KR101273953B1 - Novel compound of Sterocalpin A for Prevention and Treatment of Atherosclerosis - Google Patents
Novel compound of Sterocalpin A for Prevention and Treatment of Atherosclerosis Download PDFInfo
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- KR101273953B1 KR101273953B1 KR1020110108222A KR20110108222A KR101273953B1 KR 101273953 B1 KR101273953 B1 KR 101273953B1 KR 1020110108222 A KR1020110108222 A KR 1020110108222A KR 20110108222 A KR20110108222 A KR 20110108222A KR 101273953 B1 KR101273953 B1 KR 101273953B1
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- stereocalpin
- tnf
- cells
- atherosclerosis
- expression
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Abstract
본 발명은 스테레오칼핀 A(stereocalpin A)를 함유하는 동맥경화증 예방 또는 치료용 약학 조성물에 관한 것이다.
본 발명에 따른 스테레오칼핀 A(stereocalpin A)가 함유된 조성물은 TNF-α에 의해 유도되는 세포부착분자의 발현을 억제시킴으로써, 동맥경화증 치료 및 예방에 유용하게 사용할 수 있다. The present invention relates to a pharmaceutical composition for preventing or treating atherosclerosis containing stereocalpin A.
The composition containing stereocalpin A according to the present invention can be usefully used for the treatment and prevention of atherosclerosis by inhibiting the expression of cell adhesion molecules induced by TNF-α.
Description
본 발명은 스테레오칼핀 A를 함유하는 동맥경화증 예방 또는 치료용 조성물에 관한 것이다. The present invention relates to a composition for preventing or treating atherosclerosis containing stereocalpin A.
죽상동맥경화증(atherosclerosis)은 지방을 포함한 끈적끈적한 액체가 점차 동맥 내벽 표면에 축적되어 조직으로 도달하는 혈류를 막아 조직의 괴사를 일으키는 혈관질환으로 그 조직이 뇌나 심근일 경우 뇌졸증 및 심장마비를 유발하게 된다. 이러한 동맥경화증은 어떤 특정한 원인에 의해 발병되는 단일 질환이 아니라, 연령, 성별, 유전적 환경, 스트레스, 비만, 흡연, 음주, 불포화 지방의 섭취, 당뇨병, 다이어트 등의 다양한 원인들이 복합적으로 작용하여 일어나는 질환이다. Atherosclerosis is a vascular disease that causes sticky fluids, including fat, to accumulate on the inner wall of the arteries, blocking blood flow to the tissues, leading to necrosis of the tissues, which can lead to stroke and heart failure if the tissues are brain or myocardium. do. Arteriosclerosis is not a single disease caused by a specific cause, but is caused by a combination of various causes such as age, sex, genetic environment, stress, obesity, smoking, drinking, unsaturated fat intake, diabetes, and diet. Disease.
죽상동맥경화증의 시작은, 동맥의 혈관벽 내의 지질과 섬유요소의 축적이 특징인 염증 반응으로 뚜렷해지는 것이 특징이고, 이것은 심혈관 질병의 주요 원인이 된다. 이에, 최근들어 동맥경화는 일종의 염증성 질환으로 여겨지고 있다. 동맥경화증에서 가장 먼저 관찰되는 반응은 동맥의 내막에 존재하는 내피세포(endothelial cell)의 손상으로 인한 기능이상이다. 내피세포의 기능 이상은, 주위 자극에 대하여 가역적인 변화를 나타내는 것을 말하며 내피세포에 혈중 단핵세포(monocyte)와 혈소판의 부착이 증가하는 현상으로 나타난다. 내피세포의 손상을 일으키는 유발 물질로는 각종 사이토카인(cytokine), 박테리아 생성물, 바이러스 보체, 산소결핍 등이 알려져 있다. The onset of atherosclerosis is characterized by an inflammatory response characterized by the accumulation of lipids and fibrous elements in the vessel walls of the arteries, which is a major cause of cardiovascular disease. Recently, arteriosclerosis is considered to be a kind of inflammatory disease. The first response observed in atherosclerosis is dysfunction due to damage to endothelial cells in the lining of the arteries. Endothelial dysfunction refers to a reversible change in peripheral stimuli, which results in an increase in adhesion of blood monocytes and platelets to endothelial cells. As a cause of endothelial cell damage, various cytokines (cytokine), bacterial products, virus complement, oxygen deficiency and the like are known.
손상된 내피세포에서 발현되는 세포부착분자(cell adhesion molecule)인 세포간 부착분자-1(intracellular adhesion molecule-1; 이하, ICAM-1이라 함), 혈관 세포부착분자-1(vascular cell adhesion molecule-1; 이하 VCAM-1이라 함), E-selectin, P-selectin 등은 단핵세포의 표면에 부착되어 수용체로서의 역할을 하게 된다. 이후 세포부착분자와 결합한 단핵세포는 내피세포 사이를 투과해 동맥의 내막으로 이동하여 대식세포로 분화한다. 이 과정에 내피세포와 대식세포로부터 분비되는 M-CSF(macrophage colony stimulation factor)가 관여하며, 분화한 대식세포는 TNF-α(tumor necrosis factor-α) 등의 사이토카인이 병의 진행에 중요한 역할을 하는 것으로 보고되었다(Peter Libby, Nature, 420, 6917:868-874, 2002).Intracellular adhesion molecule-1 (hereinafter referred to as ICAM-1) and vascular cell adhesion molecule-1 (cell adhesion molecule-1) expressed in damaged endothelial cells (Hereinafter referred to as VCAM-1), E-selectin, P-selectin, etc. are attached to the surface of monocytes to act as a receptor. Monocytes that bind to the cell adhesion molecules then penetrate between the endothelial cells, migrate to the lining of the arteries, and differentiate into macrophages. In this process, macrophage colony stimulation factor (M-CSF) secreted from endothelial cells and macrophages is involved. Differentiated macrophages, such as TNF-α (tumor necrosis factor-α), play an important role in disease progression. (Peter Libby, Nature , 420, 6917: 868-874, 2002).
TNF-α(tumor necrosis factor-α)는 면역계에서 염증을 일으키는 주요 사이토카인으로 면역세포들에 의해 분비되며, 특히 자극받은 대식세포에서 다량이 분비된다. 또한, 이 사이토카인은 세포를 자극할 경우, 세포사멸을 일으키는 신호와 NF-κB의 활성을 통해서 세포 사멸을 억제하는 두 가지 모순적인 신호를 보내게 된다. 동맥경화에 있어서는 신생혈관의 생성, 혈전생성의 증가와 출혈성 괴사 등을 유발하여 죽상경화의 진행에 관여하며, 심근의 염증반응도 유도한다. Tumor necrosis factor-α (TNF-α) is a major cytokine that causes inflammation in the immune system and is secreted by immune cells, especially in stimulated macrophages. In addition, when cytokines stimulate cells, they send two contradictory signals that cause cell death and inhibit cell death through NF-κB activity. In atherosclerosis, the formation of neovascularization, increased thrombus formation and hemorrhagic necrosis are involved in the progression of atherosclerosis, and also induces inflammatory reactions of myocardium.
또한, 세포부착분자는 고혈압과 동맥경화와 같은 다양한 혈관 질환에 관여하며, 세포의 표면에 발현하여 다른 세포나 세포외기질에 부착시키는 역할을 한다. 그 중 ICAM-1과 VCAM-1은 immunoglobulin gene superfamily에 속하는 막투과성 당단백질로, 급성 또는 만성 염증상태나 질병의 유발과정에서 TNF-α 등과 같은 염증성 사이토카인에 의해 조절되며, 초기 동맥경화가 유발된 곳의 내피세포에서 발현이 증가되는 반면, 동맥경화가 유발되지 않은 부위에서는 관찰되지 않기 때문에 동맥 경화에 있어서 작용을 한다고 알려져 있다(Sharon J. Hyduk et al ., Progress in Flammation Reserah, Part 2, 141-174, 2007). 또한 ICAM-1은 leukocytes에서 발현되는 LFA-1과 같은 integrin에 결합하고, VCAM-1은 mononuclear leukocytes, eosinophils and basophils에서 발현되는 integrin VLA4와 결합하므로, 혈관벽 내로 유입된 leukocytes의 축적을 용이하게 한다. 즉, 동맥경화에 있어서, ICAM-1 및 VCAM-1은 염증성 질환을 유발시키는 주요 인자로 볼 수 있다. 이에, 상기 ICAM-1, VCAM-1 등의 세포부착분자의 발현을 억제하면 혈관 질병을 예방 또는 치료할 수 있다. In addition, cell adhesion molecules are involved in various vascular diseases such as hypertension and arteriosclerosis, and are expressed on the surface of cells to attach to other cells or extracellular matrix. Among them, ICAM-1 and VCAM-1 are transmembrane glycoproteins belonging to the immunoglobulin gene superfamily, which are regulated by inflammatory cytokines such as TNF-α during the acute or chronic inflammatory state or disease-causing process. It is known to act on atherosclerosis because of increased expression in the endothelial cells of the injured area, but not at the site where atherosclerosis is not induced (Sharon J. Hyduk et. al . , Progress in Flammation Reserah , Part 2, 141-174, 2007). In addition, ICAM-1 binds to integrins such as LFA-1 expressed in leukocytes and VCAM-1 binds to integrin VLA4 expressed in mononuclear leukocytes, eosinophils and basophils, thus facilitating the accumulation of leukocytes introduced into the vessel wall. In other words, in atherosclerosis, ICAM-1 and VCAM-1 may be regarded as major factors causing inflammatory diseases. Thus, by inhibiting the expression of cell adhesion molecules, such as ICAM-1, VCAM-1, can prevent or treat vascular diseases.
한편, 최근 들어 남극 고유생물 유래 천연물을 활용하기 위한 보고가 증가하고 있다. 남극 지역에서는 지의류가 다양하게 서식하고 있다(Smith RI. Terrestrial plant biology of the sub-Antarctic and Antarctic. In Antarctic Ecology, Laws RM eds. (London Academic Press) 1984 161-162). 지의류는, 종속영양생물인 진균류와 남조류 또는 조류 간의 공생 관계에 의하여 형성된 생물로서, 이들의 천연 산물은 화장품, 식품 또는 천연 치료약으로 사용된 바 있다 (Choudhary MI, Azizuddin, Jalil S, Atta-ur-Rahman. Bioactive phenolic compounds from a medicinal lichen, Usnea longissima. Phytochemistry 200566:2346-2350, MK. Pharmaceutically relevant metabolites from lichens. Appl Microbiol Biotechnol 200156:9-16). 그러나, 지의류 유래 천연물의 산물의 구체적인 산업적 적합성 (industrial screening), 이들 산물에 대한 치료적 잠재력은 아직 미약하다.On the other hand, in recent years, reports for utilizing natural products derived from Antarctic endemic organisms are increasing. In the Antarctic lichens and diverse habitat (Smith RI. Terrestrial plant biology of the sub-Antarctic and Antarctic. In Antarctic Ecology , Laws RM eds. (London Academic Press) 1984 161-162). Lichens are organisms formed by the symbiotic relationship between heterotrophic fungi and cyanobacteria or algae, and their natural products have been used as cosmetics, foods or natural remedies (Choudhary MI, Azizuddin, Jalil S, Atta-ur- Rahman.Bioactive phenolic compounds from a medicinal lichen, Usnea longissima.Phytochemistry 200566: 2346-2350, MK.Pharmaceutically relevant metabolites from lichens.Appl Microbiol Biotechnol 200156: 9-16). However, the specific industrial screening of products of lichen-derived natural products, and the therapeutic potential for these products is still weak.
이에, 본 발명자들은 동맥경화증 치료에 효과적인 기작인 TNF-α를 유도하는 세포부착분자의 발현을 억제하는 물질에 대하여 탐색하던 중, 남극의 지의류인 라말리나 테라브라타 (Ramalina terebrata)의 추출물로부터 분리된 스테레오칼핀 A가 TNF-α에 의해 유도되는 세포부착분자의 발현을 억제시킨다는 것을 확인하고, 본 발명을 완성하게 되었다.Therefore, the present inventors, while searching for a substance that suppresses the expression of TNF-α-induced cell adhesion molecules, an effective mechanism for treating atherosclerosis, isolated from the extract of Ramalina terebrata, an antarctic lichen The present invention was completed by confirming that stereocalpin A thus inhibited expression of cell adhesion molecules induced by TNF-α.
본 발명의 목적은 스테레오칼핀 A를 유효성분으로 함유하는 동맥경화증 예방 또는 치료용 약학 조성물을 제공하는데 있다.An object of the present invention to provide a pharmaceutical composition for preventing or treating atherosclerosis containing stereocalpine A as an active ingredient.
본 발명의 다른 목적은 스테레오칼핀 A를 함유하는 동맥경화 예방 또는 개선용 기능성 건강식품을 제공하는데 있다.Another object of the present invention to provide a functional health food for preventing or improving atherosclerosis containing stereocalpin A.
상기와 같은 목적을 달성하기 위하여, 본 발명은 다음 화학식 I로 표시되는 스테레오칼핀 A(stereocalpin A)를 유효성분으로 함유하는 동맥경화증 예방 또는 치료용 약학 조성물을 제공한다.In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating atherosclerosis containing stereocalpin A (stereocalpin A) represented by the following formula (I) as an active ingredient.
[화학식 I](I)
본 발명은 또한, 본 발명은 다음 화학식 I로 표시되는 스테레오칼핀 A(stereocalpin A)를 유효성분으로 함유하는 동맥경화증 예방 또는 개선용 기능성 건강식품을 제공한다.The present invention also provides a functional health food for preventing or improving atherosclerosis, which contains stereocalpin A represented by the following formula (I) as an active ingredient.
본 발명에 따른 스테레오칼핀 A(stereocalpin A)가 함유된 조성물은 TNF-α에 의해 유도되는 세포부착분자의 발현을 억제시킴으로써, 동맥경화증 치료 및 예방에 유용하게 사용할 수 있다. The composition containing stereocalpin A according to the present invention can be usefully used for the treatment and prevention of atherosclerosis by inhibiting the expression of cell adhesion molecules induced by TNF-α.
도 1은 스테레오칼핀 A(화학식 I의 화합물)의 농도에 따른 VSMC에 부착된 THP-1 세포를 측정한 것이다 ((A): VSMCs에 부착된 THP-1 세포를 측정한 그래프, (B): VSMCs에 부착된 THP-1 세포를 형광현미경(100x)으로 측정한 사진).
도 2는 스테레오칼핀 A(화학식 I의 화합물)의 농도에 따른 TNF-α 처리된 부착분자의 발현을 나타낸 것이다((A): ELISA를 통해 측정된 VCAM-1 및 ICAM-1의 발현율 그래프, (B): western blot assay를 이용한 VCAM-1 및 ICAM-1의 단백질 레벨을 나타낸 이미지).
도 3은 스테레오칼핀 A(화학식 I의 화합물)의 농도에 따른 TNF-α에 의해 유도된 VCAM-1 및 ICAM-1의 mRNA 발현을 나타낸 것이다((A): RT-PCR를 통해 측정된 이미지, (B): GAPDH에 대한 VCAM-1 및 ICAM-1의 발현을 측정한 그래프).
도 4는 스테레오칼핀 A(화학식 I의 화합물)의 농도에 따른 NF-κB 활성 및 IκBα저하를 측정한 것이다((A): 루시페라아제 활성 측정 그래프, (B): western blot assay를 이용한 p65 세포핵 단백질 레벨을 측정한 이미지, (C): western blot assay를 이용하여 IκBα 항체를 측정한 이미지).
도 5는 스테레오칼핀 A(화학식 I의 화합물)의 농도에 따른 pp38, p-JNK, p-ERK 및 Akt 활성을 측정한 것이다((A): western blot assay를 이용한 pp38, p-JNK, p-ERK 및 Akt 활성 측정 그래프, (B): pERK/ERK, pJNK/JNK, pp38/p38, pAKT/AKT 비율 측정 그래프)).
도 6은 스테레오칼핀 A(화학식 I의 화합물)의 농도에 따른 ROS 생성을 측정한 결과 그래프이다.1 is a measure of THP-1 cells attached to VSMC according to the concentration of stereocalpin A (compound of formula I) ((A): graph measuring THP-1 cells attached to VSMCs, (B): THP-1 cells attached to VSMCs as measured by fluorescence microscopy (100 ×).
Figure 2 shows the expression of TNF-α treated adhesion molecule according to the concentration of stereocalpin A (compound of formula I) ((A): graph of expression rate of VCAM-1 and ICAM-1 measured by ELISA, ( B): Image showing protein levels of VCAM-1 and ICAM-1 using western blot assay.
Figure 3 shows mRNA expression of VCAM-1 and ICAM-1 induced by TNF-α according to the concentration of stereocalpin A (compound of formula I) ((A): image measured via RT-PCR, (B): Graph measuring expression of VCAM-1 and ICAM-1 on GAPDH).
FIG. 4 shows NF-κB activity and IκBα decrease according to the concentration of stereocalpin A (compound I) ((A): Luciferase activity measurement graph, (B): p65 nucleus protein level using western blot assay) (C): Image of measuring IκBα antibody using western blot assay.
FIG. 5 shows the measurement of pp38, p-JNK, p-ERK and Akt activity according to the concentration of stereocalpin A (compound I) ((A): pp38, p-JNK, p- ERK and Akt activity measurement graph, (B): pERK / ERK, pJNK / JNK, pp38 / p38, pAKT / AKT ratio measurement graph)).
6 is a graph showing the results of ROS generation according to the concentration of stereocalpin A (compound of Formula I).
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 가진다. 일반적으로, 본 명세서에서 사용된 명명법 은 본 기술분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.
본 발명의 상세한 설명 등에서 사용되는 주요 용어의 정의는 다음과 같다. Definitions of main terms used in the detailed description of the present invention are as follows.
본원에서, '유효성분'이라 함은, 주(主)가 되는 성분물질로서, 본 발명에서는 TNF-α에 의해 증가되는 세포부착분자 발현을 억제시키는 기능을 나타내는 주요 성분을 의미한다.As used herein, the term "active ingredient" refers to a main ingredient exhibiting a function of inhibiting cell adhesion molecule expression increased by TNF-α as a main ingredient substance.
본원에서, '예방 또는 치료'라 함은, 동맥경화증을 치료하거나, 미리 예방하거나, 또는 동맥경화증으로 인한 건강 상태를 호전시키거나 개선시키는 모든 형태를 의미한다.As used herein, "prevention or treatment" means any form that treats, prevents, or improves or improves the state of health caused by atherosclerosis.
본원에서, '약학적으로 허용되는'이라 함은, 생리학적으로 허용되고 인간에게 투여될 때, 통상적으로 위장 장애, 현기증과 같은 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 조성물을 말한다. As used herein, "pharmaceutically acceptable" refers to a composition that, when physiologically acceptable and administered to a human, typically does not cause an allergic reaction, such as gastrointestinal disorders, dizziness, or the like.
본 발명은 일 관점에서, 스테레오칼핀 A(stereocalpin A)를 유효성분으로 함유하는 동맥경화증 예방 또는 치료용 약학 조성물에 관한 것이다. The present invention, in one aspect, relates to a pharmaceutical composition for preventing or treating atherosclerosis containing stereocalpin A as an active ingredient.
본 발명에 있어서, 스테레오칼핀 A(stereocalpin A)는 남극 지의류의 일종인 라말리나 테라브라타 (Ramalina terebrata)로부터 얻을 수 있다In the present invention, stereocalpin A can be obtained from Ramalina terebrata , a kind of antarctic lichen.
본 발명은 다음 화학식 I로 표시되는 스테레오칼핀 A(stereocalpin A)를 유효성분으로 함유하는 동맥경화증의 예방 또는 치료용 약학 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing or treating atherosclerosis containing stereocalpin A represented by the following formula (I) as an active ingredient.
[화학식 I](I)
상기 화학식 I로 표현되는 스테레오칼핀 A는 라말리나 테라브라타 (Ramalina terebrata) 추출물로부터 크로마토그래피를 이용하여 용출해 내어 분리된 것이다. Stereocalpin A represented by Formula I is isolated by eluting with chromatography from Ramalina terebrata extract.
상기 화학식 I로 표현되는 스테레오칼핀 A의 분리방법은 라말리나 테라브라타 (Ramalina terebrata)을 탄소수 3 내지 6의 알코올 및 이들의 혼합물로 구성된 군에서 선택된 용매로 추출하고, 상기 추출된 라말리나 테라브라타 (Ramalina terebrata) 추출물을 크로마토그래피 및 HPLC를 이용하여 정제함으로써 화학식 1로 표시되는 스테레오칼핀 A를 수득한다.Separation method of the stereo calpin A represented by the formula (I) is extracted from Ramalina terebrata (ramalina terebrata ) with a solvent selected from the group consisting of alcohols having 3 to 6 carbon atoms and mixtures thereof, the extracted ramalina terrabra ( Ramalina terebrata ) Purification of the extract using chromatography and HPLC yields the stereocalpin A represented by formula (1).
전술된 바와 같이, 분리된 화학식 I의 화합물은 HRESIMS, NMR, HMQC 및 HMBC 스펙트럼을 수행하였다. 스테레오칼핀 A는 백색의 가루로서, 한국 기초 과학 연구회의 Mariner ESI-MS(Perseptive Biosystem, USA)를 이용하여 HRESIMS(m/z 515.2513(M+Na)+ △-0.9mmu)을 통해 분자식을 확인한 결과, 불포화 13 등급을 나타내는 C29H36N2O5로 밝혀졌다. 이는 구조 내에 전례없는 5-하이드록시-2,4-디메틸-3-옥소-옥탄산(5-hydrozy-2,4-dimethyl-3-oxo-octanoic acid, HMDOO)를 포함하는 독특한 고리형 펩타이드인 바, 본 발명자는 본 발명 이전의 한국등록특허 제0923308호에 화학식 I의 화합물을 스테레오칼핀 A (Stereocalpin A)로 명명한 바 있다. 스테레오칼핀 A는 수개의 서로 다른 종양 세포에 대하여 종양치사 (tumoricidal) 활성을 나타낸다는 것은 상기 발명에 개시되어 있다. 하지만, 이의 기타 생물학적 활성과 분자적 메카니즘은 아직 알려진 바 없다. As described above, the isolated compounds of formula I performed HRESIMS, NMR, HMQC and HMBC spectra. Stereocalpin A is a white powder, and the molecular formula was confirmed by HRESIMS (m / z 515.2513 (M + Na) + Δ-0.9mmu) using Mariner ESI-MS (Perseptive Biosystem, USA). , C 29 H 36 N 2 O 5 indicating unsaturated 13 grade. It is a unique cyclic peptide that contains unprecedented 5-hydroxy-2,4-dimethyl-3-oxo-octanoic acid (HMDOO) in its structure. The present inventors have named the compound of Formula I as Stereocalpin A in Korean Patent No. 0923308 prior to the present invention. It is disclosed in the invention that stereocalpin A exhibits tumoricidal activity against several different tumor cells. However, its other biological activities and molecular mechanisms are not yet known.
본 발명에 있어서, 스테레오칼핀 A (Stereocalpin A)는 조성물 100 중량부에 대하여, 0.01 내지 10 중량부로 함유될 수 있다. In the present invention, Stereocalpin A may be contained in an amount of 0.01 to 10 parts by weight based on 100 parts by weight of the composition.
본 발명은, 화학식 I의 화합물 스테레오칼핀 A(Stereocalpin A)의 전처리에 의해 ICAM-1 및 VCAM-1 발현에 어떠한 영향을 미치는지 알아보기 위해 혈관 평활근 세포(이하, MOVAS-1이라 함)를 이용하여 ELISA, cell adhesion assay, RT-PCR, western blot analysis 등을 수행하였다 (실시예 1 내지 7 참조). 그 결과, TNF-α처리에 의해 증가하는 VCAM-1의 발현은, 화학식 I의 화합물 스테레오칼핀 A(stereocalpin A)의 처리에 의하여 농도 의존적으로, mRNA와 단백질 레벨 및 세포표면으로의 발현이 감소됨을 확인할 수 있었다. 또한, 다양한 스트레스에 의해 유도되어지는 세포의 신호전달 경로에서 중요한 역할을 하는 MAP kinase family와 Akt의 활성을 확인한 결과, TNF-α에 의해 증가된 인산화(phosphorylation)가 화학식 I의 화합물 스테레오칼핀 A(stereocalpin A) 처리에 의하여 농도 의존적으로 감소됨을 확인할 수 있었다.The present invention uses vascular smooth muscle cells (hereinafter referred to as MOVAS-1) to determine how pretreatment of the compound stereocalpin A of formula I affects ICAM-1 and VCAM-1 expression. ELISA, cell adhesion assay, RT-PCR, western blot analysis and the like were performed (see Examples 1 to 7). As a result, expression of VCAM-1 increased by TNF-α treatment was found to decrease the expression of mRNA and protein levels and cell surface in a concentration-dependent manner by the treatment of the compound stereocalpin A of formula (I). I could confirm it. In addition, as a result of confirming the activity of the MAP kinase family and Akt, which plays an important role in cell signaling pathways induced by various stresses, the phosphorylation increased by TNF-α is shown to be a compound of formula I It was confirmed that the concentration-dependent decrease by the stereocalpin A) treatment.
본 발명에서는 또한, MAP kinase와 Akt에 의해 활성화되고, ICAM-1 및 VCAM-1의 발현에 주요한 영향을 미치는 전사인자로 잘 알려진 NF-κB의 세포질에서 핵으로의 이동과 DNA 결합 활성은 화학식 I의 스테레오칼핀 A(stereocalpin A)의 농도가 증가함에 따라 감소됨을 western blot analysis를 통하여 확인하였다. 아울러, NF-κB의 세포질에서 핵으로의 이동에 관여하는 IκBα의 변화를 확인한 결과, 화학식 I의 화합물 스테레오칼핀 A(stereocalpin A)를 처리하는 경우, 상기 IκBα가 TNF-α에 의해 분해되는 것이 억제됨을 확인할 수 있었다. 마지막으로, TNF-α에 의한 ROS의 생성에 화학식 I의 화합물 스테레오칼핀 A(stereocalpin A)가 어떠한 영향을 미치는지 측정한 결과, 화학식 I의 화합물 스테레오칼핀 A(stereocalpin A)의 농도가 증가함에 따라 ROS의 생성이 감소되는 것을 확인할 수 있었다.In the present invention, the NF-κB cytoplasm to the nucleus and DNA binding activity, which is activated by MAP kinase and Akt and is well known as a transcription factor that has a major effect on the expression of ICAM-1 and VCAM-1, is represented by Formula I. It was confirmed by Western blot analysis that the concentration of the stereocalpin A (stereocalpin A) decreases with increasing. In addition, as a result of confirming the change of IκBα involved in the migration of NF-κB from the cytoplasm to the nucleus, the treatment of the compound stereocalpin A of formula (I) inhibits the degradation of IκBα by TNF-α. Could confirm. Finally, as a result of measuring the effect of the compound stereocalpin A of formula (I) on the production of ROS by TNF-α, as the concentration of the compound stereocalpin A of formula (I) increases It was confirmed that the production of.
즉, TNF-α에 의해 유도되는 ICAM-1 및 VCAM-1의 발현은, 화학식 I의 화합물 스테레오칼핀 A(stereocalpin A)에 의한 ROS 생성의 억제로 인하여 MAP kinse, Akt 및 NF-κB로의 신호전달이 이루어지지 않아 억제된다. 따라서, 상기 스테레오칼핀 A는 동맥경화증의 치료 및 예방에 유용하게 사용할 수 있다. In other words, the expression of ICAM-1 and VCAM-1 induced by TNF-α is signaled to MAP kinse, Akt and NF-κB due to inhibition of ROS production by the compound stereocalpin A of formula (I). This is not done and suppressed. Therefore, the stereocalpine A can be usefully used for the treatment and prevention of atherosclerosis.
본 발명의 동맥경화증 예방 또는 치료용 약학 조성물은 유효성분 이외에 추가로 약학적으로 허용 가능한 담체를 1종 이상 포함하여 제조할 수 있다. 여기서 약제학적으로 허용가능한 담체는 식염수, 멸균수, 링거액, 완충 식염수, 텍스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다.The pharmaceutical composition for preventing or treating atherosclerosis of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the active ingredient. The pharmaceutically acceptable carrier may be saline, sterile water, Ringer's solution, buffered saline, textose solution, maltodextrin solution, glycerol, ethanol and one or more of these components in combination, and if necessary, antioxidants, buffers And other conventional additives such as bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
본 발명의 약학 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구 투여(예를 들어 정맥 내, 피하, 복강 내 또는 국소에 적용)할 수 있으며, 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도 등에 따라 그 범위가 다양하다. The pharmaceutical composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the weight, age, sex and health of the patient. The range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease.
본 발명의 스테레오칼핀 A(stereocalpin A) 화합물의 일일 투여량은 약 0.1 ~ 500mg/kg(체중)이고, 바람직하게는 1 ~ 300mg/kg(체중)이며, 하루 일회 내지 수회에 나누어 투여하는 것이 더욱 바람직하다.The daily dosage of the stereocalpin A compound of the present invention is about 0.1 to 500 mg / kg (body weight), preferably 1 to 300 mg / kg (body weight), and is administered once or several times a day. desirable.
본 발명의 약학 조성물은 또한, 동맥경화의 예방 및 치료를 위하여 단독으로, 또는 수술, 방사선 치료, 호르몬 치료, 화학 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다. The pharmaceutical compositions of the present invention may also be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention and treatment of atherosclerosis.
본 발명은 또 다른 관점에서, 다음 화학식 I로 표시되는 스테레오칼핀 A(stereocalpin A)를 함유하는 동맥경화 예방 또는 개선용 기능성 건강식품에 관한 것이다.In another aspect, the present invention relates to a functional health food for preventing or improving arteriosclerosis containing stereocalpin A represented by the following formula (I).
[화학식 I](I)
본 발명에 따른 스테레오칼핀 A(stereocalpin A)를 동맥경화 예방 또는 개선의 목적으로 식품의 형태로 제조될 수 있다. 따라서 본 발명은 스테레오칼핀 A(stereocalpin A)를 유효성분으로 함유하는 식품 조성물인 건강기능식품에 관한 것이다. Stereocalpin A according to the present invention can be prepared in the form of food for the purpose of preventing or improving atherosclerosis. Therefore, the present invention relates to a health functional food that is a food composition containing stereocalpin A (stereocalpin A) as an active ingredient.
본 발명에 따른 건강 기능 식품은 식품 조성물로써, 기능성 식품(functional food), 영양 보조제(nutritional supplement), 건강식품(health food) 및 식품 첨가제(food additives) 등의 모든 형태를 포함한다. 상기 유형의 건강 기능 식품은 당업계에 공지된 통상적인 방법에 따라 다양한 형태로 제조할 수 있다. 예를 들면, 건강식품으로는 본 발명의 둥근잎유홍초 추출물 자체를 차, 쥬스 및 드링크의 형태로 제조하여 음용하도록 하거나, 과립화, 캡슐화 및 분말화하여 섭취할 수 있다. 또한, 기능성 식품으로는 음료(알콜성 음료 포함), 과실 및 그의 가공식품(예: 과일통조림, 병조림, 잼, 마말레이드 등), 어류, 육류 및 그 가공식품(예: 햄, 소시지 콘비프 등), 빵류 및 면류(예: 우동, 메밀국수, 라면, 스파게티, 마카로니 등), 과즙, 각종 드링크, 쿠키, 엿, 유제품(예: 버터, 치즈 등), 식용식물유지, 마가린, 식물성 단백질, 레토르트 식품, 냉동식품, 각종 조미료(예: 된장, 간장, 소스 등) 등에 본 발명의 스테레오칼핀 A(stereocalpin A)를 첨가하여 제조할 수 있다.The health functional food according to the present invention is a food composition, and includes all forms such as functional food, nutritional supplement, health food and food additives. Health functional foods of this type can be prepared in various forms according to conventional methods known in the art. For example, as a health food can be prepared by drinking the round leaf vinegar extract itself of the present invention in the form of tea, juice and drinks, or ingested by granulation, encapsulation and powdering. Functional foods also include beverages (including alcoholic beverages), fruits and processed foods (e.g. canned fruit, canned foods, jams, marmalade, etc.), fish, meat and processed foods (e.g. ham, sausage corned beef, etc.), Breads and noodles (e.g. udon, soba, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, malts, dairy products (e.g. butter, cheese, etc.), edible vegetable oils, margarine, vegetable protein, retort food, Frozen food, various seasonings (eg, miso, soy sauce, sauce, etc.) can be prepared by adding the stereocalpin A (stereocalpin A) of the present invention.
상기 건강 기능식품 또한, 식품조성물로써 기능성 식품, 영양보조제, 건강 식품, 식품 첨가제 등의 다양한 형태를 포함하는 것이며, 당업계에 공지된 통상적인 방법에 따라 다양한 형태, 예컨대, 앞서 언급한 스테레오칼핀 A(stereocalpin A) 자체를 차, 쥬스, 드링크의 형태로 제조하거나, 과립화, 캡슐화, 분말화 하거나, 이러한 추출물을 음료, 과실 및 가공식품, 어류, 육류 및 그 가공식품, 빵류, 면류, 조미료 등 각종 식품에 첨가하여 제조함으로써 제공될 수 있다.
The health functional food also includes a variety of forms, such as functional foods, nutritional supplements, health foods, food additives, etc. as a food composition, various forms, for example, the aforementioned stereocalpine A according to conventional methods known in the art. (stereocalpin A) itself is produced in the form of tea, juice, drink, granulated, encapsulated, powdered or these extracts are used in beverages, fruits and processed foods, fish, meat and processed foods, breads, noodles, seasonings, etc. It can be provided by adding to various foods and manufacturing.
< 실시예 > < Example >
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.
실시예Example 1: One: 라말리나Ramalina 테라브라타Terrabrata ( ( RamalinaRamalina terebrataterebrata ) 추출물의 제조) Preparation of extract
라말리나 테라브라타 (Ramalina terebrata)는 2003년 1월 남국 킹 조지 섬의 세종기지(S 62°13.3', W58°47.0') 주위의 바튼 반도(Barton Peninsular)에서 발명자 중 한 명(J.H. Yim)이 직접 채취하여 분리 동정한 지의류로서, 대조 표본 (L-5)은 한국 극지 연구 협회에 기탁하였다. Ramalina Terrabrata terebrata ) was collected and identified by one of the inventors (JH Yim) at the Barton Peninsular around King Sejong Station (S 62 ° 13.3 ', W58 ° 47.0') on King George Island in southern 2003. As lichens, a control specimen (L-5) was deposited with the Korean Polar Research Association.
채취된 라말리나 테라브라타 (Ramalina terebrata)의 건조 샘플(50g)을 24시간 동안 메탄올(1L×2)로 추출한 후, 감압 농축하여 메탄올 조추출물(crude MeOH extract) 5.9g을 수득하였다.
Harvested Ramalina Terrabrata ( Ramalina terebrata ) was extracted with methanol (1 L × 2) for 24 hours, and then concentrated under reduced pressure to obtain 5.9 g of crude crude methanol extract (crude MeOH extract).
실시예Example 2: 2: 라말리나Ramalina 테라브라타Terrabrata ( ( RamalinaRamalina terebrataterebrata ) 추출물로부터의 From extracts 스테레오칼핀Stereo Kalpin A의 분리 Separation of A
상기 실시예 1에서 제조된 라말리나 테라브라타 (Ramalina terebrata)의 메탄올 조추출물 5.9g을 Aldrich octadecyl-functionalized silica gel(C18)이 충진된 플래쉬 컬럼 크로마토그래피(flash column chromatography, 3× 15㎝)에 통과시키고, 물 400mL 당 각 20%, 40%, 60%, 70%, 80%, 90% 및 100%(v/v) 메탄올의 혼합용매를 순차적으로 주입하여 각각의 분획물을 획득하였다. Ramalina terrabrata prepared in Example 1 ( Ramalina 5.9 g of the crude methanol extract of terebrata ) was passed through flash column chromatography (3 × 15 cm) filled with Aldrich octadecyl-functionalized silica gel (C 18 ), each 20%, 40%, per 400 mL of water. Each fraction was obtained by sequentially injecting a mixed solvent of 60%, 70%, 80%, 90% and 100% (v / v) methanol.
상기 90% 메탄올로 용출된 용출액(55㎎)은 Shiseido Capcell Pak C18 column(10× 250㎜; 5㎛ 입자 크기, 유속 2㎖/min)을 사용하여 70~97% CH3CN(in 0.1% 포름산)의 농도 구배에 의하여 27분 동안 반-분취 역상 (semi-preparative reversed-phase) HPLC를 수행한 후, 254㎚ UV(Biochrom 1300 UV/Visible spectrophotometer)로 검출하여 화학식 I의 스테레오칼핀 A(stereocalpin A) 화합물(3.9mg; tR=22.4분)을 수득하였다. 이와 같이 분리된 화학식 I의 화합물 구조분석 및 물성은 한국등록특허 제0923308호에 기재되었다.
The eluate (55 mg) eluted with 90% methanol was prepared using a Shiseido Capcell Pak C18 column (10 × 250 mm; 5 μm particle size, flow rate 2 ml / min) at 70-97% CH 3 CN (in 0.1% formic acid). After performing a semi-preparative reversed-phase HPLC for 27 minutes by a concentration gradient of), it was detected by a 254 nm UV (Biochrom 1300 UV / Visible spectrophotometer) to determine the stereocalpin A of formula I (stereocalpin A). ) Compound (3.9 mg; t R = 22.4 min) was obtained. Compound structure analysis and physical properties of the formula (I) thus separated are described in Korean Patent No. 0923308.
실험예Experimental Example 1: 세포배양 1: Cell Culture
혈관평활근 세포주 MOVAS-1은 ATCC(Rockville, MD)로부터 구입하여 200mg/ml G418, 100IU/ml 페니실린, 100mg/ml 스트렙토마이신 및 10% 소의 태아혈청(FBS)(Carlsbad, CA)이 포함된 DMEM 배지(Carlsbad, CA)에 37℃, 5% CO2 조건을 유지하며 배양하였다. 계대배양하는 경우, 세포는 0.01M EDTA를 포함하는 0.125% 트립신을 사용하여 분주하였고, 본 실험에서 사용된 세포는 처음에서 여섯번까지 계대이고, 모든 실험은 단일 도너로부터 MOVAS-1의 동일 배치로 수행되었다. THP-1(ATCC), 인간골수세포주(a human myelomonocytic cell line)는 MOVAS-1의 세포부착 어세이용으로 사용되었다. 이러한 세포들은 2mM L-글루타민, 100㎍/ml 스트렙토마이신, 100IU/ml 페니실린, 10%FBS가 함유된 RPMI 1640에서 배양하였다.
Vascular smooth muscle cell line MOVAS-1 was purchased from ATCC (Rockville, MD) and contained a DMEM medium containing 200 mg / ml G418, 100 IU / ml penicillin, 100 mg / ml streptomycin and 10% fetal bovine serum (FBS) (Carlsbad, CA). (Carlsbad, CA) was incubated at 37 ℃, 5% CO 2 conditions. When passaged, cells were dispensed using 0.125% trypsin containing 0.01 M EDTA, the cells used in this experiment were passaged from first to six times, and all experiments were performed in the same batch of MOVAS-1 from a single donor It became. THP-1 (ATCC), a human myelomonocytic cell line, was used for the cell adhesion assay of MOVAS-1. These cells were cultured in RPMI 1640 containing 2 mM L-glutamine, 100 μg / ml streptomycin, 100 IU / ml penicillin, 10% FBS.
실험예Experimental Example 2: 통계학적 분석 2: statistical analysis
모든 실험결과는 평균± 표준편차로 나타내었고, 시험군 간의 통계적 유의성을 조사하기 위하여 일원배치 분산분석(one-way ANOVA)와 Bonferroni 사후검정을 p < 0.05 수준에서 실시하였다.
All experimental results were expressed as mean ± standard deviation. One-way ANOVA and Bonferroni post hoc tests were performed at p <0.05 level to investigate statistical significance between test groups.
실험예Experimental Example 3: 3: TNFTNF -α에 의해 by -α 증가되는Increased 혈관 세포부착분자 발현 및 Vascular cell adhesion molecule expression and 단핵세포Mononuclear cell 부착에 대한 For attachment 억제능Inhibition 측정 Measure
TNF-α처리에 의해 증가되는 단핵구(THP-1)의 부착에 대한 실시예 2의 화합물 스테레오칼핀 A의 영향을 측정하기 위해 세포 부착 어세이(cell adhesion assay)를 이용하였다. 세포 부착 어세이(cell adhesion assay)는 Chen C 등이 사용한 방법을 적용하였다(Chen C et al ., Cell Signal, 13:543-53, 2001). 세포 부착 어세이는 96 well plate에 MOVAS-1을 배양한 후, 다양한 농도의 실시예 2의 화합물 스테레오칼핀 A(0.1~10㎍/ml)로 2시간 동안 전처리하고, 6시간 동안 TNF-α(10ng/ml)를 첨가한 다음, 배지를 제거하고, BCECF-라벨화된 THP-1 세포(2.5×105 cells/well)를 분주하였다. 각 실험은 3회 반복하였다. 37℃에서 1시간 동안 배양 후에, microwells은 0.2ml 따듯한 배지로 두 번 세척한 다음, cytoflour 2350(Milipore, Bedford, MA)를 사용하여 형광강도를 측정하여 부착 세포수를 측정하였다. 그 결과, THP-1 단핵구의 부착이 전처리된 스테레오칼핀 A의 농도에 의존적으로 감소됨을 확인하였다 (도 1). A cell adhesion assay was used to determine the effect of the compound stereocalpin A of Example 2 on the adhesion of monocytes (THP-1) increased by TNF-α treatment. The cell adhesion assay was applied by Chen C et al. (Chen C et al ., Cell Signal , 13: 543-53, 2001). Cell attachment assay was incubated in 96 well plate MOVAS-1, and then pre-treated for 2 hours with various concentrations of compound stereocalpine A (0.1 ~ 10㎍ / ml) of Example 2, TNF-α (for 6 hours) 10 ng / ml) was added, then the medium was removed and BCECF-labeled THP-1 cells (2.5 × 10 5 cells / well) were aliquoted. Each experiment was repeated three times. After incubation at 37 ° C. for 1 hour, the microwells were washed twice with 0.2 ml warm medium, and then the number of adherent cells was determined by measuring the fluorescence intensity using cytoflour 2350 (Milipore, Bedford, MA). As a result, it was confirmed that adhesion of THP-1 monocytes was reduced depending on the concentration of pretreated stereocalpin A (FIG. 1).
또한, TNF-α에 의해 유도된 접착분자 발현에 대한 실시예 2의 화합물인 스테레오칼핀 A의 영향을 확인하기 위하여 ELISA(enzyme-linked immunosorbent assay)를 측정하였다. ELISA는 Mo SJ 등이 사용한 방법을 이용하였다(Mo SJ et al ., J. Ethnopharmacol, 109:78-86, 2007). 즉, 96 well 젤라틴이 코팅된 플레이트에 세포(2×104cell/well)을 배양한 후 37℃에서 2시간 동안 실시예 2의 화합물 스테레오칼핀 A(0.1~10㎍/ml)로 전처리하였다. 상기 전처리된 세포는 VCAM-1 및 ICAM-1의 발현을 측정하기 위해 8시간 동안 TNF-α(10ng/ml)를 포함하는 신선한 성장 배지에서 배양하였다. 배양 후에는 상기 세포를 pH 7.4의 PBS로 세척시킨 다음, 4℃에서 30분 동안 1.0% 글루타르알데히드(glutaraldehyde)로 고정화시키고, 여기에 소혈정알부민(1.0% in PBS)을 넣어 상온에서 30분 non-specific binding시킨다. 상기 세포는 ICAM-1, VCAM-1 또는 표준 대조군 항체(0.25g/ml, diluted in blocking buffer)와 함께 4℃에서 하룻밤 동안 배양하고 PBS로 세척한 다음, alkaline phosphatase-conjugated goat anti-mouse secondary antibody (1㎍/ml, diluted in PBS)와 함께 배양하였다. 상기 배양된 세포는 PBS로 세척하고, peroxidase substrate(p-nitorphenyl phosphate 1 mg/ml in 0.1 M glycin buffer, pH 10.4 containing 1 mM MgCl2, and 1 mM ZnCl2)을 첨가하였다. Molecular device microplate reader(Menlo Park, CA)를 사용하여 405nm에서 흡광도를 측정하였다. In addition, an enzyme-linked immunosorbent assay (ELISA) was measured to confirm the effect of the stereocalpin A compound of Example 2 on the expression of adhesion molecules induced by TNF-α. ELISA used the method used by Mo SJ et al. (Mo SJ et al ., J. Ethnopharmacol , 109: 78-86, 2007). That is, cells (2 × 10 4 cell / well) were incubated in 96 well gelatin-coated plates, and then pretreated with compound stereocalpin A (0.1˜10 μg / ml) of Example 2 for 2 hours at 37 ° C. The pretreated cells were incubated in fresh growth medium containing TNF-α (10 ng / ml) for 8 hours to measure expression of VCAM-1 and ICAM-1. After incubation, the cells were washed with PBS at pH 7.4, immobilized with 1.0% glutaraldehyde for 30 minutes at 4 ° C, and bovine serum albumin (1.0% in PBS) was added thereto for 30 minutes at room temperature. Non-specific binding. The cells were incubated overnight at 4 ° C. with ICAM-1, VCAM-1 or standard control antibody (0.25 g / ml, diluted in blocking buffer) and washed with PBS, followed by alkaline phosphatase-conjugated goat anti-mouse secondary antibody. (1 μg / ml, diluted in PBS). The cultured cells were washed with PBS and peroxidase substrate (p-
그 결과, ICAM-1 및 VCAM-1의 세포 표면 발현은 TNF-α에 의해 유도되어지는 것으로 나타났다 (도 2(A)). 그러나, 스테레오칼핀 A로 전처리된 샘플은 스테레오칼핀 A의 농도에 의존적으로 TNF-α에 의해 유도된 ICAM-1 및 VCAM-1 발현을 유의적으로 감소시키는 것으로 나타났다. 또한, 실시예 2의 스테레오칼핀 A는 TNF-α에 의해 유도된 ICAM-1 및 VCAM-1 단백질 수치를 현저하게 감소시켰다 (도 2(B)). 두 부착분자 발현의 유의적 감소는 스테레오칼핀 A이 10㎍/ml로 처리한 경우에서 관찰되었다. 스테레오칼핀 A가 β-actin 발현에 영향이 없는 것을 나타내나, 억제효과는 ICAM-1 및 VCAM-1에 대하여 선택적임을 알 수 있었다. 따라서, 스테레오칼핀 A는 ICAM-1 및 VCAM-1의 발현 유도를 억제시킨다는 것을 확인할 수 있었다.
As a result, cell surface expression of ICAM-1 and VCAM-1 was shown to be induced by TNF-α (FIG. 2 (A)). However, samples pretreated with stereocalpin A have been shown to significantly reduce ICAM-1 and VCAM-1 expression induced by TNF-α depending on the concentration of stereocalpin A. In addition, the stereocalpin A of Example 2 significantly reduced the levels of ICAM-1 and VCAM-1 proteins induced by TNF-α (FIG. 2 (B)). Significant decrease in the expression of both adhesion molecules was observed when stereocalpin A was treated with 10 μg / ml. Stereocalpin A showed no effect on β-actin expression, but the inhibitory effect was found to be selective for ICAM-1 and VCAM-1. Therefore, it was confirmed that stereocalpin A inhibits the expression induction of ICAM-1 and VCAM-1.
실험예Experimental Example 4: 4: 혈관세포Vascular cells 부착분자 Adhesion molecule mRNAmRNA 유도 측정 Induction measurement
TNF-α에 의해 유도되는 ICAM-1 및 VCAM-1의 mRNA 발현(전사)에 대한 실시예 2의 화합물인 스테레오칼핀 A의 영향을 측정하였다. mRNA의 유전자 전사 농도를 측정하기 위하여, 총 세포 RNA를 VSMC로부터 추출하고, ICAM-1와 VCAM-1의 특이적 프라이머를 사용하여 RT-PCR(revers transcription-polymerase chain reaction)을 수행하였다. The effect of stereocalpin A, a compound of Example 2, on mRNA expression (transcription) of ICAM-1 and VCAM-1 induced by TNF-α was measured. To measure the gene transcription concentration of mRNA, total cell RNA was extracted from VSMC and subjected to reverse transcription-polymerase chain reaction (RT-PCR) using specific primers of ICAM-1 and VCAM-1.
총 세포 RNA는 VSMC로부터 단일단계 guanidinium thiocyanate-phenol-chloroform 방법을 이용하여 추출하고, RNA의 수율과 순도는 260nm 내지 280nm에서 흡광도 비율로 측정하였다. PCR은 각각의 특이적인 cDNA를 식별하기 위하여 하기와 같은 ICAM-1와 VCAM-1의 특이적 프라이머를 사용하였다. Total cellular RNA was extracted from VSMC using single-step guanidinium thiocyanate-phenol-chloroform method, and RNA yield and purity were measured as absorbance ratio at 260nm to 280nm. PCR used the following specific primers of ICAM-1 and VCAM-1 to identify each specific cDNA.
다음과 같이 ICAM-1 특이적 프라이머를 합성하였다. ICAM-1 specific primers were synthesized as follows.
SEQ ID No. 1: sense primer, 5'-CCTGTTTCCTGCCTCTGAAG-3';SEQ ID NO. 1: sense primer, 5'-CCTGTTTCCTGCCTCTGAAG-3 ';
SEQ ID No. 2: antisense primer, 5'-GTCTGCTGAGACCCCTCTTG-3'.SEQ ID NO. 2: antisense primer, 5'-GTCTGCTGAGACCCCTCTTG-3 '.
또한, 다음과 같이 VCAM-1 특이적 프라이머를 합성하였다.In addition, VCAM-1 specific primers were synthesized as follows.
SEQ ID No. 3: sense primer, 5'-CCCAAGGATCCAGAGATTCA-3';SEQ ID NO. 3: sense primer, 5'-CCCAAGGATCCAGAGATTCA-3 ';
SEQ ID No. 4: antisense primer, 5'-TAAGGTGAGGGTGGCATTTC-3'.SEQ ID NO. 4: antisense primer, 5'-TAAGGTGAGGGTGGCATTTC-3 '.
GAPDH PCR 프라이머는 다음과 같다.GAPDH PCR primers are as follows.
SEQ ID No. 5: 5- GGTCCTCAGTGTAGCCCAAG-3 (sense); SEQ ID NO. 5: 5-GGTCCTCAGTGTAGCCCAAG-3 (sense);
SEQ ID No. 6: 5-AATGTGTCCGTCGTGGATCT-3 (antisense). SEQ ID NO. 6: 5-AATGTGTCCGTCGTGGATCT-3 (antisense).
오염물의 부재는 프라이머가 첨가되지 않은 음성 대조군 샘플의 RT-PCR assay로 확인하였다. 총 cellular RNAs는 VSMCs로부터 분리되고, VCAM-1 또는 ICAM-1 특이적 프로브를 사용하여 RT-PCR로 분석하였다. VSMCs는 2시간 동안 다양한 농도의 스테레오칼핀 A로 전처리한 다음, 4시간 동안 TNF-α로 처리하였다. Absence of contaminants was confirmed by RT-PCR assay of negative control samples without primers added. Total cellular RNAs were isolated from VSMCs and analyzed by RT-PCR using VCAM-1 or ICAM-1 specific probes. VSMCs were pretreated with stereocalpine A at various concentrations for 2 hours and then treated with TNF-α for 4 hours.
그 결과, 스테레오칼핀 A로 처리된 VSMCs는 TNF-α에 의해 유도된 VCAM-1 및 ICAM-1의 발현을 유의적으로 억제시키는 것으로 나타났고, 이러한 결과로부터 TNF-α에 의해 유도된 VCAM-1 및 ICAM-1의 발현이 스테레오칼핀 A에 의해 전사 후 단계(post-transcriptionally)에서 조절된다는 것을 확인할 수 있었다 (도 3).
As a result, VSMCs treated with stereocalpin A were found to significantly inhibit the expression of VCAM-1 and ICAM-1 induced by TNF-α, and from these results, VCAM-1 induced by TNF-α. And expression of ICAM-1 was regulated post-transcriptionally by stereocalpin A (FIG. 3).
실험예Experimental Example 5: 5: TNFTNF -α에 의해 유도된 induced by -α NFNF -κB 활성 억제 측정 -κB activity inhibition measurement
NF-κB는 부착분자 발현에서 결정적인 역할을 하는 유비퀴터스 (ubiquitous) 전사 인자이다(Angel and Karin, 1991; Beg et al., 1993). 이에, NF-κB 전사적 활성에 대한 실시예 2의 스테레오칼핀 A의 영향을 측정하였다. 상기 세포는 2시간 동안 다양한 농도의 실시예 2의 스테레오칼핀 A로 처리한 다음, 4시간 동안 TNF-α로 처리하였다. NF-κB 독립적인 전사에 대한 스테레오칼핀 A의 영향을 측정하기 위해 transcriptional activation assays를 사용하였다. 상기 transcriptional activation assays는 세포(5×105 cell/ml)를 6-well plate의 각 well에 분주하였다. 분주된 세포는 Life Technologies사(Carlsbad, CA)에서 구입한 Lipofectamine Plus를 사용하여 제작자의 프로토콜에 따라, 플라스미드, pGL3-NF-κB(Promega, Madison, WI) 및 pCMV-β-gal(Lonza, Walkersville, MD)로 주입하였다. 즉, pGL3-NF-κB 0.5㎍ 및 pCMV-β-gal 0.2㎍이 함유된 형질전환 혼합물은 Lipofectamine Plus 시약에 혼합하고, 각 세포에 첨가된 다음, 4시간 후에 상기 세포는 2시간 동안 스테레오칼핀 A에 의해 전처리하고, 여기에 4시간 동안 TNF-α로 처리하였다. 그 다음, lysis buffer[24mM Tris-HCl (pH 7.8), 2mM dithiotreitol, 2 mM EDTA, 10% glycerol, and 1% Triton X-100] 200㎕로 세포를 용해시키고, 상기 세포 용해물 10㎕는 루시페라아제 활성 어세이(Promega, Madison, WI)를 사용하여 루시페라아제 및 β-갈락토시다아제(galactosidase)의 활성을 측정하였다. 각 실험은 3회 반복 수행하였고, 측정값은 평균값으로 나타내었다. NF-κB is a ubiquitous transcription factor that plays a critical role in adhesion molecule expression (Angel and Karin, 1991; Beg et al., 1993). Thus, the influence of stereocalpin A of Example 2 on the NF-κB transcriptional activity was measured. The cells were treated with stereocalpine A of Example 2 at various concentrations for 2 hours and then with TNF-α for 4 hours. Transcriptional activation assays were used to determine the effect of stereocalpin A on NF-κB independent transcription. In the transcriptional activation assays, cells (5 × 10 5 cells / ml) were dispensed into each well of a 6-well plate. Aliquoted cells were plasmid, pGL3-NF-κB (Promega, Madison, WI) and pCMV-β-gal (Lonza, Walkersville) using Lipofectamine Plus purchased from Life Technologies (Carlsbad, CA) according to the manufacturer's protocol. , MD). That is, the transformation mixture containing 0.5 μg of pGL3-NF-κB and 0.2 μg of pCMV-β-gal was mixed with Lipofectamine Plus reagent, added to each cell, and then 4 hours later, the cells were stereocalpine A for 2 hours. Pretreated by TNF-α for 4 hours. Then, the cells were lysed with 200 µl of lysis buffer [24 mM Tris-HCl (pH 7.8), 2 mM dithiotreitol, 2 mM EDTA, 10% glycerol, and 1% Triton X-100], and 10 µl of the cell lysate was luciferase. Activity assays (Promega, Madison, WI) were used to measure the activity of luciferase and β-galactosidase. Each experiment was repeated three times, and the measured values were expressed as average values.
그 결과, 도 4(A)에 나타난 바와 같이, TNF-α의 처리는 루시페라아제 활성을 약 3.6배 증가시키는 것으로 나타났고, 상기 증가는 스테레오칼핀 A에 의해 유의적으로 감소되는 것으로 나타났다. As a result, as shown in FIG. 4 (A), treatment with TNF-α was shown to increase luciferase activity by about 3.6-fold, and the increase was significantly reduced by stereocalpin A.
NF-κB 서브유닛 p65는 많은 염증성 유전자의 전사에 중요한 활성을 수행하는 것으로 입증되었기 때문에, p65 NF-κB 단백질의 발현에 대한 실시예 2의 화합물인 스테레오칼핀 A의 영향도 측정하였다. 도 4(B)에 나타난 바와 같이, 스테레오칼핀 A로 전처리된 VSMCs는 핵분획물에 대한 p65 NF-κB 전좌(translocation)가 스테레오칼핀 A에 의해 농도의존적으로 감소되는 것으로 나타남에 따라, 스테레오칼핀 A가 TNF-α에 의해 유도된 NF-κB의 세포핵의 위치 변화(nuclear translocation)를 억제시킨다는 것을 알 수 있었다. Since the NF-κB subunit p65 has been demonstrated to play an important activity in the transcription of many inflammatory genes, the effect of stereocalpin A, a compound of Example 2, on the expression of the p65 NF-κB protein was also measured. As shown in FIG. 4 (B), VSMCs pretreated with stereocalpin A have been shown to have a concentration-dependent decrease in p65 NF-κB translocation to the nucleus fraction by stereocalpin A. It was found that it inhibits the nuclear translocation of NF-κB induced by TNF-α.
또한, 스테레오칼핀 A에 대한 TNF-α에 의해 유도되는 IκBα저하를 측정하기 위해, IκBα 단백질의 발현을 Western blot assay로 측정하였다. Western blot assay는 Cho SJ 등이 사용한 방법으로 사용하였다(Cho SJ et al ., Toxicon, 42:601-11, 2003). 즉, 세포는 실시예 2의 스테레오칼핀 A(0.1~10㎍/ml)로 2시간 동안 전처리한 다음, 30분 또는 8시간 동안 TNF-α(10ng/ml)를 함유하는 신선한 성장 배지에서 배양하였다. 상기 처리 후, 상기 세포는 PBS에서 2회 세척한 다음, 버퍼 A[10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.5 mM PMSF and Protease Inhibitor Cocktail (Sigma)] 70㎕에 현탁시키고, 얼음에서 배양하였다. 15분 후에, 0.5% Nonidet P (NP)-40을 상기 세포에 첨가시켜 세포를 용해시킨 다음, 10초 동안 와류(vortex)시켰다. 그 후, 시토졸 세포(cytosolic cell) 추출물은 4℃에서 10분 동안 1500xg으로 원심분리한 다음, 회수하였다. 회수된 세포핵들은 버퍼 C[20mM HEPES (pH 7.9), 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 25% v/v Glycerol, 0.5 mM PMSF and Protease Inhibitor Cocktail] 50㎕에 현탁시키고, 간헐적으로 교반시키면서 20분 동안 얼음에서 배양하였다. 세포핵 추출물은 4℃에서 13,000xg로 10분 동안 원심분리 한 다음 회수하였다. 단백질 농도는 기준으로 BSA을 사용하여 Bio-Rad protein assay (Bio-Rad Lab, Hercules, CA)로 측정하였다. 전체 세포 용해물(20㎍)과 세포핵 추출물(40㎍)은 각각 7.5% SDS-polyacrylamide gel에 용해시켰다. 분획된 단백질은 immobilon polyvinylidene difuride membrane(Amersham, Arlington Hights, IL)에 전기 영동하고, 적절한 항체로 측정하였다. blots은 ECL(enhanced chemoluminescence) kit(Amersham)를 사용하여 전개하였다. 모든 면역 블로팅법 실험에서 blots은 단백질 로딩용 대조군으로 안티-β-액틴으로 재실험하였다.In addition, the expression of IκBα protein was measured by Western blot assay, in order to measure the IκBα degradation induced by TNF-α on stereocalpin A. Western blot assay was used by Cho SJ et al. (Cho SJ et al ., Toxicon , 42: 601-11, 2003). That is, the cells were pretreated with stereocalpine A (0.1-10 μg / ml) of Example 2 for 2 hours, and then cultured in fresh growth medium containing TNF-α (10 ng / ml) for 30 minutes or 8 hours. . After the treatment, the cells were washed twice in PBS, then buffer A [10 mM HEPES (pH 7.9), 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM DTT, 0.5 mM PMSF and Protease Inhibitor Cocktail (Sigma)]. Suspended in 70 μl and incubated on ice. After 15 minutes, 0.5% Nonidet P (NP) -40 was added to the cells to lyse the cells and then vortex for 10 seconds. Thereafter, the cytosolic cell extract was centrifuged at 1500 × g for 10 minutes at 4 ° C., and then recovered. The recovered nuclei are suspended in 50 μl of buffer C [20 mM HEPES (pH 7.9), 1.5 mM MgCl 2 , 420 mM NaCl, 0.2 mM EDTA, 25% v / v Glycerol, 0.5 mM PMSF and Protease Inhibitor Cocktail] and intermittently. Incubate on ice for 20 minutes with stirring. Cell nucleus extract was recovered after centrifugation at 13,000xg for 10 minutes at 4 ℃. Protein concentration was measured by Bio-Rad protein assay (Bio-Rad Lab, Hercules, CA) using BSA as a reference. Total cell lysate (20 μg) and nucleus extract (40 μg) were dissolved in 7.5% SDS-polyacrylamide gel, respectively. Fractionated proteins were electrophoresed on immobilon polyvinylidene difuride membrane (Amersham, Arlington Hights, IL) and measured with appropriate antibodies. Blots were developed using an enhanced chemoluminescence kit (Amersham). In all immunoblotting experiments, blots were retested with anti-β-actin as a protein loading control.
그 결과, 도 4(C)에 나타난 바와 같이, IκBα 특정 항체를 사용한 세포 추출물에 있어서, TNF-α로 처리한 샘플이 TNF-α로 처리하지 않은 샘플에 비해 IκBα의 빠른 저하가 야기되는 것으로 나타났으나, 스테레오칼핀 A에 의해 미리 배양된 세포에서는 IκBα가 저하되지 않는 것으로 나타났다. 이러한 결과는 TNF-α에 의해 유도되는 NF-κB 활성이 억제됨을 보여주는 것이다.
As a result, as shown in FIG. 4 (C), in the cell extract using the IκBα-specific antibody, the sample treated with TNF-α was shown to cause a rapid decrease in IκBα compared to the sample not treated with TNF-α. However, it was shown that IκBα was not lowered in cells pre-cultured by stereocalpin A. These results show that NF-κB activity induced by TNF-α is inhibited.
실험예Experimental Example 6: 6: TNFTNF -α처리에 의한 by α treatment MAPMAP kinaseskinases 및 And AktAkt 영향 측정 Impact measurement
TNF-α처리에 대응되는 MAP kinases(이하 MAPK라 함)의 활성은 부착분자의 발현을 증가시키는 것으로 알려져 있다(Ju et al ., IUBMB Life, 54:293-9, 2002; Ho et al ., Immunobilolgy, 213:533-44, 2008). 이에, 스테레오칼핀 A가 TNF-α에 의한 부착분자의 발현을 방해하는지를 확인하기 위하여 p38 MAPK, ERK1/2 및 JNK kinase pathway를 측정하였다. The activity of MAP kinases (hereinafter referred to as MAPK) corresponding to TNF-α treatment is known to increase the expression of adhesion molecules (Ju et al ., IUBMB Life , 54: 293-9, 2002; Ho et al . , Immunobilolgy , 213: 533-44, 2008). Thus, p38 MAPK, ERK1 / 2 and JNK kinase pathways were measured to determine whether stereocalpin A interferes with the expression of adhesion molecules by TNF-α.
그 결과, 도 5에 나타난 바와 같이, 처리되지 않은 세포의 p38 MAPK, ERK1/2 및 JNK 키나아제의 활성 수치는 TNF-α 처리에 의해 분명히 증가되는 것으로 나타났다. 그러나, 유도된 MAPK 활성은 2시간 동안 스테레오칼핀 A의 전처리로 인해 유의적으로 감소되는 것으로 나타났다. As a result, as shown in FIG. 5, the activity levels of p38 MAPK, ERK1 / 2 and JNK kinase in untreated cells were clearly shown to be increased by TNF-α treatment. However, induced MAPK activity was found to be significantly reduced due to pretreatment of stereocalpin A for 2 hours.
또한, Akt는 TNF-α에 의해 유도되는 부착분자 발현 및 NF-κB 활성의 조절의 원인을 알려져 있다(Kang et al ., Mol Pharmacol, 69:941-9, 2006; Oh JH and Kwon TK, Int Immunopharmacol, 9(5):614-9, 2009). 이에, TNF-α에 의해 유도된 Akt 활성 억제에 대한 스테레오칼핀 A 영향에 대하여 측정하였다. In addition, Akt is known to be a cause of adhesion molecule expression induced by TNF-α and regulation of NF-κB activity (Kang et al ., Mol Pharmacol , 69: 941-9, 2006; Oh JH and Kwon TK, Int Immunopharmacol , 9 (5): 614-9, 2009). Thus, the effect of stereocalpin A on the inhibition of Akt activity induced by TNF-α was measured.
그 결과, 도 5에 나타난 바와 같이, 실시예 2의 스테레오칼핀 A로 처리된 샘플은 TNF-α에 의해 유도된 Akt 활성이 감소되는 것으로 나타남에 따라, 스테레오칼핀 A가 MAPK 및 Akt pathway의 활성 감소에 의해 TNF-α에 의해 유도된 VCAM-1 및 ICAM-1의 발현을 감소시킬 수 있음을 확인할 수 있었다.
As a result, as shown in Figure 5, the sample treated with stereocalpin A of Example 2 is shown to decrease the Akt activity induced by TNF-α, stereocalpine A reduced the activity of MAPK and Akt pathway It was confirmed that it is possible to reduce the expression of VCAM-1 and ICAM-1 induced by TNF-α.
실험예Experimental Example 7: 7: TNFTNF -α 처리에 의한 by -α treatment ROSROS 생성 측정 Produce measure
혈관세포에서 TNF-α에 의해 유도된 ROS 생성은 NF-κB을 활성화시키는 것을 알려져 있다(Yoon et al ., Biochem Biophys Res Commun, 391(1):96-101, 2010; Zhang HS and Wang SQ, Free Radic Biol Med, 40(9):1664-74, 2006). 이하에서는 TNF-α에 의해 유도된 ROS의 생성에 대한 스테레오칼핀 A의 영향을 측정하였다. ROS 측정은 산화환원 감응 형광염료인 CMH2-DCFDA(5,6-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate; acetyl ester, Molecular Probes, Eugene, OR)는 flow cytometry에 의한 세포간의 ROS 수치를 평가하는데 사용하였다. VSMCs(3×106 cells/ml)은 2시간 동안 다양한 농도의 스테레오칼핀 A로 전처리한 다음, 4시간 동안 TNF-α(10ng/ml)를 첨가시켰다. 이와 같이 처리된 세포는 37℃에서 15분 동안 CMH2-DCFDA 5μM로 착색시켰다. 상기 세포는 어두운 상태에서 얼음에서 유지시킨 다음, Becton Dickinson FACSCalibur(BD Biosciences, San Jose, CA)을 사용하여 각 샘플에서 적어도 10,000 세포를 분석하였다. ROS generation induced by TNF-α in vascular cells is known to activate NF-κB (Yoon et. al ., Biochem Biophys Res Commun , 391 (1): 96-101, 2010; Zhang HS and Wang SQ, Free Radic Biol Med , 40 (9): 1664-74, 2006). In the following, the effect of stereocalpin A on the generation of ROS induced by TNF-α was measured. ROS measurement was performed by CMH2-DCFDA (5,6-chloromethyl-2 ′, 7′-dichlorodihydrofluorescein diacetate; acetyl ester, Molecular Probes, Eugene, OR), which is a redox-sensitive fluorescent dye. Used. VSMCs (3 × 10 6 cells / ml) were pretreated with various concentrations of stereocalpin A for 2 hours and then TNF-α (10ng / ml) was added for 4 hours. Cells thus treated were stained with 5 μM of CMH2-DCFDA at 37 ° C. for 15 minutes. The cells were kept on ice in the dark and then analyzed at least 10,000 cells in each sample using Becton Dickinson FACSCalibur (BD Biosciences, San Jose, Calif.).
그 결과, 10 μg/ml의 스테레오칼핀 A를 처리한 결과, ROS의 생산이 대략 60% 감소되었다. 즉, TNF-α에 의해 유도된 ROS의 생성은 스테레오칼핀 A의 농도에 의존적이면서 유의적으로 억제되며, 스테레오칼핀 A는 ROS 생성의 억제를 통한 NF-κB 활성을 방지하고, 항산화 활성을 가짐을 확인할 수 있다 (도 7).
As a result, treatment with 10 μg / ml stereocalpine A resulted in approximately 60% reduction in ROS production. In other words, the production of ROS induced by TNF-α is significantly dependent on the concentration of stereocalpin A, and stereocalpine A prevents NF-κB activity through inhibition of ROS production and has antioxidant activity. It can be confirmed (FIG. 7).
[[ 제조예Manufacturing example 1] One] 라말리나Ramalina 테라브라타Terrabrata ( ( RamalinaRamalina terebrataterebrata ) 추출물 함유 약제의 A) extract containing pharmaceuticals 제조예Manufacturing example
일일 0.1 내지 1,000mg의 용량의 본 발명에 따른 라말리나 테라브라타 (Ramalina terebrata) 유래 스테레오칼핀 A(stereocalpin A)는 약제학적으로 통상으로 사용되는 부형제나 보조제와 혼합하여 통상의 약제학적인 방법으로 경구 또는 비경구로 투여할 수 있는 약학적 제제로 제제화하여 의약품으로 사용될 수 있다. 다음에 제제실시예로서 제제의 제조예를 예시한다.
Ramalina terrabrata according to the present invention at a dose of 0.1 to 1,000 mg daily terebrata ) derived stereocalpin A (stereocalpin A) can be used as a medicament by being formulated into a pharmaceutical preparation that can be administered orally or parenterally by conventional pharmaceutical methods by mixing with excipients or adjuvants used conventionally. Next, the preparation example of a preparation is illustrated as a preparation example.
제제실시예Formulation Example 1: 캡슐제의 제조 1: Preparation of Capsule
유효성분: 10 mg Active ingredient: 10 mg
옥수수 전분: 100mgCorn Starch: 100mg
유당: 100mgLactose: 100 mg
스테아린산 마그네슘: 2mgMagnesium Stearate: 2mg
상기 성분을 혼합한 후, 통상의 캡슐제 제조방법에 따라 50mg의 젤라틴 캡슐에 충진하여 캡슐제를 제조하였다.
After mixing the ingredients, a capsule was prepared by filling 50 mg of gelatin capsules according to a conventional capsule preparation method.
제제실시예Formulation Example 2: 정제의 제조 2: Preparation of tablets
유효성분: 10 mgActive ingredient: 10 mg
옥수수전분: 100mgCorn Starch: 100mg
유당: 100mgLactose: 100 mg
스테아린산 마그네슘: 2mgMagnesium Stearate: 2mg
상기 성분들을 혼합한 후, 통상의 정제의 제조방법에 따라 50mg의 정제로 타정한다.
After mixing the components, it is compressed into 50 mg tablets according to the conventional method for preparing tablets.
제조예Manufacturing example 3: 3: 산제의Sanje 제조 Produce
유효성분: 10 mgActive ingredient: 10 mg
유당: 1gLactose: 1 g
스테아린산 마그네슘: 2mgMagnesium Stearate: 2mg
상기 성분들을 혼합하고, 기밀포에 충진하여 산제를 제조하였다.
The above ingredients were mixed and filled in airtight cloth to prepare a powder.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.
Claims (7)
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