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Kinetic studies of the thermal degradation of sulforaphane and its hydroxypropyl-β-cyclodextrin inclusion complex
Food Research International 53 (2013) 529–533
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Food Research International journal homepage: www.elsevier.com/locate/foodres
Kinetic studies of the thermal degradation of sulforaphane and its hydroxypropyl-β-cyclodextrin inclusion complex Yuanfeng Wu a,b, Jianwei Mao b,c, Lehe Mei a,d,⁎, Shiwang Liu b,c a
Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027 Zhejiang, China School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, 310023 Zhejiang, China Zhejiang Provincial Key Lab for Chem & Bio Processing Technology of Farm Produces, Hangzhou, 310023 Zhejiang, China d School of Biotechnology and Chemical Engineering, Ningbo Institute of Technology, Zhejiang University, Ningbo, 315100 Zhejiang, China b c
a r t i c l e
i n f o
Article history: Received 5 December 2012 Accepted 25 May 2013 Keywords: Kinetics Thermal degradation Sulforaphane Hydroxypropyl-β-cyclodextrin
a b s t r a c t Sulforaphane (SF) has been of increasing interest for nutraceutical and pharmaceutical industries due to its anti-cancer effect, but it is very sensitive to changes in the temperature and pH. In the present work, pure SF and inclusion complex of SF with hydroxypropyl-β-cyclodextrin (SF/HP-β-CD) were prepared, the depletion rates of SF and SF/HP-β-CD were investigated at temperatures of 60, 75, 82 and 90 °C and pH values of 2.2, 3.0, 4.0, 5.0 and 6.0. The results showed that SF and SF/HP-β-CD were more stable at lower pH values and temperatures, and SF/HP-β-CD was more stable than SF. The SF and SF/HP-β-CD degradation was observed to follow first order kinetics, and the temperature-dependent rate constants for SF and SF/HP-β-CD inclusion complex in aqueous solution were well described by the Arrhenius equation with corresponding activation energies of 70.7 to 94.5 kJ/mol, depending on the pH values. Finally, models to describe the retention ratio of SF and SF/HP-β-CD at different pH values, depletion time and temperatures were proposed. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Sulforaphane (1-isothiocyanate-(4R)-(methylsulfinyl) butane, SF) is a compound derived from cruciferous plants. Its anticancer properties have been demonstrated both, in cancer cell lines as well as tumors in animal models. SF was initially identified as a major and probably the principal inducer of the phase II detoxification enzyme quinone reductase in Hepa 1c1c7 cell cultures (Zhang, Talalay, Cho, & Posner, 1992). SF can show its antitumor effect via anti-proliferative mechanisms, such as cell cycle arrest and apoptosis (Choi, Choi, Lee, & Choi, 2008; Choi & Singh, 2005; Gamet-Payrastre, 2006; Singh et al., 2004). Studies have shown that the administration of SF enhances natural killer cell activity and antibody-dependent cellular cytotoxicity in both normal and tumor-bearing animals (Singh et al., 2009; Thejass & Kuttan, 2006, 2007). SF is also effective at blocking the initiation and progression of carcinogenesis (Clarke, Dashwood, & Ho, 2008; Yao et al., 2008). More recently, Wiczk, Hofman, Konopa, and Herman-Antosiewicz (2012) have demonstrated that SF can target synthesis of proteins, especially short-lived ones that are indispensable for cancer cell survival. Beyond its anticancer properties, current research has also focused on the effects of SF against hyperglycemia and the damage to brain, kidney, liver, renal,
⁎ Corresponding author at: Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027 Zhejiang, China. Tel.: + 86 571 87951982; fax: + 86 571 87951358. E-mail address: [email protected] (L. Mei). 0963-9969/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodres.2013.05.017
heart, and muscle (Guerrero-Beltrán, Calderón-Oliver, Pedraza-Chaverri, & Chirino, 2012). Consumption of cruciferous vegetables such as broccoli and Brussels sprouts, which can provide SF, is linked to reduced cancer risk (Jeffery & Araya, 2009). Even though SF shows great promise as an anticancer agent, it is not very stable under normal conditions (Van Eylen, Oey, Hendrickx, & Van Loey, 2007). Complexation of SF with cyclodextrins (CD) is one way to improve its stability. CD are cyclic oligosaccharides constituted by six, seven or eight α-1, 4-linked glucose residues and are characterized by a truncated cone shape. In aqueous solution or solid state, the CD molecule forms an inner hydrophobic cavity, which provides interior hollow for appropriate guest molecules, and the formation of dynamic inclusion complexes can lead to the modification of some physical and chemical properties of the drug (Zerrouk, Ginès Dorado, Arnaud, & Chemtob, 1998). Through complexation, CD can stabilize unstable compounds. To date, a few studies have investigated the stability and thermal degradation of SF. Chiang, Pusateri, and Leitz (1998) found that SF was decomposed to other compounds due to high temperature conditions in the injection ports of GC or GC/MS equipment. Jin, Wang, Rosen, and Ho (1999) studied the thermal decomposition products of SF in aqueous solution, and a possible mechanism for the formation of these products was proposed. Van Eylen et al. (2007) studied the stability of endogenous myrosinase, SF and phenethyl isothiocyanate (PEITC), which are glucosinolate hydrolysis products in broccoli, and found that SF and PEITC from broccoli had higher thermolabile and
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Fig. 1. HSCCC chromatogram of the crude extract of broccoli seed meal in the n-hexane/ethyl acetate/methanol/water (1:5:1:5, v/v/v/v) solvent system, flow rate 1.5 ml/min, and detection at 241 nm.
pressure stability than myrosinase. Wu, Liang, Yuan, Wang, and Yan (2010) found that the stability of SF was influenced by temperature, pH and oxidants. They also compared the stability of SF and SF/ HP-β-CD inclusion complex, and concluded that the stability of the inclusion complex against heat, oxygen and alkaline conditions was enhanced compared with that of SF. However, no information is available on the kinetics of the thermal degradation of SF and its CD inclusion complex at various pH values and temperatures. In the present work, pure SF was isolated by high-speed countercurrent chromatography (HSCCC), and then HP-β-CD was used as the host compound to encapsulate the guest SF. Our efforts focused on the kinetic investigation of the thermal degradation of pure SF and SF/HP-β-CD in aqueous solutions under various conditions. The present study had the following main objectives: (1) to study the stabilities of SF and SF/HP-β-CD inclusion complex with heat treatment and under different acidic conditions, and determine the degradation rate constants, k, for SF and SF/HP-β-CD at different temperatures (60–90 °C) and pH values (2.2–6.0); (2) to use the Arrhenius equation to describe the dependence of the degradation rate constant on temperature at each pH value; (3) to develop kinetic model relating to the calculated kinetic data and to apply this model to predict the degradation of SF and SF/HP-β-CD.
2.3. Preparation of the inclusion complex of SF with HP-β-CD The inclusion complex of SF with HP-β-CD at 1:1 molar ratio was prepared by the method of Wu with some modifications (Wu et al., 2010). The accurately weighed HP-β-CD was dissolved in distilled water to get a saturated solution. Then SF solution in ethanol was slowly added and a suspension was formed. The suspension was maintained stirring at room temperature for 24 h. The obtained
2. Materials and methods 2.1. Materials and chemicals Broccoli seed was kindly provided by Taizhou Academy of Agricultural Science. HP-β-CD was purchased from Sangon Biotech Co. Ltd. (Shanghai, China). Distilled water was used throughout the study. Methanol (Tedia, USA) was HPLC grade, and all other reagents were of analytical reagent grade and were purchased from Huadong Medicine Co. Ltd. (Hangzhou, China).
2.2. Preparation of pure SF by HSCCC SF was extracted from defatted broccoli seed meal hydrolysate, then pure SF (purity > 96%) was isolated by HSCCC performed on a TBE 300A instrument (Tauto Biotech Co. Ltd., Shanghai, China) by the method of Liang with some modifications (Liang, Li, Yuan, & Vriesekoop, 2008) (Fig. 1). SF purity was checked by nuclear magnetic resonance (NMR) and high performance liquid chromatography (HPLC) methods (data not show), and it was stored below 0 °C.
Fig. 2. Plots of −ln(Ct/C0) vs water bath time, t, with 0.1 mg/ml of (a) pure SF and (b) SF/HP-β-CD at different pH values and temperatures of 60 °C (■), 75 °C (○), 82 °C (●) and 90 °C (□).
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a thermostatic water bath (Beijing Era Beili Centrifuge Co. Ltd., China) preheated to a given temperature. At regular time intervals (1 h interval), one tube of each pH was randomly taken from the water bath and rapidly cooled by plunging into an ice water bath (Hou, Qin, Zhang, Cui, & Ren, 2013). The SF contents of cooled tubes were analyzed by HPLC. All experiments were done in triplicates. 2.5. Analytical method The content of SF was analyzed by Waters e2695 HPLC system. The column employed in our experiment was a ZORBAX Eclipse XDB-C18 (4.6 × 250 mm, 5 μm). The mobile phase consisted of 20% methanol in water, changing linearly over 10 min to 60% methanol, then increasing to 100% in 2 min and maintained for 2 min to purge the column. The column oven temperature was set at 25 °C, the flow rate was 1.0 ml/min, and 10 μl samples were injected onto the column. SF was detected using a Waters 2489 detector at 241 nm (Wu et al., 2010). 2.6. Statistical analysis All experiments were done in triplicates and the results were expressed as mean values. The errors of experimental data from the mean values were expressed as standard deviation using the Microsoft Excel software for Mac 2011 and illustrated as error bars. 3. Results and discussion 3.1. Stability of SF and SF/HP-β-CD inclusion complex with heat treatment and under different acidic conditions The SF and SF/HP-β-CD concentrations during heating were plotted with a regular interval of 1 h. The overall order and rate constant for the degradation were determined using the following equation: lnr ¼ lnðC t =C 0 Þ ¼ −kt
ð1Þ
−1
t 1=2 ¼ − lnð0:5Þk
ð2Þ
Table 1 Rate constant k, regression coefficient (R2) and RMSE of SF and SF/HP-β-CD inclusion complex degradation at pH 2.2, 3.0, 4.0, 5.0 and 6.0. pH
Fig. 2 (continued).
product was filtered through 0.45 μm membrane filter and was then dried in a vacuum rotavapor to get SF/HP-β-CD inclusion complex.
2.2
3.0
2.4. Study on SF and SF/HP-β-CD inclusion complex stability The effect of pH on SF and SF/HP-β-CD inclusion complex thermal stability was studied at five pH values (2.2, 3.0, 4.0, 5.0 and 6.0) and four temperatures (60, 75, 82 and 90 °C). Citrate-phosphate buffers were prepared to provide the specified pH situation and SF or SF/ HP-β-CD inclusion complex was dissolved with 0.1 mol/L citratephosphate buffer at each pH value to get concentrations of 0.1 mg/ ml. 2 ml of SF or SF/HP-β-CD inclusion complex solution was put into a plastic tube (3 ml total volume, Thermo Fisher Scientific Inc., USA). The sample tubes (6 tubes per each pH) covered with aluminum foil were well capped to avoid evaporation and were placed in
4.0
5.0
6.0
T [°C]
60 75 82 90 60 75 82 90 60 75 82 90 60 75 82 90 60 75 82 90
Pure SF
SF/HP-β-CD inclusion complex
k [h−1]
R2
RMSE
k [h−1]
R2
RMSE
0.01 0.05 0.11 0.22 0.02 0.08 0.15 0.29 0.04 0.15 0.25 0.46 0.07 0.22 0.34 0.62 0.10 0.29 0.47 0.80
0.985 0.994 0.985 0.989 0.986 0.997 0.991 0.993 0.989 0.990 0.995 0.999 0.988 0.998 0.999 0.998 0.992 0.996 0.993 0.997
0.004 0.008 0.026 0.048 0.005 0.008 0.027 0.050 0.009 0.029 0.036 0.033 0.015 0.017 0.022 0.055 0.017 0.035 0.080 0.053
0.01 0.05 0.09 0.17 0.02 0.06 0.12 0.22 0.03 0.12 0.21 0.31 0.04 0.18 0.30 0.48 0.07 0.25 0.39 0.60
0.986 0.983 0.986 0.992 0.987 0.988 0.990 0.992 0.975 0.991 0.993 0.994 0.988 0.997 0.997 0.998 0.991 0.993 0.997 0.999
0.007 0.012 0.021 0.029 0.003 0.014 0.024 0.040 0.009 0.027 0.035 0.050 0.010 0.020 0.033 0.045 0.012 0.041 0.045 0.038
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where r is the retention value at time t; C0 is the initial SF content (mol/L); Ct is the SF content (mol/L) after t hours heating at a given temperature; t is the heating time (h); k is the degradation rate constant (h− 1); and t1/2 is the half life time (h), i.e. the time needed for 50% degradation of SF and SF/HP-β-CD. Accordingly, ln(−Ct/C0) was plotted vs t (Fig. 2) for the thermal degradation of SF and SF/HP-β-CD at temperatures between 60 and 90 °C and pH values between 2.2 and 6.0. The correlation coefficient was > 0.9 and with low root mean square of error (RMSE) in all cases (Table 1), this confirmed that the thermal degradation of SF and SF/HP-β-CD followed first order reaction kinetics with respect to temperature. The degradation rate constants (k) for SF depletion at different temperatures and pH values, estimated by linear regression of the experimental data in Fig. 2, are indicated in Table 1. At each pH value, the k values showed that the stability of SF decreased with increasing temperature. The pH also had a strong influence on the stability of SF. As seen in Table 1, the k of SF increased from 0.01 to 0.10 h− 1 when the pH was increased from 2.2 to 6.0 at 60 °C. The same trend was observed at other temperatures. Generally, at each pH value, the k value increased 2.4 to 5.0 fold, when the temperature increased with 15 °C. At pH 2.2 and 60 °C, the t1/2 of SF was 46.5 h, and at the same pH, the t1/2 at room temperature should be longer. At high temperature and pH, the t1/2 decreased sharply, for example, at pH 6.0 and 90 °C, the t1/2 was 0.9 h. This also shows the sensitivity of SF to high pH values and temperatures. Thermal degradation of SF/HP-β-CD inclusion complex also followed first order reaction kinetics, and the k values increased as the pH increased. The inclusion complex of SF had lower k values than pure SF (about 0.6–1.0 folds). This shows that SF/HP-β-CD is more stable than SF at various pH values, and that HP-β-CD can improve the stability of SF. 3.2. Dependence of rate constant on temperature From the hydrolysis rate constants, it was possible to establish a relationship between the value of k and temperature by fitting the data to the Arrhenius equation: k ¼ K 0 expð−Ea =RT Þ
ð3Þ
where Ea is the activation energy of the reaction (J/mol); R is the universal gas constant (8.314 Jmol/K); T is the absolute temperature (K); and K0 frequency factor (1/s) is a pre-exponential constant.
Fig. 3. Arrhenius plots for (a) pure SF and (b) SF/HP-β-CD inclusion complex degradation in aqueous solutions at pH 2.2(□), 3.0(●), 4.0(△), 5.0(▼) and 6.0( ).
◇
Table 2 Activation energy (Ea), lnK0, regression coefficient (R2) and RMSE of SF and SF/HP-β-CD inclusion complex degradation at pH 2.2, 3.0, 4.0, 5.0 and 6.0. pH
2.2 3.0 4.0 5.0 6.0
Pure SF
SF/HP-β-CD inclusion complex
Ea [kJ mol−1]
lnK0
R2
RMSE
Ea [kJ mol−1]
lnK0
R2
RMSE
90.6 85.4 80.8 73.4 70.7
28.4 27.0 26.0 23.8 23.2
0.994 0.998 1.000 1.000 1.000
0.080 0.039 0.010 0.016 0.004
94.5 89.6 84.2 81.1 75.1
29.5 28.2 26.9 26.2 24.5
1.000 0.999 0.987 0.995 0.991
0.019 0.031 0.107 0.064 0.081
Using Eq. (1) one can get lnðC t =C 0 Þ ¼ lnC t − lnC 0 ¼ −kt ¼ −K 0 expð−Ea =RT Þt:
ð4Þ
Activation energy was calculated from a linear relationship of lnk versus 1/T. Fig. 3 shows the Arrhenius plots for SF and SF/HP-β-CD inclusion complex degradation at pH 2.2, 3.0, 4.0, 5.0 and 6.0, whereas Ea values are gathered in Table 2. The Ea decreased from 90.6 to 70.7 kJ/mol as the pH increased. The Ea value of SF was similar to that obtained by Van Eylen et al. (2007), who reported an Ea of 89.03 kJ/mol for SF in broccoli juice. The slight difference between our and Van Eylen's results could be attributed to the presence of other substances in the juice. Jiang, Zhang, Tian, and Li (2006) reported an Ea of 59.0 kJ/mol for the reaction between pure allyl isothiocyanate (AITC) and hydroxyl/water and Neoh, Yamamoto, Ikefuji, Furuta, and Yoshii (2012) reported that the Ea values for the pure AITC, pure phenyl isothiocyanate (PITC), AITC inclusion complex, and PITC inclusion complex in paraffin were 26, 48, 44, and 54 kJ/mol, respectively. Compared with the Ea of pure SF, the Ea value of the SF/HP-β-CD inclusion complex was increased about 3.4–7.7 kJ/mol. The Ea increase observed in this study was much lower than that observed for AITC (22 kJ/mol) and PITC (10 kJ/mol) (Neoh et al., 2012). The difference may be attributed to the different modes of interactions between the functional groups of these compounds and the cavity of HP-β-CD. The complexation between AITC or PITC and RM-β-CD is 1:1 (Neoh et al., 2012), and the inclusion ratio between SF and HP-β-CD is also found to be 1:1 (Wu et al., 2010). But different from AITC and PITC, SF is quite easily dissolved in water, which can be attributed to the hydrophilic group of methylsulfinyl butane in SF. The mode of interactions between the hydrophobic benzene ring of PITC or allyl of AITC may be different from the methylsulfinyl butane of SF, the inclusion effect of SF with HP-β-CD may not be as good as AITC and PITC. This may explain
Fig. 4. The −Ea/R (a) and lnK0 (b) at different pH values of SF (●) and SF/HP-β-CD (□). Parameters of linear fitting: the slope of (1), (2), (3) and (4) were 648, 591, −1.43 and −1.26, respectively, the intercept of (1), (2), (3) and (4) were −12,264, −12,597, 31.46 and 32.15, respectively, and the R2 values of (1), (2), (3) and (4) were 0.985, 0.991, 0.978 and 0.983, respectively.
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Conflict of interest statement The authors declare no competing financial interest. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (21176220 and 31240054), Zhejiang Provincial Natural Science Foundation of China (Z13B060002) and Zhejiang Provincial Key Science and Technology Innovation Team (2009R50028). References
Fig. 5. Relation between the experimental retention ratios and the retention ratios estimated by the exponential model (Eqs. (5) and (6)), R2 was 0.985, and RMSE was 0.031.
why the stability increase of SF inclusion complex with β-CD is not as good as that of AITC or PITC. 3.3. Combined effect of temperature and pH values on the kinetics of SF and SF/HP-β-CD complex degradation The Arrhenius equation has been widely used in food research, but its applications in some fields, such as enzymatic reactions and growth rates of microorganisms remain some arguments (Peleg, Normand, & Corradini, 2012). But noticed that the Arrhenius equation could be written in a linear form (Table 2), one can develop mathematical models to predict the losses of SF and SF/HP-β-CD inclusion complex. As shown in Fig. 4, from the plots of −Ea/R and lnK0 for SF and SF/HP-β-CD as a function of pH values, one can observe that the correlation coefficient was >0.9. From the slopes and intercepts for each linear fit and Eqs. (1) and (4), we can obtain the kinetics equations for the thermal degradation of SF, Eq. (5) and the SF/HP-β-CD complex, Eq. (6): r ¼ expðð expð−1:43x þ 31:46Þ⋅ expð648x−12264Þ=T Þ⋅t Þ
ð5Þ
r ¼ expðð expð−1:26x þ 32:15Þ⋅ expð591x−12597Þ=T Þ⋅t Þ
ð6Þ
where x is the pH value (2.2–6.0). The goodness of fit of the exponential model is graphically presented in Fig. 5, where the relationship between the experimental thermal retention ratios and the thermal retention ratios estimated by the exponential model is depicted. Using this method, the retention value of SF at different pH values, depletion time and temperatures can be calculated. 4. Conclusions This research provided detailed information regarding the changes in the kinetic stability of pure SF and SF/HP-β-CD inclusion complex at different temperatures (60, 75, 82 and 90 °C) and pH values (2.2, 3.0, 4.0, 5.0 and 6.0). The temperature and pH level strongly influenced the stability of SF and SF/HP-β-CD inclusion complex, which were more stable at low pH values and temperatures. The SF/HP-β-CD inclusion complex was more stable than pure SF. Thermal degradation of SF and SF/HP-β-CD inclusion complex was adequately described by first-order model, and the Arrhenius equation could be applied to establish the relationship between the degradation rate constant and temperature at various pH values. The main dynamic parameters of SF and SF/HP-β-CD inclusion complex at various pH values were estimated. Then mathematical models were developed to predict the losses of SF and SF/HP-β-CD inclusion complex at different pH values, depletion time and temperatures.
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Food Research International journal homepage: www.elsevier.com/locate/foodres
Kinetic studies of the thermal degradation of sulforaphane and its hydroxypropyl-β-cyclodextrin inclusion complex Yuanfeng Wu a,b, Jianwei Mao b,c, Lehe Mei a,d,⁎, Shiwang Liu b,c a
Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027 Zhejiang, China School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, 310023 Zhejiang, China Zhejiang Provincial Key Lab for Chem & Bio Processing Technology of Farm Produces, Hangzhou, 310023 Zhejiang, China d School of Biotechnology and Chemical Engineering, Ningbo Institute of Technology, Zhejiang University, Ningbo, 315100 Zhejiang, China b c
a r t i c l e
i n f o
Article history: Received 5 December 2012 Accepted 25 May 2013 Keywords: Kinetics Thermal degradation Sulforaphane Hydroxypropyl-β-cyclodextrin
a b s t r a c t Sulforaphane (SF) has been of increasing interest for nutraceutical and pharmaceutical industries due to its anti-cancer effect, but it is very sensitive to changes in the temperature and pH. In the present work, pure SF and inclusion complex of SF with hydroxypropyl-β-cyclodextrin (SF/HP-β-CD) were prepared, the depletion rates of SF and SF/HP-β-CD were investigated at temperatures of 60, 75, 82 and 90 °C and pH values of 2.2, 3.0, 4.0, 5.0 and 6.0. The results showed that SF and SF/HP-β-CD were more stable at lower pH values and temperatures, and SF/HP-β-CD was more stable than SF. The SF and SF/HP-β-CD degradation was observed to follow first order kinetics, and the temperature-dependent rate constants for SF and SF/HP-β-CD inclusion complex in aqueous solution were well described by the Arrhenius equation with corresponding activation energies of 70.7 to 94.5 kJ/mol, depending on the pH values. Finally, models to describe the retention ratio of SF and SF/HP-β-CD at different pH values, depletion time and temperatures were proposed. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Sulforaphane (1-isothiocyanate-(4R)-(methylsulfinyl) butane, SF) is a compound derived from cruciferous plants. Its anticancer properties have been demonstrated both, in cancer cell lines as well as tumors in animal models. SF was initially identified as a major and probably the principal inducer of the phase II detoxification enzyme quinone reductase in Hepa 1c1c7 cell cultures (Zhang, Talalay, Cho, & Posner, 1992). SF can show its antitumor effect via anti-proliferative mechanisms, such as cell cycle arrest and apoptosis (Choi, Choi, Lee, & Choi, 2008; Choi & Singh, 2005; Gamet-Payrastre, 2006; Singh et al., 2004). Studies have shown that the administration of SF enhances natural killer cell activity and antibody-dependent cellular cytotoxicity in both normal and tumor-bearing animals (Singh et al., 2009; Thejass & Kuttan, 2006, 2007). SF is also effective at blocking the initiation and progression of carcinogenesis (Clarke, Dashwood, & Ho, 2008; Yao et al., 2008). More recently, Wiczk, Hofman, Konopa, and Herman-Antosiewicz (2012) have demonstrated that SF can target synthesis of proteins, especially short-lived ones that are indispensable for cancer cell survival. Beyond its anticancer properties, current research has also focused on the effects of SF against hyperglycemia and the damage to brain, kidney, liver, renal,
⁎ Corresponding author at: Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027 Zhejiang, China. Tel.: + 86 571 87951982; fax: + 86 571 87951358. E-mail address: [email protected] (L. Mei). 0963-9969/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodres.2013.05.017
heart, and muscle (Guerrero-Beltrán, Calderón-Oliver, Pedraza-Chaverri, & Chirino, 2012). Consumption of cruciferous vegetables such as broccoli and Brussels sprouts, which can provide SF, is linked to reduced cancer risk (Jeffery & Araya, 2009). Even though SF shows great promise as an anticancer agent, it is not very stable under normal conditions (Van Eylen, Oey, Hendrickx, & Van Loey, 2007). Complexation of SF with cyclodextrins (CD) is one way to improve its stability. CD are cyclic oligosaccharides constituted by six, seven or eight α-1, 4-linked glucose residues and are characterized by a truncated cone shape. In aqueous solution or solid state, the CD molecule forms an inner hydrophobic cavity, which provides interior hollow for appropriate guest molecules, and the formation of dynamic inclusion complexes can lead to the modification of some physical and chemical properties of the drug (Zerrouk, Ginès Dorado, Arnaud, & Chemtob, 1998). Through complexation, CD can stabilize unstable compounds. To date, a few studies have investigated the stability and thermal degradation of SF. Chiang, Pusateri, and Leitz (1998) found that SF was decomposed to other compounds due to high temperature conditions in the injection ports of GC or GC/MS equipment. Jin, Wang, Rosen, and Ho (1999) studied the thermal decomposition products of SF in aqueous solution, and a possible mechanism for the formation of these products was proposed. Van Eylen et al. (2007) studied the stability of endogenous myrosinase, SF and phenethyl isothiocyanate (PEITC), which are glucosinolate hydrolysis products in broccoli, and found that SF and PEITC from broccoli had higher thermolabile and
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Fig. 1. HSCCC chromatogram of the crude extract of broccoli seed meal in the n-hexane/ethyl acetate/methanol/water (1:5:1:5, v/v/v/v) solvent system, flow rate 1.5 ml/min, and detection at 241 nm.
pressure stability than myrosinase. Wu, Liang, Yuan, Wang, and Yan (2010) found that the stability of SF was influenced by temperature, pH and oxidants. They also compared the stability of SF and SF/ HP-β-CD inclusion complex, and concluded that the stability of the inclusion complex against heat, oxygen and alkaline conditions was enhanced compared with that of SF. However, no information is available on the kinetics of the thermal degradation of SF and its CD inclusion complex at various pH values and temperatures. In the present work, pure SF was isolated by high-speed countercurrent chromatography (HSCCC), and then HP-β-CD was used as the host compound to encapsulate the guest SF. Our efforts focused on the kinetic investigation of the thermal degradation of pure SF and SF/HP-β-CD in aqueous solutions under various conditions. The present study had the following main objectives: (1) to study the stabilities of SF and SF/HP-β-CD inclusion complex with heat treatment and under different acidic conditions, and determine the degradation rate constants, k, for SF and SF/HP-β-CD at different temperatures (60–90 °C) and pH values (2.2–6.0); (2) to use the Arrhenius equation to describe the dependence of the degradation rate constant on temperature at each pH value; (3) to develop kinetic model relating to the calculated kinetic data and to apply this model to predict the degradation of SF and SF/HP-β-CD.
2.3. Preparation of the inclusion complex of SF with HP-β-CD The inclusion complex of SF with HP-β-CD at 1:1 molar ratio was prepared by the method of Wu with some modifications (Wu et al., 2010). The accurately weighed HP-β-CD was dissolved in distilled water to get a saturated solution. Then SF solution in ethanol was slowly added and a suspension was formed. The suspension was maintained stirring at room temperature for 24 h. The obtained
2. Materials and methods 2.1. Materials and chemicals Broccoli seed was kindly provided by Taizhou Academy of Agricultural Science. HP-β-CD was purchased from Sangon Biotech Co. Ltd. (Shanghai, China). Distilled water was used throughout the study. Methanol (Tedia, USA) was HPLC grade, and all other reagents were of analytical reagent grade and were purchased from Huadong Medicine Co. Ltd. (Hangzhou, China).
2.2. Preparation of pure SF by HSCCC SF was extracted from defatted broccoli seed meal hydrolysate, then pure SF (purity > 96%) was isolated by HSCCC performed on a TBE 300A instrument (Tauto Biotech Co. Ltd., Shanghai, China) by the method of Liang with some modifications (Liang, Li, Yuan, & Vriesekoop, 2008) (Fig. 1). SF purity was checked by nuclear magnetic resonance (NMR) and high performance liquid chromatography (HPLC) methods (data not show), and it was stored below 0 °C.
Fig. 2. Plots of −ln(Ct/C0) vs water bath time, t, with 0.1 mg/ml of (a) pure SF and (b) SF/HP-β-CD at different pH values and temperatures of 60 °C (■), 75 °C (○), 82 °C (●) and 90 °C (□).
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a thermostatic water bath (Beijing Era Beili Centrifuge Co. Ltd., China) preheated to a given temperature. At regular time intervals (1 h interval), one tube of each pH was randomly taken from the water bath and rapidly cooled by plunging into an ice water bath (Hou, Qin, Zhang, Cui, & Ren, 2013). The SF contents of cooled tubes were analyzed by HPLC. All experiments were done in triplicates. 2.5. Analytical method The content of SF was analyzed by Waters e2695 HPLC system. The column employed in our experiment was a ZORBAX Eclipse XDB-C18 (4.6 × 250 mm, 5 μm). The mobile phase consisted of 20% methanol in water, changing linearly over 10 min to 60% methanol, then increasing to 100% in 2 min and maintained for 2 min to purge the column. The column oven temperature was set at 25 °C, the flow rate was 1.0 ml/min, and 10 μl samples were injected onto the column. SF was detected using a Waters 2489 detector at 241 nm (Wu et al., 2010). 2.6. Statistical analysis All experiments were done in triplicates and the results were expressed as mean values. The errors of experimental data from the mean values were expressed as standard deviation using the Microsoft Excel software for Mac 2011 and illustrated as error bars. 3. Results and discussion 3.1. Stability of SF and SF/HP-β-CD inclusion complex with heat treatment and under different acidic conditions The SF and SF/HP-β-CD concentrations during heating were plotted with a regular interval of 1 h. The overall order and rate constant for the degradation were determined using the following equation: lnr ¼ lnðC t =C 0 Þ ¼ −kt
ð1Þ
−1
t 1=2 ¼ − lnð0:5Þk
ð2Þ
Table 1 Rate constant k, regression coefficient (R2) and RMSE of SF and SF/HP-β-CD inclusion complex degradation at pH 2.2, 3.0, 4.0, 5.0 and 6.0. pH
Fig. 2 (continued).
product was filtered through 0.45 μm membrane filter and was then dried in a vacuum rotavapor to get SF/HP-β-CD inclusion complex.
2.2
3.0
2.4. Study on SF and SF/HP-β-CD inclusion complex stability The effect of pH on SF and SF/HP-β-CD inclusion complex thermal stability was studied at five pH values (2.2, 3.0, 4.0, 5.0 and 6.0) and four temperatures (60, 75, 82 and 90 °C). Citrate-phosphate buffers were prepared to provide the specified pH situation and SF or SF/ HP-β-CD inclusion complex was dissolved with 0.1 mol/L citratephosphate buffer at each pH value to get concentrations of 0.1 mg/ ml. 2 ml of SF or SF/HP-β-CD inclusion complex solution was put into a plastic tube (3 ml total volume, Thermo Fisher Scientific Inc., USA). The sample tubes (6 tubes per each pH) covered with aluminum foil were well capped to avoid evaporation and were placed in
4.0
5.0
6.0
T [°C]
60 75 82 90 60 75 82 90 60 75 82 90 60 75 82 90 60 75 82 90
Pure SF
SF/HP-β-CD inclusion complex
k [h−1]
R2
RMSE
k [h−1]
R2
RMSE
0.01 0.05 0.11 0.22 0.02 0.08 0.15 0.29 0.04 0.15 0.25 0.46 0.07 0.22 0.34 0.62 0.10 0.29 0.47 0.80
0.985 0.994 0.985 0.989 0.986 0.997 0.991 0.993 0.989 0.990 0.995 0.999 0.988 0.998 0.999 0.998 0.992 0.996 0.993 0.997
0.004 0.008 0.026 0.048 0.005 0.008 0.027 0.050 0.009 0.029 0.036 0.033 0.015 0.017 0.022 0.055 0.017 0.035 0.080 0.053
0.01 0.05 0.09 0.17 0.02 0.06 0.12 0.22 0.03 0.12 0.21 0.31 0.04 0.18 0.30 0.48 0.07 0.25 0.39 0.60
0.986 0.983 0.986 0.992 0.987 0.988 0.990 0.992 0.975 0.991 0.993 0.994 0.988 0.997 0.997 0.998 0.991 0.993 0.997 0.999
0.007 0.012 0.021 0.029 0.003 0.014 0.024 0.040 0.009 0.027 0.035 0.050 0.010 0.020 0.033 0.045 0.012 0.041 0.045 0.038
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where r is the retention value at time t; C0 is the initial SF content (mol/L); Ct is the SF content (mol/L) after t hours heating at a given temperature; t is the heating time (h); k is the degradation rate constant (h− 1); and t1/2 is the half life time (h), i.e. the time needed for 50% degradation of SF and SF/HP-β-CD. Accordingly, ln(−Ct/C0) was plotted vs t (Fig. 2) for the thermal degradation of SF and SF/HP-β-CD at temperatures between 60 and 90 °C and pH values between 2.2 and 6.0. The correlation coefficient was > 0.9 and with low root mean square of error (RMSE) in all cases (Table 1), this confirmed that the thermal degradation of SF and SF/HP-β-CD followed first order reaction kinetics with respect to temperature. The degradation rate constants (k) for SF depletion at different temperatures and pH values, estimated by linear regression of the experimental data in Fig. 2, are indicated in Table 1. At each pH value, the k values showed that the stability of SF decreased with increasing temperature. The pH also had a strong influence on the stability of SF. As seen in Table 1, the k of SF increased from 0.01 to 0.10 h− 1 when the pH was increased from 2.2 to 6.0 at 60 °C. The same trend was observed at other temperatures. Generally, at each pH value, the k value increased 2.4 to 5.0 fold, when the temperature increased with 15 °C. At pH 2.2 and 60 °C, the t1/2 of SF was 46.5 h, and at the same pH, the t1/2 at room temperature should be longer. At high temperature and pH, the t1/2 decreased sharply, for example, at pH 6.0 and 90 °C, the t1/2 was 0.9 h. This also shows the sensitivity of SF to high pH values and temperatures. Thermal degradation of SF/HP-β-CD inclusion complex also followed first order reaction kinetics, and the k values increased as the pH increased. The inclusion complex of SF had lower k values than pure SF (about 0.6–1.0 folds). This shows that SF/HP-β-CD is more stable than SF at various pH values, and that HP-β-CD can improve the stability of SF. 3.2. Dependence of rate constant on temperature From the hydrolysis rate constants, it was possible to establish a relationship between the value of k and temperature by fitting the data to the Arrhenius equation: k ¼ K 0 expð−Ea =RT Þ
ð3Þ
where Ea is the activation energy of the reaction (J/mol); R is the universal gas constant (8.314 Jmol/K); T is the absolute temperature (K); and K0 frequency factor (1/s) is a pre-exponential constant.
Fig. 3. Arrhenius plots for (a) pure SF and (b) SF/HP-β-CD inclusion complex degradation in aqueous solutions at pH 2.2(□), 3.0(●), 4.0(△), 5.0(▼) and 6.0( ).
◇
Table 2 Activation energy (Ea), lnK0, regression coefficient (R2) and RMSE of SF and SF/HP-β-CD inclusion complex degradation at pH 2.2, 3.0, 4.0, 5.0 and 6.0. pH
2.2 3.0 4.0 5.0 6.0
Pure SF
SF/HP-β-CD inclusion complex
Ea [kJ mol−1]
lnK0
R2
RMSE
Ea [kJ mol−1]
lnK0
R2
RMSE
90.6 85.4 80.8 73.4 70.7
28.4 27.0 26.0 23.8 23.2
0.994 0.998 1.000 1.000 1.000
0.080 0.039 0.010 0.016 0.004
94.5 89.6 84.2 81.1 75.1
29.5 28.2 26.9 26.2 24.5
1.000 0.999 0.987 0.995 0.991
0.019 0.031 0.107 0.064 0.081
Using Eq. (1) one can get lnðC t =C 0 Þ ¼ lnC t − lnC 0 ¼ −kt ¼ −K 0 expð−Ea =RT Þt:
ð4Þ
Activation energy was calculated from a linear relationship of lnk versus 1/T. Fig. 3 shows the Arrhenius plots for SF and SF/HP-β-CD inclusion complex degradation at pH 2.2, 3.0, 4.0, 5.0 and 6.0, whereas Ea values are gathered in Table 2. The Ea decreased from 90.6 to 70.7 kJ/mol as the pH increased. The Ea value of SF was similar to that obtained by Van Eylen et al. (2007), who reported an Ea of 89.03 kJ/mol for SF in broccoli juice. The slight difference between our and Van Eylen's results could be attributed to the presence of other substances in the juice. Jiang, Zhang, Tian, and Li (2006) reported an Ea of 59.0 kJ/mol for the reaction between pure allyl isothiocyanate (AITC) and hydroxyl/water and Neoh, Yamamoto, Ikefuji, Furuta, and Yoshii (2012) reported that the Ea values for the pure AITC, pure phenyl isothiocyanate (PITC), AITC inclusion complex, and PITC inclusion complex in paraffin were 26, 48, 44, and 54 kJ/mol, respectively. Compared with the Ea of pure SF, the Ea value of the SF/HP-β-CD inclusion complex was increased about 3.4–7.7 kJ/mol. The Ea increase observed in this study was much lower than that observed for AITC (22 kJ/mol) and PITC (10 kJ/mol) (Neoh et al., 2012). The difference may be attributed to the different modes of interactions between the functional groups of these compounds and the cavity of HP-β-CD. The complexation between AITC or PITC and RM-β-CD is 1:1 (Neoh et al., 2012), and the inclusion ratio between SF and HP-β-CD is also found to be 1:1 (Wu et al., 2010). But different from AITC and PITC, SF is quite easily dissolved in water, which can be attributed to the hydrophilic group of methylsulfinyl butane in SF. The mode of interactions between the hydrophobic benzene ring of PITC or allyl of AITC may be different from the methylsulfinyl butane of SF, the inclusion effect of SF with HP-β-CD may not be as good as AITC and PITC. This may explain
Fig. 4. The −Ea/R (a) and lnK0 (b) at different pH values of SF (●) and SF/HP-β-CD (□). Parameters of linear fitting: the slope of (1), (2), (3) and (4) were 648, 591, −1.43 and −1.26, respectively, the intercept of (1), (2), (3) and (4) were −12,264, −12,597, 31.46 and 32.15, respectively, and the R2 values of (1), (2), (3) and (4) were 0.985, 0.991, 0.978 and 0.983, respectively.
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Conflict of interest statement The authors declare no competing financial interest. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (21176220 and 31240054), Zhejiang Provincial Natural Science Foundation of China (Z13B060002) and Zhejiang Provincial Key Science and Technology Innovation Team (2009R50028). References
Fig. 5. Relation between the experimental retention ratios and the retention ratios estimated by the exponential model (Eqs. (5) and (6)), R2 was 0.985, and RMSE was 0.031.
why the stability increase of SF inclusion complex with β-CD is not as good as that of AITC or PITC. 3.3. Combined effect of temperature and pH values on the kinetics of SF and SF/HP-β-CD complex degradation The Arrhenius equation has been widely used in food research, but its applications in some fields, such as enzymatic reactions and growth rates of microorganisms remain some arguments (Peleg, Normand, & Corradini, 2012). But noticed that the Arrhenius equation could be written in a linear form (Table 2), one can develop mathematical models to predict the losses of SF and SF/HP-β-CD inclusion complex. As shown in Fig. 4, from the plots of −Ea/R and lnK0 for SF and SF/HP-β-CD as a function of pH values, one can observe that the correlation coefficient was >0.9. From the slopes and intercepts for each linear fit and Eqs. (1) and (4), we can obtain the kinetics equations for the thermal degradation of SF, Eq. (5) and the SF/HP-β-CD complex, Eq. (6): r ¼ expðð expð−1:43x þ 31:46Þ⋅ expð648x−12264Þ=T Þ⋅t Þ
ð5Þ
r ¼ expðð expð−1:26x þ 32:15Þ⋅ expð591x−12597Þ=T Þ⋅t Þ
ð6Þ
where x is the pH value (2.2–6.0). The goodness of fit of the exponential model is graphically presented in Fig. 5, where the relationship between the experimental thermal retention ratios and the thermal retention ratios estimated by the exponential model is depicted. Using this method, the retention value of SF at different pH values, depletion time and temperatures can be calculated. 4. Conclusions This research provided detailed information regarding the changes in the kinetic stability of pure SF and SF/HP-β-CD inclusion complex at different temperatures (60, 75, 82 and 90 °C) and pH values (2.2, 3.0, 4.0, 5.0 and 6.0). The temperature and pH level strongly influenced the stability of SF and SF/HP-β-CD inclusion complex, which were more stable at low pH values and temperatures. The SF/HP-β-CD inclusion complex was more stable than pure SF. Thermal degradation of SF and SF/HP-β-CD inclusion complex was adequately described by first-order model, and the Arrhenius equation could be applied to establish the relationship between the degradation rate constant and temperature at various pH values. The main dynamic parameters of SF and SF/HP-β-CD inclusion complex at various pH values were estimated. Then mathematical models were developed to predict the losses of SF and SF/HP-β-CD inclusion complex at different pH values, depletion time and temperatures.
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