小葉葡萄神經保護功能之研究
Study of the neuroprotective effects of Vitis thunbergii var. taiwaniana in vitro and in vivo
中文摘要
小葉葡萄(Vitis thunbergii var. taiwaniana，簡稱 VTT)是一台灣原生葡萄,
與其它Vitis spp.一樣在台灣是被用來當作一種民間傳統藥物或是可食用的植
物。它富含多酚類化合物(polyphenols)，特別是槲皮素(quercetin)、白藜蘆 醇(resveratrol)，這些多酚類化合物在很多研究中，被證實具有保護心血管疾 病、抗癌、抗衰老及改善腦神經退化性疾病(neurodegenerative diseases)。
以白藜蘆醇為例，它具有高度抗氧化、抗發炎的活性，並且被視為是一種植物雌 激素(phytoestrogen)，這些活性均有助於其保護腦神經細胞。在抗氧化方面，
它可直接捕捉活性氧分子(reactive oxygen species, ROS)或經由活化抗氧化 酵素，來防止神經元細胞及神經膠質細胞(glial)受到活性氧分子的攻擊；在抗發 炎方面，它可抑制經β-amyloid, LPS, cytokines 所引起神經膠質細胞(如微小 神經膠質細胞)的過度發炎反應；此外過去的研究發現 estrogen 具有優越的保 護神經功能，更年期後的婦女若能長期服用植物性雌激素，可有效降低老人失智 症的發生率，它還能對抗多種來源的氧化壓力，而白藜蘆醇具有植物雌激素的活 性，我們認為白藜蘆醇也有estrogen 類似的神經保護功能。
在本研究中，我們使用小葉葡萄的根、支幹、葉及主幹的粗萃取物，在體外、體
内試驗其抗腦神經退化的活性。首先在有血清培養的BV2 微小神經膠質細胞
中，發現支幹及葉萃取物顯著抑制iNOS 蛋白質表現及 NO 產生; 而在無血清培 養的BV2 微小膠細胞中，四種粗萃取物均可顯著抑制 iNOS 蛋白質表現及 NO 產生。在初代培養的大鼠神經細胞中，處以海人酸(kainic acid, 簡稱 KA) 發 現可引起活性氧化物的產生，並會引起神經細胞死亡。使用四種粗萃取物均可顯
著抑制活性氧化物的產生，然而只有根及支幹的萃取物可以降低KA 所引起的神
經細胞死亡。總結體外試驗，小葉葡萄粗萃取物降低微小神經膠質細胞發炎及神 經細胞死亡的活性順序分別是支幹≈葉>根>主幹。在動物試驗方面，發現給予 小葉葡萄粗萃取物，雖然不能降低小鼠發生癲癇，但顯著延後癲癇發作的時間，
其中支幹萃取物及葉萃取物效果較顯著，發作的小鼠也較少。四種小葉葡萄萃取 物，均可保護海馬迴神經細胞，其中小葉葡萄支幹萃取物，保護效果較佳。小葉 葡萄粗萃取物在動物體上保護神經的活性順序分別是支幹≈葉>根>主幹
英文摘要
Vitis thunbergii var. taiwaniana (VTT) is rich in quercetin, resveratrol, and other polyphenols that are known to protect against cardiovascular diseases and cancers, as well as to promote anti-aging effects in numerous organisms. Resveratrol has
antioxidant and anti-inflammatory activities, and acts as a phytoestrogen. All of the abilities contributes to protect against neurodegenerative diseases. In antioxidant, resveratrol can prevent neuron and glial cells from reactive oxygen species (ROS) attack by directly scavenge ROS or indirectly activate antioxidant enzymes. On the other hand, resveratrol can inhibit inflammation induced by ??-amyloid, LPS or cytokines in microglia. Previous studies have demonstrated that estrogen has an excellent function in protecting neuron, such as Alzheimer''s disease and in protecting ROS.
In this plan, we used various kinds of VTT extracts including its root, branch, leaf, and trunk to examine the protective function in neurodegenerative diseases in vivo and in vitro. We found that the branch and leaf extracts of VTT significantly inhibited iNOS protein expression and nitric oxide production in BV2 microglia when cultured in medium with 10% FBS. On the other hand, all four kinds of VTT extracts could inhibit iNOS protein expression and nitric oxide production in BV2 microglia when cultured in medium without FBS. Using primary neurons, we found that kainic acid (KA) could induce ROS production in a dose-dependent manner and finally resulted in neuron cells death. All four kinds of VTT extracts inhibited the ROS production, however only root and branch extracts were able to prevent the cell death induced by KA. The protective activity of various kinds of VTT extracts on the microglia
inflammation and neuron cells death could be ranked as follows:
branch≈leaf>root>trunk. In animal study, we found that i.p. injecting KA caused seizure, neurons cells death in hippocampus area, furthermore induced mice death in C57BL/6 mice. Administration of all four kinds of VTT extracts could delay the onset time of seizure and decrease the hippocampus neurons cells death. The potency of VTT extracts could be ranked as follows: branch≈leaf>root>trunk. However, all four kinds of VTT extracts could not decrease the number of mice death induced by KA.
