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The mechanism of sulforaphene degradation to different water contents
Food Chemistry 194 (2016) 1022–1027
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
The mechanism of sulforaphene degradation to different water contents Guifang Tian a,1, Yuan Li a,1, Li Cheng a, Qipeng Yuan a,⇑, Pingwah Tang a, Pengqun Kuang b, Jing Hu a a b
State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, PR China Shandong Provincial Engineering Technology Research Center for Lunan Chinese Herbal Medicine, Linyi University, Linyi 276000, PR China
a r t i c l e
i n f o
Article history: Received 7 May 2015 Received in revised form 25 August 2015 Accepted 25 August 2015 Available online 28 August 2015 Keywords: Sulforaphene Stability Degradation product Degradation kinetics Water content
a b s t r a c t Sulforaphene extracted from radish seeds was strongly associated with cancer prevention. However, sulforaphene was unstable in aqueous medium and at high temperature. This instability impairs many useful applications of sulforaphene. In this paper, the stability of sulforaphene (purity above 95%) during storage at 20 °C, 4 °C and 26 °C was studied. The degradation product was purified by preparative HPLC and identified by ESI/MS, NMR (1H and 13C NMR) and FTIR spectroscopy. The degradation pathway of sulforaphene was presented. Furthermore, we found that the degradation rate of sulforaphene was closely related to the water content of sulforaphene sample. The higher the water content was, the faster the sulforaphene sample degraded. A mathematical model was developed to predict the degradation constant at various water contents. It provided a guideline for industry to improve the stability of sulforaphene during preparation, application and storage. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction In recent years, natural bioactive compounds such as paclitaxel, vinblastine and navelbine have received considerable attention because of their antitumor activities (Lee, 1999). In addition, their side effects are lower than that of conventional radiotherapy and chemotherapy agents. Epidemiological studies indicated that the consumption of cruciferous vegetables including radish (Raphanus sativus L.), broccoli, cauliflower, kale and Chinese cabbage could reduce the risk of many cancers and cardiovascular diseases (Higdon, Delage, Williams, & Dashwood, 2007). It is may be due to the presence of various phytochemicals, such as glucosinolates or flavonoids (Block, Patterson, & Subar, 1992; Heimler, Vignolini, Dini, Vincieri, & Romani, 2006; Lima, Rocha, Takaki, Ramos, & Ono, 2008). However, glucosinolates are not endowed with biological activity. When cruciferous vegetables are ground or chopped, glucosinolates are catalyzed by myrosinase to release b-D-glucose and unstable thiohydroximate-O-sulfonate. The thiohydroximateO-sulfonate is then transformed into isothiocyanates (Lossen rearrangement), or nitriles and other sulfur containing chemicals by enzymatic degradation (Hanschen, Lamy, Schreiner, & Rohn, 2014; Holst & Williamson, 2004). Isothiocyanates, which are
⇑ Corresponding author at: Beijing University of Chemical Technology, West Room 314, Science and Technology Building, No. 15 North Third Ring East Road, Chaoyang District, Beijing 100029, PR China. E-mail address: [email protected] (Q. Yuan). 1 Both authors have contributed equally to this work. http://dx.doi.org/10.1016/j.foodchem.2015.08.107 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
responsible for the pungent aroma and flavor of several Brassica vegetables, have a strong anti-cancer activity against many kinds of cancers, such as pancreatic cancer (Chan, Wang, & Holly, 2005), colorectal cancer (Voorips, Goldbohm, van Poppel, et al., 2000), prostate cancer (Joseph et al., 2004), breast cancer (Ambrosone et al., 2004), lung cancer (Neuhouser et al., 2003; Voorips, Goldbohm, Verhoeven, et al., 2000). Sulforaphene (4-methylsufinyl-3-butenyl isothiocyanate), a member of isothiocyanate family derived from radish (Kuang et al., 2013), has shown a great potential as an anticancer agent (Papi et al., 2008). It was reported that sulforaphene is capable of inhibiting cell proliferation in a dose-dependent manner and inducing apoptosis in HCT-116 (Pocasap, Weerapreeyakul, & Barusrux, 2013), LoVo, and HT-29 cancer cell lines (Papi et al., 2008). Sulforaphene could also reduce the cell proliferation of human and murine erythroleukemic cells, human T-lymphoid cells, human cervix carcinoma cells and H3-T1-1 cells (Nastruzzi et al., 2000). Furthermore, sulforaphene has a remarkable antimutagenicity activity in the TA100 strain in the presence of Aroclor 1254-induced rat liver S9 (Shishu & Kaur, 2009). It was reported that the antimutagenicity activity was two times higher for sulforaphene than sulforaphane (Shishu & Kaur, 2009). These studies showed that sulforaphene has a potential as an effective cancer chemopreventive agent. However, isothiocyanates are not stable in aqueous medium and at high temperature (Kawakishi & Namiki, 1969; Uda, Ozawa, Ohshima, & Kawakishi, 1990). Due to the electrophilic carbon atom in the isothiocyanate group, the compounds tend to react
G. Tian et al. / Food Chemistry 194 (2016) 1022–1027
with nucleophiles such as hydroxyl, amino, or thiol groups, forming O-thiocarbamates, thiourea derivatives, or dithiocarbamates, respectively (Zhang & Talalay, 1994). The stability of sulforaphene during storage became a major concern for its bioactivity. It was reported that sulforaphene was susceptible to degradation at alkaline environment (Song et al., 2013). Both sulforaphene and sulforaphane (SFN) belongs to isothiocyanates. The difference is that sulforaphene has a vinylic sulfoxide functionality. Our previous study showed that sulforaphene was sensitive to heat (Tian et al., 2015) same as SFN (Jin, Wang, Rosen, & Ho, 1999; Wu, Liang, Yuan, Wang, & Yan, 2010; Wu, Zou, Mao, Huang, & Liu, 2014). The thermal degradation of sulforaphene followed the first-order reaction kinetics (Tian et al., 2015). Literature showed that SFA would decompose to N,N0 -di-(methylsulfinyl) butyl thiourea under thermal processing conditions in aqueous solution between 50 °C and 100 °C. The nonvolatile degradation product of SFN under heating was a dimer of SFN (Jin et al., 1999). Residual chemical reagents and water might be present in the sulforaphene product after separation and purification processes. For example, the residue of ethanol was about 90 ppm after freeze drying of sulforaphene. Therefore, the key factors that could affect the stability of sulforaphene should be explored. Besides the mechanism of sulforaphene degradation during storage need to be investigated. It can provide a guideline for industry to improve the stability of sulforaphene during preparation, application and storage. The object of this study was to investigate the mechanism of sulforaphene prepared by our laboratory in different water contents under various temperatures. Firstly, the main degradation product of sulforaphene during storage was collected. Second, the chemical structure of the degradation product was identified. Then the degradation pathway of sulforaphene was proposed. Finally, we proposed a linear regression for modeling the relation between the water content and the degradation rate constant during storage. 2. Materials and methods
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2.3. Separation and purification of the degradation product Sulforaphene (1 g) with the purity of 95% was placed at 26 °C in drug stability test chamber for 35 days for the degradation product formation. Afterwards, the degradation product-rich product mixed with ultra-pure water at the concentration of 15 mg/mL was subjected into the preparative HPLC system after being filtrated with 0.22 lm membrane. The preparative HPLC parameters of the experiment were as follows: the mobile phase system consisted of 17% (v/v) methanol in ultrapure water, the flow rate was 10 mL/min, the detection wavelength was 219 nm and the injection volume was 1 mL. The peak fraction of the degradation product was collected manually according to the preparative HPLC chromatogram, and then dried at 45 °C under vacuum with a rotator evaporator and the final product was freeze dried.
2.4. ESI/MS and NMR The identification of the degradation product was carried out by ESI/MS and NMR (1H and 13C NMR). The purified degradation product with the concentration of 100 ppm was detected by ESI/MS with an ion source temperature of 200 °C and a probe temperature of 25 °C. 550 lL deuterium oxide containing 20 mg of the degradation product was subjected to a Bruker high resolution AV600 NMR spectrometer at 600 MHz using tetramethylsilane (TMS) as internal standard.
2.5. Fourier transform infrared spectroscopy (FTIR) FTIR spectrum was investigated to detect the molecular structure of the purified degradation product through a NEXUS 670 FTIR spectrometer (Nicolet, USA). The degradation product was tested by thin-film method. Spectrum was acquired using air as background.
2.1. Materials Sulforaphene (purity >95%) was prepared and purified from radish seeds in our laboratory by preparative high-performance liquid chromatography and its purity and chemical structure were identified by analytical HPLC, ESI-MS and NMR (Kuang et al., 2013). Methanol and trifluoroacetic acid (TFA) used for analytical and preparative HPLC were of chromatographic grade and purchased from Fisher Scientific Co., Ltd. (Tustin, CA). Ultra-pure water was obtained by Q Millipore System (Millipore, Bedford, MA). All other reagents were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
2.6. The determination of water content in sulforaphene YT-11133F Karl-Fisher Titrator (Shanghai yutong Instrument Factory, China) was used to test water content by volumetry. 1 mL sulforaphene prepared by our lab was injected. And the samples were tested in triplicate.
2.2. The storage stability of sulforaphene About 10 mg of accurately weighed samples of sulforaphene were placed in 2 mL sample vials. After being tightly sealed, the vials were placed in three different temperatures chambers: 20 °C, 4 °C and 26 °C. The samples were taken out weekly, and dissolved in 5 mL of ultra-pure water and filtered by 0.22 lm membrane. The filtrate was tested by reversed-phase HPLC system. The mobile phase system consisted of methanol as mobile phase A and 0.02% (v/v) TFA in ultra-pure water as mobile phase B. A linear change of methanol remained from 20% to 80% in first 20 min, and then raised to 100% methanol and maintained for 2 min to purge the column. The flow rate was 1 mL/min and the detection wavelengths were 254 nm and 219 nm. The temperature of the column oven was 30 °C. All samples were tested in triplicate.
Fig. 1. The stability of sulforaphene at 20 °C, 4 °C and 26 °C.
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Fig. 2. Chromatograms of the HPLC analysis of sulforaphene and its degradation product at 26 °C (a), preparative HPLC chromatogram of the degradation product (b) and the analytical HPLC chromatogram of the purified degradation product obtained by preparative HPLC (c).
2.7. Degradation kinetics of sulforaphene in different water contents A certain amount of sulforaphene was weighed accurately. Then various amounts of water were added. The water contents of sulforaphene were 0.35%, 10%, 50% and 99.8%, respectively. About 10 mg sulforaphene of the different water contents accurately weighed were placed in 2 mL perfectly sealed sample vials. The samples were placed at 26 °C in drug stability test chamber and taken out weekly. The collected samples were dissolved in 5 mL of ultra-pure water and filtered by 0.22 lm membrane. The filtrate
was detected by reversed-phase HPLC system as described in Section 2.2. All samples were tested in triplicate. The degradation of sulforaphene could be modeled as the first-order reaction (Tian et al., 2015):
ln
C ¼ kt C0
ð1Þ
where C is the residual content of sulforaphene at time t, C0 is the initial content of sulforaphene, and k is the reaction rate constant.
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Fig. 3. The NMR spectra (D2O, 600 MHz) of the degradation product purified by preparative HPLC: 1H NMR spectrum (a) and 13C NMR spectrum (b).
3. Results and discussion 3.1. The storage stability of sulforaphene Since the stability of natural bioactive compounds during storage is related to temperature, we carefully examined the stability of sulforaphene under various temperatures: 20 °C, 4 °C and 26 °C in the aspect of its residual rate in time. As shown in Fig. 1, the residual rates of sulforaphene at the temperatures of 20 °C, 4 °C and 26 °C after 5 weeks of storage remained around 96.56 ± 0.15%, 95.18 ± 0.20% and 78.45 ± 0.28%, respectively. Variations differ greatly from each other (p < 0.01). Our findings indicated that the degradation rate of sulforaphene increased by increasing the temperature, which suggested that the relative high temperature accelerated the degradation rate, according to the molecular thermal motion (Jin et al., 1999). The results indicated that the suitable storage temperatures of sulforaphene were 20 °C or 4 °C. 3.2. The HPLC analysis of sulforaphene and its main degradation product The identification of the by-products structures from the degradation process might offer some insight of the sulforaphene degradation mechanism. As shown in Fig. 2a, compared with the blank, 21.55% of sulforaphene stored at 26 °C (retention time: 16.21 min) degraded after 35 days which is obviously shown by the apparition
Fig. 4. Proposed degradation pathway of sulforaphene.
of a new peak in the chromatogram at 10.92 min. The peak had a maximum peak area at 219 nm according to the full wavelength scanning. This peak also appeared in sulforaphene stored at 20 °C and 4 °C. These results have given evidence that the new peak represented the main degradation product of sulforaphene during storage. 3.3. Separation of the degradation product by preparative HPLC The main degradation product of sulforaphene stored for 35 days at 26 °C was collected by preparative HPLC in order to investigate its chemical structure. The eluent of the major peak (the fraction from 75 to 85 min) at 219 nm was collected and subjected to analytical HPLC (Fig. 2b). As shown in Fig. 2c, the purity of the final product was more than 95% according to analytical HPLC detection. 3.4. Identification of the degradation product To identify the chemical structure of the sulforaphene degradation product, the sample was submitted to the ESI/MS, 1H and 13C NMR analysis. The HR-ESI–MS spectrum showed that the
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Fig. 5. The FTIR spectra of the degradation product.
degradation product has a molecular formula of [C11H21N2O2S3]+ (calculated: 309.0765, found: 309.0764), which was two-folds larger than the sulforaphene molecular weight. As shown in Fig. 3, the 1H and 13C NMR data (600 MHz, D2O) were close to that of sulforaphene (Kuang et al., 2013). As compared with the chemical structure of sulforaphene, it was postulated that the degradation product was a dimer of sulforaphene monomer, which is similar to the degradation of SFN and allyl-isothiocyanate in water. SFN was converted to (methylsulfinyl) butyl-thiourea. And allyl-isothiocyanate converted to diallylthiourea in aqueous solution (Chen & Ho, 1998; Jin et al., 1999). 3.5. The proposed degradation pathway of sulforaphene The results of the ESI/MS and 1H and 13C NMR confirmed the dimer formation of sulforaphene during the storage. The proposed degradation pathway of sulforaphene in water was presented in Fig. 4. The hydroxyl groups of water in the sulforaphene product works as a nucleophilic agent which has reacted with the electrophilic carbon atom of AN@C@S groups on sulforaphene leading to the formation of R-NH2. The carbon atom of AN@C@S groups on sulforaphene could readily react with the resulting amine to generate a dimer compound (Kawakishi & Namiki, 1969). 3.6. Structure analysis of the degradation product by FTIR spectroscopy The degradation product was analyzed by the FTIR spectroscopy. The results (Fig. 5) showed that the peak at 1554 cm1 was due to NAH deformation and CAN stretching of AHNA (C@S)ANHA; the peak at 1030 cm1 was due to S@O stretching; two peaks at 3076 cm1 and 961 cm1 were due to AHC@CHA; two peaks at 2930 cm1 and 1418 cm1 were due to ACH2A; and the two peaks at 2870 and 1382 cm1 were due to CAH stretching from ACH3. The proposed structure was found consistent with the one identified by NMR. 3.7. Degradation kinetics of sulforaphene of different water contents The stability of sulforaphene was affected by the moisture content. The water content of the sulforaphene product after freezedrying was 3.447 lg/mL. A model (Eq. (1)) based on the sulforaphene degradation with different water contents was proposed. According to Fig. 6a, it was found that the degradation of sulforaphene with different water content all fit first-order kinetics with a good linearity (R2 = 0.9599–0.9995). The degradation rate constants k were 0.047, 0.086, 0.111 and 0.263 for sulforaphene
Fig. 6. The degradation kinetics of sulforaphene with different water content (a) and the correlation between water content of SFE and the degradation rate constant (b) at the temperature of 26 °C.
degradation under water contents of 0.35%, 10%, 50% and 99.8% respectively. Our findings indicated that that the higher the water content was, the faster the sulforaphene degradation occurred. By plotting the degradation rate constants as a function of water contents, we have found a linear relationship between this two constants (Fig. 6b). The coefficients of x were inversely proportional to the degradation rate constant k. The correlation at 26 °C was described by the equation y = 0.0021x 0.04 (R2 = 0.9093), in which x represented the water content of sulforaphene sample, and y represented the degradation rate constant. Obviously, there is a strong correlation between the degradation rate and the water contents. It suggests that the sulforaphene degradation may be caused by the presence of water which initiated the degradation process. This was consistent with the degradation pathway that we proposed in Fig. 4. The sulforaphene was attacked by the hydroxyl groups of water leading to a dimer formation, which is in accordance with the degradation pathway of SFN (Jin et al., 1999) and allyl-isothiocyanate in water (Chen & Ho, 1998; Kawakishi & Namiki, 1969). 4. Conclusion In this study, 4 °C and 20 °C were found to be the optimal storage temperatures for sulforaphene. The degradation product
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of sulforaphene during storage was found to be its dimer. This product was formed by the interaction between AN@C@S groups on sulforaphene and ANH2 groups which was formed by nucleophilic attack of the hydroxyl groups in water. We found a strong correlation between the degradation rate constant and the water contents. The model we developed can be applied to predict the residual rates of sulforaphene in different water contents during storage. This study provided useful information for sulforaphene storage for industrial application. Acknowledgment The authors would like to acknowledge the financial support of the National Natural Science Foundation of China (No. 21376017 and 21176018). References Ambrosone, C. B., McCann, S. E., Freudenheim, J. L., Marshall, J. R., Zhang, Y., & Shields, P. G. (2004). Breast cancer risk in premenopausal women is inversely associated with consumption of broccoli, a source of isothiocyanates, but is not modified by GST genotype. Journal of Nutrition, 134, 1134–1138. Block, G., Patterson, B., & Subar, A. (1992). Fruit, vegetables, and cancer prevention: A review of the epidemiological evidence. Nutrition and Cancer, 18, 1–29. Chan, J. M., Wang, F., & Holly, E. A. (2005). Vegetable and fruit intake and pancreatic cancer in a population-based case-control study in the San Francisco bay area. Cancer Epidemiology Biomarkers & Prevention, 14, 2093–2097. Chen, C., & Ho, C. (1998). Thermal degradation of allyl isothiocyanate in aqueous solution. Journal of Agricultural and Food Chemistry, 48, 220–223. Hanschen, Franziska S., Lamy, E., Schreiner, M., & Rohn, S. (2014). Reactivity and stability of glucosinolates and their breakdown products in foods. Angewandte Chemie International Edition, 53, 11430–11450. Heimler, D., Vignolini, P., Dini, M. G., Vincieri, F. F., & Romani, A. (2006). Antiradical activity and polyphenol composition of local Brassicaceae edible varieties. Food Chemistry, 99, 464–469. Higdon, J. V., Delage, B., Williams, D. E., & Dashwood, R. H. (2007). Cruciferous vegetables and human cancer risk: Epidemiologic evidence and mechanistic basis. Phamacological Research, 55, 224–236. Holst, B., & Williamson, G. (2004). A critical review of the bioavailability of glucosinolates and related compounds. Natural Product Reports, 21, 425–447. Jin, Y., Wang, M. F., Rosen, Rosen T., & Ho, C. T. (1999). Thermal degradation of sulforaphane in aqueous solution. Journal of Agricultural and Food Chemistry, 47, 3121–3123. Joseph, M. A., Moysich, K. B., Freudenheim, J. L., Shiedlds, P. G., Bowman, E. D., & Zhang, Y. (2004). Cruciferous vegetables, genetic polymorphisms in glutathione S-transulforaphenerases M1 and T1, and prostate cancer risk. Nutrition and Cancer, 50, 206–213. Kawakishi, S., & Namiki, M. (1969). Decomposition of allyl isothiocyanate in aqueous solution. Agricultural and Biological Chemistry, 33, 452–459.
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Kuang, P. Q., Song, D., Yuan, Q. P., Yi, R., Lv, X. H., & Liang, H. (2013). Separation and purification of sulforaphene from radish seeds using macroporous resin and preparative high-performance liquid chromatography. Food Chemistry, 136, 342–347. Lee, K. H. (1999). Anticancer drug design based on plant-derived natural products. Journal of Biomedical Science, 4(6), 236–250. Lima, G. P. P., Rocha, S. A., Takaki, M., Ramos, P. R. R., & Ono, E. O. (2008). Comparison of polyamine, phenol and flavonoid contents in plants grown under conventional and organic methods. International Journal of Food Science and Technology, 43, 1838–1843. Nastruzzi, C., Cortesi, R., Esposito, E., Menegatti, E., Leoni, O., Iori, R., & Palmieri, S. (2000). In vitro antiproliferative activity of isothiocyanates and nitriles generated by myrosinase-mediated hydrolysis of glucosinolates from seeds of cruciferous vegetables. Journal of Agricultural and Food Chemistry, 48, 3572–3575. Neuhouser, M. L., Patterson, R. E., Thornquist, M. D., Omenn, G. S., King, I. B., & Goodman, G. E. (2003). Fruits and vegetable are associated with lower lung cancer risk only in the placebo arm of the beta-carotene and retinol efficacy trial. Cancer Epidemiology Biomarkers & Prevention, 12, 350–358. Papi, A., Orlandi, M., Bartolini, G., Barillari, J., Iori, R., Paolini, M., ... Valgimigli, L. (2008). Cytotoxic and antioxidant activity of 4-methylthio-3-butenyl isothiocyanate from Raphanus sativus L. (Kaiware Daikon) sprouts. Journal of Agriculture and Food Chemistry, 56, 875–883. Pocasap, P., Weerapreeyakul, N., & Barusrux, S. (2013). Cancer preventive effect of Thai rat-tailed radish (Raphanus sativus L. var. caudatus Alef). Journal of Functional Foods, 5, 1372–1381. Shishu & Kaur, Indu Pal (2009). Inhibition of cooked food-induced mutagenesis by dietary constituents: Comparison of two natural isothiocyanates. Food Chemistry, 112, 977–981. Song, D., Liang, H., Kuang, P. Q., Tang, P. W., Hu, G. F., & Yuan, Q. P. (2013). Instability and structural change of 4-methylsulfinyl-3-butenyl isothiocyanate in the hydrolytic process. Journal of Agricultural and Food Chemistry, 61, 5097–5102. Tian, G. F., Li, Y., Yuan, Q. P., Cheng, L., Kuang, P. Q., & Tang, P. W. (2015). The stability and degradation kinetics of sulforaphene in microcapsules based on several biopolymers via spray drying. Carbohydrate Polymers, 122, 5–10. Uda, Y., Ozawa, Y., Ohshima, T., & Kawakishi, S. (1990). Identification of enolated 2thioxo-3-pyrrolidinecarbaldehyde, a new degradation product of 4-methylthio3-butenyl isothiocyanate. Agricultural and Biological Chemistry, 54, 613–617. Voorips, L. E., Goldbohm, R. A., van Poppel, G., Sturmans, F., Hermus vanden, R. J., & Brandt, P. A. (2000). Vegetable and fruit consumption and risks of colon and rectal cancer in a prospective cohort study: The Netherlands cohort study on diet and cancer. American Journal of Epidemiology, 152, 1081–1092. Voorips, L. E., Goldbohm, R. A., Verhoeven, D. T., van Poppel, G. A., Sturmans, F., & Hermus, R. J. (2000). Vegetable and fruit consumption and lung cancer risk in the Netherlands Cohort Study on diet and cancer. Cancer Causes and Control, 11, 1010–1015. Wu, H. H., Liang, H., Yuan, Q. P., Wang, T. X., & Yan, X. (2010). Preparation and stability investigation of the inclusion complex of sulforaphane with hydroxypropyl-b-cyclodextrin. Carbohydrate Polymers, 82, 613–617. Wu, Y. F., Zou, L. G., Mao, J. W., Huang, J., & Liu, S. W. (2014). Stability and encapsulation efficiency of sulforaphane microencapsulated by spray drying. Carbohydrate Polymers, 102, 497–503. Zhang, Y. S., & Talalay, P. (1994). Anticarcinogenic activities of organic isothiocyanates: Chemistry and mechanisms. Cancer Research, 54, 1976–1981.
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
The mechanism of sulforaphene degradation to different water contents Guifang Tian a,1, Yuan Li a,1, Li Cheng a, Qipeng Yuan a,⇑, Pingwah Tang a, Pengqun Kuang b, Jing Hu a a b
State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, PR China Shandong Provincial Engineering Technology Research Center for Lunan Chinese Herbal Medicine, Linyi University, Linyi 276000, PR China
a r t i c l e
i n f o
Article history: Received 7 May 2015 Received in revised form 25 August 2015 Accepted 25 August 2015 Available online 28 August 2015 Keywords: Sulforaphene Stability Degradation product Degradation kinetics Water content
a b s t r a c t Sulforaphene extracted from radish seeds was strongly associated with cancer prevention. However, sulforaphene was unstable in aqueous medium and at high temperature. This instability impairs many useful applications of sulforaphene. In this paper, the stability of sulforaphene (purity above 95%) during storage at 20 °C, 4 °C and 26 °C was studied. The degradation product was purified by preparative HPLC and identified by ESI/MS, NMR (1H and 13C NMR) and FTIR spectroscopy. The degradation pathway of sulforaphene was presented. Furthermore, we found that the degradation rate of sulforaphene was closely related to the water content of sulforaphene sample. The higher the water content was, the faster the sulforaphene sample degraded. A mathematical model was developed to predict the degradation constant at various water contents. It provided a guideline for industry to improve the stability of sulforaphene during preparation, application and storage. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction In recent years, natural bioactive compounds such as paclitaxel, vinblastine and navelbine have received considerable attention because of their antitumor activities (Lee, 1999). In addition, their side effects are lower than that of conventional radiotherapy and chemotherapy agents. Epidemiological studies indicated that the consumption of cruciferous vegetables including radish (Raphanus sativus L.), broccoli, cauliflower, kale and Chinese cabbage could reduce the risk of many cancers and cardiovascular diseases (Higdon, Delage, Williams, & Dashwood, 2007). It is may be due to the presence of various phytochemicals, such as glucosinolates or flavonoids (Block, Patterson, & Subar, 1992; Heimler, Vignolini, Dini, Vincieri, & Romani, 2006; Lima, Rocha, Takaki, Ramos, & Ono, 2008). However, glucosinolates are not endowed with biological activity. When cruciferous vegetables are ground or chopped, glucosinolates are catalyzed by myrosinase to release b-D-glucose and unstable thiohydroximate-O-sulfonate. The thiohydroximateO-sulfonate is then transformed into isothiocyanates (Lossen rearrangement), or nitriles and other sulfur containing chemicals by enzymatic degradation (Hanschen, Lamy, Schreiner, & Rohn, 2014; Holst & Williamson, 2004). Isothiocyanates, which are
⇑ Corresponding author at: Beijing University of Chemical Technology, West Room 314, Science and Technology Building, No. 15 North Third Ring East Road, Chaoyang District, Beijing 100029, PR China. E-mail address: [email protected] (Q. Yuan). 1 Both authors have contributed equally to this work. http://dx.doi.org/10.1016/j.foodchem.2015.08.107 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
responsible for the pungent aroma and flavor of several Brassica vegetables, have a strong anti-cancer activity against many kinds of cancers, such as pancreatic cancer (Chan, Wang, & Holly, 2005), colorectal cancer (Voorips, Goldbohm, van Poppel, et al., 2000), prostate cancer (Joseph et al., 2004), breast cancer (Ambrosone et al., 2004), lung cancer (Neuhouser et al., 2003; Voorips, Goldbohm, Verhoeven, et al., 2000). Sulforaphene (4-methylsufinyl-3-butenyl isothiocyanate), a member of isothiocyanate family derived from radish (Kuang et al., 2013), has shown a great potential as an anticancer agent (Papi et al., 2008). It was reported that sulforaphene is capable of inhibiting cell proliferation in a dose-dependent manner and inducing apoptosis in HCT-116 (Pocasap, Weerapreeyakul, & Barusrux, 2013), LoVo, and HT-29 cancer cell lines (Papi et al., 2008). Sulforaphene could also reduce the cell proliferation of human and murine erythroleukemic cells, human T-lymphoid cells, human cervix carcinoma cells and H3-T1-1 cells (Nastruzzi et al., 2000). Furthermore, sulforaphene has a remarkable antimutagenicity activity in the TA100 strain in the presence of Aroclor 1254-induced rat liver S9 (Shishu & Kaur, 2009). It was reported that the antimutagenicity activity was two times higher for sulforaphene than sulforaphane (Shishu & Kaur, 2009). These studies showed that sulforaphene has a potential as an effective cancer chemopreventive agent. However, isothiocyanates are not stable in aqueous medium and at high temperature (Kawakishi & Namiki, 1969; Uda, Ozawa, Ohshima, & Kawakishi, 1990). Due to the electrophilic carbon atom in the isothiocyanate group, the compounds tend to react
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with nucleophiles such as hydroxyl, amino, or thiol groups, forming O-thiocarbamates, thiourea derivatives, or dithiocarbamates, respectively (Zhang & Talalay, 1994). The stability of sulforaphene during storage became a major concern for its bioactivity. It was reported that sulforaphene was susceptible to degradation at alkaline environment (Song et al., 2013). Both sulforaphene and sulforaphane (SFN) belongs to isothiocyanates. The difference is that sulforaphene has a vinylic sulfoxide functionality. Our previous study showed that sulforaphene was sensitive to heat (Tian et al., 2015) same as SFN (Jin, Wang, Rosen, & Ho, 1999; Wu, Liang, Yuan, Wang, & Yan, 2010; Wu, Zou, Mao, Huang, & Liu, 2014). The thermal degradation of sulforaphene followed the first-order reaction kinetics (Tian et al., 2015). Literature showed that SFA would decompose to N,N0 -di-(methylsulfinyl) butyl thiourea under thermal processing conditions in aqueous solution between 50 °C and 100 °C. The nonvolatile degradation product of SFN under heating was a dimer of SFN (Jin et al., 1999). Residual chemical reagents and water might be present in the sulforaphene product after separation and purification processes. For example, the residue of ethanol was about 90 ppm after freeze drying of sulforaphene. Therefore, the key factors that could affect the stability of sulforaphene should be explored. Besides the mechanism of sulforaphene degradation during storage need to be investigated. It can provide a guideline for industry to improve the stability of sulforaphene during preparation, application and storage. The object of this study was to investigate the mechanism of sulforaphene prepared by our laboratory in different water contents under various temperatures. Firstly, the main degradation product of sulforaphene during storage was collected. Second, the chemical structure of the degradation product was identified. Then the degradation pathway of sulforaphene was proposed. Finally, we proposed a linear regression for modeling the relation between the water content and the degradation rate constant during storage. 2. Materials and methods
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2.3. Separation and purification of the degradation product Sulforaphene (1 g) with the purity of 95% was placed at 26 °C in drug stability test chamber for 35 days for the degradation product formation. Afterwards, the degradation product-rich product mixed with ultra-pure water at the concentration of 15 mg/mL was subjected into the preparative HPLC system after being filtrated with 0.22 lm membrane. The preparative HPLC parameters of the experiment were as follows: the mobile phase system consisted of 17% (v/v) methanol in ultrapure water, the flow rate was 10 mL/min, the detection wavelength was 219 nm and the injection volume was 1 mL. The peak fraction of the degradation product was collected manually according to the preparative HPLC chromatogram, and then dried at 45 °C under vacuum with a rotator evaporator and the final product was freeze dried.
2.4. ESI/MS and NMR The identification of the degradation product was carried out by ESI/MS and NMR (1H and 13C NMR). The purified degradation product with the concentration of 100 ppm was detected by ESI/MS with an ion source temperature of 200 °C and a probe temperature of 25 °C. 550 lL deuterium oxide containing 20 mg of the degradation product was subjected to a Bruker high resolution AV600 NMR spectrometer at 600 MHz using tetramethylsilane (TMS) as internal standard.
2.5. Fourier transform infrared spectroscopy (FTIR) FTIR spectrum was investigated to detect the molecular structure of the purified degradation product through a NEXUS 670 FTIR spectrometer (Nicolet, USA). The degradation product was tested by thin-film method. Spectrum was acquired using air as background.
2.1. Materials Sulforaphene (purity >95%) was prepared and purified from radish seeds in our laboratory by preparative high-performance liquid chromatography and its purity and chemical structure were identified by analytical HPLC, ESI-MS and NMR (Kuang et al., 2013). Methanol and trifluoroacetic acid (TFA) used for analytical and preparative HPLC were of chromatographic grade and purchased from Fisher Scientific Co., Ltd. (Tustin, CA). Ultra-pure water was obtained by Q Millipore System (Millipore, Bedford, MA). All other reagents were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
2.6. The determination of water content in sulforaphene YT-11133F Karl-Fisher Titrator (Shanghai yutong Instrument Factory, China) was used to test water content by volumetry. 1 mL sulforaphene prepared by our lab was injected. And the samples were tested in triplicate.
2.2. The storage stability of sulforaphene About 10 mg of accurately weighed samples of sulforaphene were placed in 2 mL sample vials. After being tightly sealed, the vials were placed in three different temperatures chambers: 20 °C, 4 °C and 26 °C. The samples were taken out weekly, and dissolved in 5 mL of ultra-pure water and filtered by 0.22 lm membrane. The filtrate was tested by reversed-phase HPLC system. The mobile phase system consisted of methanol as mobile phase A and 0.02% (v/v) TFA in ultra-pure water as mobile phase B. A linear change of methanol remained from 20% to 80% in first 20 min, and then raised to 100% methanol and maintained for 2 min to purge the column. The flow rate was 1 mL/min and the detection wavelengths were 254 nm and 219 nm. The temperature of the column oven was 30 °C. All samples were tested in triplicate.
Fig. 1. The stability of sulforaphene at 20 °C, 4 °C and 26 °C.
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Fig. 2. Chromatograms of the HPLC analysis of sulforaphene and its degradation product at 26 °C (a), preparative HPLC chromatogram of the degradation product (b) and the analytical HPLC chromatogram of the purified degradation product obtained by preparative HPLC (c).
2.7. Degradation kinetics of sulforaphene in different water contents A certain amount of sulforaphene was weighed accurately. Then various amounts of water were added. The water contents of sulforaphene were 0.35%, 10%, 50% and 99.8%, respectively. About 10 mg sulforaphene of the different water contents accurately weighed were placed in 2 mL perfectly sealed sample vials. The samples were placed at 26 °C in drug stability test chamber and taken out weekly. The collected samples were dissolved in 5 mL of ultra-pure water and filtered by 0.22 lm membrane. The filtrate
was detected by reversed-phase HPLC system as described in Section 2.2. All samples were tested in triplicate. The degradation of sulforaphene could be modeled as the first-order reaction (Tian et al., 2015):
ln
C ¼ kt C0
ð1Þ
where C is the residual content of sulforaphene at time t, C0 is the initial content of sulforaphene, and k is the reaction rate constant.
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Fig. 3. The NMR spectra (D2O, 600 MHz) of the degradation product purified by preparative HPLC: 1H NMR spectrum (a) and 13C NMR spectrum (b).
3. Results and discussion 3.1. The storage stability of sulforaphene Since the stability of natural bioactive compounds during storage is related to temperature, we carefully examined the stability of sulforaphene under various temperatures: 20 °C, 4 °C and 26 °C in the aspect of its residual rate in time. As shown in Fig. 1, the residual rates of sulforaphene at the temperatures of 20 °C, 4 °C and 26 °C after 5 weeks of storage remained around 96.56 ± 0.15%, 95.18 ± 0.20% and 78.45 ± 0.28%, respectively. Variations differ greatly from each other (p < 0.01). Our findings indicated that the degradation rate of sulforaphene increased by increasing the temperature, which suggested that the relative high temperature accelerated the degradation rate, according to the molecular thermal motion (Jin et al., 1999). The results indicated that the suitable storage temperatures of sulforaphene were 20 °C or 4 °C. 3.2. The HPLC analysis of sulforaphene and its main degradation product The identification of the by-products structures from the degradation process might offer some insight of the sulforaphene degradation mechanism. As shown in Fig. 2a, compared with the blank, 21.55% of sulforaphene stored at 26 °C (retention time: 16.21 min) degraded after 35 days which is obviously shown by the apparition
Fig. 4. Proposed degradation pathway of sulforaphene.
of a new peak in the chromatogram at 10.92 min. The peak had a maximum peak area at 219 nm according to the full wavelength scanning. This peak also appeared in sulforaphene stored at 20 °C and 4 °C. These results have given evidence that the new peak represented the main degradation product of sulforaphene during storage. 3.3. Separation of the degradation product by preparative HPLC The main degradation product of sulforaphene stored for 35 days at 26 °C was collected by preparative HPLC in order to investigate its chemical structure. The eluent of the major peak (the fraction from 75 to 85 min) at 219 nm was collected and subjected to analytical HPLC (Fig. 2b). As shown in Fig. 2c, the purity of the final product was more than 95% according to analytical HPLC detection. 3.4. Identification of the degradation product To identify the chemical structure of the sulforaphene degradation product, the sample was submitted to the ESI/MS, 1H and 13C NMR analysis. The HR-ESI–MS spectrum showed that the
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Fig. 5. The FTIR spectra of the degradation product.
degradation product has a molecular formula of [C11H21N2O2S3]+ (calculated: 309.0765, found: 309.0764), which was two-folds larger than the sulforaphene molecular weight. As shown in Fig. 3, the 1H and 13C NMR data (600 MHz, D2O) were close to that of sulforaphene (Kuang et al., 2013). As compared with the chemical structure of sulforaphene, it was postulated that the degradation product was a dimer of sulforaphene monomer, which is similar to the degradation of SFN and allyl-isothiocyanate in water. SFN was converted to (methylsulfinyl) butyl-thiourea. And allyl-isothiocyanate converted to diallylthiourea in aqueous solution (Chen & Ho, 1998; Jin et al., 1999). 3.5. The proposed degradation pathway of sulforaphene The results of the ESI/MS and 1H and 13C NMR confirmed the dimer formation of sulforaphene during the storage. The proposed degradation pathway of sulforaphene in water was presented in Fig. 4. The hydroxyl groups of water in the sulforaphene product works as a nucleophilic agent which has reacted with the electrophilic carbon atom of AN@C@S groups on sulforaphene leading to the formation of R-NH2. The carbon atom of AN@C@S groups on sulforaphene could readily react with the resulting amine to generate a dimer compound (Kawakishi & Namiki, 1969). 3.6. Structure analysis of the degradation product by FTIR spectroscopy The degradation product was analyzed by the FTIR spectroscopy. The results (Fig. 5) showed that the peak at 1554 cm1 was due to NAH deformation and CAN stretching of AHNA (C@S)ANHA; the peak at 1030 cm1 was due to S@O stretching; two peaks at 3076 cm1 and 961 cm1 were due to AHC@CHA; two peaks at 2930 cm1 and 1418 cm1 were due to ACH2A; and the two peaks at 2870 and 1382 cm1 were due to CAH stretching from ACH3. The proposed structure was found consistent with the one identified by NMR. 3.7. Degradation kinetics of sulforaphene of different water contents The stability of sulforaphene was affected by the moisture content. The water content of the sulforaphene product after freezedrying was 3.447 lg/mL. A model (Eq. (1)) based on the sulforaphene degradation with different water contents was proposed. According to Fig. 6a, it was found that the degradation of sulforaphene with different water content all fit first-order kinetics with a good linearity (R2 = 0.9599–0.9995). The degradation rate constants k were 0.047, 0.086, 0.111 and 0.263 for sulforaphene
Fig. 6. The degradation kinetics of sulforaphene with different water content (a) and the correlation between water content of SFE and the degradation rate constant (b) at the temperature of 26 °C.
degradation under water contents of 0.35%, 10%, 50% and 99.8% respectively. Our findings indicated that that the higher the water content was, the faster the sulforaphene degradation occurred. By plotting the degradation rate constants as a function of water contents, we have found a linear relationship between this two constants (Fig. 6b). The coefficients of x were inversely proportional to the degradation rate constant k. The correlation at 26 °C was described by the equation y = 0.0021x 0.04 (R2 = 0.9093), in which x represented the water content of sulforaphene sample, and y represented the degradation rate constant. Obviously, there is a strong correlation between the degradation rate and the water contents. It suggests that the sulforaphene degradation may be caused by the presence of water which initiated the degradation process. This was consistent with the degradation pathway that we proposed in Fig. 4. The sulforaphene was attacked by the hydroxyl groups of water leading to a dimer formation, which is in accordance with the degradation pathway of SFN (Jin et al., 1999) and allyl-isothiocyanate in water (Chen & Ho, 1998; Kawakishi & Namiki, 1969). 4. Conclusion In this study, 4 °C and 20 °C were found to be the optimal storage temperatures for sulforaphene. The degradation product
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of sulforaphene during storage was found to be its dimer. This product was formed by the interaction between AN@C@S groups on sulforaphene and ANH2 groups which was formed by nucleophilic attack of the hydroxyl groups in water. We found a strong correlation between the degradation rate constant and the water contents. The model we developed can be applied to predict the residual rates of sulforaphene in different water contents during storage. This study provided useful information for sulforaphene storage for industrial application. Acknowledgment The authors would like to acknowledge the financial support of the National Natural Science Foundation of China (No. 21376017 and 21176018). References Ambrosone, C. B., McCann, S. E., Freudenheim, J. L., Marshall, J. R., Zhang, Y., & Shields, P. G. (2004). Breast cancer risk in premenopausal women is inversely associated with consumption of broccoli, a source of isothiocyanates, but is not modified by GST genotype. Journal of Nutrition, 134, 1134–1138. Block, G., Patterson, B., & Subar, A. (1992). Fruit, vegetables, and cancer prevention: A review of the epidemiological evidence. Nutrition and Cancer, 18, 1–29. Chan, J. M., Wang, F., & Holly, E. A. (2005). Vegetable and fruit intake and pancreatic cancer in a population-based case-control study in the San Francisco bay area. Cancer Epidemiology Biomarkers & Prevention, 14, 2093–2097. Chen, C., & Ho, C. (1998). Thermal degradation of allyl isothiocyanate in aqueous solution. Journal of Agricultural and Food Chemistry, 48, 220–223. Hanschen, Franziska S., Lamy, E., Schreiner, M., & Rohn, S. (2014). Reactivity and stability of glucosinolates and their breakdown products in foods. Angewandte Chemie International Edition, 53, 11430–11450. Heimler, D., Vignolini, P., Dini, M. G., Vincieri, F. F., & Romani, A. (2006). Antiradical activity and polyphenol composition of local Brassicaceae edible varieties. Food Chemistry, 99, 464–469. Higdon, J. V., Delage, B., Williams, D. E., & Dashwood, R. H. (2007). Cruciferous vegetables and human cancer risk: Epidemiologic evidence and mechanistic basis. Phamacological Research, 55, 224–236. Holst, B., & Williamson, G. (2004). A critical review of the bioavailability of glucosinolates and related compounds. Natural Product Reports, 21, 425–447. Jin, Y., Wang, M. F., Rosen, Rosen T., & Ho, C. T. (1999). Thermal degradation of sulforaphane in aqueous solution. Journal of Agricultural and Food Chemistry, 47, 3121–3123. Joseph, M. A., Moysich, K. B., Freudenheim, J. L., Shiedlds, P. G., Bowman, E. D., & Zhang, Y. (2004). Cruciferous vegetables, genetic polymorphisms in glutathione S-transulforaphenerases M1 and T1, and prostate cancer risk. Nutrition and Cancer, 50, 206–213. Kawakishi, S., & Namiki, M. (1969). Decomposition of allyl isothiocyanate in aqueous solution. Agricultural and Biological Chemistry, 33, 452–459.
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