로 포함하는 염증성 질환의 예방 및 치료용 조성물에 관한 것이다. 본 발명의 패장 추출물은 지질다당체(Lipopolysaccharide)에 의해 유도되는 산화질소(NO)의 생성 및 IL-6 분비를 억제하고, 지질다당체에 의해 유도되는 iNOS와 COX 단백질 및 이들의 mRNA의 발현을 억제하는 활성을 나타내며, NF-κB 신호전달 과정을 억제함으로써 결과적으로 염증성 질환의 예방 및 치료에 뛰어난 효과를 나타낸다.The present invention relates to a composition for the prevention and treatment of inflammatory diseases comprising the extract as an active ingredient. The shellfish extract of the present invention inhibits the production of nitric oxide (NO) and the secretion of IL-6 induced by lipopolysaccharide, and suppresses the expression of iNOS and COX proteins and their mRNA induced by lipopolysaccharide. It exhibits activity and suppresses the NF-κB signaling process, resulting in an excellent effect in the prevention and treatment of inflammatory diseases.
도 1은 마우스 RAW 264.7 대식세포에서 패장의 다양한 분획물의 세포 생존능력 및 지질다당체에 의해 유도된 산화질소에 대한 효과를 확인한 그래프이다. 패널 A : 패장(Patrinia scabiosaefolia)의 다양한 분획(100 μg/ml)으로 24시간 동안 세포를 처리하고 MTT 분석법으로 세포 생존능력을 측정하였다. 각 독립된 실험 결과를 평균을 내었고, 비처리된 대조군 세포의 생존능력과 비교하여 백분율로 나타내었다. 패널 B : 아질산염(nitrite) 생성은 실험방법에 기재된 바와 같이 Griess 반응 분석법에 의해 측정하였다. 세포들을 EtOH 추출물의 상이한 농도로 2시간 동안 전처리하고, LPS(1 μg/ml)로 22시간 동안 자극하였다. 패널 C : 세포를 상이한 농도의 에틸아세테이트 분획(EA)으로 2시간 동안 전처리하고, LPS(1 μg/ml)로 22시간 동안 자극하였다. 패널 D: 세포를 상이한 농도의 부탄올 분획(BuOH)으로 2시간 동안 전처리하고, LPS(1 μg/ml)로 22시간 동안 자극하였다. 얻어진 값들은 DMEM(Dulbecco's modified Eagle's minimal essential medium)에 용해된 소디엄나이트레이트(sodium nitrate)의 표준 농도와 비교하고, 샘플처리된 세포의 조건 배지내에서의 아질산염 농도를 계산하였다. 데이터는 3차례의 독립된 실험으로부터 얻었으며, 평균±표준편차로 표시하였다. ***P < 0.001는 지질다당체를 처리한 그룹으로부터 유의한 차이를 나타내며, ###P < 0.001는 자극되지 않은 대조군으로부터 유의한 차이를 나타낸다.
도 2는 마우스 RAW 264.7 대식세포에서 지질다당체로부터 유도된 iNOS, COX-2 유전자 생성물 및 IL-6 생성에 대한 패장의 에틸 아세테이트 분획물(ethyl acetate fraction of Patrinia scabiosaefolia, EAPS)의 억제효과를 나타낸 그림이다. 패널 A: RAW 264.7 세포를 상이한 농도의 패장 에틸아세테이트 분획물(EAPS)으로 2시간 동안 전처리하고, LPS(1 μg/ml)으로 22시간 동안 자극하였다. 동량의 총단백질(25 μg/레인)을 8%(iNOS에 대해) 및 10%(COX-2에 대해) SDS-PAGE를 행하여 분리하고, iNOS 및 COX-2 단백질의 발현을 항-iNOS 및 항-COX-2 단백질 특이적 항체를 사용하여 검출하였다. β-액틴은 로딩 대조군으로 사용하였다. 패널 B: LPS-자극된 RAW 264.7 세포내에서 EAPS에 의한 iNOS 및 COX-2 mRNA 발현의 억제를 보여준다. RAW 264.7 세포는 지정된 농도의 EAPS로 2시간 동안 전처리하고, LPS(1 μg/ml)으로 20시간 동안 처리하였다. 총 RNA를 분리하고, iNOS 및 COX-2 mRNA의 발현을 RT-PCR 분석으로 검출하였다. 시료의 cDNA 초기 유사 함량에 대한 대조군으로서, GAPDH의 PCR을 수행하였다. 패널 C: 세포를 상이한 농도의 EAPA으로 2시간 동안 전처리하고, LPS(1 μg/ml)로 22시간 동안 자극하였다. 분비된 IL-6의 양은 IL-6 항체 코팅된 ELISA 키트에 의해 측정하였다. 데이터는 3차례의 독립된 실험으로부터 얻었으며, 평균±표준편차로 표시하였다. * P<0.05, ** P<0.01 및 *** P<0.001는 지질다당체를 처리한 그룹으로부터 유의한 차이를 나타내며, ###P < 0.001는 자극되지 않은 대조군으로부터 유의한 차이를 나타낸다.
도 3은 마우스 RAW 264.7 대식세포에서 EAPS에 의한 지질다당체에 의해 유도되는 NF-κB의 활성저하를 보여준다. 패널 A: 세포를 지시된 농도의 EAPS로 2시간 동안 전처리하고, LPS(1μg/ml)으로 60분간 자극하여, 핵 추출물을 준비하였다. NF-κB 결합 활성은 EMSA(electrophoretic mobility shift assay)에 의해 수행하였다. 이 실험은 3회 반복하여 유사한 결과를 얻었다. 패널 B: 트랜스펙션된 세포를 상이한 농도의 EAPS으로 2시간 동안 전처리하고, LPS(1μg/ml)으로 16시간 동안 자극하엿다. 트랜스펙션된 세포는 NF-κB 활성에 반응하여 전사 리포터로서 분비성 알카라인 포스파타아제(secretory alkaline phosphatase, SEAP)를 방출하도록 디자인하였고, 제네티신(geneticin) 내성에 대한 우성 선별 마커로서 NPT(neomycin phosphotransferase) 유전자를 포함한다. 데이터는 3차례의 독립된 실험으로부터 얻었으며, 평균±표준편차로 표시하였다. * P<0.01는 지질다당체(LPS)를 처리한 그룹으로부터 유의한 차이를 나타내며, ###P < 0.001는 자극되지 않은 대조군으로부터 유의한 차이를 나타낸다. RFU는 비교형광단위(relative fluorescence unit)를 나타낸다. 패널 C : 세포를 100μg/ml의 EAPS으로 2시간 동안 전처리하고, LPS (1μg/ml)으로 지정 시간동안 자극하였다. 세포질 및 핵 추출물을 실험방법에 기재된 바와 같이 준비하고, 단백질을 SDS-PAGE에 의해 분리한 후, 니트로셀룰로오스 멤브레인으로 전기적으로 이동시키고, 이어서, 인산-특이적 항-p65(ser276) 항체 및 항-65 항체를 사용하여 웨스턴 블롯 분석을 행하였다. Lamin B (핵 분획) 및 β-액틴(세포질 분획) 단백질을 로딩 대조군으로 사용하였다. 결과는 3회의 독립된 실험의 대푯값으로서 나타내었다. 패널 D : LPS-유도된 p38, ERK 및 JNK의 활성화에 대한 EAPS의 영향을 보여준다. 세포를 100μg/ml의 EAPS으로 2시간 동안 전처리하고, LPS(1μg/ml)으로 지정된 시간 동안 자극하였다. 전체세포 추출물을 준비하고, 단백질을 SDS-PAGE에 의해 분리한 후, 니트로셀룰로오스 멤브레인으로 전기적으로 이동시키고, 이어서, 인산-특이적 항-p38 항체, 항-ERK 항체, 및 인산 특이적-항-JNK 항체를 사용하여 웨스턴 블롯 분석을 행하였다. 동일한 멤브레인을 항-p38, 항-ERK 및 항-JNK 항체로 다시 블로팅하였다. 결과는 3회의 독립된 실험의 대푯값으로서 나타내었다.
도 4는 EAPS 처리에 따른 지질다당체 주사 마우스에서 TNF-α의 생성 저해 및 지질다당체에 의해 자극된 BALB/c 마우스의 비장세포(splenocytes)에서 IL-6 및 TNF-α 생성의 억제를 나타낸 그래프이다. 패널 A : BALB/c 마우스(그룹당 10 개체)에 25mg/kg의 LPS를 복강투여하였다. 150 mg/kg 용량의 EAPS을 24시간 시점 및 LPS 투여 2시간 전 시점에서 식도 카테터를 통해 경구 투여하였다. LPS 투여 2시간 및 4시간 시점에서 혈청을 수집하고 TNF-α 수준을 측정하였다. TNF-α는 ELISA 키트를 사용하여 측정하였다. 데이터는 그룹당 무작위 선택한 3마리 동물의 데이터로부터 얻은 평균±SD이다. 패널 B 및 패널 C : BALB/c 마우tm에서 얻은 비장세포(splenocyte)에서의 LPS-유도된 IL-6 및 TNF-α의 생성에 미치는 EAPS의 영향을 보여준다. 분리한 세포를 지시된 농도의 EAPS으로 2시간 동안 전처리하고, 1μg/ml의 LPS으로 다양한 시간 간격으로 자극하였다. 방출되는 TNF-α 및 IL-6의 양은 마우스 IL-6 또는 TNF-α ELISA 키트를 사용하여 측정하였다. 표시한 결과는 3회의 독립적인 실험의 대푯값이다.1 is a graph confirming the cell viability of various fractions of the shell in mouse RAW 264.7 macrophages and the effect on nitric oxide induced by lipopolysaccharide. Panel A: Patrinia scabiosaefolia ), the cells were treated with various fractions (100 μg/ml) for 24 hours, and cell viability was measured by MTT assay. The results of each independent experiment were averaged and expressed as a percentage compared to the viability of untreated control cells. Panel B: Nitrite production was measured by the Griess reaction assay as described in the experimental method. Cells were pretreated with different concentrations of EtOH extract for 2 hours and stimulated with LPS (1 μg/ml) for 22 hours. Panel C: Cells were pretreated with different concentrations of ethyl acetate fraction (EA) for 2 hours, and then stimulated with LPS (1 μg/ml) for 22 hours. Panel D: Cells were pretreated with different concentrations of butanol fraction (BuOH) for 2 hours and stimulated with LPS (1 μg/ml) for 22 hours. The obtained values were compared with the standard concentration of sodium nitrate dissolved in DMEM (Dulbecco's modified Eagle's minimal essential medium), and the nitrite concentration in the conditioned medium of the sample-treated cells was calculated. Data were obtained from 3 independent experiments and expressed as mean±standard deviation. *** P <0.001 represents a significant difference from the group treated with lipopolysaccharide, and ### P <0.001 represents a significant difference from the unstimulated control group.
2 is an ethyl acetate fraction of Patrinia for production of iNOS, COX-2 gene products, and IL-6 derived from lipopolysaccharides in mouse RAW 264.7 macrophages. scabiosaefolia , EAPS) is a picture showing the inhibitory effect. Panel A: RAW 264.7 cells were pretreated with different concentrations of shell ethyl acetate fraction (EAPS) for 2 hours, and stimulated with LPS (1 μg/ml) for 22 hours. The same amount of total protein (25 μg/lane) was separated by 8% (for iNOS) and 10% (for COX-2) SDS-PAGE, and the expression of iNOS and COX-2 proteins was determined by anti-iNOS and anti- -COX-2 protein specific antibody was used to detect. β-actin was used as a loading control. Panel B: Shows inhibition of iNOS and COX-2 mRNA expression by EAPS in LPS-stimulated RAW 264.7 cells. RAW 264.7 cells were pretreated with EAPS at the indicated concentration for 2 hours and treated with LPS (1 μg/ml) for 20 hours. Total RNA was isolated and the expression of iNOS and COX-2 mRNA was detected by RT-PCR analysis. As a control for the initial cDNA similar content of the sample, PCR of GAPDH was performed. Panel C: Cells were pretreated with different concentrations of EAPA for 2 hours and stimulated with LPS (1 μg/ml) for 22 hours. The amount of secreted IL-6 was measured by an IL-6 antibody coated ELISA kit. Data were obtained from 3 independent experiments and expressed as mean±standard deviation. * P <0.05, ** P <0.01 and *** P <0.001 represent a significant difference from the group treated with lipopolysaccharide, and ### P <0.001 represents a significant difference from the unstimulated control group.
Figure 3 shows the decrease in activity of NF-κB induced by lipopolysaccharides by EAPS in mouse RAW 264.7 macrophages. Panel A: Cells were pretreated with EAPS at the indicated concentration for 2 hours and stimulated with LPS (1 μg/ml) for 60 minutes to prepare a nuclear extract. NF-κB binding activity was performed by EMSA (electrophoretic mobility shift assay). This experiment was repeated three times to obtain similar results. Panel B: Transfected cells were pretreated with different concentrations of EAPS for 2 hours and stimulated with LPS (1 μg/ml) for 16 hours. Transfected cells were designed to release secretory alkaline phosphatase (SEAP) as a transcription reporter in response to NF-κB activity, and NPT ( neomycin phosphotransferase) gene. Data were obtained from 3 independent experiments and expressed as mean±standard deviation. * P <0.01 represents a significant difference from the group treated with lipopolysaccharide (LPS), and ### P <0.001 represents a significant difference from the unstimulated control group. RFU stands for relative fluorescence unit. Panel C: Cells were pretreated with 100 μg/ml EAPS for 2 hours, and stimulated with LPS (1 μg/ml) for a specified time. Cytoplasmic and nuclear extracts were prepared as described in the experimental method, proteins were separated by SDS-PAGE, and then electrically transferred to a nitrocellulose membrane, followed by phosphoric acid-specific anti-p65 (ser276) antibody and anti- Western blot analysis was performed using 65 antibodies. Lamin B (nuclear fraction) and β-actin (cytoplasmic fraction) proteins were used as loading controls. The results are presented as representative values of three independent experiments. Panel D: shows the effect of EAPS on LPS-induced activation of p38, ERK and JNK. Cells were pretreated with 100 μg/ml of EAPS for 2 hours and stimulated for the designated time with LPS (1 μg/ml). Whole cell extracts were prepared, proteins were separated by SDS-PAGE, and then electrically transferred to a nitrocellulose membrane, followed by phosphoric acid-specific anti-p38 antibodies, anti-ERK antibodies, and phosphoric acid specific-anti- Western blot analysis was performed using the JNK antibody. The same membrane was blotted again with anti-p38, anti-ERK and anti-JNK antibodies. The results are presented as representative values of three independent experiments.
4 is a graph showing inhibition of TNF-α production in lipopolysaccharide-injected mice according to EAPS treatment and inhibition of IL-6 and TNF-α production in splenocytes of BALB/c mice stimulated by lipopolysaccharide. . Panel A: BALB/c mice (10 individuals per group) were intraperitoneally administered 25 mg/kg of LPS. EAPS at a dose of 150 mg/kg was orally administered through an esophageal catheter at 24 hours and 2 hours before LPS administration. Serum was collected at 2 hours and 4 hours of LPS administration and the level of TNF-α was measured. TNF-α was measured using an ELISA kit. Data are mean±SD obtained from data of 3 randomly selected animals per group. Panels B and C: Shows the effect of EAPS on the production of LPS-induced IL-6 and TNF-α in splenocytes obtained from BALB/c Mautm. The isolated cells were pretreated with EAPS at the indicated concentration for 2 hours, and stimulated with 1 μg/ml LPS at various time intervals. The amount of released TNF-α and IL-6 was measured using a mouse IL-6 or TNF-α ELISA kit. The displayed results are representative values of three independent experiments.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .
실시예Example
재료 및 방법Materials and methods
세포 배양Cell culture
마우스 RAW 264.7 대식 세포주는 한국 세포주 은행(KCLB, 서울대학교 의과대학)에서 구입하였다. 마우스 RAW 264.7 대식세포들은 10% FBS(fetal bovine serum), 페니실린 G(penicillin G)(100 units/mL), 스트렙토마이신(streptomycine)(100 μg/mL)을 포함한 DMEM 배지(Gibco, Grand Island, NY)를 사용하여 온도 37℃, 5%의 CO2를 유지하면서 인큐베이터(incubator)에서 배양하였다. NFκB 활성을 확인한 형질전환 마우스 RAW 264.7 대식세포는 안정된 형질주입체의 유지 및 선별을 위해 500 μg/mL의 제네티신(100 μg/mL) 조건에서 배양하였다(E.J. Noh et al., Life Sci. 79:695-701(2006), K.S. Ahn et al., Life Sci. 76:2315-2328(2005)). 혈구계수기(hemocytometer) 및 트립판 블루를 이용하여 살아있는 세포의 수를 계수하였다. Mouse RAW 264.7 macrophage cell line was purchased from Korea Cell Line Bank (KCLB, Seoul National University College of Medicine). Mouse RAW 264.7 macrophages were prepared in DMEM medium (Gibco, Grand Island, NY) containing 10% fetal bovine serum (FBS), penicillin G (100 units/mL), and streptomycine (100 μg/mL). ) Was cultured in an incubator while maintaining a temperature of 37°C and 5% CO 2. Transgenic mouse RAW 264.7 macrophages having confirmed NFκB activity were cultured under 500 μg/mL geneticin (100 μg/mL) conditions for maintenance and selection of stable transfectants (EJ Noh et al., Life Sci . 79:695-701 (2006), KS Ahn et al., Life Sci . 76:2315-2328 (2005)). The number of living cells was counted using a hemocytometer and trypan blue.
실험재료Experimental material
지질다당체(lipopolysaccharide, LPS)(Escherichia coli 055:B5), FBS 및 항생제/항진균제(antibiotic-antimycotic)는 GIBCO-BRL로부터 구입하였다. 항-IκB-a(sc-371) 항체, 항-β-액틴(sc-47778) 항체 및 항-Lamin B(sc-6216) 항체는 Santa Cruz Biotechnology(Santa Cruz, CA)로부터 구입하였다. 항-COX-2 항체 및 항-iNOS 항체는 BD Biosciences(San Diego, CA)로부터 구입하였다. 인산-특이 항-p38(Thr180/Tyr180) 항체, 항-p38 항체, 인산-특이 항-ERK(Thr202/Tyr204) 항체, 항-ERK 항체, 인산-특이 항-JNK(Thr183/Tyr185) 항체, 항-JNK 항체 및 인산-특이 항-p65(Ser276) 항체는 Cell Signaling(Beverly, MA)에서 구입하였다. Lipopolysaccharide (LPS) ( Escherichia coli 055:B5), FBS and antibiotic/antimycotic were purchased from GIBCO-BRL. Anti-IκB-a (sc-371) antibody, anti-β-actin (sc-47778) antibody and anti-Lamin B (sc-6216) antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-COX-2 antibodies and anti-iNOS antibodies were purchased from BD Biosciences (San Diego, CA). Phosphoric acid-specific anti-p38 (Thr180/Tyr180) antibody, anti-p38 antibody, phosphoric acid-specific anti-ERK (Thr202/Tyr204) antibody, anti-ERK antibody, phosphate-specific anti-JNK (Thr183/Tyr185) antibody, anti -JNK antibody and phosphate-specific anti-p65 (Ser276) antibody were purchased from Cell Signaling (Beverly, MA).
식물재료Plant material
패장은 경동시장에서 구입하였으며 증거표본(voucher specimen)(D-618)은 한국과학기술원 강릉분원 식물표본실에 기탁하였다. The patch was purchased at the Gyeongdong Market, and the voucher specimen (D-618) was deposited in the Plant Specimen Office of the Gangneung Branch, Korea Advanced Institute of Science and Technology.
분리 및 식별Separation and identification
패장의 뿌리(1.5 kg)은 뜨거운 에탄올에서 4시간동안 4 차례 추출하였으며, 에탄올을 증발시켜 총 추출물(220.0 g)을 수득하였다. 이 추출물을 증류수로 현탁시킨 후 n-헥산, 메틸렌클로라이드, 에틸아세테이트 및 n-부탄올의 순서대로 분획하고 각각 증발과정을 거쳐 n-헥산(28.1 g), 메틸렌 클로라이드(8.5 g), 에틸 아세테이트(5.9 g) 및 n-부탄올(26.4 g)의 분획물을 수득하였다. The roots of the shellfish (1.5 kg) were extracted four times in hot ethanol for 4 hours, and the ethanol was evaporated to obtain a total extract (220.0 g). The extract was suspended in distilled water, fractionated in order of n-hexane, methylene chloride, ethyl acetate, and n-butanol, and evaporated to n-hexane (28.1 g), methylene chloride (8.5 g), and ethyl acetate (5.9. g) and n-butanol (26.4 g) fractions were obtained.
세포 생존능력 측정Cell viability measurement
패장의 세포독성은 MTT{3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide} 분석법을 통해 측정하였다. 마우스 RAW 264.7 대식세포를 96 웰(well) 플레이트에 웰당 1× 104 개 넣고 37℃에서 배양하였다. 세포에 패장 추출물을 50 μg/ml 농도로 처리하고 24시간 배양한 후, MTT 용액 20 μl를 각 웰에 첨가하고 37℃에서 배양하였다. 색 변화는 ELISA reader(Model 680, Bio-RAD) 570 nm 파장에서 측정하였다. The cytotoxicity of the shell was measured by MTT{3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide} assay. Mouse RAW 264.7 macrophages were placed in a 96 well plate and 1×10 4 cells per well were cultured at 37°C. Cells were treated with a 50 μg/ml concentration and cultured for 24 hours, and then 20 μl of MTT solution was added to each well and cultured at 37°C. Color change was measured at a wavelength of 570 nm in an ELISA reader (Model 680, Bio-RAD).
아질산염(Nitrite) 분석Nitrite analysis
마우스 RAW 264.7 대식세포를 24 웰 플레이트에 웰당 2× 105개 넣고 37℃에서 배양하였다. 세포에 패장 추출물을 농도별로 2시간 동안 전처리한 후, 지질다당체(LPS)를 1 μg/ml 농도로 22시간 동안 처리하였다. 배양액 내 아질산염의 축적은 Griess reagent(Sigma, St. Louis, MO)를 이용한 Griess 반응에 의해 비색적(colorimetrically)으로 측정되었다. 분석을 위하여 동량의 배양액 및 Griess reagent를 혼합하고, 소듐 아질산염 용액 표준(sodium nitrite solution standard, Sigma, St. Louis, MO)을 이용하여 흡수계수(absorption coefficient)를 보정하였다. Griess 반응 후 각 샘플의 흡광도는 ELISA reader 540 nm 파장에서 측정하였다. Mouse RAW 264.7 macrophages were placed in a 24 well plate and 2×10 5 cells per well and cultured at 37°C. After pre-treatment of the cell extract for 2 hours by concentration, lipopolysaccharide (LPS) was treated at a concentration of 1 μg/ml for 22 hours. The accumulation of nitrite in the culture medium was measured colorimetrically by Griess reaction using Griess reagent (Sigma, St. Louis, MO). For analysis, the same amount of the culture medium and Griess reagent were mixed, and the absorption coefficient was corrected using a sodium nitrite solution standard (Sigma, St. Louis, MO). After the Griess reaction, the absorbance of each sample was measured at a wavelength of 540 nm with an ELISA reader.
IL-6 분석IL-6 assay
IL-6의 양은 마우스 RAW 264.7 대식세포 배양 상등액에서 측정된 물질의 양에 의해 명시되었다. 마우스 RAW 264.7 대식세포를 24 웰 플레이트에 웰당 5× 105개를 500 μl의 배양배지와 함께 37℃에서 12시간동안 배양하였다. 세포에 다양한 시간 및 처리량의 EAPS를 지질다당체(1 μg/ml)가 포함된 또는 포함되지 않은 조건에서 전처리하였다. IL-6 생성량은 ELISA kit (R&D Systems, Minneapolis, MN)를 이용하여 제조사의 지시에 따라 측정하였다. The amount of IL-6 was specified by the amount of material measured in the mouse RAW 264.7 macrophage culture supernatant. Mouse RAW 264.7 macrophages were cultured in a 24-well plate with 5×10 5 cells per well at 37° C. for 12 hours with 500 μl of culture medium. Cells were pretreated with various times and throughputs of EAPS in conditions with or without lipopolysaccharide (1 μg/ml). IL-6 production was measured according to the manufacturer's instructions using an ELISA kit (R&D Systems, Minneapolis, MN).
RT-PCR(Reverse transcription-polymerase chain reaction) RT-PCR ( Reverse transcription-polymerase chain reaction )
TRIzol reagent(Gibco, Grand Island, NY)를 이용하여 마우스 RAW 264.7 대식세포로부터 총 RNA를 추출하였다. RNA의 농도 및 순수도는 260/280 nm에서 흡광도를 측정하여 결정하였다. iNOS에 대한 정방향 및 역방향 프라이머는 각각 5’- TCTTTGACGCTCG-GAACTGTAGCA-3’ 및 5’-CGTGAAGCCATGACCTTTCGCATT-3’이다. COX-2에 대한 정방향 및 역방향 프라이머는 각각 5’-TTGCTGTACAAGCAGT-GGCAAAGG-3’ 및 5’-AGGACAAACACCGGAGGGAATCTT-3’이다. 마우스 GAPDH mRAN 발현에 대한 정방향 및 역방향 프라이머는 각각 5’-AACTTTGGCATTGTGGAAGGGCTC-3’ 및 5’-TGGAAGAGTGG-GAGTTGCTGTTGA-3’이다. RT-PCR은 ONE-STEP RT-PCR PriMix kit(invitrogen Co., USA)를 이용하여 제조사의 지시에 따라 수행하였다. Total RNA was extracted from mouse RAW 264.7 macrophages using a TRIzol reagent (Gibco, Grand Island, NY). RNA concentration and purity were determined by measuring absorbance at 260/280 nm. The forward and reverse primers for iNOS are 5'-TCTTTGACGCTCG-GAACTGTAGCA-3' and 5'-CGTGAAGCCATGACCTTTCGCATT-3', respectively. The forward and reverse primers for COX-2 are 5'-TTGCTGTACAAGCAGT-GGCAAAGG-3' and 5'-AGGACAAACACCGGAGGGAATCTT-3', respectively. The forward and reverse primers for mouse GAPDH mRAN expression are 5'-AACTTTGGCATTGTGGAAGGGCTC-3' and 5'-TGGAAGAGTGG-GAGTTGCTGTTGA-3', respectively. RT-PCR was performed according to the manufacturer's instructions using the ONE-STEP RT-PCR PriMix kit (invitrogen Co., USA).
웨스턴 블롯 분석Western blot analysis
마우스 RAW 264.7 대식세포는 원심분리를 통해 수득하였으며, PBS로 2회 세척하고 단백질 분해효소 억제제(Roche Molecular Biochemicals, Mannheim, Germany)를 추가적으로 포함하는 추출 용해 버퍼(50 mM Tris-HCl(pH 7.5), 150 mM NaCl, 1 mM EDTA, 20 mM NaF, 0.5% NP-40, 1% Triton X-100)로 부유시켰다. 단백질 정량 후 단백질을 10% 소디움 도데실 설페이트-폴리아크릴아마이드 겔의 각 웰에 주입하여 전기영동하였다. 전기영동이 끝난 소디움 도데실 설페이트-폴리아크릴아마이드 겔은 PVDF 멤브레인(Bio-RAD)에 블로팅(blotting)하고 이 멤브레인에 5% 스킴 밀크를 포함한 TBST(Tris-buffered saline with 0.1% Tween 20) 용액에서 1시간 동안 가하여 블로킹시켰다. 반응이 끝나면 1X TBST액으로 세척한 후 5% 스킴 밀크 또는 2% 소 혈청 알부민을 포함하는 TBST에 1/1000으로 희석된 1차 항체에 담가 4℃에서 하룻밤동안 반응시켰다. 반응이 끝나면 세척 후 5% 스킴 밀크를 포함하는 TBST에 1/2000으로 희석된 HRP-결합 항-마우스 IgG 또는 항-토끼 IgG 항체에 1시간 동안 반응시켰다. 반응이 완료되면 세척 후 적당히 건조된 멤브레인에 화학발광 기질 용액(ECL, Amersham Biosciences, UK)을 첨가하여 발색시켰다. Mouse RAW 264.7 macrophages were obtained through centrifugation, washed twice with PBS and an extraction lysis buffer (50 mM Tris-HCl (pH 7.5) additionally containing a protease inhibitor (Roche Molecular Biochemicals, Mannheim, Germany)), 150 mM NaCl, 1 mM EDTA, 20 mM NaF, 0.5% NP-40, 1% Triton X-100). After protein quantification, the protein was injected into each well of a 10% sodium dodecyl sulfate-polyacrylamide gel, followed by electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel after electrophoresis was blotting on a PVDF membrane (Bio-RAD), and a solution of TBST (Tris-buffered saline with 0.1% Tween 20) containing 5% skim milk on the membrane. Blocking was performed by adding at for 1 hour. After the reaction was completed, the mixture was washed with 1X TBST solution, and then immersed in the primary antibody diluted 1/1000 in TBST containing 5% skim milk or 2% bovine serum albumin, and reacted overnight at 4°C. At the end of the reaction, after washing, HRP-binding anti-mouse IgG or anti-rabbit IgG antibody diluted 1/2000 in TBST containing 5% skim milk was reacted for 1 hour. When the reaction was completed, a chemiluminescent substrate solution (ECL, Amersham Biosciences, UK) was added to an appropriately dried membrane after washing to develop color.
세포 형질주입(transfection)Cell transfection
형질주입은 부착세포의 안정한 형질주입 방법(Behr, J.P. et al., Proc Natl Acad Sci USA 86:6982986(1989), 13. Loeffler, J.P. et al., Meth Enzymol 217:599618(1993))을 일부 수정하여 수행하였다. 6-웰 Costar plates(35 mm, Cambridge, MA)에 마우스 RAW 264.7 대식세포 웰당 3× 105개를 2 mL의 배양배지와 함께 37℃에서 하룻밤동안 배양하였다. 무혈청 배지로 세포를 2회 세척한 후, 세포에 pNF-κBSEAP-NPT(6 μg/100 μl) 및 lipofectamine(25 μg/100 μl, Life Technologies)을 포함하는 복합체를 처리하고 무혈청 배지에서 2시간 배양하였다. 그 다음, 혈청을 포함하는 배양배지로 교체하고 48시간 동안 배양하였다. 안정적으로 형질주입된 세포를 선별하기 위해 형질주입된 세포의 배양시 제네티신(200 μg/mL)을 첨가하였다. Transfection is a stable transfection method of adherent cells (Behr, JP et al., Proc Natl Acad Sci USA 86:6982986 (1989), 13.Loeffler, JP et al., Meth Enzymol 217:599618 (1993)) was performed with some modifications. Mouse RAW 264.7 macrophages 3×10 5 per well were incubated overnight at 37°C with 2 mL of culture medium on 6-well Costar plates (35 mm, Cambridge, MA). After washing the cells twice with serum-free medium, the cells were treated with a complex containing pNF-κBSEAP-NPT (6 μg/100 μl) and lipofectamine (25 μg/100 μl, Life Technologies), and 2 in serum-free medium. Incubated for hours. Then, it was replaced with a culture medium containing serum and cultured for 48 hours. Geneticin (200 μg/mL) was added when the transfected cells were cultured to select stably transfected cells.
리포터 유전자 분석법Reporter gene analysis method
리포터 효소 활성(SEAP)는 J.S. Ahmed 등에 의해 종래에 알려진 방법에 따라 측정되었다(J.S. Ahmed et al., Parasitol Res. 85:539-549(1999), K. Funakoshi et al., Digestion. 59:73-78(1998), M.J. Bottomley et al., Clin Exp Immunol. 117:171-176(1999)). 형질주입된 마우스 RAW 264.7 대식세포(1.5 × 105)를 12 웰 플레이트에 넣고 24시간 배양한 후, 새로운 배지로 교환하고 패장 추출물을 2시간 동안 전처리하였다. 그 다음, 지질다당체(1 μg/ml) 또는 지질다당체 및 패장 추출물을 16시간 동안 처리하였다. SEAP/MUP의 형광은 96-웰 플레이트 형광 강도 측정기(Molecular Devices, Gemini XS, Sunnyvale, CA)를 이용하여 측정하였다(excitation : 360 nm, light emission : 449 nm)(K.S. Ahn et al., J Dermatol Sci. 31:193-201(2003)).Reporter enzyme activity (SEAP) was measured according to a conventionally known method by JS Ahmed et al. (JS Ahmed et al., Parasitol Res . 85:539-549 (1999), K. Funakoshi et al., Digestion . 59:73 -78(1998), MJ Bottomley et al., Clin Exp Immunol . 117:171-176 (1999)). Transfected mouse RAW 264.7 macrophages (1.5 × 10 5 ) were placed in a 12-well plate and cultured for 24 hours, then replaced with a new medium, and the shellfish extract was pretreated for 2 hours. Then, lipopolysaccharide (1 μg/ml) or lipopolysaccharide and shellfish extract were treated for 16 hours. The fluorescence of SEAP/MUP was measured using a 96-well plate fluorescence intensity meter (Molecular Devices, Gemini XS, Sunnyvale, CA) (excitation: 360 nm, light emission: 449 nm) (KS Ahn et al., J Dermatol Sci . 31:193-201 (2003)).
EMSA(Electrophoretic mobility shift assay)EMSA (Electrophoretic Mobility Shift Assay)
지질다당체에 의한 NF-κB 활성을 확인하기 위해 EMSA를 수행하였다. 지질다당체가 처리된 세포(1×106 cells/ml)로부터 수득한 핵 추출물을 인간면역결핍바이러스 긴말단반복순서인 5’-TTGTTACAA GGGACTTTC CGCTG GGGACTTTC CAGGGAGGCGTGG-3’(볼드체는 NF-κB 결합위치)로부터 제작된 32P-end-labeled 45-mer double-stranded NF-κB 올리고뉴클레오타이드(15 μg의 단백질에 16 fmol의 DNA를 결합)와 37℃에서 30분간 반응시키고, 반응을 통해 형성된 DNA-단백질 복합체를 올리고뉴클레오타이드가 없는 6.6% 네이티브 폴리아크릴아마이드 겔에서 분리하였다. 돌연변이를 포함하는 이중가닥 올리고뉴클레오타이드(5’-TTGTTACAA CTCACTTTC CGCTG CTCACTTTC CAGGGAGGCGTGG-3’)는 DNA에 결합하는 NF-κB의 특이성을 확인하기 위해 사용되었다. 결합의 특이성은 표식되지 않은 올리고뉴클레오타이드와의 경쟁을 통해 평가되었다. 수퍼시프트 분석(supershift assay)을 수행하기 위해 지질다당체가 처리된 세포로부터 수득한 핵 추출물을 항-p50 항체 또는 NF-κB의 p65 소단위에 대한 항체와 37℃에서 30분간 반응시켰다. 면역전혈청(preimmune serum ,PIS)은 음성 대조군으로써 포함되었다. 건조한 겔은 Storm820로 가시화하였으며, 방사성의 밴드는 Imagequant software(Amersham, Piscataway, NJ)를 이용하여 정량화하였다. EMSA was performed to confirm the NF-κB activity by the lipopolysaccharide. Nuclear extract obtained from lipopolysaccharide-treated cells (1×10 6 cells/ml) was obtained from human immunodeficiency virus long-terminal repeat sequence of 5′-TTGTTACAA GGGACTTTC CGCTG GGGACTTTC CAGGGAGGCGTGG-3′ (bold is NF-κB binding site) The resulting 32 P-end-labeled 45-mer double-stranded NF-κB oligonucleotide (15 μg of protein bound to 16 fmol of DNA) and reacted at 37° C. for 30 minutes, and the DNA-protein complex formed through the reaction Were separated on a 6.6% native polyacrylamide gel without oligonucleotides. The double-stranded oligonucleotide containing the mutation (5'-TTGTTACAA CTCACTTTC CGCTG CTCACTTTC CAGGGAGGCGTGG-3') was used to confirm the specificity of NF-κB binding to DNA. The specificity of binding was assessed through competition with unlabeled oligonucleotides. In order to perform a supershift assay, a nuclear extract obtained from lipopolysaccharide-treated cells was reacted with an anti-p50 antibody or an antibody against the p65 subunit of NF-κB at 37° C. for 30 minutes. Preimmune serum (PIS) was included as a negative control. The dried gel was visualized with Storm820, and the radioactive band was quantified using Imagequant software (Amersham, Piscataway, NJ).
핵 및 세포질 추출물Nuclear and cytoplasmic extract
형질주입 마우스 RAW 264.7 대식세포를 60 mm 배양 플레이트에 웰당 2× 106개 넣고 37℃에서 12시간동안 배양하였다. 세포에 패장 추출물을 농도별로 2시간 동안 전처리한 후, 지질다당체를 1 μg/ml 농도로 0, 5, 15, 30 및 60분 동안 처리하였다. 세포는 원심분리를 통해 수득하였고, 아이스-콜드 PBS/인산가수분해효소 억제제로 세척하였다. 마우스 RAW 264.7 대식세포의 핵 및 세포질 추출물은 Nuclear Extract Kit(ACTIVE MOTIF, USA)를 이용하여 제조사의 지시에 따라 추출하였으며, 단백질분해효수 억제제는 추가적으로 첨가하였다. 핵 및 세포질 단백질 추출물 30 μg를 10% 소디움 도데실 설페이트-폴리아크릴아마이드 겔의 각 웰에 주입하여 전기영동하였다. Transfected mouse RAW 264.7 macrophages were placed in a 60 mm culture plate and 2×10 6 cells per well were incubated at 37°C for 12 hours. Cells were pretreated for 2 hours with a concentration of the shellfish extract, and then the lipopolysaccharide was treated at a concentration of 1 μg/ml for 0, 5, 15, 30 and 60 minutes. Cells were obtained through centrifugation and washed with ice-cold PBS/phosphatase inhibitor. Nuclear and cytoplasmic extracts of mouse RAW 264.7 macrophages were extracted according to the manufacturer's instructions using the Nuclear Extract Kit (ACTIVE MOTIF, USA), and a proteolytic potency inhibitor was additionally added. 30 μg of nuclear and cytoplasmic protein extract was injected into each well of a 10% sodium dodecyl sulfate-polyacrylamide gel, followed by electrophoresis.
비장세포(splenocytes)로부터 IL-6 및 TNF-α의 ELISA 분석ELISA analysis of IL-6 and TNF-α from splenocytes
BALB/c 마우스로부터 무균적으로 비장을 적출하고, 나일론 그물망을 통과시켜 RPMI 1640내에 단일 비장 세포 부유액을 수득하였다. RBC(red blood cell)를 용해시킨 후, 세포를 24-웰 플레이트에 웰당 2× 106개 넣어 배양하고, 다양한 농도의 EAPS를 지질다당체(1 μg/ml)가 포함된 또는 포함되지 않은 조건에서 전처리하였다. 플레이트를 원심분리하고 상등액으로부터 IL-6 및 TNF-α의 수치를 ELISA를 이용하여 측정하였다. The spleen was aseptically removed from BALB/c mice and passed through a nylon mesh to obtain a single splenic cell suspension in RPMI 1640. After dissolving RBC (red blood cells), the cells were cultured by putting 2×10 6 cells per well in a 24-well plate, and EAPS at various concentrations were added under conditions with or without lipopolysaccharide (1 μg/ml). It was pretreated. The plate was centrifuged and the levels of IL-6 and TNF-α were measured from the supernatant using ELISA.
BALB/c 마우스 혈장 내 TNF-α 측정Measurement of TNF-α in plasma of BALB/c mice
6주령 웅성 BALB/c 마우스를 NARA biotech로부터 구입하고 그룹당 10마리씩 분류하였다. TNF-α의 혈장 내 수치를 측정하기 위해, 마우스에 지질다당체(25 mg/kg)를 복강주사하였다. EAPS(300 mg/kg)는 지질다당체 주사 24시간 및 2시간전에 식도 카테터를 이용하여 입을 통해 주입하였다. 전혈 샘플은 지질다당체 주사 2시간 및 4시간 후에 안와정맥총(Retro-Orbital plexus)을 통해 수득하였고, 혈청은 TNF-α 양을 측정하기 위해 수집하였다. Six week old male BALB/c mice were purchased from NARA biotech and sorted into 10 per group. In order to measure the plasma level of TNF-α, mice were intraperitoneally injected with lipopolysaccharide (25 mg/kg). EAPS (300 mg/kg) was injected through the mouth using an
통계 분석Statistical analysis
모든 실험결과는 통계 처리하여 평균치와 표준편차를 계산하였으며, 아노바(analysis of variance)의 본페로니 교정은 데이터의 다중 비교의 통계학적 분석을 위해 사용되었다. p-값이 0.01 이하일 때, 통계학적으로 유의성이 있는 것으로 판단하였다. All experimental results were statistically processed to calculate mean values and standard deviations, and Bonferroni correction of Anova (analysis of variance) was used for statistical analysis of multiple comparisons of data. When the p -value was less than or equal to 0.01, it was judged to be statistically significant.
실험결과Experiment result
본 발명자들은 마우스 RAW 246.7 대식세포의 NF-κB 신호전달 경로에서 패장 추출물의 항염증 효과를 조사하였다. 패장의 4개 분획물 및 에탄올 추출물의 세포독성을 먼저 확인하였고, MTT 분석법을 이용하여 세포 생존능력을 측정하였다. 에탄올 추출물(EtOH), 에틸 아세테이트(EA) 및 부탄올(BuOH) 분획물은 100 μg/ml 농도에서 세포독성이 나타나지 않았다(도 1의 패널 A). The present inventors investigated the anti-inflammatory effect of shellfish extract in the NF-κB signaling pathway of mouse RAW 246.7 macrophages. The cytotoxicity of the four fractions of the shell and the ethanol extract was first confirmed, and cell viability was measured using the MTT assay. Ethanol extract (EtOH), ethyl acetate (EA) and butanol (BuOH) fractions did not show cytotoxicity at a concentration of 100 μg/ml (Panel A of FIG. 1).
패장 분획물의 지질다당체(LPS)-유도 산화질소(Nitric Oxide, NO) 생성 저해 Inhibition of Lipopolysaccharide (LPS)-induced Nitric Oxide (NO) production of shellfish fraction
패장 분획물들의 항염증 효과를 조사하기 위해, 에탄올 추출물 및 선택된 2개 분획물의 지질다당체에 의해 자극된 마우스 RAW 264.7 대식세포에서 산화질소 생성 효과를 확인하였다. 마우스 RAW 264.7 대식세포에서 지질다당체에 의해 유도된 산화질소 생성에 대한 분획물들의 효과는 Griess 반응에 의해 평가된 배양배지 내 축적 아질산염을 측정함으로써 조사되었다. 세포에 3개 분획물(100 μg/mL)을 2시간 동안 처리한 후, 지질다당체(1 μg/mL)를 22시간 동안 처리하였다. 지질다당체 처리는 산화질소의 농도를 현저히 증가시켰다. 도 1의 패널 B-D에서 보는 바와 같이, 에탄올 추출물, 에틸 아세테이트 및 부탄올 분획물을 다양한 농도(10, 30, 50, 100 μg/mL)로 전처리한 세포 및 지질다당체(1 μg/mL)를 22시간 동안 함께 처리한 세포 가운데 에탄올 추출물 및 에틸 아세테이트 분획물을 처리한 세포에서 농도 의존적으로 아질산염의 생성이 저해되는 것을 확인하였다. 다른 분획물과 비교하여 에틸 아세테이트 분획물이 지질다당체에 의해 유도된 산화질소의 억제제로서 가장 가능성이 높은 것으로 판단되어 이후 실험에 에틸 아세테이트 분획물을 사용하였다(도 1의 패널 C). In order to investigate the anti-inflammatory effect of the shell fractions, the effect of nitric oxide production in mouse RAW 264.7 macrophages stimulated by the ethanol extract and the lipopolysaccharide of the selected two fractions was confirmed. The effect of the fractions on the nitric oxide production induced by lipopolysaccharide in mouse RAW 264.7 macrophages was investigated by measuring the nitrite accumulated in the culture medium evaluated by the Griess reaction. After the cells were treated with three fractions (100 μg/mL) for 2 hours, lipopolysaccharide (1 μg/mL) was treated for 22 hours. Lipopolysaccharide treatment significantly increased the concentration of nitric oxide. As shown in panel BD of FIG. 1, cells and lipopolysaccharides (1 μg/mL) pretreated with ethanol extract, ethyl acetate, and butanol fractions at various concentrations (10, 30, 50, 100 μg/mL) for 22 hours Among the cells treated together, it was confirmed that the production of nitrite was inhibited in a concentration-dependent manner in the cells treated with the ethanol extract and the ethyl acetate fraction. Compared to other fractions, the ethyl acetate fraction was judged to be the most probable as an inhibitor of nitric oxide induced by lipopolysaccharide, so that the ethyl acetate fraction was used in the subsequent experiment (Panel C of FIG. 1).
EAPS에 의한 지질다당체-유도 iNOS 및 COX-2 단백질 발현 억제Inhibition of lipopolysaccharide-induced iNOS and COX-2 protein expression by EAPS
마우스 RAW 264.7 대식세포에서 iNOS 및 COX-2 단백질 발현에 대한 EAPS의 효과는 웨스턴 블롯 분석을 통해 측정하였다. 도 2의 패널 A에서 보는 바와 같이, 자극을 주지 않은 조건 하에서 iNOS 및 COX-2 단백질은 측정하기에 현저하게 낮은 발현율을 나타냈으나, 지질다당체(1 μg/ml)를 22시간 처리한 후에는 iNOS 및 COX-2의 발현이 현저하게 증가하였다. EAPS(10, 30, 50, 100 μg/ml)를 2시간 동안 전처리한 세포는 처리량에 의존적으로 지질다당체-유도 iNOS 및 COX-2의 발현이 현격히 억제되었다(도 2의 패널 A). EAPS(50 μg/ml) 개별 처리는 iNOS 및 COX-2의 기본적인 발현 수준에 영향을 미치지 않았다. The effect of EAPS on iNOS and COX-2 protein expression in mouse RAW 264.7 macrophages was measured by Western blot analysis. As shown in panel A of FIG. 2, iNOS and COX-2 proteins showed remarkably low expression rates to be measured under non-stimulated conditions, but after treatment with lipopolysaccharide (1 μg/ml) for 22 hours The expression of iNOS and COX-2 was markedly increased. Cells pretreated with EAPS (10, 30, 50, 100 μg/ml) for 2 hours remarkably inhibited the expression of lipopolysaccharide-induced iNOS and COX-2 depending on the throughput (Fig. 2, panel A). Individual treatment of EAPS (50 μg/ml) did not affect the basal expression levels of iNOS and COX-2.
EAPS에 의한 지질다당체-유도 iNOS 및 COX-2 mRNA 발현 억제Lipopolysaccharide-induced iNOS and COX-2 mRNA expression inhibition by EAPS
iNOS 및 COX-2의 단백질 발현이 그들의 mRNA 레벨과 유사한지 확인하기 위해 RT-PCR을 수행하였다. EAPS를 2시간 동안 전처리한 후 마우스 RAW 264.7 대식세포에 지질다당체를 22시간 동안 처리하고 RT-PCR을 통해 iNOS 및 COX-2 mRNA 레벨을 확인하였다. 그 결과, 지질다당체 처리에 따른 자극으로 iNOS 및 COX-2의 mRNA 레벨이 증가하였다. 또한, 지질다당체에 의해 자극되지 못한 마우스 RAW 264.7 대식세포에서는 mRNA이 낮아 측정할 수 없었다. RT-PCR 분석 결과는 EAPS가 지질다당체-유도 iNOS 및 COX-2의 mRNA 레벨을 처리량에 의존적으로 억제하는 것을 나타낸다(도 2의 패널 B). RT-PCR was performed to confirm that the protein expression of iNOS and COX-2 was similar to their mRNA level. After pre-treatment of EAPS for 2 hours, lipopolysaccharide was treated in mouse RAW 264.7 macrophages for 22 hours, and iNOS and COX-2 mRNA levels were confirmed through RT-PCR. As a result, the mRNA levels of iNOS and COX-2 increased due to stimulation following lipopolysaccharide treatment. In addition, in mouse RAW 264.7 macrophages that were not stimulated by lipopolysaccharide, the mRNA was low and could not be measured. RT-PCR analysis results show that EAPS inhibits the mRNA levels of lipopolysaccharide-induced iNOS and COX-2 in a throughput-dependent manner (Fig. 2, panel B).
EAPS에 의한 지질다당체-유도 IL-6 분비 억제Inhibition of lipopolysaccharide-induced IL-6 secretion by EAPS
마우스 RAW 264.7 대식세포에서 지질다당체에 의해 유도된 IL-6 생성에 대한 EAPS의 항-염증 가능성을 확인하기 위해, EAPS(10, 30, 50, 100 μg/ml)를 2시간 동안 전처리하고 지질다당체(1 μg/ml)를 22시간 동안 처리하였다. IL-6 농도는 세포 배양액의 상등액에서 ELISA 분석법을 통해 측정하였다. 그 결과, EAPS의 처리 농도에 의존적으로 IL-6의 생성이 억제되었다(도 2의 패널 C). To confirm the anti-inflammatory potential of EAPS for IL-6 production induced by lipopolysaccharide in mouse RAW 264.7 macrophages, EAPS (10, 30, 50, 100 μg/ml) was pretreated for 2 hours and lipopolysaccharide (1 μg/ml) was treated for 22 hours. IL-6 concentration was measured by ELISA assay in the supernatant of the cell culture medium. As a result, the production of IL-6 was inhibited depending on the concentration of EAPS treatment (Fig. 2, panel C).
EAPS에 의한 지질다당체-유도 NF-κB 활성 억제Inhibition of lipopolysaccharide-induced NF-κB activity by EAPS
NF-κB는 지질다당체에 대한 반응으로 활성화되는 전사인자로 iNOS 및 COX-2 유전자 발현 유도에 있어 NF-κB의 활성화는 중요한 단계이다(K.S. Ahn et al., Curr Mol Med. 7:619-637(2007)). 지질다당체에 의해 유도된 NF-κB의 활성화를 막는 EAPS의 능력을 확인하기 위해 EMSA를 수행하였다. 지질다당체를 처리하고 1시간 후, 대식세포의 핵 추출물에 대한 NF-κB의 결합은 현저하게 증가되었다. EAPS를 전처리한 세포에서, 지질다당체에 의해 유도된 NF-κB의 활성화는 처리량에 의존적으로 억제되었다(도 3의 패널 A). 또한, 인핸서(enhancer) 요소에 NF-κB 결합 위치를 포함하는 pNF-κB-SEAP-NPT 플라스미드를 마우스 RAW 264.7 대식세포에 형질주입시켜 EAPS에 의한 NF-κB 활성 억제를 조사하였다. 형광 분석법(fluorescence method assay)을 이용하여 형질주입된 마우스 RAW 264.7 대식세포에서 NF-κB 활성화 레벨을 확인하였다. 지질다당체에 의해 유도된 NF-κB 활성은 EAPS의 처리량에 의존적으로 현저하게 약화되었다(도 3의 패널 B). 이러한 데이터는 NF-κB 결합 분석법 결과와 일치한다. 지질다당체에 의해 유도된 p65의 인산화 및 p65의 핵 전좌(nuclear translocation)에 있어 EAPS의 효과는 웨스턴 블롯 분석법을 이용하여 확인하였다. 도 3의 패널 C에서 보는 바와 같이, EAPS는 p65의 전사 활성 및 핵 내로의 이동을 억제하였다. NF-κB is a transcription factor that is activated in response to lipopolysaccharide, and activation of NF-κB is an important step in inducing iNOS and COX-2 gene expression (KS Ahn et al., Curr Mol. Med . 7:619-637 (2007)). EMSA was performed to confirm the ability of EAPS to prevent activation of NF-κB induced by lipopolysaccharide. One hour after treatment with the lipopolysaccharide, the binding of NF-κB to the nuclear extract of macrophages was remarkably increased. In cells pretreated with EAPS, activation of NF-κB induced by lipopolysaccharide was inhibited in a dose-dependent manner (Fig. 3, panel A). In addition, the pNF-κB-SEAP-NPT plasmid containing the NF-κB binding site in an enhancer element was transfected into mouse RAW 264.7 macrophages to investigate the inhibition of NF-κB activity by EAPS. NF-κB activation levels were confirmed in the transfected mouse RAW 264.7 macrophages using a fluorescence method assay. The NF-κB activity induced by the lipopolysaccharide was significantly attenuated depending on the throughput of EAPS (Fig. 3, panel B). These data are consistent with the results of the NF-κB binding assay. The effect of EAPS on the phosphorylation of p65 and nuclear translocation of p65 induced by lipopolysaccharide was confirmed using Western blot analysis. As shown in panel C of FIG. 3, EAPS inhibited the transcriptional activity and migration of p65 into the nucleus.
EAPS에 의한 지질다당체-유도 p38, ERK1/2 및 JNK 인산화 억제Inhibition of lipopolysaccharide-induced p38, ERK1/2 and JNK phosphorylation by EAPS
ERK1/2 억제제(PD98059) 및 p38 억제제(SB203580)는 지질다당체에 의해 유도된 산화질소 생성을 억제하는 것으로 알려져 있다(S. Mi Jeong et al., Int Immunopharmacol. 9:319-323(2009)). 본 발명자들은 EAPS가 MAPKs 신호전달 경로를 방해하여 지질다당체에 의해 유도된 산화질소의 생성을 억제한다는 가설을 성립하였다. 이에 지질다당체에 의해 유도된 p38, ERK1/2 및 JNK의 인산화에 대한 EAPS의 효과를 확인하였다(도 3의 패널 D). 지질다당체의 처리(5-30분간)는 p38, ERK1/2 및 JNK의 인산화를 현저하게 증가시켰다. 이러한 결과는 EAPS가 마우스 RAW 264.7 대식세포에서 NF-κB 신호전달을 억제하여 지질다당체에 의해 유도된 산화질소 생성을 저해시키지만, MAPKs 경로에는 작용하지 않는다는 것을 의미한다. ERK1/2 inhibitor (PD98059) and p38 inhibitor (SB203580) are known to inhibit nitric oxide production induced by lipopolysaccharides (S. Mi Jeong et al., Int Immunopharmacol . 9:319-323 (2009)). . The present inventors established the hypothesis that EAPS inhibits the production of nitric oxide induced by lipopolysaccharides by interfering with the MAPKs signaling pathway. Accordingly, the effect of EAPS on the phosphorylation of p38, ERK1/2 and JNK induced by lipopolysaccharide was confirmed (FIG. 3, panel D). Treatment of lipopolysaccharide (5-30 minutes) significantly increased the phosphorylation of p38, ERK1/2 and JNK. These results imply that EAPS inhibits NF-κB signaling in mouse RAW 264.7 macrophages, thereby inhibiting nitric oxide production induced by lipopolysaccharides, but does not act on the MAPKs pathway.
지질다당체를 주사한 마우스에서 TNF-α의 혈장 내 농도에 대한 EAPS의 효과Effect of EAPS on the plasma concentration of TNF-α in mice injected with lipopolysaccharide
마우스의 TNF-α 혈장농도에 대한 EAPS의 억제 효과를 평가하기 위해, 지질다당체를 주사한 마우스에서 TNF-α의 혈장 내 농도에 대한 EAPS의 효과를 확인하였다. EAPS의 투여는 대조군에 비해 지질다당체에 의해 유도된 TNF-α 혈장 내 농도 증가를 약화시켰다(도 4의 패널 A). In order to evaluate the inhibitory effect of EAPS on the plasma concentration of TNF-α in mice, the effect of EAPS on the plasma concentration of TNF-α was confirmed in mice injected with lipopolysaccharide. Administration of EAPS attenuated the increase in the plasma concentration of TNF-α induced by lipopolysaccharide compared to the control group (Fig. 4, panel A).
지질다당체에To lipopolysaccharide 의해 자극된 Stimulated by 비장세포에서In splenocytes EAPS의EAPS IL-6 및 IL-6 and TNFTNF -α 분비 억제 효과 -α secretion inhibitory effect
다른 면역세포에서 EAPS의 효과에 대한 추가적인 정보를 얻기 위해, EAPS가 지질다당체에 의해 자극된 비장세포로부터 IL-6 및 TNF-α 생성을 억제하는지를 확인하였다. 비장세포는 BALB/c 마우스의 비장으로부터 수득하였다. 비장세포에 EAPS(10, 30, 50, 100 μg/ml)를 2시간 처리한 후, 다양한 시간(12, 24, 36, 48시간) 동안 지질다당체(1 μg/ml)를 처리하였다. 그 결과, IL-6 및 TNF-α 생성은 EPAS의 처리량 및 처리 시간에 의존적으로 감소하였다(도 4의 패널 B-C). In order to obtain additional information on the effect of EAPS in other immune cells, it was confirmed whether EAPS inhibits IL-6 and TNF-α production from splenocytes stimulated by lipopolysaccharides. Splenocytes were obtained from the spleen of BALB/c mice. The splenocytes were treated with EAPS (10, 30, 50, 100 μg/ml) for 2 hours, and then lipopolysaccharide (1 μg/ml) was treated for various times (12, 24, 36, 48 hours). As a result, the production of IL-6 and TNF-α decreased depending on the amount of EPAS and the treatment time (Panels B-C in FIG. 4).
고찰Review
본 발명은 패장의 에틸 아세테이트 분획이 지질다당체에 의해 자극된 마우스 RAW 264.7 대식세포에서 NF-κB 신호전달 경로를 억제함으로써 항염증 효과를 나타내는지, 또한 EAPS가 지질다당체를 주사한 마우스에서 혈장 내 TNF-α 농도에 영향을 끼치는지를 확인하였다. 최종적으로 비장세포에서 지질다당체에 의해 유도된 IL-6 및 TNF-α에 대해 억제효과를 측정하였다. 본 발명자들은 EAPS가 NF-κB p65의 인산화 및 핵 전위를 억제하는 것을 입증하였다. 결론적으로 EAPS는 마우스 RAW 264.7 대식세포에서 염증반응에 관여하는 NF-κB-의존적 유전자들(iNOS, COX-2, TNF-α 및 IL-6)의 발현을 저해한다. EAPS는 마우스 혈장 내 TNF-α 농도를 현저하게 낮추며, 비장세포에서 지질다당체에 의해 유도된 IL-6 및 TNF-α를 감소시킨다. 이러한 결과는 지질다당체에 의해 활성화된 NF-κB에 대한 EAPS의 항염증 효과를 처음으로 확인한 것이다.The present invention is to determine whether the ethyl acetate fraction of the shell exhibits an anti-inflammatory effect by inhibiting the NF-κB signaling pathway in mouse RAW 264.7 macrophages stimulated by lipopolysaccharide, and also, in mice injected with the lipopolysaccharide, EAPS has TNF in plasma. It was confirmed whether it affects the -α concentration. Finally, the inhibitory effect on IL-6 and TNF-α induced by lipopolysaccharide in splenocytes was measured. The present inventors demonstrated that EAPS inhibits phosphorylation and nuclear translocation of NF-κB p65. In conclusion, EAPS inhibits the expression of NF-κB-dependent genes (iNOS, COX-2, TNF-α and IL-6) involved in inflammatory response in mouse RAW 264.7 macrophages. EAPS significantly lowers the concentration of TNF-α in mouse plasma and decreases IL-6 and TNF-α induced by lipopolysaccharides in splenocytes. These results are the first to confirm the anti-inflammatory effect of EAPS on NF-κB activated by lipopolysaccharide.
본 발명은 EAPS가 지질다당체에 의한 NF-κB의 활성을 억제하며, 이로 인해 지질다당체 신호전달의 염증반응을 저해하는데 중요한 역할을 하는 것이라고 생각된다. 또한, 본 발명의 결과들은 EAPS가 NF-κB의 DNA 결합에 대한 직접적인 방해없이 NF-κB의 활성을 억제한다는 것을 나타낸다. 이러한 결과들은 패장의 구성성분인 올레아노닉산 및 우르솔릭산이 NF-κB 저해를 통해 TNF-α에 의해 유도된 E-셀렉틴 생성을 억제한다는 종래 연구와 일치하는 것이다(K. Takada et al., Phytomedicine. 17:1114-1119(2010)). 기본적으로 NF-κB 활성화는 IκB kinase(IKK) 활성을 필요로 하며, IKK 활성화는 IκBa의 32번째 및 36번째 세린을 인산화시켜 IκBa의 분해 및 p65의 인산화, 핵 전위를 일으킨다(S. Ghosh et al., Cell. 109:S81-96(2002)). 본 발명의 웨스턴 블롯 결과는 EAPS에 의한 NF-κB p65의 인산화 및 핵 전위의 저해를 통해 NF-κB 신호전달이 억제된다는 것을 보여준다. In the present invention, EAPS is believed to play an important role in inhibiting the inflammatory response of lipopolysaccharide signaling by inhibiting the activity of NF-κB by lipopolysaccharide. In addition, the results of the present invention indicate that EAPS inhibits the activity of NF-κB without direct interference with DNA binding of NF-κB. These results are consistent with previous studies that oleanonic acid and ursolic acid, which are constituents of shellfish, inhibit TNF-α-induced E-selectin production through NF-κB inhibition (K. Takada et al., Phytomedicine). 17:1114-1119 (2010)). Basically, NF-κB activation requires IκB kinase (IKK) activity, and IKK activation phosphorylates the 32nd and 36th serine of IκBa, causing degradation of IκBa, phosphorylation of p65, and nuclear translocation (S. Ghosh et al. ., Cell . 109:S81-96 (2002)). Western blot results of the present invention show that NF-κB signaling is inhibited through inhibition of nuclear translocation and phosphorylation of NF-κB p65 by EAPS.
iNOS에 의해 생성된 과량의 산화질소는 급성 및 만성 염증을 매개하기 때문에 산화질소는 중요한 매개체이다. 그러므로, 산화질소 생성의 억제는 염증성 질환을 예방 및 치료하는데 새로운 약리학적 전략으로써 강력하게 제안되고 있다(A.C. Tinker et al., Curr Top Med Chem. 6:77-92(2006)). 본 발명자들은 EAPS가 마우스 RAW 264.7 대식세포에서 지질다당체에 의해 자극된 산화질소 생성을 현저하게 억제시킨다는 것을 확인하였고, 이것은 패장이 항염증 요소로써 유망한 물질임을 나타낸다(C. Szabo et al., New Horiz. 3:2-32(1995)). 이러한 결과들은 패장 뿌리의 메탄올 추출물이 덱스트란 황산 나트륨(dextran sulfate sodium)에 의해 유도된 대장염을 가진 마우스에서 산화질소 생산을 억제하는 기존 연구 내용과도 일치하는 것이다(E.J. Cho et al., J Ethnopharmacol.(in Press)(2010)).Nitric oxide is an important mediator because excess nitric oxide produced by iNOS mediates acute and chronic inflammation. Therefore, inhibition of nitric oxide production has been strongly proposed as a new pharmacological strategy for preventing and treating inflammatory diseases (AC Tinker et al., Curr Top Med Chem . 6:77-92 (2006)). The present inventors confirmed that EAPS remarkably inhibits nitric oxide production stimulated by lipopolysaccharides in mouse RAW 264.7 macrophages, indicating that the shell is a promising substance as an anti-inflammatory factor (C. Szabo et al., New Horiz). 3:2-32 (1995)). These results are consistent with previous studies in which methanol extracts from the roots of shellfish inhibit nitric oxide production in mice with colitis induced by sodium dextran sulfate (EJ Cho et al., J Ethnopharmacol). .(in Press) (2010)).
게다가, 종양 프로모터, 사이토카인 및 미토겐은 다양한 세포에서 COX-2 발현을 유도한다(L.J. Crofford et al., J Clin Invest. 93:1095-1101(1994), H. Inoue et al., J Biol Chem. 270:24965-24971(1995)). COX-2는 프로스타글란딘E2(prostaglandinE2, PGE2)와 같은 프로스타글란딘의 생성을 촉진시킴으로써 염증 반응에 중요한 역할을 하는 것으로 잘 알려져 있다. 따라서, 대식세포, 표피세포 및 섬유아세포와 같이 염증과 관련이 있는 많은 세포 종류들은 COX-2 유전자를 발현할 수 있다(T. Hla et al., Ann N Y Acad Sci. 696:197-204(1993)). 본 발명자들은 EAPS가 마우스 RAW 264.7 대식세포에서 지질다당체에 의해 유도된 COX-2 단백질의 발현을 처리량에 의존적으로 억제한다는 것을 처음으로 밝혔다. 많은 연구에서 iNOS 및 COX-2 프로모터의 상류 위치에 NF-κB가 결합하는 것이 지질다당체 및 IFN-γ에 의해 유도된 iNOS 및 COX-2 유전자를 최대로 발현하는데 중요한 역할을 한다는 것이 보고되었다(K.S. Ahn et al., Curr Mol Med. 7:619-637(2007), A. Weisz et al., J Biol Chem. 269:8324-8333(1994), Q.W. Xie et al., J Exp Med. 177:1779-1784(1993), Y.M. Kim et al., Biochem Biophys Res Commun. 236:655-660(1997)). 마우스 RAW 264.7 대식세포에서 지질다당체 자극은 iNOS 및 COX-2 전사 및 번역을 유도할 수 있고, 이어 산화질소 및 PGE2 생성을 유도된다(Y.C. Liang et al., Carcinogenesis. 20:1945-1952(1999)). 이러한 점에 기초하여, 본 발명자들은 EAPS가 iNOS 및 COX-2 mRNA 발현을 저하시킴으로써 iNOS 및 COX-2의 단백질 수준을 낮춘다는 것을 확인하였다. 이러한 결과들은 EAPS가 NF-κB 활성을 저해함으로써 지질다당체에 의해 자극된 iNOS 및 COX-2 유전자의 발현을 억제한다는 것을 의미한다. 그러나, 다른 전사 요소들의 억제 가능성을 배제할 수는 없다. 따라서, EAPS는 세포에서 NF-κB 신호전달 경로 저해를 통해 잠재적으로 바이오마커들을 조절한다. In addition, tumor promoters, cytokines and mitogens induce COX-2 expression in various cells (LJ Crofford et al., J Clin Invest . 93:1095-1101 (1994), H. Inoue et al., J Biol. Chem . 270:24965-24971 (1995)). COX-2 is well known to play an important role in the inflammatory response by promoting the production of prostaglandins such as prostaglandinE2 (PGE2). Thus, many cell types associated with inflammation, such as macrophages, epidermal cells, and fibroblasts, can express the COX-2 gene (T. Hla et al., Ann NY Acad Sci . 696:197-204 (1993) )). The present inventors have revealed for the first time that EAPS inhibits the expression of COX-2 protein induced by lipopolysaccharide in mouse RAW 264.7 macrophages in a throughput-dependent manner. In many studies, it has been reported that the binding of NF-κB to the upstream position of the iNOS and COX-2 promoters plays an important role in maximally expressing iNOS and COX-2 genes induced by lipopolysaccharide and IFN-γ (KS Ahn et al., Curr Mol Med . 7:619-637 (2007), A. Weisz et al., J Biol Chem . 269:8324-8333 (1994), QW Xie et al., J Exp Med . 177:1779-1784 (1993), YM Kim et al., Biochem Biophys Res Commun . 236:655-660 (1997)). Lipopolysaccharide stimulation in mouse RAW 264.7 macrophages can induce iNOS and COX-2 transcription and translation, followed by nitric oxide and PGE2 production (YC Liang et al., Carcinogenesis . 20:1945-1952 (1999)). ). Based on these points, the present inventors confirmed that EAPS lowers the protein levels of iNOS and COX-2 by lowering iNOS and COX-2 mRNA expression. These results indicate that EAPS inhibits the expression of iNOS and COX-2 genes stimulated by lipopolysaccharides by inhibiting NF-κB activity. However, the possibility of inhibition of other transcription factors cannot be ruled out. Thus, EAPS potentially modulate biomarkers through inhibition of the NF-κB signaling pathway in cells.
TNF-α 및 IL-6는 인 비트로 및 인 비보에서 항염증 효과를 갖는 것이 알려져 있다. 이러한 사이토카인들은 주고 대식세포에 의해 분비되며, 열(fever)과 같이 지질다당체에 의해 유도된 염증반응의 내생적 조절자로서 여겨진다(R.G. Molloy et al., Br J Surg. 80:289-297(1993), M.A. West et al., J Trauma. 37:82-89; discussion 89-90(1994)). 게다가, 비장세포는 서로 다른 면역 기능을 갖는 T 림프구, B 림프구, 수지상세포 및 대식세포와 같은 다양한 세포로 구성되어있다. 대식세포는 세포성 및 체액성 면역반응을 개시 및 조절하는데 중요하다(J.S. Ahmed et al., Parasitol Res. 85:539-549(1999)). 활성화된 대식세포는 TNF-α 및 IL-6를 포함하는 프로-염증 사이토카인을 생성한다. 이러한 사이토카인들은 염증성창자병(inflammatory bowel diseases)을 포함하는 많은 병리(pathological) 조건의 급성 악화와 관련되어 있으며(K. Funakoshi et al., Digestion. 59:73-78(1998)), rheumatoid arthritis(M.J. Bottomley et al., Clin Exp Immunol. 117:171-176(1999)), vasculitis(M. Noris et al., Circulation. 100:55-60(1999)), 패혈쇼크의 병리생리학에도 관련되어 있다(P. Lauzurica et al., Eur J Immunol. 29:1890-1900(1999)). 본 발명에서 EAPS는 마우스 RAW 264.7 대식세포에서 지질다당체에 의해 자극된 IL-6 생성을 현저하게 억제하였고, 지질다당체를 주사한 마우스에서 TNF-α 및 IL-6 생성을 억제하였다. 패장 내에는 페트리노사이드(patrinoside)(H. Taguchi et al., Chem Pharm Bull(Tokyo). 22:1935-1937(1974)), 사포닌(J.S. Choi et al., Planta Med. 53:62-65(1987), B. Yan et al., Zhong Yao Cai. 22:189-190(1999)), 술파페트리노사이드 Ⅰ 및 Ⅱ(sulfapatrinosides Ⅰ 및 Ⅱ)(A. Inada et al., Chem Pharm Bull (Tokyo). 36:4269-4274(1988)), 우르솔릭산 및 올레아놀릭산(T. Nakanishi et al., Chem Pharm Bull. 41:183-186(1993), B. Yang et al., Zhong Yao Cai. 22:23-24(1999)), 트리테르페노이드 사포닌(triterpenoid saponin)(S.S. Kang et al., J Nat Prod. 60:1060-1062(1997)) 및 페트리놀리드 A(patrinolide A)(M.Y. Yang et al., Arch Pharm Res. 24:416-417(2001))를 포함하는 많은 화합물들이 있다.It is known that TNF-α and IL-6 have anti-inflammatory effects in vitro and in vivo. These cytokines are given and secreted by macrophages, and are considered as endogenous regulators of inflammatory responses induced by lipopolysaccharides, such as fever (RG Molloy et al., Br J Surg . 80:289-297 ( 1993), MA West et al., J Trauma . 37:82-89; discussion 89-90 (1994)). In addition, splenocytes are composed of various cells such as T lymphocytes, B lymphocytes, dendritic cells and macrophages with different immune functions. Macrophages are important for initiating and regulating cellular and humoral immune responses (JS Ahmed et al., Parasitol Res . 85:539-549 (1999)). Activated macrophages produce pro-inflammatory cytokines including TNF-α and IL-6. These cytokines are associated with acute exacerbation of many pathological conditions, including inflammatory bowel diseases (K. Funakoshi et al., Digestion . 59:73-78(1998)), and rheumatoid arthritis. (MJ Bottomley et al., Clin Exp Immunol . 117:171-176(1999)), vasculitis (M. Noris et al., Circulation . 100:55-60(1999)), also involved in the pathophysiology of septic shock. (P. Lauzurica et al., Eur J Immunol . 29:1890-1900 (1999)). In the present invention, EAPS remarkably inhibited IL-6 production stimulated by lipopolysaccharide in mouse RAW 264.7 macrophages, and inhibited TNF-α and IL-6 production in mice injected with lipopolysaccharide. In the shell, petrinoside (H. Taguchi et al., Chem. Pharm Bull (Tokyo) . 22:1935-1937 (1974)), saponins (JS Choi et al., Planta Med . 53:62-65 (1987), B. Yan et al., Zhong Yao Cai . 22:189-190 (1999)), sulfapatrinosides I and II (A. Inada et al., Chem. Pharm Bull (Tokyo) . 36:4269-4274 (1988)), ursolic acid and oleanolic acid (T. Nakanishi et al., Chem Pharm Bull . 41:183-186 (1993), B. Yang et al., Zhong Zhong Yao Cai . 22:23-24 (1999)), triterpenoid saponin (SS Kang et al., J Nat Prod . 60:1060-1062 (1997)) and patrinolide A (MY) There are many compounds including Yang et al., Arch Pharm Res . 24:416-417 (2001)).
본 발명에서 패장의 에틸 아세테이트 분획이 4개의 다른 분획들 가운데 항염증 활성이 가장 좋은 것을 확인하였다. 일반적으로 에틸 아세테이트 분획은 극성 화합물을 포함하고 있기 때문에 에틸 아세테이트 분획의 항염증 활성은 이러한 극성 성분에서 기인한 것일 수 있다.In the present invention, it was confirmed that the ethyl acetate fraction of shellfish had the best anti-inflammatory activity among the four other fractions. In general, since the ethyl acetate fraction contains a polar compound, the anti-inflammatory activity of the ethyl acetate fraction may be due to this polar component.
결론적으로, 본 발명의 결과는 EAPS가 지질다당체에 의해 유도된 iNOS 및 COX-2 유전자의 발현을 억제하는데 있어 강한 효과를 나타내며, 이러한 억제 효과는 NF-κB 활성을 저해함으로써 이루어지는 것임을 분명히 보여주는 것이다. EAPS는 또한 TNF-α, IL-6와 같은 프로-염증 사이토카인 및 산화질소의 생성을 억제한다. 따라서, EAPS는 패장의 주요 분획 중 하나로 염증성 질환을 예방 및 치료하는 가능성을 갖는다고 할 수 있다. In conclusion, the results of the present invention clearly show that EAPS exhibits a strong effect in inhibiting the expression of iNOS and COX-2 genes induced by lipopolysaccharide, and this inhibitory effect is achieved by inhibiting NF-κB activity. EAPS also inhibits the production of pro-inflammatory cytokines such as TNF-α and IL-6 and nitric oxide. Therefore, EAPS can be said to have the potential to prevent and treat inflammatory diseases as one of the major fractions of the shell.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto for those of ordinary skill in the art. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.
<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION, Kyung Hee University <120> Compositions for Treatment and Prevention of Inflammatory Disease Comprising Extract of Patrinia scabiosaefolia as Active Ingredient <130> PN110570 <160> 8 <170> KopatentIn 2.0 <210> 1 <211> 24 <212> DNA <213> iNOS forward primer <400> 1 tctttgacgc tcggaactgt agca 24 <210> 2 <211> 24 <212> DNA <213> iNOS reverse primer <400> 2 cgtgaagcca tgacctttcg catt 24 <210> 3 <211> 24 <212> DNA <213> COX-2 forward primer <400> 3 ttgctgtaca agcagtggca aagg 24 <210> 4 <211> 24 <212> DNA <213> COX-2 reverse primer <400> 4 aggacaaaca ccggagggaa tctt 24 <210> 5 <211> 24 <212> DNA <213> GAPDH forward primer <400> 5 aactttggca ttgtggaagg gctc 24 <210> 6 <211> 24 <212> DNA <213> GAPDH reverse primer <400> 6 tggaagagtg ggagttgctg ttga 24 <210> 7 <211> 45 <212> DNA <213> HIV long terminal repeat <400> 7 ttgttacaag ggactttccg ctggggactt tccagggagg cgtgg 45 <210> 8 <211> 45 <212> DNA <213> Double-stranded mutated oligonucleotide <400> 8 ttgttacaac tcactttccg ctgctcactt tccagggagg cgtgg 45 <110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION, Kyung Hee University <120> Compositions for Treatment and Prevention of Inflammatory Disease Comprising Extract of Patrinia scabiosaefolia as Active Ingredient <130> PN110570 <160> 8 <170> KopatentIn 2.0 <210> 1 <211> 24 <212> DNA <213> iNOS forward primer <400> 1 tctttgacgc tcggaactgt agca 24 <210> 2 <211> 24 <212> DNA <213> iNOS reverse primer <400> 2 cgtgaagcca tgacctttcg catt 24 <210> 3 <211> 24 <212> DNA <213> COX-2 forward primer <400> 3 ttgctgtaca agcagtggca aagg 24 <210> 4 <211> 24 <212> DNA <213> COX-2 reverse primer <400> 4 aggacaaaca ccggagggaa tctt 24 <210> 5 <211> 24 <212> DNA <213> GAPDH forward primer <400> 5 aactttggca ttgtggaagg gctc 24 <210> 6 <211> 24 <212> DNA <213> GAPDH reverse primer <400> 6 tggaagagtg ggagttgctg ttga 24 <210> 7 <211> 45 <212> DNA <213> HIV long terminal repeat <400> 7 ttgttacaag ggactttccg ctggggactt tccagggagg cgtgg 45 <210> 8 <211> 45 <212> DNA <213> Double-stranded mutated oligonucleotide <400> 8 ttgttacaac tcactttccg ctgctcactt tccagggagg cgtgg 45
Claims (11)
Patrinia scabiosaefolia ) as an active ingredient in an amount of 30 to 100 ug / mL obtained by fractionating the ethanol extract with ethyl acetate.
3. The composition of claim 1, wherein the extract is root extract of cabbage.
The composition of claim 1, wherein the extract inhibits nitric oxide production.
The composition of claim 1, wherein the extract inhibits the expression of an iNOS protein or an mRNA encoding the iNOS protein.
The composition of claim 1, wherein the extract inhibits the expression of a COX-2 protein or an mRNA encoding the same.
The composition of claim 1, wherein the extract inhibits the activity of NF-kB.
The composition of claim 1, wherein the extract inhibits the secretion of IL-6 (Interleukin-6).
The composition of claim 1, wherein the extract inhibits secretion of TNF-a.
(a) Patrinia a pharmaceutically effective amount of a composition comprising 30 to 100 ug / mL of a fraction obtained by fractionating an ethanol extract of scabiosaefolia with ethyl acetate as an active ingredient; And (b) a pharmaceutically acceptable carrier.
(a) Patrinia scabiosaefolia ) as an active ingredient in an amount of 30 to 100 ug / mL obtained by fractionating an ethanol extract of ethyl acetate with ethyl acetate; And (b) a pharmaceutically acceptable carrier.
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KR20010104497A (en) * | 2000-05-02 | 2001-11-26 | 한영복 | Composition for B type Hepatitis and Cirrhosis of Liver Comprising Phellodendron amurense Rupr. cortex and Patrinia scabiosaefolia FISCH. Extract |
KR20060034155A (en) * | 2004-10-18 | 2006-04-21 | 주식회사 황토생활건강 | Cosmetic composition for whitening, anti-inflammation and anti-dendruff containing yellow soil and medicine herbs extract |
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KR20010104497A (en) * | 2000-05-02 | 2001-11-26 | 한영복 | Composition for B type Hepatitis and Cirrhosis of Liver Comprising Phellodendron amurense Rupr. cortex and Patrinia scabiosaefolia FISCH. Extract |
KR20060034155A (en) * | 2004-10-18 | 2006-04-21 | 주식회사 황토생활건강 | Cosmetic composition for whitening, anti-inflammation and anti-dendruff containing yellow soil and medicine herbs extract |
Non-Patent Citations (4)
Title |
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Eu-jin Cho 외 8명. Anti-inflammatory effects of methanol extract of Patrinia scabiosaefolia in mice with ulcerative colitis. Journal of Ethnopharmacology. Vol.136, 2011년, pp. 428-435 * |
Sang-Wan Seo 외 10명. Inhibitory effect of Patrinia scabiosaefolia on acute pancreatitis. World Journal of Gastroenterology. Vol. 12, No. 7, 2006년, pp. 1110-1114 * |
송채석. Lipopolysaccharide에 의해 유발된 發熱에 있어서 敗醬의 解熱效果. 원광대학교 대학원 한의학과 박사학위논문, 2001년 * |
이준석 외 5명. 급성췌장염 유발된 흰쥐에 대한 패장의 억제 효과. 한방재활의학과학회지. Vol. 15, No. 1, 2005년, pp. 99-108 * |
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