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Preparation and physicochemical characterization of the supramolecular inclusion complex of naringin dihydrochalcone and hydroxypropyl-β-cyclodextrin
Food Research International 54 (2013) 691–696
Contents lists available at ScienceDirect
Food Research International journal homepage: www.elsevier.com/locate/foodres
Preparation and physicochemical characterization of the supramolecular inclusion complex of naringin dihydrochalcone and hydroxypropyl-β-cyclodextrin Benguo Liu a, Xiaoai Zhu b, Jie Zeng a, Jian Zhao c,⁎ a b c
School of Food Science, Henan Institute of Science and Technology, Xinxiang 453003, PR China College of Food Science, South China Agricultural University, Guangzhou 510642, PR China School of Chemical Engineering, the University of New South Wales, Sydney, NSW 2052, Australia
a r t i c l e
i n f o
Article history: Received 2 May 2013 Accepted 10 August 2013 Available online 19 August 2013 Keywords: Naringin dihydrochalcone Hydroxypropyl-β-cyclodextrin Inclusion complex Supramolecule Physicochemical property
a b s t r a c t Naringin dihydrochalcone (naringin DC) is an intense sweetener and a strong antioxidant with potential applications in many food and pharmaceutical products. However, its low water solubility impedes the realization of these applications. This study investigated the feasibility of using hydroxypropyl-β-cyclodextrin (HP-β-CD) to form a supramolecular inclusion complex with naringin DC to improve its solubility. The inclusion complex was prepared by stirring an equal molar solution of naringin DC and HP-β-CD at 30 °C for 24 h, followed by freeze-drying. The resultant complex was characterized by ultraviolet–visible spectroscopy (UV), Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), X-ray diffractometry (XRD) and proton nuclear magnetic resonance spectroscopy (1H-NMR) to determine its spectral, phase transitional and morphological properties and to elucidate its conformational structure. Results showed clearly the formation of a supramolecular complex in which the guest molecule, naringin DC, was entrapped inside the cavity of the host, HP-β-CD. The close association between naringin DC and HP-β-CD resulted in changes in some of the characteristic spectral, phase transitional and morphological properties of naringin DC. Furthermore, 1H-NMR analyses demonstrated that it was the B ring of naringin DC that was inserted into the HP-β-CD cavity to form the supramolecular complex. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Naringin dihydrochalcone (naringin DC, Fig. 1) is an intense sweetener, which is 500–700 times sweeter than sucrose and obtained by alkaline treatment of the natural flavonoid naringin (Surana, Gokhale, Rajmane, & Jadhav, 2006). As a new-style sweetening agent similar to neohesperidin dihydrochalcone, it possesses many desirable properties such as clean taste, low calorie, innocuity and safety, which allow it to be used in both food and pharmaceutical products (Surana et al., 2006; Grenby, 1991; Crosby & Beidler, 1976). Apart from being used as a sweetener, its intense sweetness is also useful for masking bitterness in foods and medicines (Gaudette & Pickering, 2013). Furthermore, naringin DC is a strong antioxidant which exhibits greater free radical scavenging capacity than its corresponding flavanone naringin (Nakamura, Watanabe, Miyake, Kohno, & Osawa, 2003). Therefore, naringin DC has the capacity to function both as a non-calorie sweetener and an antioxidant with potential applications in functional foods and beverages, nutraceuticals and pharmaceuticals. However, the poor solubility and stability of naringin DC
⁎ Corresponding author. Tel.: +61 2 93854304; fax: +61 2 93855966. E-mail address: [email protected] (J. Zhao). 0963-9969/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodres.2013.08.007
in aqueous systems severely restricts its applications in those products (Chebil et al., 2007). In recent years, using cyclodextrins to form supramolecular inclusion complexes has emerged as a promising method to improve the solubility of flavonoids. Cyclodextrins (CDs) are a family of cyclic oligosaccharides consisting of several glucopyranoses bound together by α-(1,4) glycosidic bonds to form a ring, and are obtained by enzymatic hydrolysis of starch (Palem, Chopparapu, Subrahmanyam, & Yamsani, 2012). The unique cone-shaped structure and properties of CDs confer them a remarkable capacity to entrap hydrophobic molecules to form host-guest complexes through weak intermolecular interaction with a wide variety of guests including flavonoids (Del Valle, 2004; Astray, GonzalezBarreiro, Mejuto, Rial-Otero, & Simal-Ga'ndara, 2009). In previous studies, we have investigated the formation of inclusion complexes between cyclodextrins and several flavonoids including rutin, dihydromyricetin, hesperidin and found that the formation of such supramolecular complexes not only improves the solubility of the flavonoids but also protect them from heat and radiation degradation and enhance their antioxidant activity (Liu, Li, Nguyen, & Zhao, 2012; Liu, Zhao, Liu, Zhu, & Zeng, 2012; Nguyen, Liu, Zhao, Thomas, & Hook, 2013). Of the many different types of cyclodextrins, β-CD is one of the most widely used for this purpose because its cavity size, at a diameter of 6.0–6.5 Å and of volume 265 Å3
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freeze-dried powder were dissolved in ethanol and the absorbance of the solution was measured by a model TU-1810PC scanning UV spectrophotometer (Purkinje, Beijing, China) at 281 nm and compared with a standard curve of pure naringin DC. The influence of HP-β-CD in the complex on the absorbance of naringin DC was negligible as its absorbance at UV-range was very low (see Results and discussion section). The yield of the inclusion complex was expressed as the percentage of the mass of recovered inclusion complex (freeze dried powder) over the mass of initial materials (naringin DC plus HP-β-CD). The inclusion ratio of naringin DC was calculated as the mass of naringin DC in the inclusion complex over the initial mass of naringin DC used for the complexation. 2.3. Preparation of the physical mixture of naringin DC and HP-β-CD Naringin DC (0.62 g) and HP-β-CD (1.375 g) were mixed and stirred in a small beaker (100 mL) at room temperature until a uniform mixture was formed. The obtained product was collected as the physical mixture of naringin DC and HP-β-CD. 2.4. Ultraviolet–visible spectroscopy (UV)
Fig. 1. Chemical structure of naringin DC (Crosby & Beidler, 1976).
(Del Valle, 2004), is suitable for common flavonoids with molecular weights between 200 and 800 g/mol (Wang, Cao, Sun, & Wang, 2011). Caccia et al. (1998) used β-CD to form an inclusion complex with neohesperidin dihydrochalcone while Fronza, Fuganti, Genesio, and Mele (2002) studied the structural features of β-CD complexes formed with naringin and its dihydrochalcone and aglycone derivatives. However, unmodified or substituted β-CD, i.e., β-CD with no substitutional groups on the glucopyranose units, has relatively poor water solubility itself and may be parenterally unsafe due to its nephrotoxicity (Jullian, Moyano, Yanez, & Olea-Azar, 2007). Many derivatives of β-CD, such as hydroxypropyl-β-cyclodextrin (HP-β-CD), on the other hand, offer improved water solubility and lower toxicity, and are increasingly replacing native CDs for various applications (Palem et al., 2012). The objectives of the present study were to explore the feasibility of HP-β-CD as an entrapping agent for naringin DC to form a host–guest inclusion complex, and to investigate the physicochemical characteristics of the formed supramolecule using spectral, phase transitional, X-ray diffractometric, scanning electron microscopic and proton nuclear magnetic resonance spectroscopic techniques. 2. Materials and methods 2.1. Chemicals Naringin dihydrochalcone (N 99%) was obtained from Shaanxi Huike Botanical Development Co., Ltd. (Xi'an, China). HP-β-CD (N99%, MW1375) was purchased from Seebio Biotech, Inc. (Shanghai, China). Other chemicals were of analytical grade unless stated otherwise. 2.2. Preparation of naringin DC and HP-β-CD inclusion complex Naringin DC (0.62 g, 1 mM) and HP-β-CD (1.375 g, 1 mM) were mixed in 25 mL of distilled water, stirred for 24 h at 30 °C and then filtered through a 0.45 μm membrane filter. The filtrate (25 mL) was freeze-dried (Alpha 1-4, Christ, Germany) for 72 h and the resultant powder was weighed and collected as the inclusion complex. To determine the amount of naringin DC in the complex, aliquots (10 mg) of the
UV spectra were recorded for HP-β-CD, naringin DC, their physical mixture and the inclusion complex using a TU-1810PC UV spectrophotometer (Purkinje, Beijing, China). Each sample (20–40 μg/mL) was dissolved in water at room temperature (25 ± 1 °C) and the aqueous solutions were scanned with a quartz cuvette cell (path length, 1 cm) in the wavelength range from 220 to 400 nm to obtain the UV spectra. The spectral bandwidth used was 0.1 nm and the scanning interval was 1 nm. 2.5. Fourier transform infrared spectroscopy (FT-IR) The FT-IR spectra of HP-β-CD, naringin DC, their physical mixture and the inclusion complex were collected between 4000 and 400 cm−1 on a TENSOR 27 infrared spectrophotometer (Bruker, Germany) with 256 scans at a resolution of 4 cm−1 by the KBr method. Briefly, 300 mg KBr was thoroughly mixed with 3 mg of the sample, and the mixture was compressed into a disk before being placed into the sample cell of the spectrophotometer. The transmittance data were recorded and processed by the OPUS software supplied with the instrument. 2.6. Differential scanning calorimetry (DSC) Thermal analysis was carried out for HP-β-CD, naringin DC, their physical mixture and the inclusion complex using a Q200 differential calorimeter calibrated with indium (TA Instruments, DE, USA). The samples (2 mg) were sealed in the aluminum crimp pans and heated at the rate of 10 °C/min from 30 to 300 °C in the atmosphere of nitrogen. An empty pan sealed in the same way was used as reference. The data were recorded and processed by the Universal Analysis 2000 software supplied with the instrument. 2.7. Scanning electron microscopy (SEM) Scanning electron micrographs (SEM) was performed with a Quanta 200 environmental scanning electron microscope (FEI, OR, USA). The samples were evenly distributed on SEM specimen stubs with double adhesive tape and the micrographs were obtained with an accelerating potential of 15 kV under low vacuum. 2.8. X-ray diffractometry (XRD) Monochromatic Cu Ka radiation (wavelength = 1.54056 Å) was produced by a D8 Advance X-ray diffractometer (Bruker, Germany). Samples were finely ground in an agate mortar and packed tightly in a
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rectangular aluminum cell prior to exposure to the X-ray beam. The scanning regions of the diffraction angle, 2θ, were 3–80° and radiation was detected with a proportional detector. Other instrument conditions were: step width, 0.02° of 2θ; count time, 0.15 s/step; scanning speed, 12°/min; voltage, 20 kV; current, 5 mA. 2.9. Proton nuclear magnetic resonance spectroscopy (1H-NMR) 1
H-NMR spectra of naringin DC and its complex with HP-β-CD were recorded with a 400 MHz Bruker AVANCE II spectrometer at 25 °C. The samples were dissolved in D2O and degassed by bubbling N2 directly in the NMR tubes. The chemical shifts (δ) were reported as ppm and referenced to the HOD signal. Instrumental conditions were: probe, 5 mm PABBO BB-1H Z-GRD Z824801/0050; number of scans, 8; receiver gain, 322; relaxation delay, 2.0; pulse width, 11.0; frequency, 400.2; spectral width, 8012.8. 3. Results and discussion 3.1. Preparation of naringin DC/HP-β-CD complex There are several different procedures that have been used by researchers to prepare CD inclusion complexes, including co-precipitation, neutralization, kneading, spray drying, freeze-drying, solvent evaporation, ball-milling and sealed-heating (Yamada et al., 2000). In this study, the freeze-drying method was used to prepare the inclusion complex of naringin DC and HP-β-CD, which had the advantage of minimizing potential chemical decomposition and loss of bioactivity which may occur to the flavonoid (Del Valle, 2004). A yield of 90.2% was achieved by this method with an inclusion ratio (the amount of naringin DC in the complex over the initial mass of naringin DC used) of 63.0%. 3.2. UV and FT-IR analysis The UV and FT-IR spectra of HP-β-CD, naringin DC, their physical mixture and the inclusion complex are shown in Figs. 2 and 3, respectively. As expected, the UV absorbance of HP-β-CD was extremely low and essentially flat owning to the lack of π-electrons (double bonds) in the molecule that can absorb energy in the form of ultraviolet light. The characteristic absorption peak of naringin DC was found at 281 nm, which was identical to that of the physical mixture and the complex. This demonstrated that the formation of an inclusion complex with HP-β-CD did not alter the UV absorption characteristics of naringin DC. The FT-IR spectrum of naringin DC consisted of the prominent
Fig. 2. UV spectra of HP-β-CD (1), naringin DC (2), their physical mixture (3) and inclusion complex (4).
Fig. 3. IR spectra of HP-β-CD (1), naringin DC (2), their physical mixture (3) and inclusion complex (4). Numbers indicate wavenumbers of key absorption peaks.
absorption bands of the hydroxyl group (3390 cm−1), aromatic conjugated carbonyl group (1628 cm−1), and the aromatic nucleus (1515, 1436 cm−1). The FT-IR spectrum of HP-β-CD showed prominent absorption bands at 3402 cm−1 (for O\H stretching vibrations), 2930 cm−1 (for C\H stretching vibrations) and 1155, 1084 and 1038 cm−1 (C\H, C\O stretching vibration). The IR spectrum of the physical mixture was essentially a combination of the spectra of HP-β-CD and naringin DC, showing the characteristic bands of both molecules. In contrast, in the spectrum of the inclusion complex, some of the small but characteristic absorption peaks of naringin DC between 400 and 1500 cm−1, such as the 1515 and 1436 cm−1 peaks for the aromatic nucleus and the 820 cm−1 peak for the para-substituted ring, almost totally disappeared. This was likely due to the restriction of the vibration of naringin DC molecule, suggesting that it was entrapped in the cavity of HP-β-CD molecule. 3.3. Differential scanning calorimetry Fig. 4 shows the DSC thermograms of HP-β-CD, naringin DC, their physical mixture and the inclusion complex. Consistent with
Fig. 4. DSC curves of HP-β-CD (1), naringin DC (2), their physical mixture (3) and inclusion complex (4).
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the amorphous nature of HP-β-CD, its thermogram showed a relatively flat line with a shallow trough appearing around 95 °C, similar to that reported in the literature (Hu, Zhanga, Song, Gu, & Hu, 2012). The thermogram of naringin DC showed three major peaks: one broad peak around 100 °C, probably due to loss of water, a relatively sharp peak at about 170 °C, corresponding to its melting point (Krbechek et al., 1968), and a large peak at about 220 °C, likely due to the thermal decomposition of the molecule. The thermogram of the physical mixture mainly showed the features of naringin DC, although the presence of HP-β-CD appeared to have blunted the melting peak and postponed the appearance of the thermal decomposition peak. This is most likely due to the absorption of part of the thermal energy by HP-β-CD in the mixture, with consequently greater thermal energy (higher temperature) required to cause decomposition of the molecule. In contrast, the DSC curve of the inclusion complex showed only the features of HP-β-CD, while the characteristic endothermal peaks of naringin DC totally disappeared, suggesting that the former was dispersed inside the molecules of the latter, probably in the form of an inclusion complex (Lasonder & Weringa, 1990). Similar observations of guest molecules losing their characteristic DSC peaks when formed inclusion complexes with cyclodextrins have been reported elsewhere (Wang et al., 2011; Liu, Li et al., 2012). 3.4. SEM analysis The scanning electron micrographs of HP-β-CD, naringin DC, their physical mixture and inclusion complex are shown in Fig. 5. Naringin DC existed in needle-like crystals while HP-β-CD occurred as amorphous spheres. In the micrograph of the physical mixture, the characteristic crystals of naringin DC and the amorphous spheres of HP-β-CD were found to exist side by side. In contrast, the inclusion complex appeared as irregular particles in which the original morphology of both components disappeared and tiny aggregates of amorphous pieces of irregular size were present. These images demonstrated that when
the powders of naringin DC and HP-β-CD were simply mixed together, they formed no close association and continued to exist in their original states, whereas when the solutions of the two compounds were freezedried, they formed a close association, probably in the form of inclusion complex, in which naringin DC no longer existed in the crystal state. 3.5. XRD analysis Further evidence for the formation of naringin DC/HP-β-CD complex was obtained from XRD (Fig. 6). XRD has proven to be a useful method for studying the formation of inclusion complexes between cyclodextrins and various guests, because the formation of an inclusion complex would disrupt the diffraction pattern of the guests. This would make the diffraction pattern of the complex clearly different from the superposition of the diffraction patterns of individual components (Nikolic et al., 2004). As seen in Fig. 6, the XRD pattern of naringin DC showed sharp, intense peaks that are consistent with the crystalline nature of the compound. In contrast, the XRD pattern of HP-β-CD displayed two broad peaks, confirming its amorphous nature. For the physical mixture, the diffraction pattern was essentially those of the crystalline naringin DC and the amorphous HP-β-CD superimposed over each other. The sharp peaks in the diffraction pattern indicated that the crystalline structure of naringin DC was retained in the physical mixture. In contrast, the XRD pattern of the inclusion complex was very similar to that of the amorphous HP-β-CD and did not exhibit the characteristic peaks of naringin DC, suggesting that naringin DC was molecularly dispersed in HP-β-CD with consequent loss of its crystallinity. 3.6. 1H-NMR analysis Further evidence supporting the formation of an inclusion complex between naringin DC and HP-β-CD was obtained from 1H-NMR analysis, which has proven to be extremely insightful in the study of inclusion
Fig. 5. Scanning electron micrographs of HP-β-CD (1), naringin DC (2), their physical mixture (3) and inclusion complex (4).
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Fig. 6. XRD patterns of HP-β-CD (1), naringin DC (2), their physical mixture (3) and inclusion complex (4).
complexation phenomenon between CD and flavonoids. The formation of inclusion complexes would cause a change in the chemical environment for the guest and CD molecules with resultant chemical shifts in their 1H-NMR spectra. Such chemical shifts not only could confirm the formation of guest-CD inclusion complex, but also provide valuable insight into the molecular conformation of the formed supramolecular structure, i.e. which part of the guest molecule is inserted into the CD cavity. For naringin DC, only chemical shifts in the aromatic protons were observed, which are shown in Fig. 7 while Table 1 gives values of the chemical shifts before and after complexation. Significant downward shifts were observed for the 2′/6′-H and 3′/5′-H protons in ring B, which was likely due to their interaction with the hydroxyl groups in the cavity of HP-β-CD. Only a small upward shift occurred to the
6/8-H proton in ring A, suggesting that there was probably no close association between ring A and the cavity of HP-β-CD. These chemical shifts data demonstrated clearly that naringin DC formed an inclusion complex with HP-β-CD. Furthermore, it was ring B that was most likely inserted in the cavity of HP-β-CD while ring A remained on the outside of the cavity, probably due to the obstructive effect of its large disaccharide group. 4. Conclusions In this study, we have shown that inclusion complex between naringin DC and HP-β-CD can be successfully prepared with good yield, using the freeze-drying method. Evidence obtained by UV, FT-IR,
Fig. 7. Chemical shifts of aromatic protons of naringin DC when formed an inclusion complex with HP-β-CD.
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Table 1 Chemical shift values (ppm) of naringin DC before and after complexation with HP-β-CD. Protons
δfree
δcomplex
Δδ = δcomplex − δfree
H-2′/6′ H-3′/5′ H-6/8
7.33 7.08 6.19
7.13 6.89 6.21
−0.2 −0.19 +0.02
DSC, SEM, XRD and 1H-NMR analyses demonstrated clearly the complexation process led to the formation of a supramolecular structure in which the B ring of naringin DC was inserted into the cavity of HP-β-CD. The formation of the supramolecular inclusion complex resulted in changes in some of the spectral, phase transitional and morphological properties of naringin DC. Future research could be directed at investigating the processing properties of the complex, such as stability under various processing and storage conditions, and exploring its application in food and nutraceutical products.
Acknowledgments Dr. Benguo Liu is a recipient of the Endeavour Postdoctoral Research Fellowship Award of the Australian Federal Government. The financial support provided by the National Natural Science Foundation of China (31101232 and 21166024) was greatly appreciated.
References Astray, G., Gonzalez-Barreiro, C., Mejuto, J. C., Rial-Otero, R., & Simal-Ga'ndara, J. (2009). A review on the use of cyclodextrins in foods. Food Hydrocolloids, 23, 1631–1640. Caccia, F., Dispenza, R., Fronza, G., Fuganti, C., Malpezzi, L., & Mele, A. (1998). Structure of neohesperidin dihydrochalcone/β-cyclodextrin inclusion complex: NMR, MS, and X-ray spectroscopic investigation. Journal of Agricultural and Food Chemistry, 46, 1500–1505. Chebil, L., Humeau, C., Anthoni, J., Dehez, F., Engasser, J. -M., & Ghoul, M. (2007). Solubility of flavonoids in organic solvents. Journal of Chemical and Engineering Data, 52, 1552–1556. Crosby, G. A., & Beidler, L. M. (1976). New sweeteners. CRC Critical Reviews in Food Science and Nutrition, 4, 297–323.
Del Valle, E. M. (2004). Cyclodextrins and their uses: A review. Process Biochemistry, 39, 1033–1046. Fronza, G., Fuganti, C., Genesio, E., & Mele, A. (2002). Structural features of the β-CD complexes with naringin and its dihydrochalcone and aglycone derivatives by 1H NMR. Journal of Inclusion Phenomena and Macrocyclic Chemistry, 44, 225–228. Gaudette, N. J., & Pickering, G. J. (2013). Modifying bitterness in functional food systems. Critical Reviews in Food Science and Nutrition, 53, 464–481. Grenby, T. H. (1991). Intense sweeteners for the food industry: An overview. Trends in Food Science & Technology, 2, 1–5. Hu, L., Zhanga, H., Song, W., Gu, D., & Hu, Q. (2012). Investigation of inclusion complex of cilnidipine with hydroxypropyl-β-cyclodextrin. Carbohydrate Polymers, 90, 1719–1724. Jullian, C., Moyano, L., Yanez, C., & Olea-Azar, C. (2007). Complexation of quercetin with three kinds of cyclodextrins: An antioxidant study. Spectrochimica Acta Part A, 67, 230–234. Krbechek, L., Inglett, G., Holik, M., Dowling, B., Wagner, R., & Riter, R. (1968). Dihydrochalcones, synthesis of potential sweetening agents. Journal of Agricultural and Food Chemistry, 16, 108–112. Lasonder, E., & Weringa, W. D. (1990). An NMR and DSC study of the interaction of phospholipid vesicles with some anti-inflammatory agents. Journal of Colloid and Interface Science, 139, 469–478. Liu, B., Li, W., Nguyen, T. A., & Zhao, J. (2012). Empirical, thermodynamic and quantum-chemical investigations of inclusion complexation between flavanones and (2-hydroxypropyl)-cyclodextrins. Food Chemistry, 134, 926–932. Liu, B., Zhao, J., Liu, Y., Zhu, X., & Zeng, J. (2012). Physicochemical properties of the inclusion complex of puerarin and glucosyl-β-cyclodextrin. Journal of Agricultural and Food Chemistry, 60, 12501–12507. Nakamura, Y., Watanabe, S., Miyake, N., Kohno, H., & Osawa, T. (2003). Dihydrochalcones: Evaluation as novel radical scavenging antioxidants. Journal of Agricultural and Food Chemistry, 51, 3309–3312. Nguyen, T. A., Liu, B., Zhao, J., Thomas, D. S., & Hook, J. M. (2013). An investigation into the supramolecular structure, solubility, stability and antioxidant activity of rutin/cyclodextrin inclusion complex. Food Chemistry, 136, 186–192. Nikolic, V., Stankovic, M., Kapor, A., Nikolic, L., Cvetkovic, D., & Stamenkovic, J. (2004). Allylthiosulfinate: Beta-cyclodextrin inclusion complex: Preparation, characterization and microbiological activity. Pharmazie, 59, 845–848. Palem, C. D., Chopparapu, K. S.C., Subrahmanyam, P. V. R. S., & Yamsani, M. R. (2012). Cyclodextrins and their derivatives in drug delivery: A review. Current Trends in Biotechnology and Pharmacy, 255, 255–275. Surana, S. J., Gokhale, S. B., Rajmane, R. A., & Jadhav, R. B. (2006). Non-saccharide natural intense sweeteners — An overview of current status. Natural Product Radiance, 54, 270–278. Wang, J., Cao, Y., Sun, B., & Wang, C. (2011). Physicochemical and release characterisation of garlic oil-β-cyclodextrin inclusion complexes. Food Chemistry, 127, 1680–1685. Yamada, T., Imai, T., Ouchi, K., Otagiri, M., Hirayama, F., & Uekama, K. (2000). Inclusion complex of 3,9-bis(N,N-dimethylcarbamoloxy)-5H-benzofuro[3,2-c] quinoline-6-one (KCA-098) with Heptakis(2,6-di-O-methyl)-β-cyclodextrin: Interaction and dissolution properties. Chemical and Pharmaceutical Bulletin, 48, 1264–1269.
Contents lists available at ScienceDirect
Food Research International journal homepage: www.elsevier.com/locate/foodres
Preparation and physicochemical characterization of the supramolecular inclusion complex of naringin dihydrochalcone and hydroxypropyl-β-cyclodextrin Benguo Liu a, Xiaoai Zhu b, Jie Zeng a, Jian Zhao c,⁎ a b c
School of Food Science, Henan Institute of Science and Technology, Xinxiang 453003, PR China College of Food Science, South China Agricultural University, Guangzhou 510642, PR China School of Chemical Engineering, the University of New South Wales, Sydney, NSW 2052, Australia
a r t i c l e
i n f o
Article history: Received 2 May 2013 Accepted 10 August 2013 Available online 19 August 2013 Keywords: Naringin dihydrochalcone Hydroxypropyl-β-cyclodextrin Inclusion complex Supramolecule Physicochemical property
a b s t r a c t Naringin dihydrochalcone (naringin DC) is an intense sweetener and a strong antioxidant with potential applications in many food and pharmaceutical products. However, its low water solubility impedes the realization of these applications. This study investigated the feasibility of using hydroxypropyl-β-cyclodextrin (HP-β-CD) to form a supramolecular inclusion complex with naringin DC to improve its solubility. The inclusion complex was prepared by stirring an equal molar solution of naringin DC and HP-β-CD at 30 °C for 24 h, followed by freeze-drying. The resultant complex was characterized by ultraviolet–visible spectroscopy (UV), Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), X-ray diffractometry (XRD) and proton nuclear magnetic resonance spectroscopy (1H-NMR) to determine its spectral, phase transitional and morphological properties and to elucidate its conformational structure. Results showed clearly the formation of a supramolecular complex in which the guest molecule, naringin DC, was entrapped inside the cavity of the host, HP-β-CD. The close association between naringin DC and HP-β-CD resulted in changes in some of the characteristic spectral, phase transitional and morphological properties of naringin DC. Furthermore, 1H-NMR analyses demonstrated that it was the B ring of naringin DC that was inserted into the HP-β-CD cavity to form the supramolecular complex. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Naringin dihydrochalcone (naringin DC, Fig. 1) is an intense sweetener, which is 500–700 times sweeter than sucrose and obtained by alkaline treatment of the natural flavonoid naringin (Surana, Gokhale, Rajmane, & Jadhav, 2006). As a new-style sweetening agent similar to neohesperidin dihydrochalcone, it possesses many desirable properties such as clean taste, low calorie, innocuity and safety, which allow it to be used in both food and pharmaceutical products (Surana et al., 2006; Grenby, 1991; Crosby & Beidler, 1976). Apart from being used as a sweetener, its intense sweetness is also useful for masking bitterness in foods and medicines (Gaudette & Pickering, 2013). Furthermore, naringin DC is a strong antioxidant which exhibits greater free radical scavenging capacity than its corresponding flavanone naringin (Nakamura, Watanabe, Miyake, Kohno, & Osawa, 2003). Therefore, naringin DC has the capacity to function both as a non-calorie sweetener and an antioxidant with potential applications in functional foods and beverages, nutraceuticals and pharmaceuticals. However, the poor solubility and stability of naringin DC
⁎ Corresponding author. Tel.: +61 2 93854304; fax: +61 2 93855966. E-mail address: [email protected] (J. Zhao). 0963-9969/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodres.2013.08.007
in aqueous systems severely restricts its applications in those products (Chebil et al., 2007). In recent years, using cyclodextrins to form supramolecular inclusion complexes has emerged as a promising method to improve the solubility of flavonoids. Cyclodextrins (CDs) are a family of cyclic oligosaccharides consisting of several glucopyranoses bound together by α-(1,4) glycosidic bonds to form a ring, and are obtained by enzymatic hydrolysis of starch (Palem, Chopparapu, Subrahmanyam, & Yamsani, 2012). The unique cone-shaped structure and properties of CDs confer them a remarkable capacity to entrap hydrophobic molecules to form host-guest complexes through weak intermolecular interaction with a wide variety of guests including flavonoids (Del Valle, 2004; Astray, GonzalezBarreiro, Mejuto, Rial-Otero, & Simal-Ga'ndara, 2009). In previous studies, we have investigated the formation of inclusion complexes between cyclodextrins and several flavonoids including rutin, dihydromyricetin, hesperidin and found that the formation of such supramolecular complexes not only improves the solubility of the flavonoids but also protect them from heat and radiation degradation and enhance their antioxidant activity (Liu, Li, Nguyen, & Zhao, 2012; Liu, Zhao, Liu, Zhu, & Zeng, 2012; Nguyen, Liu, Zhao, Thomas, & Hook, 2013). Of the many different types of cyclodextrins, β-CD is one of the most widely used for this purpose because its cavity size, at a diameter of 6.0–6.5 Å and of volume 265 Å3
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freeze-dried powder were dissolved in ethanol and the absorbance of the solution was measured by a model TU-1810PC scanning UV spectrophotometer (Purkinje, Beijing, China) at 281 nm and compared with a standard curve of pure naringin DC. The influence of HP-β-CD in the complex on the absorbance of naringin DC was negligible as its absorbance at UV-range was very low (see Results and discussion section). The yield of the inclusion complex was expressed as the percentage of the mass of recovered inclusion complex (freeze dried powder) over the mass of initial materials (naringin DC plus HP-β-CD). The inclusion ratio of naringin DC was calculated as the mass of naringin DC in the inclusion complex over the initial mass of naringin DC used for the complexation. 2.3. Preparation of the physical mixture of naringin DC and HP-β-CD Naringin DC (0.62 g) and HP-β-CD (1.375 g) were mixed and stirred in a small beaker (100 mL) at room temperature until a uniform mixture was formed. The obtained product was collected as the physical mixture of naringin DC and HP-β-CD. 2.4. Ultraviolet–visible spectroscopy (UV)
Fig. 1. Chemical structure of naringin DC (Crosby & Beidler, 1976).
(Del Valle, 2004), is suitable for common flavonoids with molecular weights between 200 and 800 g/mol (Wang, Cao, Sun, & Wang, 2011). Caccia et al. (1998) used β-CD to form an inclusion complex with neohesperidin dihydrochalcone while Fronza, Fuganti, Genesio, and Mele (2002) studied the structural features of β-CD complexes formed with naringin and its dihydrochalcone and aglycone derivatives. However, unmodified or substituted β-CD, i.e., β-CD with no substitutional groups on the glucopyranose units, has relatively poor water solubility itself and may be parenterally unsafe due to its nephrotoxicity (Jullian, Moyano, Yanez, & Olea-Azar, 2007). Many derivatives of β-CD, such as hydroxypropyl-β-cyclodextrin (HP-β-CD), on the other hand, offer improved water solubility and lower toxicity, and are increasingly replacing native CDs for various applications (Palem et al., 2012). The objectives of the present study were to explore the feasibility of HP-β-CD as an entrapping agent for naringin DC to form a host–guest inclusion complex, and to investigate the physicochemical characteristics of the formed supramolecule using spectral, phase transitional, X-ray diffractometric, scanning electron microscopic and proton nuclear magnetic resonance spectroscopic techniques. 2. Materials and methods 2.1. Chemicals Naringin dihydrochalcone (N 99%) was obtained from Shaanxi Huike Botanical Development Co., Ltd. (Xi'an, China). HP-β-CD (N99%, MW1375) was purchased from Seebio Biotech, Inc. (Shanghai, China). Other chemicals were of analytical grade unless stated otherwise. 2.2. Preparation of naringin DC and HP-β-CD inclusion complex Naringin DC (0.62 g, 1 mM) and HP-β-CD (1.375 g, 1 mM) were mixed in 25 mL of distilled water, stirred for 24 h at 30 °C and then filtered through a 0.45 μm membrane filter. The filtrate (25 mL) was freeze-dried (Alpha 1-4, Christ, Germany) for 72 h and the resultant powder was weighed and collected as the inclusion complex. To determine the amount of naringin DC in the complex, aliquots (10 mg) of the
UV spectra were recorded for HP-β-CD, naringin DC, their physical mixture and the inclusion complex using a TU-1810PC UV spectrophotometer (Purkinje, Beijing, China). Each sample (20–40 μg/mL) was dissolved in water at room temperature (25 ± 1 °C) and the aqueous solutions were scanned with a quartz cuvette cell (path length, 1 cm) in the wavelength range from 220 to 400 nm to obtain the UV spectra. The spectral bandwidth used was 0.1 nm and the scanning interval was 1 nm. 2.5. Fourier transform infrared spectroscopy (FT-IR) The FT-IR spectra of HP-β-CD, naringin DC, their physical mixture and the inclusion complex were collected between 4000 and 400 cm−1 on a TENSOR 27 infrared spectrophotometer (Bruker, Germany) with 256 scans at a resolution of 4 cm−1 by the KBr method. Briefly, 300 mg KBr was thoroughly mixed with 3 mg of the sample, and the mixture was compressed into a disk before being placed into the sample cell of the spectrophotometer. The transmittance data were recorded and processed by the OPUS software supplied with the instrument. 2.6. Differential scanning calorimetry (DSC) Thermal analysis was carried out for HP-β-CD, naringin DC, their physical mixture and the inclusion complex using a Q200 differential calorimeter calibrated with indium (TA Instruments, DE, USA). The samples (2 mg) were sealed in the aluminum crimp pans and heated at the rate of 10 °C/min from 30 to 300 °C in the atmosphere of nitrogen. An empty pan sealed in the same way was used as reference. The data were recorded and processed by the Universal Analysis 2000 software supplied with the instrument. 2.7. Scanning electron microscopy (SEM) Scanning electron micrographs (SEM) was performed with a Quanta 200 environmental scanning electron microscope (FEI, OR, USA). The samples were evenly distributed on SEM specimen stubs with double adhesive tape and the micrographs were obtained with an accelerating potential of 15 kV under low vacuum. 2.8. X-ray diffractometry (XRD) Monochromatic Cu Ka radiation (wavelength = 1.54056 Å) was produced by a D8 Advance X-ray diffractometer (Bruker, Germany). Samples were finely ground in an agate mortar and packed tightly in a
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rectangular aluminum cell prior to exposure to the X-ray beam. The scanning regions of the diffraction angle, 2θ, were 3–80° and radiation was detected with a proportional detector. Other instrument conditions were: step width, 0.02° of 2θ; count time, 0.15 s/step; scanning speed, 12°/min; voltage, 20 kV; current, 5 mA. 2.9. Proton nuclear magnetic resonance spectroscopy (1H-NMR) 1
H-NMR spectra of naringin DC and its complex with HP-β-CD were recorded with a 400 MHz Bruker AVANCE II spectrometer at 25 °C. The samples were dissolved in D2O and degassed by bubbling N2 directly in the NMR tubes. The chemical shifts (δ) were reported as ppm and referenced to the HOD signal. Instrumental conditions were: probe, 5 mm PABBO BB-1H Z-GRD Z824801/0050; number of scans, 8; receiver gain, 322; relaxation delay, 2.0; pulse width, 11.0; frequency, 400.2; spectral width, 8012.8. 3. Results and discussion 3.1. Preparation of naringin DC/HP-β-CD complex There are several different procedures that have been used by researchers to prepare CD inclusion complexes, including co-precipitation, neutralization, kneading, spray drying, freeze-drying, solvent evaporation, ball-milling and sealed-heating (Yamada et al., 2000). In this study, the freeze-drying method was used to prepare the inclusion complex of naringin DC and HP-β-CD, which had the advantage of minimizing potential chemical decomposition and loss of bioactivity which may occur to the flavonoid (Del Valle, 2004). A yield of 90.2% was achieved by this method with an inclusion ratio (the amount of naringin DC in the complex over the initial mass of naringin DC used) of 63.0%. 3.2. UV and FT-IR analysis The UV and FT-IR spectra of HP-β-CD, naringin DC, their physical mixture and the inclusion complex are shown in Figs. 2 and 3, respectively. As expected, the UV absorbance of HP-β-CD was extremely low and essentially flat owning to the lack of π-electrons (double bonds) in the molecule that can absorb energy in the form of ultraviolet light. The characteristic absorption peak of naringin DC was found at 281 nm, which was identical to that of the physical mixture and the complex. This demonstrated that the formation of an inclusion complex with HP-β-CD did not alter the UV absorption characteristics of naringin DC. The FT-IR spectrum of naringin DC consisted of the prominent
Fig. 2. UV spectra of HP-β-CD (1), naringin DC (2), their physical mixture (3) and inclusion complex (4).
Fig. 3. IR spectra of HP-β-CD (1), naringin DC (2), their physical mixture (3) and inclusion complex (4). Numbers indicate wavenumbers of key absorption peaks.
absorption bands of the hydroxyl group (3390 cm−1), aromatic conjugated carbonyl group (1628 cm−1), and the aromatic nucleus (1515, 1436 cm−1). The FT-IR spectrum of HP-β-CD showed prominent absorption bands at 3402 cm−1 (for O\H stretching vibrations), 2930 cm−1 (for C\H stretching vibrations) and 1155, 1084 and 1038 cm−1 (C\H, C\O stretching vibration). The IR spectrum of the physical mixture was essentially a combination of the spectra of HP-β-CD and naringin DC, showing the characteristic bands of both molecules. In contrast, in the spectrum of the inclusion complex, some of the small but characteristic absorption peaks of naringin DC between 400 and 1500 cm−1, such as the 1515 and 1436 cm−1 peaks for the aromatic nucleus and the 820 cm−1 peak for the para-substituted ring, almost totally disappeared. This was likely due to the restriction of the vibration of naringin DC molecule, suggesting that it was entrapped in the cavity of HP-β-CD molecule. 3.3. Differential scanning calorimetry Fig. 4 shows the DSC thermograms of HP-β-CD, naringin DC, their physical mixture and the inclusion complex. Consistent with
Fig. 4. DSC curves of HP-β-CD (1), naringin DC (2), their physical mixture (3) and inclusion complex (4).
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the amorphous nature of HP-β-CD, its thermogram showed a relatively flat line with a shallow trough appearing around 95 °C, similar to that reported in the literature (Hu, Zhanga, Song, Gu, & Hu, 2012). The thermogram of naringin DC showed three major peaks: one broad peak around 100 °C, probably due to loss of water, a relatively sharp peak at about 170 °C, corresponding to its melting point (Krbechek et al., 1968), and a large peak at about 220 °C, likely due to the thermal decomposition of the molecule. The thermogram of the physical mixture mainly showed the features of naringin DC, although the presence of HP-β-CD appeared to have blunted the melting peak and postponed the appearance of the thermal decomposition peak. This is most likely due to the absorption of part of the thermal energy by HP-β-CD in the mixture, with consequently greater thermal energy (higher temperature) required to cause decomposition of the molecule. In contrast, the DSC curve of the inclusion complex showed only the features of HP-β-CD, while the characteristic endothermal peaks of naringin DC totally disappeared, suggesting that the former was dispersed inside the molecules of the latter, probably in the form of an inclusion complex (Lasonder & Weringa, 1990). Similar observations of guest molecules losing their characteristic DSC peaks when formed inclusion complexes with cyclodextrins have been reported elsewhere (Wang et al., 2011; Liu, Li et al., 2012). 3.4. SEM analysis The scanning electron micrographs of HP-β-CD, naringin DC, their physical mixture and inclusion complex are shown in Fig. 5. Naringin DC existed in needle-like crystals while HP-β-CD occurred as amorphous spheres. In the micrograph of the physical mixture, the characteristic crystals of naringin DC and the amorphous spheres of HP-β-CD were found to exist side by side. In contrast, the inclusion complex appeared as irregular particles in which the original morphology of both components disappeared and tiny aggregates of amorphous pieces of irregular size were present. These images demonstrated that when
the powders of naringin DC and HP-β-CD were simply mixed together, they formed no close association and continued to exist in their original states, whereas when the solutions of the two compounds were freezedried, they formed a close association, probably in the form of inclusion complex, in which naringin DC no longer existed in the crystal state. 3.5. XRD analysis Further evidence for the formation of naringin DC/HP-β-CD complex was obtained from XRD (Fig. 6). XRD has proven to be a useful method for studying the formation of inclusion complexes between cyclodextrins and various guests, because the formation of an inclusion complex would disrupt the diffraction pattern of the guests. This would make the diffraction pattern of the complex clearly different from the superposition of the diffraction patterns of individual components (Nikolic et al., 2004). As seen in Fig. 6, the XRD pattern of naringin DC showed sharp, intense peaks that are consistent with the crystalline nature of the compound. In contrast, the XRD pattern of HP-β-CD displayed two broad peaks, confirming its amorphous nature. For the physical mixture, the diffraction pattern was essentially those of the crystalline naringin DC and the amorphous HP-β-CD superimposed over each other. The sharp peaks in the diffraction pattern indicated that the crystalline structure of naringin DC was retained in the physical mixture. In contrast, the XRD pattern of the inclusion complex was very similar to that of the amorphous HP-β-CD and did not exhibit the characteristic peaks of naringin DC, suggesting that naringin DC was molecularly dispersed in HP-β-CD with consequent loss of its crystallinity. 3.6. 1H-NMR analysis Further evidence supporting the formation of an inclusion complex between naringin DC and HP-β-CD was obtained from 1H-NMR analysis, which has proven to be extremely insightful in the study of inclusion
Fig. 5. Scanning electron micrographs of HP-β-CD (1), naringin DC (2), their physical mixture (3) and inclusion complex (4).
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Fig. 6. XRD patterns of HP-β-CD (1), naringin DC (2), their physical mixture (3) and inclusion complex (4).
complexation phenomenon between CD and flavonoids. The formation of inclusion complexes would cause a change in the chemical environment for the guest and CD molecules with resultant chemical shifts in their 1H-NMR spectra. Such chemical shifts not only could confirm the formation of guest-CD inclusion complex, but also provide valuable insight into the molecular conformation of the formed supramolecular structure, i.e. which part of the guest molecule is inserted into the CD cavity. For naringin DC, only chemical shifts in the aromatic protons were observed, which are shown in Fig. 7 while Table 1 gives values of the chemical shifts before and after complexation. Significant downward shifts were observed for the 2′/6′-H and 3′/5′-H protons in ring B, which was likely due to their interaction with the hydroxyl groups in the cavity of HP-β-CD. Only a small upward shift occurred to the
6/8-H proton in ring A, suggesting that there was probably no close association between ring A and the cavity of HP-β-CD. These chemical shifts data demonstrated clearly that naringin DC formed an inclusion complex with HP-β-CD. Furthermore, it was ring B that was most likely inserted in the cavity of HP-β-CD while ring A remained on the outside of the cavity, probably due to the obstructive effect of its large disaccharide group. 4. Conclusions In this study, we have shown that inclusion complex between naringin DC and HP-β-CD can be successfully prepared with good yield, using the freeze-drying method. Evidence obtained by UV, FT-IR,
Fig. 7. Chemical shifts of aromatic protons of naringin DC when formed an inclusion complex with HP-β-CD.
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Table 1 Chemical shift values (ppm) of naringin DC before and after complexation with HP-β-CD. Protons
δfree
δcomplex
Δδ = δcomplex − δfree
H-2′/6′ H-3′/5′ H-6/8
7.33 7.08 6.19
7.13 6.89 6.21
−0.2 −0.19 +0.02
DSC, SEM, XRD and 1H-NMR analyses demonstrated clearly the complexation process led to the formation of a supramolecular structure in which the B ring of naringin DC was inserted into the cavity of HP-β-CD. The formation of the supramolecular inclusion complex resulted in changes in some of the spectral, phase transitional and morphological properties of naringin DC. Future research could be directed at investigating the processing properties of the complex, such as stability under various processing and storage conditions, and exploring its application in food and nutraceutical products.
Acknowledgments Dr. Benguo Liu is a recipient of the Endeavour Postdoctoral Research Fellowship Award of the Australian Federal Government. The financial support provided by the National Natural Science Foundation of China (31101232 and 21166024) was greatly appreciated.
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