Fructus Alpiniae oxyphyllae (Yi zhi) is a traditional medicine used for treating diarrhea with splenic cold, polyuria, gastralgia, spontaneous salivation, kidney asthenia with enuresis, spermatorrhea and turbid urine. The Chinese name of Fructus Alpiniae oxyphyllae means beneficial for intelligence. These suggest that Fructus Alpiniae oxyphyllae is a potential candidate for treating brain disorders. In this study, the ethanol extract of Fructus Alpiniae oxyphyllae was identified to have potent neuroprotect effect and angiogenic activity using multiple bioassays. The viability and cellular damage of the PC12 cells were determined by MTT assay and LDH assay, respectively. Intracellular nitric oxide (NO) was quantified by DAF-FM diacetate staining. The expression of inducible nitric oxide synthase (iNOS) was determined using Western blot. The viability of dopaminergic (DA) neuron in zebrafish was examined by ant- tyrosine hydroxylase (TH) immunostaining. Pretreatments of AOE (3, 6, 12, 25, 50μg/ml) increased the viability of 6-OHDA-damaged PC12 cells in a dose dependent manner. Moreover, AOE exhibited significant neuroprotective activity against 6-OHDA-induced dopaminergic (DA) neuron loss in zebrafish. Mechanistic study showed that anti-oxidative (inhibition of NO production and iNOS expression in PC12 cells in vitro) activities of AOE may play a role in its neuroprotective effect in 6-OHDA-induced DA neuron damage. On the other hand, AOE induced human umbilical endothelial cells (HUVEC) proliferation and the numbers of branching points during endothelial cells capillary formation on Matrigel. Meanwhile, the vasculature of the zebrafish caudal fin regeneration reconstructed better in the AOE treatment group. These results demonstrate the neuroprotective activity and proangiogenesis effect of AOE in vivo and in vitro, which suggest that Fructus Alpiniae oxyphyllae has potential to be developed as a therapeutic agent for Parkinson’s disease and regeneration. Key words Fructus Alpiniae oxyphyllae, neuroprotective, Parkinson’s disease, angiogenesis, zebrafish, PC12, NO, iNOS, HUVEC, regenerate

益智(Fructus Alpiniae oxyphyllae) 是一味傳統的補益類中藥。根據相關資料 的記載，益智的功能主治是溫脾止瀉，攝垂涎，暖腎，固精縮尿，用於脾寒泄瀉， 口多垂涎，腎虛遺尿，小便頻數，遺精白濁。由於該味藥材的用藥部位是其果仁， 因此，又稱益智仁。僅從字面意思來理解該藥材的功能，可以得知它對智力有正 向的積極作用。 本實驗以 pheochromocytoma (PC12) 細胞、人臍靜脈內皮細胞 HUVEC 和斑 馬魚為實驗模型，探討益智仁乙醇提取物 (AOE) 在神經保護及血管新生方面的 藥效。 通過4-甲基偶氮唑藍比色法 (MTT法) 和乳酸脫氫酶釋放法 (LDH法) 檢測 PC12細胞的活力，益智仁乙醇提取物能夠劑量依賴性地提高6-羥基多巴胺 (6-OHDA) 損傷的PC12細胞的存活率，減少細胞凋亡。利用DAF-FM diacetate螢 光染色來檢測PC12細胞內的一氧化氮 (NO)，發現AOE能夠抑制6-OHDA在斑馬魚 上所引起的多巴胺神經的減少。其作用機理可能是因為益智仁能夠降低PC12細 胞內由6-OHDA刺激所產生的NO的含量，免疫印跡雜交實驗結果證實AOE能夠降 低PC12細胞內一氧化氮合成酶 (iNOS) 的蛋白表達。在斑馬魚尾鰭再生實驗 中，AOE能夠很好地促進尾鰭脈管系統的修復，推動尾鰭再生的發展。 AOE對6-OHDA誘導的PC12細胞損傷具有保護作用, 能減少6-OHDA引起 的細胞凋亡, 並且可以減少6-OHDA在斑馬魚上引起的多巴胺神經元減少, 其 作用機制可能與降低NO的產生和iNOS的表達量有關。此外，通過尾鰭再生實 驗，我們可以猜測，AOE可能是通過促進腦部損傷部位周圍的血管再生，識得其神經保護作用得以更好的發揮。 關鍵詞： 益智仁，神經保護，帕金森綜合症，血管新生，斑馬魚，PC12細胞，一氧 化氮，一氧化氮合酶，人臍靜脈內皮細胞，再生