KR100734512B1 - Composition comprising TanshinoneⅠ for treating and preventing liver disease - Google Patents
Composition comprising TanshinoneⅠ for treating and preventing liver disease Download PDFInfo
- Publication number
- KR100734512B1 KR100734512B1 KR1020060107756A KR20060107756A KR100734512B1 KR 100734512 B1 KR100734512 B1 KR 100734512B1 KR 1020060107756 A KR1020060107756 A KR 1020060107756A KR 20060107756 A KR20060107756 A KR 20060107756A KR 100734512 B1 KR100734512 B1 KR 100734512B1
- Authority
- KR
- South Korea
- Prior art keywords
- tanshinone
- hepatocytes
- damage
- oxidative
- liver
- Prior art date
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- AIGAZQPHXLWMOJ-UHFFFAOYSA-N Tanshinone I Chemical compound C1=CC2=C(C)C=CC=C2C(C(=O)C2=O)=C1C1=C2C(C)=CO1 AIGAZQPHXLWMOJ-UHFFFAOYSA-N 0.000 title claims abstract description 107
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Abstract
본 발명은 탄시논 I (Tanshinone I)을 함유하는 간질환 치료 및 예방용 조성물에 관한 것이다. 보다 구체적으로, 본 발명은 산화성 또는 비산화성 괴사 및 담즙산염에 의한 자가사멸 등의 간세포 손상을 억제하는 효능이 있는 탄시논 I을 유효성분으로 함유하는 간질환 치료 및 예방 효과가 뛰어난 약학조성물 및 건강보조식품을 제공할 수 있다.The present invention relates to a composition for treating and preventing liver disease containing tanshinone I. More specifically, the present invention provides a pharmaceutical composition and health excellent pharmaceutical treatment and prevention effect of liver disease containing tanshinone I as an active ingredient, which is effective in inhibiting hepatocellular damage such as oxidative or non-oxidative necrosis and bile salts autophagy. Supplements may be provided.
탄시논 I, 간질환, 간세포 괴사, 간세포 자가사멸, 담즙울체 Tanshinone I, liver disease, hepatocellular necrosis, hepatocellular apoptosis, cholestasis
Description
도 1은 본 발명에 따른 소수성 담즙산염 처리에 의한 간세포 DNA 단편화가 탄시논 I에 의해 억제됨을 보여준 도면이다.1 is a diagram showing that hepatocellular DNA fragmentation by hydrophobic bile salt treatment according to the present invention is inhibited by tanshinone I.
본 발명은 탄시논 I을 유효성분으로 함유하는 간질환 치료 및 예방용 조성물에 관한 것이다. 보다 구체적으로, 본 발명의 탄시논 I은 산화성 또는 비산화성 괴사 및 담즙산염에 의한 자가사멸 등의 간세포 손상을 억제하는 간질환 치료 및 예방을 위한 조성물에 관한 것이다. The present invention relates to a composition for treating and preventing liver disease containing tanshinone I as an active ingredient. More specifically, tanshinone I of the present invention relates to a composition for the treatment and prevention of liver disease that inhibits hepatocellular damage such as oxidative or non-oxidative necrosis and self-killing by bile salts.
간은 침묵의 장기로 완충작용이 탁월하여 간의 일부가 손상을 당해 그 기능을 수행하지 못하여도 남은 일부가 그 기능까지 수행함으로써 간 질환시 초기에 자각증상을 거의 느끼지 못하여 조기진단이 어렵고 말기에서야 발견되기 때문에 그 치료가 쉽지 않다. 또한 치료제도 거의없는 실정으로 우리나라에서도 연간 15,000 명 이상이 간 질환으로 사망하하는 등 조기진단의 어려움과 더불어 높은 사망률로 사회적인 문제를 야기하고 있다. The liver is a long-lasting silencer with excellent buffering effects, and some parts of the liver are damaged and unable to perform their functions. Because that is not easy to treat. In addition, there are few treatment systems, and in Korea, more than 15,000 people die of liver disease every year, causing social problems with high mortality and difficulty of early diagnosis.
간은 소화기계와 전신순환계 사이에 위치함으로써 소화기계로 들어온 생체외 물질로부터 전신을 방어하는 기능을 수행하고 있는 매우 중요한 장기이다. 일단 생체내로 들어온 생체외 물질이 간을 통과하는 까닭에 간은 영양소외에도 많은 독성물질에 노출될 위험이 다른 장기보다 많아 그만큼 손상당할 확률도 다른 장기에 비해 높다.The liver is a very important organ that is located between the digestive system and the systemic circulatory system, which functions to defend the whole body from ex vivo substances entering the digestive system. Since the extracellular substance that enters the living body passes through the liver, the liver is more at risk of being exposed to many toxic substances besides nutrients, which is more likely to be damaged than other organs.
간은 재생능력이 우수한 장기로 약간의 간세포 손상의 경우에는 충분히 정상으로 회복된다. 그러나 지속적으로 손상될 경우 간세포가 완전히 파괴되면서 파괴된 부분이 정상적으로 회복되지 못하고 결합조직이 과다축적되면서 간조직에 상흔(scar)이 형성되고 간 기능이 정상으로 회복되지 못하는 상태가 된다(Lissoos, et al., J. Clin . Gastroentero., 15(1), pp63-67, 1992). 간손상의 원인은 알콜, 담즙, 바이러스 등으로 매우 다양하지만 손상이 지속되어 만성 간질환(chronic liver disease; CLD) 상태가 되면 그 원인과는 상관없이 모두 염증상태 지속, 세포외기질(extracellular matrix; ECM)의 질적양적 변화가 유발된다. 세포수준에서의 변화로는 활성화된 간성상세포, 거식세포와 쿠퍼세포가 관여하게 되며, 분자적 수준에서는 여러 성장인자(growth factor), 사이토카인(cytokine), 케모카인(chemokine), ECM 조직체 및 구조 변화, 그리고 산화적 스트레스 관련 물질(oxidative stress-related molecule)이 주요한 역할을 담당한다. 원인 및 간섬유화 진행정도가 각기 다른 만성간질환 및 실험적 만성 간질환 모델 모두 공통적으 로 산화적 스트레스가 유발되는 것으로 보고되고 있다. 산화적 스트레스가 관측되는 간질환으로는 임상에서는 만성 HCV 감염, 알콜에 의한 간질환(alcohol liver disease), 유전적 혈색소증(genetic hemochromatosis), 윌슨씨병(Wilson's disease), 원발성 담즙성 간경변증(primary biliary cirrhosis) 및 기타 담즙울체성 간질환(cholestatic disease), 실험동물 모델로는 담관결찰(bile duct ligation) 모델, CCl4 투여(administration), 에탄올 투여(ethanol administration), 철분과부하(iron overload) 모델, 구리과부하(copper overload) 모델 등에서 관찰되고 있다. 산화적 스트레스란 산화적 스트레스 관련 물질이 세포의 산화방지제에 의한 보호(cellular antioxidant defence)를 능가하여 세포내 단백질, 지질 탄수화물, 핵산 등에 산화적 손상이 가해지는 현상이다. 세포내외로부터 유래되는 산화적 스트레스 관련 물질로는 (1)과산화수소 및 초산화 음이온(superoxide anion)과 같은 반응성 산소 중간물질(reactive oxygen intermediates; ROI), (2)지질과산화에 의한 알데히드 최종물질(aldehydic end-products) 등이 있다.The liver is an organ with good regenerative capacity, and in case of some hepatocellular damage, it is restored to normal enough. However, if the damage is continued, hepatic cells are completely destroyed, and the damaged parts cannot be recovered normally. As the connective tissue is over-accumulated, scars form in the liver tissue and liver function is not restored to normal (Lissoos, et. al., J. Clin . Gastroentero ., 15 (1) , pp 63-67, 1992). The causes of liver damage are various, such as alcohol, bile, virus, etc., but if the damage persists to chronic liver disease (CLD) status, regardless of the cause of all inflammatory conditions, extracellular matrix (extracellular matrix); Quantitative changes in ECM) are induced. At the cellular level, activated hepatic stellate cells, macrophages and Cooper cells are involved, and at the molecular level, several growth factors, cytokines, chemokines, ECM tissues and structures Change and oxidative stress-related molecules play a major role. Both chronic liver disease and experimental chronic liver disease models with different causes and progression of liver fibrosis have been reported to cause oxidative stress in common. Liver diseases for which oxidative stress is observed include clinically chronic HCV infection, alcoholic liver disease, genetic hemochromatosis, Wilson's disease, and primary biliary cirrhosis. ) And other cholestatic diseases, experimental animal models include bile duct ligation, CCl 4 administration, ethanol administration, iron overload model, copper Observations have been made in copper overload models. Oxidative stress is a phenomenon in which oxidative stress-related substances outperform cellular antioxidant defenses and cause oxidative damage to intracellular proteins, lipid carbohydrates, and nucleic acids. Oxidative stress-related substances derived from inside and outside cells include (1) reactive oxygen intermediates (ROI), such as hydrogen peroxide and superoxide anions, and (2) aldehyde final substances by lipid peroxidation. end-products).
간세포 손상은 간질환에서 일반적 현상으로 산화적 스트레스의 정도에 따라 간세포의 괴사(necrosis) 또는 자가사멸을 유도한다. 급성 간장애와 같이 산화적 스트레스가 심한 상태에서는 주요 세포 구조(미토콘드리아와 cytoskeletal protein), 거대분자(DNA, 지질, 효소단백질) 및 대사경로(metabolic pathway)가 손상되어 결국 괴사를 초래하게 된다. CLD 상태에서는 대부분 중등도의 산화적 스트 레스가 유지되는 것으로 생각되며 간섬유화 상태에서도 중등도의 산화적 스트레스가 유지되는 것으로 여겨지고 있다. Hepatocellular injury is a common phenomenon in liver disease and induces necrosis or self death of hepatocytes depending on the degree of oxidative stress. In severe oxidative stress conditions such as acute liver failure, major cellular structures (mitochondria and cytoskeletal proteins), macromolecules (DNA, lipids, enzyme proteins) and metabolic pathways are damaged and eventually lead to necrosis. It is thought that moderate oxidative stress is maintained in most CLD states, and moderate oxidative stress is maintained even in hepatic fibrosis.
산화적 스트레스가 상대적으로 약한 경우 즉, 미토콘드리아 기능을 비가역적으로 완전히 손상시킬 수 없는 경우에는 간세포가 괴사보다는 자가사멸이 유도되는 것으로 보고되고 있다. 담즙산염의 경우 여러 가지 경로로 간세포 자가사멸을 유도하는 것으로 보고되고 있으며 그중 원인으로 산화적 스트레스가 거론되고 있다 (Patel et al., Toxicol Appl . Pharmacol ., 142(1), pp116-22, 1997). Hepatic cells have been reported to induce self-killing rather than necrosis when oxidative stress is relatively weak, that is, when irreversible damage to mitochondrial function is not possible. In the case of bile salts, it has been reported to induce hepatocellular apoptosis through various pathways, among which oxidative stress is mentioned (Patel et al., Toxicol) Appl . Pharmacol ., 142 (1) , pp 116-22, 1997).
일반적으로 자가사멸은 염증반응을 유발하지 않으므로 세포의 자가사멸은 생리적 변화에 미치는 영향이 크지 않지만, 간세포 자가사멸은 이러한 일반적인 상태와는 많이 다른 것으로 보고되고 있다. 간세포 자가사멸이 심각한 경우 간세포내 여러 효소수치가 혈중 증가되며 또 탐식세포에 의한 제거가 충분하지 못한 상태가 되어 결국에는 염증반응이 유발되며 간조직의 정상적인 구조가 파괴된다. 과거에 괴사에 의해서 유도된다고 알려졌던 많은 질환들에서도 자가사멸이 중요한 역할을 하는 것으로 보고되고 있다. 알콜, 바이러스 및 담즙정체 등으로 인한 간질환에서도 심각한 간손상 및 간기능 저하는 간세포 자가사멸임이 보고되고 있다 (Gores et al., J. Hepatol ., 37(3), pp400-410, 2002).In general, self-killing does not cause an inflammatory response, so cell self-killing has little effect on physiological changes, but hepatocyte self-killing has been reported to be very different from this general condition. If hepatic cell death is severe, several enzyme levels in the hepatocytes are increased in blood, and the removal by the phagocytes is insufficient. Eventually, an inflammatory reaction is caused and the normal structure of the liver tissue is destroyed. Self-killing has been reported to play an important role in many diseases known to be induced by necrosis in the past. Severe liver damage and hepatic insufficiency have been reported in hepatic disease due to alcohol, virus and cholestasis (Gores et al., J. Hepatol ., 37 (3) , pp400-410, 2002).
담즙정체(cholestasis)는 여러 만성 진행성 간질환(chronic progressive liver disease)에서 발견되는 특징으로 결국에는 간부전(liver failure), 간경변증(cirrhosis)을 유발하며 결국 사망까지 초래한다. 담즙정체시 케노데옥시콜레이트(chenodeoxycholate)와 데옥시콜레이트(deoxycholate)와 같은 소수성 담즙산 염(hydrophobic bile salt)가 간세포내 축적됨으로써 간손상이 유도된다. 담즙정체에 의한 간손상시 임상에서의 조직관찰 결과 간세포 괴사보다 간세포의 자가사멸이 더 중요한 역할을 하는 것으로 보고되었다. 1차 배양한 간세포에 20-100μM의 글리코케노데옥시콜레이트 (glycochenodeoxycholate; GCDC)와 같은 소수성 담즙산염을 처리함으로써 간세포 자가사멸을 유도하는 생체 외 모델은 내인성 독성물질에 의한 간세포 자가사멸 연구에 유용하게 사용되고 있다. GCDC와 같은 소수성산(hydrophobic acid)은 파스 소중합체화 (Fas oligomerization)를 유도함으로써 자가사멸을 유발한다. 파스 소중합체화에 의해 프로카스파제-8 (procaspase-8)이 카스파제-8 (caspase-8)으로 분해 및 활성화되어 다운스트림 카스파제 (downstream caspase; effector caspase; caspase-3,-6,-7)를 활성화시킴으로써 자가사멸이 유도된다. 또 GCDC는 단백질 키나아제 C(PKC)의 활성 및 전위에 의해 세포내 마그네슘이 증가되어 Mg2 +-의존형 핵산 내 가수분해효소가 활성화됨으로써 DNA 분해가 유발된다고도 보고되고 있다.Cholestasis is a feature found in several chronic progressive liver diseases that eventually lead to liver failure, cirrhosis and eventually death. During bile stagnation, liver damage is induced by the accumulation of hydrophobic bile salts such as chenodeoxycholate and deoxycholate in hepatocytes. Clinical observations of hepatic injuries caused by cholestasis showed that hepatic cell death was more important than hepatic necrosis. An in vitro model that induces hepatocyte self-killing by treating 20-100 μM glycochenodeoxycholate (GCDC) with primary cultured hepatocytes is useful for the study of hepatocyte self-killing by endogenous toxins. It is used. Hydrophobic acid, such as GCDC, causes self-killing by inducing fas oligomerization. By caspase oligomerization, procaspase-8 is decomposed and activated by caspase-8, resulting in downstream caspase; effector caspase; caspase-3, -6,- Self-killing is induced by activating 7). Further GCDC is protein kinase C (PKC) increased magnesium to cells by the activation and the potential is of the Mg 2 + - has been reported that DNA degradation is caused by being within dependent nucleic acid hydrolase is active.
간조직중에는 영양물질 흡수 및 대사, 저장, 혈장단백질 합성 등과 같은 간의 주요기능을 수행하는 간세포(hepatocyte) 외에 간내 대식세포(macrophage)인 쿠퍼세포(Kupffer cell), 간 손상 시 결합조직을 과다 합성함으로써 간섬유화를 초래하는 간성상세포(hepatic stellate cell; HSC) 및 내피 세포(Endothelial cell), 오목 세포(Pit cell) 등이 존재한다. 이중 간세포는 간조직을 구성하는 전체 세포의 약 90%이상을 차지하며, 간의 주요기능을 수행하는 실질 세포(parenchymal cell)로 써 간질환시 간기능 저하는 바로 이 간세포 손상으로 인해 초래되는 것이고 간세포 손상이 지속될 경우 간섬유화와 간경화가 초래된다. 그러므로 간세포 손상 억제 및 예방은 간 질환 예방 및 치료에 있어서 가장 중요한 부분이라 할 수 있겠다. In liver tissues, hepatocytes that perform major functions of the liver, such as nutrient absorption and metabolism, storage, and plasma protein synthesis. In addition, hepatic macrophages (Kupffer cells) and over-synthesizing connective tissues during liver injury Hepatic stellate cells (HSC), endothelial cells, concave cells (Pit cells) and the like, which cause hepatic fibrosis, are present. Hepatocytes make up more than 90% of all the cells that make up the liver tissue, and are the parenchymal cells that perform the major functions of the liver. If damage persists, hepatic fibrosis and cirrhosis are caused. Therefore, the inhibition and prevention of hepatocellular damage may be the most important part in the prevention and treatment of liver disease.
이에, 본 발명자들은 간세포 손상을 효과적으로 보호 및 예방할 수 있는 효능의 물질을 찾고자 연구를 거듭한 결과, 단삼 (Salvia miltiorrhiza )으로부터 분리된 퀴노이드 디터페인(quinoid diterpene)계에 속하는 성분인 1,6-디메틸-페난트로[1,2-b]퓨란-10,11-다이온(1,6-Dimethyl-phenanthro [1,2-b]furan-10,11-dione)의 화학명을 갖는 탄시논 I (Tanshinone I)이 산화적 및 비산화적 원인에 의한 간세포 괴사 및 담즙산염에 의한 간세포의 자가사멸을 방지하여 간질환 치료 및 예방에 이용될 수 있음을 발견하고 본 발명을 완성하였다.Accordingly, the present inventors have repeatedly studied to find a substance having an effect that can effectively protect and prevent hepatocellular damage, Salvia ( salvia) 1,6-dimethyl-phenanthro [1,2- b ] furan-10,11-dione (1,6-Dimethyl-phenanthro [ 1 ], a component of the quinoid diterpene family isolated from miltiorrhiza ) Tanshinone I, which has the chemical name 1,2- b ] furan-10,11-dione), prevents hepatocellular necrosis caused by oxidative and non-oxidative causes and autogenous death of hepatocytes by bile salts. The present invention has been completed and found that it can be used for treatment and prevention.
본 발명의 목적은 산화성 또는 비산화성 괴사 및 담즙산염에 의한 자가사멸 등의 간세포 손상을 억제하는 효능이 있는 탄시논 I을 유효성분으로 함유하는 간질환 치료 및 예방을 위한 조성물을 제공하는데 있다.An object of the present invention is to provide a composition for treating and preventing liver disease containing tanshinone I, which is effective in inhibiting hepatocellular damage such as oxidative or non-oxidative necrosis and bile salts-induced death.
상기한 목적을 달성하기 위하여, 본 발명은 하기 화학식 1의 탄시논 I을 유효성분으로 함유하는 황달에 의한 간손상, 산화적 및 비산화적 스트레스에 의한 간손상 및 바이러스성 간염의 치료 및 예방을 위한 약학조성물을 제공한다.In order to achieve the above object, the present invention is for the treatment and prevention of liver damage due to jaundice containing tanshinone I of formula (1) as an active ingredient, liver damage due to oxidative and non-oxidative stress and viral hepatitis Provide a pharmaceutical composition.
본 발명에 따른 탄시논 I은 당업계 잘 알려진 방법으로 추출 또는 화학적 합성법에 의해 제조될 수 있으며, 또한 시판중인 것을 적의 선택하여 사용할 수 있으며, 그 방법 또는 물질은 특별히 한정되지 않는다. Tanshinone I according to the present invention may be prepared by extraction or chemical synthesis by methods well known in the art, and may also be selected and used commercially available, the method or material is not particularly limited.
이하, 본 발명을 보다 구체적으로 설명한다.Hereinafter, the present invention will be described in more detail.
예를 들어, 탄시논 I은 단삼으로부터 분리하거나 또는 상업적으로 시판되는 것, 즉 중국 약물생물제품 검정소(Chinese Institute for Drug and Biological Product Control, Beijing, China)에서 공급되는 시판품 (제품번호 0867-200104)을 구입하여 사용하여, 무게의 약 1내지 10배, 바람직하게는 2내지 5배의 물, 메탄올 또는 에탄올 등의 저급알코올의 극성용매, 바람직하게는 에탄올로 1 내지 12시간, 바람직하게는 2 내지 5시간동안 가열 환류 추출, 냉침, 열수추출, 초음파 추출방법, 바람직하게는 가열 환류추출을 이용하여 수득한 추출액을 여과, 감압농축하여 극성용매에 가용한 조추출물을 얻는 제 1단계;For example, tanshinone I is isolated from salvia or commercially available, that is, commercially available from the Chinese Institute for Drug and Biological Product Control (Beijing, China) (product no. 0867-200104). It is purchased and used, about 1 to 10 times the weight, preferably 2 to 5 times the water, a polar solvent of a lower alcohol such as methanol or ethanol, preferably ethanol for 1 to 12 hours, preferably 2 to A first step of filtrating and concentrating the extract obtained using a heated reflux extraction, cold soaking, hot water extraction, an ultrasonic extraction method, preferably heating reflux extraction, to obtain a crude extract available in a polar solvent;
상기의 조추출물을 물 또는 메탄올 등과 같은 극성용매 또는 이들의 혼합용매, 바람직하게는 60% 메탄올에 현탁하여, 이를 현탁액의 약 1내지 100배, 바람직 하게는 약 1내지 20배 부피의 헥산, 에틸아세테이트, 클로로포름, 디클로로메탄과 같은 비극성용매, 바람직하게는 헥산과 클로로포름을 가하여 극성용매가용층에 비극성용매 가용층을 1회 내지 10회, 바람직하게는 2회 내지 5회 분획하고 감압농축하여 비극성용매가용및 극성용매가용부를 얻는 제 2단계;The crude extract is suspended in a polar solvent such as water or methanol, or a mixed solvent thereof, preferably 60% methanol, which is about 1 to 100 times the suspension, preferably about 1 to 20 times the volume of hexane, ethyl. Nonpolar solvents such as acetate, chloroform and dichloromethane, preferably hexane and chloroform, add the nonpolar solvent soluble layer to the polar solvent soluble layer once to 10 times, preferably 2 to 5 times, and concentrated under reduced pressure to produce a nonpolar solvent. Obtaining a soluble and polar solvent soluble part;
이어서 상기 비극성용매가용부를 디클로로메탄-에틸아세테이트 혼합용매를 용출용매로 하여 실리카겔 컬럼 크로마토그래피를 수행하여 용출물을 박층 크로마토그래피(TLC 실리카 겔)의 패턴에 따라 1 내지 7개, 바람직하게는 2내지 6개의 분획으로 분리하여 그 분획물을 얻는 제 3단계 ; Subsequently, silica gel column chromatography was carried out using the dipolar methane-ethyl acetate mixed solvent as the elution solvent, and the eluate was 1 to 7, preferably 2 to 7, according to the pattern of thin layer chromatography (TLC silica gel). A third step of separating the fraction into six fractions to obtain the fraction;
상기 분획물중 2번째 분획물인 분획 2를 헥산: 에틸아세테이트 중량대비 10:1 내지 1:10 바람직하게는 7 : 1~ 1 : 7 의 배합비를 가지는 이동상을 이용하여 실리카겔 컬럼 크로마토그래피를 수행하여 1~10개, 바람직하게는 2~8개의 소분획물을 얻는 제 4단계; Fraction 2, which is the second fraction of the fraction, was subjected to silica gel column chromatography using a mobile phase having a mixing ratio of 10: 1 to 1:10, preferably 7: 1 to 1: 7, based on the weight of hexane: ethyl acetate. A fourth step of obtaining ten, preferably two to eight small fractions;
마지막으로 상기 소분획 3을 헥산: 에틸아세테이트 중량대비 15 : 1 내지 1 : 15 바람직하게는 10 : 1~ 1 : 10 의 배합비를 가지는 이동상을 이용하여 실리카겔 컬럼 크로마토그래피를 수행하여 탄시논 I 을 얻는 제 5 단계로 구성된 분리 공정을 통하여 수득할 수 있다. Finally, the subfraction 3 was subjected to silica gel column chromatography using a mobile phase having a mixing ratio of 15: 1 to 1:15, preferably 10: 1 to 1:10, to the weight of hexane: ethyl acetate to obtain tanshinone I. It can be obtained through a separation process consisting of a fifth step.
또한 추가로 통상의 분획 공정을 수행할 수도 있다(Harborne, J.B., Phytochemical methods: A guide to modern techniques of plant analysis , 3 rd Ed ., pp6-7, 1998). In addition, conventional fractionation processes can also be carried out (Harborne, JB, Phytochemical methods: A guide). to modern techniques of plant analysis , 3 rd Ed . , pp 6-7, 1998).
본 발명은 상기의 제조공정으로 얻어진 탄시논 I 화합물을 유효성분으로 함 유하는 간질환 치료 및 예방을 위한 약학조성물을 제공한다.The present invention provides a pharmaceutical composition for the treatment and prevention of liver disease containing the tanshinone I compound obtained by the above manufacturing process as an active ingredient.
본 발명에 따른 탄시논 I은 간세포에서 3차 부틸-하이드로페록사이드(tertiary-butyl hydroperoxide ; tBH)에 의한 산화적 간세포 손상 및 D-갈락토사민 (D-Galactosamine ; GalN)에 의한 비산화적 간세포 손상시, 세포 괴사의 지표인 락테이트 디하이드로게나제(Lactate dehydrogenase ; LDH)의 세포 밖으로의 유출을 탁월하게 억제하여 산화 및 비산화성 간손상으로 인한 간질환 치료 및 예방을 위한 조성물으로도 이용될 수 있다.Tanshinone I according to the present invention is oxidative hepatocyte damage by tertiary-butyl hydroperoxide (tBH) in hepatocytes and non-oxidative hepatocyte damage by D-Galactosamine (GalN). Lactate dehydrogenase (LDH), an indicator of cell necrosis, can be used as a composition for treating and preventing liver disease caused by oxidative and non-oxidative liver damage. have.
또한, 본 발명에 따른 탄시논 I 은 담즙울체시 나타나는 간세포의 자가사멸 현상을 억제하여 담즙울체에 의한 간질환 치료 및 예방을 위한 조성물으로도 이용될 수 있다.In addition, tanshinone I according to the present invention may be used as a composition for treating and preventing hepatic diseases caused by the cholestasis by inhibiting the autopopulation of hepatocytes during cholestasis.
본 발명의 간질환의 치료 및 예방용 약학조성물은, 조성물 총 중량에 대하여 상기 화합물을 0.1 내지 50 중량%로 포함한다. The pharmaceutical composition for treating and preventing liver disease of the present invention comprises 0.1 to 50% by weight of the compound based on the total weight of the composition.
본 발명의 화합물을 포함하는 약학조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다. Pharmaceutical compositions comprising the compounds of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.
본 발명의 화합물의 약학적 투여 형태는 이들의 약학적 허용 가능한 염의 형태로도 사용될 수 있고, 또한 단독으로 또는 타 약학적 활성 화합물과 결합뿐만 아니라 적당한 집합으로 사용될 수 있다. Pharmaceutical dosage forms of the compounds of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.
본 발명에 따른 화합물을 포함하는 약학조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 화합물을 포 함하는 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.The pharmaceutical compositions comprising the compounds according to the present invention may be prepared in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Can be formulated and used. Carriers, excipients and diluents that may be included in the composition comprising the compound include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose, or the like. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
본 발명의 화합물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 그러나 바람직한 효과를 위해서, 본 발명의 화합물은 1일 0.0001 내지 100mg/kg으로, 바람직하게는 0.001 내지 10mg/kg으로 투여하는 것이 좋다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다. Preferred dosages of the compounds of the present invention depend on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, but may be appropriately selected by those skilled in the art. However, for the desired effect, the compound of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 10 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.
본 발명의 화합물은 쥐, 생쥐, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁 내 경막 또는 뇌혈관 내(intracerebroventricular) 주사에 의해 투여될 수 있다. The compounds of the present invention can be administered to mammals such as mice, mice, livestock, humans, and the like by various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.
본 발명은 간질환 예방 및 개선 효과를 나타내는 탄시논 I 화합물 및 식품학적으로 허용 가능한 식품보조 첨가제를 포함하는 건강보조식품을 제공한다. 본 발명의 화합물을 첨가할 수 있는 식품으로는, 예를 들어, 각종 식품류, 껌, 차, 비타민 복합제, 분말, 과립, 정제, 캡슐 또는 음료, 건강 기능성 식품류 등이 있다. The present invention provides a dietary supplement comprising a tanshinone I compound and a foodstuff acceptable food supplement additive exhibiting an effect of preventing and improving liver disease. Examples of the food to which the compound of the present invention can be added include various foods, gums, teas, vitamin complexes, powders, granules, tablets, capsules or beverages, and health functional foods.
또한, 간질환 예방 및 개선을 위한 식품 또는 음료에 첨가될 수 있다. 이 때, 식품 또는 음료 중의 상기 화합물의 양은 전체 식품 중량의 0.01 내지 15 중량%로 가할 수 있으며, 건강 음료 조성물은 100 ㎖를 기준으로 0.02 내지 5 g, 바람직하게는 0.3 내지 1g의 비율로 가할 수 있다. It may also be added to foods or beverages for the prevention and improvement of liver disease. At this time, the amount of the compound in the food or beverage may be added at 0.01 to 15% by weight of the total food weight, the health beverage composition may be added in a ratio of 0.02 to 5 g, preferably 0.3 to 1g based on 100 ml. have.
본 발명의 건강 음료 조성물은 지시된 비율로 필수 성분으로서 상기 화합물을 함유하는 외에는 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진등) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100㎖당 일반적으로 약 1 내지 20g, 바람직하게는 약 5 내지 12g이다. The health beverage composition of the present invention is not particularly limited to other ingredients except for containing the compound as essential ingredients in the indicated proportions, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.
상기 외에 본 발명의 화합물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 본 발명의 화합물들은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 그렇게 중요하진 않지만 본 발명의 화합물 100 중량부 당 0 내지 약 20 중량부의 범위에서 선택되는 것이 일반적이다. In addition to the above, the compounds of the present invention include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the compounds of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is usually selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the compound of the present invention.
이하, 본 발명을 하기 실시예 및 실험예에 의해 상세히 설명한다. Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples.
단, 하기 실시예 및 실험예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예 및 실험예에 의해 한정되는 것은 아니다.However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.
실시예Example 1. One. 탄시논Tanshinone I의 분리 및 구조동정 I separation and structure identification
1-1. 1-1. 단삼Salvia 조추출물의Crude extract 제조 Produce
건조한 단삼 600g을 경동시장에서 구입하여 분말화한 후, 에탄올 2ℓ를 가하여 2시간 동안 가열 환류 추출하고, 진공 여과하여 상층액을 회수하였다. 이 과정을 3회 반복하여 상층액을 모은 후, 감압 농축(EYELA사, N-1000, 일본)하여 단삼 조추출물 120g을 수득하였다. 600 g of dried sweet ginseng was purchased from Kyungdong Market, powdered, 2 L of ethanol was added thereto, heated to reflux for 2 hours, and vacuum filtered to recover the supernatant. This process was repeated three times to collect the supernatant, and then concentrated under reduced pressure (EYELA Co., N-1000, Japan) to obtain 120 g of crude salvia extract.
1-2. 1-2. 단삼Salvia 비극성용매가용추출물의Nonpolar Solvent Soluble Extracts 제조 Produce
상기 실시예 1-1 단계에서 얻은 단삼 조추출물 120g을 60% 메탄올 1ℓ에 현탁시킨 후, 헥산과 클로로포름을 가각 0.8ℓ씩 가하여 2회씩 순차적으로 분배한 다음 각각의 용매 분획을 감압농축하여 헥산 가용부와 클로로포름 가용부를 얻었다. After 120 g of the crude extract of Salvia ginseng obtained in Example 1-1 was suspended in 1 L of 60% methanol, hexane and chloroform were added to each 0.8 L, and then sequentially dispensed twice. Each solvent fraction was concentrated under reduced pressure. And a chloroform soluble portion were obtained.
1-3. 1-3. 단삼Salvia 추출물로부터 From extract 탄시논Tanshinone I의 분리 I separation
상기 실시예 1-2에서 얻은 클로로포름 가용부 10.5g은 세파덱스 LH-20 컬럼에 걸은 다음 디클로로메탄 0.5ℓ와 디클로로메탄과 메탄올 25:1 혼합용매 (2.0 ℓ)로 용출하고, 상기 용출물을 박층 크로마토그래피(TLC 실리카 겔)의 패턴에 따라 4개 분획으로 나누었다. 그 중 분획 2(2.22 g)를 헥산 : 에틸아세테이트 (6 : 1, v/v) 혼합용매(1 ℓ)를 이동상으로 사용하여 실리카 겔 컬럼 크로마토그래피를 실시하였다. 실시 후 8개의 소분획물을 얻어 이 중 소분획 3 (370 mg)을 헥산 : 에틸아세테이트 (9 : 1, v/v) 혼합용매를 이동상으로 사용하여 실리카 겔 컬럼 크 로마토그래피를 실시하였다. 그 결과, 하기 물성치를 갖는 적갈색의 침상결정물의 탄시논 I (29 mg)을 분리하여 하기 실험의 시료로 사용하였다.10.5 g of the chloroform soluble part obtained in Example 1-2 was suspended on a Sephadex LH-20 column, and then eluted with 0.5 l of dichloromethane, 25: 1 mixed solvent of dichloromethane and methanol (2.0 L), and the eluate was thin. Divided into four fractions according to the pattern of chromatography (TLC silica gel). Among them, fraction 2 (2.22 g) was subjected to silica gel column chromatography using hexane: ethyl acetate (6: 1, v / v) mixed solvent (1 L) as a mobile phase. Subsequently, eight small fractions were obtained. Among them, small fraction 3 (370 mg) was subjected to silica gel column chromatography using a hexane: ethyl acetate (9: 1, v / v) mixed solvent as a mobile phase. As a result, tanshinone I (29 mg) of reddish brown needles having the following physical properties were separated and used as a sample for the following experiment.
융점 ; 232-233℃Melting point; 232-233 ℃
1H-NMR (400MHz, CDCl3) ppm; 9.22 (1H, d, J=8.9 Hz), 8.25 (1H, d, J=8.7 Hz), 7.77 (1H, d, J=8.7 Hz), 7.52 (1H, dd, J=8.9, 6.9 Hz), 7.34 (1H, d, J=6.9 Hz), 7.28 (1H, s), 2.66 (3H, br. s), 2.27 (3H, br. s) 1 H-NMR (400 MHz, CDCl 3 ) ppm; 9.22 (1H, d, J = 8.9 Hz), 8.25 (1H, d, J = 8.7 Hz), 7.77 (1H, d, J = 8.7 Hz), 7.52 (1H, dd, J = 8.9, 6.9 Hz), 7.34 (1H, d, J = 6.9 Hz), 7.28 (1H, s), 2.66 (3H, br.s), 2.27 (3H, br.s)
13C-NMR (100MHz, CDCl3) ppm; 183.55, 175.72, 161.25, 142.11, 135.26, 133.72, 132.99, 132.83, 130.73, 129.70, 128.43, 124.85, 123.23, 121.84, 120.57, 118.80, 19.93, 8.88 13 C-NMR (100 MHz, CDCl 3 ) ppm; 183.55, 175.72, 161.25, 142.11, 135.26, 133.72, 132.99, 132.83, 130.73, 129.70, 128.43, 124.85, 123.23, 121.84, 120.57, 118.80, 19.93, 8.88
(+)-ESI-MS; [M+Na]+ m/z 299, [M+H]+m/z 277(+)-ESI-MS; [M + Na] + m / z 299, [M + H] + m / z 277
참고예Reference Example 1. 흰쥐로부터 간세포 분리 및 배양 1. Isolation and Culture of Hepatocytes from Rats
새글랜(Seglen) 등의 방법 (Seglen et al., Exp . Cell . Res ., 82, pp391-398, 1973)에 따라 흰쥐로부터 간세포를 분리하였다. 흰쥐를 마취하여 복부를 절개한 후, 간문맥에 삽관하여 5% CO2 및 95% O2의 혼합기체를 불어넣은 37℃의 Ca2 +, Mg2+없는 행크의 균형염 용액(Ca2 +, Mg2 + free Hank's balanced salt solution)을 관 에 흘려보내 간 내 혈액을 제거하였다. 그 후, 0.05% Ⅳ형 콜라겐나아제(collagenase type IV) 및 1mM CaCl2 용액을 흘려보냈다. 간 조직을 몸체로부터 떼어낸 다음 간세포 현탁액을 만들어 50×g에서 2분간 원심분리하고 침전된 간세포를 취해 Ca2 +, Mg2 +-없는 행크의 균형염 용액으로 2회 세척한 다음 1형 콜라겐으로 코팅된 배양용기에 10%(v/v) 우태아 혈청(fetal bovine serum, FBS; GibcoBRL), 10-8 M 인슐린, 50 U/㎖의 페니실린 및 50 ㅅg/㎖의 스트렙토마이신(Sigma)이 첨가된 윌리암스 배지 E(Williams'Medium E; WME, GibcoBRL, USA)를 배양액으로 하여 세포수가 1×106 세포수/㎖ 되도록 한 후, 5% CO2, 37℃의 조건에서 배양하였다. 2시간 배양 후 세포를 행크의 균형염 용액으로 세척하여 붙지 않은 간세포는 제거하고 새 배양액으로 교체한 다음 5% CO2, 37℃의 조건에서 16시간동안 배양시킨 다음 실험에 사용하였다.Hepatocytes were isolated from rats according to the method of Seglen et al. (Seglen et al., Exp . Cell . Res ., 82, pp391-398, 1973). Rats were anesthetized by anesthesia, intubated into the portal vein, 5% CO 2 And Hank's balanced salt solution (Ca 2 + , Mg 2 + free Hank's balanced salt solution) without Ca 2 + and Mg 2+ at 37 ° C with a mixed gas of 95% O 2 . Removed. Thereafter, 0.05% collagen type IV (collagenase type IV) and 1 mM CaCl 2 solution were flowed. The liver tissue was removed from the body, and then made a hepatocyte suspension, centrifuged at 50 × g for 2 minutes, the precipitated hepatocytes were taken, washed twice with a balanced salt solution of Ca 2 + , Mg 2 + -free Hank, and then with type 1 collagen. 10% (v / v) fetal bovine serum (FBS; GibcoBRL), 10 -8 M insulin, 50 U / ml penicillin and 50 mg / ml streptomycin (Sigma) Cell number 1 × 10 6 using the added Williams media E (WME, GibcoBRL, USA) as a culture medium The cells were allowed to have a number of cells / ml and cultured at 5% CO 2 and 37 ° C. After 2 hours of incubation, the cells were washed with Hank's balanced salt solution to remove non-adherent hepatocytes, replaced with fresh culture, and then incubated for 16 hours under conditions of 5% CO 2 , 37 ° C and used for the experiment.
참고예Reference Example 2. 1차 배양 간세포에서 2. In primary cultured hepatocytes 간손상Liver damage 유도 및 처리 Induction and treatment
2-1. 3차 부틸 2-1. Tertiary butyl 하이드로페록사이드에On hydroperoxide 의한 by 간손상Liver damage 유도 Judo
참고예 1과 같이 분리배양한 간세포에 산화적 손상을 유발하기 위하여 1.5 mM 3차 부틸 하이드로페록사이드 (tBH, Sigma, USA)를 처리하였다. 간세포에 탄시논 I 을 함유하는 배양액을 가한 다음 1.5 mM tBH를 1.0시간 동안 처리하였다. tBH는 150 mM이 되도록 HBSS로 희석한 다음 배양액에 가하여 1.5 mM이 되도록 준비한 것을 사용하였다. 실험군은 하기 표 1과 같이 설정하였다.1.5 mM tertiary butyl hydroperoxide (tBH, Sigma, USA) was treated to induce oxidative damage to the isolated cultured hepatocytes as in Reference Example 1. A culture solution containing tanshinone I was added to the hepatocytes and then treated with 1.5 mM tBH for 1.0 hour. tBH was diluted with HBSS to 150 mM and then added to the culture solution to prepare 1.5 mM. The experimental group was set as shown in Table 1 below.
2-2. D-2-2. D- 갈락토사민(D-galactosamine)에Galactosamine (D-galactosamine) 의한 by 간손상Liver damage 유도 Judo
탄시논 I 의 간세포 보호효과가 비산화적 손상에 대해서도 유효한지 여부를 알아보기 위하여 D-갈락토사민(GalN; Sigma, USA)으로도 간세포 손상을 유도하여 그 보호효과를 확인하였다. GalN은 사람에서의 바이러스성 간염과 유사한 간 손상을 유도하는 것으로 알려져 있으며, 우리딘 뉴클레오티드 (uridine nucleotide)를 고갈시킴으로써 원형질막 단백질 (plasma membrane protein)과 RNA 합성을 저해함으로써 간세포 손상을 유발하는 것으로 알려져 있다 (Wu. et al., Hepatology , 23(2), pp359-65, 1996).To determine whether the hepatocellular protective effect of tanshinone I is effective against non-oxidative damage, D-galactosamine (GalN; Sigma, USA) was also induced to induce hepatocyte damage. GalN is known to induce hepatic damage similar to viral hepatitis in humans, and is known to cause hepatocellular damage by inhibiting plasma membrane protein and RNA synthesis by depleting uridine nucleotides. (Wu. Et al., Hepatology , 23 (2) , pp 359-65, 1996).
참고예 1에서와 같이 분리 배양한 간세포에 손상을 유발하기 위하여 30 mM D-GalN을 처리하였다. 간세포에 탄시논 I 을 함유하는 배양액을 가하여 10분 동안 전처리한 후 30 mM GalN를 가하여 24시간 동안 처리하였다. GalN는 HBSS에 용해한 다음 배양액에 가하여 30 mM이 되도록 준비한 것을 사용하였다. 실험군은 하기 표 2과 같이 설정하였다.As in Reference Example 1, 30 mM D-GalN was treated to cause damage to the cultured hepatocytes. A culture solution containing tanshinone I was added to the hepatocytes, pretreated for 10 minutes, and then treated with 24 mM GalN for 24 hours. GalN was prepared by dissolving in HBSS and then added to the culture to 30 mM. The experimental group was set as shown in Table 2 below.
2-3. 소수성 2-3. Hydrophobic 담즙산염에On bile salts 의한 간세포 자가사멸 유도 Induced Hepatocellular Apoptosis
참고예 1에서와 같이 분리 배양한 간세포에 담즙울체와 같은 조건을 형성하여 자가사멸을 유발하기 위하여, 담즙울체시 간독성을 나타내는 소수성 담즙산염인 글리코케노디옥시콜레이트(glycochenodeoxycholate ; GCDC)를 간세포에 처리한다. 간세포에 탄시논 I을 10분 전처리하고 GCDC 100 μM을 가하여 4시간 동안 배양하였다. GCDC는 HBSS에 용해한 다음 0.22 ㎛ 필터로 여과한 다음 사용하였다. 실험군은 다음과 같이 설정하였다.Hepatic cells were treated with glycochenodeoxycholate (GCDC), a hydrophobic bile salt that exhibits hepatotoxicity in bile ducts, to form autologous death by forming conditions such as bile ducts in isolated and cultured hepatocytes as in Reference Example 1. do. Hepatocytes were pretreated with tanshinone I for 10 minutes and incubated for 4 hours with 100 μM of GCDC. GCDC was used after dissolving in HBSS and filtering with a 0.22 μm filter. The experimental group was set as follows.
실험예Experimental Example 1. One. tBHtBH 산화적Oxidative 손상에 대한 Against damage 탄시논Tanshinone I의 효과 측정 Measure the effect of I
1-1. 간세포 보호활성 측정1-1. Hepatoprotective activity measurement
참고예 2-1에서와 같이 간세포에 tBH를 처리시 산화적 손상이 유도되며; 산화적 손상으로 인한 간세포 괴사과정이 진행되면서 세포막 유동성이 증대되고; 세포로부터 배양액으로 락테이트 디하이드로게나제(lactate dehydrogenase;LDH)가 흘러나오게 된다. 이 LDH 농도를 자동건조 화학 분석기 (autodry chemistry analyzer; SPOTCHEMTM SP4410, Arkray, Japan)를 이용하여 측정하였다.Oxidative damage is induced upon treatment of tBH in hepatocytes as in Reference Example 2-1; Hepatic necrosis process due to oxidative damage increases cell membrane fluidity; Lactate dehydrogenase (LDH) flows from the cell into the culture. This LDH concentration was measured using an autodry chemistry analyzer (SPOTCHEM ™ SP4410, Arkray, Japan).
간세포의 생존율을 측정하기 위하여 tBH와 탄시논 I 함유 배양액을 걷어내고 3-(4,5-디메틸티아졸-1-일)2,5-디페닐 테트라졸리움 브로마이드 (3-(4,5- dimethylthiazol-1-yl) 2,5-diphenyl tetrazolium bromide ; MTT)를 0.5 ㎎/㎖ 함유하는 배양액 중에서 2시간 동안 배양한 다음 살아있는 간세포에 의하여 생성된 포마잔 (formazan)을 디메칠술폭사이드 (dimethylsulfoxide)에 용해하여 540 nm에서의 흡광도값를 구하여 하기 표 4에 나타내었다. To determine the viability of hepatocytes, the culture medium containing tBH and tanshinone I was removed and 3- (4,5-dimethylthiazol-1-yl) 2,5-diphenyl tetrazolium bromide (3- (4,5-dimethylthiazol 1-yl) 2,5-diphenyl tetrazolium bromide (MTT) in a culture medium containing 0.5 mg / ml for 2 hours, and then formazan (formazan) produced by living hepatocytes was transferred to dimethylsulfoxide. Dissolved to obtain an absorbance value at 540 nm is shown in Table 4 below.
상기 결과로부터, 본 발명에 따른 탄시논 I은 산화적 간세포 손상 유도시 LDH 유출 억제 및 세포 사멸(cell death)을 억제하여 간세포 보호활성을 가짐을 확인할 수 있었다. From the above results, it was confirmed that tanshinone I according to the present invention has hepatocellular protective activity by inhibiting LDH efflux and cell death upon inducing oxidative hepatocyte damage.
2-2. 간세포 내 2-2. Within hepatocytes ROSROS 생성 억제 및 Generation suppression and GSHGSH 고갈 억제 활성 측정 Depletion Inhibition Activity Measurement
간세포에서의 반응성 산소종 (reactive oxygen species ; ROS)의 생성 정도를 2',7'-디클로로플루오레신 아세테이트 (2',7'-dichlorofluorecin acetate ; DCFH-DA)를 이용하여 측정하였다. DCFH-DA는 비극성 화합물(nonpolar compound)로, 세포내로 용이하게 유입(uptake)된 후 형광을 띠지 않는 DCFH로 가수분해되어 세포내에 갇히게(trap) 되며, ROS 등의 산화성 물질에 의해 형광을 띠는 2',7'-디클로로플루오레신 (2',7'-dichlorofluorescein ; DCF)으로 변환되는 특징을 갖는 물질로, 세포내 ROS의 존재 정도를 측정하는데 사용된다. 참고예 2-1에서와 같이 탄시논 I과 tBH를 처리한 간세포에 20 μM DCFH-DA를 가하여 15분 동안 배양한 다음 간세포 중 형광강도를 여기파장 485 ㎚, 방출파장 530 ㎚에서 형광광도계 (RF-5301, Shimadzu, Japan)를 이용하여 측정하였다. 각 실험군의 DCF 생성능은 2군에서의 DCF 형광강도를 100%로 하여 산출하였다. The generation of reactive oxygen species (ROS) in hepatocytes was measured using 2 ', 7'-dichlorofluorescein acetate (2', 7'-dichlorofluorecin acetate; DCFH-DA). DCFH-DA is a nonpolar compound, which is easily taken up into cells and then hydrolyzed to non-fluorescent DCFH and trapped in cells, and is fluorescent by oxidizing substances such as ROS. Substances characterized by conversion to 2 ', 7'-dichlorofluorescein (DCF) and are used to measure the presence of intracellular ROS. As in Reference Example 2-1, 20 μM DCFH-DA was added to the hepatocytes treated with tanshinone I and tBH, followed by incubation for 15 minutes, and the fluorescence intensity of the hepatocytes was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. -5301, Shimadzu, Japan). DCF generation ability of each experimental group was calculated by the DCF fluorescence intensity in group 2 as 100%.
세포내 항산화작용을 나타내는 방어 기작으로 세포내 자유라디칼(free raical )또는 과산화물을 제거함으로써 보호작용을 나타내는 GSH(환원형)(Chen HW, Chiang T, Food Chem . Toxicol, 38, pp.1089-1096, 2000)의 함유량은 착색 검정 키트(colorimetric assay kit; Bioxytech GSH-400, OXIX International, Portland, USA)를 사용하여 키트의 사용방법에 따라 측정하였으며, 측정결과를 하기 표 5에 나타내었다.It is a defense mechanism that shows intracellular antioxidant activity. GSH (reduced type) which shows protection by removing free radicals or peroxides in cells (Chen HW, Chiang T, Food Chem . Toxicol , 38 , pp. 1089-1096, 2000) was measured using a colorimetric assay kit (Bioxytech GSH-400, OXIX International, Portland, USA) according to the method of use of the kit. It is shown in Table 5 below.
상기 결과로부터, 탄시논 I은 간세포 내 산화적 손상의 원인이 되는 ROS를 유의적으로 감소시키고 세포내 GSH의 고갈을 억제하여, 간세포의 산화적 손상을 효과적으로 억제함을 확인할 수 있었다.From the above results, it was confirmed that tanshinone I significantly reduced ROS causing oxidative damage in hepatocytes and inhibited depletion of intracellular GSH, effectively inhibiting oxidative damage of hepatocytes.
실험예Experimental Example 2. 2. GalNGalN 간세포 손상에 대한 For liver cell damage 탄시논Tanshinone I의 효과 측정 Measure the effect of I
2-1. 간세포 보호활성 측정2-1. Hepatoprotective activity measurement
상기 참고예 2-2의 손상을 유발한 간세포에 탄시논 I을 처리하여 간세포로부터 배양액으로 흘러나온 락테이트 디하이드로게나제 수치 (LDH)를 자동건조 화학 분석기 (autodry chemistry analyzer; SPOTCHEMTM SP4410, Arkray, Japan)를 이용하여 측정하였다.Lactate dehydrogenase levels (LDH) flowed from the hepatocytes into the culture medium by treating tanshinone I to the hepatocytes causing the damage of Reference Example 2-2 were analyzed by an autodry chemistry analyzer (SPOTCHEM ™ SP4410, Arkray). , Japan).
간세포의 생존율을 측정하기 위하여 GalN과 탄시논 I 함유 배양액을 걷어내고 3-(4,5-디메틸티아졸-1-일)2,5-디페닐 테트라졸리움 브로마이드를 0.5 ㎎/㎖ 함유하는 배양액 중에서 2시간 동안 배양한 다음 살아있는 간세포에 의하여 생성된 포마잔을 디메칠술폭사이드에 용해하여 540nm에서의 흡광도값을 구하였다. 그 결과를 표 6에 나타내었다.In order to measure the survival rate of hepatocytes, the culture medium containing GalN and tanshinone I was removed, and the culture solution containing 0.5 mg / ml of 3- (4,5-dimethylthiazol-1-yl) 2,5-diphenyl tetrazolium bromide After incubation for 2 hours, formazan produced by living hepatocytes was dissolved in dimethylsulfoxide to obtain absorbance at 540 nm. The results are shown in Table 6.
상기 결과로부터, 본 발명에 따른 탄시논 I은 비산화적 손상에 의한 간세포 손상 유도시에도 LDH 유출 억제 및 세포 사멸을 억제하여 간세포 보호활성을 가짐을 확인할 수 있었다. From the above results, it was confirmed that tanshinone I according to the present invention had hepatocellular protective activity by inhibiting LDH efflux and cell death even when inducing hepatocellular injury by non-oxidative damage.
2-2. 간세포 내 2-2. Within hepatocytes GSHGSH 고갈 억제 활성 측정 Depletion Inhibition Activity Measurement
실험예 1-2에서와 같이 탄시논 I과 GalN를 처리한 간세포내 GSH(환원형)의 함유량을 착색 검정 키트(colorimetric assay kit; BioxytechTM GSH-400, OXIS International, Portland, USA)를 사용하여 키트의 사용방법에 따라 측정하였으며, 그 결과를 하기 표 7에 나타내었다.The content of GSH (reduced form) in hepatocytes treated with tanshinone I and GalN as in Experimental Example 1-2 was measured using a colorimetric assay kit (Bioxytech TM GSH-400, OXIS International, Portland, USA). It was measured according to the method of use of the kit, and the results are shown in Table 7 below.
상기 결과로부터, 탄시논 I은 간세포 내 GSH의 고갈을 억제하여, 간세포의 손상을 효과적으로 억제함을 확인할 수 있었다.From the above results, it was confirmed that tanshinone I inhibits the depletion of GSH in hepatocytes, thereby effectively inhibiting damage to hepatocytes.
실험예Experimental Example 3. 소수성 3. Hydrophobicity 담즙산염에On bile salts 의한 간세포 자가사멸시 When hepatocyte self-killing 탄시논Tanshinone I의 효과 측정 Measure the effect of I
3-1. 3-1. 캐스파제Caspase -3 활성에 대한 -3 for active 탄시논Tanshinone I의 보호활성 Protective activity of I
간세포 자가사멸 과정 중 활성이 증대되는 캐스파제-3의 활성은 시판되는 캐스파제-3 활성 착색 검정 키트(activity colorimetric assay kit; R&D Systems, Inc., USA)를 이용하여 참고예 2-3에서 처리한 간세포 실험군을 대상으로 측정하였다. 하기 표 8에서 보는 바와 같이, 탄시논 I은 100 μM GCDC 처리시 활성이 증대되는 캐스파제-3의 활성을 유의적으로 감소시킴을 확인할 수 있었다.The activity of caspase-3, which is increased in activity during hepatocyte self-killing, was treated in Reference Example 2-3 using a commercially available caspase-3 activity colorimetric assay kit (R & D Systems, Inc., USA). One hepatocyte experimental group was measured. As shown in Table 8, it was confirmed that tanshinone I significantly decreased the activity of caspase-3, the activity of which was increased upon 100 μM GCDC treatment.
3-2.3-2. DNADNA 단편화에 대한 For fragmentation 탄시논Tanshinone I의 보호활성 Protective activity of I
간세포내 DNA 단편화에 미치는 탄시논 I의 보호효과를 참고예 2-3에서 처리한 실험군에서 다음과 같이 실시하였다. The protective effect of tanshinone I on DNA fragmentation in hepatocytes was performed in the experimental group treated in Reference Example 2-3 as follows.
간세포 내 DNA를 시판되는 위저드 제노믹 DNA (Wizard Genomic DNA) 정량 키트 (Promega, USA)를 이용하여 추출 및 정량한 다음 30 ㎍의 DNA를 2% 아가로스 겔에서 50 mV, 40분간 전기영동하여 UV 하 DNA 단편화를 확인하였다. 그 결과를 도 1에서 나타낸 바와 같이, 100 μM GCDC 처리시 간세포의 DNA 단편화가 유도되었으나 탄시논 I과 100 μM GCDC를 같이 처리한 간세포의 경우 DNA 단편화 유도가 억제됨을 확인하였다.Hepatocellular DNA was extracted and quantified using a commercially available Wizard Genomic DNA Quantification Kit (Promega, USA), and 30 μg of DNA was electrophoresed at 50 mV for 40 minutes on a 2% agarose gel. DNA fragmentation was confirmed. As shown in FIG. 1, it was confirmed that DNA fragmentation of hepatocytes was induced upon 100 μM GCDC treatment, but induction of DNA fragmentation was inhibited in hepatocytes treated with tanshinone I and 100 μM GCDC.
이상에서 살펴본 바와 같이, 본 발명의 탄시논 I은 산화 및 비산화적 손상에 의한 간세포 손상을 유의성있게 억제하며, 소수성 담즙에 의한 간세포 자가사멸에 탁월하게 효과를 보임으로써, 간질환 치료 및 예방을 위한 약학조성물 및 건강보조식품으로 유용하게 이용될 수 있다. As described above, tanshinone I of the present invention significantly inhibits hepatocellular damage caused by oxidative and non-oxidative damage, and has an excellent effect on hepatocellular self-killing by hydrophobic bile, thereby treating and preventing liver disease. It can be usefully used as a pharmaceutical composition and dietary supplement.
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