Atanu Barik

Photophysical studies on binding of curcumin to bovine serum albumin

Abstract The excited-state photophysical properties of curcumin in the presence of bovine serum albumin (BSA) have been studied. The absorption and fluorescence changes in curcumin on binding to BSA have been followed at varying concentrations of either curcumin or BSA to determine the binding constant, which has been found to be ∼104 to 105 M−1. Stopped-flow kinetics studies suggested at least two distinct kinetic steps for the binding of curcumin to BSA. The photophysical properties of the singlet-excited state of the curcumin–BSA complex have also been studied. Whereas the absorption spectrum of curcumin is redshifted, the fluorescence spectrum of curcumin was blueshifted in the presence of BSA. The fluorescence quantum yield of curcumin on complexing with BSA was ∼0.05. Steady-state fluorescence anisotropy studies showed a significant increase in the anisotropy value of 0.37 in BSA-bound curcumin. The fluorescence decay of the curcumin–BSA complex followed a biexponential decay with fluorescence lifetimes of 413 ps (33%) and 120 ps (67%). On the basis of these complementary results, it has been concluded that curcumin shows very high binding to BSA, probably at the hydrophobic cavities inside the protein.

Quantitative cellular uptake, localization and cytotoxicity of curcumin in normal and tumor cells.

Using absorption and fluorescence spectroscopic methods, quantitative cellular uptake of curcumin, an antioxidant and anti-tumor agent from Curcuma longa, was calculated in two types of normal cells: spleen lymphocytes, and NIH3T3 and two tumor cell lines: EL4 and MCF7. Both the uptake and fluorescence intensity of curcumin were significantly higher in tumor cells compared to the normal cells. A linear dependency on the uptake was observed with treatment concentration of curcumin. Using laser confocal microscopy, intracellular localization of curcumin was monitored and the results indicated that curcumin is located both in the cell membrane and the nucleus. Sub-cellular fractionation of curcumin-loaded MCF7 cells supported the differential distribution of curcumin in membrane, cytoplasm and nuclear compartments of cell with maximum localization in the membrane. Cytotoxicity studies in different cell lines indicated that the toxicity of curcumin increased with increasing uptake.

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).

Effect of solvent polarity on the photophysical properties of coumarin-1 dye

Effects of solvent polarity on the photophysical properties of coumarin-1 (C1; 7-NEt2-4-CH3-1,2-benzopyrone) dye have been investigated using steady-state and time-resolved fluorescence measurements. In moderate to higher polarity solvents the properties like Stokes’ shifts (Δν), fluorescence quantum yields (Φf), fluorescence lifetimes (τf), radiative rate constants (kf), and nonradiative rate constants (knr) follow more or less linear correlation with the solvent polarity function Δf [={(D−1)/(2D+1)}−{(n2−1)/(2n2+1)}]. In nonpolar solvents, namely, hexane, cyclohexane, methylcyclohexane, 3-methylpentane, and decalin, however, all the above-mentioned properties show unusual deviation in comparison to the trend observed in moderate to higher polarity solvents. Thus, the Δν and knr values are unusually lower and the Φf, τf, and kf values are unusually higher in nonpolar solvents. Comparing the results of C1 with those of its lower analogue, namely, coumarin-120 (C120; 7-NH2-4-CH3-1,2-benzopyrone), it has ...

Melanin, a promising radioprotector: Mechanisms of actions in a mice model

The radioprotective effect of extracellular melanin, a naturally occurring pigment, isolated from the fungus Gliocephalotrichum simplex was examined in BALB/C mice, and the probable mechanism of action was established. At an effective dose of 50mg/kg body weight, melanin exhibited both prophylactic and mitigative activities, increasing the 30-day survival of mice by 100% and 60%, respectively, after exposure to radiation (7Gy, whole body irradiation (WBI)). The protective activity of melanin was primarily due to inhibition of radiation-induced hematopoietic damages as evidenced by improvement in spleen parameters such as index, total cellularity, endogenous colony forming units, and maintenance of circulatory white blood cells and platelet counts. Melanin also reversed the radiation-induced decrease in ERK phosphorylation in splenic tissue, which may be the key feature in its radioprotective action. Additionally, our results indicated that the sustained activation of AKT, JNK and P38 proteins in splenic tissue of melanin pre-treated group may also play a secondary role. This was also supported by the fact that melanin could prevent apoptosis in splenic tissue by decreasing BAX/Bcl-XL ratio, and increasing the expressions of the proliferation markers (PCNA and Cyclin D1), compared to the radiation control group. Melanin also reduced the oxidative stress in hepatic tissue and abrogated immune imbalance by reducing the production of pro-inflammatory cytokines (IL6 and TNFα). In conclusion, our results confirmed that fungal melanin is a very effective radioprotector against WBI and the probable mechanisms of radioprotection are due to modulation in pro-survival (ERK) signaling, prevention of oxidative stress and immunomodulation.

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.

Inhibitory property of the Piper betel phenolics against photosensitization-induced biological damages

The Piper betel phenolics, allylpyrocatechol (APC) and chavibetol (CHV), were found to protect photosensitization-mediated lipid peroxidation (LPO) of rat liver mitochondria effectively, APC being significantly more potent. The better activity of APC vis-à-vis CHV could be attributed to its higher reactivity with (1)O(2), as revealed from the rate constant values of (1)O(2) quenching by the respective phenolics. APC also prevented the detrimental effects of the Type II photosensitization-induced toxicity to mouse fibroblast L929 cells. The results suggest that APC may play an important role in protecting biological systems against damage, by eliminating (1)O(2) generated from certain endogenous photosensitizers.

DNA binding, photoactivated DNA cleavage and cytotoxic activity of Cu(II) and Co(II) based Schiff-base azo photosensitizers

A new class of Cu(II) and Co(II) complexes of azo-containing Schiff base of the type [Cu(L1)2] and [Co(L1)2], where L1=4-[(E)-{2-hydroxy-3-[(E)-(4-bromophenyl)diazenyl]benzylidene}amino]benzoic acid have been synthesized and characterized. Extension of conjugation and the presence of free carboxylic acid group of the ligand L1 increased the wavelength of the complexes from visible region to the near IR region (620-850 nm). The Cu(II) and Co(II) complexes interacted with CT-DNA via intercalative mode with the respective Kb value of 3.2×10(4) M(-1) and 2.9×10(4) M(-1) and acted as proficient photocleavers of SC pUC19 DNA in UV-A light, forming (1)O2 as the reactive oxygen species with the quantum yield of 0.38 and 0.36, respectively. Furthermore, the Cu(II) and Co(II) complexes showed photocytotoxicity toward two selected tumor cell lines MCF-7 and A549 by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) method, and the Cu(II) complex exhibits higher photocytotoxicity than Co(II) complex against each of the selected cell lines, this result is identical with their DNA binding ability order.