Live Cell Assays for the Assessment of Antioxidant Activities of Plant Extracts

Abstract

Plant extracts and pharmacopoeias represent an exceptional breeding ground for the discovery of new antioxidants. Until recently, the antioxidant activity was only measured by chemical hydrogen atom transfer (HAT) and single-electron transfer (SET) cell-free assays that do not inform about the actual effect of antioxidants in living systems. By providing information about the mode of action of antioxidants at the subcellular level, recently developed live cell assays are now changing the game. The idea of this review is to present the different cell-based approaches allowing a quantitative measurement of antioxidant effects of plant extracts. Up to date, only four different approaches have reached a certain degree of standardization: (1) the catalase-like assay using H2O2 as a stressor, (2) the cell antioxidant assay (CAA) using AAPH as a stressor and DCFH-DA as a readout, (3) the AOP1 assay which uses photoinduction to monitor and control cell ROS production, and (4) the Nrf2/ARE gene reporter system. The molecular aspects of these assays are presented in detail along with their features, drawbacks, and benefits. The Nrf2/ARE gene reporter system dedicated to indirect antioxidant effect measurement currently represents the most standardized approach with high-throughput applications. AOP1, the first technology linking a fine-tuning of cell ROS production with a quantitative signal, appears to be the most promising tool for the assessment of direct cellular ROS-scavenging effects at an industrial scale.

Cellular Antioxidant Effect of an Aronia Extract and Its Polyphenolic Fractions Enriched in Proanthocyanidins, Phenolic Acids, and Anthocyanins

“…Different cell lines (liver cells (HepG2), intestinal cells (Caco-2), and neuron-like cells (SH-SY5Y)) were used to assess and compare the antioxidant effect of the AE across a short panel of physiologically relevant cell models using the cell-based test called AOP1. This is a universal assay by which the antioxidant effect of extracts can be assessed accurately and quantitatively by determining the intracellular radical scavenging activity [ 34 , 35 , 39 , 40 ]. As shown in Table 1 and Figure 2 , AE exerted antioxidant activities in the three cell lines, but the magnitude of effect varied between cell lines.…”

“…The in vivo relevance of an antioxidant effect has been put into question, in part due to the acellular environment widely employed to characterize this function. In living organisms, antioxidant effects can be triggered directly, by quenching free radicals, or indirectly, by activating cellular enzymatic antioxidant systems via the induction or repression of transcription factors or a combination of both, something that cannot be captured with traditional antioxidant in vitro assays such as DPPH, FRAP, or ORAC [ 40 ].…”

Metabolomics-Based Profiling, Antioxidant Power, and Uropathogenic Bacterial Anti-Adhesion Activity of SP4TM, a Formulation with a High Content of Type-A Proanthocyanidins

“…The LUCS assay measures the ability of an antioxidant to neutralize the oxidative stress at the cellular level [ 52 ]. The LUCS assay is based on the production of cellular radical species following the addition in the human hepatocellular carcinoma (HepG2) cells culture medium of thiazole orange (TO), a photo-inducible fluorescent nucleic acid biosensor [ 71 ]. The approach has been standardized on high throughput 96-well plates to allow reliable statistical analyses.…”

Rosmarinus officinalis L. hexane extract: phytochemical analysis, nanoencapsulation, and in silico, in vitro, and in vivo anti-photoaging potential evaluation

“…Among the methods used, ABTS and FRAP assays are the most correlated, a finding that was formerly reported 68 , 69 . It should be noted that the antioxidant assays are preferably performed in the context of the whole organism to obtain more reliable information 70 , which is complemented in the present work by in vivo biochemical analysis.…”