Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/51807
標題: 四種類黃酮對Fe/NTA誘導S.D.大鼠及HepG2細胞氧化傷害之影響---洽體內及活體外糸統之比較
Effects of 4 kinds flavonoids on the oxidative damage in S.D. rats and HepG2 cells induced by Ferric nitrilotriacetate----comparsion between in vivo and in vitro system.
作者: 蕭學民
Sio, Hok-Man
關鍵字: 氧化傷害模式;oxidative damage model;Fe/NTA;2-nitropropane;bromobenzene;ADP-FeCl2-;Fe/NTA;2-nitropropane;bromobenzene;ADP-FeCl2-ascorbic acid;flavonoids;quercetin;rutin;hesperidin;silibinin;in vivo and in vitro
出版社: 食品科學系
摘要: 
中文總摘要
本研究分為兩大部份,第一部份是比較不同的氧化傷害模式對S.D.大鼠的氧化傷害作用,此等氧化傷害模式均已被多數學者證實可在動物模式中引起肝及/或腎毒性,本實驗亦試圖由這些模式中找出一個同時具有肝、腎毒性的氧化傷害模式。這些氧化模式分別是: (1)腹腔注射Fe/NTA( 9mg Fe/kg b.w.),並在30、60、90 、120分鐘後犧牲。(2)腹腔注射2-nitropropane( 100mg/kg b.w.) ,並在6小時或15小時後犧牲。(3)管餵Bromobenzene(9 mmol/kg b.w.),並在 17小時後犧牲。(4)連續腹腔注射ADP-FeCl2-ascorbic acid(5mg-30mg-170mg/kg b.w.)之混合物三天,並在最後一次注射後的24小時犧牲。肝、腎脂質過氧化(TBARS)、非蛋白質硫醇基(NPSH)及DNA傷害(8-OHdG)為氧化傷害的測定指標。
實驗結果發現,與各種氧化傷害模式比較,腹腔注射Fe/NTA(9mg Fe/kg b.w.)並在120分鐘後犧牲者可在大鼠體內誘導嚴重的氧化傷害。此一氧化傷害模式可顯著增加肝、腎脂質過氧化以及8-OHdG的含量,並可顯著減低肝中NPSH的含量。 由於此一模式可同時對肝、腎兩器官產生嚴重的氧化傷害作用,因此選出此一活體內的氧化傷害模式作為第二部份實驗之用。
第二部份之實驗則為探討四種類黃酮(quercetin, rutin, hesperidin, silibinin)在活體內對Fe/NTA所誘導的氧化傷害之作用。在本實驗中利用管餵的方式給予S.D.大鼠四種類黃酮 (100mg/kg b.w./日)為期一週,之後則以第一部份所選出之Fe/NTA氧化傷害模式(9 mg Fe/kg b.w.)對大鼠進行氧化傷害。另外,由於考慮到一般市面上大鼠飼料中均含有高量的維生素E,可能會對實驗結果造成干擾,並想探討類黃酮與維生素E之間在活體內是否會有相互作用,因此在本實驗中所用的飼料採取自行調配的方式,每組再細分為兩小組,其中一組餵食不含維生素E之飼料,另一組則餵食含有低量維生素E之飼料(30mg α-tocopherol acetate/kg diet)。肝、腎脂質過氧化( TBARS)、非蛋白質硫醇基(NPSH)及DNA傷害(Comet assay、8-OHdG)為氧化傷害之測定指標。
由實驗結果發現,綜觀各種氧化傷害而言:(1) 類黃酮的化學結構(例如OH基的位置及數目)會強烈影響其抗/促氧化能力。(2) 某些類黃酮的抗/促氧化性可在腎臟中表現卻無法在肝臟中表現,此一結果暗示各種類黃酮在肝臟中的含量可能比在腎臟中為低。(3) 各種類黃酮與維生素E共存時,可以表現出更好的抗氧化能力。
另外在第二部份之實驗中,亦嘗試以HepG2細胞為模式,探討各種類黃酮對Fe/NTA誘導DNA 傷害以及脂質過氧化之影響,並進行多濃度多時間的實驗,以探討活體外(HepG2)模式與活體內(S.D. rats)模式的相關性。
實驗結果發現在體外模式中類黃酮的抗/促氧化特性會隨培養濃度或培養時間而改變,另外不論是DNA傷害或脂質過氧化,活體外模式中所得到的結果與體內所得到的結果均不具有絕對的相關性。故由此一實驗可得知進行活體外實驗時需同時考慮多濃度及多個時間點,另外由實驗結果亦發現體內外模式並沒有絕對相關性。
關鍵詞: 氧化傷害模式, Fe/NTA, 2-nitropropane, bromobenzene, ADP-FeCl2-
ascorbic acid, 類黃酮, quercetin, rutin, hesperidin, silibinin, 活體內及活體外

Abstract
There were two parts in this research. In part one we compared the oxidative damage to S.D. rats by different oxidative damage models which have been described and proved to cause oxidative damage in liver and/or kidney in animal model by many investigators and try to select a model which caused oxidative damage both in liver and kidney. These oxidative models are described as follow: (1) I.p injection of Fe/NTA (9 mg Fe/kg b.w.) to rats and killed after 30,60,90,120 minutes. (2) I.p. injection of 2-nitropropane (100mg/
kg b.w.) to rats and killed after 6 or 15 hours. (3) Introduced bromobenzene (9 mmol/kg b.w.) to rats by intragastric, and killed the rats after 17 hours. (4) I.p.
injection of ADP-FeCl2-ascorbic acid (5mg-30mg-170mg/kg b.w.) to rats for 3 days, and killed the rats 24 hours after the last administration. Lipid peroxidation (TBARS), non protein SH groups (NPSH), and DNA damage (8-OHdG) in liver and kidney were determined as marker of oxidative damage.
Results showed that when compare to other oxidative models, i.p injection of Fe/NTA(9 mg Fe/kg b.w.) and killed 120 minutes after the injection caused serious oxidative damage in the rats. This oxidative model significantly increased the lipid peroxidation and 8-OHdG both in liver and kidney, it also significantly decreased the NPSH levels in liver. Since this oxidative model caused serious oxidative damage both in liver and kidney, we used it in the second part research as a sutibule oxidative model.
In part 2 we investigate the effects of 4 kinds flavonoids ( quercetin, rutin, hesperidin, silibinin ) to oxidative damage induced by Fe/NTA. S.D rats were administrated to 4 kinds of flavonoids by intragastric for 1 week (100mg/kg b.w./day), and were followed by Fe/NTA-induced oxidative damage model selected from part 1 (9mg Fe/kg b.w, 120 mins). By the consideration of high content of vit E in general laboratory diet and in order to investigate is there any interaction between vit E and flavonoids in vivo, we used the self-mixed diet, each group sub-divided into two groups and supplied with vit E-deficient diet and vit E-supplemented diet (30 mg α-tocopherol acetate/kg diet). Lipid peroxidation (TBARS), non protein SH groups (NPSH), and DNA damage (8-OHdG,Comet assay) in liver and kidney were determined as marker of oxidative damage.
The result showed that: (1) The chemical structure of flavonoids (such as the number and position of hydroxyl groups) strongly affected their antioxidant/
prooxidant activity in vivo. (2) Some antioxidant/prooxidant activity of flavonoids only found in kidney but not in liver, these results imply that the absorbtion and metabolism are different in liver and kidney. (3)When the flavonoids were co-exist with vit E, the higher antioxidant activity was result.
We also investigated the antioxidant/prooxidant activity of flavonoids on
Fe/NTA-induced DNA damage and lipid peroxidation in HepG2 cell line by
time and dose dependent experiment, and we used this result to compare with
the results in vivo for investigated the correlation between in vivo (S.D rats)
and in vitro (HepG2 cell line) systems.
Results showed that the incubation time and dose both strongly effects the antioxidant/prooxidant activity of flavonoids in HepG2 cell line, and there was no absoult correlation between in vivo (S.D rats) and in vitro (HepG2 cell line) systems. From this results we suggest that a wide range of incubation time and dose is needed for in vitro experiment and there is no absoult correlation between in vivo and in vitro.
Key words: oxidative damage model, Fe/NTA, 2-nitropropane, bromobenzene, ADP-FeCl2-ascorbic acid, flavonoids, quercetin, rutin, hesperidin, silibinin, in vivo and in vitro
URI: http://hdl.handle.net/11455/51807
Appears in Collections:食品暨應用生物科技學系

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