Plasma protein binding of dietary polyphenols to human serum albumin: A high performance affinity chromatography approach.

Herein, the protein binding rates of structurally different flavonoids to human serum albumin (HSA) were elucidated by applying the high performance affinity chromatography (HPAC). The flavonoids with hydroxyl groups on ring A showed a higher protein binding rate compared with those that there was no hydroxyl on ring A. However, the hydroxylation of ring B lowered the protein binding rate. It was also found that an additional methoxy group in flavone ring A would decrease the protein binding rate. Nevertheless, the methoxy group in flavanone ring A (position 6) and isoflavone ring B (position 4') increased the protein binding rate. Methoxy group at other positions of flavonoids slightly enhanced or no significantly affected the binding rates on human serum albumin. Hydrogenation of C2C3 double bond of flavonoids decreased the protein binding rate and had the same effect as glycosylation which decrease the protein binding rate by 5%-25%.

Importance of the hydroxyl substituents in the B-ring of plant flavonols on their preferential binding interactions with VEGF G-quadruplex DNA: Multi-spectroscopic and molecular modeling studies.

G-quadruplex (G4) structures are known to be promising anticancer drug targets and flavonols (an important class of flavonoids) are small molecules reported to possess several health-promoting properties including those of anticancer activities. In this work, we explored the interactions of the structurally related plant flavonols kaempferol (KAE; 3,5,7,4'OH flavone) and morin (MOR; 3,5,7,2',4'OH flavone) with various G4-DNA sequences along with duplex DNA using a combination of spectroscopic and molecular docking studies. Our results revealed that KAE shows preferential interaction with VEGF G4-DNA in comparison to the other G4 sequences and duplex DNA. Moreover, KAE enhances the thermal stability of VEGF G4-DNA. In contrast, MOR exhibits an appreciably weaker level of interaction with both duplex and various G4-DNAs, with no significant structural specificity. The contrasting DNA binding behaviors suggest a crucial role of the 2'OH substituent in the B-ring of flavonol moiety. While KAE is relatively planar, MOR adopts a significantly non-planar conformation attributable to steric hindrance from the additional 2'OH substituent. This small structural difference is apparently very important for the ability of KAE and MOR to interact with VEGF G4-DNA. Thus, KAE (but not MOR) appears to be an effective ligand for VEGF G4-DNA, opening up possibilities of its application for regulation of gene expression in cancer cells.

Molecular insight into the inclusion of the dietary plant flavonol fisetin and its chromophore within a chemically modified γ-cyclodextrin: Multi-spectroscopic, molecular docking and solubility studies.

We explored the encapsulation of dietary plant flavonols fisetin and its chromophore 3-hydroxyflavone, within 2-hydroxypropyl-γ-cyclodextrin (HPγ-CDx) nano-cavity in aqueous solution using multi-spectroscopic approaches and molecular docking. Upon addition of HPγ-CDx, dramatic changes occur in the intrinsic 'two color' fluorescence behavior of the fluorophores. This is manifested by significant increase in the steady state fluorescence intensities, anisotropies, average fluorescence lifetimes and rotational correlation times. Furthermore, in the CDx environment, intrinsically achiral flavonols exhibit prominent induced circular dichroism bands. These findings indicate that the flavonol molecules spontaneously enter the relatively hydrophobic, chiral environment of the HPγ-CDx nano-cavities. Molecular docking computations corroborate the spectroscopic findings, and predict selectivity in orientation of the encapsulated flavonols. HPγ-CDx inclusion increases the aqueous solubility of individual flavonols ∼100-1000 times. The present study demonstrates that the hydroxypropyl substituent in γ-CDx controls the inclusion mode of the flavonols, leading to their enhanced solubilization and altered spectral signatures.

Exploring the Interactions of the Dietary Plant Flavonoids Fisetin and Naringenin with G-Quadruplex and Duplex DNA, Showing Contrasting Binding Behavior: Spectroscopic and Molecular Modeling Approaches.

Guanine-rich sequences have the propensity to fold into a four-stranded DNA structure known as a G-quadruplex (G4). G4 forming sequences are abundant in the promoter region of several oncogenes and become a key target for anticancer drug binding. Here we have studied the interactions of two structurally similar dietary plant flavonoids fisetin and naringenin with G4 as well as double stranded (duplex) DNA by using different spectroscopic and modeling techniques. Our study demonstrates the differential binding ability of the two flavonoids with G4 and duplex DNA. Fisetin more strongly interacts with parallel G4 structure than duplex DNA, whereas naringenin shows stronger binding affinity to duplex rather than G4 DNA. Molecular docking results also corroborate our spectroscopic results, and it was found that both of the ligands are stacked externally in the G4 DNA structure. C-ring planarity of the flavonoid structure appears to be a crucial factor for preferential G4 DNA recognition of flavonoids. The goal of this study is to explore the critical effects of small differences in the structure of closely similar chemical classes of such small molecules (flavonoids) which lead to the contrasting binding properties with the two different forms of DNA. The resulting insights may be expected to facilitate the designing of the highly selective G4 DNA binders based on flavonoid scaffolds.

Ground and excited state proton transfer of the bioactive plant flavonol robinetin in a protein environment: spectroscopic and molecular modeling studies.

We performed spectroscopic and molecular modeling studies to explore the interaction of the bioactive plant flavonol robinetin (3,7,3',4',5'-OH flavone), with the carrier protein human serum albumin (HSA). Multiparametric fluorescence sensing, exploiting the intrinsic "two color" fluorescence of robinetin (comprising excited state intramolecular proton transfer (ESIPT) and charge transfer (CT) emissions) reveals that binding to HSA significantly affects the emission and excitation profiles, with strongly blue-shifted (∼29 nm) normal fluorescence and remarkable increase in the ESIPT fluorescence anisotropy (r) and lifetime (τ). Flavonol-induced HSA (tryptophan) fluorescence quenching data yield the dynamic quenching constant (KD) as 5.42 × 10(3) M(-1) and the association constant (Ks) as 5.59 × 10(4) M(-1). Time-resolved fluorescence anisotropy decay studies show dramatic (∼170 times) increase in the rotational correlation time (τ(rot)), reflecting greatly enhanced restrictions in motion of robinetin in the protein matrix. Furthermore, prominent induced circular dichroism (ICD) bands appear, indicating that the chiral environment of HSA strongly perturbs the electronic transitions of the intrinsically achiral robinetin molecule. Molecular docking calculations suggest that robinetin binds in subdomain IIA of HSA, where specific interactions with basic residues promote ground state proton abstraction and stabilize an anionic species, which is consistent with spectroscopic observations.

Michael Kasha: from photochemistry and flowers to spectroscopy and music.

A brilliant scientist and an outstanding personality who was one of the founders of modern photochemistry-Michael Kasha-is the subject of this Essay. Kasha's rule and the Kasha effect both bear his name, and he also discovered the chemical production of singlet molecular oxygen, and was a pioneer of excited-state proton transfer systems. Kasha combined his passion for chemistry and physics with that for music, photography, and botany.

Prospect of bioflavonoid fisetin as a quadruplex DNA ligand: a biophysical approach.

Quadruplex (G4) forming sequences in telomeric DNA and c-myc promoter regions of human DNA are associated with tumorogenesis. Ligands that can facilitate or stabilize the formation and increase the stabilization of G4 can prevent tumor cell proliferation and have been regarded as potential anti-cancer drugs. In the present study, steady state and time-resolved fluorescence measurements provide important structural and dynamical insights into the free and bound states of the therapeutically potent plant flavonoid fisetin (3,3',4',7-tetrahydroxyflavone) in a G4 DNA matrix. The excited state intra-molecular proton transfer (ESPT) of fisetin plays an important role in observing and understanding the binding of fisetin with the G4 DNA. Differential absorption spectra, thermal melting, and circular dichroism spectroscopic studies provide evidences for the formation of G4 DNA and size exclusion chromatography (SEC) proves the binding and 1∶1 stoichiometry of fisetin in the DNA matrix. Comparative analysis of binding in the presence of EtBr proves that fisetin favors binding at the face of the G-quartet, mostly along the diagonal loop. Time resolved fluorescence anisotropy decay analysis indicates the increase in the restrictions in motion from the free to bound fisetin. We have also investigated the fingerprints of the binding of fisetin in the antiparallel quadruplex using Raman spectroscopy. Preliminary results indicate fisetin to be a prospective candidate as a G4 ligand.

Interactions of dietary flavonoids with proteins: insights from fluorescence spectroscopy and other related biophysical studies.

In 1936, Rusznyak and Szent-Györgyi first drew attention to the therapeutically beneficial role of dietary flavonoids, which are the most common group of polyphenols ubiquitously present in plant based food and beverages. Recent years have witnessed a renascence of interest on these nutraceuticals, which, because of their high potency and low systemic toxicity, are gradually emerging as promising alternatives to conventional therapeutic drugs. There is a mounting evidence that various proteins frequently serve as targets for therapeutically important flavonoids. In this article we present perspectives exemplifying the growing potential of fluorescence spectroscopy as an exquisitely sensitive tool for noninvasive sensing of protein-flavonoid interactions at physiologically relevant concentrations, via measurements of steady state emission parameters as well as decay kinetics studies of the intrinsic fluorescence of the target (protein) and/or ligand (flavonoid). Especially, we highlight novel applications of the remarkably environment sensitive 'two color' fluorescence exhibited by many important flavonoids, which permits multiparametric and ratiometric measurements. To consolidate findings obtained via fluorescence spectroscopy, use of other relevant experimental biophysical techniques and molecular modeling have proved to be valuable and are also discussed here. Such complementary studies provide additional insights regarding the thermodynamics and conformational aspects of the protein-flavonoid interactions, together with details, at atomistic level, of the dominant noncovalent interactions involved in the docking of different flavonoids to their target proteins.

Contrasting binding of fisetin and daidzein in γ-cyclodextrin nanocavity.

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.

A CRITICAL STUDY ON THE INTERACTIONS OF HESPERITIN WITH HUMAN HEMOGLOBIN: FLUORESCENCE SPECTROSCOPIC AND MOLECULAR MODELING APPROACH.

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.

Binding and antioxidant properties of therapeutically important plant flavonoids in biomembranes: insights from spectroscopic and quantum chemical studies.

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.

Probing the interactions of hemoglobin with antioxidant flavonoids via fluorescence spectroscopy and molecular modeling studies.

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 × 10(4)M(-1) and 9.83 × 10(3)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) τ(o)/τ≈1 (where τ(o) 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.

Binding of the bioflavonoid robinetin with model membranes and hemoglobin: Inhibition of lipid peroxidation and protein glycosylation.

Recent years have witnessed burgeoning interest in plant flavonoids as novel therapeutic drugs targeting cellular membranes and proteins. Motivated by this scenario, we explored the binding of robinetin (3,7,3',4',5'-pentahydroxyflavone, a bioflavonoid with remarkable 'two color' intrinsic fluorescence properties), with egg yolk phosphatidylcholine (EYPC) liposomes and normal human hemoglobin (HbA), using steady state and time resolved fluorescence spectroscopy. Distinctive fluorescence signatures obtained for robinetin indicate its partitioning (K(p)=8.65x10(4)) into the hydrophobic core of the membrane lipid bilayer. HbA-robinetin interaction was examined using both robinetin fluorescence and flavonoid-induced quenching of the protein tryptophan fluorescence. Specific interaction with HbA was confirmed from three lines of evidence: (a) bimolecular quenching constant K(q)>>diffusion controlled limit; (b) closely matched values of Stern-Volmer quenching constant and binding constant; (c) tau(0)/tau=1 (where tau(0) and tau are the unquenched and quenched tryptophan fluorescence lifetimes, respectively). Absorption spectrophotometric assays reveal that robinetin inhibits EYPC membrane lipid peroxidation and HbA glycosylation with high efficiency.

Ground and excited state proton transfer and antioxidant activity of 7-hydroxyflavone in model membranes: absorption and fluorescence spectroscopic studies.

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.

Ground- and excited-state proton transfer and antioxidant activity of 3-hydroxyflavone in egg yolk phosphatidylcholine liposomes: absorption and fluorescence spectroscopic studies.

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.

Interaction of 7-hydroxyflavone with human serum albumin: a spectroscopic study.

Numerous recent investigations have revealed that various synthetic as well as therapeutically active natural flavonoids possess novel luminescence properties that can serve as highly sensitive monitors for exploring their interactions with relevant physiological targets. Here we report a detailed study on the interactions of the model flavone, 7-hydroxyflavone (7HF) with the plasma protein human serum albumin (HSA), employing electronic absorption, fluorescence (steady state and time resolved) and induced circular dichroism (ICD) spectroscopy. The spectral data indicate that in the protein matrix, the neutral 7HF molecules are predominantly transformed to a conjugate anion (7HFA) by a proton abstraction in the ground state. The protein (HSA) environment induces dramatic enhancements in the fluorescence emission intensity, anisotropy (r) and lifetime (tau) values, as well as pronounced changes in the fluorescence excitation and emission profiles of the fluorophore. Moreover, evidence for efficient Förster type resonance energy transfer (FRET, from tryptophan to 7HFA) is presented, from which we infer that the binding site of 7HF in HSA is proximal (estimated distance, R=23.6A) to the unique tryptophan - 214 residue present in the inter-domain (between IIA and IIIA domains) loop region of the protein. The binding constant (K=9.44x10(4)M(-1)) and the Gibbs free energy change (DeltaG=-28.33kJ/mol) for 7HFA-HSA interaction have been estimated from the emission data. Finally, the near-UV circular dichroism (CD) studies show that the electronic transitions of 7HF are strongly perturbed on binding to the chiral host (HSA), leading to the appearance of ICD bands. Implications of these results are discussed.

Effect of beta-cyclodextrin nanocavity confinement on the photophysics of robinetin.

We have studied the confinement of robinetin, a therapeutically active plant flavonol, in cyclodextrin (CDx) nanocavities, using steady state and time resolved fluorescence spectroscopy. Enhanced tautomer emission (arising from excited state intramolecular proton transfer (ESIPT)) as well as dramatically blue shifted (approximately 10 nm in beta-CDx and approximately 33 nm in SHP beta-CDx) normal fluorescence observed upon addition of the beta-CDxs indicate that robinetin readily enters the doughnut-shaped hydrophobic cavity of beta-CDx where the chromone moiety is well shielded from external hydrogen bonding perturbations. Detailed analyses of the fluorescence data (emission profile, anisotropy, decay times) indicate that robinetin forms 1:1 inclusion complexes with both natural and chemically modified beta-cyclodextrins (beta-CDx and SHP beta-CDx) with affinity constant values K=195+/-17 M(-1) and 1055+/-48 M(-1) respectively, indicating the prospective utility of SHP beta-CDx in particular as an effective drug carrier. Unlike beta-CDxs, alpha-CDxs do not form inclusion complexes with robinetin. To further characterize the robinetin/beta-CDxs complexes, circular dichroism (CD) spectroscopic studies have been performed, which reveal that incorporation of robinetin molecules in the chiral environment of the beta-CDxs strongly affects the electronic transitions of robinetin leading to the occurrence of positive induced circular dichroism (ICD) bands in the near ultra-violet (UV) region. Molecular mechanics calculations show that the inclusion complex with the chromone ring inserted into the beta-CDx cavity is most favorable, in agreement with our spectroscopic data.

Interaction of flavonoids with red blood cell membrane lipids and proteins: antioxidant and antihemolytic effects.

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.

Interactions of the plant flavonoid fisetin with macromolecular targets: insights from fluorescence spectroscopic studies.

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.

Investigations on the binding and antioxidant properties of the plant flavonoid fisetin in model biomembranes.

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.