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Spectrochimica Acta Part A 75 (2010) 1506–1510

Contents lists available at ScienceDirect

Spectrochimica Acta Part A: Molecular andBiomolecular Spectroscopy

journa l homepage: www.e lsev ier .com/ locate /saa

comparison study on the interaction of hyperoside and bovine serum albuminith Tachiya model and Stern–Volmer equation

ing Qin1, Yixuan Zhang1,2, Shulian Yan, Ling Ye ∗

chool of Chemical Biology and Pharmaceutical Sciences, Capital Medical University, Beijing 100069, PR China

r t i c l e i n f o

rticle history:eceived 23 October 2009eceived in revised form 20 January 2010ccepted 3 February 2010

a b s t r a c t

The interaction between hyperoside and bovine serum albumin (BSA) was examined by fluorescencespectroscopy at 298, 304, and 310 K. The spectroscopic data were analyzed using Tachiya model andStern–Volmer equation to determine the binding sites and apparent binding constant between hyper-

eywords:yperosideovine serum albuminluorescence quenchingachiya model

oside and BSA. For Tachiya model, both binding sites and apparent binding constants increased withthe increasing of temperature, whereas for Stern–Volmer equation, the corresponding binding constantsdecreased as temperature increasing and the binding sites were independent of temperature. The positivesign of enthalpy change (�H) and entropy change (�S) suggested that hydrophobic forces played a majorrole in the interaction. Synchronous fluorescence spectra indicated that the conformation of protein wasperturbed by the interaction of hyperoside with BSA. Moreover, the presence of metal ion affected the

hyperoside-BSA binding.

. Introduction

As a flavonoid compound, hyperoside (quercetin-3-O-ˇ-D-alactoside, Fig. 1) exhibits a multitude of biological functionsncluding anti-viral activity [1], anti-inflammatory [2], antidepres-ant [3], hepatoprotective [4,5] and gastric mucosal-protectiveffects [6]. It has been demonstrated that hyperoside possessesignificant local and central analgesic effects [7]. Furthermore,yperoside shows a protective effect against myocardial and cere-ral ischemia-reperfusion injury owing to its inhibition of freeadical production and inward flow of calcium ion [8,9]. Never-heless, it is necessary to understand the pharmacodynamics andharmacokinetics of hyperoside before clinical therapy.

The affinity of a drug toward plasma proteins is an importanteterminant of its pharmacokinetics profile affecting its ADMEabsorption, distribution, metabolism and excretion) profile andonsequently its levels of activity and toxicity [10]. Serum albu-in is the most abundant one in blood plasma and acts as a depot

nd transport protein for numerous endogenous and exogenous

ubstances [11]. Many drugs bind reversibly to serum albumin,nd this in turn alters the distribution, free concentration and theetabolism of drugs [12]. In addition, the metal ions existing in

lood plasma may affect the binding. Thus, it is of great importance

∗ Corresponding author. Tel.: +86 10 83911525; fax: +86 10 83911533.E-mail address: lingye@ccmu.edu.cn (L. Ye).

1 These authors contributed equally to this work.2 Current address: Beijing Golden Elephant Pharmacy Co., Ltd., PR China.

386-1425/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.saa.2010.02.007

© 2010 Elsevier B.V. All rights reserved.

to study the interaction between drugs and serum albumin withand without metal ions. However, the interaction between hyper-oside and serum protein at molecular level has not been reportedso far.

In this work, we selected bovine serum albumin (BSA) as themodel protein and hyperoside as a model drug to investigate thedrug-serum albumin binding reaction under physiological condi-tions. The molecular interaction was assayed by the fluorometricmethod because of its high sensitivity, rapidity and ease of imple-mentation [13]. Quenching of intrinsic fluorescence of BSA wasemployed to explore the binding of hyperoside to BSA. In addi-tion, the impact of metal ions on the hyperoside-BSA interactionwas studied as well. Some important parameters of the BSA-druginteraction such as the apparent binding constant and bindingsite were firstly acquired from Stern–Volmer equation. Tachiyamodel, which differs from Stern–Volmer equation in emphasiz-ing the difference between the number of binding sites (m) andthe number of molecules (n) actually bound to the sites [14], wasused as well to evaluate the binding parameters. The obtainedvalues from Stern–Volmer equation and Tachiya model were com-pared.

2. Materials and methods

2.1. Chemicals

Bovine serum albumin (BSA, Fraction V, essentially fatty acidfree, approximately 99%) was purchased from Sigma (USA). Hyper-oside was generously provided by the College of Pharmacy of West

Y. Qin et al. / Spectrochimica Acta Part A 75 (2010) 1506–1510 1507

CcD

2

tws(

2

wcom2

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3

3S

3

v

dhsBwo

given by adsorption rate constant = 1 − (n/m)k1 for n < m

= 0 for n > m (4)

desorption rate constant = nk2 (5)

Table 1Binding parameters obtained from the interaction of hyperoside with BSA byStern–Volmer equation.

Fig. 1. Structure of hyperoside.

hina University of Medical Sciences (Sichuan, China). All otherhemicals were of analytical reagent grade and used as received.ouble distilled water was used throughout the experiment.

.2. Apparatus

The fluorescence spectra were acquired on a RF-5301PC spec-rofluorophotometer (Shimadzu, Japan) equipped with a SB-11ater bath (Eyela) and 1.0 cm quartz cells. The UV–vis absorption

pectrum was recorded on a UV-2250 UV–vis spectrophotometerShimadzu, Japan).

.3. Procedures

5 �M or 10 �M BSA (the molecular weight of 66,000) solutionas prepared with 0.1 M pH 7.4 phosphate buffer solution (PBS)

ontaining 0.15 M NaCl. The 1.25 �M solution of hyperoside wasbtained by dissolving appropriate amount of drug in absoluteethanol. The various metal ion solutions at the concentration of

.5 mM were prepared in double distilled water.For the fluorescence quenching measurement, 10 �L hyperoside

as successively added to 2.5 mL 5 �M or 10 �M BSA solutions withr without 10 �M metal ions in a 1.0 cm quartz cell. The concen-rations of hyperoside were ranged from 5 to 40 �M and the totalccumulated volume of hyperoside was no greater than 100 �L. Flu-rescence spectra were then recorded in the range of 300–500 nmpon excitation at 280 nm. All fluorescence quenching measure-ents were performed at three temperatures (298, 304 and 310 K).

ach experiment was repeated three times to obtain an average andstandard deviation. The synchronous fluorescence spectra were

cquired by scanning simultaneously with �� = 60 nm (from 300 to00 nm) and �� = 15 nm (from 280 to 350 nm). The UV absorbancepectrum of hyperoside with concentration of 10 �M was recordedrom 300 to 500 nm at 298 K.

. Results and discussion

.1. Fluorescence quenching spectra and binding parameters bytern–Volmer equation

.1.1. Fluorescence quenching spectra of BSA with hyperosideThe fluorescence emission spectra of BSA in the presence of

arious amounts of hyperoside at 298 K are shown in Fig. 2.It was observed from Fig. 2 that the fluorescence intensity of BSA

ecreased regularly with gradual increase in the concentration of

yperoside, indicating that interaction between BSA and hypero-ide occurred. In addition, the maximum emission wavelength ofSA showed a blue shift, indicating that the chromophore of BSAas placed in a more hydrophobic environment upon the addition

f hyperoside.

Fig. 2. Fluorescence quenching spectra of BSA in the presence of different concen-trations of hyperoside at 298 K. CBSA = 10 �M. CHyp from a to i: 0 �M (a), 5 �M (b),10 �M (c), 15 �M (d), 20 �M (e), 25 �M (f), 30 �M (g), 35 �M (h) and 40 �M (i).

3.1.2. Binding constants and binding sitesWhen small molecules are bound independently to a set of

equivalent sites on a macromolecule, the equilibrium between freeand bound molecules is given by the equation [15]

log(F0 − F)

F= log Ka + n log[Q ] (1)

where Ka and n are the apparent bind constant and the number ofbinding sites per BSA, respectively. Hereby, from the intercept andslope of the plot of log(F0 − F)/F vs log[Q], we acquired the valuesof Ka and n (Table 1). The great magnitude of Ka revealed a strongbinding force between BSA and hyperoside, which was strongerthan that of isoquercitrin-BSA or kaempferol-BSA binding [16,17].In addition, the values of n were roughly 1, demonstrating that therewas one association site on BSA for hyperoside.

3.2. Interaction between BSA and hyperoside by Tachiya model

3.2.1. Binding parametersIn general, the BSA-drug binding parameters can be acquired

from Stern–Volmer equation. However, the numbers of bindingsites are assumed as the identical number of molecules actuallybound to BSA in Stern–Volmer equation. This might not always thereal case. On the contrary, Tachiya model takes into account thedifference between the number of binding sites and the number ofmolecules actually bound to BSA [14]. Therefore, the fluorescenceintensity data were further analyzed by Tachiya model.

Given BSA is combined with n quenchers, the process ofquencher binding to BSA can be divided into adsorption and des-orption

Bn + Qap− > Bn+1 (2)

Bn− > Bn−1 + Qap (3)

The corresponding rate constant of processes (2) and (3) are

T (K) Ka (M−1) n r (nm)

298 4.83 × 106 1.3626 2.55304 4.48 × 106 1.3502 2.52310 4.22 × 106 1.3283 2.45

1 Acta Part A 75 (2010) 1506–1510

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Q

508 Y. Qin et al. / Spectrochimica

here k1 and k2 are the absorption and desorption rate constant,espectively. m is the number of binding sites which is differentrom n, the number of molecules really bound to BSA. If m is fixed,hen n follows a binomial distribution [18].

n = mCn

(nav

m

)n(1 − nav

m

)m−n

(6)

here nav is the average number of quenchers bound to BSA whichs given by the following equation

av = mK[Qaq]

m + K[Qaq]

= 12

[[Q ][M]

+ m + m

K[M] −

√([Q ][M]

+ m + m

K[M]

)2

− 4m[Q ][M]

]

(7)

Here, [Qaq] is the concentration of free quenchers in aqueousolution and K = k1/k2. [M] is the concentration of BSA, and [Qaq] ishe total concentration of quenchers calculated by the equation

Q ] = nav[M] + [Qaq] (8)

ke is the rate constant for Förster energy transfer from BSA to auencher described by

e = �−1(

R0

r

)6(9)

here � is the lifetime of BSA without quenchers, R0 the Förster dis-ance. r stands for the distance between BSA and quencher. Supposehe distance between every bound quencher with BSA is the same, the fluorescence intensity is decreased and can be estimated byhe equation

= 1

1 + n(R0/r)6(10)

The relationship between fluorescence intensity of BSA in theresence and absence of quencher can be described as the followingquation

F

F0=

m∑n=0

Qn

1 + n(R0/r)6=

m∑n=0

mCn(nav/m)n(1 − nav/m)m−n

1 + n(R0/r)6(11)

[M], [Q] and F/F0 are known or can be obtained experimentally,he values of m, K and r can be estimated from two limiting cases.

When the value of [M] is constant, F/F0 changes as [Q] changes,hat is, F/F0 decreases as [Q] increases. However, it saturates at suf-ciently high [Q] when all of the binding sites on BSA are combinedith quenchers. In this limiting case, the distribution of the number

f quenchers complies with the equation

n = ı (n − m) (12)

Accordingly, the fluorescence intensity at saturation can bettained through the equation

F

F0

)saturation

= 1

1 + m(R0/r)6(13)

In another limiting case, if the value of [Q] is low enough, theverage number of quenchers bound to BSA is very small and cane evaluated using the Eq. (7). Here,

av = K [Q ]K [M] + 1

(14)

nd the number of bound quencher is

n = 1 − nav n = 0

Fig. 3. Curves of F/F0 vs the concentration of hyperoside at 298 K (a), 304 K (b), 310 K(c).

= nav n = 1 (15)

Hence, the fluorescence intensity obeys the equation

F

F0= 1 − nav

(R0/r)6

1 + (R0/r)6= 1 − (R0/r)6

1 + (R0/r)6

K [Q ]K [M] + 1

(16)

where nav is given by Eq. (14).Fig. 3 shows the curves of F/F0 vs the concentration of hyperoside

at different temperatures. The values of binding constant, bindingsites and binding distance were calculated according to Eq. (16)and the data were presented in Table 2. As shown in Table 2, theapparent binding constant between BSA and hyperoside increasedwith the increasing of temperature. In contrast, the reversed effect

was observed from Stern–Volmer equation. This was similar to thatof the interaction of trans-resveratrol with BSA [14].

It was also found that the binding sites calculated from Tachiyamodel were different from that of Stern–Volmer equation. Thenumber of binding site from Stern–Volmer equation was indepen-

Y. Qin et al. / Spectrochimica Acta Part A 75 (2010) 1506–1510 1509

Table 2Binding parameters obtained from the interaction of hyperoside with BSA by Tachiyamodel.

T (K) Ka (M−1) n r (nm)

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298 6.63 × 104 1.5550 2.86304 7.26 × 104 1.6825 3.12310 1.21 × 105 2.1909 3.77

ent of the temperature. However, the binding site from Tachiyaodel increased with the increasing of temperature. This is because

hat there is no difference between the number of binding sitesm) and the number of molecules (n) actually bound to the sites intern–Volmer equation while this difference is taken into accountn Tachiya model [14]. Moreover, the binding distance obtainedrom Tachiya model differed from that of Stern–Volmer equationn both values and the temperature dependence.

.2.2. Binding mode and nature of the binding forceThe interaction forces between drugs and biomolecules include

ydrophobic forces, Van der Waals interactions, electrostatic inter-ctions, hydrogen bonds, and so on [19]. The thermodynamicarameters enthalpy change (�H), entropy change (�S) and freenergy change (�G) relying on temperature could be calculatedo elucidate the acting force between BSA and Hyperoside. Forrug–protein interaction, �H > 0, �S > 0 is taken as evidence forydrophobic interaction; �H < 0, �S < 0 is for hydrogen bonds;H ≈ 0, �S > 0 is indicative of electrostatic force [20].If �H does not vary significantly in the temperature range stud-

ed, �H and �S can be determined simultaneously by the van’t Hoffquation

n K = −�H

RT+ �S

R(17)

here K is the binding constant obtained from the Tachiya modelnd R is the gas constant. The temperatures adopted were 298, 304nd 310 K. �H and �S were calculated through the slope and therdinate of the origin of the plot of ln K vs. 1/T, respectively. �G wasstimated from the following relationship:

G = �H − T�S (18)

The corresponding data were summarized in Table 3. Theegative sign for �G manifested that the binding process was spon-aneous, while the positive values of �H and �S showed thatydrophobic forces played a major role in the interaction. Theseere as same as that of kaempferol and isoquercitrin interactionith BSA [16,17].

.2.3. Energy transfer between BSA and hyperosideThe overlap of the UV absorption spectrum of hyperoside with

he fluorescence emission spectrum of BSA is shown in Fig. 4.ccording to Föster’s non-radiative energy transfer theory [21], theate of energy transfer depends on (1) the relative orientation of theonor and acceptor dipoles, (2) the extent overlap of fluorescence

mission spectrum of the donor with the absorption spectrum ofhe acceptor, and (3) the distance between the donor and the accep-or. By employing the efficiency of transfer, the distance betweenmall molecules and tryptophan residues of BSA can be evaluated.

able 3hermodynamic parameters of hyperoside–BSA interaction.

T (K) �G (kJ mol−1) �H (kJ mol−1) �S (J mol−1 K−1)

298 −27.403 41.22 229.73304 −28.289310 −30.166

Fig. 4. Spectral overlap between (a) BSA fluorescence spectrum and (b) hyperosideUV–vis absorbance spectrum.

The efficiency E is related not only to the distance (r) betweenthe acceptor and the donor but also to the critical energy transferdistance (R0). This relationship follows the equation

E = 1 − F

F0= R6

0

R60 + r6

(19)

where F and F0 are fluorescence intensity of BSA in the presenceand absence of hyperoside, respectively. When the transfer effi-ciency is 50%, the critical energy transfer distance, R0, is calculatedby equation

R60 = 8.8 × 10−25k2N−4˚J (20)

where k2 is the spatial orientation factor of the dipole. N and ˚ arethe refractive index of medium and the fluorescence quantum yieldof the donor, respectively. J is the overlap integral of the fluores-cence emission spectrum of the donor and the absorption spectrumof the acceptor, given by

J =∑

F(�)ε(�)�4��∑F(�)��

(21)

where F(�) is the fluorescence intensity of the fluorescent donorat wavelength �, ε(�) the molar absorption coefficient of theacceptor at wavelength �. From equations, we could calculatethat J = 3.21 × 10−13 cm3 L mol−1, R0 = 4.54 nm considering k = 2/3,N = 1.336, ˚ = 0.15 [22]. The value of distance between hyperosideand Trp-214 residues of BSA was in the range from 2 to 8 nm, whichrevealed that the energy transfer from BSA to hyperoside occurredwith high probability [23].

3.2.4. Conformational studiesTo explore the effect of hyperoside on the conformation of

BSA, synchronous fluorescence measurement was conducted. Syn-chronous fluorescence spectra can give information about themolecular environment in a vicinity of the chromophore molecules,which was obtained by scanning the monochromators of excitationand emission simultaneously while the difference between excita-tion and emission wavelength (��) was fixed. When �� = 15 nmand �� = 60 nm, the synchronous fluorescence provides the char-acteristic information of tyrosine residues and tryptophan residues,respectively [24].

Synchronous fluorescence spectra of BSA upon addition of var-

ied amounts of hyperoside were shown in Fig. 5. It could be seenthat the fluorescence intensity of tryptophan residues decreasedmore remarkably than that of tyrosine residues, which suggestedthat hyperoside mainly bound to the tryptophan residue of BSA.Furthermore, a notable red shift of maximum emission wavelength

1510 Y. Qin et al. / Spectrochimica Acta Part A 75 (2010) 1506–1510

Fig. 5. Synchronous fluorescence spectra of BSA upon addition of hyperoside at 298 K. CB

(e), 25 �M (f), 30 �M (g), 35 �M (h) and 40 �M (i).

Table 4Binding constants Ka of hyperoside-BSA complex in the presence of metal ions at298 K.

Ka (M−1) n r (nm)

BSA–hyperoside 6.36 × 104 1.5550 2.86BSA–hyperoside–K+ 1.05 × 105 2.0489 3.98

2+ 4

wam

3

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BSA–hyperoside–Cu 9.50 × 10 1.9487 3.72BSA–hyperoside–Zn2+ 6.41 × 104 1.5625 3.47BSA–hyperoside–Mg2+ 5.04 × 104 1.3404 2.43BSA–hyperoside–Ca2+ 1.00 × 105 2.0000 3.83

as observed, indicating that the conformation of BSA was alterednd the tryptophan residue was placed in a more polar environ-ent.

.2.5. Effect of metal ions on BSA–hyperoside interactionIt is well known that metal ions existing in plasma may affect

he BSA–drug interaction, which in turn changes the distribution,harmacological property and metabolism of the drug. In our work,e explored how common ions made an impact on the reaction

etween BSA and hyperoside at 298 K. As evidenced from Table 4,he binding constant between BSA and hyperoside increased inhe presence of Cu2+, Ca2+, Zn2+, K+, while decreased with theddition of Mg2+, that is, Cu2+, Ca2+, Zn2+, K+ strengthened theinding of hyperoside to BSA while Mg2+ weakened the binding.he higher binding constants might result from the formation ofompound between metal ions and hyperoside, indicating longertorage time of hyperoside in blood plasma and enhanced max-mum effectiveness of the drug [25]. The lower binding constant

ight contribute to the altering of conformation of BSA evokedy the formation of BSA–ion complex which affected the bind-

ng between BSA and hyperoside. This might lead to the need forore doses of hyperoside to achieve the desired therapeutic effect

26,27].

. Conclusions

The interaction between BSA and hyperoside was investi-ated by fluorescence spectroscopy. The binding parameters werebtained from Stern–Volmer equation and Tachiya model, respec-ively. Both binding sites and apparent binding constants asbtained from Tachiya model increased with the increasing of

emperature. However, for Stern–Volmer equation, the corre-ponding binding constants decreased as temperature increasingnd the binding sites were independent of temperature. The ther-odynamic parameters demonstrated that the binding was a

pontaneous process and hydrophobic force played an important

[[[[

SA = 10 �M. Chyperoside from a to k: 0 �M (a), 5 �M (b), 10 �M (c), 15 �M (d), 20 �M

role. Synchronous fluorescence study suggested that the confor-mation of BSA was altered upon the addition of hyperoside. Thebinding of BSA and hyperoside was strengthened in the presenceof Cu2+, Ca2+, Zn2+ and K+ while was weakened with Mg2+.

Acknowledgements

This work was sponsored by the Scientific Research Com-mon Program of Beijing Municipal Commission of Education (no.KM200810025025) and Funding Project for Academic HumanResources Development in Institutions of Higher Learning Underthe Jurisdiction of Beijing Municipality (no. PHR201007114). Wealso would like to acknowledge the use of resources at Beijing AreaMajor Laboratory of Peptide and Small Molecular Drugs.

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