KR101508294B1 - Composition for preventing or treating Hepatitis C comprising extract of Garcinia Mangostana or Gamma, alpha-mangostins - Google Patents
Composition for preventing or treating Hepatitis C comprising extract of Garcinia Mangostana or Gamma, alpha-mangostins Download PDFInfo
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- KR101508294B1 KR101508294B1 KR20130079307A KR20130079307A KR101508294B1 KR 101508294 B1 KR101508294 B1 KR 101508294B1 KR 20130079307 A KR20130079307 A KR 20130079307A KR 20130079307 A KR20130079307 A KR 20130079307A KR 101508294 B1 KR101508294 B1 KR 101508294B1
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- mangosteen
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- gamma
- virus
- hepatitis
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- A—HUMAN NECESSITIES
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A—HUMAN NECESSITIES
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
Abstract
본 발명은 망고스틴 추출물 또는, 감마 또는 알파 망고스틴을 유효성분으로 포함하는 C형 간염의 예방 또는 치료용 조성물에 관한 것이다. 본 발명에 따른 망고스틴 열매의 추출물 및 이로부터 분리한 감마 또는 알파 망고스틴은 간세포에 대한 독성이 매우 낮으면서도, HCV의 게놈의 복제를 선택적으로 저해하는 효과를 우수하게 나타냄으로써, C형 간염의 예방 또는 치료에 매우 유용하게 사용할 수 있다.The present invention relates to a composition for preventing or treating hepatitis C comprising mangosteen extract or gamma or alpha mangosteen as an active ingredient. The extract of mangosteen fruit according to the present invention and the gamma or alpha mangosteen isolated therefrom exhibit an extremely low toxicity to hepatocytes and an excellent effect of selectively inhibiting the replication of the genome of HCV, It can be very useful for prevention or treatment.
Description
본 발명은 망고스틴 추출물 또는 감마, 알파 망고스틴을 유효성분으로 포함하는 C형 간염의 예방 또는 치료용 조성물에 관한 것이다.
The present invention relates to a composition for preventing or treating hepatitis C comprising mangosteen extract or gamma or alpha mangosteen as an active ingredient.
간암과 간질환은 뇌혈관질환, 심장질환 그리고 호흡기질환과 더불어 한국인 인구 10만 명당 사망자 수가 가장 높은 4대 질환 중 하나로, 한국인의 간암과 간질환의 원인은 B형 및 C형 간염 바이러스에 의한 바이러스성 간염이 전체의 83%를 차지한다. 그 중에서도 C형 간염 바이러스는 전 세계적으로 약 1억 7천만 명의 감염자를 가지고 있는 의학적으로 매우 중요한 병원균이다. C형 간염은 1980년대 중반까지 비(非)A형 비(非)B형 수혈 후 연관간염(non-A non-B post-transfusion associated hepatitis, NANB)으로 구분되다가, 1989년도에 비로소 전혀 새로운 종류의 바이러스에 의한 감염에 의해 생기는 질병임이 밝혀지게 되었고, 그 후 이에 대한 활발한 연구와 개발이 진행되어 왔다. C형 간염 바이러스에 의한 간세포의 감염은 대부분의 경우 만성간염으로 진행되고, 15년에서 20년 정도의 장기간에 걸쳐 간경화와 간암과 같은 만성간질환으로 발전하기 때문에 치명적이며, C형 간염 바이러스(HCV, Hepatitis B Virus)에 의한 만성간염, 간경화 및 간암으로 매년 미국에서만 8000명 내지 10000명 정도가 사망하고 있는 것으로 보고된다. 특히, 대부분의 말기 C형 간염환자들의 경우 간 이식 수술을 기다리다 간 이식을 받지 못해 사망하는 경우가 대부분이며, 통계적으로 한국의 경우 전 인구 중 1.5% 정도가 C형 간염 바이러스에 감염되어 있다고 알려져 있다.Liver cancer and liver disease are one of the four major diseases with the highest number of deaths per 100,000 Koreans, along with cerebrovascular disease, heart disease and respiratory disease. Liver cancer and liver disease in Koreans are caused by hepatitis B and C virus Sexual hepatitis accounts for 83% of the total. Among them, hepatitis C virus is a very important medically important pathogen with about 170 million people worldwide. Hepatitis C was classified as non-A non-A post-transfusion associated hepatitis (NANB) after non-A type hepatitis B until the mid-1980s, The virus has been found to be a disease caused by infection by viruses, and then researches and developments have been actively conducted thereafter. Hepatocellular carcinoma of the hepatitis C virus is most likely chronic hepatitis and is fatal because it develops into chronic liver disease such as liver cirrhosis and liver cancer for a long period of 15 to 20 years. , Hepatitis B Virus), chronic hepatitis, liver cirrhosis and hepatocellular carcinoma are reported to be killed by 8,000 to 10,000 people in the United States every year. Most hepatitis C patients die from liver transplantation because they are not waiting for liver transplantation. Statistically, in Korea, about 1.5% of the total population is known to be infected with hepatitis C virus .
C형 간염 바이러스는 플라비비리데(flaviviridae)군 중 헤파시바이러스(hepacivirus)속으로 분류되는 유일한 바이러스로, 약 9600개의 핵산으로 이루어진 단일가닥(strand)의 RNA를 바이러스게놈으로 가지고 있다. 상기 RNA는 간세포 내에서 약 3000개의 아미노산으로 이루어진 다단백질(polyprotein)로 번역되고, 번역된 다단백질(polyprotein)은 간세포의 소포체에 존재하는 신호 펩티다제(signal peptidase)와 바이러스의 비구조 단백질 중 하나인 NS3 단백질분해효소(protease)에 의해 10가지의 다른 바이러스단백질로 발현된다. 발현된 바이러스단백질 중 외피 당단백질(envelope glycoprotein)인 E1, E2, 그리고 캡시드(capsid) 단백질인 코어(core)와 같은 구조 단백질들(structural proteins)은 바이러스의 입자(particle)를 만드는데 쓰이고 NS2, NS3, NS4A, NS4B, NS5A, 그리고 NS5B와 같은 비구조 단백질들(non-structural proteins)은 바이러스의 게놈을 복제하는데 필요한 바이러스게놈 복제 화합체(virus genome replication complex)를 만드는데 쓰인다. HCV는 소포체(endoplasmic reticulum)에서 발생한 막피(membrane)에서 바이러스게놈 복제화합체를 만들어서 바이러스의 RNA 게놈을 복제한다. 바이러스게놈 복제화합체는 막피에 쌓여 움푹 들어간 형태(invaginated)를 유지함으로써 외부환경에서 분리된 독립적인 구조를 만들고 이 구조 속에 바이러스의 비구조 단백질들을 발현시키고 발현된 비구조 단백질들이 바이러스의 RNA 중합효소인 NS5B 단백질과 함께 바이러스의 게놈복제공장의 역할을 한다. 하지만 이 바이러스게놈 복제화합체가 어떻게 만들어지고 유지되는지에 대해서는 잘 알려져 있지 않다. 바이러스의 비구조 단백질들 중 특히 NS4B 단백질은 간세포 내에서 바이러스의 게놈복제에 꼭 필요한 다소포구조(multi-vesicular structure)인 세포막거미줄(membranous web)을 만드는데 가장 결정적인 역할을 하는 것으로 밝혀졌다. 이 NS4B에 의해서 만들어진 세포막거미줄(membranous web)이라는 구조는 바이러스게놈 복제화합체를 만드는데 필요한 물리적인 구조를 제공한다고 믿어진다. 또한, NS5A 단백질은 바이러스게놈 복제화합체를 다른 바이러스의 비구조 단백질들을 이용하여 조립하고, 만들어진 바이러스게놈을 지방물방울(lipid droplet)이라는 구조로 옮기고, 여기서 바이러스의 코어(core) 단백질을 바이러스 게놈과 결합시킴으로써 실제적인 바이러스입자를 만드는데 중요한 역할을 한다고 알려져있다. 특히 바이러스의 비구조 단백질간의 단백질상호작용(protein-protein interaction)은 바이러스게놈 복제화합체를 만드는데 결정적인 역할을 한다고 알려져 있지만 정확하게 분자수준에서 어떤 역할을 하는지에 대해서는 아직 잘 알려져 있지 않은 상태이다. The hepatitis C virus is the only virus classified as a hepacivirus in the flaviviridae family, and has a virus genome of RNA of a strand of about 9600 nucleotides. The RNA is translated into a polyprotein of about 3,000 amino acids in the hepatocyte, and the translated polyprotein is expressed in the signal peptidase and non-structural proteins of the virus, It is expressed in 10 different viral proteins by one NS3 protease. Structural proteins such as envelope glycoproteins E1, E2, and capsid proteins are used to make virus particles, while NS2, NS3 Non-structural proteins such as NS4A, NS4B, NS5A, and NS5B are used to create the viral genome replication complex necessary to replicate the genome of a virus. HCV replicates the RNA genome of a virus by creating a viral genomic clone from the membrane of the endoplasmic reticulum. The viral genomic clones are invaginated by clumping to form an independent structure separated from the external environment, expressing the non-structural proteins of the virus in the structure, and expressing the non-structural proteins as viral RNA polymerase Together with the NS5B protein, acts as a genomic cloning site for the virus. However, it is not known how this viral genomic clone is created and maintained. NS4B proteins, among the nonstructural proteins of viruses, have been shown to play a crucial role in making the membranous web, a multi-vesicular structure that is essential for the genome replication of viruses in hepatocytes. The structure of the membrane made by NS4B is believed to provide the physical structure necessary to make viral genomic clones. In addition, the NS5A protein is constructed by assembling the viral genome replicase using non-structural proteins of other viruses, transferring the viral genome to a structure called lipid droplet, where the core protein of the virus is combined with the viral genome It is known to play an important role in creating real virus particles. In particular, protein-protein interaction between non-structural proteins of viruses is known to play a crucial role in the replication of viral genomes, but it is not yet known exactly what role they play at the molecular level.
바이러스성 질환을 치료할 수 있는 항바이러스제의 종류로는 항바이러스제의 작용메커니즘에 따라 바이러스를 간접적으로 공격하는 항바이러스제(indirect-acting antivirals, IAAs)와 바이러스를 직접적으로 공격하는 항바이러스제(direct-acting antivirals, DAAs)가 있다. 바이러스와 같은 병원성 외부미생물이 체내 침투시 체내에서 분비되는 인터페론 알파는 바이러스를 간접적으로 공격하는 대표적인 항바이러스성 내재면역 증강물질로써, 바이러스 자체를 공격하기보다는 바이러스가 기생하고 있는 숙주세포의 면역저항성을 높임으로써 바이러스의 세포 내 증식을 차단하기 때문에, 여러 가지 다른 종류의 바이러스성 질환에 효과가 있고 바이러스가 기생하고 있는 숙주세포의 단백질의 기능을 조절하여 항바이러스 활성을 나타내므로, 내성과 저항성을 가지고 있는 돌연변이 바이러스가 생길 확률이 비교적 적다. 그러나, 특정한 바이러스에 대해서만 항바이러스 활성을 나타낼 수 있는 항바이러스 특이성(antiviral specificity)이 현저하게 떨어지기 때문에, 이를 극복하기 위해서 생체 내에서 자연스럽게 분비되는 인터페론의 양보다 훨씬 더 많은 양의 인터페론을 치료목적으로 투여하게 되고, 따라서 자살충동, 우울증, 빈혈 등과 같은 심각한 부작용 및 독성이 나타나게 된다.Examples of antiviral agents that can treat viral diseases include indirect-acting antivirals (IAAs) that indirectly attack viruses and direct-acting antiviral agents that directly attack viruses , DAAs). Interferon alpha, which is secreted in the body by pathogenic external microorganisms such as viruses, is a typical antiviral immune enhancer that indirectly attacks viruses. It is an immune response of host cells parasitized with virus rather than attacking virus itself. Since it inhibits the intracellular proliferation of viruses, it is effective against various kinds of viral diseases, and since it exhibits antiviral activity by controlling the function of the protein of the host cell in which the virus is parasitic, it has resistance and resistance There is a relatively low probability that a mutant virus is present. However, since the antiviral specificity, which can only exhibit antiviral activity for a specific virus, is significantly lowered, a much higher amount of interferon than the amount of interferon that is secreted naturally in vivo to overcome it Such as suicidal thoughts, depression, anemia, and other serious side effects and toxicity.
바이러스를 직접적으로 공격하는 항바이러스제의 경우, 인터페론과 달리 바이러스의 생활사에서 꼭 필요한 바이러스 단백질들의 특수한 기능만을 선택적으로 저해함으로써 상대적으로 낮은 농도에서도 높은 항바이러스활성을 나타낸다. 또한 바이러스를 간접적으로 공격하는 항바이러스제에 비해, 독성이 적고 안전성이 높은 것이 특징이다. 인간면역결핍바이러스(human immunodeficiency virus, HIV)의 역전사효소 저해제(reverse transcriptase inhibitor)와 단백질분해효소 저해제(protease inhibitor)는 바이러스를 직접적으로 공격하는 대표적인 항바이러스제로써, 각각 바이러스의 생활사에서 필수적인 효소인 역전사효소, 단백질분해효소를 저해하여 바이러스의 세포 내 증식을 억제한다. 그러나, 바이러스가 돌연변이를 통해 항바이러스제의 타겟이 된 바이러스단백질의 아미노산서열을 변화시킴으로써 기존 항바이러스제에 대한 저항성 및 내성을 나타낼 수 있는 가능성이 상대적으로 크며, 특히, C형 간염 바이러스의 RNA 중합효소(polymerase)는 RNA게놈의 복제시 잘못 들어간 핵산을 교정하는 기능을 하는 5‘-3’ 엔도뉴클레아제(endonuclease) 활성이 없으므로, C형 간염 바이러스 게놈인 RNA가 복제되는 과정에서 쉽게 돌연변이 바이러스가 생기게 되어 바이러스를 직접적으로 공격하는 항바이러스제에 대해 저항성과 내성을 갖기가 쉽다. 현재 C형 간염치료제로 일반적으로 사용되고 있는 인터페론 알파(interferon-alpha)와 리바비린(ribavirin)은 각각 내재면역증진과 핵산생합성저해를 통해 바이러스를 간접적으로 공격하는 항바이러스제(indirect-acting antivirals, IAAs)로서, 두 약물을 함께 병용하여도 52주간의 정맥주사 및 경구투여를 통한 약물투여 후 치료 성공률(sustained virologic response, SVR)이 바이러스의 게놈형(genotype)에 따라서는 50%에도 미치지 못하여 약효가 매우 낮을 뿐만 아니라, 자살충동, 우울증, 빈혈과 같은 심각한 부작용 및 독성을 가지고 있어 많은 C형 간염환자들이 중도에 치료를 포기하고 있는 실정이다. 따라서, 상기와 같은 기존 C형 간염 바이러스에 대한 항바이러스 약물들을 대체할, 보다 효과적이면서도 안전한 약물개발이 필요하다. Antiviral agents that directly attack viruses, unlike interferon, selectively inhibit only the specific functions of viral proteins essential for the life cycle of viruses, thereby exhibiting high antiviral activity even at relatively low concentrations. Compared to antiviral agents that indirectly attack viruses, they are less toxic and have higher safety. Reverse transcriptase inhibitors and protease inhibitors of the human immunodeficiency virus (HIV) are typical antiviral agents that directly attack viruses. These viruses are known as reverse transcriptase Enzymes and proteolytic enzymes are inhibited to suppress intracellular proliferation of viruses. However, the possibility that the virus can exhibit resistance and resistance to existing antiviral agents by changing the amino acid sequence of the virus protein targeted by the antiviral agent through the mutation is relatively large, and in particular, the RNA polymerase of the hepatitis C virus polymerase) does not have 5'-3 'endonuclease activity, which is a function of correcting a wrongly inserted nucleic acid in the replication of the RNA genome. Therefore, mutant viruses are easily produced in the course of replication of RNA, the genome of hepatitis C virus And it is easy to have resistance and resistance against antiviral agents that directly attack viruses. Currently, interferon-alpha and ribavirin, commonly used as anti-hepatitis C drugs, are indirect-acting antivirals (IAAs) that indirectly attack viruses through internal immunity enhancement and inhibition of nucleic acid biosynthesis, respectively , The combination of the two drugs together resulted in a sustained virologic response (SVR) of less than 50% due to the genotype of the virus after intravenous administration of 52 weeks of intravenous administration and oral administration In addition, many serious adverse reactions such as suicidal thoughts, depression, anemia, and toxicity have caused many patients with hepatitis C to give up treatment moderately. Therefore, there is a need to develop a more effective and safe drug that replaces the existing antiviral drugs against hepatitis C virus.
한편, 망고스틴(Garcinia mangostana)은 말레이시아 원산의 무환자나무목 고추나무과에 속하는 쌍떡잎식물의 열매로 납작한 공모양의 탁구공보다 조금 큰 크기의 열매를 맺는다. 망고스틴 열매는 폴리페놀의 일종인 크산톤(xanthone)이라는 대표적인 항산화제 성분을 함유하고 있어 뛰어난 항산화력을 지닌 것으로 알려져 있으며, 동남아에서는 오래전부터 민간약으로서 염증이나 상처의 치료 등에 사용되어 온 것으로 알려져 있다. 또한 망고스틴은 항염증 효과 외에 항균, 항암 효과에 대해서도 알려져 있다.On the other hand, mangosteen (Garcinia mangostana) is a fruit of a dicotyledonous plant belonging to the rootless pepper tree of the origin of Malaysia, and a fruit of a size slightly larger than a flat ball-shaped table tennis ball is formed. Mangosteen is known to have excellent antioxidant ability because it contains a typical antioxidant component called xanthone, which is a type of polyphenol, and it has been known for a long time as a civilian medicine in Southeast Asia and has been used for the treatment of inflammation and wound . In addition to anti-inflammatory effects, mangosteen is known for its antibacterial and anticancer effects.
상기한 바와 같이, 망고스틴의 다양한 약리효과가 알려져 있지만, 망고스틴의 C형 간염에 대한 예방 또는 치료 효과에 대해서는 알려져 있지 않으며, 이에 대한 연구도 전무한 상태이다. 따라서, 망고스틴을 이용한 C형 간염 치료제의 개발의 필요성이 절실히 요구되고 있다.As described above, although various pharmacological effects of mangosteen are known, the preventive or therapeutic effect of mangosteen against hepatitis C has not been known, and there have been no studies on it. Therefore, there is an urgent need to develop a therapeutic agent for hepatitis C using mangosteen.
본 발명자들은 C형 간염의 예방 또는 치료효과를 갖는 물질을 탐색하기 위하여 연구하던 중, 망고스틴(Garcinia mangostana) 열매의 추출물과 이로부터 분리한 감마 또는 알파 망고스틴이 간세포에 대한 독성이 매우 낮으면서도, HCV의 게놈의 복제를 선택적으로 저해하는 효과를 매우 우수하게 나타내는 것을 확인하고, 본 발명을 완성하였다.
The inventors of the present invention have conducted studies to search for a substance having the preventive or therapeutic effect of hepatitis C, and found that the extract of fruit mangosteen (Garcinia mangostana) and the gamma or alpha mangosteen separated therefrom are very low in toxicity to hepatocytes , And the effect of selectively inhibiting the replication of the genome of HCV is very excellent. Thus, the present invention has been completed.
본 발명은 망고스틴 추출물 또는, 감마 또는 알파 망고스틴을 유효성분으로 포함하는 C형 간염의 예방 또는 치료용 약학적 조성물을 제공하고자 한다.The present invention provides a pharmaceutical composition for preventing or treating hepatitis C comprising mangosteen extract or gamma or alpha mangosteen as an active ingredient.
또한, 본 발명은 망고스틴 추출물 또는, 감마 또는 알파 망고스틴을 유효성분으로 포함하는 C형 간염의 예방 또는 개선용 식품 조성물을 제공하고자 한다.
The present invention also provides a food composition for preventing or ameliorating hepatitis C comprising mangosteen extract or gamma or alpha mangosteen as an active ingredient.
상기 과제를 해결하기 위하여, 본 발명은 망고스틴 추출물 또는, 감마 또는 알파 망고스틴을 유효성분으로 포함하는 C형 간염의 예방 또는 치료용 약학적 조성물을 제공한다.In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating hepatitis C comprising mangosteen extract or gamma or alpha mangosteen as an active ingredient.
또한, 본 발명은 망고스틴 추출물 또는, 감마 또는 알파 망고스틴을 유효성분으로 포함하는 C형 간염의 예방 또는 개선용 식품 조성물을 제공한다.
The present invention also provides a food composition for preventing or ameliorating hepatitis C comprising mangosteen extract or gamma or alpha mangosteen as an active ingredient.
본 발명에 따른 망고스틴 열매의 추출물 및 이로부터 분리한 감마 또는 알파 망고스틴은 간세포에 대한 독성이 매우 낮으면서도, HCV의 게놈의 복제를 선택적으로 저해하는 효과를 우수하게 나타냄으로써, C형 간염의 예방 또는 치료에 매우 유용하게 사용할 수 있다.
The extract of mangosteen fruit according to the present invention and the gamma or alpha mangosteen isolated therefrom exhibit an extremely low toxicity to hepatocytes and an excellent effect of selectively inhibiting the replication of the genome of HCV, It can be very useful for prevention or treatment.
도 1은 본 발명의 망고스틴 에탄올 추출물이 C형 간염 바이러스의 RNA 게놈 복제 및 세포생존율에 미치는 영향을 나타낸 도이다 (파란선: 세포생존율, 붉은선: 게놈복제율).
도 2는 알파 망고스틴이 C형 간염 바이러스의 RNA 게놈 복제 및 세포생존율에 미치는 영향을 나타낸 도이다 (파란선: 세포생존율, 붉은선: 게놈복제율).
도 3은 감마 망고스틴이 C형 간염 바이러스의 RNA 게놈 복제 및 세포생존율에 미치는 영향을 나타낸 도이다 (파란선: 세포생존율, 붉은선: 게놈복제율).
도 4는 C형 간염 바이러스의 게놈복제를 측정하기 위해 사용된 FL-J6/JFH-5C19Rluc2AUbi HCV 리포터바이러스의 게놈 구조를 나타낸 도이다.
도 5는 본 발명의 망고스틴의 메탄올 (T), 물 (W), 헥산 (H), 부탄올 (B), 에틸아세테이트 (E), 또는 에탄올 (EtOH) 추출물이 C형 간염 바이러스의 RNA 게놈 복제 및 세포생존율에 미치는 영향을 나타낸 도이다(파란선: 세포생존율, 붉은선: 게놈복제율).
도 6은 본 발명의 망고스틴 에탄올 추출물 (10 mg/ml)이 시간이 변함에 따라 C형 간염 바이러스의 RNA 게놈 복제 및 세포생존율에 미치는 영향을 나타낸 도이다 (붉은선: 세포생존율, 파란선: 게놈복제율).
도 7은 본 발명의 망고스틴 에탄올 추출물이 C형 간염 바이러스의 RNA 게놈 수준에 미치는 영향을 나타낸 도이다
도 8은 본 발명의 망고스틴 에탄올 추출물이 C형 간염 바이러스의 단백질 발현에 미치는 영향을 웨스턴 블롯으로 나타낸 도이다.
도 9는 본 발명의 망고스틴 에탄올 추출물이 C형 간염 바이러스의 단백질 발현에 미치는 영향을 형광염색으로 나타낸 도이다
도 10은 본 발명의 망고스틴 에탄올 추출물이 C형 간염 바이러스의 단백질 발현에 미치는 영향을 GFP가 붙어있는 바이러스의 단백질을 FACS로 분석해 나타낸 도이다
도 11은 본 발명의 감마 망고스틴이 C형 간염 바이러스의 RNA 게놈 수준에 미치는 영향을 나타낸 도이다
도 12는 본 발명의 감마 망고스틴이 C형 간염 바이러스의 단백질 발현에 미치는 영향을 웨스턴 블롯으로 나타낸 도이다.FIG. 1 shows the effect of the ethanol extract of mangosteen according to the present invention on RNA genome replication and cell viability of hepatitis C virus (blue line: cell viability, red line: genomic replication ratio).
Figure 2 shows the effect of alpha mangosteen on RNA genome replication and cell viability of hepatitis C virus (blue line: cell viability, red line: genomic replication rate).
Figure 3 shows the effect of gamma mangosteen on RNA genome replication and cell viability of hepatitis C virus (blue line: cell viability, red line: genomic replication rate).
Figure 4 shows the genomic structure of FL-J6 / JFH-5C19Rluc2AUbi HCV reporter virus used to measure genomic replication of hepatitis C virus.
FIG. 5 is a graph showing the effect of the methanol (T), water (W), hexane (H), butanol (B), ethyl acetate (E) or ethanol (EtOH) extract of mangosteen of the present invention on the RNA genome replication (Blue line: cell viability, red line: genome replication rate).
6 shows the effect of the mangosteen ethanol extract (10 mg / ml) of the present invention on the RNA genome replication and cell survival rate of hepatitis C virus as time elapses (red line: cell survival rate, blue line: Genome replication rate).
FIG. 7 is a graph showing the effect of the ethanol extract of mangosteen of the present invention on the RNA genome level of hepatitis C virus
FIG. 8 is a Western blot graph showing the effect of the mangosteen ethanol extract of the present invention on protein expression of hepatitis C virus.
FIG. 9 is a graph showing the effect of the ethanol extract of mangosteen of the present invention on protein expression of hepatitis C virus by fluorescent staining
10 shows the effect of the ethanol extract of mangosteen according to the present invention on protein expression of hepatitis C virus by FACS analysis of the protein of GFP-associated virus
Figure 11 is a graph showing the effect of the gamma mangosteen of the present invention on the RNA genome level of hepatitis C virus
FIG. 12 is a Western blot graph showing the effect of gamma-mangosteen of the present invention on protein expression of hepatitis C virus.
본 발명은 망고스틴 추출물을 유효성분으로 포함하는 C형 간염의 예방 또는 치료용 조성물을 제공한다. The present invention provides a composition for preventing or treating hepatitis C comprising mangosteen extract as an active ingredient.
또한, 본 발명은 하기 화학식 1로 표시되는 감마 망고스틴 또는 하기 화학식 2로 표시되는 알파 망고스틴을 유효성분으로 포함하는 C형 간염의 예방 또는 치료용 조성물을 제공한다.The present invention also provides a composition for preventing or treating hepatitis C comprising gamma mangosteen represented by the following formula (1) or alpha mangosteen represented by the following formula (2) as an active ingredient.
[화학식 1][Chemical Formula 1]
[화학식 2](2)
상기 조성물은 약학적 조성물 또는 식품 조성물을 포함한다.
The composition comprises a pharmaceutical composition or a food composition.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 유효성분인 망고스틴 추출물 또는, 감마 또는 알파 망고스틴은 통상적인 추출방법에 의해 얻을 수 있고, 시판되는 것을 구입해서 사용할 수 있다. 통상적인 추출 방법은 초음파 추출법, 여과법 및 환류 추출법 등을 포함할 수 있으나, 이에 한정되지 않는다.Mangosteen extract or gamma or alpha mangosteen, which is an effective ingredient of the present invention, can be obtained by a conventional extraction method, and commercially available ones can be purchased and used. Typical extraction methods include, but are not limited to, ultrasonic extraction, filtration, and reflux extraction.
본 발명의 일 실시예에서는 망고스틴 추출물을 하기와 같은 방법으로 수득하였다. In one embodiment of the present invention, a mangosteen extract was obtained in the following manner.
먼저, 망고스틴을 물로 깨끗이 세척하고 건조한 후 분쇄한다. 분쇄된 망고스틴에 물, C1~C4의 알콜 또는 물과 C1~C4의 알콜의 혼합용매를 가하고 1~5시간, 바람직하게는 약 2시간 동안 침지하고 초음파 추출법(sonication)을 이용하여 추출한다. 이때, 용매의 부피는 분쇄한 망고스틴 중량의 1~10배, 바람직하게는 2~5배로 한다. 상기 C1~C4의 알콜은 바람직하게는 메탄올 또는 에탄올 중에서 선택될 수 있다. 이후, 망고스틴 추출액을 원심분리하여 분리하고 상층액을 모아서 여과한 후, 여액을 감압 하에 농축한 다음 동결건조하여 분말 형태의 망고스틴 추출물을 얻는다. 상기 망고스틴 추출물에 증류수를 가하고 헥산, 클로로포름, 에틸아세테이트, n-부탄올로 순차 분획하여 각 용매 추출 분획물을 얻는다. 상기 망고스틴 클로로포름 분획물로부터 컬럼크로마토그래피를 이용하여 49개의 분획물을 얻는다. 이중에서 8번 분획물을 재결정하여 노란색 분말인 감마-망고스틴을 얻고, 24, 25번 분획물을 재결정하여 알파-망고스틴을 얻는다.
First, mangosteen is cleaned with water, dried and then ground. To the ground mangosteen water, C 1 ~ C 4 of was added a mixed solvent of alcohol or water and an alcohol of C 1 ~ C 4 1 ~ 5 hours, preferably dipped for about 2 hours using an ultrasonic extraction (sonication) . At this time, the volume of the solvent is 1 to 10 times, preferably 2 to 5 times the weight of the ground mangosteen. The C 1 -C 4 alcohol can be selected preferably from methanol or ethanol. Then, the mangosteen extract is separated by centrifugation, and the supernatant is collected and filtered. The filtrate is concentrated under reduced pressure and then lyophilized to obtain a powdery mangosteen extract. Distilled water is added to the above mangosteen extract and fractionated with hexane, chloroform, ethyl acetate and n-butanol in order to obtain each solvent fraction. From the mangosteen chloroform fraction, 49 fractions are obtained by column chromatography. Among them, the
본 발명에 따른 감마 또는 알파 망고스틴은 금속염, 유기 염기와의 염, 무기산과의 염, 유기산과의 염, 염기성 또는 산성 아미노산과의 염 등 약학적으로 허용가능한 염의 형태로 사용할 수 있다. 적합한 금속염으로는, 나트륨염, 칼륨염 등과 같은 알칼리 금속염; 칼슘염, 마그네슘염, 바륨염 등과 같은 알칼리 토금속염; 알루미늄염 등이 있고, 적합한 유기 염기와의 염으로는, 예를 들어, 트리메틸아민, 트리에틸아민, 피리딘, 피콜린, 2,6-루티딘, 에탄올아민, 디에탄올아민, 트리에탄올아민, 시클로헥실아민, 디시클로헥실아민, N,N-디벤질에틸렌디아민 등과의 염이 있다. 또한, 적합한 무기산과의 염의 예로는, 염산, 브롬화수소산, 질산, 황산, 인산 등과의 염이 있으며, 적합한 유기산과의 염으로는, 포름산, 아세트산, 트리플루오로아세트산, 프탈산, 푸마르산, 옥살산, 타르타르산, 말레인산, 시트르산, 숙신산, 메탄술폰산, 벤젠술폰산, p-톨루엔술폰산 등과의 염이 있다. 염기성 아미노산과의 염의 적합한 예로서는, 예를 들어, 알기닌, 라이신, 오르니틴 등과의 염이 있고, 산성 아미노산과의 염의 적합한 예로서는, 예를 들어, 아스파르트산, 글루탐산 등과의 염이 있다. 특히 바람직한 염으로는, 화합물이 그 내에 산성 관능기를 가지는 경우, 알칼리 금속염 (예컨대, 나트륨염, 칼륨염 등), 알칼리 토금속염 (예컨대, 칼슘염, 마그네슘염, 바륨염 등) 등과 같은 무기염, 및 암모늄염과 같은 유기염이 있으며, 화합물이 그 내에 염기성 관능기를 가지는 경우, 염산, 브롬화수소산, 질산, 황산, 인산 등과 같은 무기산과의 염, 아세트산, 프탈산, 푸마르산, 옥살산, 타르타르산, 말레인산, 시트르산, 숙신산, 메탄술폰산, p-톨루엔술폰산 등과 같은 유기산과의 염이 있다.Gamma or alpha mangosteen according to the present invention can be used in the form of pharmaceutically acceptable salts such as metal salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. Suitable metal salts include alkali metal salts such as sodium salts, potassium salts and the like; Alkaline earth metal salts such as calcium salts, magnesium salts, barium salts and the like; Aluminum salts and the like. Suitable salts with organic bases include, for example, salts with organic bases such as trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexyl Amine, dicyclohexylamine, N, N-dibenzylethylenediamine and the like. Examples of the salt with a suitable inorganic acid include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like. Examples of suitable salts with organic acids include formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, , Maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and the like. Suitable examples of salts with basic amino acids include salts with, for example, arginine, lysine, ornithine and the like. Suitable examples of salts with acidic amino acids include salts with, for example, aspartic acid, glutamic acid and the like. Particularly preferred salts include inorganic salts such as alkali metal salts (for example, sodium salts and potassium salts) and alkaline earth metal salts (for example, calcium salts, magnesium salts and barium salts) when the compound has an acidic functional group therein, And salts with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like, organic acids such as acetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, Succinic acid, methanesulfonic acid, p-toluenesulfonic acid and the like.
본 발명의 조성물의 유효성분인 망고스틴 추출물, 감마 또는 알파 망고스틴은 간세포에 대한 독성이 매우 낮아 안전성이 높고, C형 간염 바이러스의 RNA 게놈복제 단계에서 게놈 복제를 선택적으로 차단하는 효과가 우수하므로, 내성과 저항성을 나타낼 수 있는 돌연변이 바이러스의 발생을 억제하면서도, 적은 부작용 및 독성을 나타낸다. 따라서, 본 발명의 망고스틴 추출물, 감마 또는 알파 망고스틴은 안전하면서도 HCV에 작용특이성이 높은 C형 간염의 예방 또는 치료용 의약품 또는 건강기능식품으로 유용하게 사용될 수 있다.
The mangosteen extract, gamma or alpha mangosteen, which is an active ingredient of the composition of the present invention, is highly safe due to its low toxicity to hepatocytes and has excellent effect of selectively blocking genome replication at the RNA genome replication stage of hepatitis C virus , Exhibits few side effects and toxicity while inhibiting the generation of mutant viruses, which may show resistance and resistance. Therefore, the mangosteen extract of the present invention, gamma or alpha mangosteen can be useful as a pharmaceutical or health functional food for prevention or treatment of hepatitis C which is safe and highly specific for HCV.
본 발명의 조성물은 유효성분인 망고스틴 추출물, 감마 또는 알파 망고스틴과 함께 C형 간염의 예방 또는 치료 효과를 갖는 공지의 유효성분을 1종 이상 포함할 수 있다.The composition of the present invention may contain at least one known active ingredient having an effect of preventing or treating hepatitis C in combination with mangosteen extract, gamma or alpha mangosteen as an active ingredient.
본 발명의 조성물은, 투여를 위하여 상기 기재한 유효성분 이외에 추가로 약학적으로 허용 가능한 담체를 1종 이상 포함하여 제조할 수 있다. 약학적으로 허용 가능한 담체는 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로오스 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. 더 나아가 당 분야의 적정한 방법으로 또는 Remington's Pharmaceutical Science(최근판), Mack Publishing Company, Easton PA에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다.The composition of the present invention may further comprise at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components. If necessary, an antioxidant, , And other conventional additives such as a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using appropriate methods in the art or by the method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA.
본 발명의 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구 투여(예를 들어, 정맥 내, 피하, 복강 내 또는 국소에 적용)할 수 있으며, 투여량은 개체의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도 등에 따라 그 범위가 다양하다. 상기 망고스틴 추출물의 일일 투여량은 약 0.1~1000㎎/㎏ 이고, 감마 또는 알파 망고스틴의 일일 투여량은 약 0.001~100 mg/kg 이며, 하루 일회 내지 수회에 나누어 투여하는 것이 바람직하다.The composition of the present invention can be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the desired method, and the dose can be determined depending on the body weight, age, sex, , Diet, administration time, method of administration, excretion rate, and severity of the disease. The daily dose of the mangosteen extract is about 0.1 to 1000 mg / kg, and the daily dose of gamma or alpha mangosteen is about 0.001 to 100 mg / kg, preferably administered once a day or several times a day.
본 발명의 조성물은 C형 간염의 예방 또는 치료를 위하여 단독으로, 또는 수술, 호르몬 치료, 약물치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다.
The composition of the present invention can be used alone or in combination with methods for the prevention or treatment of hepatitis C, or using surgery, hormone therapy, drug therapy and biological response modifiers.
본 발명에서, 건강기능식품이란 질병의 예방 또는 개선, 생체방어, 면역, 병후의 회복, 노화 억제 등 생체조절기능을 가지는 식품을 말하는 것으로, 장기적으로 복용하였을 때 인체에 무해하여야 한다. 본 발명의 망고스틴 추출물, 감마 또는 알파 망고스틴은 C형 간염의 예방 또는 개선을 목적으로 건강기능식품에 첨가될 수 있다. 본 발명의 망고스틴 추출물, 감마 또는 알파 망고스틴을 식품 첨가물로 사용할 경우, 상기 망고스틴 추출물, 감마 또는 알파 망고스틴을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 혼합양은 사용 목적 (예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조 시에 본 발명의 망고스틴 추출물, 감마 또는 알파 망고스틴은 원료에 대하여 15중량 % 이하, 바람직하게는 10 중량 % 이하의 양으로 첨가된다. 그러나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다.In the present invention, the health functional food refers to a food having a biological control function such as prevention or improvement of disease, bio-defense, immunity, recovery after disease, suppression of aging, and should be harmless to human body when taken over a long period of time. The mangosteen extract, gamma or alpha mangosteen of the present invention may be added to a health functional food for the purpose of preventing or improving hepatitis C infection. When the mangosteen extract of the present invention, gamma or alpha mangosteen is used as a food additive, the above-mentioned mangosteen extract, gamma or alpha mangosteen can be directly added or used together with other food or food ingredients, Lt; / RTI > The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, the mangosteen extract, gamma or alpha mangosteen of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material, when the food or drink is produced. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.
상기 식품의 종류에는 특별한 제한은 없다. 상기 물질을 첨가할 수 있는 식품의 예로는 육류, 소시지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 수프, 음료수, 차, 드링크제, 알코올 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다.There is no particular limitation on the kind of the food. Examples of foods to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.
본 발명의 건강음료 조성물은 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 포함할 수 있다. 상술한 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드, 말토오스, 수크로오스와 같은 디사카라이드, 및 덱스트린, 사이클로덱스트린과 같은 천연 감미제나, 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 ml 당 일반적으로 약 0.01 내지 10 g, 바람직하게는 약 0.01 내지 0.1 g 이다.The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharine and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g per 100 ml of the composition of the present invention.
상기 외에 본 발명의 조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산 음료에 사용되는 탄산화제 등을 포함할 수 있다. 그 밖에 본 발명의 조성물은 천연 과일주스, 과일주스 음료 및 야채 음료의 제조를 위한 과육을 포함할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 크게 중요하진 않지만 본 발명의 조성물 100 중량부 당 0.01 내지 0.1 중량부의 범위에서 선택되는 것이 일반적이다.
In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, A carbonating agent used in a carbonated beverage, and the like. In addition, the composition of the present invention may comprise flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the ratio of such additives is not critical, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.
Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.
실시예Example 1. 본 발명의 1. The present invention 망고스틴Mangosteen 수 추출물의 제조 Preparation of water extract
먼저 망고스틴을 물로 깨끗이 세척하고 건조한 후 분쇄한다. 분쇄된 망고스틴 100g에 물 500ml를 가하여 2시간 동안 침지하고 초음파 추출법(sonication)을 이용하여 추출하였다. 이후, 망고스틴 추출액을 원심분리하여 분리하고 상층액을 모아서 여과한 후, 여액을 감압 하에 농축한 다음 동결 건조하여 분말 형태의 망고스틴 수 추출물을 얻었다.
First, mangosteen is cleaned with water, dried and then ground. To 100 g of the ground mangosteen was added 500 ml of water and immersed for 2 hours and extracted using sonication. Then, the mangosteen extract was separated by centrifugation, and the supernatant was collected and filtered. The filtrate was concentrated under reduced pressure and then lyophilized to obtain a powdery mangosteen water extract.
실시예Example 2. 본 발명의 2. The present invention 망고스틴Mangosteen 메탄올 추출물의 제조 Preparation of methanol extract
상기 실시예 1에서 물 대신 1~100%의 메탄올을 사용한 것을 제외하고는 실시예 1과 동일하게 하여 망고스틴 메탄올 추출물을 얻었다.
Mangosteen methanol extract was obtained in the same manner as in Example 1, except that 1 to 100% of methanol was used instead of water in Example 1.
실시예 3. 본 발명의 망고스틴 에탄올 추출물의 제조Example 3 Preparation of Mangosteen Ethanol Extract of the Present Invention
상기 실시예 1에서 물 대신 1~100%의 에탄올을 사용한 것을 제외하고는 실시예 1과 동일하게 하여 망고스틴 에탄올 추출물을 얻었다.
The ethanol extract of mangosteen was obtained in the same manner as in Example 1, except that 1 to 100% of ethanol was used instead of water in Example 1.
실시예 4. 본 발명의 망고스틴 추출물로부터 감마 망고스틴의 분리Example 4. Isolation of gamma mangosteen from the mangosteen extract of the present invention
음건한 망고스틴 과피(1.3 kg)를 100% 메탄올로 8 L씩 3회 초음파 추출하여 여과 및 감압농축 뒤 메탄올 추출물 401 g을 얻었다. 이중에서 164 g의 메탄올 추출물을 증류수에 현탁시킨 후 헥산, 클로로포름, 에틸아세테이트, n-부탄올로 용매 분획을 실시하여 감압농축 뒤 클로로포름 분획물(37.9 g)을 얻었다. 망고스틴 클로로포름 분획물(37.9 g)에 대하여 헥산과 에틸아세테이트 혼합용매로 gradient silica gel (593 g) open column chromatography를 진행하여 49 개의 분획물(GMC1 내지 GMC 49)로 나뉘었고, 그 중 35번 분획물(3.36 g)을 클로로포름과 아세톤 혼합용매로 gradient silica gel (120 g) MPLC를 실시하여 23 개 분획물을 얻었다. 이중에서 8번 분획물(GMC35-8, 698 mg)을 메탄올과 증류수로 재결정시키는 방법으로 노란색 분말인 감마-망고스틴을 얻었다.
The shaded mangosteen hulls (1.3 kg) were sonicated three times by 8 L each in 100% methanol, filtered and concentrated under reduced pressure to obtain 401 g of a methanol extract. Of these, 164 g of the methanol extract was suspended in distilled water, followed by solvent fractionation with hexane, chloroform, ethyl acetate and n-butanol, followed by concentration under reduced pressure to obtain a chloroform fraction (37.9 g). Mangosteen chloroform fractions (37.9 g) were fractionated into 49 fractions (GMC1 to GMC 49) by gradient silica gel (593 g) using hexane and ethyl acetate mixed solvent. Fractions 35 (3.36 g) were subjected to gradient silica gel (120 g) MPLC with a mixture of chloroform and acetone to obtain 23 fractions. Among them, the fraction 8 (GMC35-8, 698 mg) was recrystallized from methanol and distilled water to obtain a yellow powder, gamma-mangosteen.
실시예 5. 본 발명의 망고스틴 추출물로부터 알파 망고스틴의 분리Example 5 Isolation of Alpha Mangosteen from the Mangosteen Extract of the Present Invention
음건한 망고스틴 과피 (1.3 kg) 를 100% 메탄올로 8 L씩 3회 초음파 추출하여 여과 및 감압농축 뒤 메탄올 추출물 401 g을 얻었다. 이중에서 164 g 메탄올 추출물을 증류수에 현탁시킨 후 헥산, 클로로포름, 에틸아세테이트, n-부탄올로 용매 분획을 실시하여 감압농축 뒤 클로로포름 분획물(37.9 g)을 얻었다. 망고스틴 클로로포름 분획물(37.9 g)에 대하여 헥산과 에틸아세테이트 혼합용매로 gradient silica gel (593 g) open column chromatography를 진행하여 49 개의 분획물 (GMC1 내지 GMC 49)로 나뉘었고, 24, 25분획물에서 메탄올과 증류수로 재결정시키는 방법으로 알파-망고스틴을 얻었다.
The shaded mangosteen hulls (1.3 kg) were sonicated three times by 8 L each in 100% methanol, filtered and concentrated under reduced pressure to obtain 401 g of a methanol extract. Of these, 164 g of the methanol extract was suspended in distilled water, followed by solvent fractionation with hexane, chloroform, ethyl acetate and n-butanol. After concentration under reduced pressure, chloroform fraction (37.9 g) was obtained. Mangosteen chloroform fraction (37.9 g) was fractionated into 49 fractions (GMC1 to GMC 49) by gradient silica gel (593 g) with hexane and ethyl acetate mixed solvent. Alpha-mangosteen was obtained by recrystallization from distilled water.
실험예Experimental Example 1. 본 발명의 망고스틴 추출물 또는 이로부터 분리한 감마 또는 알파 망고스틴의 간세포 독성 분석 1. Hepatocellular Toxicity Analysis of Mangosteen Extract of the Present Invention or Gamma or Alpha Mangosteen Isolated therefrom
본 발명의 망고스틴 추출물 또는 이로부터 분리한 감마 또는 알파 망고스틴의 간세포 독성 여부를 확인하기 위하여, EZ-Cytox cell viability assay(Daeil Lab Service)를 사용하여 하기와 같은 실험을 수행하였다. 상기 감마, 알파 망고스틴은 동국대학교 약학대학이 소유하고 있는 소분자라이브러리의 화합물을 이용하였다(Sigma Aldrich).
The following experiments were conducted using the EZ-Cytox cell viability assay (Daeil Lab Service) to confirm the toxicity of the mangosteen extract of the present invention or gamma or alpha mangosteen separated therefrom to hepatocytes. The gamma, alpha mangosteen utilized a compound of the small molecule library owned by the College of Pharmacy, Dongguk University (Sigma Aldrich).
1-1. 1-1. 망고스틴Mangosteen 추출물의 간세포 독성 Hepatocellular toxicity of extract
96웰-플레이트에 웰당 1.7X104 내지 2.0X104개의 Huh7.5 인간 간암 세포를 첨가하고 37℃ 배양기에서 24시간 동안 배양하였다. 대조군으로는 DMSO(dimethyl sulfoxide)를, 실험군으로는 여러 농도(2.5~400μg/ml)의 망고스틴 추출물들을 상기 세포에 처리하고 72시간 동안 배양하였다. 상기 배양액을 버리고, PBS로 세척한 후, 수용성 테트라졸리움 염(tetrazolium salt)이 포함된 EZ-Cytox 시약에 세포배양액을 첨가하여 제조한 1/10(v/v) 희석액을 100 μl 첨가하고 3시간 동안 반응시켰다. 반응이 종료된 후 분광광도계를 이용하여 450nm의 파장으로 상기 세포의 흡광도를 계측하였다. DMSO 처리시의 흡광도를 100으로 기준하여, 망고스틴의 메탄올 추출물, 수 추출물 및 에탄올 추출물 처리시의 상대적인 흡광도를 계산하였다. 그 결과를 표 1 및 도 1에 나타내었다. 96 well-per well was added to 1.7X10 4 2.0X10 4 Huh7.5 of human lung cancer cells to the plate and incubated at 37 ℃ incubator for 24 hours. DMSO (dimethyl sulfoxide) was used as a control group and mangosteen extracts at various concentrations (2.5-400 μg / ml) were treated with the cells and cultured for 72 hours. The culture medium was discarded, washed with PBS, and then 100 μl of a 1/10 (v / v) dilution prepared by adding a cell culture medium to a water-soluble tetrazolium salt-containing EZ-Cytox reagent was added, Lt; / RTI > After the reaction was completed, the absorbance of the cells was measured at a wavelength of 450 nm using a spectrophotometer. The relative absorbance of methanol extract, water extract and ethanol extract of mangosteen was calculated based on the absorbance of DMSO treated at 100. The results are shown in Table 1 and Fig.
표 1 및 도 1에 나타낸 바와 같이, 망고스틴의 메탄올 또는 에탄올 추출물은 2.5~10 μg/ml의 농도에서, 망고스틴의 수 추출물은 0~200μg/ml의 범위에서 매우 우수한 세포생존율을 나타내어 간세포에 대한 독성이 거의 없으며, 안전성이 높음을 확인하였다.
As shown in Table 1 and FIG. 1, the methanol or ethanol extract of mangosteen exhibited excellent cell survival rate at a concentration of 2.5-10 μg / ml and the water extract of mangosteen at 0-200 μg / ml, It was confirmed that there was almost no toxicity and safety was high.
1-2. 감마, 알파 1-2. Gamma, alpha 망고스틴의Mangosteen 간세포 독성 Hepatocellular toxicity
감마, 알파 망고스틴의 간세포 독성을 알아보기 위하여 실험물질로 다양한 농도의 감마, 알파 망고스틴 (0.0001 내지 100μM)을 사용한 것을 제외하고는, 상기 실험예 1-1과 동일한 방법으로 실험을 수행하였다. 그 결과를 도 2 및 도 3에 나타내었다. Experiments were carried out in the same manner as in Experimental Example 1-1, except that gamma, alpha mangosteen at various concentrations of gamma, alpha mangosteen (0.0001-100 mu M) was used as an experimental material in order to examine hepatocellular toxicity. The results are shown in Fig. 2 and Fig.
도 2 및 도 3에 나타낸 바와 같이, 알파 망고스틴은 0.0001 내지 5μM의 농도에서, 감마 망고스틴은 0.0001 내지 4μM의 농도에서 매우 우수한 세포 생존율을 나타내어 간세포에 대한 독성이 거의 없으며, 안전성이 높음을 확인하였다.
As shown in FIG. 2 and FIG. 3, alpha mangosteen exhibited excellent cell viability at a concentration of 0.0001 to 5 μM, gamma mangosteen at a concentration of 0.0001 to 4 μM, so that there was almost no toxicity to hepatocytes, Respectively.
실험예Experimental Example 2. 본 발명의 망고스틴 추출물 또는 이로부터 분리한 감마 또는 알파 망고스틴의 C형 간염 바이러스 게놈 복제 저해 활성 측정 2. Measurement of hepatitis C viral genome replication inhibitory activity of the mangosteen extract of the present invention or gamma or alpha mangosteen isolated therefrom
본 발명의 망고스틴 추출물 또는 이로부터 분리한 감마 또는 알파 망고스틴의 C형 간염 바이러스 게놈 복제 저해 활성을 측정하기 위해, 하기와 같은 실험을 수행하였다.
In order to measure the hepatitis C virus genome replication inhibitory activity of the mangosteen extract of the present invention or gamma or alpha mangosteen separated therefrom, the following experiment was conducted.
2-1. C형 간염 바이러스의 게놈복제를 측정하기 위한 리포터 바이러스의 준비2-1. Preparation of reporter viruses to measure genomic replication of hepatitis C virus
C 형 간염 바이러스의 게놈복제를 측정하기 위해 유전형(genotype) 2a에 속하는 FL-J6/JFH-5C19Rluc2AUbi HCV replicon을 리포터바이러스로 사용하였다. 상기 리포터 바이러스의 게놈 구조도는 도 4에 나타내었다. To measure genomic replication of hepatitis C virus, FL-J6 / JFH-5C19Rluc2AUbi HCV replicon belonging to genotype 2a was used as a reporter virus. The genome structure of the reporter virus is shown in Fig.
도 4에 나타낸 바와 같이, 상기 리포터바이러스는 유전형(genotype) 2 에 속하는 C 형 간염 바이러스의 전체 게놈이 cDNA 상태로 포함되어있고, 바이러스의 내부리보솜진입위치(IRES, internal ribosome entry site)와 바이러스의 코어(core) 단백질 사이에 레닐라 루시퍼라제와 스스로 절단하는(self cleaving) 구족병 바이러스(foot and mouth disease virus) 2A 단백질의 Ubi 시퀀스(sequence)가 부착되어있어, 상기 FL-J6/JFH-5C19Rluc2AUbi 플라스미드를 T7 RNA 중합효소(polymerase)를 사용하여 시험관상(in-vitro)에서 전사(transcription)하여 얻은 HCV RNA를 간암세포주인 Huh7.5 세포에 주입(transfection)하면, 주입된 바이러스의 RNA가 내부 리보솜 진입위치(IRES)를 사용한 번역을 통해서 다중단백질(polyprotein)을 생산한다. 이때 생산된 다중단백질 중 레닐라 루시퍼라제는 스스로 잘려나가는 구족병 바이러스 2A 단백질의 Ubi 시퀀스의 도움을 받아 바이러스 비구조 단백질들과 분리되고, 분리된 레닐라 루시퍼라제의 활성을 측정함으로써 간접적으로 간세포내의 바이러스 RNA 게놈복제를 측정할 수 있게 된다.
As shown in FIG. 4, the reporter virus contains the entire genome of the hepatitis C virus belonging to
2-2. 망고스틴 추출물의 C형 간염바이러스 게놈 복제 저해 활성의 측정2-2. Measurement of hepatitis C virus genome replication inhibitory activity of mangosteen extract
Huh7.5 인간 간암 세포를 트립신화하여 PBS 용액으로 1.5X107 개/ml 의 세포 밀도를 갖도록 재현탁시켰다. 총 5μg의 시험관 내(in vitro) 전사된 FL-J6/JFH-5C19Rluc2AUbi RNA를 Huh7.5 간암세포가 포함된 400μl PBS 완충액과 혼합한 후 2-mm-gap cuvette(BTX)에 넣었다. BTX-830 전기천공기로 0.82kV로 99ms 동안 5회 펄스를 가하여 상기 FL-J6/JFH-5 C19Rluc2AUbi RNA를 Huh7.5 인간 간암 세포 내로 트랜스펙션하였다. 상기 전기천공을 실시한지 6시간 경과 후, 대조군으로는 DMSO를, 실험군으로는 여러 농도(2.5~400μg/ml)의 망고스틴 추출물을 각각 상기 세포에 처리하고 72시간 동안 배양하였다. 그 다음, 레닐라 루시퍼라아제 분석법을 시행하였다. Huh7.5 human liver cancer cells were trypsinized and resuspended in PBS solution to a cell density of 1.5X10 < 7 > cells / ml. A total of 5 μg of in vitro transfected FL-J6 / JFH-5C19Rluc2AUbi RNA was mixed with 400 μl PBS buffer containing Huh7.5 liver cancer cells and placed in a 2-mm-gap cuvette (BTX). The FL-J6 / JFH-5 C19Rluc2AUbi RNA was transfected into Huh7.5 human liver cancer cells by applying a pulse five times for 99ms at 0.82kV with a BTX-830 electric perforator. Six hours after the electroporation, the cells were treated with DMSO as a control group and mangosteen extracts at various concentrations (2.5-400 μg / ml) as an experimental group, and cultured for 72 hours. Then, renal luciferase assay was performed.
보다 구체적으로, 상기 물질을 처리한 세포의 배양액을 버리고 PBS로 세포가 부착되어 있는 웰을 세척한 다음, 20μl의 세포용해 완충액(cell lysis buffer)을 넣고 얼음 내에서 20분간 방치하였다. 레닐라 루시퍼라제 기질을 레닐라 루시퍼라제 완충액으로 100배 희석한 희석액을 준비하고, 상기 희석액 100 μl를 각 웰에 넣어주었다. 인테그레이션 타임을 10초로 하여 레닐라 루시퍼라제의 발광도를 측정하였다. DMSO 처리시의 발광도를 100으로 기준하여, 망고스틴 추출물 처리시의 상대적인 발광도를 계산하였다. 그 결과를 도 5 및 도 6에 나타내었다. More specifically, the culture medium of the cells treated with the substance was discarded, and the wells to which the cells were attached were washed with 20 μl of cell lysis buffer, and left in ice for 20 minutes. A diluted solution obtained by diluting a
도 5에 나타낸 바와 같이, 여러가지 망고스틴 추출물 중에서도 망고스틴 에탄올 추출물이 1~10 μg/ml의 농도에서 HCV 게놈의 복제를 저해하는 활성을 가장 우수하게 나타냄을 확인하였다. As shown in FIG. 5, it was confirmed that among the various mangosteen extracts, the mangosteen ethanol extract showed the most excellent activity of inhibiting the replication of the HCV genome at a concentration of 1 to 10 μg / ml.
또한, 도 6에 나타낸 바와 같이, 망고스틴 에탄올 추출물을 10 μg/ml의 농도로 처리한 결과, 시간의 흐름에 따라 세포 생존에는 영향을 미치지 않으면서도, HCV의 게놈 복제를 현저하게 저해함을 확인하였다.
Further, as shown in FIG. 6, when the ethanol extract of mangosteen was treated at a concentration of 10 μg / ml, it was confirmed that the genome replication of HCV was inhibited remarkably without affecting cell viability over time Respectively.
2-3. 망고스틴 추출물을 이용한 정량적 실시간 RT-PCR (qRT-PCR) 분석2-3. Quantitative RT-PCR (qRT-PCR) analysis using mangosteen extract
상기 실험예 2-2에서 우수한 HCV 게놈 복제 저해 활성을 나타낸 망고스틴 에탄올 추출물의 영향을 RNA 수준에서 확인하기 위하여, qRT-PCR을 수행하였다. 보다 구체적으로, J6/JFH RNA-감염된 Huh7.5 세포에 망고스틴 에탄올 추출물을 72시간 동안 처리하였다. 그 후, 전체 세포 RNA(Total cellular RNA)를 생산자의 지시에 따라 RNeasy mini kit (Qiagen)를 사용하여 추출하였다. 추출된 RNA의 수율은 분광광도계를 사용하여 측정하였으며, 표준 발현 유전자인 GAPDH (glyceraldehydes-3-phosphate dehydro-genase) RNA 수치 및 상대적 HCV 수치를 qRT-PCR을 통해 측정하였다. 사용된 프라이머를 하기 표 2에 나타내었으며, 그 결과를 도 7에 나타내었다. QRT-PCR was performed to confirm the effect of the mangosteen ethanol extract showing excellent HCV genome replication inhibitory activity at the RNA level in Experimental Example 2-2. More specifically, J6 / JFH RNA-infected Huh7.5 cells were treated with mangosteen ethanol extract for 72 hours. Total cellular RNA was then extracted using the RNeasy mini kit (Qiagen) according to the manufacturer's instructions. The yield of extracted RNA was measured using a spectrophotometer. GAPDH (glyceraldehydes-3-phosphate dehydrogenase) RNA level and relative HCV level were measured by qRT-PCR. The primers used are shown in Table 2, and the results are shown in Fig.
도 7에 나타낸 바와 같이, 본 발명의 망고스틴 에탄올 추출물이 1 ~10 μg/ml의 농도에서 RT-PCR을 통해서 정량한 HCV 게놈 복제를 저해하는 우수한 활성을 나타냄을 확인하였다.
As shown in FIG. 7, it was confirmed that the mangosteen ethanol extract of the present invention exhibited excellent activity inhibiting HCV genome replication quantified by RT-PCR at a concentration of 1 to 10 μg / ml.
2-4. 망고스틴 추출물을 이용한 웨스턴블롯 분석2-4. Western blot analysis using mangosteen extract
상기 실험예 2-2에서 우수한 HCV 게놈 복제 저해 활성을 나타낸 망고스틴 에탄올 추출물의 영향을 단백질 수준에서 확인하기 위하여, 웨스턴블롯을 수행하였다. Western blotting was performed to confirm the effect of the mangosteen ethanol extract showing excellent HCV genome replication inhibitory activity in Experimental Example 2-2 at the protein level.
보다 구체적으로, Bart79I RNA-감염된 Huh7.5 세포에 망고스틴 에탄올 추출물을 72시간 동안 처리하고, 상기 처리된 세포에서 전체 단백질을 분리하였다. 이를 SDS-PAGE로 분리한 후, 멤브레인으로 옮겨서 바이러스 NS5A 항체로 바이러스의 단백질 발현을 비교하였다. 그 결과를 도 8에 나타내었다. More specifically, Bart79I RNA-infected Huh7.5 cells were treated with the mangosteen ethanol extract for 72 hours and the whole protein was isolated from the treated cells. It was separated by SDS-PAGE, transferred to the membrane, and the protein expression of the virus was compared with the virus NS5A antibody. The results are shown in Fig.
도 8에 나타낸 바와 같이, 본 발명의 망고스틴 에탄올 추출물의 처리에 따라 바이러스의 단백질 발현이 감소함을 확인하였다.
As shown in FIG. 8, it was confirmed that the protein expression of virus was decreased by treatment with the mangosteen ethanol extract of the present invention.
2-5. 망고스틴 추출물을 이용한 면역형광 분석2-5. Immunofluorescence analysis using mangosteen extract
상기 실험예 2-2에서 우수한 HCV 게놈 복제 저해 활성을 나타낸 망고스틴 에탄올 추출물의 영향을 단백질 수준에서 확인하기 위하여, 면역형광 분석을 수행하였다. Immunofluorescence analysis was performed to confirm the influence of the ethanol extract of mangosteen showing excellent HCV genome replication inhibitory activity in Experimental Example 2-2 at the protein level.
보다 구체적으로, Bart79I RNA-감염된 Huh7.5 세포에 망고스틴 에탄올 추출물을 72시간 동안 처리하고, 상기 처리된 세포를 아세트알데히드로 고정한 후, 바이러스 NS5A 항체로 면역염색하였다. 그 결과를 도 9에 나타내었다. More specifically, the Bart79I RNA-infected Huh7.5 cells were treated with mangosteen ethanol extract for 72 hours, the treated cells were fixed with acetaldehyde, and immunostained with a virus NS5A antibody. The results are shown in Fig.
도 9에 나타낸 바와 같이, 본 발명의 망고스틴 에탄올 추출물의 처리에 따라 바이러스의 단백질 발현이 감소함을 확인하였다.
As shown in FIG. 9, it was confirmed that the protein expression of virus was decreased by treatment with the mangosteen ethanol extract of the present invention.
2-6. 망고스틴 추출물의 C형 간염바이러스 단백질 발현 저해 활성의 측정2-6. Measurement of inhibitory activity of hepatitis C virus protein expression of mangosteen extract
상기 실험예에서 확인한 망고스틴 추출물의 C형 간염 바이러스 게놈 복제 저해 활성이 바이러스의 단백질 발현 저해와 관계되는지를 확인하기 위하여, 유전형 1b C형 간염 바이러스의 서브지노믹 레플리콘인 Bart79I를 사용하여 망고스틴 에탄올 추출물 투여시 C형 간염 바이러스의 단백질 발현 정도를 확인하였다. 이때 바이러스의 게놈에서 발현되는 GFP (녹색형광단백질)의 형광의 양을 FACS (형광세포분석기)를 통해 측정하였다. 그 결과를 도 10에 나타내었다. In order to confirm whether the inhibitory activity of the hepatitis C virus genome of the mangosteen extract against the inhibition of the protein expression of the virus was confirmed by the use of Bart79I, a subgenomic replicon of the
도 10에 나타낸 바와 같이, 본 발명의 망고스틴 에탄올 추출물을 50 ng ~ 1 ug/ml까지 투여했을 경우, 간세포에서 발현하는 GFP 형광이 유의적으로 감소하는 것을 확인하였다. 따라서, 본 발명의 망고스틴 에탄올 추출물 투여시 바이러스의 게놈 복제 저해를 통한 바이러스 단백질 발현이 저해됨을 확인하였다.
As shown in FIG. 10, when the mangosteen ethanol extract of the present invention was administered at 50 ng to 1 ug / ml, it was confirmed that GFP fluorescence expressed in hepatocytes was significantly decreased. Therefore, it was confirmed that the virus protein expression was inhibited by the inhibition of genome replication of the virus when the mangosteen ethanol extract of the present invention was administered.
2-7. 감마 또는 알파 망고스틴의 C형 간염바이러스 게놈 복제 저해활성의 측정2-7. Gamma or alpha mangosteen to measure hepatitis C viral genome replication inhibitory activity
감마 또는 알파 망고스틴의 C형 간염바이러스 게놈 복제 저해활성을 측정하기 위하여 실험물질로 다양한 농도의 감마 또는 알파 망고스틴을 사용한 건을 제외하고는, 상기 실험예 2-2와 동일한 방법으로 실험을 수행하였다. 그 결과를 도 2 및 도 3에 나타내었다 (붉은선). Experiments were carried out in the same manner as in Experimental Example 2-2, except that gamma or alpha mangosteen was used as an experimental substance in order to measure the hepatitis C virus genome replication inhibitory activity of gamma or alpha mangosteen Respectively. The results are shown in Fig. 2 and Fig. 3 (red line).
도 2 및 도 3에 나타낸 바와 같이, 본 발명의 알파 또는 감마 망고스틴은 1~10 μM의 농도에서 HCV 게놈의 복제를 저해하는 활성을 우수하게 나타내어 HCV의 증식을 저해하는 효과가 우수함을 확인하였다.
As shown in FIG. 2 and FIG. 3, the alpha or gamma mangosteen of the present invention exhibited excellent activity of inhibiting the replication of the HCV genome at a concentration of 1 to 10 μM, thereby confirming the excellent effect of inhibiting the proliferation of HCV .
2-8. 감마 망고스틴을 이용한 정량적 실시간 RT-PCR (qRT-PCR) 분석2-8. Quantitative real-time RT-PCR (qRT-PCR) analysis using gamma-mangosteen
감마 망고스틴의 C형 간염바이러스 게놈 복제 저해활성을 측정하기 위하여 실험물질로 다양한 농도의 감마 또는 알파 망고스틴을 사용한 건을 제외하고는, 상기 실험예 2-3과 동일한 방법으로 실험을 수행하였다. 그 결과를 도 12에 나타내었다 (붉은선). Experiments were carried out in the same manner as in Experimental Example 2-3, except that gamma or alpha mangosteen was used as an experimental material in order to measure the hepatitis C viral genome replication inhibitory activity of gamma mangosteen. The results are shown in Fig. 12 (red line).
도 11에 나타낸 바와 같이, 본 발명의 감마 망고스틴이 1 ~5 μM의 농도에서 RT-PCR을 통해서 정량한 HCV 게놈의 복제를 저해하는 우수한 활성을 나타냄을 확인하였다.
As shown in FIG. 11, it was confirmed that the gamma-mangosteen of the present invention exhibited excellent activity inhibiting the replication of HCV genome quantitated by RT-PCR at a concentration of 1 to 5 μM.
2-9. 감마 망고스틴을 이용한 웨스턴블롯 분석2-9. Western blot analysis using gamma-mangosteen
감마 망고스틴의 C형 간염바이러스 게놈 복제 저해활성을 측정하기 위하여 실험물질로 다양한 농도의 감마 또는 알파 망고스틴을 사용한 건을 제외하고는, 상기 실험예 2-4와 동일한 방법으로 실험을 수행하였다. 그 결과를 도 13에 나타내었다 (붉은선). Experiments were carried out in the same manner as in Experimental Example 2-4, except that gamma or alpha mangosteen was used as an experimental material in order to measure the hepatitis C viral genome replication inhibitory activity of gamma mangosteen. The results are shown in Fig. 13 (red line).
도 12에 나타낸 바와 같이, 본 발명의 감마 망고스틴의 경우 농도가 증가함에 따라 바이러스 단백질의 양이 줄어드는 것을 확인하였다.
As shown in FIG. 12, in the case of the gamma-mangosteen of the present invention, it was confirmed that the amount of the viral protein decreased as the concentration increased.
하기에 본 발명의 조성물을 제조하기 위한 제제예를 예시한다.
Formulation examples for preparing the composition of the present invention are illustrated below.
제제예Formulation example . 약학적 제제의 제조. Preparation of pharmaceutical preparations
1. 산제의 제조1. Manufacturing of powder
망고스틴 추출물/감마, 알파 망고스틴 100mgMangosteen extract / gamma, alpha mangosteen 100mg
유당 200mgLactose 200mg
상기의 성분을 혼합하고 기밀포에 충진하여 산제를 제조하였다.
The above components were mixed and packed in airtight bags to prepare powders.
2. 정제의 제조 2. Preparation of tablets
망고스틴 추출물/감마, 알파 망고스틴 100㎎Mangosteen extract / gamma, alpha mangosteen 100mg
옥수수전분 100㎎100 mg of corn starch
유 당 100㎎100 mg of milk
스테아린산 마그네슘 2㎎2 mg of magnesium stearate
상기의 성분을 혼합한 후, 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조하였다.
After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.
3. 캡슐제의 제조3. Preparation of capsules
망고스틴 추출물/감마, 알파 망고스틴 100㎎Mangosteen extract / gamma, alpha mangosteen 100mg
옥수수전분 100㎎100 mg of corn starch
유 당 100㎎100 mg of milk
스테아린산 마그네슘 2㎎2 mg of magnesium stearate
상기의 성분을 혼합한 후, 통상의 캡슐제의 제조방법에 따라서 젤라틴 캡슐에 충전하여 캡슐제를 제조하였다.
After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.
제제예 2. 식품 조성물의 제조Formulation Example 2. Preparation of Food Composition
2-1. 건강식품의 제조2-1. Manufacture of health food
망고스틴 추출물/감마, 알파 망고스틴 100 mgMangosteen extract / gamma,
비타민 혼합물 적량Vitamin mixture quantity
비타민 A 아세테이트 70 μg Vitamin A
비타민 E 1.0 mgVitamin E 1.0 mg
비타민 B1 0.13 mgVitamin B1 0.13 mg
비타민 B2 0.15 mg0.15 mg of vitamin B2
비타민 B6 0.5 mgVitamin B6 0.5 mg
비타민 B12 0.2 μg Vitamin B12 0.2 μg
비타민 C 10 mg
비오틴 10 μg Biotin 10 μg
니코틴산아미드 1.7 mgNicotinic acid amide 1.7 mg
엽산 50 μg
판토텐산 칼슘 0.5 mgCalcium pantothenate 0.5 mg
무기질 혼합물 적량Mineral mixture quantity
황산제1철 1.75 mg1.75 mg of ferrous sulfate
산화아연 0.82 mg0.82 mg of zinc oxide
탄산마그네슘 25.3 mgMagnesium carbonate 25.3 mg
제1인산칼륨 15 mgPotassium monophosphate 15 mg
제2인산칼슘 55 mgSecondary calcium phosphate 55 mg
구연산칼륨 90 mg
탄산칼슘 100 mg
염화마그네슘 24.8 mgMagnesium chloride 24.8 mg
상기의 비타민 및 미네랄 혼합물의 조성비는 비교적 건강식품에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만, 그 배합비를 임의로 변형 실시하여도 무방하며, 통상의 건강식품 제조방법에 따라 상기의 성분을 혼합한 다음, 과립을 제조하고, 통상의 방법에 따라 건강식품 조성물 제조에 사용할 수 있다.
Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.
2-2. 건강음료의 제조2-2. Manufacture of health drinks
망고스틴 추출물/감마, 알파 망고스틴 100 mgMangosteen extract / gamma,
비타민 C 15 gVitamin C 15 g
비타민 E(분말) 100 gVitamin E (powder) 100 g
젖산철 19.75 g19.75 g of ferrous lactate
산화아연 3.5 g3.5 g of zinc oxide
니코틴산아미드 3.5 gNicotinic acid amide 3.5 g
비타민 A 0.2 gVitamin A 0.2 g
비타민 B1 0.25 gVitamin B1 0.25 g
비타민 B2 0.3 gVitamin B2 0.3 g
물 정량Water quantification
통상의 건강음료 제조방법에 따라 상기의 성분을 혼합한 다음, 약 1시간 동안 85 ℃에서 교반 가열한 후, 만들어진 용액을 여과하여 멸균된 2 l 용기에 취득하여 밀봉 멸균한 뒤 냉장 보관한 다음 본 발명의 건강음료 조성물 제조에 사용한다.The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 DEG C for about 1 hour. The solution thus prepared was filtered and sterilized in a sterilized 2 liter container, It is used in the production of the health beverage composition of the invention.
상기 조성비는 비교적 기호음료에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만 수요계층이나, 수요국가, 사용용도 등 지역적, 민족적 기호도에 따라서 그 배합비를 임의로 변형 실시하여도 무방하다.Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.
Claims (11)
A pharmaceutical composition for preventing or treating hepatitis C comprising mangosteen ethanol extract as an active ingredient.
The pharmaceutical composition for preventing or treating hepatitis C virus according to claim 1, wherein the ethanol extract of mangosteen has selective inhibitory activity of RNA genome replication of hepatitis C virus.
A food composition for preventing or ameliorating hepatitis C comprising mangosteen ethanol extract as an active ingredient.
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| KR20020011856A (en) * | 2000-08-02 | 2002-02-09 | 시게미 다께오 | Cyclooxygenase Inhibitor, and Food and Beverage containg the Same |
| KR100653948B1 (en) | 2004-04-08 | 2007-04-16 | 롯데제과주식회사 | Ikappa;B KINASE INHIBITOR AND Ikappa;B KINASE INHIBITING COMPOSITION COMPRISING THEREOF |
| KR20110122158A (en) * | 2009-02-02 | 2011-11-09 | 라일라 뉴트라슈티칼스 | Composition from sphaeranthus indicus and garcinia mangostana for the control of metabolic syndrome |
| TW201225953A (en) * | 2010-12-22 | 2012-07-01 | Ind Tech Res Inst | Pharmaceutical composition and health food for liver fibrosis, cirrhosis and hepatitis |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20020011856A (en) * | 2000-08-02 | 2002-02-09 | 시게미 다께오 | Cyclooxygenase Inhibitor, and Food and Beverage containg the Same |
| KR100653948B1 (en) | 2004-04-08 | 2007-04-16 | 롯데제과주식회사 | Ikappa;B KINASE INHIBITOR AND Ikappa;B KINASE INHIBITING COMPOSITION COMPRISING THEREOF |
| KR20110122158A (en) * | 2009-02-02 | 2011-11-09 | 라일라 뉴트라슈티칼스 | Composition from sphaeranthus indicus and garcinia mangostana for the control of metabolic syndrome |
| TW201225953A (en) * | 2010-12-22 | 2012-07-01 | Ind Tech Res Inst | Pharmaceutical composition and health food for liver fibrosis, cirrhosis and hepatitis |
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