Induction of antioxidant defenses in human HepG2 cells by a methanolic extract of hypericum perforatum cells elicited with agrobacterium tumefaciens

Abstract

Hypericum perforatum (HP) commonly known as St. John’s wort is an important medicinal plant traditionally used in several ailments including mild to moderate depression. In a recent study, elicitation of HP cells (culture of cells in suspension) with Agrobacterium tumefaciens (AT) increased significantly the production of compounds with antioxidant and antimicrobial properties [1]. The present investigation has been conducted to test the efficacy of methanolic extracts of elicited and control HP cells in protecting human hepatocytes (HepG2 cell line) upon oxidative stress. Briefly, HepG2 cells were treated with tert-butyl hydroperoxide (tert-BOOH) to induce oxidative stress, and the ability of the HP extracts to protect against cell death were measured by MTT and LDH leakage assays. The ability of these extracts to induce cellular antioxidant defenses were investigated by western blot and biochemical determinations. When HepG2 cells were co-incubated with 800 uM tert-BOOH and HP extracts (80 ug/ml), only the extract from the elicited HP cells was able to significantly prevent tert-BOOH-induced cell death. The extract from control HP cells did not show any protective effect, on the contrary, stimulated tert-BOOH toxicity. When HepG2 cells were pre-incubated for 5 h with HP extracts, followed by a period of 16 h of recovery with fresh medium, prior to incubation with tert-BOOH, only the HP extract from elicited cells significantly protected against cell death. This suggested the ability of HP extract from elicited cells to induce intracellular antioxidant defenses in HepG2 cells. That was confirmed by the induction of about 40% in the content of glutathione in HepG2 cells, whereas control extract increased only 10%. As well, only the extract from elicited HP cells were able to induce the levels of cytoprotective enzymes such as HO-1 and NQO1. In conclusion, we observed that elicitation of HP cells with AT produced bioactive compounds present in the methanolic extract able to protect HepG2 cells against oxidative stress, and also to induce intracellular antioxidant defenses of this human cell line.

Acknowledgements: ACC is supported by BI1-PTDC/QUI-BIQ/101392/2008 grant. This work is supported by FCT research grants NaturAge – PTDC/QUI-BIQ/101392/2008 and (Hyperi-Food)-PTDC/AGR-ALI/105169/2008, co-funded by the program COMPETE from QREN with co-participation from the European Community fund FEDER.

[1] Franklin et al., Phytochemistry, 2009, 70: 60-8.