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Quercetin (3,3′,4′,5,7-pentahydroxyflavone), a ubiquitous, bioactive plant flavonoid, is known to possess anti-cancer, anti-tumor, and other important therapeutic activities of significant potency and low systemic toxicity. In this communication, we report for the first time a study on the interactions of quercetin with the plasma protein human serum albumin (HSA), exploiting the intrinsic fluorescence emission properties of quercetin as a probe. Quercetin is weakly fluorescent in aqueous buffer medium, with an emission maximum at ≈538 nm. Binding of quercetin with HSA leads to dramatic enhancement in the fluorescence emission intensity and anisotropy (r), along with significant changes in the fluorescence excitation and emission profiles. The excitation spectrum suggests occurrence of efficient Fo¨rster type resonance energy transfer (FRET) from the single tryptophan-214 residue of HSA to the protein bound quercetin. The emission, excitation, and anisotropy (r=0.18 at [HSA]=30 μM) data (using the native protein) along with emission studies of quercetin using partially denatured HSA (by 8 M urea) indicate that the quercetin molecules bind at a motionally restricted site near tryptophan-214 in the interdomain cleft region of HSA. Furthermore, the binding constant (K=1.9×105 M−1) and Gibbs free energy change (ΔG0=−30.12 kJ/mol)) for quercetin–HSA interaction have been calculated from the relevant anisotropy data. Implications of these results are examined, particularly in relation to prospective applications in biomedical research. [Copyright &y& Elsevier]

Abstract: Steady state and time resolved fluorescence spectroscopy, combined with molecular modeling computations, have been used to explore the interactions of two therapeutically important flavonoids, fisetin (3,7,3′,4′-OH-flavone) and 3-hydroxyflavone (3-HF), with normal human hemoglobin (HbA). Distinctive ‘two color’ fluorescence signatures and fairly high fluorescence anisotropy (r =0.12–0.28) of fisetin and 3-HF reveal their specific interactions with HbA. Binding constants estimated from the fluorescence studies were ≈4.00×104 M−1 and 9.83×103 M−1 for fisetin and 3-HF respectively. Specific interactions with HbA were further confirmed from flavonoid-induced static quenching of the protein tryptophan fluorescence as indicated by: (a) bimolecular quenching constant K q ≫diffusion controlled limit (b) closely matched values of Stern–Volmer quenching constant and binding constant (c) τo/τ≈1 (where τo and τ are the unquenched and quenched tryptophan fluorescence lifetimes respectively). Molecular docking and electrostatic surface potential calculations reveal contrasting binding modes of fisetin and 3-HF with HbA. [Copyright &y& Elsevier]

Abstract: Serotonin is a physiologically important biogenic amine, deficiency of which leads to mental disorders such as Alzheimer’s disease, schizophrenia, infantile autism, and depression. Both β-cyclodextrin (β-CD) and its chemically substituted synthetic varieties (often possessing enhanced aqueous solubility and improved drug complexing abilities) are finding wide applications as drug delivery vehicles. Here we have studied the encapsulation of serotonin in β-CD and succinyl-2-hydroxypropyl β-cyclodextrin (SHP-β-CD) by exploiting the intrinsic serotonin fluorescence. Enhanced fluorescence emission intensity (which increases by ∼18% and 34% in β-CD and SHPβ-CD respectively) and anisotropy (r) (r =0.075 and 0.1 in β-CD and SHPβ-CD respectively) are observed in presence of the cyclodextrins. From the fluorescence data host–guest interaction with 1:1 stoichiometry is evident, the association constants (K) being 126.06M−1 and 461.62M−1 for β-CD and SHPβ-CD respectively. Additionally, molecular docking and semiempirical calculations have been carried out which provide, for the first time, detailed insights regarding the encapsulation process. In particular, it is evident that the indole ring is inserted within the β-CD cavity with the aliphatic amine side chain protruding towards the primary rim of the β-CD cavity. Docking calculations reveal that hydrogen bonding interactions are involved in the formation of the inclusion complex. Semiempirical calculations indicate that formation of the 1:1 inclusion complex is energetically favorable which is consistent with the fluorescence data. [Copyright &y& Elsevier]

Abstract: Steady state and time resolved fluorescence spectroscopy have been used to probe microenvironments of the therapeutically active intrinsically fluorescent flavonoid, 7-hydroxyflavone (7-HF), in model membranes consisting of multilamellar phosphatidylcholine liposomes. Additionally, the antioxidant effects of 7-HF against lipid peroxidation have been evaluated using spectrophotometric assay. Large Stokes shifted emissions with distinct spectroscopic signatures, are observed from the excited state proton transfer (ESPT) tautomer (which is generated by a solvent mediated mechanism) and the ground state anion of 7-HF. The neutral (7-HFN) and anionic (7-HFA) species'' appear to be located in the non-polar acyl chain and the polar head group regions of the lipid vesicles respectively. The partition coefficients of 7-HFN and 7-HFA in these vesicles have also been estimated using their intrinsic fluorescence. Anisotropy (r) versus temperature (T) measurements reveal the utility of the tautomer fluorescence anisotropy as a sensitive parameter for exploring structural changes in the membranes. Fluorescence decay kinetics studies indicate heterogeneity in the microenvironments of both 7-HFN and 7-HFA. Furthermore, we demonstrate that lipid peroxidation of the model membranes is partially arrested upon 7-HF binding, suggesting its potential usefulness as an inhibitor of peroxidative damage of cell membranes. [Copyright &y& Elsevier]

Abstract: Fisetin (3,7,3′,4′-tetrahydroxyflavone) is a bioactive plant flavonoid of immense importance as a potentially useful therapeutic drug, for various free radical mediated as well as other diseases. In a recent paper, we demonstrated the novel uses of the exquisitely sensitive intrinsic fluorescence of this compound to explore its binding characteristics in liposomal membranes [B. Sengupta, A. Banerjee, P.K. Sengupta, Investigations on the binding and antioxidant properties of the plant flavonoid fisetin in model biomembranes, FEBS Lett. 570 (2004) 77–81]. Here, we have exploited this technique to examine its interactions with relevant macromolecular targets, namely double stranded DNA (from calf thymus), and the physiologically important circulatory protein, Human Serum Albumin (HSA). In the presence of DNA dramatic changes are observed in the intrinsic fluorescence behaviour of fisetin. These, along with other relevant supporting spectroscopic data, suggest that fisetin binds intercalatively between the base pairs of DNA. From the studies on fisetin–HSA interaction, the existence of two distinct binding sites are inferred. Furthermore we present evidence for the occurrence of efficient Förster type fluorescence resonance energy transfer from tryptophan to fisetin, indicating that both binding sites of fisetin in HSA are proximal to the unique tryptophan – 214 residue present in the interdomain (between IIA and IIIA domains) loop region of the protein. [Copyright &y& Elsevier]

Abstract: Steady state and time resolved fluorescence along with anisotropy and induced circular dichroism (ICD) spectroscopy provide useful tools to observe and understand the behavior of the therapeutically important plant flavonoids fisetin and daidzein in γ-cyclodextrin (γ-CDx) nanocavity. Benesi–Hildebrand plots indicated 1:1 stoichiometry for both the supramolecular complexes. However, the mode of the binding of fisetin significantly differs from daidzein in γ-CDx, as is observed from ICD spectra which is further confirmed by docking studies. The interaction with γ-CDx proceeds mainly by the phenyl ring and partly by the chromone ring of fisetin whereas only the phenyl ring takes part for daidzein. A linear increase in the aqueous solubility of the flavonoids is assessed from the increase in the binding of the flavonoids with the γ-CDx cavity, which are determined by the gradual increase in the ICD signal, fluorescence emission as well as increase in fluorescence anisotropy with increasing (γ-CDx). This confirms γ-CDx as a nanovehicle for the flavonoids fisetin and daidzein in improving their bioavailability. [Copyright &y& Elsevier]

Abstract: Plant flavonoids are emerging as potent therapeutic drugs effective against a wide range of free radical mediated diseases. Hence their interactions with cell membranes, which generally serve as targets for lipid peroxidation, are of enormous interest. Here we report in vitro studies, via absorption and fluorescence spectroscopy, on the effects of several flavonoids (namely fisetin, quercetin, chrysin, morin, and 3-hydroxyflavone, 3-HF) in goat RBC membranes. Owing to the presence of functionally relevant membrane protein components embedded in the lipid bilayer RBC ghosts provide a more realistic system for exploring drug actions in biomembranes than simpler membrane models like phosphatidylcholine liposomes used in our previous studies [e.g. B. Sengupta, A. Banerjee, P.K. Sengupta, FEBS Lett. 570 (2004) 77–81]. Here, we demonstrate that binding of the flavonoids to the RBC membranes significantly inhibits lipid peroxidation, and at the same time enhances their integrity against hypotonic lysis. Interestingly, the antioxidant and antihemolytic activities are found to be crucially dependent on the locations of the flavonoids in the membrane matrix as revealed by fluorescence studies. Furthermore, we observe that FRET (from membrane protein tryptophans to flavonoids) occurs with significant efficiency indicating that the flavonoid binding sites lie in close proximity to the tryptophan residues in the ghost membrane proteins. [Copyright &y& Elsevier]