CN111655255B - 包含二萜类化合物的用于预防或治疗神经退行性疾病的组合物 - Google Patents
包含二萜类化合物的用于预防或治疗神经退行性疾病的组合物 Download PDFInfo
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Abstract
本发明涉及包含二萜类化合物或其药学上可接受的盐的用于预防或治疗神经退行性疾病的药学组合物。具体地，本发明的二萜类化合物将核受体相关因子1(Nurr1)蛋白激活，抑制炎症反应，由此可以预防或治疗因核受体相关因子1蛋白的活性抑制而引发的神经退行性疾病。
Description
技术领域
本发明涉及包含二萜类化合物或其药学上可接受的盐的用于预防或治疗神经退行性疾病的药学组合物。
而且，本发明涉及包含二萜类化合物或其药学上可接受的盐的用于预防或改善神经退行性疾病的健康功能食品。
背景技术
从天然物质中分离的天然生物活性物质的多种生物体调节功能为人所知，在利用天然生物活性物质的新药开发、健康功能食品等领域中的研究日益活跃。然而，天然物质中包含多种生物活性物质，各种物质的物理、化学性质不相同，即使以相同的天然物质为对象进行提取，根据提取溶剂的种类，所分离的生物活性物质的种类也不相同。而且，有报道称即使是从相同的天然物质中分离的生物活性物质，也呈现出每个种类所呈现的活性及其效果不相同的特性。因此，最近正在进行以相同的天然物质为对象而改变提取溶剂来分离以前未知的具有新的生物活性物质的研究。
神经退行性疾病(neurodegenerative diseases)与神经细胞衰退、丧失功能、以及经常性凋亡的情况下的症状有关。患有神经退行性疾病的患者在认知(cognitive)或运动(motor)能力方面有可能会遭受严重的退化，因为这些疾病大多是进行性的，最终这些患者的生活质量以及对生活的期待有可能会显著降低。
这些疾病包括帕金森病(Parkinson's Disease；PD)、阿尔茨海默氏病(Alzheimer's Disease；AD)、肌萎缩侧索硬化(amyotrophic lateral sclerosis；ALS)、亨廷顿病(Huntington's Disease；HD)、额颞痴呆(Frontotemporal Dementia)、皮质基底节变性(Cortico Basal Degeneration)、进行性核上性麻痹(progressive supranuclearpalsy；PSP)及其他疾病。
另一方面，相当数量的神经退行性疾病与核受体相关因子1蛋白有密切的关联。具体地，在美国公开专利第2009-0226401号中公开了作为神经退行性疾病的一种的帕金森病是与多巴胺神经细胞有关的疾病，还公开了在受体相关因子1被激活的情况下，将呈现出帕金森病的治疗效果。而且，在国际专利公开公告WO2010-04221号中公开了如下的方法，即，核受体相关因子1在使多巴胺激活的过程中起到必不可少的作用，将核受体相关因子1激活并调节激活多巴胺的神经传递来治疗帕金森病。
因上述核受体相关因子1的功能障碍而引发的具有代表性的神经退行性疾病为帕金森病。帕金森病以震颤、僵硬、运动缓慢和步态异常等作为主要症状，作为现代高龄化社会中重要疾病中的一种，是在大脑的黑质(substantia nigra)、纹状体(corpus striatum)部位，因称为多巴胺(dopamin)的神经递质不足而引发的慢性疾病。
作为用于治疗上述帕金森病的药物，已知L-多巴(L-dopa)制剂、多巴胺受体激动剂、抗胆碱药剂、咪多吡(Eldepryl)等，这些药物中的大部分仅起到调节症状，而并非根源性治疗，因此，需要持续服用药物。至今，作为帕金森病治疗剂制造了众多药物并得到商业化，但是，尚未开发出用于完全治疗帕金森病的根本性治疗剂。
对此，本发明人员使用多种提取及分馏溶剂，使从芫花(Daphne genkwa)的花、茎和根中获得的多种化合物激活核受体相关因子1，确认了抑制炎症反应的效果，并确认了对于因核受体相关因子1功能障碍而引发的包括帕金森病在内的多种神经退行性疾病的治疗效果优秀，完成了本发明。
本发明参照了美国公开专利第2009-0226401号及国际专利公开公告WO2010-04221号的发明内容。
发明内容
发明要解决的技术问题
本发明的目的在于提供包含二萜类化合物或其药学上可接受的盐的用于预防或治疗神经退行性疾病的药学组合物。
而且，本发明的目的在于提供包含二萜类化合物或其药学上可接受的盐的用于预防或改善神经退行性疾病的健康功能食品。
用于解决问题的方案
作为本发明的一个实施方式，本发明提供用于预防或治疗神经退行性疾病的药学组合物，其包含二萜类化合物或其药学上可接受的盐，上述二萜类化合物为选自下述化学式1至化学式16中的任一种以上。
而且，本发明提供用于预防或治疗神经退行性疾病的药学组合物，其将选自下述化学式A表示的化合物中的任一个以上作为有效成分。
本发明的二萜类化合物可以为下述化学式1至化学式16表示的化合物，具体地，可以为芫花酯丙(yuanhuafine)(化学式1)、芫花烯(genkwadaphnine)(化学式2)、芫花素H(genkwanine H)(化学式3)、芫花素M(genkwanine M)(化学式4)、芫花素K(genkwanin K)(化学式5)、芫花酯甙(yuanhuapine)(化学式6)、芫花素A(genkwanin A)(化学式7)、原苯甲酸酯2(orthobenzoate 2)(化学式8)、1,2α-二氢瑞香毒素(1,2α-dihydrodaphnetoxin)(化学式9)或芫花素I(genkwanin I)(化学式10)、acutilonine F(化学式11)、荛花因子M1(wikstroemia factor M1)(化学式12)、普罗斯左汀Q(prostratin Q)(化学式13)、芫花酯乙(yuanhuadine)(化学式14)、芫花酯丁(yuanhuatine)(化学式15)或12-O-正癸烷-2,4,6-三烯酰基-佛波醇(13)-乙酸酯(12-O-n-deca-2,4,6-trienoyl-phorbol-(13)-acetate)(化学式16)。
在以IUPAC(国际纯粹与应用化学联合会)名称进行命名时，作为上述化学式1的化合物的芫花酯丙为(2S,3aR,3cS,4aR,5S,5aS,8bR,9R,10R,10aS)-5,5a-二羟基-4a-(羟甲基)-7,9-二甲基-6-氧代-2-苯基-10a-(丙-1-烯-2-基)-3a,3b,3c,4a,5,5a,8a,9,10,10a-十氢-6H-2,8b-环氧乙烯并[2”,3”:6',7']甘菊环[5',4':3,4]苯并[1,2-d][1,3]二氧戊环-10-基乙酸酯((2S,3aR,3cS,4aR,5S,5aS,8bR,9R,10R,10aS)-5,5a-dihydroxy-4a-(hydroxymethyl)-7,9-dimethyl-6-oxo-2-phenyl-10a-(prop-1-en-2-yl)-3a,3b,3c,4a,5,5a,8a,9,10,10a-decahydro-6H-2,8b-epoxyoxireno[2”,3”:6',7']azuleno[5',4':3,4]benzo[1,2-d][1,3]dioxol-10-yl acetate)、作为化学式2的化合物的芫花烯为(2S,3aR,3cS,4aR,5S,5aS,8bR,9R,10R,10aS)-5,5a-二羟基-4a-(羟甲基)-7,9-二甲基-6-氧代-2-苯基-10a-(丙-1-烯-2-基)-3a,3b,3c,4a,5,5a,8a,9,10,10a-十氢-6H-2,8b-环氧乙烯并[2”,3”:6',7']甘菊环[5',4':3,4]苯并[1,2-d][1,3]二氧戊环-10-基苯甲酸酯((2S,3aR,3cS,4aR,5S,5aS,8bR,9R,10R,10aS)-5,5a-dihydroxy-4a-(hydroxymethyl)-7,9-dimethyl-6-oxo-2-phenyl-10a-(prop-1-en-2-yl)-3a,3b,3c,4a,5,5a,8a,9,10,10a-decahydro-6H-2,8b-epoxyoxireno[2”,3”:6',7']azuleno[5',4':3,4]benzo[1,2-d][1,3]dioxol-10-yl benzoate)、作为化学式3的化合物的芫花素H为(2S,3aR,4S,5S,6S,6aR,7S,8S,9bR,10R,11aR)-4,5,6,6a,7-五羟基-8,10-二甲基-2-苯基-11a-(丙-1-烯-2-基)十二氢-5H-2,9b-环氧乙烯并[5',4':3,4]苯并[1,2-d][1,3]二氧戊环-5-基)甲基苯甲酸酯((2S,3aR,4S,5S,6S,6aR,7S,8S,9bR,10R,11aR)-4,5,6,6a,7-pentahydroxy-8,10-dimethyl-2-phenyl-11a-(prop-1-en-2-yl)dodecahydro-5H-2,9b-epoxyazuleno[5',4':3,4]benzo[1,2-d][1,3]dioxol-5-yl)methyl benzoate)、作为化学式4的化合物的芫花素M为(2S,3aR,3cS,4aR,5S,5aR,6S,7S,8bR,9R,10aR)-5,5a,6-三羟基-7,9-二甲基-2-苯基-10a-(丙-1-烯-2-基)十二氢-4aH-2,8b-环氧乙烯并[2”,3”:6',7']甘菊环[5',4':3,4]苯并[1,2-d][1,3]二氧-4a-基)甲基苯甲酸酯((2S,3aR,3cS,4aR,5S,5aR,6S,7S,8bR,9R,10aR)-5,5a,6-trihydroxy-7,9-dimethyl-2-phenyl-10a-(prop-1-en-2-yl)dodecahydro-4aH-2,8b-epoxyoxireno[2”,3”:6',7']azuleno[5',4':3,4]benzo[1,2-d][1,3]dioxol-4a-yl)methyl benzoate)、作为化学式5的化合物的芫花素K为((2S,3S,3aR,4S,5S,6S,7R,8R,10R,10aR)-7-(苯甲酰氧基)-3,3a,4,5,6,8,10a-七羟基-2,10-二甲基-8-(丙-1-烯-2-基)十四氢苯并[e]甘菊环-5-基)甲基苯甲酸酯((2S,3S,3aR,4S,5S,6S,7R,8R,10R,10aR)-7-(benzoyloxy)-3,3a,4,5,6,8,10a-heptahydroxy-2,10-dimethyl-8-(prop-1-en-2-yl)tetradecahydrobenzo[e]azulen-5-yl)methyl benzoate)、作为化学式6的化合物的芫花酯甙为(2S,3aR,3cS,4aR,5S,5aS,7S,8bR,9R,10R,10aS)-5,5a-二羟基-4a-(羟甲基)-7,9-二甲基-6-氧代-2-苯基-10a-(丙-1-烯-2-基)十二氢-6H-2,8b-环氧乙烯并[2”,3”：6',7']甘菊环并[5',4':3,4]苯并[1,2-d][1,3]二氧戊环-10-基乙酸酯((2S,3aR,3cS,4aR,5S,5aS,7S,8bR,9R,10R,10aS)-5,5a-dihydroxy-4a-(hydroxymethyl)-7,9-dimethyl-6-oxo-2-phenyl-10a-(prop-1-en-2-yl)dodecahydro-6H-2,8b-epoxyoxireno[2”,3”:6',7']azuleno[5',4':3,4]benzo[1,2-d][1,3]dioxol-10-yl acetate)、作为化学式7的化合物的芫花素A为(2S,3aR,4S,5S,6S,6aR,7S,9bR,10R,11aR)-5-(羟甲基)-8,10-二甲基-2-苯基-11a-(丙-1-烯-2-基)-3a,3b,4,5,6,7,9a,10,11,11a-十氢-6aH-2,9b-环氧甘菊环并[5',4'：3,4]苯并[1,2-d][1,3]二氧-4,5,6,6a,7-五醇((2S,3aR,4S,5S,6S,6aR,7S,9bR,10R,11aR)-5-(hydroxymethyl)-8,10-dimethyl-2-phenyl-11a-(prop-1-en-2-yl)-3a,3b,4,5,6,7,9a,10,11,11a-decahydro-6aH-2,9b-epoxyazuleno[5',4':3,4]benzo[1,2-d][1,3]dioxole-4,5,6,6a,7-pentaol)、作为化学式8的化合物的原苯甲酸酯2为(2S,3aR,3cS,4aR,5S,5aR,6S,7S,8bR,9R,10aR)-4a-(羟甲基)-7,9-二甲基-2-苯基-10a-(丙-1-烯-2-基)十二氢-5aH-2,8b-环氧乙烯并[2”,3”：6',7']甘菊环并[5',4':3,4]苯并[1,2-d][1,3]二氧戊环-5,5a,6-三醇((2S,3aR,3cS,4aR,5S,5aR,6S,7S,8bR,9R,10aR)-4a-(hydroxymethyl)-7,9-dimethyl-2-phenyl-10a-(prop-1-en-2-yl)dodecahydro-5aH-2,8b-epoxyoxireno[2”,3”:6',7']azuleno[5',4':3,4]benzo[1,2-d][1,3]dioxole-5,5a,6-triol)及作为化学式9的化合物的1,2α-二氢瑞香毒素为(2S,3aR,3cS,4aR,5S,5aS,7S,8bR,9R,10aR)-5,5a-二羟基-4a-(羟甲基)-7,9-二甲基-2-苯基-10a-(丙-1-烯-2-基)十二氢-6H-2,8b-环氧乙烯并[2”,3”：6',7']甘菊环并[5',4':3,4]苯并[1,2-d][1,3]二氧-6-酮((2S,3aR,3cS,4aR,5S,5aS,7S,8bR,9R,10aR)-5,5a-dihydroxy-4a-(hydroxymethyl)-7,9-dimethyl-2-phenyl-10a-(prop-1-en-2-yl)dodecahydro-6H-2,8b-epoxyoxireno[2”,3”:6',7']azuleno[5',4':3,4]benzo[1,2-d][1,3]dioxol-6-one)的IUPAC名称。
在以IUPAC名称进行命名时，作为上述化学式11的化合物的acutilonineF为(2S,3aR,3cS,4aR,5S,5aS,6S,7S,8bR,9R,10aR)-5,5a-二羟基-4a-(羟甲基)-7,9-二甲基-2-苯基-10a-(丙-1-烯-2-基)十二氢-6H-2,8b-环氧乙烯并[2”,3”：6',7']甘菊环并[5',4':3,4]苯并[1,2-d][1,3]二氧-6-基(2E,4E,6E)-癸烷-2,4,6-三烯酸酯((2S,3aR,3cS,4aR,5S,5aS,6S,7S,8bR,9R,10aR)-5,5a-dihydroxy-4a-(hydroxymethyl)-7,9-dimethyl-2-phenyl-10a-(prop-1-en-2-yl)dodecahydro-6H-2,8b-epoxyoxireno[2”,3”:6',7']azuleno[5',4':3,4]benzo[1,2-d][1,3]dioxol-6-yl(2E,4E,6E)-deca-2,4,6-trienoate)、作为化学式12的化合物的荛花因子M1为(2S,3aR,3cS,4aR,5S,5aS,6S,7S,8bR,9R,10aR)-5,5a-二羟基-4a-(羟甲基)-7,9-二甲基-2-苯基-10a-(丙-1-烯-2-基)十二氢-6H-2,8b-环氧乙烯并[2”,3”：6',7']甘菊环并[5',4'：3,4]苯并[1,2-d][1,3]二氧戊环-6-基(2E,4E)-癸烷-2,4-二烯酸酯((2S,3aR,3cS,4aR,5S,5aS,6S,7S,8bR,9R,10aR)-5,5a-dihydroxy-4a-(hydroxymethyl)-7,9-dimethyl-2-phenyl-10a-(prop-1-en-2-yl)dodecahydro-6H-2,8b-epoxyoxireno[2”,3”:6',7']azuleno[5',4':3,4]benzo[1,2-d][1,3]dioxol-6-yl(2E,4E)-deca-2,4-dienoate)、作为化学式13的化合物的普罗斯左汀Q为(2S,3aR,3bS,3cS,4aR,5S,5aS,8aR,8bR,9R,10R,10aS)-5,5a-二羟基-4a-(羟甲基)-7,9-二甲基-2-((1E,3E)-壬烷-1,3-二烯-1-基)-6-氧代-10a-(丙-1-烯-2-基)-3a,3b,3c,4a,5,5a,8a,9,10,10a-十氢-6H-2,8b-环氧乙烯并[2”,3”:6',7']甘菊环并[5',4':3,4]苯并[1,2-d][1,3]二氧-10-基-乙酸酯((2S,3aR,3bS,3cS,4aR,5S,5aS,8aR,8bR,9R,10R,10aS)-5,5a-dihydroxy-4a-(hydroxymethyl)-7,9-dimethyl-2-((1E,3E)-nona-1,3-dien-1-yl)-6-oxo-10a-(prop-1-en-2-yl)-3a,3b,3c,4a,5,5a,8a,9,10,10a-decahydro-6H-2,8b-epoxyoxireno[2”,3”:6',7']azuleno[5',4':3,4]benzo[1,2-d][1,3]dioxol-10-yl acetate)、作为化学式14的化合物的芫花酯乙为(2S,3aR,3cS,4aR,5S,5aS,8bR,9R,10R,10aS)-5,5a-二羟基-4a-(羟甲基)-7,9-二甲基-6-氧代-2-苯基-10a-(丙-1-烯-2-基)十二氢-6H-2,8b-环氧乙烯并[2”,3”：6',7']甘菊环并[5',4'：3,4]苯并[1,2-d][1,3]二氧-10-基-苯甲酸酯((2S,3aR,3cS,4aR,5S,5aS,8bR,9R,10R,10aS)-5,5a-dihydroxy-4a-(hydroxymethyl)-7,9-dimethyl-6-oxo-2-phenyl-10a-(prop-1-en-2-yl)dodecahydro-6H-2,8b-epoxyoxireno[2”,3”:6',7']azuleno[5',4':3,4]benzo[1,2-d][1,3]dioxol-10-yl benzoate)、作为化学式15的化合物的芫花酯丁为(1aR,1bS,4aR,7aS,7bS,8R,9R,9aS)-9a-乙酰氧基-4a,7b-二羟基-3-(羟甲基)-1,1,6,8-四甲基-5-氧代-1a,1b,4,4a,5,7a,7b,8,9,9a-十氢-1H-环丙烷[3,4]苯并[1,2-e]甘菊环-9-基-(2E,4E)-癸烷-2,4-二烯酸酯((1aR,1bS,4aR,7aS,7bS,8R,9R,9aS)-9a-acetoxy-4a,7b-dihydroxy-3-(hydroxymethyl)-1,1,6,8-tetramethyl-5-oxo-1a,1b,4,4a,5,7a,7b,8,9,9a-decahydro-1H-cyclopropa[3,4]benzo[1,2-e]azulen-9-yl(2E,4E)-deca-2,4-dienoate)及作为化学式16的化合物的12-O-正癸烷-2,4,6-三烯酰基-巴豆醇(13)-乙酸盐为(4aR,7bS,8R,9R,9aS)-9a-乙酰氧基-4a,7b-二羟基-3-(羟甲基)-1,1,6,8-四甲基-5-氧代-1a,1b,4,4a,5,7a,7b,8,9,9a-十氢-1H-环丙烷[3,4]苯并[1,2-e]甘菊环-9-基(2E,4E,6E)-癸烷-2,4,6-三烯酸酯((4aR,7bS,8R,9R,9aS)-9a-acetoxy-4a,7b-dihydroxy-3-(hydroxymethyl)-1,1,6,8-tetramethyl-5-oxo-1a,1b,4,4a,5,7a,7b,8,9,9a-decahydro-1H-cyclopropa[3,4]benzo[1,2-e]azulen-9-yl(2E,4E,6E)-deca-2,4,6-trienoate)的IUPAC名称。
上述二萜类化合物可由所属领域技术人员通过公知的方法制备、也可通过购买市售的化合物来合成制备、还可使用极性或非极性溶剂从本发明所属技术领域公知的植物中分离纯化。具体地，上述化合物可从芫花中提取及分离。更具体地，上述化合物可从芫花植物的花、根、茎的提取物中分离。
本发明中所使用的术语“芫花(Daphne genkwa)”是指双子叶植物桃金娘目芫花科的落叶灌木。也被称为黄花树、红豆树，具有主要生长在海边附近的特性。
本发明的芫花提取物是指从芫花植物的花、根、茎中获得的提取物。具体地，上述提取物可以为用水或有机溶剂来提取芫花植物的花、根和/或茎而获得的提取物，更具体地，可以为用水、C1至C5的低级烷基醇或它们的混合溶剂进行提取而获得的提取物。上述烷基醇可以为10％、20％、30％、40％、50％、60％、70％、80％、90％或100％的烷基醇。
根据本发明的一个实施例的芫花提取物优选为用80％乙醇提取的提取物。
上述芫花提取物可以为提取物的分离物，上述分离物是指使用特定溶剂从上述芫花提取物中分离本发明的化合物而获得的活性分离物(fraction)。
根据本发明的一个实施例，对于上述获得的芫花提取物，可利用己烷、氯仿、乙酸乙酯、丁醇或蒸馏水等有机溶剂或这些的混合溶剂来分离各个溶剂的分离层而获得，利用色谱法等所属技术领域公知的分离方法来将本发明的化合物以高纯度分离纯化来制备分离物。
本发明的药学组合物可以以选自上述化学式1至化学式16的化合物中的任一种以上的化合物的形态或以其药学上可接受的盐的形态使用。
本发明中所使用的术语“药学上可接受的盐”是指保有上述化合物的期望的生物学和/或生理学活性、最小限度呈现非期望的毒物学效果的所有盐。作为盐，使用通过药学上可接受的游离酸(free acid)所形成的酸式盐。酸式盐用通常的方法制备，例如将化合物溶解在过量的酸水溶液中，使用与水混溶的有机溶剂，例如甲醇、乙醇、丙酮或乙腈使该盐沉淀来制备。可以对相同摩尔量的化合物及水中的酸或醇(例如，乙二醇单甲醚)进行加热，接着使上述混合物蒸发干燥或抽滤所析出的盐。此时，作为游离酸，可以使用无机酸和有机酸，作为无机酸，可以使用盐酸、氢溴酸、磷酸、硝酸、硫酸、酒石酸等，作为有机酸，可以使用甲烷磺酸、对甲苯磺酸、乙酸、三氟乙酸、马来酸(maleic acid)、琥珀酸、草酸、苯甲酸、酒石酸、富马酸(fumaric acid)、扁桃酸、丙酸(propionic acid)、柠檬酸(citric acid)、乳酸(lactic acid)、乙醇酸(glycollic acid)、葡萄糖酸(gluconic acid)、半乳糖醛酸、谷氨酸、戊二酸(glutaric acid)、葡萄糖醛酸(glucuronic acid)、天冬氨酸、抗坏血酸、羧酸、香草酸、氢碘酸等，但并不局限于此。
而且，可使用碱来制备药学上可接受的金属盐。碱金属盐或碱土金属盐例如可如下获得：使化合物溶解在过量的碱金属氢氧化物或碱土金属氢氧化物溶液中，过滤不溶解化合物盐之后，将剩余液体蒸发干燥来获得。此时，作为金属盐，特别是制备钠盐、钾盐或钙盐这些在制药方面适合的盐，但并不局限于此。而且，与之对应的银盐可以将碱金属盐或碱土金属盐与适当的银盐(例如，硝酸银)反应来获得。
上述化学式1至16的化合物在药学上可接受的盐只要没有另外说明，几乎包含全部有可能存在于上述化合物的酸性盐或碱性盐。例如，作为药学上可接受的盐，可包含羟基的钠、钙和钾盐等，作为氨基的其他药学上可接受的盐，可以为氢溴酸、硫酸盐、硫酸氢盐、磷酸盐、磷酸氢盐、磷酸二氢盐、乙酸盐、琥珀酸盐、柠檬酸盐、酒石酸盐、乳酸盐、扁桃酸盐、甲磺酸盐(Mesylate)及对甲苯磺酸盐(Tosilate)等，可通过所属技术领域中已知的盐的制备方法来制备。
包含本发明的二萜类化合物或其药学上可接受的盐的药学组合物可用于预防或治疗神经退行性疾病。具体地，上述神经退行性疾病可选自帕金森病(Parkinson'sDisease；PD)、阿尔茨海默氏病(Alzheimer's disease；AD)、肌萎缩侧索硬化(amyotrophiclateral sclerosis；ALS)、亨廷顿病(Huntington's disease；HD)、额颞痴呆(Fronto-Tmporal Dementia)、皮质基底节变性(Cortico Basal Degeneration)及进行性核上性麻痹(progressive supranuclear palsy；PSP)中的任一种。
根据本发明的一个实施例，确认了化学式1至16的化合物会增加核受体相关因子1蛋白的活性度(表2及图2)、抑制神经细胞中一氧化氮的生成(表4)。具体地，化学式2及8的化合物呈现出更优秀的增加核受体相关因子1蛋白活性的效果。更具体地，化学式2不仅呈现出增加核受体相关因子1蛋白活性的效果，还呈现出更优秀的抑制一氧化氮生成的活性。
而且，确认了化学式11至16的化合物会增加核受体相关因子1蛋白的活性度(表3)、抑制神经细胞中一氧化氮的生成(表5)，并抑制神经细胞中促炎性细胞因子的生成(图1)。具体地，化学式13、14及16的化合物呈现出更优秀的增加核受体相关因子1蛋白活性的效果，更具体地，化学式14不仅呈现出增加核受体相关因子1蛋白活性的效果，还呈现出更优秀的抑制一氧化氮生成的活性。
因此，选自包含本发明的化学式1至16的化合物中的任一种以上的化合物或其的盐的药学组合物可有效地用于预防或治疗神经退行性疾病，上述药学组合物所包含的化合物可以为化学式1至16的化合物中任一种以上的化合物。
本发明中所使用的术语“治疗”是指为了改变需要治疗的各人或细胞的天然过程而进行临床上的介入，治疗可在临床病理状态进行的期间进行或为了预防上述状态而进行。目标治疗效果包括预防疾病的发生或复发、缓和症状、降低疾病导致的所有直接性或间隔性病理学结果、减缓疾病进行速度、减轻或暂时缓和疾病状态、推动或改善预后。优选地，在本发明中涵盖了通过包含二萜类化合物或其药学上可接受的盐的组合物的给药来使神经退行性疾病过程好转的所有行为。而且，“预防”是指通过本发明的包含二萜类化合物或其药学上可接受的盐的组合物的给药来抑制或延迟上述神经退行性疾病的发病的所有行为。
本发明的药学组合物除包含二萜类化合物或其药学上可接受的盐作为有效成分之外，还可追加包含药学上可接受的载体。
本发明中可使用的载体的种类并不受特别的限制，只要是本发明所属技术领域中通常使用的载体则可以任意使用。作为上述载体的非限定性的例子，可以是盐水、无菌水、林格氏液、缓冲盐水、白蛋白注射液、乳糖、右旋糖、蔗糖、山梨糖醇、甘露醇、木糖醇、赤藓糖醇、麦芽糖醇、麦芽糖糊精、甘油、乙醇等。这些可以单独使用或者2种以上混合使用。
而且，本发明的药学组合物在需要的情况下，可以添加使用赋形剂、稀释剂、抗氧化剂、缓冲剂或抑菌剂等其他药学可接受的添加剂，还可附加添加使用填充剂、增量剂、湿润剂、崩解剂、分散剂、表面活性剂、粘合剂和润滑剂等。
在本发明的药学组合物中，以药学组合物的总重量为基准，可包含0.01重量％至90.00重量％的二萜类化合物或其药学上可接受的盐，优选0.01重量％至90.00重量％，更优选0.1重量％至70重量％，进一步优选0.1重量％至50重量％，但并不局限于此，可根据给药对象的状态、具体病症的种类、进行程度等以多种方式进行变更。在需要的情况下，也可整体包含药学组合物。
本发明的药学组合物可制剂化为适于口服给药或胃肠道外给药的多种剂型来使用。
作为利用本发明的药学组合物的口服给药用制剂的非限定性例子，可例举片剂(troches)、菱形锭剂(lozenge)、片剂、水性悬浮液、油性悬浮液、制剂粉末、颗粒剂、乳剂、硬胶囊、软胶囊、糖浆、酏剂等。
为了将本发明的药学组合物制剂化成口服给药用，可以使用如乳糖、蔗糖、山梨糖醇、甘露糖醇、淀粉、支链淀粉、纤维素或明胶等的结合剂、如磷酸二钙等的赋型剂、如玉米淀粉或地瓜淀粉等的崩解剂、如硬脂酸镁、硬脂酸钙、硬脂富马酸钠或聚乙二醇蜡等的润滑剂等，也可使用甜味剂、芳香剂、糖浆剂等。进而，在胶囊的情况下，除上述提及的物质之外，还可追加使用如脂肪油的液体载体等。
作为利用本发明的药学组合物的胃肠道外用制剂的非限定性例子，可以为注射剂、栓剂、呼吸道用粉剂、喷雾气雾剂、软膏剂、涂敷用粉剂、油、乳膏等。
为了将本发明的药学组合物制剂化成胃肠道外给药用制剂，可以使用灭菌水溶液、非水溶剂、悬浮制剂、油剂、冻干制剂、外用制剂等，作为上述非水性溶剂、悬浮制剂可以使用如丙二醇、聚乙二醇、如橄榄油的植物油和如油酸乙酯的可注射的酯等。
在将本发明的药学组合物制剂化成注射液的情况下，将本发明的药学组合物与稳定剂或缓冲剂在水中一同混合来制备溶液或悬浮液，并可将这些制剂化成安瓿(ampoule)或小瓶(vial)的单位给药用制剂。
在将本发明的药学组合物制剂化成气雾剂的情况下，为了使水分散的浓缩液或湿润粉末分散，可以与促进剂等添加剂一同配合。
在将本发明的药学组合物制剂化成软膏、乳膏、涂敷用粉剂、油、皮肤外用剂等的情况下，可使用动物油、植物油、蜡、石蜡、淀粉、黄蓍胶、纤维素衍生物、聚乙二醇、硅酮、膨润土、二氧化硅、滑石粉、氧化锌等作为载体使用来制剂化。
本发明的药学组合物的药学上的有效量、有效给药量根据药学组合物的制剂化方法、给药方式、给药时间和/或给药路径等会有所不同，可根据包括通过药学组合物的给药实现的反应的种类和程度、作为给药对象的个体的种类、年龄、体重、一般健康状态、疾病的症状或程度、性别、饮食、排泄、向对应客体同时或在不同时间一同使用的药物其他组合物的成分等在内的多个因素及医药领域中的类似因子改变，本发明所属技术领域的普通技术人员可轻松确定目标治疗所需的有效给药量并进行处理。
本发明的药学组合物的给药可以1天给药1次，也可分成多次给药。本发明的药学组合物可以作为单独治疗剂给药或者与其他治疗并用给药，可以与现有的治疗剂依次或同时给药。考虑到上述所有因素，在不产生副作用的前提下，可以以能够获得最大效果的量进行给药，这可以由本领域所属技术人员容易地确定。
用于治疗的“药学上的有效量”是指以可适用于医学用途的合理比例抑制或缓和疾病的充分的量，有效容量水平可根据包括个体种类及重症度、年龄、性别、药物的活性、对于药物的敏感度、给药时间、给药路径及排出比例、治疗时间、同时使用的药物在内的要素及其他医学领域公知的要素来确定。本发明的组合物可以作为单独治疗剂给药或者与其他治疗剂并用给药，还可以与现有的治疗剂依次或同时给药。而且，可以单次或多次给药。
考虑到上述所有要素，在不产生副作用的前提下，可以以能够获得最大效果的量进行给药，这点很重要，这可以由本领域所属技术人员容易地确定。例如，对于二萜类化合物或其药学上可接受的盐，药学上有效的量为0.01mg/天/体重㎏至100mg/天/体重㎏，具体地为0.1mg/天/体重㎏至10mg/天/体重㎏。
本发明的药学组合物的给药路径及给药方式可以分别独立，只要上述药学组合物可以到达目的对应部位，则可以没有特别限制地通过任意给药路径及给药方式进行。上述药学组合物可通过口服给药或胃肠道外给药方式给药。
作为本发明的药学组合物的胃肠道外给药方法，可利用静脉内给药、腹腔内给药、肌肉内给药、经皮给药或皮下给药，而且也可以利用将上述组合物涂敷、喷雾、吸入到疾病部位的方法，但并不局限于此。
即使单独使用本发明的药学组合物，也可以发挥出优秀的效果，为了增加治疗效率，还可以与放射线疗法、化学疗法等多种癌症治疗方法并用。
作为本发明的另一个实施方式，本发明提供用于预防或治疗神经退行性疾病的药学组合物，其作为包含二萜类化合物或其药学上可接受的盐的用于预防或治疗因核受体相关因子1功能障碍而诱发的疾病的药学组合物，上述二萜类化合物为选自上述化学式1至化学式16中的任一种以上。
在本发明中，“核受体相关因子1(Nurr1,nuclear receptor related1)”是指也被称为NR4A2(nuclear receptor subfamily[0037]4,group A,member 2)的已知的核受体相关1蛋白，已知其通过人类的NR4A2基因编码。并且已知其与相当量的神经退行性疾病有关。虽然，上述核受体相关因子1蛋白作为孤儿核受体(orphan nuclear receptor)，至今尚未确定针对上述蛋白的配体，但已确定上述核受体相关因子1作为属于细胞内转录因子核受体家族的蛋白质，起到在大脑中维持多巴胺系统(dopaminergic system)的核心作用。因上述核受体相关因子1功能障碍而诱发的疾病并不局限于此，包括帕金森病(Parkinson'sDisease；PD)、阿尔茨海默氏病(Alzheimer's Disease；AD)、肌萎缩侧索硬化(amyotrophiclateral sclerosis；ALS)、亨廷顿病(Huntington's Disease；HD)、额颞痴呆(Frontotemporal Dementia)、皮质基底节变性(Cortico Basal Degeneration)、进行性核上性麻痹(progressive supranuclear palsy；PSP)等神经退行性疾病和因多巴胺功能障碍而诱发的类风湿关节炎、精神分裂症、躁郁症等广泛的炎症性疾病。
作为本发明的另一个实施方式，本发明提供用于预防或改善神经退行性疾病的健康功能食品，其作为包含二萜类化合物或其药学上可接受的盐的用于预防或改善神经退行性疾病的健康功能食品，上述二萜类化合物为选自上述化学式1至化学式16中的任一种以上。
上述健康功能食品作为强调食品的身体调节功能的食品，是利用物理、生物化学、生物工程方法来作用及表达在特定目标以赋予附加价值的食品。该健康功能食品的成分进行了设计加工，以对身体充分发挥与身体防御和身体节奏的调节、疾病的防止及恢复有关的身体调节功能，可包含食品可接受的食品辅助添加剂、甜味剂或功能性原料。
在将本发明的二萜类化合物或其药学上可接受的盐用作健康功能食品(或健康功能饮料添加物)的情况下，可直接添加上述化合物或者与其他食品或食品成分一同使用，根据通常的方法适当使用。上述化合物的混合量可根据其使用目的(预防、健康或改善、治疗处理)来适当确定。
上述健康功能食品可包含多种营养剂、维生素、矿物(电解质)、合成调味剂及天然调味剂等调味剂、着色剂及增进剂(芝士、巧克力等)、果胶酸及其盐、有机酸、保护性胶体增稠剂、pH调节剂、稳定化剂、防腐剂、甘油、醇类、用于碳酸饮料的碳酸化剂等。而且，本发明的健康功能食品可包含用于制备水果及蔬菜饮料的果肉。该成分可以单独或组合使用，这种添加剂的比例一般选自相对于组合物整体重量的0.001重量份至50重量份的范围。
上述健康功能食品的种类没有特别限制。可添加上述化合物的食品为包括香肠、肉类、面包、巧克力、小吃类、糖果类、拉面、比萨、其他面类、口香糖类、包含冰淇淋类的乳制品、各种汤、饮料、茶、口服液、酒精饮料和维生素复合剂等。在剂型化成饮料的情况下，除新的乳酸菌之外，所添加的液体成分并不局限于此，如通常的饮料，可以包含多种香料或天然碳水化合物等作为追加成分。上述天然碳水化合物可以为单糖(例如，葡萄糖、果糖等)、二糖(例如，麦芽糖、蔗糖等)及多糖(例如糊精、环糊精等的一般的糖)及木糖醇、山梨糖醇、赤藓糖醇等的糖醇。
本发明提供神经退行性疾病的治疗方法，包括将包含二萜类化合物或其药学上可接受的盐作为有效成分的药学组合物给药到个体的步骤。
在本发明中，个体是指患有神经退行性疾病或发病的包括人类在内的所有动物，也可以为除人类之外的个体。通过将本发明的药学组合物对个体给药，会在神经退行性疾病的治疗中呈现出优秀的效果。
本发明提供组合物的用途，在制备用于治疗神经退行性疾病的药剂的过程中，包含二萜类化合物或其药学上可接受的盐。
本发明提供组合物，其包含用于神经退行性疾病的治疗的二萜类化合物或其药学上可接受的盐。
发明的效果
本发明的二萜化合物呈现出提高核受体相关因子1蛋白的活性、抑制神经细胞中的炎症反应的效果，由此可有效地用于因核受体相关因子1蛋白的活性被抑制而诱发的包括帕金森病在内的神经退行性疾病的预防或治疗。
附图说明
图1为确认在作为神经细胞的BV-2细胞中，本发明的化合物11至化合物16抑制炎症相关因子表达的图。图1A为通过蛋白质印迹确认抑制白细胞介素-1b(IL-1b)的表达的图。图1B为通过聚合酶链式反应(PCR)确认抑制白细胞介素-1b的mRNA表达的图。图1C为通过PCR确认抑制白细胞介素-6(IL-6)的mRNA表达的图表。图1D为通过PCR确认抑制肿瘤坏死因子-a(TNF-a)的mRNA的表达的图表。
图2为确认本发明的化合物1至10的核受体相关因子1激活效果的图表。
具体实施方式
以下，通过实施例更加详细地说明本发明。但是，这些实施例用于例示性说明本发明，本发明的范围并不局限于这些实施例。
实施例1-a：芫花提取物的制备
将4.47kg的芫花植物(Daphne genkwa)的干燥的花在40L、80％乙醇中浸渍72小时，过滤来获得液相成分。在减压条件下将获得的上述液相成分浓缩之后，制备了435g的芫花植物的花的提取物。
实施例1-b
将4.47kg的芫花(Daphne genkwa)植物的根和茎切碎之后，在12L、80％乙醇中浸渍4小时，进行过滤来分离固体成分与第一次液相成分。将上述分离的固体成分在12L、80％乙醇中再浸渍4小时，并进行过滤来获得第二次液相成分。将上述获得的第一次液相成分与第二次液相成分混合，在减压条件下浓缩上述混合物之后，对浓缩物进行冷冻干燥，制备255.1g的芫花提取物。
实施例2：利用多种溶剂从芫花提取物中分离活性成分
用2L的蒸馏水和2L的己烷、氯仿、乙酸乙酯和丁醇分别依次对在上述实施例1-a中获得的芫花植物的花的提取物进行溶剂分离。其中，对氯仿层进行减压浓缩，利用硅胶柱色谱，以氯仿与甲醇的梯度(gradient)混合溶剂(100∶0、50∶1、20∶1、10∶1、5∶1、2∶1、1∶1)洗脱氯仿分离物(17.6g)，总计获得3个分离物(Fr.C1、C2、C3)。其中，在乙醇与水的梯度混合溶液(60∶40、80∶20、100∶0)的条件下对Fr.C2(4.5g)实施反相柱色谱(ODS silicagelchromatography)来获得5个子分离物(sub-fraction)(Fr.C21、C22、C23、C24、C25)。在氯仿与丙酮的梯度混合溶剂(99∶1-95∶5)的条件下再次对Fr.C23(300mg)实施硅胶(40-63μm；4gflash column)MPLC来获得3个子分离物(sub-fraction)(Fr.C231、C232、C233)。最终将Fr.C233(140mg)通过流速5mL/分钟的55％乙腈洗脱液，进行十八烷基-硅胶高效液相色谱(ODS HPLC)，获得白色粉末形态的芫花酯丙(25.5mg，化学式1的化合物)。根据如下的NMR、MS、[α]20 D数据鉴定上述化合物的结构。
化学式1：芫花酯丙
[α]20 D+29.3(c 0.5,CHCl3)；
ESI-MS,m/z573.9[M+Na]+；
1HNMR以ppm为单位(CD3OD,400MHz)：δH 7.68(dd,J＝7.3,1.8,2H,H-3',H-7'),7.57(s,1H,H-1'),7.41-7.37(m,3H,H-4',H-5',H-6'),5.08(br,1H,H-12),5.05(s,1H,H-16a),5.02(d,J＝2.4,1H,H-16b),4.98(br,1H,H-14),4.14(s,1H,H-5),4.05(d,J＝12.3,1H,H-20a),3.96(m,1H,H-10),3.65(dd,J＝7.3,4.9,1H,H-20b),3.59(s,1H,H-8),2.54(q,J＝7.3,1H,H-11),2.00(s,3H,H-2”),1.86(s,3H,H-17),1.76(d,J＝1.1,3H,H-19),1.32(d,J＝7.3,3H,H-18)；
13CNMR以ppm为单位(CD3OD,101Hz)：δC 209.8(C-3),171.6(C-1”),160.0(C-1),145.0(C-15),138.3(C-2),137.2(C-2'),130.5(C-5'),128.9(C-4',C-6'),127.2(C-3',C-7'),119.1(C-1'),113.9(C-16),85.4(C-13),82.1(C-14),80.3(C-9),79.8(C-12),74.5(C-4),71.3(C-5),65.1(C-20),64.7(C-7),63.2(C-6),48.9(C-10),45.1(C-11),36.6(C-8),21.0(C-2”),19.1(C-18),18.7(C-17),10.0(C-19)。
用氯仿与乙醇(1∶1)的混合溶剂对Fr.C24(210mg)再次实施Sephadex LH-20柱色谱，最终以流速5mL/分钟的65％乙腈洗脱液进行十八烷基-硅胶高效液相色谱，获得白色粉末形态的芫花烯(25.0mg，化学式2的化合物)。根据如下的NMR、MS、[α]20 D数据鉴定上述化合物的结构。
化学式2：芫花烯
[α]20 D+56.7(c 0.1,CHCl3)；
ESI-MS,m/z625.5[M+Na]+；
1HNMR以ppm为单位(CD3OD,400MHz)；δH 7.98(d,J＝7.5,2H,H-3”,H-7”),7.72(m,2H,H-3',H-7'),7.61(m,1H,H-5”),7.59(s,1H,H-1),7.48(t,J＝7.7,2H,H-4”,H-6”),7.40(m,3H,H-4',H-5',H-6'),5.26(br,1H,H-12),5.21(d,J＝2.4,1H,H-14),5.13(s,1H,H-16a),5.02(s,1H,H-16b),4.13(s,1H,H-5),4.06(d,J＝12.3,1H,H-20a),3.99(m,1H,H-10),3.76(d,J＝2.3,1H,H-8),3.68(s,1H,H-7),3.66(d,J＝12.5,1H,H-20b),2.69(q,J＝7.3,1H,H-11),1.90(s,3H,H-17),1.75(s,3H,H-19),1.42(d,J＝7.3,3H,H-18)：
13CNMR以ppm为单位(CD3OD,101Hz)：δC 209.8(C-3),166.9(C-1”),160.0(C-1),144.9(C-15),138.2(C-2),137.2(C-2'),134.5(C-5”),131.1(C-2”),130.7(C-3”,C-7”),130.5(C-5'),129.7(C-4”,C-6”),128.9(C-4',C-6'),127.2(C-3',C-7'),119.1(C-1'),114.1(C-16),85.7(C-13),82.0(C-14),80.4(C-9),80.2(C-12),74.4(C-4),71.4(C-5),65.2(C-20),64.8(C-7),63.2(C-6),49.9(C-10),45.3(C-11),37.0(C-8),19.1(C-17),18.9(C-18),10.0(C-19)。
用氯仿与乙醇(1∶1)的混合溶剂对Fr.C25(130mg)实施Sephadex LH-20柱层析，最终以流速5mL/分钟的70％乙腈洗脱液进行十八烷基-硅胶高效液相色谱，获得白色粉末形态的芫花素H(4.0mg，化学式3的化合物)及芫花素M(4.0mg，化学式4的化合物)。根据如下的NMR、MS、[α]20D数据鉴定上述化合物的结构。
化学式3：芫花素H
[α]20 D+46.2(c 1.5,CHCl3)：
ESI-MS,m/z631.6[M+Na]+：
1HNMR以ppm为单位(CD3OD,400MHz)：δH 8.09(d,J＝7.4,2H,H-3”,H-7”),7.67(d,J＝9.5,2H,H-3',H-7'),7.60(t,J＝7.4,1H,H-5”),7.49(t,J＝7.6,2H,H-4”,H-6”),7.36(m,3H,H-4',H-5',H-6'),5.07(s,1H,H-16a),4.91(s,1H,H-16b),4.78(d,J＝11.0,1H,H-20a),4.62(d,J＝2.5,1H,H-14),4.61(s,1H,H-7),4.51(d,J＝11.1,1H,H-20b),4.16(d,J＝4.1,1H,H-2),3.41(s,1H,H-5),2.74(m,2H,H-10,H-11),2.67(d,J＝2.3,1H,H-8),2.34(dd,J＝14.1,8.0,1H,H-12a),1.84(s,3H,H-17),1.81-1.65(m,4H,H-1,H-2,H-12b),1.31(d,J＝6.9,3H,H-18),1.06(d,J＝6.0,3H,H-19)；
13CNMR以ppm为单位(CD3OD,176Hz)：δC 168.4(C-1”),148.3(C-15),137.9(C-2'),134.4(C-5”),131.8(C-2”),130.8(C-3”,C-7”),130.5(C-5'),129.7(C-3',C-7'),129.1(C-4”,C-6”),127.2(C-4',C-6'),118.6(C-1'),111.5(C-16),86.9(C-13),86.6(C-14),85.6(C-9),82.7(C-4),78.9(C-3),77.6(C-6),77.2(C-7),74.8(C-5),68.9(C-20),52.9(C-10),38.4(C-8),37.5(C-12),37.4(C-2),36.5(C-11),36.1(C-1),21.5(C-18),19.6(C-17),13.8(C-19)。
化学式4：芫花素M
[α]20 D-8.4(c 0.05,MeOH)：
ESI-MS,m/z613.5[M+Na]+：
1HNMR以ppm为单位(CD3OD,400MHz)：δH 8.07(d,J＝7.3,2H,H-3”,H-7”),7.69(d,J＝9.5,2H,H-3',H-7'),7.61(t,J＝7.4,1H,H-5”),7.49(t,J＝7.7,2H,H-4”,H-6”),7.35(m,3H,H-4',H-5',H-6'),5.07(s,1H,H-16a),5.06(d,J＝12.0,1H,H-20a),4.91(s,1H,H-16b),4.63(d,J＝2.6,1H,H-14),4.05(d,J＝11.8,1H,H-20b),3.83(s,1H,H-3),3.78(s,1H,H-5),3.53(s,1H,H-7),3.18(d,J＝2.6,1H,H-8),2.67(t,J＝9.0,1H,H-10),2.48(m,1H,H-11),2.31(dd,J＝14.1,8.0,1H,H-12a),1.84(s,3H,H-17),1.79-1.55(m,4H,H-1,H-2,H-12b),1.27(d,J＝6.8,3H,H-18),1.04(d,J＝5.2,3H,H-19)：
13CNMR以ppm为单位(CD3OD,176Hz)：δc 168.0(C-1”),148.5(C-15),138.2(C-2'),134.4(C-5”),131.6(C-2”),130.8(C-3”,C-7”),130.3(C-5'),129.74(C-4”,C-6”),128.9(C-3',C-7'),127.4(C-4',C-6'),118.8(C-1'),111.4(C-16),86.1(C-13),84.0(C-14),82.2(C-4),81.1(C-9),78.8(C-3),73.4(C-5),69.3(C-20),65.8(C-7),62.0(C-6),50.3(C-10),38.4(C-2),38.0(C-8),37.2(C-12),36.6(C-11),36.1(C-1),21.6(C-18),19.6(C-17),13.7(C-19)。
在乙醇与水的梯度混合溶剂(60∶40、80∶20、100∶0)的条件下对Fr.C3(4.0g)实施反相柱色谱，获得4个子分馏物(Fr.C31、C32、C33、C34)。用乙醇洗脱溶剂对Fr.C34(1.06g)实施Sephadex LH-20柱色谱，获得3个子分馏物(sub-fraction)(Fr.C341、C342、C343)。最终以流速4.5mL/分钟50％乙腈洗脱液对Fr.C341(120mg)、C342(180mg)、C343(119mg)分别进行十八烷基-硅胶高效液相色谱，从Fr.C341获得芫花素K(9.6mg，化学式5的化合物)，从Fr.C342获得芫花酯甙(79.5mg，化学式6的化合物)，从Fr.C343获得白色粉末形态的芫花素A(4.7mg，化学式7的化合物)、原苯甲酸酯2(85.6mg，化学式8的化合物)、1,2α-二氢瑞香毒素(2.9mg,化学式9的化合物)及芫花素I(2.4mg,化学式10的化合物)。根据如下的NMR、MS、[α]20 D数据鉴定上述化合物的结构。
化学式5：芫花素K
[α]20D+38.5(c 0.1,CHCl3)：
ESI-MS,m/z627.6[M+Na]+：
1HNMR以ppm为单位(CD3OD,400MHz)：δH 7.98(d,J＝7.5,2H,H-3',H-7'),7.90(d,J＝7.5,2H,H-3”,H-7”),7.57(m,2H,H-5',H-5”),7.44(d,J＝7.7,2H,H-4”,H-6”),7.42(d,J＝7.6,2H,H-4',H-6'),5.72(d,J＝6.2,1H,H-14),5.06(s,1H,H-16a),4.84(s,1H,H-16b),4.62(d,J＝13.4,1H,H-20a),4.42(d,J＝3.0,1H,H-3),4.24(d,J＝5.15,1H,H-20b),4.19,(d,J＝2.4,H-7),3.33(s,1H,H-5),3.15(d,J＝6.2,1H,H-8),2.34(m,2H,H-10,H-11),2.01(m,3H,H-2,H-1a,H-12a),1.81(s,3H,H-17),1.60(m,2H,H-1b,H-12b),1.24(d,J＝7.1,3H,H-18),1.08(d,J＝6.5,3H,H-19)：
13CNMR以ppm为单位(CDCl3,101Hz)：δC 168.3(C-1”),166.7(C-1'),148.1(C-15),133.6(C-5”),133.5(C-5”),130.2(C-3”,C-7”),130.1(C-3',C-7'),129.9(C-2”),129.4(C-2'),128.7(C-4”,C-6”),128.6(C-4',C-6'),111.6(C-16),83.8(C-4),77.9(C-9),77.4(C-6),77.3(C-14),76.0(C-7),75.5(C-3),74.5(C-13),73.2(C-5),67.4(C-20),54.6(C-10),40.6(C-8),37.9(C-12),36.6(C-2),34.9(C-1),34.4(C-11),19.5(C-18),19.0(C-17),13.5(C-19)。
化学式6：芫花酯甙
[α]20 D+28.5(c 0.05,CHCl3)：
ESI-MS,m/z565.5[M+Na]+：
1HNMR以ppm为单位(CD3OD,400MHz)：δH 7.67(m,2H,H-3',H-7'),7.37(m,3H,H-4',H-5',H-6'),5.07(s,1H,H-16a),5.07(d,J＝2.9,1H,H-12),5.04(s,1H,H-16b),4.95(d,J＝2.7,1H,H-14),4.02(s,1H,H-5),4.02(d,J＝12.1,1H,H-20a),3.61(d,J＝2.9,1H,H-8),3.60(d,J＝10.6,1H,H-20b),3.54(s,1H,H-7),3.18(dd,J＝13.3,5.8,1H,H-10),2.47(q,J＝6.9,1H,H-11),2.38(dt,J＝13.3,6.8,1H,H-1a),2.25(dt,J＝13.0,6.6,1H,H-1b),2.01(s,3H,H-2”),1.86(s,3H,H-17),1.46(d,J＝13.1,1H,H-2),1.37(d,J＝7.0,3H,H-18),1.07(d,J＝6.5,3H,H-19)：
13CNMR以ppm为单位(CD3OD,101Hz)：δC 218.9(C-3),171.6(C-1”),145.0(C-15),137.4(C-2'),130.4(C-5'),128.8(C-4',C-6'),127.1(C-3',C-5'),119.3(C-1'),113.7(C-16),84.8(C-13),82.3(C-14),80.6(C-9),79.2(C-12),77.0(C-4),70.5(C-5),65.3(C-20),64.6(C-7),63.3(C-6),44.9(C-11),44.8(C-10),43.7(C-1),36.8(C-8),34.5(C-2),21.1(C-2”),19.1(C-18),19.0(C-17),12.8(C-19)。
化学式7：芫花素A
[α]20 D+47.7(c0.1,CHCl3)：
ESI-MS,m/z505.6[M+H]+和m/z527.4[M+Na]+：
1HNMR以ppm为单位(CD3OD,400MHz)：δH 7.66(dd,J＝6.6,3.0,2H,H-3',H-7'),7.38(m,3H,H-4',H-5',H-6'),5.07(d,J＝6.5,1H,H-16a),4.91(s,1H,H-16b),4.60(d,J＝2.5,1H,H-14),4.42(s,1H,H-7),4.13(d,J＝4.6,1H,H-3),3.82(q,J＝11.0,2H,H-20),3.38(s,1H,H-5),2.71(m,2H,H-10,H-11),2.62(d,J＝2.4,1H,H-8),2.32(dd,J＝14.1,8.0,1H,H-12a),1.87(m,2H,H-1a,H-2),1.83(s,3H,H-17),1.69(m,1H,H-1b),1.62(m,2H,H-12b),1.30(d,J＝6.9,3H,H-18),1.05(d,J＝6.2,3H,H-19)：
13CNMR以ppm为单位(CD3OD,101Hz)：δC 148.3(C-15),137.9(C-15),130.6(C-5'),129.1(C-4',C-6'),127.3(C-3',C-5'),118.6(C-1'),111.5(C-16),86.8(C-13),86.5(C-14),85.4(C-9),82.8(C-4),78.5(C-7),78.3(C-6),77.5(C-3),75.0(C-5),67.6(C-20),52.9(C-10),38.5(C-2),37.5(C-8),37.4(C-12),36.5(C-11),36.0(C-1),21.5(C-17),19.8(C-18),13.8(C-19)。
化学式8：原苯甲酸酯2
[α]20 D-16.6(c 0.05,MeOH)：
ESI-MS,m/z509.2[M+Na]+：
1HNMR以ppm为单位(CD3OD,400MHz)：δH 7.69(dd,J＝6.6,2.9,2H,H-3',H-7'),7.35(m,3H,H-4',H-5',H-6'),5.06(s,1H,H-16a),4.90(s,1H,H-16b),4.56(d,J＝2.6,1H,H-14),4.02(d,J＝12.2,1H,H-20a),3.76(d,J＝2.6,1H,H-3),3.70(s,1H,H-5),3.59(d,J＝12.3,1H,H-20b),3.42(m,1H,H-7),3.07(d,J＝2.6,1H,H-8),2.67(dd,J＝12.6,5.9,1H,H-10),2.47(p,J＝6.9,1H,H-11),2.28(dd,J＝13.9,7.9,1H,H-12a),1.82(s,3H,H-17),1.69(d,J＝14.0,1H,H-1a),1.64(dd,J＝9.5,4.8,1H,H-2),1.58(m,2H,H-1b,H-12b),1.27(d,J＝6.8,3H,H-18),1.03(d,J＝5.3,3H,H-19)：
13CNMR以ppm为单位(CD3OD,101Hz)：δC 148.3(C-15),138.1(C-2'),130.1(C-5'),128.7(C-4',C-6'),127.2(C-3',C-7'),118.5(C-1'),111.2(C-16),85.8(C-13),84.0(C-14),82.1(C-9),81.0(C-2),78.6(C-4),73.7(C-5),65.7(C-20),64.9(C-7),63.2(C-6),50.0(C-10),38.2(C-2),37.8(C-8),37.0(C-12),36.5(C-11),35.9(C-1),21.4(C-17),19.5(C-18),13.6(C-19)。
化学式9：1,2α-二氢瑞香毒素
[α]20 D+6.2(c 0.001,MeOH)；
ESI-MS,m/z507.3[M+Na]+；
1HNMR以ppm为单位(CD3OD,400MHz)：δH 7.72(m,2H,H-3',H-7'),7.36(m,3H,H-4',H-5',H-6'),5.07(s,1H,H-16a),4.91(s,1H,H-16b),4.60(d,J＝2.8,1H,H-14),4.01(s,1H,H-5),3.99(d,J＝12.2,1H,H-20a),3.57(d,J＝12.2,1H,H-20b),3.44(s,1H,H-7),3.16(dd,J＝13.3,5.8,1H,H-10),3.01(d,J＝2.7,1H,H-8),2.55(m,1H,H-11),2.38(m,1H,H-12a),2.29(m,2H,H-1),1.83(s,3H,H-17),1.73(d,J＝14.0,1H,H-2),1.53(m,1H,H-12b),1.30(d,J＝6.8,3H,H-18),1.08(d,J＝6.6,3H,H-19)；
13CNMR以ppm为单位(CD3OD,101Hz)：δC 219.3(C-3),148.3(C-15),138.1(C-2'),130.4(C-5'),128.9(C-4',C-6'),127.4(C-3',C-5'),118.8(C-1'),111.5(C-16),86.0(C-13),84.1(C-14),82.0(C-9),77.2(C-4),70.9(C-5),65.5(C-20),64.9(C-7),63.3(C-6),45.8(C-10),43.9(C-8),37.9(C-1),37.2(C-2),36.7(C-12),34.7(C-11),21.6(C-17),19.6(C-18),12.9(C-19)。
化学式10：芫花素I
[α]20 D-22.8(c 0.005,MeOH)；
ESI-MS,m/z513.3[M+H]+和m/z535.3[M+Na]+；
1HNMR以ppm为单位(CD3OD,400MHz)：δH 7.69(dd,J＝7.5,1.9,2H,H-3',H-7'),7.36(m,3H,H-4',H-5',H-6'),5.09(s,1H,H-16a),4.93(s,1H,H-16b),4.72(d,J＝4.5,1H,H-3),4.64(d,J＝2.8,1H,H-14),4.10(s,1H,H-5),4.09(d,J＝12.3,1H,H-20a),3.53(d,J＝12.4,1H,H-20b),3.49(s,1H,H-7),2.93(dd,J＝13.3,6.1,1H,H-10),2.78(d,J＝2.8,1H,H-8),2.35(dd,J＝14.1,7.8,1H,H-12a),2.12(m,1H,H-11),2.04(dd,J＝11.7,6.0,1H,H-1a),1.95(dd,J＝12.0,6.6,1H,H-2),1.84(s,3H,H-17),1.82(d,J＝14.9,1H,H-12b),1.58(d,J＝12.6,1H,H-1b),1.31(d,J＝6.7,3H,H-18),1.13(d,J＝6.7,3H,H-19)：
13CNMR以ppm为单位(CD3OD,101Hz)：δC 156.7(C-21),148.0(C-15),137.8(C-2'),130.5(C-5'),128.9(C-4',C-6'),127.4(C-3',C-7'),118.8(C-1'),111.8(C-16),94.1(C-4),90.4(C-3),85.8(C-13),84.2(C-14),81.4(C-9),71.1(C-5),65.0(C-20),64.7(C-7),62.8(C-6),50.7(C-10),37.9(C-2),37.4(C-11),37.3(C-12),37.1(C-8),36.2(C-1),21.6(C-18),19.5(C-17),12.9(C-19)。
实施例2-b：利用多种溶剂从芫花提取物中分离活性成分
将上述实施例1中获得的芫花提取物溶解在200mL的蒸馏水与己烷的1∶1的混合溶剂中，分离获得己烷层。将用相同方法再实施2次而获得的己烷层进行减压浓缩，获得己烷分离物。利用硅胶柱色谱，以己烷与乙酸乙酯的梯度混合溶剂(10∶1、5∶1、2∶1、1∶1、1∶2)洗脱以此获得的己烷分离物(20g)，总计获得3个分馏物(Fr.I、II、III)。
其中，在75％乙腈的条件下对Fr.I(577mg)实施反相硅胶制备的薄层色谱(reverse phasesilica gel prep TLC)，获得活性带。以流速3mL/分钟的83％乙腈洗脱液对上述活性带进行十八烷基-硅胶高效液相色谱，分别在滞留时间(retention time)15.2分钟和18.5分钟获得白色粉末形态的acutilonine F(14.0mg，化学式11的化合物)和荛花因子M1(wikstroemia factor)(7.0mg，化学式12的化合物)。根据如下的NMR、MS、[α]20 D数据鉴定上述化合物的结构。
化学式11：
[α]20 D-32.1(c 1.3,MeOH)；
ESI-MS,m/z635.6[M+H]+,657.7[M+Na]+；
1HNMR(CD3OD,500MHz)：δH 7.70(2H,dd,J＝7.35,2.22Hz,H-3',H-7'),7.37(1H,m,H-5'),7.36(2H,m,H-4',H-6'),7.35(1H,m,H-3”),6.64(1H,dd,J＝14.82,10.75Hz,H-5”),6.34(1H,dd,J＝14.79,11.40Hz,H-4”),6.22(1H,dd,J＝15.16,10.69Hz,H-6”),6.01(1H,m,H-7”),6.00(1H,m,H-2”),5.06(1H,brs,H-16a),5.03(1H,d,J＝4.52Hz,H-3),4.90(1H,brs,H-16b),4.56(1H,d,J＝2.66Hz,H-14),3.98(1H,d,J＝12.19Hz,H-20a),3.92(1H,s,H-5),3.59(1H,d,J＝12.20Hz,H-20b),3.41(1H,brs,H-7),3.07(1H,d,J＝2.69Hz,H-8),2.83(1H,dd,J＝13.03,5.32Hz,H-10),2.59(1H,m,H-11),2.29(2H,m,H-12),2.15(2H,m,H-8”),1.82(3H,s,H-17),1.78(1H,m,H-2),1.71(2H,m,H-1),1.47(2H,m,H-9”),1.31(3H,d,J＝6.89Hz,H-18),0.99(3H,d,J＝5.77Hz,H-19),0.94(3H,t,J＝7.39Hz,H-10”)；
13CNMR以ppm为单位(CD3OD,126Hz)：δC 169.4(C,C-1”),148.5(C,C-15),147.2(CH,C-3”),143.3(CH,C-5”),141.9(CH,C-7”),138.2(C,C-2'),131.6(CH,C-6”),130.3(CH,C-5'),129.2(CH,C-4”),128.9(CH,C-4',C-6'),127.4(CH,C-3',C-7'),120.7(CH,C-2”),118.7(C,C-1'),111.4(CH2,C-16),86.1(C,C-13),84.0(CH,C-14),82.9(C,C-4),82.3(CH,C-3),82.1(C,C-9),74.1(CH,C-5),66.1(CH2,C-20),65.1(CH,C-7),63.0(C,C-6),37.9(CH,C-8),37.7(CH,C-2),37.3(CH2,C-1),37.1(CH2,C-12),36.5(CH,C-11),36.2(CH2,C-8”),23.4(CH2,C-9”),21.6(CH3,C-18),19.6(CH3,C-17),14.1(CH3,C-10”),13.8(CH3,C-19)。
化学式12：
[α]20 D+18.9(c1.0,MeOH)：
ESI-MS,m/z637.6[M+H]+,659.4[M+Na]+,635.2[M-H]-：
1HNMR(CDCl3,500MHz)：δH 7.75(2H,m,H-3',H-7'),7.36(3H,m,H-4',H-5',H-6'),7.34(1H,dd,J＝15.4和10.2,H-3”),6.21(1H,dd,J＝14.8和10.4,H-4”),5.90(1H,d,J＝15.2,H-2”),5.05(1H,brs,H-16a),4.92(1H,brs,H-16b),4.69(1H,d,J＝5.19,H-3),4.51(1H,d,J＝2.76,H-14),4.06(1H,s,H-5),3.88(1H,d,J＝12.2,H-20a),3.77(1H,d,J＝12.2,H-20b),3.44(1H,s,H-7),2.96(1H,d,J＝2.8,H-8),2.82(1H,dd,J＝13.2,5.5,H-10),2.48(1H,m,H-11),2.20(2H,重叠,H-6”),2.20(1H,重叠,H-12a),1.93(1H,m,H-1a),1.83(3H,s,H-17),1.78(1H,m,H-12b),1.73(1H,m,H-1b),1.71(1H,m,H-2),1.45(2H,m,H-7”),1.33(3H,d,J＝6.9,H-18),1.32(2H,重叠,H-9”),1.31(2H,重叠,H-8”),1.06(3H,d,J＝6.5,H-19),0.91(3H,t,J＝6.9,H-10'')：
13CNMR(CDCl3,126Hz)：δC 169.6(C,C-1”),147.4(CH,C-3”),146.8(CH,C-5”),146.7(C,C-15),136.4(C,C-2'),129.4(CH,C-5'),128.4(CH,C-4”),128.2(CH,C-4',C-6'),126.3(CH,C-3',C-7'),118.0(CH,C-2”),117.6(C,C-1'),111.4(CH2,C-16),84.5(C,C-13),82.7(CH,C-14),82.2(CH,C-3),81.7(C,C-4),80.5(C,C-9),75.0(CH,C-5),66.3(CH2,C-20),64.2(CH,C-7),60.6(C,C-6),48.9(CH,C-10),36.6(CH,C-8),36.4(CH,C-2),36.3(CH2,C-12),36.0(CH2,C-1),35.5(CH,C-11),33.3(CH2,C-6”),31.6(CH2,C-8”),28.5(CH2,C-7”),22.7(CH2,C-9”),21.1(CH3,C-18),19.4(CH3,C-17),14.2(CH3,C-10”),13.3(CH3,C-19)。
同样地，在75％乙腈的条件下对Fr.III(400mg)实施反相硅胶制备的薄层色谱(reverse phase silica gel prep TLC)，获得活性带。在CHCl3-MeOH(50∶1)的条件下对该活性带实施硅胶制备的薄层色谱(silica gel prep TLC)，在Rf0.4和0.25处获得作为两个子分馏物(sub-fraction)的Fr.III-1和Fr.III-2。以流速3mL/分钟的65％乙腈洗脱液对Fr.III-1进行十八烷基-硅胶高效液相色谱，在滞留时间17.2分钟和23.4分钟分别获得白色粉末形态的普罗斯左汀Q(2.1mg，化学式13的化合物)和芫花酯乙(4.0mg，化学式14的化合物)。用与Fr.III-1相同的方法对Fr.III-2进行十八烷基-硅胶高效液相色谱，分别在滞留时间(retention time)19分钟和21.4分钟获得白色粉末形态的芫花酯丁(4.4mg，化学式15的化合物)、12-O-正癸烷-2,4,6-三烯酰基-佛波醇(13)-乙酸酯(1.8mg，化学式16的化合物)。根据如下的NMR、MS、[α]20 D数据鉴定上述化合物的结构。
化学式13：
[α]20 D+14.1(c 0.03,MeOH)：
ESI-MS,m/z579.5[M+Na]+：
1HNM(CDCl3,500MHz)：δH 7.60(1H,s,H-1),7.24(1H,m,H-3”),6.19(1H,m,H-4”),6.15(1H,m,H-5”),5.79(1H,d,J＝15.41Hz,H-2”),5.70(1H,d,J＝4.71Hz,H-7),5.47(1H,d,J＝10.27Hz,H-12),4.03(2H,q,J＝13.04Hz,H-20),3.26(2H,重叠,H-8,H-10),2.53(2H,m,H-5),2.17(2H,重叠,H-6”),2.17(1H,重叠,H-11),2.11(3H,s,H-2'),1.78(3H,s,H-19),1.44(2H,m,H-7”),1.32(2H,m,H-9”),1.31(2H,m,H-8”),1.27(3H,s,H-16),1.22(3H,s,H-17),1.10(1H,d,J＝5.20Hz,H-14),0.91(3H,d,J＝6.93Hz,H-18),0.90(3H,t,J＝7.06Hz,H-10”)：
13CNMR以ppm为单位(CDCl3,126Hz):δC 209.1(C,C-3),174.1(C,C-1'),167.3(C,C-1”),161.0(CH,C-1),145.8(CH,C-3”),145.5(CH,C-5”),140.7(C,C-6),133.1(C,C-2),129.5(CH,C-7),128.5(CH,C-4”),119.1(CH,C-2”),78.4(C,C-9),74.0(C,C-4),68.2(CH2,C-20),65.9(C,C-13),56.4(CH,C-10),43.4(CH,C-11),39.3(CH,C-8),38.9(CH2,C-5),36.6(CH,C-14),33.2(CH2,C-6”),31.6(CH2,C-8”),28.6(CH2,C-7”),25.9(C,C-15),24.0(CH3,C-17),22.7(CH2,C-9”),21.3(CH3,C-2'),17.0(CH3,C-16),14.2(CH3,C-10”),10.3(CH3,C-19)。
化学式14：
[α]20 D+7.5(c 1.3,CH2Cl2)：
ESI-MS,m/z587.6[M+H]+和609.5[M+Na]+：
1HNMR(CDCl3,500MHz)：δH 7.58(1H,s,H-1),6.67(1H,dd,J＝15.45,10.66Hz,H-3'),6.05(1H,dD,J＝15.14,10.71Hz,H-4'),5.86(1H,m,H-5'),5.65(1H,d,J＝15.46Hz,H-2'),5.02(1H,brs,H-16a),4.99(1H,brs,H-12),4.96(1H,brs,H-16b),4.76(1H,d,J＝2.47Hz,H-14),4.26(1H,brs,H-7),3.94(1H,dd,J＝12.34,5.86Hz,H-20a),3.82(1H,m,H-10),3.80(1H,m,H-20b),3.56(1H,s,H-5),3.52(1H,d,J＝2.45Hz,H-8),2.38(1H,q,J＝7.22Hz,H-11),2.10(2H,q,J＝7.20Hz,H-6'),2.00(3H,s,H-2”),1.84(3H,s,H-17),1.80(3H,d,J＝1.31Hz,H-19),1.39(2H,dt,J＝14.26,7.28Hz,H-7'),1.30(2H,m,H-9'),1.27(2H,m,H-8'),0.89(3H,t,J＝6.93Hz,H-10')；
13CNMR以ppm为单位(CDCl3,126Hz)：δC 209.7(C,C-3),169.9(C,C-1”),160.6(CH,C-1),143.3(C,C-15),139.6(CH,C-5'),137.1(C,C-2),135.3(CH,C-3'),128.8(CH,C-4'),122.5(CH,C-2'),117.2(C,C-1'),113.5(CH2,C-16),83.9(C,C-13),80.7(CH,C-14),78.5(CH,C-12),78.3(C,C-9),72.5(C,C-4),72.2(CH,C-5),65.3(CH2,C-20),64.5(C-7),60.7(C-6),47.7(CH,C-10),44.3(CH,C-11),35.6(CH,C-8),32.9(CH2,C-6'),31.5(CH2,C-8'),28.9(CH2,C-7'),22.7(CH2,C-9'),21.4(CH3,C-2”),18.9(CH3,C-17),18.5(CH3,C-18),14.2(CH3,C-10'),10.1(CH3,C-19)。
化学式15：
[α]20 D+52.8(c 0.5,MeOH)：
ESI-MS,m/z605.5[M+H]+,627.4[M+Na]+,603.3[M-H]-：
1HNMR(CDCl3,500MHz)：δH 7.94(2H,m,H-3”,H-7”),7.75(2H,m,H-3',H-7'),7.60(1H,t,J＝7.4,H-5”),7.48(2H,m,H-4”,H-6”),7.40(3H,m,H-4',H-5',H-6'),5.42(1H,brs,H-12),5.07(1H,brs,H-16a),5.03(1H,brs,H-16b),4.99(1H,d,J＝2.8,H-14),4.10(1H,s,H-5),3.90(1H,d,J＝12.4,H-20a),3.85(1H,d,J＝12.3,H-20b),3.69(1H,d,J＝2.8,H-8),3.67(1H,brs,H-7),3.06(1H,dd,J＝13.3和5.9,H-10),2.59(1H,q,J＝6.9,H-11),2.40(1H,m,H-1a),2.28(1H,m,H-2),1.92(3H,s,H-17),1.63(1H,m,H-1b),1.51(3H,d,J＝6.9,H-18),1.12(3H,d,J＝6.6,H-19)；
13CNMR(CDCl3,126Hz)：δC 220.4(C,C-3),165.8(C,C-1”),143.2(C,C-15),135.7(C,C-2'),133.5(CH,C-5”),130.0(CH,C-5'),129.8(C,C-2”),129.7(CH,C-3”,C-7”),128.9(CH,C-4”,C-6”),128.3(CH,C-4',C-6'),126.2(CH,C-3',C-7'),118.4(C,C-1'),113.8(CH2,C-16),83.9(C,C-13),81.4(CH,C-14),79.3(C,C-9),78.7(CH,C-12),75.2(C,C-4),71.5(CH,C-5),65.3(CH2,C-20),64.5(CH,C-7),61.0(C,C-6),44.3(CH,C-11),44.2(CH,C-10),43.1(CH,C-2),36.3(CH,C-8),33.6(CH2,C-1),19.0(CH3,C-17,C-18),12.6(CH3,C-19)。
化学式16：
[α]20 D-15.1(c 0.2,CHCl3)：
ESI-MS,m/z577.5[M+Na]+,553.4[M-H]-：
1HNMR(CDCl3,500MHz)：δH 7.61(1H,s,H-1),7.28(1H,dd,J＝15.3和11.22,H-3”),6.54(1H,dd,J＝14.9和10.7,H-5”),6.23(1H,dd,J＝14.8和11.4,H-4”),6.15(1H,dd,J＝15.1和10.8,H-6”),5.95(1H,m,H-7”),5.84(1H,d,J＝15.3,H-2”),5.70(1H,d,J＝4.8,H-7),5.47(1H,d,J＝10.3,H-12),4.05(1H,d,J＝12.9,H-20a),4.00(1H,d,J＝12.9,H-20b),3.26(1H,重叠,H-10),3.26(1H,重叠,H-8),2.52(2H,m,H-5),2.17(1H,m,H-11),2.13(2H,重叠,H-8”),2.11(3H,s,H-2'),1.78(3H,d,J＝1.5,H-19),1.45(2H,dq,J＝14.6和7.3,H-9”),1.27(3H,s,H-16),1.22(3H,s,H-17),1.10(1H,d,J＝5.1,H-14),0.93(3H,t,J＝7.3,H-10”),0.91(3H,d,J＝6.4,H-18)；
13CNMR(CDCl3,126Hz)：δC 209.2(C,C-3),174.1(C,C-1'),167.2(C,C-1”),161.0(CH,C-1),145.6(CH,C-3”),141.8(CH,C-5”),141.0(CH,C-7”),140.7(C,C-6),133.1(C,C-2),130.2(CH,C-6”),129.5(CH,C-7),127.9(CH,C-4”),119.9(CH,C-2”),78.5(C,C-9),76.9(CH,C-12),74.0(C,C-4),68.2(CH2,C-20),65.9(C,C-13),56.4(CH,C-10),43.4(CH,C-11),39.3(CH,C-8),38.8(CH2,C-5),36.6(CH,C-14),35.5(CH2,C-8”),26.0(C,C-15),24.0(CH3,C-17),22.4(CH2,C-9”),21.3(CH3,C-2'),17.0(CH3,C-16),14.6(CH3,C-18),13.9(CH3,C-10”),10.3(CH3,C-19)。
实施例3：化合物对核受体相关因子1蛋白活性的效果
如上述实施例2所示，通过萤光素酶分析，确认了基于从芫花提取物中分离的二萜类化合物的浓度，核受体相关因子1蛋白的活性度。
具体地，将GLA4基因可以结合的碱基序列(5'-CTCGGAGGACAGTACTCCG-3'，序列号1)反复8次的基因结合于作为报道基因的萤光素酶来合成载体，然后向BE(2)C细胞注入(transfection)包含核受体相关因子1-外侧器官界域基因(Nurr1-LBD)的DNA和具有β-半乳糖苷酶(β-galactosidase)的DNA等3种质粒DNA。6小时后，将从上述实施例2中分离的化合物1至10按下述表1的浓度进行处理。将以此处理的细胞在37℃、5％二氧化碳培养基培养20小时后，进行萤光素酶分析。对照组使用0.1％二甲基亚砜(DMSO)，此时的活性显现为以活性度成倍增加，阳性对照组使用阿莫地喹(Amodiaquine,AQ)。
表1
分析结果如下述表2及图1所示，化学式1至10的所有化合物均使核受体相关因子1激活。具体地，在将化学式1至化学式10的所有化合物按1μM的浓度进行处理的情况下，会使核受体相关因子1激活，尤其是化学式2的化合物在0.01μM的低浓度也呈现出使核受体相关因子1的激活的优秀的效果。由此可知从芫花提取物中分离的多种化合物可使核受体相关因子1激活，同时，活性会根据化合物的结构而不同。
表2
| 浓度 | DMSO | AQ | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | DGII-2 |
| 低 | 1.0 | 0.9 | 1.0 | 2.1 | 1.2 | 1.0 | 0.9 | 1.0 | 1.2 | 1.7 | 1.7 | 1.8 | 1.1 |
| 中 | - | 1.8 | 1.5 | 2.2 | 1.4 | 1.4 | 1.2 | 1.8 | 2.6 | 2.0 | 2.4 | 1.9 | 2.6 |
| 高 | - | - | 1.7 | 1.9 | 1.4 | 1.7 | 1.9 | 2.4 | 2.0 | 1.6 | 1.5 | 1.1 | 2.2 |
分析结果如以下表1所示，确认了化合物13及14的普罗斯左汀Q及芫花酯乙在0.003μM的低浓度也会使核受体相关因子1激活，化合物16的12-O-正癸烷-2，4，6-三烯酰基-佛波醇(13)-乙酸酯与上述化合物13及14一同，会在0.03μM中明显地使核受体相关因子1激活。而且，确认了化合物11的acutilonine F、化合物12的荛花因子M1及化合物15的芫花酯丁分别在1μM、0.2μM及0.3μM处显著地使核受体相关因子1激活。由此可知从芫花提取物中分离的多种化合物可以使核受体相关因子1激活，同时，活性根据化合物的结构而不同。
表3
(*P＜0.05，**P＜0.01对比对照组的处理)
实施例4：在小神经胶质细胞BV-2细胞中的一氧化氮生成抑制活性
在小神经胶质细胞(microglia cell)中，基于炎症反应，大脑神经细胞的死亡是如老年痴呆、帕金森病的退行性大脑疾病的主要原因之一(Sarkar S et al.，Neurotoxicology，44，250-262(2014)；Bower JH et al.，Neurology，67，494-496(2006))。因此，在小神经胶质细胞中，以从上述实施例2分离的多种化合物为对象，研究作为代表性炎症因子的一氧化氮(nitric oxide，NO)的生成抑制活性。具体地，在96孔板中以5×104细胞/孔放入小神经胶质BV-2细胞，培养2日后，将脂多糖(LPS，1mg/mL)与从上述实施例2中分离的化合物一同培养24小时。利用Griess试剂在540nm处对培养上清液测定吸光度来对亚硝酸盐(nitrite)进行定量，由此研究一氧化氮的生成量。阳性对照组使用米诺环素(Minocyline)。
研究结果如下述表3所示，确认了所有化合物在低的浓度下会阻碍一氧化氮的生成。尤其是确认了化合物2的芫花烯在0.06±0.02的极低浓度呈现出一氧化氮生成的阻碍活性，确认了化合物14的芫花酯乙在1.03μM的极低浓度呈现一氧化氮生成的阻碍活性。
表4
| 化合物 | IC50(μM) |
| 米诺环素 | 21.28±0.48 |
| 化学式1(芫花酯丙) | 0.37±0.15 |
| 化学式2(芫花烯) | 0.06±0.02 |
| 化学式3(芫花素H) | 1.06±0.12 |
| 化学式4(芫花素M) | 0.18±0.04 |
| 化学式5(芫花素K) | 4.67土3.10 |
| 化学式6(芫花酯甙) | 0.25±0.06 |
| 化学式7(芫花素A) | 3.41±0.99 |
| 化学式8(原苯甲酸酯2) | 1.22±0.13 |
| 化学式9(1，2d-二氢瑞香毒素) | 1.60±0.37 |
| 化学式10(芫花素I) | 7.79±0.91 |
表5
实施例5：在小神经胶质BV-2细胞中的促炎细胞因子生成抑制活性
研究化合物对小神经胶质细胞中作为代表性促炎因子的白细胞介素-1b、白细胞介素-6及肿瘤坏死因子a的生成抑制活性。在96孔板中以1×105细胞/孔放入小神经胶质BV-2细胞，将脂多糖(1mg/mL)与化合物一同培养5小时。在各个孔中回收细胞，实施蛋白质印迹和实时聚合酶链式反应(real-time PCR)。
具体地，通过蛋白质印迹研究白细胞介素-1b蛋白的表达量。使用兔抗白细胞介素-1b(rabbit anti-IL-1b)[细胞信号(Cell Signaling)(美国马萨诸塞州丹佛斯(Danvers，MA，USA))；1∶1000])作为一次抗体，使用小鼠抗肌动蛋白(mouseanti-actin)(Sigma 1∶5000)作为对照组(control)。使用horseradish peroxidase-conjugated anti-mouse或anti-rabbit immunoglobulinG(IgG)antibody(美国纽约州皮斯卡塔维安玛西亚(Amersham，Piscataway，NY，USA))作为二次抗体，通过增强性化学发光底物(enhanced-chemiluminescent substrate(Amersham))观察显色。
同时，通过实时定量(Real-time quantatitive)聚合酶链式反应分析白细胞介素一1b、白细胞介素-6、肿瘤坏死因子-a的mRNA表达量。所有鼠的细胞因子和甘油醛-3-磷酸脱氢酶(GAPDH)的引物都购买自Invitrogen，将细胞因子mRNA表达量归一化(normalized)成甘油醛-3-磷酸脱氢酶mRNA表达量来求出。
蛋白质印迹及聚合酶链式反应进行结果如图1A及1B所示，确认了在所有化合物的给药组中，白细胞介素-1b的表达量减少。同时，聚合酶链式反应进行结果如图1C及1D所示，确认了在所有化合物的给药组中，白细胞介素-6及肿瘤坏死因子-a的表达量也减少。
在本说明书中，只要是本领域所属技术人员则可以充分认识、推测的内容，则省略其详细记载，除在本说明书中记载的具体示例之外，在不变更本发明的技术思想或必要结构的范围内，可进行多种变形。因此，本发明也可以按与在本说明书中具体说明并例示的方法不同的方式实施，本领域所属技术人员可以理解这些事项。
产业上的可利用性
如上所述，本发明涉及包含二萜类化合物或其药学上可接受的盐的用于预防或治疗神经退行性疾病的药学组合物，其呈现出抑制神经细胞中的炎症反应的效果，由此，可有效地用于因核受体相关因子1蛋白的活性被抑制而诱发的包括帕金森病在内的神经退行性疾病的预防及治疗。
序列表(Free text)
序列1
DNA
人工序列(Artificial Sequence)
GLA4结合基因(GLA4 bindinggene)
ctcggaggac agtactccg
Claims (5)
1.二萜类化合物或其药学上可接受的盐在制备用于预防或治疗帕金森病的药学组合物中的用途，所述二萜类化合物为选自下述化学式2、化学式13和化学式14中的任一种以上：
化学式2：
2.根据权利要求1所述的用途，其中，所述二萜类化合物为化学式2、化学式13或化学式14所示的化合物。
3.根据权利要求1所述的用途，其中，所述二萜类化合物为化学式2所示的化合物。
4.根据权利要求1所述的用途，其中，所述二萜类化合物为化学式14所示的化合物。
5.根据权利要求1所述的用途，其中，所述二萜类化合物为从芫花提取物中分离的化合物。
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| PCT/KR2018/013990 WO2019098699A1 (ko) | 2017-11-15 | 2018-11-15 | 다이터펜계 화합물을 포함하는 신경퇴행성 질환 예방 또는 치료용 조성물 |
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