Abstract
The natural flavonoid quercetin is known to activate the transcription factor Nrf2, which regulates the expression of cytoprotective enzymes such as heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1). In this study, a novel semisynthetic flavonoid 7-O-galloylquercetin (or quercetin-7-gallate, 3) was prepared by direct galloylation of quercetin, and its effect on the Nrf2 pathway was examined. A luciferase reporter assay showed that 7-O-galloylquercetin, like quercetin, significantly activated transcription via the antioxidant response element in a stably transfected human AREc32 reporter cell line. In addition, 7-O-galloylquercetin caused the accumulation of Nrf2 and induced the expression of HO-1 at both the mRNA and protein levels in murine macrophage RAW264.7 cells. The induction of HO-1 by 7-O-galloylquercetin was significantly suppressed by N-acetyl-l-cysteine and SB203580, indicating the involvement of reactive oxygen species and p38 mitogen-activated protein kinase activity, respectively. HPLC/MS analyses also showed that 7-O-galloylquercetin was not degalloylated to quercetin, but it was conjugated with glucuronic acid and/or methylated in RAW264.7 cells. Furthermore, 7-O-galloylquercetin was found to increase the protein levels of Nrf2 and HO-1, and also the activity of NQO1 in murine hepatoma Hepa1c1c7 cells. Taken together, we conclude that 7-O-galloylquercetin increases Nrf2 activity and induces Nrf2-dependent gene expression in RAW264.7 and Hepa1c1c7 cells.
Highlights
IntroductionBeing inspired by the fact that the Nrf activation by catechins (flavanols) positively depends on the presence of a galloyl moiety in the molecule [20], we have previously prepared galloyl esters of quercetin and of its natural derivative, the flavanonol taxifolin (2,3-dihydroquercetin), and examined their effect on the Nrf pathway in murine macrophage RAW264.7 cells
Since the activation of NF-E2 p45related factor 2 (Nrf2) may be mediated through its phosphorylation [28], we investigated, using pharmacological inhibitors, whether mitogen-activated protein kinases (MAPKs) including p38 MAPKs, extracellular signal-regulated kinases (ERKs), and c-Jun N-terminal kinases (JNKs) could play a role in the cell response to 3
This is supported by the findings that 3 i) activates the antioxidant response element in AREc32 reporter cells, ii) induces the accumulation of Nrf2 in RAW264.7 and Hepa1c1c7 cells, iii) induces the expression of heme oxygenase-1 (HO-1) at both the mRNA and protein levels in RAW264.7 cells, and iv) increases the activity of NAD(P)H:quinone oxidoreductase 1 (NQO1) and protein levels of HO-1 and GCLM in Hepa1c1c7 cells
Summary
Being inspired by the fact that the Nrf activation by catechins (flavanols) positively depends on the presence of a galloyl moiety in the molecule [20], we have previously prepared galloyl esters of quercetin and of its natural derivative, the flavanonol taxifolin (2,3-dihydroquercetin), and examined their effect on the Nrf pathway in murine macrophage RAW264.7 cells. The same study found that 7-O-galloyltaxifolin was readily oxidized to 7-O-galloylquercetin (Fig. 1) in RAW264.7 cells [11] Since it was unclear which of these two molecules was responsible for the biological response, we decided to prepare 7-O-galloylquercetin and to investigate its effect on the Nrf pathway in vitro
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