- Research article
- Open Access
Free radicals produced by the oxidation of gallic acid: An electron paramagnetic resonance study
© Eslami et al 2010
Received: 12 April 2010
Accepted: 5 August 2010
Published: 5 August 2010
Gallic acid (3,4,5-trihydroxybenzoic acid) is found in a wide variety of plants; it is extensively used in tanning, ink dyes, as well as in the manufacturing of paper. The gallate moiety is a key component of many functional phytochemicals. In this work electron paramagnetic spectroscopy (EPR) was used to detect the free radicals generated by the air-oxidation of gallic acid.
We found that gallic acid produces two different radicals as a function of pH. In the pH range between 7-10, the spectrum of the gallate free radical is a doublet of triplets (aH = 1.00 G, aH = 0.23 G, aH = 0.28 G). This is consistent with three hydrogens providing hyperfine splitting. However, in a more alkaline environment, pH >10, the hyperfine splitting pattern transforms into a 1:2:1 pattern (aH (2) = 1.07 G). Using D2O as a solvent, we demonstrate that the third hydrogen (i.e. aH = 0.28 G) at lower pH is a slowly exchanging hydron, participating in hydrogen bonding with two oxygens in ortho position on the gallate ring. The pKa of this proton has been determined to be 10.
This simple and novel approach permitted the understanding of the prototropic equilibrium of the semiquinone radicals generated by gallic acid, a ubiquitous compound, allowing new insights into its oxidation and subsequent reactions.
Gallic acid (3,4,5-trihydroxybenzoic acid), found in a variety of plants, is extensively used in tanning, ink dyes, as well as in the manufacturing of paper . In addition, the gallate moiety is a key component of many foods and drinks, e.g. there are two gallate moieties in the important polyphenol, (-)-epi-gallocatechin-3-gallate (EGCG); this and related polyphenols are responsible for the antioxidant, anticarcinogenic, and antiviral properties of some of the most widely consumed beverages in the world, such as green tea [2, 3]. The three aromatic phenoxyl groups of gallic acid are prone to oxidation with the formation of hydrogen peroxide, quinones, and semiquinones . We have observed the formation of two distinct semiquinones formed upon the oxidation of gallic acid. Here we have investigated the nature of these two different radicals.
Gallic acid (3,4,5-trihydroxybenzoic acid, CAS No.: 149-91-7) was obtained from Sigma, USA; sodium hydroxide was from Fisher. All experiments were carried out in 100 mM potassium phosphate. The final concentration of gallic acid was 1 mM, unless noted otherwise. All solutions above neutral pH (alkaline solutions) were prepared by adjusting with 1 M sodium hydroxide solution.
EPR spectroscopy was done using a Bruker EMX spectrometer equipped with a high-sensitivity cavity and an Aqua-X sample holder. Spectra were obtained at room temperature (24-27°C). Typical EPR parameters were as follows: 3510 G center field; 10 G sweep width; 9.852 GHz microwave frequency; 20 mW power; receiver gain varied as needed; modulation frequency of 100 kHz; modulation amplitude of 0.10 G; conversion time of 40.96 ms; and time constant of 20.48 ms with 20 X-scans for each 1024 point spectrum. Spectral simulations of EPR spectra were performed using the WinSim program developed at the NIEHS by Duling .
In the experiments to determine the second pKa of the gallate free radical 5.0 mL of 10 mM gallic acid (final concentration) was spiked with various amounts of 1 M NaOH to obtain EPR spectra of the gallate free radical at different pH-values. For these experiments, EPR spectra were obtained at room temperature. Typical EPR parameters were as follows: 3510 G center field; 10 G sweep width; 9.852 GHz microwave frequency; 1.282 mW power; 1 × 104 receiver gain; modulation frequency of 100 kHz; modulation amplitude of 0.20 G; conversion time of 40.96 ms; time constant of 40.96 ms with 5 X-scans for each 1024 point spectrum. Quantitation was done using double integration of spectra. With spectra having poor S/N, line height was used to estimate relative concentrations of a species using the results of the double integration of a spectrum of that same species with good S/N as a benchmark.
Results and discussion
Thus, under the two pH conditions (9 and 11) of Figure 1, the carboxylic acid as well as a phenolic-OH of the semiquinone will be ionized; the proposed structures of these radicals are as presented in Figure 1. The 1:2:1 splitting of the species observed at high pH is consistent with hyperfine splittings due to the two identical ring protons. However, we propose that the third hydrogen splitting in the spectrum observed at lower pH is due to a slowly exchanging proton; this proton is participating in hydrogen bonding with two oxygens in ortho position on the gallate ring. A rapidly exchanging proton would produce no hydrogen splittings. However, the third proton splitting could also arise as a result of oligimerization .
Typically one would expect an isosbestic point in such a plot as would be seen in a species distribution diagram of a simple titration of a weak mono-acid with a weak mono-base. However, there is not a one-to-one correspondence in species concentration at pH values <10 compared to pH values >10; the intensity of the EPR signal of the fully ionized species increases dramatically in the more alkaline environment. The highest concentration of the fully ionized radical species present at high pH (≈13) is about 1000 times greater than the singly ionized species seen at lower pH (≈9). This increased signal is due in part to the fact that at higher pH the rate of disproportionation of semiquinone radicals decreases [4, 14]. In addition, as the pH increases the rate of air-oxidation increases, thus the lack of a one-to-one correspondence for the concentrations of the two different gallate radicals. Thus, the second pKa of the gallate free radical is 10.
In a study by Oniki and Takahoma of the oxidation of gallic acid, it was proposed that the EPR spectrum of the gallate radical observed at ≅ pH 10 was due to covalent bond formation in the gallate solution . Our observation of a slowly exchanging hydron may explain in part their observations.
Previously, there were no experimental data available to determine the second pKa of the free radical derived from gallic acid. However, Jovanovic determined the two pKa values of free radical derived from methyl gallate to be 4.4 and 9.2 ; these pKa's for the free radical derived from gallic acid are 5.0 and 10. This is consistent with the lower pKa values of two acidic OH-groups on the ring of methyl gallate (8.0 and 11.6) compared to gallic acid (8.7 and 12.4) . That the differences in the pKa values of the reduced species and their radicals are each 0.6 and 0.8 is striking. This suggests a linear offset in the electron density on the phenoxyl oxygens will occur in a gallate moiety upon oxidation of the hydroquinone to a semiquinone radical, parallel to that seen upon the one-electron oxidation of para-hydrobenzoquinones .
In conclusion, we have demonstrated that the third proton splitting of the gallate radical observed in the pH range of 7-10 is a slowly exchanging hydron; this proton is mostly likely participating in hydrogen-bonding with two oxygens in ortho position on the gallate ring thereby bringing asymmetry to the radical. We found the second pKa of the gallate free radical to be 10.
This work was supported by NIH grants P42ES013661 and R01GM073929. ACE was supported in part by The University of Iowa Graduate College.
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