Contrasting binding of fisetin and daidzein in γ-cyclodextrin nanocavity

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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.

Graphical abstract

Induced circular dichroism of fisetin (A) and daidzein (C) in γ-CDx matrix. B: Lowest energy docked complex of fisetin (green stick) and daidzein (cyan stick representation) with γ-CDx cavity (yellow line) representations.

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Highlights

► Fisetin/γ-CDx and daidzein/γ-CDx inclusion complexes were prepared and contrasting binding modes were explored. ► Steady state spectroscopic analyses confirmed the supramolecular interaction of complexes. ► 1:1 Stoichiometry of the complexes of flavonoid-γ-CDx was determined from ICD spectra. ► Increased fluorescence anisotropy and lifetime assures increased solubility of the flavonoids. ► Molecular docking reveals hydrogen bonding primarily dictates the inclusion complex formation.

Introduction

Various bioactive flavonoids [1], [2] have come into prominence as alternative therapeutic drugs in the past few decades, due to their important therapeutic activities with high potency and low systemic toxicity, which is marked by an explosive growth of research in this area [1], [2], [3], [4], [5], [6], [7], [8], [9], [10]. Many recent studies, both in vivo and in vitro, have established that flavonoids are powerful antioxidants effective against a wide range of free radical mediated and other diseases including various types of cancers, tumors, diabetes mellitus, atherosclerosis, ischemia, neuronal degeneration, cardiovascular ailments, and AIDS [3], [4], [5], [6], [7], [8]. The flavonol fisetin (3,3′,4′,7-tetrahydroxyflavone, Scheme 1) is a flavonoid present in a number of commonly eaten foods, such as strawberries, vegetables, nuts, and wine [9], and has been reported to protect nerve cells from oxidative stress-induced death [10] and promote the differentiation of nerve cells [11]. Fisetin is also found to inhibit Aβ fibril formation in vitro [12], [13]. In vivo, fisetin has recently been shown to possess interesting anticancer activity in lung carcinoma [14], prostate tumours [15], and human embryonal carcinoma [16] in mice. Another flavonoid of contemporary interest is the isoflavone daidzein (7-hydroxy-3(-4′-hydroxyphenyl)chromen-4-one, Scheme 1), commonly found in legumes [17]. Daidzein has a wide spectrum of physiological and pharmacological functions including antiestrogenic [18], [19], anticancer [20], [21], anti-inflammatory [22], cardioprotective [23] and enzyme-inhibitory effects [18], [19]. Another important and interesting feature of fisetin and other structurally related flavonoids, is their dual fluorescence behavior [7], [24], [25], [26], which shows remarkable sensitivity to the surrounding microenvironment serving as exquisitely sensitive fluorescent molecular probes.

Despite the vast importance of the bioflavonoids as therapeutic drugs for the treatment of cancer and other diseases, their administration in vivo remains problematic partly due to their poor water solubility [27], [28], [29], [30]. Encapsulation of the drugs into polymer-based nanoparticles appears to markedly help the oral delivery of flavonoids in three ways: (1) by increasing solubility, (2) by protecting the drug from degradation in the gastrointestinal tract, and (3) by virtue of their unique absorption mechanism through the lymphatic system and first-pass metabolism in the liver [30]. Hence, to improve the solubility of fisetin and daidzein, thereby improving their in vivo administration, we chose γ-cyclodextrin [28], [30] as a suitable molecular carrier to achieve a better bioavailability.

Cyclodextrins are capable of enhancing the solubility, dissolution rate and membrane permeability [28], [29] of such drugs. Cyclodextrins (CDxs) are cyclic oligosaccharides which consist of (1,4)-linked α-d-glucopyranose units and are produced by enzymatic degradation of starch by cyclodextrin glycosyltransferase (CGTase) [31], [32]. The properties of the natural cyclodextrins (CDxs), their complexes and derivatives seem to be surprising because the 7-membered β-CDx (with cavity diameter 0.6–0.66 nm) is the least soluble (at 25 °C solubility in water is 18.5 mg/cm3), the 6-membered α-CDx (0.47–0.53 nm) is more soluble (solubility in water is 130 mg/cm3), and the 8-membered γ-CDx (0.75–0.83) (Scheme 2) attains the highest solubility (solubility in water is 300 mg/cm3) [31], [32]. The aim of the present study is therefore to characterize γ-CDx as the nanovehicle of fisetin and daidzein that could be suitable for parenteral administration.

Section snippets

Experimental

Fisetin, daidzein and γ-cyclodextrin are purchased from Sigma–Aldrich Chemical Company and used as obtained. The solvents used are of spectroscopic grade and checked for any absorbing and/or fluorescent impurities. The final concentration of fisetin and daidzein are kept in the order of 10−6 M and methanol/ethanol concentrations are below 1% v/v. Stock γ-CDx solutions are prepared by dissolving requisite amount of cyclodextrin powder in deionized water. To prepare each solution for spectroscopic

Steady state fluorescence spectroscopy

Fig. 1A and B present the fluorescence emission and excitation spectra and Fig. 1C presents the absorption spectra of fisetin with increasing concentration of γ-cyclodextrin. It is evident that addition of γ-cyclodextrin induces dramatic changes in the emission behavior of fisetin. In aqueous medium, the fluorescence spectra of fisetin exhibits strong overlap between the normal and tautomer emission bands [24]. With the addition of the γ-cyclodextrin, dual fluorescence behavior is observed. The

Summary and concluding remarks

Complexation of molecules to cyclodextrin occurs through a non-covalent interaction between the molecule and the CDx cavity. Dramatic changes are evident from the steady state and time resolved fluorescence as well as circular dichroism spectroscopic studies of fisetin and daidzein in CDx microenvironment when compared to that in water. However, the mode of binding varies remarkably for these two different kinds of flavonoids as is depicted through ICD, fluorescence anisotropy and docking

Acknowledgements

BSG likes to acknowledge the financial support from NIH/NCMHHD/RIMI Grant # 1P20MD002725 and research support from HBCU-UP Grant, NSF ID: 0811638 and Epscor Grant MS-EPS-0903787, at Tougaloo College. PKS gratefully acknowledges CSIR, India for the award of a grant under CSIR Emeritus scientist scheme, sanction no. 21(0864)/11/EMR-II. We thank Professor Abhijit Chakrabarti of Structural Genomics Division of SINP for letting us use the circular dichroism spectrometer and Mr. Subrata Das of the

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