Enhancement of Solubility and Bioavailability of Quercetin by Inclusion

Article DOI: 10.1002/bkcs.11192

K. H. Park et al.

BULLETIN OF THE KOREAN CHEMICAL SOCIETY

Enhancement of Solubility and Bioavailability of Quercetin by Inclusion Complexation with the Cavity of Mono-6-deoxy-6aminoethylamino-β-cyclodextrin Kyeong Hui Park,†,k Jae Min Choi,‡,k Eunae Cho,‡ Daham Jeong,† Vijay Vilas Shinde,‡ Hyungsup Kim,§ Youngjin Choi,¶ and Seunho Jung†,‡,* †

Department of Systems Biotechnology, Microbial Carbohydrate Resource Bank (MCRB), Center for Biotechnology Research in UBITA (CBRU), Konkuk University, Seoul 05029, Republic of Korea. *E-mail: [email protected] ‡ Institute for Ubiquitous Information Technology and Applications (UBITA), Center for Biotechnology Research in UBITA (CBRU), Konkuk University, Seoul 05029, Republic of Korea § Department of Organic and Nano System Engineering, Konkuk University, Seoul 05029, Republic of Korea ¶ Department of Food Science and Technology, BioChip Research Center, Hoseo University, Asan 31499, Republic of Korea Received May 19, 2017, Accepted June 16, 2017 Quercetin (QUE) has a wide range of health benefits; however, its application is limited due to its low solubility. We synthesized mono-6-deoxy-6-aminoethylamino-β-cyclodextrin (Et-β-CD) to overcome this limitation. The solubility of QUE was increased 35.1-fold compared with QUE, by its complexation with Et-β-CD. Changes in physicochemical properties following successful complexation were investigated using field emission scanning electron microscope, differential scanning calorimetry, and Fourier transform infrared spectroscopy. The complexation behavior of QUE and Et-β-CD in aqueous solution was monitored by 1H nuclear magnetic resonance (NMR), 2D rotating frame nuclear overhauser, and diffusion-ordered spectroscopy. The molecular docking simulation showed that the oblong shaped QUE is suitable for the formation of a stable complex with a characteristic cavity of Et-β-CD. Furthermore, the antioxidant activity and photostability of QUE was also improved after its complexation with Et-β-CD. From these results, we suggest that the characteristic elliptical cavity of Et-β-CD can be utilized for other hardly soluble drug molecules, which can expand the development of drug delivery systems. Keywords: Quercetin, Mono-6-deoxy-6-aminoethylamino-β-cyclodextrin, Solubilization, Antioxidant, Photostability

Introduction Quercetin (QUE) is one of the most abundant bioflavonoids in the human diet.1 QUE is a 3,5,7,30 ,40 pentahydroxyflavone that belongs to the flavonol class. QUE has been extensively studied over the years,2–5 and is well-known as an antioxidant molecule that can be used in the preservation of the foods.6 QUE has many beneficial properties including cardiovascular protection,7 anti-ulcer effects,8 anti-allergy,9 antiviral,10 anti-inflammatory,11 and antitumor activities.12 Despite the potential uses of QUE, its low aqueous solubility limits its applications. Various methods for improving drug solubility, such as the micronization of drug molecules,13 drug salt formation,14 co-solvency with alcohol or surfactants,15 and complexation with host molecules have been reported.16,17 Especially, host–guest complexation has gained much k

These authors contributed equally to this work.

Bull. Korean Chem. Soc. 2017

attention in the areas of chemistry, physics, biology, and materials science.18 In host–guest complexation, various non-covalent interactions are involved, including hydrogen bonds, van der Waals forces, electrostatic interactions, and hydrophobic interactions.18 Cyclodextrins (CD) are widely used as host molecules that are produced through the treatment of amylose with CD glucosyltransferase. They are cyclic α(1-4)-linked glucans, most commonly composed of six, seven, or eight glucose units known as α-, β-, and γ-CD, respectively. CDs have a truncated cone shape with narrow and wide rims, and guest molecules can enter the cavity and form inclusion complexes with these host molecules via non-covalent interactions.19 Although the applications of β-CD are widely used in the pharmaceutical industry, they are limited by the poor water solubility of β-CD itself.20 In addition, the main factor in host–guest complexation, is the shape and size of the CD cavity. During complex formation, the guest is positioned within the cavity of the host CD,

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and the complex is formed through a dimensional fit between the host cavity and the guest drug. The rigidity and the limited cavity size of β-CDs was often the obstacle in capturing large drug molecules.21 To overcome these limitations, we synthesized mono-6-deoxy-6-aminoethylamino-β-cyclodextrin (Et-β-CD), and utilized it as the solubilization agent for the hardly soluble flavonoid QUE. In our previous study, we showed the effect of the monoaminoethylamine substitution on the degree of distortion in the conformation of the circular cavity. We proposed that Et-β-CD could host a three-dimensionally extended oblong shaped guest molecule, within a newly created oval-shaped wide rim.22 The inclusion complexation between β-CD derivatives and QUE has been previously investigated.23–26 The binding mode of complexation between QUE and β-CD, 2hydroxypropyl-β-CD (HP-β-CD) or sulfobutyl ether-β-CD (SBE-β-CD) in a buffer solution at pH 3.0 was examined by proton nuclear magnetic resonance spectroscopy (1H NMR).27 Another study investigated the thermodynamic parameters of complexes formed between QUE and HPβ-CD, SBE-β-CD, and heptakis-(2,6-di-O-methyl)-β-CD (DM-CD).6 The host–guest binding mode of the complex was proposed using solid and liquid high-resolution NMR spectroscopy. It was reported that the complexation occurs in the aromatic ring A (bearing the two phenolic hydroxyl groups in a meta position to each other) rather than in aromatic the ring B (bearing the two phenolic hydroxyl groups in an ortho position to each other).28 However, to the best of our knowledge, there are no comprehensive reports on the binding mode between the poorly soluble QUE molecules and host molecules using inclusion complexation. In our study, the complex of QUE with Et-β-CD was analyzed using various NMR spectroscopy analyses, such as 1H NMR spectroscopy, 2D rotating frame nuclear Overhauser spectroscopy (ROESY), and diffusion-ordered spectroscopy (DOSY). The results of the different NMR spectroscopy analyses indicated that the protons at B ring of QUE are included in Et-β-CD, which suggests that the wider rim of Et-β-CD can effectively capture whole QUE structures. The ROESY-driven molecular docking study also showed that the molecular shape of QUE led to the formation a stable complex with Et-β-CD via strong hydrogen bonding. The improvement in the bioavailability of QUE with Et-β-CD complex was also confirmed by a photostability, and oxygen radical absorbance capacityfluorescein (ORAC-FL) assay. Furthermore, the physicochemical changes in the properties of QUE after complexation were measured using scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and Fourier-transform infrared (FT-IR) spectroscopy. The successful complexation of QUE with Et-β-CD led to a 35.1-fold increase in the solubility of QUE. Based on these results, we propose that Et-β-CD can be potentially utilized as a host for other hardly soluble guest drugs. Bull. Korean Chem. Soc. 2017

BULLETIN OF THE KOREAN CHEMICAL SOCIETY Experimental

Materials QUE dehydrate, 2-Hydroxypropyl-β-CD (HP-β-CD, Mn 1,460 Da, 0.8 molar substitution), Heptakis-(2,6-di-Omethyl)-β-CD (DM-β-CD, Mn 1,331.36 Da), 2,20 -Azobis (2-methylpropionamidine) dihydrochloride (AAPH), and fluorescein were purchased from Sigma-Aldrich Chemicals Co. (St. Louis, Mo, USA). β-CD (>95.0% [High Performance Liquid Chromatography HPLC], Mw 1134.99 Da), 1-(p-toluenesulfonyl) imidazole, ethylenediamine, and 6hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were obtained from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). SBE-β-CD (total degree of substitution = 6–7; Captisol®) was purchased from Cydex, Inc. (Lenexa, KS, USA). Deionized water was produced with a Milli-Q system from Millipore (Saint-Quentin-enYvelines, France). All other chemicals used in the study were of analytical grade, and were used as received. Synthesis of Mono-6-deoxy-6-aminoethylamino-β-cyclodextrin. Et-β-CD was synthesized according to a reported procedure.29 Briefly, Et-β-CD synthesis was started from mono-6-O-p-toluenesulfonyl-β-CD (Tosyl-β-CD) synthesis. Tosyl β-CD was obtained from the Microbial Carbohydrate Resource Bank (MCRB) at Konkuk University, Korea. Tosyl-β-CD was synthesized according to a previously reported method.30 Tosyl-β-CD was dissolved in ethylenediamine at 75 C under nitrogen for 12 h. The mixtures were evaporated to a constant volume in vacuo, and precipitated with acetone. The resultant samples were purified on a CM-Sephadex C-25 column with 500 mM NH4HCO3 solution, and distilled water was used as the eluent. The separated samples were desalted using a Bio-Gel P-2 column. MALDI-TOF MS: m/z 1177 [M + H]+. 1H NMR (600 MHz, 60 % d6-DMSO/D2O): δ4.82 (s, 7H, H1), 3.683.56 (m, 28H, H3, H5 and H6), 3.38-3.56 (m, 14H, H2 and H4), 2.86-2.60 (br, 4H, H60 and H7), and 2.59-2.53 (m. 2H, H8). 13C NMR (600 MHz, 60 % d6-DMSO/D2O): δ102.54 (C1), 84.13 (C40 ), 82.22 (C4), 73.65 (C3), 72.70 (C2), 71.11 (C5), 60.76 (C6), 49.32 (C60 ), and 48.51 (C7, C8). Preparation of the Inclusion Complex. The inclusion complexes of QUE with Et-β-CD were prepared by freezedrying at a 1:1 molar ratio. Equimolar concentrations of QUE and Et-β-CD were added to distilled water, and then stirred at 25 C for 24 h in the dark. After reaching equilibrium, the samples were filtered through a PTFE 0.20 μm filter (Advantec MFS, Inc., Dublin, CA, USA) to remove uncomplexed QUE. The filtered solution was freeze-dried. Preparation of the Physical Mixture. The physical mixture was prepared by grinding QUE and Et-β-CD in a molar ratio of 1:1. Phase Solubility Studies. Phase solubility studies were performed according to the methods reported by Higuchi


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Solubilization of Querctin by Complex

and Conners.31 An excess amount of QUE (3 mM) was added to an aqueous solution containing an increasing amount of β-CD, HP-β-CD, DM-β-CD, SBE-β-CD, and Etβ-CD (from 0 to 5 mM). The mixtures were stirred in the dark at 25 C for 24 h. After filtration, the solubilized QUE was measured using a UV–vis spectrophotometer (UV 2450, Shimadzu Corporation, Kyoto, Japan) at a wavelength of 254 nm. Experiments were performed in triplicate. The apparent stability constants (k1:1) of the inclusion complex were calculated based on the phase solubility diagrams (PSD) according to the following equation: k1:1 =

slope S0 ð1− slopeÞ

ð1Þ

The solubilization efficiency (Se) was derived as the increment in the apparent solubility of QUE in the presence of host cyclic oligosaccharides (Shost) with respect to the solubility of QUE in the absence of CD (S0): Se =

Shost S0

ð2Þ

Differential Scanning Calorimetry. DSC analyses of QUE, Et-β-CD, physical mixtures of QUE/Et-β-CD, and of the QUE/Et-β-CD inclusion complex were carried out using the Q600 SDT (TA Instrument, New Castle, DE, USA). The operating conditions were a scanning rate of 10 C/min in a temperature range of 30–350 C, under a nitrogen flow of 100 mL/min. Fourier-transform Infrared Spectra. FT-IR spectra of QUE, Et-β-CD, the QUE/Et-β-CD physical mixtures, and the QUE/Et-β-CD inclusion complex were recorded using VERTEX 80 V (Bruker GmbH, Karlsruhe, Germany) in the scanning range of 500–4000 cm−1. The resolution was 4 cm−1. Samples were prepared in KBr disks using hydrostatic pressure. Field Emission Scanning Electron Microscopy (FESEM). FE-SEM was recorded using a Hitachi S-4700 (Hitachi High-Technologies Corporation, Tokyo, Japan). The powdered samples were fixed on a brass stub using double-sided adhesive carbon tape. The fixed samples were coated with a thin layer of gold. The images were recorded at an excitation voltage of 5 kV. NMR Spectroscopy 1 H NMR Spectroscopy. The 1H NMR experiments were performed using a Bruker Avance 600 MHz spectrometer (Bruker GmbH, Karlsruhe, Germany) in a D2O solvent, at 25 C. Rotating Frame Nuclear Overhauser Spectroscopy. The 2D 1H-1H ROESY spectrum of the inclusion complex of QUE with Et-β-CD was recorded using 256/2048 complex data points, and a pulse train to attain a spin-lock field with a mixing time of 700 ms for the complex. The NMR Bull. Korean Chem. Soc. 2017


analyses were carried out on the Bruker Avance 600 MHz spectrometer in D2O solvent at 25 C. Molecular Modeling. The structural model for Et-β-CD was taken from a previous study on Et-β-CD, using a molecular modeling tool.22 In brief, a mixed torsional/lowmode sampling method was applied to the conformational search module in the Maestro software (ver. 2016-1, Schrodinger Inc.). The final Et-β-CD conformation was used as the host structure for molecular docking using QUE as the guest molecule. The β-CD model that was used for the crystallographic structure was obtained from Protein Data Bank (PDB id 3CGT). Ligand-flexible molecular docking simulations of QUE were conducted using the Glide tool32 for each CD. The initial coordinate of the QUE was prepared using the molecular builder tool in the Maestro software. The molecular grid was defined for each β-CD and Et-β-CD using the Receptor Grid Generation tool implemented in Glide. For both CD models, a cubic grid was configured with a box size of 20 Å. Molecular docking was performed using the extra precision (XP) mode, and the Glide XP 5.0 scoring function to estimate the accurate binding mode and data. In this docking mode, an energy window of 2.5 kcal/mol and a distance-dependent dielectric constant (ε = 1) was applied during pose sampling. To generate the initial docked pose of QUE upon β-CD and Et-β-CD binding, a maximum of 100 000 poses was considered for the initial phase of docking, and the best 3,000 poses were kept for the energyminimization process. The final docked structures were recorded, and up to 10 poses were retained. Photostability Studies. QUE photostability was measured under UVA irradiation (H125-BL, 125 W, Interlight, Hammond, IN, USA), at a maximum emission wavelength of ca. 360 nm. The QUE and QUE/ Et-β-CD samples were prepared in an ethanol/water (15/85 v/v) solution. The sample, consisting of 100 μM of QUE solution was located 10 cm from the light source, and the path length of the quartz cells was 1 cm. The samples were exposed to UVA radiation at different time intervals over 24 h. Each sample was tested in triplicate. Then, the samples were analyzed using a UV-2450 spectrophotometer (UV 2450, Shimadzu Corporation, Kyoto, Japan). Antioxidant Activity. The antioxidant activities of free QUE, β-CD, HP-β-CD, DM-β-CD, SBE-β-CD, Et-β-CD, and QUE complexed with CD derivatives were analyzed using the ORAC-FL assay.33 The ORAC assay was performed in a fluorescence microplate reader (Gemini EM; Molecular Devices, Sunnyvale, CA, USA) using clear bottom black 96-well polystyrene microplate. Fluorescence was read through the clear bottom, at an excitation wavelength of 485 nm, with an emission filter of 528 nm. The reaction was performed in 75 mM sodium phosphate buffer (pH 7.4). The volume of the final reaction mixture was 200 μL per well. Next, 100 μL of fluorescein (3 nM, final concentration) and 70 μL of the samples (17.5 μM, final concentration) were added to the wells and incubated at



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37 C. After 30 min, 30 μL of AAPH solution (127 mM, final concentration) in phosphate buffer (pH 7.4) was quickly added to the mixture. The microplate was placed in the reader immediately, and the fluorescence was recorded every 1.5 min for 2 h. The blank was prepared with fluorescein and AAPH with buffer. Trolox was used as the reference standard; the inhibition capacity was expressed as Trolox equivalents (μM), and was quantified by integrating the area under the curve (AUC). All reaction mixtures were prepared in triplicate. The results were expressed as μM of Trolox equivalents, by using the Trolox calibration curve. The area under the fluorescence decay curve was calculated using the following Eq. (3): AUC = 1 +

iX = 120 i=0

fi f0

ð3Þ

where, f0 is the initial fluorescence read at 0 min, and fi is the fluorescence read at time i. The net AUC was determined by subtracting the AUC of the blank from that of the sample. The relative ORAC value (Trolox equivalents) was calculated using the Eq. (4):

AUCsample − AUCblank ½Trolox × ORAC value = ðAUCTrolox − AUCblank Þ ½sample

ð4Þ

Figure 1. Chemical sketches of (a) mono-6-deoxy-6-aminoethylamino-β-cyclodextrin (Et-β-CD) and (b) quercetin (QUE).

where AUCsample is the AUC in the presence of the tested compounds; AUCblank is the AUC in the absence of the tested compounds; AUCTrolox is the area under curve in the presence of Trolox, and [Trolox] and [sample] are the molar concentrations of Trolox and the tested compounds, respectively.34

correlated with the C7 and C8 carbons. These structural analyses indicate that one ethylenediamine residue was successfully substituted at the C6 position in the native β-CD. Previous study of ours, the stable hydrogen bondings between ethylenediamine moiety and the primary hydroxyls of the β-CD can cause the distortion of the circular cavity to a novel induced oval-shaped one inside of the Et-β-CD which proved by the 2D-NOESY NMR analysis and molecular modeling study. The cross peaks between the protons H40 , H5, and H50 of Et-β-CD and the protons of modified ethylenediamine moiety (H60 and H8) of Et-β-CD were appeared in 2D-NOESY NMR analysis. Also the molecular docking study result based on the NOESY spectroscopic analysis, stable hydrogen bonding between diamine moiety and the primary hydroxyls of the Et-β-CD can cause distortion from its circular form to the an oval-shaped one.22

Results and Discussion Characterization of Et-β-CD. The synthesized Et-β-CD (Figure 1(a)) was confirmed using the heteronuclear single quantum coherence (HSQC) spectrum (Figure S1, Supporting information). In Figure S1, F2 represents the 1H NMR spectrum, where the H1–H6 protons of the glucopyranose units are assigned at 4.82, 3.38, 3.66, 3.33, 3.55, and 3.66 ppm, respectively. The substituted ethylene protons of H7 and H8 appeared at 2.67 and 2.61 ppm. Furthermore, the 2:7 integral ratios of the H8 protons to the H1 protons confirmed the mono-functionalization of β-CD. F1 indicates the 13C NMR chemical shifts of Et-β-CD, which were assigned as C1 and C4 at 102.12 and 81.94 ppm, respectively. The 13C NMR chemical shifts of C2, C3, and C5 were observed at 72.70, 73.65, and 71.11 ppm, respectively. The substituted C7 and C8 carbons appeared at 49.32 ppm. More specifically, the substituted C60 carbon was shifted upfield to 49.32 ppm, compared with the C6 carbon (60.43 ppm) with the free OH group, at which point the H60 resonances attached to the C60 carbon were correlated. The newly observed H7 and H8 resonances were also Bull. Korean Chem. Soc. 2017

Phase Solubility Study. The solubility enhancement of QUE (Figure 1(b)) using host cyclic oligosaccharides was studied in aqueous solution at 25 C, based on the method of Higuchi and Connors.35 In the PSD in Figure 2, QUE in the Et-β-CD complex displayed an AL-type slope, and the solubility of QUE increased linearly as a function of the Etβ-CD concentration. This indicated that the QUE/Et-β-CD complex is formed with a 1:1 stoichiometry. The stability constants of QUE with various cyclic oligosaccharides were calculated with Eq. (1). The stability constants of the complexes are listed in Table 1. The order of the stability



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Figure 2. Phase solubility diagrams of QUE as a function of Et-β-CD ( ), sulfobutyl ether-β-CD (SBE-β-CD) ( ), heptakis(2,6-di-O-methyl)-β-CD (DM-β-CD) ( ), 2-hydroxypropyl-β-CD (HP-β-CD) ( ) and β-CD ( ) in water at 25 C.

constants of QUE complexes was Et-β-CD > SBE-β-CD > DM-β-CD > HP-β-CD > β-CD. The stability constant of QUE/Et-β-CD had the highest value at 9234.0 M−1 compared with the stability values for other cyclic oligosaccharides. This value was 2.3-fold higher than the previously reported stability constant of QUE/SBE-β-CD.27,36 The solubility of QUE was enhanced up to 35.1-fold by complexation with 5 mM Et-β-CD, whereas the β-CD and β-CD derivatives led to more moderate increases in the aqueous solubility of QUE. This tendency suggests that the molecular size of QUE and the different cavity size of the CD derivatives can affect the binding affinity when forming the inclusion complex. The steric effect depends on the size of the CD relative to the size of the guest molecule. This is a major factor that can influence the formation of the inclusion complex. If the cavity in the CD derivative is improperly sized for QUE, CDs cannot capture QUE perfectly.37 Therefore, the low stability constants of SBE-β-CD, DMβ-CD, HP-β-CD, and β-CD complexes are possibly due to the CD cavities being too small to capture QUE molecules. Differential Scanning Calorimetry. DSC is a useful technique for the characterization of solid state interactions between a host and a guest molecule.38 When a guest


molecule is embedded in the CD cavity, its phase transition points, such as its melting point, boiling and sublimation points shift or disappear, if they occur within the temperature range where the CD cavity is stable.39 The thermal behaviors of QUE, Et-β-CD, the QUE/Et-β-CD physical mixture, and the QUE/Et-β-CD complex were investigated by DSC (Figure 3). The sharp endothermic peak in the DSC curve of QUE was observed at 324.9 C, which corresponds to the melting point of QUE. In the thermogram, Et-β-CD exhibited a broad endothermic peak at 65.15 C, which originated from the dehydration process. In the DSC spectrum of the physical mixtures of QUE/Et-β-CD, the endothermic peaks of QUE appeared shifted at 308.0 C; this shift might be attributed to the solid–solid interaction.40 However, in the DSC curve of the QUE/Et-β-CD complexes, the endothermic peaks corresponding to the free QUE disappeared. These results indicated that QUE successfully formed complexes with Et-β-CD, which changed their physical properties. FT-IR Spectroscopic Analysis. The vibrational changes of QUE and the QUE/Et-β-CD complex were monitored by FT-IR spectroscopy.41 The changes in intensity and shape of the FT-IR absorption peak, or the shift in the peaks can provide sufficient information about the successful formation of a host–guest complex.42 The FT-IR spectra of QUE, Et-β-CD, the QUE/Et-β-CD physical mixture, and the QUE/Et-β-CD complex are shown in Figure 4. All the observed IR absorption peaks are summarized in Table 2. The characteristic peaks of QUE (Figure 4, black line) were observed, including the C–O–C stretching at 1089 cm−1, C–O stretching around 1261 cm−1, C = O stretching at 1660 cm−1, and aromatic C = C stretching at 1614 cm−1, whereas these characteristic peaks were absent in the FT-IR spectra of the QUE/Et-β-CD complex (Figure 4 blue line), indicating that the environment of QUE was modified. The

Table 1. The stability constants and solubilization efficiencies of QUE complexes with CD derivatives. Host

k (M−1)

Solubilization efficiency (Se)

β-CD HP-β-CD DM-β-CD SBE-β-CD Et-β-CD

167.5 404.9 473.3 3744.9 9234.0

1.6 4.3 4.5 19.9 35.1


Figure 3. Differential scanning calorimetry (DSC) curves of QUE (black), Et-β-CD (red), the QUE/Et-β-CD physical mixture (green), and the QUE/Et-β-CD complex (blue).



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QUE/Et-β-CD, which further confirmed the formation of the complex.

Figure 4. (a) FT-IR spectra of QUE (black), Et-β-CD (red), the QUE/Et-β-CD physical mixture (green), and the QUE/Et-β-CD complex (blue). Spectra were acquired between 4000 and 400 cm−1.

FT-IR spectrum of the physical mixture contained absorption peaks with reduced intensity levels, and a slightly shifted position to that of the native QUE at 1080, 1261, 1652, and 1615 cm−1, respectively, which indicates that the simple mixture of QUE and Et-β-CD does not change the physical properties of QUE. FE-SEM Analysis. FE-SEM was used to study the morphological characteristics of the inclusion complex.43,44 The SEM images of QUE, Et-β-CD, the QUE/Et-β-CD physical mixture, and the QUE/Et-β-CD complex are shown in Figure 5. QUE appeared as needle-shaped crystalline structure (Figure 5(a)), Et-β-CD as irregular planar particles (Figure 5(b)), and the physical mixture of QUE/Et-β-CD clearly showed the surface morphologies of both the needle-shaped QUE and the planar shaped Etβ-CD (Figure 5(c)). However, the QUE/Et-β-CD complex in Figure 5(d) appeared as amorphous particles that were quite different from the sizes and shapes of both QUE and

NMR Spectroscopy of the Complex. The 1H NMR spectra are valuable to evaluate the non-covalent interactions at a molecular level.45 Because of the extremely poor water solubility of QUE, the specific proton peaks of QUE did not appear in the spectrum (Figure 6(a)).46 The assignments of the proton signals of QUE/Et-β-CD are shown in Figure 6(b). The evaluation of the QUE/Et-β-CD complexes by 1H NMR proved the presence of the specific protons of QUE molecule and implied a significant solubility enhancement for QUE/Et-β-CD relative to that of native QUE (Figure 6(b)). The chemical shifts of QUE in the 1H NMR spectra can prove the formation of inclusion complexes.47,48 The inclusion complex was also analyzed in detail by 2D NMR spectroscopy (Figure 7). The NOE peaks appear when two protons are closely located within a distance of 5 Å.49 The NOE cross-peaks from the correlation between CIP and Et-β-CD provide conformational information on the complex model. In Figure 7, clear cross-peaks were observed between the He0 , Hf0 , and Hb0 protons of QUE at 6.83, 7.33, and 7.39 ppm, and the inner cavity H3, H6, and H5 protons of Et-β-CD were observed at 3.82 and 3.92 ppm, respectively. These results indicate that the protons at B ring of QUE were embedded in the cavity of Etβ-CD whereas protons at A ring are probably located outer part of cavity. Furthermore, those results were quite different from the previous study on the inclusion complex of QUE with HP-β-CD, where the complexation occurred only in the aromatic ring A of QUE rather than other the parts of QUE.21 Those are probably due to the morphological difference of each cavity of the host CDs. Partly, primary amines at Et-β-CD can undergo nucleophilic addition with ketones of QUE to give carbinolamines which then dehydrate to result in substituted imines,50,51 and the possibility of imine formation can be proved by 1H, 13C-HMBC, NMR,52 FT-IR,53 and Raman spectroscopy.54 However, the major component in solution is attributed to the complex as shown in cross peaks between B ring of QUE and inner

Table 2. FT-IR absorption bands for QUE, Et-β-CD, the QUE/Et-β-CD physical mixture, and the QUE/Et-β-CD complex. Assignment ν (N–H) ν (O–H) ν (C–O–C) ν (C–O) ν (C–H) ν (C=O) δ (O–H) ν (C–C(=O)–C) C=C aromatic ring


Que (cm−1)

Et-β-CD (cm−1)

QUE/Et-β-CD physical mixture (cm−1)

QUE/Et-β-CD complex (cm−1)

— 3322 1089 1261

1652 3402 1080 1031 2929 — — — —

1653 3368 1080 1031, 1261 2929 1652 1385 1323 1615

1636 3420 1074 1031 2929 — — — —

1660 1381 1319 1614



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Figure 5. Field emission scanning electron microscope image of (a) QUE, (b) Et-β-CD, (c) the QUE/Et-β-CD physical mixture, and (d) the QUE/Et-β-CD complex with a 1:1 molar ratio (×2000 magnification, bar = 10 μm).

cavity of Et-β-CD (Figure 7), and it is accordance with the molecular modeling data of the complex in Figure 8. Molecular Modeling. The molecular features of the 1:1 host–guest interactions between Et-β-CD and QUE were characterized using docking simulations. Figure 8 shows the highest scoring poses of QUE for both β-CD and Etβ-CD. The QUE molecule formed a typical inclusion complex with the β-CD. The dihydroxyphenyl group of the QUE was oriented towards the secondary rim of the β-CD, whereas the trihydroxy-chromenone moiety was located on

primary face of the CD. One hydroxyl group of the catechol moiety formed a weak hydrogen bond with the 20 -OH of the β-CD. Therefore, the driving force for the inclusion complexation between QUE and β-CD is a favorable van der Waals attraction with the hydrophobic inner-cavity of β-CD. QUE showed different bonding for Et-β-CD, compared with regular β-CD. Because the ethylenediamine moiety located on the primary face is able to cause macrocyclic distortion, the Et-β-CD has a characteristic oval shape blocked by a bulky primary rim.22 In contrast to the

Figure 6. The partial 1H NMR spectra of (a) QUE and (b) the QUE/Et-β-CD complex in a solution of D2O solvent and (c) the chemical structure of QUE. Bull. Korean Chem. Soc. 2017



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(a)

(b)

b’ f’

e’ H 2N

OH

6

H3, H4 H6 H2 H4’ H7H6’H8 H6’

2.5

NH

8 5 4

4.0

HOD

4.5

F1 Chemical Shift (ppm)

H5

3.5

3 2

O OH

H1

OH c' b' i

A

f e OH

7.5

7.0 F2 Chemical Shift (ppm)

6.5

a O

h

HO

O

OH

HO

(c)

g

5.0

O

5' 1 4' 3 2

1

HO

3.0

6'

7

O

b

a'

d

OH

e' f'

C j

d'

B

c

OH

O

6.0

Figure 7. (a) The partial ROESY spectra of the QUE/Et-β-CD complex in D2O solvent at 25 C with a mixing time of 700 ms (600 MHz, 25 C, spectra recorded in D2O; signals indicate residual HDO).

complex with β-CD, the dihydroxyphenyl group of QUE was oriented towards the primary face of the Et-β-CD. This model reflects the ROESY-driven experimental result, in which the dihydroxyphenyl moiety interacted mainly with the inner cavity protons of the Et-β-CD. In this computational result, the catechol hydroxyl groups of QUE formed double hydrogen bonds with a nitrogen atom of the ethylenediamine moiety in Et-β-CD. The trihydroxychromenone moiety of QUE interacted with the secondary face of Et-β-CD. The molecular shape of QUE is suitable for the formation of a stable complex with the oval-shaped Et-β-CD via strong hydrogen bonding. Binding energetics also demonstrated that QUE and Et-β-CD can form a stable inclusion complex. The calculated docking score of QUE for β-CD and Et-β-CD was −5.39 and −5.93 kcal/mol, respectively. This energy-difference was well-correlated with the experimentally determined sixfold increase in the solubility constant for Et-β-CD.

Figure 8. The highest-scoring docked poses of QUE with β-CD (left), and Et-β-CD (right) as reported by molecular docking simulations. (a) side-view, (b) top-view, and (c) molecular surface areas for β-CD and Et-β-CD. The carbon atoms of CDs are colored in green and the QUE atoms are marked in yellow. All nonpolar hydrogen atoms are omitted for clarity. Bull. Korean Chem. Soc. 2017

Photostability Studies. To measure the photostability of the QUE complex, the ethanol solution of the inclusion complex and QUE were exposed to UVA at 360 nm (Figure 9). At the initiation stage, the free QUE, and the QUE/Et-β-CD complexes showed a similar decreasing tendency; however, after 6 h, a marked difference between these two samples was observed. At this stage, the free QUE was rapidly degraded compared to the complexed QUE. After 18 h of UV exposure, 75% free QUE remained, whereas about 90% of QUE was retained in the QUE/Et-β-CD complex. These results showed that the photostability of QUE increased via its inclusion in the complex with Et-β-CD, which confirms that the QUE, which is entirely captured in the Et-β-CD cavity, is protected from UV irradiation.



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Figure 9. Photodegradation kinetics of QUE, and QUE/Et-CD derivatives, upon UVA irradiation. The mean SD is obtained from three independent experiments (n = 2).

Antioxidant Activity. The effect of the inclusion of QUE in a complex with Et-β-CD on its antioxidant activity was investigated using the ORAC-FL assay (Figure 10). This method evaluated the ability of antioxidants to protect fluorescein from damage by free radicals, and consisted of measuring the decrease in fluorescein following oxidative damage produced by the peroxyl radical from AAPH.55 QUE contains many phenolic hydroxyl groups that can donate hydrogen atoms to this radical.56 Because of these structural features, QUE showed peroxyl and hydroxyl radical-scavenging activity in the ORAC assay.57 Figure 10 shows the antioxidant activity of the QUE and QUE complexes with CD derivatives. The QUE/Et-β-CD complex has the highest ORAC value among QUE complexes. The QUE/Et-β-CD complex almost doubled the antioxidant activity of QUE. Conversely, the QUE/β-CD, QUE/ HPβ-CD, and QUE/DM-β-CD complexes showed lower antioxidant activity than that of QUE alone, and the antioxidant activity of QUE/SBE-β-CD was slightly higher than that of QUE. This effect on the antioxidant activity of QUE may be due to the formation of the inclusion complex of QUE with Et-β-CD, resulting in an enhancement in the solubility of QUE. Therefore, QUE was more effectively oxidized by AAPH, which prevented fluorescein oxidation.


Figure 10. Radical scavenging activity (Trolox equivalents, μM) of QUE alone, and of the QUE/β-CD, QUE/ HP-β-CD, QUE/DMβ-CD, QUE/SBE-β-CD, and QUE/Et-β-CD complexes. The results represent the mean SD of values obtained from three measurements (n = 3).

QUE with Et-β-CD was monitored by 2D ROESY spectroscopy. The results showed that the protons at B ring of QUE were included in the cavity of Et-β-CD. In the ROESY-driven molecular docking simulation result, QUE formed a stable complex with the oval-shaped Et-β-CD via strong hydrogen bonding. The effect of the complexation of QUE with Et-β-CD on its antioxidant activity was determined using the ORAC-FL assay, and the QUE/Et-β-CD complex showed the highest antioxidant effect. Our results also demonstrated that a greater complexation of QUE with Et-β-CD was achieved, when compared to that with β-CD and β-CD derivatives. Furthermore, the QUE/Et-β-CD complex enhanced the solubility of QUE up to 35.1-fold, compared to the natural solubility of QUE. From these results, we suggest that the characteristic elliptical cavity of Etβ-CD can be utilized for other oblong nonpolar drug molecules, which can expand the development of drug delivery systems. Acknowledgments. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2015R1D1A1A01058686) and the Bio & Medical Technology Development Program of the NRF funded by the Korean Government, MSIP (NRF-2015M3A9B8031831) SDG. Supporting Information. Additional supporting information is available in the online version of this article.

Conclusion In the present study, the solubility of QUE was successfully enhanced through the formation of an inclusion complex with Et-β-CD. This inclusion complex was analyzed by UV–vis, 1H, and DOSY NMR spectroscopy, DSC, FT-IR spectroscopy, and SEM. The complexation behavior of Bull. Korean Chem. Soc. 2017

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