The increased incidences, mortality rate and economic impact of noncommunicable diseases (e.g. high blood pressure and diabetes) associated with oxidative stress, have led to the higher demand for antioxidant supplements for their prevention. The use of naturally occurring antioxidants is becoming a more attractive option due to the health risks associated with synthetic antioxidants. Phenolic compounds from plants have been shown to have antioxidant properties with the potential to be used as substitutes to synthetic antioxidants. However, monomeric phenolic compounds have several short comings such as low bioavailability, poor solubility, and low antioxidant capacity while some have pro-oxidant properties at high concentrations. Hence there has been increasing research focused on the biotransformation of these phenolic antioxidants through enzymatic oligomerisation to higher molecular weight compounds with improved antioxidant capacity and stability. Of the investigated enzymes, laccases have shown the most promise owing to their green catalytic properties. Their reaction mechanism involves the use of molecular oxygen as a co- substrate in oxidising phenolic compounds to corresponding radicals, with water as the only by- product. This study focused on the synthesis of antioxidants with enhanced antioxidant capacity using a laccase from Trametes pubescens as biocatalyst. To establish the potential of the phenolic compounds for use as substrates for the coupling reactions, a preliminary screening process was done. Guaiacol, caffeic acid, vanillic acid, eugenol, catechol, gallic acid, ferulic acid and quercetin hydrate were identified as suitable substrates for the laccase enzyme. However, only products from eugenol, coumaric acid and quercetin could be isolated, hence coupling reactions were carried out using these substrates in monophasic systems. Reaction products were monitored using thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Purification was carried out using preparative TLC and characterisation using liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). The antioxidant capacities of reaction products were determined using ABTS (2,2’-Azinobis 3- ethylbenzthiazoline-6-sulfonic acid), DPPH (2,2-diphenyl-1-picrylhydrazyl) and FRAP (ferric- reducing antioxidant power) assays. Quercetin hydrate oxidation produced one product which was purified and characterised. The product had an Rf of 0.68, tR 13.567 and m/z 601 in negative mode, indicating that it was a dimeric form of quercetin. Oxidation of ρ-coumaric acid resulted in the production of two products designated P1 (Rf 0.47) and P2 (Rf 0.42). Further characterisation was done on product P2 since product P1 could not be successfully purified. P2 had a retention time of 11.295 and m/z 325, indicating that it was a dimer of ρ-coumaric. The ρ-coumaric dimer had an enhanced antioxidant capacity, approximately 2-fold, 3-fold and 6-fold higher compared to the substrate, as demonstrated by the ABTS, DPPH and FRAP assays, respectively. A symmetrical 5-5 eugenol dimer (m/z 325, [M] =326), bis-eugenol, was produced from eugenol oxidation. Maximum product formation (50% yield) was obtained in a monophasic system with 40% v/v dioxane as co-solvent after incubation for 18 h. The bis- eugenol dimer had an improved antioxidant capacity of up to three and four times that of eugenol as demonstrated by the ABTS and DPPH assays, respectively. In conclusion, two dimers with high antioxidant capacity were successfully produced, purified and characterised. The study has demonstrated the potential of the T. pubescens laccase as a catalyst for the synthesis of phenolic compounds with enhanced antioxidant capacity.
Effect of drying method on the phenolic content and antioxidant capacity of spearmint