ABTS/PP Decolorization Assay of Antioxidant Capacity Reaction Pathways
Abstract
:1. Introduction
2. ABTS/PP Abundance Statistics
3. ABTS/PP Basic Chemistry
4. Reaction Stoichiometry
5. Reaction Pathways with Adducts Formation
- (1)
- As ABTS•+ was generated in the ABTS/laccase system, but not ABTS/PP, one could argue that it could hypothetically influence the observed ABTS•+-antioxidant reaction and subsequent ABTS•+ depletion. There are many precedents when certain antioxidants reveal different antioxidant capacities against the same model radical but a different radical-generating system, for example in the ABTS/metmyoglobin/H2O2 assay, the TEAC of quercetin and cyanidin was 4.72 and 4.4 [76,77], whereas in the ABTS/PP assay it was 3.03 and 2.48 [1], respectively. Additionally, laccase can act as an oxidizing agent alone, not to mention that the combination of laccase with ABTS or other mediators shows a higher oxidation ability than laccase or ABTS•+ separately [78,79,80,81,82]. Nevertheless, these concerns seem to be unfounded, as the same researchers observed these ABTS•+ degradation products again in their next report when applying the ABTS/PP system [83].
- (2)
- The time of reaction was 1–2 h, which is much more than the 4–6 min in the original TEAC assay, and thus there was much more time for ABTS•+ cleavage.
- (3)
- The fact that polyphenol was added “dropwise” to the concentrated ABTS/laccase mixture, which also differs from the original design of ABTS/PP assay, did not seem to matter either, as generally the same situation C(ABTS•+) > C(antioxidant) at any given time was reproduced.
- (1)
- The principal possibility of ABTS•+ degradation and ABTS-antioxidant adduct formation was demonstrated for the first time in the ABTS/PP assay.
- (2)
- The extent to which this degradation and adduct formation influenced the final TEAC was not obvious due to the modified experiment design and no quantitative estimations.
6. Reaction Pathways without Adduct Formation
7. Conclusions
- (1)
- Some antioxidants can form adducts with ABTS•+, whereas others can undergo oxidation without coupling with ABTS•+. Thus, coupling with ABTS•+ is a specific reaction for certain groups of antioxidants, apparently at least of phenolic nature. Establishing the structural features that determine the direction of antioxidant interaction with ABTS•+ is important for future understanding and interpretation of antioxidant capacity measurements.
- (2)
- Adduct-free oxidation pathways are substrate-specific and can be influenced both by ABTS•+ radical specific features or by the radical-initiator system. However, they seem to be reliably consistent with the results obtained when other oxidants are applied instead of ABTS•+.
- (3)
- The coupling reaction can occur with phenolic compounds, and the coupling adduct can be the principal product as well as undergoing further oxidative degradation, which might depend on the antioxidant/ABTS•+ ratio and ABTS•+ generation methodology.
- (4)
- Further oxidative degradation of the coupling product results from the oxidative cleavage between the two nitrogen-linked benzothiazole rings. This leads to hydrazindyilidene-like and imine-like adduct formation. 3-Ethyl-2-oxo-1,3-benzothiazoline-6-sulfonate can presumably witness a hydrazindyilidene-like adduct formation pathway, whereas 3-ethyl-2-imino-1,3-benzothiazoline-6-sulfonate testifies to the imine-like adduct formation pathway.
- (5)
- The extent that the coupling reaction contributes to the reaction between antioxidants and ABTS•+ (e.g., kinetics and stoichiometry) is unclear due to the lack of quantitative estimation of their formation, and sometimes this may be quite considerable.
Author Contributions
Funding
Conflicts of Interest
Abbreviations
ABTS | 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt |
AFMK | N1-acetyl-N2-formyl-5-methoxykynuramine |
c3OHM | cyclic 3-hydroxymelatonin |
CEP | concerted electron–proton transfer |
COSY | correlation spectroscopy |
CUPRAC | cupric reducing antioxidant capacity |
DAD | diode-array detection |
DPPH | 2,2-diphenyl-1-picrylhydrazyl |
ESI-HRMS | high resolution electrospray ionization mass spectrometry |
ESI-MS | electrospray ionization mass spectrometry |
ET-PT | electron transfer–proton transfer |
FRAP | ferric reducing antioxidant power |
FKBP | fujimycin binding protein |
GBG | guaiacylglycerol-β-guaiacyl ether |
GBGox | oxidation products of guaiacylglycerol-β-guaiacyl ether |
GSH | glutathione |
GSSG | glutathione disulfide |
HAT | hydrogen atom transfer |
HMBC | heteronuclear multiple bond correlation |
HSQC | heteronuclear single quantum coherence |
HPLC-ECD | high performance liquid chromatography, electrochemical detection |
HPLC-UV | high performance liquid chromatography, ultraviolet detection |
IC50 | concentration which leads to 50% inhibition |
IR | infrared spectroscopy |
MS-MS | tandem mass spectrometry |
NI | negative mode in electrospray ionization mass spectrometry |
NMR | nuclear magnetic resonance |
ORAC | oxygen radical absorbance capacity |
PBS | phosphate buffer solution |
PDA | photodiode array |
PI | positive mode in electrospray ionization mass spectrometry |
PP | potassium persulfate |
QTOF-MS | quadrupole time of flight mass spectrometer |
RP-UHPLC | reversed-phase ultrahigh-performance liquid chromatography |
SET | single electron transfer |
SIMS | secondary ion mass spectrometry |
SPLET | sequential proton loss electron transfer |
TEAC | trolox equivalent antioxidant capacity |
TLC | thin layer chromatography |
UV-VIS | ultraviolet–visible spectroscopy |
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Method | References of Basic Publications | Citation Rate 1 | Frequency of Abbreviation Use (Keywords Used) |
---|---|---|---|
TEAC | [1,2,16] | 13,220 | 3772 2 (“TEAC” or “Trolox equivalent antioxidant capacity”) since 1993 |
ABTS/PP decolorization assay | [1] | 9845 | 10,109 (“ABTS antioxidant” or “ABTS antiradical” or “ABTS radical scavenging”) since 1988 |
DPPH | [17,18,19,20] | 11,177 | 35,136 (“DPPH antioxidant” or “DPPH antiradical” or “DPPH radical scavenging”) since 1955 |
FRAP | [21,22,23] | 11,040 | 9492 (“FRAP” or “Ferric reducing antioxidant power”) 3 since 1994 |
Folin–Ciocalteu | [24,25,26,27] | 7630 | 2803 (“Folin–Ciocalteu antioxidant”) since 1976 |
ORAC | [28,29,30,31,32,33] | 3478 | 3619 (“ORAC” or “Oxygen radical absorbance capacity”) since 1993 |
CUPRAC | [34,35,36] | 1260 | 685 (“CUPRAC” or “Cupric ion reducing antioxidant capacity”) since 2004 |
λ(ABTS•+), nm | Extinction Coefficient, ε | Reference |
---|---|---|
415 nm | 36,000 L∙mol−1∙cm−1 in water | [43,44] |
414 nm | 31,100 l mol−1 cm−1 in water (sodium phosphate buffer, pH 7.5) 33,630 L∙mol−1∙cm−1 in ethanol | [3,45] |
734 nm | 15,000 L∙mol−1∙cm−1 in water 16,000 L∙mol−1∙cm−1 in ethanol | [1] |
730 nm | 12,947 l mol−1 cm−1 in water (sodium phosphate buffer, pH 7.5) 14,750 L∙mol−1∙cm−1 in ethanol | [3,45] |
Antioxidant | The Calculated Number of ABTS•+ Molecules Reduced by One Molecule of Antioxidant 1 | ||
---|---|---|---|
Name | Formula | Decolorization Assay | Lag-Time Assay |
Trolox | 2.4 | 1.7 | |
Quercetin | 12.0 | 4.7 | |
Morin | 6.9 | 3.3 | |
Rutin | 6.6 | 2.8 | |
Taxifolin | 5.9 | 2.7 | |
Apigenin | 5.3 | ND | |
Naringenin | 4.6 | ND | |
Glutathione | 2.7 | 0.8 | |
α-Tocopherol | 1.9 | 1.9 |
Antioxidant | Antioxidant Concentration Needed to Inhibit 50% of ABTS•+, µM | The Ratio Antioxidant/ABTS•+ |
---|---|---|
Trolox | 11.0 | 1:4 |
Glutathion | 8.4 | 1:6 |
Taxifolin | 4.1 | 1:11 |
Quercetin | 1.8 | 1:26 |
α-Tocopherol | 12.4 | 1:4 |
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Ilyasov, I.R.; Beloborodov, V.L.; Selivanova, I.A.; Terekhov, R.P. ABTS/PP Decolorization Assay of Antioxidant Capacity Reaction Pathways. Int. J. Mol. Sci. 2020, 21, 1131. https://doi.org/10.3390/ijms21031131
Ilyasov IR, Beloborodov VL, Selivanova IA, Terekhov RP. ABTS/PP Decolorization Assay of Antioxidant Capacity Reaction Pathways. International Journal of Molecular Sciences. 2020; 21(3):1131. https://doi.org/10.3390/ijms21031131
Chicago/Turabian StyleIlyasov, Igor R., Vladimir L. Beloborodov, Irina A. Selivanova, and Roman P. Terekhov. 2020. "ABTS/PP Decolorization Assay of Antioxidant Capacity Reaction Pathways" International Journal of Molecular Sciences 21, no. 3: 1131. https://doi.org/10.3390/ijms21031131