K. Indira Priyadarsini

Effect of Solvent on the Excited‐state Photophysical Properties of Curcumin¶

Abstract Photophysical properties of curcumin, 1,7-bis-(4-hydroxy-3-methoxy phenyl)-1,6-heptadiene-2,5-dione, a pigment found in the rhizomes of Curcuma longa (turmeric) have been studied in different kinds of organic solvent and also in Triton X-100 aqueous micellar media using time-resolved fluorescence and transient absorption techniques having pico and nanosecond time resolution, in addition to steady-state absorption and fluorescence spectroscopic techniques. Steady-state absorption and fluorescence characteristics of curcumin have been found to be sensitive to the solvent characteristics. Large change (Δμ = 6.1 Debye) in dipole moments due to photoexcitation to the excited singlet state (S1) indicates strong intramolecular charge transfer character of the latter. Curcumin is a weakly fluorescent molecule and the fluorescence decay properties in most of the solvents could be fitted well to a double-exponential decay function. The shorter component having lifetime in the range 50–350 ps and percent contribution of amplitude more than 90% in different solvents may be assigned to the enol form, whereas the longer component, having lifetime in the range 500–1180 ps with less than 10% contribution may be assigned to the di-keto form of curcumin. Our nuclear magnetic resonance study in CDCl3 and dimethyl sulfoxide-D6 also supports the fact that the enol form is present in the solution by more than about 95% in these solvents. Excited singlet (S1) and triplet (T1) absorption spectrum and decay kinetics have been characterized by pico and nanosecond laser flash photolysis. Quantum yield of the triplet is low (ϕT ≤ 0.12). Both the fluorescence and triplet quantum yields being low (ϕf + ϕT < 0.18), the photophysics of curcumin is dominated by the energy relaxation mechanism via the internal conversion process.

Free Radical Reactions of Curcumin in Membrane Models

Free radical reactions of curumin, a lipid soluble antioxidant from turmeric (Curcuma longa), have been studied with a variety of oxidants using TX 100 micelle as a model membrane. The phenoxyl radicals of curcumin generated by one electron oxidizing azide radicals in acetonitrile-water mixture and TX 100 micelles show very similar spectral behavior. However, in membrane models the radical lifetimes and the molar extinction coefficients are significantly different from the homogeneous solutions. Micellized curcumin reacts with haloperoxyl radicals, superoxide, and lipid peroxyl radicals with rate constants of 5 X 10(3), 4.6 X 10(4), and 5.3 X 10(5) M-1s-1, respectively. Curcumin derived phenoxyl radicals decay by radical-radical reactions in homogeneous solutions, while in the micelles, radical decay is mostly first order when the average occupancy of the micelle is less than 1. Implications of these results in evaluating curcumin as an antioxidant is discussed.

Free radical scavenging ability and antioxidant efficiency of curcumin and its substituted analogue.

Free radical reactions of curcumin and its ethoxy substituted derivative (C1) 1,7-bis-(4-hydroxy-3-ethoxy phenyl)-1,6-heptadiene-3,5-dione have been studied using a pulse radiolysis technique in homogeneous aqueous-organic solutions like acetonitrile-water and isopropanol-water mixtures, as well as in neutral TX-100 and cationic CTAB micellar solutions. The phenoxyl radicals of curcumin or C1 were generated by one-electron transfer to several oxidants like N(3)(.), Br(2)(-.), CCl(3)O(2)(.), glutathione radicals which exhibit absorption from a 300-600-nm wavelength region with the maximum at 490-500 nm. Other important properties of the phenoxyl radicals such as extinction coefficient, radical lifetime and their formation and decay rate constants were also determined in these systems. The antioxidant property of curcumin and C1 were estimated in terms of their ability to inhibit the lipid peroxidation in liposomes and also in terms of trolox equivalent antioxidant capacity (TEAC). The results were compared with alpha-tocopherol.

Pyrroloquinoline-quinone: a reactive oxygen species scavenger in bacteria

Transgenic Escherichia coli expressing pyrroloquinoline‐quinone (PQQ) synthase gene from Deinococcus radiodurans showed superior survival during Rose Bengal induced oxidative stress. Such cells showed significantly low levels of protein carbonylation as compared to non‐transgenic control. In vitro, PQQ reacted with reactive oxygen species with rate constants comparable to other well known antioxidants, producing non‐reactive molecular products. PQQ also protected plasmid DNA and proteins from the oxidative damage caused by γ‐irradiation in solution. The data suggest that radioprotective/oxidative stress protective ability of PQQ in bacteria may be consequent to scavenging of reactive oxygen species per se and induction of other free radical scavenging mechanism.

Fluorescence spectroscopic studies on binding of a flavonoid antioxidant quercetin to serum albumins

Binding of quercetin to human serum albumin (HSA) was studied and the binding constant measured by following the red-shifted absorption spectrum of quercetin in the presence of HSA and the quenching of the intrinsic protein fluorescence in the presence of different concentrations of quercetin. Fluorescence lifetime measurements of HSA showed decrease in the average lifetimes indicating binding at a location, near the tryptophan moiety, and the possibility of fluorescence energy transfer between excited tryptophan and quercetin. Critical transfer distance (Ro) was determined, from which the mean distance between tryptophan-214 in HSA and quercetin was calculated. The above studies were also carried out with bovine serum albumin (BSA).

Reactions of superoxide radicals with curcumin: probable mechanisms by optical spectroscopy and EPR.

Reactions of superoxide-crown ether complex with curcumin have been studied in acetonitrile. Optical absorption spectra showed that curcumin on reaction with superoxide forms a blue color intermediate absorbing at 560 nm, which subsequently decayed in a few hours with the development of the absorption band corresponding to the parent curcumin. The regeneration was 100% at low superoxide concentrations (1:1, or 1:2 or 1:3 of curcumin:superoxide) but reduced to 60% at high superoxide concentration (>1:5). The regeneration of curcumin is confirmed by HPLC analysis. Stopped-flow studies in acetonitrile following either the decay of parent curcumin at 420 nm or formation of 560 nm absorption have been used to determine the rate constant for the reaction of superoxide with curcumin. EPR studies confirmed the disappearance of characteristic superoxide signal in presence of curcumin with the formation of new featureless signal with g=2.0067. Based on these studies it is concluded that at low superoxide concentrations curcumin effectively causes superoxide dismutation without itself undergoing any chemical change. At higher concentrations of superoxide, curcumin inhibits superoxide activity by reacting with it.

Selenium-containing amino acids are targets for myeloperoxidase-derived hypothiocyanous acid: determination of absolute rate constants and implications for biological damage

Elevated MPO (myeloperoxidase) levels are associated with multiple human inflammatory pathologies. MPO catalyses the oxidation of Cl−, Br− and SCN− by H2O2 to generate the powerful oxidants hypochlorous acid (HOCl), hypobromous acid (HOBr) and hypothiocyanous acid (HOSCN) respectively. These species are antibacterial agents, but misplaced or excessive production is implicated in tissue damage at sites of inflammation. Unlike HOCl and HOBr, which react with multiple targets, HOSCN targets cysteine residues with considerable selectivity. In the light of this reactivity, we hypothesized that Sec (selenocysteine) residues should also be rapidly oxidized by HOSCN, as selenium atoms are better nucleophiles than sulfur. Such oxidation might inactivate critical Sec-containing cellular protective enzymes such as GPx (glutathione peroxidase) and TrxR (thioredoxin reductase). Stopped-flow kinetic studies indicate that seleno-compounds react rapidly with HOSCN with rate constants, k, in the range 2.8×103–5.8×106 M−1·s−1 (for selenomethionine and selenocystamine respectively). These values are ~6000-fold higher than the corresponding values for H2O2, and are also considerably larger than for the reaction of HOSCN with thiols (16-fold for cysteine and 80-fold for selenocystamine). Enzyme studies indicate that GPx and TrxR, but not glutathione reductase, are inactivated by HOSCN in a concentration-dependent manner; k for GPx has been determined as ~5×105 M−1·s−1. Decomposed HOSCN did not induce inactivation. These data indicate that selenocysteine residues are oxidized rapidly by HOSCN, with this resulting in the inhibition of the critical intracellular Sec-dependent protective enzymes GPx and TrxR.

Reactions of reactive oxygen species (ROS) with curcumin analogues: Structure–activity relationship

Abstract Three curcumin analogues viz., bisdemethoxy curcumin, monodemethoxy curcumin, and dimethoxycurcumin that differ at the phenolic substitution were synthesized. These compounds have been subjected for free radical reactions with DPPH radicals, superoxide radicals (O2•−), singlet oxygen (1O2) and peroxyl radicals (CCl3O2•) and the bimolecular rate constants were determined. The DPPH radical reactions were followed by stopped-flow spectrometer, 1O2 reactions by transient luminescence spectrometer, and CCl3O2• reactions using pulse radiolysis technique. The rate constants indicate that the presence of o-methoxy phenolic OH increases its reactivity with DPPH and CCl3O2•, while for molecules lacking phenolic OH, this reaction is very sluggish. Reaction of O2•− and 1O2 with curcumin analogues takes place preferably at β-diketone moiety. The studies thus suggested that both phenolic OH and the β-diketone moiety of curcumin are involved in neutralizing the free radicals and their relative scavenging ability depends on the nature of the free radicals.

Differential antioxidant/pro-oxidant activity of dimethoxycurcumin, a synthetic analogue of curcumin

Abstract Dimethoxycurcumin (Dimc), a metabolically stable analogue of curcumin, is under investigation as an anti-tumour agent. Recently a number of studies have been performed on Dimc in this laboratory and also by others. In the present article, all these results have been summarized and wherever possible compared with those of curcumin. Rate constant for reactions of Dimc with superoxide radicals was comparable with that of curcumin, while its reaction with peroxyl radicals was much slower. These results were further supported by the observations on the scavenging of basal ROS levels in lymphocytes and evaluation of antioxidant activities. In line with the earlier reports on curcumin, Dimc was a pro-oxidant and generated ROS in tumour cells. Both curcumin and Dimc were non-toxic to lymphocytes, while exhibiting comparable cytotoxicity to tumour cells. Additionally, these compounds showed higher uptake in tumour cells than in normal lymphocytes. Fluorescence studies on both the compounds revealed their binding to genomic DNA, similar sub-cellular distribution and nuclear localization. All these studies suggested that methylation of the phenolic-OH group in curcumin, although decreasing the antioxidant activity marginally, showed comparable pro-oxidant activity, making it a promising anti-tumour agent.

Delayed activation of PKCδ and NFκB and higher radioprotection in splenic lymphocytes by copper (II)–Curcumin (1:1) complex as compared to curcumin

A mononuclear 1:1 copper complex of curcumin had been found to be superior to curcumin in its anti‐oxidant properties. This paper describes the radio‐protective effects of the complex in splenic lymphocytes from swiss mice. The complex was found to be very effective in protecting the cells against radiation‐induced suppression of glutathione peroxidase, catalase and superoxide dismutase (SOD) activities. Both curcumin and the complex protected radiation‐induced protein carbonylation and lipid peroxidation in lymphocytes with the complex showing better protection than curcumin. It also showed better overall protection by decreasing the radiation‐induced apoptosis. The kinetics of activation of PKCδ and NFκB after irradiation in presence or absence of these compounds was looked at to identify the molecular mechanism involved. The modulation of irradiation‐induced activation of PKCδ and NFκB by curcumin and the complex was found different at later time periods although the initial response was similar. The early responses could be mere stress responses and the activation of crucial signaling factors at later time periods may be the determinants of the fate of the cell. In this study this delayed effect was observed in case of complex but not in case of curcumin. The delayed effect of the complex along with the fact that it is a better free radical scavenger must be the reason for its better efficacy. The complex was also found to be less cytotoxic then curcumin at similar concentration. J. Cell. Biochem. 102: 1214–1224, 2007.