本論文利用斑馬魚進行活體毒性及抗氧化能力測試，作為篩選黃酮衍生物防護UVB能力的平台。首先，以100 mJ/cm2的UVB照射三天大的斑馬魚幼魚，每半小時照射一次共六次，發現最易觀察鰭傷害的變化是腹鰭。再加入一系列黃酮衍生物預防UVB所造成的傷害，以生物存活統計分析其毒性，並以抗氧化能力測試比較其防護UVB的能力，發現morin跟chrysin具有良好防護UVB的效果。進一步藉由理論與實驗結合的藥物開發方法—定量結構活性關係（Quantitative Structure-Activity Relationship. QSAR）分析，分別建立黃酮衍生物的毒性模型及抗氧化性模型，驗證QSAR模型分析與斑馬魚活體實驗的結果相符，並推測出黃酮衍生物在3’與4’位置的取代基若為氨基則具有良好的抗氧化能力。本研究結果顯示，結合斑馬魚活體實驗與QSAR分析，可提供快速篩選及推測驗證新化合物毒性及防護UVB能力的平台。

To screen UV-protective flavonoid derivatives with higher efficacy and less toxicity, this study used zebrafish as a whole-organism platform to examine the toxicity and antioxidant activity of flavonoid derivatives. AT first, 3-dpf zebrafish were exposed to 100 mJ/cm2 of UVB given six times separated by 30 min intervals. Fin malformation phenotypes are the most evident consequences after exposure to UVB, pelvic fin seemed to be the most sensitive target after UVB exposure. The next, a series of flavonoid derivatives were next examined for their toxicity and antioxidant activity. “Morin” and “Chrysin” showed better UV-protective activities among these tested flavonoid derivatives. Furthermore, Quantitative Structure-Activity Relationship (QSAR) analysis was used to establish both toxicity and antioxidant activity models for flavonoid derivatives. The theoretical antioxidant activities predicted by QSAR models were consistent with the experimental data obtained from zebrafish. Using QSAR models to predict a series of flavonoid derivatives, our data suggested that the amino substituents at positions 3’ and 4’ of the A ring may play important roles in the antioxidant activity. 

In conclusion, this study demonstrated that combining zebrafish and QSAR can provide a useful platform to quickly evaluate the toxicity and antioxidant activity of UV-protective compounds.

目錄
中文摘要……………………………………..…………I
英文摘要…………………………………….………... II
目錄………………………………………………….. IV
圖表目錄…………….……………………….……..VIII
文獻……………………………………………………41
附錄……………………………………………………72

第一章	前言	1
1.1	UV對皮膚的傷害	1
1.1.1	UV簡介	1
1.1.2	UV造成皮膚傷害	1
1.2	皮膚傷害、DNA傷害跟氧化作用之間的關係	2
1.2.1	紫外線造成DNA傷害	2
1.2.2	氧化作用	4
1.3	類黃酮簡介	5
1.4	類黃酮分類跟結構	5
1.5	類黃酮的抗氧化	7
1.6	類黃酮抗UV	10
1.7	QSAR簡介	11
1.8	斑馬魚作為模式物種的優勢	13
1.9	實驗目的	14
第二章	材料與方法	15
2.1	實驗魚－斑馬魚隻飼養與胚胎收集（DANIO RERIO）	15
2.2	紫外線曝露	16
2.3	藥物的取得跟治癒	16
2.4	類黃酮(FLAVONOID)和黃酮衍生物半致死濃度浸泡	17
2.5	鰭損傷的觀察紀錄	18
2.6	TUNEL 分析細胞凋亡	18
2.7	顯微鏡使用與照相系統及影像處理	20
2.8	活性氧之偵測	20
2.9	統計分析	21
2.10	輔助藥物設計（COMPUTER AIDED DRUG DESIGN）	21
第三章	結果	22
3.1	生物模型的建立	22
3.1.1	經由適當的UVB照射後，魚鰭的萎縮是最明顯的變化	22
3.1.2	綠茶萃取物會增加魚鰭回復的速率	23
3.1.3	保護魚鰭受到紫外線的傷害是預防勝於治療，腹鰭的表現最具有差異	23
3.1.4	利用EGCG可預防UVB在腹鰭誘導出的傷害	24
3.1.5	綠茶萃取物和EGCG的預防，可以保護斑馬魚幼體免受UVB誘導出的傷害，是藉由防止細胞凋亡的發生	25
3.2	鑑定黃酮藥物抗UVB誘導傷害	26
3.2.1	使用黃酮、黃烷酮跟查爾酮比較，黃酮有較好的預防UVB能力	26
3.2.2	黃酮衍生物產生ROS的比較跟半致死濃度的關係	27
3.2.3	抗氧化能力較好的黃酮衍生物Morin跟Chrysin其預防UV的能力相對也強	28
3.3	利用 QSAR來分析黃酮結構所產生的毒性跟抗氧化性	28
3.3.1	建立模型分析	28
3.3.2	新結構分析	30
3.4	驗證模擬預測值	35
3.5	總結	35
第四章	討論	36
4.1	斑馬魚幼體可以被當作篩選預防UV藥物的模式物種	36
4.2	使用光修復機制來治癒UV對於魚鰭傷害	36
4.3	表現型的優勢和篩選	37
4.4	利用黃酮的抗氧化力來預防斑馬魚皮膚上UVB傷害	38
4.5	預測毒性低跟抗氧化的黃酮新結構	38
4.6	利用斑馬魚當作防曬藥物的模式平台	39

圖表目錄
FIG.1.對UVB的暴露劑量以及預防治療實驗設計	48
FIG.2:經由UVB照射後表現型跟劑量之間的關係	49
FIG.3. KAPLAN-MEIER統計分析各魚鰭的畸形率(D,E,F,G:PF,VF,CF,DF)在UVB照射下，控制組跟綠茶預防跟治癒的比較曲線圖	50
FIG.4.KAPLAN-MEIER統計分析圖，分析腹鰭在EGCG預防下經過UVB照射後的畸形率	51
FIG.5.UVB暴露後在魚鰭上所造成的細胞凋亡現象，在腹鰭的位置使用TUNEL法，深褐色的點為細胞凋亡位置	52
FIG.6. KAPLAN-MEIER統計分析各魚鰭的畸形率在UVB照射下，(A)控制組跟類黃酮生物預防的比較曲線圖	53
FIG.7.UVB暴露後在魚鰭上所造成的細胞凋亡現象，在腹鰭的位置使用TUNEL法，深褐色的點為細胞凋亡位置	54
FIG.8.黃酮衍生物ROS，經過2,7-DICHLOROFLUORESCIN DIACETATE (H2DCF-DA)染劑處理後相對UVB控制組螢光強度倍率	55
FIG.9. KAPLAN-MEIER統計分析各魚鰭的畸形率在UVB照射下，控制組跟黃酮衍生物預防的比較曲線圖	56
FIG.10.各個黃酮衍生物的結構圖跟抗氧化能力模擬值	57
FIG.11.推測具有高抗氧化能力的黃酮衍生物結構圖	58
TABLE.1.使用KAPLAN-MEIER法，統計分析各個魚鰭組別	59
TABLE.2.COX PROPORTIONAL HAZARDS REGRESSION 統計臀鰭、腹鰭、背鰭在UVB+綠茶的預防跟治療分析	60
TABLE.3.COX PROPORTIONAL HAZARDS REGRESSION 統計腹鰭在UVB+綠茶的預防	61
TABLE.4.COX PROPORTIONAL HAZARDS REGRESSION 統計腹鰭在UVB+類黃酮的預防	62
TABLE.5.COX PROPORTIONAL HAZARDS REGRESSION 統計腹鰭在UVB+黃酮不同濃度的預防	63
TABLE.6.COX PROPORTIONAL HAZARDS REGRESSION 統計腹鰭在UVB+黃酮衍生物不同濃度的預防	64
TABLE.7.黃酮衍生物各組別的半致死濃度PPM(LC50)、抗氧化能力和跟照射UVB回復腹鰭的相對能力	65
TABLE.8.QSAR計算毒性的各個高相關性方程式	66
TABLE.9.QSAR計算抗氧化的各個高相關性方程式	67
TABLE.10.QSAR計算黃酮衍生物半致死濃度	68
TABLE.11.QSAR計算黃酮衍生物抗氧化性	69
TABLE.12.QSAR計算推測黃酮衍生物半致死濃度	70
TABLE.13.QSAR計算推測黃酮衍生物抗氧化性	71
TABLE.14.黃酮衍生物比較抗氧化能力，主要位置取代基分配圖	72

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