Inclusion complexes of quercetin with three β-cyclodextrins derivatives at physiological pH: Spectroscopic study and antioxidant activity

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 115 (2013) 854–860

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Inclusion complexes of quercetin with three b-cyclodextrins derivatives at physiological pH: Spectroscopic study and antioxidant activity Min Liu a,⇑, Lina Dong a, Aiju Chen a, Yan Zheng a, Dezhi Sun a, Xu Wang b, Bingquan Wang c a

School of Chemistry and Chemical Engineering, Liaocheng University, Liaocheng 252059, China Zhejiang Medical College, Hangzhou 310053, China c School of Pharmacrutical, Liaocheng University, Liaocheng 252059, China b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 The inclusion interaction of quercetin

with three kinds of b-CDs were investigated.  The thermodynamic parameters of inclusion process were obtained.  The inclusion interactions were enthalpy-driven processes. 1  H NMR provided conclusive evidence regarding the formation of complexation.  The antioxidant activity of quercetin and its inclusion complexes were researched.

a r t i c l e

i n f o

Article history: Received 11 March 2013 Received in revised form 27 May 2013 Accepted 2 July 2013 Available online 10 July 2013 Keywords: Quercetin b-Cyclodextrin Phase-solubility Fluorescence spectra NMR Antioxidant activity

a b s t r a c t Properties of the inclusion complexes of quercetin (QUE) with sulfobutyl ether-b-cyclodextrin (SBE-b-CD), hydroxypropyl-b-cyclodextrin (HP-b-CD), and methylated-b-cyclodextrin (M-b-CD) in tris–HCl buffer solutions of pH 7.40 were investigated. The stoichiometry and thermodynamic parameters for the complexation process (stability constants K, Gibbs free energy change DG, enthalpy change DH and entropy change DS) were determined using phase-solubility and fluorescence spectra analysis. The thermodynamic studies indicated that the inclusion reactions between QUE and the three b-CDs are enthalpy-driven processes. Proton nuclear magnetic resonance spectroscopy indicated that B-ring, C-ring, and part of A-ring of QUE interact with the cavity of b-CDs. The antioxidant activity of QUE and its inclusion complexes were determined by the scavenging of stable radical DPPH. The results showed that the complexed QUE/CDs were more effective than free QUE, with the QUE/SBE-b-CD complex as the best form. Ó 2013 Elsevier B.V. All rights reserved.

Introduction Quercetin (QUE), 3,5,7,30 ,40 - pentahydroxyflavone, is one of the most abundant flavonoids present in fruits and vegetables [1]. Onion ranked highest in quercetin content in a survey of 28 ⇑ Corresponding author. Address: School of Chemistry and Chemical Engineering, Liaocheng University, Liaocheng 252059, Shandong Province, China. Tel.: +86 635 8230612; fax: +86 635 8239880. E-mail address: [email protected] (M. Liu). 1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.07.008

vegetables and nine fruits [2]. Like many flavonoids, QUE is best known for its antioxidant activity [3]; it could help preserve food quality by preventing the oxidative deterioration of lipids. Numerous studies have shown that QUE has many other biological activities that are potentially beneficial to health. Reported health benefits of QUE include cardiovascular protection [4], anti-ulcer effects [5], anti-allergy [6], antiviral [7], anti-inflammatory [8] and antitumor activities [9]. Therefore, QUE has emerged as a major bioactive ingredient with potential applications in many functional foods and pharmaceutical products. However, a major problem

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related to this phytochemical is its poor water solubility, which severely restricts its bioavailability and absorption upon oral administration. The limitation could be overcome by the formation of inclusion complexes with cyclodextrins (CDs). The applications of CDs in the pharmaceutical industry as additives and drug-complexing agents have recently developed rapidly. b-cyclodextrin (b-CD) possesses a rigid cone-shaped structure, which is a cyclic (a-1, 4)-linked oligosaccharide composed of seven a-D-glucopyranose units. The structure can be represented as a hydrophilic outer surface and a hydrophobic inner cavity [10,11]. The hydrophobic cavity of CDs enables them to trap hydrophobic molecules to form host–guest complexes. Thus, CDs have been utilized in food and pharmaceutical industries and has also been used for analytical purposes [12–14]. However, the application of unmodified or unsubstituted b-CD in the pharmaceutical field is limited owing to its poor water solubility and nephrotoxicity [15]. Therefore, modified and relatively safe b-CDs have been synthesized and used in parenteral formulations, such as sulfobutyl ether-b-cyclodextrin (SBE-b-CD) and hydroxypropyl-b-cyclodextrin (HP-b-CD) [16,17]. The inclusion complexation between CDs and insoluble QUE has been studied. Zheng et al. determined the mode of complexation between QUE and b-CD, HP-b-CD or SBE-b-CD in a pH 3.0 buffer solution by phase solubility study and proton nuclear magnetic resonance spectroscopy (1H NMR) analysis [18]. Jullian and co-workers studied the complexation of QUE with b-CD, HPb-CD or SBE-b-CD in water and determined the effect of the complexation process on their antioxidant capacity [19]. However, to the best of our knowledge, no reports exist on the inclusion complexation between poorly soluble QUE molecules and different b-CD derivatives at physiological pH (pH 7.40). The acid–base balance is an important factor for the adjustment of the internal environment in the human body. Many diseases, such as cancer, are closely correlated with acidic pH. Therefore, the bioavailability of drugs, including QUE, in the living body is affected by pH value. The aim of this study was to investigate the complexation of QUE with SBE-b-CD, HP-b-CD, and methylated-b-cyclodextrin (M-b-CD) at physiological pH to improve solubility. This study also aimed to determine the effect of the complexation process on their antioxidant capacity. Phase-solubility analysis, fluorescence spectra, and NMR spectroscopy were employed to determine the stoichiometry, the thermodynamic parameters for the complexation process, and the characterization of the complexation. In addition, the antioxidant activity of the three complexes was determined by the scavenging of the stable free-radical DPPH.

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Phase-solubility measurements Phase-solubility measurements were performed according to the method of Higuchi and Connors [20]. Excess amounts of QUE were added into 5 mL of Tris–HCl buffer solutions (50 mM, pH = 7.40, containing 0.05 M NaCl to maintain ionic strength) containing increasing amounts of HP-b-CD, M-b-CD, and SBE-bCD (from 0 mM to 10 mM). The resulting mixture was equilibrated in an electro-heating standing-temperature cultivator for 24 h at four temperatures (25, 30, 35, and 40 °C). This equilibration time was sufficient for the QUE concentration to reach saturation because no significant concentration changes were observed at 24, 36, 48, and 72 h. The flasks were covered with aluminum foil to minimize photochemical degradation. After equilibrium was reached, suspensions were filtered through a 0.45 lm cellulose acetate membrane filter to remove undissolved solids. An aliquot from each flask was adequately diluted and analyzed on a HP 8453 ultraviolet–visible (UV–vis) spectrophotometer (Hewlett–Packard Company, USA) connected to a CS501-SP Thermo Bath unit (Chongqing Huida Experimental Equipment Co., Ltd., China) kept at 25 °C. The absorbances were measured in triplicate at 373 nm.

Fluorimetric measurements Steady-state fluorescence measurements were performed on an LS 55 spectrofluorimeter (Perkin Elmer, USA). The emission spectrum was recorded in 440 nm to 590 nm intervals at a fixed excitation wavelength of 396 nm. Fluorescence intensities of QUE in the presence or absence of CDs at 526 nm were used to calculate the thermodynamic parameters. Sample concentrations of CDs ranged from 0 mM up to 10 mM, whereas those of QUE were kept at 0.05 mM.

1

H NMR measurements 1

H NMR experiments were performed on a Mercury Plus 400 MHz NMR spectrometer (Varian, Inc., USA). QUE and CDs were dissolved in D2O/DMSO (V:V = 6:4) solution because of the very low solubility of QUE in D2O. TMS was employed as internal standard. The addition of DMSO does not significantly alter the basic model of interaction [21].

Antioxidant activity measurements Experimental section Materials QUE (purity > 99%) was purchased from the National Institute for Food and Drug Control (Beijing, China). SBE-b-CD (total degree of substitution = 6.8), HP-b-CD (average molecular weight = 1460), and M-b-CD (average molecular weight = 1310) were supplied by Shandong Binzhou Zhiyuan Bio-Technology Co., Ltd. (Binzhou, China), Aldrich (St. Louis, MO, USA), and J&K Chemical (Beijing, China), respectively. DPPH (2,2-di (4-tert-octylphenyl)-1-picrylhydrazyl) was purchased from J&K Chemical (Beijing, China). Tris-(hydroxymethyl) aminomethane (Tris, purity P 99%) was obtained from Beijing Biodee Biotechnology Co., Ltd. (Beijing, China). Dimethyl sulfoxide-d6 (DMSO) containing 0.03% (v/v) tetra methyl silane (TMS) was also from Aldrich (St. Louis, MO, USA). Other reagents were all of analytical grade. Deionized and double-distilled water were used throughout the study.

The antioxidant activity was measured by the scavenging of the stable free-radical DPPH, which showed a characteristic absorbance peak at 517 nm in ethanol. The addition of an antioxidant resulted in a decrease in the absorbance proportional to the concentration and antioxidant activity of the compound itself. An ethanolic solution of the radical DPPH was prepared and protected from light. The linear relationship (R2 > 0.997) between radical concentration and absorbance was used as the standard curve. QUE, QUE/SBE-b-CD, QUE/HP-b-CD or QUE/M-b-CD samples of different concentrations in Tris–HCl buffer solutions were added to 60 lM DPPH ethanolic solution. The decrease in absorbance was determined at 517 nm every 15 min until the reaction reached a plateau. The exact DPPH concentration in the reaction medium was calculated from the standard curve. All measurements were performed in triplicate. Antioxidant activity was defined as the amount of antioxidant necessary to decrease the initial DPPH concentration by 50% (EC50). The smaller the EC50, the more efficient the antioxidant is.

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Results and discussion Phase-solubility measurements Fig. 1 shows the phase-solubility diagrams of QUE with three different types of b-CDs in Tris–HCl buffer solutions of pH 7.40. The solubility of QUE linearly increased as a function of SBE-bCD, HP-b-CD, and M-b-CD concentrations. This linear relationship

is a feature of typical AL type complexes, indicating a 1:1 molecular complex formation for the three b-CDs [22,23]. The stability constant (K) is a useful index to estimate the binding strength of host–guest and the changes in the physico-chemical properties of the guest in the complex. The K values of the complexation can be calculated from the phase-solubility diagrams according to the following equation [24]:

K¼

slope S0 ð1  slopeÞ

ð1Þ

where S0 is the solubility of QUE in the absence of CDs and slope indicates the corresponding slope of the phase-solubility diagrams. The values of K are summarized in Table 1. As shown in Table 1, the stability constants of complexes determined from the three b-CD derivatives followed the rank order SBE-b-CD > HP-b-CD > M-b-CD. Jullian et al. [19] and Zheng et al. [18] reported that QUE shows stronger binding with SBE-b-CD than with HP-b-CD [18,19]. This discrepancy may be explained as follows: SBE-b-CD and HP-b-CD are hydrophilic CDs, whereas M-bCD is a hydrophobic CD. For QUE/SBE-b-CD complexation, the stronger ion–dipole interactions between the anions of the outer surface of SBE-b-CD and the hydroxyl groups of QUE lead to a stronger complexation. However, the hydrophobic–hydrophilic interactions between the methyl group of the outer surface of Mb-CD and the hydroxyl groups of QUE are unfavorable to the complexation process. Interestingly, the stability constants of QUE complexes reported in different media are significantly different. Pralhad and Rajendrakumar calculated the stability constants of QUE/HP-b-CD (532 ± 142 M1, 25 °C) in water by phase-solubility method [25]. Zheng et al. reported on the stability constants of QUE/SBE-b-CD (25340 ± 500 M1, 24 °C) and QUE/HP-b-CD (11048 ± 142 M1, 24 °C) at pH 3 using the phase-solubility method [18]. Moreover, Jullian and his coworkers obtained the stability constants of QUE/SBE-b-CD (4032 M1, 30 °C) and QUE/HP-b-CD (1419 M1, 30 °C) in water using the same method [19]. The discrepancy indicates that different buffer aqueous media and temperatures have strong impacts on the complexation process.

Table 1 Stability constants and thermodynamic parameters of QUE inclusion complexations with different CDs at different temperatures obtained by phase-solubility analysis and two processing methods of fluorescence spectra analysis. SBE-b-CD K/(L mol1)

DH/(kJ mol1) DS/(J mol1 K1)

DG/(kJ mol1)

Fig. 1. Phase-solubility diagrams of QUE/b-CD in tris–HCl buffer solutions of pH 7.40 at different temperatures: j (T = 25 °C); d (T = 30 °C); N (T = 35 °C); . (T = 40 °C). (a) SBE-b-CD; (b) HP-b-CD; (c) M-b-CD.

HP-b-CD

25 °C (phase-solubility analysis) 5608 ± 82 3110 ± 38 25 °C (double-reciprocal plot) 4962 ± 24 3616 ± 28 25 °C (nonlinear regression analysis) 5680 ± 26 4000 ± 22 30 °C (phase-solubility analysis) 3627 ± 48 2291 ± 22 35 °C (phase-solubility analysis) 2589 ± 24 1734 ± 10 40 °C (phase-solubility analysis) 1864 ± 12 1298 ± 8 Phase-solubility analysis 56.85 ± 4.38 45.40 ± 1.74 Phase-solubility analysis 119.06 ± 14.34 85.37 ± 5.69 25 °C (phase-solubility analysis) 21.40 ± 0.02 19.94 ± 0.03 30 °C (phase-solubility analysis) 20.66 ± 0.03 19.50 ± 0.02 35 °C (phase-solubility analysis) 20.13 ± 0.02 19.11 ± 0.01 40 °C (phase-solubility analysis) 19.61 ± 0.02 18.66 ± 0.02

M-b-CD 1378 ± 17 1935 ± 14 2137 ± 9 1095 ± 9 855 ± 5 669 ± 3 37.79 ± 1.04 66.54 ± 3.40 17.92 ± 0.03 17.64 ± 0.02 17.30 ± 0.01 16.94 ± 0.01

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Hydroxyl groups of QUE molecules are sequentially dissociated into anions with the elevation of pH values [26]. The QUE anions exhibit much weaker binding with CD derivatives due to their enhanced hydrophilic character. Thus, the stability constants of QUE/SBE-b-CD (5599 ± 89 M1, 25 °C) and QUE/HP-b-CD (3145 ± 41 M1, 25 °C) at pH 7.40 are much smaller than those obtained by Zheng et al. at pH 3. Therefore, investigation on the inclusion complexation between QUE and the different b-CD derivatives at physiological pH and different temperatures is important. The integrated form of van’t Hoff equation (Eq. (2)) was employed to obtain more thermodynamic information by calculating relevant thermodynamic parameters for the complexation process, i.e., Gibbs free energy change (DG), enthalpy change (DH), and entropy change (DS) [27].

ln K ¼ 

DH DS þ RT R

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molecules (positive contribution to DS) [30]. A negative entropy change indicates that the first effect is more pronounced. Therefore, the inclusion reactions are concluded as enthalpy-driven processes based on the large negative values of DH and the slightly negative values of DS.

Fluorescence spectra The fluorescence spectra of QUE (0.05 mM) in pH 7.40 buffer solutions with varying concentrations of the three kinds of b-CDs (SBE-b-CD, HP-b-CD, and M-b-CD) were recorded. The exemplary

ð2Þ

The van’t Hoff plots (i.e., ln K versus 1/T) for the complexes are linear. The relevant thermodynamic parameters are listed in Table 1. The replacement and removal of solvation CDs and water molecule of the guest are the essence of the formation of the host– guest inclusion complex between CDs and the guest molecule in aqueous solutions. The process includes the release of enthalpyrich water from the hydrophobic cavity of CDs to bulk mass water and the replacement of the vacated site with the low-enthalpy guest molecule [28]. The main factors used to subserve the formation of the complexation are electrostatic interactions, van der Waals interactions, hydrophobic interactions, hydrogen bonding with the OH groups at the periphery of the cavity, exclusion of cavity-bound high-energy water, and electric charge transfer, among others [29]. The formation of complexation is often accompanied by evident heat quantity change, which is the resultant effect of the indicated interactions. The negative values of DH indicate that the formation of host– guest inclusion complexes is exothermic, whereas the negative values of DG reflect a spontaneous process. The largest negative value of DH for QUE/SBE-b-CD complex may be attributed to the strong ion–dipole interaction (negative contribution to DH), which is consistent with the conclusion obtained from stability constant K. The data in Table 1 show that the standard entropy changes (DS) are also negative. The two opposite effects are generally accepted to contribute to DS during the process of complexation. The first effect is the loss of rotational and translational degrees of freedom of the free guest and the host molecules (negative contribution to DS). The second effect is the release of the highly ordered solvent molecules surrounding the host and guest

Fig. 2. Fluorescence emission spectra of QUE in absence and presence of different bCDs (1.0  103 mol L1) in tris–HCl buffer solutions of pH 7.40. (a) QUE; (b) QUE/ SBE-b-CD; (c) QUE/M-bCD; (d) QUE/HP-bCD.

Fig. 3. Nonlinear curve-fitting plots of the variation in fluorescence intensity as a function of b-CD concentration. inset: the double-reciprocal plots of the same data. (a) SBE-b-CD; (b) HP-b-CD; (c) M-b-CD.

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fluorescence spectra shown in Fig. 2 corresponds to the concentration of 1.0  103 mol L1 for the three b-CDs. As shown in Fig. 2, the addition of CDs produces a significantly increase in the fluorescence intensity of quercetin with a bathochromic shift. The emission maxima of quercetin were shifted from 526 nm to 535 nm, 541 nm and 543 nm in the presence of SBE-b-CD, HP-b-CD and M-b-CD, respectively. The bathochromic shift has been detected in the inclusion complexation of b-CD and its derivatives [31– 33]. The larger red shift may be due to the hydrogen bonding interaction between the hydroxyl groups of QUE and the hydroxyl groups present in the outer rim of the three b-CD derivatives [31]. In addition, when QUE is entrapped in the CDs cavity, this microenvironment with smaller polarity and stronger rigidity would restrict the freedom of guest molecules and increase the fluorescence intensity [34,35]. The stoichiometry and stability constants can be obtained using the modified Benesi–Hildebrand equation (double-reciprocal plot) [36]:

1 1 1 ¼ þ F  F 0 F 1  F 0 K½F 1  F 0 ½Hn0

ð3Þ

where [H]0 signifies the initial concentrations of CDs. F and F0 represent the fluorescence intensities of QUE in the presence and absence of total added CD concentration, respectively. F1 is the fluorescence intensity of QUE with the highest concentration of CDs. K is the binding constant for QUE/CD complex, and n represents the stoichiometry of the formed complex. The double-reciprocal plots of SBE-b-CD, HP-b-CD, and M-b-CD for n = 1 are shown in Fig. 2a–c (inset), respectively. For the three CDs, the n = 1 plots exhibited good linearity, whereas the n = 2 plots (data not shown) deviated from the linearity. From these results, QUE can be inferred to form 1:1 inclusion complexes with the three substituted CDs, which coincided with the results obtained by phase-solubility measurement. The stability constants can be estimated by dividing the intercept by the slope of the double-reciprocal plots. The stability constants of the complexation at 25 °C are provided in Table 1. The nonlinear regression is a more precise method for the determination of complex stability constant [37]. The equation of nonlinear regression is as follows: 8 sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi9    2 = 1< 1 1 DF ¼ DeF ½H0 þ ½G0 þ  4De2F ½H0 ½G0  De2F ½H0 þ ½G0 þ ; 2: K K ð4Þ

where DF is the change of fluorescence intensity (at 526 nm) with the addition of different concentrations of CDs, i.e., DF = F  F0. DeF is the change in molar fluorescence intensity, which can be obtained from nonlinear regression analysis together with the stability constant K using MATLAB 7.01. The curve-fitting plots for SBEb-CD, HP-b-CD, and M-b-CD are shown in Fig. 2a–c, respectively. The figures showed good curve-fitting plots, which verified the 1:1 complex stoichiometry between QUE and the three kinds of bCDs. The calculated results are summarized in Table 1. Although the data used in the two processing approaches are the same, evident disparities still exist in the calculated values of K due to the hypothesis of [H]0  [G]0 in the double-reciprocal plot. However, the general rank order and order of magnitude of stability constants are consistent, that is, the rank order of stability constants is SBE-b-CD > HP-b-CD > M-b-CD, which is consistent with that obtained by the method of phase-solubility. 1

H NMR

NMR spectroscopy is a powerful tool for the investigation of the formation of complexation. Generally, UV–vis and fluorescence spectra analysis only indicate that the entire guest molecule or a part of it is included by the host. Consequently, NMR spectroscopy is required to supply more conclusive evidence regarding the relative orientation of the guest in the hydrophobic cavity of the host (i.e., the co-conformation phenomenon). The chemical shifts of CDs are not reliable for NMR characterization because of their degree and position of substitution [18]. Therefore, only the proton chemical shifts of the guest were considered in this study. The 1H NMR spectra of QUE in the absence and presence of CDs are presented in Fig. 3. Each proton position of QUE was assigned according to previous reports [18]. Fig. 4 shows the molecular structure of QUE and the detailed change of the 1H chemical shifts (DD) of QUE. The data were calculated using the equation:

Dd ¼ dðcomplexationÞ  dðguestÞ

ð5Þ

where d(complexation) and d(guest) are the 1H chemical shift of the complexation and the guest, respectively. The average of the triplicate values for each sample was its final result. As shown in Figs. 3 and 4, the presence of CDs caused an upfield shift (H20 , H60 , and H50 ) and a downfield shift (H8 and H6) in the protons. Although the addition of DMSO may reduce the binding strength of the QUE/b-CD complex, a general trend was revealed

Fig. 4. Chemical shifts of aromatic protons of QUE in the absence and presence of different b-CDs in D2O/DMSO (V:V = 6:4) solution.

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groups, which in turn makes the hydrogen donation by the hydroxyl groups of QUE becoming easier. In contrast, the hydrophobic methyl groups present in the outer rim of M-b-CD impair the hydrogen-bonding interaction between QUE and M-b-CD. This is unfavorable to the hydrogen-donating ability of QUE. So the antioxidant activity of QUE/SBE-b-CD complex is stronger than that of QUE/M-b-CD, which is consistent with the binding ability of the three b-CD derivatives. Therefore, we can conclude that the antioxidant property of QUE/b-CD complex is closely related their mode of binding.

Conclusions

Fig. 5. Chemical shifts change of aromatic protons of quercetin in the presence and absence of b-CDs in D2O/DMSO (V:V = 6:4) solution. h (QUE/SBE-b-CD); j (QUE/ HP-b-CD); P (QUE/M-b-CD). inset: molecular structure of QUE.

by the change in the chemical shift. On one hand, the chemical shift change of H60 is largest, whereas that of H6 is smallest, suggesting that B-ring, C-ring, and part of A-ring interact with the hydrophobic cavity of CDs. On the other hand, the chemical shift change of SBE-b-CD is most prominent compared with the other two CDs. The results are in good agreement with those of UV–vis and fluorescence spectra analyses. Antioxidant activity of quercetin in free and complex form Fig. 5 shows a comparison of the antioxidant activity of QUE, QUE/SBE-b-CD, QUE/HP-b-CD, and QUE/M-b-CD as measured by the DPPH scavenging capacity assays. The order of the calculated EC50 is QUE (47.6 g kg1) > QUE/M-b-CD (44.6 g kg1) > QUE/HP-bCD (13.7 g kg1) > QUE/SBE-b-CD (12.8 g kg1), which indicates that the complexed QUE/CDs are more effective than free QUE, with the QUE/SBE-b-CD complex as the best form. The DPPH radical-scavenging ability of an antioxidant is believed to be closely associated with its hydrogen-donating ability [38]. The increase in the antioxidant activity of QUE in complex form with CDs is probably attributed to the enhancement of its hydrogen-donating capacity caused by complexation. When QUE is complexed with CDs, one or more intermolecular hydrogen bonds form between QUE and CDs, which weaken the intramolecular hydrogen bonds of QUE. In turn, the hydrogen-donating ability of QUE is improved (see Fig. 6). The strongly binding between QUE and SBE-b-CD weakens the covalent bonds between hydrogen and oxygen in the hydroxyl

Fig. 6. The calculated values of EC50 for free QUE and its complexes with SBE-b-CD, HP-b-CD, and M-b-CD.

The inclusion complexes formed between QUE and the three bCDs (SBE-b-CD, HP-b-CD, and M-b-CD) in tris–HCl buffer solutions of pH 7.40 were studied. The inclusion process altered the spectral features of QUE, as reflected by UV–vis, fluorescence spectra, and 1 H NMR. The stoichiometry and the thermodynamic parameters (K, DG, DH, and DS) for the complexation process were determined using phase-solubility analysis. Bathochromic shifts were observed in the presence of CDs in the fluorescence spectra. The stoichiometry and K obtained by double-reciprocal plot and nonlinear regression employed in the fluorescence spectra analysis were in accordance with those obtained by phase-solubility analysis. 1H NMR results indicated that the B-ring, C-ring, and part of the A-ring of QUE were most likely inserted into the cavity of the CDs. Antioxidant activity analysis showed that the formation of an inclusion complex enhanced the antioxidant capacity of QUE, and QUE/ SBE-b-CD complex was the most effective form. Acknowledgments Natural Science Foundation of China is thanked for Funding (No. 21103079). This work was also supported by Innovation Program for Graduate Education of Shandong Province (SDYC 10044/LCUYZ 10008), Tai-Shan Scholar Research Fund of Shandong Province, and Natural Science Foundation of Zhejiang Province (LQ12B03001). References [1] R. Apak, K. Güçlü, B. Demirata, M. Özyürek, S.E. Çelik, B. Bektasog˘lu, K. Isıl Berker, D. Özyurt, Molecules 12 (2007) 1496–1547. [2] M.G.L. Hertog, P.C.H. Hollman, M.B. Katan, J. Agri. Food Chem. 40 (1992) 2379– 2383. [3] S. Karakaya, S. Nehir EL, Food Chem. 66 (1999) 289–292. [4] J.V. Formica, W. Regelson, Food Chem. Toxicol. 33 (1995) 1061–1080. [5] F. Sánchez de Medina, J. Gárlvez, M. González, A. Zarzuelo, K.E. Barrett, Life Sci. 61 (1997) 2049–2055. [6] A. Kahraman, N. Erkasap, T. Köken, M. Serteser, F. Aktepe, S. Erkasap, Toxicology 183 (2003) 133–142. [7] E. Ohnishi, H. Bannai, Antivir. Res. 22 (1993) 327–331. [8] T. Guardia, A.E. Rotelli, A.O. Juarez, L.E. Pelzer, Il Farmaco 56 (2001) 683–687. [9] R. Khonkarn, S. Mankhetkorn, W.E. Hennink, S. Okonogi, Eur. J. Pharm. Biopharm. 79 (2011) 268–275. [10] C. Tablet, M. Hillebrand, Spectrochim. Acta A 70 (2008) 740–748. [11] S. Yousuf, D. Radhika, I.V.M.V. Enoch, M. Easwaran, Spectrochim. Acta A 98 (2012) 405–412. [12] A.A. Abdel-Shafi, S.S. Al-Shihry, Spectrochim. Acta A 72 (2009) 533–537. [13] M. El-Kemarya, S. Sobhya, S. El-Dalyb, A.A. Abdel-Shafi, Spectrochim. Acta A 79 (2011) 1904–1908. [14] A.A. Abdel-Shafi, Spectrochim. Acta A 66 (2007) 1228–1236. [15] M.E. Brewster, T. Loftsson, Adv. Drug Deliver. Rev. 59 (2007) 645–666. [16] V.J. Stella, R.A. Rajewski, Pharm. Res. 14 (1997) 556–567. [17] C. Tablet, L. Minea, L. Dumitrache, M. Hillebrand, Spectrochim. Acta A 92 (2012) 56–63. [18] Y. Zheng, I.S. Haworth, Z. Zuo, M.S.S. Chow, A.H.L. Chow, J. Pharm. Sci. 94 (2005) 1079–1089. [19] C. Jullian, L. Moyano, C. Yañez, C. Olea-Azar, C. Spectrochim. Acta A 67 (2007) 230–234. [20] T. Higuchi, K.A. Connors, Adv. Anal. Chem. Instrum. 4 (1965) 117–212. [21] E. Junquera, D. Ruiz, E. Aicart, J. Colloid Interface Sci. 216 (1999) 154–160. [22] J.H. Shi, Y.F. Zhou, Spectrochim. Acta A 83 (2011) 570–574.

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