Biocompatibility and Oxidative Stress Profiling of Laccase-Catalyzed Conversion Products of Biomass-Derived Phenolics

The safety profile for bio-derived phenols post-oxidation and their related antioxidant/redox potential remain largely under-explored. Oxidation by fungi, in terms of environmental impacts via fungal oxidation by enzymes, remains an attractive strategy under milder conditions, since it is one route by which many naturally occurring lignocellulosic phenols are modified; thus, an immediate need still exists for characterizing the effects that these modified phenolic compounds may have.

We examined four different biomass-derived phenolics-vanillin, ferulic acid, syringaldehyde and guaiacol-that were oxidized with fungal laccase and characterized their effects on normal human lung fibroblasts and levels of cellular oxidative stress. Laccase activity was evaluated via the ABTS method and through simple observation and UV-Vis spectroscopic scanning of the phenolics in question, and compared with the untreated version of each phenolic. In addition to assessing the cytotoxic effect and oxidative stress generated by the phenols alone, an ELISA-based measurement assay was used to investigate the relative abundance of malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and reduced glutathione (GSH) in the human normal lung fibroblast cell line under varying treatment regimes, complemented by phase-contrast microscopy. Scores integrating the biomarkers were analyzed via clustering, PCA, radar and Pearson correlation analyses, to discern distinct trends in antioxidant potential after laccase conversion.

Each of the four tested phenolics demonstrated the presence of laccase activity, leading to substantial differences in visible appearance compared with the control and characteristic absorbance shifts at differing wavelengths from the original molecule. Cell viability dropped dramatically as phenol concentration was increased and the untreated phenolics resulted in diminished confluence and induced greater levels of oxidative damage, from guaiacol and syringaldehyde. Laccase treatment resulted in higher MTT reduction activity and improved cellular morphology compared with the corresponding untreated phenolic compounds. Untreated phenols induced the highest levels of MDA, while decreasing SOD, CAT, GPx and GSH levels. Post-oxidation with laccase, there were lower amounts of lipid peroxidation, along with improved levels of antioxidant activity compared with the control phenol. Multi-technique analyses show clear distinctness between the untreated and laccase-converted phenolic groups. Clustering with multivariate techniques separated all cell groups in line with control samples, grouping the laccase-converted treatments towards the middle and displaying an inverse relationship between MDA and the antioxidant markers.

Laccase conversion markedly decreases the adverse effects that bio-derived phenols have on normal cell viability and induces fewer detrimental effects on the cellular redox balance. This is a critical discovery in terms of finding greener methods by which to upgrade bio-derived substances as we research these lignocellulosic phenols. By employing ELISA-based measurements along with multiple analysis techniques, we present a suitable paradigm for studying biological effects in all bio-based goods intended for pharmaceuticals, packaging materials, nutraceuticals or a host of different applications.