Curcumin derivatives and Aß-fibrillar aggregates: An interactions' study for diagnostic/therapeutic purposes in neurodegenerative diseases.

Several neurodegenerative diseases, like Alzheimer's (AD), are characterized by amyloid fibrillar deposition of misfolded proteins, and this feature can be exploited for both diagnosis and therapy design. In this paper, structural modifications of curcumin scaffold were examined in order to improve its bioavailability and stability in physiological conditions, as well as its ability to interfere with ß-amyloid fibrils and aggregates. The acid-base behaviour of curcumin derivatives, their pharmacokinetic stability in physiological conditions, and in vitro ability to interfere with Aß fibrils at different incubation time were investigated. The mechanisms governing these phenomena have been studied at atomic level by means of molecular docking and dynamic simulations. Finally, biological activity of selected curcuminoids has been investigated in vitro to evaluate their safety and efficiency in oxidative stress protection on hippocampal HT-22 mouse cells. Two aromatic rings, π-conjugated structure and H-donor/acceptor substituents on the aromatic rings showed to be the sine qua non structural features to provide interaction and disaggregation activity even at very low incubation time (2h). Computational simulations proved that upon binding the ligands modify the conformational dynamics and/or interact with the amyloidogenic region of the protofibril facilitating disaggregation. Significantly, in vitro results on hippocampal cells pointed out protection against glutamate toxicity and safety when administered at low concentrations (1 µM). On the overall, in view of its higher stability in physiological conditions with respect to curcumin, of his rapid binding to fibrillar aggregates and strong depolymerizing activity, phtalimmide derivative K2F21 appeared a good candidate for both AD diagnostic and therapeutic purposes.

Excited state dynamics of bis-dehydroxycurcumin tert-butyl ester, a diketo-shifted derivative of the photosensitizer curcumin.

Bis-dehydroxycurcumin tert-butyl ester (K2T23) is a derivative of the natural spice curcumin. Curcumin is widely studied for its multiple therapeutic properties, including photosensitized cytotoxicity. However, the full exploitation of curcumin phototoxic potential is hindered by the extreme instability of its excited state, caused by very efficient non radiative decay by means of transfer of the enolic proton to the nearby keto oxygen. K2T23 is designed to exhibit a tautomeric equilibrium shifted toward the diketo conformers with respect to natural curcumin. This property should endow K2T23 with superior excited-state stability when excited in the UVB band, i.e., in correspondence of the diketo conformers absorption peaks, making this compound an interesting candidate for topical photodynamic therapy of, e.g., skin tumors or oral infections. In this work, the tautomeric equilibrium of K2T23 between the keto-enolic and diketo conformers is assessed in the ground state in several organic solvents by UV-visible absorption and by nuclear magnetic resonance. The same tautomeric equilibrium is also probed in the excited-state in the same environments by means of steady-state fluorescence and time-correlated single-photon counting measurements. These techniques are also exploited to elucidate the excited state dynamics and excited-state deactivation pathways of K2T23, which are compared to those determined for several other curcuminoids characterized in previous works of ours. The ability of K2T23 in photosensitizing the production of singlet oxygen is compared with that of curcumin.

In vitro study on potential pharmacological activity of curcumin analogues and their copper complexes.

Curcumin and its derivatives have attracted great interest in the prevention and treatment of Alzheimer's disease, thanks both to the ability to hinder the formation of amyloid-beta (Aß) aggregates and the ability to bind Cu (II) ion. In this article, we explore the ability of curcumin derivatives of K2T series to affect amyloid Aß1-40 aggregation. These derivatives were obtained by introducing the t-butyl ester group through a methylenic spacer on the central carbon atom of the ß-diketo moiety of curcumin frame. The studied curcuminoids were demonstrated to inhibit Aß1-40 fibrillization at substoichiometric concentrations with IC50 value near that of curcumin. In addition, the antioxidant properties and DNA interaction of their Cu(II) complexes is evaluated. The structure of Cu(II)-K2T31 complex is also proposed on the basis of DFT calculation.

SiO2-CaO-P2O5 Bioactive Glasses: A Promising Curcuminoids Delivery System.

In this paper, we report the study of the loading and the release of curcuminoids by bioactive glasses (BG) and mesoporous bioactive glasses (MBG). Through a detailed spectroscopic study, it was possible to determine the amount and the type of molecules released in water and in simulated body fluid (SBF). In particular, curcumin and K2T21 show a good ability to be released in di-keto and keto-enolic form, depending from the pH. However, after 24 h, the amount of pristine curcumin release is very low with a consequent increment of degradation products derived by curcuminoids. The presence of -OH groups on curcuminoids is a fundamental pre-requisite in order to obtain a high loading and release in polar solution such as water and SBF. The substrate on which we loaded the drugs does not seem to affect significantly the loading and the release of the drugs. The environment, instead, affects the release: for all the drugs, the release in SBF, buffered at pH of 7.4, is slightly worse than the release in water (basic pH values).

Antibacterial phototoxic effects of synthetic asymmetric and glycosylated curcuminoids in aqueous formulations: studies on curcumin and curcuminoids. LIV.

The aim of this study was to evaluate the in vitro phototoxic potential of synthetic asymmetric and glycosylated curcuminoids on planktonic model bacteria by counting the colony forming units. The Gram-positive Enterococcus faecalis and the Gram-negative Escherichia coli were exposed to aqueous solutions of the curcuminoids (⩽2.5 µM) in the presence or absence of selected pharmaceutical excipients (Pluronic F127, PEG 400 and HPγCD) in combination with a low irradiation dose (5 J/cm(2); λmax: 450 nm) of constant irradiance and time. All the asymmetric curcuminoids, but only one of the glycosylated curcuminoids demonstrated substantial phototoxic effect on E.faecalis (⩾4.7 log reduction). Only two of the asymmetric curcuminoids showed a moderate to low phototoxic effect on the more persistent E.coli. This study emphasized that aromatic hydroxyl substituents in the para-position are important to maintain the phototoxic potential of curcuminoids independent of molecular symmetry. Glycosylation of the aromatic substituents resulted in a substantial loss in phototoxicity towards planktonic bacteria, an apparent change in the non-radiative S1-decay process and a weaker interaction with Pluronic F127 compared to the non-glycosylated curcuminoids. The selected excipients Pluronic F127, PEG 400 and HPγCD strongly influenced the phototoxic potential of the unsymmetrical, non-glycosylated compounds.

Curcumin derivatives as metal-chelating agents with potential multifunctional activity for pharmaceutical applications.

Curcuminoids represent new perspectives for the development of novel therapeutics for Alzheimer's disease (AD), one probable mechanism of action is related to their metal complexing ability. In this work we examined the metal complexing ability of substituted curcuminoids to propose new chelating molecules with biological properties comparable with curcumin but with improved stability as new potential AD therapeutic agents. The K2T derivatives originate from the insertion of a -CH2COOC(CH3)3 group on the central atom of the diketonic moiety of curcumin. They retain the diketo-ketoenol tautomerism which is solvent dependent. In aqueous solution the prevalent form is the diketo one but the addition of metal ion (Ga(3+), Cu(2+)) causes the dissociation of the enolic proton creating chelate complexes and shifting the tautomeric equilibrium towards the keto-enol form. The formation of metal complexes is followed by both NMR and UV-vis spectroscopy. The density functional theory (DFT) calculations on K2T21 complexes with Ga(3+) and Cu(2+) are performed and compared with those on curcumin complexes. [Ga(K2T21)2(H2O)2](+) was found more stable than curcumin one. Good agreement is detected between calculated and experimental (1)H and (13)C NMR data. The calculated OH bond dissociation energy (BDE) and the OH proton dissociation enthalpy (PDE), allowed to predict the radical scavenging ability of the metal ion complexed with K2T21, while the calculated electronic affinity (EA) and ionization potential (IP) represent yardsticks of antioxidant properties. Eventually theoretical calculations suggest that the proton-transfer-associated superoxide-scavenging activity is enhanced after binding metal ions, and that Ga(3+) complexes display possible superoxide dismutase (SOD)-like activity.

Synthesis and characterization of (68)Ga-labeled curcumin and curcuminoid complexes as potential radiotracers for imaging of cancer and Alzheimer's disease.

Curcumin (CUR) and curcuminoids complexes labeled with fluorine-18 or technetium-99m have recently shown their potential as diagnostic tools for Alzheimer's disease. Gallium-68 is a positron-emitting, generator-produced radionuclide, and its properties can be exploited in situ in medical facilities without a cyclotron. Moreover, CUR showed a higher uptake in tumor cells compared to normal cells, suggesting potential diagnostic applications in this field. In spite of this, no studies using labeled CUR have been performed in this direction, so far. Herein, (68)Ga-labeled complexes with CUR and two curcuminoids, namely diacetyl-curcumin (DAC) and bis(dehydroxy)curcumin (bDHC), were synthesized and characterized by means of experimental and theoretical approaches. Moreover, a first evaluation of their affinity to synthetic ß-amyloid fibrils and uptake by A549 lung cancer cells was performed to show the potential application of these new labeled curcuminoids in these diagnostic fields. The radiotracers were prepared by reacting (68)Ga(3+) obtained from a (68)Ge/(68)Ga generator with 1 mg/mL curcuminoids solutions. Reaction parameters (precursor amount, reaction temperature, and pH) were optimized to obtain high and reproducible radiochemical yield and purity. Stoichiometry and formation of the curcuminoid complexes were investigated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, NMR, ultraviolet-visible, and fluorescence spectroscopy on the equivalent (nat)Ga-curcuminoids (nat = natural) complexes, and their structure was computed by theoretical density functional theory calculations. The analyses evidenced that CUR, DAC, and bDHC were predominantly in the keto-enol form and attested to Ga(L)2(+) species formation. Identity of the (68)Ga(L)2(+) complexes was confirmed by coelution with the equivalent (nat)Ga(L)2(+) complexes in ultrahigh-performance liquid chromatography analyses.(68)Ga(CUR)2(+), (68)Ga(DAC)2(+), and (68)Ga(bDHC)2(+) were highly (87 ± 4, 90 ± 1%) and moderately (48 ± 2%), respectively, retained by synthetic ß-amyloid fibrils in vitro. All the Ga-curcuminoid complexes showed an uptake in A549 lung cancer cells, at least equivalent to the respective free curcuminoids, confirming potential applications as cancer-detecting radiotracers.

Metal binding ability of curcumin derivatives: a theoretical vs. experimental approach.

Theoretical calculations employing DFT at the B3LYP/6-311G++** level are used to investigate the tautomeric equilibrium in curcumin derivatives. The solvent effect is evaluated using the CPCM continuum solvation method. The results are compared with experimental data obtained from the X-ray crystal structure of K2A23 and UV-vis data. The KE tautomer is more stable in a vacuum and in the solid state, while in water the DK tautomer reaches a population of 90%. In agreement with spectroscopic data, theoretical calculations predict a slight prevalence of the DK form in non-aqueous solvent systems. The ability to chelate metal ions [Fe(3+), Ga(3+) and Cu(2+)] is then explored by means of (1)H, (13)C NMR and UV-Vis spectroscopy. From the calculation of the overall stability constants of metal complexes and (1)H NMR titrations with Ga(3+), it is clear that the more stable species has a 1 : 2 M/L molar ratio. The curcuminoid coordinates the metal ion through the keto-enol function in the dissociated form; in addition 2D (1)H (13)C NMR experiments suggest the involvement of carboxylic oxygen in metal coordination it was found in the solid state for the complex [Ga(K2A33)2]PF6. The rate of the complexation reaction is strongly influenced by the type of substituent on the aromatic ring of the curcuminoid (K2A33 ≈ K2A23 ≫ K2A21). In addition DPPH assay evidences how antioxidant ability of curcumin derivatives is mainly due to the presence of a phenolic group and metal coordination by a keto-enolic moiety does not affect it, especially for K2A21.

bis-Dehydroxy-Curcumin triggers mitochondrial-associated cell death in human colon cancer cells through ER-stress induced autophagy.

BACKGROUND: The activation of autophagy has been extensively described as a pro-survival strategy, which helps to keep cells alive following deprivation of nutrients/growth factors and other stressful cellular conditions. In addition to cytoprotective effects, autophagy can accompany cell death. Autophagic vacuoles can be observed before or during cell death, but the role of autophagy in the death process is still controversial. A complex interplay between autophagy and apoptosis has come to light, taking into account that numerous genes, such as p53 and Bcl-2 family members, are shared between these two pathways. METHODOLOGY/PRINCIPAL FINDINGS: In this study we showed a potent and irreversible cytotoxic activity of the stable Curcumin derivative bis-DeHydroxyCurcumin (bDHC) on human colon cancer cells, but not on human normal cells. Autophagy is elicited by bDHC before cell death as demonstrated by increased autophagosome formation -measured by electron microscopy, fluorescent LC3 puncta and LC3 lipidation- and autophagic flux -measured by interfering LC3-II turnover. The accumulation of poly-ubiquitinated proteins and ER-stress occurred upstream of autophagy induction and resulted in cell death. Cell cycle and Western blot analyses highlighted the activation of a mitochondrial-dependent apoptosis, which involves caspase 7, 8, 9 and Cytochrome C release. Using pharmacological inhibitions and RNAi experiments, we showed that ER-stress induced autophagy has a major role in triggering bDHC-cell death. CONCLUSION/SIGNIFICANCE: Our findings describe the mechanism through which bDHC promotes tumor selective inhibition of proliferation, providing unequivocal evidence of the role of autophagy in contrasting the proliferation of colon cancer cells.

Excited-state dynamics of bis-dehydroxycurcumin carboxylic acid, a water-soluble derivative of the photosensitizer curcumin.

Bis-dehydroxycurcumin carboxylic acid (K2A23) is a synthetic curcuminoid designed to exhibit enhanced water solubility and photosensitizing potential with respect to natural curcumin. In this work, the tendency of the compound to form intra- and intermolecular hydrogen bonds in the ground state is studied by UV-visible absorption and by nuclear magnetic resonance (NMR). The excited-state dynamics of the drug are probed in different environments by means of time-correlated single-photon counting measurements and related to its hydrogen bonding affinity in the excited state.

Newly synthesized curcumin derivatives: crosstalk between chemico-physical properties and biological activity.

New curcumin analogues (ester and acid series) were synthesized with the aim to improve the chemical stability in physiological conditions and potential anticancer activity. Cytotoxicity against different tumorigenic cell lines (human ovarian carcinoma cells -2008, A2780, C13*, and A2780/CP, and human colon carcinoma cells HCT116 and LoVo) was tested to evaluate cellular specificity and activity. Physico-chemical properties such as acidity, lipophilicity, kinetic stability, and free radical scavenging activity were investigated to shed light on the structure-activity relationship and provide new attractive candidates for drug development. Most of ester derivatives show IC(50) values lower than curcumin and exhibit selectivity against colon carcinoma cells. Especially they are extremely active after 24 h exposure showing enhanced inhibitory effect on cell viability. The best performances of ester curcuminoids could be ascribed to their high lipophilicity that favors a greater and faster cellular uptake overcoming their apparently higher instability in physiological condition.

New synthetic glucosyl-curcuminoids, and their (1)H and (13)C NMR characterization, from Curcuma longa L.

Turmeric extracts, among which curcumin and bis-demethoxycurcumin, are by far known for their therapeutic activities. In this study we propose easy and low cost synthetic pathways in order to obtain glucosyl-curcuminoids, safe and water soluble potential drugs and dyes, which may be implied in different fields ranging from pharmacology to food chemistry. The complete (1)H and (13)C NMR characterization of naturally occurring curcumin, bis-demethoxycurcumin and new synthetic glucosyl-curcuminoids is reported.

Synthesis, cytotoxic and combined cDDP activity of new stable curcumin derivatives.

New curcumin derivatives are synthesized in order to improve chemical properties of curcumin. The aromatic ring glycosylation of curcumin provides more water-soluble compounds with a greater kinetic stability which is a fundamental feature for drug bioavailability. The glycosylation reaction is quite simple, low cost, with high yield and minimum waste. NMR data show that the ability of curcumin to coordinate metal ion, in particular Ga(III), is maintained in the synthesized products. Although the binding of glucose to curcumin reduces the cytotoxicity of the derivatives towards cisplatin (cDDP)-sensitive and -resistant human ovarian carcinoma cell lines, the compounds display a good selectivity since they are much less toxic against non-tumourigenic Vero cells. The combination of cDDP with the most active glycosyl-curcuminoid drug against both cDDP-sensitive and -resistant as well as against Vero cell lines is tested. The results show an improvement of cDDP efficacy with higher selectivity towards cancer cells than non-cancer cells. These studies indicate the need for developing new valid components of drug treatment protocols to cDDP-resistant cells as well.

Synthesis and characterization of glucosyl-curcuminoids as Fe3+ suppliers in the treatment of iron deficiency.

The Fe(3+) chelating ability of some curcumin glucosyl derivatives (Glc-H; Glc-OH; Glc-OCH(3)) is tested by means of UV and NMR study. The pK(a) values of the ligands and the overall stability constants of Fe(3+) and Ga(3+) complexes are evaluated from UV spectra. The only metal binding site of the ligand is the beta-diketo moiety in the keto-enolic form; the glucosyl moiety does not interact with metal ion but it contributes to the stability of metal/ligand 1:2 complexes by means of hydrophilic interactions. These glucosyl derivatives are able to bind Fe(3+) in a wide pH rage, forming complex species thermodynamically more stable than those of other ligands commonly used in the treatment of iron deficiency. In addition they demonstrate to have a poor affinity for competitive biological metal ions such as Ca(2+). All ligands and their iron complexes have a good lypophilicity (log P > -0.7) suggesting an efficient gastrointestinal absorption in view of their possible use as iron supplements in oral therapy. The ligand molecules are also tested for their antioxidant properties in "ex vivo" biological system.

Synthesis, chemical and biological studies on new Fe(3+)-glycosilated beta-diketo complexes for the treatment of iron deficiency.

A simple synthetic pathway to obtain glycosilated beta-diketo derivatives is proposed. These compounds show a good iron(III) affinity therefore we may suggest the use of their Fe(3+)-complexes as oral iron supplements in the treatment of anaemia. The glycosilated compounds (6-GlcH, 6-GlcOH and 6-GlcOCH(3)) are characterized by means of spectroscopic (UV, (1)H and (13)C NMR) and potentiometric techniques; they have a good water solubility, are kinetically stable in physiological condition (t(1/2)>100h) and show a low cytotoxicity also in high concentrations (IC(50)>400 microM). They are able to bind Fe(3+) ion in acid condition (pH approximately 2) forming complex species thermodynamically more stable than those of other ligands commonly used in the treatment of iron deficiency. The iron complexes show also a good kinetic stability both in acidic and physiological pH and have a good lypophilicity (logP>-0.7) that suggests an efficient gastrointestinal absorption in view of their possible use in oral therapy. In addition they demonstrate a poor affinity for competitive biological metal ion such as Ca(2+), and in particular 6-GlcOCH(3) is able to inhibit lipid peroxidation.

TG-DTA, DRIFT and NMR characterisation of humic-like fractions from olive wastes and amended soil.

The purpose of the present study is to investigate, by means of thermogravimetric analysis (TG) and differential thermal analysis (DTA), diffuse reflectance infrared Fourier transform (DRIFT) and 2D nuclear magnetic resonance (NMR) spectroscopies, the structural features of the humic-like fraction (HLF) from olive pulp (OP), its effluents originated from the fermentation processes for hydrogen (EH2) and methane production (ECH4) and humic acid (HA) from soil amended with each of these materials. A considerable structural modification emerged between the HLF, in particular from the ECH4 effluent, which was characterised by a high content of polyphenolic and polypeptidic substances. The short-term amendment trial with OP and EH2 indicated that no chemical or structural changes in soil HA appeared. In contrast, the amendment with ECH4 substantially influenced the chemical and structural composition of soil HA. The structural interpretation performed by 2D NMR indicated the presence of aliphatic and aromatic protons while the sugar-like content and O-CH3 groups decreased with respect to the soil control HA. It emerges from this study that olive wastes contain stabilised humic-like material that may be recycled as an amendment in areas where olive trees are cultivated.

Synthesis and characterization of new beta-diketo derivatives with iron chelating ability.

Here we report the synthesis, the characterization and a theoretical study on new glycosylated phenyl substituted beta-diketones; two classes of compounds are obtained according to the condensation reaction: central and side derivatives. Their iron(III) chelating ability is tested by means of UV-visible (UV-vis), potentiometric and NMR techniques. The conformation of central derivatives does not allow any metal chelation, while side derivatives bind iron(III) through the beta-dioxo moiety. The glycosyl moiety does not interact with metal ion but it helps to stabilize metal/ligand (1/3) complexes by means of hydrophylic interactions. The pK(a) of the ligands and the stability constants of their Fe(III) complexes are evaluated by means of UV-vis spectroscopy and potentiometry. A comparison with other iron-chelating agents, on the basis of lipophilicity and the pFe(III), is finally reported.

Co-ordination of transition metal ions by galactaric acid: a potentiometric and spectroscopic study.

A solution study on the ability of galactaric acid [GalaH(2), HOOC(CH)(4)COOH] in the complexation of biological metal ions such as Co(II) and Ni(II) and toxic metal ions such as Cd(II), Pb(II) and Hg(II), is reported. The stability constants of the complex species are determined by means of potentiometric measurements. Galactaric acid behaves as chelate ligand through carboxylic oxygen and alpha-hydroxy group towards Co(II) and Ni(II), while in the Pb(II) and Cd(II) containing system it co-ordinates the metal ion with carboxylic oxygen and two alcoholic hydroxy groups. The prevailing species at acidic or neutral pH is [MGala] which is also isolated in the solid state and characterized by means of IR spectroscopy. On increasing pH, the [MGalaH(-1)](-) species is also formed where the co-ordinated OH group undergoes deprotonation in all metal ion complexes except those with Hg(II), where the co-ordination of hydroxide ion is suggested as the precipitation of the metal hydroxide occurs at pH 7.

Removal of cadmium ion by means of synthetic hydroxyapatite.

The reaction behaviour of synthetic hydroxyapatite [Ca10(PO4)6(OH)2] (HAP) toward cadmium ion was investigated for the Cd/Ca molar ratio in the range 1-0.005, by means of ions, pH measurements and XRD, SEM, IR techniques. The reaction behaviour between HAP and cadmium ion could be explained by a formation of an amorphous phase and/or a sorption mechanism.

Binding ability of sialic acid towards biological and toxic metal ions. NMR, potentiometric and spectroscopic study.

The binary complexes of 5-amino-3,5-dideoxy-D-glycero-D-galactononulosic acid (NANA), commonly called N-acetyl neuraminic acid, formed with biological metal ions such as Co(II) and Cu(II) and toxic metal ions such as Cd(II) and Pb(II) were investigated in aqueous solution by means of potentiometry, UV and NMR spectroscopy. The corresponding ternary systems with 2,2'-bipyridine were studied in aqueous solution by potentiometry and UV spectroscopy. NANA co-ordinates all metal ions, in both binary and ternary systems through the carboxylic group (protonated or deprotonated according to pH), pyranosidic ring oxygen and glycerol chain alcoholic hydroxy groups. The prevailing species in the pH range 2-7 are of [M(NANA)(2)] type, and their stability constants are greater than those of simple carboxylate complexes. Above pH 7, the species [M(NANA)(2)OH](-) are also formed, but they do not prevent the precipitation of metal hydroxides. This work provides information on the solution state chemistry of NANA in the presence of bivalent metal ions; its great affinity for the toxic metals Cd(II) and Pb(II), near physiological conditions, and the relatively high stability of the complex species found may also account for the mechanism of toxicity.