臺灣博碩士論文加值系統

以欖仁樹落葉為材料，分別使用乙醇、丙酮、正己烷、超臨界二氧化碳(SC-CO2)萃取及熱水萃取方式進行萃取，再依Ames test以Salmonella typhimurium TA98和TA100為測試菌株，探討欖仁樹落葉萃取物之抗致突變性時，結果顯示在0.05-0.5 mg/plate的添加濃度範圍，對TA98和TA100菌株不具毒性及致突變性，但具有抗致突變性，且皆隨濃度的增加而增加，其中以SC-CO2萃取物的抗致突變性最佳。SC-CO2萃取物(0.5mg/plate)對於需要S-9活化的間接致突變劑(B[a]P ; 2μg/plate)的抗致突變效果為75.5%，而對於不需S-9活化之直接突變劑(MNNG ; 5μg/plate)的抗致突變效果為40.6%。由此可知欖仁葉SC-CO2萃取物中確實含有抗致突變物質，經GC-MS分析鑑定後，得知鯊烯(squalene)為其主要的活性成分，再經Ames test測試後，得知squalene的抗致突變機制可能為吸附於菌體表面而阻礙突變劑進入菌體內，而降低突變的機率。另外，SC-CO2萃取物對Huh 7 cell和Chang liver cell的毒殺效果較squalene佳，同時也具有較佳的選擇性毒殺肝癌細胞作用。在抗氧化性方面，分別採樣測定欖仁樹嫩葉、成熟葉、老化葉和落葉SC-CO2萃取物中squalene的含量時，其含量分別為65、210、451、1499μg/g powder，顯示squalene的含量會隨著葉子成熟度的增加而增加。各種萃取物對於抑制共軛雙烯氫過氧化物(CDHP)生成的能力及清除DPPH自由基的能力比較，依序為落葉＞老化葉＞成熟葉＞嫩葉。另外種子萃取物雖未測得squalene，但其對於抑制CDHP生成的能力與老化葉近似，表示其含有其他抗氧化成分。綜合本研究之結果顯示，欖仁葉SC-CO2萃取物具有良好之抗致突變性和抗氧化性。

The absicissed leaves of Terminalia catappa were extracted with ethanol, acetone, n-hexane, supercritical CO2 (SC-CO2) and boiling water, respectively and the extracts were subjected to Ames test using Salmonella typhimurium TA98 and TA100 as testing strains, neither toxicity nor mutagenicity of the extracts was detected up to a dose of 0.5mg/plate. SC-CO2 extracts showed the strongest antimutagenicity among all types of extracts. At the dose of 0.5mg/plate of SC-CO2 extracts, the mutagenicity of B[a]P (with S-9) was 75.5% of inhibition while 40.6% of the mutagenicity of MNNG (without S-9) was inhibited. After subjecting to GC-MS analysis, the active compound of squalene was presumptively identified and was rich in the SC-CO2 extracts of T. catappa leaves. When squalene was applied in mutagenicity test, a dose-response relationship was observed. When the SC-CO2 extracts and squalene were applied to the test of cytotoxicity, the growth of the Huh 7 and Chang liver cells were significantly inhibited by SC-CO2 extracts rather than by squalene. In comparison, the SC-CO2 extracts exhitited a higher cytotoxic effect on hepatoma cells (Huh 7) rather than on normal liver cells (Chang liver). When the freeze-dried immature, mature, senescent and abscisic leaves were subjected to SC-CO2 extraction and HPLC quantitation, squalene contents were 65, 210, 451 and 1499 μg/g powder, respectively. The results showed that the squalene content increased with an increase of leaf maturity. When the extracts were applied for antioxidative characterization by supplementation in an iron/ascorbate system with linoleic acid, in a pork-fat storage system or in a free radical scavenging system with DPPH, the extracts of leaves exhibited potent antioxidative and DPPH scavenging activities and the activities increased with an increase of leaf maturity. Squalene was not detected in the extracts of T. catappa seeds while the extracts exhibited a significant antioxidative activity. In conclusion, the SC-CO2 extracts of T. catappa leaves exerted potent antimutagenicity and antioxidative activities.

中文摘要………………………………………………………………… Ⅰ
英文摘要………………………………………………………………… Ⅱ
壹、 前言………………………………………………………………… 1
貳、 文獻整理…………………………………………………………… 3
一、 癌症的危害…………………………………………………… 3
二、 環境因子與癌症……………………………………………… 3
(一)、增加癌症發生之因素…………………………………. 4
(二)、化學致癌作用之機制………………………………….. 8
三、 抗致突變成分………………………………………………… 13
四、 抗致突變物/抗致癌物之作用機制…………………………… 17
五、 致突變性檢測方法……………………………………………. 21
六、 Salmonella typhimurium TA98和TA100 的DNA 序列之特
異性…………………………………….………………………. 22
七、 肝癌之防治……………………………………………………. 23
八、 氧化與疾病……………………………………………………. 23
(一)、活性氧與自由基……………………………………….. 23
(二)、氧化壓力……………………………………………….. 24
(三)、抗氧化防禦系統……………………………………….. 27
(四)、膳食抗氧化物對致癌作用之影響…………………….. 29
九、超臨界二氧化碳萃取之簡介…………………………………. 33
十、欖仁葉的植物特徵……………………………………………. 35
十一、欖仁葉的研究概述…………………………………………. 37
參、 欖仁葉的抗致突變性
一、材料與方法…………………………………………………… 40
(一)、原料……………………………………………………….. 40
(二)、化學試藥……………………………………………………. 40
(三)、試驗菌株……………………………………………………. 40
(四)、試驗細胞株…………………………………………………. 40
(五)、一般成分分析……………………………………………… 40
1、水分………………………………………………………… 40
2、脂質………………………………………………………… 41
3、粗蛋白質…………………………………………………… 41
4、灰分………………………………………………………… 41
5、粗纖維……………………………………………………… 42
6、無氮化合物……………………….………………………… 42
(六)、溶劑萃取物的製備………………………………………… 42
(七)、水萃取物的製備…………………………………………… 42
(八)、超臨界二氧化碳萃取物的製備…………………………… 43
(九)、欖仁葉萃取物抗致突變性之研究………………………… 43
1、毒性試驗……………………………………………………. 43
2、致突變性試驗………………………………………………. 43
3、抗致突變性試驗……………………………………………. 44
(十)、欖仁葉萃取物抗致突變性成分之測定……………………. 45
1、超臨界二氧化碳(SC-CO2)萃取物成分之分析與鑑定……. 45
2、Squalene含量的測定………………………………………. 45
(十一)、抗致突變成分之抗致突變反應機制…………………… 45
1、生物抗致突變性…………………………………………… 46
2、阻礙試驗…………………………….……………………… 46
3、抑制致突變劑………………………………………………. 47
4、抑制突變劑代謝活化物…………………………………….. 48
5、使S-9失活………………………………………………….… 49
(十二)、毒殺人類肝癌細胞之測定………………………………… 50
1、細胞培養方法………………………………………………….. 50
2、細胞活性定量(Acid phosphatase assay)………………………. 50
(十三)數據之統計分析……………………………………………… 51
二、結果與討論………………………………………………………… 52
(一)、欖仁葉萃取物對Salmonella typhimurium TA98 和TA100
之抗致突變性…………………………………………………… 52
(二)、欖仁葉超臨界二氧化碳萃取物之氣相層析圖與組成分……. 65
(三)、Squalene之抗致突變機制……………………………………. 72
1、生物抗致突變劑……………………………………………….. 72
2、阻礙作用……………………………………………………….. 75
3、抑制致突變劑…………………..…………………………….. 75
4、抑制突變劑的代謝活化物之作用…………………………….. 78
5、使S-9失活…………………………………………..………… 84
(四)、Squalene對人類肝癌細胞生長的影響………………………. 84
三、結論………………………………………………………………… 91
肆、 欖仁葉的抗氧化特性
一、材料與方法……………………………………………………………. 92
(一)、材料…………………………………………………………….. 92
(二)、超臨界二氧化碳萃取………………………………………… 92
(三)、Squalene含量的測定………………………………………… 92
(四)、清除DPPH自由基能力之測定……………………………… 92
(五)、在亞麻油酸/鐵/維生素C系統中的抗氧化能力之測定……. 92
(六)、抑制豬油貯存時生成共軛雙烯氫過氧化物(CDHP)能力
之測定………………………………………………………………… 93
(七)、數據之統計分析………………………………………………. 93
二、結果與討論………………………………………………………….... 94
(一)、不同成熟度欖仁樹葉和種子的超臨界二氧化碳萃取率及其
squalene之含量…………….………………………………………... 94
(二)、不同成熟度欖仁樹葉和種子的超臨界二氧化碳萃取物對
DPPH自由基的清除能力…………………………………………… 97
(三)、不同成熟度欖仁樹葉和種子的超臨界二氧化碳萃取物在
亞麻油酸/鐵離子/維生素C測試系統中的抗氧化能力…………… 99
(四)、不同成熟度欖仁樹葉和其種子的超臨界二氧化碳萃取物抑
制共軛雙烯氫過氧化物生成的能力…………….………….…….… 102
三、結論…………………………………………………………….……… 106
伍、參考文獻……………………………………………………….…..……. 107
List of Tables
Table 1. 行政院衛生署90年十大死亡原因……………………………… 2
Table 2. Categories of foods with most prominent chemopreventive
compounds…………………………………….……………………. 15
Table 3. Conditions associated with oxidative damage……………….……. 25
Table 4. Oxidant and antioxidant balance…………………………… …..….. 28
Table 5. Inhibition of chemical carcinogenesis by antioxidants……………. 31
Table 6. Applications of supercritical fluid extraction……………...………... 36
Table 7. Proximate composition of Terminalia catappa leaves…………… . 53
Table 8. Extraction yields from Terminalia catappa leaves with various
solvents…………………………………………………………..…. 54
Table 9. Cytotoxicity of various solvent extracts of Terminalia catappa leaves
toward Salmonella typhimurium TA98 and TA100………………… 55
Table 10. Mutagenicity of various solvent extracts of Terminalia catappa
leaves toward Salmonella typhimurium TA98 and TA100…………. 57
Table 11. Antimutagenicity of various solvent extracts of Terminalia catappa
leaves against the mutagenicity of MNNG toward Salmonella
typhimurium TA98 and TA100…………………………………….... 58
Table 12. Antimutagenicity of various solvent extracts of Terminalia catappa
leaves against the mutagenicity of B[a]P toward Salmonella
typhimurium TA98 and TA100……………………………………... 59
Table 13. Presumptive identification of the comprising components in the SC-
CO2 extracts (extracted at 2000 psi and 40 oC) of the abscisic leaves
of Terminalia catappa by GC-MS analysis and library scanning….. 67
Table 14. The antimutagenic mechanism of squalene against the mutagenicity
of B[a]P toward Salmonella typhimurium TA98 and TA100………… 88
Table 15. The supercritical CO2 extraction yields and squalene content
(extracted at 3000 psi and 40 oC) of the immature, mature,
senescent, abscisic leaves and seeds of Terminalia catappa ……..… 96
List of Figures
Figure 1. Structures of some aflatoxins……………………………………….. 6
Figure 2. Metabolic activation of genotoxic chemical carcinogens…………... 10
Figure 3. DNA adduct formation with activated benzo[a]pyrene……………… 12
Figure 4. The classes of the antimutagenic mechanisms………………………. 18
Figure 5. Interaction of active B[a]P and ellagic acid………………………….. 20
Figure 6. Commonly used synthetic antioxidants……………………………… 32
Figure 7. Phase diagram of carbon dioxide ………………………………….. 34
Figure 8. Antimutagenicity of supercritical CO2 extracts of the abscisic leaves
of Terminalia catappa against the mutagenicity of MNNG toward
Salmonella typhimurium TA98……………………………………….. 60
Figure 9. Antimutagenicity of supercritical CO2 extracts of the abscisic leaves
of Terminalia catappa against the mutagenicity of MNNG toward
Salmonella typhimurium TA100………………………………………. 61
Figure 10. Antimutagenicity of supercritical CO2 extracts of the abscisic leaves
of Terminalia catappa against the mutagenicity of B[a]P toward
Salmonella typhimurium TA98………………………….……………. 63
Figure 11. Antimutagenicity of supercritical CO2 extracts of the abscisic leaves
of Terminalia catappa against the mutagenicity of B[a]P toward
Salmonella typhimurium TA100…………………………………...…. 64
Figure 12. Gas chromatogram of the supercritical CO2 extracts (extracted
at 2000psi and 40℃) of the abscisic leaves of T. catappa and the
separated compounds were identified by GC-MS analysis and
library scanning……………………………………………………..… 66
Figure 13. HPLC chromatograms of the supercritical CO2 extracts before and
after spiking with an authentic standard. A: 50 µg/mL of the supercritical
CO2 extracts, B: spiked with an equal volume of authentic squalene
(50 µg/mL)……………………………………………...……...….… 68
Figure 14. Antimutagenicity of squalene against the mutagenicity of MNNG
toward Salmonella typhimurium TA98 and TA100………………..… 70
Figure 15 Antimutagenicity of squalene against the mutagenicity of B[a]P
toward Salmonella typhimurium TA98 and TA100……………..…… 71
Figure 16. Bioantimutagenicity of squalene against the mutagenicity of
MNNG toward Salmonella typhimurium TA98 and TA100……..…. 73
Figure 17. Bioantimutagenicity of squalene against the mutagenicity of B[a]P
toward Salmonella typhimurium TA98 and TA100…………..…….. 74
Figure 18. Antimutagenicity of preincubation of Salmonella typhimurium
TA98 and TA100 with squalene against the mutagenicity of MNNG.…. 76
Figure 19. Antimutagenicity of preincubation of Salmonella typhimurium
TA98 and TA100 with squalene against the mutagenicity of B[a]P.…… 77
Figure 20. Antimutagenicity of preincubation of MNNG with squalene against
the mutagenicity of MNNG toward Salmonella typhimurium TA98 and
TA100…………………………………………………………………. 79
Figure 21. Change of UV spectrum in association with the interaction of
MNNG and squalene……..……………………………..…..……….... 80
Figure 22. Antimutagenicity of preincubation of B[a]P with squalene against
the mutagenicity of B[a]P toward Salmonella typhimurium TA98 and
TA100……………………………………………………………..…... 81
Figure 23. Change of UV spectrum in association with the interaction of B[a]P
and squalene……..…………………………………………………..… 82
Figure 24. Antimutagenicity of preincubation of activated-B[a]P with squalene
against the mutagenicity of B[a]P toward Salmonella typhimurium
TA98 and TA100……………………………………...…….…………. 83
Figure 25. Change of UV spectrum in association with the interaction of
activated B[a]P and squalene…………………..………………..…….. 85
Figure 26. Antimutagenicity of preincubation of S-9 mixture with squalene
against the mutagenicity of B[a]P toward Salmonella typhimurium
TA98 and TA100 ………...……………………………..……………... 86
Figure 27. Change of UV spectrum in association with the interaction of S-9
and squalene…………………………….…………………..…………. 87
Figure 28. The cell viability of Chang liver (C) and Huh 7 (H) cell exposured
to squalene (SQ) and supercritical CO2 extracts (E) for 24h……..…… 90
Figure 29. Standard curve of squalene determined with HPLC …………....… 95
Figure 30. The scavenging activities of the supercritical CO2 extracts of
immature (I), mature (M), senescent (S) and abscisic (A) leaves
and seeds (SD) of Terminalia catappa, butylated hydroxy toluene
(BHT) and squalene (SQ) determined with DPPH radical……….…... 98
Figure 31. The antioxidative potency of the supercritical CO2 extracts of
immature (I), mature (M), senescent (S) and abscisic (A) leaves
and seeds of Terminalia catappa, butylated hydroxy toluene (BHT)
and squalene (SQ) determined with linoleic acid in an iron/ascorbate
system………………………………………………………………… 101
Figure 32. Changes of CDHP contents during storage at 60oC for 6 days of the
pork-patty oils supplemented with the supercritical CO2 extracts of
immature (I), mature (M), senescent (S) and abscisic (A) leaves and
seeds (SD) of Terminalia catappa, butylated hydroxy toluene (BHT)
and squalene (SQ) determined with pork-fat storage method………... 103

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