KR19990047905A - Hepatitis C Therapeutics with Ogapi Extract - Google Patents
Hepatitis C Therapeutics with Ogapi Extract Download PDFInfo
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- KR19990047905A KR19990047905A KR1019970066473A KR19970066473A KR19990047905A KR 19990047905 A KR19990047905 A KR 19990047905A KR 1019970066473 A KR1019970066473 A KR 1019970066473A KR 19970066473 A KR19970066473 A KR 19970066473A KR 19990047905 A KR19990047905 A KR 19990047905A
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- extract
- hepatitis
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- hcv
- protease
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Abstract
본 발명은 오갈피(Acanthopanacis cortex) 속 나무의 추출물을 포함하는 C형 간염 치료제에 관한 것이다.The present invention relates to a hepatitis C therapeutic agent comprising an extract of the genus Acanthopanacis cortex .
보다 상세하게는 오갈피 속 나무의 뿌리, 줄기 및 가지 부분의 껍질을 증류수로 끓이거나 유기용매로 냉침시켜 얻은 추출물을 유효 활성성분으로 함유하는 C형 간염 치료제에 관한 것으로서, 본 발명의 오가피 추출물은 C형 간염 단백질 분해효소에 대한 강한 저해활성을 나타내므로 C형 간염 치료제로 유용하게 사용될 수 있을 뿐만 아니라 각종 식음료에 포함되어 사용될 수 있다.More specifically, the present invention relates to a hepatitis C therapeutic agent containing an extract obtained by boiling the bark of the roots, stems and branches of the genus Ogalpi with distilled water or cooling with an organic solvent as an active ingredient. Since it exhibits a strong inhibitory activity against hepatitis protease, it can be usefully used as a therapeutic agent for hepatitis C and can be used in various food and beverages.
Description
본 발명은 오갈피(Acanthopanacis cortex) 나무의 추출물을 포함한 C형 간염 치료제에 관한 것으로, 보다 상세하게는 오갈피 속 나무의 뿌리, 줄기 및 가지 부분의 껍질을 증류수로 끓이거나 유기용매로 냉침시켜 얻은 추출물을 포함한 C형 간염 치료제에 관한 것이다.The present invention relates to a hepatitis C therapeutic agent comprising an extract of the Acanthopanacis cortex tree, and more particularly, to extracts obtained by boiling the bark of roots, stems and branches of the genus Agalpi in distilled water or cooling them with an organic solvent. It relates to a hepatitis C treatment including.
C 형 간염 바이러스(Hepatitis C Virus; 이하, "HCV"라 약칭한다)는 비-A형 또는 비-B형 간염을 유발하는 것으로 알려진 간염의 주요 원인이 되는 바이러스로서, 사람에 침입하여 급성 또는 만성 간염을 일으킬 뿐만 아니라 간경화 또는 간암으로의 이행에도 관여한다고 한다(Alter, H. J. et al.,N. Eng. J. Med,321, p.1494-1500,1989). HCV는 전 세계 인구의 1% 가 감염되어 있는 것으로 보고된 바 있으며, 새로이 HCV에 감염되는 환자의 수가 매년 약 백만명 정도에 이르는 것으로 추정되며, 특히 아프리카, 일본 및 한국에는 전체 인구의 1.5∼2.0%가 만성 보균자인 것으로 알려져 있다(Purcell,FEMS Microbial. Rev, 14, p.181-192,1994; van der Poel,Hepatitis C Virus, 1994 :H. W. Reesink ed.,Current Studies in Hematology and Blood Transfusion,p.137-193; Alter, H. J., A. J. Zuckerman ed.,Viral Hepatitis and Liver Disease, p.537-542,1988).Hepatitis C Virus (abbreviated as "HCV") is a virus that is a major cause of hepatitis known to cause non-A or non-B hepatitis. Not only does it cause hepatitis, but it is also involved in cirrhosis or transition to liver cancer (Alter, HJ et al., N. Eng. J. Med , 321 , p. 1494-1500, 1989 ). HCV has been reported to be infected with 1% of the world's population, with an estimated 1 million new cases of HCV infected each year, with 1.5-2.0% of the total population in Africa, Japan and Korea. Is known to be a chronic carrier (Purcell, FEMS Microbial. Rev, 14 , p . 181-192, 1994 ; van der Poel, Hepatitis C Virus, 1994 : HW Reesink ed., Current Studies in Hematology and Blood Transfusion, p. 137-193; Alter, HJ, AJ Zuckerman ed., Viral Hepatitis and Liver Disease , p.537-542, 1988 ).
HCV의 감염에 의한 간염이 B형 간염 바이러스(Hepatitis B virus; 이하, HBV라 약칭한다)의 감염에 의해 발생되는 B형 간염과 구별되는 주요한 특징은 C형 간염에서 만성간염으로 진행될 가능성이 HBV 감염의 경우에 비하여 훨씬 높다는 것이다. 즉, HCV에 감염된 환자들 중 약 40-50% 는 간염이 진전되어 만성 간염 환자가 되고, 약 20% 정도는, 더욱 진전되어 간경변 또는 간암 환자가 되는 것으로 알려져 있다 (Dienstag, J. L.,Semin. Liver. Dis, 6 ,p.67-81,1986).The main feature that distinguishes hepatitis from HCV infection from hepatitis B caused by infection with Hepatitis B virus (hereinafter referred to as HBV) is the possibility of progression from hepatitis C to chronic hepatitis HBV infection. It is much higher than in the case of. That is, about 40-50% of HCV-infected patients develop hepatitis and become chronic hepatitis patients, and about 20% become more advanced and become cirrhosis or liver cancer patients (Dienstag, JL, Semin. Liver Dis, 6, p. 67-81, 1986 ).
수혈에 의해 비-A형 또는 비-B형 간염 바이러스에 감염된 간염환자의 과반수 이상이 만성간염으로 악화되고, 비-A형 또는 비-B형 간염 환자의 약 10∼20% 는 간경변에 걸리게 된다.Transfusion causes more than half of hepatitis patients infected with non-A or non-B hepatitis virus to become chronic hepatitis, and about 10-20% of patients with non-A or non-B hepatitis develop cirrhosis .
HBV에 감염되지 않고 간경변 또는 간암을 앓는 환자의 항-HCV 항체 형성율은 대조군(HBV 감염의 경우)보다 훨씬 높다고 알려져 있는데(Ikeda et al.,Hepatology,1993,vol. 18, 47∼53), 이 조사에 따르면 HCV 감염환자의 75% 는 발병된지 약 15년 후에는 간암에 걸리게 된다고 한다. 이는 HBV 감염의 경우에 나타나는 27% 의 간암 진행율 보다 훨씬 높은 것으로서, 지금까지 알려진 어떤 발암물질보다도 HCV 감염이 간암과 밀접한 상관 관계를 갖고 있다는 것을 알려준다.Anti-HCV antibody formation rates in patients without HBV infection and with cirrhosis or liver cancer are known to be much higher than controls (for HBV infection) (Ikeda et al., Hepatology , 1993 , vol. 18 , 47-53). According to the survey, 75% of people with HCV will develop liver cancer about 15 years after the disease. This is much higher than the 27% hepatocarcinoma progression seen with HBV infection, suggesting that HCV infection is more closely correlated with liver cancer than any carcinogen known to date.
현재 HCV에 대해서는 진단시약이 개발되어 수혈에 의한 감염은 어느 정도 방지가 가능하게 되었지만, 아직 백신이 개발되어 있지 않아서 수혈 이외의 경로를 통해서 감염될 위험성은 여전히 존재한다. 환자가 HCV에 감염되었는지를 알기 위해서는 감염된지 6개월 이상의 시간이 지나야만 진단시약으로 항체 감지가 가능하기 때문에 감염 후 6개월 이내의 혈액은 정상혈액으로 오인되어 수혈에 사용될 위험성이 있다. 또한, HCV 감염자의 약 10% 는 HCV 항체가 발견되지 않아 이러한 환자의 혈액도 정상혈액으로 오인되어 수혈에 사용될 수 있다는 위험성을 안고 있다.Currently, diagnostic reagents have been developed for HCV to prevent some of the infections caused by blood transfusions, but there is still a risk of infection through routes other than blood transfusions since vaccines have not been developed. In order to know whether a patient is infected with HCV, antibodies can be detected with a diagnostic reagent only 6 months or more after the infection. Therefore, blood within 6 months of infection may be mistaken for normal blood and used for transfusion. In addition, about 10% of people with HCV have a risk that no HCV antibody can be found and thus blood from such patients may be mistaken for normal blood and used for transfusion.
HCV의 만성적 감염을 효과적으로 치료하기 위한 치료방법은 아직까지 개발되어 있지 않고, 거의 유일하게 α-인터페론을 HCV의 치료제로 투여하는 것이 시행되고 있다. 그러나, 이 치료법은 최근들어 인터페론-내성 서열(Interferon resistance sequence)을 갖는 HCV에 대해서는 효과가 없는 것으로 나타나고 있어 새로운 형태의 치료제의 개발이 요구되고 있는 실정이다. 새로운 형태의 HCV 치료제는 AIDS 바이러스와 같은 RNA 바이러스의 치료제로서 단백질 분해효소 혹은 RNA 중합효소와 같은 단백질에 대한 저해제를 이용하는 것이 가장 효과적일 것이다.Therapies for effectively treating chronic infection of HCV have not been developed yet, and the only administration of α-interferon as a therapeutic agent for HCV has been carried out. However, these therapies have recently been shown to be ineffective against HCV having an Interferon resistance sequence, requiring the development of new types of therapeutics. The new form of HCV therapy would be most effective with inhibitors of proteins such as proteases or RNA polymerases for the treatment of RNA viruses such as the AIDS virus.
HCV는 비-A형 또는 비-B형 간염 환자의 혈장을 침팬지에 접종시켜 분리함으로써 회수할 수 있다(Bradley, D. W. et al.,Gatroenterology, 88, p.773-779,1988). 이렇게 얻어진 HCV는 9400 염기로 이루어진 양성가닥 리보핵산 형태의 유전자를 가지고 있으며, 이로부터 3010-3033 개의 아미노산으로 이루어지는 다단백질(polyprotein)을 만들어 낸다 (Choo, Q. L. et al., Science, 244, p.359-362,1989; Choo, Q. L. et al.,PNAS, 88,p.2451-2455,1991). 상기와 같은 유전자 구조를 고려할 때 HCV는 플래비바이러스(flaviviruses) 또는 페스티바이러스(pestiviruses)와 유사하므로, HCV는 플래비비리대(Flaviviridae)에 속하는 새로운 속 (genus)으로 분류되었다(van Doorn,Review. J. Med. Virol, 43,p.345-356,1994).HCV can be recovered by inoculating the chimpanzees with plasma from non-Hepatitis A or non-B hepatitis patients (Bradley, DW et al., Gatroenterology, 88 , p.773-779, 1988 ). The HCV thus obtained has a gene in the form of a positive strand ribonucleic acid consisting of 9400 bases, from which a polyprotein consisting of 3010-3033 amino acids is produced (Choo, QL et al., S cience, 244 , p). 359-362, 1989 ; Choo, QL et al., PNAS, 88, p.2451-2455, 1991 ). Given this genetic structure, HCV is similar to flaviviruses or pestiviruses, so HCV has been classified as a new genus belonging to Flaviviridae (van Doorn, Review). J. Med. Virol, 43, p.345-356, 1994 ).
HCV의 다단백질은 아미노 말단으로부터 코어-E1-E2-NS2-NS3-NS4A-NS4B -NS5A-NS5B 의 순서로 구성되어 있다. 즉, 다단백질상의 아미노 말단에는 바이러스의 뉴클레오캡시드(nucleocapsid)와 외피(envelope)의 구성요소인 구조 단백질(core, E1, E2)이 존재하고, 그의 카복시 말단 쪽에는 HCV가 증식하는데 필수적인 비구조 단백질들(NS2, NS3, NS4A, NS4B, NS5A, NS5B)이 위치하고 있다. 이 다단백질은 세포내 시그날 펩티다제(signal peptidase) 및 바이러스에 존재하는 단백질 분해효소인 NS2-3 단백질 및 NS3 단백질이 작용하여 상기 전구체 다단백질의 특정 부위를 절단함으로써 9개의 숙성된 바이러스 단백질을 만들어 낸다. 구체적으로 NS3 단백질은 NS3 단백질과 NS4A 단백질 사이, NS4A 단백질과 NS4B 단백질 사이, NS4B 단백질과 NS5A 단백질 사이, NS5A 단백질과 NS5B 단백질 사이에 존재하는 4개의 절단 부위에 작용하는 것으로 밝혀져 있다(Hijikata et al.,PNAS,88,p.5547-5551,1991;Bartenschlarger et al.,J. Virol, 68,p.5045-5055,1994; Grakoui et al.,J. Virol, 67, p.1385-1395,1993; Tomei et al.,J. Virol, 67, p.4017-4026,1993). 또한 이러한 NS3 단백질은 조효소인 NS4A 단백질과 결합하고 결합한 조효소와 상호 작용하여 그의 효소 활성이 증가한다고 보고되어 있다(Shimizu, Y. et al.,J. Virol, 70,p.127-132,1996; Love R. A. et al.,Cell. 87, p.331-342,1996).The polyprotein of HCV consists of the core-E1-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B from the amino terminal. That is, at the amino terminus of the polyprotein, there are structural proteins (cores, E1 and E2), which are components of the nucleocapsid and envelope of the virus, and at the carboxy terminus thereof, the non-structural essential for HCV proliferation. Proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B) are located. The polyprotein is a protein peptide peptidase and NS2-3 protein and NS3 protein, which is present in the virus, acts to cleave specific sites of the precursor polyprotein, thereby removing nine mature viral proteins. Make it up Specifically, the NS3 protein has been found to act on four cleavage sites existing between NS3 protein and NS4A protein, between NS4A protein and NS4B protein, between NS4B protein and NS5A protein, and between NS5A protein and NS5B protein (Hijikata et al. , PNAS , 88, p.5547-5551, 1991; Bartenschlarger et al., J. Virol, 68, p.5045-5055, 1994 ; Grakoui et al., J. Virol, 67 , p.1385-1395, 1993 Tomei et al., J. Virol, 67 , p. 4017-4026, 1993 ). In addition, these NS3 proteins have been reported to increase their enzymatic activity by binding to the coenzyme and the coenzyme bound to the coenzyme NS4A protein (Shimizu, Y. et al., J. Virol, 70, p. 127-132, 1996 ; Love RA et al., Cell. 87 , p . 331-342, 1996 ).
NS3 단백질은 그의 아미노 말단쪽 1/3 지점까지에는 단백질 분해 활성이 있고, 나머지 부분에는 헬리케이즈(Helicase) 활성이 있어서 시험관 내(in vitro)에서 NS3 단백질을 따로 발현시켰을 때 각각의 활성이 나타난다는 것이 확인되었다(D, Souza et al,J. Gen. Virol. 76,p.1729-1736,1993). 57번째 아미노산이 히스티딘(histidine), 81번째 아미노산이 아스파테이트(aspartate), 그리고 139번째 아미노산이 세린(serine)인 NS3 단백질은 전형적인 트립신-유사 세린 단백질 분해효소(Trypsin-like serine proteinase)에서 나타나는 활성부위(catalytic motif)를 가지며, 이러한 특성은 지금까지 발견된 수 많은 HCV의 변종에서 상기 아미노산 서열을 분석한 결과 일치하게 나타나는 것으로 확인되었다.NS3 protein has proteolytic activity up to one-third of its amino terminus and helicase activity in the remainder, so that each activity occurs when NS3 protein is expressed separately in vitro . (D, Souza et al, J. Gen. Virol. 76, p . 1729-1736, 1993 ). NS3 protein with 57th amino acid histidine, 81th amino acid aspartate, and 139th amino acid serine is the activity found in typical Trypsin-like serine proteinase. It has a site (catalytic motif), and this property has been found to appear consistently by analyzing the amino acid sequence in a number of variants of HCV discovered so far.
그러나, NS3 단백질은 1) 헬리케이즈 단백질과 연결되어 있고, 2) 아연(Zn)과 같은 2가 금속 이온이 필요하며, 3) 분해효소 활성을 나타내는데 54개의 아미노산으로 구성된 NS4A 펩타이드가 주요인자로 관여하여 NS3 단백질과 서로 작용하여 분해효소 활성을 갖는 단백질 구조를 형성(Hijikata et al.,J. Virol. 67, p.4665-4675,1993)하는데 도움을 준다는 점에서 트립신-유사 세린 단백질 분해효소와는 다르다.However, NS3 protein is 1) linked to helicase protein, 2) divalent metal ions such as zinc (Zn), and 3) NS4A peptide consisting of 54 amino acids is involved as a major factor in degrading enzyme activity. And trypsin-like serine protease in that it interacts with the NS3 protein to help form a protein structure with degrading enzyme activity (Hijikata et al., J. Virol. 67 , p.4665-4675, 1993 ). Is different.
NS3 폴리펩타이드 중에서 아미노 말단의 약 180개의 아미노산으로 구성된 단백질도 분해효소 활성을 지닌다고 밝혀졌으며(Raffaele D. F. et al.,Biochem. 35,p.13282-13287,1996), NS4A 펩타이드의 존재 유무에 따라 그 활성도의 차이가 크게 난다는 것이 알려졌다. 뿐만아니라 NS3의 아미노 말단 180개 정도의 아미노산만을 발현시켜 정제한 단백질이 낮은 농도로 존재할 때는 NS4A 펩타이드가 약 1000∼2000배 높은 농도로 존재해야만 최대 활성도(Vmax)가 나타나는 것으로 밝혀졌다. 즉, NS3 단백질과 NS4A가 저농도로 존재할 때에는 NS3 및 NS4A의 복합 단백질의 활성구조도 저농도로 존재하기 때문에 전체 반응속도가 느려지며, NS4A 펩타이드가 고농도로 존재하면 복합 단백질 분해효소가 대부분 활성 구조로 존재하기 때문에 반응속도가 빨라진다.Among NS3 polypeptides, proteins composed of about 180 amino acids at the amino terminus were also found to have degrading enzyme activity (Raffaele DF et al., Biochem. 35, p.13282-13287, 1996 ), depending on the presence or absence of the NS4A peptide. It is known that there is a large difference in activity. In addition, when the protein purified by expressing only about 180 amino-terminal amino acids of NS3 is present at a low concentration, the maximum activity (V max ) appears only when the NS4A peptide is present at a concentration of about 1000-2000 times higher. That is, when NS3 protein and NS4A are present in low concentration, the overall reaction rate is slowed because the active structure of the complex protein of NS3 and NS4A is also present in low concentration. When the NS4A peptide is present in high concentration, the complex protease is present in most active structures. Because the reaction rate is faster.
54개 아미노산의 NS4A 펩타이드 중에서도 21∼34번째 아미노산이 단백질 분해효소의 활성구조를 형성하는데 가장 중요한 역할을 하는 것으로 알려져 있어 활성구조를 갖는 단백질을 얻기 위해서 NS3 단백질의 일부 또는 전부분에 NS4 펩타이드 일부 또는 전부분을 차례대로 융합하여 발현시킴으로써 활성구조를 갖는 복합 단백질 분해효소를 제조하려는 시도가 계속되었다. 또한, 단백질의 3차 구조 형성 또는 수용성을 증가시키기 위해서 NS3 단백질의 아미노 말단쪽에 글루타티온 S-트랜스퍼라아제(GST) 또는 말토오스 결합 단백질(maltose binding protein)과 같은 이종 단백질을 융합시키거나(Akiko Mori et al.,FEBS Letter 378, p.37-42,1996), 아미노 말단 혹은 카르복시 말단쪽에 히스티딘 표지(Histidine tag)를 붙이기도 하였다. 그러나, 어떤 경우에서도 NS3 단백질과 NS4A 펩타이드의 배열은 HCV 게놈상에 나타난 순서대로 융합되었다.Among the 54 amino acid NS4A peptides, the 21st to 34th amino acids are known to play the most important role in forming the active structure of protease. Attempts have been made to produce complex proteases with active structures by fusion and expression of all parts in sequence. In addition, a heterologous protein, such as glutathione S-transferase (GST) or maltose binding protein, is fused to the amino terminus of the NS3 protein (Akiko Mori et. al., FEBS Letter 378 , p. 37-42, 1996 ), and a histidine tag was attached at the amino or carboxy terminus. In either case, however, the arrangement of NS3 protein and NS4A peptides were fused in the order in which they appear on the HCV genome.
김(J. L. Kim) 등은 NS3 단백질 분해효소와 NS4A 펩타이드가 활성 구조를 이룬 복합 단백질의 결정구조를 밝힘으로써 NS4A 펩타이드가 NS3 단백질 상에 어떠한 위치에 접근할 때 활성구조 형성에 기여하는지를 보여주었는데 (J. L. Kim et al.,Cell 87, p.343-355,1996), 그 활성 구조를 자세히 살펴보면 실제 NS4A 펩타이드가 NS3 단백질의 아미노 말단 부위와 더 인접한 곳에 위치하고 있다. 본 발명자들은 이러한 사실로부터 NS4A 펩타이드의 일부를 원래의 HCV 게놈 서열과는 반대로 181개의 아미노산으로 구성된 NS3 단백질 분해효소에 약 1000∼2000 배의 NS4A 펩타이드를 첨가하였을 때 나타나는 활성도를 지닐 수 있는 신규한 융합 단백질을 합성하고 특허출원한 바 있으며(대한민국 특허출원 제 97-59161호), 또한, 상기한 기출원된 발명의 실시예에 의해 정제한 단백질 분해효소를 사용하고 또한 이 분해효소의 기질이 되는 GST-NS5AB-UB 단백질을 합성하였고, 적절한 완충조건 하에서 반응하여 약 20개의 C-말단 아미노산이 탈리됨을 측정하여 빠른 시간 내에 단백질 분해 효소의 활성을 측정할 수 있는 방법을 개발하였다.Kim et al. Revealed the crystal structure of a complex protein in which an NS3 protease and an NS4A peptide form an active structure, and showed that the NS4A peptide contributes to the formation of an active structure when the position approaches on the NS3 protein (JL). Kim et al., Cell 87 , p. 343-355, 1996 ), the active structure of the NS4A peptide is located closer to the amino terminal region of the NS3 protein. From this fact, the inventors have found that a novel fusion that can have some of the activity that occurs when a portion of the NS4A peptide is added to the NS3 protease consisting of 181 amino acids in contrast to the original HCV genome sequence is about 1000-2000 times the NS4A peptide. GST has been synthesized and patented (Korean Patent Application No. 97-59161), and GST is also used as a substrate of the protease which is purified by the above-described embodiment of the invention. -NS5AB-UB protein was synthesized, and a method for measuring proteolytic enzyme activity in a short time by measuring the detachment of about 20 C-terminal amino acids by reacting under appropriate buffer conditions was developed.
한편, 본 발명에 사용하는 오갈피 속의 식물로는 한국에 자생하거나 재배되는 오갈피나무(Acanthopanax sessiliflorusSeeman), 지리산 오갈피나무(Acanthopanax chiisanensisNakai), 섬 오갈피나무(Acanthopanax koreanumNakai), 털 오갈피나무(Acanthopanax rufinerveNakai), 서울 오갈피나무 (Acanthopanax seoulenseNakai), 가시 오갈피나무(Acanthopanax senticosusHarms) 및 오가나무(Acanthopanax sieboldianumMakino) 등과 중국에서 자생되거나 재배되고 있는 오갈피 속의 나무(Acanthopanax gracilistylusSmith) 등이 있다. 오가피는 상기한 나무의 근피 및 수피를 말하는 것으로, 오래전부터 한방에서는 강장제로 이용되어 왔으며, 강장뿐 아니라 소염, 진통, 거풍습(祛風濕) 및 활혈(活血) 등의 효능이 있어, 마비통증, 요통, 수종, 창종, 각기 및 근골 위약 등의 질환을 치료하는데 사용되어 온 약재이다 (김재길,원색 천연약물 대사전, 상권, p.261,1984; 한 대석,생약학, p.121,1989;중약 대사전, 상해 과학기술 출판사 소학관편, 2권p.793).On the other hand, the plants of the genus of the genus used in the present invention are Agalthopanax ( Acanthopanax sessiliflorus Seeman) grown in Korea or cultivated in Korea ( Acanthopanax chiisanensis Nakai), the island of the Agalthophyte ( Acanthopanax koreanum Nakai), the hairy Ogalpi ( Acanthopanax rufinerve Nakai), Acanthopanax seoulense Nakai, Acanthopanax senticosus Harms, and Acanthopanax sieboldianum Makino, and Acanthopanax gracilistylus Smith, which are native or grown in China. Ogapi refers to the root and bark of the above-mentioned trees, and has been used as a tonic in Korean medicine for a long time. As well as tonic, it has the effects of anti-inflammatory, analgesic, swelling and blood loss, active paralysis, It is used to treat diseases such as back pain, edema, changjong, respectively, and a placebo on musculoskeletal medicine (gimjaegil, primary natural drug Dictionary, commercial, p.261, 1984; Oishi one, pharmacognosy, p.121, 1989; Encyclopedia Chinese medicine , Shanghai Science and Technology Publishing House, Vol . 2, p.793).
이미 오가피의 성분 및 그의 생리활성에 관한 연구가 활발히 진행되어 리그난, 테르페노이드, 쿠마린, 플라보노이드 및 페닐프로파노이드계 화합물 등이 분리되어 각각의 성분들에 대한 약리효과가 보고되고 있다. 또한, 오가피에서 나타나는 강장 및 강정 등의 약효를 음료에 도입하려는 연구도 보고되고 있으나(대한민국 특허공고 제 91-7606; 대한민국 특허공고 제 94-618), 상기한 문헌들 및 연구 보고들에서 오가피의 C형 간염에 대한 치료효과는 전혀 보고된 바 없다.Algae's components and its physiological activity have been actively studied, and lignans, terpenoids, coumarins, flavonoids, and phenylpropanoid compounds have been separated, and pharmacological effects on the respective components have been reported. In addition, studies have been reported to introduce medicinal effects such as tonic and gangjeong appearing in Ogapi (Korean Patent Publication No. 91-7606; Korean Patent Publication No. 94-618). No therapeutic effect has been reported for hepatitis C.
이에 본 발명자들은 천연 약재로부터 HCV의 치료제를 얻기 위해 노력하던 중, 기존에 마비통증, 요통, 수종, 창종, 각기 및 근골 위약 등의 질환을 치료하는데 널리 사용되어 온 약재인 오가피의 증류수 추출물 및 유기용매 추출물이 HCV의 단백질 분해효소에 대해 강한 저해활성을 나타낸다는 사실을 알아내어 본 발명을 완성하였다.Therefore, the present inventors have been trying to obtain a HCV therapeutic agent from natural medicines, while distilled water extracts and organic extracts of Ogapi, which have been widely used for treating diseases such as numb pain, low back pain, swelling, swelling, and musculoskeletal The present invention was completed by finding that the solvent extract exhibits strong inhibitory activity against the protease of HCV.
본 발명의 목적은 오갈피 속 나무의 추출물을 유효 활성성분으로 함유하는 C형 간염 치료제를 제공하는 것이다.An object of the present invention is to provide a hepatitis C therapeutic agent containing the extract of the genus Agpipi as an active ingredient.
도 1은 본 발명의 오가피 증류수 추출물의 단백질 분해효소 저해활성을 SDS-폴리아크릴아미드 젤 전기영동(SDS-PAGE)을 수행하여 나타낸 것이다. Figure 1 shows the protease inhibitory activity of the distilled distilled water extract of the present invention by performing SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
레인 1:기질Lane 1: Substrate
레인 2: 기질+단백질 분해효소Lane 2: Substrate + Protease
레인 3: 표준 분자량 단백질Lane 3: standard molecular weight protein
레인 4∼8: 기질+단백질 분해효소+오가피 증류수 추출액Lanes 4 to 8: Substrate + Protease + Organic Distilled Water Extract
최종농도: 333(㎍/㎖), 166(㎍/㎖), 83(㎍/㎖), 42(㎍/㎖), 21(㎍/㎖).Final concentrations: 333 (μg / mL), 166 (μg / mL), 83 (μg / mL), 42 (μg / mL), 21 (μg / mL).
레인 9∼13: 기질+단백질 분해효소+결명자 증류수 추출액Lanes 9-13: Substrate + Protease + Declarator Distilled Water Extract
최종농도: 333(㎍/㎖), 166(㎍/㎖), 83(㎍/㎖), 42(㎍/㎖), 21(㎍/㎖).Final concentrations: 333 (μg / mL), 166 (μg / mL), 83 (μg / mL), 42 (μg / mL), 21 (μg / mL).
도 2는 본 발명의 오가피 메탄올 추출액의 단백질 분해효소 저해활성을 SDS-폴리아크릴아미드젤 전기영동(SDS-PAGE)을 수행하여 나타낸 것이다. Figure 2 shows the protease inhibitory activity of the methanol extract of the present invention by performing SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
레인 1:기질Lane 1: Substrate
레인 2: 기질+단백질 분해효소Lane 2: Substrate + Protease
레인 3: 표준 분자량 단백질Lane 3: standard molecular weight protein
레인 4∼8: 기질+단백질 분해효소+오가피 메탄올 추출액Lanes 4 to 8: Substrate + Protease + Ogapi Methanol Extract
최종농도: 333(㎍/㎖), 166(㎍/㎖), 83(㎍/㎖), 42(㎍/㎖), 21(㎍/㎖).Final concentrations: 333 (μg / mL), 166 (μg / mL), 83 (μg / mL), 42 (μg / mL), 21 (μg / mL).
레인 9∼13: 기질+단백질 분해효소+결명자 메탄올 추출액Lanes 9-13: Substrate + Protease + Declarator Methanol Extract
최종농도: 333(㎍/㎖), 166(㎍/㎖), 83(㎍/㎖), 42(㎍/㎖), 21(㎍/㎖).Final concentrations: 333 (μg / mL), 166 (μg / mL), 83 (μg / mL), 42 (μg / mL), 21 (μg / mL).
상기와 같은 목적을 달성하기 위하여, 본 발명에서는 오갈피 속 나무의 뿌리, 줄기 및 가지 부분의 껍질을 증류수로 끓이거나 유기용매로 냉침시켜 얻은 추출물을 포함한 C형 간염 치료제를 제공한다. 본 발명의 C형 간염 치료제는 각종 식음료에 포함되어 사용될 수 있다.In order to achieve the above object, the present invention provides a hepatitis C therapeutic agent comprising an extract obtained by boiling the bark of the root, stem and branch of the genus Galgalpi with distilled water or cooled by an organic solvent. Hepatitis C therapeutic agent of the present invention can be used in a variety of food and beverages.
이하, 본 발명을 상세히 설명하면 다음과 같다.Hereinafter, the present invention will be described in detail.
본 발명은 C형 간염의 치료에 사용될 수 있는 오가피 추출물을 제공한다. 본 발명은 증류수를 이용한 오가피 추출물과 유기용매를 이용한 오가피 추출물을 제공한다.The present invention provides an extract of Ogapi that can be used for the treatment of hepatitis C. The present invention provides an organic extract using an organic solvent and an organic extract.
우선, 증류수를 이용한 오가피의 추출액은 먼저, 잘게 썰은 오가피에 5∼10배(부피/질량)의 증류수를 넣고 2∼3시간 정도 끓인 다음 글래스 필터(glass filter)가 달린 감압 여과기에 여과보조제로 셀라이트를 적당량 채운후 감압하에서 증류수 추출물을 여과한다. 이렇게 하여 얻은 순수한 증류수 추출액을 냉동 건조하여 농축 추출액을 얻는다.First, the extract of Ogapi using distilled water was first put into 5 ~ 10 times (volume / mass) of distilled water in finely sliced Ogapi and boiled for 2 ~ 3 hours, then the cell was used as a filter aid in a vacuum filter equipped with a glass filter. After filling the appropriate amount of light, distilled water extract is filtered under reduced pressure. The pure distilled water extract thus obtained is freeze-dried to obtain a concentrated extract.
상기에서 얻은 본 발명의 오가피 추출물을 이용하여 추출액을 적절한 농도로 증류수에 녹여 HCV 단백질 분해효소 저해도를 측정한다.Using the Ogapi extract of the present invention obtained above, the extract is dissolved in distilled water at an appropriate concentration to measure the degree of inhibition of HCV protease.
또한, 유기용매를 이용한 오가피의 추출액은 먼저 분쇄기를 이용하여 오가피를 분말로 만든 다음 5∼10배(부피/질량)의 유기용매를 넣은 후 교반기로 교반하면서 16∼24시간 동안 추출하거나, 교반하지 않은 상태로 4∼7일 동안 냉침하여 추출한다. 이때 유기용매로는 메탄올 등을 사용하는 것이 바람직하다. 추출한 후 원심분리기를 이용하여 원심분리하여 오가피 분말을 제거하고 증류수 추출액을 여과하는 것과 동일한 방법으로 여과하여 순수한 추출액을 얻으며, 이 추출액은 감압 회전농축기를 이용하여 농축한다. 농축된 추출액은 적절한 농도로 디메틸설폭사이드(DMSO) 등의 유기용매에 녹인 후 HCV 단백질 분해효소 저해 역가를 측정한다.In addition, the extract of organic skin using organic solvent is first made into powder by using a grinder, and then 5-10 times (volume / mass) of organic solvent is added and then extracted for 16 to 24 hours with stirring with a stirrer or not stirred. Extract by chilling for 4 to 7 days in the absence. At this time, it is preferable to use methanol or the like as the organic solvent. After extraction, centrifugation is performed using a centrifuge to remove the organ powder, and the distilled water extract is filtered in the same manner as the filtrate to obtain a pure extract. The extract is concentrated using a vacuum concentrator. The concentrated extract was dissolved in an organic solvent such as dimethyl sulfoxide (DMSO) at an appropriate concentration, and then measured for HCV protease inhibition titer.
상기에서 분리·정제한 오가피 추출물은 SDS-폴리아크릴아미드 젤 전기영동(이하, "SDS-PAGE"라 약칭한다)을 수행하여 HCV 단백질 분해효소 저해도를 측정한다.The isolated and purified Ogapi extract is subjected to SDS-polyacrylamide gel electrophoresis (hereinafter abbreviated as "SDS-PAGE") to measure the degree of HCV protease inhibition.
상기한 오가피 추출물에 의한 HCV 단백질 분해효소 저해도를 측정하면, 오가피 추출물이 포함되지 않은 시료에서는 단백질 분해효소의 작용에 의해 기질로부터 아미노산이 떨어져 나가게 되며, 이를 SDS-PAGE를 이용하여 분석하면 분자량이 작아진 새로운 밴드가 나타난다. 반면에 오가피 추출물이 첨가된 시료에서는 첨가된 오가피 추출물의 농도에 따라 저해정도가 결정되어 분리현상의 정도를 기대할 수 있으며, 이로부터 짧은 시간내에 저해활성 정도를 육안으로 또는 덴시토미터 등을 사용하여 판별할 수 있다(도 1및도 2참조).When measuring the degree of inhibition of HCV protease by the above-mentioned Ogapi extract, the amino acid is separated from the substrate by the action of the protease in the sample that does not contain the Ogapi extract, the molecular weight is analyzed by using SDS-PAGE A new band appears smaller. On the other hand, in the sample to which the Ogapi extract was added, the degree of inhibition was determined according to the concentration of the added Ogapi extract, and the degree of separation could be expected. From this, the degree of inhibition activity was visually determined using a densitometer or the like. Can be determined (see FIGS . 1 and 2 ).
본 발명의 C형 간염 치료제는 오가피 추출물의 한 성분을 단독으로 순수하게 분리하거나 다성분 혼합체로 분리하여 이를유효성분으로 각각 함유할 수 있으며, 여기에 오가피 추출물에 약제학적으로 허용되는 담체, 희석제, 충전제, 응집방지제 및 향미제 등의 부형제를 첨가하여 경구제제, 피하주사제 또는 정맥주사제 등의 약제학적 제제로 제조될 수 있다.The hepatitis C therapeutic agent of the present invention may be separated into pure components alone or separated into multi-component mixtures alone or contained as an active ingredient, and the pharmaceutically acceptable carrier, diluent, Excipients such as fillers, anticoagulants and flavors can be added to prepare pharmaceutical preparations such as oral preparations, subcutaneous injections or intravenous injections.
이하, 실시예에 의하여 본 발명의 오가피 추출물을 포함하는 C형 간염 치료제를 상세히 설명하기로 한다. 단 하기 실시예는 본 발명을 예시하는 것일 뿐 본 발명이 실시예에 의하여 한정되는 것은 아니다.Hereinafter, the hepatitis C therapeutic agent containing the ogapi extract of the present invention will be described in detail by Examples. However, the following examples are only for exemplifying the present invention, and the present invention is not limited by the examples.
〈실시예 1〉증류수를 이용한 오가피 추출물의 제조.Example 1 Preparation of Ogapi Extract Using Distilled Water.
전기 약탕기(대웅제약에서 시판)에 증류수 500㎖를 넣은 후 한약상에서 구입한 잘게 썰어진 오가피 50g을 넣고 2시간 30분 동안 끓인 뒤 정치시키고 오가피 고형물을 제거한 후 상징액을 얻었다. 글래스 필터가 달린 감압여과기에 여과 보조제로 셀라이트를 1/3 정도 채운 뒤 상기의 상징액을 부은 후 감압하에서 여과하여 불용성 물질이 제거된 증류수 추출액을 얻었다. 얻어진 추출액은 10㎎/㎖의 농도로 증류수에 녹여 HCV 단백질 분해효소 저해도를 측정하였다.500ml of distilled water was added to an electric bath (commercially available from Daewoong Pharm.), And 50g of chopped chopped oranges, which were purchased from Chinese medicine, boiled for 2 hours and 30 minutes, left to stand, and the supernatant was removed. After filling the celite with a filter aid in a vacuum filter equipped with a glass filter about 1/3, the supernatant was poured and filtered under reduced pressure to obtain a distilled water extract from which an insoluble substance was removed. The obtained extract was dissolved in distilled water at a concentration of 10 mg / ml to measure the degree of inhibition of HCV protease.
〈실시예 2〉유기용매를 이용한 오가피 추출물의 제조.Example 2 Preparation of Ogapi Extract Using Organic Solvents
먼저 오가피를 분쇄기를 이용하여 미세한 분말이 될때까지 분쇄한 뒤 시험관에 분말 1g과 메탄올 10㎖를 넣어 마개로 봉한 다음 교반기에서 20시간 동안 교반하면서 추출한 후 3000rpm의 속도로 오가피 고형물을 원심분리하여 제거하였다. 얻어진 추출 상징액은 상기 실시예 1에서의 증류수 추출물을 여과하는 방법과 동일하게 여과하여 불순물을 완전히 제거하여 오가피 추출물을 얻었다. 이 추출액은 감압 회전 농축기를 이용하여 농축한 후 농축액을 10㎎/㎖의 농도로 디메틸설폭사이드(DMSO)에 녹여 HCV의 단백질 분해효소 저해 역가 측정에 사용하였다.First, the scallop was pulverized to a fine powder by using a pulverizer. Then, 1 g of powder and 10 ml of methanol were put into a test tube, sealed with a stopper, extracted with stirring for 20 hours in a stirrer, and then the scabies were removed by centrifugation at a speed of 3000 rpm. . The obtained extract supernatant was filtered in the same manner as the method of filtering the distilled water extract in Example 1 to completely remove impurities to obtain an extract of Ogapi. The extract was concentrated using a reduced pressure rotary concentrator, and the concentrate was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mg / ml and used for measuring protease inhibitory titer of HCV.
〈실험예〉 오가피 추출물에 의한 HCV 단백질 분해효소 저해도 측정Experimental Example Measurement of HCV Protease Inhibition by Ogapi Extract
오가피 추출물에 의한 단백질 분해효소의 활성도 측정은 다음과 같은 단계로 실시하였다.Determination of the activity of protease by the extract of Ogapi was carried out in the following steps.
먼저, 본 출원인에 의해 기출원된 방법(대한민국 특허출원 제 97-59161호참조)으로 정제된 단백질 분해효소를 얻고 본 출원인에 의해 기출원된 단백질 분해효소 활성도 측정방법에 기재된 방법에 따라 정제된 GST-5A5B-Ubep2-2 기질 단백질을 얻었다. GST-5A5B-Ubep2-2 기질 단백질은 20개의 아미노산 5A/5B 펩타이드의 아미노 말단에 GST 단백질이 붙어 있으며, 카르복시 말단에는 10개의 유비퀴틴 단백질 유래 아미노산을 함유하고 있어서 기질이 단백질 분해효소에 의해 특이적으로 분해되면 20개의 아미노산이 본래의 기질 단백질로부터 분리되며 이를 SDS-PAGE로 관찰하여 활성 정도를 측정할 수 있다. 적합한 활성도를 측정하기 위해 50mM 트리스(Tris), 10mM DTT, 2% 챕스{CHAPS : 3-[(3-cholamidopropyl)- dimethylammonio]-1-propane-sulfonate} 및 37.5% 글리세롤(glycerol)이 포함된 pH 7.5의 완충용액 [이하, "시험 완충용액(assay buffer)"이라 지칭한다]에 기질 10㎕(농도: 500㎍/㎖)과 C형 단백질 분해효소 10㎕(농도: 8㎍/㎖)로 구성된 20㎕의 용액을 넣고 1시간 동안 상온에서 반응시켰다. 여기에 반응을 완결시키기 위하여 3X SDS-PAGE 견본 완충용액(sampling buffer)를 15㎕씩 넣고 80℃에서 5분 동안 방치한 후 이중 20㎕씩을 취하여 13.5% 의 SDS-PAGE을 이용하여 전개시켰다. 오가피 추출물이 특이하게 HCV의 단백질 분해효소 활성을 저해하는 것을 알아내기 위한 대조군으로는 결명자 열매를 상기 실시예 1 또는 2의 방법과 동일하게 처리하여 얻은 결명자 증류수 추출액과 결명자 메탄올 추출액을 사용하였다.First, purified according to the process described in the Applicant issued the original method by (Republic of Korea Patent Application No. 97-59161 call reference) the protease method for measuring the gain issued by the applicant the original protease activity by purified GST -5A5B-Ubep2-2 substrate protein was obtained. GST-5A5B-Ubep2-2 substrate protein has a GST protein attached to the amino terminus of 20 amino acids 5A / 5B peptide, and contains 10 ubiquitin protein-derived amino acids at the carboxy terminus. Upon digestion, 20 amino acids are separated from the original substrate protein and can be observed by SDS-PAGE to determine their activity. PH with 50 mM Tris, 10 mM DTT, 2% chaps {CHAPS: 3-[(3-cholamidopropyl) -dimethylammonio] -1-propane-sulfonate} and 37.5% glycerol to determine the appropriate activity 7.5 buffer solution (hereinafter referred to as "assay buffer") consisting of 10 µl of substrate (concentration: 500 µg / ml) and 10 µl of type C protease (concentration: 8 µg / ml) 20 μl of solution was added and reacted at room temperature for 1 hour. To complete the reaction, 15 μl of 3X SDS-PAGE sample buffer was added thereto, and the resultant was allowed to stand at 80 ° C. for 5 minutes, followed by 20 μl of the sample, which was developed using 13.5% of SDS-PAGE. As a control to find out that the extract of Ogapi specifically inhibits the protease activity of HCV, the C. distilled water extract and C. ethanol methanol extract obtained by treating the C. locust fruit in the same manner as in Example 1 or 2 above were used.
상기한 오가피 추출물에 의한 HCV 단백질 분해효소 저해도 측정 실험 결과, 오가피 추출물 즉, 단백질 분해효소에 대한 저해제가 없는 대조군의 경우에는 단백질 분해효소의 작용에 의해 기질로부터 20개의 아미노산이 떨어져 나가며 따라서 분자량이 대략 2000 정도 작아진 새로운 밴드가 나타났으나, 오가피 추출물이 첨가된 경우에는 첨가된 오가피 추출물의 농도에 따라 저해정도가 결정되어 오가피 추출물의 농도가 클수록, 즉 저해정도가 클수록 분리가 적게 되었음을 알 수 있었다(도 1및도 2참조).As a result of measuring the inhibition of HCV protease by the above-mentioned organ extract, 20 amino acids are separated from the substrate by the action of the protease in the case of the control group without the inhibitor of the organ extract, that is, the protease. Although a new band of about 2000 was shown, the degree of inhibition was determined according to the concentration of the added Ogapi extract, and the higher the concentration of the Ogapi extract, that is, the higher the degree of inhibition, the less the separation. (See FIGS . 1 and 2 ).
즉, 오가피의 증류수 추출물액 및 유기용매 추출액이 HCV 단백질 분해효소에 대한 강한 저해효과를 갖는 것으로 밝혀져 오가피의 추출물이 효과적인 간염 치료제로서 유용하게 사용될 수 있음을 알 수 있었다.That is, it was found that the distilled water extract and the organic solvent extract of Ogapi had a strong inhibitory effect on HCV protease, and thus the extract of Ogapi could be usefully used as an effective hepatitis therapeutic agent.
이상에서 살펴본 바와 같이, 오가피의 증류수 추출물 및 유기용매 추출물이 HCV 단백질 분해효소에 대한 강한 저해 효과를 나타내었으며, 따라서 본 발명의 오가피의 추출물을 유효성분으로 포함하는 조성물은 효과적인 C형 간염 치료제로 유용하게 사용될 수 있다. 또한, 본 발명의 오가피 추출물은 각종 식음료에 포함되어 다양하게 C형 간염 치료제로서 사용될 수 있다.As described above, the distilled water extract and the organic solvent extract of Ogapi showed a strong inhibitory effect on HCV protease, and thus the composition comprising the Ogapi extract of the present invention as an active ingredient is useful as an effective hepatitis C therapeutic agent. Can be used. In addition, the ogapi extract of the present invention can be used in various hepatitis C therapeutic agents included in various food and beverages.
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