KR20030007105A - A composition for the protection and regeneration of nerve cells containing the extract of coptidis rhizoma - Google Patents
A composition for the protection and regeneration of nerve cells containing the extract of coptidis rhizoma Download PDFInfo
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- KR20030007105A KR20030007105A KR1020020040110A KR20020040110A KR20030007105A KR 20030007105 A KR20030007105 A KR 20030007105A KR 1020020040110 A KR1020020040110 A KR 1020020040110A KR 20020040110 A KR20020040110 A KR 20020040110A KR 20030007105 A KR20030007105 A KR 20030007105A
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- extract
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- neuronal
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Abstract
Description
본 발명은 황련추출물을 함유하는 신경세포의 보호, 성장촉진 및 재생용 조성물에 관한 것이다. 또한 본 발명은 황련추출물을 함유하는 신경질환 또는 신경손상 질환의 예방 및 치료에 유용한 약학 및 기능성 식품 조성물에 관한 것이다.The present invention relates to a composition for the protection, growth promotion and regeneration of nerve cells containing sulfur extract. The present invention also relates to pharmaceutical and functional food compositions useful for the prevention and treatment of neurological diseases or neuroinjury disorders containing sulfur extract.
본 발명에 따르면 다양한 종류의 퇴행성 신경질환, 허혈성 신경질환 또는 신경손상 질환의 예방 및 치료제와 학습능력 향상제로 매우 유용하게 사용될수 있다.According to the present invention, it can be very useful as a preventive and therapeutic agent for various types of degenerative neurological diseases, ischemic neurological diseases or neuroinjury diseases and as an improvement in learning ability.
신경세포와 신경세포간의 연결을 시냅스(synapse)라고 하며, 평균적으로 신경세포 1개는 다른 신경 세포와 약 1000-5000개의 시냅스를 이루며 인간의 뇌에는 적어도 1011개 정도의 신경세포가 있을 것으로 추정된다. 따라서 뇌내에는 적어도 1014개 이상의 무수한 시냅스 연결이 있을 것이며 이 수 많은 신경망을 통해서 인간의 모든 복잡하고 다양한 기능, 즉 사고, 인식, 기억, 학습, 행동과정이 수행되고 있는 것이다. 이러한 많은 시냅스의 연결은 신경세포의 생존에 필수적인 요건이며 각 신경세포간의 연결에 따른 특수한 기능이 수행되므로써 다양하고 무한한 고차원의 뇌 기능이 인간에게서 표출되게 되는 것이다. 시냅스의 주체인 신경세포의 특징은 손상을 받으면 대부분은 재생하지 않는 것으로 알려져 있다. 따라서 손상신경 조직 또는 아직 확실하게 규명되어 있지 않은 만성퇴행성 질환을 치료할 방법에 대한 발상과 시도들이 다방면에서 이루어지고 있다. 1940 년대에 Hamburger와 Levi-Montalcini들이 Chick embryo limb의 분화과정에서 운동신경세포 생존에 결정적인 미지의 물질을 발견하고 그 근거로 제기한 신경성인자 가설(neurotrophic factor hypothesis)을 바탕으로 NGF(nerve growth factor)가 발견되었고, 그 후에 뇌로부터 유도된 BDNF(brain-derived neurotrophic factor), NT-3(neurotrophic factor-3) 등의 신경성인자(neurotrophic factor)들이 발견되었다. 아울러 각 분화신경세포 집단의 생존에 어떤 종류의 신경성인자들의 공급이 필수적인 지에 대해서도 일부 형질전환 동물실험상에서 밝혀져 있다. 물론 신경세포의 생존에는 각종 뉴로트로핀(neurotrophin)들 만이 관여하는 것은 아니고 여러 종류의 싸이토카인(cytokine)들도 참여하고 있음이 밝혀지고 있다. 신경세포의 생존에는 그 외에 또 다른 많은 요인들이 관여하겠지만 뉴로트로핀이나 싸이토카인의 공급이 중단되거나 이 인자들을 받아들이는 수용체들이 세포에서 발현되지 않을 경우에 세포는 사망하게 되고 이러한 인자들이 공급될 때 세포는 생존 할 수 있는 것이다. 신경세포가 죽음에 이르는 형태는 일반 다른 세포의 경우처럼 세포자멸사(apoptosis)와 괴사(necrosis)로 대별된다. 이들은 서로 다른 형태학적, 분자생물학적 특징을 나타낸다. 축색돌기(axon)가 절단되면(axotomy) 세포체가 붙어있는 부분과 시냅스를 형성하고 있던 말단부분이 두 부분으로 분리된다. 축색돌기의 절단은 표적 세포체로부터 공급받는 여러 고유한 단백질 인자의 단절로 인한 시냅스의 변성 뿐 만 아니라, 축색돌기가 절단된 신경세포에 접촉하고 있는 시냅스들의 대부분이 탈리되는 현상(synaptic detachment)이 일어난다. 즉 재생이 성공적으로 이루어진다면 세포가 생존 할 수 있겠지만 대부분의 신경세포는 이로 인해 결국 세포자멸사의 형태로 사망한다. 사망한 신경세포의 자리는 흔히 말초신경계에서는 신경교세포(glia cell)로, 중추신경계에서는 미세교세포(microglia) 또는 신경별교세포(astrocyte)로 대치되는 과정(synaptic stripping)이 진행된다. 또한 손상의 정도에 따라 여기에 단핵세포(monocyte), 대식세포(macrophage)등의 면역계 세포들이 참여할 수 도 있다. 신경세포의 물리적인 손상을 포함하여 급성 신경독성, 급 만성 신경장애, 간질, 치매의 기전은 너무나 많은 방면에서 이론이 제기되고 있지만 이들 질환의 궁극적인 공통무대는 특정 신경 세포들과 그 지지 조직이다. 이 세포들이 수직 및 수평 방향으로 뻗고 있는 수많은 수상돌기들과 축색돌기들의 연결, 이들로 이루어지는 수많은 신경회로의 이상이 신호전달계의 이상을 초래하고 다양한 뇌신경계질환으로 나타나는 것이다. 현재 흥분성 신경전달물질인 글루타메이트(glutamate)에 반응하는 글루타메이트성 신경회로는 급 만성 뇌신경질환 발병에 가장 주목받고 있는 신경회로이다.The connection between neurons and neurons is called a synapse. On average, one neuron makes up about 1000-5000 synapses with other neurons, and there are at least 10 11 neurons in the human brain. do. Therefore, there are at least 10 14 innumerable synaptic connections in the brain, and through this many neural networks, all the complex and diverse functions of human beings are performed, such as thinking, perception, memory, learning, and behavior. Many of these synaptic connections are essential to the survival of neurons, and by performing specific functions according to the connections between neurons, various and infinitely high-level brain functions are expressed in humans. The characteristics of nerve cells, which are the main agents of synapses, are known to not regenerate most when damaged. Therefore, ideas and attempts to treat injured nerve tissue or chronic degenerative diseases that have not yet been clearly identified have been made in various fields. Based on the neurotrophic factor hypothesis, Hamburger and Levi-Montalcini discovered an unknown substance critical for motor neuron survival in the differentiation of chicken embryo limbs in the 1940s, and based on the neurotrophic factor hypothesis, NGF (nerve growth factor) After that, neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and neurotrophic factor-3 (NT-3) derived from the brain were found. In addition, some types of neurological factors are essential in the survival of each differentiated neuronal cell population. Of course, neurotrophic survival is not only involved in the various neurotrophin (neurotrophin) but also various types of cytokines (cytokine) is found to participate. Many other factors may be involved in the survival of neurons, but if the supply of neurotrophin or cytokine is interrupted or the receptors that accept these factors are not expressed in the cell, the cell dies and the cells are supplied when these factors are supplied. Is to survive. Neuronal death is largely divided into apoptosis and necrosis, as is the case with other cells. They exhibit different morphological and molecular biological characteristics. When the axon is cut (axotomy), the part where the cell body is attached and the terminal part that forms the synapse are separated into two parts. Cleavage of axons results in synaptic detachment as well as degeneration of synapses due to the disruption of several unique protein factors supplied from the target cell body, as well as the removal of most of the synapses in contact with the axons. . In other words, if regeneration is successful, cells can survive, but most neurons eventually die in the form of apoptosis. The place of dead neurons is often replaced by glia cells in the peripheral nervous system and by microglia or astrocytes in the central nervous system (synaptic stripping). In addition, depending on the degree of damage, immune system cells such as monocytes (macrophage) may participate. The mechanisms of acute neurotoxicity, sudden chronic neuropathy, epilepsy, and dementia, including physical damage to neurons, are being discussed in too many ways, but the ultimate common stage for these diseases is specific neurons and their supporting tissues. . The connection of numerous dendrites and axons, and the neural circuits that these cells extend in the vertical and horizontal directions, causes abnormalities in the signaling system and manifests as various neurological diseases. Glutamate neural circuits that respond to the current excitatory neurotransmitter glutamate are the ones that are attracting the most attention for the development of sudden chronic neurological disease.
포유동물의 뇌는 신경줄기세포(neuronal stem cell)의 분열과 분화 및 생존과 사멸(apoptosis) 그리고 시냅스 형성 등 일련의 과정을 거쳐 체계적인 신경회로망(neural network)을 발생(development)함으로서 복잡한 기능을 수행할 수 있게 된다. 동물의 성체시기에도 뇌신경세포에서는 신경성장에 필요한 많은 물질을 생산하여 신경축색(axon)과 수상돌기(dendrite)가 성장하게 되고 새로운 학습과 기억을 할 때마다 시냅스 연결과 신경회로망(neural network)을 끊임없이 재구성(synaptic remodeling)하므로 성체에서도 계속 분화(differentiation)하고 있다고 할 수 있다. 신경세포는 세포분화하고 시냅스를 형성하는 과정에서 신경성장인자와 같은 표적유래 생존인자(target-derived survival factor)를 받지 못하면 세포사멸하며 스트레스와 세포독성물질(cytotoxic agent)에 의한 세포사멸은 퇴행성뇌질환의 주요원인이 된다. 또한 말초신경이 손상되었을 때는 신경 상해 부위의 뒤쪽 축색은 월러변성(Wallerian degeneration)으로 알려진 과정에 의해 퇴화되고 신경의 세포체는 축색의 성장(axonal regrowth)을 다시 시작하며 슈반세포는 분열한 후 생존과 사멸에 의해 표적신경을 결정하고 다시 분화하는 등, 발생과정을 다시 거쳐 재생하게 된다.Mammalian brains perform complex functions by developing systematic neural networks through a series of processes, including division and differentiation of neuronal stem cells, survival and apoptosis, and synapse formation. It becomes possible. Even in adulthood, brain nerve cells produce many substances necessary for nerve growth, which leads to the growth of axons and dendrites, and to synaptic connections and neural networks as new learning and memories are developed. The remodeling (synaptic remodeling) is constantly differentiation in adults. When a neuron does not receive a target-derived survival factor such as a neuronal growth factor in the process of cell differentiation and synapse formation, apoptosis occurs and apoptosis caused by stress and cytotoxic agents is a degenerative brain. It is a major cause of disease. Also, when the peripheral nerve is damaged, the posterior axon of the nerve injury site is degenerated by a process known as Wallerian degeneration, the nerve cell body resumes axonal regrowth, and Schwann cells divide and survive. By killing, the target nerve is determined and differentiated again.
최근에는 성체에도 신경줄기세포가 있다는 것이 확인되고 있으며 줄기세포가 성체의 뇌에서 발생분화하는 과정은 바로 재생(regeneration)과정이라 할 수 있다(Johansson, C. B., Momma S., Clarke D. L., Risling M., Lendahl U., and Frisen J. (1999) Identification of a neural stem cell in the adult mammalian central nervous system,Cell96, 25-34). 신경줄기세포는 주로 측뇌실(lateral ventricle)과 맞닿는 줄무늬체(striatum)의 부뇌실 구역(subventricular zone)에서 발견되고 또한 해마(hippocampus)의 치상회(dentate gyrus)에 있는 subgranular zone에서 줄기세포들이 분열하여 과립세포(granule cell)로 되며 이 세포들이 분화하여 기능을 가질 수 있다(van Praag et al.,Nature415, 1031-1034 (2002). 따라서 신경줄기세포의 발생과 분화를 증진하면 신경재생을 촉진할 수 있다.Recently, it has been confirmed that neural stem cells exist in adults, and the process of stem cell development in the adult brain is a regeneration process (Johansson, CB, Momma S., Clarke DL, Risling M., Lendahl U., and Frisen J. (1999) Identification of a neural stem cell in the adult mammalian central nervous system, Cell 96, 25-34). Neural stem cells are found mainly in the subventricular zone of the striatum, which contacts the lateral ventricle, and also in the subgranular zone in the dentate gyrus of the hippocampus, causing stem cells It is a granule cell and these cells can differentiate and function (van Praag et al., Nature 415, 1031-1034 (2002). Thus, promoting the development and differentiation of neural stem cells can promote neuronal regeneration. have.
포유동물의 뇌 발생과정에서는 생성된 신경세포의 반 이상이 세포사멸 한다. 또한 신경세포는 신경계 질환에서 뿐 아니라 정상 성인의 뇌에서도 매일 많은 숫자가 죽어가며 노화하는 신경계에서는 말할 나위도 없다(Yuan and Yankner,Nature.407, 802-809 (2000). 따라서, 신경세포의 세포사멸을 극복하는 것은 중추신경계의 퇴행성 뇌질환 뿐 아니라 척수손상 및 말초신경계 손상 등 거의 모든 신경계 질환에서 중요한 문제가 된다. 최근 유럽에서는 퇴행성뇌질환 환자 특히 파킨슨씨병 환자의 경우 태아에서 얻은 신경줄기세포를 이식하는 것이 임상적으로 적용되고 있는데 이식 후에 환자는 매우 빨리 회복되어 행동이 좋아지지만 3달 후면 대부분의 이식된 세포가 사멸하여 다시 이식해야 하는 것이 문제이다(Olanow C. W., Kordower J. H., Freeman T. B. (1996) Fetal nigral transplantation as a therapy for Parkinson's disease.Trends Neurosci.19, 102-109.) 신경계에서 장기적 생존을 증진하기 위해서는 결국 이식된 세포가 그 조직에 적합한 종류의 신경세포로 분화하여 표적세포와 시냅스를 만들고 전기적 신호전달에 참여하여 표적세포로부터 계속해서 생존인자를 공급받아야 가능하다.In mammalian brain development, more than half of the generated neurons die. In addition, neurons die every day in the brain of normal adults, as well as in neurological diseases, not to mention in the aging nervous system (Yuan and Yankner, Nature. 407, 802-809 (2000). Overcoming death is an important problem in almost all neurological diseases, including spinal cord injury and peripheral nervous system damage, as well as degenerative brain diseases of the central nervous system, etc. Recently, in Europe, patients with degenerative brain diseases, especially Parkinson's disease, have transplanted neural stem cells from the fetus. It is clinically applied, and after transplantation, patients recover very quickly and improve their behavior, but after three months most transplanted cells are killed and need to be transplanted again (Olanow CW, Kordower JH, Freeman TB (1996)). fetal nigral transplantation as a therapy for Parkinson 's disease. Trends Neurosci. 19, 102-109.) long-term survival in the nervous system In order to promote it is possible to end the implanted cells should be continuously supplied to survival factors from the target cell to create and participate in target cells and synapses electrical signaling to differentiate into the appropriate type of neural cells in the tissue.
신경세포 사멸에 대한 연구는 분화된 신경세포에서는 많이 연구되어왔으나 신경줄기세포에서는 연구 보고가 거의 없으며 신경세포 사멸을 극복할 수 있는 물질에 대한 연구도 아직 미비하다.The research on neuronal cell death has been studied in differentiated neurons, but there are few reports on neural stem cells, and there are still few studies on substances that can overcome neuronal cell death.
신경줄기세포는 분열하여 자기 자신과 또는 분화할 세포를 만드는데 이때 세포분열이 잘못되었거나 필요 없는 세포는 세포자연사(cell death)하게 되고 생존(survival)하게 된 세포는 앞으로 어떤 세포로 분화할 것인지 운명을 결정하게 된다. 신경세포로 분화하는 신경전구세포(neuronal precursor 또는 neuroblast)는 그 장소에 적합한 신경전달물질을 분비하는 세포로 분화하게 되고 신경교세포로 분화하는 신경교 전구세포(glial precursor)는 신경별교세포와 희소돌기아교세포(oligodendrocyte)로 분화한다. 이들은 신경세포를 보조하는 세포로 신경별교세포는 기계적 대사적으로 지지하고 성체 뇌의 70-80%를 이루며, 희소돌기아교세포는 신경축색을 둘러싸서 신경전달을 빠르게 하는 지방물질인 미엘린(myelin)을 생산한다. 태아와 성체의 중추신경계 신경줄기세포는 뇌조직 내의 삼차원적 환경과 그들이 전달받는 신호의 종류에 따라 세 가지 종류 뇌세포를 모두 만들 수 있다.Neural stem cells divide to form cells that will differentiate themselves or to differentiate cells that have poor or no need for cell division and will die, and which cells will become surviving. Done. Neuronal precursor cells (neuronal precursors or neuroblasts) that differentiate into neurons are differentiated into cells that secrete neurotransmitters suitable for the site. Glial precursors that differentiate into glial cells are glial precursors and oligodendrocytes. Differentiate into oligodendrocytes. These are the cells that support nerve cells, and neuroglial cells support mechanical metabolism and make up 70-80% of the adult brain, while oligodendrocytes surround my nerve axons, myelin, a fatty substance that accelerates neurotransmission. To produce. Fetal and adult central nervous system neural stem cells can produce all three types of brain cells, depending on the three-dimensional environment within the brain tissue and the types of signals they receive.
중추신경계 줄기세포로 세 가지 종류가 보고되었는데 이들 모두 설치류 성체 뇌에 존재하고 인간의 성체 뇌에도 존재할 것으로 보인다. 한 종류는 뇌실 구역과 부뇌실 구역으로 알려진 뇌실(ventricle)과 맞닿는 부위의 뇌조직에 존재한다. 뇌실은 뇌척수액(cerebrospinal fluid)이 흐르는 공간이다. 태아 발생과정에서 뇌실 주변 조직은 세포 분열이 매우 활발히 일어나는 부위이고 성체에도 이 부위에 줄기세포가 존재하나 조직이 훨씬 작아져 있다. 줄기세포가 존재하는 두번째 부위는 사람에서는 아직 발견되지 않은 부위로 측뇌실과 후각망울(olfactory bulb)를 연결하는 길다란 틈을 이루는 부위(rostral migratory stream)이고, 세번째 부위는 기억의형성에 관계하는 뇌 부위인 해마로 성체 쥐와 사람의 뇌에서 존재한다.Three types of central nervous system stem cells have been reported, all of which appear to be present in rodent adult brains and in human adult brains. One type exists in the brain tissue at the site of contact with the ventricles, known as ventricular and paraventricular zones. The ventricles are spaces through which cerebrospinal fluid flows. During fetal development, the tissues around the ventricles are very active in cell division, and stem cells are present in the adult but much smaller. The second site where stem cells are present has not yet been found in humans. It is the rostral migratory stream that connects the lateral ventricles and olfactory bulbs. The third site is the brain region involved in the formation of memory. The hippocampus is present in the adult rat and human brain.
해마에서 줄기세포가 있는 부위는 치상회(dentate gyrus)의 subgranular zone이다. 쥐에서 BrdU(bromodeoxyuridine)로 분열하는 세포를 표지했을 때 표지된 세포의 반 정도가 치상회의 과립세포(granule cell)로 분화하고 15%는 신경교세포로 분화하며 나머지는 확실한 표현형을 갖지 않는다.The site of stem cells in the hippocampus is the subgranular zone of the dentate gyrus. In rats that divide with brodoxyuridine (BrdU), half of the labeled cells differentiate into granule cells of the dentate gyrus, 15% differentiate into glial cells, and the rest do not have a clear phenotype.
사람과 쥐의 치상회에서 BrdU표지된 일부 세포들은 신경세포 표지자인 NeuN, neuron-specific enolase, 또는 calbindin 등을 발현하며 이들 신경 유사세포들은 치상회의 과립세포와 형태적으로 유사하였다. 다른 BrdU 표지된 세포들은 신경별교세포 표지자인 GFAP를 발현하였다. 최근 연구에서는 진단 목적으로 BrdU를 주입한 다섯 암 환자(57-72세)의 뇌조직에서 BrdU 표지된 세포를 조사한 결과 가장 나이 많은 환자의 뇌에서 가장 많은 수의 BrdU 표지된 세포가 발견되었으며 이는 해마에서의 신경세포 형성이 생애 마지막까지도 계속된다는 것을 의미한다.Some BrdU-labeled cells in the dentate gyrus of humans and rats express neuronal markers such as NeuN, neuron-specific enolase, or calbindin. Other BrdU labeled cells expressed glial cell marker GFAP. In a recent study, BrdU-labeled cells were examined in brain tissues of five cancer patients (57-72 years) who were injected with BrdU for diagnostic purposes and found that the largest number of BrdU-labeled cells were found in the brains of the oldest patients. Neuronal cell formation in Esau continues until the end of life.
신경성장인자들은 포유동물의 신경발생과정에서 신경줄기세포의 분열·분화와 사멸의 운명결정, 신경과 신경교세포로의 운명결정, 그리고 시냅스 형성에 관여하는 것으로 알려져 있다.Nerve growth factors are known to be involved in the division and differentiation of neural stem cells and the fate of apoptosis, the fate of neurons and glial cells, and the formation of synapses in mammalian neurogenesis.
Fibroblast growth factors(FGF)는 neuroectoderm과 mesoderm-derived cell의 분열을 촉진하는 성장인자로 발견되어 여러 종류가 있으며 특히 isoelectric point에 따라 acidic FGF(aFGF), basic FGF(bFGF)로 나눈다. FGF receptor 중low-affinity binding site는 membrane-associated proteoglycan이고 FGF가 high-affinity binding site에 결합하는데 필수적이다. High-affinity receptor들은 거의 모두가 receptor-tyrosine kinase이고 FGF가 결합하면 dimer를 이루어 tyrosine을 autophosphorylation시키고 신호를 전달하는 것이 3T3 fibroblast와 platelet에서 알려졌다. FGF 수용체는 4개의 유전자로부터 alternative splicing에 의해 다양한 transcript로 발현되며, 각 type의 수용체는 한가지 이상의 FGF family member와 binding할 수 있으며 ligand binding specificity는 receptor type 및 splicing form에 의해서 결정된다. FGF는 mitogen으로써의 활성 뿐 아니라, cell differentiation을 일으키며, PC12 세포에 처리하면 NGF를 처리했을 경우와 같이 neuronal phenotype을 갖는 세포로 분화하게 한다.Fibroblast growth factors (FGF) are found as growth factors that promote the division of neuroectoderm and mesoderm-derived cells, and are classified into acidic FGF (aFGF) and basic FGF (bFGF) according to the isoelectric point. The low-affinity binding site of the FGF receptor is a membrane-associated proteoglycan and is essential for FGF binding to the high-affinity binding site. Almost all high-affinity receptors are receptor-tyrosine kinase, and when FGF binds, it is known in 3T3 fibroblast and platelet to form dimer, autophosphorylation and signal transduction of tyrosine. FGF receptors are expressed in various transcripts by alternative splicing from four genes, and each type of receptor can bind to one or more FGF family members, and ligand binding specificity is determined by receptor type and splicing form. FGF not only acts as a mitogen, but also causes cell differentiation. When treated with PC12 cells, FGF differentiates into cells with neuronal phenotype as in the case of NGF treatment.
FGF 수용체의 신호전달계는 많이 연구되어 있지 않으나 해마의 일차 배양 세포와 PC12세포에 처리하였을 때 tyrosine phosphorylation이 증가하며 mitogen-activated protein kinase(MAP kinase)인 p42 MAP kinase(ERK2)와 p44 MAP kinase(ERK1)가 활성화된다. 또한, bFGF가 c-fos와 같은 transcription factor를 유도하는 것도 알려졌다. FGF는 발생 중인 흰쥐 뇌의 1차 신경세포 배양에서 해마와 대뇌피질신경의 생존과 neurite outgrowth를 증가시키며, glutamate에 의한 excitotoxicity를 감소시키는 것이 발견되었다. 배양된 FGF 수용체의 mRNA는 성체의 뇌에서 많이 발견되며 특히 발생 중인 쥐의 뇌와 해마의 1차 배양된 신경세포에서도 발견되었다. Xenopus의 망막 시신경 세포가 발생하는 중에 FGF는 이들 신경의 생존을 증진시키며 특히 FGF의 발현이 매우 짧은 기간동안 급격히 증가하는 것이알려져 있다.Although the signaling system of FGF receptor is not studied much, tyrosine phosphorylation increases when treated in primary culture cells and PC12 cells of hippocampus, and p42 MAP kinase (ERK2) and p44 MAP kinase (ERK1), which are mitogen-activated protein kinase (MAP kinase) ) Is activated. It is also known that bFGF induces transcription factors such as c-fos. FGF was found to increase the survival and neurite outgrowth of hippocampus and cerebral cortex nerves in primary neuronal cultures of developing rat brain, and to reduce glutamate-induced excitotoxicity. Cultured FGF receptor mRNA was found in adult brains, especially in developing rat brain and hippocampal primary neurons. During the development of Xenopus retinal optic nerve cells, FGF enhances the survival of these nerves, and it is known that the expression of FGF increases rapidly in a very short period of time.
해마의 pyramid 신경세포가 발생하기 시작하는 E16에 신경간세포를 1차배양하고 신경성장인자인 FGF를 처리하면 세포의 분열이 증가하고 분화하여 약 30%가 신경세포로, 나머지는 glia 세포로 발달한다. McKay group은 PDGF를 처리하면 약 80%가 신경세포로 분화하도록 운명이 제한되고 신경세포로 분화하여 neuronal marker를 발현하며, 또한 FGF와 EGF를 처리한 후 CNTF를 처리하면 astrocyte로 분화하며 thyroid hormone T3는 oligodendrocyte로의 분화를 촉진한다고 발표하였다. 이는 PDGF가 원시신경세포 발생의 매우 초기에 neurotrophic factor로 작용하여 neuronal cell fate를 결정하는 것을 의미한다. 우리는 해마의 원시신경세포주(HiB5)에 PDGF와 FGF를 처리하였을 때 세포사멸이 억제되었고 신경세포 또는 glia로 분화하는 cell fate에 영향을 주는 것을 발견하였다.( Kwon, Y. Kim (1997) Expression of brain-derived neutrophic factor mRNA stimulated by basic fibroblast grwoth factor adn platelet-derived growth factor in rat hippocampal cell line,Mol. Cells7, 320-325.).The primary culture of neural stem cells on E16, which begins to develop pyramid neurons in the hippocampus, and treatment with FGF, a nerve growth factor, increase cell division and differentiate, resulting in about 30% development of neurons and the rest of glia cells. . McKay group is limited to about 80% of PDGF treatment to differentiate into neurons, differentiates into neurons to express neuronal markers, and after treatment with FGF and EGF, CNTF treatment to differentiate into astrocytes and thyroid hormone T3 Announced that it promotes differentiation into oligodendrocytes. This means that PDGF acts as a neurotrophic factor at an early stage of primitive neuronal cell development and determines neuronal cell fate. We found that treatment of PDGF and FGF in the hippocampal primitive neuronal cell line (HiB5) inhibited apoptosis and affected cell fate that differentiates into neurons or glia (Kwon, Y. Kim (1997) Expression of brain-derived neutrophic factor mRNA stimulated by basic fibroblast grwoth factor adn platelet-derived growth factor in rat hippocampal cell line, Mol. Cells 7, 320-325.).
신경성장인자들은 신경간세포의 분열개시, 분열한 세포의 수를 사멸과정으로 조절하는 동시에 분화를 시작하도록 하며, 표적유래성장인자로서 정향 이동된 세포의 생존과 잘못된 방향으로 이동된 세포의 사멸을 유도하여 시냅스전 신경세포의 생존을 조절하는 한편 새로운 시냅스 형성과 재구성을 조절한다. 또한 성체의 신경 생존과 시냅스 가소성 및 신경손상 시 재생하는 과정에서도 유사한 기능을 가질 것으로 추측된다. 인간의 경우 중추신경계는 물론 말초신경계도 재생이 어려워 퇴행성 뇌질환 뿐만 아니라 현대사회의 산업재해 및 교통사고, 전쟁불구자 등이 누적되면서 사회문제화 되고 있으므로 신경계의 재생에 관한 연구에 많은 관심이 집중되고 있다.Nerve growth factors initiate the division of neural stem cells, control the number of dividing cells into a process of killing and initiate differentiation, and induce the survival of oriented cells and the death of cells migrated in the wrong direction as target-derived growth factors. It regulates the survival of presynaptic neurons while also regulating new synapse formation and reconstitution. In addition, the neuronal survival of the adult, synaptic plasticity and neuronal damage during regeneration process is expected to have a similar function. In the case of humans, the central nervous system as well as the peripheral nervous system is difficult to regenerate, so as not only degenerative brain diseases but also industrial accidents, traffic accidents, and war-deficiency in modern society are accumulating social problems, so much attention has been focused on the regeneration of nervous systems. .
한편 황련(黃連,Coptidis rhizoma,Coptis japonica, Makino속 식물의 뿌리)은On the other hand, yellow lotuses ( Coptidis rhizoma , Coptis japonica , and the roots of Makino plants)
적어도 3000년 이전부터 중국의학에서 사용되어 왔다It has been used in Chinese medicine since at least 3000 years.
한방에서 건위·진정·소염·항균·해열 등의 효능이 있어 소화불량·위염·장염·복통·구토·이질·정신불안·하혈·화상 등의 치료에 오래 전부터 처방되어 왔다. 종래의 황련에 대한 연구결과들을 분류하면 항염증성, 소염기능, 당뇨병 치료기능, 항종양기능, 면역증강성, 부정맥조절기능, 신장보호 기능 등의 약리작용에 관한 연구들이다. (Birdsall & Kelly, 1977).It has long been prescribed for the treatment of dyspepsia, gastritis, enteritis, abdominal pain, vomiting, dysentery, mental anxiety, bleeding, and burns because it has the effects of dry stomach, soothing, anti-inflammatory, antipyretic, etc. in oriental medicine. Classification of the results of the conventional researches on the anti-inflammatory, anti-inflammatory, anti-inflammatory function, diabetes treatment function, anti-tumor function, immuno-enhancing, arrhythmia control function, pharmacological action such as kidney protection functions. (Birdsall & Kelly, 1977).
미합중국특허 제4608258호에는 황련이 설사치료제로서의 효과가 있음을 개시하였으며,U.S. Patent No. 4608258 discloses that rhubarb has an effect as a diarrhea treatment.
미합중국특허 제5916555호에는 황련을 포함하는 생약조성물이 당뇨병치료제로서의 효과가 있음을 개시하였다.U.S. Patent No. 5916555 discloses that the herbal composition containing the yellow lotus has the effect of treating diabetes.
그 외에도 고지혈증 흰쥐를 이용한 황련의 항고지혈 효과 연구, 기관지평활근 이완 효과 등에 관한 연구가 있으나, 황련추출물이 뇌신경계 질환에 사용된 바가 없다.더구나 신경세포돌기(neurite)에 대한 신생, 성장촉진, 방어, 재생 작용에 대한 효과는 아직 보고된 바 없다.In addition, there have been studies on antihyperlipidemic effect and the bronchial smooth muscle relaxation effect of H. coli in hyperlipidemic rats. However, L. extract has not been used for the neurological diseases of the brain. However, no effect on regenerative action has been reported.
선진 사회의 문제 중의 하나는 고령화로 인한 여러 가지 만성, 난치성 질환들로 인한 사회적, 경제적 부담일 것이다. 그 중 신경 계통의 질환은 뇌기능 연구에 대한 신경과학의 전반적인 연구가 아직 부진한 상태이므로 각종 만성 신경계 질환의 치료 약제개발 또한 난관에 처해 있다.One of the problems of advanced societies will be the social and economic burden of various chronic and intractable diseases caused by aging. Among the diseases of the nervous system, the overall research of neuroscience about the study of brain function is still in a poor state, and the development of therapeutic drugs for various chronic nervous system diseases is also in trouble.
본 발명의 목적은 황련추출물을 함유하는 신경세포의 보호, 성장촉진 및 재생용 약학 및 식품 조성물을 제공하는 것이다. 본 발명의 다른 목적은 신경질환 또는 신경손상 질환의 예방 및 치료에 유용한 황련추출물을 함유하는 약학 및 기능성 식품 조성물을 제공하는 것이다. 본 발명의 또 다른 목적은 황련추출물을 함유하는 다양한 종류의 퇴행성 신경질환, 허혈성 신경질환 또는 사고 등에 의한 신경손상 질환의 예방 및 치료제와 학습능력 향상제를 제공하는 것이다.It is an object of the present invention to provide pharmaceutical and food compositions for the protection, growth promotion and regeneration of nerve cells containing rhubarb extract. It is another object of the present invention to provide a pharmaceutical and functional food composition containing rhubarb extract useful for the prevention and treatment of neurological or neurological damage diseases. Still another object of the present invention is to provide an agent for preventing and treating neuroinjury diseases caused by degenerative neurological diseases, ischemic neurological diseases or accidents, and the improvement of learning ability, which contains the extract of the rye.
도 1은 분화된 신경세포주인 인간 신경아세포종(neuroblastoma) SH-SY5Y에서 혈청제거 스트레스에 의해 유발되는 세포사멸에 대한 황련추출물의 보호 효과를 나타내는 그래프이다.FIG. 1 is a graph showing the protective effect of Rhizome extract against apoptosis induced by desorption stress in human neuroblastoma SH-SY5Y, a differentiated neuronal cell line.
도 2는 흰쥐 신경릉(neural crest)에서 유래한 신경세포암종인 pheochromocytoma cell(PC12) 세포에 혈청제거(serum deprivation) 스트레스를 주고 이에 대한 황련추출물의 세포사멸 방어 효과를 나타내는 그래프이다.Figure 2 is a graph showing the anti-apoptotic protection effect of the rhubarb extract to give a serum deprivation stress to the pheochromocytoma cell (PC12) cells, a neuronal carcinoma derived from neural crest of rats.
도 3은 분화된 신경세포주인 인간 신경아세포종(neuroblastoma) SH-SY5Y에서 산화적 스트레스에 의해 유발되는 세포사멸에 대한 황련추출물의 보호 효과를 나타내는 그래프이다.Figure 3 is a graph showing the protective effect of the barberry extract against apoptosis induced by oxidative stress in human neuroblastoma SH-SY5Y, a differentiated neuronal cell line.
도 4는 흰쥐 유래 신경줄기세포인 HiB5에 황련추출물을 처리한 후, 세포분화 유도효과를 공초점 현미경(confocal microscope)으로 촬영한 사진이다. bFGF(basic fibroblast growth factor)를 처리한 상태를 bFGF+로, 처리하지 않은 상태를 bFGF-로 나타내었다.FIG. 4 is a photograph taken after treatment of the barberry extract to HiB5, a rat-derived neural stem cell, using a confocal microscope. FIG. The state treated with bFGF (basic fibroblast growth factor) was expressed as bFGF + and the state not treated with bFGF-.
도 5는 분화된 신경세포주인 인간 신경아세포종(human neuroblastoma) SH-SY5Y를 사용하여 신경돌기(neurite) 재생에 대한 황련추출물의 효과를 공초점 현미경으로 촬영한 사진이다. 레티노산(retinoic acid)는 SH-SY5Y의 신경돌기 분화를 일으키는 양성대조군이다.Figure 5 is a photograph taken by confocal microscopy of the effect of the Rhizome extract on neurite regeneration using human neuroblastoma SH-SY5Y, a differentiated neuronal cell line. Retinoic acid is a positive control group that causes neurite differentiation of SH-SY5Y.
도 6a 내지 6c는 본 발명의 황련추출물을 각각 20, 60, 200 ㎍/㎖로 18일간 투여 한 후 나타난 PC12 세포의 형태학적 변화를 보여주는 사진으로서, 뚜렷한 신경세포돌기(neurite)의 생성을 보여주고 있다.Figure 6a to 6c is a photograph showing the morphological changes of the PC12 cells after 18 days of administration of the rhubarb extract of the present invention at 20, 60, 200 ㎍ / ㎖, respectively, showing the production of distinct neuronal processes (neurite) have.
도 7은 NGF (Nerve Growth Factor. 50 ng/㎖)를 투여한 후 3일이 경과된 PC12 세포의 신경세포돌기(neurite)의 생성을 보여주는 사진이다.Figure 7 is a photograph showing the generation of neurons (neurite) of PC12 cells 3 days after administration of NGF (Nerve Growth Factor. 50 ng / ㎖).
도 8a 내지 도 8b는 황련추출물(120 ㎍/㎖)을 기간별(도 8a: 4일 동안 처치한 군, 도 8b: 10일 동안 처치한 군)로 PC12 세포의 배양액에 처리하여 생성되는 신경세포돌기의 길이를 측정하여 막대그래프로 나타낸 것이다.Figures 8a to 8b is a neuronal projection produced by treating the culture of PC12 cells with a barberry extract (120 ㎍ / ㎖) by period (Fig. 8a: group treated for 4 days, Figure 8b: group treated for 10 days) Measure the length of the bar graph.
도 9a는 PC12 세포에서 황련추출물 처치에 의해 나타난 NGF mRNA의 발현을 보여주는 사진으로서 정상군에 비해 황련추출물 처치군에서 현저히 높은 NGF 발현도를 보여주고 있다. 도9b는 mRNA의 정량에 있어 대조군으로 사용되는 GAPDH mRNA 발현 양상을 보여주고 있다.Figure 9a is a picture showing the expression of NGF mRNA by the treatment of rhubarb extract in PC12 cells showing a significantly higher NGF expression in the rhubarb extract treatment group than the normal group. Figure 9b shows the expression of GAPDH mRNA used as a control in the quantification of mRNA.
도 10a 및 10b는 생후 7일된 흰쥐의 정상 대뇌 절편의 TUNEL 염색을 보여주고 있다. 도 10b는 도 10a의 네모부분을 확대한 사진(배율: X400)이다.10A and 10B show TUNEL staining of normal cerebral sections of 7-day-old rats. FIG. 10B is an enlarged photograph (magnification: X400) of the square of FIG. 10A.
도 11a 및 11b는 MK-801 (0.5 mg/kg)을 생후 7일된 흰쥐에 복강 주사한 1일 후에, 대뇌절편상에서 볼 수 있는 신경세포의 사망양상을 보여주는 사진이다. 도 11a는 대뇌의 전체 coronal 절편을 보여준다(까맣게 나타나는 세포들은 핵 내의 DNA가 분절된 세포에만 양성으로 염색되는 apoptosis 검색법, 즉 TUNEL법에 양성반응을 보이는 세포들이다). 도 11b는 도 11a의 네모부분을 확대한 사진(배율: X400)으로서 apoptosis로 사망한 세포들을 보여준다.11a and 11b are photographs showing the death pattern of neurons seen on cerebral sections after 1 day of intraperitoneal injection of MK-801 (0.5 mg / kg) in 7-day-old rats. Figure 11a shows the entire coronal section of the cerebrum (the cells that appear black are cells that are positive for apoptosis detection, ie TUNEL, in which DNA in the nucleus is stained positively only in the segmented cells). FIG. 11B is an enlarged photograph (magnification: X400) of the square of FIG. 11A, showing cells killed by apoptosis.
도 12a 및 12b는 본 발명의 황련추출물(20 mg/kg)만을 생후 7일된 흰쥐에 복강내 주사한 후 3일 째 적출한 대뇌 절편의 TUNEL 염색사진으로 황련추출물 자체는 신경세포사를 유발하지 않음을 보여주고 있다. 도 12b는 도 12a의 네모부분을 확대한 사진(배율: X400)이다.12A and 12B are TUNEL staining photographs of cerebral slices extracted three days after intraperitoneal injection into rats of 7 days old only when the stratus extract of the present invention (20 mg / kg) does not induce neuronal cell death. Is showing. FIG. 12B is an enlarged photograph (magnification: X400) of the square of FIG. 12A.
도 13a 및 13b는 본 발명의 황련추출물(20 mg/kg)을 생후 7일된 흰쥐의 복강에 3일 동안 전처치한 후 MK-801 (0.5 mg/kg)을 복강주사한 실험군의 대표사진으로서, MK-801 (0.5 mg/kg)에 의해 유도된 신경세포자멸사의 정도가 본 발명의 황련추출물에 의해서 현저하게 억제됨을 보여주고 있다. 도 13b는 도 13a의 네모부분을 확대한 사진이다(배율: X400).13A and 13B are representative photographs of the experimental group in which MK-801 (0.5 mg / kg) was intraperitoneally injected after pretreatment of the rye extract of the present invention (20 mg / kg) in the abdominal cavity of 7-day-old rats for 3 days. The degree of neuronal apoptosis induced by MK-801 (0.5 mg / kg) is shown to be significantly suppressed by the sulfur extract of the present invention. FIG. 13B is an enlarged photograph of the square of FIG. 13A (magnification: X400).
도 14a 및 14b는 생후 7일된 흰쥐의 복강에 MK-801 (0.5 mg/kg)을 주사하여 신경세포 자멸사를 유도한 후 본 발명의 황련추출물을(20 mg/kg)을 6일 동안 복강 주사한 후 적출한 대뇌절편 사진으로서, MK-801 (0.5 mg/kg)에 의해 유도된 신경세포자멸사의 정도가 본 발명의 황련추출물에 의해서 현저하게 억제됨을 보여주고 있다. 도 14b는 도 14a의 네모부분을 확대한 사진이다(배율: X400).Figures 14a and 14b is injected into the abdominal cavity of 7 days old rats MK-801 (0.5 mg / kg) to induce neuronal apoptosis after intraperitoneal injection of the barberry extract of the present invention (20 mg / kg) for 6 days The post-extracted cerebral slice photograph shows that the degree of neuronal apoptosis induced by MK-801 (0.5 mg / kg) is significantly suppressed by the rye extract of the present invention. 14B is an enlarged photograph of the square of FIG. 14A (magnification: X400).
도 15는 어린 흰쥐의 대뇌절편상에서 MK-801 (0.5 mg/kg)에 의해 사망하는 신경세포자멸사의 정도(TUNEL positive cell numbers)가 황련추출물에 의해서 억제되는 양상을 정량적으로 나타낸 그림이다.FIG. 15 is a quantitative diagram showing that the degree of neuronal apoptosis (TUNEL positive cell numbers) killed by MK-801 (0.5 mg / kg) in the cerebral sections of young rats is inhibited by the L. extract.
도 16a는 본 발명의 황련추출물을 용량별로 생후 7일된 흰쥐에 1일(lane 1, 2, 3) 또는 3일(lane 4, 5, 6) 동안 복강 주사한 후에 대뇌 조직에서 발현되는 항고사 유전자인 bcl-2 mRNA의 발현양상을 RT-PCR 방법으로 조사한 것으로써 정상군에 비하여 그 발현이 증가함을 보여주는 사진이다.Figure 16a is an anti-apoptotic gene expressed in cerebral tissue after intraperitoneal injection of the extract of the present invention for 7 days of age by the dose per 1 day (lane 1, 2, 3) or 3 days (lane 4, 5, 6) Phosphorus expression of bcl-2 mRNA was investigated by RT-PCR method.
도 17은 치매가 유도된 흰쥐의 뇌조직을 헤마톡실린(hematocylin)으로 염색하여 황련추출물의 신경재생 효과를 현미경으로 촬영한 사진이다.Figure 17 is a photograph of the brain tissue of rats induced by dementia stained with hematoxylin (hematocylin), the neuronal regeneration effect of the rhubarb extract under a microscope.
도 18은 치매가 유도된 흰쥐의 뇌조직을 신경특이적표지자인 calbindin항체로 형광염색하여 황련추출물의 신경재생 효과를 공초점 현미경으로 촬영한 사진이다.FIG. 18 is a photograph taken by confocal microscopy of the neuronal regeneration effect of the rhubarb extract by fluoresceining dementia-induced brain tissue with a calbindin antibody, a neurospecific marker.
도 19는 말초신경인 좌골신경이 손상된 흰쥐에 황련추출물을 복강주사한 후 1주일뒤의 신경재생효과를 공초점 현미경으로 촬영한 사진이다(200배율)19 is a photograph taken by confocal microscopy of the neuronal regeneration effect after one week after intraperitoneal injection of the rat sciatic nerve injured peripheral nerve (200 magnification)
beta-tubulin isotypeIII(붉은색)로 염색한 사진의 흰선은 길이가 300um가 넘는 축색을 나타내며, MBP(myelin binding protein, 녹색)항체로 염색한 사진상의 화살표(arrowhead)는 퇴화된 미엘린조각을 나타낸다.The white line on the photo stained with beta-tubulin isotype III (red) shows axons of more than 300 μm in length, and the arrowhead on the photo stained with myelin binding protein (MBP) antibody shows degenerated myelin fragments.
도 20은 좌골신경이 손상된 흰쥐에 황련추출물을 복강주사한 후 2주일뒤의 신경재생효과를 공초점 현미경으로 촬영한 사진이다(200배율)20 is a photograph taken with a confocal microscope of the nerve regeneration effect after two weeks after the abdominal injection of rhubarb extract in rats with sciatic nerve (200 magnification)
beta-tubulin isotypeIII(붉은색)로 염색한 사진의 흰선은 길이가 300um가 넘는 축색을 나타내며, MBP(myelin binding protein, 녹색)항체로 염색한 사진상의 화살표(arrow)는 길이가 200um가 넘는 슈반세포의 재생된 미엘린수초를 나타낸다.The white line in the picture stained with beta-tubulin isotype III (red) shows axons of more than 300 μm in length, and the arrow on the picture stained with myelin binding protein (MBP) antibody shows Schwann cells over 200 μm in length. Represents the regenerated myelin sheath of.
도 21은 좌골신경이 손상된 흰쥐에 황련추출물을 복강주사한 후 4주일뒤의 신경재생효과를 공초점 현미경으로 촬영한 사진이다(200배율)21 is a photograph taken by confocal microscopy of the neuronal regeneration effect after 4 weeks after the abdominal injection of rhubarb extract in rats with sciatic nerve (200 magnification)
beta-tubulin isotypeIII(붉은색)로 염색한 사진의 흰선은 길이가 300um가 넘는 축색을 나타내며, MBP(myelin binding protein, 녹색)항체로 염색한 사진상의 화살표는 길이가 200um가 넘는 미엘린을 나타낸다. 길고 굵은 축색과 미엘린 수초가 많아졌음을 확인 할 수 있다. 미엘린이 분화하여 재성장한 축색을 둘러싸면 두 항체표지자의 염색이 겹쳐 노란색으로 보이기도 한다.The white line on the picture stained with beta-tubulin isotype III (red) shows axons of more than 300 μm in length, and the arrow on the picture stained with myelin binding protein (MBP) antibody shows myelin over 200 μm in length. Long, thick axons and myelin sheaths are more common. When the myelin differentiates and regrows the axon, the staining of the two antibody markers may appear yellow.
도 22는 도19을 400배율로 촬영한 사진이다.22 is a photograph taken at 400 magnification of FIG. 19.
도 23은 도20를 400배율로 촬영한 사진이다.23 is a photograph taken at 400 magnification of FIG. 20.
도 24는 도21을 400배율로 촬영한 사진이다.24 is a photograph taken at 400 magnification of FIG. 21.
도 25는 신경근접합부 재형성 과정에서 황련추출물의 신경재생효과를 나타낸 사진이다. 대조군에서 신경말단은 하나의 근섬유에 도달하여 있으나 다른 섬유에 퍼지지 못한 상태이고 황련추출물을 투여한 경우 모든 근섬유에 도달하여 신경근 접합부를 형성한 것으로 보인다.Figure 25 is a photograph showing the neuronal regeneration effect of the rhubarb extract during the neuromuscular junction remodeling process. In the control group, the nerve endings reached one muscle fiber but did not spread to the other fiber, and when the rhubarb extract was administered, all the nerve fibers reached to form the nerve root junction.
본 발명은 황련추출물의 신경세포 보호 및 성장촉진 및 신경세포 재생용 조성물에 관한 것으로, 본 발명은 신경계의 물리적 손상 및 퇴행성, 허혈성 뇌신경 손상, 말초신경 손상에 대한 예방 및 치료제로 매우 유용하게 이용 될 수 있다.The present invention relates to a composition for nerve cell protection and growth promotion and neuronal regeneration of the rhubarb extract, the present invention is very useful as a preventive and therapeutic agent for physical damage and degeneration of the nervous system, ischemic cranial nerve damage, peripheral nerve damage Can be.
본 발명에서는 체외(in vitro) 배양된 신경줄기세포와 신경세포주의 스트레스 모델(혈청제거 및 산화적 스트레스) 등을 통한 황련추출물의 신경세포보호능 및 분화능, 그리고 신경독성약물 처리를 통한 화학적 뇌신경세포사멸 동물모델과 치매 동물모델들을 통해 황련추출물의 신경보호효과를 체내(in vivo)실험으로 조사하였고 아울러 말초신경손상 동물모델을 통해 말초신경재생능을 확인하였다. 체외실험에서는 인간의 신경아세포종(human neuroblastoma ; SH-SY5Y), 흰쥐의 신경줄기세포(rat neuronal stem cell ; HiB5), PC12 세포 등 다양한 세포주에 황련추출물을 처리함으로써 혈청제거와 강력한 산화제 처리에 의한 세포사멸스트레스로부터의 황련추출물의 세포사멸억제능, 신경줄기세포와 신경세포에 대한 분화 및 재생 촉진여부 등을 종합적으로 실험하였고 일반적인 퇴행성질환의 공통점인 신경세포사멸 현상을 구현하기 위해 MK-801을 처리하여 쥐의 뇌에 전반적인 세포사멸을 유도한 동물모델 뿐만 아니라, 이보테닉산(ibotenic acid) 투여로 인한 해마 부위 뇌세포 사멸을 구현한 치매 동물모델을 통해 다양한 신경세포사멸 스트레스 병인에 대한 황련추출물의 세포사멸억제 효과를 확인하였다. 아울러, 좌골신경 파쇄 동물모델을 통해 황련추출물의 손상된 말초신경 재생효능도 조사하였다.In the present invention, in vitro cultured neuronal stem cells and neuronal cell protection and differentiation ability of the rhubarb extract through the stress model (serum removal and oxidative stress), such as in vitro culture, and chemical brain neuronal cell death through neurotoxic drug treatment The neuroprotective effect of rhubarb extract was investigated by in vivo experiment in animal model and dementia animal model, and peripheral neuronal regeneration was confirmed in peripheral nerve injury animal model. In vitro experiments were carried out to treat various cell lines such as human neuroblastoma (SH-SY5Y), rat neuronal stem cell (HiB5), PC12 cells, etc., to remove apoptosis by serum removal and strong oxidant treatment. To examine the apoptosis inhibitory ability of stress, and to promote differentiation and regeneration of neural stem cells and neurons from stress, MK-801 was treated to treat neuronal cell death, which is common to general degenerative diseases. Inhibition of apoptosis of rhubarb extract against various neuronal cell death stress pathologies not only in animal models that induce apoptosis in the brain, but also in dementia animal models that induce hippocampal brain cell death due to ibotenic acid administration. The effect was confirmed. In addition, impaired peripheral nerve regeneration effects of rhubarb extracts were investigated using a sciatic nerve crushing animal model.
상기 실험들을 통해 황련추출물의 신경세포사멸억제능, 신경줄기세포를 포함한 신경세포 분화 및 재생 효과를 확인하였다.The experiments confirmed the neuronal cell death inhibitory effect, neuronal differentiation and regeneration effects including nerve stem cells.
따라서, 본 발명의 황련추출물은 단일 혹은 이를 포함한 복합제제 등의 다양한 형태로 신경계발달장애나 치매를 포함한 일반적인 퇴행성 뇌질환 등의 신경계질환, 그리고 교통사고 등으로 인한 신경계 손상을 치료하고 예방하는 데 유용할 것으로 판단된다.Therefore, the extract of the present invention is useful in treating and preventing neurological disorders such as neurodevelopmental disorders such as neurodevelopmental disorders or general degenerative brain diseases including dementia, and traffic accidents in various forms, such as single or complex preparations including the same. I think it will.
이하 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
1. 황련추출물의 제조1. Manufacturing of Lactobacillus Extract
본 발명의 황련추출물은 통상적인 방법에 따라 제조가 가능하다. 본 발명에서는 건조된 황련의 뿌리를 10 - 20 mesh size의 크기로 균질화 한 다음 환류냉각기가 부착된 둥근플라스크에 넣은 후, 열수추출 또는 에탄올 추출하였다.The sulfur extract of the present invention can be prepared according to a conventional method. In the present invention, the roots of the dried yellow lotus are homogenized to a size of 10-20 mesh size, and then placed in a round flask with a reflux condenser, followed by hot water extraction or ethanol extraction.
2. 다양한 신경관련 세포들에서 스트레스에 대한 황련추출물의 세포사멸 방어 효과2. Apoptosis-protective Effect of Rhizome Extract Against Stress in Various Neuronal Cells
1) 혈청제거 스트레스 상태에서 SH-SY5Y 세포와 PC12 세포의 생존에 미치는 영향1) Effect on Survival of SH-SY5Y Cells and PC12 Cells in Serum Removal Stress
뇌에서 기원하는 인간 신경아세포종 세포인 SH-SY5Y를 이용하여 뇌졸중 유사 세포모델을 만들기 위해 혈청이 제거된 상태에서 세포를 배양하고 황련추출물을 처리한 뒤 세포생존율을 MTT 분석법으로 측정하여 세포 손상에 대한 보호 효과를 확인하였다. SH-SY4Y 세포의 분화인자로서 세포생존력을 향상시키는 레티노산(retinoic acid)을 양성 대조군으로서 사용하였다. 혈청 제거 3시간 전에황련추출물을 처리한 후 화학조성 배양액(chemically defined media) N2에서 2일 간 세포배양하면서 신경재생을 유도하였다.To produce a stroke-like cell model using SH-SY5Y, a human neuroblastoma cell originated from the brain, the cells were cultured with serum removed and treated with Lactobacillus extract and the cell viability was measured by MTT assay. The protective effect was confirmed. Retinoic acid, which enhances cell viability as a differentiation factor of SH-SY4Y cells, was used as a positive control. Three hours before serum removal, the extract was treated with sulfur and then cultured for 2 days in chemically defined media N2 to induce neuronal regeneration.
그 결과 황련추출물을 처리하였을 때 대조군에 비해 1.7배의 세포생존률을 보였다.As a result, when treated with rhubarb extract showed 1.7 times the cell survival rate compared to the control.
따라서, 황련추출물은 세포사멸 방어, 생존 증진, 신경보호 효과가 있음을 알 수 있다.Therefore, it can be seen that the extract of L. aureus has apoptosis defense, survival improvement, and neuroprotective effect.
또한 흰쥐 PC12 세포에 혈청 제거 스트레스를 주고 황련추출물을 처리한 뒤 세포 손상에 대한 보호 효과를 측정하였다. 양성 대조군으로는 생존 증진 및 분화를 유발하는 발생인자인 NGF를 처리하였다. 그 결과 황련추출물은 대조군에 비해 1.5배로 세포생존률이 증가하였다.In addition, serum PC12 cells were subjected to serum removal stress, and treatment of H. extract was measured for protection against cell damage. The positive control group was treated with NGF, a developmental factor that induces survival and differentiation. As a result, H. extract increased cell viability by 1.5 times compared to the control.
따라서 황련추출물은 PC12 세포에서도 세포사멸을 방어하는 신경보호 효과를 가지는 것으로 판단된다.Hence, it can be concluded that L. extract has a neuroprotective effect against apoptosis in PC12 cells.
2) 산화적 스트레스상태에서 SH-SY5Y세포의 생존에 미치는 영향2) Effect on SH-SY5Y Cell Survival Under Oxidative Stress
퇴행성 뇌질환은 신경세포의 세포사멸이 원인이며 세포사멸의 공통적 기전으로 산화적 스트레스를 들 수 있다. H2O2는 강력한 산화제로서 세포에 산화적 스트레스를 유발하므로 뇌졸중 및 치매 등 퇴행성뇌질환의 세포모델로 공통적으로 사용된다. 따라서 본 발명에서도 SH-SY5Y세포에 H2O2를 처리하여 세포 손상을 주었으며 황련추출물은 H2O2를 처리하기 3시간 전에 세포배양액에 넣어 세포보호 효과를 조사하였다.Degenerative brain disease is caused by apoptosis of nerve cells and oxidative stress is a common mechanism of cell death. H 2 O 2 is a powerful oxidant and causes oxidative stress in cells, so it is commonly used as a cell model for degenerative brain diseases such as stroke and dementia. Therefore, in the present invention, SH-SY5Y cells were treated with H 2 O 2 to injure the cells, and the L. extract was examined in the cell culture solution for 3 hours before H 2 O 2 treatment.
그 결과 황련추출물은 산화적 스트레스에 대해 2배 이상의 세포 보호 효과를 보임이 관찰되었다. 따라서 신경세포주의 스트레스 모델인 산화적 스트레스상태에서 황련추출물은 신경아세포종 세포의 세포사멸을 방어하고 생존을 증진하는 효과가 있음을 알 수 있다.As a result, it was observed that the extract of L. oleracea showed two times more protective effect against oxidative stress. Therefore, it can be seen that in the oxidative stress state, which is a stress model of neuronal cell line, the extract of L. aeruginosa protects apoptosis of neuroblastoma cells and enhances survival.
3. 다양한 신경 관련세포들의 분화에 미치는 황련추출물의 재생효과3. Regeneration Effect of Lactobacillus Extract on Differentiation of Various Neuronal Cells
신경세포가 분화하고 신경돌기(neurite)가 재생하는데 황련추출물의 효과를 신경줄기세포(HiB5)와 분화된 신경세포인 신경아세포종(SH-SY5Y), 그리고 PC12 세포를 통해 조사하였다.The effects of Rhizome extract on the differentiation of neurons and regeneration of neurites were investigated through neural stem cells (HiB5), neuroblastomas (SH-SY5Y), and PC12 cells.
1) 분화 유도 효과1) Differentiation Induction Effect
먼저 신경줄기세포에서 황련추출물이 세포분화를 유도하는지 조사하기 위해 HiB5세포를 분화시작조건에서 1일간 배양한 후 황련추출물을 처리하고 다시 2일간 배양한 뒤, 신경돌기 성장을 관찰하였다. 양성 대조군으로 같은 조건에서 bFGF를 처리하여 신경세포로의 분화를 유도하였다.First, in order to investigate whether rhus extract induces cell differentiation in neural stem cells, HiB5 cells were cultured for 1 day at differentiation starting conditions, treated with rhus extract, and then cultured again for 2 days, and then neurite growth was observed. BFGF was treated under the same conditions as a positive control to induce differentiation into neurons.
그 결과, 황련추출물을 처리한 경우도 신경성장인자인 bFGF를 처리한 경우와 마찬가지로 신경세포로의 분화가 유도되어 세포체(cell body)가 작아지고 신경축색(axon)과 수상돌기(dendrite)의 전구체인 신경돌기가 신장하여 대부분 그 길이가 세포체의 2배이상 이었다. 따라서 신경줄기세포에 황련추출물을 처리한 경우 신경세포로의 분화가 유도됨을 알 수 있다.As a result, the treatment of rhubarb extract also induced differentiation into neurons as in the case of treatment with bFGF, a nerve growth factor, resulting in a smaller cell body and precursors of axons and dendrites. Pharyngeal neurites were elongated and their length was more than twice the length of cell bodies. Therefore, it can be seen that differentiation into neurons is induced when rhus extracts are treated on neural stem cells.
2) 신경재생 효과2) nerve regeneration effect
신경세포의 신경돌기(neurite) 재생에 황련추출물이 효과가 있는지 조사하기 위해 SH-SY5Y와 PC12 세포를 사용하여 같은 방법으로 조사하였다. 신경돌기 성장을 유도하는 레티노산과 NGF는 각 세포들에서 양성 대조군으로 사용되었다. 그 결과, 두 세포 모두에서 황련추출물은 양성 대조군과 동일한 양상의 신경돌기 재생 효과를 보였다.In order to investigate the effect of rhubarb extract on neuronal neurite regeneration, SH-SY5Y and PC12 cells were used in the same way. Retinoic acid and NGF, which induce neurite growth, were used as positive controls in each cell. As a result, the rhubarb extract showed the same neurite regeneration effect as the positive control in both cells.
한편, PC12 세포에서 황련추출물 처치에 의한 NGF mRNA의 발현양상을 조사한 결과, 이를 처리하지 않은 대조군보다 황련추출물을 처리한 실험군에서 현저히 높은 NGF mRNA가 발현됨을 확인할 수 있었다. 이는 PC12 세포에 황련추출물이 나타내는 생물학적 효과가 신경성장인자처럼 직접 그리고/혹은 간접적인 작용기전에 의함을 암시한다.On the other hand, as a result of investigating the expression pattern of NGF mRNA by treatment with rhubarb extract in PC12 cells, it was confirmed that significantly higher NGF mRNA was expressed in the experimental group treated with the rhubarb extract than the control group without treatment. This suggests that the biological effects of rhubarb extract on PC12 cells are due to direct and / or indirect mechanisms of action, such as neuronal growth factors.
4. MK-801 동물모델에서 뇌신경사멸에 대한 황련추출물의 신경재생 및 방어효과4. Neuronal Regeneration and Defense Effects of Rhizome Extracts on Cerebral Nerve Death in MK-801 Animal Models
황련추출물을 단독 투여한 어린 흰쥐의 뇌에서는 MK-801로 상해를 입은 어린 흰쥐 뇌의 신경세포들에 비하여 TUNEL 검색에서 양성반응을 보이는 고사신경세포를 발견할 수 없었으며, 황련추출물은 MK-801에 의해 일어나는 세포사멸을 현저히 억제하였다. 또한 황련추출물 투여에 의하여 주요 항-세포사멸(anti-apoptosis) 유전자인 bcl-2 mRNA가 대뇌조직에서 증가함을 관찰할 수 있었다.In the brains of young rats treated with Lactobacillus extract alone, there was no detectable dead neural cells in the TUNEL test compared to the neurons of the Ml-801 injured rat brain. Significantly inhibited apoptosis. In addition, it was observed that bcl-2 mRNA, a major anti-apoptosis gene, was increased in cerebral tissues by the administration of the extract.
현재까지 세포사에 관하여 알려진 바에 의하면(Helmreich, 2001), 신경세포성장인자의 작용기전은 크게 1) 세포사 지령유전자(death effector gene) 발현을 저지하던가 2) bcl-2 또는 bcl-xL 등의 세포 생존지지유전자(survival promoting gene)발현을 증가시키는데 있다고 본다. 그러므로 황련추출물은 신경성장인자와 유사한 신경세포 성장기능을 가지고 있으며, 분자적 작용기전으로는 대표적인 항-세포사멸 단백질인 Bcl-2의 생산을 증가시킴으로써 신경세포사멸을 효율적으로 억제 할 수 있는 것으로 사료된다.As far as is known about cell death (Helmreich, 2001), the mechanism of action of neuronal growth factors is largely 1) inhibiting the expression of death effector genes or 2) cell survival such as bcl-2 or bcl-xL. It is believed to increase the expression of survival promoting genes. Therefore, L. extract has neuronal growth function similar to that of nerve growth factor, and it can effectively suppress neuronal cell death by increasing the production of Bcl-2, a representative anti-cell death protein. do.
5. 치매동물모델에서 황련추출물의 신경재생 효과5. Neuronal Regeneration Effects of Lactobacillus Extracts in Dementia Animal Models
알츠하이머 환자에서 초기에 발견되는 공통되는 특징은 기억손상이며 학습과 기억을 담당하는 대뇌변연계(limbic system)에서도, 단기기억과 장기기억의 형성에 관여하는 해마는 전뇌(forebrain)와 함께 환자의 뇌에서 가장 퇴행이 심각하고 노인성 반점(senile plaques)이 많이 발견되는 부위이다. 특히 해마의 CA1 부위와 entorhinal cortex에서 신경세포의 퇴행이 두드러진다. 기저전뇌(basal forebrain)의 콜린성 뉴런(cholinergic neuron)들은 해마로 뻗어있고 표적유래 신경성인자(target-derived neurotrophic factor)인 NGF와 BDNF에 의해 생존이 유지되므로 알츠하이머병에서 심각한 퇴행을 일으킨다.A common feature found early in Alzheimer's patients is memory impairment, and even in the limbic system responsible for learning and memory, the hippocampus involved in the formation of short-term and long-term memory, along with the forebrain, in the brain of the patient. It is the most severe degenerative site and many senile plaques are found. In particular, neuronal degeneration is prominent in the CA1 region of the hippocampus and the entorhinal cortex. Cholinergic neurons in the basal forebrain extend to the hippocampus and are maintained by NGF and BDNF, which are target-derived neurotrophic factors, causing severe degeneration in Alzheimer's disease.
그동안 많은 연구실에서 알츠하이머병의 원인적인 치료를 위해 발병인자를 연구하여 노인성 반점의 주성분인 베타-아밀로이드(b-amyloid)나 세포사멸하는 신경세포내에서 발견되는 신경섬유덩어리(neurofibrillary tangle)에서 많이 발견되는 tau 단백질의 비정상적인 인산화와 apoE4 등이 관련된다는 실험적 증거들이 제시되어 왔으나 관련유전자가 매우 많고 환자에서 공통되는 발병원인이나 유전자변이 등은 발견되지 않고 있다. 따라서 그동안 사용되어온 치료제는 알츠하이머병 환자에서 현저하게 줄어들어 있는 콜린(choline)성 신경전달계 활성을 높여주기 위해 아세틸콜린 에스테라제(acetylcholine esterase) 억제제 또는 아세틸콜린 에스테라제 합성전구체를 투여하거나, 신경세포의 energy 대사를 개선시키는 약물등을 투여한다. 이러한 방법들은 일시적으로 증세를 경감시켜줄 뿐이므로 알츠하이머병의 진행을 늦추거나 원인적 치료를 위해 신경세포의 생존을 증가시키는 신경조절인자(neurotrophin) 또는 신경세포자극물질이 연구되고 있다. 신경성장인자는 많이 주목받고 있으나 그동안 임상적으로 시도되었던 NGF는 콜린성 뉴런에서 일부 효과가 나타나기도 하였으나(Knusel B. and Gao H.,Life Sci. 58:2019-2027(1996); Lapchak P.A., Exp Neurol 124:16-20(1993)) 큰 효과는 보지 못하였다(Neve et al., Brain Res Mol Res. 1996. 39(1-2) : 185-197(1996)). 따라서 신경세포의 사멸을 막아주고 생존과 재생을 증진하거나 신경줄기세포의 생존과 분화를 증진하는 물질을 선별하는 것이 필요하다.Many laboratories have studied pathogens for the causative treatment of Alzheimer's disease and have been found in beta-amyloid, a major component of senile plaques, or in neurofibrillary tangles found in apoptotic neurons. Experimental evidence has been suggested that abnormal phosphorylation of tau protein is associated with apoE4, but there are many related genes and no common causes or gene mutations have been found in patients. Therefore, the therapeutic agents used in the past have been administered acetylcholine esterase inhibitors or acetylcholine esterase synthetic precursors to increase choline neurotransmitter activity, which is significantly reduced in patients with Alzheimer's disease, Inject drugs to improve energy metabolism. Since these methods only temporarily reduce symptoms, neurotrophin or neuronal stimulants are being studied to slow the progression of Alzheimer's disease or increase the survival of neurons for causative treatment. NGF has attracted much attention, but clinically attempted NGF has shown some effects on cholinergic neurons (Knusel B. and Gao H., Life Sci . 58: 2019-2027 (1996); Lapchak PA, Exp. Neurol 124: 16-20 (1993)) did not see significant effects (Neve et al., Brain Res Mol Res. 1996. 39 (1-2): 185-197 (1996)). Therefore, it is necessary to select a substance that prevents the death of neurons, promotes survival and regeneration, or promotes the survival and differentiation of neural stem cells.
본 발명에서는 학습과 기억을 담당하는 해마에 있어서 알츠하이머병에 의한 신경퇴행의 진전을 늦추고 신경재생을 촉진하기 위하여 황련추출물의 세포사멸 방어 및 신경 재생 효과를 치매동물 모델에서 확인하였다.In the present invention, in order to slow the progression of neurodegeneration caused by Alzheimer's disease in the hippocampus responsible for learning and memory, and to promote neuronal regeneration, the anti-apoptotic defense and neuronal regeneration of the Rhizome extract was confirmed in a dementia animal model.
즉, 본 발명은 치매 모델로 kanate 유도체인 이보테닉산(ibotenic acid)을 흰쥐 성체 뇌의 entorhinal cortex에 stereotaxic frame을 사용하여 미세주입(microinjection)하는 동물 모델을 사용하였다.That is, the present invention used an animal model of microinjection of kanate derivative ibotenic acid into the entorhinal cortex of rat adult brain using a stereotaxic frame as a dementia model.
그 결과 해마와 entorhinal cortex에서 calbindin-positive neruon이 2주 후 30-40%로 감소하였으며 4주 후 일부 회복되는 경향을 보였다. 해마의 CA1 부위보다 치상회(dentate gyrus) 부위의 세포들은 좀더 천천히 감소하는 경향을 보였다. 그러나 황련추출물을 주입한 경우에는 calbindin-positive neruon의 생존율이 증가하였는데 특히 CA1 부위의 추상세포(pyramidal cell)의 경우 약 2배 이상 증가하였고 치상회의 과립세포(granule cell)는 약 1.5배 증가하였다. 죽어 가는 신경세포의 생존을 증진하는 것은 결국 재생을 촉진하는 것이 되므로 황련추출물은 치매동물모델에서 세포사멸로부터 신경세포를 보호하고 신경재생 효과가 있는 것으로 판단된다.As a result, calbindin-positive neruon in the hippocampus and entorhinal cortex decreased to 30-40% after 2 weeks and partially recovered after 4 weeks. Cells in the dentate gyrus site tended to decrease more slowly than the CA1 site in the hippocampus. However, the injection rate of the rhubarb extract increased the survival rate of calbindin-positive neruon. Especially, pyramidal cells of CA1 region increased more than 2 times and granule cells of dentate gyrus increased about 1.5 times. Since the survival of dying neurons will eventually promote regeneration, it can be concluded that L. extracts have neuroprotective effects and protect neurons from apoptosis in animal models of dementia.
6. 황련추출물에 의한 말초신경계 좌골신경(sciatic nerve) 재생효과6. Regeneration Effect of Peripheral Nervous System Sciatic Nerve by Husband Extract
인간의 경우 중추신경계는 물론 말초신경계도 재생이 어려워 퇴행성 뇌질환 뿐만 아니라 현대사회의 산업재해 및 교통사고, 전쟁불구자 등이 누적되면서 사회문제화 되고 있으므로 신경계의 재생에 관한 연구에 많은 관심이 집중되고 있다.In the case of humans, the central nervous system as well as the peripheral nervous system is difficult to regenerate, so as not only degenerative brain diseases but also industrial accidents, traffic accidents, and war-deficiency in modern society are accumulating social problems, so much attention has been focused on the regeneration of nervous systems. .
말초신경계가 발생하고 재생하는 과정에서 슈반세포는 중요한 역할을 한다. 배아가 발생하는 동안에 신경릉(neural crest)에서 기원하는 슈반세포는 축색이 점유하게될 곳을 따라 미리 분열하므로 말초 신경계의 축색이 슈반세포에 의존하여 성장하게된다. 특히 슈반세포는 영양인자(trophic factors)를 생산하여 신경의 생존과 신경돌기 성장을 조절한다. 또한 신경은 축색에서 뉴레글린(neuregulin)을 분비하여 슈반세포의 생존을 증진하고 축색과 슈반세포의 비율을 조절하며 이때 축색의 영향을 받지 못한 슈반세포는 사멸한다. 발생후기에 슈반세포는 미엘린 수초를 생산하여 축색을 감싸서 슈반세포의 분화가 완성된다.Schwann cells play an important role in the development and regeneration of the peripheral nervous system. During embryonic development, Schwann cells originating from the neural crest divide in advance along where the axons will occupy, so that the axons of the peripheral nervous system grow depending on the Schwann cells. Schwann cells, in particular, produce trophic factors that regulate neuronal survival and neurite growth. In addition, nerves secrete neuroregulin from axons to enhance the survival of Schwann cells, control the ratio of axons to Schwann cells, and kill Schwann cells unaffected by axons. In the late stage of development, Schwann cells produce myelin sheaths and wrap the axons to complete the differentiation of Schwann cells.
신경발생이 끝난 성체에서, 말초신경이 다친 경우에는 그 상해 부위에서 신경말단 쪽의 축색부분(distal stump)은 월러퇴행(Wallerian degeneration)이라고 알려진 과정을 통해 점차 퇴화한다. 이때 상해 부위로부터 세포체 쪽의 축색(proximal stump)은 재성장을 시작한다. 상해 부위의 신경말단 쪽의 축색부분에서는 퇴화한 축색과 수초물질을 제거하고 상해 부위로부터 세포체 쪽의 축색부분은 축색이 새롭게 자랄 수 있도록 환경을 조성한다(Kwon, Y. Kim, Bhattacharyya, W.V., Cheon, K., Stiles, C.D., and Pomeroy, S.L. (1997) Activation of ErbB2 during Wallerian degeneration of sciatic nerve,J. Neurosci.17, 8293-8299; Joung, I., Kim, H.S., Hong, J.S., Kwon, H., and Kwon, Y.K. (2000) Effective gene transfer into regenerating sciatic nerves by adenoviral vectors: potentials for gene therapy of peripheral nerve injury.Mol. Cells10, 540-545).In adult neurogenesis, when the peripheral nerves are injured, the distal stump at the nerve end at the site of injury gradually degenerates through a process known as Wallerian degeneration. At this point, the proximal stump from the injury site begins to regrow. Axons in the nerve endings of the injured area remove degenerated axons and myelin material, and axons in the cell body from the injured area create a new environment for axons to grow (Kwon, Y. Kim, Bhattacharyya, WV, Cheon). , K., Stiles, CD, and Pomeroy, SL (1997) Activation of ErbB2 during Wallerian degeneration of sciatic nerve, J. Neurosci. 17, 8293-8299; Joung, I., Kim, HS, Hong, JS, Kwon, H., and Kwon, YK (2000) Effective gene transfer into regenerating sciatic nerves by adenoviral vectors: potentials for gene therapy of peripheral nerve injury.Mol . Cells 10, 540-545).
신경이 다친 후 슈반세포는 즉시 급격한 분열을 하는데 이것은 축색과의 접촉 상실이나 축색이 분비하는 성장인자에 의해 촉진되는 것으로 생각되어왔다. 축색이 재성장하는 동안에 슈반세포가 축색에 접촉하면 다시 분화하여 수초를 재생한다. 세포 표면에서의 상호작용 이외에도 슈반세포는 멀리서도 재생하는 축색에 영향을 줄 수 있다. 신경이 절단된 후 1cm만큼 떨어져 있어도 축색은 distal stamp를 향해 재생한다. 이 때의 축색의 정향운동은 distal stump에 살아있는 슈반세포가 존재해야 일어난다.Schwann cells immediately divide rapidly after nerve injury, which is thought to be promoted by loss of contact with axons or growth factors secreted by axons. If Schwann cells come into contact with axons during axon regrowth, they differentiate and regenerate myelin. In addition to interactions at the cell surface, Schwann cells can affect regenerating axons from a distance. The axon regenerates toward the distal stamp even if the nerve is cut 1 cm away. At this time, the axon's crust movement occurs only when living Schwann cells are present in the distal stump.
말초신경계의 재생과정에서는 먼저 슈반세포가 절단된 축색으로부터 분리되어 다시 분열능력을 얻게되는 탈분화(dedifferentiation) 과정, 상처부위로 부터 신경세포의 축색이 다시 자라나오는 과정, 그리고 다시 자라나온 축색을 슈반세포가 미에린 수초로 감싸는 재분화(redifferentiation) 과정과 축색이 근육까지 자라서 다시 근육세포에 신경근 접합부(neuromuscular junction)를 만드는 과정으로 크게 나눌 수 있다.In the regeneration process of the peripheral nervous system, Schwann cells are first separated from the cut axons and dedifferentiation process is obtained, and the axons of nerve cells are regrown from the wound site, and the axons are regrown. The process of redifferentiation, which is wrapped in a myelin sheath, and the process of axon growth up to the muscle, and then the creation of neuromuscular junctions in the muscle cells can be divided into two types.
본 발명에서는 말초신경계에서 가장 신경섬유가 많이 지나가는 좌골신경을 사용하여 그 재생과정에서 황련추출물이 축색돌기의 재성장, 미엘린 수초의 재생, 근육세포에 신경근 접합부를 만드는 과정에서 재생을 촉진하는지 조사하였다.In the present invention, using the sciatic nerve, which passes the most nerve fibers in the peripheral nervous system, it was investigated whether rhubarb extract promotes regeneration in the process of regrowth of axons, myelin myelin regeneration, and neuromuscular junctions in muscle cells.
본 발명에서는 흰쥐의 좌골신경에 PBS(phosphate-buffered saline) 또는 황련추출물을 복강주사한 뒤 봉합하여 1주, 2주와 4주 뒤에 신경재생 정도를 관찰하였다.In the present invention, the abdominal injection of PBS (phosphate-buffered saline) or rhubarb extract in the sciatic nerve of the rat was sutured to observe the degree of nerve regeneration after 1, 2 and 4 weeks.
그 결과 4주 후에는 300㎛가 넘는 축색의 수가 2배 이상 증가하였다. 미엘린은 1주 후부터 200㎛가 넘는 미엘린 수가 2배 이상되고 4주에는 3배에 달하였다. 따라서,황련추출물은 말초신경 재생과정에서 축색돌기의 성장과 미엘린 수초의 재생을 촉진함을 알 수 있다.As a result, after 4 weeks, the number of axons exceeding 300 µm increased more than twice. Myelin has more than doubled myelin number more than 200 μm from 1 week and tripled at 4 weeks. Therefore, it can be seen that sulfur extract promotes axon growth and myelin regeneration during peripheral nerve regeneration.
또한 신경근접합부에서 신경말단의 재생에 영향을 주는지 보기 위해 수술 4주 후에 좌골신경에 연결된 뒤다리 근육을 분리하여 관찰 한 결과 수술 4주 후에 대조군에서는 신경말단이 염색되었으나 근육 한부위에 모여 있고 아직 근섬유 각각에 퍼져 하나씩의 신경근접합부를 형성하지 못했으나 황련추출물을 주사한 경우에는 신경말단이 근섬유에 퍼져 있는 것이 관찰되었다In addition, 4 weeks after surgery, the hind limb muscles connected to the sciatic nerve were separated and examined in the neuromuscular junction to see if it affected the nerve endings. It did not form a single neuromuscular junction, but the injection of the rhubarb extract showed that the nerve endings were spread over the muscle fibers.
따라서 황련추출물은 말초신경 재생과정에서 축색돌기의 성장, 미엘린 수초의 재생과 신경근접합부를 형성하기 위한 신경말단 재생을 촉진하는 것으로 판단된다.Hence, it is believed that the extract of L. oleracea promotes axon growth, myelin myelin regeneration and nerve ending regeneration to form neuromuscular junctions during peripheral nerve regeneration.
7. 신경재생과정에서 신경성장인자와 황련추출물의 역할7. The Role of Nerve Growth Factor and Rhubarb Extract in Neural Regeneration
신경성장인자들은 신경줄기세포의 분열개시, 분열한 세포의 수를 사멸과정으로 조절하는 동시에 분화를 시작하도록 하며, 표적유래성장인자로서 정향 이동된 세포의 생존과 잘못된 방향으로 이동된 세포의 사멸을 유도하여 시냅스전 신경세포의 생존을 조절하는 한편 새로운 시냅스 형성과 재구성을 조절한다. 또한 성체의 신경 생존과 시냅스 가소성 및 신경손상 시 재생하는 과정에서도 유사한 기능을 가질 것으로 추측된다. 황련추출물은 신경줄기세포의 분화를 유도하고 세포사멸을 방어하며 신경돌기 분화를 촉진하므로 직접 그리고/또는 간접적인 작용기전을 통해 신경성장인자와 유사한 기능을 수행하는 것으로 사료된다.Nerve growth factors initiate the division of neural stem cells, control the number of dividing cells into a process of killing and initiate differentiation, and induce the survival of the crusted cells and the death of cells moved in the wrong direction as target-derived growth factors. It regulates the survival of presynaptic neurons while also regulating new synapse formation and reconstitution. In addition, the neuronal survival of the adult, synaptic plasticity and neuronal damage during regeneration process is expected to have a similar function. It is thought that the extract of L. oleculum is similar to nerve growth factor through direct and / or indirect mechanism of action because it induces differentiation of neural stem cells, prevents cell death and promotes neurogenesis.
한편 황련추출물의 독성과 부작용을 조사하기 위해 흰쥐를 이용한 생체실험결과, 황련추출물은 일반적인 급성독성 및 간 기능에 부작용을 나타내지 않았고, 과다한 글루타메이트(glutamate)에 의한 흥분성 신경독성에 대해서도 현저한 억제효과를 보이는 것으로 나타났다.On the other hand, the results of biological experiments using rats to investigate the toxicity and side effects of rhubarb extract showed no adverse effects on general acute toxicity and liver function. Appeared.
본 발명에 따른 황련추출물의 투여용량은 통상적으로 1일 100-150mg/60kg(체중) 정도, 1일 2-3회 투여하는 것이 바람직하며 환자의 연령, 성별, 체중, 환자의 중증도, 건강상태에 따라 증감될 수 있다.The dosage of the sulfur extract according to the present invention is generally administered about 100-150mg / 60kg (body weight) per day, 2-3 times a day, and the age, sex, weight, severity, and health of the patient are preferred. Can be increased or decreased accordingly.
황련추출물은 통상적인 방법에 따라 적절한 담체 또는 부형제와 혼합하거나 희석제로 희석하여 사용 할 수도 있다. 적합한 담체, 부형제 및 희석제의 예로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 상기 조성물에 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제, 방부제 등을 추가로 포함할 수 있다. 본 발명의 조성물은 포유 동물에 투여된 후 활성 성분의 신속, 또는 지연된 방출을 제공할 수 있도록 당 업계에 잘 알려진 방법을 사용하여 제형화 될 수 있다. 제형은 정제, 분말, 환제,새세이(sachet), 엘릭서(elixir), 현탁액, 에멀젼, 용액, 시럽, 에어로졸, 연질 또는 경질 젤라틴 캅셀, 멸균 주사용액, 멸균 분말 등의 형태일 수 있다. 본 발명의 조성물은 경구, 경피, 피하, 정맥 또는 근육을 포함한 여러 경로를 통해 투여될 수 있다. 본 발명의 황련추출물은 각종 신경계 질환의 예방 및 치료 목적으로 약학제제로 제조되거나 식품 또는 음료에 첨가될 수 있다. 또한 본 발명은 치매, 만성 간질, 중풍, 허혈성 뇌질환, 파킨슨병(Parkinson's disease), 알츠하이머병(Alzheimer's disease)등의 퇴행성 뇌질환의 치료효과를 나타내는 의약품 또는 식품(각종 식품류, 음료, 껌, 차, 비타민 복합제, 건강보조식품류)으로 제공될 수 있다.Lactobacillus extract may be used by mixing with a suitable carrier or excipient or diluting with a diluent according to conventional methods. Examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The composition may further include fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like. Compositions of the present invention may be formulated using methods well known in the art to provide rapid or delayed release of the active ingredient after administration to a mammal. The formulations may be in the form of tablets, powders, pills, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders and the like. The compositions of the present invention can be administered via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular. The rhubarb extract of the present invention may be prepared as a pharmaceutical preparation or added to food or beverage for the purpose of preventing and treating various nervous system diseases. In addition, the present invention is a drug or food (various foods, drinks, gum, tea, etc.) that exhibits a therapeutic effect of dementia, chronic epilepsy, stroke, ischemic brain disease, Parkinson's disease, Alzheimer's disease and other degenerative brain diseases. , Vitamin complexes, dietary supplements).
이하 본 발명을 실시예에 의하여 보다 구체적으로 설명하겠으나, 이들 실시예에 의해 본 발명의 범위가 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited by these Examples.
실시예 1. 본 발명의 일반적인 실험방법Example 1 General Experimental Method of the Present Invention
1) 신경세포주 배양1) Neuronal cell culture
흰쥐 배아의 해마에서 기원하는 HiB5 세포는 온도 민감성(temperature sensitive) SV40 T-항원(antigen)을 레트로바이러스로 감염시켜(retrovirus infection) 만들었으므로 34oC에서 배양할 때는 세포분열하나 쥐의 정상체온인 39oC로 옮기면 분열을 멈춘다. 여기에 bFGF(20ng/ml)를 넣어주면 생존이 증가하고 신경세포로 분화하여 신경세포 표지분자를 발현한다. 배지는 DMEM에 10% FBS(fetal bovine serum), 페니실린/스트렙토마이신, 글루타민, sodium pyruvate(0.11g/L)를 첨가하여 사용하고, 39oC에서 분화시 에는 무혈청 배지(serum-free media)인 N2 (DMEM/F12, 인슐린, 트랜스페린, Putreseine, BSA 포함 ;Botten Stein & Sato., 1979)에 피루베이트(pyruvate)를 첨가하여 사용한다. PC12와 SH-SY5Y 세포의 배양은 10% FBS를 첨가한 DMEM으로 배양하고 분화를 위해서는 무혈청 배지에 NGF나 레티노산을 처리한다.HiB5 cells originating from the hippocampus of rat embryos were made by retrovirus infection of temperature sensitive SV40 T-antigens, resulting in cell division when cultured at 34 o C. Transfer to 39 o C to stop cleavage. Adding bFGF (20ng / ml) increases survival and differentiates into neurons to express neuronal marker molecules. Medium was used by adding 10% FBS (fetal bovine serum), penicillin / streptomycin, glutamine, sodium pyruvate (0.11 g / L) to DMEM, and serum-free media when differentiated at 39 o C. Pyruvate is added to phosphorus N2 (including DMEM / F12, insulin, transferrin, Putreseine, BSA; Botten Stein & Sato., 1979). Cultures of PC12 and SH-SY5Y cells were incubated with DMEM with 10% FBS and treated with NGF or retinoic acid in serum-free medium for differentiation.
2) 신경세포주의 MTT 분석2) MTT analysis of neuronal cell line
혈청제거 및 산화적 스트레스에 의한 세포손상의 정도를 MTT 분석법으로 평가한다. MTT 분석을 위해 세포를 7.5×103개로 심고 N2 배지에서 신경독(neurotoxin)이나 신경성장인자를 첨가하고 분화시킨 후 0.1 부피의 4mg/ml MTT를 넣고 37℃에서 3-4시간 보존한 뒤 100ul의 용해버퍼(solubilization buffer)를 넣고 다시 24시간 보존하여 ELISA 분석기로 O.D.값을 측정한다.The extent of cell damage due to deserialization and oxidative stress is assessed by MTT assay. For MTT analysis, cells were planted in 7.5 × 10 3 cells, added neurotoxin or nerve growth factor in N2 medium and differentiated. Then, 0.1 volume of 4mg / ml MTT was added and stored at 37 ° C. for 3-4 hours. The solubilization buffer was added and stored again for 24 hours to measure the OD value with an ELISA analyzer.
3) 면역조직화학적 방법(Immunohistochemistry)3) Immunohistochemistry
해당 조직을 4% 파라포름알데히드(paraformaldehyde)로 고정하고 40 um로 동결박절(cryosection)하여 신경 혹은 슈반세포 표지자 항체와 FITC로 표지된 이차항체로 염색한 후, 공초점 현미경으로 관찰한다. 신경세포표지 항체로 세포를 염색하기 위해 배양된 세포를 4% 파라포름알데히드로 20분간 고정한 후 0.5% NP-40로 5분간 삼투하고, 1% BSA 용액을 사용하여 30분간 차단(blocking)과정을 거친다. 일차항체로 4℃에서 약 12시간 반응한 후, FITC로 표지된 이차항체 또는 rhodamin(TRITC)으로 표지된 이차항체로 1시간 다시 반응하고 고정하여 공초점 현미경으로 관찰하였다.The tissues were fixed with 4% paraformaldehyde and cryosectioned to 40 um, stained with nerve or Schwann cell marker antibodies and FITC-labeled secondary antibodies, followed by confocal microscopy. In order to stain the cells with the neuronal cell labeled antibody, the cultured cells were fixed for 20 minutes with 4% paraformaldehyde, followed by osmosis with 0.5% NP-40 for 5 minutes, and 30 minutes blocking with 1% BSA solution. Rough After reacting with the primary antibody at 4 ° C. for about 12 hours, the reaction was fixed for 1 hour again with a secondary antibody labeled with FITC or a secondary antibody labeled with rhodamin (TRITC), and observed by confocal microscopy.
4) 흰쥐의 좌골신경 파쇄(scatic nerve crush)4) Strict nerve crush in rats
Sprague-Dawley 흰쥐(male 약 200g)를 pentobarbital(50mg/kg)로 마취시킨 후 왼쪽 좌골(sciatic notch)를 노출시키고, 좌골신경에서 동맥을 제외하고 신경섬유를 자르거나 #9 혈관봉합사로 양쪽을 묶고 가운데를 홍채 절제 가위(iridectomy scissors)로 자른다. 또는 파쇄모델에서는 파쇄용 집게를 사용하여 신경섬유를 두 번 완벽하게 파쇄한다. 신경 손상 후 여러 시간대(6시간, 1일, 3일, 7일, 14일, 21일, 28일)에서 proximal stump, distal stump를 각각 얻어 손상 및 재생상태를 조사한 후 실험에 적용한다. 오른쪽 좌골신경을 대조군로 이용하였다.Sprague-Dawley rats (about 200 g) were anesthetized with pentobarbital (50 mg / kg), exposed to the left sciatic notch, cut nerve fibers from the sciatic nerve except the arteries, or tied to both sides with # 9 angiospores. Cut the middle with iridectomy scissors. Or in a crush model, crushing forceps are used to completely break a nerve fiber twice. After the nerve injury, proximal and distal stumps were obtained at various time periods (6 hours, 1 day, 3 days, 7 days, 14 days, 21 days, and 28 days). The right sciatic nerve was used as a control.
실시예 2. 황련 추출물의 제조Example 2. Preparation of Rhubarb Extract
(1) 열수추출물의 제조(1) Preparation of hot water extract
1) 서울 경동약령시에서 5년근 황련을 구입하였다.1) A five-year-old Hwang Ryun was purchased from Gyeongdong Yangnyeongsi, Seoul.
2) 황련 뿌리를 증류수로 1차 세정한 후 상온의 그늘에서 혹은 40℃ 이하로 맞추어진 건조기에서 24시간 동안 충분히 건조하여 잡질을 제거한다.2) The roots of the yellow lotus are first washed with distilled water, and then dried thoroughly in a shade at room temperature or in a drier set to 40 ° C. or lower for 24 hours to remove miscellaneous impurities.
3) 임의의 크기로 약재를 절단한 후, 이산화규소(Silicagel, 흡습제)가 채워진 데시케이터에서 다시 24시간 건조한 후 10∼20 mesh size의 크기로 균질화 한다.3) After cutting the medicine to an arbitrary size, dry it again in a desiccator filled with silicon dioxide (Silicagel, moisture absorbent) for 24 hours and homogenize to 10-20 mesh size.
4) 균질화된 황련 뿌리 1kg을 취하여 환류냉각기가 부착된 3L 용량의 둥근플라스크에 넣은 후, 열수 추출을 위하여 증류수 1L를 가한다.4) Take 1 kg of homogenized rhubarb root and place it in a 3 L round flask equipped with a reflux condenser and add 1 L of distilled water to extract hot water.
5) 황련 뿌리와 증류수를 잘 섞은 후 100℃에서 3시간 동안 가열한다.5) Mix the roots of the yellow lotus and distilled water well and heat at 100 ℃ for 3 hours.
6) 열수 추출 과정 후에 추출액을 상온까지 냉각한 후에 100mesh로 여과하여 8 Brix농도의 추출물 7.5L를 얻었다.6) After the hot water extraction process, the extract was cooled to room temperature and filtered through 100mesh to obtain 7.5L of extract of 8 Brix concentration.
7) 이 추출물을 60배 희석하여 0.2Brix(고형분 0.2%)로 하여 사용하였다.7) The extract was diluted 60-fold and used as 0.2Brix (0.2% of solid content).
(2) 에탄올추출물의 제조(2) Preparation of Ethanol Extract
1) 서울 경동약령시에서 5년근 황련을 구입하였다.1) A five-year-old Hwang Ryun was purchased from Gyeongdong Yangnyeongsi, Seoul.
2) 황련 뿌리를 증류수로 1차 세정한 후 상온의 그늘에서 혹은 40℃ 이하로 맞추어진 건조기에서 24시간 동안 충분히 건조하여 잡질을 제거한다.2) The roots of the yellow lotus are first washed with distilled water, and then dried thoroughly in a shade at room temperature or in a drier set to 40 ° C. or lower for 24 hours to remove miscellaneous impurities.
3) 임의의 크기로 약재를 절단한 후, 이산화규소(Silicagel, 흡습제)가 채워진 데시케이터에서 다시 24시간 건조한 후 10∼20 mesh size의 크기로 균질화 한다.3) After cutting the medicine to an arbitrary size, dry it again in a desiccator filled with silicon dioxide (Silicagel, moisture absorbent) for 24 hours and homogenize to 10-20 mesh size.
4) 균질화된 황련 뿌리 2kg을 취하여 환류냉각기가 부착된 3L 용량의 둥근플라스크에 넣은 후, 추출을 위하여 에탄올 20L를 가한다음 100℃에서 3시간 동안 가열한다.4) Take 2 kg of homogenized rhubarb root and place it in a 3 L round flask equipped with a reflux condenser, add 20 L of ethanol for extraction, and heat at 100 ° C for 3 hours.
6) 추출 과정 후 추출액을 상온까지 냉각한 후에 100mesh로 여과하여 4 Brix농도의 추출물 17L를 얻었다.6) After the extraction process, the extract was cooled to room temperature, filtered through 100mesh to obtain 17L of extract of 4 Brix concentration.
7) 이를 진공농축하여 에탄올을 제거하고 정제수를 가하여 20 Brix농도의 농축액을 만든다음, 0.2%로 희석하여 실험에 사용하였다.7) The resultant was concentrated in vacuo to remove ethanol, and purified water was added to make a concentrate of 20 Brix concentration, diluted to 0.2% and used for the experiment.
실시예 3. 다양한 신경관련 세포들에서 스트레스에 대한 황련추출물의 세포사멸 방어 효과Example 3 Apoptosis Defense Effect of Rhizome Extract Against Stress in Various Neuronal Cells
1) 혈청제거 스트레스 상태에서 SH-SY5Y 세포와 PC12 세포의 생존에 미치는 영향1) Effect on Survival of SH-SY5Y Cells and PC12 Cells in Serum Removal Stress
SH-SY5Y를 이용하여 뇌졸중 유사 세포모델을 만들기 위해 혈청이 제거된 상태에서 세포를 배양하고 황련추출물을 처리한 뒤 세포생존율을 MTT 분석법으로 측정하여 세포 손상에 대한 보호 효과를 확인하였다. 분화인자 레티노산은 SH-SY5Y의 분화를 유발하는 발생인자로서 처리하여 분화양성 대조군으로서 사용하였고 황련추출물을 30-100 ug/ml 농도로 혈청 제거 3 시간 전에 처리한 후 화학조성 배양액 N2에서 2일 간 배양하면서 신경재생을 유도하였다(표1, 도1 참조).In order to make a stroke-like cell model using SH-SY5Y, the cells were cultured with serum removed, treated with L. extract, and the cell viability was measured by MTT assay to confirm the protective effect against cell damage. Differentiation factor Retinoic acid was used as a differentiation-positive control by treating SH-SY5Y as a developmental factor that induces differentiation, and treated with rhododendron extract at 30-100 ug / ml concentration 3 hours before serum removal for 2 days in chemical composition N2. Neuronal regeneration was induced while incubating (Table 1, see FIG. 1).
[ 표 1]TABLE 1
황련추출물을 60 ug/ml의 농도로 처리하였을 때 대조군에 비해 1.5배의 세포생존률을 보였으며 100 ug/ml에서는 약간 감소하였다. 따라서, 황련추출물은 세포사멸 방어, 생존 증진, 그리고 신경보호 효과를 가진다.When the extract was treated with a concentration of 60 ug / ml, cell viability was 1.5 times higher than that of the control group and was slightly decreased at 100 ug / ml. Thus, L. extract has apoptosis defense, survival enhancement, and neuroprotective effects.
또한 흰쥐의 부신종양에서 기원하는 PC12 세포에 혈청 제거 스트레스를 주고 황련추출물을 처리한 뒤 세포 손상에 대한 보호 효과를 측정하였다. NGF를 생존 증진 및 분화를 유발하는 발생인자로서 처리하여 양성 대조군으로서 사용하였고 황련추출물을 30-100 ug/ml 농도로 처리한 후 2일간 배양하면서 신경재생을 유도하였다(표2, 도2 참조).In addition, PC12 cells derived from adrenal tumors in rats were subjected to serum removal stress and treatment with Rhus extracts, and the protective effect against cell damage was measured. NGF was treated as a positive control factor to induce survival and differentiation, and was used as a positive control. After treatment with 30-100 ug / ml concentration, the extract was incubated for 2 days (see Table 2, Figure 2). .
[표 2]TABLE 2
황련추출물을 60-100 ug/ml의 농도로 처리하였을 때 대조군에 비해 세포생존률이 약 1.5배 증가하였다. 이는 황련추출물이 PC12 세포에서도 세포사멸을 방어하는 신경보호 효과를 가지는 것을 보여준다.When the L. extract was treated at a concentration of 60-100 ug / ml, the cell survival rate was increased about 1.5 times compared to the control. This suggests that the extract of L. aureus has a neuroprotective effect against apoptosis in PC12 cells.
2) 산화적 스트레스 상태에서 SH-SY5Y 세포의 생존에 미치는 영향2) Effect on the Survival of SH-SY5Y Cells Under Oxidative Stress
SH-SY5Y세포에 H2O2를 30분간 처리하여 세포 손상를 주었으며 황련추출물을 H2O2를 처리하기 3시간 전에 30-100 ug/ml 농도로 배양액에 넣어 보호 효과를 조사하였다(표3, 도3 참조).SH-SY5Y cells were treated with H 2 O 2 for 30 minutes to injure the cells, and the protective effect was investigated by adding rhubarb extract to the culture solution at a concentration of 30-100 ug / ml 3 hours before H 2 O 2 treatment (Table 3, 3).
[표 3]TABLE 3
실험결과 황련추출물은 최적농도 60-100 ug/ml에서 산화적 스트레스에 대해 2배 이상의 세포 보호 효과를 보임이 관찰되었다.Experimental results showed that the extract of L. oleracea showed more than twice the cell protection against oxidative stress at the optimal concentration of 60-100 ug / ml.
따라서 신경세포주의 스트레스 모델인 산화적 스트레스 상태에서 황련추출물은 신경아세포종 세포의 사멸을 방어하고 생존을 증진하는 효과를 보인다.Therefore, in the oxidative stress state, which is a stress model of neuronal cell lines, the extract of L. oleracea protects against neuroblastoma cell death and enhances survival.
실시예 4. 다양한 신경 관련세포들의 분화에 미치는 황련추출물의 재생효과Example 4 Regeneration Effect of Lactobacillus Extract on Differentiation of Various Neuronal Cells
1) 분화 유도 효과1) Differentiation Induction Effect
먼저 신경줄기세포에서 황련추출물이 세포분화를 유도하는지 조사하기 위해 HiB5 세포를 분화시작조건에서 1일간 배양한 후, 황련추출물을 100 ug/ml 농도로 처리하고 다시 2일간 배양한 뒤 신경특이적 표지분자로 면역염색(immunostaining)하고 공초점 현미경을 사용하여 신경돌기의 성장을 관찰하였다. 양성 대조군으로 위와 동일한 조건에서 bFGF(20ng/ml)를 처리하여 신경세포로의 분화를 유도하였다. 신경세포의 분화정도는 신경 특이적 표지분자인 항-신경세사(anti-neurofilament) 항체와 FITC로 표지된 이차항체(녹색)를 사용하여 신경돌기를 염색하고 세포핵은 프로피디움 요오드(propidium iodide, 적색)로 이중염색하였다.First, in order to investigate whether rhubarb extract induces cell differentiation in neural stem cells, HiB5 cells were cultured for 1 day at differentiation starting conditions, then the rhubarb extract was treated at 100 ug / ml concentration and cultured again for 2 days, followed by neurospecific marker molecules. Immunostaining was performed and the growth of neurites was observed using confocal microscopy. As a positive control, differentiation into neurons was induced by treatment with bFGF (20ng / ml) under the same conditions as above. The degree of differentiation of neurons was measured using anti-neurofilament antibodies, which are neuron-specific marker molecules, and secondary antibodies (green) labeled with FITC, and the nucleus was stained with propidium iodide (red). Double staining).
도4에서 보는 바와 같이 bFGF를 처리하면 신경세포로의 분화가 유도되어 세포체가 작아지고 축색돌기와 수상돌기의 전구체인 신경돌기가 나와 길이가 길어진다. 황련추출물을 처리한 경우에도 이와 동일한 세포형태 변화를 보이면서 신경세포 분화가 유도되었다(도4 참조).As shown in FIG. 4, the treatment of bFGF induces differentiation into neurons, resulting in a decrease in cell body and lengthening of neurites, which are precursors of axons and dendrites. Neuronal differentiation was induced even when the extract was treated with the same morphology (see Figure 4).
2) 신경재생 효과2) nerve regeneration effect
신경세포의 신경돌기 재생에 황련추출물이 효과가 있는지 조사하기 위해 분화된 신경세포주인 SH-SY5Y와 PC12 세포를 사용하여 조사하였다. 도5과 도7은 양성 대조군으로서 각각의 세포에 처리된 레티노산(50uM 농도로 처리)과 NGF(50ng/ml 농도로 처리)에 의해 신경돌기가 신장된 것을 보여준다. 황련추출물을 처리했을 때(SH-SY5Y 세포에는 100 ug/ml, PC12 세포에는 20-200 ug/ml 농도로 처리), 두 세포 모두에서 양성 대조군과 마찬가지로 신경돌기가 신장되었으며 세포체 길이의 3배 이상 되는 신경돌기를 가지는 세포수의 비율도 약 1.5배 많아졌다(도5, 도6a-6c 참조).In order to investigate whether rhus extracts are effective in regenerating neurites, we investigated SH-SY5Y and PC12 cells, which are differentiated neurons. 5 and 7 show that neurites were elongated by retinoic acid (treated at 50 uM) and NGF (treated at 50 ng / ml) treated with each cell as a positive control. When treated with Lactobacillus extract (treated at 100 ug / ml for SH-SY5Y cells and 20-200 ug / ml for PC12 cells), the neurites were elongated as well as the positive control in both cells and more than three times the length of the cell body. The ratio of the number of cells having the neurites to be increased also about 1.5 times (see Figs. 5 and 6A-6C).
특히, 황련추출물에 의한 PC12 세포의 신경돌기 생성양상은 효과 지속기간이 상당히 긴 것으로 나타났는데, NGF로 분화된 PC12 세포는 2주 이상 지속되기 어려운 반면에 황련추출물로 분화된 PC12 세포는 30일 이상 지속되었다.In particular, the neurite outgrowth of PC12 cells by the rhubarb extract showed a very long duration of effect. PC12 cells differentiated with NGF were difficult to last for more than two weeks, whereas PC12 cells differentiated with rhus extract were more than 30 days. Lasted.
한편, PC12 세포에서 황련추출물 처치에 의한 NGF mRNA의 발현양상을 조사한 결과, 이를 처리하지 않은 대조군보다 황련추출물을 처리한 실험군에서 현저히 높은 NGF mRNA가 발현됨을 확인할 수 있었다. 이는 PC12 세포에 황련추출물이 나타내는 생물학적 효과가 신경성장인자처럼 직접 그리고/혹은 간접적인 작용기전에 의함을 암시한다.On the other hand, as a result of investigating the expression pattern of NGF mRNA by treatment with rhubarb extract in PC12 cells, it was confirmed that significantly higher NGF mRNA was expressed in the experimental group treated with the rhubarb extract than the control group without treatment. This suggests that the biological effects of rhubarb extract on PC12 cells are due to direct and / or indirect mechanisms of action, such as neuronal growth factors.
실시예 5. PC12 세포에서의 신경세포돌기 정량 비교Example 5. Comparison of Neuronal Dendritic Quantities in PC12 Cells
PC12 세포의 배양액에 황련추출물 120 ㎍/㎖처치군, 생리식염수 처치군, NGF 50 ng/ml 처치군을 2주 이상 배양하면서 생성되는 신경세포돌기의 길이를 측정하였다. 신경세포돌기의 생성을 신경세포 분화지표로 간주하여 분화지수를 다음과 같이 산출하였다. 신경세포돌기 신장을 볼 수 없는 세포를 0, 발현된 신경세포돌기 길이가 세포체의 직경 미만이면 1, 발현된 신경세포돌기 길이가 세포체 직경 정도이면 2, 신경세포돌기 길이가 세포체 직경의 1배 이상 2배 이하이면 3, 신경세포돌기 길이가 세포체 직경의 2배 이상 또는 다른 세포의 신경세포돌기와 시냅스를 형성하면 4의 점수로 계산하였다. 분화지수는 각 약물 처치군의 microculture well에서 200개의 분화세포를 1단위로 하여 5개의 영상을 통계 처리하였다. 그 결과는 도8a 및 도8b로서 황련추출물 처치군은 생리식염수 처치군에 비하여 신경돌기생성이 현저히 우수하였다.In the culture medium of PC12 cells, the length of neuronal projections generated by culturing 120 µg / ml treatment group, physiological saline treatment group, and NGF 50 ng / ml treatment group for two weeks or longer was measured. The differentiation index was calculated as follows. 0 for cells that cannot see neural protuberance elongation, 1 if the expressed neuronal protuberance is less than the diameter of the cell body, 2 if the expressed neuronal protuberance is about the diameter of the cell body, and 2 or more times the neuronal protuberance diameter 2 or less times, the number of neuronal projections was calculated as a score of 4 or more than twice the diameter of the cell body or when forming synapses with neuronal processes of other cells. Differentiation index was statistically processed five images using 200 differentiation cells as one unit in the microculture well of each drug treatment group. As a result, as shown in Figs. 8a and 8b, the treatment group of rhubarb extract was significantly superior in neurogenesis compared to the physiological saline treatment group.
실시예 6. PC12 세포에서의 NGF mRNA 및 GAPDH mRNA 발현양상 비교Example 6 Comparison of NGF mRNA and GAPDH mRNA Expression in PC12 Cells
PC12 세포 배양액에 황련추출물 120 ㎍/㎖처치군, 생리식염수 처치군, NGF 50 ng/ml 처치군을 2주이상 배양한 후, NGF mRNA 및 대조군인 GAPDH mRNA 발현양상을 RT-PCR법으로 조사하였다.After incubating 120 µg / ml treatment group, physiological saline treatment group and NGF 50 ng / ml treatment group in PC12 cell culture medium for 2 weeks or longer, the expression patterns of NGF mRNA and GAPDH mRNA as a control group were investigated by RT-PCR method. .
1) Total RNA 분리1) Total RNA Isolation
조직 100 mg당 TRI Reagent(Molecular Research Center Inc., USA) 1 ㎖를 넣고 균질화 시킨 후 상온에 10분간 방치하였다. 균질액 1 ㎖당 0.1 ㎖의 BCP (Sigma, USA)를 넣고 1분간 잘 혼합하고 4℃에 10분간 방치하였다가 4℃에서 12,000 rpm으로 15분간 원심분리한 후 상층액을 취하여 차가운 isopropanol을 넣고 -20℃에서 16시간 이상 방치하였다. 그 후 4℃에서 12,000 rpm으로 15분간 원심분리하여 얻은 RNA 침천물을 DEPC (diethylpyrocarbonate) 처리된 차가운 75% 에탄올로 세척하고 SpeedVac을 이용하여 건조시켰다. 건조시킨 RNA를 DEPC 처리된 증류수를 넣어 녹인 후 260 nm 파장에서 분광광도계로 농도 및 순도를 측정하고 -20℃에 보관하여 사용하였다.1 ml of TRI Reagent (Molecular Research Center Inc., USA) per 100 mg of tissue was homogenized and left to stand at room temperature for 10 minutes. Add 0.1 ml of BCP (Sigma, USA) per 1 ml of homogenate, mix well for 1 minute, leave at 4 ° C for 10 minutes, centrifuge at 12,000 rpm for 15 minutes at 4 ° C, take supernatant and add cold isopropanol- It was left to stand at 20 degreeC for 16 hours or more. Thereafter, the RNA precipitate obtained by centrifugation at 12,000 rpm for 15 minutes at 4 ° C. was washed with cold 75% ethanol treated with DEPC (diethylpyrocarbonate) and dried using SpeedVac. The dried RNA was dissolved in DEPC treated distilled water, and the concentration and purity were measured using a spectrophotometer at 260 nm wavelength and stored at -20 ° C.
2) cDNA 합성(Reverse Transcription: RT)2) cDNA synthesis (Reverse Transcription: RT)
Total RNA 2 ㎍에 대하여 4.0 ㎕의 5X RT buffer, 1.0 ㎕의 oligo(dT16) (100 pmoles/㎕), 4 ㎕의 10 mM dNTPs (Promega, USA), 0.5 ㎕의 RNasin (40 Units/㎕, Promega, USA), 1.0 ㎕의 MMLV reverse transcriptase (200 units/㎕, Promega, USA)를 혼합하고 반응액의 전체부피가 30 ㎕되게 DEPC 처리된 증류수를 첨가하였다. 이 반응액을 DNA thermal cycler (Perkin Elmer 2400, USA)에서 42℃에서 1시간동안 반응시켜 cDNA를 합성하였다.4.0 μl of 5X RT buffer, 1.0 μl of oligo (dT 16 ) (100 pmoles / μl), 4 μl of 10 mM dNTPs (Promega, USA), 0.5 μl of RNasin (40 Units / μl, for 2 μg of total RNA) Promega, USA), 1.0 μl of MMLV reverse transcriptase (200 units / μl, Promega, USA) were mixed and DEPC treated distilled water was added so that the total volume of the reaction solution was 30 μl. The reaction solution was reacted with a DNA thermal cycler (Perkin Elmer 2400, USA) at 42 ° C. for 1 hour to synthesize cDNA.
3) 중합효소연쇄반응 (Polymerse Chain Reaction: PCR)3) Polymerase Chain Reaction (PCR)
RT 산물 1 ㎕ 기준으로 sense 및 antisense primers (각 10 pmoles), 1 ㎕의 10 mM dNTPs, 2 ㎕의 10X buffer (20 mM Tris-Cl, 1.5 mM MgCl2, 25 mM KCl, 0.1 ㎎/㎖ gelatin, pH 8.4), 1 unit의TaqDNA polymerase (Promege, USA)를 첨가하여 반응액의 전체부피가 25 ㎕가 되게 증류수를 첨가하여 DNA thermal cycler (Perkin Elmer 2400, USA)를 사용하여 중합효소연쇄반응을 실시하였다.Sense and antisense primers (10 pmoles each), 1 μl of 10 mM dNTPs, 2 μl of 10X buffer (20 mM Tris-Cl, 1.5 mM MgCl 2 , 25 mM KCl, 0.1 mg / ml gelatin, pH 8.4), 1 unit of Taq DNA polymerase (Promege, USA) was added, and distilled water was added so that the total volume of the reaction solution was 25 μl. The polymerase chain reaction was performed using a DNA thermal cycler (Perkin Elmer 2400, USA). Was carried out.
4) 전기영동 및 분석4) Electrophoresis and Analysis
증폭된 PCR 산물 10 ㎕를 1.5 % agarose gel에 전기영동하여 gel documentation system (Bio-Rad Lab, USA)을 이용하여 밀도를 측정, 비교하였다.10 μl of the amplified PCR product was electrophoresed on 1.5% agarose gel, and the density was measured and compared using a gel documentation system (Bio-Rad Lab, USA).
그 결과는 도 9a 및 도 9b로서 황련추출물 처치군은 생리식염수 처치군에 비하여 현저히 높은 NGF 발현도를 보여주고 있다.As a result, as shown in Figs. 9a and 9b, the treatment group of the barberry extract showed a significantly higher NGF expression level than the physiological saline treatment group.
실시예 7. MK-801모델에 의한 황련추출물의 뇌 신경세포재생 및 방어효과 확인Example 7 Confirmation of Brain Neuronal Regeneration and Protective Effect of Lactobacillus Extract by MK-801 Model
MK-801은 동물실험에서 주사 후 10∼30분만에 혈청 및 뇌조직에 최고치에 도달하고 흰쥐에서 반감기는 1.9시간으로 추정되고 있다(Vezzani et al, 1989). Olney등은 어린 흰쥐(생후 7-8일)에 MK-801을 투여하여 NMDA 수용체를 억제하면(2-3시간 정도) 감수성이 높은 NMDA성 신경세포들이 고사(apoptosis)의 경로를 거쳐 사멸하며 부위에 따라 다르지만 사망하는 신경세포의 수는 전체의 12∼26%에 이른다는 것을 발견하였다(Ikonomidou et al, 1999).In animal experiments, MK-801 peaks in serum and brain tissue 10-30 minutes after injection, and the half-life in rats is estimated to be 1.9 hours (Vezzani et al, 1989). Olney et al., When MK-801 is administered to young rats (7-8 days after birth) to inhibit NMDA receptors (2-3 hours), highly sensitive NMDA neurons die through the path of apoptosis. The number of neurons that died, depending on the human body, was found to be 12-26% of the total (Ikonomidou et al, 1999).
본 발명에 따른 황련추출물의 신경세포보호기능은 생후 7일된 어린 흰쥐에서 MK-801로 유도된 신경세포 고사모델을 이용하여 검정하였다.Neuroprotective function of the extract of the rhubarb extract according to the present invention was assayed using MK-801 induced neuronal apoptosis model in young rats 7 days old.
구체적으로 어린 흰쥐를 5개 그룹 즉, 가) 생리식염수 단독투여군, 나) MK-801(0.5 mg/kg) 단독투여군, 다) 황련추출물(20 mg/kg) 단독투여군, 라) 황련추출물(20 mg/kg) 전처치 후 MK-801(0.5 mg/kg) 투여군, 마) MK-801(0.5 mg/kg) 전 처치후 황련추출물(20 mg/kg) 투여군으로 나누고 모든 약물은 복강 주사하였다.Specifically, the young rats were divided into five groups, namely, a) saline alone administration group, b) MK-801 (0.5 mg / kg) alone administration, c) nasturtium extract (20 mg / kg) alone administration, and d) humus extract (20). mg / kg) pretreatment MK-801 (0.5 mg / kg) administration group e) MK-801 (0.5 mg / kg) pretreatment group after the treatment with nasturtium extract (20 mg / kg) administration group and all drugs were intraperitoneally injected.
뇌신경세포의 관찰방법은 실험동물을 마취 하에서 희생시키고 뇌를 적출하여 포르마린에 고정한 후 조직절편을 제작하고, 그 조직절편에서 TUNEL(terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) 기법(Gavrieliet al, 1992)을 이용하였으며 광학현미경(Olympus BX 50)으로 1.25배 및 400배로 촬영하였다. 실험결과는 하기와 같다.Cerebral nerve cells were observed under anesthesia, the brains were extracted, the brains were fixed and fixed in formalin, and then tissue sections were prepared. ) Was taken at 1.25 and 400 times with an optical microscope (Olympus BX 50). The experimental results are as follows.
1) 생리식염수 단독투여군1) Saline alone group
도10a 및 도10b는 생후 7일된 흰쥐의 정상 대뇌 절편의 TUNEL 염색을 보여주고 있다.10A and 10B show TUNEL staining of normal cerebral sections of 7-day-old rats.
2) MK-801 단독투여군2) MK-801 administration group
MK-801 (0.5 mg/kg)만을 생후 7일된 흰쥐에 복강 주사한 1일 후에, 대뇌절편의 신경세포의 사멸양상을 확인하였다.One day after intraperitoneal injection of MK-801 (0.5 mg / kg) into 7-day-old rats, neuronal death of cerebral sections was confirmed.
도11a 및 11b는 대뇌 조직의 관상 절편(coronal section)을 보여준다. 까맣게 나타나는 세포들은 핵 내의 DNA가 분절된 세포에만 양성으로 염색되는 세포사멸 검색법, 즉 TUNEL법에 양성반응을 보이는 세포들이다.11A and 11B show coronal sections of cerebral tissue. Cells that appear black are cells that are positive for apoptosis detection (TUNEL), in which the DNA in the nucleus is stained positively only in the segmented cells.
3) 황련추출물 단독투여군3) alone administration of rhubarb extract
본 발명의 황련추출물(20 mg/kg)만을 생후 7일된 흰쥐에 복강내 주사한 후 3일 째 적출하여 대뇌 절편을 TUNEL 염색사진으로 관찰하였다. 황련추출물 자체는 신경세포사를 유발하지 않음을 보여주고 있다(도12a 및 도12b).Only the rhubarb extract (20 mg / kg) of the present invention was intraperitoneally injected into 7-day-old rats after 3 days of extraction, and the cerebral sections were observed by TUNEL staining. Rhubarb extract itself has not been shown to induce neuronal death (Figs. 12a and 12b).
4) 황련추출물 전처치 후 MK-801 투여군4) MK-801-administered group after pretreatment of rhubarb extract
황련추출물(20 mg/kg)을 생후 7일된 흰쥐의 복강에 3일 동안 전처치한 후 MK-801 (0.5 mg/kg)을 복강주사한 후 상기의 방법으로 대뇌절편을 관찰하였다. 그결과 MK-801에 의해 유도된 신경세포자멸사의 정도가 본 발명의 황련추출물에 의해서 현저하게 억제됨을 확인 할 수 있었다(도13a 및 13b 참조).After treatment with rye extract (20 mg / kg) in the abdominal cavity of 7-day-old rats for 3 days, MK-801 (0.5 mg / kg) was intraperitoneally injected and cerebral sections were observed by the above method. As a result, it was confirmed that the degree of neuronal apoptosis induced by MK-801 is significantly suppressed by the sulfur extract of the present invention (see FIGS. 13A and 13B).
5) MK-801(0.5 mg/kg) 전 처치후 황련추출물(20 mg/kg) 투여군5) MK-801 (0.5 mg / kg) group after treatment with rhubarb extract (20 mg / kg)
생후 7일된 흰쥐의 복강에 MK-801 (0.5 mg/kg)을 주사하여 신경세포 자멸사를 유도한 후 본 발명의 황련추출물을(20 mg/kg)을 6일 동안 복강 주사한 후 적출한 대뇌절편을 관찰하였다. 그 결과 MK-801에 의해 유도된 신경세포자멸사의 정도가 본 발명의 황련추출물에 의해서 현저하게 억제됨을 보여주고 있다(도14a 및 14b).Induced neuronal apoptosis by injecting MK-801 (0.5 mg / kg) into the abdominal cavity of 7-day-old rats, followed by intraperitoneal injection of the barberry extract of the present invention (20 mg / kg) for 6 days Was observed. The results show that the degree of neuronal apoptosis induced by MK-801 is significantly suppressed by the barberry extract of the present invention (FIGS. 14A and 14B).
실시예 8. 흰쥐 대뇌에서의 신경세포자멸사 정량비교Example 8 Quantitative Comparison of Neuronal Apoptosis in Rat Cerebral
어린 흰쥐의 대뇌절편상에서 MK-801(0.5 mg/kg) 단독 투여군, 황련추출물(20 mg/kg) 단독 6일 투여군, 황련추출물(20 mg/kg)을 3일 동안 전처치한 후 MK-801 (0.5 mg/kg)을 투여한 군, MK-801(0.5 mg/kg)을 투여한 후 황련추출물(20 mg/kg)을 6일간 투여한 군을 대상으로 MK-801에 의해 사망하는 신경세포자멸사의 정도(TUNEL positive cell numbers)가 황련추출물에 의해서 억제되는 양상을 정량적으로 나타내었다(도15). 정량방법은 TUNEL 양성 고사신경세포 수를 각 군당 12 마리의 대뇌절편의 동일 면적에서 세어서 평균하여 표시하였다.MK-801 (0.5 mg / kg) alone, Lactobacillus extract (20 mg / kg) alone for 6 days, and Lactobacillus extract (20 mg / kg) were pretreated for 3 days on cerebral sections of young rats. Neuronal cells killed by MK-801 in a group administered with (0.5 mg / kg), MK-801 (0.5 mg / kg) and then administered with Lactobacillus extract (20 mg / kg) for 6 days. The degree of suicide (TUNEL positive cell numbers) was shown to be quantitatively suppressed by the sulfur extract (Fig. 15). In the quantitative method, the number of TUNEL-positive dead nerve cells was counted and averaged in the same area of 12 cerebral sections in each group.
실시예 9. 흰쥐 대뇌에서의 bcl-2 mRNA 및 GAPDH mRNA 발현양상 비교Example 9 Comparison of bcl-2 mRNA and GAPDH mRNA Expression in Rat Brain
생후 7일된 흰쥐에 본 발명의 황련추출물을 각각 25 mg/kg, 50 mg/kg, 100 mg/kg씩 복강 주사한 후 대뇌 조직에서 발현되는 항-세포사멸 유전자인 bcl-2 mRNA의 발현양상을 RT-PCR법으로 조사하였다. 그 결과 정상군에 비하여 bcl-2 mRNA의 발현이 증가였다. 대조군으로는 GAPDH mRNA의 발현양상을 RT-PCR 방법으로 조사하였다(도16a 및 16b).The 7-day-old rats were intraperitoneally injected with 25 mg / kg, 50 mg / kg, and 100 mg / kg of the rhubarb extract of the present invention, respectively, to express the expression of bcl-2 mRNA, an anti-apoptotic gene expressed in cerebral tissue. It was investigated by RT-PCR method. As a result, the expression of bcl-2 mRNA was increased compared to the normal group. As a control group, expression patterns of GAPDH mRNA were examined by RT-PCR method (FIGS. 16A and 16B).
실시예 10. 치매동물모델에서 황련추출물의 신경재생 효과.Example 10 Neuronal Regeneration Effect of Lactobacillus Extract in Animal Model for Dementia
본 발명에서는 치매 모델로 kanate 유도체인 이보테닉산을 흰쥐 성체 뇌의 entorhinal cortex에 stereotaxic frame을 사용하여 미세주입하는 동물 모델을 사용하였다. 치매에서 심각한 퇴행을 보이는 entorhinal cortex에 이보테닉산을 주입하면 이 부위로 뻗어 있는 CA부위의 추상세포과 치상회의 과립세포들이 함께 세포사멸하므로 화학적 병변(chemical lesion) 모델로 사용된다.In the present invention, an animal model in which the kanate derivative ibothenic acid is injected into the entorhinal cortex of the rat adult brain using a stereotaxic frame is used as a dementia model. The injection of ivoonic acid into the entorhinal cortex, which shows severe degeneration in dementia, kills the abstract cells in the CA region and granulocytes of the dentate gyrus, which are used as a chemical lesion model.
먼저 흰쥐 성체를 깊이 마취하고 머리를 stereotaxic frame을 사용하여 뇌의 위치를 고정한 뒤 entorhinal cortex부위에 이보테닉산을 1.5 ul (1mg/ml) 주입하여 치매모델을 만들고, 황련추출물을 같은 부위에 주입하였다. 수술 2주 후에 뇌 절편를 얻어 헤마톡실린(hematocylin)으로 대조염색(counter stain)하여 세포사멸 및 방어효과를 관찰하였다. 형광염색한 뒤 세포수 측정에 사용된 부위를 사각형 상자로 표시하였다(도17). 또한 신경특이적표지자인 calbindin항체로 형광염색하고 신경세포의 생존율을 조사하였다(도18).First, the adult rats were deeply anesthetized, the head was fixed using a stereotaxic frame, and then 1.5 ul (1 mg / ml) of ibothenic acid was injected into the entorhinal cortex to make a dementia model. . Two weeks after the operation, brain sections were obtained and counterstained with hematocylin to observe cell death and protective effects. After fluorescence staining, the site used for cell number measurement was marked with a rectangular box (FIG. 17). In addition, fluorescence staining with a calbindin antibody that is a neurospecific marker was investigated the survival rate of neurons (Fig. 18).
도 18에서 보는 바와 같이 이보테닉산만을 주입한 경우에는 해마와 entorhinal cortex에서 calbindin-positive neruon이 2주 후 30-40%로 감소하였으며 4주 후 일부 회복되는 경향을 보였다. 해마의 CA1 부위보다 치상회 부위의 세포들은 좀더 천천히 감소하는 경향을 보였다. 그러나 황련추출물을 주입한 경우에는 calbindin-positive neruon의 생존율이 증가하였는데 특히 CA1 부위의 추상세포(pyramidal cell)의 경우 약 2배 이상 증가하였고 치상회의 과립세포는 약 1.5배 증가하였다. 그리고 entorhinal cortex에서는 크게 증가하지 않았으나 통계적으로 유의하였다. 그러므로, 베르베리은 치매동물모델에서 세포사멸로부터 신경세포를 보호하고 신경재생 효과가 있는 것으로 판단된다.As shown in FIG. 18, calbindin-positive neruon in the hippocampus and the entorhinal cortex was reduced to 30-40% after 2 weeks and partially recovered after 4 weeks. Cells in the dentate gyrus region tended to decrease more slowly than the CA1 region of the hippocampus. The survival rate of calbindin-positive neruon was increased in the injection of H. extract, especially in the pyramidal cells of CA1 region, and the granulocytes in the dentate gyrus increased 1.5 times. The entorhinal cortex did not increase significantly but was statistically significant. Therefore, berberri is believed to protect neurons from apoptosis and have neuronal regeneration in dementia animal models.
실시예 11. 황련추출물에 의한 말초신경계 좌골신경(sciatic nerve) 재생효과Example 11 Peripheral Nervous System Sciatic Nerve Regeneration Effect
흰쥐를 깊이 마취하고 좌골신경을 좌골에서 노출하여 파쇄하고 PBS 또는 황련추출물을 쥐몸무게 0.1kg당 2mg되게 복강주사한 뒤 봉합하여 1주, 2주와 4주 뒤에 신경재생 정도를 관찰하였다. 동물을 관류(perfusion)하고 수술 부위 뒤쪽(distal stump)의 좌골신경을 얻어 7-10um로 동결박절하고 축색표지자인 beta-tubulin isotypeIII (cy3, red)와 슈반세포의 분화(미엘린)표지자 MBP(myelin binding protein, cy2, green) 항체로 이중염색한 뒤 공초점 현미경으로 관찰하여 300um보다 긴 축색과 200um보다 긴 미엘린 수를 측정하였다(도19-도24 참조).Rats were deeply anesthetized, exposed to the sciatic nerve at the sciatic bone, broken, and intraperitoneally injected with PBS or rhododendron extract at 2 mg per 0.1 kg of rat weight, and then sutured to observe the degree of nerve regeneration after 1, 2 and 4 weeks. The animals were perfused and obtained with a sciatic nerve at the back of the surgical site (frozen) and frozen at 7-10um. Beta-tubulin isotypeIII (cy3, red), axon marker, and Schwann cell differentiation (myelin) marker, MBP (myelin) binding protein, cy2, green) and double staining with an antibody and observed by confocal microscopy to determine the number of axons longer than 300um and myelin number longer than 200um (see Fig. 19-24).
수술 1주 후에는 distal stump에서 축색과 미엘린이 퇴화하고 있으므로 퇴화된 미엘린조각들이 염색되었다(화살표, 도19와 도22 참조). 수술 2주까지 황련추출물의 효과는 크지 않았으나 4주 후에는 300um가 넘는 축색(도 21, 흰선)의 수가 2배 이상 증가하였다. 미엘린은 1주 후부터 200um가 넘는 미엘린 수가 2배 이상되고 4주에는 3배에 달하였다.One week after surgery, the degenerated myelin fragments were stained because the axons and myelin deteriorated in the distal stump (see arrows, Figs. 19 and 22). Up to 2 weeks after the operation, the effect of the rhubarb extract was not significant, but after 4 weeks, the number of axons (Fig. 21, white line) exceeding 300um increased more than twice. Myelin has more than doubled the number of myelin more than 200um after one week and tripled in four weeks.
따라서, 황련추출물은 말초신경 재생과정에서 축색돌기의 성장과 미엘린 수초의 재생을 촉진함을 알 수 있다.Therefore, it can be seen that the extract of the Rhododendron promotes the growth of axons and the myelin sheath during peripheral nerve regeneration.
또한 신경근 접합부에서 신경말단의 재생에 영향을 주는지 보기 위해 수술 4주 후에 좌골신경에 연결된 뒷다리 근육을 분리하여 동결박절하고 신경근접합부를 신경표지자인 beta-tubulin isotypeIII와 신경세사(neurofilament)로 염색하였다. 수술 4주 후에 대조군에서는 신경말단이 염색되었으나 근육 한 부위에 모여 있고 아직 근섬유 각각에 퍼져 하나씩의 신경근접합부를 형성하지 못했으나 황련추출물을 주사한 경우에는 신경말단이 근섬유에 퍼져 있는 것이 관찰되었다(도25).Four weeks after surgery, the hind limb muscles connected to the sciatic nerve were isolated and cryo-dissected and the neuromuscular junctions were stained with beta-tubulin isotype III and neurofilament. Four weeks after the operation, the nerve endings were stained in the control group, but they were gathered in one part of the muscle and still spread to each of the muscle fibers, which did not form one neuromuscular junction. 25).
따라서 황련추출물은 말초신경 재생과정에서 축색돌기의 성장, 미엘린 수초의 재생과 신경근접합부를 형성하기 위한 신경말단 재생을 촉진하는 것으로 보인다.Hence, it can be seen that the extract of R. oleracea promotes axon growth, myelin myelin regeneration, and nerve ending regeneration to form neuromuscular junctions during peripheral nerve regeneration.
상기 한 바와 같이, 본 발명에 따른 황련추출물을 함유하는 조성물은 신경세포의 축색돌기(axon) 및 수상돌기(dendrite)를 재생시킴으로써 신경세포 손상에 대한 보호작용과 성장촉진작용 및 신경세포의 재생작용이 있다. 따라서 본 발명의 조성물은 다양한 종류의 퇴행성 신경질환 또는 신경손상 질환의 예방 및 치료에 효과가 있으며, 특히 치매, 파킨슨병, 알츠하이머병, 간질, 중풍, 허혈성 뇌질환과 말초신경 손상 등의 예방 및 치료에 매우 유용하다.As described above, the composition containing the rhubarb extract according to the present invention protects nerve cell damage and promotes growth and nerve cell regeneration by regenerating axons and dendrites of nerve cells. There is this. Therefore, the composition of the present invention is effective in the prevention and treatment of various types of degenerative neurological diseases or neuroinjury diseases, and in particular, the prevention and treatment of dementia, Parkinson's disease, Alzheimer's disease, epilepsy, stroke, ischemic brain disease and peripheral nerve damage. Very useful for
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