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研究生:陳心怡
研究生(外文):Shin-Yi Chen
論文名稱:香椿萃取物對小鼠抗氧化系統之影響及主要活性成分分析
論文名稱(外文):Effects of Different Extracts of Toona sinensis on the Antioxidant Activities in Mice and Its Active Components
指導教授:張素瓊張素瓊引用關係
指導教授(外文):Sue-Joan Chang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生命科學系碩博士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:162
中文關鍵詞:抗氧化成份分析香椿
外文關鍵詞:Toona sinensisactive componentantioxidant activity
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文獻記載香椿為具有保健功效,然而至今對香椿功效的科學研究仍有限。本研究以體外試驗探討不同香椿萃取物 (TSL-2, TSL-2P 與 TSL-6) 之抗氧化特性及其成份分析,結果發現 TSL-2 總酚類含量 (580.49 ± 3.41 μg/mg) 及類黃酮含量 (309.62 ± 10.78 μg/mg) 最高,約有 50.30 % 沒食子酸、1.40 % 槲黃素、2.49 % 的芸香苷、1.30 % 兒茶素以及 0.19 % 維生素 C,亦具有最高還原能力、清除 DPPH 自由基能力 (IC50 = 4.18±0.12 μg/ml) 及總抗氧化能力 (6.54 ± 0.03 μM Trolox),然而 TSL-2 螯合亞鐵離子能力最低,並沒有達到抑制50 %,無法估算IC50。TSL-2P 則約有 22.53 % 沒食子酸、1.45 % 槲黃素、2.20 % 芸香苷、0.66 % 沒食子酸乙酯及 0.20 % 維生素 C,其還原能力、清除 DPPH 自由基能力 (IC50 = 4.62 ±0.23 μg/ml) 及總抗氧化能力 (6.33 ± 0.04 μM Trolox) 則次於 TSL-2,TSL-2P 螯合亞鐵離子能力 (IC50 = 230.22 ± 45.92 μg/ml) 則次於 TSL-6。TSL-6 約有 7.85 % 沒食子酸、0.92 % 槲黃素、1.17 % 芸香苷及 0.36 % 維生素 C,其還原能力、清除 DPPH自由基能力 ( IC50 = 16.22 ± 4.15 μg/ml) 及總抗氧化能力 (3.08 ± 0.01 μM Trolox) 最低,螯合亞鐵離子的能力 (IC50 = 49.62 ± 6.1 μg/ml) 則為最高。
本研究另以動物模式探討香椿萃取物的抗氧化作用。結果顯示 TSL-2顯著降低睪丸中 SOD、CAT 及 GR 活性、提升 GPx 及 GST 活性、提升大腦中 GST 活性、降低 GSSG 濃度且使 GSH/GSSG 比值顯著提升,此外亦提升小腦中 MDA 濃度。TSL-2P 則能顯著提升肝臟中 GPx 活性、GSH 濃度且降低 MDA 濃度和 ROS 含量、腎臟中 ROS 含量、睪丸中 GR 活性及 ROS 含量,並且提升大腦中 GST 活性且降低 ROS 含量、提升小腦中 GST 活性及 GSH/GSSG 比值。TSL-6 顯著降低肝臟中 MDA 含量,降低睪丸中 GR 活性且提升 GSH 濃度,提升大腦內 CAT 及 GST 活性。
本研究進一步利用高壓液相層析儀 (HPLC) 將香椿萃取物進行分劃,所得之冷凍乾燥物處理HepG2細胞後進行抗氧化作用分析。實驗結果顯示,TSL-2-Fr-6 會顯著增加 HepG2 細胞中 MDA 濃度。其餘分劃部分並無顯著作用。TSL-2P-Fr-2、TSL-2P-Fr-5、TSL-2P-Fr-6分別顯著增加 HepG2 細胞中 GPx 活性及總抗氧化能力、SOD 活性及GST 活性,TSL-2P-Fr-5 亦降低 HepG2 細胞中 MDA 濃度。TSL-6-Fr-2、TSL-6-Fr-3 皆顯著增加 HepG2 細胞中 GST 活性。TSL-6-Fr-1、TSL-6-Fr-2、TSL-6-Fr-3 亦同時降低 HepG2 細胞中 MDA 濃度。
將這些具有抗氧化活性物質送測 LC-MS 比對標準品之結果發現, TSL-2P-Fr-6、TSL-6-Fr-1 及 TSL-6-Fr-3 比對到沒食子酸、沒食子酸甲酯,TSL-6-Fr-1 及 TSL-6-Fr-3 比對到沒食子酸乙酯。TSL-2P-Fr-6 及 TSL-6-Fr-3 亦分別比對到芸香苷及槲黃素。
綜合以上結果發現,TSL-2 雖含有最多總酚類、類黃酮及沒食子酸,但僅在還原及清除 DPPH 試驗具有良好效果,在體內反而可能造成睪丸及小腦的促氧化作用。此外,細胞實驗亦發現TSL-2-Fr-6 會促進 HepG2 的脂質過氧化現象。TSL-2P 在體外和體內皆有良好抗氧化能力,推測為槲黃素在 TSL-2P 中協同沒食子酸一同扮演抗氧化的角色,並回復沒食子酸對睪丸及小腦的氧化損傷。此外,細胞實驗亦發現 TSL-2P-Fr-6 亦顯著增加 HepG2 細胞中 GST 活性,並鑑定出含有沒食子酸、槲黃素及沒食子酸甲酯。TSL-6可提升睪丸和大腦的抗氧化酵素活性,達到提升保護的功效,推測 TSL-6 主要活性成份為單寧酸而非沒食子酸,在體外可有效清除螯合亞鐵離子,達到抗氧化保護效果。TSL-2、TSL-2P 與 TSL-6 皆有抗氧化功效,但其中以 TSL-2P 與 TSL-6 能達到較佳之保護功效,未來將針對這些有效分劃物繼續進行抗氧化機制之探討。
Chinese herb Toona sinensis (TS) was reported to be a functional food for enhancement of health in Chinese ancient books. However, the scientific data for its functionality is limited. In this study, the antioxidative activities of TSL (Toona sinensis leave) extracts revealed that TSL-2 had the highest DPPH radical scavenging activity ( IC50 = 4.18±0.12 μg/ml ). IC50 of DPPH radical scavenging activities of TSL-2P and TSL-6 were 4.62 ± 0.23 and 16.22 ± 4.15 μg/ml, respectively. The tendency in reducing power was TSL-2 > TSL-2P > TSL-6. The tendency in total antioxidant capacity was TSL-2 > TSL-2P > TSL-6. The ferrous ion chelating activity of TSL-2 (32.68 %) was too low to obtain the IC50 while the IC50 of TSL-2P and TSL-6 was 230.22 ± 45.92 and 49.62 ± 6.1 μg/ml, respectively. Total phenol contents of TSL-2, TSL-2P and TSL-6 were 580.49 ± 3.41, 551.98 ± 9.74 and 255.85 ± 2.99 μg/mg, respectively. The flavonoid contents of TSL-2, TSL-2P and TSL-6 were 309.62 ± 10.78 μg/ml, 154.05 ± 2.69 μg/ml and 111.7 ± 0.33μg/ml respectively. The major active compounds in different Toona sinensis extracts showed that TSL-2 contained 50.30 % gallic acid, 1.40 % quercetin, 2.49 % rutin, 1.30 % catechin and 0.19 % vitamin C. in addition, TSL-2P contained 22.53 % gallic acid, 1.45 % quercetin, 2.2 % rutin, 0.66 % ethyl gallate and 0.2 % vitamin C. While TSL-6 contained 7.85 % gallic acid, 0.92 % quercetin, 1.17 % rutin and 0.36 % vitamin C.
Furthermore, the antioxidative activities of TSLs in mice fed with TSL-2, TSL-2P, TSL-6 for 8 weeks were investigated. Results showed that TSL-2 significantly decreased SOD, CAT, GR enzyme activities and GSSG concentration in testes and cerebrum, respectively. However, GPx enzyme activities were increased in testes and cerebrum, while GST enzyme activities and GSH/GSSG ratio were found increased in cerebrum. Meanwhile TSL-2 significantly increased MDA level in cerebellum.
TSL-2P increased GPx enzyme activity, GSH concentration in liver, but MDA content were decreased. ROS content in liver, kidney, testes and cerebrum was found to be lower than that in healthy controls. Increased of GST enzyme activity were revealed in cerebrum and cerebellum. Meanwhile GSH/GSSG ratio was found increased in cerebellum.
TSL-6 significantly decreased MDA content in liver and GR enzyme activity, GSH concentration in liver and testes, respectively. However CAT and GST enzyme activities in cerebrum were found to be higher in TSL-6 group.
The antioxidative components in TSLs separated by HPLC system were furher investigated. The results showed that TSL-2P-Fr-2 significantly increased GPx enzyme activity and total antioxidant capacity. In addition, TSL-2P-Fr-5, TSL-2P-Fr-6, TSL-6-Fr-2 and TSL-6-Fr-3 significantly increased SOD and GST enzyme activities, respectively. TSL-6-Fr-1 significantly decreased MDA content. These bioactive fractionations were analyzed by LC-MS and results showed that TSL-2P-Fr-6, TSL-6-Fr-1 and TSL-6-Fr-3 both contained gallic acid and methyl gallate. Ethyl gallate were found in TSL-6-Fr-1 and TSL-6-Fr-3. However, only rutin and quercetin were found in TSL-2P-Fr-6 and TSL-6-Fr-3, respectively.
In conclusion, TSL-2 contained the most abundant total phenol, flavonoids and gallic acid. TSL-2 was found to benefit antioxidant effect in reducing power and DPPH test. But TSL-2 significant induced oxidative stress in testes and cerebellum. Meanwhile, TSL-2-Fr-6 was induced lipid pro-oxidation in HepG2 cell.
TSL-2P had beneficial antioxidant effect both in vitro and in vivo. Quercetin in TSL-2P was synergistic with gallic acid to elevate antioxidant effect in liver, kidney and cerebrum, and also recover the pro-oxidant effect by gallic acid in testes and cerebellum. Meanwhile, TSL-2P-Fr-6 was increased GST enzyme activity in HepG2 cell and contained gallic acid, quercetin and methyl gallate.
TSL-6 only had beneficial at ferrous ion chelating activity in vitro and had no significant effect and significant increase testes and cerebrum antioxidant ability. Besides gallic acid, quercetin and methyl gallate which were identified, tannic acid was suspect to be a possible component in TSL-6 to reveal ferrous ion chelating activity in vitro without pro-oxidant effect.
TSL-2, TSL-2P and TSL-6 both had antioxidant capacities, but TSL-2P and TSL-6 were revealed better protect effects. The role of bioactive fractionations in the mechanism of antioxidant system will be investigated in the future.
目 錄
頁數
中文摘要----------------------------------------------------------------------------------I
英文摘要--------------------------------------------------------------------------------III
誌謝--------------------------------------------------------------------------------------VI
目錄------------------------------------------------------------------------------------VIII
表目錄------------------------------------------------------------------------------------X
圖目錄----------------------------------------------------------------------------------XII
第一章:前言----------------------------------------------------------------------------1
第二章:文獻探討----------------------------------------------------------------------3
自由基簡介------------------------------------------------------------------------------------3
生物體抗氧化系統簡介---------------------------------------------------------------------5
香椿簡介------------------------------------------------------------------------------------- 14
第三章:實驗方法與步驟--------------------------------------------------------------------19
實驗藥品-----------------------------------------------------------------------------------19
實驗儀器-----------------------------------------------------------------------------------22
實驗架構------------------------------------------------------------------------------------24
實驗方法------------------------------------------------------------------------------------29
第四章:結果-----------------------------------------------------------------------------------42
一、香椿萃取物抗氧化能力之體外實驗--------------------------------------------42
(一)、香椿萃取物 (TSL-2、TSL-2P 與 TSL-6) 清除 DPPH 之能力--------42
(二)、香椿萃取物 (TSL-2、TSL-2P 與 TSL-6) 螯合亞鐵離子之能力-------42
(三)、香椿萃取物 (TSL-2、TSL-2P 與 TSL-6) 清除超氧陰離子之能力----43
(四)、香椿萃取物 (TSL-2、TSL-2P 與 TSL-6) 之還原能力-------------------43
(五)、香椿萃取物 (TSL-2、TSL-2P 與 TSL-6) 之總抗氧化能力-------------43
(六)、香椿萃取物 (TSL-2、TSL-2P 與 TSL-6) 之多酚類含量----------------44
(七)、香椿萃取物 (TSL-2、TSL-2P 與 TSL-6) 之類黃酮含量----------------44
(八)、香椿萃取物 (TSL-2、TSL-2P 與 TSL-6) 之沒食子酸、槲黃素、芸香苷、兒茶素、沒食子酸乙酯、維生素 C含量--------------------------------44
二、香椿萃取物抗氧化能力之體內實驗--------------------------------------------47
(一)、餵食沒食子酸或香椿萃取物 (TSL-2、TSL-2P 與 TSL-6) 八週後對小鼠體重之影響---------------------------------------------------------------------47
(二)、餵食沒食子酸或香椿萃取物 (TSL-2、TSL-2P 與 TSL-6) 八週後對小鼠攝食量及飲水量之影響------------------------------------------------------47
(三)、餵食沒食子酸或香椿萃取物 (TSL-2、TSL-2P 與 TSL-6) 八週後對小鼠肝臟、腎臟、睪丸、大腦及小腦平均重量與體重比例之影響---------------------------------------------------------------------------------------47
(四)、餵食沒食子酸或香椿萃取物 (TSL-2、TSL-2P 與 TSL-6) 八週後對小鼠肝臟、腎臟、睪丸、大腦及小腦抗氧化酵素活性及指標之影響---------------------------------------------------------------------------------------48
三、不同香椿萃取物生物活性成份鑑定--------------------------------------------59
(一)、利用HPLC將香椿萃取物 (TSL-2、TSL-2P 與 TSL-6) 進行分劃-----59
(二)、不同濃度之Trolox、AAPH、沒食子酸、槲黃素與香椿萃取物 ( TSL-2、TSL-2P、TSL-6 )之分劃物對HepG2存活的影響---------------------------60
(三)、處理Trolox、AAPH、槲黃素、沒食子酸與香椿萃取物 ( TSL-2、TSL-2P、TSL-6 )之分劃物對HepG2抗氧化酵素活性及指標的影響---------------------------------------------------------------------------------------62
四、香椿萃取物活性成份鑑定---------------------------------------------------------66
(一)、不同香椿萃取物 ( TSL-2、TSL-2P與TSL-6 )之分劃物送測LC-MS比對標準品之結果---------------------------------------------------------------66
(二)、不同香椿萃取物 ( TSL-2、TSL-2P與TSL-6 )之分劃物送測NMR之結果---------------------------------------------------------------------------------66
第五章 討論------------------------------------------------------------------------------------68
一、香椿萃取物抗氧化能力之體外實驗--------------------------------------------68
二、香椿萃取物抗氧化能力之體內實驗--------------------------------------------72
三、不同香椿萃取物生物活性成份鑑定--------------------------------------------77
第六章 結論------------------------------------------------------------------------------------81
第七章 參考文獻------------------------------------------------------------------------------82
表------------------------------------------------------------------------------------------------- 96
圖------------------------------------------------------------------------------------------------119
自述----------------------------------------------------------------------------------159
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