Antioxidative and Apoptotic Properties of Polyphenolic Extracts from Edible Part of Artichoke (Cynarascolymus L.) on Cultured Rat Hepatocytes and on Human Hepatoma Cells

Abstract

Cultured rat hepatocytes and human hepatoma HepG2 cells were used to evaluate the hepatoprotective properties of polyphenolic extracts from the edible part of artichoke (AE). The hepatocytes were exposed to H₂O₂generated in situ by glucose oxidase and were treated with either AE, or pure chlorogenic acid (ChA) or with the well known antioxidant, N, N′-diphenyl-p-phenilenediamine (DPPD). Addition of glucose oxidase to the culture medium caused depletion of intracellular glutathione (GSH) content, accumulation of malondialdehyde (MDA) in the cultures, as a lipid peroxidation indicator, and cell death. These results demonstrated that AE protected cells from the oxidative stress caused by glucose oxidase, comparable to DPPD. Furthermore, AE, as well as ChA, prevented the loss of total GSH and the accumulation of MDA. Treatment of HepG2 cells for 24 h with AE reduced cell viability in a dose-dependent manner, however, ChA had no prominent effects on the cell death rate. Similarly, AE rather than ChA induced apoptosis, measured by flow cytometric analysis of annexin and by activation of caspase-3, in HepG2 cells. Our findings indicate that AE had a marked antioxidative potential that protects hepatocytes from an oxidative stress. Furthermore, AE reduced cell viability and had an apoptotic activity on a human liver cancer cell line.

ACKNOWLEDGMENTS

We thank Dr. Pier Giorgio Natali, Dr. Anna Maria Mileo and Dr. Lucia Monaco for critical reading of the manuscript and helpful comments. We thank SAFU technicians (Regina Elena Institute Rome Italy) for excellent assistance and Lara Palomba for her technical assistance in preparing the manuscript. This study was supported by grants from MiPAF, Italy.

Notes

*The results represent the means ± SD of three independent extractions and are expressed as mg L^{− 1}of chlorogenic acid (for mono- and di-caffeoylquinic acids) or luteolin-7-glucoside and apigenin-7-glucoside (for luteolin and apigenin glycosides, respectively).

*Cultured hepatocytes were treated with 0.1 U/ml glucose oxidase alone or glucose oxidase plus 1 μ M DPPD or glucose oxidase plus 1mM ChA or glucose oxidase plus 1 mM AE. After 60 minutes the content of MDA in the cultures was measured and the percentage of the dead cells was determined (numbers in parentheses). The results are the means ± SD of the determinations on three separate cultures.