Effect of β-cyclodextrin nanocavity confinement on the photophysics of robinetin

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Journal of Photochemistry and Photobiology B: Biology 89 (2007) 88–97

Effect of b-cyclodextrin nanocavity confinement on the photophysicsof robinetin

Anwesha Banerjee, Kaushik Basu, Pradeep K. Sengupta *

Biophysics Division, Saha Institute of Nuclear Physics, 1/AF, Bidhannagar, Kolkata 700 064, India

Received 28 March 2007; received in revised form 14 August 2007; accepted 13 September 2007Available online 19 September 2007

Abstract

We have studied the confinement of robinetin, a therapeutically active plant flavonol, in cyclodextrin (CDx) nanocavities, using steadystate and time resolved fluorescence spectroscopy. Enhanced tautomer emission (arising from excited state intramolecular proton trans-fer (ESIPT)) as well as dramatically blue shifted (�10 nm in b-CDx and �33 nm in SHP b-CDx) normal fluorescence observed uponaddition of the b-CDxs indicate that robinetin readily enters the doughnut-shaped hydrophobic cavity of b-CDx where the chromonemoiety 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 b-cyclodextrins(b-CDx and SHP b-CDx) with affinity constant values K = 195 ± 17 M�1 and 1055 ± 48 M�1 respectively, indicating the prospectiveutility of SHP b-CDx in particular as an effective drug carrier. Unlike b-CDxs, a-CDxs do not form inclusion complexes with robinetin.To further characterize the robinetin/b-CDxs complexes, circular dichroism (CD) spectroscopic studies have been performed, whichreveal that incorporation of robinetin molecules in the chiral environment of the b-CDxs strongly affects the electronic transitions ofrobinetin leading to the occurrence of positive induced circular dichroism (ICD) bands in the near ultra-violet (UV) region. Molecularmechanics calculations show that the inclusion complex with the chromone ring inserted into the b-CDx cavity is most favorable, inagreement with our spectroscopic data.� 2007 Elsevier B.V. All rights reserved.

Keywords: Robinetin; b-Cyclodextrins; Excited state intramolecular proton transfer (ESIPT); Fluorescence spectroscopy; Induced circular dichroism(ICD); Molecular mechanics

1. Introduction

Flavonols and related natural products of the flavonoidgroup are ubiquitous in higher plants [1]. Recent interest inflavonols stems from mainly two different contexts ofintrinsic importance. First, flavonols are among the bestknown molecular systems exhibiting excited state intramo-lecular proton transfer (ESIPT) and dual fluorescencebehavior [2,3]. Thus they can serve as useful models formechanistic studies on ESIPT and related photophysical

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doi:10.1016/j.jphotobiol.2007.09.001

* Corresponding author. Tel.: +91 033 23370379x3508; fax: +91 03323374637.

E-mail addresses: [email protected], [email protected] (P.K. Sengupta).

aspects. Secondly, such compounds are found to possessnovel therapeutic properties of high potency and low sys-temic toxicity. Interest on this latter aspect dates back to1936, when Rusznyak and Szent-Gyorgyi [4] first drewattention to the therapeutically beneficial role of dietaryflavonoids. Recent years have witnessed revitalized atten-tion in this area with an explosive growth of research onvarious bioactive flavonols possessing a broad range oftherapeutic properties (effective against cancers, tumors,allergies, cardiac problems, inflammation, AIDS, etc.) [5,6].

Earlier papers from our laboratory emphasized the useof the highly sensitive fluorescence properties of flavonolsto probe their interactions with various physiologically rel-evant intracellular targets, e.g., proteins [7–9], DNA [9],membranes [10,11], and membrane mimetic model systems

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mailto:pradeepsinp@yahoo.

A. Banerjee et al. / Journal of Photochemistry and Photobiology B: Biology 89 (2007) 88–97 89

[12]. In a recent report, we focused attention on the ESIPTand dual emission behaviour of some representative flavo-nols in motionally constrained cyclodextrin (CDx) nano-cavities [13]. Discriminating spectroscopic signatures,useful for characterizing the flavonol–CDx complexes,were obtained. As cyclodextrins (internal cavity diametervarying between 0.45 and 0.8 nm, depending on the num-ber of a-D-glucose units present in the cyclic oligomer)are capable of encapsulating a wide range of compounds[14–20], their application as efficient drug delivery vehiclesis becoming increasingly popular in the pharmaceuticalindustry [17,18]. In this respect the prospects of usingcyclodextrins as drug delivery vehicles for flavonoids (formany of which the bioavailibilty is often severely restrictedby their intrinsically poor water solubility) is especiallyattractive [21–24], since encapsulation in such nanocavitiescan effectively enhance their hydrosolubility, thereby lead-ing to improved bioavailability. The toxicity aspect of suchdrug delivery agents has been the topic of much interest.Various studies suggest that native as well as substitutedcyclodextrin derivatives are toxicologically benign andwell-tolerated in animal species [25].

Robinetin (3,7,3 0,4 0,5 0-OH flavone, structure shown inScheme 1) is a naturally occurring flavonol which is gainingincreasing attention for its interesting biological and relatedtherapeutic (e.g. antioxidant, anti-cancer, anti-leishmanialand anti-mutagenetic) properties [26–29]. Previous studiesfrom our laboratory demonstrated interesting uses of rob-inetin as a sensitive optical probe (by exploiting its ESIPTand dual fluorescence properties) for investigating the localenvironments in reverse micelles [12]. In the present studythe encapsulation of robinetin, inside the nano-cavities ofnatural (a- and b-) and chemically modified cyclodextrins(SHP b-CDx) has been investigated utilizing the exquisitelysensitive dual emission properties of the flavonol as probe. Inaddition to fluorescence (steady state and time resolved),induced circular dichroism (ICD) spectroscopy and molecu-lar mechanics calculations have been employed to explorethe formation and structure of the flavonol–CDx complexes.

Scheme 1. (a) Robinetin and (

2. Materials and methods

2.1. Materials

Robinetin was a product of K and K Laboratories and3HF was purchased from Aldrich Chemical Company.Fisetin, b-cyclodextrin (b-CDx), succinyl-(2-hydroxypro-pyl)-b-cyclodextrin (SHP b-CDx) (degree of substitution(DS) = 4) and a-cyclodextrin (a-CDx) were obtained fromSigma. All the cyclodextrins used in this work are fairly sol-uble in water. Stock CDx solutions (5–12 mM) were pre-pared by dissolving requisite amounts in quartz distilledwater. Concentrated stock solutions of the flavonol wereprepared in spectrograde methanol (E. Merck) from whichrequisite aliquots were added to the aqueous b-CDx solu-tions. The final concentrations of flavonols was in the order�10�5 M, and that of methanol was <1%.

2.2. Methods

Steady state electronic absorption and fluorescence spec-tra were recorded with a Cecil 7500 spectrophotometer,and Perkin–Elmer LS-55 spectrofluorometers, respectively.The fluorescence spectra were corrected for the wavelengthdependence of the sensitivity of the apparatus. The relativefluorescence quantum yields for robinetin emission weremeasured under different CDx concentrations using qui-nine sulfate (in 0.1 M H2SO4, U = 0.53) as the quantumyield standard. If Ux and Us are the quantum yields of agiven fluorophore species ‘x’ and the standard ‘s’, respec-tively, then

/x ¼ /s

F x

F s

� �n2

x

n2s

� �As

Ax

� �ð1Þ

where Fx, Fs are the wavelength integrated emission inten-sities of the two samples; As, Ax are the optical densities attheir corresponding wavelengths of excitation and nx, ns aretheir refractive indices, respectively, [30]. The fluorescenceanisotropy (r) values were obtained using the expression

b) b-cyclodextrin (b-CDx).

Fig. 1. Fluorescence emission spectra of robinetin (kexc = 360 nm) in thepresence of increasing concentrations of (a) b-CDx (0! 12 mM), robine-tin (15 lM) and (b) SHP b-CDx (0! 5 mM), robinetin (21 lM).

90 A. Banerjee et al. / Journal of Photochemistry and Photobiology B: Biology 89 (2007) 88–97

r ¼ IVV � GIVH

IVV þ 2GIVH

ð2Þ

where IVV and IVH are the vertically and horizontallypolarized components of probe emission with excitationby vertically polarized light at the respective wavelengthand G is the sensitivity factor of the detection system [30].

Fluorescence decay measurements were performed usingan Edinburgh Instruments nanosecond time correlated sin-gle photon counting setup with a 370 nm nanosecond diodelaser excitation source (IBH, UK, nanoLED-03) having apulse FWHM �1.2–1.4 ns. Data analysis was carried outby a deconvolution method using a non-linear least squarefitting programme and fitted with a multi exponential decayfunction, F(t) =

Pi Ai exp (�t/si),

Pi Ai = 1 where Ai and si

represent the amplitudes and time constants, respectively, ofthe individual components in multiexponential decay pro-files. The goodness of fit was estimated by using v2 values.For time resolved fluorescence measurements robinetin con-centration was kept at 25 lM and that of b-CDxs was 5 mM.

Circular dichroism (CD) spectra were recorded on a Jas-co J-600 spectropolarimeter, using a rectangular cuvettewith 1 cm pathlength. The spectra were accumulated threetimes with a bandwidth of 1.0 nm and a scanning step of0.1 nm at a scan speed of 50 nm/min. Induced CD spectrawere obtained as the CD of flavone–CDx mixture minusthe CD of CDx alone. For CD measurements robinetinconcentration was kept at 50 lM.

All spectral measurements were carried out at ambienttemperature (298 K).

2.3. Molecular mechanics studies

The calculations were performed for the 1:1 inclusioncomplexes of the flavonol and b-CDx using molecularmechanics methods incorporated in HYPERCHEM 7.5[31]. The molecule was built on screen and fully optimizedin vacuo using MM+ force field. No cut-offs were used andgeometry optimization was carried out to an energy con-vergence of 0.01 kcal A�1 mol–1 with the Polak–Ribiereconjugate gradient algorithm.

Low energy structures for the 1:1 inclusion complexes ofthe flavonol with b-CDx were obtained in vacuo by follow-ing a docking procedure as described in our earlier work[13]. Two distinct inclusion orientations of the flavonolmolecules were considered, (I) with the chromone ring,and (II) with the phenyl ring, respectively inserted intothe b-CDx cavity. The stabilities of the inclusion complexesformed were judged from their energy of formation values(DE) calculated as the difference in the total energies of thecomplex and those of the free flavonol (guest) and b-CDx(host), respectively. The most stable complex among allthe configurations corresponds to the greatest negativevalue of DE. Solvation studies were then performed usinga combination of MM+ and AMBER force fields in a peri-odic box of dimensions 25 A · 25 A · 25 A consisting of517 water molecules.

3. Results and discussion

3.1. Excited state intramolecular proton transfer (ESIPT)

of robinetin in b-CDxs

Fig. 1a and b depict the fluorescence emission spectra ofrobinetin obtained in b-CDx and SHP b-CDx, respectively.On addition of b-CDxs conspicuous dual fluorescencebehavior is observed with appearance of a green emissionband �530 nm and a high energy band in the blue-violetregion. While the latter has been assigned to the S1 (pp*)! S0 normal (non-proton-transferred) emission, thelarge Stokes shifted green fluorescence band (red shiftedby �8 nm in b-CDx and �13 nm in SHP b-CDx relative


to that in aqueous medium) is attributable to emissionfrom a tautomer species generated by an excited state pro-ton transfer (ESIPT) process occurring along the internalH-bond (i.e. C(4) = OÆÆÆHO–C(3)) of the molecules) [2].This is in sharp contrast to the situation observed in aque-ous solution where a broad single fluorescence band isobserved, which can be attributed to the strong overlapbetween the normal and tautomer emissions. The ESIPTtautomer emission is highly sensitive to external hydro-gen-bonding perturbation, which can compete with theintramolecular H-bond formation, leading to decrease intautomer emission yield. Thus the remarkably enhancedESIPT tautomer fluorescence observed upon addition ofthe b-CDxs indicates incorporation of robinetin into rela-tively hydrophobic environments where the chromone moi-ety (which is the part of the molecule mainly relevant to theESIPT process) is well shielded from the water molecules.

Interestingly, the normal fluorescence of robinetinexhibits large blue shifts in presence of the b-CDxs(�10 nm in b-CDx and �33 nm in SHP b-CDx, Table 1).For robinetin, the highly sensitive dependence of the nor-mal fluorescence energy on solvent polarity has been previ-ously noted, and interpreted in terms of solvent dipolarreorientation mechanism [12]. In a motionally constrainedenvironment (such as inside the cyclodextrin nanocavity)dipolar reorientation is hindered, resulting in a dramaticblue shift of the fluorescence band. The enhanced tautomeremission together with the strongly blue shifted normalfluorescence band indicates that the guest molecule experi-ences a relatively hydrophobic viscous environment, inpresence of b-CDxs. The possibility of uncomplexed rob-inetin molecules contributing towards the overall spectracannot be ruled out, but considering the remarkablyenhanced tautomer emission it can be inferred that major-ity of the flavonol molecules are complexed with the b-CDxs and safely shielded from the external environment.

Fig. 2a and b show the relative quantum yields (U) plot-ted as a function of the b-CDx concentrations. Markedincrease in the quantum yields are observed on incorpora-tion inside the b-CDx cavities. This may be related to thefact that encagement in the CDx nanocavities could resultin reduction of the non-radiative pathways. Further it isnoteworthy that the yields obtained in the SHP b-CDx sys-tem are significantly higher than those obtained in b-CDx.

The ratio of the intensities of the green (II) and blue-vio-let emission (III) bands, II/III, is an especially useful param-eter for monitoring the enhancement in relative yield of theESIPT tautomer emission and provides a convenient indi-

Table 1Steady state fluorescence emission parameters of robinetin in b-CDxs

Medium Wavelength (nm) II/I

kabsmax kem

max (Blue-Violet) kemmax (Green)

Water 358.6 486 520 –b-CDx 354.7 476 528 1.8SHP b-CDx 359.6 453 533 2.6

cator of the hydrophobicity of the microenvironment offlavonols as emphasized in our previous studies [9,13]. Ascan be seen in Fig. 2c and d, the II/III increases dramati-cally on incorporation into the b-CDxs, gradually levelingoff at high CDx concentrations.

We also noted characteristic changes in the excitationspectra (monitored in the green emission region) on incor-poration into the b-CDx nanocavities (Fig. 3a). In particu-lar, pronounced blue shifts of the excitation maxima arenoted (�15 nm for SHP b-CDx and �8 nm for b-CDx),as compared to that in aqueous media. Such blue shiftsindicate conspicuous changes in the microenvironment ofthe flavonol molecules on encagement inside the b-CDxs.In addition, the fluorescence excitation spectra of robinetinencapsulated in b-CDxs have been recorded at differentemission wavelengths (Fig. 3b). We observe that in aque-ous medium, the excitation spectra of robinetin is indepen-dent of the emission wavelength (data not shown).However on incorporation into the b-CDxs, there is a blueshift in the excitation maximum as the emission wavelengthis varied from 450 nm to 550 nm. This suggests that a mix-ture of species (probably differing in their hydrogen bond-ing nature) is present. This could arise due to the presenceof several H-bond donor/acceptor substituents on the host(b-CDx) and guest (robinetin) molecules. Thus the robine-tin emission arises from a heterogeneous population offluorophore molecules encapsulated in the b-CDxs, differ-ing in their hydrogen bonding characteristics.

3.2. Determination of binding stoichiometry and affinityconstants

The binding stoichiometry and the affinity constantswere estimated from the fluorescence data by using themodified Benesi–Hildebrand (BH) equation [32]:

1

DF¼ 1

DF max

þ 1

Ka½CDx�n� �

1

DF max

� �ð3Þ

where DF = Fx � F0, Fx and F0 represent the fluorescenceintensities of the flavonols in the presence and absence oftotal added b-CDx concentrations respectively. DFmax isthe maximum change in fluorescence intensity, Ka is theaffinity constant for robinetin/b-CDx complex, and n rep-resents the stoichiometry of the complex formed.

Typical double reciprocal plots for n = 1 are shown inFig. 4a and b for robinetin/b-CDx and robinetin/SHP b-CDx complexes respectively. In case of both the b-CDxs,the n = 1 plots exhibit good linearity whereas the n = 2

II r g (cP) K (M�1) at 298 K DG0 (kJ mol�1)

0.085 – – –92 0.206 2.19 195 ± 17 �13.0695 0.202 1.98 1055 ± 48 �17.25

Fig. 2. Plots of relative quantum yields (a, b) and II/III ratio (c, d) of robinetin in presence of b-CDx and SHP b-CDx.


plots (data not shown) deviate from linearity. From theseresults we can infer that robinetin forms 1:1 inclusion com-plexes with both substituted and native b-CDxs.

Non-linear least-squares regression (NLR) analysis wasused to obtain more precise values of the affinity constants.The affinity constant values estimated from the linear BHplots (described above) were used as an initial guess in aniterative NLR fit using the equation

DF ¼ KaDF max½CDx�n

1þ Ka½CDx�n ð4Þ

which is obtained by rearranging Eq. (3). Fig. 4a and b, in-sets show the non-linear curve fits obtained for the b-CDxand SHP b-CDx systems respectively. The Ka values wereobtained from the NLR fits to be 195 ± 17 M�1 and

1055 ± 48 M�1 for robinetin/b-CDx and robinetin/SHPb-CDx complexes respectively. This shows that the bindingaffinities of the flavonol in the SHP b-CDx system are sig-nificantly higher than that in the native b-CDx suggestingthe use of SHP b-CDx as an effective drug carrier providinggood bioavailibility. It is noteworthy that the Gibbs freeenergy change (DG0) values (Table 1) are negative for boththe complexation cases indicating spontaneous binding ofrobinetin with the b-CDxs.

A comment is in order regarding the higher affinity con-stant (Ka) of the flavonol for the substituted CDx com-pared to the native one. When a substituted form of CDxis employed for encapsulation two factors affect the processof complexation. The guest molecule faces increased stericcrowding, which disfavors the complexation, whereas the

Fig. 3. (a) Fluorescence excitation spectra of robinetin (kem = 525 nm) inwater (- - - -), 10 mM b-CDx (––) and 5 mM SHP b-CDx (-ÆÆ-ÆÆ-). (b)Fluorescence excitation spectra of robinetin in 10 mM b-CDx and 5 mMSHP b-CDx (inset) recorded at kem (1) 450 nm, (2) 480 nm, (3) 520 nm, (4)530 nm and (5) 550 nm.

Fig. 4. Double reciprocal plots (Eq. (3)) for robinetin complexed with (a)b-CDx and (b) SHP b-CDx for 1:1 binding stoichiometries. Insets: non-linear curve fits (Eq. (4)) for complexation of robinetin with (a) b-CDx and(b) SHP b-CDx, considering 1:1 binding stoichiometry. Measurementswere made at T = 298 K.


presence of substituents offer larger number of interactionsites for the incoming guest. Clearly these factors operatein opposite directions. Depending upon the nature of sub-stituents and degree of substitution one of these two factorswould tend to dominate over the other [33]. In the presentcase, the low degree of substitution (DS = 4) and largernumber of interactions outweighs the enhanced stericrepulsion, which can account for higher Ka values. Theability of b-CDxs to form inclusion complexes is relatedto the intrinsic solubility of the host molecule in water.The remarkably larger inclusion constants for SHP b-CDx may be explained by the fact that the chemically mod-ified cyclodextrins are endowed with suitable functionalgroups which improve the solubility and flexibility of cyclo-dextrins to a greater degree compared to the native b-CDx.

3.3. Fluorescence anisotropy measurements and

determination of microviscosity

Fluorescence anisotropy (r) measurements were per-formed since this parameter serves as a useful indicator ofthe rigidity of the local environment of the fluorophore andalso for monitoring ligand binding to macromolecular sys-tems [30]. The anisotropy values are low in fluid solutionswhere the fluorophore molecules can freely rotate andincrease in motionally constrained environments. Typicalfluorescence anisotropy data for the ESIPT tautomer fluores-cence of robinetin is displayed in Fig. 5. Upon complexationwith cyclodextrins the anisotropy values become consider-ably higher than that in fluid solutions (Table 1). This indi-cates that the flavonol molecules are firmly incorporated in


the motionally constrained central cavities of the cyclo-dextrins where rotational diffusion is restricted.

Fluorescence anisotropy can also give estimates of themicroviscosity (g) of the environment of the fluorophore.Microviscosity, at a definite temperature, is often estimatedby comparing the fluorescence anisotropy of a fluorophorein a specific environment with those of the probe in solventsof known viscosity. To have the relative measure of micro-viscosity in CDx environments, fluorescence anisotropiesof robinetin in the b-CDx solutions under saturating condi-tions were compared with the values in glycerol–water mix-tures of different compositions at 298 K [34]. The anisotropyvalue suggests the average environment around the probemolecules upon incorporation into b-CDx and SHP b-CDx corresponds to �24% and �22% glycerol–water mix-ture (mass percentage), respectively (Fig. 6). By using thecalibration curve based on available data [35], the effectivemicroviscosity values in these environments were estimatedas �2.088 cP and 1.857 cP.

Fig. 5. Variation of fluorescence anisotropy (r) of robinetin withincreasing concentrations of (a) b-CDx (kexc = 360 nm, kem = 525 nm)and (b) SHP b-CDx (kexc = 360 nm, kem = 545 nm).

3.4. Emission behaviour of flavonols in presence of a-CDx

Fig. 7 presents a comparison of the emission behaviourof robinetin along with that of fisetin (a flavonol of relatedinterest which has close similarities in its chemical structurewith robinetin, the latter having an extra OH group in theB ring) in presence of b- and a-CDxs. In case of both rob-inetin and fisetin, encapsulation into the b-CDx cavity ischaracterized by the appearance of a strong green tautomerfluorescence band and a blue-violet normal emission, asdescribed earlier (Section 3.1 and Ref. [13]). However theemission profiles obtained in aqueous media and on addi-tion of a-CDxs, are similar suggesting that the flavonolsare not encapsulated in the a-CDx cavities, presumablybecause of the smaller cavity size. These observations lendfurther credence to our proposal that enhanced ESIPT tau-tomer emission is compatible with the picture that thechromone moiety is encaged in the b-CDx cavities, thusshielded from the water molecules so that external H-bond-ing interference on the ESIPT process is minimized. Thisdemonstrates that ESIPT can serve as a reliable probe toidentify the mode of entry of flavonols into the b-CDx cav-ities. Thus in view of the enhanced tautomer emissionobserved in this case, the possibility of entry of flavonolsinto b-CDx cavity through the phenyl ring (rather thanthe chromone), as was suggested in a recent study [24],seems to be less likely.

3.5. Time resolved fluorescence studies of robinetin in b-CDxs

Time resolved fluorescence studies were performed toexamine how encapsulation in the CDx cavities influencesthe ESPT tautomer emission decay kinetics of the flavonol.The results are presented in Table 2. Biexponentialdecay behavior is observed with a predominant (>80%

Fig. 6. Calibration plot of fluorescence anisotropy (r) of robinetin as afunction of glycerol–water mixture. Anisotropy values in the CDxenvironment are indicated to find the equivalent condition in terms ofglycerol–water mixture.

Fig. 7. Fluorescence emission behavior of (a) robinetin and (b) fisetin in a- and b-cyclodextrins. The spectrum recorded in aqueous medium is alsoincluded.


contribution) short lifetime species (s1 = 0.41 ns (in b-CDx); s1 = 0.60 ns (in SHP b-CDx)). In addition, a minorcomponent with a much longer lifetime (s2 > 2 ns) is alsopresent. Since the emission yield in water is extremelylow, thereby precluding lifetime measurements to be car-ried out, we chose methanol, a typical homogeneous proticsolvent as a basis for comparison. Consistent with an ear-lier study from our laboratory [12], the decay kinetics isfound to be biexponential in methanol in contrast to polaraprotic solvents where strictly single exponential decayoccurs. The biexponential decay in hydrogen bonddonor/acceptor (HBA-D) solvents such as methanol isattributed to the presence of two types of H-bonded com-plexes of robinetin with the solvent molecules [12]. Suchbiexponential decay character of the ESPT tautomer emis-sion was previously reported for different benzimidazolederivatives by Dogra and co-workers. This was rational-ized in terms of differences in solvation of the tautomer[36]. It is noteworthy, that the microenvironment near theedge of the cyclodextrin cavity resembles the propertiesof a binary aqueous solvent (e.g., EtOH:water) [37–39]while the interior of the CDx cavity is similar in polarityto oxygenated solvents such as EtOH, dioxane, isopropylether, and ethylene glycol [33]. Thus, while the edge of

Table 2Fluorescence decay parameters of robinetin for ESIPT tautomer emissionin different media

Medium s1 (ns) A1 s2 (ns) A2 v2

Methanol 0.32 ± 0.010 0.880 1.48 ± 0.005 0.12 1.098b-CDx 0.407 ± 0.015 0.82 3.10 ± 0.025 0.18 1.311SHP b-CDx 0.597 ± 0.014 0.868 2.12 ± 0.037 0.132 1.257

kexc = 360 nm, kemmax ¼ 550 nm.

the cyclodextrin cavity can participate in H-bonding withsubstituents of the guest molecule, the CDx cavity presentsa comparatively more hydrophobic environment. We pre-sume that the two lifetime components observed for thetautomer species of robinetin, arise from populations dif-fering in the extent of H-bonding with the microenviron-ment. Furthermore we note that both the lifetimecomponents of the cyclodextrin encapsulated robinetinmolecules (Ref. Table 2) have significantly higher valuescompared to those observed in methanol (wheres1 = 0.32 ns, s2 = 1.48 ns), suggesting that the robinetinmolecules experience relatively hydrophobic microenviron-ments within the CDx cavities where non-radiative decayprocesses are reduced (this is also reflected in increasedquantum yield values upon addition of the b-CDxs).

3.6. Induced circular dichroism (ICD) studies of flavonols in

a- and b-CDxs

To further characterize the robinetin/b-CDxs com-plexes, circular dichroism (CD) spectroscopic studies havebeen performed. When a molecule, which is intrinsicallyoptically inactive, binds to a chiral host, the dissymmetricenvironment of the host induces optical activity in the guestmolecule. This results in appearance of induced circulardichroism bands in the absorption region of the guest[40–42]. Occurrence of ICD bands therefore provides a sen-sitive spectroscopic characterization of such host–guestinteractions. Fig. 8 displays the ICD spectra of robinetin,along with those of two other flavonols of related interest(namely 3-hydroxyflavone and fisetin) in b-CDxs. Flavo-nols being achiral, do not show any circular dichroism sig-nals intrinsically. However on addition of b-CDxs positivecircular dichroism signals are induced in the near-UV

Fig. 8. Induced CD spectra of robinetin, 3-hydroxyflavone and fisetinupon incorporation into b-CDx.


region indicating complexation of the flavonols with b-CDxs. The UV-absorption spectrum of flavones is charac-terized by two principal absorption bands designated asBand I (300–380 nm) and Band II (240–285 nm) [43]. BandI is attributed to light absorption in the cinnamoyl (B + C)portion of the molecule, while Band II is associated withthe benzoyl moiety (A + C). These bands are believed tobe essentially p–p* in nature [43]. The observation ofICD signals in this region, corresponding to the electronictransitions, is a further indication that the flavonols experi-

Fig. 9. Axial and equatorial views of low energy structures calculated for th

ence strong interactions with the chiral skeleton of the b-CDx nanocavities. The above studies were extended to a-CDxs also (data not shown), where no such occurrenceof ICD was noted, consistent with the lack of complexationwith the latter.

3.7. Molecular mechanics studies

Molecular mechanics calculations were performed inorder to gain insight into the thermodynamic and struc-tural features of the 1:1 complexes of robinetin with b-CDx. Judging from the energy of formation values of theinclusion complexes formed (see Fig. 9), it is evident thatthe inclusion complex with the chromone ring inserted intothe b-CDx cavity and the phenyl ring exposed (complexesI) is more stable (compared to complex II, where the phe-nyl ring is inside the cavity). Such an arrangement of theflavonols inside the hydrophobic b-CDx cavity, where thechromone ring is favorably shielded from the bulk water,could explain the enhanced tautomer emission as well asblue shifts in the normal emission. Moreover on solvatingthe structures, it is found that when robinetin is insertedthrough the chromone group, the 3 0 hydroxyl proton andoxygen forms two intermolecular bonds with the O andH atoms of the secondary hydroxyls at the b-CDx rim(H-bonds indicated as dotted lines in Fig. 9). In addition,the O1 atom of robinetin in also capable of forming anintermolecular hydrogen bond with b-CDx, in such a con-figuration. On the other hand, the entry of the guest mole-cule via the phenyl ring (complex II) leads to the formation

e complexes I (a and c) and II (b and d) between b-CDx and robinetin.


of only two intermolecular hydrogen bonds viz., the 3 0-hydroxyl oxygen with a primary hydroxyl and 3-hydroxyloxygen with a secondary hydroxyl of b-CDx respectively.This indicates that complex I will be more stabilized thancomplex II. However our results are in variance with arecently published report by Guzzo et al. [24] where itwas proposed that the inclusion of the flavonol, fisetin(3,7,3 0,4 0-OH flavone) inside the b-CDx cavity is preferen-tially via the phenyl group.

4. Concluding remarks

We expect that the present research would stimulate fur-ther investigations to fruitfully exploit the exquisitely sensi-tive fluorescence emission properties of therapeuticallyactive flavonols to investigate their interaction with CDxbased drug delivery vehicles.

Acknowledgements

P.K.S. is grateful to Professor Michael Kasha for thekind gift of the robinetin sample. A.B. thanks the CSIR,India for a Senior Research fellowship (CSIR GrantAward No. 9/489 (44)/2002-EMR-I). We thank our col-league Mr. Sudip Chaudhuri (for his interest and valuablesuggestions), Prof. N. Chattopadhyay, Jadavpur Univer-sity and his students, Dr. A. Mallik and Mr. A. Chakr-aborty (for access to the fluorescence lifetime setup andkind help) and Mr. J. Guin, PD laboratory, Bose Institute(for his valuable assistance in connection with the CircularDichroism measurements).

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