Dietary polyphenols and their role in free radical scavenging: A comprehensive evaluation of antioxidant mechanisms

Introduction

Populations consuming Mediterranean diets rich in olive oil, red wine, and fresh produce have long enjoyed lower rates of cardiovascular disease, cancer, and neurodegeneration. Polyphenolic compounds abundant in this diet have emerged as key molecular mediators of health benefits, primarily through their antioxidant and anti-inflammatory properties [1] . Polyphenols constitute a diverse class of secondary plant metabolites characterised by multiple phenolic hydroxyl groups. Major dietary subclasses include flavonoids (quercetin, catechin), phenolic acids (gallic acid, caffeic acid), stilbenes (resveratrol), and tannins (ellagic acid), with total daily intake estimated at 500-1000 mg depending on food choices [2] . The antioxidant activity of polyphenols derives from their ability to donate hydrogen atoms or electrons to free radicals, effectively terminating oxidative chain reactions that would otherwise damage cellular components including lipids, proteins, and DNA [3] . Reactive Oxygen Species (ROS) including superoxide anion, hydroxyl radical, and hydrogen peroxide are continuously generated during normal cellular metabolism. While cells possess endogenous antioxidant defences, oxidative stress arises when ROS production exceeds scavenging capacity, leading to molecular damage implicated in ageing and chronic disease pathogenesis [4] . Multiple assay methodologies have been developed to quantify antioxidant capacity. The DPPH assay measures hydrogen atom donation to a stable nitrogen radical, while ABTS assesses electron transfer to a cation radical [5] . FRAP quantifies reducing power through iron reduction, and ORAC measures peroxyl radical scavenging in a more physiologically relevant context. No single assay captures the full spectrum of antioxidant activity, necessitating multiassay approaches. Beyond direct radical scavenging, polyphenols exert antioxidant effects through metal chelation, preventing iron-and copper-catalysed Fenton reactions. The present research aimed to systematically characterise eight dietary polyphenols using four complementary assay methodologies to establish structureactivity relationships and mechanistic understanding.

Biochemical basis of polyphenol antioxidant activity

The antioxidant mechanism of polyphenols primarily involves Hydrogen Atom Transfer (HAT) or Single Electron Transfer (SET) reactions, with the dominant pathway depending on compound structure and reaction environment. In HAT mechanisms, the polyphenol donates a hydrogen atom directly to a free radical, forming a relatively stable phenoxyl radical that does not propagate oxidative damage. The SET mechanism involves electron donation followed by proton transfer, achieving the same net result through a stepwise process. The stability of the resulting phenoxyl radical critically determines antioxidant effectiveness. Ortho-dihydroxyl (catechol) arrangements, as found in quercetin and catechin, enable intramolecular hydrogen bonding that stabilises the semiquinone intermediate formed upon one-electron oxidation. This stabilisation explains the generally superior activity of catechol-containing polyphenols compared to mono-hydroxylated analogues. Gallic acid, with its trihydroxyl substitution pattern, can donate three hydrogen atoms sequentially, accounting for its exceptional activity in assays measuring total reducing capacity. Metal chelation represents a distinct antioxidant mechanism wherein polyphenols bind transition metal ions, particularly iron and copper, that would otherwise catalyse hydroxyl radical formation through Fenton chemistry. Chelation sites typically involve adjacent hydroxyl groups or carbonylhydroxyl arrangements that provide bidentate coordination to metal centres.

Material and Methods

This in vitro antioxidant research was conducted at the Laboratory of Nutritional Biochemistry, Instituto Atlântico de Ciências da Saúde, Porto, Portugal, between November 2021 and September 2022. Polyphenol standards including quercetin (≥95% purity), (+)-catechin hydrate (≥98%), gallic acid (≥97.5%), trans-resveratrol (≥99%), caffeic acid (≥98%), ellagic acid (≥95%), and ferulic acid (≥99%) were obtained from Sigma-Aldrich Portugal. L-Ascorbic acid (≥99%) served as reference standard. DPPH radical scavenging was assessed following the method of Brand-Williams with modifications. DPPH solution (100 μM in methanol) was mixed with test compounds at concentrations ranging from 0.5 to 100 μM and incubated at room temperature for 30 minutes. Absorbance was measured at 517 nm. ABTS radical cation was generated by reacting ABTS (7 mM) with potassium persulfate (2.45 mM) for 16 hours. Test compounds were mixed with ABTS solution and absorbance measured after 6 minutes at 734 nm. FRAP assay followed the method of Benzie and Strain using acetate buffer/TPTZ/FeCl3 reagent, monitoring absorbance at 593 nm for 30 minutes at 37°C. ORAC assay employed fluorescein as oxidisable substrate and AAPH as peroxyl radical generator, monitoring fluorescence decay for 90 minutes. All experiments were performed in triplicate on three independent occasions (n=9). IC50 values were calculated by nonlinear regression. Statistics: one-way ANOVA, Tukey post-hoc, Pearson correlations, PCA (p<0.05).

Results

Discussion

Gallic acid's exceptional antioxidant activity reflects its trihydroxyl substitution pattern, enabling sequential donation of three hydrogen atoms and explaining its superior performance in reducing assays like FRAP. The ortho-dihydroxyl arrangements in quercetin and catechin stabilise phenoxyl radicals through intramolecular hydrogen bonding, consistent with their high DPPH and ABTS activities. The correlation between assays (r = 0.67-0.89) indicates that while different methodologies capture overlapping information, each provides unique mechanistic insights. Metal chelation by quercetin through its 3hydroxy-4-keto arrangement represents an additional antioxidant mechanism beyond direct scavenging. The principal component analysis revealing two major factors (electron donation capacity and hydrogen atom transfer ability) explains 84% of the total variance, suggesting that polyphenol antioxidant activity can be largely understood through these two mechanistic dimensions. Compounds excelling in one dimension may show moderate performance in the other, explaining why no single compound consistently ranks highest across all assays. The ORAC assay, measuring peroxyl radical scavenging, provides the most physiologically relevant assessment, as peroxyl radicals are the predominant chaincarrying species in lipid peroxidation. Quercetin's superior ORAC performance likely reflects both its radical scavenging capacity and its ability to partition into lipophilic environments where peroxyl radicals propagate. These findings support multi-assay approaches for comprehensive antioxidant characterisation and provide quantitative benchmarks for dietary polyphenols.

Conclusion

These findings provide quantitative benchmarks for polyphenol antioxidant capacity and mechanistic understanding supporting dietary recommendations for oxidative stress-related disease prevention. Gallic acid and quercetin demonstrate highest potency with distinct mechanistic profiles involving both direct radical scavenging and metal chelation.

References

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