Antioxidant Activity and Phenolic Compound Identification and Quantification in Western Australian Honeys

This study reports on the total phenolic content and antioxidant activity as well as the phenolic compounds that are present in Calothamnus spp. (Red Bell), Agonis flexuosa (Coastal Peppermint), Corymbia calophylla (Marri) and Eucalyptus marginata (Jarrah) honeys from Western Australia. The honey's total phenolic content (TPC) was determined using a modified Folin-Ciocalteu assay, while their total antioxidant activity was determined using FRAP and DPPH assays. Phenolic constituents were identified using a High Performance Thin-Layer Chromatography (HTPLC)-derived phenolic database, and the identified phenolic compounds were quantified using HPTLC. Finally, constituents that contribute to the honeys' antioxidant activity were identified using a DPPH-HPTLC bioautography assay. Based on the results, Calothamnus spp. honey (n = 8) was found to contain the highest (59.4 ± 7.91 mg GAE/100 g) TPC, followed by Eucalyptus marginata honey (50.58 ± 3.76 mg GAE/100 g), Agonis flexuosa honey (36.08 ± 4.2 mg GAE/100 g) and Corymbia calophylla honey (29.15 ± 5.46 mg GAE/100 g). In the FRAP assay, Calothamnus spp. honey also had the highest activity (9.24 ± 1.68 mmol Fe²⁺/kg), followed by Eucalyptus marginata honey (mmol Fe²⁺/kg), whereas Agonis flexuosa (5.45 ± 1.64 mmol Fe²⁺/kg) and Corymbia calophylla honeys (4.48 ± 0.82 mmol Fe²⁺/kg) had comparable FRAP activity. In the DPPH assay, when the mean values were compared, it was found that Calothamnus spp. honey again had the highest activity (3.88 ± 0.96 mmol TE/kg) while the mean DPPH antioxidant activity of Eucalyptus marginata, Agonis flexuosa, and Corymbia calophylla honeys were comparable. Kojic acid and epigallocatechin gallate were found in all honeys, whilst other constituents (e.g., m-coumaric acid, lumichrome, gallic acid, taxifolin, luteolin, epicatechin, hesperitin, eudesmic acid, syringic acid, protocatechuic acid, t-cinnamic acid, o-anisic acid) were only identified in some of the honeys. DPPH-HPTLC bioautography demonstrated that most of the identified compounds possess antioxidant activity, except for t-cinnamic acid, eudesmic acid, o-anisic acid, and lumichrome.