KR101274182B1 - Compositions comprising an extract of Peanut Sprout for prevention or treatment of neurodegenerative diseases - Google Patents
Compositions comprising an extract of Peanut Sprout for prevention or treatment of neurodegenerative diseases Download PDFInfo
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- KR101274182B1 KR101274182B1 KR1020100124109A KR20100124109A KR101274182B1 KR 101274182 B1 KR101274182 B1 KR 101274182B1 KR 1020100124109 A KR1020100124109 A KR 1020100124109A KR 20100124109 A KR20100124109 A KR 20100124109A KR 101274182 B1 KR101274182 B1 KR 101274182B1
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- extract
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- peanut
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- hme
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Abstract
본 발명은 퇴행성 신경질환의 치료 및 예방에 효과를 갖는 땅콩나물 추출물 조성물에 관한 것으로, 더욱 구체적으로 땅콩나물 추출물을 유효성분으로 함유하는 퇴행성 신경질환 치료 및 예방용 조성물에 관한 것이다.
본 발명에 따르면, 땅콩나물의 부위별로 전체(whole), 머리(head) 및 줄기(stem) 부분의 추출물을 제조할 수 있으며, 각 추출물들을 독성이 유도된 신경세포에 처리하는 경우 신경세포 보호 효과를 나타내었으며, 특히 땅콩나물 머리(head) 부분의 추출물(HME 디에틸 에테르(diethyl ether) 분획물)이 그 효과가 가장 높았다. 또한 상기 땅콩나물 머리(head) 부분 추출물(HME 디에틸 에테르(diethyl ether) 분획물)을 처리한 후 아팝토시스(apoptosis)를 측정해본 결과, 본 발명의 추출물 처리에 의해 아팝토시스가 억제됨을 확인하였다. 따라서 본 발명의 땅콩나물 추출물은 신경세포 보호 효과가 탁월하므로, 본 발명의 땅콩나물 추출물을 이용한 퇴행성 신경질환 치료 및 예방용 조성물로써의 사용이 기대된다.The present invention relates to a peanut sprout extract composition having an effect on the treatment and prevention of neurodegenerative diseases, and more particularly to a composition for treating and preventing degenerative neurological diseases containing peanut sprout extract as an active ingredient.
According to the present invention, extracts of whole, head and stem portions can be prepared for each part of peanut sprouts, and when the extracts are treated to toxic-induced neurons, neuroprotective effect is obtained. In particular, the extract of the head part of peanut sprouts (HME diethyl ether fraction) had the highest effect. In addition, after measuring the apoptosis (apoptosis) after treatment of the head part extract (HME diethyl ether fraction) of the peanut sprouts, it was confirmed that the apoptosis is suppressed by the extract treatment of the present invention It was. Therefore, since the peanut sprout extract of the present invention has an excellent neuronal protective effect, it is expected to be used as a composition for treating and preventing degenerative neurological diseases using the peanut sprout extract of the present invention.
Description
본 발명은 퇴행성 신경질환의 치료 및 예방에 효과를 갖는 땅콩나물 추출물 조성물에 관한 것으로, 더욱 구체적으로 땅콩나물 추출물을 유효성분으로 함유하는 퇴행성 신경질환 치료 및 예방용 조성물에 관한 것이다.
The present invention relates to a peanut sprout extract composition having an effect on the treatment and prevention of neurodegenerative diseases, and more particularly to a composition for treating and preventing degenerative neurological diseases containing peanut sprout extract as an active ingredient.
최근 들어 사회, 경제적 발달로 인해 생활수준이 향상되고, 과학기술과 의료기술이 발전함에 따라 인간의 평균 수명이 증가하여 점차 인구구조가 고령화 사회가 되면서 노화 및 노화와 관련된 각종 퇴행성 신경질환이 전체 사망원인의 압도적 우위를 차지함에 따라 사회적인 문제로의 관심이 증가하고 있다 [Emerit J., et al., Biomed. Pharmacother. 2004, 58, 39-46; Kadomura K., et al., Biosci Biotechmol. Biochem. 2007, 71, 2029-2033]. 퇴행성 신경질환은 정상적인 노화의 과정과는 달리 비정상적인 신경세포의 죽음에 의하여 뇌나 척수에 이상이 생겨 인지능력, 보행능력, 운동능력이 감소하게 되는 질환이다. 이러한 퇴행성 질환의 주된 원인이 활성산소(free radical, oxygen radical)에 기인한다는 것이 인정됨에 따라 활성산소를 조절할 수 있는 항산화제의 개발과 연구에 더 많은 관심이 집중되고 있다 [Sagara Y., et al., Free. Radic. Biol. Med. 1998, 24, 1375-1389; Yoon M. Y., et al., J. Korean Soc. Appl. Chem. 2007, 50, 63-67; Jeong G. S., et al., Biol. Pharm. Bull. 2009, 32, 945-949]. 활성산소는 미토콘드리아내의 전자전달체 및 백혈구 세포의 활성화 등 정상적인 세포기능을 유지하는데 중요한 역할을 담당하는 세포내 신호전달 물질로 작용한다. 그러나 활성산소들은 불안정하고 산화력이 높아 생체물질과 쉽게 반응하기 때문에 인체 내에서 제거되지 못하면 산화적 스트레스(oxidative stress)를 유발하게 되며 [Jeong E. J., et al., Natural Product Sciences 2008, 14, 156-160; Parfenova H., et al., Am J. Physiol. Cell. Physiol. 2006, 290, 1399-1410], 이러한 산화적 스트레스는 지질과산화를 유도하고 단백질, 세포막 및 DNA 등을 손상시켜 세포의 노화와 변형을 유도함으로써 뇌졸중, 알츠하이머병, 파킨슨병 등 다양한 퇴행성 신경질환을 유발하게 된다 [Ha J. S. and S. S. Park, Neuroscience letters 2006, 393, 165-169; Yoon M. Y., et al., Food Sci. Biotechnol. 2007, 16, 858-862].In recent years, social and economic development has improved the standard of living, and as science and medical technology have developed, the average life expectancy of humans has increased, and the population structure has become an aging society. With the overwhelming dominance of the causes, interest in social issues is increasing [Emerit J., et al., Biomed . Pharmacother . 2004, 58, 39-46; Kadomura K., et al., Biosci Biotechmol . Biochem . 2007, 71, 2029-2033. Neurodegenerative diseases, unlike normal aging processes, are caused by abnormal neuronal cell death due to abnormal neuronal cell death, cognitive ability, gait ability, exercise ability is reduced. As it is recognized that the main cause of this degenerative disease is caused by free radicals and oxygen radicals, more attention is being focused on the development and research of antioxidants that can control free radicals [Sagara Y., et al. ., Free . Radic . Biol . Med . 1998, 24, 1375-1389; Yoon MY, et al., J. Korean Soc . Appl . Chem . 2007, 50, 63-67; Jeong GS, et al., Biol . Pharm . Bull . 2009, 32, 945-949]. Free radicals act as intracellular signaling substances that play an important role in maintaining normal cellular functions such as the activation of electron transporters and white blood cells in the mitochondria. However, because free radicals are unstable and highly oxidizing, they react easily with biological materials, and if not removed in the body, they cause oxidative stress. [Jeong EJ, et al., Natural Product Sciences 2008, 14, 156-160; Parfenova H., et al., Am J. Physiol . Cell . Physiol . 2006, 290, 1399-1410], these oxidative stresses induce lipid peroxidation and damage proteins, membranes and DNA, leading to aging and transformation of cells, leading to a variety of neurodegenerative diseases such as stroke, Alzheimer's disease and Parkinson's disease. [Ha JS and SS Park, Neuroscience letters 2006, 393, 165-169; Yoon MY, et al., Food Sci . Biotechnol . 2007, 16, 858-862.
인체 내에는 SOD(superoxide dismutase), catalase, 글루타치온 과산화효소(glutathione peroxidase) 등의 항산화 효소와 글루타치온(glutathione), 요산(uric acid) 등의 여러 가지 비효소적 항산화 물질이 있어 활성산소의 축적이나 해로운 산화작용을 막아주게 된다. 그 중 글루타치온 과산화효소(glutathione peroxidase)는 GSH를 생산하여 우리 몸의 균형을 이루어주게 되는데 [Murphy T. H., et al., Neuron. 2, 1547-1558], 이때 GSH는 글라이신(glycine), 글루타메이트(glutamate) 및 시스테인(cysteine)의 세 가지 아미노산이 결합된 단백질로 간과 세포에서 활성산소 및 각종 독성물질을 제거하여 세포손상을 방지하고 면역력을 높이는 역할을 한다 [Shin A. Y., et al., J. Neurosci. 2006, 26, 10514-10523; Milatovic D., et al., Toxicol. Appl. Pharm. 2009, 240, 219-225]. 이러한 GSH를 이루고 있는 아미노산 중 글루타메이트(glutamate)는 중추신경계 시냅스의 15 ~ 20 %를 차지하는 중심적인 흥분성 신경전달물질로 작용하지만, 뇌 속의 과다한 축적은 체내의 항산화 시스템의 기능을 저하시킴으로 인해 산화계와 항산화계의 불균형을 초래하여 신경을 과도하게 흥분시킴으로 인해 흥분독성(excitotoxicity)을 야기하게 된다 [Kim M. H., et al., J. Neurosci. 2008, 28, 10852-10863; Penugonda S., et al., Toxicol. Appl. Pharm. 2006, 216, 197-205; Federico H., et al., J. Neurochem. 2007, 100, 736-746]. 흥분독성은 세포의 사멸을 일으키게 되어 알츠하이머병, 파킨슨병과 같은 여러 퇴행성 신경질환을 유발하게 된다 [Choi B. H., et al., J. Cell. Sci. 2005, 119, 1329-1340; Lim C. S., et al., Biol. Pharm. Bull. 2006, 29, 1212-1216]. 상기와 같은 퇴행성 신경질환을 치료하기 위해서 항산화물 처리, 세포이식, 외과적 수술 등 다양한 치료법이 제시되고 있지만 대부분이 위험요소와 부작용을 가지고있어 각종 천연자원으로부터 보다 안전하고 신경세포 보호효과가 뛰어난 치료제의 개발이 요구된다.There are many non-enzymatic antioxidants such as SOD (superoxide dismutase), catalase, glutathione peroxidase, glutathione, uric acid, etc. It prevents oxidation. Among them, glutathione peroxidase produces GSH and balances the body [Murphy TH, et al., Neuron . 2, 1547-1558], wherein GSH is a protein that combines three amino acids, glycine, glutamate and cysteine, and removes free radicals and various toxic substances from liver and cells to prevent cell damage. Plays a role in enhancing immunity [Shin AY, et al., J. Neurosci . 2006, 26, 10514-10523; Milatovic D., et al., Toxicol . Appl . Pharm . 2009, 240, 219-225. Glutamate (glutamate) among the amino acids that make up GSH acts as a central excitatory neurotransmitter, which accounts for 15-20% of the central nervous system synapse, but excessive accumulation in the brain decreases the function of the antioxidant system in the body Imbalance in the system leads to excessive excitability of the nerve, resulting in excitotoxicity [Kim MH, et al., J. Neurosci . 2008, 28, 10852-10863; Penugonda S., et al., Toxicol . Appl . Pharm . 2006, 216, 197-205; Federico H., et al., J. Neurochem . 2007, 100, 736-746. Excitotoxicity causes cell death, leading to several degenerative neurological diseases such as Alzheimer's disease and Parkinson's disease [Choi BH, et al., J. Cell . Sci . 2005, 119, 1329-1340; Lim CS, et al., Biol . Pharm . Bull . 2006, 29, 1212-1216. In order to treat such neurodegenerative diseases, various treatments such as antioxidant treatment, cell transplantation, and surgical surgery have been proposed, but most of them have risk factors and side effects, which are safer from various natural resources and have superior neuronal protection effects. Development is required.
한편, 땅콩나물은 땅콩을 콩나물처럼 싹을 띄운 것으로 항산화 효과가 있는 레스베라트롤(resveratrol)과 숙취해소에 좋은 아스파라긴(asparagine)이 많이 함유되어 있다. 또한 콩나물에 비해 비타민이 10 배 이상 높으며, 양질의 불포화 지방산이 많아 심혈관 질환과 심근경색에 좋은 것으로 보고되어 있다. 그러나 땅콩나물에 대한 약리학적 연구는 아직 초보적인 단계에 불과하며, 특히 신경세포 보호 효과와 관련된 연구는 수행된 바가 없다.On the other hand, peanut sprouts are sprouting peanuts as bean sprouts and contain antioxidant resveratrol and asparagine, which is good for hangover. In addition, vitamins are more than 10 times higher than soybean sprouts and high quality unsaturated fatty acids have been reported to be good for cardiovascular disease and myocardial infarction. However, pharmacological studies on peanut sprouts are still in their infancy, and no studies on neuroprotective effects have been conducted.
이에, 본 발명자들은 상기 종래기술들의 문제점들을 극복하기 위하여 예의 연구 노력한 결과, 땅콩나물 추출물을 제조하였으며, 상기 땅콩나물 추출물을 독성이 유도된 신경세포에 적용하는 경우, 신경세포를 효과적으로 보호할 수 있음을 확인하고, 본 발명을 완성하게 되었다.
Thus, the present inventors have made a peanut sprout extract as a result of earnest research to overcome the problems of the prior art, when applying the peanut sprout extract to the neurons induced toxicity, can effectively protect the nerve cells It was confirmed that the present invention was completed.
따라서 본 발명의 주된 목적은 신경세포 보호효과를 갖는 땅콩나물 추출물 조성물을 제공하는데 있다.Therefore, the main object of the present invention is to provide a peanut sprout extract composition having a neuronal protective effect.
본 발명의 다른 목적은 상기 땅콩나물 추출물을 이용한 퇴행성 신경질환 치료 및 예방용 조성물을 제공하는데 있다.
Another object of the present invention to provide a composition for treating and preventing degenerative neurological diseases using the peanut sprouts extract.
본 발명의 한 양태에 따르면, 본 발명은 땅콩나물 추출물을 유효성분으로 함유하는 퇴행성 신경질환 치료 및 예방용 조성물을 제공한다.According to one aspect of the invention, the present invention provides a composition for the treatment and prevention of neurodegenerative diseases comprising peanut sprouts extract as an active ingredient.
본 발명의 상기 땅콩나물은 땅콩을 재배하여 마치 콩나물과 같이 싹을 띄운 것을 특징으로 한다. 예컨대, 땅콩을 약 18 ~ 23 ℃에서 3 ~ 10 일간 수경재배하면, 마치 콩나물처럼 싹을 띄워 머리 부분과 줄기(뿌리) 부분으로 나누어진다.The peanut sprouts of the present invention is characterized by cultivating peanuts, as if sprouting sprouts. For example, when peanuts are hydroponicly grown at about 18 to 23 ° C. for 3 to 10 days, they sprout like a bean sprout and are divided into a head part and a stem part.
본 발명의 조성물에 있어서, 상기 땅콩나물 추출물은 추출물을 제조할 수 있는 어떠한 부위도 가능하나, 바람직하게는 땅콩나물의 머리(head) 부분의 추출물인 것을 특징으로 한다.In the composition of the present invention, the peanut sprout extract may be any site from which the extract can be prepared, preferably an extract of the head part of the peanut sprout.
본 발명의 구체적인 실시예에서는 땅콩나물로부터 전체(whole), 머리(head) 및 줄기(stem)로부터 추출물을 제조하였으며, 그 중 가장 효과가 좋은 머리(head) 부분의 땅콩나물 추출물을 본 발명에 이용하였다.In a specific embodiment of the present invention, an extract was prepared from whole, head and stem from peanut sprouts, and the most effective head peanut extract of the head was used in the present invention. It was.
구체적으로 본 발명의 실시예에서 확인한 바와 같이, 세포독성이 유도된 신경세포에 각각의 땅콩나물 추출물을 처리한 후, 신경세포 보호 효과를 관찰하는 경우, 땅콩나물 머리(head) 부분의 추출물을 처리하였을 때 신경세포 보호 효과가 가장 높았다.Specifically, as confirmed in the embodiment of the present invention, after treating each peanut sprout extract to the cytotoxicity-induced neurons, when the neuronal protective effect is observed, the extract of the head of the peanut sprouts (head) treatment The neuroprotective effect was the highest.
본 발명의 조성물에 있어서, 상기 땅콩나물 추출물은 땅콩나물로부터 다양한 추출방법, 예컨대 용매추출(solvent extraction), 증류(hydrodistillation) 및 수증기 증류(steam distillation) 추출 등의 방법을 사용할 수 있으나, 바람직하게는 알코올 용매추출을 이용하여 추출하는 것을 특징으로 하며, 보다 바람직하게는 에탄올, 메탄올과 같은 탄소수 1 내지 4의 저급알코올 용매로 추출되는 것을 특징으로 한다.In the composition of the present invention, the peanut sprout extract may be a variety of extraction methods, such as solvent extraction (solvent extraction, hydrodistillation) and steam distillation (steam distillation) extraction from peanut sprouts, but preferably It is characterized in that the extraction using alcohol solvent extraction, more preferably characterized in that extracted with a lower alcohol solvent having 1 to 4 carbon atoms such as ethanol, methanol.
본 발명의 구체적인 실시예에서는 메탄올(methanol)을 가하여 추출한 후 여과 및 농축하여 추출물을 얻어내었다.In a specific embodiment of the present invention, methanol and methanol were added, followed by filtration and concentration to obtain an extract.
본 발명의 조성물에 있어서, 상기 땅콩나물 추출물은 8,13-디하이드록시-9,11-옥타데카디에노익 애시드(8,13-dihydroxy-9,11-octadecadienoic acid)를 함유하는 것을 특징으로 한다.In the composition of the present invention, the peanut sprout extract is characterized in that it contains 8,13-dihydroxy-9,11-octadecadienoic acid (8,13-dihydroxy-9,11-octadecadienoic acid). .
본 발명의 다른 양태에 따르면, 본 발명은 8,13-디하이드록시-9,11-옥타데카디에노익 애시드(8,13-dihydroxy-9,11-octadecadienoic acid)를 유효성분으로 함유하는 퇴행성 신경질환 치료 및 예방용 조성물을 제공한다.According to another aspect of the invention, the present invention is a degenerative nerve containing 8,13-dihydroxy-9,11-octadecadienoic acid (8,13-dihydroxy-9,11-octadecadienoic acid) as an active ingredient Provided are compositions for treating and preventing diseases.
본 발명의 상기 퇴행성 신경질환의 치료 및 예방은 신경세포의 아팝토시스(apoptosis)를 억제함으로써 수행되는 것을 특징으로 한다.The treatment and prevention of the neurodegenerative disease of the present invention is characterized by being performed by inhibiting apoptosis of neurons.
상기 아팝토시스(apoptosis)는 다세포생물에서의 programmed cell death를 일컫는 용어로 세포의 죽음에 관련되어 능동적으로 일어나는 세포의 죽음과정 중 하나이다.Apoptosis is a term for programmed cell death in multicellular organisms and is one of cell death processes actively occurring in relation to cell death.
상기 퇴행성 신경질환은 중추신경계의 신경세포에 퇴행성 변화가 나타나면서 운동 및 감각기능의 손상, 기억, 학습, 연산 추리 등의 고차원적인 기능의 저해와 같은 여러 가지 증상을 유발하는 질환으로써, 인체 내에서 산화계와 항산화계의 불균형의 초래로 신경이 과도하게 흥분되어 흥분독성이 야기되고 상기 흥분독성이 세포 사멸을 일으키게 되면서 발생된다. 본 발명의 땅콩나물 추출물은 이러한 신경세포의 세포사멸을 억제시킴으로써 퇴행성 신경질환을 치료 및 예방할 수 있다.The neurodegenerative disease is a disease causing degenerative changes in nerve cells of the central nervous system and causing various symptoms such as impairment of high-order functions such as impairment of motor and sensory functions, memory, learning, and computational reasoning, and in the human body. The imbalance between the oxidative and antioxidant systems causes the nerves to be excessively excited to cause excitatory toxicity, which is caused by cell death. Peanut sprout extract of the present invention can treat and prevent neurodegenerative diseases by inhibiting apoptosis of these nerve cells.
구체적으로 본 발명의 실시예에서 확인한 바와 같이, 독성을 유도시킨 신경세포에 본 발명의 땅콩나물 추출물을 처리하는 경우 아팝토시스(apoptosis)가 효과적으로 억제되는 것을 확인하였다.Specifically, as confirmed in the embodiment of the present invention, it was confirmed that the apoptosis (apoptosis) is effectively suppressed when the peanut sprout extract of the present invention is treated to the nerve cells induced toxicity.
본 발명의 상기 퇴행성 신경질환은 신경세포의 아팝토시스와 관련된 어떤 신경질환도 포함하나, 바람직하게는 알츠하이머병(Alzheimer's disease), 파킨슨병(Parkinson's disease), 루게릭병(Lou Gehrig disease), 헌팅톤병(Huntington's disease), 다발성 경화증(Multiple sclerosis), 뇌졸중(stroke) 또는 치매(dementia)인 것을 특징으로 한다.The neurodegenerative diseases of the present invention include any neurological diseases related to apoptosis of neurons, but are preferably Alzheimer's disease, Parkinson's disease, Lou Gehrig disease, Huntington's disease ( Huntington's disease, multiple sclerosis, stroke or dementia.
본 발명의 퇴행성 신경질환 치료 및 예방용 조성물이 약제로 이용되기 위해서는 약제학적 분야에서 공지된 방법에 의해 제조될 수 있으며, 이 경우 유효성분으로서의 본 발명의 추출물 이외에 약제학적으로 허용되는 담체를 포함한다. 본 발명의 약제학적 조성물에 포함되는 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약제학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약제학적으로 허용되는 담체 및 제제는 Remington's Pharmaceutical Sciences(19th ed., 1995)에 상세히 기재되어 있다.In order to use the composition for treating and preventing degenerative neurological diseases of the present invention, it may be prepared by a method known in the pharmaceutical field, in which case it includes a pharmaceutically acceptable carrier in addition to the extract of the present invention as an active ingredient. . Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, silicic acid Calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oils, and the like. It is not. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).
본 발명의 약제학적 조성물은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 또는 부형제를 이용하여 제제화됨으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 분말제, 과립제, 정제, 캅셀제 또는 주사제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다. 이들은 비경구 투여(예컨대, 정맥 내, 피하, 복강 내 또는 국소에 적용)하거나 경구 투여될 수 있다.The pharmaceutical compositions of the present invention may be prepared in unit dose form by being formulated with a pharmaceutically acceptable carrier or excipient according to methods which can be easily carried out by those skilled in the art. It can be prepared by incorporation into a multi-dose container. The formulation may be in the form of a solution, suspension or emulsion in oil or medium, or may be in the form of extracts, powders, granules, tablets, capsules or injections, and may further comprise dispersants or stabilizers. They may be parenterally administered (eg, applied intravenously, subcutaneously, intraperitoneally or topically) or orally.
본 발명의 약제학적 조성물의 적합한 투여량은 제제화 방법, 투여방식, 환자의 연령, 체중, 성, 건강상태, 질병 증상의 정도, 음식, 투여시간, 투여방법 및 반응 감응성과 같은 요인들에 의해 적절히 선택될 수 있으며, 바람직하게는 성인기준 1일 당 0.01 ~ 100 mg이 투여될 수 있다.
Appropriate dosages of the pharmaceutical compositions of the present invention may be appropriately determined by such factors as formulation method, mode of administration, age, weight, sex, health condition, degree of disease symptom, food, time of administration, method of administration and response to reaction. It may be selected, preferably 0.01 to 100 mg per day of adult can be administered.
본 발명의 한 양태에 따르면, 본 발명은 신경세포 보호 효과를 갖는 땅콩나물 추출물을 유효성분으로 함유하는 퇴행성 신경질환의 치료 및 예방을 위한 건강기능식품을 제공한다.According to an aspect of the present invention, the present invention provides a health functional food for the treatment and prevention of degenerative neurological diseases containing peanut sprout extract having an effect of protecting nerve cells as an active ingredient.
본 발명에서 정의되는 상기 “건강기능식품”은 건강기능식품에 관한 법률 제6727호에 따른 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 의미하며, “기능성”이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻을 목적으로 섭취하는 것을 의미한다.The term "health functional food" as defined in the present invention means a food manufactured and processed using raw materials or ingredients having functional properties useful to the human body according to Act No. 6767 of the Health Functional Food Act. It means ingestion for the purpose of obtaining useful effects on health use such as nutrient control or physiological action on the structure and function of the human body.
본 발명의 퇴행성 신경질환의 치료 및 예방을 위한 건강기능식품은 조성물 총 중량에 대하여 상기 땅콩나물 추출물을 0.01 내지 95 %, 바람직하게는 1 내지 80 % 중량백분율로 포함한다.The dietary supplement for the treatment and prevention of neurodegenerative diseases of the present invention comprises the peanut sprout extract in an amount of 0.01 to 95%, preferably 1 to 80% by weight, based on the total weight of the composition.
또한 상기 건강기능식품은 퇴행성 신경질환의 치료 및 예방을 위한 목적으로 정제, 캅셀, 분말, 과립, 액상, 환 등의 형태인 건강기능식품으로 제조 및 가공이 가능하다. 예컨대, 상기 정제 형태의 건강기능식품은 그대로 또는 부형제, 결합제, 붕해제 또는 다른 첨가제를 넣어 고르게 섞은 것을 적당한 방법으로 과립상으로 한 다음, 활택제 등을 넣어 압축성형하여 조제하거나 정제 형태의 건강기능식품을 그대로 또는 부형제, 결합제, 붕해제 또는 다른 적당한 첨가제를 넣어 고르게 섞은 것을 직접 압축성형하여 만들거나 또는 미리 만든 과립에 건강기능식품을 그대로 혹은 적당한 첨가제를 넣어 고르게 섞은 다음 압축성형하여 조제하거나 건강기능식품에 부형제, 결합제 또는 다른 적당한 첨가제를 넣어 고르게 섞은 분말을 용매로 습윤시키고, 습윤된 분말을 저압으로 틀에 넣어서 성형한 후, 적당한 방법으로 건조하여 조제한다. 또한 상기 정제 형태의 건강기능식품에 필요에 따라 교미제 등을 넣을 수 있으며, 적당한 제피제로 제피 가능하다.In addition, the health functional food is manufactured and processed as a health functional food in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of the treatment and prevention of degenerative neurological diseases. For example, the health functional food in the form of tablets may be granulated in a suitable manner as it is or evenly mixed with an excipient, a binder, a disintegrating agent or other additives, and then compressed into a glidant or the like to prepare a health function. It is made by directly compressing the food as it is or by adding the excipient, binder, disintegrating agent or other suitable additives, or by direct compression molding, or by mixing the health functional foods as it is or by adding the appropriate additives and mixing them evenly, Excipients, binders or other suitable additives are added to the food and the mixed powder is wetted with a solvent, the wet powder is molded into a mold at low pressure, and then dried and prepared by a suitable method. In addition, the tablet can be put into the nutraceutical, such as if necessary in the health functional food, it is possible to peel with a suitable epidermal.
상기 캅셀 형태의 건강기능식품 중 경질캅셀제는 보통 캅셀에 건강기능식품 또는 건강기능식품에 적당한 부형제 등을 고르게 섞은 것 또는 적당한 방법으로 입상으로 한 것 또는 입상으로 한 것에 적당한 제피제로 제피한 것을 그대로 또는 가볍게 성형하여 충전하여 조제하며, 연질캅셀제는 보통 캅셀에 건강기능식품 또는 건강기능식품에 적당한 부형제 등을 넣은 것을 젤라틴 등 적당한 캅셀기제에 글리세린 또는 소르비톨 등을 넣어 소성을 높인 캅셀기제로 피포하여 일정한 형상으로 성형하여 조제하며, 필요에 따라 상기 캅셀기제에 착색료, 보존료 등을 첨가할 수 있다.Among the health functional foods in the form of capsules, the hard capsules are usually prepared by mixing the capsules evenly with the health functional foods or excipients suitable for the health functional foods, granulated by a suitable method, or granulated with a suitable epidermal agent as it is or Soft capsules are prepared by filling them, and soft capsules are usually filled with capsules containing glycerin or sorbitol in a capsule form containing gelatin or appropriate excipients suitable for health functional foods or health functional foods. It is molded and prepared, and a coloring agent, a preservative, etc. can be added to the said capsule base as needed.
환 형태의 건강기능식품은 보통 건강기능식품에 부형제, 결합제, 붕해제 등을 고르게 섞은 다음 적당한 방법으로 구상으로 성형하여 조제하며, 필요에 따라 백당이나 다른 적당한 제피제로 제피를, 또는 전분, 탈크 또는 적당한 물질로 환의를 입힐 수도 있다.Ring-shaped dietary supplements are usually prepared by mixing excipients, binders, disintegrants, etc. evenly in a dietary supplement and then forming them into spherical forms in a suitable manner. You can also be greeted with a suitable substance.
과립 형태의 건강기능식품은 보통 건강기능식품을 그대로 또는 건강기능식품에 부형제, 결합제, 붕해제 등을 넣어 고르게 섞은 다음 적당한 방법으로 입상으로 만들고, 될 수 있는 대로 입자를 고르게 한 것이며, 필요에 따라 착향료, 교미제 등을 넣을 수 있다.Functional foods in the form of granules are usually mixed with the functional food as it is or by adding excipients, binders, disintegrating agents, etc., evenly and then granulated in a suitable way, and as uniformly as possible particles. Flavoring agent, copulation agent, etc. can be added.
본 발명의 상기 부형제, 결합제, 붕해제, 활택제, 교미제, 착향료 등에 대한 용어 정의는 당업계에 공지된 문헌에 기대된 것으로 그 기능 등이 동일 내지 유사한 것들을 포함한다 [대한약전 해설편, 문성사, 한국약학대학협의회, 제 5 개정판, p33-48, 1989].
The term definitions of the excipients, binders, disintegrants, lubricants, copulation agents, flavoring agents, etc. of the present invention are expected in the literature known in the art and include those having the same or similar functions. Sacrament, Korean Pharmacy University Council, 5th Revision, p33-48, 1989].
이상 설명한 바와 같이, 본 발명에 따르면 땅콩나물의 부위별로 전체(whole), 머리(head) 및 줄기(stem) 부분의 추출물을 제조할 수 있다.As described above, according to the present invention, extracts of whole, head and stem portions of peanut sprouts can be prepared.
상기 실시예에서 살펴본 바와 같이, 상기 땅콩나물의 추출물들을 독성이 유도된 신경세포에 처리하는 경우 신경세포 보호 효과를 나타내었으며, 특히 땅콩나물 머리(head) 부분의 추출물(HME 디에틸 에테르(diethyl ether) 분획물)이 그 효과가 가장 높았다. 또한 상기 땅콩나물 머리(head) 부분 추출물(HME 디에틸 에테르(diethyl ether) 분획물)을 처리한 후 아팝토시스(apoptosis)를 측정해본 결과, 본 발명의 추출물 처리에 의해 아팝토시스가 억제됨을 확인하였다. 따라서 본 발명의 땅콩나물 추출물은 신경세포 보호 효과가 탁월하므로, 본 발명의 땅콩나물 추출물을 이용한 퇴행성 신경질환 치료 및 예방용 조성물로써의 사용이 기대된다.
As described in the above examples, the extracts of the peanut sprouts showed neuroprotective effects when treated with neurons induced by toxicity, in particular the extract of the head of the peanut sprouts (HME diethyl ether). Fractions) had the highest effect. In addition, after measuring the apoptosis (apoptosis) after treatment of the head part extract (HME diethyl ether fraction) of the peanut sprouts, it was confirmed that the apoptosis is suppressed by the extract treatment of the present invention It was. Therefore, since the peanut sprout extract of the present invention has an excellent neuronal protective effect, it is expected to be used as a composition for treating and preventing degenerative neurological diseases using the peanut sprout extract of the present invention.
도 1은 땅콩나물 머리(head) 부분 추출물(HME)의 용매 분획물의 제조단계 모식도를 나타낸 것이다.
도 2은 본 발명의 땅콩나물 추출물의 처리에 따른 신경세포의 MTT reduction assay 측정 결과를 나타낸 것이다.
도 3은 땅콩나물 머리(head) 부분 추출물(HME)의 각 용매 분획물의 처리에 따른 신경세포의 MTT reduction assay 측정 결과를 나타낸 것이다.
도 4는 땅콩나물 머리 부분 추출물(HME)로부터 유효성분의 분리 및 정제 모식도를 나타낸 것이다.
도 5은 분리 및 정제된 유효성분의 HPLC 측정 결과를 나타낸 것이다.
도 6는 PSE-1의 구조 결정을 위한, HRESI-Mass 측정 결과를 나타낸 것이다.
도 7는 PSE-1의 구조 결정을 위한, 자외선 흡수 스펙트럼(UV absorbance spectrum) 측정 결과를 나타낸 것이다.
도 8의 (A)는 PSE-1의 구조 결정을 위한, 1H-NMR 측정 결과를 나타낸 것이며, (B)는 13C-NMR 측정 결과를 나타낸 것이다.
도 9은 구조 분석을 통해 얻어낸 PSE-1의 구조를 나타낸 것이다.
도 10a는 땅콩나물 머리(head) 부분 추출물(HME)의 처리에 따른 신경세포의 LDH release assay 측정 결과를 나타낸 것이며, 도 10b는 땅콩나물 머리(head) 부분 추출물(HME)의 처리에 따른 신경세포의 형태학적 변화를 측정한 결과이다.
도 11은 땅콩나물 머리(head) 부분 추출물(HME)의 처리에 따른 신경세포의 sub-G1기의 세포수를 측정한 결과를 나타낸 것이다.Figure 1 shows a schematic diagram of the preparation step of the solvent fraction of peanut head extract (HME) head fraction (HME).
Figure 2 shows the results of MTT reduction assay measurement of neurons according to the treatment of the peanut sprouts extract of the present invention.
Figure 3 shows the results of MTT reduction assay measurement of neurons according to the treatment of each solvent fraction of peanut sprout head (HME) fraction extract (HME).
Figure 4 shows a schematic diagram of the separation and purification of the active ingredient from the peanut sprouts head extract (HME).
Figure 5 shows the HPLC measurement results of the separated and purified active ingredient.
Figure 6 shows the HRESI-Mass measurement results for the structure determination of PSE-1.
FIG. 7 illustrates UV absorbance spectrum measurement results for determining the structure of PSE-1.
8 (A) shows the 1 H-NMR measurement result for determining the structure of PSE-1, and (B) shows the 13 C-NMR measurement result.
9 shows the structure of PSE-1 obtained through structural analysis.
Figure 10a shows the results of LDH release assay measurement of neurons according to the treatment of the peanut head extract (HME), Figure 10b shows the nerve cells according to the treatment of peanut head extract (HME) It is the result of measuring the morphological change of.
Figure 11 shows the results of measuring the number of cells of the sub-G1 phase of neurons according to the treatment of the head part extract (HME) peanut sprouts.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.
Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.
실시예Example 1. 시약 및 실험재료 1. Reagents and Experimental Materials
본 발명에 사용된 땅콩나물은 부산대학교 원예생명과학과로부터 제공받아 추출 및 분획하여 실험에 사용하였다. 신경세포주 배양을 위해 필요한 DMEM(Dulbecco's Modified Eagle's Medium), FBS(fetal bovine serum), HS(horse serum) 및 HAT supplement 등은 Gibco-BRL(Grand Island, NT, USA)에서 구입하였다. 또한 신경세포 보호 효과 실험에 사용된 L-glutamic acid(monosodium salt hydrate), MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), 아스코르빈산(ascorbic acid), DMSO(dimethyl sulfoxide)는 Sigma chemical co.(st. Louis, MO, USA)의 제품을 구입하여 사용하였으며 그 외 실험에 사용된 용매 및 시약은 모두 일급 이상의 등급을 사용하였다.
Peanut sprouts used in the present invention were supplied from the Department of Horticultural Life Science, Pusan National University, extracted and fractionated and used for experiments. Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), horse serum (HS) and HAT supplements required for neuronal cell culture were purchased from Gibco-BRL (Grand Island, NT, USA). In addition, L-glutamic acid (monosodium salt hydrate), MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl tetrazolium bromide), and ascorbic acid, which were used in the neuroprotective effect experiments Dimethyl sulfoxide (DMSO) was purchased from Sigma Chemical Co. (st. Louis, MO, USA), and the solvents and reagents used in the other experiments all used first-grade or higher grades.
실시예Example 2. 땅콩나물의 추출 및 2. Extraction of Peanut Sprouts 분획물Fraction 제조 Produce
본 발명의 땅콩나물 추출물을 제조하기 위해, 먼저 부위별 추출물을 제조하였다. 땅콩나물을 전체(whole), 머리(head) 및 줄기(stem) 부분으로 나누었다. 상기 각각의 부위별 시료 5 g에 메탄올 100 ml을 가하여 상온에서 3일 동안 정치시켜 추출한 후, 여과지(Advantage, Tokyo, Japan)로 여과하였다. 상기 여과된 추출액은 회전감압농축기(EYELA N-1000, Tokyo, Japan)를 이용하여 40 ℃에서 감압 농축하여 추출물을 얻어내었다. 땅콩나물의 부위별로 전체(whole) 추출물은 WME, 머리(head) 부분 추출물은 HME, 줄기(stem) 부분 추출물은 SME라 명명하였다.In order to prepare the peanut sprout extract of the present invention, first, an extract for each part was prepared. Peanut sprouts were divided into whole, head and stem portions. 100 ml of methanol was added to 5 g of the sample for each site, and the mixture was left to stand for 3 days at room temperature, followed by extraction, followed by filtration with filter paper (Advantage, Tokyo, Japan). The filtered extract was concentrated under reduced pressure at 40 ℃ using a rotary pressure reducer (EYELA N-1000, Tokyo, Japan) to obtain an extract. For each part of the peanut sprouts, the whole extract was named WME, the head part extract was HME, and the stem part extract was named SME.
이후, HME에서 다음 단계의 분획물을 얻기 위해 용매의 극성을 달리하여 순차적으로 용매분획을 얻었다. 즉 헥산(hexane)과 물을 동일 비율로 분획 추출하여 헥산 층을 분리하였으며, 동일한 방법으로 디에틸 에테르(diethyl ether), 에틸 아세테이드(ethyl acetate) 및 물(water) 층으로 분획하여 얻어진 각각의 용매 분획물을 감압 농축시켜 분획물을 얻었다. 각 추출물 및 분획물은 10 mg/ml로 DMSO에 녹여 적당한 농도로 희석하고, 1 % DMSO가 되도록 처리하였다. 상기 HME 용매 분획물의 제조단계 모식도는 도 1에 나타내었다.
Then, solvent fractions were obtained sequentially by varying the polarity of the solvent in order to obtain a fraction of the next step in HME. That is, the hexane layer was separated by fractional extraction of hexane and water in the same ratio, and each obtained by dividing into diethyl ether, ethyl acetate and water layers in the same manner. The solvent fractions of were concentrated under reduced pressure to give a fraction. Each extract and fractions were dissolved in DMSO at 10 mg / ml, diluted to the appropriate concentration, and treated to 1% DMSO. A schematic diagram illustrating the preparation of the HME solvent fraction is shown in FIG. 1.
실시예 3. 세포 배양Example 3. Cell Culture
본 발명의 땅콩나물 추출물의 신경세포 보호 효과를 알아보기 위해, 한국생명공학연구원으로부터 세포주 hybridoma N18-RE-105 세포를 분양받아 본 발명에 사용하였다. 상기 세포의 배양을 위해, DMEM 배지에 10 % FBS, 5 % HS 및 HAT supplement(1×)를 첨가하여 사용하였으며, 95 %의 습도가 유지되는 37 ℃, 5 % CO2 인큐베이터(incubator)(MCO-18AIC, Sanyo, Osaka, Japan)에서 배양하였다.
In order to investigate the neuronal protective effect of the peanut sprout extract of the present invention, the cell line hybridoma N18-RE-105 cells were distributed from the Korea Research Institute of Bioscience and Biotechnology and used in the present invention. For culturing the cells, 10% FBS, 5% HS and HAT supplement (1 ×) were added to DMEM medium, and 37 ° C., 5% CO 2 incubator (MCO) was maintained at 95% humidity. -18AIC, Sanyo, Osaka, Japan).
실시예 4. MTT reduction assayExample 4. MTT reduction assay
본 발명의 땅콩나물 추출물의 신경세포 보호 효과를 알아보기 위해, MTT reduction assay를 수행하였다. 먼저, 상기 실시예 4에서 배양한 세포주를 96-웰 플레이트(96-well plate)에 1×105 cells/ml의 농도로 100 μl씩 분주하여 24 시간 동안 37 ℃, 5 % CO2 인큐베이터(incubator)에서 배양한 후, 상기 실시예 2에서 메탄올 추출 후 농축하여 얻어낸 각각의 땅콩나물 부위별 추출물 WME, HME 및 SME을 DMSO에 녹여 각각 10, 50, 100 μg/ml의 농도로 제조하여 N18-RE-105 세포주에 처리하였다. 이후, 30 분 동안 배양한 후, 20 mM 글루타메이트(glutamate)를 처리하여 24 시간 배양하고, 각 웰(well)에 PBS 완충용액에 녹인 MTT(5 mg/ml) 용액을 10 μl씩 첨가하여 1 시간 동안 다시 반응시켰다. 반응이 완료되면 포르마잔(formazan)의 형성을 확인한 후, 배지를 완전히 제거하고 웰(well) 바닥에 형성된 포르마잔을 녹이기 위해 100 μl의 DMSO를 첨가한 후, ELISA reader(Model 680, BioRad, USA)를 이용하여 540 nm에서 흡광도를 측정하였다. 이때 본 발명의 땅콩나물 추출물을 처리하지 않고 배양시킨 정상군 세포를 100 %로 하였을 때의 상대적인 세포성장 억제율로 나타내었으며, 글루타메이트(glutamate)만을 처리한 대조군과 비교하였다. 측정 결과는 도 2에 나타내었다.In order to investigate the neuroprotective effect of the peanut sprout extract of the present invention, an MTT reduction assay was performed. First, the cell lines cultured in Example 4 were dispensed in 100 μl at a concentration of 1 × 10 5 cells / ml in a 96-well plate at 37 ° C., 5% CO 2 incubator for 24 hours. ), WME, HME and SME for each extract of peanut sprouts obtained by concentration after methanol extraction in Example 2 were dissolved in DMSO and prepared at concentrations of 10, 50, and 100 μg / ml, respectively, and N18-RE. Treatment with -105 cell lines. Then, after incubation for 30 minutes, incubated for 24 hours by treatment with 20 mM glutamate (glutamate), 1 well by adding 10 μl of MTT (5 mg / ml) solution dissolved in PBS buffer in each well (hour) Reaction again. When the reaction was completed, after confirming the formation of formazan, the medium was completely removed and 100 μl of DMSO was added to dissolve the formazan formed at the bottom of the well, followed by ELISA reader (Model 680, BioRad, USA). The absorbance at 540 nm was measured using). At this time, the relative cell growth inhibition rate when the normal group of cells cultured without the peanut sprout extract of the present invention was 100%, was compared with the control group treated with glutamate only. The measurement results are shown in FIG. 2.
도 2에 나타낸 것과 같이, 글루타메이트(glutamate)로 유도된 세포독성으로부터 본 발명의 땅콩나물 추출물의 처리에 따른 신경세포 보호 효과를 관찰하였다. 상기 땅콩나물 추출물을 처리하지 않고 글루타메이트(glutamate)만 처리한 대조군의 세포 생존율은 29.8 %로 나타난 반면, 세포독성이 유도된 세포에 각각의 땅콩나물 추출물을 농도별로 각 세포주에 처리한 경우, 세포 생존율이 WME(도 2의 (A))의 경우 37.2, 39.3, 47.7 %, HME(도 2의 (B))의 경우 38.3, 48.1, 62.9 % 였으며, SME(도 2의 (C))의 경우 38.9, 42.2, 43.2 %로 모두 농도 의존적으로 생존율이 증가함을 보였다. 특히, HME를 100 μg/ml의 농도로 처리하는 경우 세포 생존율이 62.9 %로 가장 높은 신경세포 보호 효과를 확인할 수 있었다.
As shown in Figure 2, the effect of neuronal cell protection according to the treatment of peanut sprout extract of the present invention from glutamate-induced cytotoxicity was observed. The cell survival rate of the control group treated with glutamate alone without the peanut sprouts extract was 29.8%, while the cell viability was obtained when each peanut sprout extract was treated in each cell line by concentration in cytotoxicly induced cells. 37.2, 39.3, 47.7% for this WME (FIG. 2A), 38.3, 48.1, 62.9% for HME (FIG. 2B), and 38.9 for SME (FIG. 2C). , 42.2 and 43.2% showed increased survival rate. In particular, when HME is treated at a concentration of 100 μg / ml, the cell survival rate was 62.9%, confirming the highest neuronal protective effect.
실시예 5. 땅콩나물 추출물(HME) 분획물의 특성 분석Example 5 Characterization of Peanut Sprout Extract (HME) Fraction
상기 실시예 2에서 메탄올 추출 후 농축하여 얻은 HME 추출물을 용매의 극성을 달리하여 얻은 HME의 용매(헥산(hexane), 디에틸 에테르(diethyl ether), 에틸 아세테이트(ethyl acetate), 물(water)) 분획물들이 신경세포에 미치는 영향을 알아보기 위해, N18-RE-105 세포주에 각각의 분획물을 농도별(10, 50, 100 μg/ml)로 처리하여 실시예 4와 동일한 방법으로 MTT reduction assay를 수행하였다. 측정 결과는 도 3에 나타내었다.HME extract obtained by concentrating after extracting methanol in Example 2 solvent of hexane (hexane, diethyl ether, ethyl acetate, water) obtained by varying the polarity of the solvent In order to examine the effect of fractions on neurons, MTT reduction assay was performed in the same manner as in Example 4 by treating each fraction in concentrations (10, 50, 100 μg / ml) in the N18-RE-105 cell line. It was. The measurement results are shown in FIG. 3.
도 3에 나타낸 것과 같이, N18-RE-105 세포주에 각각의 용매 분획물을 농도별(10, 50, 100 μg/ml)로 처리한 경우 디에틸 에테르(도 3의 (B)) 분획물이 40.6, 46.5, 52.5 %로 가장 높은 활성을 보였다. 다른 용매 분획물(헥산(도 3의 (A))의 경우 40.9, 41.1, 46.4 %, 에틸 아세테이트(도 3의 (C))의 경우 38.8, 38.0, 42.1 %, 물(도 3의 (D))의 경우 38.1, 37.8, 44.4 %)에서도 활성을 보이긴 했으나, 디에틸 에테르(diethyl ether) 분획물과 비교하는 경우 활성이 약한 것을 확인할 수 있었다. 또한 활성은 농도 의존적으로 증가하였다. 이러한 결과로 신경세포를 보호하는 본 발명의 땅콩나물 추출물의 특성은 대체로 비극성의 성질을 가지는 것으로 사료된다.
As shown in FIG. 3, when the solvent fractions of the N18-RE-105 cell lines were treated at different concentrations (10, 50, 100 μg / ml), the fraction of diethyl ether ((B) of FIG. 3) was 40.6, 46.5 and 52.5% showed the highest activity. 40.9, 41.1, 46.4% for other solvent fractions (hexane (FIG. 3)), 38.8, 38.0, 42.1% for ethyl acetate (FIG. 3C), water (FIG. 3D) In the case of 38.1, 37.8, 44.4%) was also active, but when compared to the diethyl ether fraction it was confirmed that the activity is weak. In addition, activity increased concentration-dependently. As a result, the properties of the peanut sprout extract of the present invention, which protects nerve cells, are considered to have generally nonpolar properties.
실시예 6. 땅콩나물 머리부분 추출물(HME)로부터 유효성분 분리 및 정제Example 6 Separation and Purification of Active Ingredients from Peanut Sprout Head Extract (HME)
상기 실시예 2에서 메탄올 추출 후 농축하여 얻은 HME 추출물로부터 극성 및 비극성 용매 조건에 따라 헥산(hexane), 디에틸 에테르(diethyl ether), 에틸 아세테이트(ethyl acetate), 물(water) 순으로 용매 분획하여 얻은 각각의 분획물의 활성을 알아보기 위해, 각각 농도를 달리하여 대표적인 세포생존률을 측정하는 방법인 MTT reduction assay를 실시한 결과, HME 디에틸 에테르(diethyl ether) 분획물에서 가장 높은 활성을 확인할 수 있었다 (실시예 5).Solvent fractionation in the order of hexane, diethyl ether, ethyl acetate, water in accordance with the polar and non-polar solvent conditions from the HME extract obtained by concentration after methanol extraction in Example 2 In order to determine the activity of each of the obtained fractions, the MTT reduction assay, a method of measuring the typical cell viability at different concentrations, was performed. As a result, the highest activity was confirmed in the HME diethyl ether fraction. Example 5).
따라서 가장 높은 활성을 보였던 HME 디에틸 에테르(diethyl ether) 분획물을 실리카 겔(silica gel) TLC를 이용하여 상기 분획물에 대한 특성을 규명하였으며, 실리카 겔 컬럼 크로마토그래피(silica gel column chromatography)를 이용하여 활성물질을 분리 및 정제하였다. 이후 분리된 물질이 단일물질인지 확인하기 위해 HPLC로 확인하였다 (HPLC 조건: Column; HAISIL 100Å C18 5μm (250×4.6 mm), Detection; UV absorption at 210 nm, Eluent; 70% EtOH, Flow rate; 1 ml/min). 활성물질의 분리 및 정제 과정의 모식도는 도 4에 나타내었다.Therefore, the most active HME diethyl ether fractions were characterized by silica gel TLC, and the fractions were characterized, and silica gel column chromatography was used for the activity. The material was separated and purified. It was then confirmed by HPLC to confirm that the separated material was a single material (HPLC condition: Column; HAISIL 100IL C18 5μm (250 × 4.6 mm), Detection; UV absorption at 210 nm, Eluent; 70% EtOH, Flow rate; 1 ml / min). A schematic diagram of the separation and purification of the active material is shown in FIG. 4.
HPLC 결과는 도 5에 나타내었으며, 210 nm에서 70 %의 메탄올로 이동상을 흘렸을 때 약 5 분경에 활성물질의 peak를 확인하였다. 이 물질을 PSE-1이라고 명명하였다.
HPLC results are shown in Figure 5, the peak of the active material was confirmed in about 5 minutes when flowing the mobile phase with 70% methanol at 210 nm. This material was named PSE-1.
이후, 단일물질로 확인된 활성물질 PSE-1의 구조 분석을 수행하였다.Thereafter, structural analysis of the active substance PSE-1 identified as a single substance was performed.
먼저, PSE-1의 분자량 및 분자식 결정을 위해 HRESI-Mass(Beckman Coulter, USA) 분석을 수행하였다. 그 결과, 도 6에 나타낸 바와 같이 positive mode[M+Na]+ 에서 335의 분자이온의 peak가 나타났으며, negative mode[M-Na]- 에서 311의 분자이온 peak가 검출되었다. 따라서 상기의 질량 스펙트럼(Mass spectrum) 결과로부터 PSE-1의 분자량은 312.23, 분자식은 C18H32O4로 결정되었다.First, HRESI-Mass (Beckman Coulter, USA) analysis was performed to determine molecular weight and molecular formula of PSE-1. As a result, as shown in FIG. 6, a peak of 335 molecular ions was observed in the positive mode [M + Na] + , and a peak of 311 was detected in the negative mode [M-Na] − . Therefore, the molecular weight of PSE-1 was determined to be 312.23, and the molecular formula of C 18 H 32 O 4 was obtained from the mass spectrum.
그리고 PES-1의 자외선 흡수 스펙트럼(Beckman Coulter, USA)을 분석한 결과, 도 7에 나타낸 바와 같이 230 nm(aqueous acetonitrile)에서 최대 흡수파장을 보이는 것을 확인할 수 있다.As a result of analyzing the ultraviolet absorption spectrum (Beckman Coulter, USA) of PES-1, it can be seen that the maximum absorption wavelength at 230 nm (aqueous acetonitrile) as shown in FIG.
다음으로 PSE-1의 화학구조 규명을 위해 1H-NMR(Varian, USA) 및 13C-NMR(Varian, USA)로 구조를 분석하였다. 그 결과, 1H-NMR에서는 olefin proton, methylene proton 및 methyl proton이 검출 되었다 (도 8의 (A)). 따라서 긴 aliphatic series가 있을 것이라 예측할 수 있다. 또한 13C-NMR에서도 1H-NMR의 결과와 마찬가지로 olefin carbon, methylene carbon, methyl carbon 및 ester가 검출 되었다 (도 8의 (B)). 상기의 결과들은 1H-NMR과 13C-NMR이 개연성이 있다는 것을 의미하는 스펙트럼이며, PSE-1의 구조가 지방산 유도체라는 것을 알 수 있다. 상기 NMR 결과는 하기 표 1에 자세히 나타내었다.
Next, the structure of PSE-1 was analyzed by 1 H-NMR (Varian, USA) and 13 C-NMR (Varian, USA). As a result, olefin protons, methylene protons and methyl protons were detected in 1 H-NMR (Fig. 8 (A)). So we can expect to have a long aliphatic series. In addition, olefin carbon, methylene carbon, methyl carbon and ester were detected in 13 C-NMR as well as the result of 1 H-NMR (FIG. 8 (B)). The above results are spectra indicating that 1 H-NMR and 13 C-NMR are probabilistic, and it can be seen that the structure of PSE-1 is a fatty acid derivative. The NMR results are shown in detail in Table 1 below.
[표 1. 1H-NMR, 13C-NMR 측정 결과]Table 1. 1 H-NMR, 13 C-NMR measurement results
상기의 스펙트럼 분석들을 통해 구조를 동정해본 결과, PSE-1은 8,13-디하이드록시-9,11-옥타데카디에노익 애시드(8,13-dihydroxy-9,11-octadecadienoic acid)로 동정되었다. 상기 물질의 구조는 도 9에 나타내었다.
As a result of identifying the structure through the above spectral analysis, PSE-1 was identified as 8,13-dihydroxy-9,11-octadecadienoic acid (8,13-dihydroxy-9,11-octadecadienoic acid). . The structure of the material is shown in FIG. 9.
실시예Example 7. 7. LDHLDH releaserelease assayassay 및 세포의 형태분석 And morphology analysis of cells
LDH(lactate dehydrogenase) release assay는 세포 괴사율을 측정할 수 있는 colorimetric 방법으로, 세포배양시 괴사된 세포는 LDH를 배지로 방출하는데 이때 방출된 LDH에 colorless tetrazolium salt를 넣으면 환원된 형태의 colored formazan을 형성한다.The lactate dehydrogenase (LDH) release assay is a colorimetric method for measuring cell necrosis rate. Cell cultures release necrotic cells into the medium when the cells are cultured. Form.
본 발명의 LDH 방출 특성은 상기 실시예 5에서 신경세포 보호에 가장 활성이 좋은 HME 디에틸 에테르(diethyl ether) 분획물 처리에 따른 LDH 방출 특성을 분석하였으며, 이때 LDH release assay kit를 사용하여 LDH 방출을 측정하였다. 먼저, 세포주를 1×105 cells/ml로 맞춘 후, 100 μl씩 96-웰 플레이트(96-well plate)에 분주하여 CO2 인큐베이터(incubator)에서 24 시간 동안 배양하였다. 이후, HME 디에틸 에테르(diethyl ether) 분획물을 10, 50, 100 μg/ml의 농도로 세포주에 처리하여 30 분 동안 배양한 후, 다시 20 mM 글루타메이트(glutamate)를 처리하여 24 시간 동안 배양하였다. 배양이 완료되면, 배양액을 새로운 96-웰 플레이트에 50 μl 분주하고, LDH reagent를 50 μl를 첨가하여 반응시킨 후, 반응이 완료되면 1 N HCl을 100 μl 첨가하여 반응을 중지시킨다. 그 후, 살아남은 세포의 LDH 측정을 위해 남은 배양액을 제거하고, 0.5 % Triton X-100 용액을 50 μl 첨가하여 40 rpm으로 10 분 동안 shaking 시키고, 같은 방법으로 LDH reagent를 첨가하여 반응시킨다. 반응이 완료되면 반응 정지액을 넣은 뒤, 각각을 540 nm에서 흡광도를 측정하였다. LDH에 의한 세포독성의 백분율은 배양액과 살아있는 세포에서 유리된 총 LDH에 대한 배양액으로부터 유리된 LDH의 값으로 계산하여 무처리 군과 비교한 값을 나타내었다. 측정 결과는 도 10a에 나타내었다.LDH release characteristics of the present invention was analyzed the LDH release characteristics according to the treatment of HME diethyl ether fractions most active in neuronal cell protection in Example 5, wherein the LDH release assay kit using LDH release assay kit Measured. First, the cell lines were adjusted to 1 × 10 5 cells / ml, and 100 μl of the cells were dispensed into 96-well plates and incubated for 24 hours in a CO 2 incubator. Subsequently, HME diethyl ether fractions were treated with cell lines at concentrations of 10, 50, 100 μg / ml, incubated for 30 minutes, and then incubated for 24 hours with 20 mM glutamate. When the incubation is completed, 50 μl of the culture solution is added to a new 96-well plate, and 50 μl of LDH reagent is added to the reaction, and when the reaction is completed, the reaction is stopped by adding 100 μl of 1 N HCl. Afterwards, the remaining culture solution is removed for LDH measurement of surviving cells, 50 μl of 0.5% Triton X-100 solution is added and shaken at 40 rpm for 10 minutes, and the reaction is performed by adding LDH reagent in the same manner. After the reaction was completed, the reaction stopper was added, and absorbance was measured at 540 nm. The percentage of cytotoxicity by LDH was calculated as the value of LDH released from the culture to the total LDH released from the culture and living cells compared to the untreated group. The measurement results are shown in FIG. 10A.
도 10a에 나타낸 것과 같이, 글루타메이트(glutamate)만 처리한 대조군의 경우 정상군에 비해 LDH의 방출 양이 69.1 % 정도인 반면, 본 발명의 땅콩나물 추출물(HME 디에틸 에테르 분획물)을 처리하는 경우 처리 농도(10, 50, 100 μg/ml)에 따라 51.0, 48.1, 47.3 %로 농도 의존적으로 LDH의 방출이 감소하였다. 이러한 결과는 본 발명의 땅콩나물 머리(head)부분 추출물(HME)이 글루타메이트(glutamate)에 의해 유발된 신경세포의 손상을 강력하게 억제하는 것을 알 수 있다.
As shown in Figure 10a, the control group treated only with glutamate (glutamate) is about 69.1% of the release amount of LDH compared to the normal group, while treating the peanut sprout extract (HME diethyl ether fraction) of the present invention The concentration-dependent release of LDH decreased to 51.0, 48.1 and 47.3% depending on the concentration (10, 50, 100 μg / ml). These results suggest that the peanut head extract (HME) of the present invention strongly inhibits the damage of nerve cells caused by glutamate.
다음으로 HME가 신경세포(N18-RE-105 세포주)의 형태학적 변화에 미치는 영향을 알아보기 위해, 먼저 6-웰 플레이트(6-well plate)에 2×105 cells/ml로 2 ml씩 첨가하여 24 시간 동안 배양한 후, 본 발명의 HME 디에틸 에테르(diethyl ether) 분획물을 50, 100 μg/ml의 농도로 처리하였다. 이후, 20 mM의 글루타메이트(glutamate)에 24 시간 노출시킨 후, 광학현미경(phase-contrast microscope, Nikon, Japan)을 이용하여 세포의 형태변화를 관찰하였다. 관찰 결과는 도 10b에 나타내었다.Next, to determine the effect of HME on the morphological changes of neurons (N18-RE-105 cell line), first add 2 ml of 2 × 10 5 cells / ml to a 6-well plate. After culturing for 24 hours, the HME diethyl ether fraction of the present invention was treated at a concentration of 50, 100 μg / ml. Then, after 24 hours exposure to 20 mM glutamate (glutamate), the morphological changes of the cells were observed using a light microscope (phase-contrast microscope, Nikon, Japan). The observation result is shown in FIG. 10B.
도 10b에 보이는 것과 같이, 아무것도 처리하지 않은 정상군(ⓐ)에서는 건강하게 뻗은 신경돌기의 모습을 관찰할 수 있지만, 글루타메이트(glutamate)를 처리한 대조군(ⓑ)은 산화적 스트레스로 인하여 신경돌기가 거의 소멸된 양상을 보임으로써 신경세포의 형태학적으로 큰 변화가 유도된 것을 확인할 수 있다. 그러나 본 발명의 땅콩나물 추출물(HME 디에틸 에테르 분획물)을 농도(50(ⓒ), 100(ⓓ) μg/ml)별로 처리하는 경우, 농도 의존적으로 신경세포 손상정도가 거의 회복되어 있음을 확인할 수 있다. 따라서 본 발명의 땅콩나물 머리(head)부분 추출물(HME)은 글루타메이트(glutamate)에 의한 신경세포의 손상을 억제 또는 보호하는 효과가 있는 것으로 사료된다.
As shown in Figure 10b, in the normal group (ⓐ) that did not process anything, you can observe the appearance of healthy neurites, but the control group (ⓑ) treated with glutamate (glutamate) is due to oxidative stress The morphological changes of neurons were induced by showing almost disappearing patterns. However, when the peanut sprout extract (HME diethyl ether fraction) of the present invention was treated for each concentration (50 (ⓒ), 100 (ⓓ) μg / ml), the degree of nerve cell damage was almost recovered in a concentration-dependent manner. have. Therefore, the peanut head extract (HME) of the present invention is considered to have an effect of inhibiting or protecting the damage of nerve cells by glutamate.
실시예 8. Flow cytometric analysisExample 8 Flow cytometric analysis
본 발명의 땅콩나물 추출물(HME)의 아팝토시스(apoptosis) 억제 효과를 알아보기 위해, 신경세포(N18-RE-105 세포주)에 HME 디에틸 에테르(diethyl ether) 분획물을 처리하여 flow cytometer로 apoptotic cell을 나타내는 sub-G1기의 세포수를 측정하였다.In order to examine the apoptosis (apoptosis) inhibitory effect of the peanut sprouts extract (HME) of the present invention, HME diethyl ether fractions were treated to neurons (N18-RE-105 cell line) by apoptotic flow cytometer. The cell number of the sub-G1 phase representing the cell was measured.
먼저, N18-RE-105 세포주를 6 cm 디쉬(dish)에 5×104 cell/dish로 세포를 분주하여 24 시간 동안 배양한 후, 본 발명의 땅콩나물 추출물(HME)을 50, 100 μg/ml의 농도로 처리하였다. 이후, 세포를 24 시간 동안 배양시키고, 부유 세포 및 트립신(trypsin)을 처리한 세포들을 모아서 4 ℃에서 2,000 rpm으로 5 분간 원심분리 하였다. 원심분리 완료 후, 에탄올 1 ml을 넣고, 4 ℃에서 세포를 고정시켰다. 다시 4 ℃에서 2,000 rpm으로 2 분간 원심분리하고, PBS 용액으로 수세한 후, propidium iodide/RNase staining buffer(BD pharmingenTM)를 500 μg/ml 첨가하고, 빛을 차단한 상태로 상온에서 30 분간 염색(staining)시킨 후, flow cytometry caliber(Beckman coulter epics XL)를 이용하여 세포주기를 분석하였다. 분석 결과는 도 11에 나타내었다.First, the N18-RE-105 cell line was cultured for 24 hours by dispensing cells at 5 × 10 4 cells / dish in a 6 cm dish, and then 50, 100 μg / of peanut sprout extract (HME) of the present invention. Treated at a concentration of ml. Thereafter, the cells were incubated for 24 hours, suspended cells and trypsin-treated cells were collected and centrifuged at 2,000 rpm for 5 minutes at 4 ° C. After completion of centrifugation, 1 ml of ethanol was added and the cells were fixed at 4 ° C. Centrifuged again at 2,000 rpm for 2 minutes at 4 ° C, washed with PBS solution, 500 μg / ml of propidium iodide / RNase staining buffer (BD pharmingen TM ) was added, and stained for 30 minutes at room temperature while blocking light. After staining, the cell cycle was analyzed using flow cytometry caliber (Beckman coulter epics XL). The analysis results are shown in FIG. 11.
도 11에 나타낸 것과 같이, 글루타메이트(glutamate)만을 처리한 대조군의 sub-G1기의 apoptotic cell은 58.5 %로 나타나는 반면, 글루타메이트(glutamate)로 인해 세포독성이 유도된 세포에 본 발명의 HME 디에틸 에테르(diethyl ether) 분획물을 농도별로 처리하는 경우 apoptotic cell이 각각 23.1, 9.1 %로 나타났다. 이러한 결과는 땅콩나물 머리(head)부분의 추출물(HME)이 글루타메이트(glutamate)에 의해 유도되는 아팝토시스(apoptosis)를 억제하여 신경세포를 보호하는 효과를 나타내는 것을 의미한다.
As shown in FIG. 11, the apoptotic cells of the sub-G1 phase of the control group treated with glutamate were shown as 58.5%, whereas the HME diethyl ether of the present invention was expressed in cells induced by cytotoxicity due to glutamate. Treatment of (diethyl ether) fractions by concentration showed 23.1 and 9.1% apoptotic cells, respectively. These results indicate that the extract of the head of peanut sprouts (HME) exhibits an effect of protecting neurons by inhibiting apoptosis induced by glutamate.
이하, 제제예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 본 발명의 땅콩나물 추출물을 함유하는 조성물은 아래와 같은 제형으로 제조될 수 있으며, 아래의 제제예는 본 발명을 예시하는 것일 뿐, 이에 의해 본 발명의 내용이 제한되는 것은 아니다.
Hereinafter, the present invention will be described in more detail with reference to formulation examples. The composition containing the peanut sprouts extract of the present invention can be prepared in the following formulation, the following formulation example is only to illustrate the present invention, whereby the content of the present invention is not limited.
제제예Formulation example 1. 정제의 제조 1. Preparation of tablets
본 발명의 땅콩나물 추출물 200 mg, 유당 100 mg, 전분 100 mg, 스테아린산 마그네슘 적량을 통상의 제조방법에 따라 상기의 성분을 혼합하고 타정하여 정제를 제조한다.
Peanut sprout extract 200 mg of the present invention,
제제예Formulation example 2. 2. 액제의Liquid 제조 Produce
본 발명의 땅콩나물 추출물 1000 mg, 설탕 20 g, 이성화당 20 g, 레몬향 적량을 혼합한 후, 정제수를 가하여 전체 1000 ml로 맞춘다. 이후, 통상의 제조방법에 따라 상기의 성분을 혼합한 다음, 갈색병에 충전하고 멸균시켜 액제를 제조한다.
제제예Formulation example 3. 캅셀제의 제조 3. Preparation of capsules
본 발명의 땅콩나물 추출물 300 mg, 결정성 셀룰로오스 3 mg, 락토오스 14.8 mg, 마그네슘 스테아레이트 0.2 mg을 통상의 캅셀제 제조방법에 따라 상기의 성분을 혼합하고 젤라틴 캅셀에 충전하여 캅셀제를 제조한다.
300 mg of peanut sprout extract of the present invention, 3 mg of crystalline cellulose, 14.8 mg of lactose, and 0.2 mg of magnesium stearate are mixed according to a conventional capsule preparation method and filled into gelatin capsules to prepare a capsule.
Claims (9)
A composition for treating and preventing neurodegenerative diseases comprising 8,13-dihydroxy-9,11-octadecadienoic acid (8,13-dihydroxy-9,11-octadecadienoic acid) as an active ingredient.
The neurodegenerative disease according to claim 5, wherein the 8,13-dihydroxy-9,11-octadecadienoic acid is extracted from peanut sprouts. Therapeutic and prophylactic compositions.
The composition for treating and preventing degenerative neurological diseases according to claim 5, wherein the treatment and prevention of the neurodegenerative diseases are performed by inhibiting apoptosis of neurons.
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| KR101403964B1 (en) * | 2012-08-29 | 2014-06-13 | 농업회사법인(주)장수채 | Extract method of peanut sprout increased resveratrol content |
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