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Host-guest inclusion systems of daidzein with 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) and sulfobutyl ether-β-cyclodextrin (SBE-β-CD): Preparation, binding behaviors and water solubility
Journal of Molecular Structure 1118 (2016) 307e315
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Host-guest inclusion systems of daidzein with 2-hydroxypropyl-bcyclodextrin (HP-b-CD) and sulfobutyl ether-b-cyclodextrin (SBE-bCD): Preparation, binding behaviors and water solubility Yinghui Deng a, b, Yanhua Pang c, Yafei Guo a, Yufeng Ren a, Fen Wang a, Xiali Liao a, *, Bo Yang a, ** a b c
Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China Qujing Medical College, Qujing 655000, China Armed Police Yunnan Corps and Qujing Detachment, Qujing 655000, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 November 2015 Received in revised form 8 April 2016 Accepted 13 April 2016 Available online 19 April 2016
Daidzein is an isoflavone of naturally abundance existing in plants and foods which has attracted much attention for its significant benefits on human health. However, its application was severely limited by its poor solubilities, instability and low bioavailability. To overcome these drawbacks, inclusion complexes of daidzein with two cyclodextrin (CD) derivatives, i.e., 2-hydropropyl-b-cyclodextrin (HP-b-CD) and sulfobutyl ether-b-cyclodextrin (SBE-b-CD) were prepared and characterized both in solution and solid state by 1D and 2D NMR, XRD, SEM and elemental analyses. Fluorescence spectroscopy and the Job plot were used to demonstrate a mainly 1:1 inclusion mode between daidzein and CDs. Their thermal stabilities were evaluated with TG and DSC experiments. Moreover, water solubility of daidzein was significantly improved by inclusion complexation with CDs. These results might suggest valuable approaches to developments of new pharmaceutical formulations of daidzein. © 2016 Elsevier B.V. All rights reserved.
Keywords: Daidzein 2-Hydroxypropyl-b-cyclodextrin (HP-b-CD) Sulfobutyl ether-b-cyclodextrin (SBE-b-CD) Inclusion complex Water solubility
1. Introduction Daidzein (40 ,7-dihydroxy-isoflavone, Fig. 1) is an isoflavone of natural abundance mainly from leguminous plants and soybean products. It possesses many biological activities which are important to human health such as prevention of cardiovascular diseases [1] and menopausal symptoms [2], antidiabetes [3,4], antioxidant activities [5e8], bone protection [9,10] and antitumor activities, especially to human hepatic [11], prostate [12], and colon cancer cells [13]. Furthermore, daidzein has exhibited varies estrogendepended health benefits through its metabolite S-equol produced by intestinal bacteria [14,15]. However, the application of daidzein has been severely hindered by its low water solubility, poor stabilities, poor oral absorption, unfavorable metabolism, uterine estrogenicity and low bioavailability [16,17]. Cyclodextrins (CDs) are a series of cyclic oligosaccharides consisting of D-glucose units linked by a-1,4-glucosidic bonds, usually
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (X. Liao), [email protected] (B. Yang). http://dx.doi.org/10.1016/j.molstruc.2016.04.040 0022-2860/© 2016 Elsevier B.V. All rights reserved.
produced from starch in the presence of cyclodextrin glucosyl transferase (CGTase). The most common CDs are a-, b-, and g-CD, which consists of 6, 7 and 8 D-glucose monomers respectively. CDs have many virtues such as solubilization, stabilization, removing odors, enhancing bioavailability and donating targeted-delivering properties of drugs [18,19]. They have hydrophobic central cavities and hydrophilic external walls, which endow them with the salient ability of forming inclusion complexes with numerous guest molecules. In our group, the preparation and properties of inclusion complexes of CDs with natural products including mangiferin [20], 3-tigloyl-azadirachtol [21], artemether [22,23], scutellarein [24], norathyriol [25] were successfully conducted in recent years. In most cases, the inclusion complexation of CDs significantly enhanced water solubility, stability and bioavailability of the guest compounds. b-CD is the most frequently used one owing to its readily availability and cheapness among CDs. However, pharmaceutical application of native b-CD is usually hampered by its relatively low water solubility [26] and hemolytic effects [27,28]. Thus b-CD derivatives such as methyl-b-cyclodextrin (M-b-CD), 2hydroxypropyl-b-cyclodextrin (HP-b-CD) and sulfobutyl ether-b-
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Fig. 1. The structure of daidzein.
cyclodextrin (SBE-b-CD) were developed to overcome such drawbacks of b-CD [29e31]. Particularly, the latter two have underwent rigorous safety tests and been applied to several drugs and lots of pre- and clinical chemical entities [32]. They are both chemically modified on the hydroxyl groups of b-CD (Fig. 2) and have superior pharmaceutical properties such as excellent water solubility and much lower toxicities. Recently, conjugates [33,34] and complexes [35e39] of daidzein with biocompatible carbohydrate units were established to improve the physiochemical properties of daidzein. However, SBE-b-CD has not been used except the reported b- and g-CD, M-b-CD and HP-b-CD, and it was not so extensively studied when concerning HP-b-CD. In this paper, we reported the preparation and extensive characterization of inclusion complexes of daidzein with HP-b-CD and SBE-b-CD. The binding behaviors, thermal stability and water solubility of the inclusion complexes were elucidated. This could provide a useful approach to novel daidzein-based formulations with higher thermal stability, water solubility and bioavailability. 2. Materials and experimental 2.1. Materials Daidzein (MW: 254.24, >98%) was purchased from Aladdin Industrial Corporation in Shanghai, China. 2-Hydroxypropyl-bcyclodextrin (HP-b-CD, average MW: 1541) and sulfobutyl ether-bcyclodextrin (SBE-b-CD, average MW: 1451) were purchased from Xi'an Deli Biochem Co. Ltd in Shaanxi, China. Other chemical reagents were of analytical grade. All experiments were carried out using ultrapure water. 2.2. Preparation of solid inclusion complexes and physical mixtures of daidzein/HP-b-CD and daidzein/SBE-b-CD Solid inclusion complexes of daidzein/HP-b-CD and daidzein/ SBE-b-CD were prepared by suspension method [40]. Daidzein (50.85 mg, 0.2 mmol) was added to an aqueous solution (15 mL) of HP-b-CD (154.12 mg, 0.1 mmol) or SBE-b-CD (145.11 mg, 0.1 mmol),
Fig. 3. Job plots for daidzein/HP-b-CD (A) and daidzein/SBE-b-CD (B) systems at lem468 nm in pH 10.5 buffer.
and the obtained suspension was stirred vigorously at 70 C for 5 h in the dark. Then it was cooled to room temperature, filtered with a 0.45 mm Millipore membrane. The filtrate was evaporated under reduced pressure and the solid residue was dried in vacuo to give the solid inclusion complex of daidzein/HP-b-CD (85% yield) or daidzein/SBE-b-CD (76% yield). Other methods such as suspension in water or water-ethanol at room temperature or by Alonso's method [41] could only give trace of expected solid inclusion complexes as monitored by NMR analysis. Physical mixtures were obtained by mixing the powders of daidzein and HP-b-CD (or SBE-b-CD) in a 1:1 M ratio in an agate
Fig. 2. The structure of HP-b-CD and SBE-b-CD.
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Fig. 4. Fluorescence emission spectra of daidzein (3.0 105 mol/L) containing various concentrations of CDs (from a to h: 0.0 102, 0.2 102, 0.4 102, 0.6 102, 0.8 102, 1.0 102, 1.2 102, 1.5 102, 2.0 102 mol/L of CDs); emission at 468 nm. (A) daidzein/HP-b-CD; (B) daidzein/SBE-b-CD.
with Cu-Ka radiation (k ¼ 1.5460 Å, 40 kV, 100 mA), at a scanning rate of 5 /min. Powder samples were mounted on a vitreous sample holder and were scanned with a step size of 2q ¼ 0.02 between 2q ¼ 5 and 70 .
mortar. 2.3. Stoichiometry The stoichiometry of the inclusion complexation of daidzein with CDs in solution was determined by Job's method [42e47]. The Job plot was determined with fluorescence spectra data obtained in a pH 10.5 buffer solution on a Shimadzu RF-5301PC, with a (1 cm 1 cm 4 cm) quartz cell. The total molar concentration of daidzein ([daidzein]0) and CDs ([CD]0) was kept constant (3.0 105 mol/L), while the molar fraction of daidzein varied from 0.1 to 0.9. The stoichiometry of the solid inclusion complexes was also evaluated by elemental analysis. It was determined on a PE 2400 series II elemental analyzer.
2.7. Thermal analysis Differential scanning calorimetry (DSC) and thermogravimetric (TG) measurements were performed on a 2960 SDT V3.0F instrument, and 3e3.5 mg of each sample was heated at a rate of 10 C/ min from room temperature to 400 C under dynamic nitrogen atmosphere at a flow rate of 70 mL/min.
2.4. Spectral titration The procedure of spectral titration was carried out as following: CD (1.0 102 mol/L) and daidzein (3.0 105 mol/L) solution were conducted with a Na2CO3eNaHCO3 (pH 10.5) buffer solution. In a 10 mL colorimetric tube, 1.0 mL daidzein solution (3.0 105 mol/L) and varied amounts of CD (0.0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.5 and 2.0 mL of 1.0 102 mol/L) were added in turn. The mixed solution was diluted to the mark with the buffer solution, ultrasonically vibrated for 30 min at room temperature, and the fluorescence spectra were measured at lex/lem 385/468 nm. 2.5. 1D and 2D NMR H NMR spectrum of daidzein, CDs (HP-b-CD and SBE-b-CD) and their inclusion complexes were obtained on a Bruker Avance DRX spectrometer (500 MHz) at 298 K in D2O-DMSO-d6 (1:1, v:v) or in D2O using tetramethylsilane (TMS) as an internal reference. The ROESY experiment was also run on the same instrument in D2O. Samples were equilibrated for at least 24 h before measurements. 1
2.6. Powder X-ray diffraction (XRD) The XRD patterns were obtained using PHI 5000 VersaProbe II
Table 1 Stability constant of daidzein/HP-b-CD and daidzein/SBE-b-CD complex. Inclusion complexation
KS
logKS
DG
R
Daidzein/HP-b-CD Daidzein/SBE-b-CD
2415.7 504.6
3.3824 2.7025
19.309 15.428
0.9933 0.9668
Fig. 5. 1H NMR spectra of daidzein, CDs and their includion complexes at 25 C. (a) daidzein (in DMSO-d6); (b) HP-b-CD (in D2O); (c) daidzein/HP-b-CD complex (in D2O); (d) daidzein/SBE-b-CD complex (in D2O); (e) SBE-b-CD (in D2O).
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Table 2 Chemical shifts changes of H-3 and H-5 protons between CDs and their inclusion complexes. Protons
HP-b-CD
Daidzein/HP-b-CD
SBE-b-CD
Daidzein/SBE-b-CD
H-3 H-5
3.90 3.75
3.93 3.74
3.86 3.78
3.84 3.77
2.8. Scanning electron microscopy (SEM) The powders were previously fixed on a brass stub using double-sided adhesive tape and then were made electrically conductive by coating with a thin layer of gold (approximately 300Å) in a vacuum for 30 s and at 30 W. The pictures were taken at an excitation voltage of 15, 20 or 30 kV and a magnification of 1080, 1200, 1400 or 2000.
2.9. Solubilization test Water solubilities of daidzein/CDs complexes were determined by the method of preparation of their saturated solutions. An excess amount of complex was placed in water (2 mL, ca. pH 7.0), sheltered from light and the mixture was vigorously stirred for 1 h at 25 ± 2 C. The solution was then filtered on a 0.45 mm Millipore membrane, and the filtrate was evaporated under reduced pressure to dryness. The solid obtained was weighed.
3. Results and discussion 3.1. Stoichiometry The stoichiometry of daidzein/CD complexes in solution was obtained from the Job plot with fluorescence spectroscopy. As showed in Fig. 3(A), the plot of daidzein/HP-b-CD showed a maximal peak at the molar fraction of 0.5, which indicated a 1:1 inclusion complexation between the host (HP-b-CD) and the guest (daidzein). However, a shoulder at about 0.66 was observed in addition to the maxima at 0.5 concerning the inclusion complex of daidzein/SBE-b-CD from Fig. 3(B), which indicated mixed hostguest stoichiometries of 2:1 and 1:1 in solution of this complex. The stoichiometry of solid inclusion complexes of daidzein/CDs was determined by elemental analysis. The results were as follows, where the former numbers of water molecules were determined by TG analysis (Part 3.5): (1) daidzein/HP-b-CD complex: Anal. Calc. for (C63H112O42) (C15H10O4)*3.01(H2O): C, 50.53%; H, 6.91%; Found: C, 49.58%; H, 7.27%. (2) daidzein/SBE-b-CD complex: Anal. Calc. for (C56H97O33S3) (C15H10O4)*3.22(H2O): C, 49.97%; H, 5.59%; S, 5.6%, Found: C, 47.1%; H, 6.02%; S, 4.9%.
3.2. Spectral titration As the Job plot indicated the 1:1 and 2:1 stoichiometries for the
Table 3 Chemical shifts changes of protons of daidzein before and after complexation with CDs in D2O/DMSO-d6 (1:1, v:v): (a) daidzein; (b) daidzein/HP-b-CD; (c) daidzein/SBE-b-CD..
daidzein daidzein/HP-b-CD daidzein/SBE-b-CD
H-2
H-5
H-20 , 60
H-6
H-8
H-30 , 50
8.32 8.22 8.31
8.12 8.08 8.14
7.465 7.465 7.49
7.11 7.02 7.12
7.07 6.93 7.07
6.99 6.97 7.01
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Fig. 6. ROESY spectrum of (A) daidzein/HP-b-CD and (B) daidzein/SBE-b-CD complex in D2O.
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Fig. 7. Possible inclusion mode of the daidzein/CDs inclusion complex: (A) daidzein/HP-b-CD; (B) daidzein/SBE-b-CD.
inclusion complexation of daidzein/CDs, the equilibrium of complexation could be demonstrated by Eq. (1).
½daidzein þ n½CD⇔ ½daidzein,½CDn
(1)
where n ¼ 1 or 2. The quantitative investigation of the binding behaviors of host CDs with daidzein was conducted in carbonate buffer solution by means of fluorescence spectroscopy (Fig. 4). From the fluorescence intensity changes induced by the stepwise increased addition of the host, the stability constant of complex (Ks) was determined. The Ks was calculated for each host-guest system (1:1) from the nonlinear squares fitting to Eq. (2):
Ks ¼
½daidzein$CD DF=Dε ¼ ½daidzein½CD ½CD0 DF=Dε ½daidzein0 DF=Dε (2)
where [CD]0 and [daidzein]0 referred to the total concentration of CDs and daidzein, respectively, and Dε represented the proportionality coefficient, which could be taken as a sensitivity factor for fluorescence intensity changes. Eq. (2) could be achieved by Eq. (3):
DF ¼
SBE-b-CD, which suggested that daidzein had strong binding abilities with both HP-b-CD and SBE-b-CD. Moreover, the DG and R of inclusion complexes were also obtained. In the repeated measurements, the KS values were reproducible within an error of ±5%. 3.3.
1
H NMR and 2D NMR analyses
The information provided by NMR spectroscopy could be used to establish inclusion modes. Herein, the 1H NMR spectra of daidzein, CDs and their complexes were recorded and compared as showed in Fig. 5. As we know, H-3 and H-5 protons of CDs are located in the interior cavity while H-3 and H-5 protons are near the secondary and primary face, respectively. Significant changes of chemical shifts of H-3 and H-5 protons were observed when the two host molecules formed inclusion complexes with daidzein (Table 2). Upfield shifts of H-5 of complexes daidzein/HP-b-CD (0.01 ppm) and daidzein/SBE-b-CD (0.01 ppm) and H-3 of daidzein/SBE-b-CD (0.02 ppm) emerged, while there was also a downfield shift of H-3 of complex daidzein/HP-b-CD (0.03 ppm). These information suggested stable inclusion complexes formed between daidzein and the two CDs. In order to evaluate the changes of chemical shifts of protons of
ffi qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 ðDεÞ ½CD0 þ ½daidzein0 þ 1=Ks ± ðDεÞ2 ½CD0 þ ½daidzein0 þ 1=Ks 4ðDεÞ2 ½CD0 ½daidzein0 2
As illustrated in Fig. 4, the fluorescence intensity of daidzein was increased along with the stepwise addition of HP-b-CD (Fig. 4 A) or SBE-b-CD (Fig. 4 B). These data suggested that stable complexes were formed between CDs and daidzein. As CDs cavity could furnish a hydrophobic surrounding environment for the included chromophore, this altered microenvironment could provide favorable polarity and acid/base equilibrium for enhanced quantum efficiencies [48]. The stability constant for host-guest system was obtained by using a nonlinear least-squares curve-fitting method [49]. Excellent fits between the experimental and calculated data were also showed in Fig. 4. For each host examined, the plot of F as a function of [CD]0 gave an excellent fit, verifying the validity of the 1:1 complex stoichiometry as deduced above. The Ks of daidzein/CDs complexes were calculated as summarized in Table 1, up to 2415.7 M1 for HP-b-CD and 504.6 M1 for
(3)
daidzein before and after complexation with CDs, 1H NMR spectra of native daidzein and inclusion complexes with CDs were recorded in a mixed solvent of D2O/DMSO-d6 (1:1, v:v) as listed in Table 3. Comparing with that of H-3 and H-5 of CDs, variations of chemical shifts of protons of daidzein seemed more noticeable. In order to disclose the possible inclusion mode of daidzein/CDs complexes, their two-dimensional (2D) NMR analyses were carried out. It is well known that the 2D NMR spectroscopy provides important information about the spatial proximity between host and guest molecules via observation of intermolecular dipolar cross-correlations. Two protons which are closely located in space could induce a nuclear overhauser effect (NOE) cross-correlation between them in NOESY or ROESY spectrum. The ROESY spectra of daidzein/HP-b-CD complex (Fig. 6(A)) showed obvious correlations between H-2, 5, 20 , 30 , 50 and 60 protons of daidzein and H-5 of
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Fig. 10. DSC thermograms: (a) daidzein; (b) HP-b-CD; (c) daidzein/HP-b-CD complex; (d) SBE-b-CD; (e) daidzein/SBE-b-CD complex. Fig. 8. XRD patterns (1): (a) daidzein; (b) HP-b-CD; (c) physical mixture of daidzein/ HP-b-CD (1:1 M ratio); (d) daidzein/HP-b-CD complex.
HP-b-CD. And for daidzein/SBE-b-CD (Fig. 6 (B)), appreciable correlations between H-2, 8, 20 , 30 , 50 and 60 protons of daidzein and both H-3 and H-5 of SBE-b-CD. These results indicated different arrangement patterns of daidzein in the CD cavity for these two
CDs. Based on these observations, together with the 1:1 inclusion stoichiometry deduced by Job plot, the possible inclusion modes of daidzein with HP-b-CD and SBE-b-CD were illustrated in Fig. 7.
3.4. Powder X-ray diffraction analysis XRD analyses of daidzein, HP-b-CD, SBE-b-CD, their physical mixtures and inclusion complexes were conducted. As depicted in Fig. 8, daidzein (Fig. 8 a) displayed intense and sharp peaks which clearly indicated crystal morphologies, while the XRD patterns of HP-b-CD (Fig. 8 b) were amorphous. Simple overlaps of native patterns were observed for physical mixtures of daidzein/HP-b-CD (Fig. 8 c). In contrast, the inclusion complexes of daidzein/HP-b-CD (Fig. 8 d) showed amorphous patterns similar to that of HP-b-CD.
Fig. 9. XRD patterns (2): (a) daidzein; (b) SBE-b-CD; (c) physical mixture of daidzein/ SBE-b-CD (1:1 M ratio); (d) daidzein/SBE-b-CD complex.
Fig. 11. TG curves: (a) daidzein; (b) HP-b-CD; (c) daidzein/HP-b-CD complex; (d) SBEb-CD; (e) daidzein/SBE-b-CD complex.
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Fig. 12. Scanning electron microphotographs: (A) daidzein, (B) HP-b-CD, (C) daidzein and HP-b-CD physical mixture (1:1 M ratio), (D) daidzein/HP-b-CD inclusion complex.
Similar patterns were obtained in the case of SBE-b-CD (Fig. 9), which provided strong evidences of inclusion complexation between daidzein and both CDs. 3.5. Thermal analysis The thermal properties of the daidzein/CDs complexes were investigated by DSC and TG analyses. DSC reveals some information about the solid state interactions between CDs and guest molecules. As shown in Fig. 10, daidzein, CDs and their complexes all had a broad endothermic peak between 50 and 100 C which might come from the release of water molecules. Daidzein (Fig. 10 a) had a sharp endothermic peak at 337.6 C but it disappeared on the DSC curves of the daidzein/CDs inclusion complexes (Fig. 10 c & e). The broad endothermic peaks of HP-b-CD and SBE-b-CD shifted from 73.2, 78.9 C to 82.5 and 96.6 C respectively, which indicated that the dehydration process was changed after formation of inclusion complexes. These results further confirmed the formation of the daidzein/CDs inclusion complexes. The TG analysis of the solid inclusion complexes was also conducted (Fig. 11). It showed that native daidzein decomposed at ca. 350 C (a), and the decomposition temperatures of its solid complexes with HP-b-CD and SBE-b-CD both fell to ca. 320 C (c) and 250 C (e), respectively, which somewhat displayed similar curve shapes to that of their corresponding hosts. From the TG curves, the weight loss percentage of HP-b-CD/daidzein complex from dehydration was 3.78% (c), while it was 8.43% for HP-b-CD (b). Similarly, it was 3.89% for SBE-b-CD/daidzein (e), compared to 10.28% for SBEb-CD (d). The results indicated that water molecules in CD cavity were partially replaced by daidzein to form the inclusion complexes.
3.6. Scanning electron microphotographs The SEM experiments were conducted to visualize the surface texture of inclusion complexes of daidzein with CDs. The SEM analyses of daidzein, HP-b-CD and their physical mixture and inclusion complex were shown in Fig. 10. Daidzein (Fig. 12A) had a rodlike crystal structure, when HP-b-CD displayed spherical crystals with cavities. Simple hybrids of rod-like and spherical crystals were observed in the physical mixture (Fig. 12 C). In contrast, the inclusion complex (Fig. 12 D) appeared in the form of compact and homogeneous plate-like crystal particles in which the original morphology of both components disappeared, which verified the formation of the daidzein/HP-b-CD inclusion complex. 3.7. Solubilization The results showed that the water solubility of daidzein/HP-bCD and daidzein/SBE-b-CD complexes, compared with that of native daidzein (ca. 8.31 mg/mL), were remarkably increased to 12.25 mg/mL and 10.68 mg/mL, respectively. The significant promotion on water solubility of daidzein by inclusion complexation with CDs would benefit to the pharmaceutical utilization of daidzein. 4. Conclusions In summary, daidzein/HP-b-CD and daidzein/SBE-b-CD inclusion complexes were successfully prepared and were characterized by spectral titration, NMR, XRD, DSC and SEM. The binding behaviors of daidzein with CDs were extensively elucidated. The stability constants were also obtained. The water solubility of
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daidzein was significantly improved by inclusion complexation with CDs. This indicated that inclusion complexes of daidzein/HPb-CD and daidzein/SBE-b-CD might be novel promising formulations of daidzein for its further pharmaceutical applications. Acknowledgments This work was financially supported by National Natural Science Foundation of China (No. 21362016) and Kunming University of Science and Technology. The authors would also like to thank Mr. Congtao Yu and Mr. Pin Lv for assistance on NMR and elemental analyses. References [1] N. Gottstein, B.A. Ewins, C. Eccleston, G.P. Hubbard, I.C. Kavanagh, A.M. Minihane, P.D. Weinberg, G. Rimbach, Brit. J. Nutr. 89 (2003) 607. [2] D.A. Tonetti, Y. Zhang, H. Zhao, S.B. Lim, A.I. Constantinou, Nutr. Cancer 58 (2007) 222. [3] S.H. Cheong, K. Furuhashia, K. Ito, M. Nagaoka, T. Yonezawa, Y. Miura, K. Yagasaki, J. Nutr. Biochem. 25 (2014) 136. [4] M. Park, J. Ju, M. Park, J. Han, Eur. J. Pharmacol. 712 (2013) 48. [5] S.E. Kulling, D.M. Honig, T.J. Simat, M. Metzler, J. Agric. Food Chem. 48 (2000) 4963. [6] Y. Ungar, O.F. Osundahunsi, E. Shimoni, J. Agric. Food Chem. 51 (2003) 4394. [7] K. Nakagawa, J. Adachi, M.C. Wong, Y. Ueno, Kobe J. Med. Sci. 52 (2006) 141. €hrdanz, S. Ohler, Q.H. Tran-Thi, R. Kahl, J. Nutr. 132 (2002) 370. [8] E. Ro [9] D. Somjen, S. Katzburg, F. Kohen, B. Gayer, E. Livne, J. Cell Biochem. 103 (2008) 1826. [10] K.D. Setchell, E. Lydeking-Olsen, Am. J. Clin. Nutr. 78 (2003) 593s. [11] H.J. Park, Y.K. Jeon, D.H. You, M.J. Nam, Food Chem. Toxicol. 60 (2013) 542. [12] X. Dong, W. Xu, R.A. Sikes, C. Wu, Food Chem. 141 (2013) 1923. [13] S.R. Lepri, L.C. Zanelatto, P.B. da Silva, D. Sartori, L.R. Ribeiro, M.S. Mantovani, Hum. Cell 27 (2014) 78. [14] K.D. Setchell, C. Clerici, J. Nutr. 140 (2010) 1355S. [15] C. Atkinson, C.L. Frankenfeld, J.W. Lampe, Exp. Biol. Med. 230 (2005) 155. [16] K.D. Setchell, N.M. Brown, P. Desai, L. Zimmer-Nechemias, B.E. Wolfe, W.T. Brashear, A.S. Kirschner, A. Cassidy, J.E. Heubi, J. Nutr. 131 (2001) 1362S. [17] Y. Ungar, O.F. Osundahunsi, E. Shimoni, J. Agric. Food Chem. 51 (2003) 4394. [18] J. Szejtli, Chem. Rev. 98 (1998) 1743. [19] G. Crini, Chem. Rev. 114 (2014) 10940.
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Journal of Molecular Structure journal homepage: http://www.elsevier.com/locate/molstruc
Host-guest inclusion systems of daidzein with 2-hydroxypropyl-bcyclodextrin (HP-b-CD) and sulfobutyl ether-b-cyclodextrin (SBE-bCD): Preparation, binding behaviors and water solubility Yinghui Deng a, b, Yanhua Pang c, Yafei Guo a, Yufeng Ren a, Fen Wang a, Xiali Liao a, *, Bo Yang a, ** a b c
Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China Qujing Medical College, Qujing 655000, China Armed Police Yunnan Corps and Qujing Detachment, Qujing 655000, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 November 2015 Received in revised form 8 April 2016 Accepted 13 April 2016 Available online 19 April 2016
Daidzein is an isoflavone of naturally abundance existing in plants and foods which has attracted much attention for its significant benefits on human health. However, its application was severely limited by its poor solubilities, instability and low bioavailability. To overcome these drawbacks, inclusion complexes of daidzein with two cyclodextrin (CD) derivatives, i.e., 2-hydropropyl-b-cyclodextrin (HP-b-CD) and sulfobutyl ether-b-cyclodextrin (SBE-b-CD) were prepared and characterized both in solution and solid state by 1D and 2D NMR, XRD, SEM and elemental analyses. Fluorescence spectroscopy and the Job plot were used to demonstrate a mainly 1:1 inclusion mode between daidzein and CDs. Their thermal stabilities were evaluated with TG and DSC experiments. Moreover, water solubility of daidzein was significantly improved by inclusion complexation with CDs. These results might suggest valuable approaches to developments of new pharmaceutical formulations of daidzein. © 2016 Elsevier B.V. All rights reserved.
Keywords: Daidzein 2-Hydroxypropyl-b-cyclodextrin (HP-b-CD) Sulfobutyl ether-b-cyclodextrin (SBE-b-CD) Inclusion complex Water solubility
1. Introduction Daidzein (40 ,7-dihydroxy-isoflavone, Fig. 1) is an isoflavone of natural abundance mainly from leguminous plants and soybean products. It possesses many biological activities which are important to human health such as prevention of cardiovascular diseases [1] and menopausal symptoms [2], antidiabetes [3,4], antioxidant activities [5e8], bone protection [9,10] and antitumor activities, especially to human hepatic [11], prostate [12], and colon cancer cells [13]. Furthermore, daidzein has exhibited varies estrogendepended health benefits through its metabolite S-equol produced by intestinal bacteria [14,15]. However, the application of daidzein has been severely hindered by its low water solubility, poor stabilities, poor oral absorption, unfavorable metabolism, uterine estrogenicity and low bioavailability [16,17]. Cyclodextrins (CDs) are a series of cyclic oligosaccharides consisting of D-glucose units linked by a-1,4-glucosidic bonds, usually
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (X. Liao), [email protected] (B. Yang). http://dx.doi.org/10.1016/j.molstruc.2016.04.040 0022-2860/© 2016 Elsevier B.V. All rights reserved.
produced from starch in the presence of cyclodextrin glucosyl transferase (CGTase). The most common CDs are a-, b-, and g-CD, which consists of 6, 7 and 8 D-glucose monomers respectively. CDs have many virtues such as solubilization, stabilization, removing odors, enhancing bioavailability and donating targeted-delivering properties of drugs [18,19]. They have hydrophobic central cavities and hydrophilic external walls, which endow them with the salient ability of forming inclusion complexes with numerous guest molecules. In our group, the preparation and properties of inclusion complexes of CDs with natural products including mangiferin [20], 3-tigloyl-azadirachtol [21], artemether [22,23], scutellarein [24], norathyriol [25] were successfully conducted in recent years. In most cases, the inclusion complexation of CDs significantly enhanced water solubility, stability and bioavailability of the guest compounds. b-CD is the most frequently used one owing to its readily availability and cheapness among CDs. However, pharmaceutical application of native b-CD is usually hampered by its relatively low water solubility [26] and hemolytic effects [27,28]. Thus b-CD derivatives such as methyl-b-cyclodextrin (M-b-CD), 2hydroxypropyl-b-cyclodextrin (HP-b-CD) and sulfobutyl ether-b-
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Fig. 1. The structure of daidzein.
cyclodextrin (SBE-b-CD) were developed to overcome such drawbacks of b-CD [29e31]. Particularly, the latter two have underwent rigorous safety tests and been applied to several drugs and lots of pre- and clinical chemical entities [32]. They are both chemically modified on the hydroxyl groups of b-CD (Fig. 2) and have superior pharmaceutical properties such as excellent water solubility and much lower toxicities. Recently, conjugates [33,34] and complexes [35e39] of daidzein with biocompatible carbohydrate units were established to improve the physiochemical properties of daidzein. However, SBE-b-CD has not been used except the reported b- and g-CD, M-b-CD and HP-b-CD, and it was not so extensively studied when concerning HP-b-CD. In this paper, we reported the preparation and extensive characterization of inclusion complexes of daidzein with HP-b-CD and SBE-b-CD. The binding behaviors, thermal stability and water solubility of the inclusion complexes were elucidated. This could provide a useful approach to novel daidzein-based formulations with higher thermal stability, water solubility and bioavailability. 2. Materials and experimental 2.1. Materials Daidzein (MW: 254.24, >98%) was purchased from Aladdin Industrial Corporation in Shanghai, China. 2-Hydroxypropyl-bcyclodextrin (HP-b-CD, average MW: 1541) and sulfobutyl ether-bcyclodextrin (SBE-b-CD, average MW: 1451) were purchased from Xi'an Deli Biochem Co. Ltd in Shaanxi, China. Other chemical reagents were of analytical grade. All experiments were carried out using ultrapure water. 2.2. Preparation of solid inclusion complexes and physical mixtures of daidzein/HP-b-CD and daidzein/SBE-b-CD Solid inclusion complexes of daidzein/HP-b-CD and daidzein/ SBE-b-CD were prepared by suspension method [40]. Daidzein (50.85 mg, 0.2 mmol) was added to an aqueous solution (15 mL) of HP-b-CD (154.12 mg, 0.1 mmol) or SBE-b-CD (145.11 mg, 0.1 mmol),
Fig. 3. Job plots for daidzein/HP-b-CD (A) and daidzein/SBE-b-CD (B) systems at lem468 nm in pH 10.5 buffer.
and the obtained suspension was stirred vigorously at 70 C for 5 h in the dark. Then it was cooled to room temperature, filtered with a 0.45 mm Millipore membrane. The filtrate was evaporated under reduced pressure and the solid residue was dried in vacuo to give the solid inclusion complex of daidzein/HP-b-CD (85% yield) or daidzein/SBE-b-CD (76% yield). Other methods such as suspension in water or water-ethanol at room temperature or by Alonso's method [41] could only give trace of expected solid inclusion complexes as monitored by NMR analysis. Physical mixtures were obtained by mixing the powders of daidzein and HP-b-CD (or SBE-b-CD) in a 1:1 M ratio in an agate
Fig. 2. The structure of HP-b-CD and SBE-b-CD.
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Fig. 4. Fluorescence emission spectra of daidzein (3.0 105 mol/L) containing various concentrations of CDs (from a to h: 0.0 102, 0.2 102, 0.4 102, 0.6 102, 0.8 102, 1.0 102, 1.2 102, 1.5 102, 2.0 102 mol/L of CDs); emission at 468 nm. (A) daidzein/HP-b-CD; (B) daidzein/SBE-b-CD.
with Cu-Ka radiation (k ¼ 1.5460 Å, 40 kV, 100 mA), at a scanning rate of 5 /min. Powder samples were mounted on a vitreous sample holder and were scanned with a step size of 2q ¼ 0.02 between 2q ¼ 5 and 70 .
mortar. 2.3. Stoichiometry The stoichiometry of the inclusion complexation of daidzein with CDs in solution was determined by Job's method [42e47]. The Job plot was determined with fluorescence spectra data obtained in a pH 10.5 buffer solution on a Shimadzu RF-5301PC, with a (1 cm 1 cm 4 cm) quartz cell. The total molar concentration of daidzein ([daidzein]0) and CDs ([CD]0) was kept constant (3.0 105 mol/L), while the molar fraction of daidzein varied from 0.1 to 0.9. The stoichiometry of the solid inclusion complexes was also evaluated by elemental analysis. It was determined on a PE 2400 series II elemental analyzer.
2.7. Thermal analysis Differential scanning calorimetry (DSC) and thermogravimetric (TG) measurements were performed on a 2960 SDT V3.0F instrument, and 3e3.5 mg of each sample was heated at a rate of 10 C/ min from room temperature to 400 C under dynamic nitrogen atmosphere at a flow rate of 70 mL/min.
2.4. Spectral titration The procedure of spectral titration was carried out as following: CD (1.0 102 mol/L) and daidzein (3.0 105 mol/L) solution were conducted with a Na2CO3eNaHCO3 (pH 10.5) buffer solution. In a 10 mL colorimetric tube, 1.0 mL daidzein solution (3.0 105 mol/L) and varied amounts of CD (0.0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.5 and 2.0 mL of 1.0 102 mol/L) were added in turn. The mixed solution was diluted to the mark with the buffer solution, ultrasonically vibrated for 30 min at room temperature, and the fluorescence spectra were measured at lex/lem 385/468 nm. 2.5. 1D and 2D NMR H NMR spectrum of daidzein, CDs (HP-b-CD and SBE-b-CD) and their inclusion complexes were obtained on a Bruker Avance DRX spectrometer (500 MHz) at 298 K in D2O-DMSO-d6 (1:1, v:v) or in D2O using tetramethylsilane (TMS) as an internal reference. The ROESY experiment was also run on the same instrument in D2O. Samples were equilibrated for at least 24 h before measurements. 1
2.6. Powder X-ray diffraction (XRD) The XRD patterns were obtained using PHI 5000 VersaProbe II
Table 1 Stability constant of daidzein/HP-b-CD and daidzein/SBE-b-CD complex. Inclusion complexation
KS
logKS
DG
R
Daidzein/HP-b-CD Daidzein/SBE-b-CD
2415.7 504.6
3.3824 2.7025
19.309 15.428
0.9933 0.9668
Fig. 5. 1H NMR spectra of daidzein, CDs and their includion complexes at 25 C. (a) daidzein (in DMSO-d6); (b) HP-b-CD (in D2O); (c) daidzein/HP-b-CD complex (in D2O); (d) daidzein/SBE-b-CD complex (in D2O); (e) SBE-b-CD (in D2O).
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Table 2 Chemical shifts changes of H-3 and H-5 protons between CDs and their inclusion complexes. Protons
HP-b-CD
Daidzein/HP-b-CD
SBE-b-CD
Daidzein/SBE-b-CD
H-3 H-5
3.90 3.75
3.93 3.74
3.86 3.78
3.84 3.77
2.8. Scanning electron microscopy (SEM) The powders were previously fixed on a brass stub using double-sided adhesive tape and then were made electrically conductive by coating with a thin layer of gold (approximately 300Å) in a vacuum for 30 s and at 30 W. The pictures were taken at an excitation voltage of 15, 20 or 30 kV and a magnification of 1080, 1200, 1400 or 2000.
2.9. Solubilization test Water solubilities of daidzein/CDs complexes were determined by the method of preparation of their saturated solutions. An excess amount of complex was placed in water (2 mL, ca. pH 7.0), sheltered from light and the mixture was vigorously stirred for 1 h at 25 ± 2 C. The solution was then filtered on a 0.45 mm Millipore membrane, and the filtrate was evaporated under reduced pressure to dryness. The solid obtained was weighed.
3. Results and discussion 3.1. Stoichiometry The stoichiometry of daidzein/CD complexes in solution was obtained from the Job plot with fluorescence spectroscopy. As showed in Fig. 3(A), the plot of daidzein/HP-b-CD showed a maximal peak at the molar fraction of 0.5, which indicated a 1:1 inclusion complexation between the host (HP-b-CD) and the guest (daidzein). However, a shoulder at about 0.66 was observed in addition to the maxima at 0.5 concerning the inclusion complex of daidzein/SBE-b-CD from Fig. 3(B), which indicated mixed hostguest stoichiometries of 2:1 and 1:1 in solution of this complex. The stoichiometry of solid inclusion complexes of daidzein/CDs was determined by elemental analysis. The results were as follows, where the former numbers of water molecules were determined by TG analysis (Part 3.5): (1) daidzein/HP-b-CD complex: Anal. Calc. for (C63H112O42) (C15H10O4)*3.01(H2O): C, 50.53%; H, 6.91%; Found: C, 49.58%; H, 7.27%. (2) daidzein/SBE-b-CD complex: Anal. Calc. for (C56H97O33S3) (C15H10O4)*3.22(H2O): C, 49.97%; H, 5.59%; S, 5.6%, Found: C, 47.1%; H, 6.02%; S, 4.9%.
3.2. Spectral titration As the Job plot indicated the 1:1 and 2:1 stoichiometries for the
Table 3 Chemical shifts changes of protons of daidzein before and after complexation with CDs in D2O/DMSO-d6 (1:1, v:v): (a) daidzein; (b) daidzein/HP-b-CD; (c) daidzein/SBE-b-CD..
daidzein daidzein/HP-b-CD daidzein/SBE-b-CD
H-2
H-5
H-20 , 60
H-6
H-8
H-30 , 50
8.32 8.22 8.31
8.12 8.08 8.14
7.465 7.465 7.49
7.11 7.02 7.12
7.07 6.93 7.07
6.99 6.97 7.01
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Fig. 6. ROESY spectrum of (A) daidzein/HP-b-CD and (B) daidzein/SBE-b-CD complex in D2O.
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Fig. 7. Possible inclusion mode of the daidzein/CDs inclusion complex: (A) daidzein/HP-b-CD; (B) daidzein/SBE-b-CD.
inclusion complexation of daidzein/CDs, the equilibrium of complexation could be demonstrated by Eq. (1).
½daidzein þ n½CD⇔ ½daidzein,½CDn
(1)
where n ¼ 1 or 2. The quantitative investigation of the binding behaviors of host CDs with daidzein was conducted in carbonate buffer solution by means of fluorescence spectroscopy (Fig. 4). From the fluorescence intensity changes induced by the stepwise increased addition of the host, the stability constant of complex (Ks) was determined. The Ks was calculated for each host-guest system (1:1) from the nonlinear squares fitting to Eq. (2):
Ks ¼
½daidzein$CD DF=Dε ¼ ½daidzein½CD ½CD0 DF=Dε ½daidzein0 DF=Dε (2)
where [CD]0 and [daidzein]0 referred to the total concentration of CDs and daidzein, respectively, and Dε represented the proportionality coefficient, which could be taken as a sensitivity factor for fluorescence intensity changes. Eq. (2) could be achieved by Eq. (3):
DF ¼
SBE-b-CD, which suggested that daidzein had strong binding abilities with both HP-b-CD and SBE-b-CD. Moreover, the DG and R of inclusion complexes were also obtained. In the repeated measurements, the KS values were reproducible within an error of ±5%. 3.3.
1
H NMR and 2D NMR analyses
The information provided by NMR spectroscopy could be used to establish inclusion modes. Herein, the 1H NMR spectra of daidzein, CDs and their complexes were recorded and compared as showed in Fig. 5. As we know, H-3 and H-5 protons of CDs are located in the interior cavity while H-3 and H-5 protons are near the secondary and primary face, respectively. Significant changes of chemical shifts of H-3 and H-5 protons were observed when the two host molecules formed inclusion complexes with daidzein (Table 2). Upfield shifts of H-5 of complexes daidzein/HP-b-CD (0.01 ppm) and daidzein/SBE-b-CD (0.01 ppm) and H-3 of daidzein/SBE-b-CD (0.02 ppm) emerged, while there was also a downfield shift of H-3 of complex daidzein/HP-b-CD (0.03 ppm). These information suggested stable inclusion complexes formed between daidzein and the two CDs. In order to evaluate the changes of chemical shifts of protons of
ffi qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 ðDεÞ ½CD0 þ ½daidzein0 þ 1=Ks ± ðDεÞ2 ½CD0 þ ½daidzein0 þ 1=Ks 4ðDεÞ2 ½CD0 ½daidzein0 2
As illustrated in Fig. 4, the fluorescence intensity of daidzein was increased along with the stepwise addition of HP-b-CD (Fig. 4 A) or SBE-b-CD (Fig. 4 B). These data suggested that stable complexes were formed between CDs and daidzein. As CDs cavity could furnish a hydrophobic surrounding environment for the included chromophore, this altered microenvironment could provide favorable polarity and acid/base equilibrium for enhanced quantum efficiencies [48]. The stability constant for host-guest system was obtained by using a nonlinear least-squares curve-fitting method [49]. Excellent fits between the experimental and calculated data were also showed in Fig. 4. For each host examined, the plot of F as a function of [CD]0 gave an excellent fit, verifying the validity of the 1:1 complex stoichiometry as deduced above. The Ks of daidzein/CDs complexes were calculated as summarized in Table 1, up to 2415.7 M1 for HP-b-CD and 504.6 M1 for
(3)
daidzein before and after complexation with CDs, 1H NMR spectra of native daidzein and inclusion complexes with CDs were recorded in a mixed solvent of D2O/DMSO-d6 (1:1, v:v) as listed in Table 3. Comparing with that of H-3 and H-5 of CDs, variations of chemical shifts of protons of daidzein seemed more noticeable. In order to disclose the possible inclusion mode of daidzein/CDs complexes, their two-dimensional (2D) NMR analyses were carried out. It is well known that the 2D NMR spectroscopy provides important information about the spatial proximity between host and guest molecules via observation of intermolecular dipolar cross-correlations. Two protons which are closely located in space could induce a nuclear overhauser effect (NOE) cross-correlation between them in NOESY or ROESY spectrum. The ROESY spectra of daidzein/HP-b-CD complex (Fig. 6(A)) showed obvious correlations between H-2, 5, 20 , 30 , 50 and 60 protons of daidzein and H-5 of
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Fig. 10. DSC thermograms: (a) daidzein; (b) HP-b-CD; (c) daidzein/HP-b-CD complex; (d) SBE-b-CD; (e) daidzein/SBE-b-CD complex. Fig. 8. XRD patterns (1): (a) daidzein; (b) HP-b-CD; (c) physical mixture of daidzein/ HP-b-CD (1:1 M ratio); (d) daidzein/HP-b-CD complex.
HP-b-CD. And for daidzein/SBE-b-CD (Fig. 6 (B)), appreciable correlations between H-2, 8, 20 , 30 , 50 and 60 protons of daidzein and both H-3 and H-5 of SBE-b-CD. These results indicated different arrangement patterns of daidzein in the CD cavity for these two
CDs. Based on these observations, together with the 1:1 inclusion stoichiometry deduced by Job plot, the possible inclusion modes of daidzein with HP-b-CD and SBE-b-CD were illustrated in Fig. 7.
3.4. Powder X-ray diffraction analysis XRD analyses of daidzein, HP-b-CD, SBE-b-CD, their physical mixtures and inclusion complexes were conducted. As depicted in Fig. 8, daidzein (Fig. 8 a) displayed intense and sharp peaks which clearly indicated crystal morphologies, while the XRD patterns of HP-b-CD (Fig. 8 b) were amorphous. Simple overlaps of native patterns were observed for physical mixtures of daidzein/HP-b-CD (Fig. 8 c). In contrast, the inclusion complexes of daidzein/HP-b-CD (Fig. 8 d) showed amorphous patterns similar to that of HP-b-CD.
Fig. 9. XRD patterns (2): (a) daidzein; (b) SBE-b-CD; (c) physical mixture of daidzein/ SBE-b-CD (1:1 M ratio); (d) daidzein/SBE-b-CD complex.
Fig. 11. TG curves: (a) daidzein; (b) HP-b-CD; (c) daidzein/HP-b-CD complex; (d) SBEb-CD; (e) daidzein/SBE-b-CD complex.
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Fig. 12. Scanning electron microphotographs: (A) daidzein, (B) HP-b-CD, (C) daidzein and HP-b-CD physical mixture (1:1 M ratio), (D) daidzein/HP-b-CD inclusion complex.
Similar patterns were obtained in the case of SBE-b-CD (Fig. 9), which provided strong evidences of inclusion complexation between daidzein and both CDs. 3.5. Thermal analysis The thermal properties of the daidzein/CDs complexes were investigated by DSC and TG analyses. DSC reveals some information about the solid state interactions between CDs and guest molecules. As shown in Fig. 10, daidzein, CDs and their complexes all had a broad endothermic peak between 50 and 100 C which might come from the release of water molecules. Daidzein (Fig. 10 a) had a sharp endothermic peak at 337.6 C but it disappeared on the DSC curves of the daidzein/CDs inclusion complexes (Fig. 10 c & e). The broad endothermic peaks of HP-b-CD and SBE-b-CD shifted from 73.2, 78.9 C to 82.5 and 96.6 C respectively, which indicated that the dehydration process was changed after formation of inclusion complexes. These results further confirmed the formation of the daidzein/CDs inclusion complexes. The TG analysis of the solid inclusion complexes was also conducted (Fig. 11). It showed that native daidzein decomposed at ca. 350 C (a), and the decomposition temperatures of its solid complexes with HP-b-CD and SBE-b-CD both fell to ca. 320 C (c) and 250 C (e), respectively, which somewhat displayed similar curve shapes to that of their corresponding hosts. From the TG curves, the weight loss percentage of HP-b-CD/daidzein complex from dehydration was 3.78% (c), while it was 8.43% for HP-b-CD (b). Similarly, it was 3.89% for SBE-b-CD/daidzein (e), compared to 10.28% for SBEb-CD (d). The results indicated that water molecules in CD cavity were partially replaced by daidzein to form the inclusion complexes.
3.6. Scanning electron microphotographs The SEM experiments were conducted to visualize the surface texture of inclusion complexes of daidzein with CDs. The SEM analyses of daidzein, HP-b-CD and their physical mixture and inclusion complex were shown in Fig. 10. Daidzein (Fig. 12A) had a rodlike crystal structure, when HP-b-CD displayed spherical crystals with cavities. Simple hybrids of rod-like and spherical crystals were observed in the physical mixture (Fig. 12 C). In contrast, the inclusion complex (Fig. 12 D) appeared in the form of compact and homogeneous plate-like crystal particles in which the original morphology of both components disappeared, which verified the formation of the daidzein/HP-b-CD inclusion complex. 3.7. Solubilization The results showed that the water solubility of daidzein/HP-bCD and daidzein/SBE-b-CD complexes, compared with that of native daidzein (ca. 8.31 mg/mL), were remarkably increased to 12.25 mg/mL and 10.68 mg/mL, respectively. The significant promotion on water solubility of daidzein by inclusion complexation with CDs would benefit to the pharmaceutical utilization of daidzein. 4. Conclusions In summary, daidzein/HP-b-CD and daidzein/SBE-b-CD inclusion complexes were successfully prepared and were characterized by spectral titration, NMR, XRD, DSC and SEM. The binding behaviors of daidzein with CDs were extensively elucidated. The stability constants were also obtained. The water solubility of
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daidzein was significantly improved by inclusion complexation with CDs. This indicated that inclusion complexes of daidzein/HPb-CD and daidzein/SBE-b-CD might be novel promising formulations of daidzein for its further pharmaceutical applications. Acknowledgments This work was financially supported by National Natural Science Foundation of China (No. 21362016) and Kunming University of Science and Technology. The authors would also like to thank Mr. Congtao Yu and Mr. Pin Lv for assistance on NMR and elemental analyses. References [1] N. Gottstein, B.A. Ewins, C. Eccleston, G.P. Hubbard, I.C. Kavanagh, A.M. Minihane, P.D. Weinberg, G. Rimbach, Brit. J. Nutr. 89 (2003) 607. [2] D.A. Tonetti, Y. Zhang, H. Zhao, S.B. Lim, A.I. Constantinou, Nutr. Cancer 58 (2007) 222. [3] S.H. Cheong, K. Furuhashia, K. Ito, M. Nagaoka, T. Yonezawa, Y. Miura, K. Yagasaki, J. Nutr. Biochem. 25 (2014) 136. [4] M. Park, J. Ju, M. Park, J. Han, Eur. J. Pharmacol. 712 (2013) 48. [5] S.E. Kulling, D.M. Honig, T.J. Simat, M. Metzler, J. Agric. Food Chem. 48 (2000) 4963. [6] Y. Ungar, O.F. Osundahunsi, E. Shimoni, J. Agric. Food Chem. 51 (2003) 4394. [7] K. Nakagawa, J. Adachi, M.C. Wong, Y. Ueno, Kobe J. Med. Sci. 52 (2006) 141. €hrdanz, S. Ohler, Q.H. Tran-Thi, R. Kahl, J. Nutr. 132 (2002) 370. [8] E. Ro [9] D. Somjen, S. Katzburg, F. Kohen, B. Gayer, E. Livne, J. Cell Biochem. 103 (2008) 1826. [10] K.D. Setchell, E. Lydeking-Olsen, Am. J. Clin. Nutr. 78 (2003) 593s. [11] H.J. Park, Y.K. Jeon, D.H. You, M.J. Nam, Food Chem. Toxicol. 60 (2013) 542. [12] X. Dong, W. Xu, R.A. Sikes, C. Wu, Food Chem. 141 (2013) 1923. [13] S.R. Lepri, L.C. Zanelatto, P.B. da Silva, D. Sartori, L.R. Ribeiro, M.S. Mantovani, Hum. Cell 27 (2014) 78. [14] K.D. Setchell, C. Clerici, J. Nutr. 140 (2010) 1355S. [15] C. Atkinson, C.L. Frankenfeld, J.W. Lampe, Exp. Biol. Med. 230 (2005) 155. [16] K.D. Setchell, N.M. Brown, P. Desai, L. Zimmer-Nechemias, B.E. Wolfe, W.T. Brashear, A.S. Kirschner, A. Cassidy, J.E. Heubi, J. Nutr. 131 (2001) 1362S. [17] Y. Ungar, O.F. Osundahunsi, E. Shimoni, J. Agric. Food Chem. 51 (2003) 4394. [18] J. Szejtli, Chem. Rev. 98 (1998) 1743. [19] G. Crini, Chem. Rev. 114 (2014) 10940.
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