Light Emission from the Fe2+-EGTA-H2O2 System: Possible Application for the Determination of Antioxidant Activity of Plant Phenolics

Abstract

Oxidative reactions can result in the formation of electronically excited species that undergo radiative decay depending on electronic transition from the excited state to the ground state with subsequent ultra-weak photon emission (UPE). We investigated the UPE from the Fe2+-EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid)–H2O2 system with a multitube luminometer (Peltier-cooled photon counter, spectral range 380 to 630 nm). The UPE of 92.6 µmol/L Fe2+—185.2 µmol/L EGTA—2.6 mmol/L H2O2 reached 4319 ± 755 relative light units during 2 min measurement and was about seven times higher (p < 0.001) than the UPE of incomplete systems (Fe2+-H2O2, EGTA-H2O2) and medium alone. Substitution of Fe2+ with Cr2+, Co2+, Mn2+ or Cu2+ as well as of EGTA with EDTA (ethylenediaminetetraacetic acid) or citrate completely abolished UPE. Experiments with ROS scavengers revealed the dependence of UPE on hydroxyl radicals suggesting occurrence of oxidative attack and cleavage of the ether bond in EGTA backbone structure and formation of triplet excited carbonyl groups with subsequent light emission. Plant phenolics (ferulic, chlorogenic and caffec acids) at concentration 87 µmol/L and ascorbate at 0.46 mmol/L inhibited UPE by 90 ± 4%, 90 ± 5%, 97 ± 2% and 92 ± 1%, respectively. Quenching of UPE from Fe2+-EGTA-H2O2 system can be used for evaluation of antioxidant activity of phytochemicals.

Light emission from the Fe2+–EDTA–ascorbic acid–H2O2 system strongly enhanced by plant phenolic acids

“…Catalase and SOD were dissolved in PBS to an activity of 10 U/μL. Stock solution of EGTA (100 mmol/L, pH 8.0) was prepared as previously described and 10 mmol/L EGTA working solution was obtained by appropriate dilution with water.…”

“…The chemiluminescence was measured with a multitube luminometer (AutoLumat Plus LB 953, Berthold Technologies, Bad Wildbad, Germany) equipped with a Peltier‐cooled photon counter (spectral range from 380 to 630 nm, working temperature 8°C) as previously described . Briefly, 20 μL of 5 mmol/L FeSO 4 solution was added to the tube (Lumi Vial Tube, 5 mL, 12 × 75 mm, Berthold Technologies) containing 910 μL of PBS.…”

“…Fenton reagent [solution of hydrogen peroxide (H 2 O 2 ) with iron(II) ion (Fe 2+ )] and chelate‐modified Fenton's reagent [e.g. solution of Fe 2+ , EGTA (ethylene glycol‐bis(β‐aminoethyl ether)‐ N , N , N ′, N ′‐tetraacetic acid) with H 2 O 2 ] are used to study • OH‐induced peroxidative processes and were reported to emit light . Fenton reagent generates UPE via singlet oxygen (O 2 ( 1 ∆ g )) formation and its decay with three characteristic emission bands at 1270, 703 and 634 nm .…”

“…In the case of the Fe 2+ ‐EGTA–H 2 O 2 system another source of photon emission was proposed. EGTA undergoes the • OH‐induced oxidative attack and cleavage of the ether bond in its backbone structure and formation of the triplet excited carbonyl groups ( 3 (R = C) * ) with subsequent light emanation . UPE from Fe 2+ ‐EGTA–H 2 O 2 system was strongly inhibited by • OH scavengers [mannitol, dimethyl sulfoxide (DMSO)], ascorbic acid (AA) and selected plant phenolic acids .…”

“…EGTA undergoes the • OH‐induced oxidative attack and cleavage of the ether bond in its backbone structure and formation of the triplet excited carbonyl groups ( 3 (R = C) * ) with subsequent light emanation . UPE from Fe 2+ ‐EGTA–H 2 O 2 system was strongly inhibited by • OH scavengers [mannitol, dimethyl sulfoxide (DMSO)], ascorbic acid (AA) and selected plant phenolic acids . Another, even more frequently used chelate‐modified Fenton system is a combination of Fe 2+ , EDTA (ethylenediaminetetraacetic acid), AA and H 2 O 2.…”