Original contribution
Role of phenolic O-H and methylene hydrogen on the free radical reactions and antioxidant activity of curcumin

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Abstract

To understand the relative importance of phenolic O-H and the CH-H hydrogen on the antioxidant activity and the free radical reactions of Curcumin, (1,7-bis[4-hydroxy-3-methoxyphenyl]-1,6-heptadiene-3,5-dione), biochemical, physicochemical, and density functional theory (DFT) studies were carried out with curcumin and dimethoxy curcumin (1,7-bis[3, 4-dimethoxy phenyl]-1,6-heptadiene-3,5-dione). The antioxidant activity of these compounds was tested by following radiation-induced lipid peroxidation in rat liver microsomes, and the results suggested that at equal concentration, the efficiency to inhibit lipid peroxidation is changed from 82% with curcumin to 24% with dimethoxy curcumin. Kinetics of reaction of (2,2′-diphenyl-1-picrylhydrazyl) DPPH, a stable hydrogen abstracting free radical was tested with these two compounds using stopped-flow spectrometer and steady state spectrophotometer. The bimolecular rate constant for curcumin was found to be ∼1800 times greater than that for the dimethoxy derivative. Cyclic voltammetry studies of these two systems indicated two closely lying oxidation peaks at 0.84 and 1.0 V vs. SCE for curcumin, while only one peak at 1.0 V vs. SCE was observed for dimethoxy curcumin. Pulse radiolysis induced one-electron oxidation of curcumin and dimethoxy curcumin was studied at neutral pH using N3 radicals. This reaction with curcumin produced phenoxyl radicals absorbing at 500 nm, while in the case of dimethoxy curcumin a very weak signal in the UV region was observed. These results suggest that, although the energetics to remove hydrogen from both phenolic OH and the CH2 group of the β-diketo structure are very close, the phenolic OH is essential for both antioxidant activity and free radical kinetics. This is further confirmed by DFT calculations where it is shown that the −OH hydrogen is more labile for abstraction compared to the −CH2 hydrogen in curcumin. Based on various experimental and theoretical results it is definitely concluded that the phenolic OH plays a major role in the 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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