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Plant flavonoids are emerging as novel therapeutic drugs for free radical mediated diseases, for which cell membranes mainly serve as targets for lipid peroxidation and related deleterious effects. Screening and characterization of these ubiquitous, therapeutically potent polyphenolic compounds require a clear understanding regarding their binding and possible locations in membranes, as well as quantitative estimates of relevant parameters such as partition coefficients, antioxidant and radical scavenging capacities. In this article we present perspectives emphasizing novel uses of the exquisitely sensitive 'two color' intrinsic fluorescence of plant flavonoids (which arise due to highly efficient photoinduced excited state intramolecular proton transfer (ESIPT) reactions) to explore their binding to model biomembranes consisting of phosphatidylcholine liposomes. Extension of such studies to natural biomembranes of relevant interest is also exemplified. Spectrophotometric assays reveal that typical mono- as well as poly-hydroxy substituted flavonoids have remarkable inhibitory actions on lipid peroxidation, and are significantly more potent antioxidants (2.5-4 times higher) compared to the reference compound Trolox (an water soluble derivative of vitamin E). The structure-activity relationships emerging from such studies are consistent with theoretical predictions based on quantum chemical computations., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

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.

Excited-state intramolecular proton transfer (ESIPT) and dual luminescence behaviour of 3-hydroxyflavone (3-HF) have been utilized to monitor its binding to liposomal membranes prepared from egg yolk phosphatydilcholine (EYPC). Additionally, absorption spectrophotometric assay has been performed to evaluate the antioxidant activity of 3-HF against lipid peroxidation in this membrane system. When 3-HF molecules are partitioned into EYPC liposomes, a weak long-wavelength absorption band with lambda(abs)(max) approximately 410 nm appears in addition to the principal absorption at approximately lambda(abs)(max) = 345 nm. Selective excitation of the 410 nm band produces the characteristic emission (lambda(em)(max) approximately 460 nm) of the ground-state anionic species, whereas excitation at the higher energy absorption band leads to dual emission with predominatly ESIPT tautomer fluorescence (lambda(em)(max) = 528 nm). Both ESIPT tautomer and the anionic species exhibit fairly high fluorescence anisotropy (r) values (r = 0.122 and 0.180, respectively). Biexponential fluorescence decay kinetics are observed for the ESIPT tautomer as well as the ground-state anionic forms, indicating heterogeneity in the microenvironments of the corresponding emitting species. Furthermore, we demonstrate that lipid peroxidation of EYPC liposomes is significantly inhibited upon 3-HF binding, suggesting that 3-HF can be potentially useful as an inhibitor of peroxidative damage of cell membranes., ((c) 2008 John Wiley & Sons, Ltd.)

Plant flavonoids are emerging as potent therapeutic drugs for free radical mediated diseases, for which cell membranes generally serve as targets for lipid peroxidation and related deleterious effects. Screening and characterization of these ubiquitous, therapeutically potent polyphenolic compounds, require a clear understanding regarding their incorporation and possible location in membranes, as well as quantitative estimates of their antioxidative and radical scavenging capacities. Here, we demonstrate the novel use of the intrinsic fluorescence characteristics of the plant flavonoid fisetin (3,3',4',7-OH flavone) to explore its binding and site(s) of solubilisation in egg lecithin liposomal membranes. Spectrophotometric assays have been used to obtain quantitative estimates of its antioxidative capacity. Furthermore, our quantum mechanical semi-empirical calculations provide a quantitative measure for the free radical scavenging activity of fisetin from the OH (at 3,3', 4', 7 positions of the molecule)-bond dissociation enthalpies. Implications of these findings are discussed.

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: Hesperitin, a ubiquitous bioactive flavonoid abundant in citrus fruits is known to possess antioxidant, anti-carcinogenic, hypolipidemic, vasoprotective and other important therapeutic properties. Here we have explored the interactions of hesperitin with normal human hemoglobin (HbA), using steady state and time resolved fluorescence spectroscopy, far UV circular dicroism (CD) spectroscopy, combined with molecular modeling computations. Specific interaction of the flavonoid with HbA is confirmed from flavonoid-induced static quenching which is evident from steady state fluorescence as well as lifetime data. Both temperature dependent fluorescence measurements and molecular docking studies reveal that apart from hydrogen bonding and van der Waals interactions, electrostatic interactions also play crucial role in hesperitin-HbA interactions. Furthermore, electrostatic surface potential calculations indicate that the hesperitin binding site in HbA is intensely positive due to the presence of several lysine and histidine residues. [Copyright &y& Elsevier]