| 摘要: | 研究目的 目前已知感染B肝病毒引起肝癌比例比未感染正常人高出100倍，因此探討有效對抗並治療肝炎病毒引起的癌症是刻不容緩的課題。研究指出癌症的發生初期是由發炎反應所引起，而慢性發炎則是惡性腫瘤生長迅速導致癌症發生之主因。許多中草藥具有抗癌、抗病毒、鎮痛與免疫調節功效，但其作用機轉仍不明確。臺灣由於地理環境獨特，特有種植物的比例達26.2 %，許多具有藥用資源開發之潛力。本計畫針對臺灣特有菊科蒿屬植物之抗癌、抗病毒、鎮痛、抗發炎與免疫調節功效予以評估，並嘗試以全合成方式合成有效成分，利用代謝質體分析方法分辨三種特有蒿屬植物。 研究方法 以具B型肝炎病毒基因之肝癌細胞株以細胞培養模式對蒿屬植物所含之有效成分對肝癌、B型肝炎病毒之療效和機轉進行評估。利用醋酸扭體試驗評估細葉山艾乙醇抽提物是否具有鎮痛作用，再以福馬林舔足試驗辨別其鎮痛作用在周邊或中樞，並以λ-角叉菜膠誘導小鼠足蹠腫脹試驗探討其抗發炎作用之機轉，再分析其肝臟組織中超氧歧化?、發炎足蹠組織中的脂質過氧化指標產物丙二醛、腫瘤壞死因子-α、組織介素IL-6之含量變化。以樹狀細胞TNF-與IL-6評估是否具有免疫調節作用，評估與LPS合併是否可減少其釋出。利用優化全合成方式合成兩種類黃酮並且評估其抗肝炎病毒機轉。利用HPLC-MS配合主成分分析分辨三種蒿屬植物之代謝物差異。 結果與討論 許多蒿屬植物萃取物皆具有抗B型肝炎病毒之活性，在細葉山艾萃取物中發現具顯著抑制B型肝炎病毒DNA複製之天然成分，進一步分析其對B型肝炎病毒基因啟動子活性發現對B肝病毒X基因啟動子有顯著促進作用。此促進作用對病毒生合成機制甚至影響病毒顆粒之釋放是否有影響還有待進一步分析。細葉山艾分離之p-hydroxyacetophenone可能透過抑制病毒顆粒從細胞分泌之途徑而間接抑制肝炎病毒之產生，而chlorogenic acid可能為另一具有抗病毒之成分。體內模式評估鎮痛抗發炎部分發現細葉山艾乙醇抽提物具有鎮痛及抗發炎作用，其作用機轉可能與提升肝臟中抗氧化酵素SOD活性、減少足蹠組織中MDA的含量、組織介素IL-6及TNF-α之濃度進而達到抗發炎之作用有關。蒿屬植物水萃物具有促進TNF-與IL-6增加並在合併後可降低LPS引起之釋出。在全合成類黃酮arcapillin與eupatolitin方面，藉由改變起始物與新穎合成步驟達到高產率與簡易合成之目的，可以提供研究藥理機轉之用。此外，利用代謝質體研究方式發現可以明確分辨細葉山艾、高山艾與山艾，與藥理活性比對後發現一些分子離子峰與斷裂片，正嘗試鑑定結構中。
Aim The relative risk for HBV carriers to developing hepatocellular carcinoma is more than 100 times compared with uninfected people. Therefore the searching of effective treatment to HBV infection becomes an important medical task. Many studies indicate that inflammation in the initial stage will possibly lead to cancer formation. A lot of herbal medicines possess a variety of activities including anticancer, anti-virus, analgesia, anti-inflammation and imunoregulation, however, their underlying mechanisms are still veiled. Due to unique geographic environment of Taiwan, the percentage of endemic species is 26.2 % in plants from which many of them are with potential in discovery of medicinal resources. This project aimed to evaluate the previous activities for endemic Artemisia plants by using in vitro and in vivo models. Moreover, total synthesis to flavonoids found in some Artemisia plants including arcapillin and eupatolitin to provide with sufficient amount for subsequent pharmacological testing was also finished. Moreover, we make efforts to differentiate between close Artemisia species including A. morrisonensis, A. oligocarpa and A. kawakamii via metabolomics approaches. Method The extracts/pure compounds from Artemisia plants were all tested for their ability against HepG2 2.2.15 cell, anti-HBV together with acting mechanism. The analgesic effects exerted centrally or peripherally by the ethanolic extract of A. morisonensis (Am) were evaluated by inducing writhing caused by acetic acid and formalin test. The anti-inflammatory effects were tested with the extract of Am by inducing edema with λ-carrageenan. In order to understand the possible underlying mechanisms, further testing to SOD, MDA, IL-6 and TNF- activities were then performed. The effects to TNF- and IL-6 were assessed alone or in combination with LPS in dendritic cell for Artemisia extracts. We make endeavor to optimize the total synthetic steps to obtain enough amounts of flavonoids, arcapillin and eupatolitin for advanced studies. Besides, by applying metabolomics, three close Artemisia species were differentiated via LC-MS methodology. Results & Discussion Several ethanolic/aqueous extracts from Artemisia genus demonstrated anti-HBV activity. In A. morrisonensis, some partially-purified fractions were tested and showed potent suppressive effect on hepatitis B viral DNA replication. Promoter-reporter assay revealed specific fractions will promote the HB X gene activation, however the mechanism of this activation remains elucidated. The identified constituent, p-hydroxyacetophenone causes the inhibition to HBV propagation by inhibiting the secretion of HBV particle out of the hepatocyte. Chlorogenic acid might be another constituent responsible for the activity. The extract from Am (100, 500 mg/kg, p.o.) showed significantly analgesic and anti-inflammatory effects in vivo. The possible mechanism of analgesia might be related to AA and PGs associated with enhancement of the anti-oxidative enzyme in liver for cleaning the free radical and diminish the damage of lipid peroxidation in inflammatory paw tissue. The other mechanism perhaps associated with decreasing IL-6 and TNF-α concentration for the inhibition activated. The aqueous extract of Artemisia species showed stimulatory effects to TNF-/IL-6 whereas demonstrating inhibitory effects upon combining with LPS in dendritic cell. Through selecting appropriate starting materials and optimized synthetic steps, we successfully finished the total synthesis of arcapillin and eupatolitin for studying mechanisms. PCA analysis to LC-MS data proved to be excellent approach to differentiate A. morisonensis, A. oligocarpa and A. kawakamii which are morphologically close. Some molecular/fragment ions found in MS were identified and confirmation of their corresponding structure is currently being undertaken. |
