KR101555987B1 - Neuroprotective composition comprising the extracts or fractions of Vitex agnus-castus L. as an active ingredient - Google Patents
Neuroprotective composition comprising the extracts or fractions of Vitex agnus-castus L. as an active ingredient Download PDFInfo
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- KR101555987B1 KR101555987B1 KR1020120126822A KR20120126822A KR101555987B1 KR 101555987 B1 KR101555987 B1 KR 101555987B1 KR 1020120126822 A KR1020120126822 A KR 1020120126822A KR 20120126822 A KR20120126822 A KR 20120126822A KR 101555987 B1 KR101555987 B1 KR 101555987B1
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- glutamate
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- brain
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Abstract
본 발명은 정조목 (Vitex agnus - castus L.) 추출물 또는 이의 분획물을 유효성분으로 함유하는 신경보호용 조성물에 관한 것으로, 더 구체적으로는 본 발명의 정조목 추출물 또는 이의 분획물이 뇌세포의 특성을 지닌 N18-RE-105 세포주에 글루타메이트(glutamate) 독성 억제 활성에 따른 뇌신경세포의 보호효과 및 낮은 세포독성을 가짐으로써, 신경보호용 약학적 조성물, 뇌질환 예방 및 치료용 약학적 조성물, 또는 상기 목적의 건강식품으로 유용하게 사용될 수 있다.The present invention relates to a Vitex agnus - castus The present invention relates to a neuroprotective composition containing an extract or fraction thereof as an active ingredient. More particularly, the present invention relates to a neuroprotective composition comprising an extract of the present invention or a fraction thereof, wherein the N18-RE-105 cell line has glutamate ) Protective effect of neuronal cells according to toxic inhibitory activity and low cytotoxicity, it can be usefully used as a pharmaceutical composition for neuroprotection, a pharmaceutical composition for preventing and treating brain diseases, or a health food for the above purpose.
Description
본 발명은 정조목(Vitex agnus-castus L.) 추출물 또는 이의 분획물을 유효성분으로 함유하는 신경 보호용 조성물 또는 뇌질환 예방 및 치료용 약학적 조성물에 관한 것이다.
The present invention relates to a neuroprotective composition containing Vitex agnus-castus L. extract or a fraction thereof as an active ingredient or a pharmaceutical composition for preventing or treating brain diseases.
글루타메이트(glutamate)는 중추신경계(CNS)에서 주된 흥분성 신경전달물질(excitatory neuro-transmitter)로 작용하며 대사성 수용기(metabotropic receptor)와 이온성 수용기(ionotropic receptor)들(NMDA-, quisqualate-, kainate-type)과 결합하여 여러 가지 신경 생리학적 효과를 나타낸다. 신경세포 외로 방출된 글루타메이트는 시냅스 전 말단(presynaptic terminal)으로 직접 재흡수되거나 나트륨 이온-의존적 고친화성 수송체(Na+-dependent high affinity transporter)에 의해 성상세포(astrocyte)(교질세포(glial cell))내로 흡수된 후, ATP를 요구하는 글루타민 합성효소(glutamine synthase)에 의해 글루타민(glutamine)으로 전환되어 시냅스 전 말단으로 이동하게 된다.Glutamate acts as a major excitatory neurotransmitter in the central nervous system (CNS) and acts as a metabotropic receptor and an ionotropic receptor (NMDA-, quisqualate-, kainate-type ) And exhibits various neurophysiological effects. The outside neurons release glutamate or absorbed directly re presynaptic terminals (presynaptic terminal) sodium ion-dependent high-affinity transporter (Na + -dependent high affinity transporter) stellate cells (astrocyte) (glial cells (glial cell) by ), And then converted to glutamine by glutamine synthase, which requires ATP, to move to the front end of the synapse.
이러한 시냅스 전 말단과 성상세포의 작용으로 세포 외부의 글루타메이트 농도는 0.3 M의 저농도로 유지되게 된다. 그러나, 뇌가 뇌허혈(ischemia), 저산소증(hypoxia), 발작(seizure), 저혈당증(hypoglycemia), 뇌외상(trauma) 등에 걸리면 글루타메이트 수송에 필요한 에너지 공급이 부족하게 되어, 세포 외 글루타메이트 농도는 정상상태의 10,000배인 3 mM 정도로 증가하며, 이로 인해 중추신경 세포가 죽게 된다. 이러한 글루타메이트에 의한 신경 세포사는 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 세포구성성분의 파괴에 의한 것임이 밝혀짐으로써 이를 억제할 수 있는 자유 라디칼 제거제(free radical scavenger)와 같은 지질과산화 저해물질은 뇌세포를 보호할 수 있는 새로운 치료소재로서 매우 중요한 의미가 있다.
These synaptic terminals and the action of astrocytes cause the glutamate concentration outside the cells to be maintained at a low concentration of 0.3 M. However, when the brain is subjected to ischemia, hypoxia, seizure, hypoglycemia, trauma, etc., the energy supply required for glutamate transportation becomes insufficient, and the extracellular glutamate concentration is reduced to a normal state 3,000, which is about 10,000 times, which causes the central nerve cell to die. These glutamate-induced neuronal cell deaths are called glutamate-induced neurotoxicity and are closely related to the oxidative stress caused by the generation of free radicals in the cells. Therefore, it has been clarified that the death of neurons by glutamate toxicity is caused by the destruction of cell components such as lipids, sugars, nucleic acids and proteins due to free radicals generated in cells excessively, and thus a free radical scavenger free radical scavenger) are very important as a new therapeutic agent for protecting brain cells.
글루타메이트의 세포 외 농도 증가는 뇌허혈, 뇌졸중, 뇌외상, 저산소증, 저혈당증, 발작 등의 급성질환뿐만 아니라, 헌팅턴 병(Huntington's disease, HD), 파킨슨 병(Parkinson's disease, PD), 근위축성 측색 경화증(amyotrophic lateral sclerosis, ALS), 알츠하이머병(Alzheimer's disease, AD)과 같은 만성질환에도 관여하는 것으로 알려져있다. NIH의 조사 결과에 의하면 급성질환의 하나인 뇌졸중(stroke)은 미국인의 세 번째 사망원인이며, 성인불구의 가장 큰 원인이 되고 있다. 매년 50만 명의 환자가 발생하며, 이중 30%는 사망하고 20 ~ 30%는 영구 불구자가 되고 나머지는 신체 불구, 신체마비, 시력감퇴, 기억상실 등의 고통을 받고 있다. 또한 우리나라에서도 암에 이어서 사망 원인 2위의 중요한 질병으로 대두하여, 이러한 질병에 대한 치료제가 강력히 요구되고 있다.
Increased extracellular concentrations of glutamate are associated with 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 sclerosis lateral sclerosis, ALS), and Alzheimer's disease (AD). NIH research shows that stroke, one of the acute illnesses, is the third leading cause of death in the United States and is the leading cause of adult disability. About half a million patients die each year, of which 30% die, 20-30% become permanent cramps, and the rest suffer physical disabilities, body paralysis, vision loss, and memory loss. Also, in Korea, cancer is the second most important cause of death following cancer, and therefore there is a strong demand for a treatment for such diseases.
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 신경 세포사를 야기하지는 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, resulting in excitotoxic damage. On the other hand, in metabolic receptors, glutamate does not cause direct neuronal death, unlike ionic receptors.
이온성 수용기에 글루타메이트가 결합하여 일으키는 신경 세포사는 Na+와 Cl- 의존성과 Ca2 + 의존성의 두 가지로 나눌 수 있다. Na+와 Cl- 의존성 신경 세포사는 글루타메이트에 노출된 후, 수 분 안에 일어나며 감극 물질(depolarizing agent)을 처리한 것과 유사한 신경 세포사를 보인다. 글루타메이트가 쿼스퀄산 수용기(quisqualate receptor)를 자극하여 Na+이온이 세포 내로 과량 유입되며, Na+이온의 유입과 함께 Cl-, H2O가 세포 내부로 들어와 세포가 팽창(swelling)하여 결국 용해(lysis)된다. Neuronal cell death caused by binding of glutamate to ionic receptors can be divided into Na + , Cl - dependent and Ca 2 + dependent. Na + and Cl - dependent neuronal cells develop within a few minutes after exposure to glutamate and show neuronal cell death similar to the depolarizing agent treatment. Glutamate query Squelch acid receptor (quisqualate receptor) to stimulate the Na + ions and the excess flows into the cell, with the influx of Na + ions Cl -, finally dissolved in H 2 O is entered into the cells, cell expansion (swelling) lt; / RTI >
Ca2 + 의존성 신경 세포사는 딜레이드 신경 손상(delayed neuronal damage)로 글루타메이트 처리 후 수 시간이 지나서 일어나며, Ca2 + 이온투과담체(ionophore)인 A23187을 처리하였을 때와 유사하다. Na+이온의 유입은 세포막의 탈분극(depolarization)을 야기하며 이로 인해 전압 민감성 있는 Ca2 + 채널(voltage sensitive Ca2 + channel, VSCC)과 Mg2 +에 의해서 닫혀 있던 NMDA 수용체 Ca2 + 채널이 열리게 된다. 이로 인해 다량의 Ca2 +이 세포 내로 유입되게 된다. 이렇게 유입된 Ca2 + ion은 포스포리파아제(phospholipase A2, PLA2), 산화질소 합성효소(nitric oxide synthetase, NOS), 프로테아제(protease), 엔도뉴클레아제(endonuclease)등의 Ca2 + 의존성 효소들을 활성화한다. 특히 PLA2는 인지질(phospholipid)를 분해하여 아라키돈산(arachidonic acid)을 생성시키며, 아라키돈산의 대사과정 중에 과산화물(superoxide)과 같은 자유 라디칼이 발생한다. Ca + 2-dependent neuronal cell death delay DE nerve injury (delayed neuronal damage) to occur after a few hours after glutamate treatment, similar to when the processing of A23187 Ca 2 + ion permeable carrier (ionophore). Influx of Na + ions opens the NMDA receptor Ca 2 + channel which is closed by causing depolarization (depolarization) of the cell membrane, which causes the voltage-sensitive Ca 2 + channels (voltage sensitive Ca 2 + channel, VSCC) and Mg 2 + with do. This causes a large amount of Ca + 2 are to be introduced into the cell. The thus introduced Ca 2 + ion is Phospholipase (phospholipase A 2, PLA 2) , nitric oxide synthase (nitric oxide synthetase, NOS), Protease (protease), endonuclease (endonuclease) Ca 2 + dependence of such Energize enzymes. In particular, PLA 2 degrades phospholipids to produce arachidonic acid, and free radicals such as superoxide are produced during the metabolism of arachidonic acid.
또한, 아라키돈산과 과산화물은 시냅스전 뉴런(presynaptic neuron)에서의 글루타메이트 분비를 촉진시켜 세포 외의 글루타메이트 농도를 더욱 증가시킨다. 크산틴산화효소(Xanthine oxidase)의 작용으로 과산화물들이 생성되며, NOS의 작용으로 생성된 산화 질소(nitric oxide)는 과산화물과 반응하여 페록시나이트라이트(peroxynitrite)를 거쳐 반응성이 큰 히드록실 라디칼(hydroxyl radical)이 생성된다. 즉, PLA2, NOS, 크산틴 산화효소(xanthine oxidase) 등의 효소 작용 결과 세포 내에 자유 라디칼이 과다하게 생성되어 이로 인한 산화 스트레스(oxidative stress)로 인하여 DNA, 단백질(protein), 지질(lipid) 등이 비선택적으로 파괴되어 신경세포가 죽게 되며 또한 엔도뉴클레아제(endonuclease)의 작용으로 세포자멸사(apoptosis)가 일어나기도 한다.
In addition, arachidonic acid and peroxides promote glutamate release in presynaptic neurons, further increasing the extracellular glutamate concentration. Peroxides are formed by the action of xanthine oxidase. Nitric oxide produced by the action of NOS reacts with peroxides to pass through peroxynitrite and reacts with hydroxyl radicals (hydroxyl radical is generated. In other words, enzymatic action of PLA 2 , NOS, xanthine oxidase, etc., results in excess radicals in the cells, resulting in oxidative stress resulting in DNA, protein, lipid, And nonselective destruction of the nerve cells, and also endonuclease (endonuclease) by the action of apoptosis may occur.
이러한 글루타메이트의 독성기작을 연구하기 위하여 글루타메이트 독성에 민감한 N18-RE-105 세포주가 선택되었다. N18-RE-105 세포주는 마우스 신경모세포종 클론(mouse neuroblastoma clone) N18TG-2 와 18일된 피셔 랫(Fisher rat)의 배아의 신경 망막 세포(embryonic neural retina)의 세포융합에 의해서 만들어졌다. To investigate the toxic mechanism of this glutamate, the N18-RE-105 cell line sensitive to glutamate toxicity was selected. The N18-RE-105 cell line was generated by cell fusion of embryonic neural retina of mouse neuroblastoma clone N18TG-2 and 18-day-old Fisher rat embryos.
N18-RE-105 세포주는 해마(hippocampus)에 존재하는 글루타메이트 수용기(glutamate receptor)와 유사한 수용기가 있고, 세포 내 자유 라디칼의 축적을 통한 산화적 손상에 의한 세포사멸을 일으키는 딜레이드 칼슘 의존성 세포 사멸(delayed calcium dependent cell death)를 보이며, 이는 글루타메이트 독성에 의한 실제 뇌신경 세포사멸과 매우 유사한 특징을 가진다. 따라서, 이러한 특성으로 인해 상기 N18-RE-105 세포주는 시험관 내 허혈 모델(in vitro ischemic model)및 글루타메이트 독성 기작에 이용된다. The N18-RE-105 cell line has a receptor similar to the glutamate receptor present in the hippocampus and has the ability to inhibit the production of delocalized calcium dependent cell death resulting in oxidative damage through accumulation of intracellular free radicals delayed calcium dependent cell death, which is very similar to actual brain cell death due to glutamate toxicity. Therefore, due to these properties, the N18-RE-105 cell line is used for an in vitro ischemic model and a glutamate toxicity mechanism.
N18-RE-105 세포주에서의 글루타메이트 독성은 글루타메이트에 의한 시스테인 흡수(cystein uptake)의 저해에서 기인하는 것으로 알려져 있다. 시스테인은 세포 내에서 글루타시온(glutathione)을 구성하는 중요한 아미노산이다. N18-RE-105 세포에서는 상기 시스테인과 글루타메이트는 세포막을 통하여 서로 연관하면서 서로 반대방향으로 수송된다. 세포 외의 글루타메이트 농도가 증가하여 시스테인이 세포내로 유입되지 않으면 글루타시온의 농도가 감소하게 되며 세포 내에 자유 라디칼이 축적되어 결국 신경세포는 죽게 된다. 이러한 사실은 또한 배아의 대뇌피질성 신경(embryonic cortical neuron)의 초대 배양(primary culture)에서도 확인되어 쿼스퀄산-형 글루타메이트 독성(quisqualate-type glutamate toxicity)은 주로 산화 스트레스(oxidative stress)에 의한 것임이 시사되었다.
Glutamate toxicity in the N18-RE-105 cell line is known to result from the inhibition of cysteine uptake by glutamate. Cysteine is an important amino acid that constitutes glutathione in cells. In N18-RE-105 cells, the cysteine and glutamate are transported in opposite directions relative to each other through the cell membrane. If the extracellular glutamate concentration increases and the cysteine does not enter the cell, the concentration of glutathione decreases and free radicals accumulate inside the cell, eventually causing the neuron to die. This fact is also confirmed in the primary culture of the embryonic cortical neurons of the embryo, and the quisqualate-type glutamate toxicity is mainly due to oxidative stress. .
이상과 같이 글루타메이트가 뇌졸중(stroke), 뇌외상(trauma) 등과 같은 급성 뇌신경질환의 원인이 되며, 또한 PD, AD, HD와 같은 만성 뇌질환에서도 글루타메이트 독성에 의한 산화적 스트레스가 그 원인 중 하나라고 알려져 있다. 이에 따라 글루타메이트 독성에 의한 산화적 스트레스를 억제 또는 완화할 수 있는 지질과산화 억제물질과 같은 항산화 활성물질은 급성, 만성의 뇌신경 질환의 치료제로 기대된다. 이에 따라 in vivo에 가까운 조직(tissue) 또는 배양세포를 이용하여 글루타메이트 독성 억제물질을 탐색하면 지금까지 알려진 표적에 작용하는 물질뿐만 아니라, 새로운 신경세포 보호기작을 갖는 새로운 물질의 창출도 가능하리라 기대된다.
As described above, glutamate causes acute cranial nerve diseases such as stroke and trauma, and oxidative stress due to glutamate toxicity is one of the causes in chronic brain diseases such as PD, AD and HD It is known. Accordingly, antioxidant active substances such as lipid peroxidation inhibiting substances that can suppress or alleviate oxidative stress due to glutamate toxicity are expected to be therapeutic agents for acute and chronic neurological diseases. Therefore, searching for inhibitors of glutamate toxicity using tissues or cultured cells close to in vivo is expected to create new substances with new neuronal cell protection mechanisms as well as substances acting on known targets .
정조목(Vitex agnus-castus L.)과 관련된 기존의 특허 및 문헌 보고는 정조목에서 추출물을 수득하는 방법, 정조목 추출물의 항산화 활성, 정조목 열매 추출물(저용량 120 mg/d)은 FSH 감소, LH 증가를 통해 에스트로젠 감소, 프로제스테론과 프로락틴 증가를 유발하고 고용량(480 mg/d)에서는 프로락틴을 억제하는 것으로 보이며, 도파민 수용체(D2) 작용제의 역할에 대해 보고되고 있다. 또한 월경 전 증후군, 폐경기 증후군, 자궁내막증 등에 효과가 있으며, 이 밖에도 항염증 활성이 있는 것으로 보고되어 있다. 그러나 뇌 신경세포 보호 활성에 관하여는 아직까지 보고된 바가 없다.
Existing patents and literature reports related to Vitex agnus-castus L. have shown that the method of obtaining extracts from Jungjo tree, the antioxidant activity of mungbean tree extract, the extract of Jungjo tree (low dose 120 mg / d) Increased LH leads to estrogen deprivation, progesterone and prolactin elevation, and high dose (480 mg / d) inhibition of prolactin and the role of dopamine receptor (D2) agonists has been reported. It is also effective in premenstrual syndrome, menopausal syndrome, and endometriosis, and has also been reported to have anti-inflammatory activity. However, there has been no report on the activity of cranial nerve cell protection.
글루타메이트에 의해서 야기되는 뇌졸중이 세계적으로 심각한 질병으로 대두하면서 이에 대한 치료제 개발이 활발히 진행되고 있다. 특히, 활성산소가 글루타메이트 독성에 의한 신경 세포사의 주된 최종 원인 물질이기 때문에 새로운 자유 라디칼 제거제 또는 지질과산화 억제제와 같은 항산화 물질에 대한 연구가 활발히 진행되어 왔다. Upjohn사가 개발한 21-아미노 스테로이드(21-amino steroid)의 아민 단위(amine unit)와 비타민 E(vitamin E)의 테트라메틸-크로만 링(tetramethyl-chroman ring)으로 구성된 지질과산화 저해물질 U78517F 화합물은 in vivo 실험에서 강한 뇌졸중 치료효과를 보인다고 보고되었으며, 벤조푸란(benzofuran)을 기본골격으로 하는 항산화 활성물질 IRFI-016은 뇌허혈후 재환류시 뇌세포의 손상을 크게 억제하는 효과를 나타낸다고 보고되었다. 또한 활성산소 소거물질로 알려진 퀴논(quinonoe)계의 이데베논(idebenone)은 N18-RE-105 세포에서의 글루타메이트 세포독성을 강력하게 저해하는데, in vivo 실험에서도 뇌동맥경화증 및 뇌혈관 장애성 치매에 대하여 치료효과를 나타내어, 현재 일본에서 심장수술 후, 장기이식 후의 뇌대사 부활제로서 임상시험중인 물질로 알려져 있다. 1996년 6월에 일본 Takeda사에서 개발한 과산화 저해제인 TAK-218 화합물은 활성산소와 비정상적인 자유 도파민(free dopamine)을 소거함으로써 신경보호(neuroprotective)와 항-허혈(anti-ischemic) 효과를 보인다고 보고되었다.BACKGROUND ART A stroke caused by glutamate is becoming a worldwide serious disease, and development of a therapeutic agent for it is actively under way. Particularly, active oxygen has been actively studied for antioxidants such as novel free radical scavengers or lipid peroxidation inhibitors since it is the main ultimate source of neuronal death due to glutamate toxicity. Upjohn's U78517F compound, a lipid peroxidation inhibitor composed of an amine unit of 21-amino steroid and a tetramethyl-chroman ring of vitamin E, It has been reported that IRFI-016, which is a basic skeleton of benzofuran, significantly inhibits damage of brain cells in cerebral ischemia after reperfusion. In addition, quinonone idebenone, known as the active oxygen scavenging agent, strongly inhibits glutamate cytotoxicity in N18-RE-105 cells. In vivo experiments, it is also known that cerebrovascular atherosclerosis and cerebrovascular dementia It is known as a substance that is currently under clinical trials in Japan as a brain metabolism activator after cardiac surgery and after organ transplantation. The TAK-218 compound, a peroxidase inhibitor developed by Takeda in Japan in June 1996, showed neuroprotective and anti-ischemic effects by eliminating free oxygen and abnormal free dopamine .
아울러, 글루타메이트 독성과 관련하여, 글루타메이트로 독성을 유발한 생쥐의 해마 유래 세포주인 HT22 세포주를 대상으로 백두산 자생식물 추출물이 세포생존율에 대한 증가 여부를 관찰하여 뇌 세포 보호 활성을 평가한 논문이 보고된바 있다(생약학회지, Kor. J. Pharmacogn. 39(3) : 213217 (2008)). 또한, 국내외에서 자생하고 있는 435가지의 약용식물 추출물을 대상으로 뇌신경 세포계 융합세포(hybridoma) N18-RE-105 세포주를 이용하여 신경세포 보호효과를 갖는 천연물의 탐색을 시도한 결과, 제비꽃(Viola mandshurica)으로부터 강력한 신경세포 보호효과를 확인하고 이에 대한 내용이 등록된바 있다(대한민국 등록특허 10-0982022). 아울러, 둥근마 조추출물 및 비극성 용매 가용추출물이 글루타메이트 및 과산화수소 신경 독성에 의한 세포사멸을 차단하여 뇌신경 세포 보호활성에 유의적인 효과를 확인한바 있다(대한민국 등록특허 10-0776347). 그러나, 정조목 추출물 또는 이의 분획물의 뇌신경 세포 보호활성에 관하여는 아직까지 보고된 바가 전혀 없다.
In addition, in relation to glutamate toxicity, a study evaluating brain cell protective activity by observing an increase in cell survival rate of a plant extract of Pseudomonas aeruginosa from HT22 cell line, a hippocampus-derived cell line of mice induced by glutamate toxicity, was reported (Journal of Pharmacological Sciences, Kor. J. Pharmacogn. 39 (3): 213217 (2008)). In addition, 435 medicinal plant extracts which are naturally grown at home and abroad were searched for natural products having nerve cell protection effect by using neural cell line hybridoma N18-RE-105 cell line. As a result, Viola mandshurica ), and the contents thereof have been registered (Korean Patent No. 10-0982022). In addition, the round maturation extract and the non-polar solvent soluble extract blocked the apoptosis caused by glutamate and hydrogen peroxide neurotoxicity, thus confirming the significant effect on neuronal cell protection activity (Korean Patent No. 10-0776347). However, there has been no report on the neuronal cell protective activity of the mungbean tree extract or its fractions.
이에, 본 발명자들은 신경세포 보호효과를 갖는 천연물질을 개발하기 위해 노력하는 중, 뇌신경세포에 대한 글루타메이트 독성억제 활성을 갖는 정조목 (Vitex agnus - castus L.) 추출물 또는 이의 분획물을 스크리닝을 하였고, 특히 glutamate 독성에 민감한 뇌신경세포의 특징을 지닌 신경세포 융합(neuronal hybridoma) 세포인 N18-RE-105 세포주 대하여 정조목 추출물 또는 이의 분획물의 글루타메이트 독성억제 활성을 조사하여 신경세포를 보호하는 활성이 매우 뛰어나다는 것을 확인하였다. 따라서 본 발명의 정조목 추출물 또는 이의 분획물이 신경세포에 대하여 글루타메이트 독성을 억제하고, 세포독성이 거의 없으므로 신경보호용 조성물 또는 뇌질환 예방 및 치료용 조성물의 유효성분으로 유용하게 사용될 수 있음을 밝힘으로써, 본 발명을 완성하였다.
Accordingly, the present inventors have made efforts to develop a natural substance having a nerve cell protection effect, and have found that a compound having a glutamate toxicity-Vitex agnus - castus L.) extracts or fractions thereof were screened. In particular, the N18-RE-105 cell line, which is a neuronal hybridoma cell having characteristics of neuronal cells sensitive to glutamate toxicity, has a glutamate toxicity inhibitory activity And that the activity of protecting neurons was excellent. Accordingly, the present invention provides a neuroprotective agent or a composition for preventing or treating brain diseases, which is effective for inhibiting glutamate toxicity to nerve cells and having little cytotoxicity, Thus completing the present invention.
본 발명의 목적은 정조목(Vitex agnus-castus L.) 추출물 또는 이의 분획물을 유효성분으로 함유하는 신경보호용 조성물 및 뇌질환 예방 및 치료용 약학적 조성물을 제공하는 것이다.
It is an object of the present invention to provide a nerve-protecting composition containing Vitex agnus-castus L. extract or a fraction thereof as an active ingredient, and a pharmaceutical composition for preventing and treating brain diseases.
상기 목적을 달성하기 위하여, 본 발명은 정조목(Vitex agnus-castus L.) 추출물 또는 이의 분획물을 유효성분으로 함유하는 신경보호용 약학적 조성물을 제공한다.In order to achieve the above object, the present invention provides a pharmaceutical composition for neuroprotection comprising an extract of Vitex agnus-castus L. or a fraction thereof as an active ingredient.
또한, 본 발명은 정조목 추출물 또는 이의 분획물을 유효성분으로 함유하는 뇌질환 예방 및 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing and treating brain diseases, which comprises an extract of Orthoptera, or a fraction thereof, as an active ingredient.
또한, 본 발명은 정조목 추출물 또는 이의 분획물을 유효성분으로 함유하는 신경보호용 건강식품을 제공한다.In addition, the present invention provides a health food for neuroprotection comprising an extract of Orthodox japonica or a fraction thereof as an active ingredient.
아울러, 본 발명은 정조목 추출물 또는 이의 분획물을 유효성분으로 함유하는 뇌질환 예방 및 개선용 건강식품을 제공한다.
In addition, the present invention provides a health food for preventing or ameliorating brain diseases, which comprises an extract of Orthoptera, or a fraction thereof, as an active ingredient.
본 발명은 정조목(Vitex agnus-castus L.) 추출물 또는 이의 분획물은 신경세포의 독성을 유의적으로 억제하는 효과가 뛰어나고, 세포독성이 없어 안전하므로, 상기 정조목 추출물 또는 이의 분획물을 신경보호용 조성물 또는 뇌질환 예방 및 치료용 조성물의 유효성분으로 유용하게 이용할 수 있다.
The present invention is based on the finding that the Vitex agnus-castus L. extract or its fractions are excellent in the effect of significantly inhibiting the toxicity of nerve cells and are safe from lack of cytotoxicity, Or a composition for preventing and treating brain diseases.
도 1은 글루타메이트(glutamate)로 매개 된 흥분독성(excitotoxic)의 과정을 나타내는 도이다.
VSCC; 전압-민감성 칼슘 채널(voltage-sensitive calcium channel),
NOS; 산화 질소 합성효소(nitric oxide synthase),
PLA2; 포스포리파아제 A2(phospholipase A2),
AA; 아라키돈산(arachidonic acid),
DG; 디아실글리세롤(diacyl glycerol),
Glut; 글루타메이트(glutamate) 및
IP3; 이노시톨 3인산염(inositol triphosphate).
도 2는 정조목(Vitex agnus-castus L.) 추출물의 N18-RE-105 신경세포 보호활성 확인한 결과를 나타내는 도이다.
A. 글루타메이트를 처리하지 않은 N18-RE-105 세포주.
B. 글루타메이트(20 mM)를 처리한 N18-RE-105 세포주.
C. 글루타메이트(20 mM)와 정조목 추출물 45 /를 처리한 N18-RE-105 세포주.
도 3은 정조목 추출물 및 이의 분획물의 N18-RE-105 신경 세포주에 대한 글루타메이트 독성억제 효과를 나타내는 도이다.
(-) Glut: 글루타메이트(20 mM)를 처리하지 않은 N18-RE-105 세포주,
(+) Glut: 글루타메이트(20 mM)를 처리한 N18-RE-105 세포주.
Vitex Ext: 정조목 추출물;
CHCl3 Ext: 정조목 추출물의 클로로포름 분획물;
EtOAC Ext: 정조목 추출물의 에틸아세테이트 분획물; 및
BuOH Ext: 정조목 추출물의 부탄올 분획물.
도 4는 정조목 분획물을 제조하는 과정을 나타내는 도이다.1 is a diagram illustrating the process of glutamate mediated excitotoxic.
VSCC; Voltage-sensitive calcium channel,
NOS; Nitric oxide synthase,
PLA 2 ; Phospholipase A 2 (phospholipase A 2),
AA; Arachidonic acid,
DG; Diacyl glycerol,
Glut; Glutamate and
IP 3 ; Inositol triphosphate.
FIG. 2 is a graph showing the results of confirming N18-RE-105 neuronal cell protection activity of Vitex agnus-castus L. extract. FIG.
A. N18-RE-105 cell line without glutamate treatment.
B. N18-RE-105 cell line treated with glutamate (20 mM).
C. N18-RE-105 cell line treated with glutamate (20 mM) and Calendula wood extract 45 /.
FIG. 3 is a graph showing the inhibitory effect of glutamate toxicity on the N18-RE-105 neuronal cell line of the crude extract and its fractions.
(-) Glut: N18-RE-105 cell line not treated with glutamate (20 mM)
(+) Glut: N18-RE-105 cell line treated with glutamate (20 mM).
Vitex Ext: Cedarwood extract;
CHCl 3 Ext: Chloroform fraction of mature wood extracts;
EtOAC Ext: Ethyl acetate fractions of mulberry tree extract; And
BuOH Ext: Butanol fraction of mulberry tree extract.
FIG. 4 is a view showing a process for producing a crude woody fraction; FIG.
이하, 본 발명을 상세히 설명한다.
Hereinafter, the present invention will be described in detail.
본 발명은 정조목(Vitex agnus - castus L.) 추출물 또는 이의 분획물을 유효성분으로 함유하는 신경보호용 약학적 조성물을 제공한다.The present invention relates to a Vitex agnus - castus L.) extract or a fraction thereof as an active ingredient.
상기 정조목 추출물은 하기의 단계들을 포함하는 제조방법에 의해 제조되는 것이 바람직하나 이에 한정되지 않는다:The crude extract is preferably, but not exclusively, prepared by a process comprising the steps of:
1) 정조목에 추출용매를 가하여 추출하는 단계;1) extracting an extractant by adding an extraction solvent to the mixture;
2) 단계 1)의 추출물을 여과하는 단계; 및2) filtering the extract of step 1); And
3) 단계 2)의 여과한 추출물을 감압농축한 후 건조하는 단계; 및3) concentrating the filtered extract of step 2) under reduced pressure and drying; And
4) 단계 3)의 정조목 추출물을 추가적으로 유기용매로 추출하여 정조목 분획물을 제조하는 단계. 4) Extracting the crude extract of step 3) with an organic solvent to prepare a fraction of the purified water.
상기 방법에 있어서, 단계 1)의 정조목은 재배한 것 또는 시판되는 것 등 제한 없이 사용할 수 있다. 상기 정조목은 잎, 줄기 또는 뿌리가 모두 이용가능하며, 이에 한정되지 않는다.In the above method, the seedling of step 1) may be cultivated or commercially available. The tubular tree may be a leaf, a stem, or a root, but is not limited thereto.
상기 방법에 있어서, 상기 단계 1)의 상기 추출용매는 물, 알코올 또는 이들의 혼합물을 사용하는 것이 바람직하다. 상기 알코올로는 C1 내지 C2 저급 알코올을 이용하는 것이 바람직하며, 저급 알코올로는 또는 메탄올을 이용하는 것이 바람직하다. 추출방법으로는 진탕추출, Soxhlet 추출 또는 환류추출을 이용하는 것이 바람직하나 이에 한정되지 않는다. 상기 추출용매를 건조된 정조목 분량에 2 내지 20배 첨가하여 추출하는 것이 바람직하다. 추출온도는 20 내지 50℃인 것이 바람직하나 이에 한정하지 않는다. 또한, 추출시간은 10 내지 48시간인 것이 바람직하며, 24시간이 더욱 바람직하나 이에 한정하지 않는다. 아울러, 추출 회수는 3 내지 5회인 것이 바람직하며, 3회 반복 추출하는 것이 더욱 바람직하나 이에 한정되는 것은 아니다. In the above method, it is preferable to use water, an alcohol or a mixture thereof in the extraction solvent in the step 1). As the alcohol, it is preferable to use a C 1 to C 2 lower alcohol, and it is preferable to use methanol as the lower alcohol. As the extraction method, it is preferable to use shaking extraction, Soxhlet extraction or reflux extraction, but it is not limited thereto. It is preferable to extract the extraction solvent by 2 to 20 times the amount of the dried preparation. The extraction temperature is preferably 20 to 50 DEG C, but is not limited thereto. The extraction time is preferably 10 to 48 hours, more preferably 24 hours, but not always limited thereto. In addition, the extraction number is preferably 3 to 5 times, more preferably 3 times, but not limited thereto.
상기 방법에 있어서, 단계 3)의 감압농축은 진공감압농축기 또는 진공회전증발기를 이용하는 것이 바람직하나 이에 한정하지 않는다. 또한, 건조는 감압건조, 진공건조, 비등건조, 분무건조 또는 동결건조하는 것이 바람직하나 이에 한정하지 않는다.In the above method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in step 3), but it is not limited thereto. The drying is preferably performed under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, but not always limited thereto.
상기 방법에 있어서, 단계 3)의 감압농축은 진공감압농축기 또는 진공회전증발기를 이용하는 것이 바람직하나 이에 한정하지 않는다. 또한, 건조는 감압건조, 진공건조, 비등건조, 분무건조 또는 동결건조하는 것이 바람직하나 이에 한정하지 않는다.In the above method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in step 3), but it is not limited thereto. The drying is preferably performed under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, but not always limited thereto.
상기 방법에 있어서, 단계 4)의 유기용매는 n-헥산, 클로로포름, 에틸아세테이트 또는 부탄올인 것이 바람직하나, 이에 한정하지 않는다. 상기 분획물은 정조목 추출물을 물에 현탁시킨 후 n-헥산, 클로로포름, 에틸아세테이트, 부탄올 및 물로 순차적으로 계통 분획하여 수득한 n-헥산 분획물, 클로로포름 분획물, 에틸아세테이트 분획물, 부탄올 분획물 또는 물 분획물인 것이 바람직하나, 이에 한정하지 않는다. 상기 분획물은 상기 정조목 추출물로부터 분획 과정을 1 내지 5회, 바람직하게는 3회 반복하여 수득할 수 있고, 분획 후 감압 농축하는 것이 바람직하나 이에 한정하지 않는다
In the above method, the organic solvent in step 4) is preferably n-hexane, chloroform, ethyl acetate or butanol, but is not limited thereto. The fraction is a n-hexane fraction, a chloroform fraction, an ethyl acetate fraction, a butanol fraction, or a water fraction obtained by suspending a crude extract in water and sequentially fractionating the fraction with n-hexane, chloroform, ethyl acetate, butanol and water But is not limited thereto. The fractions can be obtained by repeating the fractionation process from the crude extract to 1 to 5 times, preferably 3 times, followed by fractionation and concentration under reduced pressure, but it is not limited thereto
본 발명의 구체적인 실시예에서, 본 발명자들은 정조목을 이용하여 정조목 추출물 및 이의 분획물을 제조하였다(도 4참조). In a specific embodiment of the present invention, the present inventors have prepared a crude tree extract and fractions thereof using a planting tree (see FIG. 4).
또한, 본 발명자들은 정조목 추출물 또는 이의 분획물의 신경세포 보호효과를 확인하기 위하여, 고농도의 글루타메이트를 처리한 N18-RE-105 세포주에 본 발명의 정조목 추출물 또는 이의 분획물을 처리한 결과, 세포사멸을 유의적으로 억제하여 신경세포 보호활성이 우수한 것을 확인하였다(도 2 참조).Further, in order to examine the protective effect of neuronal cells on the extracts or fractions thereof, the inventors of the present invention treated the N18-RE-105 cell line treated with high-concentration glutamate, or the fractions thereof, (Fig. 2). As shown in Fig.
또한, 글루타메이트 독성을 50% 억제하는 정조목 추출물 또는 이의 분획물의 농도(IC50)를 확인한 결과, 정조목 추출물의 농도는 17.1 ㎍/㎖로서 양성대조군이 비타민 E의 21.5 ㎍/㎖와 보다 높은 신경세포 보호활성을 나타내었고, 정조목 클로로포름 분획물 및 에틸아세테이트 분획물은 각각 14.9 ㎍/㎖ 및 15.1 ㎍/㎖로 높은 신경세포 보호활성을 나타내는 것을 확인하였다(표 1 참조).In addition, the concentration (IC 50 ) of the caterpillar fungus extract or its fraction (50%) inhibiting glutamate toxicity was found to be 17.1 ㎍ / ㎖, and the positive control group had 21.5 ㎍ / Cell protective activity, and the purified chloroform fraction and ethyl acetate fraction showed high neuronal cell protection activity of 14.9 ㎍ / ㎖ and 15.1 ㎍ / ㎖, respectively (see Table 1).
또한, 본 발명자들은 정조목 추출물 또는 이의 분획물의 농도에 따른 글루타메이트 독성억제효과를 확인하기 위하여, N18-RE-105 신경 세포주에 정조목 추출물 및 이의 분획물의 최종농도(13.6, 22.7, 및 45.5 ㎍/㎖)로 처리하였을 때, 농도 의존적으로 글루타메이트에 의한 N18-RE-105의 세포사멸을 억제하는 것을 확인하였다. 특히, 클로로포름 분획물 22.7 ㎍/㎖을 처리한 세포들은 글루타메이트에 의한 신경세포 사멸이 완전히 억제되어 100%의 세포가 살아있는 매우 뛰어난 신경세포 보호활성이 관찰되었다(도 3 참조).In order to confirm the inhibitory effect of glutamate toxicity according to the concentration of the extracts or their fractions, the final concentration (13.6, 22.7, and 45.5 占 퐂 / ml) of the crude extract and its fractions in the N18- Ml), it was confirmed that the cell death of N18-RE-105 by glutamate was inhibited in a concentration-dependent manner. In particular, the cells treated with 22.7 / / ml of the chloroform fraction were completely inhibited from glutamate-induced neuronal cell death, and 100% of the cells were observed to exhibit excellent neuronal cell protection activity (see FIG. 3).
또한, 본 발명자들은 정조목 추출물 또는 이의 분획물의 세포독성을 확인한 결과, 정조목 추출물 또는 이의 분획물은 활성유효농도보다 약 5배 높은 80 ㎍/㎖의 고농도에서도 세포독성이 나타나지 않는 것을 확인하였다.Furthermore, the present inventors confirmed that cytotoxicity of the extracts of Cryptomeria japonica or its fractions was not cytotoxic even at a high concentration of 80 μg / ml, which is about 5 times higher than the effective concentration of the Cryptomeria japonica extract or its fractions.
따라서, 본 발명의 정조목 추출물 또는 이의 분획물은 신경세포의 독성을 유의적으로 억제하는 효과가 뛰어나고, 세포 독성이 없어 인체에 안전함으로 신경보호용 약학적 조성물의 유효성분으로 유용하게 사용될 수 있다.
Accordingly, the extract of the present invention or its fractions thereof is excellent in the effect of significantly inhibiting the toxicity of neurons, and is safe for human body because of its cytotoxicity, so that it can be effectively used as an active ingredient of a pharmaceutical composition for neuroprotection.
본 발명의 정조목 추출물 또는 이의 분획물을 함유하는 조성물은 상기 성분에 추가로 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 함유할 수 있다. The composition containing the modified extract of the present invention or a fraction thereof may further contain one or more active ingredients showing the same or similar functions in addition to the above components.
본 발명의 조성물은 약제학적으로 허용 가능한 첨가제를 더 포함할 수 있으며, 이때 약제학적으로 허용 가능한 첨가제로는 전분, 젤라틴화 전분, 미결정셀룰로오스, 유당, 포비돈, 콜로이달실리콘디옥사이드, 인산수소칼슘, 락토스, 만니톨, 엿, 아라비아고무, 전호화전분, 옥수수전분, 분말셀룰로오스, 히드록시프로필셀룰로오스, 오파드라이, 전분글리콜산나트륨, 카르나우바 납, 합성규산알루미늄, 스테아린산, 스테아린산마그네슘, 스테아린산알루미늄, 스테아린산칼슘, 백당, 덱스트로스, 소르비톨 및 탈크 등이 사용될 수 있다. 본 발명에 따른 약제학적으로 허용 가능한 첨가제는 상기 조성물에 대해 0.1 ~ 90 중량부 포함되는 것이 바람직하나 이에 한정되는 것은 아니다.The composition of the present invention may further comprise a pharmaceutically acceptable additive, wherein pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose Starch glycolate, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, calcium stearate, , White sugar, dextrose, sorbitol and talc. The pharmaceutically acceptable additives according to the present invention are preferably included in the composition in an amount of 0.1 to 90 parts by weight, but are not limited thereto.
즉, 본 발명의 조성물은 실제 임상 투여 시에 경구 및 비경구의 여러 가지 제형으로 투여될 수 있는데, 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형 제제는 정조목 추출물 또는 이의 분획물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(Calcium carbonate), 수크로스(Sucrose), 락토오스(Lactose) 또는 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용될 수 있다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제 및 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함될 수 있다. 비수성용제, 현탁용제로는 프로필렌글리콜(Propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.That is, the composition of the present invention can be administered in various formulations of oral and parenteral administration at the time of actual clinical administration. In the case of formulation, a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, . ≪ / RTI > Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, Sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions and syrups, and various excipients such as wetting agents, sweetening agents, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are commonly used simple diluents . Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.
본 발명의 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구 투여할 수 있으며, 비경구 투여시 피하주사, 정맥주사 또는 근육내 주사 주입방식을 선택하는 것이 바람직하다. The composition of the present invention may be administered orally or parenterally, depending on the intended method. For parenteral administration, subcutaneous injection, intravenous injection, or intramuscular injection may be selected.
투약 단위는, 예를 들면 개별 투약량의 1, 2, 3 또는 4배를 함유하거나 또는 1/2, 1/3 또는 1/4배를 함유할 수 있다. 개별 투약량은 구체적으로 유효 약물이 1회에 투여되는 양을 함유하며, 이는 통상 1일 투여량의 전부, 1/2, 1/3 또는 1/4배에 해당한다. 본 발명의 조성물의 유효용량은 0.0001 ~ 10 g/kg일 수 있고, 구체적으로 0.001 ~ 1 g/kg일 수 있으며, 하루 1 ~ 6회 투여될 수 있다. The dosage unit may contain, for example, 1, 2, 3 or 4 times the individual dose or may contain 1/2, 1/3 or 1/4 times the dose. The individual dosages specifically include amounts in which the active drug is administered in a single dose, which usually corresponds to the full, half, one-third, or one-fourth of the daily dose. The effective dose of the composition of the present invention may be 0.0001 to 10 g / kg, specifically 0.001 to 1 g / kg, and may be administered 1 to 6 times a day.
그러나, 투여 경로, 질병의 중증도, 성별, 체중, 연령 등에 따라서 증감될 수 있으므로 상기 투여량이 어떠한 방법으로도 본 발명의 범위를 한정하는 것은 아니다.
However, the dosage may not be limited in any way because it may be increased or decreased depending on route of administration, severity of disease, sex, weight, age, and the like.
또한, 본 발명은 정조목 추출물 또는 이의 분획물을 유효성분으로 함유하는 뇌질환 예방 및 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing and treating brain diseases, which comprises an extract of Orthoptera, or a fraction thereof, as an active ingredient.
상기 정조목 추출물은 하기의 단계들을 포함하는 제조방법에 의해 제조되는 것이 바람직하나 이에 한정되지 않는다:The crude extract is preferably, but not exclusively, prepared by a process comprising the steps of:
1) 정조목에 추출용매를 가하여 추출하는 단계;1) extracting an extractant by adding an extraction solvent to the mixture;
2) 단계 1)의 추출물을 여과하는 단계; 및2) filtering the extract of step 1); And
3) 단계 2)의 여과한 추출물을 감압농축한 후 건조하는 단계; 및3) concentrating the filtered extract of step 2) under reduced pressure and drying; And
4) 단계 3)의 정조목 추출물을 추가적으로 유기용매로 추출하여 정조목 분획물을 제조하는 단계. 4) Extracting the crude extract of step 3) with an organic solvent to prepare a fraction of the purified water.
상기 방법에 있어서, 단계 1)의 정조목은 재배한 것 또는 시판되는 것 등 제한 없이 사용할 수 있다. 상기 정조목은 잎, 줄기 또는 뿌리가 모두 이용가능하며, 이에 한정되지 않는다.In the above method, the seedling of step 1) may be cultivated or commercially available. The tubular tree may be a leaf, a stem, or a root, but is not limited thereto.
상기 방법에 있어서, 상기 단계 1)의 상기 추출용매는 물, 알코올 또는 이들의 혼합물을 사용하는 것이 바람직하다. 상기 알코올로는 C1 내지 C2 저급 알코올을 이용하는 것이 바람직하며, 저급 알코올로는 또는 메탄올을 이용하는 것이 바람직하다. 추출방법으로는 진탕추출, Soxhlet 추출 또는 환류추출을 이용하는 것이 바람직하나 이에 한정되지 않는다. 상기 추출용매를 건조된 정조목 분량에 2 내지 20배 첨가하여 추출하는 것이 바람직하다. 추출온도는 20 내지 50℃인 것이 바람직하나 이에 한정하지 않는다. 또한, 추출시간은 10 내지 48시간인 것이 바람직하며, 24시간이 더욱 바람직하나 이에 한정하지 않는다. 아울러, 추출 회수는 3 내지 5회인 것이 바람직하며, 3회 반복 추출하는 것이 더욱 바람직하나 이에 한정되는 것은 아니다. In the above method, it is preferable to use water, an alcohol or a mixture thereof in the extraction solvent in the step 1). As the alcohol, it is preferable to use a C 1 to C 2 lower alcohol, and it is preferable to use methanol as the lower alcohol. As the extraction method, it is preferable to use shaking extraction, Soxhlet extraction or reflux extraction, but it is not limited thereto. It is preferable to extract the extraction solvent by 2 to 20 times the amount of the dried preparation. The extraction temperature is preferably 20 to 50 DEG C, but is not limited thereto. The extraction time is preferably 10 to 48 hours, more preferably 24 hours, but not always limited thereto. In addition, the extraction number is preferably 3 to 5 times, more preferably 3 times, but not limited thereto.
상기 방법에 있어서, 단계 3)의 감압농축은 진공감압농축기 또는 진공회전증발기를 이용하는 것이 바람직하나 이에 한정하지 않는다. 또한, 건조는 감압건조, 진공건조, 비등건조, 분무건조 또는 동결건조하는 것이 바람직하나 이에 한정하지 않는다.In the above method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in step 3), but it is not limited thereto. The drying is preferably performed under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, but not always limited thereto.
상기 방법에 있어서, 단계 3)의 감압농축은 진공감압농축기 또는 진공회전증발기를 이용하는 것이 바람직하나 이에 한정하지 않는다. 또한, 건조는 감압건조, 진공건조, 비등건조, 분무건조 또는 동결건조하는 것이 바람직하나 이에 한정하지 않는다.In the above method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in step 3), but it is not limited thereto. The drying is preferably performed under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, but not always limited thereto.
상기 방법에 있어서, 단계 4)의 유기용매는 n-헥산, 클로로포름, 에틸아세테이트 또는 부탄올인 것이 바람직하나, 이에 한정하지 않는다. 상기 분획물은 정조목 추출물을 물에 현탁시킨 후 n-헥산, 클로로포름, 에틸아세테이트, 부탄올 및 물로 순차적으로 계통 분획하여 수득한 n-헥산 분획물, 클로로포름 분획물, 에틸아세테이트 분획물, 부탄올 분획물 또는 물 분획물인 것이 바람직하나, 이에 한정하지 않는다. 상기 분획물은 상기 정조목 추출물로부터 분획 과정을 1 내지 5회, 바람직하게는 3회 반복하여 수득할 수 있고, 분획 후 감압 농축하는 것이 바람직하나 이에 한정하지 않는다.In the above method, the organic solvent in step 4) is preferably n-hexane, chloroform, ethyl acetate or butanol, but is not limited thereto. The fraction is a n-hexane fraction, a chloroform fraction, an ethyl acetate fraction, a butanol fraction, or a water fraction obtained by suspending a crude extract in water and sequentially fractionating the fraction with n-hexane, chloroform, ethyl acetate, butanol and water But is not limited thereto. The fraction may be obtained by repeating the fractionation process 1 to 5 times, preferably 3 times, and the fraction may be fractionated and concentrated under reduced pressure. However, the fraction is not limited thereto.
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 것이 바람직하나 이에 한정되지 않는다.The brain diseases include stroke, paralysis, dementia, Alzheimer's disease, Huntington's disease, Pick's disease, Creutzfeld-Jakob disease, thrombosis, em-bolism, transient ischemic but is not limited to, any one selected from the group consisting of stroke, attack, lacune, stroke, cerebral hemorrhage, cerebral infarction, head trauma, cerebral circulatory metabolic disturbance and brain function coma.
본 발명의 정조목 추출물 또는 이의 분획물은 신경세포의 독성을 유의적으로 억제하는 효과가 뛰어나고, 세포 독성이 없어 인체에 안전하므로 뇌질환 예방 및 치료용 약학적 조성물의 유효성분으로 유용하게 사용될 수 있다.
The present invention is useful as an effective ingredient of a pharmaceutical composition for the prevention and treatment of cerebral diseases because it has excellent effect of significantly inhibiting the toxicity of neurons and has no cytotoxicity and is safe for human body .
또한, 본 발명은 정조목 추출물 또는 이의 분획물을 유효성분으로 함유하는 신경보호용 건강식품을 제공한다.In addition, the present invention provides a health food for neuroprotection comprising an extract of Orthodox japonica or a fraction thereof as an active ingredient.
상기 정조목 추출물은 하기의 단계들을 포함하는 제조방법에 의해 제조되는 것이 바람직하나 이에 한정되지 않는다:The crude extract is preferably, but not exclusively, prepared by a process comprising the steps of:
1) 정조목에 추출용매를 가하여 추출하는 단계;1) extracting an extractant by adding an extraction solvent to the mixture;
2) 단계 1)의 추출물을 여과하는 단계; 및2) filtering the extract of step 1); And
3) 단계 2)의 여과한 추출물을 감압농축한 후 건조하는 단계; 및3) concentrating the filtered extract of step 2) under reduced pressure and drying; And
4) 단계 3)의 정조목 추출물을 추가적으로 유기용매로 추출하여 정조목 분획물을 제조하는 단계. 4) Extracting the crude extract of step 3) with an organic solvent to prepare a fraction of the purified water.
상기 방법에 있어서, 단계 1)의 정조목은 재배한 것 또는 시판되는 것 등 제한 없이 사용할 수 있다. 상기 정조목은 잎, 줄기 또는 뿌리가 모두 이용가능하며, 이에 한정되지 않는다.In the above method, the seedling of step 1) may be cultivated or commercially available. The tubular tree may be a leaf, a stem, or a root, but is not limited thereto.
상기 방법에 있어서, 상기 단계 1)의 상기 추출용매는 물, 알코올 또는 이들의 혼합물을 사용하는 것이 바람직하다. 상기 알코올로는 C1 내지 C2 저급 알코올을 이용하는 것이 바람직하며, 저급 알코올로는 또는 메탄올을 이용하는 것이 바람직하다. 추출방법으로는 진탕추출, Soxhlet 추출 또는 환류추출을 이용하는 것이 바람직하나 이에 한정되지 않는다. 상기 추출용매를 건조된 정조목 분량에 2 내지 20배 첨가하여 추출하는 것이 바람직하다. 추출온도는 20 내지 50℃인 것이 바람직하나 이에 한정하지 않는다. 또한, 추출시간은 10 내지 48시간인 것이 바람직하며, 24시간이 더욱 바람직하나 이에 한정하지 않는다. 아울러, 추출 회수는 3 내지 5회인 것이 바람직하며, 3회 반복 추출하는 것이 더욱 바람직하나 이에 한정되는 것은 아니다. In the above method, it is preferable to use water, an alcohol or a mixture thereof in the extraction solvent in the step 1). As the alcohol, it is preferable to use a C 1 to C 2 lower alcohol, and it is preferable to use methanol as the lower alcohol. As the extraction method, it is preferable to use shaking extraction, Soxhlet extraction or reflux extraction, but it is not limited thereto. It is preferable to extract the extraction solvent by 2 to 20 times the amount of the dried preparation. The extraction temperature is preferably 20 to 50 DEG C, but is not limited thereto. The extraction time is preferably 10 to 48 hours, more preferably 24 hours, but not always limited thereto. In addition, the extraction number is preferably 3 to 5 times, more preferably 3 times, but not limited thereto.
상기 방법에 있어서, 단계 3)의 감압농축은 진공감압농축기 또는 진공회전증발기를 이용하는 것이 바람직하나 이에 한정하지 않는다. 또한, 건조는 감압건조, 진공건조, 비등건조, 분무건조 또는 동결건조하는 것이 바람직하나 이에 한정하지 않는다.In the above method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in step 3), but it is not limited thereto. The drying is preferably performed under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, but not always limited thereto.
상기 방법에 있어서, 단계 3)의 감압농축은 진공감압농축기 또는 진공회전증발기를 이용하는 것이 바람직하나 이에 한정하지 않는다. 또한, 건조는 감압건조, 진공건조, 비등건조, 분무건조 또는 동결건조하는 것이 바람직하나 이에 한정하지 않는다.In the above method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in step 3), but it is not limited thereto. The drying is preferably performed under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, but not always limited thereto.
상기 방법에 있어서, 단계 4)의 유기용매는 n-헥산, 클로로포름, 에틸아세테이트 또는 부탄올인 것이 바람직하나, 이에 한정하지 않는다. 상기 분획물은 정조목 추출물을 물에 현탁시킨 후 n-헥산, 클로로포름, 에틸아세테이트, 부탄올 및 물로 순차적으로 계통 분획하여 수득한 n-헥산 분획물, 클로로포름 분획물, 에틸아세테이트 분획물, 부탄올 분획물 또는 물 분획물인 것이 바람직하나, 이에 한정하지 않는다. 상기 분획물은 상기 정조목 추출물로부터 분획 과정을 1 내지 5회, 바람직하게는 3회 반복하여 수득할 수 있고, 분획 후 감압 농축하는 것이 바람직하나 이에 한정하지 않는다.In the above method, the organic solvent in step 4) is preferably n-hexane, chloroform, ethyl acetate or butanol, but is not limited thereto. The fraction is a n-hexane fraction, a chloroform fraction, an ethyl acetate fraction, a butanol fraction, or a water fraction obtained by suspending a crude extract in water and sequentially fractionating the fraction with n-hexane, chloroform, ethyl acetate, butanol and water But is not limited thereto. The fraction may be obtained by repeating the fractionation process 1 to 5 times, preferably 3 times, and the fraction may be fractionated and concentrated under reduced pressure. However, the fraction is not limited thereto.
본 발명의 정조목 추출물 또는 이의 분획물은 신경세포의 독성을 유의적으로 억제하는 효과가 뛰어나고, 세포 독성이 없어 인체에 안전하므로 신경보호용 건강식품의 유효성분으로 유용하게 사용될 수 있다.
The extract of the present invention of the present invention or its fractions is excellent in the effect of significantly inhibiting the toxicity of nerve cells and is safe to the human body because of its cytotoxicity, so that it can be effectively used as an active ingredient of a health food for nerve protection.
본 발명의 건강식품은 정조목 추출물 또는 이의 분획물을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다.The health food of the present invention can be used as it is, or can be used in combination with other food or food ingredients, and can be suitably used according to conventional methods.
상기 건강식품의 종류에는 특별한 제한은 없다. 상기 정조목 추출물 또는 이의 분획물을 첨가할 수 있는 식품의 예로는 육류, 소시지, 빵, 초콜릿, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다.There is no particular limitation on the kind of the health food. Examples of the food to which the abovementioned mulberry tree extract or its fractions can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, , Tea, a drink, an alcoholic beverage, and a vitamin complex, 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 함유할 수 있다. 상술한 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드, 말토스, 슈크로스와 같은 디사카라이드, 및 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드, 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 감미제로서는 타우마틴, 스테비아 추출물과 같은 천연 감미제나, 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 당 일반적으로 약 0.01 ~ 0.04 g, 바람직하게는 약 0.02 ~ 0.03 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. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 of the composition of the present invention.
상기 외에 본 발명의 건강식품은 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. 그 밖에 천연 과일주스, 과일주스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 혼합하여 사용할 수 있다. 이러한 첨가제의 비율은 크게 중요하진 않지만 본 발명의 조성물 100 중량부 당 0.01 ~ 0.1 중량부의 범위에서 선택되는 것이 일반적이다.
In addition to the above, the health food of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, , A carbonating agent used in carbonated drinks, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not critical, but 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.
또한, 본 발명은 정조목 추출물 또는 이의 분획물을 유효성분으로 함유하는 뇌질환 예방 및 개선용 건강식품을 제공한다.In addition, the present invention provides a health food for prevention and improvement of brain diseases, which comprises the extract of Orthoptera, or a fraction thereof, as an active ingredient.
상기 정조목 추출물은 하기의 단계들을 포함하는 제조방법에 의해 제조되는 것이 바람직하나 이에 한정되지 않는다:The crude extract is preferably, but not exclusively, prepared by a process comprising the steps of:
1) 정조목에 추출용매를 가하여 추출하는 단계;1) extracting an extractant by adding an extraction solvent to the mixture;
2) 단계 1)의 추출물을 여과하는 단계; 및2) filtering the extract of step 1); And
3) 단계 2)의 여과한 추출물을 감압농축한 후 건조하는 단계; 및3) concentrating the filtered extract of step 2) under reduced pressure and drying; And
4) 단계 3)의 정조목 추출물을 추가적으로 유기용매로 추출하여 정조목 분획물을 제조하는 단계. 4) Extracting the crude extract of step 3) with an organic solvent to prepare a fraction of the purified water.
상기 방법에 있어서, 단계 1)의 정조목은 재배한 것 또는 시판되는 것 등 제한 없이 사용할 수 있다. 상기 정조목은 잎, 줄기 또는 뿌리가 모두 이용가능하며, 이에 한정되지 않는다.In the above method, the seedling of step 1) may be cultivated or commercially available. The tubular tree may be a leaf, a stem, or a root, but is not limited thereto.
상기 방법에 있어서, 상기 단계 1)의 상기 추출용매는 물, 알코올 또는 이들의 혼합물을 사용하는 것이 바람직하다. 상기 알코올로는 C1 내지 C2 저급 알코올을 이용하는 것이 바람직하며, 저급 알코올로는 또는 메탄올을 이용하는 것이 바람직하다. 추출방법으로는 진탕추출, Soxhlet 추출 또는 환류추출을 이용하는 것이 바람직하나 이에 한정되지 않는다. 상기 추출용매를 건조된 정조목 분량에 2 내지 20배 첨가하여 추출하는 것이 바람직하다. 추출온도는 20 내지 50℃인 것이 바람직하나 이에 한정하지 않는다. 또한, 추출시간은 10 내지 48시간인 것이 바람직하며, 24시간이 더욱 바람직하나 이에 한정하지 않는다. 아울러, 추출 회수는 3 내지 5회인 것이 바람직하며, 3회 반복 추출하는 것이 더욱 바람직하나 이에 한정되는 것은 아니다. In the above method, it is preferable to use water, an alcohol or a mixture thereof in the extraction solvent in the step 1). As the alcohol, it is preferable to use a C 1 to C 2 lower alcohol, and it is preferable to use methanol as the lower alcohol. As the extraction method, it is preferable to use shaking extraction, Soxhlet extraction or reflux extraction, but it is not limited thereto. It is preferable to extract the extraction solvent by 2 to 20 times the amount of the dried preparation. The extraction temperature is preferably 20 to 50 DEG C, but is not limited thereto. The extraction time is preferably 10 to 48 hours, more preferably 24 hours, but not always limited thereto. In addition, the extraction number is preferably 3 to 5 times, more preferably 3 times, but not limited thereto.
상기 방법에 있어서, 단계 3)의 감압농축은 진공감압농축기 또는 진공회전증발기를 이용하는 것이 바람직하나 이에 한정하지 않는다. 또한, 건조는 감압건조, 진공건조, 비등건조, 분무건조 또는 동결건조하는 것이 바람직하나 이에 한정하지 않는다.In the above method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in step 3), but it is not limited thereto. The drying is preferably performed under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, but not always limited thereto.
상기 방법에 있어서, 단계 3)의 감압농축은 진공감압농축기 또는 진공회전증발기를 이용하는 것이 바람직하나 이에 한정하지 않는다. 또한, 건조는 감압건조, 진공건조, 비등건조, 분무건조 또는 동결건조하는 것이 바람직하나 이에 한정하지 않는다.In the above method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in step 3), but it is not limited thereto. The drying is preferably performed under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, but not always limited thereto.
상기 방법에 있어서, 단계 4)의 유기용매는 n-헥산, 클로로포름, 에틸아세테이트 또는 부탄올인 것이 바람직하나, 이에 한정하지 않는다. 상기 분획물은 정조목 추출물을 물에 현탁시킨 후 n-헥산, 클로로포름, 에틸아세테이트, 부탄올 및 물로 순차적으로 계통 분획하여 수득한 n-헥산 분획물, 클로로포름 분획물, 에틸아세테이트 분획물, 부탄올 분획물 또는 물 분획물인 것이 바람직하나, 이에 한정하지 않는다. 상기 분획물은 상기 정조목 추출물로부터 분획 과정을 1 내지 5회, 바람직하게는 3회 반복하여 수득할 수 있고, 분획 후 감압 농축하는 것이 바람직하나 이에 한정하지 않는다.In the above method, the organic solvent in step 4) is preferably n-hexane, chloroform, ethyl acetate or butanol, but is not limited thereto. The fraction is a n-hexane fraction, a chloroform fraction, an ethyl acetate fraction, a butanol fraction, or a water fraction obtained by suspending a crude extract in water and sequentially fractionating the fraction with n-hexane, chloroform, ethyl acetate, butanol and water But is not limited thereto. The fraction may be obtained by repeating the fractionation process 1 to 5 times, preferably 3 times, and the fraction may be fractionated and concentrated under reduced pressure. However, the fraction is not limited thereto.
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 것이 바람직하나 이에 한정되지 않는다.The brain diseases include stroke, paralysis, dementia, Alzheimer's disease, Huntington's disease, Pick's disease, Creutzfeld-Jakob disease, thrombosis, em-bolism, transient ischemic but is not limited to, any one selected from the group consisting of stroke, attack, lacune, stroke, cerebral hemorrhage, cerebral infarction, head trauma, cerebral circulatory metabolic disturbance and brain function coma.
그 결과, 본 발명의 정조목 추출물 또는 이의 분획물은 신경세포의 독성을 유의적으로 억제하는 효과가 뛰어나고, 세포 도성이 없어 안전하므로 뇌질환 예방 및 개선용 건강식품으로 유용하게 사용될 수 있다.
As a result, the extract of the present invention of the present invention or its fractions is excellent in the effect of significantly inhibiting the toxicity of neurons, and is safe because it has no cytotoxicity, so that it can be effectively used as a health food for prevention and improvement of brain diseases.
이하, 본 발명을 실시예 및 제조예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail with reference to Examples and Production Examples.
단, 하기 실시예 및 제조예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예 및 제조예에 한정되는 것은 아니다.
However, the following examples and preparative examples are merely illustrative of the present invention, and the present invention is not limited to the following examples and preparative examples.
<< 실시예Example 1> 1> 정조목Maternity neck 추출물 제조 Extract preparation
한국생명공학연구원의 해외식물추출물은행(대전)으로부터 입수한 정조목의 건조된 잎 40 g을 메탄올 수용액(메탄올 80%, 물 20%) 400 ml로 추출한다음 감압건조하여 정조목 추출물 2.8 g을 제조하였다.
Extracted 40 g of the dried leaves of Chungjo-jin from Korea Plant Biotechnology Research Institute (Daejeon) were extracted with 400 ml of methanol aqueous solution (
<< 실시예Example 2> 2> 정조목Maternity neck 추출물의 Extract 분획물Fraction 제조 Produce
상기 <실시예 1>에서 제조된 정조목 추출물 2.8 g 을 물 1 리터에 현탁시키고, n-헥산, 클로로폼(CHCl3), 에틸아세테이트(EtOAc), 부탄올(C4H9OH)을 각 용매 500 미리리터(ml)를 사용하여 순차적으로 추출하여 클로로포름 분획물 0.7 g, 에틸아세테이트 분획물 0.7 g, 부탄올 분획물 0.6 g을 제조하였다.
2.8 g of the crude extract obtained in Example 1 was suspended in 1 liter of water and then n-hexane, chloroform (CHCl 3 ), ethyl acetate (EtOAc) and butanol (C 4 H 9 OH) And then sequentially extracted with 500 milliliters (ml) to prepare 0.7 g of chloroform fraction, 0.7 g of ethyl acetate fraction and 0.6 g of butanol fraction.
<실시예 3> 정조목 추출물과 이의 분획물의 뇌신경세포 보호활성 확인Example 3 Confirmation of Protective Activity of Cranberry Tree Extract and its Fractions on Cranial Cells
<3-1> N18-RE-105 세포 배양<3-1> N18-RE-105 cell culture
N18-RE-105 세포주는 둘베코스 변형된 이글스 배지(Dulbesco's modified Eagle's medium;DMEM)에서 HAT 첨가물(supplement)(0.1 mM hypoxanthin, 0.04 mM aminopterin, 0.14 mM thymidine)와 열처리한 신생아소혈청(fetal calf serum;FCS) 10%를 넣은 배지를 사용하여 배양하였다. 5% CO2, 37℃의 조건의 세포배양기에서 배양했으며 매 2일 마다 계대배양하였다. N18-RE-105 세포의 보관은 DMEM 배지에 동결보존제(10% DMSO)와 20% 혈청(serum)을 넣은 배지를 사용하였으며, -75℃에서 24시간 둔 후 액체질소(liquid nitrogen)에서 보관하였다.
The N18-RE-105 cell line was cultured in Dulbesco's modified Eagle's medium (DMEM) supplemented with HAT supplement (0.1 mM hypoxanthin, 0.04 mM aminopterin, 0.14 mM thymidine) and heat-treated fetal calf serum ; FCS) 10%. 5% CO 2 , 37 ° C, and subcultured every 2 days. N18-RE-105 cells were maintained in liquid nitrogen at -75 ° C for 24 hours using a medium containing cryopreservation (10% DMSO) and 20% serum in DMEM medium .
<3-2> N18-RE-105 세포에 대한 글루타메이트(glutamate) 독성억제 효과 <3-2> Inhibitory effect of glutamate toxicity on N18-RE-105 cells
상기 <실시예 1> 및 <실시예 2>에서 제조한 정조목 추출물과 이의 분획물의 뇌신경세포 보호활성을 확인하기 위하여, N18-RE-105 세포주를 T-25 플라스크에서 48시간 배양한 후 트립신(trypsin) 용액(3 ml)을 사용하여 배양기에서 2분간 배양시켜 세포를 플라스크(flask)에서 분리하였다. 그 후, 배지 1 ml을 넣어 트립신을 중화시킨 후, 1000 rpm에서 3분간 원심분리한 다음 상등액을 제거한 후, 세포를 배지로 현탁하고 헤모사이토메터(Hemocytometer)를 이용하여 세포수를 계산하여 well 당 세포수가 5 X 104개가 되도록 96-웰 마이크로플레이트에 100 ㎕씩 분주했다. 세포를 5% CO2 , 37℃에서 24시간 배양한 후 인산완충액(Phosphate Buffered Saline, PBS)에 녹인 글루타메이트(400 mM) 용액 5 ㎕와 DMSO에 녹인 정조목 추출물 또는 이의 분획물을 PBS 용액으로 10배 희석하여 0.3 mg/ml, 0.5 mg/ml 및 1.0 mg/ml 농도의 시료를 제조한 다음 각 농도별 정조목 추출물 또는 이의 분획물 용액 5 ㎕를 처리하였다. 이것을 배양기에서 24시간 배양한 다음 시료의 글루타메이트 독성억제활성을 통한 뇌신경세포 보호활성을 위상차 현미경(phase contrast microscope)을 이용해 관찰하였다. The N18-RE-105 cell line was cultured in a T-25 flask for 48 hours to examine the neuronal cell protection activity of the extract of the present invention and the fractions thereof prepared in Examples 1 and 2, trypsin solution (3 ml) for 2 minutes in an incubator to separate the cells in a flask. Thereafter, 1 ml of the medium was neutralized to neutralize trypsin, centrifuged at 1000 rpm for 3 minutes, the supernatant was removed, the cells were suspended in the medium, and the number of cells was calculated using a hemocytometer, 100 [mu] L of the solution was dispensed into 96-well microplates so that the number of cells was 5 X 10 4 . Cells a 5% CO 2, 10-fold and incubated for 24 hours at 37 ℃ the tank neck extracts or fractions thereof dissolved in glutamate (400 mM) solution 5 ㎕ and DMSO was dissolved in phosphate buffer (Phosphate Buffered Saline, PBS) in PBS solution After dilution, samples of 0.3 mg / ml, 0.5 mg / ml and 1.0 mg / ml were prepared and treated with 5 ㎕ of the extracts or their fractions. After incubation in an incubator for 24 hours, the protective activity of the sample against glutamate toxicity was observed using a phase contrast microscope.
그 결과, 도 2에 나타난 바와 같이, 본 발명의 정조목 추출물 또는 이의 분획물은 글루타메이트의 세포독성을 억제하여 신경세포를 보호하는 것을 확인하였다. 특히, N18-RE-105 세포에 글루타메이트를 처리하지 않은 대조군에 비하여 글루타메이트를 처리한 세포들에서는 팽윤(swelling)과 수포(blebbing) 및 용해(lysis)가 일어났고 세포가 사멸하여 약 40%의 세포만이 살아있으나, 45 ㎍/㎖의 정조목 추출물이 첨가된 세포들은 세포의 모양이 변하지 않았으며, 사멸이 줄어들어 80% 이상의 세포가 살아있음을 확인하였다.As a result, as shown in FIG. 2, it was confirmed that the extract of the present invention or fractions thereof protects nerve cells by inhibiting cytotoxicity of glutamate. In particular, in the glutamate-treated cells, swelling, blebbing, and lysis occurred in N18-RE-105 cells compared with the control without glutamate, However, the cells with the 45 ㎍ / ㎖ of the crude extracts showed no change in the shape of the cells and less than 80% of the cells were alive.
또한, 신경융합(Neuronal hybridoma) 세포인 N18-RE-105 세포주에 대한 글루타메이트 독성을 50% 억제하는 정조목 추출물의 농도(IC50)는 17.1 ㎍/㎖로서 양성대조군인 비타민 E의 21.5 ㎍/㎖보다 높은 신경세포 보호 활성을 나타내었다. 정조목 추출물을 유기용매를 사용하여 추출한 분획물의 신경세포 보호활성을 측정한 결과 클로로포름 추출 분획물과 에틸아세테이트 추출 분획물이 각각 14.9, 15.1 ㎍/㎖로서 뛰어난 신경세포 보호활성을 나타내었다.
In addition, the concentration (IC 50 ) of the mungbean tree extract which inhibits the glutamate toxicity by 50% of the N18-RE-105 cell line which is a neuronal hybridoma cell is 17.1 ㎍ / ㎖, and 21.5 ㎍ / ㎖ of the positive control vitamin E Showed higher neuronal cell protective activity. As a result of measuring the neuronal cell protection activity of the fractions extracted with the organic solvent, the chloroform - extracted fraction and the ethyl acetate - extracted fraction showed excellent neuronal cell protection activity as 14.9 and 15.1 ㎍ / ㎖, respectively.
<실시예 4> 정조목 추출물 또는 이의 분획물의 신경세포주에 대한 글루타메이트 독성 억제활성Example 4 Glutamate Toxicity Inhibitory Activity on Neuronal Cell Lines of Orthoptera sp. Extract or its Fraction
N18-RE-105 세포를 웰(well)당 세포수가 5 X 104개가 되도록 96-웰 마이크로플레이트에 100 ㎕씩 분주했다. 세포를 5% CO2, 37℃에서 24시간 배양한 후, DMSO에 녹인 정조목 추출물 또는 이의 분획물을 PBS 용액으로 10배 희석하여 만든 1.0 mg/ml, 0.5 mg/ml 및 0.3 mg/ml 농도의 정조목 추출물 또는 이의 분획물 용액 5 ㎕를 처리한 다음 30분 후 인산완충액(Phosphate Buffered Saline, PBS)에 녹인 글루타메이트(400 mM) 용액 5 를 처리하였다. 이것을 배양기에서 24시간 배양한 다음, 가수분해효소 분석법(EZ-Cytox assay (dehydrogenase assay))(대일 렙서비스, 한국)으로 살아있는 세포를 측정하여 정량하였다. EZ-Cytox assay는 시료처리 후 24시간 동안 배양한 세포에 플레이트 웰(plate well)당 EZ-Cytox assay reagent 10 ㎕를 처리한 후 배양기 내에서 3시간 동안 배양한 다음 반응 배양액 70 ㎕씩을 새로운 96-웰 플레이트에 옮긴 후, 마이크로플레이트 리더(miroplate reader, Molecular devices사)로 450 nm에서의 흡광도를 측정하였다. 측정된 흡광도를 하기 [수학식 1]을 이용하여 세포 생존율(cell viability)를 산출하였으며, 그 산출된 결과 값을 다시 하기 [수학식 2]를 이용하여 세포보호 활성을 산출하였다.
100 μl of N18-RE-105 cells were dispensed into 96-well microplates so that the number of cells per well was 5 × 10 4 cells. Cells were cultured in 5% CO 2 at 37 ° C for 24 hours. The cells were treated with 1.0 mg / ml, 0.5 mg / ml, and 0.3 mg / ml of Tryptophan extract dissolved in DMSO or its fractions diluted 10-fold with PBS 5 μl of the crude extract or its fraction solution was treated and then treated with glutamate (400 mM) solution 5 dissolved in phosphate buffer (PBS) for 30 minutes. After incubation in an incubator for 24 hours, living cells were quantitated by the hydrolysis enzyme assay (EZ-Cytox assay (dehydrogenase assay)) (Daeilrep Service, Korea). In the EZ-Cytox assay, 10 μl of the EZ-Cytox assay reagent was treated per plate well for 24 hours after the sample treatment, and then cultured for 3 hours in the incubator. Then, 70 μl of the reaction solution was added to the 96- After transferring to a well plate, the absorbance at 450 nm was measured with a microplate reader (Molecular devices, Inc.). The cell viability was calculated using the following formula (1), and the calculated cell viability was calculated using the following formula (2).
상기 A는 각 웰(well)의 흡광도; 및A is the absorbance of each well; And
Ac는 글루타메이트(Glutamate)를 처리하지 않은 웰(well)의 흡광도이다.
Ac is the absorbance of a well not treated with glutamate.
상기 Vc는 글루타메이트(Glutamate)를 처리하지 않은 웰(well)의 세포생존율이고;Vc is the cell survival rate of a well not treated with glutamate (Glutamate);
Vg는 글루타메이트(Glutamate)를 처리한 웰(well)의 세포생존율이고; 및Vg is the cell survival rate of a well treated with Glutamate; And
Vs는 글루타메이트(Glutamate) 및 시료를 처리한 웰(well)의 세포생존율이다.
Vs is the cell survival rate of glutamate and well treated with the sample.
그 결과, 도 3에 나타난 바와 같이, 정조목 추출물과 이의 분획물이 농도 의존적으로 세포의 글루타메이트에 의한 N18-RE-105 세포의 사멸을 억제하여 우수한 뇌신경세포 보호활성을 나타냈다. 특히, 클로로포름 분획물을 23 ㎍/㎖처리한 세포들에서는 글루타메이트에 의한 신경세포의 사멸이 완전히 억제되어 100%의 세포가 살아 있는 매우 뛰어난 신경세포 보호활성이 관찰되었다.
As a result, as shown in FIG. 3, the extracts of Cynomolgus thunbergii and its fractions inhibited the death of N18-RE-105 cells by glutamate in cells in a concentration-dependent manner and showed excellent neuronal cell protection activity. Particularly, in the cells treated with chloroform fraction at 23 / / ml, glutamate - induced neuronal cell death was completely inhibited, and 100% cell - viable neuronal cell protective activity was observed.
<실시예 5> 정조목 추출물 또는 이의 분획물의 세포독성 확인<Example 5> Cytotoxicity of purified water extract or fraction thereof
정조목 추출물 또는 이의 분획물의 세포독성을 확인하기 위하여, 5, 10, 20 및 50 및 80 ㎍/㎖을 처리하고 글루타메이트(Glutamate)를 처리하지 않은 것을 제외하고 상기 <실시예 3>과 동일한 방법으로 세포독성을 확인하였다. In order to confirm the cytotoxicity of the mungbean tree extract or its fractions, 5, 10, 20, 50 and 80 占 퐂 / ml were treated and glutamate was not treated, Cytotoxicity was confirmed.
그 결과, 정조목 추출물 및 이의 분획물은 활성 유효농도의 5배가 넘는 80 ㎍/㎖의 고농도에서도 세포독성이 나타내지 않은 것을 확인하였다.
As a result, it was confirmed that the citrus tree extract and its fractions did not show cytotoxicity even at a high concentration of 80 / / ml exceeding 5 times the effective effective concentration.
<< 제조예Manufacturing example 1> 약학적 제제의 제조 1> Preparation of pharmaceutical preparations
<1-1> <1-1> 산제의Sanje 제조 Produce
본 발명의 정조목 추출물 2 g2 g of the crude tree extract of the present invention
유당 1 gLactose 1 g
상기의 성분을 혼합하고 기밀포에 충진하여 산제를 제조하였다.
The above components were mixed and packed in airtight bags to prepare powders.
<1-2> 정제의 제조<1-2> Preparation of tablets
본 발명의 정조목 추출물 100 ㎎100 mg of the crude tree extract of the present invention
옥수수전분 100 ㎎
유 당 100 ㎎100 mg of milk
스테아린산 마그네 2 ㎎Magnesium stearate 2 mg
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.
<1-3> 캡슐제의 제조≪ 1-3 > Preparation of capsules
본 발명의 정조목 추출물 100 ㎎100 mg of the crude tree extract of the present invention
옥수수전분 100 ㎎
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.
<1-4> 환의 제조 ≪ 1-4 >
본 발명의 정조목 추출물 1 g1 g < RTI ID = 0.0 >
유당 1.5 gLactose 1.5 g
글리세린 1 gGlycerin 1 g
자일리톨 0.5 g0.5 g of xylitol
상기의 성분을 혼합한 후, 통상의 방법에 따라 1환 당 4 g이 되도록 제조하였다.
After mixing the above components, they were prepared so as to be 4 g per one ring according to a conventional method.
<1-5> 과립의 제조<1-5> Preparation of granules
본 발명의 정조목 추출물 150 ㎎150 mg of the crude tree extract of the present invention
대두추출물 50 ㎎Soybean extract 50 mg
포도당 200 ㎎200 mg of glucose
전분 600 ㎎600 mg of starch
상기의 성분을 혼합한 후, 30% 에탄올 100 을 첨가하여 섭씨 60 ℃에서 건조하여 과립을 형성한 후 포에 충진하였다.
After mixing the above components, 100% of 30% ethanol was added and the mixture was dried at 60 ° C to form granules, which were then filled in a capsule.
<< 제조예Manufacturing example 2> 건강식품의 제조 2> Manufacture of health food
본 발명의 정조목 추출물을 포함하는 건강식품들을 다음과 같이 제조하였다.The health foods containing the extract of the present invention of the present invention were prepared as follows.
<2-1> 밀가루 식품의 제조<2-1> Production of flour food
본 발명의 정조목 추출물 0.5~5.0 중량부를 밀가루에 첨가하고, 이 혼합물을 이용하여 빵, 케이크, 쿠키, 크래커 및 면류를 제조하였다.
0.5-5.0 parts by weight of the crude extract of the present invention was added to wheat flour, and the mixture was used to prepare bread, cake, cookies, crackers and noodles.
<2-2> <2-2> 스프soup 및 육즙( And juicy ( graviesgravies )의 제조)
본 발명의 정조목 추출물 0.1~5.0 중량부를 스프 및 육즙에 첨가하여 건강 증진용 육가공 제품, 면류의 수프 및 육즙을 제조하였다.
0.1 to 5.0 parts by weight of the extract of the present invention was added to soups and juices to prepare health-enhancing meat products, noodle soups and juices.
<2-3> 그라운드 <2-3> Ground 비프(ground beef)의Beef 제조 Produce
본 발명의 정조목 추출물 10 중량부를 그라운드 비프에 첨가하여 건강 증진용 그라운드 비프를 제조하였다.
10 parts by weight of the extract of the present invention was added to ground beef to prepare ground beef for health promotion.
<2-4> 유제품(<2-4> Dairy products ( dairydairy productsproducts )의 제조)
본 발명의 정조목 추출물 5~10 중량부를 우유에 첨가하고, 상기 우유를 이용하여 버터 및 아이스크림과 같은 다양한 유제품을 제조하였다.
5 to 10 parts by weight of the extract of the present invention were added to milk and various dairy products such as butter and ice cream were prepared using the milk.
<2-5> <2-5> 선식의Solar 제조 Produce
현미, 보리, 찹쌀, 율무를 공지의 방법으로 알파화시켜 건조시킨 것을 배전한 후 분쇄기로 입도 60 메쉬의 분말로 제조하였다.Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh.
검정콩, 검정깨, 들깨도 공지의 방법으로 쪄서 건조시킨 것을 배전한 후 분쇄기로 입도 60 메쉬의 분말로 제조하였다.Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer.
본 발명의 정조목 추출물을 진공 농축기에서 감압농축하고, 분무, 열풍건조기로 건조하여 얻은 건조물을 분쇄기로 입도 60 메쉬로 분쇄하여 건조분말을 얻었다.The crude extract of the present invention was concentrated under reduced pressure in a vacuum concentrator, dried by spraying and drying in a hot air drier, and pulverized to a size of 60 mesh with a pulverizer to obtain a dry powder.
상기에서 제조한 곡물류, 종실류 및 본 발명의 정조목 추출물을 다음의 비율로 배합하여 제조하였다.The above-prepared cereals, seeds and the extract of the present invention were prepared in the following proportions.
곡물류(현미 30 중량부, 율무 15 중량부, 보리 20 중량부),(30 parts by weight of brown rice, 15 parts by weight of yulmu, 20 parts by weight of barley)
종실류(들깨 7 중량부, 검정콩 8 중량부, 검정깨 7 중량부),Seeds (7 parts by weight of perilla, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds)
본 발명의 정조목 추출물(3 중량부),(3 parts by weight) of the present invention,
영지(0.5 중량부),(0.5 part by weight),
지황(0.5 중량부)
(0.5 parts by weight)
<< 제조예Manufacturing example 3> 음료의 제조 3> Manufacturing of beverage
<3-1> <3-1> 건강음료의Health drink 제조 Produce
액상과당(0.5%), 올리고당(2%), 설탕(2%), 식염(0.5%), 물(75%)과 같은 부재료와 본 발명의 본 발명의 정조목 추출물 5 g을 균질하게 배합하여 순간 살균을 한 후 이를 유리병, 패트병 등 소포장 용기에 포장하여 제조하였다.
(5 g) of the present invention of the present invention was homogeneously mixed with a supplementary material such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5% After sterilization, they were packaged in small containers such as glass bottles and plastic bottles.
<3-2> 야채 주스의 제조<3-2> Preparation of vegetable juice
본 발명의 정조목 추출물 5 g을 토마토 또는 당근 주스 1,000 ㎖에 가하여 야채 주스를 제조하였다.
5 g of the crude tree extract of the present invention was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice.
<3-3> 과일 주스의 제조<3-3> Production of fruit juice
본 발명의 정조목 추출물 1 g을 사과 또는 포도 주스 1,000 ㎖에 가하여 과일 주스를 제조하였다.
1 g of the crude tree extract of the present invention was added to 1,000 ml of apple or grape juice to prepare fruit juice.
Claims (12)
A cerebral disease selected from the group consisting of stroke, thrombosis, embolism, head trauma, brain circulatory metabolic disorder, and brain functional coma, which contains the active ingredient (s) A pharmaceutical composition for prevention and treatment.
The method according to claim 5, wherein the extract is extracted with water, a C 1 to C 2 lower alcohol or a mixture thereof as a solvent. The method of claim 5, wherein the extract is selected from the group consisting of stroke, thrombosis, embolism, head trauma, , Cerebral circulation metabolic disorder, and brain functional coma.
7. The method of claim 6, wherein the lower alcohol is selected from the group consisting of stroke, thrombosis, embolism, head trauma, brain circulatory metabolic disorders, and brain function coma, wherein the lower alcohol is ethanol or methanol. Or a pharmaceutically acceptable salt thereof.
[Claim 5] The method according to claim 5, wherein the fraction is prepared by further extracting a crude extract from the group selected from the group consisting of hexane, chloroform, ethyl acetate and butanol. Head trauma, cerebral circulatory metabolic disorders, and brain functional coma.
A cerebral disease selected from the group consisting of stroke, thrombosis, embolism, head trauma, brain circulatory metabolic disorder, and brain functional coma, which contains the active ingredient (s) Health foods for prevention and improvement.
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| US20050129783A1 (en) | 2001-04-19 | 2005-06-16 | Mccleary Edward L. | Composition and method for treatment of neurophysiological conditions and maintenance of neurophysiological health |
| US20050220900A1 (en) * | 2002-02-15 | 2005-10-06 | Michael Popp | Use of extracts containing phytoestrogen selectively modulating estrogen-receptor-beta |
| US20100151059A1 (en) * | 2007-08-29 | 2010-06-17 | Max Zeller Sohne Ag | Use of vitex agnus castus extracts for preparing a medicament |
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| US20050129783A1 (en) | 2001-04-19 | 2005-06-16 | Mccleary Edward L. | Composition and method for treatment of neurophysiological conditions and maintenance of neurophysiological health |
| US20050220900A1 (en) * | 2002-02-15 | 2005-10-06 | Michael Popp | Use of extracts containing phytoestrogen selectively modulating estrogen-receptor-beta |
| US20100151059A1 (en) * | 2007-08-29 | 2010-06-17 | Max Zeller Sohne Ag | Use of vitex agnus castus extracts for preparing a medicament |
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