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

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.     

Antioxidants and cataract

Abstract

The major causes for cataract formation are free radicals, and these free radicals are neutralized by the presence of endogenous antioxidants in the eye. Using xenobiotics, it has been confirmed that free radicals mediate the formation of cataract. Two cataract model-selenite model and the diabetic cataract model-have been developed to study the pathophysiology of cataract formation due to free radicals and the role of antioxidants during the process of cataractogenesis. This review focuses on natural compounds with antioxidant properties that could actually be applied as an interventional strategy on a large scale and are also relatively inexpensive. A brief overview of plants with antioxidant properties that in addition possess potential anti-cataract properties has been discussed. In addition to plants, three natural compounds (curcumin, vitamin C and vitamin E), on which a lot of data exist showing anti-cataract and antioxidant activities, have also been discussed. These antioxidants can be supplemented in the diet for a better defence against free radicals. Studies on vitamin C and vitamin E have proved that they are capable of preventing lipid peroxidation, thereby preventing the generation of free radicals, but their efficacy as anti-cataract agent is questionable. Unlike vitamins C and E, curcumin is well established as an anti-cataract agent, but the issue of curcumin bioavailability is yet to be addressed. Nanotechnology proves to be a promising area in increasing the curcumin bioavailability, but still a lot more research needs to be done before the use of curcumin as an effective anti-cataract agent for humans.     

Curcumin-induced inhibition of cellular reactive oxygen species generation: Novel therapeutic implications

There is evidence for increased levels of circulating reactive oxygen species (ROS) in diabetics, as indirectly inferred by the findings of increased lipid peroxidation and decreased antioxidant status. Direct measurements of intracellular generation of ROS using fluorescent dyes also demonstrate an association of oxidative stress with diabetes. Although phenolic compounds attenuate oxidative stress-related tissue damage, there are concerns over toxicity of synthetic phenolic antioxidants and this has considerably stimulated interest in investigating the role of natural phenolics in medicinal applications. Curcumin (the primary active principle in turmeric,Curcuma longa Linn.) has been claimed to represent a potential antioxidant and antiinflammatory agent with phytonutrient and bioprotective properties. However there are lack of molecular studies to demonstrate its cellular action and potential molecular targets. In this study the antioxidant effect of curcumin as a function of changes in cellular ROS generation was tested. Our results clearly demonstrate that curcumin abolished both phorbol-12 myristate-13 acetate (PMA) and thapsigargin-induced ROS generation in cells from control and diabetic subjects. The pattern of these ROS inhibitory effects as a function of dose-dependency suggests that curcumin mechanistically interferes with protein kinase C (PKC) and calcium regulation. Simultaneous measurements of ROS and Ca²⁺ influx suggest that a rise in cytosolic Ca²⁺ may be a trigger for increased ROS generation. We suggest that the antioxidant and antiangeogenic actions of curcumin, as a mechanism of inhibition of Ca²⁺ entry and PKC activity, should be further exploited to develop suitable and novel drugs for the treatment of diabetic retinopathy and other diabetic complications.     

Pro-oxidant, anti-oxidant and cleavage activities on DNA of curcumin and its derivatives demethoxycurcumin and bisdemethoxycurcumin.

Curcumin, a naturally occurring phytochemical responsible for the colour of turmeric shows a wide range of pharmacological properties including antioxidant, anti-inflammatory and anti-cancer effects. We have earlier shown that curcumin in the presence of Cu(II) causes strand cleavage in DNA through generation of reactive oxygen species, particularly the hydroxyl radical. Thus, curcumin shows both antioxidant as well as pro-oxidant effects. In order to understand the chemical basis of various biological properties of curcumin, we have studied the structure-activity relationship between curcumin and its two naturally occurring derivatives namely demethoxycurcumin (dmC) and bisdemethoxycurcumin (bdmC). Curcumin was found to be the most effective in the DNA cleavage reaction and a reducer of Cu(II) followed by dmC and bdmC. The rate of formation of hydroxyl radicals by the three curcuminoids also showed a similar pattern. The relative antioxidant activity was examined by studying the effect of these curcuminoids on cleavage of plasmid DNA by Fe(II)-EDTA system (hydroxyl radicals) and the generation of singlet oxygen by riboflavin. The results indicate that curcumin is considerably more active both as an antioxidant as well as an oxidative DNA cleaving agent. The DNA cleavage activity is the consequence of binding of Cu(II) to various sites on the curcumin molecule. Based on the present results, we propose three binding sites for Cu(II). Two of the sites are provided by the phenolic and methoxy groups on the two benzene rings and the third site is due to the presence of 1,3-diketone system between the rings. Furthermore, both the antioxidant as well as pro-oxidant effects of curcuminoids are determined by the same structural moieties.     

Curcumin and its synthetic analogue dimethoxycurcumin differentially modulates antioxidant status of normal human peripheral blood mononuclear cells

Curcumin is a polyphenol derived from the herb Curcuma longa, which has been extensively studied in terms of its antitumour, antioxidant, and chemopreventive activity as well as various other effects. In the present work we compared curcumin with its synthetic analogue dimethoxycurcumin (dimc) in terms of its antioxidant enzyme-modulating effects in human peripheral blood mononuclear cells (PBMC). We found that these compounds modulate antioxidant enzymes differentially. Both curcumin and dimethoxycurcumin effected a decrease in lipid peroxidation status in PBMC, however, curcumin had better activity in this regard. An increase in the activity of catalase was seen in the case of curcumin-treated PBMC, whereas dimc increased catalase activity significantly to almost twofold level. Real time-polymerase chain reaction (RT-PCR) analysis revealed significant up-regulation of catalase at mRNA level post treatment with curcumin as well as dimc, however, dimc had better activity in this regard. Glutathione reductase (GR) activity and reduced glutathione levels increased in the case of peripheral blood mononuclear cells (PBMC) treated with curcumin, however, the trend was reversed with dimethoxycurcumin where, both glutathione reductase activity and reduced glutathione levels were significantly reduced. RT-PCR analysis of glutathione reductase mRNA levels showed decrease in mRNA levels post treatment with dimethoxycurcumin (dimc) further corroborating GR enzyme assay results, however, we could not obtain significant result post curcumin treatment. NFkB reporter assay and western blot analysis of nuclear as well as cytosolic fractions of NFkB revealed that curcumin inhibits NFkB activation whereas inhibition was much less with dimc. It has been reported that curcumin and dimc exerts differential cytotoxicity in normal and tumour cells and the reason for this had been attributed to the differential uptake of these compounds by normal cells and tumour cells. Based on our results we propose that differential modulation of antioxidant enzymes via NFkB pathway could be the reason behind differential cytotoxicity of dimc as well as curcumin in normal cells and tumour cells in addition to differential uptake of these compounds as reported previously.     

Intracellular ROS protection efficiency and free radical-scavenging activity of curcumin

Curcumin has many pharmaceutical applications, many of which arise from its potent antioxidant properties. The present research examined the antioxidant activities of curcumin in polar solvents by a comparative study using ESR, reduction of ferric iron in aqueous medium and intracellular ROS/toxicity assays. ESR data indicated that the steric hindrance among adjacent big size groups within a galvinoxyl molecule limited the curcumin to scavenge galvinoxyl radicals effectively, while curcumin showed a powerful capacity for scavenging intracellular smaller oxidative molecules such as H₂O₂, HO^•, ROO^•. Cell viability and ROS assays demonstrated that curcumin was able to penetrate into the polar medium inside the cells and to protect them against the highly toxic and lethal effects of cumene hydroperoxide. Curcumin also showed good electron-transfer capability, with greater activity than trolox in aqueous solution. Curcumin can readily transfer electron or easily donate H-atom from two phenolic sites to scavenge free radicals. The excellent electron transfer capability of curcumin is because of its unique structure and different functional groups, including a β-diketone and several π electrons that have the capacity to conjugate between two phenyl rings. Therfore, since curcumin is inherently a lipophilic compound, because of its superb intracellular ROS scavenging activity, it can be used as an effective antioxidant for ROS protection within the polar cytoplasm.     

Trolox enhances curcumin's cytotoxicity through induction of oxidative stress

Curcumin, a natural polyphenol in the spice turmeric, has been found to exhibit anticancer activity. Although curcumin is generally considered an antioxidant, it is also able to elicit apoptosis through the generation of ROS, thereby functioning as a pro-oxidant in cancer cells. The present study investigated the effects of antioxidant pretreatment on curcumin-induced cytotoxicity in the human cancer cell lines A2780, MCF-7, and MDA-MB-231. Cytotoxicity was enhanced by trolox, vitamin C or vitamin E; trolox, a water soluble vitamin E derivative, was the most potent. The combination of curcumin (10 μM) and trolox (10-50 μM) induced apoptosis of cancer cells as evidenced by PARP cleavage and caspase-3 activation. Furthermore, expression of the pro-apoptotic protein Bad was up-regulated and expression of the anti-apoptotic proteins Bcl-2 and Bcl-xl was down-regulated in cells that had been treated with trolox plus curcumin. ROS generation was detected in curcumin-treated cells and was significantly enhanced when cells were treated with trolox plus curcumin. Exogenous catalase or SOD1 did not alter cytotoxicity, while over-expression of either catalase or SOD1 did, pointing to the importance of intracellular hydrogen peroxide generation in cell killing. In conclusion, we demonstrated for the first time that antioxidants such as trolox can potentiate cancer cell killing by curcumin, a finding which may help in the development of novel drug combination therapies.     

Differing features of proteins in membranes may result in antioxidant or prooxidant action: opposite effects on lipid peroxidation of alcohol dehydrogenase and albumin in liposomal systems

SUMMARY

The influence of 3 thiol-containing compounds, bovine serum albumin (fatty acid free: BSA), glutathione (GSH) and yeast alcohol dehydrogenase (YADH) on lipid peroxidation in multilamellar liposomes, prepared from ox-brain phospholipid, was investigated. Thiol-compounds were added either before liposome formation, or after liposome formation; and their effects compared to a positive control. Bovine serum albumin (BSA), an acidic hydrophilic protein, displays a small, concentration dependent, antioxidant effect when added to preformed liposomes. A much larger antioxidant effect was observed when the BSA was entrapped inside the liposome, by adding BSA just prior to liposome preparation. In contrast, a Zn²⁺ containing redox enzyme, YADH, a basic hydrophobic membrane-associating protein, displays a large pro-oxidant effect at much lower concentrations especially when entrapped inside the liposome. This was observed also with GSH; but per mole of -SH, YADH was about 18 times as powerful a pro-oxidant perhaps because of structural changes to the membrane. Oxidized glutathione and N-acetylcysteine were also pro-oxidant (cysteine and cystine showed little effect). Formation of thiyl radicals may occur in the presence of iron ions with these pro-oxidant sulphur-containing compounds. Partial protection against lipid peroxidation was observed with EDTA, desferrioxamine and protoporphyrin (IX), potent iron-chelating agents.     

Effects of cytoprotective antioxidants on lymphocytes from representative mitochondrial neurodegenerative diseases

Two new aza analogues of the neuroprotective agent idebenone have been synthesized and characterized. Their antioxidant activity, and ability to augment ATP levels have been evaluated in several different cell lines having suboptimal mitochondrial function. Both compounds were found to be good ROS scavengers, and to protect the cells from oxidative stress induced by glutathione depletion. The compounds were more effective than idebenone in neurodegenerative disease cells. These novel pyrimidinol derivatives were also shown to augment ATP levels in coenzyme Q₁₀-deficient human lymphocytes. The more lipophilic side chains attached to the pyrimidinol redox core in these compounds resulted in less inhibition of the electron transport chain and improved antioxidant activity.     

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

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 approximately 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 (*)N(3) 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 CH(2) group of the beta-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 -CH(2) 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.     

In Vitro Protective Effect and Antioxidant Mechanism of Resveratrol Induced by Dapsone Hydroxylamine in Human Cells

Dapsone (DDS) hydroxylamine metabolites cause oxidative stress- linked adverse effects in patients, such as methemoglobin formation and DNA damage. This study evaluated the ameliorating effect of the antioxidant resveratrol (RSV) on DDS hydroxylamine (DDS-NHOH) mediated toxicity in vitro using human erythrocytes and lymphocytes. The antioxidant mechanism was also studied using in-silico methods. In addition, RSV provided intracellular protection by inhibiting DNA damage in human lymphocytes induced by DDS-NHOH. However, whilst pretreatment with RSV (10–1000 μM significantly attenuated DDS-NHOH-induced methemoglobinemia, but it was not only significantly less effective than methylene blue (MET), but also post-treatment with RSV did not reverse methemoglobin formation, contrarily to that observed with MET. DDS-NHOH inhibited catalase (CAT) activity and reactive oxygen species (ROS) generation, but did not alter superoxide dismutase (SOD) activity in erythrocytes. Pretreatment with RSV did not alter these antioxidant enzymes activities in erythrocytes treated with DDS-NHOH. Theoretical calculations using density functional theory methods showed that DDS-NHOH has a pro-oxidant effect, whereas RSV and MET have antioxidant effect on ROS. The effect on methemoglobinemia reversion for MET was significantly higher than that of RSV. These data suggest that the pretreatment with resveratrol may decrease heme-iron oxidation and DNA damage through reduction of ROS generated in cells during DDS therapy.     

Oestrogenic compounds and oxidative stress (in human sperm and lymphocytes in the Comet assay)

Reactive oxygen species (ROS) are produced by a wide variety of chemicals and physiological processes in which enzymes catalyse the transfer of electrons from a substrate to molecular oxygen. The immediate products of such reactions, superoxide anion radicals and hydrogen peroxide can be metabolised by enzymes such as superoxide dismutase (SOD) and catalase (CAT), respectively, and depending on its concentration by Vitamin C (Vit C). Under certain circumstances the ROS form highly reactive hydroxyl radicals. We examined human sperm and lymphocytes after treatment with six oestrogenic compounds in the Comet assay, which measures DNA damage, and observed that all caused damage in both cell types. The damage was diminished in nearly all cases by catalase, and in some instances by SOD and Vit C. This response pattern was also seen with hydrogen peroxide. This similarity suggests that the oestrogen-mediated effects could be acting via the production of hydrogen peroxide since catalase always markedly reduced the response. The variable responses with SOD indicate a lesser involvement of superoxide anion radicals due to SOD-mediated conversion of superoxide to hydrogen peroxide generally causing a lower level of DNA damage than other ROS. The variable Vit C responses are explained by a reduction of hydrogen peroxide at low Vit C concentrations and a pro-oxidant activity at higher concentrations. Together these data provide evidence that inappropriate exposure to oestrogenic compounds could lead to free-radical mediated damage. It is believed that the observed activities were not generated by cell free cell culture conditions because increased responses were observed over and above control values when the compounds were added, and also increasing dose-response relationships have been found after treatment with such oestrogenic compounds in previously reported studies.     

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.     

Dietary zeolite supplementation reduces oxidative damage and plaque generation in the brain of an Alzheimer's disease mouse model

AimOxidative stress is considered one of the main events that lead to aging and neurodegeneration. Antioxidant treatments used to counteract oxidative damage have been associated with a wide variety of side effects or at the utmost to be ineffective. The aim of the present study was to investigate the antioxidant property of a natural mineral, the tribomechanically micronized zeolite (MZ).Main methodsCell death and oxidative stress were assessed in retinoic acid differentiated SH-SY5Y cells, a neuronal-like cell line, after a pro-oxidant stimulus. In vivo evaluation of antioxidant activity and amyloidogenic processing of beta amyloid have been evaluated in a transgenic model of aging related neurodegeneration, the APPswePS1dE9 transgenic mice (tg mice) after a five-month long period of water supplementation with MZ.Key findingsThe study showed that 24 h of cell pretreatment with MZ (1) protected the cells by radical oxygen species (ROS)-induced cell death and moreover (2) induced a reduction of the mitochondrial ROS production following a pro-oxidant stimulation. Looking for an antioxidant effect of MZ in vivo, we found (3) an increased activity of the endogenous antioxidant enzyme superoxide dismutase (SOD) in the hippocampus of tg mice and (4) a reduction in amyloid levels and plaque load in MZ treated tg mice compared to control tg mice.SignificanceOur results suggest MZ as a novel potential adjuvant in counteracting oxidative stress and plaque accumulation in the field of neurodegenerative diseases.     

Transport of liposomal and albumin loaded curcumin to living cells: an absorption and fluorescence spectroscopic study

Curcumin, a lipid soluble antioxidant, exhibits solvent and medium sensitive absorption and fluorescence properties. Using such changes, the average binding constants of curcumin to phosphatidylcholine (PC) liposomes and human serum albumin (HSA) were estimated to be 2.5 x 10(4) M(-1) and 6.1 x 10(4) M(-1) respectively. From the studies on temperature dependent fluorescence anisotropy of liposomal curcumin and its fluorescence quenching by acrylamide and iodide, it was concluded that curcumin is located in the gel phase of the liposomes. Similarly from the studies on quenching of tryptophan fluorescence in HSA by curcumin, it was found to be in the same domain as that of tryptophan. Both liposomal and HSA vehicles were examined for the transfer of curcumin to spleen lymphocyte cells, EL4 lymphoma cell line and compared with aqueous DMSO vehicles. From these studies it was found that liposomal vehicle is capable of loading more curcumin in to cells than HSA or aqueous-DMSO, and lymphoma cells show preferential uptake of curcumin to lymphocytes. The fluorescence of curcumin in EL4 lymphoma cells was found to be significantly higher as compared to the lymphocytes. The present study demonstrates a simple and quantitative method of estimation of curcumin delivered to cells by different vehicles using absorption and fluorescence spectroscopy.     

Curcumin induces concentration-dependent alterations in mitochondrial function through ROS in C2C12 mouse myoblasts

Curcumin exhibits antioxidant properties in normal cells where the uptake is low, unlike in tumor cells where uptake is high and curcumin increases reactive oxygen species (ROS) production and cell death. Mitochondria are the main source and primary target of cellular ROS. We hypothesized that curcumin would regulate cellular redox status and mitochondrial function, depending on cell sensitivity and/or curcumin concentration in normal cells. We examined the differences between low and high concentrations of curcumin, with specific attention focused on ROS levels, mitochondrial function, and cell viability in mouse C2C12 myoblast under normal and simulated conditions of diabetes. Cells incubated with high concentrations of curcumin (10–50 μM) resulted in decreased cell viability and sustained robust increases in ROS levels. Mechanistic studies showed that increased ROS levels in cells incubated with 20 μM curcumin induced opening of mitochondrial permeability transition pores and subsequent release of cytochrome c, activation of caspases 9 and 3/7, and apoptotic cell death. Low concentrations of curcumin (1–5 μM) did not affect cell viability, but induced a mild increase in ROS levels, which peaked at 2 hr after the treatment. Incubation with 5 μM curcumin also induced ROS-dependent increases in mitochondrial mass and membrane potential. Finally, pretreatment with 5 μM curcumin prevented high glucose-induced oxidative cell injury. Our study suggests that mitochondria respond differentially depending on curcumin concentration-dependent induction of ROS. The end result is either cell protection or death. Curcumin may be an effective therapeutic target for diabetes and other mitochondrial diseases when used in low concentrations.     

Potential antitumour and pro-oxidative effects of (E)-methyl 2-(7-chloroquinolin-4-ylthio)-3-(4-hydroxyphenyl) acrylate (QNACR)

Context: Characterization of the pro-oxidant activity of QNACR.

Objectives: Reactive oxygen species (ROS) induce cellular damage and represent unique opportunities to kill malignant cells. In this study, we synthesized and evaluated the new compound, (E)-methyl 2-(7-chloroquinolin-4-ylthio)-3-(4-hydroxyphenyl) acrylate (QNACR) as potential pro-oxidative agent against breast cancer.

Methods: Oxidative stress biomarkers such as ROS, thiobarbuturic acid reactive species (TBARs) and different antioxidant enzyme activities were determined in cell lysates.

Results: QNACR showed cytotoxic and more selective effects to tumour MCF7 cells (IC₅₀ < 25 µM) compared to antitumour controls, inducing ROS and TBARs parallel to inhibitions of catalase (CAT), glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH). Longer exposures to QNACR triggered adaptive effects increasing the overall activities of CAT, glutathione reductase, G6PDH and 6PGDH, but eventually the adaptation changes faded and cells died.

Conclusion: QNACR led to remarkable modifications in the oxidative status of tumour cells, proposing this compound as potential alternative for antitumour therapy.     

Role of oxidative stress in the antitumoral action of a new vanadyl(IV) complex with the flavonoid chrysin in two osteoblast cell lines: relationship with the radical scavenger activity

The new complex [VO(chrysin)₂EtOH]₂ (VOchrys) has been synthesized and thoroughly characterized. Fourier transform IR, UV–vis, diffuse reflectance, and EPR spectroscopies as well as elemental analysis and thermal measurements were performed. In solution, different species could be detected by EPR spectroscopy as a function of the ligand-to-metal ratio. The stoichiometry of the chelate complex formed at pH 5 was also determined by spectrophotometric titrations. Since flavonoids are natural antioxidant compounds, the antioxidant capacity of chrysin and its vanadyl(IV) complex was investigated using different radicals. Chrysin and its complex were not able to diminish the level of superoxide and 1,1-diphenyl-2-picrylhydrazyl radicals to a great extent. In contrast, they were strong scavengers for 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt radical cations and OH· radicals with a greater potency for VOchrys. Taking into account their selective antioxidant properties, we investigated the bioactivity of these compounds in two osteoblast-like cells in culture. Chrysin and VOchrys caused an inhibition of cell proliferation in MC3T3E1 normal osteoblasts and UMR106 tumor cells in a dose-response manner, with a greater effect in the latter cell line. The generation of reactive oxygen species (ROS) was evaluated in both cell lines and a correlation could be established between the antiproliferative effects of chrysin and the increase in the ROS levels. The complex did not generate types of ROS that can be detected by the dihydrorhodamine 123 technique so the antiproliferative effect may be attributed to the formation of other radicals such as superoxide, which is not detected by this probe. The morphological alterations were in agreement with these changes.     

Differential apoptotic and redox regulatory activities of curcumin and its derivatives

We have synthesized different bioconjugates of curcumin, which were tested for their pro- and antioxidant properties. In the present study five representative derivatives of curcumin, i.e., 4,4'-di-(O-acetyl) curcumin, 4,4'-di-(O-glycinoyl) curcumin, 4,4'-di-(O-glycinoyl-di-N-piperoyl) curcumin, 4,4'-di-(O-piperoyl) curcumin, and 4,4'-(O,O-cystinoyl)-3,3'-dimethoxydiphenyl-1,6-heptadiene-3,5-dione, were used for testing their apoptotic potential on tumor cells. Dipiperoyl and diglycinoyl derivatives showed higher apoptotic activity at lower concentrations, whereas diacetyl curcumin had slightly lower apoptotic activity on tumor cells. On the other hand, diglycinoyl-dipiperoyl and cystinoyl heptadiene derivatives had lost their apoptotic potential significantly. The apoptotic activity of these derivatives correlated very well with the generation of ROS by the tumor cells, whereas GSH levels remained unaltered. Our studies also indicate downregulation of Bcl-2 and participation of caspase-3 in the apoptotic death of tumor cells.