KR101735784B1 - Composition for Anti-inflammation Using an Extract of Trigonostemon reidioides - Google Patents
Composition for Anti-inflammation Using an Extract of Trigonostemon reidioides Download PDFInfo
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- KR101735784B1 KR101735784B1 KR1020150084414A KR20150084414A KR101735784B1 KR 101735784 B1 KR101735784 B1 KR 101735784B1 KR 1020150084414 A KR1020150084414 A KR 1020150084414A KR 20150084414 A KR20150084414 A KR 20150084414A KR 101735784 B1 KR101735784 B1 KR 101735784B1
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Abstract
본 발명은 트리고노스테몬 레이디오이데스 추출물을 이용한 항염증용 조성물을 개시한다. 상기 트리고노스테몬 레이디오이데스 추출물은 LPS(lipopolyssacharide)로 자극된 마우스 대식세포주(RAW 264.7 cells)에서 NO 생성 억제 활성을 보이고, 염증성 사이토카인(TNF-α, IL-1β, IL-6)의 생성 억제 활성을 보인다.The present invention discloses a composition for antiinflammation using Trigonostemmonadioides extract. The extract of Trigonostemmonadioides showed NO production inhibitory activity in LPS (lipopolyssaccharide) stimulated mouse macrophage line (RAW 264.7 cells), and the production of inflammatory cytokines (TNF-α, IL-1β, IL-6) Inhibitory activity.
Description
본 발명은 트리고노스테몬 레이디오이데스(Trigonostemon reidioides) 추출물을 이용한 항염증용 조성물에 관한 것이다.
The present invention relates to the use of Trigonostemon reidioides extract of the present invention.
염증은 물리적인 외상, 유해한 화학물질, 박테리아, 곰팡이, 바이러스에 의한 감염이나 생체 내 대사산물 중의 자극성 물질에 의하여 야기되는 병리적 상태에 대응하여 나타나는 국소적인 생체의 방어 반응이다. 염증은 손상된 조직과 이동하는 세포(migrating cells)로부터 생산되는 다양한 염증 매개 인자에 의하여 촉발된다. 염증 반응 시에는 염증 부위에 혈장이 축적되어 세균이 분비한 독성을 희석시키며, 혈류가 증가하고, 홍반, 통증, 부종, 발열 등의 증상이 수반되게 된다. 정상적인 경우에 생체는 염증 반응을 통하여 발병 요인을 중화시키거나 제거하고 상한 조직을 재생시켜서 정상적인 구조와 기능을 회복시키지만, 그렇지 못한 경우에는 만성 염증과 같은 질병 상태로 진행되기도 한다. Inflammation is a local defense of the body that responds to pathological conditions caused by physical trauma, harmful chemicals, bacteria, fungi, viruses, or irritants in vivo metabolites. Inflammation is triggered by various inflammatory mediators produced from damaged tissues and migrating cells. During the inflammation reaction, plasma accumulates on the inflamed area, diluting the toxicities secreted by the bacteria, increasing the blood flow, accompanied by symptoms such as erythema, pain, edema and fever. In normal cases, the organism neutralizes or eliminates the cause of inflammation through the inflammation reaction, regenerates the upper tissue and regenerates the normal structure and function, but if not, the disease state such as chronic inflammation also proceeds.
최근 분자생물학의 발달로 분자적 수준에서 염증 반응에 대한 많은 연구가 이루어져 있다. Recent advances in molecular biology have resulted in a number of studies on the inflammatory response at the molecular level.
염증 반응에는 다양한 생화학적 현상이 관여하지만 특히 대식세포(macrophage)는 화학적 자극 등에 의하여 산화질소(NO)와 여러 염증성 사이토카인을 생성하여 염증반응에서 중요한 역할을 한다고 알려져 있다(Ito T., et al., Curr Drug Traget Inflamm Allergy, 2(3):257-265, 2003). 산화질소의 합성효소(nitric oxide synthase, NOS)는 L-아르기닌(L-arginine)으로부터 산화질소를 합성하는데, eNOS(내피성 NOS)와 nNOS(신경성 NOS) 및 iNOS(유도성 NOS) 세 가지 이성질체가 존재하며, 이 중 iNOS는 대식세포에서 TNF-α, IL-1β, IL-6, IL-8, IL-12 등의 사이토카인, LPS(lipopolysaccharide)와 같은 세균의 내독소 등에 의한 전사인자인 NF-κB의 활성화를 매개로 발현되어 과량의 산화질소(NO)를 생성한다. 대식세포에서 iNOS에 의해 과다 생성된 산화질소는 수퍼옥사이드(superoxide)와 반응하여 퍼옥시니트라이트(peroxynitrite)를 형성하고 이는 강력한 산화제로 작용하여 세포에 손상을 입힘으로써 염증과 암을 포함한 다양한 병리적 과정에 관여한다(Gupta SC et al., Exp Biol Med., 236:658-671, 2011; Riehemann et al., FEBS Lett., 442:89-94, 1999;Stamleret al., Science, 258:1898-1902, 1992). Although various biochemical phenomena are involved in the inflammatory reaction, macrophages are known to play an important role in the inflammatory reaction by producing NO (NO) and inflammatory cytokines by chemical stimulation (Ito T., et al Curr Drug Traget Inflamm Allergy, 2 (3): 257-265, 2003). Nitric oxide synthase (NOS) synthesizes nitric oxide from L-arginine. It is composed of three isomers: eNOS (endothelial NOS), nNOS (neurogenic NOS), and iNOS (inductive NOS) And iNOS is a transcription factor of macrophages such as cytokines such as TNF-α, IL-1β, IL-6, IL-8 and IL-12 and endotoxins such as LPS (lipopolysaccharide) It is expressed through activation of NF-κB and produces excess NO (NO). Nitric oxide, which is produced by iNOS in macrophages, reacts with superoxide to form peroxynitrite, which acts as a strong oxidizing agent and damages the cells, resulting in a variety of pathologies including inflammation and cancer (Gupta SC et al., Exp Biol Med., 236: 658-671, 2011; Riehemann et al., FEBS Lett., 442: 89-94, 1999; Stamler et al., Science, 258: 1898 -1902, 1992).
염증 반응의 유도물질들 중 하나인 LPS(lipopolysaccharide)는 그람 음성 세균의 외막(outer membrane)에 존재하는 엔도톡신(endotoxin)의 일종으로 TLR4(toll-like receptor 4)와 결합하여 전사인자인 NF-κB를 활성화시킴으로써 대식세포에서 iNOS의 발현을 촉발시켜 NO 생성을 유도할 뿐 아니라 TNF-α, IL-1β, IL-6 등의 염증성 사이토카인의 유전자를 활성화시켜 염증 반응을 유도한다(Dobrovolskaia MA, et al., J Immunol., 170:508-19, 2003; Ji Y, et al., Cell Physiol Biochem., 25:631-640, 2010). 또한 다른 연구에서도 TNF-α, IL-1β, IL-6 등의 염증성 사이토카인은 류마티스 관절염(Jang C. H. et al., Rheumatology, 2006, 45(6):703-710), 섬유근통(Hernandez M. E. et. al., BMC Res. Notes., 2010, 3(1):156), 쇼그렌 증후군(Baturone R. et. al., Scand J Rheumatol., 2009, 38(5):386-389) 등에서 염증 반응을 유도하는 중요한 인자로 보고되어 있다.Lipopolysaccharide (LPS), one of the inducers of inflammatory response, is a type of endotoxin present in the outer membrane of gram-negative bacteria and binds to TLR4 (toll-like receptor 4) Activates iNOS expression in macrophages to induce NO production, as well as to activate inflammatory cytokines such as TNF-α, IL-1β and IL-6 (Dobrovolskaia MA, et. al., J Immunol., 170: 508-19, 2003; Ji Y, et al., Cell Physiol Biochem., 25: 631-640, 2010). In other studies, inflammatory cytokines such as TNF-α, IL-1β and IL-6 have been reported to be associated with rheumatoid arthritis (Jang CH et al., Rheumatology, 2006, 45 (6): 703-710), fibromyalgia (Hernandez ME et. (BMC Res. Notes., 2010, 3 (1): 156) and Sjögren's syndrome (Baturone R. et al., Scand J Rheumatol., 2009, 38 (5): 386-389) Which is known to be an important factor for induction.
이러한 연구 결과는 NO 생성을 억제하거나 TNF-α, IL-1β, IL-6 등의 염증성 사이토카인의 생성을 억제하는 약물이 유효한 항염증제로서의 가능성을 시사한다(Karin M. et al., Cold Spring Harb Perspect Biol., 1, pp1-14, 2009).These findings suggest that inhibition of NO production Drugs that inhibit the production of inflammatory cytokines such as TNF-α, IL-1β, and IL-6 suggest the possibility of being an effective anti-inflammatory agent (Karin M. et al., Cold Spring Harb Perspect Biol., 1, , 2009).
현재 항염증제로서 널리 사용되고 있는 비스테로이드성 소염제(non-steroidal anti-inflammatory drugs, NSAIDS)는 위장관 장애, 간장애, 신장애 등의 심각한 부작용을 야기한다고 알려져 있다(Rainsford KD., Subcell biochem., 42:3-27, 2007; Guruprasad P. Aithal.,Rheumatology., 7:139-150, 2011; Praveen P. N. Rao et al.,Pharmaceuticals., 3:1530-1549, 2010).Non-steroidal anti-inflammatory drugs (NSAIDS), which are now widely used as anti-inflammatory drugs, are known to cause serious side effects such as gastrointestinal disorders, hepatic impairment, and renal failure (Rainsford KD., Subcell biochem., 42: 3 -27, 2007, Guruprasad P. Aithal., Rheumatology., 7: 139-150, 2011, Praveen PN Rao et al., Pharmaceuticals., 3: 1530-1549, 2010).
따라서 항염 활성을 가지면서 부작용이 적고 효과가 지속적인 새로운 약물의 개발이 여전히 필요하다고 할 수 있다. 특히 천연물은 독성이 적은 장점을 가진다.Therefore, it is still necessary to develop new drugs that have anti-inflammatory activity, have fewer side effects, and continue to be effective. Especially, natural products have the advantage of low toxicity.
트리고노스테몬 레이디오이데스(Trigonostemon reidioides)는 캄보디아, 베트남, 태국, 미얀마, 라오스 등이 원산지인 식물로 그 뿌리 추출물은 전통적으로 거담제(expectorant), 완하제(laxative) 등으로 사용되어 왔으며(Journal of Natural Products, 53(5):1148-1151, 1990), 이로부터 분리된 레디오사이드(rediocide) B 내지 E가 살충제의 후보 물질로서 제안된 바 있다(J Nat Prod. 2004 Feb;67(2):228-31). 그러나 트리고노스테몬 레이디오이데스의 항염 활성에 대해서는 현재까지 제안된 바 없다. Trigonostemon reidioides is a plant originating in Cambodia, Vietnam, Thailand, Myanmar and Laos. Root extracts have traditionally been used as expectorants and laxatives (Journal of Natural Products, 53 (5): 1148 -1151, 1990), and rediocides B to E isolated therefrom have been proposed as candidates for insecticides (J Nat Prod. 2004 Feb; 67 (2): 228-31). However, the anti-inflammatory activity of Trigonostemmonadioides has not been proposed to date.
본 발명은 트리고노스테몬 레이디오이데스 추출물의 항염 활성을 개시한다.
The present invention discloses the anti-inflammatory activity of Trigonostemmonadioides extract.
본 발명의 목적은 트리고노스테몬 레이디오이데스 추출물을 이용한 항염증용 조성물을 제공하는 데 있다.It is an object of the present invention to provide a composition for anti-inflammation using Trigonostemnemalidioides extract.
본 발명의 구체적인 목적은 트리고노스테몬 레이디오이데스 추출물을 이용한 항염증용 약제학적 조성물을 제공하는 데 있다.It is a specific object of the present invention to provide a pharmaceutical composition for anti-inflammation using Trigonostemmonadioides extract.
본 발명의 다른 구체적인 목적은 트리고노스테몬 레이디오이데스 추출물을 이용한 항염증용 식품 조성물을 제공하는 데 있다.Another specific object of the present invention is to provide an anti-inflammatory food composition using Trigonostemmonadioides extract.
본 발명의 또 다른 구체적인 목적은 트리고노스테몬 레이디오이데스 추출물을 이용한 항염증용 화장품 조성물을 제공하는 데 있다.
It is another specific object of the present invention to provide a cosmetic composition for anti-inflammation using Trigonostemmonadioides extract.
본 발명자들은 아래의 실시예 및 실험예에서 확인되는 바와 같이, 트리고노스테몬 레이디오이데스 추출물이 LPS(lipopolysaccharide)로 자극된 마우스 대식세포주(RAW 264.7 cells)에서 NO 생성 억제 활성을 보이고, 염증성 사이토카인(TNF-α, IL-1β 및 IL-6)의 생성 억제 활성을 보임을 확인할 수 있었다. As shown in the following Examples and Experimental Examples, the present inventors have found that the extract of Trigonostemmonoidesis shows NO production inhibitory activity in mouse macrophage cell line (RAW 264.7 cells) stimulated with LPS (lipopolysaccharide), and the inflammatory cytokine (TNF-α, IL-1β and IL-6).
전술한 바를 고려할 때, 본 발명은 트리고노스테몬 레이디오이데스 추출물을 유효성분으로 포함하는 항염증용 조성물로 파악할 수 있다.In view of the foregoing, the present invention can be understood as an anti-inflammatory composition comprising Trigonostemal radiodi extract as an active ingredient.
본 명세서에서, "트리고노스테몬 레이디오이데스 추출물"이란 추출 대상인 트리고노스테몬 레이디오이데스 줄기, 잎, 열매, 꽃, 뿌리 등을 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 분획물, 상기 조추출물을 실리카겔 등이 충진된 칼럼에 흡착시킨 후 소수성 용매, 친수성 용매 또는 이들의 혼합 용매를 이동상으로 하여 얻은 분획물을 포함하는 의미이다. 또한 상기 추출물의 의미에는 동결건조, 진공건조, 열풍건조, 분무건조 등의 방식으로 추출 용매가 제거된 농축된 액상의 추출물 또는 고형상의 추출물이 포함된다. 바람직하게는 추출용매로서 물, 에탄올 또는 이들의 혼합 용매를 사용하여 얻어진 추출물, 더 바람직하게는 추출용매로서 물과 에탄올의 혼합 용매를 사용하여 얻어진 추출물을 의미한다.In the present specification, the term " Trigonostomonadhiosidus extract "refers to an extract of Trigonostemmonadioides, a leaf, a fruit, a flower, a root and the like, which is a target to be extracted, with water, a lower alcohol having 1 to 4 carbon atoms such as methanol, ethanol, (DMF), dimethylsulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or a mixture thereof. The solvent is preferably selected from the group consisting of methylene chloride, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, Refers to an extract obtained by leaching using a mixed solvent, an extract obtained by using a supercritical extraction solvent such as carbon dioxide or pentane, or a fraction obtained by fractionating the extract, and the extraction method includes considering the polarity, extraction degree, and preservation degree of the active substance Any method such as cooling, reflux, heating, ultrasonic irradiation, supercritical extraction, and the like can be applied. In the case of the fractionated extract, the crude extract is suspended in a specific solvent, and the fractions obtained by mixing and leaving with a solvent having a different polarity are adsorbed on a column packed with silica gel or the like, and a hydrophobic solvent, a hydrophilic solvent or a mixture thereof Quot; means a fraction obtained by using a solvent as a mobile phase. Also, the meaning of the extract includes a concentrated liquid extract or a solid extract in which the extraction solvent is removed by a method such as freeze drying, vacuum drying, hot air drying, spray drying and the like. Preferably an extract obtained by using water, ethanol or a mixed solvent thereof as an extraction solvent, and more preferably an extract obtained by using a mixed solvent of water and ethanol as an extraction solvent.
또 본 명세서에서 "유효성분"이란 단독으로 목적하는 활성을 나타내거나 또는 그 자체는 활성이 없는 담체와 함께 활성을 나타낼 수 있는 성분을 의미한다.In the present specification, the term " active ingredient "alone means an ingredient which exhibits the desired activity or which can exhibit activity together with a carrier which is itself inactive.
또 본 명세서에서, "항염증"은 아래에서 정의되는 염증성 질환의 개선(증상의 경감), 치료, 그러한 질환의 발병 억제 또는 지연을 포함하는 의미이다.As used herein, "anti-inflammatory" is meant to include improvement of an inflammatory disease (alleviation of symptoms), treatment, inhibition or delay of onset of such a 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 감염 또는 자가면역에 대한 국부적 또는 전신적 생체 방어 반응으로 특정되는 염증 반응이 일으키는 병리적 증상으로서 정의될 수 있다. 이러한 염증 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-1β의 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, 편두통, 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, 류마티스성 관절염, 강직성 척추염, 호지킨병, 췌장염, 결막염, 홍채염, 공막염, 포도막염, 피부염(아토피성 피부염 포함), 습진, 다발성 경화증 등이 포함될 것이다. In the present specification, the term "inflammatory disease" means an inflammatory reaction that is determined by an external physical or chemical stimulus or an infection of an external infectious source such as bacteria, fungi, viruses, various allergenic substances, or a local or systemic defense against autoimmunity Which can be defined as a pathological symptom. Such an inflammatory reaction may include activation of various inflammatory mediators and enzymes associated with immune cells (e.g., iNOS, COX-2, etc.), secretion of inflammatory mediators (e.g., secretion of NO, TNF-a, IL-6, IL- It involves a series of complex physiological responses such as fluid infiltration, cell migration, and tissue destruction, and is manifested externally by symptoms such as erythema, pain, edema, fever, loss or loss of specific function of the body. The inflammatory disease may be acute, chronic, ulcerative, allergic or necrotic, so long as it is included in the definition of inflammatory diseases as above, it may be acute, chronic, ulcerative, allergic, 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, Inflammatory bowel syndrome, inflammatory pain, migraine headache, headache, back pain, fibromyalgia, fascia disease, viral infection (e.g., C type infection), bacterial infection, fungal infection, burn, wound due to surgical or dental surgery, Rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, iritis, scleritis, uveitis, dermatitis (including atopic dermatitis), eczema, multiple sclerosis, etc. Will be included.
본 발명의 항염증용 조성물은 그 유효성분을 용도, 제형, 배합 목적 등에 따라 치료를 의도하는 염증성 질환의 개선 활성 등을 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 그러한 병리적 증상의 발병 억제/지연을 유도할 수 있는 유효성분의 양을 말한다. 이러한 유효량은 당업자의 통상의 능력 범위 내에서 실험적으로 결정될 수 있다.The anti-inflammatory 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 which is intended to be treated according to the purpose of use, formulation, blending purpose, etc., Will be determined within the range of 0.001 wt% to 15 wt% based on the total weight of the composition. As used herein, the term "effective amount" refers to the amount of active ingredient capable of inducing the improvement, treatment, or inhibition / delay of onset of an inflammatory disease or the onset of such pathological condition 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 항염증용 조성물은 구체적인 양태에 있어서, 식품 조성물로 파악할 수 있다.The composition for anti-inflammation of the present invention can be identified as a food composition in a specific embodiment.
본 발명의 식품 조성은 기능성 음료 등의 건강 기능 식품, 건강 보조 식품, 특수 영양 보충용 식품으로 제조될 수 있으며, 식품의 형태는 차, 쥬스, 탄산음료, 이온음료 등의 음료류, 우유, 요구루트 등의 가공 유류, 껌류, 떡, 한과, 빵, 과자, 면 등의 식품류, 산제, 정제, 캡슐제 등의 건강 기능 식품 제제류 등이 될 수 있다.The food composition of the present invention can be manufactured as health functional foods such as functional beverages, health supplements, special nutrition supplements, etc. The forms of foods include beverages such as tea, juice, carbonated beverage, ionic drink, milk, Food products such as processed oil, gum, rice cake, Korean lamb, bread, confectionery, cotton, etc., powders of health functional foods such as powders, tablets and capsules.
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.
생리활성 물질로서는 카테킨, 에피카테킨, 갈로카테킨, 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 본 발명의 식품 조성물은 향미나 기호성을 향상시키고 여타의 기능성을 부가하기 위하여 천연물 유래의 분말 또는 추출물을 포함할 수 있는데, 선지 분말 또는 추출물, 콩나물 분말 또는 추출물, 조개 분말 또는 추출물, 굴 분말 또는 추출물, 산미나리 분말 또는 추출물, 무 즙 또는 추출물, 오이 즙 또는 추출물, 부추 즙 또는 추출물, 시금치 즙 또는 추출물, 연근 즙 또는 추출물, 칡 즙 또는 추출물, 솔잎 즙 또는 추출물, 인삼 즙 또는 추출물, 백화사설초 분말 또는 추출물, 감초 분말 또는 추출물, 갈화 분말 또는 추출물, 갈근 분말 또는 추출물, 사인 분말 또는 추출물, 박 분말 또는 추출물, 생강 분말 또는 추출물, 대추 분말 또는 추출물, 인진 분말 또는 추출물, 지구자 분말 또는 추출물, 수비계 분말 또는 추출물, 백출 분말 또는 추출물, 저령 분말 또는 추출물, 진피(진귤의 껍질) 분말 또는 추출물, 구기자 분말 또는 추출물, 녹차 분말 또는 추출물, 오미자 분말 또는 추출물, 헛개나무 분말 또는 추출물, 지치 분말 또는 추출물, 노근 분말 또는 추출물, 계피 분말 또는 추출물, 데커시놀 등이 예시될 수 있다. 이러한 추출물은 추출 대상을 혼합하여 얻어질 수도 있다.In addition, the food composition of the present invention may contain a powder or extract derived from a natural product in order to improve flavor or palatability and to impart other functionalities, and may be a pregelatinized powder or extract, a soybean powder or extract, a shell powder or extract, Extracts, juice or extracts of ginseng, extracts of ginseng, extracts of ginseng, extracts of ginseng, extracts of ginseng, extracts of ginseng, extracts of ginseng, extracts of ginseng juice or extracts, extracts of ginseng juice or extracts, extracts of ginseng juice or extracts, extracts of spinach juice or extracts, lotus juice or extracts, Powder or extract, licorice powder or extract, granular powder or extract, granular powder or extract, sine powder or extract, pistachio powder or extract, ginger powder or extract, jujube powder or extract, Water-based powder or extract, dried powder or extract, Or extract, dermis (crustacea bark) powder or extract, ginger powder or extract, green tea powder or extract, omija powder or extract, hinoki powder or extract, dirt powder or extract, egg yolk powder or extract, cinnamon powder or extract, Cyano and the like can be exemplified. Such an extract may be obtained by mixing the object to be extracted.
본 발명의 항염증용 조성물은 다른 구체적인 양태에 있어서, 화장료 조성물로 파악할 수 있다.In another specific embodiment, the composition for anti-inflammation of the present invention can be identified as a cosmetic composition.
본 발명의 화장료 조성물은 그 유효성분 이외에 화장료 조성물에 통상적으로 이용되는 성분들, 예컨대, 안정화제, 용해화제, 계면활성제, 비타민, 색소 및 항료와 같은 통상적인 보조제 및 담체를 포함할 수 있다. The cosmetic composition of the present invention may contain, in addition to its active ingredient, conventional additives and carriers such as stabilizers, solubilizers, surfactants, vitamins, pigments and antioxidants, which are commonly used in cosmetic compositions.
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 클렌징, 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 can be prepared into any of the formulations conventionally produced in the art and can be used in the form of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a 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, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .
본 발명의 제형이 파우더 또는 스프레이인 경우에는 담체 성분으로서 락토오스, 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 a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in the case of spray, additionally chlorofluorohydrocarbons, propane / Propane 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 an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.
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.In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.
본 발명의 제형이 계면-활성제 함유 클렌징인 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, 식물성유, 라놀린 유도체 또는 에톡실화 글리세롤 지방산 에스테르 등이 이용될 수 있다.When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component is selected from aliphatic alcohol sulfates, aliphatic alcohol ether sulfates, sulfosuccinic acid monoesters, isethionates, imidazolinium derivatives, methyltaurate, sarcosinate, fatty acid amides Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.
본 발명의 화장료 조성물은 항염증 활성을 나타내는 그 유효성분을 포함하는 것을 제외하고는 당업계에 통상적으로 행하여지는 화장료 조성물의 제조방법에 따라 제조할 수 있다.The cosmetic composition of the present invention can be produced according to a method for producing a cosmetic composition which is conventionally performed in the art, except that it contains an active ingredient exhibiting anti-inflammatory activity.
본 발명의 항염증용 조성물은 다른 구체적인 양태에 있어서 약제학적 조성물로 파악될 수 있다.The anti-inflammatory composition of the present invention can be identified as a pharmaceutical composition in another specific embodiment.
본 발명의 약제학적 조성물은 유효성분 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 a form suitable for oral use (tablets, suspensions, granules, emulsions, capsules, syrups, etc.), parenteral formulations (sterile injectable aqueous or non- Ointments, ointments, solutions, creams, ointments, gels, lotions, patches, and the like).
상기에서 "약제학적으로 허용되는" 의미는 유효성분의 활성을 억제하지 않으면서 적용(처방) 대상이 적응 가능한 이상의 독성을 지니지 않는다는 의미이다.The term "pharmaceutically acceptable" as used herein means that the application (subject) does not have the above-mentioned toxicity that is adaptable without inhibiting the activity of the active ingredient.
약제학적으로 허용되는 담체의 예로서는 락토오스, 글루코오스, 슈크로오스, 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 등을 들 수 있으며 이러한 담체는 본 발명의 약제학적 조성물의 제형에 따라 적당한 것을 하나 이상 선택하여 사용할 수 있다. 약제학적으로 허용되는 적합한 담체와 제제에 대해서는 문헌[Remington's Pharmaceutical Sciences(19th ed., 1995)]을 참조할 수 있다. 본 발명의 약제학적 조성물은 유화제(예컨대 TWEENS), 습윤제(예컨대 라우릴 황산 나트륨), 착색제, 풍미제, 안정화제, 보존제, 물, 식염수, 인산염 완충 용액 등을 추가로 포함할 수 있다. Examples of the pharmaceutically acceptable carrier include lactose, glucose, sucrose, starch (e.g., corn starch, potato starch, etc.), cellulose, derivatives thereof (e.g., sodium carboxymethyl cellulose, ethylcellulose), malt, gelatin, talc, (E.g., peanut oil, cottonseed oil, sesame oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin and the like), alginic acid, and the like. Depending on the formulation of the pharmaceutical composition of the present invention, one or more suitable ones may be selected and used. For suitable pharmaceutically acceptable carriers and formulations see Remington ' s Pharmaceutical Sciences (19th ed., 1995). The pharmaceutical composition of the present invention may further comprise an emulsifier (e.g., TWEENS), a wetting agent (e.g., sodium lauryl sulfate), a coloring agent, a flavoring agent, a stabilizer, a preservative, water, saline, a phosphate buffer solution and the like.
부형제도 본 발명의 약제학적 조성물의 제형에 따라 적합한 것을 선택하여 사용할 수 있는데, 예컨대 나트륨 카르복시메틸 셀룰로오스, 메틸 셀룰로오스, 히드로프로필메틸셀룰로오스, 알긴산 나트륨, 폴리비닐피롤리돈 등의 현탁제나 분산제 등을 들 수 있다. The excipient may be selected according to the formulation of the pharmaceutical composition of the present invention. For example, suspending agents such as sodium carboxymethyl cellulose, methyl cellulose, hydropropyl methyl cellulose, sodium alginate, polyvinyl pyrrolidone, .
본 발명의 약제학적 조성물은 경구 또는 비경구로 투여될 수 있으며, 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 pharmaceutical composition of the present invention can be administered orally or parenterally, and its daily dose is usually in the range of 0.001 to 150 mg / kg body weight, and can 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 It should not be understood.
전술한 바와 같이, 본 발명에 따르면 트리고노스테몬 레이디오이데스 추출물을 이용한 항염증용 조성물을 제공할 수 있다. As described above, according to the present invention, it is possible to provide an anti-inflammatory composition using Trigonostemmonadioides extract.
본 발명의 항염증용 조성물은 염증성 질환의 개선 등의 용도로 식품, 화장품, 약품 등으로 제품화될 수 있다.
The anti-inflammatory composition of the present invention can be commercialized into foods, cosmetics, medicines, etc. for the purpose of improving inflammatory diseases and the like.
도 1은 트리고노스테몬 레이디오이데스 추출물의 LPS로 자극된 마우스 대식세포주(RAW 264.7 cells)에서의 NO 생성 억제 활성을 보여주는 결과이다.
도 2는 트리고노스테몬 레이디오이데스 추출물의 LPS로 자극된 마우스 대식세포주(RAW 264.7 cells)에서의 세포독성 결과를 보여주는 결과이다.
도 3은 트리고노스테몬 레이디오이데스 추출물의 LPS로 자극된 마우스 대식세포주(RAW 264.7 cells)에서의 염증성 사이토카인(TNF-α, IL-1β 및 IL-6)의 생성 억제 활성을 보여주는 결과이다.Fig. 1 shows the results of the inhibition of NO production in LPS-stimulated mouse macrophage line (RAW 264.7 cells) of Trigonostomonadhiides extract.
Figure 2 shows the results of cytotoxicity of LPS-stimulated mouse macrophage cell line (RAW 264.7 cells) of Trigonostomene radiosides extract.
FIG. 3 shows the results showing the activity of inhibiting the production of inflammatory cytokines (TNF-α, IL-1β and IL-6) in LPS-stimulated mouse macrophage cell line (RAW 264.7 cells) of Trigonostomonadhiides extract.
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 > > 트리고노스테몬Trigo Nostemona 레이디오이데스Lady O'Deis 추출물의 제조 Preparation of extract
트리고노스테몬 레이디오이데스(Trigonostemon reidioides)의 잎 분쇄물 100 g에 70% 에탄올 1 L를 가하여 상온에서 24시간 1회 반복추출 후 여과지로 여과하였다. 얻어진 70% 에탄올 여액을 가압 농축 후 동결 건조하여 트리고노스테몬 레이디오이데스 추출물을 얻었다.
1 L of 70% ethanol was added to 100 g of leaf ground powder of Trigonostemon reidioides , and the mixture was repeatedly extracted once at room temperature for 24 hours and then filtered with a filter paper. The resulting 70% ethanol filtrate was concentrated under pressure, and then lyophilized to obtain a trinhosynthomonadioides extract.
<< 실험예Experimental Example > > 트리고노스테몬Trigo Nostemona 레이디오이데스Lady O'Deis 추출물의 항염증 활성 실험 Anti-inflammatory activity of extracts
<실험예 1> NO assay 및 세포독성 평가( MTT assay) Experimental Example 1 NO assay and cytotoxicity assay ( MTT assay)
마우스 대식세포인 RAW 264.7 cells를 10 % FBS DMEM 배지를 넣고 37℃, 5% CO2의 조건에서 배양하였다. RAW 264.7 대식세포를 96 well plate에 5×104cells/well의 세포 수로 분주하고 24시간 후 실시예의 시료를 농도별로 37℃, 5% CO2의 조건에서 1시간 우선 처리한 뒤, LPS(lipopolyssacharide)를 처리하여 24시간 동안 배양하였다. Mouse macrophages RAW 264.7 cells were cultured in a 10% FBS DMEM medium at 37 ° C and 5% CO 2 . RAW 264.7 macrophages were seeded at a density of 5 × 10 4 cells / well in a 96-well plate. After 24 hours, the samples of the present invention were first treated for 1 hour at 37 ° C. and 5% CO 2 concentration and then treated with lipopolyssaccharide ) And cultured for 24 hours.
삭제delete
- Nitric Oxide (NO) 측정방법 : 1M NaNO2(sodium nitrite)를 표준곡선으로 0, 1.56uM, 3.12uM, 6.25uM, 12.5uM, 25uM, 50uM, 100uM 이 되도록 희석하여 기준값을 잡고 1% sulfanilamide와 0.1% N-(1-naphtyl) ethylenediamine dihydrochloride reagent를 1:1로 섞은 후, 1:1로 섞은 reagent와 시료 처리 후 24시간 배양된 배지 상등액을 1:1로 섞어주고 10분간 상온에 보관한 후 540nm에서 흡광도를 측정하여 대조군 대비 NO 생성 억제능을 측정하였다. 양성대조군으로는 N-monomethyl-L-arginine(L-NMMA)를 사용하였다.- Nitric Oxide (NO) method: 1M NaNO 2 (sodium nitrite) was diluted to 0, 1.56uM, 3.12uM, 6.25uM, 12.5uM, 25uM, 50uM, and 100uM using standard curves. After 1: 1 mixture of 0.1% N- (1-naphtyl) ethylenediamine dihydrochloride reagent and 1: 1 mixture of 1: 1 reagent and culture supernatant for 24 hours after sample treatment, the mixture was kept at room temperature for 10 minutes Absorbance was measured at 540 nm and the inhibitory effect of NO production on the control group was measured. N-monomethyl-L-arginine (L-NMMA) was used as a positive control.
- 세포독성 측정 밥법 (MTT assay) : 5mg/ml MTT solution을 배지에 100 ul/well로 넣고 37℃, 5% CO2 incubator에 넣어 2시간 배양 후 배지를 제거하고 DMSO를 100 ul/well로 넣었다. Shaker에서 15분간 용해시키고, ELISA reader를 이용해 540 nm에서 흡광도를 측정하였다. - Cytotoxicity assay (MTT assay): 5 μl of MTT solution was added to the medium at 100 μl / well and incubated at 37 ° C in a 5% CO 2 incubator for 2 hours. The medium was removed and DMSO was added at 100 μl / well . Shaker for 15 minutes and absorbance was measured at 540 nm using an ELISA reader.
NO 생성 억제 결과를 [도 1]에 나타내었고, MTT assay 결과를 [도 2]에 나타내었다.The NO production inhibition results are shown in Fig. 1, and the MTT assay results are shown in Fig.
실시예의 시료(트리고노스테몬 레이디오이데스 추출물, NIBR No. 87)는 농도 의존적으로 NO 생성 억제 활성을 보였으며, 세포독성은 거의 나타내지 않았다.The sample of the Example (Trigonostemmonadioides extract, NIBR No. 87) exhibited a concentration-dependent inhibition of NO production and showed almost no cytotoxicity.
<실험예 2> 염증성 사이토카인의 생성 억제 활성 실험( RT - PCR ) Experimental Example 2: Inhibitory activity against the production of inflammatory cytokines ( RT - PCR )
마우스 대식세포인 RAW 264.7 cells를 60 mm plate에 1.6×106cell/well의 세포 수로 분주하고 37℃, 5% CO2 조건에서 24시간 동안 배양한다. 실시예의 시료(NIBR No. 87)를 농도별로 1시간 동안 우선 처리하고 LPS (lipopolyssacharide)를 처리하여 24시간 후, PBS(phosphate buffered saline)로 세척하고 Trizol과 chloroform 을 처리하여 RNA를 분리하였다. 분리된 RNA를 이용하여 정량 후 역전사 효소인 SuperscriptⅢ(Invitrogen)를 사용하여 역전사 연쇄중합반응을 통해 cDNA를 합성하였다. 합성된 cDNA를 주형으로 하여 중합효소 연쇄반응을 통해 94℃ 45초, 55℃ 45초, 72℃ 1분 30초를 설정하여 35 cycle의 PCR 조건으로 DNA를 증폭시켰다. 유전자의 발현 억제 효과를 agarose gel 상에서 비교하였다. 사용한 primer는 아래의 [표 1]과 같고 대조군 유전자는 GAPDH를 사용하였다. 양성 대조군으로는 dexamethasone을 사용하였다.Mouse macrophages RAW 264.7 cells are seeded on a 60 mm plate at a cell number of 1.6 × 10 6 cells / well and incubated for 24 hours at 37 ° C and 5% CO 2 . The sample of the example (NIBR No. 87) was pretreated for 1 hour by concentration, treated with LPS (lipopolysaccharide), washed with PBS (phosphate buffered saline) for 24 hours, and treated with Trizol and chloroform to isolate RNA. CDNA was synthesized by reverse transcription polymerase chain reaction using Superscript III (Invitrogen), a reverse transcriptase. Using the synthesized cDNA as a template, DNA was amplified by 35 cycles of PCR under the conditions of 94 ° C. for 45 seconds, 55 ° C. for 45 seconds, and 72 ° C. for 1 minute and 30 seconds through a polymerase chain reaction. The gene expression inhibitory effect was compared on agarose gel. The primers used were shown in Table 1 below and the control gene was GAPDH. As a positive control, dexamethasone was used.
결과를 [도 3]에 나타내었다. [도 3]을 참조하여 보면 실시예의 시료(NIBR No. 87)는 농도 의존적으로 염증성 사이토카인(TNF-α, IL-1β, IL-6)의 생성을 억제한다는 것을 알 수 있다.The results are shown in Fig. 3, it can be seen that the sample of the example (NIBR No. 87) inhibits the production of inflammatory cytokines (TNF-α, IL-1β, IL-6) in a concentration-dependent manner.
Claims (6)
상기 트리고노스테몬 레이디오이데스 잎 추출물은 트리고노스테몬 레이디오이데스 잎을 물과 에탄올의 혼합용매로 추출하여 얻어진 것을 특징으로 하는
항염증용 조성물.
Trigonosynthostatic extracts containing an extract of Leiden oides as an active ingredient,
Wherein the Trigonostemmoniaeidis leaf extract is obtained by extracting Trigonostemmonadioides leaf with a mixed solvent of water and ethanol
Composition for antiinflammation.
상기 조성물은 약제학적 조성물인 것을 특징으로 하는 항염증용 조성물.
The method according to claim 1,
Wherein the composition is a pharmaceutical composition.
상기 조성물은 식품 조성물인 것을 특징으로 하는 항염증용 조성물.
The method according to claim 1,
Wherein the composition is a food composition.
상기 조성물은 화장료 조성물인 것을 특징으로 하는 항염증용 조성물.
The method according to claim 1,
Wherein the composition is a cosmetic composition.
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