葉綠素衍生物對誘發型DNA損傷之保護效應

TMUIR > College of Pharmacy > School of Pharmacy > Dissertation/Thesis > Item 987654321/4328

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Title:	葉綠素衍生物對誘發型DNA損傷之保護效應 Preventive Effects of Chlorophyll Derivatives on Induced-type DNA Damage
Authors:	許青雲 Ching-Yun Hsu
Contributors:	藥學系(博士班)
Keywords:	葉綠素脫鎂脫植醇葉綠素脫鎂醇葉綠素黃麴毒素 Chlorophyll Pheophorbide Chlorophyllide Aflatoxin
Date:	2009
Issue Date:	2009-08-31 14:17:37 (UTC+8)
Abstract:	葉綠素是植物體中含量最多最廣泛的色素。因此本研究主要探討四種葉綠素的水萃取物，包括脫植醇葉綠素 a 與b、脫鎂脫植醇葉綠素a 與b，對於淋巴球細胞中DNA氧化損傷的抗氧化作用；以及對於黃麴毒素所誘導肝細胞DNA損傷後的抗基因突變作用。第一部分，將淋巴球細胞分別與四種葉綠素衍生物5~50µM共同培養後，再將其暴露在10 、 50µM的過氧化氫使其誘導氧化傷害。利用單細胞膠體電泳法(彗星分析)觀察DNA氧化斷裂的情形，以及氧化代謝產物8-OHdG。結果顯示：四種葉綠素衍生物對10 µM的過氧化氫誘導淋巴球細胞氧化傷害都具有保護的作用；但是在50 µM的過氧化氫的誘導下，脫鎂脫植醇葉綠素a 與b對淋巴球細胞氧化傷害仍有保護的效果。第二部份，將小鼠肝臟細胞株分別暴露在四種葉綠素衍生物5~50µM共同培養後，再給與5、10 ng/mL黃麴毒素誘導肝細胞的DNA損傷。利用ELISA 測量黃麴毒素與DNA結合的產物結果顯示：四種葉綠素衍生物對於黃麴毒素誘導肝細胞的DNA損傷都具有保護的作用，推測葉綠素的結構具有捕捉分子的能力，可螯合黃麴毒素進而減少黃麴毒素對肝細胞基因的損傷。此外，在排除分子捕捉能力的研究中發現，脫鎂脫植醇葉綠素a 與b對於肝細胞DNA的損傷仍具有保護的效果，且可以調節肝細胞中麩胱甘肽轉硫酶的活性，進而增加肝臟中對黃麴毒素的解毒功能。 Chlorophylls (Chls) are the most abundant natural plant pigments. Four chlorophyll derivatives, including chlorophyllide a and b (Chlide a and b) and pheophorbide a and b (Pho a and b), were investigated for their in vitro antioxidative capacities and anticytotoxicity properties to the cell DNA damage. First, the antioxidative effects of four chlorophyll derivatives on hydrogen peroxide (H2O2)-induced strand breaks and oxidative damage were evaluated in human lymphocyte. Lymphocytes exposed to H2O2 at a concentration of 10 and 50µM revealed an increased frequency of DNA single-strand breaks (ssbs; as measured by the comet assay) and also the level of oxidized nucleoside (as measured by 8-hydroxy-2-deoxyguanosine; 8-OHdG). All Chls reduced the level of DNA ssbs and 8-OHdG within human lymphocytes following exposure to 10µM H2O2. Only Pho a and b were able to decrease DNA ssbs and 8-OHdG following treatment of lymphocytes with 50µM H2O2, in a concentration-dependent fashion. It was demonstrated herein that Pho a and b, were more antioxidative than others. We applied DPPH free-radical scavenge assays in vitro, and also got similar results. Pho a and b were higher ability on scavenging capacities than others. In the second part, the inhibitory effects of four chlorophyll derivatives on aflatoxin B1 (AFB1)-DNA adduct formation, and on the modulation of hepatic glutathione S-transferase (GST) were evaluated in murine hepatoma (Hepa-1) cells. Enzyme-linked immunosorbent assay (ELISA) showed that pretreatment with Chlide or Pho significantly reduced the formation of AFB1-DNA adducts, and that Pho was the most potent inhibitor. However, wash-out prior to adding AFB1 totally eliminated inhibition by Childe and partially eliminated inhibition by Pho, indicating that the inhibitory effect of Chlide, and to some extent Pho, was mediated through direct trapping of AFB1. Furthermore, spectrophotometric analysis showed that Pho treatment could increase GST activity in Hepa-1 cells. These observations indicate that the chlorophyll derivatives studied may attenuate AFB1-induced DNA damage in the Hepa-1 cell by direct trapping of AFB1. Pho provided additional protection not only by direct trapping, but also by increasing GST activity against hepatic AFB1 metabolites. We conclude that water extract Chls are able to enhance cells’ ability to resist H2O2-induced oxidative damage and (AFB1)-DNA adduct formation, especially for Pho a and b.
Data Type:	thesis
Appears in Collections:	[School of Pharmacy] Dissertation/Thesis

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