Development of bacterial cell-based system for intracellular antioxidant activity screening assay using green fluorescence protein (GFP) reporter

Abstract

The novel bacterial cell-based assay was developed for evaluating the intracellular antioxidant activity. The genetically engineered Escherichia coli strains harboring the fusions of sodA::gfp and fumC::gfp were constructed and applied as reporters in response to cellular superoxide stress. Using this assay, twelve pure compounds and three Thai medicinal plants were investigated for intracellular antioxidant activity in comparison with conventional chemical-based assays; 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide dismutase (SOD) activity assays. Both strains demonstrated that quercetin and α- tocopherol exhibited the most potent and significant antioxidant activity with more than 60% reduction of intracellular superoxide. These compounds also showed high DPPH radical scavenging activity. Interestingly, gallic, caffeic and protocatechuic acids had the most significant DPPH radical scavenging and SOD-like activities but with moderate to weak intracellular antioxidant activity. Our hypothesis was that the lower intracellular antioxidant activity possibly occurs due to poor permeability of compounds into biological membrane based on their structures. Moreover, our results demonstrated that intracellular antioxidant activity of three plant extracts well correlated to results from DPPH assay. Our bacterial-based assay is simple, reproducible, very specific and applicable as an alternative screening tool for assessing the activity of compounds and plant extracts affecting cellular oxidative stress.

Key words: Bacterial cell-based assay, antioxidant activity, oxidative stress, superoxide dismutase, fumarase, green fluorescence protein (GFP) reporter, plant extracts.