Original contributionRole of phenolic O-H and methylene hydrogen on the free radical reactions and antioxidant activity of curcumin
Introduction
Curcumin (1,7-bis [4-hydroxy 3-methoxy phenyl]-1,6-heptadiene-3,5-dione) is a natural phenol found as a major pigment in the Indian spice turmeric (Scheme 1). Curcumin shows remarkable pharmacological activity, including antiinflammatory, anticarcinogenic, and antioxidant activity 1, 2, 3, 4, 5, 6. It acts as a lipoxygenase substrate and also an inhibitor of cycloxygenase enzymes [7, 8. It is considered as a potential chemopreventive agent and the clinical trials in this direction are in different stages 9, 10, 11. One of the most important factors considered to be responsible for all the activity of curcumin is its ability to scavenge reactive oxygen and nitrogen free radicals 5, 6, 12, 13, 14, 15, 16. It is a diferuloyl methane having two o-methoxy phenolic OH groups attached to the α, β-unsaturated β-diketone (heptadiene-dione) moiety. The free radical scavenging activity of curcumin can arise either from the phenolic OH group or from the CH2 group of the β-diketone moiety. A reactive free radical can undergo electron transfer or abstract H-atom from either of these two sites. Recently there are some contradictory reports published in the literature supporting the two different sites of attack for the free radicals. Many initial reports based on pulse radiolysis and other biochemical methods attributed the antioxidant activity to the phenolic OH group 14, 15, 16, 17. But Jovanovic et al. indicated that hydrogen abstraction from the methylene CH2 group is responsible for the remarkable antioxidant activity of curcumin 18, 19. Later, Barclay et al., by following the inhibition of styrene oxidation by a number of curcumin derivatives, suggested that the H atom from the phenolic OH is responsible for the antioxidant activity [20]. In this article we describe applying both experimental and theoretical tools that the phenolic OH is mainly responsible for the activity of curcumin. The experimental studies include comparison of the effect of curcumin and its synthetic derivative dimethoxy curcumin (structure given in Scheme 1) on the inhibition of lipid peroxidation, kinetics of reaction with DPPH (2,2′-diphenyl-1-picrylhydrazyl), cyclic voltammetry, and pulse radiolytic oxidation and identification of the transients. In dimethoxy curcumin, the phenolic OH is blocked on both the rings and the β-diketo structure is intact, therefore it could be used to identify the contribution of the β-diketone group in its overall activity. For the theoretical calculations, a hybrid density functional theory (DFT) study has been performed to calculate the structure, stability, and atomic charges of curcumin and the radicals generated by H-abstraction reactions from phenolic OH and −CH2 groups. Bond dissociation energies have also been calculated at B3LYP level of theory for the respective H atom elimination paths and reported.
Section snippets
Chemicals
Curcumin, 2,2′-diphenyl-1-picrylhydrazyl (DPPH), thiobarbituric acid (TBA), butylated hydroxytoluene (BHT), and 2,2′-azinobis (3-ethylbenthiazoline-6-sulphonate) (ABTS−2) were purchased from Aldrich/Sigma Chemicals, St. Louis, MO, USA. Spectrograde methanol, DMSO, and chloroform from Spectro Chem. (Mumbai, India) were used as received. All other reagents used were of highest available purity. Nitrous oxide (N2O) and oxygen (O2) gases obtained from Indian Oxygen Ltd., Mumbai, were of IOLAR grade
Results and discussion
Curcumin has three ionizable protons and thus three protonation constants, reported in the literature. The three pKa values of 8.54, 9.3, and 10.69 have been attributed by Borsari et al. to the equilibria for the deprotonation from the enolic proton and the two phenolic protons, respectively [33]. Earlier, Jovanovic et al. reported two pKa values of 8.55 and 10.41 [18]. We have determined pKa of dimethoxy curcumin as 9.4. For this study, absorption spectral changes in 10 μM dimethoxy curcumin
Conclusions
The antioxidant activity, hydrogen donating capacity, and redox properties of curcumin and its dimethoxy derivative have been systematically studied to throw light on the mechanism behind the superb antioxidant activity of curcumin. The lipid peroxidation studies in microsomes have suggested that under identical conditions, curcumin shows much greater ability to inhibit lipid peroxidation than its dimethoxy counterpart, confirming that the phenolic OH is essential for the antioxidant activity.
Abbreviations
ABTS−2—2,2′-azinobis (3-ethylbenthiazoline-6-sulphonate)
BHT—butylated hydroxytoluene
CR1—carbon centered radical
DFT—density functional theory
DPPH—2,2′-diphenyl-1-picrylhydrazyl
PR1—phenoxyl radical
SCE—standard calomel electrode
TBA—thiobarbituric acid
TBARS—thiobarbituric acid reactive substances
Acknowledgements
The authors are thankful to Dr T. Mukherjee and Dr J. P. Mittal of Chemistry group for encouragement and support. The computer center of Bhabha Atomic Research Centre is gratefully acknowledged for providing the computational facility and Dr. G. Venkateshwaran for help in cyclic voltametry measurements.
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