KR101583399B1 - -- Pharmaceutical composition comprising asarinin or --sesamine for the treatment or prevention of Parkinsons disease - Google Patents
-- Pharmaceutical composition comprising asarinin or --sesamine for the treatment or prevention of Parkinsons disease Download PDFInfo
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- KR101583399B1 KR101583399B1 KR1020130125910A KR20130125910A KR101583399B1 KR 101583399 B1 KR101583399 B1 KR 101583399B1 KR 1020130125910 A KR1020130125910 A KR 1020130125910A KR 20130125910 A KR20130125910 A KR 20130125910A KR 101583399 B1 KR101583399 B1 KR 101583399B1
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- KR
- South Korea
- Prior art keywords
- sesamin
- ohda
- dopamine
- asarinin
- induced
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Abstract
본 발명은 아사리닌(asarinin) 또는 (-)-세사민((-)-sesamin)의 신규한 의약적 용도로서 파킨슨병의 예방 또는 치료에 관한 것이다.
본 발명에 의한 아사리닌 또는(-)- 세사민은 도파민 생합성 증가작용 및 6-OHDA-유도 세포독성 작용에 대한 방어작용을 나타내며, PC12 세포중의 PKA-CREB 활성화에 의하여 도파민 생합성 효소인 티로신 하이드록시라제(tyrosine hydroxylase, TH) 활성이 유도되어 도파민 생합성 증가작용을 나타내고, p38MAPK-JNK 신호전이 및 ERK-Bad-BAX 신호전이를 매개한 caspase-3 활성 저해작용을 유도하여 6-OHDA-유도 세포독성 방어작용을 나타냄으로서 도파민 신경계의 보호작용과 관련하여 파킨슨병(Parkinson's disease)과 같은 신경퇴행성 질환 치료제의 개발에 유용하게 이용될 수 있다.The present invention relates to the prophylactic or therapeutic treatment of Parkinson's disease as a novel medical use of asarinin or (-) - sesamin.
The asarinine or (-) - sesamin according to the present invention shows a protective action against dopamine biosynthesis-enhancing action and 6-OHDA-induced cytotoxic action, and by PKA-CREB activation in PC12 cells, the dopamine biosynthesis enzyme tyrosine hydroxyl Induced 6-OHDA-induced cytotoxicity by induction of tyrosine hydroxylase (TH) activity and induction of dopamine biosynthesis, caspase-3 activity mediated by p38 MAPK-JNK signal transduction and ERK-Bad- And may be useful for the development of therapeutic agents for neurodegenerative diseases such as Parkinson ' s disease in relation to the protective action of the dopamine nervous system by showing defensive action.
Description
본 발명은 리그난 화합물의 일종인 아사리닌 또는 (-)-세사민의 신규의 의약적 용도인 파킨슨병 치료 및 예방용 약학조성물에 관한 것이다.
The present invention relates to a pharmaceutical composition for the treatment and prevention of Parkinson's disease, which is a novel medicinal use of lignan compound, asaryin or (-) - sesamin.
파킨슨병은 대뇌 기저핵(basal ganglia)내 흑질(substantia nigra, pars compacta)의 도파민 신경세포(dopaminergic neuron)가 서서히 퇴행성 변화를 일으켜 선조체(corpus striatum)의 신경전달 물질인 도파민의 결핍으로 생겨나며, 선조체 전체의 70-80% 이상이 소실되면 진전(tremer), 서동(bradykinesia), 경직(rigidity) 등의 특징적인 운동장애를 나타내는 질환이다. 도파민 신경계의 도파민의 결핍은 신경세포사에 의한 도파민 생합성 효소 티로신 하이드록시라제(tyrosine hydroxylase, TH), 방향족 L-아미노산 디카복실라제(aromatic L-amino acid decarboxylase, AADC)의 활성 감소가 원인으로 알려져 있다.
Parkinson's disease is caused by the degeneration of dopaminergic neurons in the substantia nigra (pars compacta) of the basal ganglia, which is a neurotransmitter of the corpus striatum causing a gradual degenerative change, (70-80%) of the total weight loss is a disease characterized by tremor, bradykinesia, and rigidity. Dopamine deficiency in the dopaminergic system is known to be caused by a decrease in activity of dopamine biosynthesis enzymes tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) by neuronal cell death .
파킨슨병의 병인론에 관해서는 유전적인 요인, 산화 스트레스(oxidative stress), 유전적 민감성, 세포의 괴사 등을 포함한 다양한 내적, 외적발병 원인들이 거론되고 있으나 아직 정확한 기전은 밝혀져 있지 않다(Neil et al., Medicine, 36, 630-635, 2008; Yacoubian and Standaert, Biochim. Biophys. Acta, 1792, 676-687, 2009; Chen et al., Parkinson's disease, pp. 1033-1044, in Pharmacotherapy 8th Ed,, Eds., DiPiro et al., 2008). 파킨슨병에서 나타나는 흑질의 도파민 신경세포는 질병이 진행되는 기간에 비례하여 계속 소실되는 것으로 알려져 있으며(Chen et al., Parkinson's disease, pp. 1033-1044, in Pharmacotherapy 8th Ed,, Eds., DiPiro et al., 2008), 현재까지 이러한 지속적인 신경세포의 손실을 막을 수 있는 뚜렷한 방법은 알려져 있지 않다. 도파민 신경세포가 소실되고 이로 인한 선조체의 도파민 결핍이 임상증상을 초래하므로 신경세포의 소실을 방지 또는 지연시키는 신경세포 보호 작용에 관한 연구, 도파민 신경계의 활성화 및 효능 증가를 유도하는 치료제의 개발에 관한 연구가 활발하게 진행되고 있다(Yacoubian and Standaert, Biochim. Biophys. Acta, 1792, 676-687, 2009; Chen et al., Parkinson's disease, pp. 1033-1044, in Pharmacotherapy 8th Ed,, Eds., DiPiro et al., 2008). 최근에는 줄기세포를 이용한 동물모델에 대한 연구가 진행되고 있으나, 실용화 단계에는 이르지 못하고 있다.
The pathogenesis of Parkinson's disease has been addressed by various internal and external causes including genetic factors, oxidative stress, genetic susceptibility, and cell necrosis, but no precise mechanisms have been elucidated (Neil et al. Et al., Parkinson's disease, pp. 1033-1044, in Pharmacotherapy 8th Ed, Eds, < RTI ID = 0.0 > DiPiro et al., 2008). It is known that dopamine neurons in the black color appearing in Parkinson's disease continue to disappear in proportion to the duration of disease progression (Chen et al., Parkinson's disease, pp. 1033-1044, in Pharmacotherapy 8th Ed, Eds., DiPiro et al., 2008), so far there is no known method for preventing such sustained neuronal loss. Studies on the neuronal cell protection function preventing or delaying the disappearance of neuronal cells due to the disappearance of dopaminergic neurons and the resulting dopaminergic depression of the resulting striatum, and development of a therapeutic agent for inducing activation and potency of dopaminergic system (Yacoubian and Standaert, Biochim. Biophys. Acta, 1792, 676-687, 2009; Chen et al., Parkinson's disease, pp. 1033-1044, in Pharmacotherapy 8th Ed, Eds., DiPiro et al., 2008). In recent years, studies on animal models using stem cells have been carried out, but they have not reached the practical stage.
파킨슨병의 치료법으로는 약물요법, 수술요법, 운동요법 등이 이용되고 있으며, 현재 가장 널리 쓰이고 있는 약물은 L-DOPA(Levodopa, 레보도파) 요법이며, L-DOPA 요법은 투여된 L-DOPA가 혈액-뇌 관문(blood-brain barrier)을 통과하여 뇌중에서 도파민으로 전환되어 작용하는 것으로서 파킨슨병의 증상(운동장애 등)을 호전시키는 좋은 효과가 있으나, 도파민은 혈액-뇌 관문을 통과할 수 없기 때문에 L-DOPA 요법은 흑질의 도파민 신경세포의 변성을 막는 데에는 아무런 효과가 없어 완치를 위한 치료제로 이용될 수 없다(Chen et al., Parkinson's disease, pp. 1033-1044, in Pharmacotherapy 8th Ed,, Eds., DiPiro et al., 2008).
The most widely used drug is L-DOPA (Levodopa), and L-DOPA therapy is a treatment for Parkinson's disease. - It passes through the blood-brain barrier and is converted into dopamine in the brain. It has a good effect of alleviating the symptoms of Parkinson's disease (movement disorder, etc.), but since dopamine can not pass through the blood-brain barrier L-DOPA therapy has no effect in blocking the denaturation of dopamine dopamine neurons in the substantia nigra and can not be used as a therapeutic for cures (Chen et al., Parkinson's disease, pp. 1033-1044, in Pharmacotherapy 8th Ed, Eds DiPiro et al., 2008).
L-DOPA 장기 투여는 L-DOPA 치료에 대한 효능이 점점 없어지게 되고 증상은 악화되는 경과를 나타내며(Chen et al., Parkinson's disease, pp. 1033-1044, in Pharmacotherapy 8th Ed,, Eds., DiPiro et al., 2008), 이는 장기간의 L-DOPA 투여에 의하여 생성된 활성산소(reactive oxygen species, ROS)에 의한 산화스트레스-유도 세포독성(dopamine neuronal cell death, apoptosis; 신경세포사)을 일으켜 증상이 악화되기 때문으로 보고되고 있다(Basma et al., J. Neurochem., 64, 825-832, 1995). L-DOPA 요법은 L-DOPA의 투여용량 및 기간 등의 복약지도, 증상의 모니터링 등이 이루어져야 한다. L-DOPA 요법시에는 L-DOPA의 도파민으로의 전환을 방지하기 위하여 카비도파(carbidopa), 벤세라지드(benserazide) 등 AADC 저해제와의 병용요법이 시행되고 있는 실정이다.
Long-term administration of L-DOPA indicates progressive loss of efficacy for L-DOPA treatment and worsening of symptoms (Chen et al., Parkinson's disease, pp. 1033-1044, in Pharmacotherapy 8th Ed, Eds., DiPiro (ROS) induced by oxidative stress-induced cytotoxicity (dopamine neuronal cell death, apoptosis) caused by long-term administration of L-DOPA. (Basma et al., J. Neurochem., 64, 825-832, 1995). L-DOPA therapy should be monitored, such as dose and duration of administration of L-DOPA, and symptom monitoring. In L-DOPA therapy, combination therapy with AADC inhibitors such as carbidopa and benserazide has been performed to prevent the conversion of L-DOPA to dopamine.
따라서 파킨슨병에 대하여, L-DOPA 요법을 보완하거나 대체할 수 있는 새로운 치료제-도파민 생합성 유도 작용, L-DOPA 요법의 부작용 및 신경세포사의 개선 작용의 개발이 필요하며 이와 관련한 연구가 활발히 진행되고 있다.
Therefore, it is necessary to develop a new therapeutic agent for Parkinson's disease that can supplement or replace L-DOPA therapy - induction of dopamine biosynthesis, adverse effects of L-DOPA treatment, and improvement of neuronal cell death, and studies related thereto have been actively conducted .
파킨슨병의 치료와 관련해 L-DOPA 요법을 대체할 수 있는 새로운 치료제로서 주목받는 분야는 생약 추출물 및 조성물 분야이다. 대한민국 특허등록 제1068561호에는 계지, 지모 및 양강의 생약 혼합 추출물의 파킨슨병의 치료 효능이 있는 것으로 보고되어 있으며, 대한민국 특허등록 제1130030호에는 천마추출물을 유효성분으로 하는 조성물의 효능에 대해서 개시되어 있고, 대한민국 특허등록 제1118371호에는 로가닌 및 그 염의 효능으로 파킨슨병의 치료용도가 개시되어 있으나, 아직까지 실용화 단계에는 이르지 못하고 있다.
The field of herbal medicine extracts and compositions has attracted attention as a new therapeutic agent that can replace L-DOPA therapy in the treatment of Parkinson's disease. Korean Patent Registration No. 1068561 reports the therapeutic effect of Parkinson's disease of herbal medicine mixed extracts of Ganoderma lucidum, Gamma and Yang River. Korean Patent Registration No. 1130030 discloses the efficacy of a composition containing an extract of Ganoderma lucidum as an active ingredient Korean Patent Registration No. 1118371 discloses a therapeutic use of Parkinson's disease by the efficacy of roganin and its salts, but has not reached the practical stage yet.
한편, PC12 세포주는 쥐의 부신 크롬성 세포종(rat adrenal pheochromocytoma)에서 유래한 것으로, 도파민 생합성(dopamine biosynthesis) 및 유리(release), 세포독성(neurotoxicity), 세포 증식/분화(proliferation, differentiation/neurite-like formation) 등의 연구모델로 응용되고 있으며, in vitro 파킨슨병 모델로 응용되고 있다(Tischler et al., J. Neurochem., 40, 364-370, 1983). 상기의 연구는 PC12 세포주 이외에 사람의 신경모세포종(human neuroblastoma) 세포, MN9D 세포, 쥐의 1차 중뇌 세포 배양(rat primary midbrain cell cultures)을 이용하여 수행되고 있다.
On the other hand, the PC12 cell line is derived from rat adrenal pheochromocytoma, and is characterized by dopamine biosynthesis and release, neurotoxicity, proliferation, differentiation / neurite- like formation, and has been applied as an in vitro Parkinson's disease model (Tischler et al., J. Neurochem., 40, 364-370, 1983). The above studies are performed using human neuroblastoma cells, MN9D cells, and rat primary midbrain cell cultures in addition to the PC12 cell line.
도파민 생합성과 관련하여, 카테콜아민(catecholamines)은 도파민, 노르에피네프린(norepinehrine, 노르아드레날린), 에피네프린(epinephrine, 아드레날린)을 말하는데, 카테콜아민의 생합성 경로는 티로신(tyrosine)에서 L-DOPA, 도파민, 노르에피네프린, 에피네프린의 순이다. 각 단계에는 TH, AADC, 도파민 β-하이드록실라제(dopamine β-hydroxylase), 페닐에탄올아민 N-메틸트란스퍼라제(phenylethanolamine N-methyltransferase)의 효소가 관여하고 있다. 또한 도파민의 대사과정에는 모노아민 옥시다아제(monoamine oxidase) 및 카테콜 O-메틸트랜스퍼라제(catechol O-methyltransferase)가 관여하고 있으며, 도파민의 주요 대사산물은 3,4-디하이드록시아세트산(3,4-dihydroxyphenylacetic acid, DOPAC) 및 호모바닐닉산(homovanillic acid, HVA)이 알려져 있다. 도파민 생합성 효소 TH는 cyclic AMP, Ca2+ 농도, PKA, PKC, CREB 등에 의하여 활성화되며 도파민 생합성이 유도되어 세포내 도파민 함량이 증가하게 된다(Joh et al, PNAS, USA, 75, 4744-4748, 1978; Kim et al., J. Biol. Chem., 268, 15688-15695, 1993).
In relation to dopamine biosynthesis, catecholamines refer to dopamine, norepinehrine, and epinephrine. The biosynthetic pathway of catecholamines is the tyrosine, L-DOPA, dopamine, norepinephrine, Epinephrine. At each step, enzymes of TH, AADC, dopamine β-hydroxylase, and phenylethanolamine N-methyltransferase are involved. In addition, the metabolism of dopamine involves monoamine oxidase and catechol O-methyltransferase, and the major metabolites of dopamine are 3,4-dihydroxyacetic acid (3,4 -dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA). The dopamine biosynthesis enzyme, TH, is activated by cyclic AMP, Ca 2+ concentration, PKA, PKC, CREB, and dopamine biosynthesis, leading to increased intracellular dopamine content (Joh et al., PNAS, USA, 75, 4744-4748, Kim et al., J. Biol. Chem., 268, 15688-15695, 1993) .
6-OHDA는 MPTP와 더불어 신경독성 물질로 뇌(brain)로의 투여에 의하여 파킨슨병의 증상을 나타내며, 파킨슨병의 동물 모델(6-OHDA-유도 PD rat/mouse model)로 사용되고 있다(Blum et al., Neurobiol., 65, 135-172, 2001). 6-OHDA는 PC12 세포주, SK-N-BE(2)C human neuroblastoma 세포주, MN9D(dopaminergic cells) 세포주 등에서 세포독성을 나타내고 있다(Choi et al., J. Neurosci. Res., 57, 84-94, 1999; Rodriguez-Blanco et al., J. Neurochem., 107, 127-140, 2008; Lin et al., J. Neurosci. Res., 86, 108-117, 2008).
6-OHDA is a neurotoxic substance in addition to MPTP, which is used as an animal model of Parkinson's disease (6-OHDA-induced PD rat / mouse model) by administration to the brain (Blum et al Neurobiol., 65, 135-172, 2001). 6-OHDA shows cytotoxicity in PC12 cell line, SK-N-BE (2) C human neuroblastoma cell line and MN9D (dopaminergic cells) cell line (Choi et al., J. Neurosci. Res., 57, 84-94 Lin et al., J. Neurosci. Res., 86, 108-117, 2008).
최근 파킨슨병 동물모델(6-OHDA-lesioned rat model)에 대한 장기간 L-DOPA 투여는 6-OHDA의 생성이 유도되며, 이는 중금속(Fe)의 존재하에 촉진되고 있음이 보고되고 있다(Maharaj et al., Brain Res., 1063, 180-186, 2005). 6-OHDA는 산화제)(Fe2+,H2O2)의 존재하에 비효소학적 반응(non-enzymatic reaction)으로 도파민(dopamine)에서 생합성 되며, PC12 세포중에서 6-OHDA로부터 발생되는 퀴논(quinone), 과산화물음이온(superoxide anion)과 같은 ROS를 생성하고, 과산화수소(H2O2)는 세포내(influx)로 유입되어 산화스트레스에 의하여 선택적으로 도파민(dopamine) 신경계 세포사(세포독성)를 나타낸다(Saito et al., Free Rad. Biol. Med. 42, 675-685. 2007).
Recently, long-term administration of L-DOPA to a 6-OHDA-lesioned rat model has been reported to induce the production of 6-OHDA, which is promoted in the presence of heavy metals (Fe) (Maharaj et al Brain Res., 1063, 180-186, 2005). 6-OHDA is biosynthesized in dopamine by a non-enzymatic reaction in the presence of an oxidizing agent (Fe 2+ , H 2 O 2 ) and quinone generated from 6-OHDA in PC12 cells ) And peroxidic anion (superoxide anion), and hydrogen peroxide (H 2 O 2 ) is introduced into influx, which selectively induces dopamine neuronal cell death (cytotoxicity) by oxidative stress Saito et al., Free Rad Biol., 42, 675-685, 2007).
세포독성의 발현과 관련하여, 6-OHDA에 의한 일시적인 ERK(transient ERK; Extracelluar Signal-Regulated Kinases) 활성화는 Bad(Ser112)의 인산화, 지속적인 ERK(sustained ERK) 활성화는 Bad(Ser155) 인산화를 유도한다. 현재까지 Ser112 부위(Bad(Ser112))와 Ser155 부위(Bad(Ser155)) 모두 세포 생존(cell survival)에 관여하고 있음이 보고되고 있었으나, 최근 Bad(Ser155)는 산화스트레스에 의한 세포독성 유도시에 활성화 되고 있음이 보고되고 있다(Jin et al., Neurosci., 170, 390-398, 2010). 그러므로 ERK 활성화 및 Bad(Ser155)의 활성화는 6-OHDA를 비롯한 ROS-유도 인자에 의해 미토콘드리아(mitochondria)에서 세포독성이 유도되고 있음을 제시한다.
Regarding the expression of cytotoxicity, activation of transient ERK (transient ERK) by extracellular signal-regulated kinases (6-OHDA) induces phosphorylation of Bad (Ser112), activation of sustained ERK (sustained ERK) induces Bad (Ser155) phosphorylation . Recently, it has been reported that both Ser112 (Bad (Ser112)) and Ser155 (Bad (Ser155)) are involved in cell survival. Recently, Bad (Ser155) (Jin et al., Neurosci., 170, 390-398, 2010). Therefore, activation of ERK and activation of Bad (Ser155) suggests that cytotoxicity is induced in mitochondria by ROS-inducing factors including 6-OHDA.
그러므로 도파민 생합성 증가작용을 유도하는 생리활성 물질 및 6-OHDA-유도 세포독성을 저해하는 생리활성 물질은 파킨슨병의 예방 및 치료제로 응용될 수 있음을 알 수 있다.
Therefore, physiologically active substances that induce dopamine biosynthesis-enhancing action and physiologically active substances that inhibit 6-OHDA-induced cytotoxicity can be applied as preventive and therapeutic agents for Parkinson's disease.
세신은 족두리풀의 근경으로 다년생 초본이다. 세신 추출물은 methyleugenol, asaryketone, cineol, safrole, limonene, eucarvone 등의 정유화합물, 산 아마이드(acid amide) 화합물, 아사리닌(asarinin), 세사민(sesamin) 등의 리그난(lignan) 화합물, higenamine을 포함한 알카로이드 등을 함유하고 있으며, 체온강하 작용, 진경 작용, 항히스타민 작용, 강심 작용을 나타내어 국소마취, 진통, 해열, 진해, 거담 및 이뇨 작용의 목적으로 응용되고 있다(생약학, pp. 158, 2013, 생약학교재편찬위원회, 서울; Zhou RH, Resource Science of Chinese Medicinal Materials, pp. 202-211, Beijing: China Medical Amp, Pharmaceutical Sciences Press, 1993; Zhu Y, Chinese Materia Medica, Chemistry, Pharmacology and Application, pp. 66-69, Beijing, People's Health Publisher, 1998).
Seeshin is a perennial herb that is a rootstock of the bamboo shoots. The extracts of Seisin include refined compounds such as methyleugenol, asaryketone, cineol, safrole, limonene and eucarvone, acid amide compounds, lignan compounds such as asarinin and sesamin, alkaloids including higenamine And has been used for the purpose of local anesthesia, analgesia, fever, shinhae, genomic and diuretic action because it exhibits body temperature lowering action, antipyretic action, antihistaminic action, and cardiac action (Genetics, pp. 158, 2013, Zhu Y, Chinese Materia Medica, Chemistry, Pharmacology and Application, pp. 66-69, pp. 202-211, Beijing: China Medical Amp, Pharmaceutical Sciences Press, 1993; , Beijing, People's Health Publisher, 1998).
또한, 대한민국 특허등록 제0538476호에는 세신 추출물은 기억력 증진작용/뇌세포 보호작용이 보고되어 있을 뿐, 중추신경퇴행성 질환과 관련한 효능은 아직 알려진 바 없으며, 세신 성분인 아사리닌(asarinin)은 콜레스테롤 감소, 항 알러지 작용, 항암효과 등에 대해 그 효능이 알려져 있으며, (-)-세사민((-)-sesamin)은 항암작용, 비만 및 노화 예방, 항산화 작용 등에 대해 그 효능이 알려져 있다.
In addition, Korean Patent Registration No. 0538476 discloses that the extract of Seixin has memory enhancing action / protective effect on brain cells. However, its efficacy in relation to the central nervous system degenerative disease is not known yet. Asarinin, (-) - sesamin has been known for its anticancer activity, prevention of obesity and aging, and antioxidant activity.
본 발명자는 PC12 세포중의 도파민 생합성 증가작용을 유도하는 생리활성 물질 및 6-OHDA-유도 세포독성을 저해하는 생리활성 물질을 연구하던 중에 세신으로부터 분리하여 얻은 아사리닌(asarinin) 및 (-)-세사민((-)-sesamin)이 도파민 생합성 증가작용 및 6-OHDA-유도 세포독성 을 저해하는 생리활성을 갖는 것을 확인하고, 본 발명을 완성하였다.
The present inventors have studied physiologically active substances that induce dopamine biosynthesis-enhancing action in PC12 cells and physiologically active substances that inhibit 6-OHDA-induced cytotoxicity. Among them, asarinin and (-) - It has been confirmed that sesamin ((-) - sesamin) has a physiological activity that inhibits dopamine biosynthesis increasing action and 6-OHDA-induced cytotoxicity, thus completing the present invention.
본 발명은 (-)-세사민 또는 아사리닌의 신규한 의약적 용도를 제공하는 것을 목적으로 한다.
It is an object of the present invention to provide novel medicinal uses of (-) - sesamin or asarinine.
상기 목적을 달성하기 위하여, 본 발명은 아사리닌(asarinin) 또는 (-)-세사민((-)-sesamin)의 신규한 의약적 용도로서 파킨슨병 예방 또는 치료용 약학조성물을 제공한다.
In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating Parkinson's disease as a novel medical use of asarinin or (-) - sesamin.
본 발명에 따른 아사리닌 또는 (-)-세사민은 리그난 화합물의 일종으로 하기 화학식으로 표시되는 구조식으로 나타낼 수 있다.The asarinin or (-) - sesamin according to the present invention is a kind of lignan compound and can be represented by the following structural formula.
본 발명의 아사리닌 또는 (-)-세사민 화합물은 세신(Asiasari radix)으로부터 분리될 수 있으며, 세신으로부터 활성을 나타낸 식물의 활성용매 분획에 대하여 silica gel, sephadex HL-20등의 각종 담체를 이용하여 column chromatography를 통하여 활성 추적 분리법(activity-guided fractionation and isolation) 및 HPLC-UV-Vis diode array detector 법을 이용하여 분리할 수 있다.
The asarinin or (-) - sesamin compound of the present invention can be isolated from Asiasari radix, and the active solvent fraction of the plant exhibiting activity from the sesquin was treated with various carriers such as silica gel, sephadex HL-20 column can be separated by activity-guided fractionation and isolation and HPLC-UV-Vis diode array detector method.
본 발명은 아사리닌 또는 (-)-세사민 화합물에 대하여 시험모델로서 PC12 세포주를 이용하여 도파민(dopamine) 생합성 증가작용 및 6-하이드록시도파민(6-hydroxydopamine, 6-OHDA) 유도 세포독성 방어작용에 대한 활성을 검토하여 파킨슨병(Parkinson′s disease)의 예방 또는 치료에 이용될 수 있는 지 여부를 확인한 것이다.
The present invention relates to a method for inhibiting 6-hydroxydopamine (6-OHDA) -induced cytotoxic action against dopamine biosynthesis and an action to increase dopamine biosynthesis by using a PC12 cell line as a test model for an asarinin or (-) - And to determine whether it can be used to prevent or treat Parkinson's disease.
PC12 세포중에 L-DOPA(50-100 μM, 독성농도 범위)를 12-48 시간 전처치하는 경우 세포내 도파민 함량은 증가한다. L-DOPA는 산화스트레스(oxidative stress)에 의한 세포독성 작용을 나타내며 이로 인하여 48 시간 전처치에서는 L-DOPA의 독성작용에 의하여 도파민 함량이 24 시간 전처치보다 적게 나타난다(Jin et al., Eur. J. Pharmacol., 591, 88-95, 2008). 또한 L-DOPA(25-50 μM, 비독성 농도) 범위에서는 12-24 시간 전처치 시간에서 세포내 도파민 함량이 증가한다(Jin et al., Eur. J. Pharmacol., 591, 88-95, 2008).
Intracellular dopamine content increases when L-DOPA (50-100 μM, toxic concentration range) is pre-treated 12-12 hours in PC12 cells. L-DOPA exhibits cytotoxic effects due to oxidative stress, resulting in less dopamine content than 24 hours pretreatment due to the toxic action of L-DOPA at 48 hours pre-treatment (Jin et al., Eur. J. Pharmacol., 591, 88-95, 2008). In the range of L-DOPA (25-50 μM, nontoxic concentration), the intracellular dopamine content is increased at pre-treatment time of 12-24 hours (Jin et al., Eur. J. Pharmacol., 591, 88-95, 2008).
본 발명의 아사리닌(asarinin) 및 (-)-세사민((-)-sesamin)은 도파민 함량 증가작용, TH 인산화(활성화)의 증가작용, PKA-CREB 활성화 작용을 나타내었으며, 아사리닌(asarinin) 및 세사민(sesamin)이 PKA-CREB 활성화 유도작용를 매개하여 효소 TH의 활성화가 일어나 도파민 함량이 증가한 것으로 사료된다.
The asarinin and (-) - sesamin (-) - sesamin of the present invention exhibited dopaminergic activity, increased TH phosphorylation, PKA-CREB activation, And sesamin mediated the PKA-CREB activation induction, suggesting that dopamine content increased due to activation of the enzyme TH.
또한, PC12 세포중에 6-OHDA(50-100 μM, 12-36 h) 전처치는 세포독성 작용을 나타는데, 아사리닌(asarinin) 및 (-)-세사민((-)-sesamin)은 6-OHDA-유도 세포독성작용에 대한 방어작용을 나타내었다.
In addition, pretreatment of 6-OHDA (50-100 μM, 12-36 h) in PC12 cells showed cytotoxic effects, while asarinin and (-) - OHDA-induced cytotoxic effects.
따라서, 본 발명은 아사리닌(asarinin) 및 (-)-세사민((-)-sesamin)이 도파민 신경세포의 모델인 PC12 세포중에서 도파민 생합성 증가작용 및 6-OHDA-유도 세포독성의 방어작용을 나타냄으로서, 파킨슨병의 예방 및 치료제의 개발에 응용될 수 있는 자료를 제시하여 주고 있다.
Thus, the present invention demonstrates that dopamine agonists such as asarinin and (-) - sesamin ((-) - sesamin) increase dopamine biosynthesis and defense of 6-OHDA-induced cytotoxicity in dopamine neuronal cell model PC12 cells , Which provides data that can be applied to the development of preventive and therapeutic agents for Parkinson's disease.
또한 본 발명에서는 아사리닌 및 (-)-세사민이 도파민(dopamine) 생합성 증가작용 및 6-hydroxydopamine(6-OHDA)-유도 세포독성 방어작용에 대한 활성을 가짐에 착안하여, 기존의 파킨슨병 치료제인 L-DOPA와 (-)-세사민 또는 아사리닌을 병용하여 투여하였을 경우, 그 작용효과가 더욱 상승됨을 확인하였다.
Further, in the present invention, attention is paid to the fact that asarinin and (-) - sesamin have an activity to increase dopamine biosynthesis and 6-hydroxydopamine (6-OHDA) -induced cytotoxic defense action, When the combination of L-DOPA and (-) - sesamin or asarinin was administered, the effect was further enhanced.
즉, PC12 세포중에 L-DOPA(25-50 μM)를 처치할 경우(12-48 시간) 세포내 도파민 함량이 증가하는데, (-)-세사민((-)-sesamin) 또는 아사리닌(asarinin) 투여(전처리) 후 L-DOPA와 병용투여하는 경우, L-DOPA 단독투여에 비해 도파민 함량 증가작용, PKA 활성화 작용,TH 활성화 작용, CREB 활성화 작용이 더욱 증대됨을 알 수 있었다.
In other words, when PC-12 cells are treated with L-DOPA (25-50 μM) (12-48 hours), the intracellular dopamine content is increased. (-) - sesamin or (asarinin) In the case of administration of L-DOPA after the administration (pretreatment), it was found that dopamine content increase, PKA activation, TH activation and CREB activation were further enhanced compared to L-DOPA alone.
본 발명은 실제 임상 투여 시에 경구 및 비경구의 여러 가지 제형으로 투여될 수 있으며, 가장 바람직한 투여 경로는 경구투여이다. 또한, 제제화 할 경우에는 일반적으로 사용되는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다.
The present invention can be administered in various forms of oral and parenteral administration in actual clinical administration, and the most preferable administration route is oral administration. When formulating the composition, it is prepared using a commonly used diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient.
더불어, 본 발명에 따른 추출물의 투여량 또는 복용량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도에 따라 그 범위가 다양하며, 성인 기준으로 투여량 0.1 mg/kg 내지 1000 mg/kg을 1회 내지 수회에 나누어 복용하는 것이 바람직하다.
In addition, the dose or dose of the extract according to the present invention varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of disease, Preferably 0.1 mg / kg to 1000 mg / kg, once or several times.
본 발명은 아사리닌(asarinin) 및 (-)-세사민((-)-sesamin) 화합물의 신규한 의약적 용도로서 파킨슨병의 예방 및 치료용 약학조성물을 제공한다.
The present invention provides pharmaceutical compositions for the prevention and treatment of Parkinson's disease as a novel medicinal use of asarinin and (-) - sesamin compounds.
본 발명에 따른 아사리닌(asarinin) 및 (-)-세사민((-)-sesamin) 화합물은 도파민 생합성 증가작용 및 6-OHDA-유도 세포독성 방어작용을 나타냄으로써, 파킨슨병의 예방 및 치료제로 유용하며, L-DOPA 요법과 병용시 L-DOPA 효능의 증대 및 부작용 경감을 기대할 수 있다.
The asarinin and (-) - sesamin compounds according to the present invention exhibit a dopamine biosynthesis enhancing action and a 6-OHDA-induced cytotoxic defense action, thereby being useful as a preventive and therapeutic agent for Parkinson's disease D-dimer, L-DOPA, and L-DOPA.
도 1은 아사리닌(asarinin) 투여로 PC12 세포내의 도파민 함량이 증가하는 효능을 나타낸 것으로서, (A)는 아사리닌 투여시의 도파민 함량 증가를 그래프로 나타낸 그림이고, (B)는 아사리닌과 L-DOPA과의 병용투여시의 도파민 함량 증가를 그래프로 나타낸 그림이다.
도 2는 (-)-세사민((-)-sesamin)의 투여로 PC12 세포내의 도파민 함량이 증가하는 효능을 나타낸 것으로서, (A)는 (-)-세사민 투여시의 도파민 함량 증가를 그래프로 나타낸 그림이고, (B)는 (-)-세사민과 L-DOPA과의 병용투여시의 도파민 함량 증가를 그래프로 나타낸 그림이다.
도 3은 아사리닌(asarinin) 및 (-)-세사민((-)-sesamin)의 투여로 PKA 인산화(phosphorylation)를 유도하는 효능을 나타낸 것으로서, (A)는 아사리닌 또는 L-DOPA의 단독 투여시 또는 병용투여시의 PKA 인산화 정도를 그래프와 immunoblotting 법에 의한 측정으로 나타낸 그림이고, (B)는 (-)-세사민 또는 L-DOPA의 단독 투여시 또는 병용투여시의 PKA 인산화 정도를 그래프와 immunoblotting 법에 의한 측정으로 나타낸 그림이다.
도 4는 아사리닌(asarinin) 및 (-)-세사민((-)-sesamin)의 투여로 TH 인산화(phosphorylation) 를 유도하는 효능을 나타낸 것으로서, (A)는 아사리닌 또는 L-DOPA의 단독 투여시 또는 병용투여시의 TH 인산화 정도를 그래프와 immunoblotting 법에 의한 측정으로 나타낸 그림이고, (B)는 (-)-세사민 또는 L-DOPA의 단독 투여시 또는 병용투여시의 TH 인산화 정도를 그래프와 immunoblotting 법에 의한 측정으로 나타낸 그림이다.
도 5는 아사리닌(asarinin) 및 (-)-세사민((-)-sesamin)의 투여로 CREB 인산화(phosphorylation) 를 유도하는 효능을 나타낸 것으로서, (A)는 아사리닌 또는 L-DOPA의 단독 투여시 또는 병용투여시의 CREB 인산화 정도를 그래프와 immunoblotting 법에 의한 측정으로 나타낸 그림이고, (B)는 (-)-세사민 또는 L-DOPA의 단독 투여시 또는 병용투여시의 CREB 인산화 정도를 그래프와 immunoblotting 법에 의한 측정으로 나타낸 그림이다.
도 6은 아사리닌 투여시 PC12 세포내 6-OHDA-유도 세포독성 방어작용이 일어나는 효능을 나타낸 것으로서, (A)는 6-OHDA와 아사리닌(부피비 2:1)을 함께 투여시 cell viability의 감소 정도를 그래프로 나타낸 그림이며, (B)는 6-OHDA와 아사리닌(부피비 4:1)을 함께 투여시 cell viability의 감소 정도를 그래프로 나타낸 그림이고, (C)는 6-OHDA와 아사리닌을 함께 투여해 24시간 배양시 cell viability의 변화 정도를 그래프로 나타낸 그림이다.
도 7은 (-)-세사민 투여시 PC12 세포내 6-OHDA-유도 세포독성 방어작용이 일어나는 효능을 나타낸 것으로서, (A)는 6-OHDA와 (-)-세사민(부피비 2:1) 투여시 cell viability의 감소 정도를 그래프로 나타낸 그림이며, (B)는 6-OHDA와 (-)-세사민(부피비 4:1)을 함께 투여시 cell viability의 감소 정도를 그래프로 나타낸 그림이고, (C)는 6-OHDA와 (-)-세사민을 함께 투여해 24시간 배양시 cell viability의 변화 정도를 그래프로 나타낸 그림이다.
도 8은 아사리닌(asarinin) 및 (-)-세사민((-)-sesamin)의 투여로 6-OHDA-유도 NK 활성화 저해작용이 일어나는 효능을 그래프와 immunoblotting 법에 의한 측정으로 나타낸 그림이다.
도 9는 아사리닌 및 (-)-세사민의 투여로 PC12 세포내 6-OHDA-유도 p38 MAPK 활성화 저해작용이 일어나는 효능을 그래프와 immunoblotting 법에 의한 측정으로 나타낸 그림이다.
도 10은 아사리닌 및 (-)-세사민의 투여로 PC12 세포내 6-OHDA-유도 Bax 활성화 저해작용이 일어나는 효능을 그래프와 immunoblotting 법에 의한 측정으로 나타낸 그림이다.
도 11은 아사리닌 및 (-)-세사민의 투여로 발생하는 PC12 세포내 ERK 활성화 및 6-OHDA-유도 ERK 활성화에 대한 영향을 그래프와 immunoblotting 법에 의한 측정으로 나타낸 그림이다.
도 12는 아사리닌 및 (-)-세사민의 투여로 발생하는 PC12 세포내 Bad(Ser112) 활성화 및 6-OHDA-유도 Bad(Ser112) 활성화 감소작용에 대한 영향을 그래프와 immunoblotting 법에 의한 측정으로 나타낸 그림이다.
도 13은 아사리닌 및 (-)-세사민의 투여로 발생하는 PC12 세포내 caspase-3 활성에 대한 영향을 그래프와 immunoblotting 법에 의한 측정으로 나타낸 그림이다.
도 14는 아사리닌 및 (-)-세사민의 24시간의 전처치 로 발생하는 PC12 세포내 세포독성에 대한 영향을 cell viability에 대한 그래프로 나타낸 그림이다.
도 15는 6-hydroxydopamine(6-OHDA)-유도 파킨슨병(Parkinson's disease, PD) 동물 모델에서 (-)-세사민 투여에 의한 도파민(dopamine) 신경세포의 보호작용을 나타낸 것으로, (A)는 (-)-세사민 투여시 도파민 신경세포의 보호작용에 대한 뇌중의 TH-immnunoactivity의 염색을 나타낸 그림이며, (B)는 TH-면역양성(immunopositive) 신경세포의 수를 나타낸 그림이다.
도 16은 6-OHDA-유도 PD 동물 모델에서 (-)-세사민 투여에 의한 뇌의 도파민 신경세포내의 도파민(A), 노르에피네프린(norepinephrine)(B), DOPAC(C), homovanillic acid(HVA)(D)의 함량을 나타낸 그림이다.FIG. 1 is a graph showing an increase in dopamine content in PC12 cells by administration of asarinin, (A) a graph showing an increase in dopamine content during administration of asarrin, (B) -DOPA is a graph showing the increase of dopamine content in combination with DOPA.
FIG. 2 shows the effect of increasing the dopamine content in PC12 cells by administration of (-) - sesamin ((-) - sesamin), wherein (A) (B) is a graph showing the increase in dopamine content when (-) - sesamin is used in combination with L-DOPA.
FIG. 3 shows the effect of inducing PKA phosphorylation by administration of asarinin and (-) - sesamin ((-) - sesamin), wherein (A) is a single administration of asarinin or L- (B) is the graph showing the degree of PKA phosphorylation at the time of administration of (-) - sesamin or L-DOPA alone or in combination with the graph and the immunoblotting method. Fig. 3 shows the results obtained by immunoblotting.
FIG. 4 shows the effect of inducing TH phosphorylation by administration of asarinin and (-) - sesamin ((-) - sesamin), wherein (A) is a single administration of asarinin or L- (B) shows the degree of TH phosphorylation at the time of administration of (-) - sesamin or L-DOPA alone or in combination with the graph and the immunoblotting method. Fig. 3 shows the results obtained by immunoblotting.
FIG. 5 shows the effect of inducing CREB phosphorylation by administration of asarinin and (-) - sesamin ((-) - sesamin), wherein (A) is a single dose of asarinin or L-DOPA (B) shows the level of CREB phosphorylation in the case of administration of (-) - sesamin or L-DOPA alone or in combination with a graph and immunoblotting method. Fig. 3 shows the results obtained by immunoblotting.
FIG. 6 shows the effect of 6-OHDA-induced cytotoxic defense in PC12 cells upon administration of asarrinin. (A) shows a decrease in cell viability when 6-OHDA and asarinin (2: 1) (B) is a graph showing the degree of decrease in cell viability when 6-OHDA and asarinin (volume ratio 4: 1) are administered together, and (C) , Which is a graph showing the degree of change in cell viability when cultured for 24 hours.
FIG. 7 shows the effect of 6-OHDA-induced cytotoxic defense in PC12 cells upon administration of (-) - sesamin. (A) shows the effect of 6-OHDA and (-) - (B) is a graph showing the degree of decrease in cell viability when 6-OHDA and (-) - sesamin (4: 1) are administered together, and (C) Is a graph showing the degree of change in cell viability during 24-hour incubation with 6-OHDA and (-) - sesamin.
FIG. 8 is a graph showing the effect of 6-OHDA-induced NK activation inhibition by the administration of asarinin and (-) - sesamin ((-) - sesamin) by a graph and immunoblotting assay.
FIG. 9 is a graph showing the effect of 6-OHDA-induced p38 MAPK activation inhibition in PC12 cells by the administration of asarinine and (-) - sesamin in a graph and immunoblotting assay.
FIG. 10 is a graph showing the effect of 6-OHDA-induced Bax activation inhibition in PC12 cells by the administration of asarinine and (-) - sesamin by a graph and immunoblotting assay.
FIG. 11 is a graph showing the effect of ERK activation and 6-OHDA-induced ERK activation in PC12 cells induced by administration of asarinine and (-) - sesamin by a graph and immunoblotting assay.
Figure 12 shows the effect of reducing (Ser112) activation and 6-OHDA-induced Bad (Ser112) activation in PC12 cells induced by administration of asarinine and (-) - sesamin by the graph and immunoblotting It is a picture.
FIG. 13 is a graph showing the effect of caspase-3 on caspase-3 activity in PC12 cells induced by administration of asarinine and (-) - sesamin by a graph and immunoblotting assay.
FIG. 14 is a graph showing the cell viability of PC12 cells induced by 24-hour pretreatment with asarinine and (-) - sesamin in cell cytotoxicity.
FIG. 15 shows the protective action of dopamine neurons by (-) - sesamin administration in an animal model of 6-hydroxydopamine (6-OHDA) -induced Parkinson's disease (Parkinson's disease, PD) -) -The TH-immnunoactivity staining of the cerebral cortex against the protective action of dopamine neurons in case of sesamin administration. (B) The number of TH-immunopositive neurons.
Figure 16 shows that dopamine (A), norepinephrine (B), DOPAC (C), and homovanillic acid (HVA) in the dopaminergic neurons of the brain by (-) - sesamin administration in 6-OHDA- (D).
이하, 본 발명을 실시예에 의하여 상세히 설명한다. 단, 하기의 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기의 실시예에 의하여 한정되는 것은 아니다.
Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.
<실시예 1> ≪ Example 1 > 아사리닌 및 (-)-세사민의 도파민 생합성 증가 및 촉진작용Increase and promotion of dopamine biosynthesis of asarrinin and (-) - sesamin
PC12 세포중에 L-DOPA(50-100 μM, 독성농도 범위)를 12-48 시간 전처치하는 경우 세포내 도파민 함량은 증가한다. L-DOPA는 산화스트레스(oxidative stress)에 의한 세포독성 작용을 나타내며 이로 인하여 48 시간 전처치에서는 L-DOPA의 독성작용에 의하여 도파민 함량이 24 시간 전처치보다 적게 나타난다(Jin et al., Eur. J. Pharmacol., 591, 88-95, 2008). 또한 L-DOPA(25-50 μM, 비독성 농도) 범위에서는 12-24 시간 전처치 시간에서 세포내 도파민 함량이 증가한다(Jin et al., Eur. J. Pharmacol., 591, 88-95, 2008).
Intracellular dopamine content increases when L-DOPA (50-100 μM, toxic concentration range) is pre-treated 12-12 hours in PC12 cells. L-DOPA exhibits cytotoxic effects due to oxidative stress, resulting in less dopamine content than 24 hours pretreatment due to the toxic action of L-DOPA at 48 hours pre-treatment (Jin et al., Eur. J. Pharmacol., 591, 88-95, 2008). In the range of L-DOPA (25-50 μM, nontoxic concentration), the intracellular dopamine content is increased at pre-treatment time of 12-24 hours (Jin et al., Eur. J. Pharmacol., 591, 88-95, 2008).
PC12 세포주는 HS 및 FBS 함유한 RPMI1640 배지상에서 상법에 준하여 배양하였다. PC12 세포 중의 도파민 함량은 형광시약 DPE를 이용하는 HPLC-형광검출기법을 이용하여 측정하였다(Zhang et al., Neuropharmacol., 62, 2218-2225, 2012).The PC12 cell line was cultured on RPMI1640 medium containing HS and FBS according to the conventional method. The dopamine content in PC12 cells was measured using HPLC-fluorescence detection technique using fluorescent reagent DPE (Zhang et al., Neuropharmacol., 62, 2218-2225, 2012).
1) 아사리닌(asarinin)에 의한 도파민 함량 증가 1) Increased dopamine content by asarinin
아사리닌 10, 25, 50μM을 각각 PC12 세포주에 투여하고 24시간 후 HPLC-형광검출기법을 이용하여 PC12 세포 중의 도파민 함량을 측정하여 도 1(A)에 나타내었다. 도 1(A)로부터 세포내 도파민 함량은 대조군에 비하여 각각 123%, 165%, 133%의 유의적으로 증가하였음을 알 수 있다.
10, 25 and 50 μM of asarrinin were added to the PC12 cell line, respectively. After 24 hours, the dopamine content in the PC12 cells was measured using the HPLC-fluorescence detection technique, and the result was shown in FIG. 1 (A), the intracellular dopamine content was significantly increased by 123%, 165%, and 133%, respectively, as compared with the control group.
또한, 아사리닌과 L-DOPA를 병용투여하고 PC12 세포 중의 도파민 함량을 측정하여 도 1(B)에 나타내었다. 도 1(B)에서 알 수 있는 바와 같이, L-DOPA 25 및 50μM을 각각 단독투여시, PC12 세포내에서 도파민 함량은 증가하는데(약 145% 및 150%), 아사리닌(25 및 50 μM) 투여 1시간 후 L-DOPA(25 μM)와 병용투여(24시간) 하는 경우 세포내 도파민 함량은 L-DOPA(25 μM) 단독 투여군에 비하여 유의성 있게 증가하였고(각각 약 52% 및 26% 증가), 아사리닌(25 및 50 μM)과 L-DOPA(50 μM)과의 병용투여는 L-DOPA 단독 투여군(50 μM)에 비하여 도파민 함량은 유의성 있게 증가하였다(약 78% 및 47% 증가).
In addition, dopamine content in PC12 cells was measured by administering asarinin and L-DOPA in combination, and is shown in Fig. 1 (B). As shown in FIG. 1 (B), dopamine content increases (about 145% and 150%) in PC12 cells when L-
상기 결과로부터 아사리닌의 전처치는 PC12 세포내 도파민 함량 증가작용을 나타내며, L-DOPA-유도 도파민 함량 증가작용에 대한 상승·촉진작용을 나타냄을 알 수 있다.
From the above results, it can be seen that pretreatment of asarinin shows an action of increasing dopamine content in PC12 cells, and shows an action of increasing or promoting the action of increasing L-DOPA-induced dopamine content.
2) (-)-세사민((-)-sesamin)에 의한 도파민 함량 증가 2) Increased dopamine content by (-) - sesamin ((-) - sesamin)
(-)-세사민 10, 25, 50μM을 각각 PC12 세포주에 투여하여 24시간 후 HPLC-형광검출기법을 이용하여 PC12 세포 중의 도파민 함량을 측정하여 도 2(A)에 나타내었다. 도 2(A)로부터 세포내 도파민 함량은 대조군에 비하여 각각 142%, 181%, 132%의 유의적으로 증가함을 알 수 있다.
(-) -
또한, (-)-세사민과 L-DOPA를 병용투여하고 PC12 세포 중의 도파민 함량을 측정하여 도 2(B)에 나타내었다. 도 2(B)에서 알 수 있는 바와 같이, (-)-세사민(50 μM) 투여 1시간 후 L-DOPA(25 μM)와 병용투여(24시간) 하는 경우 세포내 도파민 함량은 L-DOPA(25 μM) 단독 투여군에 비하여 유의성 있게 증가하였으며(약 104% 증가), (-)-세사민(50 μM)과 L-DOPA(50μM)를 병용 투여하는 경우 L-DOPA(50 μM) 단독 투여군에 비하여 유의성 있게 증가하였다(약 120% 증가).
In addition, (-) - sesamin and L-DOPA were administered concomitantly, and the content of dopamine in PC12 cells was measured and shown in FIG. 2 (B). As shown in FIG. 2 (B), the intracellular dopamine content of L-DOPA (25 μM) was significantly lower than that of L-DOPA (25 μM) (50 μM) and L-DOPA (50 μM) were significantly higher than those of L-DOPA alone (50 μM) alone (About 120% increase).
상기 결과로부터 (-)-세사민의 전처치는 PC12 세포내 도파민 함량 증가작용을 나타내며, L-DOPA-유도 도파민 함량 증가작용에 대한 상승·촉진작용을 나타내는 것으로 사료된다.
From the above results, pretreatment of (-) - sesamin shows an effect of increasing the dopamine content in PC12 cells and showing an action of increasing or promoting the action of increasing L-DOPA-induced dopamine content.
<실시예 2> ≪ Example 2 > 아사리닌 및 (-)-세사민의 PKA 인산화(phosphorylation) 작용PKA phosphorylation of asarinin and (-) - sesamin
아사라닌 및 (-)-세사민에 의한 세포내 PKA 인산화 증가작용을 확인하기 위하여 phosphor-PKA 항체를 이용한 immunoblotting 법으로 PKA 인산화를 측정하여 도 3(A) 및 도 3(B)에 나타내었다.
PKA phosphorylation was measured by immunoblotting using phosphor-PKA antibody in order to confirm intracellular PKA phosphorylation activity by asaranine and (-) - sesamin, as shown in FIGS. 3 (A) and 3 (B).
1) 아사리닌의 PKA 인산화 작용 1) PKA phosphorylation of asarinin
아사리닌(25 μM, 투여시간 1 시간)은 PC12 세포내 PKA 활성화(인산화, phosphor-PKA)를 유도하였으며(약 1.4 배 증가), 아사리닌(25 μM)과 L-DOPA(50 μM)와 병용 투여하는 경우 PKA 활성화는 L-DOPA(50 μM) 단독 투여군에 비하여 유의적으로 증가하였다(약 2.3배 증가)(도 3A).
Asarinin (25 μM, 1 hour of dosing) induced PKA activation (phosphorylation PKA) in PC12 cells (approximately 1.4-fold increase) and was co-administered with asarinin (25 μM) and L-DOPA PKA activation was significantly increased (about 2.3-fold increase) compared to L-DOPA (50 μM) alone (FIG. 3A).
2) (-)-세사민의 PKA 인산화 작용 2) (-) - PKA phosphorylation of sesamin
(-)-세사민(25 μM, 투여시간 1 시간)은 PC12 세포내 PKA 활성화(인산화)를 유도하였으며(약 1.6 배 증가), (-)-세사민(25 μM)과 L-DOPA(50 μM)와 병용 투여하는 경우 PKA 활성화는 L-DOPA(50 μM) 단독 투여군에 비하여 유의적으로 증가하였다(약 2.4배 증가)(도 3B).
(25 μM), L-DOPA (50 μM), and (-) - sesamin (25 μM, one hour of administration time) induced PKA activation PKA activation was significantly increased (about 2.4-fold increase) compared to L-DOPA (50 μM) alone (FIG. 3B).
상기 결과로부터 아사리닌 및 (-)-세사민의 전처치는 PC12 세포내 PKA 인산화(p-PKA)를 유도하여 TH 활성화를 유도하고 도파민 함량 증가작용을 나타내는 것을 알 수 있다.
From the above results, it can be seen that pretreatment of asarinin and (-) - sesamin induces PKA phosphorylation (p-PKA) in PC12 cells, inducing TH activation and increasing dopamine content.
<실시예 3> ≪ Example 3 > 아사리닌 및 (-)-세사민의 TH 인산화(phosphorylation) 작용 TH phosphorylation of asarinin and (-) - sesamin
아사리닌 또는 (-)-세사민의 효소 TH 활성화(인산화, phosphorylation) 작용을 검토하기 위하여 아사리닌 또는 (-)-세사민을 전처치한 다음 12 시간 후 PC12 세포내 TH 인산화 함량을 측정하였다. TH 인산화는 phosphor-TH 항체를 이용한 immunoblotting 법(Park et al., Toxicol. Sci., 128, 247-257, 2012)으로 측정하여 도 4(A) 및 도 4(B)에 나타내었다.
To investigate the enzyme TH activation (phosphorylation) of asarinin or (-) - sesamin, TH phosphorylation content in PC12 cells was measured 12 hours after pretreatment with asarrinin or (-) - sesamin. TH phosphorylation was measured by immunoblotting method using phosphor-TH antibody (Park et al., Toxicol. Sci., 128, 247-257, 2012) and is shown in Figs. 4 (A) and 4 (B).
1) 아사리닌의 TH 인산화 작용 1) TH phosphorylation of asarinin
아사리닌(25 μM)의 단독 투여는 대조군에 비하여 TH 인산화가 증가하였으며(약 1.4 배), 아사리닌(25 μM)과 L-DOPA(50 μM)의 병용투여는 L-DOPA(50 μM) 단독 투여군에 비하여 TH 인산화가 약 2.4 배 증가하였다(도 4A).
(50 μM) alone or in combination with asarrinin (25 μM) increased the TH phosphorylation (about 1.4-fold) compared with the control group. L-DOPA The TH phosphorylation was increased about 2.4 times as compared with the administration group (Fig. 4A).
2) (-)-세사민의 TH 인산화 작용 2) TH phosphorylation of (-) - sesamin
또한 (-)-세사민(25 μM)의 단독 투여(전처치 12 시간)는 대조군에 비하여 TH 인산화가 증가하였으며(약 2.1 배), (-)-세사민(25 μM)과 L-DOPA(50 μM)의 병용투여는 L-DOPA(50 μM) 단독 투여군에 비하여 TH 인산화가 약 2.5 배 증가하였다(도 4B).
In addition, (-) - sesamin (25 μM) and L-DOPA (50 μM, respectively) increased TH phosphorylation (about 2.1 times) ) Increased the TH phosphorylation by about 2.5-fold compared to the L-DOPA (50 [mu] M) alone group (Fig. 4B).
상기 결과로부터 아사리닌 및 (-)-세사민의 전처치는 PC12 세포내 TH 인산화(p-TH)에 의한 TH 활성화를 유도하여 도파민 함량 증가작용을 나타내는 것을 알 수 있다.
From the above results, it can be seen that pretreatment of asarinine and (-) - sesamin induces TH activation by TH phosphorylation (p-TH) in PC12 cells, indicating that dopamine content is increased.
<실시예 4> <Example 4> 아사리닌 및 (-)-세사민의 CREB 인산화(phosphorylation) 작용CREB phosphorylation of asarinine and (-) - sesamin
아사리닌 및 (-)-세사민의 CREB(cyclic AMP-response element binding protein) 활성화(인산화, phosphorylation) 작용을 검토하기 위하여 아사리닌 및 (-)-세사민 전처치한 다음 1 시간후 PC12 세포내 CREB 인산화 함량을 측정하여 도 5(A) 및 도 5(B)에 나타내었다. CREB 인산화는 phosphor-CREB 항체를 이용한 Western blotting 법으로 측정하였다(Park et al., Toxicol. Sci., 128, 247-257, 2012).
In order to examine the action of CREB (cyclic AMP-response element binding protein) activation (phosphorylation) of asarrinin and (-) - sesamin, CREB phosphorylation in
1) 아사리닌의 CREB 인산화 작용 1) CREB phosphorylation of asarinin
아사리닌(25 μM)의 단독 투여는 대조군에 비하여 CREB 인산화가 증가하였으며(약 1.4 배), 아사리닌(25 μM)과 L-DOPA(50 μM)의 병용투여는 L-DOPA(50 μM) 단독 투여군에 비하여 TH 인산화가 약 1.9 배 증가하였다(도 5A).
(50 μM) alone or in combination with asarrinin (25 μM) increased CREB phosphorylation (about 1.4-fold) compared with the control group, and L-DOPA The TH phosphorylation was increased about 1.9 times as compared with the administration group (Fig. 5A).
2) (-)-세사민의 CREB 인산화 작용 2) CREB phosphorylation of (-) - sesamin
또한 (-)-세사민(25 μM)의 단독 투여(전처치 1 시간)는 대조군에 비하여 CREB 인산화가 증가하였으며(약 1.4 배), (-)-세사민(25 μM)과 L-DOPA(50 μM)의 병용투여는 L-DOPA(50 μM) 단독 투여군에 비하여 TH 인산화가 약 2.0 배 증가하였다(도 5B).
In addition, CREB phosphorylation (1.4 times), (-) - sesamin (25 μM) and L-DOPA (50 μM) were increased in the single treatment (1 hour pretreatment) ) Increased the TH phosphorylation about 2.0-fold as compared to the L-DOPA (50 μM) alone group (FIG. 5B).
이와 같은 결과는 아사리닌 및 (-)-세사민의 전처치는 PC12 세포내 CREB 인산화에 의한 TH 인산화(p-TH)를 유도하여 도파민 함량 증가작용을 나타내는 것을 의미한다.
These results suggest that pretreatment with asarinine and (-) - sesamin induces TH phosphorylation (p-TH) by CREB phosphorylation in PC12 cells, thereby increasing dopamine content.
<실시예 5> ≪ Example 5 > 아사리닌 및 (-)-세사민의 6-OHDA-유도 세포독성 방어작용6-OHDA-induced cytotoxic defense of asarinin and (-) - sesamin
아사리닌 및 (-)-세사민이 PC12 세포 중에 미치는 세포독성 작용을 측정하여 각각 도 6(A), (B) 및 (C), 도 7(A), (B) 및 (C)에 나타내었다. 세포독성은 미토콘드리아 손상에 의한 세포사를 측정하는 MTT 법에 의하여 시행하였다(570 nm 흡광도 측정)(Park et al., Toxicol. Sci., 128, 247-257, 2012).
The cytotoxic effects of asarinin and (-) - sesamin in PC12 cells were measured and are shown in FIGS. 6 (A), (B) and (C) . Toxicol. Sci., 128, 247-257, 2012) were used to measure cytotoxicity by MTT assay (570 nm absorbance measurement) for measuring cell death by mitochondrial damage.
1) 아사리닌의 6-OHDA-유도 세포독성 방어작용 1) 6-OHDA-induced cytotoxic defense of asarinin
6-OHDA(50-100 μM)를 투여시 세포독성을 나타내는데, 아사리닌(25 μM)을 함께 투여하여 24/36시간 배양하는 경우 cell viability가 각각 약 11-15% 및 19-27% 증가하여 6-OHDA(50-100 μM)-유도 세포독성 작용에 대항하는 세포독성 작용에 대한 보호작용을 나타내었다(도 6A 및 B). 아사리닌(10-50 μM)은 24시간 배양하는 경우 6-OHDA(100 μM)-유도 세포독성 작용에 대하여 농도-의존적으로 세포독성 방어작용을 나타내었다(도 6C).
Cell viability was increased by about 11-15% and 19-27%, respectively, when 24-36-hour incubation with asarinin (25 μM) was performed with 6-OHDA (50-100 μM) 6-OHDA (50-100 [mu] M) -induced cytotoxic action (Fig. 6A and B). Asarinin (10-50 [mu] M) showed a cytotoxic defense action in a concentration-dependent manner on the 6-OHDA (100 [mu] M) -induced cytotoxic effect when cultured for 24 hours (Fig. 6C).
2) (-)-세사민의 6-OHDA-유도 세포독성 방어작용 2) (-) - 6-OHDA-induced cytotoxic defense of sesamin
(-)-세사민(25 μM)을 6-OHDA(50-100 μM)와 함께 투여하여 24/36시간 배양하는 경우 cell viability가 각각 약 15-24% 및 20-38% 증가하여 6-OHDA(50-100 μM)-유도 세포독성 작용에 대한 보호작용을 나타내었다(도 7A 및 B). 또한 (-)-세사민(10-50 μM)은 24시간 배양하는 경우 6-OHDA(100 μM)-유도 세포독성 작용에 대하여 농도-의존적으로 세포독성 방어작용을 나타내었다(약 26-35% 세포사 방어작용)(도 7C).
Cell viability was increased by approximately 15-24% and 20-38%, respectively, when 24-36 hours of culture with (-) - sesamin (25 μM) with 6-OHDA (50-100 μM) 50-100 [mu] M) -induced cytotoxic action (Fig. 7A and B). In addition, (-) - sesamin (10-50 μM) showed a cytotoxic effect on cytotoxic effects of 6-OHDA (100 μM) -induced cytotoxicity when cultured for 24 hours (about 26-35% cell death Defense) (Fig. 7C).
<실시예 6> ≪ Example 6 > 아사리닌 및 (-)-세사민의 6-OHDA-유도 세포독성 방어작용: JNK 활성화 저해작용6-OHDA-Induced Cytotoxic Action of Asarinine and (-) - Sesamine: Inhibition of JNK Activation
독성농도의 6-OHDA 전처치는 산화스트레스에 의하여 PC12 세포내에 c-Jun N-termianl kinase1/2(JNK1/2) 활성화(인산화)를 유도하고 JNK1/2 활성화는 세포사 과정을 진행하여 세포사(apoptosis)를 일으킨다(Jin et al., Neurosci., 170, 390-398, 2010).
The toxic concentration of 6-OHDA pretreatment induces c-Jun N-termianl kinase1 / 2 (phosphorylation) activation (phosphorylation) in PC12 cells by oxidative stress and JNK1 / 2 activation leads to apoptosis ) (Jin et al., Neurosci., 170, 390-398, 2010).
JNK1/2 활성화(인산화)의 측정은 JNK1/2(Thr 183/Thr 185)의 인산화를 Western blotting 법에 의하여 측정하였다(Park et al., Toxicol. Sci., 128, 247-257, 2012).
JNK1 / 2 activation (phosphorylation) was measured by Western blotting of the phosphorylation of JNK1 / 2 (Thr 183 / Thr 185) (Park et al., Toxicol. Sci., 128, 247-257, 2012).
아사리닌(25 μM) 및 (-)-세사민(25 μM)은 6-OHDA(100 μM)-유도 JNK 활성화 작용에 대하여 유의적인 저해작용을 나타내었다(전처치 12시간). 이는 아사리닌 및 (-)-세사민이 6-OHDA 세포독성 작용에 대한 방어작용이 있음을 나타낸 것이다(도 8).
Asarinin (25 μM) and (-) - sesamin (25 μM) showed significant inhibitory effects on 6-OHDA (100 μM) -induced JNK activation (pre-treatment 12 hours). This indicates that asarinine and (-) - sesamin have a protective action against 6-OHDA cytotoxic action (FIG. 8).
<실시예 7> ≪ Example 7 > 아사리닌 및 (-)-세사민의 6-OHDA-유도 세포독성 방어작용: p38 MAPK 활성화 저해작용6-OHDA-Induced Cytotoxic Defense of Asarinin and (-) - Sesamin: Inhibition of p38 MAPK Activation
독성농도의 6-OHDA 전처치는 산화스트레스에 의하여 PC12 세포내에 p38 mitogen-activated protein kinase(p38 MAPK) 활성화(인산화)를 유도하고 p38 MAPK 활성화는 세포사 과정을 진행하여 세포사(apoptosis)를 일으킨다(Jin et al., Neurosci., 170, 390-398, 2010).
The toxic concentration of 6-OHDA pretreatment induces p38 mitogen-activated protein kinase (p38 MAPK) activation (phosphorylation) in PC12 cells by oxidative stress and p38 MAPK activation leads to apoptosis et al., Neurosci., 170, 390-398, 2010).
p38MAPK 활성화(인산화)의 측정은 phosphor-p38MAPK를 측정하는 Western blotting 법에 의하여 측정하였다(Jin et al., Neurosci., 170, 390-398, 2010).
Measurements of p38 MAPK activation (phosphorylation) were measured by Western blotting to determine phosphor-p38 MAPK (Jin et al., Neurosci., 170, 390-398, 2010).
아사리닌(25 μM) 및 (-)-세사민(25 μM)은 6-OHDA(100 μM)-유도 p38 MAPK 활성화 작용에 대하여 유의적인 저해작용을 나타내었다(전처치 12시간). 그러므로 아사리닌 및 (-)-세사민이 6-OHDA-유도 세포독성 작용에 대한 방어작용이 있음을 알 수 있다(도 9).
Asarinin (25 μM) and (-) - sesamin (25 μM) showed significant inhibitory effects on 6-OHDA (100 μM) -induced p38 MAPK activation (pre-treatment 12 hours). Thus, it can be seen that asarinin and (-) - sesamin have a protective action against 6-OHDA-induced cytotoxic effects (Fig. 9).
<실시예 8> ≪ Example 8 > 아사리닌 및 (-)-세사민의 6-OHDA-유도 세포독성 방어작용: Bax 활성화 저해작용6-OHDA-Induced Cytotoxic Action of Asarinine and (-) - Sesamin: Bax Activation Inhibitory Activity
독성농도의 6-OHDA 전처치는 산화스트레스에 의하여 PC12 세포내에 Bax 활성화(인산화)를 유도하고 BAX 활성화는 세포사 과정을 진행하여 세포사(apoptosis)를 일으킨다(Gomez-Lazrano et al., J. Neurochem., 104, 1599-1612, 2008).
The toxic concentration of 6-OHDA pretreatment induces Bax activation (phosphorylation) in PC12 cells by oxidative stress, and BAX activation promotes apoptosis by progressing the cell death process (Gomez-Lazrano et al., J. Neurochem. , 104, 1599-1612, 2008).
Bax 활성화(인산화)의 측정은 phosphor-Bax를 측정하는 Western blotting 법에 의하여 측정하였다(Gomez-Lazrano et al., J. Neurochem., 104, 1599-1612, 2008)
Bax activation (phosphorylation) was measured by Western blotting to determine phosphor-Bax (Gomez-Lazrano et al., J. Neurochem., 104, 1599-1612, 2008)
아사리닌(25 μM) 및 (-)-세사민(25 μM)은 6-OHDA(100 μM)-유도 Bax 활성화 작용에 대하여 유의적인 저해작용을 나타내었다(전처치 12시간). 그러므로 아사리닌 및 (-)-세사민이 6-OHDA 세포독성 작용에 대한 방어작용이 있음을 알 수 있다(도 10).
Asarinin (25 μM) and (-) - sesamin (25 μM) showed significant inhibitory effects on 6-OHDA (100 μM) -induced Bax activation (pre-treatment 12 hours). Therefore, it can be seen that asarinine and (-) - sesamin have a protective action against 6-OHDA cytotoxic action (FIG. 10).
<실시예 9> ≪ Example 9 > 아사리닌 및 (-)-세사민의 6-OHDA-유도 세포독성 방어작용: ERK1/2 활성화 조절작용6-OHDA-Induced Cytotoxic Defense of Asarinine and (-) - Sesamin: ERK1 / 2 Regulation of Activation
6-OHDA의 비독성 농도(20 μM)는 PC12 세포내에서 일시적 extracellular signal-regulated kinase(ERK1/2)의 활성화(transient ERK phosphorylation, 0.5-1 시간)를 유도하여 세포의 생존을 유도하며(sell survival), 6-OHDA의 독성 농도범위(50-100 μM)는 지속적 ERK 활성화(sustained ERK phosphorylation, 1-6 시간)를 유도하며 세포는 형태학적 변화를 거쳐 세포사 과정(cell death)으로 이행한다. 그러므로 지속적 ERK 활성화의 저해작용은 세포의 세포사를 방어할 수 있음이 제시되고 있다(Park et al., Toxicol. Sci., 128, 247-257, 2012).
The non-toxic concentration of 6-OHDA (20 μM) induces transient ERK phosphorylation (0.5-1 h) of transient extracellular signal-regulated kinase (ERK1 / 2) in PC12 cells The toxic concentration range (50-100 μM) of 6-OHDA induces sustained ERK phosphorylation (1-6 hours), and the cells undergo morphological changes to cell death. Thus, inhibition of sustained ERK activation has been suggested to protect cells from cell death (Park et al., Toxicol. Sci., 128, 247-257, 2012).
ERK1/2 활성화(인산화)의 측정은 ERK1/2(Thr 202/Tyr 204) 인산화[phosphor-ERK1/2 (Thr 202/Tyr 204)]를 측정하는 Western blotting 법에 의하여 측정하였다(Park et al., Toxicol. Sci., 128, 247-257, 2012).
ERK1 / 2 activation (phosphorylation) was measured by Western blotting to determine ERK1 / 2 (Thr 202 / Tyr 204) phosphorylation [phosphor-ERK1 / 2 (Thr 202 / Tyr 204)] (Park et al. , Toxicol. Sci., 128, 247-257, 2012).
아사리닌(25 μM) 및 (-)-세사민(25 μM)의 단독 처치는 일시적 ERK 활성화를 유도하며(cell survival)(도 12), 6-OHDA(100 μM) 처치에 의한 지속적 ERK 활성화에 대하여 ERK 활성화의 저해작용을 나타내었다(3-6 시간)(도 11). 그러므로 아사리닌 및 (-)-세사민이 ERK 활성화 유도 작용에 의한 6-OHDA 세포독성 작용에 대한 방어작용이 있음을 알 수 있다.
The single treatment of asarinin (25 μM) and (-) -seminin (25 μM) induces transient ERK activation (FIG. 12) and the sustained ERK activation by treatment with 6-OHDA (100 μM) ERK activation (3-6 hours) (Figure 11). Therefore, it can be seen that asarinine and (-) - sesamin protect against 6-OHDA cytotoxic action by ERK activation induction.
<실시예 10> ≪ Example 10 > 아사리닌 및 (-)-세사민의 6-OHDA-유도 세포독성 방어작용: Bad(Ser112) 활성화 저해작용6-OHDA-Induced Cytotoxic Action of Asarinine and (-) - Sesamin: Bad (Ser112) Inhibition Activity
Bad 인산화 부위는 Ser112 부위(Bad(Ser112))와 Ser155 부위(Bad(Ser155))가 알려져 있으며, 모두 세포 생존(cell survival)에 관여하고 있음이 보고되고 있다. 그러나 최근 Bad(Ser155)는 산화스트레스에 의한 세포독성 유도시에 활성화 되고 있음이 보고되고 있다(Jin et al., Neurosci., 170, 390-398, 2010). 그러므로 아사리닌 및 세사민이 Bad(Ser112) 활성화에 대한 영향을 검토하였다.
Bad phosphorylation sites are known to be Ser112 (Bad (Ser112)) and Ser155 (Bad (Ser155)), all of which are involved in cell survival. Recently, Bad (Ser155) has been reported to be activated upon induction of cytotoxicity by oxidative stress (Jin et al., Neurosci., 170, 390-398, 2010). Therefore, the effects of asarinin and sesamin on Bad (Ser112) activation were examined.
Bad 활성화(인산화)의 측정은 Ser112 부위(Bad(Ser112))와 Ser155 부위(Bad(Ser155))의 인산화를 측정하는 Western blotting 법에 의하여 측정하였다(Jin et al., Neurosci., 170, 390-398, 2010).
The measurement of Bad activation (phosphorylation) was measured by Western blotting to measure the phosphorylation of the Ser112 region (Bad (Ser112)) and the Ser155 region (Bad155) (Jin et al., Neurosci. 398, 2010).
아사리닌(25 μM) 및 (-)-세사민(25 μM)의 단독처치는 Bad(Ser112) 활성화를 유도하였으며, 6-OHDA(100 μM)-유도 Bad(Ser112) 활성화의 감소작용에 대하여 저해작용을 나타내었다(전처치 1 시간)(도 12). 그러므로 아사리닌 및 (-)-세사민이 6-OHDA-유도 세포독성 작용에 대한 저해작용이 있음을 알 수 있다.
The single treatment of asarinin (25 μM) and (-) - sesamin (25 μM) induced Bad (Ser112) activation and inhibited 6-OHDA (100 μM) -induced Bad (Ser112) (
<실시예 11> ≪ Example 11 > 아사리닌 및 (-)-세사민의 6-OHDA-유도 세포독성 방어작용: Caspase-3 활성 저해작용6-OHDA-Induced Cytotoxic Action of Asarinine and (-) - Sesamin: Caspase-3 Inhibitory Activity
Caspase-3의 활성은 세포사(apoptosis) 과정에서 활성화되어 세포사를 유도한다(Jin et al., Neurosci., 170, 390-398, 2010). 그러므로 6-OHDA 세포독성 작용에 대한 아사리닌 및 세사민의 caspase-3 활성에 대한 영향을 검토하였다.
The activity of caspase-3 is activated during apoptosis and induces apoptosis (Jin et al., Neurosci., 170, 390-398, 2010). Therefore, we examined the effects of asarinin and sesamin on caspase-3 activity on 6-OHDA cytotoxicity.
Caspase-3 및 cleaved caspase-3의 활성화(인산화)는 caspase-3 및 cleaved caspase-3(Asp 175)의 인산화를 Western blotting 법에 의하여 측정하였다(Jin et al., Neurosci., 170, 390-398, 2010).
Activation (phosphorylation) of caspase-3 and cleaved caspase-3 was measured by Western blotting of caspase-3 and cleaved caspase-3 (Asp 175) (Jin et al., Neurosci., 170, 390-398 , 2010).
아사리닌(25 μM) 및 (-)-세사민(25 μM)의 단독처치는 caspase-3 활성에는 영향을 주지 않았으나, 6-OHDA(100 μM) 전처치는 caspase-3 활성을 증가시켜 세포사를 유도하였으며(전처치 12 시간), 아사리닌 및 (-)-세사민의 병용처치(농도 각 25 μM))는 6-OHDA-유도 caspase 활성 증가작용에 대하여 저해작용을 나타내었다(도 13). 그러므로 아사리닌 및 (-)-세사민이 6-OHDA-유도 세포독성 작용에 대한 저해작용이 있음을 알 수 있다.
The single treatment of asarinin (25 μM) and (-) - sesamin (25 μM) did not affect caspase-3 activity, but pretreatment with 6-OHDA (100 μM) increased caspase- (
<실시예 12> ≪ Example 12 > 아사리닌 및 (-)-세사민의 PC12 세포주에 대한 세포독성 작용Cytotoxic effect of asarinin and (-) - sesamin on PC12 cell line
아사리닌은 전처치 24 시간 후, MTT 방법에 의한 PC12 세포중의 세포생존율(cell vuability)을 검토한 결과 250 μM 이상의 농도에서 세포독성을 나타내었다(250 μM, 16% 감소; 500 μM, 28% 감소)(도 14A). 또한 (-)-세사민은 전처치 24 시간 후 PC12 세포중의 세포생존율을 검토한 결과 250 μM 농도에서는 세포독성을 나타내지 않았으며(8% 감소), 500 μM 에서는 세포독성을 나타내었다(15% 감소)(도 14B).
Asarinin showed cytotoxicity (250 μM, 16% reduction; 500 μM, 28%) at a concentration of 250 μM or more after 24 hours of pretreatment and examined cell viability in PC12 cells by MTT method (Fig. 14A). In addition, (-) - sesamin did not show cytotoxicity at the concentration of 250 μM (8% decrease) and cytotoxicity at 500 μM (15% decrease) ) (Fig. 14B).
<실시예 13> ≪ Example 13 > 동물시험Animal test
PD 동물 모델의 작성은 대표적으로 6-OHDA의 뇌실투여법이 사용되고 있다(Blum et al., Neurobiol., 65, 135-172, 2001).
PD animal models are typically prepared by the ventricular administration of 6-OHDA (Blum et al., Neurobiol., 65, 135-172, 2001).
(-)-세사민 및 아사리닌은 PC12 세포중의 도파민 생합성 증가 작용 및 6-OHDA-유도 세포독성에 대한 저해작용을 나타내었으며, 활성의 정도는 두 화합물 모두 유의적으로 동등하게 나타났으나, 도파민 생합성 증가 작용은 (-)-세사민이 미약하지만 강하게 나타났다. 그러므로 (-)-세사민을 이용하여 PD 동물모델에 대한 뇌중 도파민 신경계의 보호작용을 검토하였다. (-)-세사민의 투여농도는 30 mg/kg을 선택하였으며, 이는 예비실험과 문헌을 참조하였다(Fujikawa et al., Biol Pharm Bull 28:169-172, 2005).
(-) - sesamin and asarinin showed inhibitory effects on dopamine biosynthesis and 6-OHDA-induced cytotoxicity in PC12 cells, and the degree of activity was significantly similar in both compounds, whereas dopamine (-) - sesamin was weak but strong. Therefore, we examined the protective effect of the brain dopamine neuronal system on PD animal models using (-) - sesamin. The dose of (-) - sesamin was selected to be 30 mg / kg, which was referred to preliminary experiments and literature (Fujikawa et al., Biol Pharm Bull 28: 169-172, 2005).
1) PD 모델의 작성 1) Creating a PD model
6-OHDA-유도 PD 동물모델은 백서(rats)의 뇌실부위에 6-OHDA를 투여하여 작성하였다(Schober, Cell. Tissue Res. 318, 215-224, 2004). 실험동물(백서, 웅성, Sprague-Dawley rat, 250-300 g)을 마취한 다음(Zoletil 50 사용, 100 mg/kg, i.p.; Vibac, Carros, 프랑스), 뇌정위 수술기구(Stereotaxic apparatus, KOPF instrument, USA)를 사용하여 동물을 고정한 후, 한쪽 부위의 흑질 치밀부위에 6-OHDA(8 μg/2 μl, 1 μl/min)를 투여하여 작성하였다(위치: bregma에서 앞 -5.3 mm, 중앙선에서 +1.9 mm 옆, 경막에서 -7.5 mm 깊이)(Paxinos and Watson, The rat brain in stereotaxic coordinates. Academic Press, Australia, 1986). PD 모델의 작성은 6-OHDA 투여 2주간으로 하였다.
The 6-OHDA-induced PD animal model was prepared by administering 6-OHDA to the ventricular zone of rats (Schober, Cell. Tissue Res. 318, 215-224, 2004). The animals were anesthetized (
2) L-DOPA 및 (-)-세사민의 투여 2) Administration of L-DOPA and (-) - sesamin
6-OHDA-유도 PD 동물모델에 대한 (-)-세사민(30 mg/kg)의 투여는 6-OHDA 투여 2주간 후에 1일1회(10:00 a.m.) 28 일간 투여 하였다(대조군, 0.9% saline 투여), L-DOPA(15 mg/kg, i.p.; benserazide, 15 mg/kg/day, i.p., 병용)의 투여는 세사민 투여 2 시간 후 1일 1회 28일간 투여 하였다. The administration of (-) - sesamin (30 mg / kg) to 6-OHDA-induced PD animal models was administered for 28 days once a day (10:00 am) after 2 weeks of 6-OHDA administration (control, 0.9% saline), L-DOPA (15 mg / kg, ip; benserazide, 15 mg / kg / day, ip) was administered for 2 days after ceceamine administration for 28 days once a day.
3) TH-면역조직화학(TH-immunohistochemistry) 염색 3) TH-immunohistochemistry staining
적출한 뇌는 관류고정(4% paraformaldehyde/50 mM phosphate 완충용액)법에 의하여 고정하고, 동결박편을 제조한 다음, TH-면역조직화학 염색은 일차 TH 항체(rabbit anti-TH antibody, 1:1000)와 이차 항체를 이용하여 시행하였다. 흑질 치밀부위의 TH-면역조직화학의 염색성 측정은 광학현미경(x 200 배, Zeiss Axiolab, Jena, Germany)을 사용하여 측정하였고, TH-면역양성 신경세포(TH-immunopositive cell; dopamine 신경세포)의 수는 흑질에서 일정면적 내의 염색 세포수를 계수하여 시행하였다(Lee et al., Neurosci., 72, 641-653, 1996).
The isolated brain was fixed with perfusion fixation (4% paraformaldehyde / 50 mM phosphate buffer solution) and the TH-immunohistochemical staining was performed using a primary TH antibody (rabbit anti-TH antibody, 1: 1000 ) And secondary antibody. The TH-immunohistochemical staining of the denuded area of the thymus was measured using an optical microscope (× 200 magnification, Zeiss Axiolab, Jena, Germany) and TH-immunopositive cells (dopamine neurons) (Lee et al., Neurosci., 72, 641-653, 1996). The number of stained cells within a certain area of black cells was counted.
도 15에서 알 수 있는 바와 같이, 6-OHDA-유도 PD 동물모델(도 A-III, B-III)에 대한 (-)-세사민(30 mg/kg)의 투여(28일간)는 6-OHDA-유도 세포 독성 작용을 유의적으로 저해하였다(뇌신경세포 보호작용)(도 15, A-VI, B-VI). 또한 6-OHDA-유도 PD 동물모델(도 15, A-III, B-III)에 대한 L-DOPA(15 mg/kg) 처치는 미약한 뇌신경세포 보호작용(도 15, A-V, B-V)을 나타내었으며, 이러한 작용은 (-)-세사민(30 mg/kg)의 투여에 의하여 더욱 유의적으로 증가하였다(도 15, A-VII, B-VII).
As can be seen in FIG. 15, administration of (-) - sesamin (30 mg / kg) to a 6-OHDA-induced PD animal model (Figures A-III, B- -Induced cytotoxic effect (neuronal cell protection function) (Fig. 15, A-VI, B-VI). In addition, L-DOPA (15 mg / kg) treatment on 6-OHDA-induced PD animal models (Fig. 15, A-III, B-III) showed weak brain cervical cytoprotection , And this effect was significantly increased by the administration of (-) - sesamin (30 mg / kg) (Fig. 15, A-VII, B-VII).
4) Dopamine, norepinephrine, DOPAC 및 HVA 함량 측정 4) Measurement of Dopamine, norepinephrine, DOPAC and HVA content
뇌시료(선조체-흑질 부위)에 대한 dopamine 및 norepinephrine 함량은 내부표준 isoproterenol(1 nmol/ml, 100 μl)을 이용한 HPLC 법에 의하여 측정하였다(Choi et al., Molecules. 15, 2814-2518, 2010; Lee et al., Biochem. Pharmacol., 66, 1787-1795, 2003). 또한 시료중의 DOPAC 및 HVA의 함량은 시료에 HClO4(300 μ1)를 가한 다음 균질화 하고 원심분리(50,000×g, 4°C, 15분)후 상등액은 여과시킨 후 여과액(50 μl)을 HPLC-전기화학검출기(ECD)를 이용하여 정량하였다(Choi et al., Molecules 18, 4342-4356, 2013).
The concentration of dopamine and norepinephrine in brain samples (striatum-black region) was measured by HPLC using internal standard isoproterenol (1 nmol / ml, 100 μl) (Choi et al., Molecules . 15 , 2814-2518, 2010 Lee et al., Biochem. Pharmacol., 66, 1787-1795, 2003). The amount of DOPAC and HVA in the sample was determined by adding HClO 4 (300 μl) to the sample, homogenizing the mixture, centrifuging (50,000 × g, 4 ° C, 15 min) (Choi et al., Molecules 18, 4342-4356, 2013) using an HPLC-electrochemical detector (ECD).
도 16에서 알 수 있는 바와 같이, 6-OHDA-유도 PD 동물모델은 dopamine 신경세포의 사멸에 의하여 뇌중의 dopamine, norepinephrine, DOPAC 및 HVA의 함량은 감소하였으며(도 16 A-D, III; 투여 14일후), 또한 28일 이후에는 더욱 dopamine 신경계는 퇴행되어 뇌중의 dopamine, norepinephrine, DOPAC 및 HVA의 함량은 현저히 감소하였다(도 16 A-D, IV). 그러나 6-OHDA-유도 PD 동물모델(도 16 A-D, 그룹 III)에 대한 (-)-세사민(30 mg/kg, 28일간)의 투여에 의하여 뇌중의 dopamine, norepinephrine, DOPAC 및 HVA의 함량은 유의적으로 증가하였다(뇌신경세포 보호작용)(도 16, A-D, VI). 또한 6-OHDA-유도 PD 동물모델(도 16, A-D, III)에 대한 L-DOPA(15 mg/kg) 처치는 미약한 뇌신경세포 보호작용(도 16, A-D, V)을 나타내어, 뇌중의 dopamine, norepinephrine, DOPAC 및 HVA의 함량이 증가하였으며, 이러한 작용은 (-)-세사민(30 mg/kg)의 투여에 의하여 더욱 유의적으로 증가하였다(도 16, A-D, VII).
As can be seen from FIG. 16, the dopamine, norepinephrine, DOPAC and HVA contents of the brain were decreased due to the death of dopamine neurons in the animal model of 6-OHDA-induced PD (Fig. 16 AD, III; , And after 28 days, the dopamine nervous system was further degenerated, and the content of dopamine, norepinephrine, DOPAC and HVA in brain was significantly decreased (FIG. 16 AD, IV). However, the contents of dopamine, norepinephrine, DOPAC and HVA in the brain were decreased by administration of (-) - sesamin (30 mg / kg, 28 days) to the 6-OHDA-induced PD animal model (Cranial nerve cell protection function) (Fig. 16, AD, VI). In addition, the treatment of L-DOPA (15 mg / kg) with 6-OHDA-induced PD animal models (Fig. 16, AD and III) showed weak brain cervical cytoprotection (Fig. 16, AD, V) , norepinephrine, DOPAC and HVA were increased. This effect was further increased by the administration of (-) - sesamin (30 mg / kg) (FIG. 16, AD, VII).
상기 결과로부터, (-)-세사민은 6-OHDA-유도 PD 동물모델에서 6-OHDA-유도 신경세포 독성작용에 대한 도파민 신경세포 보호작용과, L-DOPA 효능에 대한 증가작용을 나타내었다. 이는 (-)-세사민이 파긴슨병의 증상 예방효능과 L-DOPA의 치료효능의 증가작용이 있음을 나타내고 있다.
From the above results, (-) - sesamin exhibited an effect on dopamine neuron cytoprotection and L-DOPA potency against 6-OHDA-induced neuronal cytotoxicity in 6-OHDA-induced PD animal models. This indicates that (-) - sesamin inhibits the symptom of the disease caused by the disease and increases the therapeutic efficacy of L-DOPA.
따라서, 본 발명에 따른 아사리닌 및 (-)-세사민은 PKA-CREB 활성화 유도작용에 의한 도파민 생합성 효소 TH의 활성화를 매개하여 도파민 생합성 증가작용과 L-DOPA-유도 도파민 생합성 작용에 대한 증가작용을 나타내며, p38MAPK-JNK 신호전이 저해작용 및 ERK-Bad-Bax 신호전이 활성화를 통한 caspase-3 활성 저해작용을 매개하여 6-OHDA-유도 세포독성 작용을 저해함을 알 수 있어, 파킨슨병 치료 및 예방과 관련한 치료제 개발 분야에 유용하게 응용할 수 있다.
Therefore, the asarinin and (-) - sesamin according to the present invention mediate the activation of the dopamine biosynthesis enzyme TH by the PKA-CREB activation inducing action, thereby increasing the dopamine biosynthesis-increasing action and the L-DOPA-induced dopamine biosynthesis , And inhibits 6-OHDA-induced cytotoxic effects by mediating caspase-3 activity inhibition through p38 MAPK-JNK signal transduction inhibition and ERK-Bad-Bax signal transduction activation. Thus, treatment and prevention of Parkinson's disease The present invention can be applied to a therapeutic agent development field.
또한, 본 발명에 따른 아사리닌 및 (-)-세사민은 L-DOPA를 병용투여하는 경우, L-DOPA 단독투여에 비해 도파민 함량 증가작용, PKA 인산화(phosphorylation) 작용,TH 인산화(phosphorylation) 작용, CREB 인산화(phosphorylation) 작용이 증대됨을 알 수 있어, 파킨슨병 치료 및 예방과 관련한 치료제로 유용하게 개발될 수 있다.In addition, asparagine and (-) - sesamin according to the present invention have a dopamine content increasing action, a PKA phosphorylation action, a TH phosphorylation action, The phosphorylation of CREB is increased, and thus it can be usefully developed as a therapeutic agent for the treatment and prevention of Parkinson's disease.
Claims (3)
A pharmaceutical composition for preventing or treating Parkinson's disease, comprising asarinin or (-) - sesamin (L-DOPA) and L-DOPA as an active ingredient.
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