Food Chemistry 184 (2015) 99–104
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Preparation and characterization of inclusion complex of benzyl isothiocyanate extracted from papaya seed with b-cyclodextrin Wenzhao Li ⇑, Xiaoyu Liu, Qingfeng Yang, Ning Zhang, Yideng Du, Huaping Zhu Key Laboratory of Food Nutrition and Safety (Tianjin University of Science & Technology), Ministry of Education, Tianjin 300457, China
a r t i c l e
i n f o
Article history: Received 17 November 2014 Received in revised form 17 February 2015 Accepted 16 March 2015 Available online 28 March 2015 Keywords: Papaya seed Benzyl isothiocyanate b-Cyclodextrin Inclusion complex characterization
a b s t r a c t The inclusion complex of benzyl isothiocyanate (BITC), extracted from papaya seed with b-cyclodextrin (b-CD), was prepared. Different analytical techniques, such as Fourier transform infrared spectroscopy, thermal analysis, X-ray diffractometry, particle size distribution analysis and 1H Nuclear magnetic resonance analysis, were used to investigate the characterization of the inclusion complex (BITC-b-CD). All these approaches indicated that the inclusion complex was capable of being formed. The inclusion complex exhibited different spectroscopic and thermodynamic features and properties from BITC, and we deduced the possible inclusion modes for BITC-b-CD. The calculated apparent stability constant of the BITC-b-CD was 600.8 l/mol, and the aqueous solubility of BITC was indistinctively improved by phase solubility studies. The results illustrated that b-CD was a proper excipient for increasing the stability and controlled release of BITC. Thus, b-CD complexation technology would be a promising approach, in expanding the application of BITC as a food antibacterial agent. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Papaya seed has been used as vermifuge in India, Central and South America for centuries, but more of them were wasted during papaya processing (Fahey, Zalcmann, & Talalay, 2001). Williams, Pun, Chaliha, Scheelings, and O’Hare (2013) pointed out that papaya is one of the few tropical fruit species to contain a glucosinolate. Since most glucosinolate-containing plants possess 4– 5 glucosinolates, of which 1–2 predominate, the difference is in papaya seed, benzyl glucosinolate appears to be the sole glucosinolate present (Williams et al., 2013). Even more unusually, papaya contains a related secondary metabolite not normally found in any glucosinolate-containing species. Papaya seed extraction has been reported to have anthelmintic properties (Kermanshai, McCarry, Rosenfeld, Summer, & Weretilnyk, 2001), antimicrobial activity (Radulovic´, Dekic´, & Stojanovic´–Radic´, 2012), antifungal activity (Chávez-Quintal, Gonzalez-Flores, Rodriguez-Buenfil, & Galleqos-Tintoré, 2011), cell cycle arrest and initiation of apoptosis, inhibition of cell proliferation (Prashar, Siddiqui, & Singh, 2012), the potential to reduce the risk of various types of cancers due, at least in part, to benzyl glucosinolate. However, it is not clear whether all the activities are ⇑ Corresponding author at: No. 29, 13th Avenue, Tianjin Economic and Technological Development Area (TEDA), Tianjin 300457, China. Tel./fax: +86 22 60912514. E-mail address: [email protected] (W. Li). http://dx.doi.org/10.1016/j.foodchem.2015.03.091 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
due to one or more compounds present in these seed preparations, thus high efficient extraction and purification method are necessary to get sufficient quantity of purified benzyl glucosinolate for the investigation of real activity compounds. Fortunately, we have demonstrated previously that endogenous myrosinase in Carica Papaya seed could be utilized to convert benzyl glucosinolate to benzyl isothiocyanate (BITC) and arrived at the optimum conditions. Despite having excellent biological features, BITC is volatile and not thermostailised during food processing and storage, which does not fit well as a food antibacterial agent. These problems can be addressed by complexation with cyclodextrins (CDs) in aqueous solutions. CDs is a general term for a series of cyclic oligosaccharides consisting of (a-1,4)-linked a-D-glucopyranose units with a hydrophobic interior cavity and a hydrophobic exterior surface (Szejtli, 1998). The most commonly used CDs are a-, band c-CDs consisting of 6, 7 and 8 a-D-glucopyranose units, respectively, which can form inclusion complexes with a variety of organic compounds (Karathanos, Mourtzinos, Yannakopoulou, & Andrikopoulos, 2007). Among these, b-CD has minimal solubility in water, thus is most likely to precipitate crystals, and because of its high encapsulation efficiency, suitable cavity dimensions and low cost, it is most commonly used (Sancho, Gasull, Blanco, & Castro, 2011). Beta-cyclodextrin (b-CD) has a cylindrical structure, which ends with a hydrophilic external surface, while the internal cylinder is hydrophobic, some suitable size and shape of
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W. Li et al. / Food Chemistry 184 (2015) 99–104
the drug molecule (e.g., halo, volatile oil) are included in the cyclic structure by van der Waals forces. These features usually enhance food additives solubility in an aqueous solution and nonvolatility, widely used in food processing and food safety (Hu et al., 2012; Budryn et al., 2013; Wang, Luo, & Xiao, 2014; Song, Li, Du, & Wang, 2014). However, to the best of our knowledge, there is no scientific study on the inclusion complex of BITC with b-CDs. In this paper, the BITC extracted from papaya seed was included by b-CD and the characterizations of the complex were investigated by FT-IR, thermal analysis, XRD, particle size distribution analysis and 1H Nuclear magnetic resonance analysis. Meanwhile, phase solubility of BITC-b-CD complex was also studied. This study can be useful for the development of an active and intelligent packing material functionalist with naturally-occurring antimicrobial. 2. Materials and methods 2.1. Materials Carica papaya seed were supplied by Hainan Standard BioTechnique Co. Ltd., and were collected at Hainan province of China. Beta-cyclodextrin (b-CD) was obtained from Zibo Qianhui Biotechnology Co., Ltd. All other chemicals and reagents were of analytical grade. 2.2. Preparation of BITC By our previous studies, endogenous myrosinase in Carica Papaya seed was used to convert benzyl glucosinolate to benzyl isothiocyanate, the extraction conditions were as follows: seed powder particle size 90–120 lm, sample-to-solvent ratio1:20 (w/ v), pH of buffer solution 4.8, enzymolysis temperature 40 °C, enzymolysis time 27 min. The formed BITC was extracted by simultaneous distillation and extraction method for 2 h with dichloromethane as solvent and purified by silica gel chromatography. The purified BITC was kept under 4 °C in refrigerator for further study.
UV at 247 nm. Through the standard curve, the content was calculated. Reproducibility was checked by running the sample in triplicate. The inclusion efficiency procedure was calculated using the following equation:
IEð%Þ ¼ 100 ðmeasured BITC contentÞ=theoretical BITC content ð2Þ
2.5. Fourier transform infrared spectroscopy (FT-IR) The FT-IR spectra of BITC, b-CD and BITC-b-CD between 4000 cm1 and 400 cm1 were obtained using a Bruker Vector22 FT-IR spectrophotometer (Germany). The procedure consisted of intermixing sample (1 mg) together with KBr (150 mg), grinding them into fine powder and tablet compressing 30–60 s under 0.8–1.0 Mpa press, which should be well done for assays. The spectrum was obtained, corrected automatically by the built-in software of the spectrophotometer. 2.6. Thermal analysis The enhancement in heat stability of the BITC by inclusion with b-CD was evaluated by thermo-gravimetry (TG) and differential scanning calorimetry (DSC). TG analysis was carried out for BITC, b-CD, and the inclusion complex (BITC-b-CD) with a Q50 TGA (TA, USA). DSC analysis was performed by a DSC 1 (Mettler Toledo, Switzerland). Each prepared dried powder (5–8 mg) was heated in a crimped aluminum pan for DSC, and 9 ± 0.5 mg samples for thermo-gravimetry (TG) in an uncovered graphite crucible. All samples were heated from room temperature to 350 °C at 10 °C/ min under a nitrogen flow of 40 ml/min. The mass loss and heat flow in the sample were recorded as a function of temperature with reference to an empty pan. Reproducibility was checked by running the sample in triplicate. 2.7. X-ray diffractometry (XRD)
2.3. Preparation of BITC-b-CD complex The inclusion complex of BITC with b-CD was prepared by freeze dying. b-CD (4.0 g) was completely dissolved in 100 ml of water and kept at 40 °C. BITC anhydrous ethanol solution (0.5 ml, v:v = 1:1) was slowly blended into the prepared saturated solution of b-CD, and the mixture was intermittently reacted in ultrasonic homogenization and magnetic stirring for 3 h at 40 °C. The resulting solution was frozen at 80 °C and subsequently freeze dried for 24 h. 2.4. Determination of inclusion efficiency A certain concentration of BITC hexane solution was analysed by ultraviolet spectrophotometery (752PC, Tianjin Guanze Instrument CO., Ltd, China) from 200 nm to 400 nm, and the maximum absorption wavelength of BITC was 247 nm. Then the standard curve of the absorbance value (A) and the content of BITC (C, ll/ml) was established, the regression equation was:
A ¼ 11:688C 0:0045 R2 ¼ 0:9992
ð1Þ
The total content of BITC, in the obtained complexes, was determined by the extraction method according to Falcão et al. (2011) with modification. The sample was performed by mixing 10 mg of the lyophilized complex with 2 ml hexane, ultrasonic bath at 100 W for 20 min, and finally centrifugation at 10,000 rpm for 10 min. The supernatant obtained was immediately detected by
XRD patterns were obtained with a TD-3500 X-ray diffractometer (Dandong Tongda Science and Technology CO., Ltd, China) using Cu Ka radiation (30 kV, 20 mA), at a scanning rate of 8°/min. The samples were mounted on a sample holder and measured in the 2h angle range from 10° to 50° with a step size of 2h = 0.04°. 2.8. Particle size distribution In order to study the changes of the average particle size and polydispersity indexes (PDI) of b-CD, before and after embedding, a BI-200SM laser light scattering instrument (Brookhaven, USA) was used to measure these. Previously, 1 mg/ml of b-CD and the inclusion complex aqueous solutions were prepared, respectively. After homogeneous solutions were reached by ultrasonic vibration, samples were filtered through a 0.22 lm hydrophilic membrane filter, and then were examined. 2.9. 1H Nuclear magnetic resonance (1H NMR) All NMR experiments were carried out in Dimethyl sulfoxide (DMSO). Tetramethylsilane (TMS) was used as an internal standard. The 1H spectra of b-CD and the inclusion complex (BITC-bCD) were obtained using a Bruker Avance III spectrometer at 400 MHz and 298 K. Chemical shifts (d) are expressed in ppm relative to TMS.
W. Li et al. / Food Chemistry 184 (2015) 99–104
2.10. Phase solubility of BITC-b-CD complex As described in previous literature, phase solubility studies were performed according to the method reported by Higuchi and Connors (1965). Excess amounts of BITC anhydrous ethanol solution (20 ll, v:v = 1:1) were added to 10 ml of distilled water containing varying concentrations of b-CD (1–10 mM) in the 15 ml screw-cap tube. The mixture was placed in an ultrasonic bath and shaken for 3 h at 30 °C, and stabilization for 24 h at the room temperature in the dark. After equilibrium was reached, the aqueous solutions were withdrawn, using a syringe, and samples were filtered through a 0.45 lm hydrophilic membrane filter. The concentration of BITC in the filtrate was determined at 243 nm by an Agilent 8453E UV–visible spectroscopy (Australia). All samples were prepared in triplicate. The phase solubility profiles were obtained by plotting the BITC concentration against b-CD concentration. The apparent stability constant (Kc) of BITC and b-CD complex can be calculated from the phase solubility diagrams, according to the following equation:
101
(Nunes & Mercadante, 2007). The mixture was intermittently reacted in ultrasonic homogenization and magnetic stirring, which might be a better way to form inclusion complexes. The uncoated BITC settled down to the bottom and gathered. Intermittent ultrasonic homogenization promotes the emulsification degree of BITC and increases the contact area, which can improve the inclusion efficiency. Thus, higher inclusion efficiency provides feasibility for further application. 3.2. FT-IR spectra analysis
3. Results and discussion
The infrared (FT-IR) spectra of BITC, b-CD and their complex (BITC-b-CD) are displayed clearly in Fig. 1. Due to vibration of the N@C@S group, the IR spectra of BITC-b-CD could be characterized by the broad peak at 2140–2040 cm1. Moreover, there are three sharp absorption peaks between 1345 cm1 and 1600 cm1 in virtue of benzene ring skeleton vibration (Fig. 1a). The spectrum of b-CD manifests remarkable peaks ranged from 3540 cm1 to 3230 cm1, because of hydroxyl groups stretching vibration. We can observe the prominent absorption bands at 2926 cm1 (C–H vibration) and 1027 cm1 for C–O group vibration (Fig. 1b). By comparison, however, the FT-IR spectrum of BITC-b-CD was not completely congruent with that of b-CD (Fig. 1c). Although characteristic benzene peaks vanish in the spectrum of BITC-b-CD, the vibration bands of N@C@S groups are still existed. Therefore, in consideration of the above factors, we might suggest that the N@C@S groups exposed out the cavity of b-CD, while benzene ring of BITC has been entrapped in it.
3.1. Inclusion efficiency analysis
3.3. Thermal analysis
The inclusion efficiency of BITC was 86.4% ± 2.57. Previous reports, about the inclusion of red bell pepper pigments in b-CD by magnetic stirring and ultrasonic, resulted in inclusion efficiencies 52.95% ± 9.39 and 62.43% ± 12.89, respectively (Lidiane, Nicolly, Valéria, Deborah, & Kátia, 2014). Such studies about the inclusion efficiency of carotenoids in b-CD only by magnetic stirring were 48.96% (Chen, Chen, Guo, Li, & Li, 2007) and 50%
The thermal properties of b-CD, BITC, their inclusion complex (BITC-b-CD) were investigated using TG and DSC tests, the results are exhibited in Fig. 2. The thermogram of b-CD showed a significant thermal peak from 100 °C to 160 °C, indicating release of water molecules process in the DSC curves (Fig. 2ii). However, in the DSC curves of BITC-b-CD, an obviously wider band was viewed (Fig. 2iii), which was due to volatilization of uncoated BITC and a
Kc ¼
k S0 ð1 kÞ
ð3Þ
where k is the slope of the straight line, obtained from the initial segment of the phase solubility line, and S0 is the intrinsic solubility of BITC in distilled water in the absence of b-CD.
Fig. 1. FT-IR spectra of BITC (a), b-CD (b) and BITC-b-CD (c).
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Fig. 3. Powder X-ray diffraction spectra of b-CD (a) and BITC-b-CD (b).
had apparent sharp peaks at 2h of 11.8°, 16.9°, 17.4°, 17.7°, 18° and 21.1°, was clearly distinct from that of b-CD and exhibited another crystal form. As BITC is not solid at room temperature, the corresponding XRD patterns cannot be obtained. However, through comparative analysis of existing data, we considered that the crystal structure of b-CD and inclusion complex were different which indicated the formation of BITC and b-CD inclusion complex. 3.5. Particle size distribution analysis
Fig. 2. DSC curves of BITC (i), b-CD (ii) and BITC-b-CD (iii). TG curves of BITC (iv), BITC-b-CD (v) and b-CD (vi).
change in the substance structure after inclusion complex formation between BITC and b-CD. The TG curve of b-CD (Fig. 2vi) showed a presence of a slope near 300 °C generally attributed to the initiation of b-CD decomposition, DSC curve corroborated with this result, showing a one step of endothermic event up to 300 °C. BITC belongs to volatile material and quickly losses mass from 80 °C to 165 °C as we can see from Fig. 2iv. In fact, phase mass loss of the inclusion complex emerged an obvious slope between 140 °C and 300 °C due to the influence of b-CD, slowed down its volatilization. BITC exhibited an endothermic peak between 250 °C and 270 °C that may be related to boiling, this peak was not present in the DSC scan of the inclusion complex and appeared an endothermic peak (near 280 °C) with the b-CD decomposition, because of intermolecular force. A similar study was reported by Adriana et al. (2015). Therefore it is evident that the b-CD cavity provides protection against volatilization of BITC in the encapsulated constituents.
3.4. XRD analysis Many relevant research has reported that XRD analysis was a useful method for the detection of the inclusion complex in powder (Chen et al., 2011; Wang, Cao, Sun, & Wang, 2011; Srinivasan, Stalin, & Sivakumar, 2012). The XRD patterns of b-CD and BITC-b-CD are illustrated in Fig. 3. As shown in Fig. 3a, b-CD has intense and sharp peaks at 2h of 12.9°, 13.5°, 18.2°, 18.7°, and 19.7°, as well as several minor peaks at 2h of 10.9°, 14.2°, 15.6° and 24°, which confirm the crystalline nature of the compound. The XRD of BITC-b-CD (Fig. 3b), by comparison, which
The average inclusion complex diameters and PDIs are listed in Table 1. PDI is a measure of the uniformity of particle size. A value close to zero (
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Preparation and characterization of inclusion complex of benzyl isothiocyanate extracted from papaya seed with b-cyclodextrin Wenzhao Li ⇑, Xiaoyu Liu, Qingfeng Yang, Ning Zhang, Yideng Du, Huaping Zhu Key Laboratory of Food Nutrition and Safety (Tianjin University of Science & Technology), Ministry of Education, Tianjin 300457, China
a r t i c l e
i n f o
Article history: Received 17 November 2014 Received in revised form 17 February 2015 Accepted 16 March 2015 Available online 28 March 2015 Keywords: Papaya seed Benzyl isothiocyanate b-Cyclodextrin Inclusion complex characterization
a b s t r a c t The inclusion complex of benzyl isothiocyanate (BITC), extracted from papaya seed with b-cyclodextrin (b-CD), was prepared. Different analytical techniques, such as Fourier transform infrared spectroscopy, thermal analysis, X-ray diffractometry, particle size distribution analysis and 1H Nuclear magnetic resonance analysis, were used to investigate the characterization of the inclusion complex (BITC-b-CD). All these approaches indicated that the inclusion complex was capable of being formed. The inclusion complex exhibited different spectroscopic and thermodynamic features and properties from BITC, and we deduced the possible inclusion modes for BITC-b-CD. The calculated apparent stability constant of the BITC-b-CD was 600.8 l/mol, and the aqueous solubility of BITC was indistinctively improved by phase solubility studies. The results illustrated that b-CD was a proper excipient for increasing the stability and controlled release of BITC. Thus, b-CD complexation technology would be a promising approach, in expanding the application of BITC as a food antibacterial agent. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Papaya seed has been used as vermifuge in India, Central and South America for centuries, but more of them were wasted during papaya processing (Fahey, Zalcmann, & Talalay, 2001). Williams, Pun, Chaliha, Scheelings, and O’Hare (2013) pointed out that papaya is one of the few tropical fruit species to contain a glucosinolate. Since most glucosinolate-containing plants possess 4– 5 glucosinolates, of which 1–2 predominate, the difference is in papaya seed, benzyl glucosinolate appears to be the sole glucosinolate present (Williams et al., 2013). Even more unusually, papaya contains a related secondary metabolite not normally found in any glucosinolate-containing species. Papaya seed extraction has been reported to have anthelmintic properties (Kermanshai, McCarry, Rosenfeld, Summer, & Weretilnyk, 2001), antimicrobial activity (Radulovic´, Dekic´, & Stojanovic´–Radic´, 2012), antifungal activity (Chávez-Quintal, Gonzalez-Flores, Rodriguez-Buenfil, & Galleqos-Tintoré, 2011), cell cycle arrest and initiation of apoptosis, inhibition of cell proliferation (Prashar, Siddiqui, & Singh, 2012), the potential to reduce the risk of various types of cancers due, at least in part, to benzyl glucosinolate. However, it is not clear whether all the activities are ⇑ Corresponding author at: No. 29, 13th Avenue, Tianjin Economic and Technological Development Area (TEDA), Tianjin 300457, China. Tel./fax: +86 22 60912514. E-mail address: [email protected] (W. Li). http://dx.doi.org/10.1016/j.foodchem.2015.03.091 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
due to one or more compounds present in these seed preparations, thus high efficient extraction and purification method are necessary to get sufficient quantity of purified benzyl glucosinolate for the investigation of real activity compounds. Fortunately, we have demonstrated previously that endogenous myrosinase in Carica Papaya seed could be utilized to convert benzyl glucosinolate to benzyl isothiocyanate (BITC) and arrived at the optimum conditions. Despite having excellent biological features, BITC is volatile and not thermostailised during food processing and storage, which does not fit well as a food antibacterial agent. These problems can be addressed by complexation with cyclodextrins (CDs) in aqueous solutions. CDs is a general term for a series of cyclic oligosaccharides consisting of (a-1,4)-linked a-D-glucopyranose units with a hydrophobic interior cavity and a hydrophobic exterior surface (Szejtli, 1998). The most commonly used CDs are a-, band c-CDs consisting of 6, 7 and 8 a-D-glucopyranose units, respectively, which can form inclusion complexes with a variety of organic compounds (Karathanos, Mourtzinos, Yannakopoulou, & Andrikopoulos, 2007). Among these, b-CD has minimal solubility in water, thus is most likely to precipitate crystals, and because of its high encapsulation efficiency, suitable cavity dimensions and low cost, it is most commonly used (Sancho, Gasull, Blanco, & Castro, 2011). Beta-cyclodextrin (b-CD) has a cylindrical structure, which ends with a hydrophilic external surface, while the internal cylinder is hydrophobic, some suitable size and shape of
100
W. Li et al. / Food Chemistry 184 (2015) 99–104
the drug molecule (e.g., halo, volatile oil) are included in the cyclic structure by van der Waals forces. These features usually enhance food additives solubility in an aqueous solution and nonvolatility, widely used in food processing and food safety (Hu et al., 2012; Budryn et al., 2013; Wang, Luo, & Xiao, 2014; Song, Li, Du, & Wang, 2014). However, to the best of our knowledge, there is no scientific study on the inclusion complex of BITC with b-CDs. In this paper, the BITC extracted from papaya seed was included by b-CD and the characterizations of the complex were investigated by FT-IR, thermal analysis, XRD, particle size distribution analysis and 1H Nuclear magnetic resonance analysis. Meanwhile, phase solubility of BITC-b-CD complex was also studied. This study can be useful for the development of an active and intelligent packing material functionalist with naturally-occurring antimicrobial. 2. Materials and methods 2.1. Materials Carica papaya seed were supplied by Hainan Standard BioTechnique Co. Ltd., and were collected at Hainan province of China. Beta-cyclodextrin (b-CD) was obtained from Zibo Qianhui Biotechnology Co., Ltd. All other chemicals and reagents were of analytical grade. 2.2. Preparation of BITC By our previous studies, endogenous myrosinase in Carica Papaya seed was used to convert benzyl glucosinolate to benzyl isothiocyanate, the extraction conditions were as follows: seed powder particle size 90–120 lm, sample-to-solvent ratio1:20 (w/ v), pH of buffer solution 4.8, enzymolysis temperature 40 °C, enzymolysis time 27 min. The formed BITC was extracted by simultaneous distillation and extraction method for 2 h with dichloromethane as solvent and purified by silica gel chromatography. The purified BITC was kept under 4 °C in refrigerator for further study.
UV at 247 nm. Through the standard curve, the content was calculated. Reproducibility was checked by running the sample in triplicate. The inclusion efficiency procedure was calculated using the following equation:
IEð%Þ ¼ 100 ðmeasured BITC contentÞ=theoretical BITC content ð2Þ
2.5. Fourier transform infrared spectroscopy (FT-IR) The FT-IR spectra of BITC, b-CD and BITC-b-CD between 4000 cm1 and 400 cm1 were obtained using a Bruker Vector22 FT-IR spectrophotometer (Germany). The procedure consisted of intermixing sample (1 mg) together with KBr (150 mg), grinding them into fine powder and tablet compressing 30–60 s under 0.8–1.0 Mpa press, which should be well done for assays. The spectrum was obtained, corrected automatically by the built-in software of the spectrophotometer. 2.6. Thermal analysis The enhancement in heat stability of the BITC by inclusion with b-CD was evaluated by thermo-gravimetry (TG) and differential scanning calorimetry (DSC). TG analysis was carried out for BITC, b-CD, and the inclusion complex (BITC-b-CD) with a Q50 TGA (TA, USA). DSC analysis was performed by a DSC 1 (Mettler Toledo, Switzerland). Each prepared dried powder (5–8 mg) was heated in a crimped aluminum pan for DSC, and 9 ± 0.5 mg samples for thermo-gravimetry (TG) in an uncovered graphite crucible. All samples were heated from room temperature to 350 °C at 10 °C/ min under a nitrogen flow of 40 ml/min. The mass loss and heat flow in the sample were recorded as a function of temperature with reference to an empty pan. Reproducibility was checked by running the sample in triplicate. 2.7. X-ray diffractometry (XRD)
2.3. Preparation of BITC-b-CD complex The inclusion complex of BITC with b-CD was prepared by freeze dying. b-CD (4.0 g) was completely dissolved in 100 ml of water and kept at 40 °C. BITC anhydrous ethanol solution (0.5 ml, v:v = 1:1) was slowly blended into the prepared saturated solution of b-CD, and the mixture was intermittently reacted in ultrasonic homogenization and magnetic stirring for 3 h at 40 °C. The resulting solution was frozen at 80 °C and subsequently freeze dried for 24 h. 2.4. Determination of inclusion efficiency A certain concentration of BITC hexane solution was analysed by ultraviolet spectrophotometery (752PC, Tianjin Guanze Instrument CO., Ltd, China) from 200 nm to 400 nm, and the maximum absorption wavelength of BITC was 247 nm. Then the standard curve of the absorbance value (A) and the content of BITC (C, ll/ml) was established, the regression equation was:
A ¼ 11:688C 0:0045 R2 ¼ 0:9992
ð1Þ
The total content of BITC, in the obtained complexes, was determined by the extraction method according to Falcão et al. (2011) with modification. The sample was performed by mixing 10 mg of the lyophilized complex with 2 ml hexane, ultrasonic bath at 100 W for 20 min, and finally centrifugation at 10,000 rpm for 10 min. The supernatant obtained was immediately detected by
XRD patterns were obtained with a TD-3500 X-ray diffractometer (Dandong Tongda Science and Technology CO., Ltd, China) using Cu Ka radiation (30 kV, 20 mA), at a scanning rate of 8°/min. The samples were mounted on a sample holder and measured in the 2h angle range from 10° to 50° with a step size of 2h = 0.04°. 2.8. Particle size distribution In order to study the changes of the average particle size and polydispersity indexes (PDI) of b-CD, before and after embedding, a BI-200SM laser light scattering instrument (Brookhaven, USA) was used to measure these. Previously, 1 mg/ml of b-CD and the inclusion complex aqueous solutions were prepared, respectively. After homogeneous solutions were reached by ultrasonic vibration, samples were filtered through a 0.22 lm hydrophilic membrane filter, and then were examined. 2.9. 1H Nuclear magnetic resonance (1H NMR) All NMR experiments were carried out in Dimethyl sulfoxide (DMSO). Tetramethylsilane (TMS) was used as an internal standard. The 1H spectra of b-CD and the inclusion complex (BITC-bCD) were obtained using a Bruker Avance III spectrometer at 400 MHz and 298 K. Chemical shifts (d) are expressed in ppm relative to TMS.
W. Li et al. / Food Chemistry 184 (2015) 99–104
2.10. Phase solubility of BITC-b-CD complex As described in previous literature, phase solubility studies were performed according to the method reported by Higuchi and Connors (1965). Excess amounts of BITC anhydrous ethanol solution (20 ll, v:v = 1:1) were added to 10 ml of distilled water containing varying concentrations of b-CD (1–10 mM) in the 15 ml screw-cap tube. The mixture was placed in an ultrasonic bath and shaken for 3 h at 30 °C, and stabilization for 24 h at the room temperature in the dark. After equilibrium was reached, the aqueous solutions were withdrawn, using a syringe, and samples were filtered through a 0.45 lm hydrophilic membrane filter. The concentration of BITC in the filtrate was determined at 243 nm by an Agilent 8453E UV–visible spectroscopy (Australia). All samples were prepared in triplicate. The phase solubility profiles were obtained by plotting the BITC concentration against b-CD concentration. The apparent stability constant (Kc) of BITC and b-CD complex can be calculated from the phase solubility diagrams, according to the following equation:
101
(Nunes & Mercadante, 2007). The mixture was intermittently reacted in ultrasonic homogenization and magnetic stirring, which might be a better way to form inclusion complexes. The uncoated BITC settled down to the bottom and gathered. Intermittent ultrasonic homogenization promotes the emulsification degree of BITC and increases the contact area, which can improve the inclusion efficiency. Thus, higher inclusion efficiency provides feasibility for further application. 3.2. FT-IR spectra analysis
3. Results and discussion
The infrared (FT-IR) spectra of BITC, b-CD and their complex (BITC-b-CD) are displayed clearly in Fig. 1. Due to vibration of the N@C@S group, the IR spectra of BITC-b-CD could be characterized by the broad peak at 2140–2040 cm1. Moreover, there are three sharp absorption peaks between 1345 cm1 and 1600 cm1 in virtue of benzene ring skeleton vibration (Fig. 1a). The spectrum of b-CD manifests remarkable peaks ranged from 3540 cm1 to 3230 cm1, because of hydroxyl groups stretching vibration. We can observe the prominent absorption bands at 2926 cm1 (C–H vibration) and 1027 cm1 for C–O group vibration (Fig. 1b). By comparison, however, the FT-IR spectrum of BITC-b-CD was not completely congruent with that of b-CD (Fig. 1c). Although characteristic benzene peaks vanish in the spectrum of BITC-b-CD, the vibration bands of N@C@S groups are still existed. Therefore, in consideration of the above factors, we might suggest that the N@C@S groups exposed out the cavity of b-CD, while benzene ring of BITC has been entrapped in it.
3.1. Inclusion efficiency analysis
3.3. Thermal analysis
The inclusion efficiency of BITC was 86.4% ± 2.57. Previous reports, about the inclusion of red bell pepper pigments in b-CD by magnetic stirring and ultrasonic, resulted in inclusion efficiencies 52.95% ± 9.39 and 62.43% ± 12.89, respectively (Lidiane, Nicolly, Valéria, Deborah, & Kátia, 2014). Such studies about the inclusion efficiency of carotenoids in b-CD only by magnetic stirring were 48.96% (Chen, Chen, Guo, Li, & Li, 2007) and 50%
The thermal properties of b-CD, BITC, their inclusion complex (BITC-b-CD) were investigated using TG and DSC tests, the results are exhibited in Fig. 2. The thermogram of b-CD showed a significant thermal peak from 100 °C to 160 °C, indicating release of water molecules process in the DSC curves (Fig. 2ii). However, in the DSC curves of BITC-b-CD, an obviously wider band was viewed (Fig. 2iii), which was due to volatilization of uncoated BITC and a
Kc ¼
k S0 ð1 kÞ
ð3Þ
where k is the slope of the straight line, obtained from the initial segment of the phase solubility line, and S0 is the intrinsic solubility of BITC in distilled water in the absence of b-CD.
Fig. 1. FT-IR spectra of BITC (a), b-CD (b) and BITC-b-CD (c).
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W. Li et al. / Food Chemistry 184 (2015) 99–104
Fig. 3. Powder X-ray diffraction spectra of b-CD (a) and BITC-b-CD (b).
had apparent sharp peaks at 2h of 11.8°, 16.9°, 17.4°, 17.7°, 18° and 21.1°, was clearly distinct from that of b-CD and exhibited another crystal form. As BITC is not solid at room temperature, the corresponding XRD patterns cannot be obtained. However, through comparative analysis of existing data, we considered that the crystal structure of b-CD and inclusion complex were different which indicated the formation of BITC and b-CD inclusion complex. 3.5. Particle size distribution analysis
Fig. 2. DSC curves of BITC (i), b-CD (ii) and BITC-b-CD (iii). TG curves of BITC (iv), BITC-b-CD (v) and b-CD (vi).
change in the substance structure after inclusion complex formation between BITC and b-CD. The TG curve of b-CD (Fig. 2vi) showed a presence of a slope near 300 °C generally attributed to the initiation of b-CD decomposition, DSC curve corroborated with this result, showing a one step of endothermic event up to 300 °C. BITC belongs to volatile material and quickly losses mass from 80 °C to 165 °C as we can see from Fig. 2iv. In fact, phase mass loss of the inclusion complex emerged an obvious slope between 140 °C and 300 °C due to the influence of b-CD, slowed down its volatilization. BITC exhibited an endothermic peak between 250 °C and 270 °C that may be related to boiling, this peak was not present in the DSC scan of the inclusion complex and appeared an endothermic peak (near 280 °C) with the b-CD decomposition, because of intermolecular force. A similar study was reported by Adriana et al. (2015). Therefore it is evident that the b-CD cavity provides protection against volatilization of BITC in the encapsulated constituents.
3.4. XRD analysis Many relevant research has reported that XRD analysis was a useful method for the detection of the inclusion complex in powder (Chen et al., 2011; Wang, Cao, Sun, & Wang, 2011; Srinivasan, Stalin, & Sivakumar, 2012). The XRD patterns of b-CD and BITC-b-CD are illustrated in Fig. 3. As shown in Fig. 3a, b-CD has intense and sharp peaks at 2h of 12.9°, 13.5°, 18.2°, 18.7°, and 19.7°, as well as several minor peaks at 2h of 10.9°, 14.2°, 15.6° and 24°, which confirm the crystalline nature of the compound. The XRD of BITC-b-CD (Fig. 3b), by comparison, which
The average inclusion complex diameters and PDIs are listed in Table 1. PDI is a measure of the uniformity of particle size. A value close to zero (
