KR20210007266A - Endophytic Fungus Neosartorya fischeri JS-0553, Extracts Thereof, Fischerin Compound Isolated Therefrom, and Neuroprotective Composition Comprising the Same - Google Patents
Endophytic Fungus Neosartorya fischeri JS-0553, Extracts Thereof, Fischerin Compound Isolated Therefrom, and Neuroprotective Composition Comprising the Same Download PDFInfo
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- KR20210007266A KR20210007266A KR1020190083408A KR20190083408A KR20210007266A KR 20210007266 A KR20210007266 A KR 20210007266A KR 1020190083408 A KR1020190083408 A KR 1020190083408A KR 20190083408 A KR20190083408 A KR 20190083408A KR 20210007266 A KR20210007266 A KR 20210007266A
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Abstract
Description
본 발명은 식물내생균 네오사르토리야 피쳐리 JS-0553, 상기 균주의 추출물, 이로부터 분리된 피쳐린 화합물, 및 이를 함유하는 신경보호용 조성물에 관한 것이다.The present invention relates to a plant endophyte neosartoriya fetiri JS-0553, an extract of the strain, a featurelin compound isolated therefrom, and a composition for neuroprotection containing the same.
글루타메이트(Glutamate)는 중추신경계(CNS)에서 주요 흥분성 신경전달물질(excitatory neuro-transmitter)로 작용하며 대사성 수용기(metabotropic receptor)와 이온성 수용기들(ionotropic receptor, NMDA-, quisqualate-, kainate-type)과 결합하여 여러 가지 신경 생리학적 효과를 나타낸다. 신경세포 외로 방출된 글루타메이트는 시냅스 전말단(presynaptic terminal)으로 직접 재흡수되거나 성상세포(astrocytes) 내로 흡수된 후, 글루타민 합성효소(glutamine synthase)에 의해 글루타민(glutamine)으로 전환되어 시냅스 전 말단으로 이동하게 된다. Glutamate acts as a major excitatory neuro-transmitter in the central nervous system (CNS), metabotropic receptors, and ionic receptors (ionotropic receptors, NMDA-, quisqualate-, kainate-type). Combined with, it exhibits several neurophysiological effects. Glutamate released to the outside of nerve cells is directly reabsorbed into the presynaptic terminal or absorbed into astrocytes, and then converted into glutamine by glutamine synthase and transferred to the presynaptic terminal. Is done.
이러한 시냅스 전 말단과 성상세포의 작용으로 세포 외부의 글루타메이트 농도는 0.3μM의 저농도로 유지되게 된다. 그러나, 뇌허혈(ischemia), 저산소증(hypoxia), 발작(seizure), 저혈당증(hypoglycemia), 뇌외상(trauma) 등의 병변이 일어나면 글루타메이트 수송에 필요한 에너지 공급이 부족하게 되고, 세포 외 글루타메이트 농도는 정상상태의 10,000배인 3mM 정도로 증가하게 되며, 이로 인해 중추신경 세포가 사멸하게 된다. 이러한 글루타메이트 농도 증가에 의한 신경세포사멸은 글루타메이트로 유도된(glutamate-induced) 신경독성(neurotoxicity)으로 불리고 있으며, 세포 내의 자유 라디칼(free radical)의 발생에 의한 산화 스트레스(oxidative stress)와 매우 밀접한 관계가 있다. 따라서, 글루타메이트 독성에 의한 신경세포의 사멸이 세포 내에 과량으로 발생한 자유 라디칼에 의한 지질, 당, 핵산, 단백질 등의 세포 구성성분의 파괴에 의한 것임이 밝혀짐으로써 이를 억제할 수 있는 자유 라디칼 제거제(free radical scavenger)와 같은 지질 과산화 저해물질은 뇌세포를 보호할 수 있는 새로운 치료 소재로서 매우 중요한 의미가 있다. The concentration of glutamate outside the cell is maintained at a low concentration of 0.3 μM by the action of the pre-synaptic terminal and astrocytes. However, when lesions such as cerebral ischemia, hypoxia, seizure, hypoglycemia, and brain trauma occur, the supply of energy necessary for glutamate transport is insufficient, and the extracellular glutamate concentration is in a normal state. It increases to about 3mM, which is 10,000 times as much as that, resulting in the death of central nerve cells. Neuronal cell death caused by the increase in glutamate concentration is called glutamate-induced neurotoxicity, and is closely related to oxidative stress caused by the generation of free radicals in cells. There is. Therefore, it was found that the death of neurons due to glutamate toxicity was caused by the destruction of cellular components such as lipids, sugars, nucleic acids, proteins, etc. by excessive free radicals generated in the cell, and thus a free radical scavenger ( Lipid peroxidation inhibitors such as free radical scavenger) have very important significance as a new therapeutic material that can protect brain cells.
글루타메이트의 세포 외 농도 증가는 뇌허혈, 뇌졸중, 뇌외상, 저산소증, 저혈당증, 발작 등의 급성질환뿐만 아니라, 헌팅턴병(Huntington's disease, HD), 파킨슨병(Parkinson's disease, PD), 근위축성 측색경화증(amyotrophic lateral sclerosis, ALS), 알츠하이머병(Alzheimer's disease, AD)과 같은 만성질환에도 관여하는 것으로 알려져 있다. 미국 국립보건원(NIH)의 조사 결과에 의하면 급성질환의 하나인 뇌졸중(stroke)은 미국인의 세 번째 사망원인이며, 성인 불구의 가장 큰 원인이 되고 있다. 매년 50만 명의 환자가 발생하며, 이 중 30%는 사망하고 20~30%는 영구 장애인이 되고 나머지는 신체 불구, 신체 마비, 시력감퇴, 기억상실 등의 고통을 받고 있다. 또한, 우리나라에서도 암에 이은 사망원인 2위의 중요한 질병으로 대두되어, 이러한 질병에 대한 치료제가 강력히 요구되고 있다. Increased extracellular concentrations of glutamate include acute diseases such as cerebral ischemia, stroke, brain trauma, hypoxia, hypoglycemia, and seizures, as well as Huntington's disease (HD), Parkinson's disease (PD), amyotrophic lateral sclerosis. It is also known to be involved in chronic diseases such as sclerosis (ALS) and Alzheimer's disease (AD). According to the National Institutes of Health (NIH) survey, stroke, one of the acute diseases, is the third cause of death in Americans, and the leading cause of adult disability. 500,000 patients occur every year, of which 30% die, 20-30% become permanently disabled, and the rest suffer from physical disability, paralysis, vision loss, and memory loss. In addition, it has emerged as the second most important disease that causes death following cancer in Korea, and treatments for these diseases are strongly required.
글루타메이트는 이온성 수용기(ionotropic receptor), 대사성 수용기 (metabotropic receptor)의 두 가지 수용기(receptor)와 결합한다. 이온성 수용기는 이온 채널(ion channel)과 연관되어 있으며, 신경세포에 독립적으로 작용하여 흥분독성 손상(excitotoxic damage)을 주게 된다. 반면, 대사성 수용기의 경우는 이온성 수용기와는 달리 글루타메이트가 직접적인 신경세포사멸을 일으키지는 않는다. Glutamate binds to two receptors: an ionotropic receptor and a metabotropic receptor. Ionic receptors are associated with ion channels and act independently on nerve cells to inflict excitotoxic damage. On the other hand, in the case of metabolic receptors, glutamate does not cause direct neuronal cell death unlike ionic receptors.
이온성 수용기에 글루타메이트가 결합하여 일으키는 신경세포사멸은 Na+ 및 Cl- 이온 의존성과 Ca2 + 이온 의존성의 두 가지로 나눌 수 있다. Na+ 및 Cl- 이온 의존성 신경세포사멸은 글루타메이트에 노출된 후, 수 분 안에 일어나며 감극 물질(depolarizing agent)을 처리한 것과 유사한 신경세포사멸을 보인다. 글루타메이트가 쿼스퀄산 수용기(quisqualate receptor)를 자극하여 Na+ 이온이 세포 내로 과량 유입되며, Na+ 이온의 유입과 함께 Cl- 이온과 물(H2O) 입자가 세포 내부로 들어와 세포가 팽창(swelling)하여 결국 용해(lysis)하게 된다. Neuronal cell death caused by the binding of glutamate to the ionic receptor is Na + and It can be divided into two types: Cl - ion dependence and Ca 2 + ion dependence. Na + and Cl - ion-dependent neuronal death occurs within minutes after exposure to glutamate and shows neuronal death similar to that treated with a depolarizing agent. Glutamate stimulates the quisqualate receptor, causing excessive Na + ions to enter the cell, and with the influx of Na + ions, Cl - ions and water (H 2 O) particles enter the cell, causing the cell to expand ( swelling) and eventually lysis.
Ca2 + 이온 의존성 신경세포사멸은 딜레이드 신경 손상(delayed neuronal damage)으로 글루타메이트 처리 후 수 시간이 지나서 일어나며, Ca2 + 이온 투과담체(ionophore)인 A23187을 처리하였을 때와 유사하다. Na+ 이온의 유입은 세포막의 탈분극(depolarization)을 일으키며, 이로 인해 전압 민감성 있는 Ca2 + 채널(voltage sensitive Ca2 + channel, VSCC)과 Mg2 + 이온에 의해서 닫혀 있던 NMDA 수용체 Ca2 + 채널이 열리게 된다. 이로 인해 다량의 Ca2 + 이온이 세포 내로 유입되게 된다. 이렇게 유입된 Ca2 + 이온은 포스포리파아제(phospholipase A2, PLA2), 산화질소 합성효소(nitric oxide synthetase, NOS), 프로테아제(protease), 엔도뉴클레아제(endonuclease) 등의 Ca2 + 의존성 효소들을 활성화한다. 특히 PLA2 포스포리파아제 효소는 인지질(phospholipid)을 분해하여 아라키돈산(arachidonic acid)을 생성시키며, 아라키돈산의 대사과정 중에 과산화물(superoxide)과 같은 자유 라디칼이 발생하게 된다. Ca 2 + ion-dependent neuronal death is delayed neuronal damage, which occurs several hours after glutamate treatment, and is similar to that of A23187, a Ca 2 + ionophore. Influx of Na + ions causes a depolarization (depolarization) of the cell membrane, which is because of the voltage-sensitive Ca 2 + channels (voltage sensitive Ca 2 + channel, VSCC) and NMDA receptor Ca 2 + channel which is closed by a Mg 2 + ions Will be opened. As a result, a large amount of Ca 2 + ions are introduced into the cell. The Ca 2 + ions introduced in this way contain Ca 2 + dependent enzymes such as phospholipase (phospholipase A2, PLA2), nitric oxide synthetase (NOS), protease, and endonuclease. Activate. In particular, PLA2 phospholipase enzyme decomposes phospholipids to produce arachidonic acid, and free radicals such as superoxides are generated during the metabolism of arachidonic acid.
또한, 아라키돈산과 과산화물은 시냅스전 뉴런(presynaptic neuron)에서의 글루타메이트 분비를 촉진시키고, 세포 외 글루타메이트 농도를 더욱 증가시킨다. 크산틴산화효소(Xanthine oxidase)의 작용으로 과산화물들이 생성되며, NOS 효소의 작용으로 생성된 산화질소(nitric oxide)는 과산화물과 반응하여 페록시나이트라이트(peroxynitrite)를 거쳐 반응성이 큰 히드록실라디칼(hydroxyl radical)이 생성된다. 즉, PLA2, NOS, 크산틴산화효소(xanthine oxidase) 등의 효소작용 결과 세포 내에 자유 라디칼이 과다하게 생성되고 이로 인한 산화 스트레스(oxidative stress)로 인하여 DNA, 단백질(protein), 지질(lipid) 등이 비선택적으로 파괴되어 신경세포가 죽게 되며, 또한 엔도뉴클레아제(endonuclease) 효소의 작용으로 세포자멸사(apoptosis)가 일어나기도 한다. In addition, arachidonic acid and peroxide promote glutamate secretion in presynaptic neurons and further increase the extracellular glutamate concentration. Peroxides are produced by the action of xanthine oxidase, and the nitric oxide produced by the action of the NOS enzyme reacts with the peroxide and passes through peroxynitrite, which is highly reactive hydroxyl radical. hydroxyl radical) is produced. In other words, as a result of enzyme action such as PLA2, NOS, and xanthine oxidase, free radicals are generated excessively in cells, and due to oxidative stress, DNA, protein, lipid, etc. This non-selective destruction causes nerve cells to die, and also, apoptosis occurs due to the action of endonuclease enzymes.
이상과 같이 글루타메이트가 뇌졸증(stroke), 뇌외상(trauma) 등과 같은 급성 뇌신경 질환의 원인이 되며, 또한 헌팅턴병, 파킨슨병, 알츠하이머병과 같은 만성 뇌질환에서도 글루타메이트 독성에 의한 산화적 스트레스가 그 원인 중 하나라고 알려져 있다. 이에 따라 글루타메이트 독성에 의한 산화적 스트레스를 억제 또는 완화시킬 수 있는 지질과산화 억제물질과 같은 항산화 활성물질은 급성, 만성의 뇌신경 질환의 치료제로 기대된다. As described above, glutamate is the cause of acute neurological diseases such as stroke and trauma, and also in chronic brain diseases such as Huntington's disease, Parkinson's disease and Alzheimer's, oxidative stress caused by glutamate toxicity is one of the causes. It is known as. Accordingly, antioxidant active substances such as lipid peroxidation inhibitors capable of suppressing or alleviating oxidative stress caused by glutamate toxicity are expected as therapeutics for acute and chronic neurological diseases.
글루타메이트에 의해서 야기되는 뇌졸중이 세계적으로 심각한 질병으로 대두되면서 이에 대한 치료제 개발이 활발히 진행되고 있다. 특히, 활성산소가 글루타메이트 독성에 의한 신경세포사멸의 주된 최종 원인 물질이기 때문에 새로운 자유라디칼 제거제 또는 지질과산화 억제제와 같은 항산화 물질에 대한 연구가 활발히 진행되고 있다. As stroke caused by glutamate has emerged as a serious disease worldwide, the development of therapeutic agents for this is actively progressing. In particular, research on antioxidants such as new free radical scavengers or lipid peroxidation inhibitors has been actively conducted because reactive oxygen species is the main final causative agent of neuronal cell death due to glutamate toxicity.
한편, 갯방풍(Glehnia littoralis)은 해방풍, 빈방풍, 해사삼 등으로 불리우며, 쌍떡잎식물 산형목 산형과에 속하는 다년생의 초본으로 우리나라를 비롯하여 남태평양 지역의 해안지역이나 절벽의 바위틈에 서식한다. 전체에 흰색 털이 나고 뿌리는 모래 속에 깊이 뻗으며 높이는 약 20cm 정도이다. 잎은 두껍고 윤이 나며 끝이 뭉뚝하고, 줄기는 낮고 매우 짧으며 꽃은 흰색으로 6월 내지 8월에 피고 복산형 꽃차례로 줄기 끝에 난다. 갯방풍의 퓨라노코우마린(furanocoumarin) 계열 성분들은 항종양, 항균, 항류마티스 등의 효능이 있는 것으로 보고되고 있다.On the other hand, Glehnia littoralis is a perennial herb belonging to the dicotyledonous umbel family, and it is called liberal wind, binbangpung, and maritimesam, and it lives in the coastal areas of the South Pacific region including Korea or in the rocks of cliffs. White hairs appear on the whole, and the roots extend deep in the sand, and the height is about 20cm. The leaves are thick and shiny, the ends are blunt, the stems are low and very short, and the flowers are white and bloom from June to August, and bloom at the end of the stem in a double-lobed inflorescence. The furanocoumarin-based ingredients of Gaetbangpung are reported to have antitumor, antibacterial, and antirheumatic effects.
다른 한편, 갯방풍의 뿌리 부분은 뇌졸중의 치료제로 사용되어왔다. 갯방풍의 식물 화학에 관한 이전 보고서에 따르면, 퓨라노코우마린(furanocoumarin), 폴리아세틸렌(polyacetylene), 플라보노이드(flavonoid), 리그난(lignan), 모노테르페노이드 글리코시드(monoterpenoid glycoside) 및 에센셜 오일(essential oil)과 같은 다양한 생체 활성 화합물이 포함되어 있음이 밝혀진 바 있다.On the other hand, the root part of Gaetbangpung has been used as a treatment for stroke. According to a previous report on the phytochemicals of the phytochemicals of the canopy, furanocoumarin, polyacetylene, flavonoids, lignans, monoterpenoid glycosides and essential oils ( It has been found to contain various bioactive compounds such as essential oil).
그럼에도 불구하고, 신경보호 효과를 나타내는 생체 활성 물질이 갯방풍 자체에서 확인되지 않았으므로 본 발명자들은 갯방풍에 존재하는 식물내생균에 주목하게 되었다. Nevertheless, since a bioactive substance exhibiting a neuroprotective effect was not identified in Gaetbangpung itself, the present inventors paid attention to plant endophytes present in Gaetbangpung.
식물과 관련된 미생물은 흔히 그들의 생애주기에서 기주 식물의 조직 내 및/또는 세포 내로 콜로니를 형성하나 식물체에 부정적인 영향을 미치지 않는 미생물로 정의되는 식물내생균(endophytes)으로 불린다. 식물내생균은 기주 식물과 상호 작용하여 기주식물을 병원성 침입으로부터 보호하고 기주식물로부터 영양을 얻는 것으로 알려져 있다. 또한, 식물내생균은 의학, 농업 등의 산업 분야에서 잠재적으로 사용될 수 있는 생리활성물질 또는 새로운 화합물의 큰 원천이 될 수 있는 것으로 여겨져 왔다.Plant-related microorganisms are often referred to as endophytes, defined as microorganisms that form colonies within the tissues and/or cells of the host plant in their life cycle but do not negatively affect the plant body. Plant endophytes are known to interact with host plants to protect host plants from pathogenic invasion and to obtain nutrition from host plants. In addition, it has been believed that plant endophytes can be a large source of bioactive substances or new compounds that can potentially be used in industrial fields such as medicine and agriculture.
네오사르토리야 피쳐리(Neosartorya fischeri) 균주는 갯방풍(G. littoralis)에서 분리된 식물내생균으로, 네오사르토리야 피쳐리 균주와 신경보호 활성과의 상관관계는 아직까지 알려진 바 없다. Neosartorya fischeri strain is an endogenous plant isolated from G. littoralis, and the correlation between Neosartorya fischeri strain and neuroprotective activity has not been known yet.
상기와 같은 배경하에서, 본 발명자들은 갯방풍에서 분리된 식물내생균인 네오사르토리야 피쳐리(Neosartorya fischeri) 균주의 배양액과 이로부터 분리된 피쳐린(Fischerin) 화합물이 신경세포에 독성이 없고, 글루타메이트 농도 증가에 따른 신경세포사멸을 방지하며, 매우 우수한 신경보호 활성이 있음을 확인하고, 본 발명을 완성하게 되었다.Under the above background, the present inventors believe that the culture medium of the neosartorya fischeri strain, which is a plant endoprobiotic isolated from Gaetbangpung, and the Fischerin compound isolated therefrom are not toxic to neurons, It prevents neuronal cell death due to an increase in glutamate concentration, confirms that it has very excellent neuroprotective activity, and completes the present invention.
따라서, 본 발명의 주된 목적은 글루타메이트 농도 증가에 따른 신경세포의 사멸을 방지하고, 매우 우수한 신경보호 활성이 있는 네오사르토리야 피쳐리(Neosartorya fischeri) 균주의 배양액과 이로부터 분리된 피쳐린(Fischerin) 화합물을 유효성분으로 하는 신경보호용 조성물을 제공하는 데 있다.Therefore, the main object of the present invention to prevent neuronal death due to glutamate concentration increase, and very excellent neuroprotective activity neo Sartori's feature Li (Neosartorya in fischeri ) to provide a composition for neuroprotection comprising a culture medium of a strain and a Fischerin compound isolated therefrom as an active ingredient.
본 발명의 다른 목적은 네오사르토리야 피쳐리(Neosartorya fischeri) 균주를 배양하여 신경보호 활성이 있는 피쳐린(Fischerin) 화합물을 생산하는데 있다. Another object of the present invention is Neosartorya featureri ( Neosartorya fischeri ) is to produce a neuroprotective activity of the feature line (Fischerin) compound by culturing the strain.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.Other objects and advantages of the present invention will become more apparent by the following detailed description, claims and drawings.
본 발명의 한 양태에 따르면, 본 발명은 식물내생균 네오사르토리야 피쳐리 JS-0553 균주의 배양액 또는 이로부터 분리된 피쳐린(fischerin) 화합물을 유효성분으로 함유하는 신경보호용 약학적 조성물을 제공한다.According to one aspect of the present invention, the present invention provides a pharmaceutical composition for neuroprotection containing as an active ingredient a culture medium of the plant endoproliferative bacteria Neosartoriya Fechiri JS-0553 strain or a fischerin compound isolated therefrom. do.
본 발명자들은 천연에서 분리되고, 우수한 신경보호 활성을 갖는 물질을 찾고자 여러 가지 천연소재들을 검색한 결과, 갯방풍 식물내생균 배양액 추출물 또는 분획물과, 이로부터 분리된 피쳐린 화합물이 신경세포의 글루타메이트에 의한 세포사멸을 억제하여 뇌 신경세포 보호 활성을 갖고, 병리학적 조건에서 뇌 신경세포 내의 활성산소종(ROS) 및 칼슘 이온의 농도를 저해하여 산화 스트레스를 매우 효과적으로 감소시키고, 글루타메이트에 의해 증가된 ERK, JNK 및 p38 단백질의 인산화를 감소시켜 MAPKs 효소에 대한 지속적인 인산화 반응을 억제하여, 뇌신경 질환의 예방 및 치료에 효과적으로 활용할 수 있음을 확인하였고, 또한 안전성에도 문제가 없음을 확인하였다. The present inventors searched for a variety of natural materials to find a substance that is isolated from nature and has excellent neuroprotective activity. As a result, extracts or fractions of the endobacteriaceae culture broth, and a featurelin compound isolated therefrom were applied to glutamate of nerve cells. It has a protective activity of brain neurons by inhibiting apoptosis caused by apoptosis, inhibits the concentration of reactive oxygen species (ROS) and calcium ions in brain neurons under pathological conditions, very effectively reducing oxidative stress, and ERK increased by glutamate. , By reducing the phosphorylation of JNK and p38 proteins, it was confirmed that it can be effectively used in the prevention and treatment of cranial nerve diseases by inhibiting the continuous phosphorylation reaction to the MAPKs enzyme, and it was also confirmed that there is no problem in safety.
또한, 상기 식물내생균 네오사르토리야 피쳐리 JS-0553 균주는 수탁번호 수탁번호 KACC83025BP로 부다페스트 조약 하의 국제기탁기관인 국립농업과학원 농업유전자원정보센터에(KACC)에 국제기탁하여 수탁번호 KACC83025BP를 부여받았다. In addition, the plant endophytes Neosartoriya fetiri JS-0553 strain is assigned the accession number KACC83025BP by making an international deposit to the National Institute of Agricultural Sciences Agricultural Genetic Resource Information Center (KACC), an international depository institution under the Budapest Treaty, with the accession number KACC83025BP. received.
본 발명의 신경보호용 약학적 조성물에서, 상기 추출물은 균주의 균사체 또는 배양액을 추출하여 사용할 수 있다. 본 발명에서 사용되는 용어, "추출물"은 균사체 또는 배양액의 추출 처리에 의하여 얻어지는 추출액, 상기 추출액의 희석액이나 농축액, 상기 추출액을 건조하여 얻어지는 건조물, 상기 추출액의 조정제물이나 정제물, 또는 이들의 혼합물 등, 추출액 자체 및 추출액을 이용하여 형성 가능한 모든 제형의 추출물을 포함한다. 본 발명의 상기 추출물은 바람직하게 추출 후 건조 분말 형태로 제조되어 사용될 수 있다.In the neuroprotective pharmaceutical composition of the present invention, the extract may be used by extracting the mycelium or culture medium of the strain. As used in the present invention, the term "extract" refers to an extract obtained by extraction treatment of mycelium or culture solution, a dilution or concentrate of the extract, a dried product obtained by drying the extract, a prepared product or purified product of the extract, or a mixture thereof And the like, the extract itself, and extracts of all formulations that can be formed using the extract. The extract of the present invention may be preferably prepared and used in the form of a dry powder after extraction.
또한, 본 발명의 용어 "분획물"은 다양한 구성성분을 포함하는 혼합물로부터 특정성분 또는 특정 그룹을 분리하는 분획방법에 의하여 얻어진 결과물을 의미한다. 본 발명에서는 바람직하게는 식물내생균 네오사르토리야 피쳐리 JS-0553 균주 배양액을 실리카겔을 충진한 칼럼에 [n-hexane/acetone/MeOH]의 혼합 용매로 용리시켜 열린 칼럼 크로마토그라피 방법으로 분획한 결과물일 수 있으며, 극성 용매 분획물과 비극성 용매 분획물을 모두 포함하며, 구체적으로는 실리카겔을 충진한 칼럼에 [n-hexane/acetone/MeOH]의 혼합 용매로 용리시켜 열린 칼럼 크로마토그라피를 실시하여 총 9개의 분획을 얻은 후, 분획 G에 대하여 C18로 충진한 칼럼에 [H2O/MeOH]의 혼합용매의 비율(75:25 → 0:100)로 용리시켜 얻은 20개의 소분획 중 11번째 소분획일 수 있다. In addition, the term "fraction" of the present invention means a result obtained by a fractionation method for separating a specific component or a specific group from a mixture containing various components. In the present invention, preferably, the culture solution of the plant endophytes Neosartoriya Fechiri JS-0553 strain was fractionated by an open column chromatography method by eluting with a mixed solvent of [n-hexane/acetone/MeOH] on a column filled with silica gel. It may be a result, and includes both a polar solvent fraction and a non-polar solvent fraction. Specifically, a column filled with silica gel was eluted with a mixed solvent of [n-hexane/acetone/MeOH] to perform open column chromatography for a total of 9 After obtaining fractions, the eleventh fraction of the 20 small fractions obtained by eluting with a mixed solvent of [H 2 O/MeOH] in a column filled with C18 with respect to fraction G (75:25 → 0:100) I can.
본 발명에서, 상기 배양액 추출물, 분획물, 또는 피쳐린 화합물은 뇌 신경세포의 세포자멸사(apoptosis) 또는 괴사(necrosis)를 억제하여 세포사멸(cell death)를 방지하는 효과가 있어 신경보호에 효과가 있는 것을 특징으로 한다. 본 발명에서 용어 "신경보호"는 신경세포를 사멸로부터 보호하여, 신경세포의 사멸에 의한 신경 퇴행의 효과를 방지하는 것을 의미한다. In the present invention, the culture medium extract, fraction, or featurelin compound is effective in preventing apoptosis by inhibiting apoptosis or necrosis of brain neurons, thereby preventing cell death. It features. In the present invention, the term "neuroprotection" means to protect nerve cells from death, thereby preventing the effect of nerve degeneration by the death of nerve cells.
본 발명의 신경보호용 약학적 조성물에서, 상기 피쳐린(fischerin) 화합물은 하기 화학식 1의 화학식을 갖는다.In the neuroprotective pharmaceutical composition of the present invention, the fischerin compound has the formula of the following formula (1).
[화학식 1][Formula 1]
본 발명의 다른 양태에 따르면, 본 발명은 식물내생균 네오사르토리야 피쳐리 JS-0553 균주의 배양액 또는 이로부터 분리된 피쳐린(fischerin) 화합물을 유효성분으로 함유하는 뇌질환 예방 및 치료용 약학적 조성물을 제공한다.According to another aspect of the present invention, the present invention is a pharmaceutical for preventing and treating brain diseases containing as an active ingredient the culture medium of the plant endoproliferative bacteria Neosartoriya Peitiri JS-0553 strain or a fischerin compound isolated therefrom. Provide the appropriate composition.
본 발명의 뇌질환 예방 및 치료용 약학적 조성물에서, 상기 식물내생균 네오사르토리야 피쳐리 JS-0553 균주는 수탁번호 KACC83025BP로 기탁된 네오사토리아 속 균주인 것을 특징으로 한다.In the pharmaceutical composition for preventing and treating brain diseases of the present invention, the plant endogenous bacteria Neosartoriya fetiri JS-0553 strain is characterized in that it is a strain of the genus Neosartoria deposited with accession number KACC83025BP.
본 발명의 뇌질환 예방 및 치료용 약학적 조성물에서, 상기 피쳐린(fischerin) 화합물은 하기 화학식 1의 화학식을 갖는 것을 특징으로 한다.In the pharmaceutical composition for preventing and treating brain diseases of the present invention, the fischerin compound is characterized by having the formula of the following formula (1).
[화학식 1][Formula 1]
본 발명의 뇌질환 예방 및 치료용 약학적 조성물에서, 상기 뇌질환은 뇌졸중, 중풍, 치매, 알츠하이머병, 헌팅턴병, 피크(Pick)병, 크로이츠펠트-야콥(Creutzfeld-Jakob)병, 혈전증(thrombosis), 색전증(em-bolism), 일과성 허혈 발작(transient ischemic attack), 소경색(lacune), 뇌졸중, 뇌일혈, 뇌경색, 두부손상(head trauma), 뇌순환대사장애 및 뇌 기능혼수로 구성된 군으로부터 선택되는 어느 하나일 수 있다.In the pharmaceutical composition for preventing and treating brain diseases of the present invention, the brain diseases are stroke, stroke, dementia, Alzheimer's disease, Huntington's disease, Pick's disease, Creutzfeld-Jakob's disease, thrombosis. , Em-bolism, transient ischemic attack, lacune, stroke, cerebral congestion, cerebral infarction, head trauma, cerebral circulatory metabolic disorder and cerebral coma. It can be either.
본 발명의 일 구체예에서, 상기 약학 조성물은 환제, 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽제, 액제, 주사제, 또는 경피 투여제의 제형으로 제제화될 수 있다. In one embodiment of the present invention, the pharmaceutical composition may be formulated as a pill, powder, granule, tablet, capsule, suspension, emulsion, syrup, liquid, injection, or transdermal dosage form.
본 발명의 신경보호용 약학적 조성물 또는 뇌질환 예방 및 치료용 약학적 조성물은 약제학적으로 허용되는 담체를 더 포함할 수 있다. 본 발명의 약제학적 조성물에 포함되는 약제학적으로 허용되는 담체는 제제화에 통상적으로 이용되는 것으로서, 락토오스, 덱스트로오스, 수크로오스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로오스, 폴리비닐피롤리돈, 셀룰로오스, 물, 시럽, 메틸 셀룰로오스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되지 않는다. The neuroprotective pharmaceutical composition or the pharmaceutical composition for preventing and treating brain diseases of the present invention may further include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used for formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, silicic acid. Calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto. Does not.
본 발명의 신경보호용 약학적 조성물 또는 뇌질환 예방 및 치료용 약학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약제학적으로 허용되는 담체 및 첨가제는 Remington's Pharmaceutical Sciences (19th ed., 1995)에 상세히 기재되어 있다.The neuroprotective pharmaceutical composition or the pharmaceutical composition for preventing and treating brain diseases of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and additives are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
본 발명의 신경보호용 약학적 조성물 또는 뇌질환 예방 및 치료용 약학적 조성물은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 본 발명의 신경보호용 약학적 조성물 또는 뇌질환 예방 및 치료용 약학적 조성물의 제형은 환제, 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽제, 액제, 주사제, 또는 경피 투여제를 포함하나, 이에 한정되지 않는 약제학적 제제에 적합한 제형일 수 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다. The neuroprotective pharmaceutical composition or the pharmaceutical composition for preventing and treating brain diseases of the present invention is a pharmaceutically acceptable carrier and/or according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention belongs. Alternatively, it may be prepared in a unit dosage form by formulation using an excipient, or may be prepared by incorporating it into a multi-dose container. Formulations of the neuroprotective pharmaceutical composition or the pharmaceutical composition for preventing and treating brain diseases of the present invention include pills, powders, granules, tablets, capsules, suspensions, emulsions, syrups, solutions, injections, or transdermal administration, It may be a formulation suitable for pharmaceutical formulations, but not limited thereto, and may additionally include a dispersing agent or a stabilizer.
본 발명의 신경보호용 약학적 조성물 또는 뇌질환 예방 및 치료용 약학적 조성물은 신경세포 손상 또는 사멸로부터 신경을 보호하기 위하여, 유효성분으로서 성인을 기준으로 1일 총 투여량은 약 0.001 내지 약 20mg/체중 kg/일을 1회에 또는 임의로 수회 나누어서 투여할 수 있다. 상기 투여량은 신경세포 손상을 유발한 질병의 종류, 질병의 진행 정도, 투여 경로, 환자의 성별, 나이, 체중 등을 고려하여 결정될 수 있다. The neuroprotective pharmaceutical composition or the pharmaceutical composition for preventing and treating brain diseases of the present invention is an active ingredient in order to protect the nerve from damage or death, and the total daily dose based on an adult is about 0.001 to about 20 mg/day. The body weight kg/day can be administered once or optionally dividedly several times. The dosage may be determined in consideration of the type of disease that caused nerve cell damage, the degree of progression of the disease, the route of administration, the sex, age, and weight of the patient.
본 발명의 또 다른 양태에 따르면, 본 발명은 식물내생균 네오사르토리야 피쳐리 JS-0553 균주의 배양액 또는 이로부터 분리된 피쳐린(fischerin) 화합물을 유효성분으로 함유하는 신경보호용 건강식품을 제공한다. According to another aspect of the present invention, the present invention provides a neuroprotective health food containing as an active ingredient the culture medium of the plant endoproliferative bacteria Neosartoriya Fechiri JS-0553 strain or a fischerin compound isolated therefrom. do.
본 발명의 또 다른 양태에 따르면, 본 발명은 식물내생균 네오사르토리야 피쳐리 JS-0553 균주의 배양액 또는 이로부터 분리된 피쳐린(fischerin) 화합물을 유효성분으로 함유하는 뇌질환 예방 및 개선용 건강식품을 제공한다. According to another aspect of the present invention, the present invention is for the prevention and improvement of brain diseases containing the culture medium of the plant endophyte neosartoriya fetiri JS-0553 strain or a fischerin compound isolated therefrom as an active ingredient Provide healthy food.
상기 식물내생균 네오사르토리야 피쳐리 JS-0553 균주, 그 배양액, 추출물 및 피쳐린 화합물에 대해서는 상기에서 설명한 바와 같으며, 과도한 설명을 피하기 위해 공통된 내용은 반복 기재에 따른 명세서의 과도한 복잡성을 피하기 위하여, 그 기재를 생략한다.The plant endophytes Neosartoriya Fechiri JS-0553 strain, its culture solution, extract, and featurelin compound are as described above, and in order to avoid undue description, common content is to avoid excessive complexity of the specification due to repeated description. For that purpose, the description is omitted.
본 발명의 조성물을 건강기능식품 첨가물로 사용할 경우, 상기 조성물을 그대로 첨가하거나 다른 건강기능식품 또는 건강기능식품 성분과 함께 사용할 수 있고, 통상적인 방법에 따라 적절하게 사용할 수 있다. 유효성분의 혼합량은 사용 목적에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조 시에 본 발명의 조성물은 원료에 대하여 바람직하게는 15 중량부 이하, 보다 바람직하게는 10 중량부 이하의 양으로 첨가할 수 있다. 그러나, 건강 조절 및 위생을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있으며, 안정성 면에서 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용할 수 있다. When the composition of the present invention is used as a health functional food additive, the composition may be added as it is or used with other health functional food or health functional food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use. In general, when preparing food or beverage, the composition of the present invention may be added in an amount of preferably 15 parts by weight or less, more preferably 10 parts by weight or less based on the raw material. However, in the case of long-term intake for the purpose of health control and hygiene, the amount may be less than the above range, and there is no problem in terms of stability, so the active ingredient may be used in an amount above the above range.
본 발명의 건강기능식품의 종류에는 특별한 제한은 없다. 상기 조성물을 첨가할 수 있는 건강기능식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올 음료 및 비타민 복합제 등이 있고, 통상적인 의미에서의 건강기능식품을 모두 포함할 수 있으며, 동물을 위한 사료로 이용되는 식품을 포함할 수 있다.There is no particular limitation on the kind of the health functional food of the present invention. Examples of health functional foods to which the composition can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, There are drinks, alcoholic beverages, and vitamin complexes, and may include all health functional foods in the usual sense, and may include foods used as feed for animals.
또한, 본 발명의 건강기능식품 조성물이 음료의 형태로 사용될 경우에는 통상의 음료와 같이 여러 가지 감미제, 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상기 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드, 말토스, 수크로스와 같은 디사카라이드, 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드, 및 자일리톨, 소르비톨, 에리트리톨과 같은 당알콜일 수 있다. 상기 천연 탄수화물의 비율은 이에 제한되지는 않으나, 본 발명의 조성물 100 ㎖ 당 바람직하게는 약 0.01 내지 0.04g, 보다 바람직하게는 0.02 내지 0.03g일 수 있다. 상기 감미제는 타우마틴, 스테비아 추출물과 같은 천연 감미제 및 사카린, 아스파르탐과 같은 합성 감미제일 수 있다.In addition, when the health functional food composition of the present invention is used in the form of a beverage, it may contain various sweetening agents, flavoring agents, natural carbohydrates, and the like as an additional component, like a normal beverage. The natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The ratio of the natural carbohydrate is not limited thereto, but may be preferably about 0.01 to 0.04 g, more preferably 0.02 to 0.03 g per 100 ml of the composition of the present invention. The sweetener may be a natural sweetener such as taumatin and stevia extract, and a synthetic sweetener such as saccharin and aspartame.
상기 외에 본 발명의 건강기능식품 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, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 천연 과일쥬스, 과일쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다.In addition to the above, the health functional food composition of the present invention includes 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 , Alcohol, carbonated beverages, etc. may contain. In addition, it may contain flesh for the manufacture of natural fruit juice, fruit juice beverage and vegetable beverage.
본 발명의 또 다른 양태에 따르면, 본 발명은 식물내생균 네오사르토리야 피쳐리 JS-0553 균주(KACC83025BP)를 배양하는 단계; 및 상기 식물내생균 네오사르토리야 피쳐리 JS-0553 균주(수탁번호 KACC83025BP)의 배양액에서 피쳐린(fischerin) 화합물을 분리하는 단계;를 포함하는 피쳐린 화합물의 생산방법을 제공한다. 상기 균주는 신경보호와 뇌질환 예방 및 치료를 위한 배양액을 생산할 수 있으며, 상기 배양액의 추출물 또는 분획물이나, 여기서 분리한 피쳐린 화합물을 이용하여 신경보호용 조성물, 또는 뇌질환 예방 및 치료를 위한 조성물을 생산할 수 있다. 상기 식물내생균 네오사르토리야 피쳐리 JS-0553 균주에 대해서는 상기에서 설명한 바와 같다.According to another aspect of the present invention, the present invention comprises the steps of culturing the plant endoproliferative bacteria Neosartoriya Peitiri JS-0553 strain (KACC83025BP); And separating the fischerin compound from the culture medium of the plant endophytes Neosartoriya Fechiri JS-0553 strain (accession number KACC83025BP). The strain may produce a culture solution for neuroprotection and prevention and treatment of brain diseases, and a composition for neuroprotection or a composition for preventing and treating brain diseases using an extract or fraction of the culture solution or a featurelin compound isolated here Can be produced. The plant endogenous bacteria Neosartoriya Fechiri JS-0553 strain is as described above.
이상 설명한 바와 같이, 본 발명의 식물내생균 네오사르토리야 피쳐리 JS-0553 균주의 추출물 및 이로부터 분리된 피쳐린 화합물은 농도 의존적으로 뇌 신경세포의 글루타메이트에 의한 세포사멸을 억제하여 뇌 신경세포 보호 활성을 갖는다. As described above, the extract of the plant endophytosis Neosartoriya fetiri JS-0553 strain of the present invention and the featurelin compound isolated therefrom suppresses apoptosis by glutamate of brain neurons in a concentration-dependent manner, Has protective activity.
또한, 본 발명의 식물내생균 네오사르토리야 피쳐리 JS-0553 균주의 추출물 및 이로부터 분리된 피쳐린 화합물은 병리학적 조건에서 뇌 신경세포 내의 활성산소종(ROS) 및 칼슘 이온의 농도를 저해하여 산화 스트레스를 매우 효과적으로 감소시키고, 글루타메이트에 의해 증가된 ERK, JNK 및 p38 단백질의 인산화를 감소시켜 MAPKs 효소에 대한 지속적인 인산화 반응을 억제함으로써 뇌신경 질환의 치료 및 예방에 효과적으로 사용될 수 있다. In addition, the extract of the plant endophytes Neosartoriya pici JS-0553 strain of the present invention and the featurelin compound isolated therefrom inhibit the concentration of reactive oxygen species (ROS) and calcium ions in brain neurons under pathological conditions. Thus, it can be effectively used for the treatment and prevention of cranial nerve diseases by very effectively reducing oxidative stress and inhibiting the persistent phosphorylation reaction to MAPKs enzyme by reducing the phosphorylation of ERK, JNK and p38 proteins increased by glutamate.
또한, 본 발명의 조성물은 천연상태에서 존재하는 화합물로 인체에 매우 안전할 뿐만 아니라, 안정성도 매우 탁월하다.In addition, the composition of the present invention is a compound existing in a natural state and is not only very safe for the human body, but also has excellent stability.
도 1은 본 발명의 식물내생균 네오사르토리야 피쳐리(Neosartorya fischeri) JS-0553 균주에서 분리된 [화합물 1] 내지 [화합물 9]의 구조를 나타낸 그림이다.
도 2는 본 발명의 피쳐린 화합물[화합물 8]과 글루타메이트를 처리한 후 HT22 세포의 세포 생존능력을 측정하여 나타낸 그래프이다(mean ± SEM; (**) p < 0.001 글루타메이트 단독 처리군과 비교).
도 3은 본 발명의 피쳐린 화합물[화합물 8]과 글루타메이트를 처리한 후 HT22 세포 내의 활성산소종(도 3a 및 3b)과 칼슘 이온(도 5c 및 5d)의 농도를 측정하여 나타낸 그래프이다(mean ± SEM; (**) p < 0.001 글루타메이트 단독 처리군과 비교).
도 4는 본 발명의 피쳐린 화합물[화합물 8]과 글루타메이트를 처리한 후, HT22 세포 내의 아넥신-V-양성 세포(apoptotic 세포를 의미)를 촬영한 사진(도 4a)과; 세포사멸(apoptotic)된 세포의 비율을 측정하여 나타낸 그래프(도 4b)와; ERK, JNK 및 p38 단백질의 인산화 정도를 측정하기 위해 수행한 western blotting 사진(도 4c)과; ERK, JNK 및 p38 단백질의 인산화 정도를 비교하여 나타낸 그래프(도 4d 내지 도 4f)이다(mean ± SEM; (**) p < 0.001 글루타메이트 단독 처리군과 비교).
도 5는 본 발명의 식물내생균 네오사르토리야 피쳐리(Neosartorya fischeri) JS-0553 균주를 영양배지상에서 배양한 균총의 모습이다(배지는 Czapek Yeast Autolysate agar (CYA)와 Malt extract agar (MEA)를 사용하였고, 포자현탁액을 접종하여 25℃에서 7일간 배양한 후 균총의 모습을 앞면(Front)과 뒷면(Rear)으로 나누어 기록하였음).
도 6은 본 발명의 식물내생균 네오사르토리야 피쳐리(Neosartorya fischeri) JS-0553 균주의 분생포자(conidia)의 생성모습을 촬영한 사진이다(주머니모양(vesicle)의 분생자경(conidiophore)의 끝에만 한겹의 phialide가 만들어지고 그 위에서 분생포자가 만들어지는 것이 관찰됨).
도 7은 본 발명의 식물내생균 네오사르토리야 피쳐리(Neosartorya fischeri) JS-0553 균주의 종동정을 위해 계통유연관계를 분석한 계통수 그림이다.FIG. 1 is a diagram showing the structure of [Compound 1] to [Compound 9] isolated from the plant endophytes Neosartorya fischeri JS-0553 strain of the present invention.
Figure 2 is a graph showing the measurement of the cell viability of HT22 cells after treatment with the featurelin compound [Compound 8] of the present invention and glutamate (mean ± SEM; (**) p <0.001 compared to glutamate alone treatment group) .
FIG. 3 is a graph showing the concentration of reactive oxygen species (FIGS. 3A and 3B) and calcium ions (FIGS. 5C and 5D) in HT22 cells after treatment with the featurelin compound [Compound 8] and glutamate of the present invention (mean ± SEM; (**) p <0.001 compared to glutamate alone treatment group).
Fig. 4 is a photograph of annexin-V-positive cells (meaning apoptotic cells) in HT22 cells after treatment with the featurelin compound [Compound 8] of the present invention and glutamate (Fig. 4A); A graph showing by measuring the proportion of apoptotic cells (FIG. 4B); Western blotting photographs (Fig. 4c) performed to measure the degree of phosphorylation of ERK, JNK and p38 proteins; It is a graph showing a comparison of the degree of phosphorylation of ERK, JNK, and p38 proteins (Figs. 4D to 4F) (mean ± SEM; (**) p <0.001 compared to glutamate alone treatment group).
Figure 5 is a state of the colonies cultured on a nutrient medium of the plant endophytes Neosartorya fischeri JS-0553 strain of the present invention (medium is Czapek Yeast Autolysate agar (CYA) and Malt extract agar (MEA)) Was used, and the spore suspension was inoculated and cultured at 25° C. for 7 days, and the appearance of the colony was divided into front and rear, and recorded).
Figure 6 is a photograph taken of the generation of conidia of the plant endophytes Neosartorya fischeri JS-0553 strain of the present invention (at the end of the conidiophore of the vesicle) Only one layer of phialide is formed and conidia are observed on top of it).
7 is a phylogenetic tree diagram analyzing the phylogenetic relationship for the seed identification of the plant endophytes Neosartorya fischeri JS-0553 strain of the present invention.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.Hereinafter, the present invention will be described in more detail through examples. Since these examples are for illustrative purposes only, the scope of the present invention is not to be construed as being limited by these examples.
실시예 1. 실험 재료 및 방법Example 1. Experimental materials and methods
1-1. 실험 균주1-1. Experimental strain
식물내생균 네오사르토리야 피쳐리(Neosartorya fischeri) JS-0553은 국립생물자원관이 분리 및 동정한 균주를 사용하였으며, 동 균주는 국립생물자원관(NIBRGR0000118808)에 보관하여 사용하였다. The plant endosperm Neosartorya fischeri JS-0553 was a strain isolated and identified by the National Institute of Biological Resources, and the strain was stored and used in the National Institute of Biological Resources (NIBRGR0000118808).
1-2. 시약 및 기기1-2. Reagents and instruments
선광도는 Jasco P-2000 polarimeter로 측정하였다. IR 실험은 Agilent Cary 630 Fourier transform infrared spectrometer 기기를 사용하여 측정하였다. CD (Circular dichroism)는 Jasco J-1100 spectrometer로 측정하였다. HR-ESI-TOF-MS (High-resolution electrospray ionization time-of-flight mass spectrometry)는 Waters 사의 Q-TOF micromass spectrometer를 이용하여 측정하였다.The optical rotation was measured with a Jasco P-2000 polarimeter. IR experiments were measured using an Agilent Cary 630 Fourier transform infrared spectrometer. CD (Circular dichroism) was measured with a Jasco J-1100 spectrometer. HR-ESI-TOF-MS (High-resolution electrospray ionization time-of-flight mass spectrometry) was measured using a Waters Q-TOF micromass spectrometer.
단일 화합물의 구조를 규명하기 위하여 NMR(Nuclear magnetic resonance)은 Varian NMR spectrometer(1H, 500 MHz; 13C, 125 MHz) 기기를 이용하여 측정하였고, NMR chemical shift value는 part per million(ppm) 단위로 나타내었다. Preparative HPLC 기기는 996 PDA detector가 장착된 Waters Millipore 600 시스템을 사용하였고, 칼럼은 Phenomenex 사의 Luna 5μm C18(2) column(250mm × 10cm)을 사용하였다. Column chromatography 기기용 고정상은 Merck 사의 silica gel (Kieselgel 60, 70-230 mesh)을 사용하였다. TLC(Thin-layer chromatography) 플레이트(plate)는 머크(Merck)사의 silica gel 60 F254 및 RP-18 F254S를 사용하였고 결과 확인은 UV detector 기기를 이용하여 관찰하였으며, 10% aqueous H2SO4 시약을 이용한 발색을 병행하여 확인하였다.In order to identify the structure of a single compound, NMR (Nuclear magnetic resonance) was measured using a Varian NMR spectrometer (1H, 500 MHz; 13C, 125 MHz), and the NMR chemical shift value is expressed in parts per million (ppm). Done. A waters Millipore 600 system equipped with a 996 PDA detector was used as a preparative HPLC instrument, and a
1-3. 균주 분리1-3. Strain isolation
식물내생균 네오사르토리야 피쳐리(Neosartorya fischeri) JS-0553 균주는 2011년 10월 대한민국 제주도 제주시 조천읍 바닷가에서 채집한 갯방풍(Glehnia littoralis)의 줄기로부터 분리하였다. The plant endogenous fungus Neosartorya fischeri JS-0553 strain was isolated from the stems of Glehnia littoralis collected on the beach in Jocheon-eup, Jeju-si, Jeju-do, Korea in October 2011.
채집한 식물 시료는 흐르는 수돗물로 표면에 붙은 흙먼지를 씻고 물기를 닦은 후, 식물내생균 분리에 사용하였다. 먼저 잎 조직은 0.5×0.5cm 크기의 단편으로 작게 조각낸 후, 2% 차아염소산나트륨 용액에 1분 동안 살균하고, 다시 70% 에탄올에 1분간 표면 살균한 후, 다시 멸균된 증류수에 1분간 세척하고, 균주 분리용 배지에 치상하였다. 균주분리용 배지는 MEA(malt extract agar, Difco) 배지에 50ppm 로즈 벵갈(Rose Bengal), 50ppm 카나마이신(Kanamycin), 50ppm 스트렙토마이신(Streptomycin)을 첨가하여 조제하였다. 멸균한 잎 조직을 상기 조제된 배지에 치상한 후, 22℃의 배양기에서 7일 동안 배양하였다.The collected plant samples were used for separating plant endoprobiotics after washing and wiping off dirt adhering to the surface with flowing tap water. First, the leaf tissue is cut into small pieces of 0.5×0.5cm size, sterilized in 2% sodium hypochlorite solution for 1 minute, surface sterilized again in 70% ethanol for 1 minute, and then washed again in sterilized distilled water for 1 minute. Then, it was placed on a medium for strain isolation. A medium for strain isolation was prepared by adding 50 ppm Rose Bengal, 50 ppm Kanamycin, and 50 ppm Streptomycin to MEA (malt extract agar, Difco) medium. After placing the sterilized leaf tissue on the prepared medium, it was cultured for 7 days in an incubator at 22°C.
이렇게 배양된 균주를 PDA(potato dextrose agar) 배지에 옮겨 배양하였다. The cultured strain was transferred to a PDA (potato dextrose agar) medium and cultured.
1-4. 균주 배양 및 추출1-4. Strain culture and extraction
상기 배양된 균주는 PDA 배지를 이용하여 상온에서 7일 동안 배양하여 접종원을 준비하였다. 500mL erlenmeyer flask(10ea) 80g의 현미와 120ml의 증류수를 넣어 멸균한 후, PDA 배지에서 배양된 균주를 1.0x1.0 cm 크기의 조각으로 잘라 접종하여 상온에서 30일 동안 배양하였다. 배양된 배지에 에틸아세테이드(EtOAc)를 가하여, 3회 반복하여 추출하였고, 추출물을 감압농축 하여 1.7 g의 추출물을 얻었다.The cultured strain was cultured for 7 days at room temperature using PDA medium to prepare an inoculum. After sterilization by adding 80g of brown rice and 120ml of distilled water in a 500mL erlenmeyer flask (10ea), the strain cultured in PDA medium was cut into pieces of 1.0x1.0 cm in size and inoculated and incubated at room temperature for 30 days. Ethyl acetate (EtOAc) was added to the cultured medium, extraction was repeated three times, and the extract was concentrated under reduced pressure to obtain 1.7 g of an extract.
1-5. 화합물의 분리정제1-5. Separation and purification of compounds
상기 추출물을 실리카겔을 충진한 칼럼에 [n-hexane/acetone/MeOH]의 혼합 용매의 비율(19:1:0, 9:1:0, 6.2:1:0, 5.6:1:0, 4:1:0.1, 3:1:0.1, 0:0:1)로 용리시켜 열린 칼럼 크로마토그라피를 실시하여 총 9개의 분획을 얻었다(분획 A 내지 I). The ratio of the mixed solvent of [n-hexane/acetone/MeOH] to the column packed with silica gel (19:1:0, 9:1:0, 6.2:1:0, 5.6:1:0, 4: 1 :0.1, 3:1:0.1, 0:0:1), followed by open column chromatography to obtain a total of 9 fractions (fractions A to I).
이렇게 수득한 분획들 중 E 분획에 대하여 실리카겔로 충진한 칼럼에 [n-hexane/EtOAc]의 혼합 용매의 비율(100:0 → 75:25)로 용리시켜 [화합물 5](10.5 mg) 및 [화합물 6](8.5 mg)을 얻었다. 또한, 분획 F에 대하여 실리카겔을 충진한 칼럼에[n-hexane/acetone/MeOH]의 혼합 용매의 비율(19:1:0, 9:1:0, 7.3:1:0, 5.6:1:0, 4:1:0, 0:1:0, 0:0:1)로 용리시켜 10개의 소분획을 얻었다(소분획 F1 내지 F10). 소분획들 중 소분획 F3에 대하여 실리카겔로 충진한 칼럼에 [CHCl3/acetone/MeOH]의 혼합용매의 비율(1:0:0, 90:1:0, 70:1:0, 0:1:0, 0:0:1)로 용리시켜 [화합물 2] (27.0mg) 및 [화합물 4](3.5 mg)를 얻었고, 소분획 F5에 대하여 실리카겔을 충진한 칼럼에 [CHCl3/MeOH]의 혼합용매의 비율(95:1, 90:1, 65:1, 60:1, 0:1)로 용리시켜 [화합물 7](13.5mg)을 얻었다. 분획 G에 대하여 C18로 충진한 칼럼에 [H2O/MeOH]의 혼합용매의 비율 (75:25 → 0:100)로 용리시켜 20개의 소분획을 얻었다(소분획 G1 내지 G20). 소분획들 중 소분획 G7로부터 [H2O/acetonitrile]의 혼합용매의 비율(70:30 → 50:50)을 사용한 예비(preparative) HPLC를 통하여 화합물 3(1.5 mg)을 얻었고, 소분획 G10에 대하여 [H2O/acetonitrile]의 혼합용매의 비율(50:50 → 25:75)을 사용한 예비(preparative) HPLC를 통하여 [화합물 9](2.7mg)를 얻었으며, 소분획 G11로부터 [H2O/acetonitrile]의 혼합용매 비율(30:70 → 10:90)을 사용한 예비(preparative) HPLC를 통하여 [화합물 1](15.0 mg) 및 [화합물 8](4.5 mg)을 얻었다.Of the fractions thus obtained, the E fraction was eluted at a ratio of a mixed solvent of [n-hexane/EtOAc] (100:0 → 75:25) to a column filled with silica gel, and [Compound 5] (10.5 mg) and [ Compound 6] (8.5 mg) was obtained. In addition, the ratio of the mixed solvent of [n-hexane/acetone/MeOH] to the column filled with silica gel for fraction F (19:1:0, 9:1:0, 7.3:1:0, 5.6:1:0) , 4:1:0, 0:1:0, 0:0:1) to obtain 10 small fractions (small fractions F1 to F10). The ratio of the mixed solvent of [CHCl 3 /acetone/MeOH] to the column filled with silica gel with respect to the small fraction F3 among the small fractions (1:0:0, 90:1:0, 70:1:0, 0:1 :0, 0:0:1) to obtain [Compound 2] (27.0mg) and [Compound 4] (3.5 mg), and the column filled with silica gel for the small fraction F5 of [CHCl 3 /MeOH] It was eluted at the ratio of the mixed solvent (95:1, 90:1, 65:1, 60:1, 0:1) to obtain [Compound 7] (13.5mg). Fraction G was eluted at a ratio of a mixed solvent of [H 2 O/MeOH] to a column filled with C18 (75:25 → 0:100) to obtain 20 small fractions (small fractions G1 to G20). Compound 3 (1.5 mg) was obtained through preparative HPLC using a mixed solvent ratio (70:30 → 50:50) of [H 2 O/acetonitrile] from small fraction G7 among small fractions, and small fraction G10 [Compound 9] (2.7mg) was obtained through preparative HPLC using a mixed solvent ratio of [H 2 O/acetonitrile] (50:50 → 25:75), and [H [Compound 1] (15.0 mg) and [Compound 8] (4.5 mg) were obtained through preparative HPLC using a mixed solvent ratio (30:70 → 10:90) of 2 O/acetonitrile].
상기 [화합물 1] 내지 [화합물 9]의 구조를 동정하여 [도 1]에 나타내었다. 이 중 [화합물 1]은 천연물에서 새롭게 분리된 신규 화합물로 밝혀졌고, [화합물 2]는 [sartorypyrone A], [화합물 3]은 [cyclotryprostatin B], [화합물 4]는 [fumitremorgin B], [화합물 5]는 fumitremorgin A, [화합물 6]은 [aszonalenin], [화합물 7]은 [acetylaszonalenin], [화합물 8]은 [fischerin], 그리고 [화합물 9]는 [pyripyropene A]로 확인되었다. The structures of [Compound 1] to [Compound 9] were identified and shown in [Fig. 1]. Among them, [Compound 1] was found to be a new compound newly isolated from natural products, [Compound 2] was [sartorypyrone A], [Compound 3] was [cyclotryprostatin B], [Compound 4] was [fumitremorgin B], [Compound 5] was identified as fumitremorgin A, [Compound 6] was [aszonalenin], [Compound 7] was [acetylaszonalenin], [Compound 8] was [fischerin], and [Compound 9] was [pyripyropene A].
1-6. 식물내생균 네오사르토리야 피쳐리 JS-0553 균주의 염기서열 분석1-6. Sequencing Analysis of the Plant Endogenous Bacteria Neosartoriya Fechiri JS-0553 Strain
상기 식물내생균 네오사르토리야 피쳐리 JS-0553 균주부터 DNA를 분리하였다. 분리된 DNA를 ITS1F 및 LR5 프라이머를 이용하여 ITS(Internal Transcribed Spacer)부터 large subunit of ribosomal DNA의 D1, D2 부위(LSU)의 서열을 증폭하였고, ITS1F, ITS3, ITS4, LR0R, LR5 등 5개의 프라이머를 이용하여 ITS와 LSU 염기서열을 확보하였다(Gardes and Bruns, 1993; White et al., 1990). 이 서열들은 NCBI의 GenBank에 ITS는 MN121342번, LSU는 MN121342번으로 등록되었다.DNA was isolated from the plant endophytes Neosartoriya Fechiri JS-0553 strain. The isolated DNA was amplified from ITS (Internal Transcribed Spacer) using ITS1F and LR5 primers to the sequence of D1 and D2 regions (LSU) of large subunit of ribosomal DNA, and five primers including ITS1F, ITS3, ITS4, LR0R, LR5, etc. ITS and LSU sequences were obtained using (Gardes and Bruns, 1993; White et al., 1990). These sequences were registered in NCBI's GenBank as MN121342 for ITS and MN121342 for LSU.
확보된 염기서열은 서열분석(Sequencher) 프로그램을 이용하여 고품질 영역을 확보하고 조립하여 컨센서스 서열(consensus sequence)을 확정하고, NCBI의 뉴클레오타이드 데이터베이스를 대상으로 BLAST 검색을 수행하였다. 다중정렬 및 계통유연관계 분석은 MEGA 프로그램을 이용하였다.The secured nucleotide sequence was obtained by securing and assembling a high-quality region using a sequencing program to determine a consensus sequence, and a BLAST search was performed on NCBI's nucleotide database. The MEGA program was used to analyze multiple alignment and systematic relevance.
ITS 염기서열 정보를 바탕으로 수행한 NCBI Blast 검색에서, 식물내생균 JS-0553 균주는 네오사르토리야(완전세대명; 불완전세대명은 아스퍼질러스) 속 균주들의 서열과 상동성이 높은 것으로 나타났다. 이에, 네오사르토리야가 속한 아스퍼질러스 속 Fumigati 섹션에 속한 종들의 표준균주에 대한 ITS/LSU 서열을 대상으로 다중정렬한 후 계통유연관계를 분석하였다. 그 결과, JS-0553 균주는 네오사르토리야 피처리와 동일 서열을 가지며 같은 분지에 위치하였다.In the NCBI Blast search performed based on the ITS sequence information, it was found that the endophytes JS-0553 strain had high sequence and homology with the strains of the genus Neosartoriya (complete generation name; incomplete generation name Aspergillus). Accordingly, the ITS/LSU sequences for the standard strains of the species belonging to the Aspergillus genus Fumigati section to which Neosartoriya belongs were subjected to multiple alignments and analyzed for phylogenetic relationship. As a result, the JS-0553 strain had the same sequence as the neosartolia target and was located in the same branch.
또한, 상기 JS-0553 균주를 배양하였을 때 배지위에 하얀색의 자낭구(cleistothecia)가 가득 만들어지고, 기낭(vesicle) 형태의 분생자경(conidiophore)의 끝쪽에만 있는 한겹의 phialide에 포자가 만들어지는 형태적 특징을 보여 Neosartorya fischeri로 동정하였다. 이 균주는 국립생물자원관의 야생생물유전자원은행(NIBRGR0000118808)과 국립농업과학원 농업유전자원정보센터에 기탁번호(KACC83025BP)로 기탁하였다. In addition, when the JS-0553 strain is cultivated, white cleistothecia is made full on the medium, and spores are created in a single layer of phialide located only at the end of a vesicle-shaped conidiophore. Was identified as Neosartorya fischeri. This strain was deposited with the National Institute of Biological Resources' Wild Biological Genetic Resources Bank (NIBRGR0000118808) and the Agricultural Genetic Resource Information Center of the National Institute of Agricultural Science under the deposit number (KACC83025BP).
실시예 2. 신경세포 사멸 억제효과 검증Example 2. Verification of inhibitory effect on neuronal cell death
전술한 바와 같이, 글루타메이트는 흥분성 신경 전달 물질로 잘 알려져 있으며, 뇌의 다양한 생물학적 기능뿐만 아니라 급성 뇌 손상(acute brain insults) 및 신경 퇴행성 질환(neurodegenerative diseases)에 있어서 신경세포의 사멸에 기여한다. As described above, glutamate is well known as an excitatory neurotransmitter, and contributes to the death of neurons in acute brain insults and neurodegenerative diseases as well as various biological functions of the brain.
본 발명에서는 내분비 대사 산물로부터 강력한 신경보호제를 발견하기 위한 연구의 일환으로, 식물내생균 네오사르토리야 피쳐리 JS-0553의 배양액로부터 분리된 9 개의 화합물의 생리학적 활성에 대해 평가하였다. In the present invention, as part of a study to discover a potent neuroprotective agent from endocrine metabolites, the physiological activities of nine compounds isolated from the culture medium of the plant endophytes Neosartoriya piceri JS-0553 were evaluated.
화합물들의 신경보호 효과를 평가하기 위해, HT22 세포(immortalized mouse hippocampal cell)를 웰 당 1 x 104 세포 밀도로 96-웰 플레이트 상에 플레이팅하고 24시간 동안 부착시켰다. 세포 생존능은 제조자의 지시에 따라 EZ-CyTox 세포 생존력 분석 키트를 사용하여 측정하였다. 실험은 HT22 세포에 [화합물 1] 내지 [화합물 9]와, 5mM 글루타메이트를 동시에 투여하고 24시간 동안 배양하였다. 이후, HT22 세포에 10μL의 EZ-CyTox 시약으로 처리하고 30분 동안 배양하였다. 광학 밀도는 E-Max 마이크로 플레이트 판독기 (Molecular Devices, Sunnyvale, CA, U.S.A.)로 450 nm에서 측정하였다. 비교 평가를 위하여 [화합물 1] 내지 [화합물 9]를 투여하지 않고, 5mM 글루타메이트만을 투여한 군을 대조군으로 사용하였다. To evaluate the neuroprotective effects of the compounds, HT22 cells (immortalized mouse hippocampal cells) were plated on a 96-well plate at a density of 1 x 10 4 cells per well and adhered for 24 hours. Cell viability was measured using the EZ-CyTox cell viability assay kit according to the manufacturer's instructions. In the experiment, [Compound 1] to [Compound 9] and 5 mM glutamate were simultaneously administered to HT22 cells and cultured for 24 hours. Thereafter, HT22 cells were treated with 10 μL of EZ-CyTox reagent and incubated for 30 minutes. Optical density was measured at 450 nm with an E-Max micro plate reader (Molecular Devices, Sunnyvale, CA, USA). For comparative evaluation, a group to which [Compound 1] to [Compound 9] was not administered and only 5 mM glutamate was administered was used as a control group.
그 결과, 흥미롭게도, 글루타메이트 처리로 인해 감소한 HT22 세포의 세포 생존능력을 본 발명의 피쳐린 화합물[화합물 8]이 다른 화합물들에 비해 유의하게 증가시키는 것을 확인할 수 있었다(도 2 참조). 또한, 글루타메이트에 의해 감소된 HT22 세포의 생존능력이 매우 낮은 농도의 피쳐린 화합물의 투여로 인해 유의하게 회복됨을 확인할 수 있었고, 피쳐린 화합물이 농도 의존적으로 뇌 신경세포의 글루타메이트에 의한 세포사멸을 억제하여 뇌 신경세포 보호 활성을 나타내는 것을 확인할 수 있었다. As a result, interestingly, it was confirmed that the featurelin compound [Compound 8] of the present invention significantly increased the cell viability of HT22 cells decreased due to glutamate treatment compared to other compounds (see FIG. 2). In addition, it was confirmed that the viability of HT22 cells reduced by glutamate was significantly recovered by administration of a very low concentration of the featurelin compound, and the featurelin compound inhibited apoptosis by glutamate in brain neurons in a concentration-dependent manner. Thus, it was confirmed that it exhibits a protective activity of brain neurons.
실시예 3. 신경세포 내부의 활성산소종 생성 억제 효과 검증Example 3. Verification of the inhibitory effect on the production of reactive oxygen species inside nerve cells
병리학적 조건에서 신경세포의 세포사멸에서 산화 스트레스가 가장 중요한 요소로 간주된다. 높은 농도의 글루타메이트는 HT22 세포에서 세포 내 활성산소종(ROS) 농도를 상당하게 증가시키게 된다. 따라서, 본 발명자들은 피쳐린 화합물이 글루타메이트에 의해 유도된 세포 내 활성산소종의 증가를 감소시킬 수 있는지 확인하는 실험을 수행하였다.In pathological conditions, oxidative stress is considered the most important factor in neuronal cell death. High concentrations of glutamate significantly increase the intracellular reactive oxygen species (ROS) concentration in HT22 cells. Therefore, the present inventors performed an experiment to confirm whether the featurelin compound can reduce the increase of intracellular reactive oxygen species induced by glutamate.
ROS 검출 시약으로 세포 투과성 2', 7'- 디클로로 디히드로 플루오르신 디 아세테이트(H2DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate)를 사용하여 세포 내 활성산소종의 양을 측정하였다. HT22 세포를 2.5 및 5μM 피쳐린 화합물의 존재 또는 부재 하에 5mM 글루타메이트로 처리하고 8시간 동안 배양하였다. 세포를 2', 7'- 디클로로 디히드로 플루오르신 디 아세테이트로 30분 동안 인큐베이션한 다음 PBS 버퍼로 3회 세척하였다. 이어서 형광 현미경 판독기(SPARK 10M, Tecan, M
그 결과, 글루타메이트 단독 처리된 대조군과 비교할 때, 본 발명의 피쳐린 화합물이 처리된 군에서는 세포 내 활성산소종의 농도가 유의하게 감소하였고, 그 감소 효과가 글루타메이트 무처리군 수준으로 확연하게 감소하는 것을 확인할 수 있었다(도 3a 및 3b 참조). 상기와 같은 결과를 통해 본 발명의 피쳐린 화합물은 병리학적 조건에서 산화 스트레스를 매우 효과적으로 감소시킴으로써 신경세포의 세포사멸을 방지하며, 이를 통해 우수한 신경보호 효과가 있음을 확인할 수 있었다. As a result, compared to the control group treated with glutamate alone, the concentration of reactive oxygen species in the cell was significantly reduced in the group treated with the featurelin compound of the present invention, and the reduction effect was significantly reduced to the level of the glutamate-free group. It could be confirmed (see Figs. 3a and 3b). Through the results as described above, it was confirmed that the featurelin compound of the present invention prevents apoptosis of nerve cells by very effectively reducing oxidative stress under pathological conditions, and thus has an excellent neuroprotective effect.
실시예Example 4. 신경세포 내부의 칼슘 이온농도 감소 효과 검증 4. Verification of the effect of reducing calcium ion concentration inside nerve cells
칼슘 이온(Ca2 +)은 세포, 특히 뇌 기능에 관여하는 뉴런의 2차 메신저 역할을 수행한다. 그러나 과도한 수준의 칼슘 이온(Ca2 +) 농도 상승은 신경세포의 사멸을 초래한다. 본 실험에서는 글루타메이트에 의한 세포 내 칼슘 이온(Ca2+) 농도 증가에 피쳐린 화합물이 미치는 영향을 평가하였다. Calcium ions (Ca 2 + ) act as secondary messengers of cells, especially neurons, which are involved in brain function. However, an excessive increase in the calcium ion (Ca 2 + ) concentration causes the death of nerve cells. In this experiment, the effect of the Feline compound on the increase of the intracellular calcium ion (Ca 2+ ) concentration by glutamate was evaluated.
칼슘 이온 농도는 칼슘 이온에 대한 막투과성 형광시약(membrane-permeable fluorescent indicator)인 Fluo-4 AM(Invitrogen, Eugene, OR, U.S.A.)을 이용하여 분석하였다. HT22 세포를 5mM 글루타메이트 및 피쳐린 화합물(2.5 및 5μM)로 처리하여, 글루타메이트에 8시간 동안 노출시킨 후, 2.5μM Fluo-4 AM 형광시약을 처리하여 30분간 배양한 다음, PBS 버퍼로 3회 세척하였다. Fluo-4 AM에 대한 형광 이미지는 형광 현미경(IX71)을 사용하여 촬영하였고, ImageJ 소프트웨어(미국 메릴랜드 주, 베데스다 국립연구소)를 사용하여 분석하였다.Calcium ion concentration was analyzed using Fluo-4 AM (Invitrogen, Eugene, OR, U.S.A.), a membrane-permeable fluorescent indicator for calcium ions. HT22 cells were treated with 5 mM glutamate and featurelin compounds (2.5 and 5 μM), exposed to glutamate for 8 hours, treated with 2.5 μM Fluo-4 AM fluorescent reagent, incubated for 30 minutes, and washed 3 times with PBS buffer. I did. Fluorescence images for Fluo-4 AM were taken using a fluorescence microscope (IX71), and analyzed using ImageJ software (Bedesda National Laboratory, Maryland, USA).
그 결과, 본 발명의 피쳐린 화합물 처리가 글루타메이트에 의하여 증가된 세포 내 칼슘 이온(Ca2 +) 농도를 유의하게 감소하는 것을 확인할 수 있었다(도 5c 및 5d 참조). 이러한 결과는 피쳐린 화합물이 글루타민 처리에 의해 세포 내 칼슘 이온의 증가를 효과적으로 방지하여 신경세포 보호에 효과적임을 시사한다.As a result, it could be confirmed that the treatment of the featurelin compound of the present invention significantly reduced the intracellular calcium ion (Ca 2 + ) concentration increased by glutamate (see FIGS. 5C and 5D). These results suggest that the featurelin compound is effective in protecting neurons by effectively preventing the increase of calcium ions in cells by treatment with glutamine.
실시예 5. 신경세포의 apoptotic cell death 억제 효과 검증Example 5. Verification of the inhibitory effect of neurons on apoptotic cell death
글루타메이트를 HT22 세포에 투여하면, 조기 및 후반에 세포자멸사(apoptosis) 및 괴사(necrosis)를 통해 세포사멸(cell death)이 유도된다. 본 발명에서는 피쳐린 화합물의 뇌 신경세포의 세포사멸 억제 효과를 직접적으로 평가하기 위해 아래와 같은 실험을 수행하였다. When glutamate is administered to HT22 cells, cell death is induced through apoptosis and necrosis in the early and late stages. In the present invention, the following experiment was performed in order to directly evaluate the effect of the featurelin compound on inhibiting apoptosis of brain neurons.
HT22 세포에 5mM 글루타메이트와 2.5 또는 5μM 농도의 피쳐린 화합물을 처리한 후 12 시간 동안 배양한 후 HT22 세포를 수확하였다. 수확된 세포를 annexin-V- 결합 완충액(Invitrogen)으로 세척한 다음 Alexa-Fluor-488 결합 아넥신 V(Invitrogen)로 20분 동안 배양하였고, propidium iodide 염료로 염색하였다. 형광 이미지는 Tali-Image-based cytometer(Invitrogen)를 사용하여 촬영하였고, Annexin-V 양성 세포는 TaliPCApp(버전 1.0)로 분석하였다.HT22 cells were treated with 5 mM glutamate and a featurelin compound at a concentration of 2.5 or 5 μM, and cultured for 12 hours, and then HT22 cells were harvested. The harvested cells were washed with annexin-V-binding buffer (Invitrogen), then incubated with Alexa-Fluor-488-binding annexin V (Invitrogen) for 20 minutes, and stained with propidium iodide dye. Fluorescence images were taken using a Tali-Image-based cytometer (Invitrogen), and Annexin-V positive cells were analyzed with TaliPCApp (version 1.0).
그 결과, HT22 세포에서 글루타메이트 단독 처리군에 비해 피쳐린 화합물을 처리한 군에서는 아넥신-V-양성 세포(apoptotic 세포를 의미)가 현저히 감소한 것으로 관찰되었다(도 4a 참조). 정량 분석 결과, 글루타메이트 처리군(65.45 %)에서 세포사멸(apoptotic)된 세포의 비율은 2.5 및 5μM 농도의 피쳐린 화합물 처리군에서 각각 38.43% 및 36.21%로 유의하게 감소한 것으로 관찰되었다(도 4b 참조).As a result, it was observed that in HT22 cells, annexin-V-positive cells (meaning apoptotic cells) significantly decreased in the group treated with the featurelin compound compared to the group treated with glutamate alone (see FIG. 4A). As a result of quantitative analysis, it was observed that the percentage of apoptotic cells in the glutamate-treated group (65.45%) significantly decreased to 38.43% and 36.21% in the 2.5 and 5 μM concentrations of Feerin compound-treated groups, respectively (see FIG. 4B). ).
상기 결과는 피쳐린 화합물이 글루타메이트 농도 증가에 의해 유도된 신경세포의 세포사멸적(apoptotic) 세포사멸(cell death)을 효과적으로 억제하여 뇌신경 질환의 치료 및 예방에 효과적임을 시사한다.The above results suggest that the featurelin compound is effective in the treatment and prevention of cranial nerve diseases by effectively inhibiting apoptotic cell death of neurons induced by an increase in glutamate concentration.
실시예 6. MAPKs 효소 활성 억제 효과 검증Example 6. MAPKs enzyme activity inhibition effect verification
MAPKs(mitogen-activated protein kinases) 효소는 글루타메이트 유도 산화 스트레스의 주요 인자로 보고되고 있다. 일반적으로 MAPKs 효소는 뇌 신경세포의 생존 및 사망 모두에서 중요한 역할을 수행하는 것으로 잘 알려져 있다. 그러나, 글루타메이트 독성 환경에서 ERK, p-38, 및 JNK 단백질을 포함하는 MAPKs 효소는 정상적인 세포 환경과 비교하여 지속적으로 활성화되고, 이로 인해 신경세포의 세포사멸을 유도하는 것으로 알려져 있다. 따라서, 본 발명자들은 글루타메이트에 의해 유발된 MAPKs 효소의 지속적인 활성 유지 현상에 대한 피쳐린 화합물의 영향을 평가하기 위해 ERK, p-38, 및 JNK 단백질의 인산화 정도를 측정하였다. MAPKs (mitogen-activated protein kinases) enzymes have been reported as major factors in glutamate-induced oxidative stress. In general, MAPKs enzymes are well known to play an important role in both survival and death of brain neurons. However, in a glutamate toxic environment, MAPKs enzymes including ERK, p-38, and JNK proteins are continuously activated compared to the normal cellular environment, thereby inducing apoptosis of neurons. Accordingly, the present inventors measured the degree of phosphorylation of ERK, p-38, and JNK proteins in order to evaluate the effect of the featurelin compound on the phenomenon of sustained activity maintenance of the MAPKs enzyme induced by glutamate.
먼저, HT22 세포를 8시간 동안 5mM 글루타메이트로 처리한 후 수확하였다. 이어서, 포스파타아제(phosphatase) 저해제(1mM Na3VO4 및 1mM NaF)와 단백분해효소(proteinase) 억제제 칵테일을 첨가한 RIPA(radioimmunoprecipitation assay) 완충액[pH 7.4, 150mM NaCl, 5mM EDTA, 1% 트리톤 X-100, 0.5% SDS 및 0.5 % sodium deoxycholate]으로 HT22 세포를 용해시켰다. 용해된 세포에서 획득한 단백질을 PAGE(polyacrylamide gel electrophoresis)로 분리한 다음, 폴리비닐리덴 디플루오르 멤브레인(Merck Millipore, Darmstadt, Germany)으로 옮겼다. 비지방 우유 용액(Non-fat milk solution)[트리스 완충 식염수 및 Tween 20(TBST) 중 5%]을 사용하여 항체의 비특이적 결합을 차단하였다. 모든 항체는 Cell Signaling(Danvers, MA, U.S.A.)에서 구입하여 사용하였다. 상기 멤브레인을 IgG(horseradish peroxidase conjugated rabbit immunoglobulin G)(IgG, Cell Signaling) 항체와 함께 배양한 후, SuperSignal West Femto Maximum Sensitivity Substrate(Thermo Scientific, Rockford, IL, U.S.A.)와 반응시켰다. 면역 반응성 밴드의 이미지는 Fusion Solo 화학 발광 시스템(PEQLAB Biotechnologie GmbH, Erlangen, Germany)을 사용하여 획득하였고, 데이터는 ImageJ 소프트웨어를 사용하여 분석하였다.First, HT22 cells were harvested after treatment with 5mM glutamate for 8 hours. Subsequently, a phosphatase inhibitor (1mM Na 3 VO 4 and 1mM NaF) and a proteinase inhibitor cocktail added to RIPA (radioimmunoprecipitation assay) buffer [pH 7.4, 150mM NaCl, 5mM EDTA, 1% Triton] X-100, 0.5% SDS and 0.5% sodium deoxycholate] were used to lyse HT22 cells. The protein obtained from the lysed cells was separated by PAGE (polyacrylamide gel electrophoresis), and then transferred to a polyvinylidene difluorine membrane (Merck Millipore, Darmstadt, Germany). Non-fat milk solution (5% in Tris buffered saline and Tween 20 (TBST)) was used to block non-specific binding of the antibody. All antibodies were purchased and used from Cell Signaling (Danvers, MA, USA). The membrane was incubated with an IgG (horseradish peroxidase conjugated rabbit immunoglobulin G) (IgG, Cell Signaling) antibody, and then reacted with a SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Rockford, IL, USA). Images of the immunoreactive bands were acquired using a Fusion Solo chemiluminescence system (PEQLAB Biotechnologie GmbH, Erlangen, Germany), and the data were analyzed using ImageJ software.
그 결과, 글루타메이트에 의해 증가된 ERK, JNK 및 p38 단백질의 인산화는 본 발명의 피쳐린 화합물의 처리에 의해 유의적으로 감소하는 것을 확인할 수 있었다(도 4c 내지 4e 참조). ERK, JNK 및 p38 단백질의 인산화 억제는 MAPKs 효소에 대한 지속적인 인산화 반응의 억제로 귀결되며, 이러한 메카니즘이 글루타메이트에 의해 유발된 뇌 신경세포의 세포사멸에 대한 피쳐린 화합물의 핵심 약리 기전이 될 수 있음을 시사한다.As a result, it was confirmed that the phosphorylation of ERK, JNK and p38 proteins increased by glutamate was significantly reduced by treatment with the featurelin compound of the present invention (see FIGS. 4C to 4E). Inhibition of phosphorylation of ERK, JNK, and p38 proteins results in the suppression of the persistent phosphorylation reaction to the MAPKs enzyme, and this mechanism may be a key pharmacological mechanism of the Fethiline compound for glutamate-induced brain neuronal apoptosis. Suggests.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments and are not intended to limit the scope of the present invention to those of ordinary skill in the art. Therefore, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.
Claims (11)
A pharmaceutical composition for neuroprotection containing as an active ingredient the culture medium of the plant endophytes Neosartoriya Fechiri JS-0553 strain or a fischerin compound isolated therefrom.
상기 식물내생균 네오사르토리야 피쳐리 JS-0553 균주는 수탁번호 KACC83025BP로 기탁된 네오사토리아 속 균주인 것을 특징으로 하는 신경보호용 약학적 조성물.
The method of claim 1,
The neuroprotective pharmaceutical composition, characterized in that the plant endogenous bacteria Neo Sartoriya Peitiri JS-0553 strain is a Neosatoria genus strain deposited with accession number KACC83025BP.
상기 추출물은 균주의 균사체 또는 배양액을 추출한 것을 특징으로 하는 신경보호용 약학적 조성물.
The method of claim 1,
The extract is a neuroprotective pharmaceutical composition, characterized in that the mycelium or culture medium of the strain is extracted.
상기 피쳐린(fischerin) 화합물은 하기 화학식 1의 화학식을 갖는 것을 특징으로 하는 신경보호용 약학적 조성물.
[화학식 1]
The method of claim 1,
The fischerin compound is a neuroprotective pharmaceutical composition, characterized in that it has the formula of the following formula (1).
[Formula 1]
A pharmaceutical composition for the prevention and treatment of brain diseases containing the culture medium of the plant endophytes neosartoriya fetiri JS-0553 strain or a fischerin compound isolated therefrom as an active ingredient.
상기 식물내생균 네오사르토리야 피쳐리 JS-0553 균주는 수탁번호 KACC83025BP로 기탁된 네오사토리아 속 균주인 것을 특징으로 하는 뇌질환 예방 및 치료용 약학적 조성물.
The method of claim 5,
The plant endogenous bacteria Neosartoriya featureri JS-0553 strain is a pharmaceutical composition for preventing and treating brain diseases, characterized in that the strain of the genus Neosatoria deposited with accession number KACC83025BP.
상기 피쳐린(fischerin) 화합물은 하기 화학식 1의 화학식을 갖는 것을 특징으로 하는 뇌질환 예방 및 치료용 약학적 조성물.
[화학식 1]
The fischerin compound is a pharmaceutical composition for preventing and treating brain diseases, characterized in that it has the formula of Formula 1.
[Formula 1]
상기 뇌질환은 뇌졸중, 중풍, 치매, 알츠하이머병, 헌팅턴병, 피크(Pick)병, 크로이츠펠트-야콥(Creutzfeld-Jakob)병, 혈전증(thrombosis), 색전증(em-bolism), 일과성 허혈 발작(transient ischemic attack), 소경색(lacune), 뇌졸중, 뇌일혈, 뇌경색, 두부손상(head trauma), 뇌순환대사장애 및 뇌 기능혼수로 구성된 군으로부터 선택되는 어느 하나인 것을 특징으로 하는 뇌질환 예방 및 치료용 약학적 조성물.
The method of claim 5,
The brain diseases are stroke, stroke, dementia, Alzheimer's disease, Huntington's disease, Pick's disease, Creutzfeld-Jakob's disease, thrombosis, em-bolism, transient ischemic attack. attack), small infarction (lacune), stroke, cerebral hemorrhage, cerebral infarction, head trauma, cerebral circulatory metabolic disorder and brain dysfunction coma, characterized in that any one selected from the group consisting of brain disease prevention and treatment pharmaceutical Ever composition.
Neuroprotective health food containing as an active ingredient the culture medium of the plant endophytes Neosartoriya Fechiri JS-0553 strain or a fischerin compound isolated therefrom.
A health food for preventing and improving brain diseases containing the culture medium of the plant endophytes Neosartoriya Peitiri JS-0553 strain or a fischerin compound isolated therefrom as an active ingredient.
상기 식물내생균 네오사르토리야 피쳐리 JS-0553 균주(수탁번호 KACC83025BP)의 배양액에서 피쳐린(fischerin) 화합물을 분리하는 단계;를 포함하는 피쳐린 화합물의 생산방법.Cultivating the plant endophytes Neosartoriya Fechiri JS-0553 strain (accession number KACC83025BP); And
Separating the fischerin compound from the culture medium of the plant endophytes Neosartoriya Fechiri JS-0553 strain (accession number KACC83025BP); a method for producing a featurelin compound comprising a.
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