Protection by Chrysanthemum zawadskii extract from liver damage of mice caused by carbon tetrachloride is maybe mediated by modulation of QR activity

Abstract

Our previous study demonstrated that methanolic extract of Chrysanthemum zawadskii Herbich var. latilobum Kitamura (Compositae) has the potential to induce detoxifying enzymes such as NAD(P)H:(quinone acceptor) oxidoreductase 1 (EC 1.6.99.2) (NQO1, QR) and glutathione S-transferase (GST). In this study we further fractionated methanolic extract of Chrysanthemum zawadskii and investigated the detoxifying enzyme-inducing potential of each fraction. The fraction (CZ-6) shown the highest QR-inducing activity was found to contain (+)-(3S,4S,5R,8S)-(E)-8-acetoxy-4-hydroxy-3-isovaleroyloxy-2-(hexa-2,4-diynyliden)-1,6-dioxaspiro [4,5] decane and increased QR enzyme activity in a dose-dependent manner. Furthermore, CZ-6 fraction caused a dose-dependent enhancement of luciferase activity in HepG2-C8 cells generated by stably transfecting antioxidant response element-luciferase gene construct, suggesting that it induces antioxidant/detoxifying enzymes through antioxidant response element (ARE)-mediated transcriptional activation of the relevant genes. Although CZ-6 fraction failed to induce hepatic QR in mice over the control, it restored QR activity suppressed by CCl(4) treatment to the control level. Hepatic injury induced by CCl(4) was also slightly protected by pretreatment with CZ-6. In conclusion, although CZ-6 fractionated from methanolic extract of Chrysanthemum zawadskii did not cause a significant QR induction in mice organs such as liver, kidney, and stomach, it showed protective effect from liver damage caused by CCl(4).

Keywords: (+)-(3S,4S,5R,8S)-(E)-8-acetoxy-4-hydroxy-3-isovaleroyloxy-2-(hexa-2,4-diynyliden)-1,6-dioxaspiro[4,5]decane; Chrysanthemum zawadskii; Quinone reductase; carbon tetrachloride-induced liver injury; detoxifying enzymes.

Fig. 1

Structure of (+)-(3S,4S,5R,8S)-(E)-8-acetoxy-4-hydroxy-3-isovaleroyloxy-2-(hexa-2,4-diynyliden)-1,6-dioxaspiro[4,5]decane isolated from Chrysanthemum zawadskii

Fig. 2

Induction of quinone reductase by CZ-6 fraction in Hepa1c1c7 (A) and BPRc1 cells. After culturing 48 h in alpha-MEM containing 10% FBS, cells were exposed to various doses of CZ-6 fraction for another 24 h and subjected to QR assay.

Fig. 3

Dose-dependent increase of reporter luciferase activity in HepG2-C8 cells by CZ-6 fraction. HepG2-C8 cells stably transfected with pARE-TI-luciferase construct were exposed to various doses of CZ-6 and assayed for luciferase activity using luminometer.

Fig. 4

>Effect of treatment of carbon tetrachloride and CZ-6 fraction on body (A) and organ (B) weights of mice. Mice weighing 20-30 g were insulted with CCl₄ twice (7^th and 9^th days) in a week with and without CZ-6, and weighed for body and organs on 11^th day after initiating the study.

Fig. 5

Effect of CCl₄ and CZ-6 fraction on QR activities in mouse organs. Mice weighing 20-30 g were insulted with CCl₄ twice (7^th and 9^th days) in a week with and without CZ-6, and assayed for QR activities of tissues on 11^th day after initiating the study.

Fig. 6

Liver histology in mice after CCl₄ and CZ-6 treatments. The method for the administration of CZ-6 and induction of liver injury are described in 'Materials and Methods' section. The liver sections obtained from the mice were subjected to hematoxylin-eosin staining. Arrows indicate necrotic areas.