Stephen Joseph Orena 1, Jennifer Owen 1, Fuxia Jin 2, Morgan Fabian 2, Nicholas Gillitt 2 and Steven Zeisel 1 (2016). The Total Phenolics Content of Fruit & Vegetable Extracts Does Not Correlate with Activation of the Anti-Oxidant Response in Human Cells. The FASEB Journal, 30(1).
1. Nutrition Research Institute, UNC Chapel Hill, Kannapolis, NC
2. Dole Nutrition Research Laboratory, Dole Food Company, Kannapolis, NC
Anti-oxidants in foods are traditionally thought to act by trapping free radicals thereby preventing cellular damage to organelles, membranes and DNA. However the structures and properties of many of these molecules suggest they are poor free radical scavengers and may even function as weak pro-oxidants (electrophiles) that can activate the NRF2/anti-oxidant response element (ARE) pathway in cells. Traditional approaches for identifying anti-oxidant molecules in foods, such as total phenolics (TP) content or oxygen radical absorption capacity (ORAC) do not measure the capacity to activate AREs.
Fruits and vegetables were homogenized, extracted with acidified ethanol, lyophilized, and resuspended in growth medium. The TP content and ORAC activity were measured for each extract. Extracts were screened for activation of the anti-oxidant response element (ARE)-Firefly luciferase reporter in transiently transfected human IMR-32 neuroblastoma cells treated for 5 hours. Firefly luciferase was normalized to constitutively expressed Renilla luciferase with tertiary-butylhydroquinone (tBHQ) as a positive control. Relationships between TP and ORAC with ARE activity were determined. Extracts identified as active in the ARE reporter screen were then tested for activation of anti-oxidant genes in human peripheral blood mononuclear cells (PBMCs). After a 5 hour exposure, total RNA was purified and RT-PCR performed to assess expression of the anti-oxidant genes NAD(P)H:quinone oxidoreductase 1 (NQO1), glutamate cysteine ligase modifier subunit (GCLM), thioredoxin reductase 1 (TXNRD1), and glutathione reductase (GSR). Relationships between TP and gene expression activity were determined.
107 of 134 extracts tested significantly activated the ARE luciferase reporter from 1.2 to 58 fold above that of the solvent control (P<0.05) in human IMR-32 cells. ARE activity, TP content, and ORAC ranked higher in peels versus associated flesh. Despite this relation, ARE activity did not correlate with TP content (Spearman rho=0.05, P=0.6) and only modestly but negatively correlated with ORAC (Spearman rho=−0.2, P<0.01). Many extracts activated the ARE more than predicted by the TP content or ORAC. Active extracts from the ARE screen significantly increased expression of anti-oxidant genes in PBMCs. As with the ARE reporter, TP content of the extracts did not correlate with extract-induced expression of NQO1 (Spearman rho=−0.3, P=0.3), GCLM (Spearman rho=−0.2, P=0.5), TXNRD1 (Spearman rho=−0.1, P=0.8) and GSR (Spearman rho=−0.3, P=0.3).
These data suggest that measuring the endogenous anti-oxidant gene expression response is likely a better predictor of the anti-oxidant benefits of fruits and vegetables than TP content or ORAC.