KR101794924B1 - Method for Isolating Isorhamnetin for Prevention or Treatment of Non-alcoholic Fatty Liver Disease derived from Salicornia SPP. - Google Patents
Method for Isolating Isorhamnetin for Prevention or Treatment of Non-alcoholic Fatty Liver Disease derived from Salicornia SPP. Download PDFInfo
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- KR101794924B1 KR101794924B1 KR1020160078354A KR20160078354A KR101794924B1 KR 101794924 B1 KR101794924 B1 KR 101794924B1 KR 1020160078354 A KR1020160078354 A KR 1020160078354A KR 20160078354 A KR20160078354 A KR 20160078354A KR 101794924 B1 KR101794924 B1 KR 101794924B1
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- KR
- South Korea
- Prior art keywords
- isorhamnetin
- liver
- fraction
- fatty liver
- hepatocyte
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Abstract
Description
본 발명은 퉁퉁마디 유래의 이소람네틴을 유효성분으로 함유하는 비알코올성 지방간 질환 예방 또는 치료용 약학 조성물에 관한 것으로서 더욱 상세하게는 간세포내 염증반응을 억제하고 간세포 보호활성을 가짐으로써 비알코올성 지방간으로 촉발되는 간염, 간섬유화, 간경화 등의 간질환 예방 또는 치료용 약학 조성물에 관한 것이다. The present invention relates to a pharmaceutical composition for the prevention or treatment of nonalcoholic fatty liver disease, which comprises isopramenin derived from a Tochimantim, as an active ingredient. More particularly, the present invention relates to a pharmaceutical composition for preventing or treating nonalcoholic fatty liver disease To a pharmaceutical composition for preventing or treating liver diseases such as hepatitis, hepatic fibrosis and cirrhosis.
2014년 통계청 사망원인통계 결과를 보면 한국인의 3대 사망원인인 암, 심장질환, 혈관질환은 전체 사망자의 47.4%로 거의 절반이며, 다음으로 폐렴, 당뇨병 및 간질환으로 사망에 이른다고 보고되어 있다. 특히 간질환으로 6,635명이 사망하였고, 이는 인구 10만명당 13.1%를 차지하는 것이다. 지방간은 간 내 과도한 지방(주로 중성지방)이 쌓여서 발생되는데 일반적으로 간 무게의 5% 이상 지방이 축적되면 지방간으로 진단한다 (국가건강정보포털 의학정보). 간 기능 이상으로 병원을 방문한 환자의 대부분(60~80%)이 지방간이며, 전체 인구의 20~30%가 지방간이라는 연구가 있을 정도로 지방간은 매우 흔한 질병이다. According to statistics by the National Statistical Office (NSO) in 2014, cancer, heart disease, and vascular disease, which are the three leading causes of death in Korea, account for almost half of all deaths, accounting for 47.4%, followed by pneumonia, diabetes and liver disease. In particular, 6,635 deaths from liver disease accounted for 13.1% per 100,000 population. Fatty liver is caused by accumulation of excess fat in the liver (mainly triglycerides). Generally, when more than 5% of liver weight is accumulated, it is diagnosed as fatty liver (National Health Information Portal Medical Information). Fatty liver disease is a very common disease so that most (60-80%) of the patients who visited the hospital due to abnormal liver function are fatty liver and 20-30% of the total population is fatty liver.
또한, 국내성인 10명중 3명은 지방간 증상을 갖고 있는 것으로 나타났으며, 2014년 삼성서울병원 소화기 내과에서 지방간 내원 환자 비율을 조사한 결과, 전체 연령대 중 30대가 35.8%, 40대 34.3%, 50대 30.6%, 60대 24.6%, 20대 24.1% 순으로 나타나 30대 연령의 지방간 비율이 가장 높은 것으로 조사되었다. 대한간학회가 1988년부터 2014년까지 건강검진을 받은 성인 75만 명의 정보를 분석한 결과, 당뇨병 환자의 70%, 고혈압 환자의 52%, 대사증후군 환자의 38%가 지방간을 앓고 있는 것으로 나타나, 간 관련 치료제 시장뿐만 아니라 건강기능성 식품시장에도 간 기능성 개선 제품에 대한 관심과 소비자의 요구도가 높아지고 있는 실정이다.In addition, 3 out of 10 Korean adults showed fatty liver disease. In 2014, the proportion of patients in the provincial department of the digestive system of Samsung Seoul Hospital was 35.8% in the 30s, 34.3% in the 40s, %, 60 vs 24.6% and 20 vs 24.1%, respectively. The Korean Hepatology Association analyzed information on 750,000 adults who had undergone medical screening from 1988 to 2014, indicating that 70% of diabetic patients, 52% of hypertensive patients, and 38% of metabolic syndrome patients had fatty liver, In addition to the liver-related therapeutic market, the health functional food market is also experiencing a growing interest in consumer-oriented products and consumer needs.
지방간은 과음으로 인한 알코올성 지방간과 술과 관계없이 고지방식이로 인한 비만 등과 연관되어 발생되는 비알코올성 지방간 (Non-alcoholic fatty liver disease, NAFLD)으로 나눌 수 있으며, 비알코올성 지방간의 15%는 지방간염 및 간섬유화로, 지방간염의 5%는 간경화 또는 간암으로 이행된다. 또한 약물과다복용과 혈관 내 총콜레스테롤 및 중성지방의 양이 정상치 이상으로 증가된 고지혈증의 경우에도 간의 해독기능과 지방분해 기능을 저하시켜 비알코올성 지방간(NAFLD)이 유발되는 것으로 알려져 있다. The fatty liver can be divided into non-alcoholic fatty liver disease (NAFLD), which is caused by excessive alcohol intake and alcohol related to obesity due to high fat diet, and 15% of nonalcoholic fat liver fatty liver disease And liver fibrosis, 5% of fatty liver infections are cirrhotic or liver cancer. It is also known that hyperlipidemia, in which the drug overdose and the amount of total cholesterol and triglyceride in the blood vessels are increased to above normal levels, also causes the non-alcoholic fatty liver (NAFLD)
비알코올성 지방간 질환 예방 및 치료로 처방되는 약제들인 젬피브로질(gemfibrozil)이나 아토바스타틴(atorvastatin)과 같은 스타틴(statin) 계열 약물은 근육에서 미토콘드리아에 손상을 준다는 보고가 있어 단순 지방간 치료에 사용하기에는 적합하지 않는 것으로 알려졌다. 비알코올성 지방간의 대표적 질환들인 간염증 및 간섬유화의 발생기전 중 하나가 간세포 내 지방산 산화로 인한 활성산소가 염증관련 신호전달 경로의 중심 전사인자인 NFkB를 활성화시켜 관련 염증 유전자들인 COX-2 와 iNOS의 합성을 야기시킨다. It has been reported that statin drugs such as gemfibrozil and atorvastatin, which are drugs prescribed for the prevention and treatment of nonalcoholic fatty liver disease, cause damage to mitochondria in muscle, It is said to be unsuitable. One of the mechanisms of liver inflammation and hepatic fibrosis, which is a representative disease of nonalcoholic fatty liver, is that active oxygen induced by fatty acid oxidation in hepatocytes activates NFkB, a central transcription factor of inflammation-related signaling pathway, and induces related inflammatory genes, COX-2 and iNOS . ≪ / RTI >
비타민 E(토코페롤)와 비타민 C(아스코르브산)가 지방간 예방과 지방간염 환자들의 간 기능 수치 및 조직검사에서도 어느 정도 효과가 있으며, 간세포 내에 자연적으로 존재하는 항산화물인 글루타치온(glutathione, GSH)의 전구물질들인 여러 가지 약제들 (N-acetylcysteine, S-adenosyl-methionine [SAM])이 간내 글루타치온을 증가시키는 항산화작용으로 지방간 치료에 사용할 수 있는 것으로 알려져 있다. 간내 글로타치온(GSH)은 중심합성 효소인 감마 글루타밀시스테인 라이에이스(γ-glutamylcysteine lyase, GCL)로부터 합성되고 글루타치온리덕테이스(glutathione reductase, GR)의 작용으로 산화형인 GSSG로부터 환원되어 생성된다. 글루타치온리덕테이스(glutathione reductase, GR)와 감마 글루타밀시스테인 라이에이스(γ-glutamylcysteine lyase, GCL)와 같은 간세포내 글루타치온 합성효소의 증가는 세포 항상성 유지와 세포 보호기전의 대표적인 전사인자인 Nrf2의 활성화를 통하여 진행된다. 따라서, 염증인자인 NFkB를 활성화를 억제하고 세포보호 및 방어성 Nrf2 전사인자를 활성화시킬 수 있는 천연물 유래의 항산화성 성분들도 지방간염 질환의 예방과 치료에 효능을 보일 수 있음을 시사한다. 따라서, 부작용이 없고 저렴하면서도 치료 효과가 우수한 새로운 비알콜성 지방간의 예방 및 치료를 위한 천연물 유래 약물개발이 필요한 실정이다. Vitamin E (Tocopherol) and Vitamin C (ascorbic acid) are effective in prevention of fatty liver and hepatic function and histologic examination of patients with hepatitis, and the precursor of glutathione (GSH), an antioxidant naturally present in hepatocytes (N-acetylcysteine, S-adenosyl-methionine [SAM]) is known to be an antioxidant that increases glutathione in the liver and can be used in the treatment of fatty liver. Glutathione (GSH) in the liver is synthesized from γ-glutamylcysteine lyase (GCL), a central synthetic enzyme, and is produced by reduction from oxidized form of GSSG by the action of glutathione reductase (GR) . The increase of glutathione synthetase in hepatocytes such as glutathione reductase (GR) and γ-glutamylcysteine lyase (GCL) has been shown to be involved in cell homeostasis and activation of Nrf2, a typical transcription factor Lt; / RTI > Therefore, it is suggested that antioxidative components derived from natural materials that can inhibit activation of NFkB, an inflammatory factor, and activate cell protective and protective Nrf2 transcription factors may also be effective in the prevention and treatment of fatty liver disease. Therefore, there is a need to develop drugs derived from natural products for the prevention and treatment of new non-alcoholic fatty liver which has no side effects and is inexpensive and has excellent therapeutic effect.
한국공개특허 제2015-0103847호는 갈화추출물을 유효성분으로 함유하는 비알콜성 지방간 질환 예방 또는 치료용 조성물에 관한 것으로서, 갈화추출물은 지방축적, 지질 축적으로 의한 간조직의 변형 등 비알콜성 지방간으로 인한 병리학적소견을 완화시키고, 혈중 지질 함량을 감소시킬 수 있으며, 지방세포 전사조절인자인 PPARγ 및 C/EBPα와 지방산 생합성을 조절하는 전사인자인 SREBP-1의 발현을 억제할 수 있다고 개시하였으나, 유효성분의 분리는 시도되지 않았다. Korean Patent Laid-Open Publication No. 2015-0103847 relates to a composition for preventing or treating a non-alcoholic fatty liver disease containing an extract of Garlic as an active ingredient, wherein the garlic extract is selected from the group consisting of non-alcoholic fatty liver such as fat accumulation, , It is possible to reduce the lipid content of the blood and to inhibit the expression of SREBP-1, a transcription factor controlling fatty acid biosynthesis and PPARγ and C / EBPα, which are adipocyte transcription regulators, , The separation of the active ingredient was not attempted.
한국등록특허 제0867320호에서는 13종의 한약재의 수추출물을 이용한 간섬유화(간경화)의 예방 또는 치료용 조성물에 관한 것으로서, Dimethylnitrosoamine(DMN) 첨가 식이를 통하여 간섬유화를 유발시키고, 간손상 지표로 혈중 빌리루빈, AST, ALT 조사 및 간조직내 지질과산화물질인 MDA 함량을 분석하였으나, 유효성분에 관한 연구는 시도되지 않았다.Korean Patent No. 0867320 discloses a composition for preventing or treating liver fibrosis (liver cirrhosis) using water extracts of 13 kinds of herb medicines. The composition is used to induce liver fibrosis through a diet supplemented with dimethylnitrosoamine (DMN) Bilirubin, AST, ALT and LDA content of lipid peroxidation in liver tissue were analyzed, but no studies on active ingredients were attempted.
한편, 퉁퉁마디(Salicornia europaea)는 우리나라 서해안 갯벌에서 대량 서식하고 있는 대표적인 염생식물로서, 염 저항성이 강하여 NaCl 200mM을 첨가한 사경재배에서도 성장감소 없이 생육이 가능하며, 생육을 위하여 염류를 절대적으로 필요로 하는 절대 염생식물(Obligatory halophyte or True halophyte)이다. 퉁퉁마디는 나트륨 외에도 마그네슘, 칼륨, 철 등의 미네랄과 풍부한 아미노산을 다량 함유하여 우리나라에서는 채소, 약초, 발효식품 등으로 이용하였고, 유럽에서는 샐러드, 식초의 재료 등으로 이용하고 있다.On the other hand, Salicornia europaea ( Salicornia europaea ) is a representative algae plant that is abundant in the coastal waters of the west coast of Korea. It is resistant to salt, and can grow without reduction in growth even in the four-sided cultivation with 200 mM NaCl added. (Obligatory halophyte or True halophyte). In addition to sodium, it also contains minerals such as magnesium, potassium and iron, and a large amount of amino acids, which are used in vegetables, herbs and fermented foods in Korea, and in salads and vinegar materials in Europe.
절대 염생식물에서 삼투조절에 기여하는 용질들은 삼투저항 유기물질로 알려졌는데 세포내 축적되는 대표적인 삼투저항 물질들로는 아미노산 proline, glycin betaine과 같은 onium 화합물, 당알코올류들인 mannitol, sorbitol, ononitol 등과 단당류 trehalose 등으로 알려지고 있다.Solutes that contribute to osmotic control in absolute halophytes are known as osmotic resistance organics. Typical osmotic agents that accumulate in cells include onium compounds such as amino acids proline and glycin betaine, mannitol, sorbitol, ononitol and monosaccharide trehalose .
퉁퉁마디는 오랫동안 민간약으로 시력저하, 소화불량, 위장병, 간염, 신장병 등에 사용되어 왔고, 항당뇨, 항암, 항고혈압, 항고지혈증, 피부미백 활성 등에 대한 연구만 이루어졌을 뿐 아직까지 그 생리활성 유효성분에 대한 연구가 많이 이루어지지 않았다.It has been used for a long time as a folk remedy for visual loss, digestion failure, gastrointestinal illness, hepatitis, kidney disease, antidiabetic, anti-cancer, anti-hypertension, anti-hyperlipemia and skin whitening activity. There is not much research on this.
한국 등록특허 제0468073호는 항당뇨 활성을 갖는 함초 추출물 또는 이로부터 분리된 이소람네틴-3-O-β-D-글루코시드를 포함하는 조성물을 개시하였고, 한국 등록특허 제1212612호는 퉁퉁마디로부터 분리된 이소쿼시트린 6''-오-메칠옥살레이트 (isoquercitrin 6''-O-methyloxalate)가 항산화활성 및 과산화물 생성억제활성을 갖는다고 개시한 바 있다.Korean Patent No. 0468073 discloses a composition comprising a green tea extract having anti-diabetic activity or isorhamnetin-3-O -? - D-glucoside isolated therefrom, Korean Patent No. 1212612 discloses a composition comprising (Isoquercitrin 6 " -O-methyloxalate) isolated from the cell line has antioxidant activity and an activity of inhibiting peroxidation.
이에, 본 발명자들은 상기 문제점을 해결하기 위하여 예의 노력한 결과, 퉁퉁마디 추출물 및 이로부터 분리된 이소람네틴(isorhamnetin)이 비알코올성 지방간 질환 예방 및 치료능을 갖는다는 것을 확인하고, 본 발명을 완성하게 되었다.As a result of intensive efforts to solve the above problems, the inventors of the present invention have found that extracts of Toyochimonoides and isorhamnetin isolated therefrom have the ability to prevent and treat nonalcoholic fatty liver disease and to complete the present invention .
본 발명의 목적은 천연물인 퉁퉁마디 유래의 비알코올성 지방간 질환 예방 또는 치료용 약학조성물을 제공하는데 있다.It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of nonalcoholic fatty liver disease originating from a natural substance,
본 발명의 다른 목적은 식품원료인 퉁퉁마디 유래의 비알코올성 지방간 질환 예방 또는 개선용 식품 조성물을 제공하는데 있다.Another object of the present invention is to provide a food composition for prevention or improvement of non-alcoholic fatty liver disease originating from Tungsten mushroom which is a raw material of food.
본 발명의 다른 목적은 퉁퉁마디로부터 비알코올성 지방간 질환 예방 또는 치료능을 갖는 이소람네틴(isorhamnetin)을 분리하는 방법을 제공하는데 있다.Another object of the present invention is to provide a method for isolating isorhamnetin from non-alcoholic bean curd refuse having the ability to prevent or treat non-alcoholic fatty liver disease.
상기 목적을 달성하기 위하여, 본 발명은 퉁퉁마디(Salicornia SPP.) 유래의 이소람네틴(isorhamnetin)을 유효성분으로 함유하는 비알코올성 간질환 예방 또는 치료용 약학 조성물을 제공한다.In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating nonalcoholic liver disease, which comprises isorhamnetin derived from Salicornia spp . As an active ingredient.
본 발명에 있어서, 상기 비알코올성 지방간은 비알코올성 간염증, 비알코올성 간섬유화 및 비알코올성 간경화로 구성된 군으로부터 선택되는 것을 특징으로 한다.In the present invention, the non-alcoholic fatty liver is selected from the group consisting of non-alcoholic liver inflammation, non-alcoholic liver fibrosis and non-alcoholic liver cirrhosis.
본 발명은 퉁퉁마디(Salicornia SPP.) 유래의 이소람네틴(isorhamnetin)을 유효성분으로 함유하는 비알코올성 지방간 질환 예방 또는 개선용 식품 조성물을 제공한다.The present invention provides a food composition for preventing or ameliorating nonalcoholic fatty liver disease comprising isorhamnetin derived from Salicornia spp . As an active ingredient.
본 발명은 또한, (a) 퉁퉁마디 추출물을 얻는 단계; (b) 상기 퉁퉁마디 추출물을 용매로 분획하는 단계; (c) 상기 퉁퉁마디 용매 분획물을 크로마토그래피로 분획하여 간세포내 염증반응 억제능 및 간세포 보호활성을 갖는 크로마토그래피 분획물을 얻는 단계; 및 (d) 상기 크로마토그래피 분획물을 고속액체크로마토그래피(HPLC)를 이용하여 이소람네틴을 분리하는 단계를 포함하는 것을 특징으로 하는 퉁퉁마디(Salicornia SPP.) 유래 이소람네틴(isorhamnetin)의 분리방법을 제공한다.The present invention also relates to a process for the preparation of a composition comprising: (a) obtaining an extract of Trichoderma nodule; (b) fractionating the extract of Tungsten mulberry with a solvent; (c) fractionating the fractions of Chrysanthemum mildew solvent by chromatography to obtain a chromatographic fraction having inhibitory effect on hepatocyte inflammatory reaction and hepatocyte protective activity; And (d) isolating isorhamnetin using high performance liquid chromatography (HPLC) on said chromatographic fraction. The method of isolating isorhamnetin from Salicornia SPP . .
본 발명에 있어서, 상기 퉁퉁마디 용매 분획물은 퉁퉁마디 추출물을 순차적으로 헥산, 클로로포름, 에틸아세테이트, 부탄올 및 물로 분획하는 것을 특징으로 한다.In the present invention, it is preferable that the extract of Tungtung nodule is fractionated with hexane, chloroform, ethyl acetate, butanol and water in order.
본 발명에 있어서, 상기 크로마토그래피 분획물을 얻는 단계는 상기 용매 분획물을 흡착성 디아이온 HP-20 컬럼에 로딩시킨 후, 아세톤으로 용출시켜 간세포내 염증반응 억제능 및 간세포 보호활성을 갖는 제 1분획물을 획득하고, 이를 실리카겔 컬럼에 로딩시킨 후, 클로로포름 이동상에 메탄올을 순차적으로 추가하면서 용출시켜 간세포내 염증반응 억제능 및 간세포 보호활성을 갖는 제 2분획물을 획득하고, 이를 LH-20 컬럼에 로딩시킨 후, 메탄올로 용출시켜 간세포내 염증반응 억제능 및 간세포 보호활성을 갖는 제 3분획물을 획득하는 단계를 포함하는 것을 특징으로 한다. 상기 고속액체크로마토그래피(HPLC)는 메탄올과 물의 혼합액을 이동상으로 하여 수행되는 것을 특징으로 한다.In the present invention, in the step of obtaining the chromatographic fraction, the solvent fraction is loaded on an adsorptive Diaion HP-20 column, and then eluted with acetone to obtain a first fraction having an inhibitory effect on the inflammatory reaction in the hepatocyte and hepatocyte protective activity , And loaded on a silica gel column. Then, methanol was sequentially added to the mobile phase of chloroform to elute the second fraction, which had an inhibitory effect on the inflammatory reaction in the hepatocyte and hepatocyte protective activity. The second fraction was loaded on an LH-20 column, And obtaining a third fraction having an inhibitory effect on the inflammatory response in the hepatocyte and a hepatocyte protective activity. The high performance liquid chromatography (HPLC) is characterized in that a mixture of methanol and water is used as a mobile phase.
본 발명에 따른 퉁퉁마디(Salicornia SPP.) 유래의 이소람네틴(isorhamnetin)은 간세포내 대표적 염증마커들인 iNOS와 COX-2 단백질 발현을 농도의존적으로 저해할 뿐 만 아니라 산화스트레스에 대항하는 대표적인 세포보호 전사인자인 Nrf2 활성화를 통한 글루타치온 합성 효소들(GR, GCL)을 증가시킴으로써, 간염증, 간섬유화, 간경화 등의 비알코올성 지방간 질환의 예방 및 치료에 이용될 수 있다. Isorhamnetin derived from Salicornia spp . According to the present invention not only inhibits iNOS and COX-2 protein expression, which are typical inflammation markers in hepatocytes, in a concentration-dependent manner, but also exhibits typical cellular protection against oxidative stress By increasing the glutathione synthetase (GR, GCL) through Nrf2 activation, which is a transcription factor, it can be used for the prevention and treatment of nonalcoholic fatty liver diseases such as liver inflammation, liver fibrosis and cirrhosis.
도 1은 퉁퉁마디 추출물로부터 분리된 분획물 및 화합물 1(이소람네틴)의 HPLC 크로마토그램이다 (A: 분획물(SEW-EA-B2), B: 분획물(SEW-EA-B2-F2), C: 분획물(SEW-L-17)에서 정제된 화합물 1).
도 2는 이소람네틴 화합물의 ESI-MS, 1H-NMR 및 13C-NMR 스펙트럼이다 (A: 이소람네틴 화학식, B: ESI-MS, C: 1H-NMR, D: 13C-NMR).
도 3은 고지방식이를 통한 C57BL/6J 마우스 비만 유도를 나타낸 결과이다.
도 4는 고지방 및 정제염 식이군과 고지방 및 퉁퉁마디 추출물 식이군에서 적출된 마우스 간들의 무게와 간손상지표인 혈중 ALT 수준을 비교 측정한 결과이다(A: 마우스 간 성상 사진, B: 마우스 간 무게, C: 마우스 혈중 ALT).
도 5는 고지방 및 정제염 식이군과 고지방 및 퉁퉁마디 추출물 식이군에서의 간조직내 비알코올성 지방간 질환 지표인 염증 및 섬유화 수준을 비교 측정한 결과이다(A: 마우스 간 조직 H&E 염색 사진 및 Masson's trichrome 염색 사진, B: Steatosis, Inflammation, Ballooning 결과, C: NAFLD (비알콜성 지방간 질환) 척도 점수).
도 6은 고지방 및 정제염 식이군과 고지방 및 퉁퉁마디 추출물 식이군의 간조직내 염증 및 간섬유화 마커들(Il1b, Cxcl2, Ccl2, Cd68, Col1a1)의 mRNA 발현을 측정한 결과이다.
도 7은 퉁퉁마디 추출물에서 단리된 이소람네틴의 LPS로 자극된 인간 간세포주인 HepG2 세포에서 유도되는 아질산염(NO) 방출량 억제능(A) 및 iNOS 단백질 발현 저해능(B)을 확인한 결과이다.
도 8은 퉁퉁마디 추출물에서 단리된 이소람네틴의 LPS로 자극된 인간 간세포주인 HepG2 세포에서 유도되는 COX-2 단백질 발현 저해능(A)과 핵내에서의 NF-kB 단백질 발현 저해능(B)을 확인한 결과이다.
도 9는 퉁퉁마디 추출물에서 단리된 이소람네틴이 t-BHP가 처리된 HepG2 세포에서 생성되는 ROS의 생성과 세포사멸을 억제하고(A) 세포내 ROS 소거를 관찰한 형광현미경 사진(B)이다.
도 10은 퉁퉁마디 추출물에서 단리된 이소람네틴이 처리된 간세포 내에서의 글루타치온(GSH)의 함량(A) 및 글루타치온 합성효소(GR, GCL)의 발현능(B)을 확인한 결과이다.(SEW-EA-B2), B: fraction (SEW-EA-B2-F2), C: fraction (SEW-EA-B2) Compound (1) purified in fraction (SEW-L-17).
Figure 2 is a isopropyl person netin compounds ESI-MS, 1 H-NMR and 13 C-NMR spectrum is (A: iso person netin formula, B: ESI-MS, C : 1 H-NMR, D: 13 C-NMR ).
FIG. 3 shows the results of induction of C57BL / 6J mouse obesity via high fat diet.
FIG. 4 shows the results of a comparison between the weight of mouse liver extracted from the high fat and purified diet group and the high fat and tung fungus extract group, and the serum ALT level, which is an index of liver damage (A: mouse liver, B: mouse liver weight , C: mouse blood ALT).
FIG. 5 is a comparative measurement of the level of inflammation and fibrosis, which is a non-alcoholic fatty liver disease index in liver tissues in the high fat and purified diet group and the high fat and non-fat extract group (A: mouse hepatic tissue H & E staining and Masson's trichrome staining B: Steatosis, Inflammation, Ballooning results, C: NAFLD (non-alcoholic fatty liver disease) scoring score).
FIG. 6 shows the results of measurement of mRNA expression of inflammatory and hepatic fibrosis markers ( Il1b, Cxcl2, Ccl2, Cd68, Col1a1 ) in the liver tissues of the high fat and refined salt diet group and the high fat and noodle group extract diet groups.
FIG. 7 shows the results of confirming the inhibitory effect of nitric oxide (NO) release (A) and the inhibitory effect on the expression of iNOS protein (B) induced by human hepatocyte HepG2 cells stimulated with LPS of isolated isorhamnetin.
FIG. 8 shows the inhibition of COX-2 protein expression (A) and the inhibitory effect of NF-kB protein expression in the nucleus (B) induced by human hepatocyte-host HepG2 cells stimulated with LPS of isolated isorhamnetin to be.
FIG. 9 is a fluorescence microscopic photograph (B) showing isolametin isolated from the extract of Tongtung nodule inhibiting the production of ROS and the apoptosis of HepG2 cells treated with t-BHP (A) and observing the intracellular ROS elimination .
FIG. 10 shows the results of confirming the content (A) of glutathione (GSH) and the expression ability (B) of glutathione synthetase (GR, GCL) in hepatocytes treated with isorhamnetin isolated from the extract of Tungtung nodule.
본 발명에서는 아직까지 널리 연구가 이루어지지 않은 퉁퉁마디로부터 간세포내 염증반응 억제능 및 간세포 보호활성이 우수한 활성물질을 분리하고자 하였다.In the present invention, we have sought to isolate active substances having excellent inhibitory activity against hepatocyte inflammatory response and hepatocyte-secreting activity,
본 발명에서는, 퉁퉁마디 추출물 및 이로부터 분리한 이소람네틴이 간세포내 염증반응 억제능 및 간세포 보호능을 가지고 있다는 것을 확인하였다.In the present invention, it was confirmed that the extract of Toyochu-municipal noodles and isorhamnetin isolated therefrom have an inhibitory effect on hepatocyte inflammatory response and a hepatocyte-protective ability.
즉, 본 발명의 일 실시예에서는 퉁퉁마디 열수 추출물이 고지방식이와 정제염 식이에서 촉발되는 비알코올성 지방간으로 인한 간손상, 간염 및 간섬유화 과정을 효과적으로 억제한다는 것을 확인할 수 있었으며, 퉁퉁마디 열수 추출물을 용매 분획, 크로마토그래피 분획 및 고속액체크로마토그래피(HPLC)를 수행하여 분리한 이소람네틴이 간세포내 염증 대표적 염증마커들인 iNOS와 COX-2의 단백질 발현을 농도의존적으로 저해할 뿐 만 아니라 산화스트레스에 대항하는 대표적인 세포보호 전사인자인 Nrf2 활성화를 통한 글루타치온 합성 효소들(GR, GCL)을 증가시킨다는 것을 확인할 수 있었다.That is, in one embodiment of the present invention, it was confirmed that the extract of Tungtung-madi extract effectively inhibited liver damage, hepatitis and hepatic fibrosis caused by non-alcoholic fatty liver induced by high-fat diet and purified diet, Isolametin isolated by solvent fraction, chromatographic fractionation and high performance liquid chromatography (HPLC) not only inhibited protein expression of iNOS and COX-2, which are representative inflammation markers of hepatocyte inflammation, but also oxidative stress (GR, GCL) through the activation of Nrf2, a typical cytoprotective transcription factor, which is a counterproductive agent.
따라서, 본 발명은 퉁퉁마디(Salicornia SPP.) 유래의 이소람네틴(isorhamnetin)을 유효성분으로 함유하는 비알코올성 지방간 질환 예방 또는 치료용 약학 조성물에 관한 것이다.Accordingly, the present invention relates to a pharmaceutical composition for preventing or treating nonalcoholic fatty liver disease comprising isorhamnetin derived from Salicornia spp . As an active ingredient.
본 발명의 비알코올성 지방간 질환 예방 또는 치료용 약학 조성물은 유효성분인 이소람네틴(isorhamnetin) 이외에 약제학적으로 적합하고 생리학적으로 허용되는 보조제를 사용하여 제조될 수 있으며, 상기 보조제로는 부형제, 붕해제, 감미제, 결합제, 피복제, 팽창제, 윤활제, 활택제, 향미제 등이 사용될 수 있다.The pharmaceutical composition for preventing or treating nonalcoholic fatty liver disease of the present invention may be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to isorhamnetin as an active ingredient, Release agents, sweeteners, binders, coating agents, swelling agents, lubricants, lubricants, and flavoring agents.
상기 약학 조성물은 투여를 위해서 유효성분인 이소람네틴(isorhamnetin) 이외에 추가로 약제학적으로 허용 가능한 담체를 1종 이상 포함하여 약학 조성물로 제제화할 수 있다. 제제 형태는 과립제, 산제, 정제, 피복정, 캡슐제, 좌제, 액제, 시럽, 즙, 현탁제, 유제, 점적제, 주사 가능한 액제 등이 될 수 있다. 예를 들어, 정제 또는 캡슐제의 형태로의 제제화를 위해, 유효성분은 에탄올, 글리세롤, 물 등과 같은 경구, 무독성의 약제학적으로 허용 가능한 불활성 담체와 결합될 수 있다. 또한 필요에 따라 적합한 결합제, 윤활제, 붕해제, 발색제 등이 포함될 수 있다. 적합한 결합제로는 이에 제한되는 것은 아니나, 녹말, 젤라틴, 글루코스 또는 베타-락토오스와 같은 천연 당, 옥수수 감미제, 아카시아, 트래커캔스 또는 소듐올레이트와 같은 천연 및 합성 검, 소듐 스테아레이트, 마그네슘 스테아레이트, 소듐 벤조에이트, 소듐 아세테이트, 소듐 클로라이드 등이 사용될 수 있다. 붕해제로는 이에 제한되는 것은 아니나, 녹말, 메틸 셀룰로스, 아가, 벤토니트, 잔탄 검 등이 사용될 수 있다. 액상 용액으로 제제화를 위해 허용 가능한 약제학적 담체로는, 멸균 및 생체에 적합한 것으로 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사용액, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 등이 사용될 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 통상의 첨가제를 첨가할 수도 있다.In addition to isorhamnetin, which is an active ingredient for administration, the pharmaceutical composition may further comprise at least one pharmaceutically acceptable carrier and may be formulated into a pharmaceutical composition. Formulations may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops, injectable solutions, and the like. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Further, if necessary, suitable binders, lubricants, disintegrants, coloring agents and the like may be included. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, Sodium benzoate, sodium acetate, sodium chloride and the like can be used. Examples of the disintegrating agent include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, and the like. Sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and the like may be used as the pharmaceutical carrier which is acceptable for formulation into a liquid solution And if necessary, usual additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added.
또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. 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 염생식물인 퉁퉁마디에서 분리한 이소람네틴(isorhamnetin)을 포함하여 독성 및 부작용이 거의 없으며, 따라서 비알콜성 지방간 질환의 예방 또는 치료와 간기능 개선의 목적으로 장기간 복용 시에도 안심하고 사용할 수 있다.The pharmaceutical composition of the present invention contains isorhamnetin which is isolated from a wild plant, Tongtung nodule, and has little toxicity and side effects. Therefore, for the purpose of preventing or treating non-alcoholic fatty liver disease and improving liver function, Can be used with confidence.
또한 본 발명에 따른 퉁퉁마디(Salicornia SPP.) 유래의 이소람네틴(isorhamnetin)을 유효성분으로 함유하는 비알코올성 지방간 질환 예방 또는 치료용 약학 조성물은 연구자, 수의사, 의사 또는 기타 임상에 의해 생각되는 조직계, 동물 또는 인간에서 생물학적 또는 의학적 반응을 유도하는 유효 성분 또는 약학적 조성물의 양, 즉 치료되는 질환 또는 장애의 증상의 완화를 유도하는 양인 치료상 유효량으로 투여할 수 있다. 본 발명의 이소람네틴(isorhamnetin)을 유효성분으로 함유하는 조성물에 대한 치료상 유효 투여량 및 투여횟수는 원하는 효과에 따라 변화될 것임은 당업자에게 자명하다. 그러므로, 투여될 최적의 투여량은 당업자에 의해 쉽게 결정될 수 있으며, 질환의 종류, 질환의 중증도, 조성물에 함유된 유효성분 및 다른 성분의 함량, 제형의 종류, 및 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여시간, 투여 경로 및 조성물의 분비율, 치료기간, 동시 사용되는 약물을 비롯한 다양한 인자에 따라 조절될 수 있다. Also Salicornia (Salicornia according to the invention SPP.) Nonalcoholic fatty liver disease, for the prevention or treatment a pharmaceutical composition researcher, veterinarian, medical doctor or other clinical the jojikgye thought by animal or a biological or medical response in a human containing isopropyl person netin (isorhamnetin) of origin, as an active ingredient Or an amount of the pharmaceutical composition, i. E., A therapeutically effective amount, which is an amount that induces the reduction of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dose and the number of administrations of a composition containing isorhamnetin of the present invention as an active ingredient will vary depending on the desired effect. Thus, the optimal dosage to be administered can be readily determined by those skilled in the art and will vary with the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, The age, body weight, sex, diet, time of administration, route of administration and fraction of the composition, duration of treatment, concurrent medication, and the like.
또한 본 발명은 다른 관점에서, 퉁퉁마디(Salicornia SPP.) 유래의 이소람네틴(isorhamnetin)을 유효성분으로 함유하는 비알코올성 지방간 질환 예방 또는 개선용 식품 조성물에 관한 것이다.In another aspect, the present invention relates to a food composition for preventing or ameliorating a nonalcoholic fatty liver disease comprising isorhamnetin derived from Salicornia spp . As an active ingredient.
상기 식품 조성물은 유효성분인 이소람네틴(isorhamnetin)을 함유하는 것 이외에 통상의 식품 조성물에 사용되는 여러 향미제, 천연 탄수화물 등이 추가로 포함될 수 있다.In addition to containing isorhamnetin as an active ingredient, the food composition may further include various flavors, natural carbohydrates and the like used in ordinary food compositions.
상기 천연 탄수화물로는 모노사카라이드(예를 들어, 포도당, 과당 등), 디사카라이드(예를 들어, 말토스, 슈크로스 등), 폴리사카라이드(예를 들어, 덱스트린, 시클로덱스트린) 등과 같은 통상적인 당이나 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이 사용될 수 있다. 상기 향미제로는 천연 향미제(예를 들어, 타우마틴 등), 스테비아 추출물(예를 들어, 레바우디오시드 A, 글리시르히진 등), 합성 향미제(예를 들어, 사카린, 아스파르탐 등) 등을 사용할 수 있다.Examples of the natural carbohydrate include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin. Conventional sugars and sugar alcohols such as xylitol, sorbitol and erythritol can be used. Examples of the flavoring agent include natural flavoring agents (e.g., tauromine), stevia extracts (e.g., rebaudioside A and glycyrrhizin), synthetic flavors (e.g., saccharin, aspartame, ) Can be used.
본 발명의 식품 조성물은 상기 약학 조성물과 동일한 방식으로 제제화되어 기능성 식품으로 이용할 수도 있으며, 각종 식품에 첨가제 형태로 첨가할 수도 있다. 본 발명의 조성물을 첨가할 수 있는 식품으로는 음료류, 육류, 초코렛, 식품류, 과자류, 피자, 라면, 기타 면류, 껌류, 사탕류, 아이스크림류, 알코올 음료류, 비타민 복합제, 건강보조식품류 등이 있다.The food composition of the present invention may be formulated in the same manner as the above-described pharmaceutical composition and used as a functional food or may be added to various foods in the form of an additive. Foods to which the composition of the present invention can be added include beverages, meat, chocolates, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes and health supplement foods.
또한 상기 식품 조성물은 유효성분인 이소람네틴(isorhamnetin) 외에 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, 탄산음료에 사용되는 탄산화제 등을 더 첨가될 수 있다. 그밖에 본 발명의 식품 조성물은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수도 있다.In addition to the isorhamnetin, which is an effective ingredient, the food composition can also contain flavorings such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates such as cheese and chocolate, An acid and its salt, alginic acid and its salt, an organic acid, a protective colloid thickener, a pH adjusting agent, a stabilizer, a preservative, a glycerin, an alcohol, and a carbonating agent used in a carbonated drink. In addition, the food composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks.
상기 식품 조성물의 형태 중 건강기능식품이라 함은 건강기능식품에 관한 법률 제6727호에 따른 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 말하며, 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻을 목적으로 섭취하는 것을 의미한다.The health functional food in the form of the above food composition refers to the food prepared and processed using raw materials or ingredients having useful functions in accordance with Law No. 6727 on Health Functional Foods, It is meant to be ingested for the purpose of obtaining a beneficial effect for health use such as controlling nutrients or physiological action.
상기 건강기능식품은 통상의 식품 첨가물을 포함할 수 있으며, 식품 첨가물로서의 적합 여부는 다른 규정이 없는 한, 식품의약품안전청에 승인된 식품 첨가물 공전의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정한다.The above health functional foods may contain conventional food additives and, unless otherwise specified, conform to the general requirements of the Food Additives Ordinance approved by the Korea Food and Drug Administration, Judge by standards.
상기 '식품 첨가물 공전'에 수재된 첨가물로는 케톤류, 글리신, 구연산칼슘, 니코틴산, 계피산 등의 화학적 합성물; 감색소, 감초추출물, 결정셀룰로오스, 고량색소, 구아검 등의 천연첨가물; L-글루타민산나트륨제제, 면류첨가알칼리제, 보존료제제, 타르색소제제 등의 혼합제제류; 등이 있다.The additives added to the above-mentioned 'food additives' include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as persimmon extract, licorice extract, crystalline cellulose, high color pigment and guar gum; Mixed preparations such as a sodium L-glutamate preparation, a noodle-added alkaline agent, a preservative agent, and a tar coloring agent; .
상기 건강기능식품은 정제, 캅셀, 분말, 과립, 액상, 환 등의 형태로 제조 및 가공할 수 있다. 정제 형태의 건강기능식품은 본 발명의 유효성분인 이소람네틴(isorhamnetin)을 부형제, 결합제, 붕해제 및 다른 첨가제와 혼합한 혼합물을 통상의 방법으로 과립화한 다음, 활택제 등을 넣어 압축성형하거나, 상기 혼합물을 직접 압축 성형할 수 있다. 또한 상기 정제 형태의 건강기능식품에는 필요에 따라 교미제 등이 첨가될 수도 있다. 캅셀 형태의 건강기능식품 중 경질 캅셀제는 통상의 경질 캅셀에 본 발명의 유효성분인 이소람네틴(isorhamnetin)을 부형제 등의 첨가제와 혼합한 혼합물을 충진하여 제조할 수 있으며, 연질 캅셀제는 이소람네틴(isorhamnetin)을 부형제 등의 첨가제와 혼합한 혼합물을 젤라틴과 같은 캅셀제에 충진하여 제조할 수 있다. 상기 연질 캅셀제에는 필요에 따라 글리세린 또는 소르비톨 등의 가소제, 착색제, 보존제 등이 첨가될 수도 있다. 환 형태의 건강기능식품은 본 발명의 유효성분인 이소람네틴(isorhamnetin)과 부형제, 결합제, 붕해제 등을 혼합한 혼합물을 기존에 공지된 방법으로 성형하여 조제할 수 있으며, 필요에 따라 백당이나 다른 제피제로 제피할 수 있으며, 또는 전분, 탈크와 같은 물질로 표면을 코팅할 수도 있다. 과립 형태의 건강기능식품은 본 발명의 유효성분인 이소람네틴(isorhamnetin)과 부형제, 결합제, 붕해제 등을 혼합한 혼합물을 기존에 공지된 방법으로 입상으로 제조할 수 있으며, 필요에 따라 착향제, 교미제 등이 첨가될 수 있다.The health functional food may be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and circles. The health functional food in the form of tablets may be prepared by granulating a mixture of isorhamnetin (isorhamnetin), an active ingredient of the present invention, with an excipient, a binder, a disintegrant and other additives by a conventional method, Alternatively, the mixture can be directly compression molded. In addition, a mating agent or the like may be added to the above-described healthful functional food in the form of tablet. The hard capsule of the capsule-type health functional food can be prepared by filling a mixture of isomerhenin, an active ingredient of the present invention, with an additive such as an excipient into a normal hard capsule, and the soft capsule is isarramine (isorhamnetin) with excipients such as excipients and the like, into a capsule such as gelatin. A plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, and the like may be added to the soft capsule. The ring-shaped health functional food can be prepared by molding a mixture of isorhamnetin, an active ingredient of the present invention, an excipient, a binder, a disintegrant, and the like by a conventionally known method, It can be applied to other skin preparations, or the surface can be coated with materials such as starch, talc. The granular health functional food may be prepared by granulating a mixture of isorhamnetin, an active ingredient of the present invention with an excipient, a binder, a disintegrant, etc., in a conventional manner, , A mating agent, and the like may be added.
본 발명에 있어서, 비알코올성 지방간은 비알코올성 간염증, 비알코올성 간섬유화, 비알코올성 간경화 등을 예시할 수 있으며, 퉁퉁마디(Salicornia SPP.) 유래의 이소람네틴(isorhamnetin)은 비알코올성 지방간 질환 억제능 이외에 유리라디컬 및 활성산소 소거, 지질과산화 억제 등의 강한 항산화활성과 산화스트레스에 대한 세포보호능이 있으므로 식품, 화장품, 의약품 및 동물의약품과 기능성 사료 등의 원료로 널리 이용할 수 있다.In the present invention, nonalcoholic fatty liver may be exemplified by nonalcoholic liver inflammation, nonalcoholic liver fibrosis, nonalcoholic liver cirrhosis, and isorhamnetin derived from Salicornia spp . Inhibits nonalcoholic fatty liver disease disease In addition, there is strong antioxidant activity such as free radical and active oxygen scavenging, inhibition of lipid peroxidation, and cell protection against oxidative stress, and thus it can be widely used as a raw material for foods, cosmetics, pharmaceuticals, animal drugs and functional feeds.
본 발명은 또 다른 관점에서, (a) 퉁퉁마디 추출물을 얻는 단계; (b) 상기 퉁퉁마디 추출물을 용매로 분획하는 단계; (c) 상기 퉁퉁마디 용매 분획물을 크로마토그래피로 분획하여 간세포내 염증반응 억제능 및 간세포 보호활성을 갖는 크로마토그래피 분획물을 얻는 단계; 및 (d) 상기 크로마토그래피 분획물을 고속액체크로마토그래피(HPLC)를 이용하여 이소람네틴을 분리하는 단계를 포함하는 것을 특징으로 하는 퉁퉁마디(Salicornia SPP.) 유래 이소람네틴(isorhamnetin)의 분리방법에 관한 것이다.In another aspect, the present invention relates to a method for preparing a composition comprising: (a) (b) fractionating the extract of Tungsten mulberry with a solvent; (c) fractionating the fractions of Chrysanthemum mildew solvent by chromatography to obtain a chromatographic fraction having inhibitory effect on hepatocyte inflammatory reaction and hepatocyte protective activity; And (d) isolating isorhamnetin using high performance liquid chromatography (HPLC) on said chromatographic fraction. The method of isolating isorhamnetin from Salicornia SPP . .
상기 퉁퉁마디 추출물은 (a) 퉁퉁마디를 세척 및 세절한 후 무염수에 넣은 후 열수 추출; (b) 열수 추출액 여과; (c) 여과 잔사를 무염수에 넣은 후, 100℃에서 2~3시간 재추출; (d) 재추출액 및 1차 열수추출액을 농축; (e) 농축물을 고형분 함량 대비 1~5%의 활성탄으로 정제; (f) 건조(동결 또는 분무)함으로써 획득할 수 있다. 상기 무염수는 증류수 또는 수도수를 사용할 수 있고, 무염수는 퉁퉁마디 부피의 1~5배, 바람직하게는 1~3배 사용할 수 있으며, 열수 추출은 약 100℃에서 4~6시간 수행하는 것이 바람직하다.The extracts of Tungtung nodule are (a) washed and finely divided and then put into salt-free water and subjected to hot water extraction; (b) hot water extraction filtration; (c) The filtration residue was re-extracted at 100 ° C for 2 to 3 hours after putting it in an aqueous salt solution; (d) concentrating the re-extracted liquid and the primary hot water extract; (e) purifying the concentrate with 1 to 5% of activated carbon relative to the solid content; (f) drying (freezing or spraying). The non-saline water may be distilled water or tap water, and the non-saline water may be used in an amount of 1 to 5 times, preferably 1 to 3 times the volume of the tap water, and the hot water extraction is performed at about 100 ° C for 4 to 6 hours desirable.
상기 퉁퉁마디 용매 분획물은 유기용매의 극성에 따라 용해되는 화합물들을 분리하는 통상의 방법으로 획득할 수 있으며, 본 발명에서는 먼저 추출한 퉁퉁마디 열수 추출물에 순차적으로 헥산, 클로로포름, 에틸아세테이트, 부탄올 및 물로 분획하여 헥산 분획물, 클로로포름 분획물, 에틸아세테이트 분획물, 부탄올 분획물 및 물 분획물을 획득하였다. In the present invention, the extract of Tungtung nodule is firstly fractionated with hexane, chloroform, ethyl acetate, butanol and water, Hexane fraction, chloroform fraction, ethylacetate fraction, butanol fraction and water fraction were obtained.
상기 크로마토그래피 분획물을 얻는 단계는 디아이온 HP-20 컬럼 크로마토그래피, 실리카겔 컬럼 크로마토그래피 및 LH-20 컬럼크로마토그래피를 수행하는 단계를 포함한다. The step of obtaining the chromatographic fraction comprises performing Diaion HP-20 column chromatography, silica gel column chromatography and LH-20 column chromatography.
상기 디아이온 HP-20 컬럼 크로마토그래피의 용출용매는 70% 아세톤, 실리카겔 컬럼 크로마토그래피의 용출용매는 클로로포름 이동상에 메탄올을 순차적으로 추가하여 이용하며, LH-20 컬럼크로마토그래피의 용출용매는 메탄올을 이용하는 것이 바람직하다.The elution solvent for the Diaion HP-20 column chromatography was 70% acetone. The elution solvent for the silica gel column chromatography was methanol, which was sequentially added to the chloroform mobile phase. The elution solvent for the LH-20 column chromatography was methanol .
상기 고속액체크로마토그래피(HPLC)는 메탄올과 물의 혼합액을 이동상으로 하여 수행될 수 있는데, 예를 들면 HPLC 등급의 메탄올과 3차 증류수를 이용한 그래디언트 조건, 보다 상세히는 메탄올 : 3차 증류수의 비율이 45:55에서 순차적으로 100:0으로 변환시키면서, 1~2㎖/분의 유속으로 흐르게 하여 수행할 수 있다. The high-performance liquid chromatography (HPLC) can be carried out using a mixture of methanol and water as a mobile phase. For example, gradient conditions using methanol-grade methanol and tertiary distilled water, more specifically, methanol: : 55 to 100: 0 sequentially, and at a flow rate of 1 to 2 ml / min.
[실시예][Example]
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로, 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 only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.
실시예 1: 퉁퉁마디 추출물로부터 유효성분 화합물 1의 분리Example 1: Isolation of the
1-1. 퉁퉁마디 열수 추출물의 제조1-1. Preparation of hot water extract
세척하고, 세절된 퉁퉁마디 전초 10kg을 초고속 진공농축수 추출기(COSMOS660, ㈜경서 E & P, 한국)로 무염수 20ℓ를 첨가하여 100±5℃에서 5시간 동안 열수 추출하였다. 다음으로 추출액을 여과시킨 후, 잔사에 10ℓ의 물을 첨가하고 동일조건에서 2시간 재추출 후, 먼저 얻은 추출액과 혼합한 후 80~90℃에서 염도 20%, 고형분 40%로 농축하였다. 끝으로 농축물을 고형분 대비 1~5%의 활성탄으로 정제한 후 동결건조하여 열수 추출물을 제조하였다.And 10 kg of the crushed nodule outpost was subjected to hot water extraction at 100 ± 5 ° C for 5 hours by adding 20 L of non-salted water to an ultra-high vacuum concentrated water extractor (COSMOS 660, Kyung-Seo E & P, Korea). Next, the extract was filtered, and then 10 liters of water was added to the residue. The extract was re-extracted for 2 hours under the same conditions, and then mixed with the extract. The concentrate was then concentrated at 80 to 90 ° C to a salt content of 20% and a solid content of 40%. Finally, the concentrate was purified with 1 ~ 5% of activated carbon based on solid content and then lyophilized to prepare a hot water extract.
1-2. 용매 분획물 획득1-2. Acquisition of solvent fractions
실시예 1-1에서 얻은 퉁퉁마디 열수추출물 374g을 1ℓ의 물에 현탁시킨 후 3ℓ의 에탄올을 가하고 4℃에서 하룻밤 정치하여 생긴 고분자 침전물을 원심분리(4℃, 10000rpm, 30분) 및 감압필터 여과로 제거하고, 여과액을 감압건조하여 에탄올을 완전 제거한 액을 증류수 2ℓ에 재용해 시켰다. 374 g of the extract of Tungtung Nodi obtained in Example 1-1 was suspended in 1 L of water, 3 L of ethanol was added and the mixture was allowed to stand overnight at 4 DEG C. The resulting polymer precipitate was centrifuged (4 DEG C, 10000 rpm, 30 minutes) , And the filtrate was dried under reduced pressure to remove completely the ethanol. The solution was redissolved in 2 L of distilled water.
다음으로 5ℓ 부피의 분액깔대기에 2ℓ의 n-헥산을 혼합하여 헥산층과 수층으로 2회 분획하였다. 상기 수층에 2ℓ의 클로로포름을 첨가하여 클로로포름층과 수층으로 2회 분획하고, 수층에 다시 에틸아세테이트를 첨가하고 에틸아세테이트층과 수층으로 2회 분획하고, 최종적으로 수층에 n-부탄올을 2ℓ 가하여 부탄올층과 수층으로 2회 분획하였다. Next, 2 L of n-hexane was mixed with a 5-L separatory funnel, and the mixture was divided into two fractions, hexane layer and water layer. 2 L of chloroform was added to the water layer, and the mixture was divided into two portions with chloroform layer and water layer. Ethyl acetate was further added to the water layer, and the mixture was divided into two portions with ethyl acetate layer and water layer. Finally, 2 L of n-butanol was added to the aqueous layer, And water layer.
다음으로 각각의 분획층들을 감압건조하여 각 유기용매를 제거하고 동결건조시켜, 헥산 분획물(0.8g), 클로로포름 분획물(0.9g), 에틸아세테이트 분획물(3.4g), 부탄올 분획물(9.8g) 및 물 분획물(16.1g)을 제조하였다.Next, each of the fraction layers was dried under reduced pressure to remove each organic solvent and lyophilized to obtain 0.8 g of hexane fraction, 0.9 g of chloroform fraction, 3.4 g of ethyl acetate fraction, 9.8 g of butanol fraction, The fractions (16.1 g) were prepared.
1-3. 컬럼크로마토그래피 정제1-3. Column chromatography purification
실시예 1-2에서 제조된 유기용매 분획물 중 항산화활성과 간세포보호 활성능이 우수한 에틸아세테이트 분획물(SEW-EA) (3.4g)을 50㎖ 알칼리수 (pH 10)에 용해시켜 흡착성 디아이온 HP-20 레진이 충진된 첫 번째 컬럼 (3.5 × 40㎝) 상단부위에 흡착시킨 후 증류수, 70% 메탄올 및 70% 아세톤으로 순차적으로 용출시켜 3개의 분획물들을 획득하였다. 그중 항산화활성과 간세포 보호 활성이 우수한 아세톤 용출획분(SEW-EA-B2) 1.5g을 극성 실리카겔이 충진된 두번째 컬럼 (2.8 × 40㎝)에서 메탄올을 농도구배로 증가시킨 클로로포름 용매, 보다 상세하게 클로로포름 : 메탄올의 비율이 100:0에서 70:30으로 순차적으로 변환시키면서, 유속 0.3㎖/분의 속도로 용출시켜 350㎖씩 5개의 분획물을 획득하였다. 그리고 그중 항산화활성과 간세포 보호 활성이 우수한 획분(SEW-EA-B2-F2, 432㎎)을 감압건조 후 2㎖의 메탄올에 용해하고, 저분자용 겔여과 세파덱스 (Sephadex) LH-20이 충진된 세 번째 컬럼 (2 × 33㎝)에 도입한 다음 95% 메탄올(유속 0.2㎖/분)을 흘러보내면서 30㎖씩 분획하여 총 20개의 분획물(SEW-L-1 ~ SEW-L-20)을 획득하였다. 최종적으로 상기 20개의 분획물 중 항산화활성과 간세포 보호활성이 가장 우수한 SEW-L-17 획분을 감압농축, 동결건조하여 68㎎을 수득하였다. The ethyl acetate fraction (SEW-EA) (3.4 g) having excellent antioxidative activity and hepatocyte protective activity among the organic solvent fractions prepared in Example 1-2 was dissolved in 50 ml of alkaline water (pH 10) to obtain an adsorbable diaion HP-20 The resin was adsorbed on the upper part of the first column (3.5 × 40 cm) filled with water, and then sequentially eluted with distilled water, 70% methanol and 70% acetone to obtain three fractions. 1.5 g of an acetone elution fraction (SEW-EA-B2) having excellent antioxidative activity and hepatocyte protective activity was dissolved in a chloroform solvent in which methanol was increased to a concentration gradient in a second column (2.8 x 40 cm) filled with polar silica gel, : Methanol was sequentially converted from 100: 0 to 70:30, and eluted at a flow rate of 0.3 ml / min to obtain five fractions of 350 ml each. The fraction (SEW-EA-B2-F2, 432 mg) having excellent antioxidative activity and hepatocyte protective activity was dissolved in 2 ml of methanol after drying under reduced pressure, and gel filtration with low molecular weight Sephadex LH-20 20 fractions (SEW-L-1 to SEW-L-20) were obtained by fractionation in 30 ml portions while flowing 95% methanol (flow rate 0.2 ml / min) . Finally, SEW-L-17 fraction having the highest antioxidative activity and hepatocyte protective activity among the 20 fractions was concentrated under reduced pressure and lyophilized to obtain 68 mg.
1-4. 고속액체크로마토그라피(HPLC) 정제1-4. High Performance Liquid Chromatography (HPLC) Purification
실시예 1-3에서 수득된 SEW-L-17 분획물 68㎎을 2㎖의 HPLC 용 메탄올에 용해한 후 0.22㎛ 필터로 여과한 다음 고속액체크로마토그라피를 이용하여 항산화성과 간세포보호활성이 강한 단일물질을 분리하였다. 분석용 HPLC는 졸박스 이클립스 C18 분석 컬럼 (Zorbax Eclips, 5㎛, 4.5 × 250㎜, Agilent)이 장착된 모델(1260 Infinity, Agilent, USA)을 사용하였고, 고속분취 액체크로마토그라피는 일본 YMC사의 프렙용 컬럼(Triart C18, 20㎜ × 150㎜, 5㎛, YMC, Japan)이 장착한 모델(Multiple Preparative HPLC(LC-forte/R, YMC, Japan)을 사용하였다. 이동상 용매 조건은 메탄올과 3차 증류수를 이용한 그래디언트 조건에서 1~3㎖/분의 유속으로 흐르게 하여 분석하였으며, Agilent, 1200 DAD 검출기(detector) 또는 YMC-YUV-3400 UV 검출기를 사용하였으며, 두 파장영역 (260 및 330 nm)의 흡수도를 이용하여 화합물들을 순수분획한 결과 머무름시간 13.5 분에서 화합물 1(32mg)을 수득할 수 있었다. 도 1은 퉁퉁마디 정제 분획물인 SEW-EA-B2, SEW-EA-B2-F2, 및 SEW-L-17의 분석용 HPLC 크로마토그램과 화합물 1의 자외선 흡수 파장 스펙트럼이다. 68 mg of the SEW-L-17 fraction obtained in Example 1-3 was dissolved in 2 ml of methanol for HPLC, filtered through a 0.22 μm filter, and then subjected to high performance liquid chromatography to obtain a single substance having high antioxidative and hepatocyte protective activity Respectively. Analytical HPLC was performed using a model equipped with a Solbox Eclipse C18 analytical column (Zorbax Eclips, 5 μm, 4.5 × 250 mm, Agilent) (1260 Infinity, Agilent, USA) (Preparative HPLC (LC-forte / R, YMC, Japan) equipped with a double column (Triart C18, 20 mm × 150 mm, 5 μm, YMC, Japan) was used. Flow rate of 1 ~ 3ml / min was performed under gradient conditions using distilled water. Agilent, 1200 DAD detector or YMC-YUV-3400 UV detector was used and the two wavelength regions (260 and 330 nm) Compound 1 (32 mg) was obtained at a retention time of 13.5 minutes as a result of pure fractionation of the compounds using absorbance. Figure 1 shows SEW-EA-B2, SEW-EA-B2-F2, The analytical HPLC chromatogram of SEW-L-17 and the ultraviolet absorption wavelength spectrum of
실시예 2: 퉁퉁마디 추출물로부터 분리된 화합물 1의 구조분석Example 2: Structural analysis of
실시예 1-4에서 분리된 화합물 1의 자외선 최대 흡수대 및 시약 첨가에 따른 흡수변이(adsorption shift)는 각 시료를 메탄올에 1㎎/㎖의 농도로 용해시킨 후 자외선 분광기(Genesys 10S UV-VIS spectrophotometer, Thermo Scientific, USA)를 이용하여 190-500nm 영역내에서 측정하였다. 이때, 시약첨가에 따른 자외선 흡수대의 변화를 관찰하기 위하여 AlCl3 , NaOH, NaOAC 등을 사용하였다.The absorption maxima of
화합물의 분자량 결정을 위하여 전자포말이온화 (ESI) 질량분석기 (LC-ESI mass spectrometer, AGILENT 1100, USA Micromass Quattro II)로 포지티브 및 네거티브 스캔을 실시하고, 하이 레조루션(High resolution) MS를 측정하였다. Positive and negative scans were performed with an electronic foam ionization (ESI) mass spectrometer (LC-ESI mass spectrometer, AGILENT 1100, USA Micromass Quattro II) to determine the molecular weight of the compounds and high resolved MS was measured.
핵자기공명(NMR)분석은 화합물 1(10㎎)을 완전 건조하여 DMSO-d 6(0.5㎖)에 용해한 후 5㎜ NMR 튜브에 주입하고 제올 모델 기종 (JNM-ECA 600, Jeol, Japan)으로 분석 하였으며, 1H-NMR은 600MHz로, 13C-NMR은 150MHz로 각각 측정하였다.Compound 1 (10 mg) was completely dried, dissolved in DMSO- d 6 (0.5 ml), injected into a 5 mm NMR tube, and analyzed with a Zeol model (JNM-
상기와 같이 측정한 결과, 화합물 1은 이소람네틴(isorhamnetin)으로 동정되었고 물리화학적 성질은 다음과 같다.As a result of the measurement as described above,
(1) 분자식 : C16H12O7 (1) Molecular formula: C 16 H 12 O 7
(2) 분자량 : 316, ESI-MS: m/z 315.2 [M-H], m/z 631.2 [M+M-H], m/z 317.0 [M+H] (도 2B)(2) Molecular weight: 316, ESI-MS: m / z 315.2 [M-H], m / z 631.2 [M +
(2) 녹는점 : 392℃(2) Melting point: 392 DEG C
(3) 성상 : 황색분말(3) Appearance: yellow powder
(4) 용해성 : 메탄올, 에탄올, 에틸아세테이트, 클로로포름에 용해되고 물에 불용성(4) Solubility: soluble in methanol, ethanol, ethyl acetate, chloroform and insoluble in water
(5) TLC 발색 : FeCl3(양성), 브로모크레졸그린 (음성), UV light(양성, 황녹색), 아니린디페닐라민(음성), 앤티모니(음성), 드라겐돌프(음성) (5) TLC color development: FeCl 3 (positive), bromocresol green (negative), UV light (positive, yellow green), anilindiphenylamine (negative), antimony (negative)
(6) 자외선 최대 흡수파장 영역 (메탄올, nm) : 253, 267sh, 306sh, and 370, (+NaOH) : 273, 287sh, 326sh, 390, (+AlCl3) : 253, 267sh, 306sh, and 423 (+AlCl3 + HCl) : 253, 267sh, 306sh, and 370 ; (+NaOAc + H3BO3) 254, 306, 382(6) UV absorption maximum wavelength region (MeOH, nm): 253, 267sh, 306sh, and 370, (+ NaOH): 273, 287sh, 326sh, 390, (+ AlCl 3): 253, 267sh, 306sh, and 423 (+ AlCl 3 + HCl): 253, 267sh, 306sh, and 370; (+ NaOAc + H 3 BO 3 ) 254, 306, 382
(7) 1H 및 13C-NMR : 1H NMR (600 MHz, DMSO-d 6) δ ppm: 2.49 (1H, d, 3-H), 3.83 (3H, s, OCH3), 6.18 (1H, d, 6-H), 6.47 (1H, d, 8-H), 6.93 (1H, d, 5′-H), 7.68 (1H, m, 6′-H), 7.74 (1H, d, 2′-H), 9.43 (1H, s, OH), 9.75 (1H, s, OH), 10.76 (1H, s, OH), 12.46 (1H, s, 5-OH) (도 2C), 13C-NMR (150MHz, DMSO-d 6) δppm : 148.81(2-C), 135.86(3-C), 175.90(4-C), 156.17(5-C), 8.21(6-C), 163.94(7C), 93.60(8-C), 160.94(9-C), 103.05(10-C), 122.00(1′-C), 111.65(2′-C), 146.-63(3′-C), 147.37(4′-C), 115.55(5′-C), 121.72(6′-C), 55.73 (OCH3) (도 2D)(7) 1 H and 13 C-NMR: 1 H NMR (600 MHz, DMSO- d 6) δ ppm: 2.49 (1H, d, 3H), 3.83 (3H, s, OCH 3), 6.18 (1H (1H, d, 6H), 6.74 (1H, d, 8H) '-H), 9.43 (1H, s, OH), 9.75 (1H, s, OH), 10.76 (1H, s, OH), 12.46 (1H, s, 5-OH) ( Fig. 2C), 13 C- NMR (150 MHz, DMSO- d 6 )? Ppm: 148.81 (2-C), 135.86 (3-C), 175.90 (4-C), 156.17 , 93.60 (8-C), 160.94 (9-C), 103.05 (10-C), 122.00 (1'-C), 111.65 (4'-C), 115.55 (5'-C), 121.72 (6'-C), 55.73 (OCH3)
(8) 화학구조식(8) Chemical formula
실시예 3: 퉁퉁마디 추출물의 고지방식이와 정제염 식이 유도 비알코올성 지방간의 억제효능 확인 Example 3: Determination of inhibitory effect of non-alcoholic fatty liver induced by high-fat diet and refined salt diet
3-1. C57BL/6J 마우스의 고지방식이 급여로 인한 비만 유도3-1. High-fat diet of C57BL / 6J mice induced obesity by feeding
C57BL/6J 수컷 7주령 마우스 36 마리를 무작위로 다음과 같이 두 그룹으로 나누고 24주 동안 각 사료를 급여하여 비만 및 인슐린 저항성을 유도하였다. 저지방식이 그룹(LFD, D12450B Rodent Diet with 10 kcal% fat; Research Diets, n=5) 및 고지방식이 그룹(HFD, D12492 Rodent Diet with 60 kcal% fat; Research Diets, n=31)에 24주 동안 각각 LFD와 HFD를 섭취시키고 평균체중을 비교하였을 때 LFD 섭취군(40.5 ± 1.8g)에 비해 HFD 섭취군(52.4 ± 0.5g)의 체중이 유의적으로 높게 나타났다 (P < 0.05)(도 3). 36 C57BL / 6J male 7-week-old mice were randomly divided into two groups as follows to induce obesity and insulin resistance by feeding each feed for 24 weeks. (HFD, D12492 Rodent Diet with 60 kcal% fat; Research Diets, n = 31) were divided into two groups: LFD, D12450B, Rodent Diet with 10 kcal% ( P <0.05) in the HFD-fed group (52.4 ± 0.5g) than in the LFD-fed group (40.5 ± 1.8g) when the LFD and HFD were consumed and the mean body weight was compared ).
고지방식이 급여로 비만이 잘 유도되었음을 확인하였으므로, 고지방식이 유도 비만쥐를 대상으로 고지방식이와 정제염 식이를 통한 비알코올성 지방간 유도 실험을 진행하게 되었다. The results showed that obese rats were well induced by high fat diet method. Therefore, non - alcohol fatty liver induction experiment was conducted in high fat diet induced rats by high fat diet and refined salt diet.
3-2. 퉁퉁마디 추출물의 고지방식와 정제염 섭취로 유도된 간손상 개선 효능 평가3-2. Evaluation of hepatic injury-improving efficacy induced by high-fat diet and refined salt ingestion
고지방식이는 미국 Research Diets 社에서 구입한 D12492 (Rodent Diet with 60 kcal% fat)이며, 정제염은 국내 ㈜한주소금에서 구입하였다. 소금 농도 설정은 WHO에서 권장하는 성인 하루 섭취량(나트륨 2g 또는 소금 5g)을 기준으로, 선행문헌에 따라 이를 쥐 체적률로 환산하여 계산하였으며, 소금 함량이 쥐 사료의 약 1%가 성인의 하루 권장 소금섭취량과 같게 하고, 이를 각각 NaCl 함량 기준으로 계산하였을 시, 1-1에서 제조된 퉁퉁마디 열수추출물(NaCl 함량: 약 56.41%)은 고지방식이의 약 1.77%, 한주소금(정제염, NaCl 함량:99.52%)은 고지방식이의 약 1.00%이며, 이를 바탕으로 사료를 조제하여 실험을 진행하였다. The high-fat diet was D12492 (Rodent Diet with 60 kcal% fat) purchased from Research Diets, USA. The salt concentration was calculated based on the adult daily intake recommended by the WHO (2 g sodium or 5 g salt) according to the previous literature, and the salt content was calculated as about 1% (NaCl content: about 56.41%) was about 1.77% of that of the high-fat diet, and that of one-week salt (refined salt, NaCl content : 99.52%) was about 1.00% of the high - fat diet, and the experiment was conducted based on this.
또한, 고지방식이를 총 36주간 급여하였을 시, 지방간염증이 뚜렷하게 유발된다는 선행문헌에 따라 고지방식이를 24주간 투여한 후, 고지방과 소금을 섞은 식이를 13주간 추가 투여하여 총 37주간 실험을 진행하였다. 부검 후, 장기 무게를 분석한 결과 고지방식이와 정제염을 섭취한 군(HFD + purified salt PS)은 고지방식이만을 섭취한 군에 비해 간 무게가 유의적으로 증가하였고, 고지방식이와 퉁퉁마디 추출물을 섭취한 군(HFD + Salicornia salt, SS)에서는 간 무게가 고지방식이와 정제염을 섭취한 군보다 유의적으로 낮아(P < 0.05) 퉁퉁마디 추출물이 지방간 발생 억제효능이 있다는 것을 확인할 수 있었다(도 4A 및 4B).In addition, according to the previous literature that when the high fat diet was fed for a total of 36 weeks, fatty liver inflammation was evidently induced. The high fat diet was administered for 24 weeks, followed by the high fat and salt diet for 13 weeks. . As a result of analyzing organ weights after the autopsy, liver weight was significantly higher in the group fed high fat diet and purified salt (HFD + purified salt PS) than in the high fat diet group, In liver (HFD + Salicornia salt, SS) group, liver weight was significantly lower (P <0.05) than that of high fat diet and refined salt intake group. (Figs. 4A and 4B).
또한, 부검 후 얻은 혈장을 분석하여, 간세포가 손상을 받았을 때 혈중으로 방출되는 효소인 ALT (alanine aminotransferase)의 수치를 확인한 결과, 고지방식이와 정제염을 섭취한 군은 고지방식이만을 섭취한 군에 비해 ALT 수준이 유의적으로 증가되었고, 고지방식이와 퉁퉁마디 추출물을 섭취한 군에서는 ALT 수준이 고지방식이와 정제염을 섭취한 군보다 유의적으로 낮음 (P < 0.05)을 확인할 수 있었다(도 4C).In addition, analysis of the plasma obtained after autopsy showed that alanine aminotransferase (ALT), an enzyme released into the blood when hepatocytes were damaged, was found to be higher in the high fat diet group And ALT levels were significantly lower in the high fat diet group than in the high fat diet group ( P <0.05) ( p <0.05) 4C).
3-3. 퉁퉁마디 추출물의 비알콜성 지방간 질환(NAFLD)개선 효능의 조직학적 평가3-3. Histological evaluation of non-alcoholic fatty liver disease (NAFLD)
퉁퉁마디 추출물의 비알콜성 지방간 질환 개선 효능을 조직학적으로 확인하기 위하여, 부검 후 얻은 간 조직을 대상으로 H&E 염색 및 Masson's trichrome 염색을 수행하고, 지방간증을 분석하였다. In order to histologically confirm the non-alcoholic fatty liver disease improvement effect of the extract of Tungtung nodule, H & E staining and masson's trichrome staining were performed on liver tissue obtained after autopsy, and the fat test was analyzed.
도 5에 나타난 바와 같이, 고지방식이와 퉁퉁마디 추출물을 섭취한 군(HFD + Salicornia salt, SS)이 고지방식이와 정제염을 섭취한 군(HFD + purified salt PS)에 비해 지방간증이 억제되는 효능을 보였다. 즉, H&E 염색을 바탕으로 진행한 steatosis scoring 항목에서는 고지방식이와 퉁퉁마디 추출물을 섭취한 군(HFD + Salicornia salt, SS)이 고지방식이와 정제염을 섭취한 군(HFD + purified salt PS)보다 지방간증을 저해시킴을 확인하였으며 (P < 0.094), 간내 염증(inflammation)도 유의적으로 억제됨을 확인하였다. (HFD + Salicornia salt, SS) supplemented with high-fat diet and high-fat diet (HFD + purified-salt PS) Showed efficacy. In the steatosis scoring category based on H & E staining, HFD + Salicornia salt (SS), a high fat diet group and HFD + purified salt PS group, ( P <0.094), and inflammation of the liver was also significantly inhibited.
또한, Steatosis, Inflammation 및 Ballooning을 종합하여 NAFLD (비알콜성 지방간 질환) 개선효능을 scoring을 하였을 때, 퉁퉁마디 추출물이 고지방식이와 정제염 식사로 촉발되는 비알코올성 지방간증을 억제하는 효능이 있다는 것을 확인할 수 있었다. In addition, when combined with Steatosis, Inflammation, and Ballooning, scoring of NAFLD (nonalcoholic fatty liver disease) improving efficacy shows that the extract of Tungtung nodule inhibits nonalcoholic lipidemia triggered by high fat diet and refined salt diet I could confirm.
참고로 scoring 기준은 다음과 같다.For reference, the scoring criteria are as follows.
- 간섬유화 진행 점수: 0=섬유화가 10% 이하로 진행된 간, 1=10~33% 섬유화가 진행된 간, 2=33~66% 섬유화가 진행된 간, 3=66% 이상 섬유화가 진행된 간- Liver fibrosis progression score: 0 = Liver with fibrosis less than 10%, 1 = Liver with 10 to 33% fibrosis, 2 = 33 to 66% Liver with fibrosis, 3 =
- 간소엽내 염증 진행 점수: 0=200배 필드 현미경 하에서 관찰되는 병소가 없을 때, 1=200배 필드 현미경 하에서 관찰되는 병소가 2개 이하일 때, 2=200배 필드 현미경 하에서 관찰되는 병소가 2개에서 4개 사이일 때, 3=200배 필드 현미경 하에서 관찰되는 병소가 4개 이상일 때- Score of progression of inflammation in the lobular lobe: 0 = 200 times when there is no lesion observed under field microscope, 1 = 200 times under field microscope, 2 = 200 times under field microscope. 4 to 4, 3 = 200 times Under field microscope, there are more than 4 lesions observed
3-4. 퉁퉁마디 추출물의 간조직 내 염증 및 섬유화 마커의 mRNA 발현에 미치는 영향 평가3-4. Evaluation of the effects of extracts of Tungtung nodule on inflammation and fibrosis marker mRNA expression in liver tissue
조직내 염증 및 간섬유화 마커들(Il1b, Cxcl2, Ccl2, Cd68, Col1a1)의 mRNA 발현을 다음과 같이 측정하였다. 부검 후 얻은 간조직으로부터 RNA 분리 킷 (Ambion, RNA isolation kit, Life Technologies, Gaithersburg, MD, USA)을 이용하여 RNA를 추출하고, 이를 NanoDrop ND-2000 (Thermo Fisher Scientific, Waltham, MA, USA)에서 정량하고, 1st strand c-DNA 합성 킷(Takara Bio Inc., Otsu, Japan)을 이용하여 cDNA를 합성하였다. mRNA 발현은 SYBR Green Master Mix(BioRad, Hercules, CA, USA) 프라이머들과 합성된 c-DNA 2㎕를 사용하여 qRT-PCR(quantitative real-time polymerase chain reaction)를 실시하여 확인 하였다. MRNA expression of inflammatory and hepatic fibrosis markers (Il1b, Cxcl2, Ccl2, Cd68, Col1a1) in tissues was measured as follows. RNA was extracted from liver tissues obtained after autopsy using an RNA isolation kit (Ambion, RNA isolation kit, Life Technologies, Gaithersburg, MD, USA), and the RNA was extracted with NanoDrop ND-2000 (Thermo Fisher Scientific, Waltham, MA, USA) And cDNA was synthesized using 1 st strand c-DNA synthesis kit (Takara Bio Inc., Otsu, Japan). mRNA expression was confirmed by qRT-PCR (quantitative real-time polymerase chain reaction) using SYBR Green Master Mix (BioRad, Hercules, CA, USA) primers and 2 μl of synthesized c-DNA.
PCR은 증폭반응 전 프라이머들은 95℃에서 3분간 변성시키고, 44 사이클로 95℃에서 10분간, 60℃에서 30분, 72℃ 30분간 반응조건으로 44회 연속반응시켰다. PCR 반응물내 mRNA 함량은 CFX Connect™ Real-Time PCR Detection System (BioRad, Hercules, CA, USA)에서 측정하였다. 사용된 프라이머들은 다음과 같다. The primers before denaturation were denatured at 95 ° C for 3 minutes, and reacted for 44 cycles at 95 ° C for 10 minutes, 60 ° C for 30 minutes, and 72 ° C for 30 minutes. The mRNA content in the PCR reaction was measured using the CFX Connect ™ Real-Time PCR Detection System (BioRad, Hercules, Calif., USA). The primers used are as follows.
서열번호 1: Ccl2 forward (5′-CCA CTC ACC TGC TGC TAC TCA T-3′)SEQ ID NO: 1: Ccl2 forward (5'-CCA CTC ACC TGC TGC TAC TCA T-3 ')
서열번호 2: Ccl2 reverse (5′-TGG TGA TCC TCT TGT AGC TCT CC-3′)SEQ ID NO: 2: Ccl2 reverse (5'-TGG TGA TCC TCT TGT AGC TCT CC-3 ')
서열번호 3: Il1b forward (5′-GGA GAA CCA AGC AAC GAC AAA ATA-3′)SEQ ID NO: 3: Il1b forward (5'-GGA GAA CCA AGC AAC GAC AAA ATA-3 ')
서열번호 4: Il1b reverse (5′-TGG GGA ACT CTG CAG ACT CAA AC-3′)SEQ ID NO: 4: Il1b reverse (5'-TGG GGA ACT CTG CAG ACT CAA AC-3 ')
서열번호 5: Cd68 forward (5′-ACC TGC TCA ACA TCA TGA AGG-3′)SEQ ID NO: 5: Cd68 forward (5'-ACC TGC TCA ACA TCA TGA AGG-3 ')
서열번호 6: Cd68 reverse (5′-AGA TGG AGC TAT GCA GGT GG-3′) SEQ ID NO: 6: Cd68 reverse (5'-AGA TGG AGC TAT GCA GGT GG-3 ')
서열번호 7: Cxcl2 forward (5′-CCA AGG GTT GAC TTC AAG AAC-3′)SEQ ID NO: 7: Cxcl2 forward (5'-CCA AGG GTT GAC TTC AAG AAC-3 ')
서열번호 8: Cxcl2 reverse (5′-AGC GAG GCA CAT CAG GTA CG-3′)SEQ ID NO: 8: Cxcl2 reverse (5'-AGC GAG GCA CAT CAG GTA CG-3 ')
서열번호 9: Col1a1 forward(5′-GCT CCT CTT AGG GGC CAC T-3′)SEQ ID NO: 9: Col1a1 forward (5'-GCT CCT CTT AGG GGC CAC T-3 ')
서열번호 10: Col1a1 reverse (5′-CCA CGT CTC ACC ATT GGG G-3′) SEQ ID NO: 10: Col1a1 reverse (5'-CCA CGT CTC ACC ATT GGG G-3 ')
도 6에 나타난 바와 같이, 고지방식이와 정제염을 섭취한 군(HFD + purified salt PS)에 비하여 고지방식이와 퉁퉁마디 추출물을 섭취한 군(HFD + Salicornia salt, SS)은 염증관련 마커(cxcl2, il1b)의 발현과 섬유화관련 마커(col1a1)의 발현을 유의적으로 저해한다는 것을 확인할 수 있었다.(HFD + salicornia salt, SS), which consumed high-fat diet extract of Wanchungfumi extract (HFD + purified salt PS), showed higher levels of inflammation related markers ( cxcl2 , il1b ) and the expression of the fibrosis-related marker ( col1a1 ).
실시예 4: 이소람네틴의 효능 확인 Example 4: Efficacy of isorhamnetin
4-1. 항산화 활성 조사4-1. Antioxidant activity investigation
실시예 2의 퉁퉁마디 추출물로부터 유효성분으로 분리 동정된 이소람네틴의 항산화 활성을 확인하기 위하여 DPPH 유리 라디컬에 대한 소거능, 활성산소 소거능 및 지질과산화 저해능 평가를 수행하였다. 이때 대조군으로 비알코올성 지방간 예방과 치료에 사용되는 항산화 비타민인 알파-토코페롤(비타민 E)과 간세포 글루타치온의 전구물질인 N-acetylcystein(NAC)을 사용하였다. In order to confirm the antioxidative activity of isorhamnetin identified as an active ingredient from the extract of Tungtung nodule in Example 2, scavenging activity, active oxygen scavenging ability and lipid peroxidation inhibiting activity of DPPH free radical were evaluated. At this time, alpha-tocopherol (vitamin E) and N-acetylcystein (NAC), a precursor of hepatocyte glutathione, which are antioxidant vitamins used for prevention and treatment of nonalcoholic fatty liver disease were used as a control group.
4-1-1 : 유리라디컬 소거능 측정4-1-1: Measurement of glass radical scavenging ability
항산화 활성은 불로이스의 방법(Chen, et. al., 1999. J. Agric. Food Chem. 47. 2226-2228)에 준하여 1,1-디페닐-2-피크릴 하이드라질(1,1-diphenyl-2-picryl hydrazyl, DPPH, Sigma Co., USA)을 이용하여 측정하였다. 즉, DPPH 5㎎을 에탄올 50㎖에 녹여 DPPH 용액을 만든 후 96-well microplate에 180㎕를 가하고 시료를 0~100㎍/㎖의 농도로 첨가하고, 5초 동안 혼합한 후 30분 동안 실온에서 반응시키고, 517㎚에서 시료를 가하지 않은 대조군에 대한 흡광도 감소를 유리라디컬소거 활성(%)으로 나타내었다. 50%의 유리라디컬을 소거하는데 필요한 물질의 농도를 IC50 값으로 나타낼 수 있으며, 이 값이 낮을수록 항산화능이 강함을 의미한다.The antioxidant activity was determined according to the method of Bullois (Chen et al., 1999. J. Agric. Food Chem. 47. 2226-2228), 1,1-diphenyl-2-picrylhydrazide (1,1- diphenyl-2-picryl hydrazyl, DPPH, Sigma Co., USA). DPPH solution was prepared by dissolving 5 mg of DPPH in 50 ml of ethanol, adding 180 μl to 96-well microplate, adding 0-100 μg / ml of the sample, mixing for 5 seconds, And the absorbance reduction for the control group to which no sample was added at 517 nm was expressed as the free radical scavenging activity (%). The concentration of the substance required to eliminate 50% of the free radicals can be represented by the IC 50 value, which means that the lower the value, the stronger the antioxidant capacity.
상기 표 1에 나타난 바와 같이, 퉁퉁마디에서 분리한 이소람네틴은 알파-토코페롤보다 약 4배, N-아세틸시스테인보다 약 1.87배 강한 유리라디컬 소거능이 있음을 확인하였다. As shown in Table 1, it was confirmed that isorhamnetine isolated from Tungtung Mardi had about 4 times stronger than α-tocopherol and about 1.87 times stronger than N-acetylcysteine.
4-1-2 : 활성산소 소거능 측정4-1-2: Measurement of reactive oxygen scavenging ability
활성산소(superoxide anion) 소거능 측정은 잔틴/잔틴옥시다제 (xanthin/xanthin oxidase) 효소반응에 의한 활성산소 발생계를 이용하여 활성산소에 의한 니트로블루 테트라졸리움 (nitroblue tetrazolim, NBT)의 산화에 의한 광흡수도 (530㎚) 변화를 이용하여 측정하고, 50%의 활성산소를 소거하는데 필요한 물질의 농도를 표 2에 IC50 값으로 나타내었다. The superoxide anion scavenging activity was measured by the oxidation of nitroblue tetrazolium (NBT) by active oxygen using an active oxygen generating system by xanthine / xanthine oxidase enzyme reaction. The concentration of the substance required to remove 50% of the active oxygen is measured using the absorbance (530 nm) change and is shown in Table 2 as the IC 50 value.
상기 표 2에 나타난 바와 같이, 퉁퉁마디에서 분리한 이소람네틴은 알파-토코페롤보다 약 9배 이상, N-아세틸시스테인보다 약 3.5배 강한 활성산소 소거능이 있음을 확인하였다. As shown in Table 2, it was confirmed that isorhamnetin isolated from Tungsten maddy had about 9 times stronger than alpha-tocopherol and about 3.5 times stronger than that of N-acetylcysteine.
4-1-3 : 지질과산화 저해능 측정4-1-3: Measurement of lipid peroxidation inhibition ability
지질과산화 저해능 측정은 적출된 흰쥐 간세포를 초원심분리 (77000g, 60분, Hitachi RP 30) 하여 얻어진 마이크로좀을 지질원으로 하여 Fe2+ 아스코르베이트계에 의한 지질과산화를 유도하고 생성된 말론디알데히드 (malonaldehyde, MDA)를 티오바비트린산 (thiobarbituric acid, TBA)과 반응시켜 시료에 의해 감소된 MDA양을 정량 환산하였다. 시험관에 시료가 함유된 간 마이크로좀 용액 200㎕와 소듐도데실슬폰염(sodium dodesyl sulfate, SDS)용액 225㎕를 가하고 5초간 진 탕 혼합한 후, 20%의 아세트산, 75㎕의 증류수, 1.2% TBA 용액 1㎖씩 가하여 30분간 가온 하였다. 실온에서 30분 냉각 후에 3000rpm에서 20분간 원심분리하여 상등액을 532㎚에서 흡광도를 측정하여 시료를 첨가하지 않은 대조군에 대한 지질 과산화 저해활성(%)을 구하고, 50%의 활성산소를 소거하는데 필요한 물질의 농도를 IC50 값으로 나타내었다.The lipid peroxidation inhibition assay was performed by inducing lipid peroxidation by the Fe 2+ ascorbate system using microsomes obtained by ultracentrifugation (77000 g, 60 min, Hitachi RP 30) of the extracted rat hepatocytes as a lipid source, Aldehyde (MDA) was reacted with thiobarbituric acid (TBA) to quantitatively reduce the amount of MDA reduced by the sample. 200 μl of hepatic microsomal solution containing a sample in a test tube and 225 μl of sodium dodecyl sulfate (SDS) solution were added and mixed for 5 seconds with shaking. Then, 20% acetic acid, 75 μl of distilled water, 1.2% TBA 1 ml of the solution was added and the mixture was heated for 30 minutes. After cooling at room temperature for 30 minutes, the mixture was centrifuged at 3000 rpm for 20 minutes, and the supernatant was measured for absorbance at 532 nm to determine the lipid peroxidation inhibitory activity (%) of the control group to which no sample was added. Was expressed by IC 50 value.
상기 표 3에 나타난 바와 같이, 퉁퉁마디에서 분리한 이소람네틴은 알파-토코페롤보다 약 2.2배, N-아세틸시스테인보다 약 1.65배 강한 지질과산화 저해능이 있음을 확인하였다. As shown in Table 3 above, it was confirmed that isorhamnetine isolated from Tungtung Mardi had a lipid peroxidation potency about 2.2 times higher than that of alpha-tocopherol and about 1.65 times higher than N-acetylcysteine.
4-2. 4-2. LPS로By LPS 자극된 Human Stimulated Human HepG2HepG2 세포에서 퉁퉁마디에서 분리된 이소람네틴 유효성분의 농도의존적인 NO 방출량 Concentration-dependent NO emission of isopramenine active ingredient isolated from Tungsten nodules in cells 억제능Inhibition 및 And iNOSiNOS 단백질 발현 Protein expression 저해능Low performance 측정 Measure
퉁퉁마디에서 분리한 이소람네틴의 간세포내 항염증 효능을 분석하기 위하여 간세포 염증 유발 인자인 LPS(100ng/㎖)로 자극된 human HepG2 세포에서 생성되는 아질산염(NO)이 이소람네틴의 농도에 의존적으로 저해되는지 확인하였다. 아질산염(NO)의 측정은 Griess 시약을 이용한 NO assay를 사용하였다. In order to analyze the anti-inflammatory effect of isorhamnetin isolated from Tungtung maddy, the nitrite (NO) produced in human HepG2 cells stimulated with LPS (100 ng / ml), a hepatocyte inflammation inducer, . Nitrite (NO) was measured by NO assay using Griess reagent.
그 결과, 도 7A에 나타난 바와 같이, HepG2 세포에서 LPS에 의해서 유도되는 아질산염(NO)은 25.13 ± 0.75μM로 대조군으로 사용한 3.35 ± 0.52μM에 비하여 약 7.5배의 증가를 보였으나, 이소람네틴을 농도별(10, 25, 50μM)로 처리 한 결과 각각 20.59 ± 0.68μM, 12.08 ± 0.46μM, 5.01 ± 0.09μM로 아질산염(NO)의 양이 농도 의존적으로 감소하는 것을 확인하였다. As a result, as shown in FIG. 7A, the nitrite (NO) induced by LPS in HepG2 cells was increased by about 7.5-fold compared with the control group of 3.35 ± 0.52 μM at 25.13 ± 0.75 μM, The concentration of nitrite (NO) was decreased by 20.59 ± 0.68μM, 12.08 ± 0.46μM and 5.01 ± 0.09μM, respectively, depending on the concentrations (10, 25 and 50μM)
또한, 이소람네틴이 농도 의존적으로 아질산염(NO)의 합성 유도 효소인 iNOS의 단백질 발현을 저해하는 것을 확인하고자 Immunoblotting 실험을 실시하였다. 즉 iNOS 단백질의 발현 양상을 분석하기 위하여 이소람네틴과 LPS를 동시에 HepG2 세포에 처리하고 18시간 동안 배양한 다음, 세포를 라이시스(lysis)시키고, 단백질을 정량(Bradford assay, BioRad Laborotories, Hercules, CA)하였다. 다음으로 동일한 양(20㎍)의 단백질을 12% SDS-PAGE 겔(Min Bio-Rad, Hets, UK)에 로딩하고, 전기영동을 실시하였다. 분리된 단백질은 셀룰로스 막(nitrocelluose membrane, Hybond-C Extra, Amersham)에 트랜스퍼하고, iNOS에 대한 1차 항체(Santa Cruz Biotechnology, Santa Cruz, CA)를 결합시킨 후, 2차 항체(HRP-conjugated goat ant-rabbit immunoglobulin G antibody, Sigma)를 가하였다. iNOS 단백질 발현은 β-actin과 함께 chemiluminescence detection kit(Amersham Bioscience)을 이용하여 확인하였다. Immunoblotting experiments were carried out to confirm that isoramicin inhibits protein expression of iNOS, a nitrate (NO) synthesis inducer, in a concentration-dependent manner. In order to analyze the expression pattern of iNOS protein, isolametin and LPS were simultaneously treated with HepG2 cells and cultured for 18 hours. Then, cells were lysed, and proteins were quantitated (Bradford assay, BioRad Laborotories, Hercules, CA). Next, the same amount (20 μg) of the protein was loaded on a 12% SDS-PAGE gel (Min Bio-Rad, Hets, UK) and subjected to electrophoresis. The separated proteins were transferred to a nitrocellulose membrane (Hybond-C Extra, Amersham), bound to a primary antibody for iNOS (Santa Cruz Biotechnology, Santa Cruz, Calif.) And then incubated with a secondary antibody (HRP-conjugated goat anti-rabbit immunoglobulin G antibody, Sigma). iNOS protein expression was confirmed by β-actin and chemiluminescence detection kit (Amersham Bioscience).
도 7B에 나타난 바와 같이, LPS 처리로 세포내 증폭된 iNOS 단백질은 이소람네틴 처리에 의하여 농도의존적으로 감소됨을 확인할 수 있었고, 특히 50μM 이소람네틴은 LPS에 의해 증폭된 iNOS 단백질 발현을 LPS 처리 전과 같은 수준으로 억제시킨다는 것을 확인할 수 있었다. As shown in FIG. 7B, it was confirmed that the iNOS protein amplified intracellularly by LPS treatment was decreased in a concentration-dependent manner by isorhamnetin treatment. In particular, 50 μM isorhamnetin showed iNOS protein expression amplified by LPS, As shown in Fig.
4-3. Human HepG2세포에서 퉁퉁마디에서 분리된 이소람네틴 성분의 NF-kB와 COX-2 단백질 발현 저해능 측정4-3. Measurement of NF-kB and COX-2 Protein Expression of Isolametin Components Isolated from Tung Mun in Human HepG2 Cells
비알코올성 지방간에서 촉발되는 간염증에 대한 이소람네틴의 억제 효능을 분석하기 위하여 간세포 염증 유발 인자인 LPS(100ng/㎖)로 자극된 human HepG2 세포에서 과발현되는 COX-2와 NF-kB 단백질의 발현이 이소람네틴에 의하여 저해되는지 확인하였다. 염증생성에 관여하는 효소인 COX-2(cyclooxygenase type 2)는 세포 내 염증을 유발하는 물질인 Prostaglandin E2가 생성되게 하는 효소로서 COX-2 단백질 발현은 대표적인 염증인자의 바이오마커로 활용된다.Expression of COX-2 and NF-kB overexpressed in human HepG2 cells stimulated with LPS (100 ng / ml), a hepatocyte inflammation inducer, in order to analyze the inhibitory effect of isorhamnetin on liver inflammation induced by nonalcoholic fatty liver Were inhibited by isorhamnetin. COX-2 (cyclooxygenase type 2), an enzyme involved in the production of inflammation, is an enzyme that produces Prostaglandin E 2 , an intracellular inflammatory substance. COX-2 protein expression is used as a biomarker of a typical inflammatory factor.
세포내 산화스트레스를 주거나 염증유발 요인 즉 LPS와 같은 물질로 세포를 자극하면 COX-2 효소가 증가되므로, 이소람네틴의 간세포 내 함염증 효과를 확인하고자, HepG2 세포에 LPS(100ng/㎖) 및 이소람네틴을 농도별로 처리하고 18시간 배양하였다. 배양 후 각 세포의 단백질을 정량한 후 Western blot을 시행하여 COX-2 단백질의 발현양상을 확인하고 그 결과를 도 8A에 나타내었다. In order to examine the effect of isorhamnetin on hepatocyte inflammation, HepG2 cells were treated with LPS (100 ng / ml) and LPS (100 ng / ml) to induce oxidative stress in the cells or stimulate the cells with inflammatory factors such as LPS. Isolametin was treated for each concentration and cultured for 18 hours. After culturing, the protein of each cell was quantified and Western blot was performed to confirm the expression pattern of COX-2 protein. The result is shown in FIG. 8A.
COX-2 유전자 promoter 영역에 활성화로 인산화된 NFkB fragment인 p65가 결합하는 부위가 있어서, 산화스트레스나 염증성 자극으로 인한 COX-2 유전자의 합성이 NF-kB 활성화로 일어나는 것으로 알려진 바 있다. 따라서 LPS 처리로 활성화되는 NFkB를 핵내 phospho-p65 단백질 발현 양을 통하여 확인하고, 이소람네틴이 NFkB의 활성화 또한 억제하는지 확인하고자 HepG2 세포에 LPS(100ng/㎖) 및 이소람네틴을 농도별로 처리하고 18시간 배양한 후에 각 세포의 핵을 분리한 후, 핵내 phospho-p65의 발현 양을 Western blotting으로 확인하였다. The COX-2 gene promoter region is known to have NF-kB activation due to oxidative stress or inflammatory stimuli, as there is a region where p65 binds as an activation-phosphorylated NFkB fragment. Therefore, NFkB activated by LPS treatment was confirmed by the expression of phospho-p65 protein in the nucleus. To confirm whether isorhamnetin also inhibited NFkB activation, HepG2 cells were treated with LPS (100 ng / ml) and isorhamnetin After incubation for 18 hours, the nuclei of each cell were separated and the expression of phospho-p65 in the nucleus was confirmed by Western blotting.
HepG2 세포에서 핵을 분리하기 위하여 4℃에서 Nuclear extraction kit(Active Motif Carlsbad, CA, USA)를 이용하였으며, 분리한 핵은 라이시스한 후, 고속원심분리(21,000g, 30분)하여 상등액을 취하고 단백질 정량 후 전기영동을 실시하고 그 결과를 도 8B에 나타내었다.To separate the nuclei from HepG2 cells, a nuclear extraction kit (Active Motif, Carlsbad, Calif., USA) was used at 4 ° C. The separated nuclei were lysed and centrifuged at high speed (21,000 g, 30 min) After protein quantification, electrophoresis was carried out and the results are shown in FIG. 8B.
도 8B에 나타난 바와 같이, 이소람네틴은 농도의존적으로 NF-kB 활성화를 억제함을 확인할 수 있었다.As shown in FIG. 8B, it was confirmed that isorhamnetin inhibits NF-kB activation in a concentration-dependent manner.
4-4. t-BHP로 처리된 Human HepG2세포에서 퉁퉁마디에서 분리된 이소람네틴 유효성분의 농도의존적인 ROS 생성 및 세포사멸 억제능 측정4-4. Concentration-dependent inhibition of ROS production and cell death by the active ingredient of isorhamnetin isolated from Tungtung nodule in human HepG2 cells treated with t-BHP
비알코올성 지방간 질환의 대표적 기전 중의 하나로써, 고지방식이에 의한 간지방의 축적으로 유발되는 지방산화 중에 생성되는 활성산소들이 염증을 유발시키거나, 산화스트레스로 인한 직접적인 독성이 세포괴사를 야기하는 것으로 알려져 있다. 따라서 HepG2 세포에 직접적으로 ROS를 생성시켜는 강력한 과산화물인 t-BHP(tertiary butyl hydroperoxide)를 처리할 때 유발되는 세포괴사를 이소람네틴이 세포내 생성되는 ROS를 소거함과 동시에 억제할 수 있는지를 실험하였다.One of the representative mechanisms of nonalcoholic fatty liver disease is that active oxygen produced during fatty oxidation caused by accumulation of liver fat by high fat diet causes inflammation or direct toxicity due to oxidative stress causes cell necrosis It is known. Therefore, it is possible to directly detect ROS in HepG2 cells and to confirm whether or not isarramine is capable of eliminating and suppressing intracellular ROS produced by the treatment of t-BHP (tertiary butyl hydroperoxide), which is a strong peroxide, Respectively.
이소람네틴을 농도별로 처리한 세포와 이소람네틴을 전처리하지 않은 세포에 동일하게 1mM의 t-BHP를 가하고 18시간 후에 세포내 생성된 ROS 함량과 세포의 괴사정도를 확인하였다. 6-well 플레이트에서 배양된 HepG2 세포에 t-BHP를 처리하였을 때 생성된 세포내 ROS 함량은 DCFH(2', 7'-chlorofluorescein) 형광 probe를 이용하여 ROS에 의하여 전환되는 DCFH-DA(2', 7'-chlorofluorescein diacetate)의 형광세기를 형광측정계(TECAN, San Jose, CA)로 485~530㎚ 사이의 파장에서 측정하였다. B cells treated with isorhamnetin at the concentration and 1 mM t-BHP were added to the cells not pretreated with isorhamnetine. After 18 hours, intracellular ROS content and cell necrosis were observed. The intracellular ROS content of the HepG2 cells treated with t-BHP on 6-well plates was determined by DCFH-DA (2 ', 7'-chlorofluorescein) , 7'-chlorofluorescein diacetate) was measured with a fluorescence meter (TECAN, San Jose, Calif.) At a wavelength between 485 and 530 nm.
세포괴사로 인한 세포 생존률(cell viability)은 MTT assay를 이용하였다. 즉, 6-well에 HepG2 세포를 배양한 후 이소람네틴을 농도별로 18시간동안 처리한 후 이소람네틴이 포함된 배지성분을 제거하고, 세포를 PBS로 세척한 다음 1mM의 t-BHP를 첨가하였다. Cell viability of cell necrosis was determined by MTT assay. That is, HepG2 cells were cultured in 6-wells, treated with isorhamnetin for 18 hours at a concentration, the medium containing isorhamnetin was removed, the cells were washed with PBS, and 1 mM t-BHP was added Respectively.
도 9A로부터, t-BHP만 처리된 HepG2 세포는 세포괴사로 인하여 세포생존률이 20.2% ± 0.88로 현저히 떨어졌으나, 전처리로 이소람네틴이 첨가된 배지의 세포들은 이소람네틴 처리농도의 비례하여 세포생존률이 점점 증가하여, 이소람네틴의 50μM 처리시에는 거의 100%에 가까운 세포 생존률을 나타내어, 이소람네틴에 의한 산화스트레스 유도 세포괴사 억제효능을 확인할 수 있었다. From FIG. 9A, the cell survival rate of HepG2 cells treated with t-BHP alone was significantly decreased to 20.2% ± 0.88 due to cell necrosis. However, cells of the medium supplemented with isorhamnetin by the pretreatment showed a The survival rate gradually increased, and the cell survival rate was nearly 100% at the 50 μM treatment of isorhamnetin, and the isoxanthin-induced inhibition of oxidative stress induced cell necrosis was confirmed.
또한, t-BHP만 처리된 HepG2 세포에서는 세포내 급격히 ROS가 증가되었으나, 이소람네틴이 처리된 세포에서는 세포내 ROS의 생성이 농도의존적으로 억제되어 50μM 처리시에는 t-BHP 처리 전까지의 수준으로 낮아진 것을 확인할 수 있었다. 도 9B의 사진은 세포내 ROS를 형광현미경 촬영한 것이다. In addition, ROS was rapidly increased in the cells treated with t-BHP alone, but the production of intracellular ROS was suppressed in a concentration-dependent manner in the cells treated with isorhamnetine, and at the level of 50 μM, It was confirmed that it was lowered. 9B is a fluorescence microscope photograph of the intracellular ROS.
4-5. Human HepG2 세포에서 이소람네틴의 Nrf2 전사인자 활성화를 통한 글루타치온 합성 효소(GR, GCL) 단백질 유도 및 세포내 GSH 함량 향상능 측정4-5. Measurement of glutathione synthetase (GR, GCL) protein induction and intracellular GSH content enhancement by activation of Nrf2 transcription factor of isorhamnetin in human HepG2 cells
이소람네틴의 간세포 염증완화 및 세포보호 효과의 기전을 연구하고자 이소람네틴을 세포에 처리하였을 때 세포내 글루타치온(GSH)과 글루타치온 합성효소들(GR, GCL) 그리고 이들 효소 단백질의 합성을 일으키는 전사인자인 Nrf2 활성화 여부를 확인하고자 하였다. 간세포 보호인자로 알려진 글루타치온(GSH)의 함량은 글루타치온 합성효소인 글루타치온리덕테이스(GR)와 감마글루타밀시스테인 라이에이스(GCL)의 단백질 발현량 간의 밀접한 상관성이 있으며, 최근 글루타치온과 글루타치온의 전구물질인 N-acetylsystein은 지방간 치료에 활용되고 있다. (GSH) and glutathione synthetases (GR, GCL) and the transcription of these enzyme proteins when isorhamnetin was treated with cells to study the mechanisms of hepatocyte inflammation mitigation and cytoprotective effects of isorhamnetin And Nrf2, respectively. The content of glutathione (GSH), known as a hepatocyte protective factor, is closely related to the amount of protein expressed by glutathione reductase (GR) and gamma glutamylcysteine ly ace (GCL), and recently, the precursor of glutathione and glutathione N-acetylsystein is used in the treatment of fatty liver.
HepG2 세포에 이소람네틴을 농도별(0, 2.5, 5, 10, 25, 50, 100μM)로 18시간 처리하고 세포내 글루타치온(GSH) 함량을 측정하고, 그 결과를 도 10A에 나타내었다. HepG2 cells were treated with isorhamnetin at different concentrations (0, 2.5, 5, 10, 25, 50, 100 μM) for 18 hours and the intracellular glutathione (GSH) content was measured. The results are shown in FIG. 10A.
도 10A에 나타난 바와 같이, 이소람네틴은 농도의존적으로 세포내 글루타치온(GSH) 함량을 증가시키며, 특히 50μM 이상의 농도로 처리하였을 때 대조군 대비 2배 이상으로 글루타치온을 증가시킨다는 것을 확인할 수 있었다.As shown in FIG. 10A, it was confirmed that isorhamnetine increases intracellular glutathione (GSH) content in a concentration-dependent manner, and that glutathione increases more than twice as much as that of the control group when treated with a concentration of 50 μM or more.
따라서 이소람네틴에 의한 HepG2 세포에서의 글루타치온 증가가 이소람네틴에 의한 Nrf2 전사인자 활성과 Nrf2의 downstream gene들인 phase II 유전자 활성화에 따른 글루타치온 합성효소들인 GCL 과 GR의 단백질 발현에 의한 것인지를 확인하고자 이소람네틴을 농도별(0, 2.5, 5, 10, 25, 50, 100μM)로 18시간 처리한 후 웨스턴 블롯팅(Western Blotting)을 수행하고 그 결과를 도 10B에 나타내었다. 웨스틴 블롯팅을 위하여 이소람네틴이 처리된 세포와 대조군 세포들을 라이시스(lysis)시키고, 단백질을 정량(Bradford assay, BioRad Laborotories, Hercules, CA)하였다. 다음으로 동일한 양(20㎍)의 단백질을 12% SDS-PAGE 겔(Min Bio-Rad, Hets, UK)에 로딩하고, 전기영동을 실시하였다. 분리된 단백질은 셀룰로스 막(nitrocelluose membrane, Hybond-C Extra, Amersham)에 트랜스퍼하고, GR과 GCL에 대한 1차 항체(Santa Cruz Biotechnology, Santa Cruz, CA)를 결합시킨 후, 2차 항체(HRP-conjugated goat ant-rabbit immunoglobulin G antibody, Sigma)를 가하였다. GCL 및 GR 단백질 발현은 β-actin과 함께 chemiluminescence detection kit(Amersham Bioscience)을 이용하여 확인한 결과 이소람네틴은 농도의존적으로 글루타치온 합성효소들인 GCL 과 GR 발현을 증가시킨다는 것을 확인할 수 있었다. Therefore, we investigated whether the increase of glutathione in HepG2 cells by isorhamnetin is due to the Nrf2 transcription factor activity by isorhamnetin and the protein expression of glutathione synthetase, GCL and GR, by the phase II gene activation, the downstream genes of Nrf2 Western blotting was carried out after treatment of isorhamnetin with concentration (0, 2.5, 5, 10, 25, 50, 100 μM) for 18 hours and the results are shown in FIG. 10B. Cells treated with isorhamnetin and control cells were lysed for Westin blotting and proteins were quantified (Bradford assay, BioRad Laborotories, Hercules, CA). Next, the same amount (20 μg) of the protein was loaded on a 12% SDS-PAGE gel (Min Bio-Rad, Hets, UK) and subjected to electrophoresis. The separated proteins were transferred to a nitrocelluose membrane (Hybond-C Extra, Amersham), bound to a primary antibody against GR and GCL (Santa Cruz Biotechnology, Santa Cruz, Calif.), conjugated goat anti-rabbit immunoglobulin G antibody, Sigma). GCL and GR protein expression was confirmed by β-actin and chemiluminescence detection kit (Amersham Bioscience). It was confirmed that isorhamnetin increases GCL and GR expression, which are glutathione-synthesizing enzymes, in a concentration-dependent manner.
도 10B에 나타난 바와 같이, 이소람네틴에 의한 Nrf2 전사인자의 활성화는 핵내 존재하는 Nrf2 단백질 발현량으로 확인할 수 있었다. 이소람네틴은 10μM 이상의 농도부터 점차 Nrf2 전사인자의 핵내 이동을 통한 활성화를 시킴을 알 수 있었고, 이소람네틴의 농도의존적으로 일어나는 Nrf2활성화는 GCL과 GR의 발현과 유사한 경향을 나타내었다.As shown in FIG. 10B, the activation of Nrf2 transcription factor by isorhamnetine was confirmed by the expression amount of Nrf2 protein present in the nucleus. It was found that isorhamnetin was activated by intranuclear transfer of Nrf2 transcription factor at a concentration of 10 μM or more, and Nrf2 activation induced by isorhamnetin was similar to GCL and GR expression.
따라서 이소람네틴에 의한 HepG2세포내 글루타치온 증가는 Nrf2 전사인자 활성화에 따른 GCL 및 GR 효소단백질의 합성을 통하여 일어나며, 이는 간세포내 지방산산화에 의한 산화스트레스를 감소시켜 지방간세포내 염증완화와 치료에 효과적일 수 있음을 시사한다. Therefore, the increase of glutathione in hepG2 cells by isorhamnetin is caused by the synthesis of GCL and GR enzyme proteins by activation of Nrf2 transcription factor, which is effective for relieving inflammation and treatment of fatty liver cells by reducing oxidative stress caused by fatty acid oxidation in hepatocytes .
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시 양태일 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 thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
<110> Phyto Corporation <120> Pharmaceutical Composition for Prevention or Treatment of Non-alcoholic Fatty Liver Disease Containing Isorhamnetin derived from Salicornia europaea <130> P16117 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Ccl2 forward primer <400> 1 ccactcacct gctgctactc at 22 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Ccl2 reverse primer <400> 2 tggtgatcct cttgtagctc tcc 23 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Il1b forward primer <400> 3 ggagaaccaa gcaacgacaa aata 24 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Il1b reverse primer <400> 4 tggggaactc tgcagactca aac 23 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Cd68 forward primer <400> 5 acctgctcaa catcatgaag g 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Cd68 reverse primer <400> 6 agatggagct atgcaggtgg 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Cxcl2 forward primer <400> 7 ccaagggttg acttcaagaa c 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Cxcl2 reverse primer <400> 8 agcgaggcac atcaggtacg 20 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Col1a1 forward primer <400> 9 gctcctctta ggggccac 18 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Col1a1 reverse primer <400> 10 ccacgtctca ccattgggg 19 <110> Phyto Corporation <120> Pharmaceutical Composition for Prevention or Treatment of Non-alcoholic Fatty Liver Disease Containing Isorhamnetin derived from Salicornia europaea <130> P16117 <160> 10 <170> KoPatentin 3.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Ccl2 forward primer <400> 1 ccactcacct gctgctactc at 22 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Ccl2 reverse primer <400> 2 tggtgatcct cttgtagctc tcc 23 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Il1b forward primer <400> 3 ggagaaccaa gcaacgacaa aata 24 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Il1b reverse primer <400> 4 tggggaactc tgcagactca aac 23 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Cd68 forward primer <400> 5 acctgctcaa catcatgaag g 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Cd68 reverse primer <400> 6 agatggagct atgcaggtgg 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Cxcl2 forward primer <400> 7 ccaagggttg acttcaagaa c 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Cxcl2 reverse primer <400> 8 agcgaggcac atcaggtacg 20 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Col1a1 forward primer <400> 9 gctcctctta ggggccac 18 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Col1a1 reverse primer <400> 10 ccacgtctca ccattgggg 19
Claims (7)
(b) 상기 퉁퉁마디 추출물을 용매로 분획하는 단계;
(c) 상기 퉁퉁마디 용매 분획물을 크로마토그래피로 분획하여 간세포내 염증반응 억제능 및 간세포 보호활성을 갖는 크로마토그래피 분획물을 얻는 단계; 및
(d) 상기 크로마토그래피 분획물을 고속액체크로마토그래피(HPLC)를 이용하여 이소람네틴을 분리하는 단계를 포함하는 것을 특징으로 하는 퉁퉁마디(Salicornia SPP.) 유래 이소람네틴(isorhamnetin)의 분리방법.
(a) obtaining an extract of Tungsten mulberry;
(b) fractionating the extract of Tungsten mulberry with a solvent;
(c) fractionating the fractions of Chrysanthemum mildew solvent by chromatography to obtain a chromatographic fraction having inhibitory effect on hepatocyte inflammatory reaction and hepatocyte protective activity; And
(d) isolating isorhamnetin using high performance liquid chromatography (HPLC) on said chromatographic fraction. The method of isolating isorhamnetin from Salicornia spp .
5. The method according to claim 4, wherein the extract of Tungtung nodule is obtained by isolating isorhamnetin from Salicornia spp ., Characterized in that the extract of Tungtung nodule is sequentially fractionated with hexane, chloroform, ethyl acetate, butanol and water. Way.
5. The method of claim 4, wherein the step of obtaining the chromatographic fraction comprises: loading the solvent fraction into an adsorbable Diaion HP-20 column; then eluting the column with acetone to obtain a first fraction having inhibitory activity against inflammation in hepatocytes and hepatocyte- And then loaded on a silica gel column. Then, methanol was sequentially added to the mobile phase of chloroform to elute the second fraction, which was then loaded on an LH-20 column, To obtain a third fraction having an inhibitory effect on the inflammatory reaction in the hepatocyte and hepatocyte protective activity. The high-performance liquid chromatography (HPLC) is Salicornia (Salicornia SPP.) Method of separating iso-derived person netin (isorhamnetin), characterized in that is carried out with a mobile phase of methanol and water mixture.
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