天麻水萃物活性成分之抗憂鬱功效及其分子機制的探討

Abstract

重鬱症 (Major depressive disorder, MDD) 的問題普遍存在於世界各國。目前，全球約有超過 3.5 億的人口患有 MDD，世界衛生組織預測 MDD 在 2030 年將成為全球高負擔疾病的第一名。然而，現今西醫的治療方式，常會帶來許多病人無法接受的副作用，故從自然界尋找替代西藥的素材是迫切急需的課題。天麻 (Gastrodia elata Blume) 是中醫食療中用於作養心安神的藥材，近年研究發現具有改善多種神經退化性疾病的功效。本研究擬以天麻水萃物 (water extract of G. elata Bl, WGE) 之活性成分天麻苷 (gastrodin, GAS) 與天麻苷元 (4-hydroxybenzyl alcohol, HBA) 為素材，探討經強迫游泳試驗 (forced swimming test, FST) 誘導類憂鬱之大鼠是否藉由降低腦部單胺類系統與調節神經細胞骨架重組達到抗憂鬱效果。另外，進一步以 HPLC 建立GAS 分析方法確效以利天麻原料的管理。在動物實驗方面，六週齡 Sprague Dawley (SD) 雄性大鼠經連續 21 天分別管灌餵食 WGE (500 mg/kg bw)、GAS (100 mg/kg bw) 和 HBA (100 mg/kg bw)，以 FST 誘導類憂鬱行為。在行為分析結果顯示 WGE、GAS 和 HBA 皆可顯著降低因絕望所導致的不泳動行為 (immobility time)。腦部單胺類濃度以 HPLC 進行分析，結果顯示三種樣品皆可降低腦部血清素和多巴胺的代謝速率。由於臨床研究發現憂鬱症患者除了與單胺類濃度異常具有高度關聯性外，神經可塑性 (neuronal plasticity) 亦扮演重要角色。因此，本研究後續以西方墨點法探討 WGE、GAS 和 HBA 對神經細胞骨架重組 (neuronal cytoskeletal remodeling) 相關路徑──Slit-Robo 之影響，結果顯示各處理組皆可顯著降低海馬迴中負調節因子 Slit1 和 RhoA 蛋白表現，同時提升正調節因子 CRMP2 和 PFN1 蛋白表現，進而提高神經可塑性。在細胞實驗方面，以人類寡樹突細胞瘤 (Human oligodendroglioma, Hs683) 驗證 Slit-Robo 路徑，此細胞株分別與 WGE (0.5 mg/ml)、GAS (50、100 和 200 μM) 和 HBA (50、100 和 200 μM) 共培養 24 小時，以西方墨點法分析證實 WGE、GAS 和 中高劑量之 HBA (100 和 200 μM) 可直接降低 Slit1 蛋白表現，此現象呼應海馬迴西方墨點法的分析結果。化學分析實驗方面，以 GAS 確效之 HPLC 方法分析加工方式、萃取溶劑、產地及變種對 GAS 含量之差異，結果顯示熱水萃取之烏天麻 (G. elata f. glauca S. Chow) 切片可得到較紅天麻 (G. elata Bl. f. elata) 高的 GAS 含量。綜上所述，本研究證實 GAS 是 WGE 中抗憂鬱活性成分，藉由降低單胺類代謝與調節神經細胞骨架重組達到此功效，並建立 GAS 確效以利往後調節憂鬱情緒保健食品與抗憂鬱用藥開發之原料篩選。

Major depression disorder (MDD) has generally been found in humans worldwide. More than 350 million people suffer from MDD. The World Health Organization (WHO) predicts that MDD will be the first global burden disease in 2030. Due to undesired side effects of depression treatment, patients with depression tend to avoid medical therapies. Therefore, it is important to discover an alternative therapy from natural sources. Gastrodia elata Blume (Tian-ma) is a common Chinese medicinal herb for mind tranquilizing. Recently, some studies have reported that G. elata Bl. could be applied to ameliorate various neurodegenerative disorders. The aim of this study is to investigate the active compounds, gastrodin (GAS) and 4-hydroxybenzyl alcohol (HBA) of the water extract of G. elata Bl. (WGE), on antidepressive-like activity in rats and explore monoamine metabolism and modulated cytoskeleton remodeling-related protein expression in the Slit-Robo pathway after forced swimming test (FST). Furthermore, the analysis method of GAS using HPLC was developed and validated for source of G elata Blume identification. In vivo studies, the male Sprague Dawley rats were oral gavage with 500 mg/kg bw of WGE, 100 mg/kg bw of GAS, and 100 mg/kg bw of HBA for 21 days. It was observed that the immobility time of WGE, GAS, and HBA groups were markedly decreased in FST. Thus the depressive-like behavior could be reversed. WGE, GAS, and HBA restored higher turnover rate of serotonin and dopamine compared to control group. Due to neuronal plasticity correlates with depression, Western blot analysis was applied to Slit-Robo pathway, the neuronal cytoskeletal remodeling related protein. It was found that WGE, GAS, and HBA significantly down-regulated Slit-Robo pathway in the hippocampus that promote neuroplasticity. In vitro studies, Human oligodendroglioma (Hs683) was co-culture with WGE (0.5 mg/ml), GAS (50, 100, and 200 μM), and HBA (50, 100, and 200 μM) for 24 hours. The result indicated that WGE, GAS (50, 100, and 200 μM), and HBA (100 and 200 μM) notable down-regulated the protein expression of Slit1 directly. The validation method using HPLC was applied to investigate the quantities of GAS from preparation processes, sources and phenotypes. The results showed that the slices of Gastrodia elata f. glauca S. Chow under water extraction contained the quantity of GAS higher than G. elata Bl. f. elata. In conclusion, these results suggest that GAS provides for WGE exerting antidepressive-like effects through decreasing monoamine metabolism and down-regulating Slit-Robo pathway. Furthermore, the validation method can be used as the source of G. elata Bl. screening for health food development.